Sample records for gaba rho-1 subunit

  1. Brain distribution and molecular cloning of the bovine GABA rho1 receptor.

    PubMed

    Rosas-Arellano, Abraham; Ochoa-de la Paz, Lenin David; Miledi, Ricardo; Martínez-Torres, Ataúlfo

    2007-03-01

    GABA(C) receptors were originally found in the mammalian retina and recent evidence shows that they are also expressed in several areas of the brain, including caudate nucleus, brain stem, pons and corpus callosum. In this study, plasma membranes from the caudate nucleus were microinjected into X. laevis oocytes. This led the oocyte plasma membrane to incorporate functional bicuculline-resistant, Cl(-) conducting bovine GABA receptors, similar to those of the retina. Immunolocalization of the GABA rho1 subunit revealed its expression in bovine neurons in the head of the caudate as well as in the olive, cuneiform and reticular nuclei of the brain stem. The same antibodies failed to show expression in the callosum and pons, where the GABA rho1 mRNA was previously detected. The cloned GABA rho1 sequence predicts a protein with 473 amino acids and 74-93% similarity to other GABA rho1 subunits. Oocytes injected with the cDNA express a non-desensitizing, homomeric receptor with a GABA EC(50)=6.0 microM and a Hill coefficient of 1.8. The results confirm the presence of GABA(C) receptor mRNAs in several areas of the mammalian brain and show that some of these areas express functional GABA rho1 receptors that have the classic GABA(C) receptor characteristics.

  2. Evidence that GABA ρ subunits contribute to functional ionotropic GABA receptors in mouse cerebellar Purkinje cells

    PubMed Central

    Harvey, Victoria L; Duguid, Ian C; Krasel, Cornelius; Stephens, Gary J

    2006-01-01

    Ionotropic γ-amino butyric acid (GABA) receptors composed of heterogeneous molecular subunits are major mediators of inhibitory responses in the adult CNS. Here, we describe a novel ionotropic GABA receptor in mouse cerebellar Purkinje cells (PCs) using agents reported to have increased affinity for ρ subunit-containing GABAC over other GABA receptors. Exogenous application of the GABAC-preferring agonist cis-4-aminocrotonic acid (CACA) evoked whole-cell currents in PCs, whilst equimolar concentrations of GABA evoked larger currents. CACA-evoked currents had a greater sensitivity to the selective GABAC antagonist (1,2,5,6-tetrahydropyridin-4-yl)methylphosphinic acid (TPMPA) than GABA-evoked currents. Focal application of agonists produced a differential response profile; CACA-evoked currents displayed a much more pronounced attenuation with increasing distance from the PC soma, displayed a slower time-to-peak and exhibited less desensitization than GABA-evoked currents. However, CACA-evoked currents were also completely blocked by bicuculline, a selective agent for GABAA receptors. Thus, we describe a population of ionotropic GABA receptors with a mixed GABAA/GABAC pharmacology. TPMPA reduced inhibitory synaptic transmission at interneurone–Purkinje cell (IN–PC) synapses, causing clear reductions in miniature inhibitory postsynaptic current (mIPSC) amplitude and frequency. Combined application of NO-711 (a selective GABA transporter subtype 1 (GAT-1) antagonist) and SNAP-5114 (a GAT-(2)/3/4 antagonist) induced a tonic GABA conductance in PCs; however, TPMPA had no effect on this current. Immunohistochemical studies suggest that ρ subunits are expressed predominantly in PC soma and proximal dendritic compartments with a lower level of expression in more distal dendrites; this selective immunoreactivity contrasted with a more uniform distribution of GABAA α1 subunits in PCs. Finally, co-immunoprecipitation studies suggest that ρ subunits can form complexes

  3. Expression of gamma-aminobutyric acid rho 1 and rho 1 Delta 450 as gene fusions with the green fluorescent protein.

    PubMed

    Martinez-Torres, A; Miledi, R

    2001-02-13

    The functional characteristics and cellular localization of the gamma aminobutyric acid (GABA) rho 1 receptor and its nonfunctional isoform rho 1 Delta 450 were investigated by expressing them as gene fusions with the enhanced version of the green fluorescent protein (GFP). Oocytes injected with rho 1-GFP had receptors that gated chloride channels when activated by GABA. The functional characteristics of these receptors were the same as for those of wild-type rho 1 receptors. Fluorescence, because of the chimeric receptors expressed, was over the whole oocyte but was more intense near the cell surface and more abundant in the animal hemisphere. Similar to the wild type, rho 1 Delta 450-GFP did not lead to the expression of functional GABA receptors, and injected oocytes failed to generate currents even after exposure to high concentrations of GABA. Nonetheless, the fluorescence displayed by oocytes expressing rho 1 Delta 450-GFP was distributed similarly to that of rho 1-GFP. Mammalian cells transfected with the rho 1-GFP or rho 1 Delta 450-GFP constructs showed mostly intracellularly distributed fluorescence in confocal microscope images. A sparse localization of fluorescence was observed in the plasma membrane regardless of the cell line used. We conclude that rho 1 Delta 450 is expressed and transported close to, and perhaps incorporated into, the plasma membrane. Thus, rho 1- and rho 1 Delta 450-GFP fusions provide a powerful tool to visualize the traffic of GABA type C receptors.

  4. Structural determinants for antagonist pharmacology that distinguish the rho1 GABAC receptor from GABAA receptors.

    PubMed

    Zhang, Jianliang; Xue, Fenqin; Chang, Yongchang

    2008-10-01

    GABA receptor (GABAR) types C (GABACR) and A (GABAAR) are both GABA-gated chloride channels that are distinguished by their distinct competitive antagonist properties. The structural mechanism underlying these distinct properties is not well understood. In this study, using previously identified binding residues as a guide, we made individual or combined mutations of nine binding residues in the rho1 GABACR subunit to their counterparts in the alpha1beta2gamma2 GABAAR or reverse mutations in alpha1 or beta2 subunits. The mutants were expressed in Xenopus laevis oocytes and tested for sensitivities of GABA-induced currents to the GABAA and GABAC receptor antagonists. The results revealed that bicuculline insensitivity of the rho1 GABACR was mainly determined by Tyr106, Phe138 and Phe240 residues. Gabazine insensitivity of the rho1 GABACR was highly dependent on Tyr102, Tyr106, and Phe138. The sensitivity of the rho1 GABACR to 3-aminopropyl-phosphonic acid and its analog 3-aminopropyl-(methyl)phosphinic acid mainly depended on residues Tyr102, Val140, FYS240-242, and Phe138. Thus, the residues Tyr102, Tyr106, Phe138, and Phe240 in the rho1 GABACR are major determinants for its antagonist properties distinct from those in the GABAAR. In addition, Val140 in the GABACR contributes to 3-APA binding. In conclusion, we have identified the key structural elements underlying distinct antagonist properties for the GABACR. The mechanistic insights were further extended and discussed in the context of antagonists docking to the homology models of GABAA or GABAC receptors.

  5. Decreased agonist sensitivity of human GABA(A) receptors by an amino acid variant, isoleucine to valine, in the alpha1 subunit.

    PubMed

    Westh-Hansen, S E; Rasmussen, P B; Hastrup, S; Nabekura, J; Noguchi, K; Akaike, N; Witt, M R; Nielsen, M

    1997-06-25

    Recombinant human GABA(A) receptors were investigated in vitro by coexpression of cDNAs coding for alpha1, beta2, and gamma2 subunits in the baculovirus/Sf-9 insect cell system. We report that a single amino acid exchange (isoleucine 121 to valine 121) in the N-terminal, extracellular part of the alpha1 subunit induces a marked decrease in agonist GABA(A) receptor ligand sensitivity. The potency of muscimol and GABA to inhibit the binding of the GABA(A) receptor antagonist [3H]SR 95531 (2-(3-carboxypropyl)-3-amino-6-(4-methoxyphenyl)pyridazinium bromide) was higher in receptor complexes of alpha1(ile 121) beta2gamma2 than in those of alpha1(val 121) beta2gamma2 (IC50 values were 32-fold and 26-fold lower for muscimol and GABA, respectively). The apparent affinity of the GABA(A) receptor antagonist bicuculline methiodide to inhibit the binding of [3H]SR 95531 did not differ between the two receptor complex variants. Electrophysiological measurements of GABA induced whole-cell Cl- currents showed a ten-fold decrease in the GABA(A) receptor sensitivity of alpha1 (val 121) beta2gamma2 as compared to alpha1(ile 121) beta2gamma2 receptor complexes. Thus, a relatively small change in the primary structure of the alpha1 subunit leads to a decrease selective for GABA(A) receptor sensitivity to agonist ligands, since no changes were observed in a GABA(A) receptor antagonist affinity and benzodiazepine receptor binding.

  6. Linking GABA(A) receptor subunits to alcohol-induced conditioned taste aversion and recovery from acute alcohol intoxication.

    PubMed

    Blednov, Y A; Benavidez, J M; Black, M; Chandra, D; Homanics, G E; Rudolph, U; Harris, R A

    2013-04-01

    GABA type A receptors (GABA(A)-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABA(A)-R subunits1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011). All together, they indicate that aversive property of ethanol is dependent on ethanol action on α2-containing GABA(A)-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABA(A)-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 (-/-) and α3 (-/Y) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. Copyright © 2012 Elsevier Ltd. All rights reserved.

  7. Fragrant dioxane derivatives identify beta1-subunit-containing GABAA receptors.

    PubMed

    Sergeeva, Olga A; Kletke, Olaf; Kragler, Andrea; Poppek, Anja; Fleischer, Wiebke; Schubring, Stephan R; Görg, Boris; Haas, Helmut L; Zhu, Xin-Ran; Lübbert, Hermann; Gisselmann, Günter; Hatt, Hanns

    2010-07-30

    Nineteen GABA(A) receptor (GABA(A)R) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of beta1-subunit-containing GABA(A)Rs is unknown. Here we report the discovery of a new structural class of GABA(A)R positive modulators with unique beta1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed alpha1betaxgamma2L (x-for 1,2,3) GABA(A)R FDD were 6 times more potent at beta1- versus beta2- and beta3-containing receptors. Serine at position 265 was essential for the high sensitivity of the beta1-subunit to FDD and the beta1N286W mutation nearly abolished modulation; vice versa the mutation beta3N265S shifted FDD sensitivity toward the beta1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to beta1-negative cerebellar Purkinje neurons. Immunostaining for the beta1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by beta1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of beta1-containing GABA(A)Rs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABA(A)Rs.

  8. Proton modulation of recombinant GABAA receptors: influence of GABA concentration and the β subunit TM2–TM3 domain

    PubMed Central

    Wilkins, Megan E; Hosie, Alastair M; Smart, Trevor G

    2005-01-01

    Regulation of GABAA receptors by extracellular pH exhibits a dependence on the receptor subunit composition. To date, the molecular mechanism responsible for the modulation of GABAA receptors at alkaline pH has remained elusive. We report here that the GABA-activated current can be potentiated at pH 8.4 for both αβ and αβγ subunit-containing receptors, but only at GABA concentrations below the EC40. Site-specific mutagenesis revealed that a single lysine residue, K279 in the β subunit TM2–TM3 linker, was critically important for alkaline pH to modulate the function of both α1β2 and α1β2γ2 receptors. The ability of low concentrations of GABA to reveal different pH titration profiles for GABAA receptors was also examined at acidic pH. At pH 6.4, GABA activation of αβγ receptors was enhanced at low GABA concentrations. This effect was ablated by the mutation H267A in the β subunit. Decreasing the pH further to 5.4 inhibited GABA responses via αβγ receptors, whereas those responses recorded from αβ receptors were potentiated. Inserting homologous β subunit residues into the γ2 subunit to recreate, in αβγ receptors, the proton modulatory profile of αβ receptors, established that in the presence of β2H267, the mutation γ2T294K was necessary to potentiate the GABA response at pH 5.4. This residue, T294, is homologous to K279 in the β subunit and suggests that a lysine at this position is an important residue for mediating the allosteric effects of both acidic and alkaline pH changes, rather than forming a direct site for protonation within the GABAA receptor. PMID:15946973

  9. Gamma-aminobutyric acid (GABA) stimulates pancreatic cancer growth through overexpressing GABAA receptor pi subunit.

    PubMed

    Takehara, Akio; Hosokawa, Masayo; Eguchi, Hidetoshi; Ohigashi, Hiroaki; Ishikawa, Osamu; Nakamura, Yusuke; Nakagawa, Hidewaki

    2007-10-15

    Gamma-aminobutyric acid (GABA) functions primarily as an inhibitory neurotransmitter in the mature central nervous system, and GABA/GABA receptors are also present in nonneural tissues, including cancer, but their precise function in nonneuronal or cancerous cells has thus far been poorly defined. Through the genome-wide cDNA microarray analysis of pancreatic ductal adenocarcinoma (PDAC) cells as well as subsequent reverse transcription-PCR and Northern blot analyses, we identified the overexpression of GABA receptor pi subunit (GABRP) in PDAC cells. We also found the expression of this peripheral type GABAA receptor subunit in few adult human organs. Knockdown of endogenous GABRP expression in PDAC cells by small interfering RNA attenuated PDAC cell growth, suggesting its essential role in PDAC cell viability. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRP-expressing PDAC cells, but not GABRP-negative cells, and GABAA receptor antagonists inhibited this growth-promoting effect by GABA. The HEK293 cells constitutively expressing exogenous GABRP revealed the growth-promoting effect of GABA treatment. Furthermore, GABA treatment in GABRP-positive cells increased intracellular Ca2+ levels and activated the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/Erk) cascade. Clinical PDAC tissues contained a higher level of GABA than normal pancreas tissues due to the up-regulation of glutamate decarboxylase 1 expression, suggesting their autocrine/paracrine growth-promoting effect in PDACs. These findings imply that GABA and GABRP could play important roles in PDAC development and progression, and that this pathway can be a promising molecular target for the development of new therapeutic strategies for PDAC.

  10. d Subunit-Containing GABA[subscript A] Receptor Prevents Overgeneralization of Fear in Adult Mice

    ERIC Educational Resources Information Center

    Zhang, Wen-Hua; Zhou, Jin; Pan, Han-Qing; Wang, Xiao-Yang; Liu, Wei-Zhu; Zhang, Jun-Yu; Yin, Xiao-Ping; Pan, Bing-Xing

    2017-01-01

    The role of d subunit-containing GABA[subscript A] receptor (GABA[subscript A](d)R) in fear generalization is uncertain. Here, by using mice with or without genetic deletion of GABA[subscript A](d)R and using protocols in which the conditioned tone stimuli were cross presented with different nonconditioned stimuli, we observed that when the two…

  11. Effect of progesterone on the expression of GABA(A) receptor subunits in the prefrontal cortex of rats: implications of sex differences and brain hemisphere.

    PubMed

    Andrade, Susie; Arbo, Bruno D; Batista, Bruna A M; Neves, Alice M; Branchini, Gisele; Brum, Ilma S; Barros, Helena M T; Gomez, Rosane; Ribeiro, Maria Flavia M

    2012-12-01

    Progesterone is a neuroactive hormone with non-genomic effects on GABA(A) receptors (GABA(A)R). Changes in the expression of GABA(A)R subunits are related to depressive-like behaviors in rats. Moreover, sex differences and depressive behaviors have been associated with prefrontal brain asymmetry in rodents and humans. Thus, our objective was to investigate the effect of progesterone on the GABA(A)R α1 and γ2 subunits mRNA expression in the right and left prefrontal cortex of diestrus female and male rats exposed to the forced swimming test (FST). Male and female rats (n = 8/group) were randomly selected to receive a daily dose of progesterone (0·4 mg·kg⁻¹) or vehicle, during two complete female estrous cycles (8-10 days). On the experiment day, male rats or diestrus female rats were euthanized 30 min after the FST. Our results showed that progesterone significantly increased the α1 subunit mRNA in both hemispheres of male and female rats. Moreover, there was an inverse correlation between depressive-like behaviors and GABA(A)R α1 subunit mRNA expression in the right hemisphere in female rats. Progesterone decreased the GABA(A)R γ2 mRNA expression only in the left hemisphere of male rats. Therefore, we conclude that the GABA(A) system displays an asymmetric distribution according to sex and that progesterone, at lower doses, presents an antidepressant effect after increasing the GABA(A) R α1 subunit expression in the right prefrontal cortex of female rats. Copyright © 2012 John Wiley & Sons, Ltd.

  12. Expression of specific ionotropic glutamate and GABA-A receptor subunits is decreased in central amygdala of alcoholics.

    PubMed

    Jin, Zhe; Bhandage, Amol K; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R; Birnir, Bryndis

    2014-01-01

    The central amygdala (CeA) has a role for mediating fear and anxiety responses. It is also involved in emotional imbalance caused by alcohol abuse and dependence and in regulating relapse to alcohol abuse. Growing evidences suggest that excitatory glutamatergic and inhibitory γ-aminobutyric acid-ergic (GABAergic) transmissions in the CeA are affected by chronic alcohol exposure. Human post-mortem CeA samples from male alcoholics (n = 9) and matched controls (n = 9) were assayed for the expression level of ionotropic glutamate and GABA-A receptors subunit mRNAs using quantitative real-time reverse transcription-PCR (RT-qPCR). Our data revealed that out of the 16 ionotropic glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA1 and GluA4; one kainate receptor subunit GluK2; one NMDA (N-methyl-D-aspartate) receptor subunit GluN2D and one delta receptor subunit GluD2 were significantly decreased in the CeA of alcoholics. In contrast, of the 19 GABA-A receptor subunits, only the mRNA encoding the α2 subunit was significantly down-regulated in the CeA of the alcoholics as compared with control subjects. Our findings imply that the down-regulation of specific ionotropic glutamate and GABA-A receptor subunits in the CeA of alcoholics may represent one of the molecular substrates underlying the new balance between excitatory and inhibitory neurotransmission in alcohol dependence.

  13. Expression of specific ionotropic glutamate and GABA-A receptor subunits is decreased in central amygdala of alcoholics

    PubMed Central

    Jin, Zhe; Bhandage, Amol K.; Bazov, Igor; Kononenko, Olga; Bakalkin, Georgy; Korpi, Esa R.; Birnir, Bryndis

    2014-01-01

    The central amygdala (CeA) has a role for mediating fear and anxiety responses. It is also involved in emotional imbalance caused by alcohol abuse and dependence and in regulating relapse to alcohol abuse. Growing evidences suggest that excitatory glutamatergic and inhibitory γ-aminobutyric acid-ergic (GABAergic) transmissions in the CeA are affected by chronic alcohol exposure. Human post-mortem CeA samples from male alcoholics (n = 9) and matched controls (n = 9) were assayed for the expression level of ionotropic glutamate and GABA-A receptors subunit mRNAs using quantitative real-time reverse transcription-PCR (RT-qPCR). Our data revealed that out of the 16 ionotropic glutamate receptor subunits, mRNAs encoding two AMPA [2-amino-3-(3-hydroxy-5-methyl-isoxazol-4-yl)propanoic acid] receptor subunits GluA1 and GluA4; one kainate receptor subunit GluK2; one NMDA (N-methyl-D-aspartate) receptor subunit GluN2D and one delta receptor subunit GluD2 were significantly decreased in the CeA of alcoholics. In contrast, of the 19 GABA-A receptor subunits, only the mRNA encoding the α2 subunit was significantly down-regulated in the CeA of the alcoholics as compared with control subjects. Our findings imply that the down-regulation of specific ionotropic glutamate and GABA-A receptor subunits in the CeA of alcoholics may represent one of the molecular substrates underlying the new balance between excitatory and inhibitory neurotransmission in alcohol dependence. PMID:25278838

  14. γ-Amino-butyric acid (GABA) receptor subunit and transporter expression in the gonad and liver of the fathead minnow (Pimephales promelas).

    PubMed

    Biggs, Katie; Seidel, Jason S; Wilson, Alex; Martyniuk, Christopher J

    2013-09-01

    γ-Amino-butyric acid (GABA) is the major inhibitory neurotransmitter in the vertebrate central nervous system. GABA receptors and synthesizing enzymes have also been localized to peripheral tissues including the liver, oviduct, uterus and ovary of mammals but the distribution and role of GABA in peripheral tissues of fish has not been fully investigated. The objectives of this study were to (1) determine if mRNA encoding GABA synthesizing enzymes (glutamic acid decarboxylase 65 and 67; gad65 and gad67), GABA transporters, and GABAA receptor subunits are localized to liver and gonad of fathead minnow (Pimephales promelas) (FHM) (2) investigate the effects of GABA on ovarian 17β-estradiol (E2) production, and (3) measure transcript responses in the ovary after in vitro incubation to GABA. Real-time PCR assays were developed for gad65, gad67, vesicular GABA transporter (vgat) and GABA transporter 1 (gat1), and select GABAA receptor subunits (gabra1, gabra5, gabrb1, gabrb2, gabrg1, gabrg2). All transcripts were localized to the brain as expected; however transcripts were also detected in the liver, ovary, and testis of FHMs. In the female liver, gad65 mRNA was significantly higher in expression compared to the male liver. Transcripts for gad67 were the highest in the brain>gonad>liver and in the gonads, gad67 was significantly higher in expression than gad65 mRNA. In the liver and gonad, the relative abundance of the subunits followed a general trend of gabrb1>gabrb2=gabrg1=gabrg2>gabra1=gabra5. To explore the effects of GABA in the ovary, tissue explants from reproductive female FHMs were treated with GABA (10(-10), 10(-8) and 10(-6)M) for 12h. GABA had no significant effect on 17β-estradiol production or on mRNA abundance for genes involved in ovarian steroidogenesis (e.g., 11βhsd, cyp17, cyp19a). There was a significant decrease in estrogen receptor 2a (esr2a) mRNA with 10(-10)M GABA. This study begins to investigate the GABA system in non-neural tissues of

  15. Non-neuronal, slow GABA signalling in the ventrobasal thalamus targets δ-subunit-containing GABAA receptors

    PubMed Central

    Jiménez-González, Cristina; Pirttimaki, Tiina; Cope, David W; Parri, H R

    2011-01-01

    The rodent ventrobasal (VB) thalamus contains a relatively uniform population of thalamocortical (TC) neurons that receive glutamatergic input from the vibrissae and the somatosensory cortex, and inhibitory input from the nucleus reticularis thalami (nRT). In this study we describe γ-aminobutyric acid (GABA)A receptor-dependent slow outward currents (SOCs) in TC neurons that are distinct from fast inhibitory postsynaptic currents (IPSCs) and tonic currents. SOCs occurred spontaneously or could be evoked by hypo-osmotic stimulus, and were not blocked by tetrodotoxin, removal of extracellular Ca2+ or bafilomycin A1, indicating a non-synaptic, non-vesicular GABA origin. SOCs were more common in TC neurons of the VB compared with the dorsal lateral geniculate nucleus, and were rarely observed in nRT neurons, whilst SOC frequency in the VB increased with age. Application of THIP, a selective agonist at δ-subunit-containing GABAA receptors, occluded SOCs, whereas the benzodiazepine site inverse agonist β-CCB had no effect, but did inhibit spontaneous and evoked IPSCs. In addition, the occurrence of SOCs was reduced in mice lacking the δ-subunit, and their kinetics were also altered. The anti-epileptic drug vigabatrin increased SOC frequency in a time-dependent manner, but this effect was not due to reversal of GABA transporters. Together, these data indicate that SOCs in TC neurons arise from astrocytic GABA release, and are mediated by δ-subunit-containing GABAA receptors. Furthermore, these findings suggest that the therapeutic action of vigabatrin may occur through the augmentation of this astrocyte–neuron interaction, and highlight the importance of glial cells in CNS (patho) physiology. PMID:21395866

  16. GABA stimulates human hepatocellular carcinoma growth through overexpressed GABAA receptor theta subunit

    PubMed Central

    Li, Yue-Hui; Liu, Yan; Li, Yan-Dong; Liu, Yan-Hong; Li, Feng; Ju, Qiang; Xie, Ping-Li; Li, Guan-Cheng

    2012-01-01

    AIM: To investigate the function of gamma-aminobutyric acid (GABA) and gamma-aminobutyric acid A receptor θ subunit (GABRQ) in hepatocellular carcinoma (HCC). METHODS: Semiquantitative polymerase chain reaction was used for detecting the expression of GABRQ receptor among HCC cell line HepG2, normal liver cell line L-02, non-malignant Chang’s liver cells, 8 samples of HCC tissues and paired non-cancerous tissues. HepG2 cells were treated with GABA at serial concentrations (0, 1, 10, 20, 40 and 60 μmol/L), and their proliferating abilities were analyzed with the methyl thiazolyl tetrazolium assay, cell cycle analysis and tumor implanted in nude mice. Small interfering RNA was used for knocking down the endogenous GABRQ in HepG2. Proliferating abilities of these cells treated with or without GABA were analyzed. RESULTS: We identified the overexpression of GABRQ in HCC cell lines and half of the tested HCC tissues. Knockdown of endogenous GABRQ expression in HepG2 attenuated HCC cell growth, suggesting its role in HCC cell viability. We studied the effect of GABA in the proliferation of GABRQ-positive cell lines in vitro and in vivo, and found that GABA increased HCC growth in a dose-dependent manner. Notably, the addition of GABA into the cell culture medium promoted the proliferation of GABRQ-expressing HepG2 cells, but not GABRQ-knockdown HepG2 cells, which means that GABA stimulates HepG2 cell growth through GABRQ. CONCLUSION: GABRQ play important roles in HCC development and progression and could be a promising molecular target for the development of new diagnostic and therapeutic strategies of HCC. PMID:22690081

  17. The morphological and chemical characteristics of striatal neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the rat.

    PubMed

    Waldvogel, H J; Kubota, Y; Trevallyan, S C; Kawaguchi, Y; Fritschy, J M; Mohler, H; Faull, R L

    1997-10-01

    The distribution, morphology and chemical characteristics of neurons immunoreactive for the alpha1-subunit of the GABA(A) receptor in the striatum of the basal ganglia in the rat brain were investigated at the light, confocal and electron microscope levels using single, double and triple immunohistochemical labelling techniques. The results showed that alpha1-subunit immunoreactive neurons were sparsely distributed throughout the rat striatum. Double and triple labelling results showed that all the alpha1-subunit-immunoreactive neurons were positive for glutamate decarboxylase and immunoreactive for the beta2,3 and gamma2 subunits of the GABA(A) receptor. Three types of alpha1-subunit-immunoreactive neurons were identified in the striatum on the basis of cellular morphology and chemical characteristics. The most numerous alpha1-subunit-immunoreactive neurons were medium-sized, aspiny neurons with a widely branching dendritic tree. They were parvalbumin-negative and were located mainly in the dorsolateral regions of the striatum. Electron microscopy showed that these neurons had an indented nuclear membrane, typical of striatal interneurons, and were surrounded by small numbers of axon terminals which established alpha1-subunit-immunoreactive synaptic contacts with the soma and dendrites. These cells were classified as type 1 alpha1-subunit-immunoreactive neurons and comprised 75% of the total population of alpha1-subunit-immunoreactive neurons in the striatum. The remaining alpha1-subunit-immunoreactive neurons comprised of a heterogeneous population of large-sized neurons localized in the ventral and medial regions of the striatum. The most numerous large-sized cells were parvalbumin-negative, had two to three relatively short branching dendrites and were designated type 2 alpha1-subunit-immunoreactive neurons. Electron microscopy showed that the type 2 neurons were characterized by a highly convoluted nuclear membrane and were sparsely covered with small axon

  18. Downregulation of GABA[Subscript A] Receptor Protein Subunits a6, ß2, d, e, ?2, ?, and ?2 in Superior Frontal Cortex of Subjects with Autism

    ERIC Educational Resources Information Center

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rustan, Oyvind G.; Rooney, Robert J.; Thuras, Paul D.

    2014-01-01

    We measured protein and mRNA levels for nine gamma-aminobutyric acid A (GABA[subscript A]) receptor subunits in three brain regions (cerebellum, superior frontal cortex, and parietal cortex) in subjects with autism versus matched controls. We observed changes in mRNA for a number of GABA[subscript A] and GABA[subscript B] subunits and overall…

  19. GABA(C) receptors: a molecular view.

    PubMed

    Enz, R

    2001-08-01

    In the central nervous system inhibitory neurotransmission is primarily achieved through activation of receptors for gamma-aminobutyric acid (GABA). Three types of GABA receptors have been identified on the basis of their pharmacological and electrophysiological properties. The predominant type, termed GABA(A), and a recently identified GABA(C) type, form ligand-gated chloride channels, whereas GABA(B) receptors activate separate cation channels via G proteins. Based on their homology to nicotinic acetylcholine receptors, GABA(C) receptors are believed to be oligomeric protein complexes composed of five subunits in a pentameric arrangement. To date up to five different GABA(C) receptors subunits have been identified in various species. Recent studies have shed new light on the biological characteristics of GABA(C) receptors, including the chromosomal localization of its subunit genes and resulting links to deseases, the cloning of new splice variants, the identification of GABA(C) receptor-associated proteins, the identification of domains involved in subunit assembly, and finally structure/function studies examining functional consequences of introduced mutations. This review summarizes recent data in view of the molecular structure of GABA(C) receptors and presents new insights into the biological function of this protein in the retina.

  20. Identification and cloning of a gamma 3 subunit splice variant of the human GABA(A) receptor.

    PubMed

    Poulsen, C F; Christjansen, K N; Hastrup, S; Hartvig, L

    2000-05-31

    cDNA sequences encoding two forms of the GABA(A) gamma 3 receptor subunit were cloned from human hippocampus. The nucleotide sequences differ by the absence (gamma 3S) or presence (gamma 3L) of 18 bp located in the presumed intracellular loop between transmembrane region (TM) III and IV. The extra 18 bp in the gamma 3L subunit generates a consensus site for phosphorylation by protein kinase C (PKC). Analysis of human genomic DNA encoding the gamma 3 subunit reveals that the 18 bp insert is contiguous with the upstream proximal exon.

  1. Somato-synaptic variation of GABA(A) receptors in cultured murine cerebellar granule cells: investigation of the role of the alpha6 subunit.

    PubMed

    Mellor, J R; Wisden, W; Randall, A D

    2000-07-10

    Electrophysiological investigation of cultured cerebellar murine granule cells revealed differences between the GABA(A) receptors at inhibitory synapses and those on the cell body. Specifically, mIPSCs decayed more rapidly than cell body receptors deactivated, the mean single channel conductance at the synapse (32 pS) was greater than that at cell body (21 pS) and only cell body receptors were sensitive to Zn(2+) (150 microM), which depressed response amplitude by 82+/-5% and almost doubled the rate of channel deactivation. The GABA(A) receptor alpha6 subunit is selectively expressed in cerebellar granule cells. Although concentrated at synapses, it is also found on extrasynaptic membranes. Using a mouse line (Deltaalpha6lacZ) lacking this subunit, we investigated its role in the somato-synaptic differences in GABA(A) receptor function. All differences between cell body and synaptic GABA(A) receptors observed in wild-type (WT) granule cells persisted in Deltaalpha6lacZ cells, thus demonstrating that they are not specifically due to the cellular distribution of the alpha6 subunit. However, mIPSCs from WT and Deltaalpha6lacZ cells differed in both their kinetics (faster decay in WT cells) and underlying single channel conductance (32 pS WT, 25 pS Deltaalpha6lacZ). This provides good evidence for a functional contribution of the alpha6 subunit to postsynaptic GABA(A) receptors in these cells. Despite this, deactivation kinetics of mIPSCs in WT and Deltaalpha6lacZ granule cells exhibited similar benzodiazepene (BDZ) sensitivity. This suggests that the enhanced BDZ-induced ataxia seen in Deltaalpha6lacZ mice may reflect physiological activity at extrasynaptic receptors which, unlike those at synapses, display differential BDZ-sensitivity in WT and Deltaalpha6lacZ granule cells (Jones, A.M., Korpi, E.R., McKernan, R.M., Nusser, Z., Pelz, R., Makela, R., Mellor, J.R., Pollard, S., Bahn, S., Stephenson, F.A., Randall, A.D., Sieghart, W., Somogyi, P., Smith, A.J.H., Wisden

  2. Immunocytochemical Mapping of an RDL-Like GABA Receptor Subunit and of GABA in Brain Structures Related to Learning and Memory in the Cricket Acheta domesticus

    PubMed Central

    Strambi, Colette; Cayre, Myriam; Sattelle, David B.; Augier, Roger; Charpin, Pierre; Strambi, Alain

    1998-01-01

    The distribution of putative RDL-like GABA receptors and of γ-aminobutyric acid (GABA) in the brain of the adult house cricket Acheta domesticus was studied using specific antisera. Special attention was given to brain structures known to be related to learning and memory. The main immunostaining for the RDL-like GABA receptor was observed in mushroom bodies, in particular the upper part of mushroom body peduncle and the two arms of the posterior calyx. Weaker immunostaining was detected in the distal part of the peduncle and in the α and β lobes. The dorso- and ventrolateral protocerebrum neuropils appeared rich in RDL-like GABA receptors. Staining was also detected in the glomeruli of the antennal lobe, as well as in the ellipsoid body of the central complex. Many neurons clustered in groups exhibit GABA-like immunoreactivity. Tracts that were strongly immunostained innervated both the calyces and the lobes of mushroom bodies. The glomeruli of the antennal lobe, the ellipsoid body, as well as neuropils of the dorso- and ventrolateral protocerebrum were also rich in GABA-like immuno- reactivity. The data demonstrated a good correlation between the distribution of the GABA-like and of the RDL-like GABA receptor immunoreactivity. The prominent distribution of RDL-like GABA receptor subunits, in particular areas of mushroom bodies and antennal lobes, underlines the importance of inhibitory signals in information processing in these major integrative centers of the insect brain. PMID:10454373

  3. Fragrant Dioxane Derivatives Identify β1-Subunit-containing GABAA Receptors*

    PubMed Central

    Sergeeva, Olga A.; Kletke, Olaf; Kragler, Andrea; Poppek, Anja; Fleischer, Wiebke; Schubring, Stephan R.; Görg, Boris; Haas, Helmut L.; Zhu, Xin-Ran; Lübbert, Hermann; Gisselmann, Günter; Hatt, Hanns

    2010-01-01

    Nineteen GABAA receptor (GABAAR) subunits are known in mammals with only a restricted number of functionally identified native combinations. The physiological role of β1-subunit-containing GABAARs is unknown. Here we report the discovery of a new structural class of GABAAR positive modulators with unique β1-subunit selectivity: fragrant dioxane derivatives (FDD). At heterologously expressed α1βxγ2L (x-for 1,2,3) GABAAR FDD were 6 times more potent at β1- versus β2- and β3-containing receptors. Serine at position 265 was essential for the high sensitivity of the β1-subunit to FDD and the β1N286W mutation nearly abolished modulation; vice versa the mutation β3N265S shifted FDD sensitivity toward the β1-type. In posterior hypothalamic neurons controlling wakefulness GABA-mediated whole-cell responses and GABAergic synaptic currents were highly sensitive to FDD, in contrast to β1-negative cerebellar Purkinje neurons. Immunostaining for the β1-subunit and the potency of FDD to modulate GABA responses in cultured hypothalamic neurons was drastically diminished by β1-siRNA treatment. In conclusion, with the help of FDDs we reveal a functional expression of β1-containing GABAARs in the hypothalamus, offering a new tool for studies on the functional diversity of native GABAARs. PMID:20511229

  4. Activation of RhoA by Lysophosphatidic Acid and Gα12/13 Subunits in Neuronal Cells: Induction of Neurite Retraction

    PubMed Central

    Kranenburg, Onno; Poland, Mieke; van Horck, Francis P. G.; Drechsel, David; Hall, Alan; Moolenaar, Wouter H.

    1999-01-01

    Neuronal cells undergo rapid growth cone collapse, neurite retraction, and cell rounding in response to certain G protein–coupled receptor agonists such as lysophosphatidic acid (LPA). These shape changes are driven by Rho-mediated contraction of the actomyosin-based cytoskeleton. To date, however, detection of Rho activation has been hampered by the lack of a suitable assay. Furthermore, the nature of the G protein(s) mediating LPA-induced neurite retraction remains unknown. We have developed a Rho activation assay that is based on the specific binding of active RhoA to its downstream effector Rho-kinase (ROK). A fusion protein of GST and the Rho-binding domain of ROK pulls down activated but not inactive RhoA from cell lysates. Using GST-ROK, we show that in N1E-115 neuronal cells LPA activates endogenous RhoA within 30 s, concomitant with growth cone collapse. Maximal activation occurs after 3 min when neurite retraction is complete and the actin cytoskeleton is fully contracted. LPA-induced RhoA activation is completely inhibited by tyrosine kinase inhibitors (tyrphostin 47 and genistein). Activated Gα12 and Gα13 subunits mimic LPA both in activating RhoA and in inducing RhoA-mediated cytoskeletal contraction, thereby preventing neurite outgrowth. We conclude that in neuronal cells, LPA activates RhoA to induce growth cone collapse and neurite retraction through a G12/13-initiated pathway that involves protein-tyrosine kinase activity. PMID:10359601

  5. Interaction of Myosin Phosphatase Target Subunit (MYPT1) with Myosin Phosphatase-RhoA Interacting Protein (MRIP): A Role of Glutamic Acids in the Interaction.

    PubMed

    Lee, Eunhee; Stafford, Walter F

    2015-01-01

    Scaffold proteins bind to and functionally link protein members of signaling pathways. Interaction of the scaffold proteins, myosin phosphatase target subunit (MYPT1) and myosin phosphatase-RhoA interacting protein (MRIP), causes co-localization of myosin phosphatase and RhoA to actomyosin. To examine biophysical properties of interaction of MYPT1 with MRIP, we employed analytical ultracentrifugation and surface plasmon resonance. In regard to MRIP, its residues 724-837 are sufficient for the MYPT1/MRIP interaction. Moreover, MRIP binds to MYPT1 as either a monomer or a dimer. With respect to MYPT1, its leucine repeat region, LR (residues 991-1030) is sufficient to account for the MYPT1/MRIP interaction. Furthermore, point mutations that replace glutamic acids 998-1000 within LR reduced the binding affinity toward MRIP. This suggests that the glutamic acids of MYPT1 play an important role in the interaction.

  6. Variation in GABA-A subunit gene copy number in an autistic patient with mosaic 4 p duplication (p12p16).

    PubMed

    Kakinuma, Hiroaki; Ozaki, Mamoru; Sato, Hitoshi; Takahashi, Hiroaki

    2008-09-05

    Autism has been associated with chromosomal aberrations, including duplications at chromosome 4, and the identification of genetic factors contributing to the etiology of this disease is the focus of much research. Here we report a Japanese girl with mosaic of chromosome 4p duplication, mos 46,XX,dup(4)(p12p16)[54]/46,XX[6], who was diagnosed with autism at 3 years of age. Fluorescence in situ hybridization (FISH) with probes covering the region spanning a cluster of the gamma aminobutyric acid A (GABA-A) receptor subunit genes in the proximal short arm of chromosome 4 demonstrated total three signals for the GABRG1, GABRA4, and GABRA2 genes, but only two signals for GABRB1. This suggests that aberrant copy number of the GABA-A receptor subunit genes may contribute to the etiology of autism in this patient. 2007 Wiley-Liss, Inc.

  7. Antiseizure Activity of Midazolam in Mice Lacking δ-Subunit Extrasynaptic GABA(A) Receptors.

    PubMed

    Reddy, Sandesh D; Younus, Iyan; Clossen, Bryan L; Reddy, Doodipala Samba

    2015-06-01

    Midazolam is a benzodiazepine anticonvulsant with rapid onset and short duration of action. Midazolam is the current drug of choice for acute seizures and status epilepticus, including those caused by organophosphate nerve agents. The antiseizure activity of midazolam is thought to result from its allosteric potentiation of synaptic GABA(A) receptors in the brain. However, there are indications that benzodiazepines promote neurosteroid synthesis via the 18-kDa cholesterol transporter protein (TSPO). Therefore, we investigated the role of neurosteroids and their extrasynaptic GABA(A) receptor targets in the antiseizure activity of midazolam. Here, we used δ-subunit knockout (DKO) mice bearing a targeted deletion of the extrasynaptic receptors to investigate the contribution of the extrasynaptic receptors to the antiseizure activity of midazolam using the 6-Hz and hippocampus kindling seizure models. In both models, midazolam produced rapid and dose-dependent protection against seizures (ED50, 0.4 mg/kg). Moreover, the antiseizure potency of midazolam was undiminished in DKO mice compared with control mice. Pretreatment with PK11195 [1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinolinecarboxamide], a TSPO blocker, or finasteride, a 5α-reductase neurosteroid inhibitor, did not affect the antiseizure effect of midazolam. The antiseizure activity of midazolam was significantly reversed by pretreatment with flumazenil, a benzodiazepine antagonist. Plasma and brain levels of the neurosteroid allopregnanolone were not significantly greater in midazolam-treated animals. These studies therefore provide strong evidence that neurosteroids and extrasynaptic GABA(A) receptors are not involved in the antiseizure activity of midazolam, which mainly occurs through synaptic GABA(A) receptors via direct binding to benzodiazepine sites. This study reaffirms midazolam's use for controlling acute seizures and status epilepticus. Copyright © 2015 by The American Society for

  8. L-type Calcium Channel Blockers Enhance Trafficking and Function of Epilepsy-associated α1(D219N) Subunits of GABA(A) Receptors.

    PubMed

    Han, Dong-Yun; Guan, Bo-Jhih; Wang, Ya-Juan; Hatzoglou, Maria; Mu, Ting-Wei

    2015-09-18

    Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory ion channels in the mammalian central nervous system and play an essential role in regulating inhibition-excitation balance in neural circuits. The α1 subunit harboring the D219N mutation of GABAA receptors was reported to be retained in the endoplasmic reticulum (ER) and traffic inefficiently to the plasma membrane, leading to a loss of function of α1(D219N) subunits and thus idiopathic generalized epilepsy (IGE). We present the use of small molecule proteostasis regulators to enhance the forward trafficking of α1(D219N) subunits to restore their function. We showed that treatment with verapamil (4 μM, 24 h), an L-type calcium channel blocker, substantially increases the α1(D219N) subunit cell surface level in both HEK293 cells and neuronal SH-SY5Y cells and remarkably restores the GABA-induced maximal chloride current in HEK293 cells expressing α1(D219N)β2γ2 receptors to a level that is comparable to wild type receptors. Our drug mechanism study revealed that verapamil treatment promotes the ER to Golgi trafficking of the α1(D219N) subunits post-translationally. To achieve that, verapamil treatment enhances the interaction between the α1(D219N) subunit and β2 subunit and prevents the aggregation of the mutant protein by shifting the protein from the detergent-insoluble fractions to detergent-soluble fractions. By combining (35)S pulse-chase labeling and MG-132 inhibition experiments, we demonstrated that verapamil treatment does not inhibit the ER-associated degradation of the α1(D219N) subunit. In addition, its effect does not involve a dynamin-1 dependent endocytosis. To gain further mechanistic insight, we showed that verapamil increases the interaction between the mutant protein and calnexin and calreticulin, two major lectin chaperones in the ER. Moreover, calnexin binding promotes the forward trafficking of the mutant subunit. Taken together, our data indicate that

  9. [Schizophrenia and cortical GABA neurotransmission].

    PubMed

    Hashimoto, Takanori; Matsubara, Takuro; Lewis, David A

    2010-01-01

    Individuals with schizophrenia show disturbances in a number of brain functions that regulate cognitive, affective, motor, and sensory processing. The cognitive deficits associated with dysfunction of the dorsolateral prefrontal cortex result, at least in part, from abnormalities in GABA neurotransmission, as reflected in a specific pattern of altered expression of GABA-related molecules. First, mRNA levels for the 67-kilodalton isoform of glutamic acid decarboxylase (GAD67), an enzyme principally responsible for GABA synthesis, and the GABA membrane transporter GAT1, which regulates the reuptake of synaptically released GABA, are decreased in a subset of GABA neurons. Second, affected GABA neurons include those that express the calcium-binding protein parvalbumin (PV), because PV mRNA levels are decreased in the prefrontal cortex of subjects with schizophrenia and GAD67 mRNA is undetectable in almost half of PV-containing neurons. These changes are accompanied by decreased GAT1 expression in the presynaptic terminals of PV-containing neurons and by increased postsynaptic GABA-A receptor alpha2 subunit expression at the axon initial segments of pyramidal neurons. These findings indicate decreased GABA synthesis/release by PV-containing GABA neurons and compensatory changes at synapses formed by these neurons. Third, another subset of GABA neurons that express the neuropeptide somatostatin (SST) also appear to be affected because their specific markers, SST and neuropeptide Y mRNAs, are decreased in a manner highly correlated with the decreases in GAD67 mRNA. Finally, mRNA levels for GABA-A receptor subunits for synaptic (alpha1 and gamma2) and extra-synaptic (delta) receptors are decreased, indicating alterations in both synaptic and extra-synaptic GABA neurotransmission. Together, this pattern of changes indicates that the altered GABA neurotransmission is specific to PV-containing and SST-containing GABA neuron subsets and involves both synaptic and extra

  10. Epigenetic regulation of dorsal raphe GABA(B1a) associated with isolation-induced abnormal responses to social stimulation in mice.

    PubMed

    Araki, Ryota; Hiraki, Yosuke; Nishida, Shoji; Kuramoto, Nobuyuki; Matsumoto, Kinzo; Yabe, Takeshi

    2016-02-01

    In isolation-reared mice, social encounter stimulation induces locomotor hyperactivity and activation of the dorsal raphe nucleus (DRN), suggesting that dysregulation of dorsal raphe function may be involved in abnormal behaviors. In this study, we examined the involvement of dorsal raphe GABAergic dysregulation in the abnormal behaviors of isolation-reared mice. We also studied an epigenetic mechanism underlying abnormalities of the dorsal raphe GABAergic system. Both mRNA and protein levels of GABA(B1a), a GABA(B) receptor subunit, were increased in the DRN of isolation-reared mice, compared with these levels in group-reared mice. In contrast, mRNA levels for other GABAergic system-related genes (GABA(A) receptor α1, β2 and γ2 subunits, GABA(B) receptor 1b and 2 subunits, and glutamate decarboxylase 67 and 65) were unchanged. Intra-DRN microinjection of 0.06 nmol baclofen (a GABA(B) receptor agonist) exacerbated encounter-induced hyperactivity and aggressive behavior, while microinjection of 0.3 nmol phaclofen (a GABA(B) receptor antagonist) attenuated encounter-induced hyperactivity and aggressive behavior in isolation-reared mice. Furthermore, microinjection of 0.06 nmol baclofen elicited encounter-induced hyperactivity in group-reared mice. Neither baclofen nor phaclofen affected immobility time in the forced swim test and hyperactivity in a novel environment of isolation reared mice. Bisulfite sequence analyses revealed that the DNA methylation level of the CpG island around the transcription start site (TSS) of GABA(B1a) was decreased in the DRN of isolation-reared mice. Chromatin immunoprecipitation analysis showed that histone H3 was hyperacetylated around the TSS of GABA(B1a) in the DRN of isolation-reared mice. These findings indicate that an increase in dorsal raphe GABA(B1a) expression via epigenetic regulation is associated with abnormal responses to social stimulation such as encounter-induced hyperactivity and aggressive behavior in isolation

  11. Functional reorganization of visual cortex maps after ischemic lesions is accompanied by changes in expression of cytoskeletal proteins and NMDA and GABA(A) receptor subunits.

    PubMed

    Zepeda, Angelica; Sengpiel, Frank; Guagnelli, Miguel Angel; Vaca, Luis; Arias, Clorinda

    2004-02-25

    Reorganization of cortical representations after focal visual cortex lesions has been documented. It has been suggested that functional reorganization may rely on cellular mechanisms involving modifications in the excitatory/inhibitory neurotransmission balance and on morphological changes of neurons peripheral to the lesion. We explored functional reorganization of cortical retinotopic maps after a focal ischemic lesion in primary visual cortex of kittens using optical imaging of intrinsic signals. After 1, 2, and 5 weeks postlesion (wPL), we addressed whether functional reorganization correlated in time with changes in the expression of MAP-2, GAP-43, GFAP, GABA(A) receptor subunit alpha1 (GABA(A)alpha1), subunit 1 of the NMDA receptor (NMDAR1), and in neurotransmitter levels at the border of the lesion. Our results show that: (1) retinotopic maps reorganize with time after an ischemic lesion; (2) MAP-2 levels increase gradually from 1wPL to 5wPL; (3) MAP-2 upregulation is associated with an increase in dendritic-like structures surrounding the lesion and a decrease in GFAP-positive cells; (4) GAP-43 levels reach the highest point at 2wPL; (5) NMDAR1 and glutamate contents increase in parallel from 1wPL to 5wPL; (6) GABA(A)alpha1 levels increase from 1wPL to 2wPL but do not change after this time point; and (7) GABA contents remain low from 1wPL to 5wPL. This is a comprehensive study showing for the first time that functional reorganization correlates in time with dendritic sprouting and with changes in the excitatory/inhibitory neurotransmission systems previously proposed to participate in cortical remodeling and suggests mechanisms by which plasticity of cortical representations may occur.

  12. Insights into GABA receptor signalling in TM3 Leydig cells.

    PubMed

    Doepner, Richard F G; Geigerseder, Christof; Frungieri, Monica B; Gonzalez-Calvar, Silvia I; Calandra, Ricardo S; Raemsch, Romi; Fohr, Karl; Kunz, Lars; Mayerhofer, Artur

    2005-01-01

    Gamma-aminobutyric acid (GABA) is an emerging signalling molecule in endocrine organs, since it is produced by endocrine cells and acts via GABA(A) receptors in a paracrine/autocrine fashion. Testicular Leydig cells are producers and targets for GABA. These cells express GABA(A) receptor subunits and in the murine Leydig cell line TM3 pharmacological activation leads to increased proliferation. The signalling pathway of GABA in these cells is not known in this study. We therefore attempted to elucidate details of GABA(A) signalling in TM3 and adult mouse Leydig cells using several experimental approaches. TM3 cells not only express GABA(A )receptor subunits, but also bind the GABA agonist [(3)H]muscimol with a binding affinity in the range reported for other endocrine cells (K(d) = 2.740 +/- 0.721 nM). However, they exhibit a low B(max) value of 28.08 fmol/mg protein. Typical GABA(A) receptor-associated events, including Cl(-) currents, changes in resting membrane potential, intracellular Ca(2+) or cAMP, were not measurable with the methods employed in TM3 cells, or, as studied in part, in primary mouse Leydig cells. GABA or GABA(A) agonist isoguvacine treatment resulted in increased or decreased levels of several mRNAs, including transcription factors (c-fos, hsf-1, egr-1) and cell cycle-associated genes (Cdk2, cyclin D1). In an attempt to verify the cDNA array results and because egr-1 was recently implied in Leydig cell development, we further studied this factor. RT-PCR and Western blotting confirmed a time-dependent regulation of egr-1 in TM3. In the postnatal testis egr-1 was seen in cytoplasmic and nuclear locations of developing Leydig cells, which bear GABA(A) receptors and correspond well to TM3 cells. Thus, GABA acts via an atypical novel signalling pathway in TM3 cells. Further details of this pathway remain to be elucidated. Copyright (c) 2005 S. Karger AG, Basel.

  13. Molecular and functional differences in voltage-activated sodium currents between GABA projection neurons and dopamine neurons in the substantia nigra

    PubMed Central

    Ding, Shengyuan; Wei, Wei

    2011-01-01

    GABA projection neurons (GABA neurons) in the substantia nigra pars reticulata (SNr) and dopamine projection neurons (DA neurons) in substantia nigra pars compacta (SNc) have strikingly different firing properties. SNc DA neurons fire low-frequency, long-duration spikes, whereas SNr GABA neurons fire high-frequency, short-duration spikes. Since voltage-activated sodium (NaV) channels are critical to spike generation, the different firing properties raise the possibility that, compared with DA neurons, NaV channels in SNr GABA neurons have higher density, faster kinetics, and less cumulative inactivation. Our quantitative RT-PCR analysis on immunohistochemically identified nigral neurons indicated that mRNAs for pore-forming NaV1.1 and NaV1.6 subunits and regulatory NaVβ1 and Navβ4 subunits are more abundant in SNr GABA neurons than SNc DA neurons. These α-subunits and β-subunits are key subunits for forming NaV channels conducting the transient NaV current (INaT), persistent Na current (INaP), and resurgent Na current (INaR). Nucleated patch-clamp recordings showed that INaT had a higher density, a steeper voltage-dependent activation, and a faster deactivation in SNr GABA neurons than in SNc DA neurons. INaT also recovered more quickly from inactivation and had less cumulative inactivation in SNr GABA neurons than in SNc DA neurons. Furthermore, compared with nigral DA neurons, SNr GABA neurons had a larger INaR and INaP. Blockade of INaP induced a larger hyperpolarization in SNr GABA neurons than in SNc DA neurons. Taken together, these results indicate that NaV channels expressed in fast-spiking SNr GABA neurons and slow-spiking SNc DA neurons are tailored to support their different spiking capabilities. PMID:21880943

  14. Long-term administration with levonorgestrel decreases allopregnanolone levels and alters GABA(A) receptor subunit expression and anxiety-like behavior.

    PubMed

    Porcu, Patrizia; Mostallino, Maria Cristina; Sogliano, Cristiana; Santoru, Francesca; Berretti, Roberta; Concas, Alessandra

    2012-08-01

    Fluctuations in the concentrations of the neuroactive steroid allopregnanolone are thought to influence γ-amino-butyric acid type A (GABA(A)) receptor gene expression and function. Long-term treatment with ethinyl estradiol (EE) plus levonorgestrel (LNG), two of the most widely used steroids in the hormonal contraceptive pill, decreases allopregnanolone levels in rat cerebral cortex and plasma, alters GABA(A) receptor expression and induces anxiety-like behavior. We evaluated which component of the hormonal contraceptive pill is responsible for the aforementioned changes. Female rats were injected subcutaneously (s.c.) with EE (0.030 mg) or LNG (0.125 mg) once a day for 4 weeks. Compared to the respective vehicle-treated control groups, EE decreased cerebral cortical levels of allopregnanolone, progesterone and pregnenolone by 76, 72 and 33%, respectively and hippocampal levels by 52, 56 and 50%, respectively. Likewise, LNG decreased cerebral cortical levels of allopregnanolone, progesterone and pregnenolone by 75, 68 and 33%, respectively, and hippocampal levels by 55, 65 and 60%, respectively. Administration of LNG, but not EE, increased the abundance of the γ2 subunit peptide in cerebral cortex and hippocampus by 38 and 59%, respectively. Further, LNG, but not EE, decreased the time spent and the number of entries into the open arms of the elevated plus maze by 56 and 43%, respectively, an index of anxiety-like behavior. These results suggest that alterations in GABA(A) receptor subunit expression and anxiety-like behavior induced by long-term treatment with combined EE/LNG appear to be caused by LNG. Given that both EE and LNG decrease allopregnanolone levels in a similar manner, these results further suggest that changes in allopregnanolone levels are not associated with GABA(A) receptor expression. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. GABA receptor subunit distribution and FMRP-mGluR5 signaling abnormalities in the cerebellum of subjects with schizophrenia, mood disorders, and autism

    PubMed Central

    Fatemi, S. Hossein; Folsom, Timothy D.

    2016-01-01

    Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. GABAergic receptor abnormalities have been documented in several major psychiatric disorders including schizophrenia, mood disorders, and autism. Abnormal expression of mRNA and protein for multiple GABA receptors has also been observed in multiple brain regions leading to alterations in the balance between excitatory/inhibitory signaling in the brain with potential profound consequences for normal cognition and maintenance of mood and perception. Altered expression of GABAA receptor subunits has been documented in Fragile X mental retardation 1 (FMR1) knockout mice, suggesting that loss of its protein product, fragile X mental retardation protein (FMRP), impacts GABAA subunit expression. Recent postmortem studies from our laboratory have shown reduced expression of FMRP in brains of subjects with schizophrenia, bipolar disorder, major depression, and autism. FMRP acts as a translational repressor and, under normal conditions, inhibits metabotropic glutamate receptor 5 (mGluR5)-mediated signaling. In fragile X syndrome (FXS), absence of FMRP is hypothesized to lead to unregulated mGluR5 signaling, ultimately resulting in the behavioral and intellectual impairments associated with this disorder. Our laboratory has identified changes in mGluR5 expression in autism, schizophrenia, and mood disorders. In the current review article, we discuss our postmortem data on GABA receptors, FMRP, and mGluR5 levels and compare our results with other laboratories. Finally, we discuss the interactions between these molecules and the potential for new therapeutic interventions that target these interconnected signaling systems. PMID:25432637

  16. GABA receptor subunit distribution and FMRP-mGluR5 signaling abnormalities in the cerebellum of subjects with schizophrenia, mood disorders, and autism.

    PubMed

    Fatemi, S Hossein; Folsom, Timothy D

    2015-09-01

    Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the brain. GABAergic receptor abnormalities have been documented in several major psychiatric disorders including schizophrenia, mood disorders, and autism. Abnormal expression of mRNA and protein for multiple GABA receptors has also been observed in multiple brain regions leading to alterations in the balance between excitatory/inhibitory signaling in the brain with potential profound consequences for normal cognition and maintenance of mood and perception. Altered expression of GABAA receptor subunits has been documented in fragile X mental retardation 1 (FMR1) knockout mice, suggesting that loss of its protein product, fragile X mental retardation protein (FMRP), impacts GABAA subunit expression. Recent postmortem studies from our laboratory have shown reduced expression of FMRP in the brains of subjects with schizophrenia, bipolar disorder, major depression, and autism. FMRP acts as a translational repressor and, under normal conditions, inhibits metabotropic glutamate receptor 5 (mGluR5)-mediated signaling. In fragile X syndrome (FXS), the absence of FMRP is hypothesized to lead to unregulated mGluR5 signaling, ultimately resulting in the behavioral and intellectual impairments associated with this disorder. Our laboratory has identified changes in mGluR5 expression in autism, schizophrenia, and mood disorders. In the current review article, we discuss our postmortem data on GABA receptors, FMRP, and mGluR5 levels and compare our results with other laboratories. Finally, we discuss the interactions between these molecules and the potential for new therapeutic interventions that target these interconnected signaling systems. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Cell wall integrity modulates RHO1 activity via the exchange factor ROM2.

    PubMed Central

    Bickle, M; Delley, P A; Schmidt, A; Hall, M N

    1998-01-01

    The essential phosphatidylinositol kinase homologue TOR2 of Saccharomyces cerevisiae controls the actin cytoskeleton by activating a GTPase switch consisting of RHO1 (GTPase), ROM2 (GEF) and SAC7 (GAP). We have identified two mutations, rot1-1 and rot2-1, that suppress the loss of TOR2 and are synthetic-lethal. The wild-type ROT1 and ROT2 genes and a multicopy suppressor, BIG1, were isolated by their ability to rescue the rot1-1 rot2-1 double mutant. ROT2 encodes glucosidase II, and ROT1 and BIG1 encode novel proteins. We present evidence that cell wall defects activate RHO1. First, rot1, rot2, big1, cwh41, gas1 and fks1 mutations all confer cell wall defects and suppress tor2(ts). Second, destabilizing the cell wall by supplementing the growth medium with 0.005% SDS also suppresses a tor2(ts) mutation. Third, disturbing the cell wall with SDS or a rot1, rot2, big1, cwh41, gas1 or fks1 mutation increases GDP/GTP exchange activity toward RHO1. These results suggest that cell wall defects suppress a tor2 mutation by activating RHO1 independently of TOR2, thereby inducing TOR2-independent polarization of the actin cytoskeleton and cell wall synthesis. Activation of RHO1, a subunit of the cell wall synthesis enzyme glucan synthase, by a cell wall alteration would ensure that cell wall synthesis occurs only when and where needed. The mechanism of RHO1 activation by a cell wall alteration is via the exchange factor ROM2 and could be analogous to signalling by integrin receptors in mammalian cells. PMID:9545237

  18. Immunochemical Localization of GABAA Receptor Subunits in the Freshwater Polyp Hydra vulgaris (Cnidaria, Hydrozoa).

    PubMed

    Concas, A; Imperatore, R; Santoru, F; Locci, A; Porcu, P; Cristino, L; Pierobon, P

    2016-11-01

    γ-aminobutyric acid (GABA) receptors, responding to GABA positive allosteric modulators, are present in the freshwater polyp Hydra vulgaris (Cnidaria, Hydrozoa), one of the most primitive metazoans to develop a nervous system. We examined the occurrence and distribution of GABA A receptor subunits in Hydra tissues by western blot and immunohistochemistry. Antibodies against different GABA A receptor subunits were used in Hydra membrane preparations. Unique protein bands, inhibited by the specific peptide, appeared at 35, 60, ∼50 and ∼52 kDa in membranes incubated with α3, β1, γ3 or δ antibodies, respectively. Immunohistochemical screening of whole mount Hydra preparations revealed diffuse immunoreactivity to α3, β1 or γ3 antibodies in tentacles, hypostome, and upper part of the gastric region; immunoreactive fibers were also present in the lower peduncle. By contrast, δ antibodies revealed a strong labeling in the lower gastric region and peduncle, as well as in tentacles. Double labeling showed colocalization of α3/β1, α3/γ3 and α3/δ immunoreactivity in granules or cells in tentacles and gastric region. In the peduncle, colocalization of both α3/β1 and α3/γ3 immunoreactivity was found in fibers running horizontally above the foot. These data indicate that specific GABA A receptor subunits are present and differentially distributed in Hydra body regions. Subunit colocalization suggests that Hydra GABA receptors are heterologous multimers, possibly sub-serving different physiological activities.

  19. GABA, its receptors, and GABAergic inhibition in mouse taste buds

    PubMed Central

    Dvoryanchikov, Gennady; Huang, Yijen A; Barro-Soria, Rene; Chaudhari, Nirupa; Roper, Stephen D.

    2012-01-01

    Taste buds consist of at least three principal cell types that have different functions in processing gustatory signals — glial-like Type I cells, Receptor (Type II) cells, and Presynaptic (Type III) cells. Using a combination of Ca2+ imaging, single cell RT-PCR, and immunostaining, we show that γ-amino butyric acid (GABA) is an inhibitory transmitter in mouse taste buds, acting on GABA-A and GABA-B receptors to suppress transmitter (ATP) secretion from Receptor cells during taste stimulation. Specifically, Receptor cells express GABA-A receptor subunits β2, δ, π, as well as GABA-B receptors. In contrast, Presynaptic cells express the GABA-Aβ3 subunit and only occasionally GABA-B receptors. In keeping with the distinct expression pattern of GABA receptors in Presynaptic cells, we detected no GABAergic suppression of transmitter release from Presynaptic cells. We suggest that GABA may serve function(s) in taste buds in addition to synaptic inhibition. Finally, we also defined the source of GABA in taste buds: GABA is synthesized by GAD65 in Type I taste cells as well as by GAD67 in Presynaptic (Type III) taste cells and is stored in both those two cell types. We conclude that GABA is released during taste stimulation and possibly also during growth and differentiation of taste buds. PMID:21490220

  20. GABA, its receptors, and GABAergic inhibition in mouse taste buds.

    PubMed

    Dvoryanchikov, Gennady; Huang, Yijen A; Barro-Soria, Rene; Chaudhari, Nirupa; Roper, Stephen D

    2011-04-13

    Taste buds consist of at least three principal cell types that have different functions in processing gustatory signals: glial-like (type I) cells, receptor (type II) cells, and presynaptic (type III) cells. Using a combination of Ca2+ imaging, single-cell reverse transcriptase-PCR and immunostaining, we show that GABA is an inhibitory transmitter in mouse taste buds, acting on GABA(A) and GABA(B) receptors to suppress transmitter (ATP) secretion from receptor cells during taste stimulation. Specifically, receptor cells express GABA(A) receptor subunits β2, δ, and π, as well as GABA(B) receptors. In contrast, presynaptic cells express the GABA(A) β3 subunit and only occasionally GABA(B) receptors. In keeping with the distinct expression pattern of GABA receptors in presynaptic cells, we detected no GABAergic suppression of transmitter release from presynaptic cells. We suggest that GABA may serve function(s) in taste buds in addition to synaptic inhibition. Finally, we also defined the source of GABA in taste buds: GABA is synthesized by GAD65 in type I taste cells as well as by GAD67 in presynaptic (type III) taste cells and is stored in both those two cell types. We conclude that GABA is an inhibitory transmitter released during taste stimulation and possibly also during growth and differentiation of taste buds.

  1. Structure and functional interaction of the extracellular domain of human GABA[subscript B] receptor GBR2

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Geng, Yong; Xiong, Dazhi; Mosyak, Lidia

    2012-10-24

    Inhibitory neurotransmission is mediated primarily by GABA. The metabotropic GABA{sub B} receptor is a G protein-coupled receptor central to mammalian brain function. Malfunction of GABA{sub B} receptor has been implicated in several neurological disorders. GABA{sub B} receptor functions as a heterodimeric assembly of GBR1 and GBR2 subunits, where GBR1 is responsible for ligand-binding and GBR2 is responsible for G protein coupling. Here we demonstrate that the GBR2 ectodomain directly interacts with the GBR1 ectodomain to increase agonist affinity by selectively stabilizing the agonist-bound conformation of GBR1. We present the crystal structure of the GBR2 ectodomain, which reveals a polar heterodimericmore » interface. We also identify specific heterodimer contacts from both subunits, and GBR1 residues involved in ligand recognition. Lastly, our structural and functional data indicate that the GBR2 ectodomain adopts a constitutively open conformation, suggesting a structural asymmetry in the active state of GABA{sub B} receptor that is unique to the GABAergic system.« less

  2. Low concentrations of ethanol do not affect radioligand binding to the delta-subunit-containing GABAA receptors in the rat brain.

    PubMed

    Mehta, Ashok K; Marutha Ravindran, C R; Ticku, Maharaj K

    2007-08-24

    In the present study, we investigated the co-localization pattern of the delta subunit with other subunits of GABA(A) receptors in the rat brain using immunoprecipitation and Western blotting techniques. Furthermore, we investigated whether low concentrations of ethanol affect the delta-subunit-containing GABA(A) receptor assemblies in the rat brain using radioligand binding to the rat brain membrane homogenates as well as to the immunoprecipitated receptor assemblies. Our results revealed that delta subunit is not co-localized with gamma(2) subunit but it is associated with the alpha(1), alpha(4) or alpha(6), beta(2) and/or beta(3) subunit(s) of GABA(A) receptors in the rat brain. Ethanol (1-50 mM) neither affected [(3)H]muscimol (3 nM) binding nor diazepam-insensitive [(3)H]Ro 15-4513 (2 nM) binding in the rat cerebellum and cerebral cortex membranes. However, a higher concentration of ethanol (500 mM) inhibited the binding of these radioligands to the GABA(A) receptors partially in the rat cerebellum and cerebral cortex. Similarly, ethanol (up to 50 mM) did not affect [(3)H]muscimol (15 nM) binding to the immunoprecipitated delta-subunit-containing GABA(A) receptor assemblies in the rat cerebellum and hippocampus but it inhibited the binding partially at a higher concentration (500 mM). These results suggest that the native delta-subunit-containing GABA(A) receptors do not play a major role in the pharmacology of clinically relevant low concentrations of ethanol.

  3. Systematic study of association of four GABAergic genes: glutamic acid decarboxylase 1 gene, glutamic acid decarboxylase 2 gene, GABA(B) receptor 1 gene and GABA(A) receptor subunit beta2 gene, with schizophrenia using a universal DNA microarray.

    PubMed

    Zhao, Xu; Qin, Shengying; Shi, Yongyong; Zhang, Aiping; Zhang, Jing; Bian, Li; Wan, Chunling; Feng, Guoyin; Gu, Niufan; Zhang, Guangqi; He, Guang; He, Lin

    2007-07-01

    Several studies have suggested the dysfunction of the GABAergic system as a risk factor in the pathogenesis of schizophrenia. In the present study, case-control association analysis was conducted in four GABAergic genes: two glutamic acid decarboxylase genes (GAD1 and GAD2), a GABA(A) receptor subunit beta2 gene (GABRB2) and a GABA(B) receptor 1 gene (GABBR1). Using a universal DNA microarray procedure we genotyped a total of 20 SNPs on the above four genes in a study involving 292 patients and 286 controls of Chinese descent. Statistically significant differences were observed in the allelic frequencies of the rs187269C/T polymorphism in the GABRB2 gene (P=0.0450, chi(2)=12.40, OR=1.65) and the -292A/C polymorphism in the GAD1 gene (P=0.0450, chi(2)=14.64 OR=1.77). In addition, using an electrophoretic mobility shift assay (EMSA), we discovered differences in the U251 nuclear protein binding to oligonucleotides representing the -292 SNP on the GAD1 gene, which suggests that the -292C allele has reduced transcription factor binding efficiency compared with the 292A allele. Using the multifactor-dimensionality reduction method (MDR), we found that the interactions among the rs187269C/T polymorphism in the GABRB2 gene, the -243A/G polymorphism in the GAD2 gene and the 27379C/T and 661C/T polymorphisms in the GAD1 gene revealed a significant association with schizophrenia (P<0.001). These findings suggest that the GABRB2 and GAD1 genes alone and the combined effects of the polymorphisms in the four GABAergic system genes may confer susceptibility to the development of schizophrenia in the Chinese population.

  4. Breath-hold black-blood T1rho mapping improves liver T1rho quantification in healthy volunteers.

    PubMed

    Wáng, Yì Xiáng J; Deng, Min; Lo, Gladys G; Liang, Dong; Yuan, Jing; Chen, Weitian

    2018-03-01

    Background Recent researches suggest that T1rho may be a non-invasive and quantitative technique for detecting and grading liver fibrosis. Purpose To compare a multi-breath-hold bright-blood fast gradient echo (GRE) imaging and a single breath-hold single-shot fast spin echo (FSE) imaging with black-blood effect for liver parenchyma T1rho measurement and to study liver physiological T1rho value in healthy volunteers. Material and Methods The institutional Ethics Committee approved this study. 28 healthy participants (18 men, 10 women; age = 29.6 ± 5.1 years) underwent GRE liver T1rho imaging, and 20 healthy participants (10 men, 10 women; age = 36.9 ± 10.3 years) underwent novel black-blood FSE liver T1rho imaging, both at 3T with spin-lock frequency of 500 Hz. The FSE technique allows simultaneous acquisition of four spin lock times (TSLs; 1 ms, 10 ms, 30 ms, 50msec) in 10 s. Results For FSE technique the intra-scan repeatability intraclass correlation coefficient (ICC) was 0.98; while the inter-scan reproducibility ICC was 0.82 which is better than GRE technique's 0.76. Liver T1rho value in women tended to have a higher value than T1rho values in men (FSE: 42.28 ± 4.06 ms for women and 39.13 ± 2.12 ms for men; GRE: 44.44 ± 1.62 ms for women and 42.36 ± 2.00 ms for men) and FSE technique showed liver T1rho value decreased slightly as age increased. Conclusion Single breath-hold black-blood FSE sequence has better scan-rescan reproducibility than multi-breath-hold bright-blood GRE sequence. Gender and age dependence of liver T1rho in healthy participants is observed, with young women tending to have a higher T1rho measurement.

  5. Oxytocin modulates GABAAR subunits to confer neuroprotection in stroke in vitro.

    PubMed

    Kaneko, Yuji; Pappas, Colleen; Tajiri, Naoki; Borlongan, Cesar V

    2016-10-21

    Oxytocin protects against ischemia-induced inflammation and oxidative stress, and is associated with GABA (γ-aminobutyric acid, an inhibitory neurotransmitter) signaling transduction in neurons. However, the molecular mechanism by which oxytocin affords neuroprotection, especially the interaction between oxytocin receptor and GABA A receptor (GABA A R), remains to be elucidated. Primary rat neural cells were exposed to oxytocin before induction of experimental acute stroke model via oxygen-glucose deprivation-reperfusion (OGD/R) injury. Pretreatment with oxytocin increased cell viability, decreased the cell damage against oxidative stress, and prevented the release of high mobility group box1 during OGD/R. However, introduction of oxytocin during OGD/R did not induce neuroprotection. Although oxytocin did not affect the glutathione-related cellular metabolism before OGD, oxytocin modulated the expression levels of GABA A R subunits, which function to remove excessive neuronal excitability via chloride ion influx. Oxytocin-pretreated cells significantly increased the chloride ion influx in response to GABA and THIP (δ-GABA A R specific agonist). This study provides evidence that oxytocin regulated GABA A R subunits in affording neuroprotection against OGD/R injury.

  6. Zolpidem modulation of phasic and tonic GABA currents in the rat dorsal motor nucleus of the vagus

    PubMed Central

    Gao, Hong; Smith, Bret N.

    2010-01-01

    Zolpidem is a widely prescribed sleep aid with relative selectivity for GABAA receptors containing α1–3 subunits. We examined the effects of zolpidem on the inhibitory currents mediated by GABAA receptors using whole-cell patch-clamp recordings from DMV neurons in transverse brainstem slices from rat. Zolpidem prolonged the decay time of mIPSCs and of muscimol-evoked whole-cell GABAergic currents, and it occasionally enhanced the amplitude of mIPSCs. The effects were blocked by flumazenil, a benzodiazepine antagonist. Zolpidem also hyperpolarized the resting membrane potential, with a concomitant decrease in input resistance and action potential firing activity in a subset of cells. Zolpidem did not clearly alter the GABAA receptor-mediated tonic current (Itonic) under baseline conditions, but after elevating extracellular GABA concentration with nipecotic acid, a non-selective GABA transporter blocker, zolpidem consistently and significantly increased the tonic GABA current. This increase was suppressed by flumazenil and gabazine. These results suggest that α1–3 subunits are expressed in synaptic GABAA receptors on DMV neurons. The baseline tonic GABA current is likely not mediated by these same low affinity, zolpidem-sensitive GABAA receptors. However, when the extracellular GABA concentration is increased, zolpidem-sensitive extrasynaptic GABAA receptors containing α1–3 subunits contribute to the Itonic. PMID:20226798

  7. Virus-mediated swapping of zolpidem-insensitive with zolpidem-sensitive GABA(A) receptors in cortical pyramidal cells.

    PubMed

    Sumegi, Mate; Fukazawa, Yugo; Matsui, Ko; Lorincz, Andrea; Eyre, Mark D; Nusser, Zoltan; Shigemoto, Ryuichi

    2012-04-01

    Recently developed pharmacogenetic and optogenetic approaches, with their own advantages and disadvantages, have become indispensable tools in modern neuroscience. Here, we employed a previously described knock-in mouse line (GABA(A)Rγ2(77I)lox) in which the γ2 subunit of the GABA(A) receptor (GABA(A)R) was mutated to become zolpidem insensitive (γ2(77I)) and used viral vectors to swap γ2(77I) with wild-type, zolpidem-sensitive γ2 subunits (γ2(77F)). The verification of unaltered density and subcellular distribution of the virally introduced γ2 subunits requires their selective labelling. For this we generated six N- and six C-terminal-tagged γ2 subunits, with which cortical cultures of GABA(A)Rγ2(−/−) mice were transduced using lentiviruses. We found that the N-terminal AU1 tag resulted in excellent immunodetection and unimpaired synaptic localization. Unaltered kinetic properties of the AU1-tagged γ2 ((AU1)γ2(77F)) channels were demonstrated with whole-cell patch-clamp recordings of spontaneous IPSCs from cultured cells. Next, we carried out stereotaxic injections of lenti- and adeno-associated viruses containing Cre-recombinase and the (AU1)γ2(77F) subunit (Cre-2A-(AU1)γ2(77F)) into the neocortex of GABA(A)Rγ2(77I)lox mice. Light microscopic immunofluorescence and electron microscopic freeze-fracture replica immunogold labelling demonstrated the efficient immunodetection of the AU1 tag and the normal enrichment of the (AU1)γ2(77F) subunits in perisomatic GABAergic synapses. In line with this,miniature and action potential-evoked IPSCs whole-cell recorded from transduced cells had unaltered amplitudes, kinetics and restored zolpidem sensitivity. Our results obtained with a wide range of structural and functional verification methods reveal unaltered subcellular distributions and functional properties of γ2(77I) and (AU1)γ2(77F) GABA(A)Rs in cortical pyramidal cells. This transgenic–viral pharmacogenetic approach has the advantage that it

  8. 2-Aminoethyl Methylphosphonate, a Potent and Rapidly Acting Antagonist of GABA A-ρ1 Receptors

    DOE PAGES

    Xie, A.; Yan, J.; Yue, L.; ...

    2011-08-02

    All three classes of receptors for the inhibitory neurotransmitter GABA (GABAR) are expressed in the retina. This study investigated roles of GABAR, especially GABA(C)R (GABA(A)-rho), in retinal signaling in vivo by studying effects on the mouse electroretinogram (ERG) of genetic deletion of GABA(C)R versus pharmacological blockade using receptor antagonists. Brief full-field flash ERGs were recorded from anesthetized GABA(C)R(-/-) mice, and WT C57BL/6 (B6) mice, before and after intravitreal injection of GABA(C)R antagonists, TPMPA, 3-APMPA, or the more recently developed 2-AEMP; GABA(A)R antagonist, SR95531; GABA(B)R antagonist, CGP, and agonist, baclofen. Intravitreal injections of TPMPA and SR95531 were also made in Brownmore » Norway rats. The effect of 2-AEMP on GABA-induced current was tested directly in isolated rat rod bipolar cells, and 2-AEMP was found to preferentially block GABA(C)R in those cells. Maximum amplitudes of dark (DA) and light-adapted (LA) ERG b-waves were reduced in GABA(C)R(-/-) mice, compared to B6 mice, by 30-60%; a-waves were unaltered and oscillatory potential amplitudes were increased. In B6 mice, after injection of TPMPA (also in rats), 3-APMPA or 2-AEMP, ERGs became similar to ERGs of GABA(C)R(-/-) mice. Blockade of GABA(A)Rs and GABA(B)Rs, or agonism of GABA(B)Rs did not alter B6 DA b-wave amplitude. The negative scotopic threshold response (nSTR) was slightly less sensitive in GABA(C)R(-/-) than in B6 mice, and unaltered by 2-AEMP. However, amplitudes of nSTR and photopic negative response (PhNR), both of which originate from inner retina, were enhanced by TPMPA and 3-APMPA, each of which has GABA(B) agonist properties, and further increased by baclofen. The finding that genetic deletion of GABA(C)R, the GABA(C)R antagonist 2-AEMP, and other antagonists all reduced ERG b-wave amplitude, supports a role for CABA(C)R in determining the maximum response amplitude of bipolar cells contributing to the b-wave. GABA(C)R antagonists

  9. A Negative Allosteric Modulator for α5 Subunit-Containing GABA Receptors Exerts a Rapid and Persistent Antidepressant-Like Action without the Side Effects of the NMDA Receptor Antagonist Ketamine in Mice

    PubMed Central

    Nelson, Mackenzie E.; Krimmel, Samuel R.; Georgiou, Polymnia; Gould, Todd D.

    2017-01-01

    Abstract New antidepressant pharmacotherapies that provide rapid relief of depressive symptoms are needed. The NMDA receptor antagonist ketamine exerts rapid antidepressant actions in depressed patients but also side effects that complicate its clinical utility. Ketamine promotes excitatory synaptic strength, likely by producing high-frequency correlated activity in mood-relevant regions of the forebrain. Negative allosteric modulators of GABA-A receptors containing α5 subunits (α5 GABA-NAMs) should also promote high-frequency correlated electroencephalogram (EEG) activity and should therefore exert rapid antidepressant responses. Because α5 subunits display a restricted expression in the forebrain, we predicted that α5 GABA-NAMs would produce activation of principle neurons but exert fewer side effects than ketamine. We tested this hypothesis in male mice and observed that the α5 GABA-NAM MRK-016 exerted an antidepressant-like response in the forced swim test at 1 and 24 h after administration and an anti-anhedonic response after chronic stress in the female urine sniffing test (FUST). Like ketamine, MRK-016 produced a transient increase in EEG γ power, and both the increase in γ power and its antidepressant effects in the forced swim test were blocked by prior administration of the AMPA-type glutamate receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX). Unlike ketamine, however, MRK-016 produced no impairment of rota-rod performance, no reduction of prepulse inhibition (PPI), no conditioned-place preference (CPP), and no change in locomotion. α5 GABA-NAMs, thus reproduce the rapid antidepressant-like actions of ketamine, perhaps via an AMPA receptor (AMPAR)-dependent increase in coherent neuronal activity, but display fewer potential negative side effects. These compounds thus demonstrate promise as clinically useful fast-acting antidepressants. PMID:28275719

  10. RNA editing of the GABAA receptor α3 subunit alters the functional properties of recombinant receptors

    PubMed Central

    Nimmich, Mitchell L.; Heidelberg, Laura S.; Fisher, Janet L.

    2009-01-01

    RNA editing provides a post-transcriptional mechanism to increase structural heterogeneity of gene products. Recently, the α3 subunit of the GABAA receptors has been shown to undergo RNA editing. As a result, a highly conserved isoleucine residue in the third transmembrane domain is replaced with a methionine. To determine the effect of this structural change on receptor function, we compared the GABA sensitivity, pharmacological properties and macroscopic kinetics of recombinant receptors containing either the edited or unedited forms of the α3 subunit along with β3 and γ2L. Editing substantially altered the GABA sensitivity and deactivation rate of the receptors, with the unedited form showing a lower GABA EC50 and slower decay. Comparable effects were observed with a mutation at the homologous location in the α1 subunit, suggesting a common role for this site in regulation of channel gating. Except for the response to GABA, the pharmacological properties of the receptor were unaffected by editing, with similar enhancement by a variety of modulators. Since RNA editing of the α3 subunit increases through development, our findings suggest that GABAergic neurotransmission may be more effective early in development, with greater GABA sensitivity and slower decay rates conferred by the unedited α3 subunit. PMID:19367790

  11. Quality components and antidepressant-like effects of GABA green tea.

    PubMed

    Teng, Jie; Zhou, Wen; Zeng, Zhen; Zhao, Wenfang; Huang, Yahui; Zhang, Xu

    2017-09-20

    Gamma (γ)-aminobutyric acid (GABA) green tea, with high GABA content, is a kind of special green tea. The goals of this study are to analyze the changes in quality components of green tea during anaerobic treatment, and to investigate whether or not the extract of GABA present in green tea can prevent depression or improve the depressive state of animals. Results showed that GABA content in green tea had increased significantly after anaerobic treatment. The contents of tea polysaccharides, total free amino acids, and water extracts were also increased whereas tea polyphenols were reduced. More importantly, the extract of GABA green tea could alleviate mouse depression and stress from desperate environments through the forced swim test (FST), tail suspension test (TST), mRNA and protein expression levels of GABA A receptors. Therefore, these results indicate that GABA green tea may have a health effect on prevention and alleviation of depression, and it works on the GABAergic neurotransmission of mouse cerebral cortex via up-regulating expression of the GABA A receptor α1 subunit, thus ameliorating depression.

  12. Regulated Localization Is Sufficient for Hormonal Control of Regulator of G Protein Signaling Homology Rho Guanine Nucleotide Exchange Factors (RH-RhoGEFs)*

    PubMed Central

    Carter, Angela M.; Gutowski, Stephen; Sternweis, Paul C.

    2014-01-01

    The regulator of G protein signaling homology (RH) Rho guanine nucleotide exchange factors (RhoGEFs) (p115RhoGEF, leukemia-associated RhoGEF, and PDZ-RhoGEF) contain an RH domain and are specific GEFs for the monomeric GTPase RhoA. The RH domains interact specifically with the α subunits of G12 heterotrimeric GTPases. Activated Gα13 modestly stimulates the exchange activity of both p115RhoGEF and leukemia-associated RhoGEF but not PDZ-RhoGEF. Because all three RH-RhoGEFs can localize to the plasma membrane upon expression of activated Gα13, cellular localization of these RhoGEFs has been proposed as a mechanism for controlling their activity. We use a small molecule-regulated heterodimerization system to rapidly control the localization of RH-RhoGEFs. Acute localization of the proteins to the plasma membrane activates RhoA within minutes and to levels that are comparable with activation of RhoA by hormonal stimulation of G protein-coupled receptors. The catalytic activity of membrane-localized RhoGEFs is not dependent on activated Gα13. We further show that the conserved RH domains can rewire two different RacGEFs to activate Rac1 in response to a traditional activator of RhoA. Thus, RH domains act as independent detectors for activated Gα13 and are sufficient to modulate the activity of RhoGEFs by hormones via mediating their localization to substrate, membrane-associated RhoA. PMID:24855647

  13. The role of GABA(A) receptors in the development of alcoholism.

    PubMed

    Enoch, Mary-Anne

    2008-07-01

    Alcoholism is a common, heritable, chronic relapsing disorder. GABA(A) receptors undergo allosteric modulation by ethanol, anesthetics, benzodiazepines and neurosteroids and have been implicated in the acute as well as the chronic effects of ethanol including tolerance, dependence and withdrawal. Medications targeting GABA(A) receptors ameliorate the symptoms of acute withdrawal. Ethanol induces plasticity in GABA(A) receptors: tolerance is associated with generally decreased GABA(A) receptor activation and differentially altered subunit expression. The dopamine (DA) mesolimbic reward pathway originating in the ventral tegmental area (VTA), and interacting stress circuitry play an important role in the development of addiction. VTA GABAergic interneurons are the primary inhibitory regulators of DA neurons and a subset of VTA GABA(A) receptors may be implicated in the switch from heavy drinking to dependence. GABA(A) receptors modulate anxiety and response to stress; important elements of sustained drinking and relapse. The GABA(A) receptor subunit genes clustered on chromosome 4 are highly expressed in the reward pathway. Several recent studies have provided strong evidence that one of these genes, GABRA2, is implicated in alcoholism in humans. The influence of the interaction between ethanol and GABA(A) receptors in the reward pathway on the development of alcoholism together with genetic and epigenetic vulnerabilities will be explored in this review.

  14. Differential binding of RhoA, RhoB, and RhoC to protein kinase C-related kinase (PRK) isoforms PRK1, PRK2, and PRK3: PRKs have the highest affinity for RhoB.

    PubMed

    Hutchinson, Catherine L; Lowe, Peter N; McLaughlin, Stephen H; Mott, Helen R; Owen, Darerca

    2013-11-12

    Protein kinase C-related kinases (PRKs) are members of the protein kinase C superfamily of serine-threonine kinases and can be activated by binding to members of the Rho family of GTPases via a Rho-binding motif known as an HR1 domain. Three tandem HR1 domains reside at the N-terminus of the PRKs. We have assessed the ability of the HR1a and HR1b domains from the three PRK isoforms (PRK1, PRK2, and PRK3) to interact with the three Rho isoforms (RhoA, RhoB, and RhoC). The affinities of RhoA and RhoC for a construct encompassing both PRK1 HR1 domains were similar to those for the HR1a domain alone, suggesting that these interactions are mediated solely by the HR1a domain. The affinities of RhoB for both the PRK1 HR1a domain and the HR1ab didomain were higher than those of RhoA or RhoC. RhoB also bound more tightly to the didomain than to the HR1a domain alone, implicating the HR1b domain in the interaction. As compared with PRK1 HR1 domains, PRK2 and PRK3 domains bind less well to all Rho isoforms. Uniquely, however, the PRK3 domains display a specificity for RhoB that requires both the C-terminus of RhoB and the PRK3 HR1b domain. The thermal stability of the HR1a and HR1b domains was also investigated. The PRK2 HR1a domain was found to be the most thermally stable, while PRK2 HR1b, PRK3 HR1a, and PRK3 HR1b domains all exhibited lower melting temperatures, similar to that of the PRK1 HR1a domain. The lower thermal stability of the PRK2 and PRK3 HR1b domains may impart greater flexibility, driving their ability to interact with Rho isoforms.

  15. Multiple roles for the Na,K-ATPase subunits, Atp1a1 and Fxyd1, during brain ventricle development

    PubMed Central

    Chang, Jessica T.; Lowery, Laura Anne; Sive, Hazel

    2012-01-01

    Formation of the vertebrate brain ventricles requires both production of cerebrospinal fluid (CSF), and its retention in the ventricles. The Na,K-ATPase is required for brain ventricle development, and we show here that this protein complex impacts three associated processes. The first requires both the alpha subunit (Atp1a1) and the regulatory subunit, Fxyd1, and leads to formation of a cohesive neuroepithelium, with continuous apical junctions. The second process leads to modulation of neuroepithelial permeability, and requires Atp1a1, which increases permeability with partial loss of function and decreases it with overexpression. In contrast, fxyd1 overexpression does not alter neuroepithelial permeability, suggesting that its activity is limited to neuroepithelium formation. RhoA regulates both neuroepithelium formation and permeability, downstream of the Na,K-ATPase. A third process, likely to be CSF production, is RhoA-independent, requiring Atp1a1, but not Fxyd1. Consistent with a role for Na,K-ATPase pump function, the inhibitor ouabain prevents neuroepithelium formation, while intracellular Na+ increases after Atp1a1 and Fxyd1 loss of function. These data include the first reported role for Fxyd1 in the developing brain, and indicate that the Na,K-ATPase regulates three aspects of brain ventricle development essential for normal function - formation of a cohesive neuroepithelium, restriction of neuroepithelial permeability, and production of CSF. PMID:22683378

  16. Distinctive and selective route of PI3K/PKCα-PKCδ/RhoA-Rac1 signaling in osteoclastic cell migration.

    PubMed

    Kim, Jin-Man; Kim, Mi Yeong; Lee, Kyunghee; Jeong, Daewon

    2016-12-05

    Cell migration during specialized stages of osteoclast precursors, mononuclear preosteoclasts, and multinucleated mature osteoclasts remain uncertain. M-CSF- and osteopontin-induced osteoclastic cell migration was inhibited by function-blocking monoclonal antibodies specific to the integrin αv and β3 subunits, suggesting that integrin αvβ3 mediates migratory signaling induced by M-CSF and osteopontin. M-CSF and osteopontin stimulation was shown to regulate two branched signaling processes, PI3K/PKCα/RhoA axis and PI3K/PKCδ/Rac1 axis. Interestingly, inactivation of RhoA or Rac1 blocked preosteoclast and mature osteoclast migration but not osteoclast precursor migration in a transwell-based cell migration assay. Moreover, the inhibitory effect on preosteoclast and mature osteoclast migration induced by Rac1 inactivation was more effective than that by RhoA inactivation. Collectively, our findings suggest that osteoclast precursor migration depends on PI3K/PKCα-PKCδ signaling mediated via integrin αvβ3 bypassing RhoA and Rac1, whereas preosteoclast and mature osteoclast migration relies on PI3K/PKCα-PKCδ/RhoA-Rac1 axis signaling mediated via integrin αvβ3 with increased dependency on PKCδ/Rac1 signaling route as differentiation progresses. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  17. PKC/CREB pathway mediates the expressions of GABAA receptor subunits in cultured hippocampal neurons after low-Mg2+ solution treatment.

    PubMed

    Wu, Guofeng; Yu, Jinpeng; Wang, Likun; Ren, Siying; Zhang, Yixia

    2018-02-01

    To investigate the potential effects of the PKC/CREB pathway on the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons using a model of epilepsy that employed conditions of low magnesium (Mg 2+ ). A total of 108 embryonic rats at the age of 18 embryonic days (E18)prepared from adult female SD rats were used as experimental subjects. Primary rat hippocampal cultures were prepared from the embryonic 18 days rats. The cultured hippocampal neurons were then treated with artificial cerebrospinal fluid containing low Mg 2+ solutions to generate a low Mg 2+ model of epilepsy. The low Mg 2+ stimulation lasted for 3 h and then returned to in maintenance medium for 20 h. The changes of the GABA A receptor subunit α1, γ2, δ were observed by blocking or activating the function of the CREB. The quantification of the GABA A receptor subunit α1, γ2, δ and the CREB were determined by a qRT-PCR and a Western blot method. After the neurons were exposed to a low-Mg 2+ solution for 3 h, GABA A receptor mRNA expression markedly increased compared to the control, and then gradually decreased. In contrast, CREB mRNA levels exhibited a dramatic down-regulation 3 h after terminating low-Mg 2+ treatment, and then peaked at 9 h. Western blot analyses verified that staurosporine suppressed CREB phosphorylation (p-CREB). The mRNA expression of GABA A receptor subunit α1 increased only in the presence of staurosporine, whereas the expressions of subunits γ2 and δ significantly increased in the presence of either KG-501 or staurosporine. Furthermore, phorbol 12-myristate 13-acetate (PMA) decreased the expressions of GABA A subunits α1, γ2, and δ when administered alone. However, the administration of either KG-501 or staurosporine reversed the inhibitory effects of PMA. The PKC/CREB pathway may negatively regulate the expressions of GABA A receptor subunits α1, γ2, and δ in cultured hippocampal neurons in low Mg 2+ model of

  18. Expression of functional receptors by the human γ-aminobutyric acid A γ2 subunit

    PubMed Central

    Martínez-Torres, Ataúlfo; Miledi, Ricardo

    2004-01-01

    γ-Aminobutyric acid A (GABAA) receptors are heteromeric membrane proteins formed mainly by various combinations of α, β, and γ subunits; and it is commonly thought that the γ2 subunit alone does not form functional receptors. In contrast, we found that cDNA encoding the γ2L subunit of the human GABAA receptor, injected alone into Xenopus oocytes, expressed functional GABA receptors whose properties were investigated by using the two-microelectrode voltage-clamp technique. GABA elicited desensitizing membrane currents that recovered after a few minutes' wash. Repetitive applications of GABA induced a “run-up” of GABA currents that nearly doubled the amplitude of the first response. The GABA currents inverted direction at about -30 mV, indicating that they are carried mainly by Cl- ions. The homomeric γ2L receptors were also activated by β-alanine > taurine > glycine, and, like some types of heteromeric GABAA receptors, the γ2L receptors were blocked by bicuculline and were potentiated by pentobarbital and flunitrazepam. These results indicate that the human γ2L subunit is capable of forming fully functional GABA receptors by itself in Xenopus oocytes and suggest that the roles proposed for the various subunits that make up the heteromeric GABAA receptors in situ require further clarification. PMID:14981251

  19. Decreased surface expression of the δ subunit of the GABAA receptor contributes to reduced tonic inhibition in dentate granule cells in a mouse model of fragile X syndrome.

    PubMed

    Zhang, Nianhui; Peng, Zechun; Tong, Xiaoping; Lindemeyer, A Kerstin; Cetina, Yliana; Huang, Christine S; Olsen, Richard W; Otis, Thomas S; Houser, Carolyn R

    2017-11-01

    While numerous changes in the GABA system have been identified in models of Fragile X Syndrome (FXS), alterations in subunits of the GABA A receptors (GABA A Rs) that mediate tonic inhibition are particularly intriguing. Considering the key role of tonic inhibition in controlling neuronal excitability, reduced tonic inhibition could contribute to FXS-associated disorders such as hyperactivity, hypersensitivity, and increased seizure susceptibility. The current study has focused on the expression and function of the δ subunit of the GABA A R, a major subunit involved in tonic inhibition, in granule cells of the dentate gyrus in the Fmr1 knockout (KO) mouse model of FXS. Electrophysiological studies of dentate granule cells revealed a marked, nearly four-fold, decrease in tonic inhibition in the Fmr1 KO mice, as well as reduced effects of two δ subunit-preferring pharmacological agents, THIP and DS2, supporting the suggestion that δ subunit-containing GABA A Rs are compromised in the Fmr1 KO mice. Immunohistochemistry demonstrated a small but statistically significant decrease in δ subunit labeling in the molecular layer of the dentate gyrus in Fmr1 KO mice compared to wildtype (WT) littermates. The discrepancy between the large deficits in GABA-mediated tonic inhibition in granule cells in the Fmr1 KO mice and only modest reductions in immunolabeling of the δ subunit led to studies of surface expression of the δ subunit. Cross-linking experiments followed by Western blot analysis demonstrated a small, non-significant decrease in total δ subunit protein in the hippocampus of Fmr1 KO mice, but a four-fold decrease in surface expression of the δ subunit in these mice. No significant changes were observed in total or surface expression of the α4 subunit protein, a major partner of the δ subunit in the forebrain. Postembedding immunogold labeling for the δ subunit demonstrated a large, three-fold, decrease in the number of symmetric synapses with

  20. Association between the GABA(A) receptor alpha5 subunit gene locus (GABRA5) and bipolar affective disorder.

    PubMed

    Papadimitriou, G N; Dikeos, D G; Karadima, G; Avramopoulos, D; Daskalopoulou, E G; Vassilopoulos, D; Stefanis, C N

    1998-02-07

    Genetic factors seem to play an important role in the pathogenesis of affective disorder. The candidate gene strategies are being used, among others, to identify the genes conferring vulnerability to the disease. The genes coding for the receptors of gamma-aminobutyric acid (GABA) have been proposed as candidates for affective disorder, since the GABA neurotransmitter system has been implicated in the pathogenesis of the illness. We examined the possible genetic association between the GABA(A) receptor alpha5 subunit gene locus (GABRA5) on chromosome 15 and affective disorder, in 48 bipolar patients (BP), 40 unipolar patients (UP), and 50 healthy individuals, age- and sex-matched to the patients. All patients and controls were unrelated Greeks. Diagnoses were made after direct interviews according to the DSM-IV and ICD-10 criteria. For the genotyping, a dinucleotide (CA) repeat marker was used. The polymerase chain reaction (PCR) products found were nine alleles with lengths between 272 and 290 base pairs (bp). The distribution of allelic frequencies of the GABRA5 locus differed significantly between BP patients and controls with the 282-bp allele found to be associated with BP affective disorder, while no such difference was observed between the groups of UP patients and controls nor between the two patient groups. The presence or absence of the 282-bp allele in the genotype of BP patients was not shown to influence the age of onset and the overall clinical severity, but was found to be associated with a preponderance of manic over depressive episodes in the course of the illness.

  1. Erbb4 Deletion from Medium Spiny Neurons of the Nucleus Accumbens Core Induces Schizophrenia-Like Behaviors via Elevated GABAA Receptor α1 Subunit Expression.

    PubMed

    Geng, Hong-Yan; Zhang, Jing; Yang, Jian-Ming; Li, Yue; Wang, Ning; Ye, Mao; Chen, Xiao-Juan; Lian, Hong; Li, Xiao-Ming

    2017-08-02

    Medium spiny neurons (MSNs), the major GABAergic projection neurons in the striatum, are implicated in many neuropsychiatric diseases such as schizophrenia, but the underlying mechanisms remain unclear. We found that a deficiency in Erbb4 , a schizophrenia risk gene, in MSNs of the nucleus accumbens (NAc) core, but not the dorsomedial striatum, markedly induced schizophrenia-like behaviors such as hyperactivity, abnormal marble-burying behavior, damaged social novelty recognition, and impaired sensorimotor gating function in male mice. Using immunohistochemistry, Western blot, RNA interference, electrophysiology, and behavior test studies, we found that these phenomena were mediated by increased GABA A receptor α1 subunit (GABA A R α1) expression, which enhanced inhibitory synaptic transmission on MSNs. These results suggest that Erbb4 in MSNs of the NAc core may contribute to the pathogenesis of schizophrenia by regulating GABAergic transmission and raise the possibility that GABA A R α1 may therefore serve as a new therapeutic target for schizophrenia. SIGNIFICANCE STATEMENT Although ErbB4 is highly expressed in striatal medium spiny neurons (MSNs), its role in this type of neuron has not been reported previously. The present study demonstrates that Erbb4 deletion in nucleus accumbens (NAc) core MSNs can induce schizophrenia-like behaviors via elevated GABA A receptor α1 subunit (GABA A R α1) expression. To our knowledge, this is the first evidence that ErbB4 signaling in the MSNs is involved in the pathology of schizophrenia. Furthermore, restoration of GABA A R α1 in the NAc core, but not the dorsal medium striatum, alleviated the abnormal behaviors. Here, we highlight the role of the NAc core in the pathogenesis of schizophrenia and suggest that GABA A R α1 may be a potential pharmacological target for its treatment. Copyright © 2017 the authors 0270-6474/17/377450-15$15.00/0.

  2. Decreased GABA receptor in the cerebral cortex of epileptic rats: effect of Bacopa monnieri and Bacoside-A.

    PubMed

    Mathew, Jobin; Balakrishnan, Savitha; Antony, Sherin; Abraham, Pretty Mary; Paulose, C S

    2012-02-24

    Gamma amino butyric acid (GABA), the principal inhibitory neurotransmitter in the cerebral cortex, maintains the inhibitory tones that counter balances neuronal excitation. When this balance is perturbed, seizures may ensue. In the present study, alterations of the general GABA, GABAA and GABAB receptors in the cerebral cortex of the epileptic rat and the therapeutic application of Bacopa monnieri were investigated. Scatchard analysis of [3H]GABA, [3H]bicuculline and [3H]baclofen in the cerebral cortex of the epileptic rat showed significant decrease in Bmax (P < 0.001) compared to control. Real Time PCR amplification of GABA receptor subunits such as GABAAά1, GABAAγ, GABAAδ, GABAB and GAD where down regulated (P < 0.001) in epileptic rats. GABAAά5 subunit and Cyclic AMP responsible element binding protein were up regulated. Confocal imaging study confirmed the decreased GABA receptors in epileptic rats. Epileptic rats have deficit in radial arm and Y maze performance. Bacopa monnieri and Bacoside-A treatment reverses epilepsy associated changes to near control suggesting that decreased GABA receptors in the cerebral cortex have an important role in epileptic occurrence; Bacopa monnieri and Bacoside-A have therapeutic application in epilepsy management.

  3. Fast detection of extrasynaptic GABA with a whole-cell sniffer.

    PubMed

    Christensen, Rasmus K; Petersen, Anders V; Schmitt, Nicole; Perrier, Jean-François

    2014-01-01

    Gamma-amino-butyric acid (GABA) is the main inhibitory transmitter of the brain. It operates by binding to specific receptors located both inside and outside synapses. The extrasynaptic receptors are activated by spillover from GABAergic synapses and by ambient GABA in the extracellular space. Ambient GABA is essential for adjusting the excitability of neurons. However, due to the lack of suitable methods, little is known about its dynamics. Here we describe a new technique that allows detection of GABA transients and measurement of the steady state GABA concentration with high spatial and temporal resolution. We used a human embryonic kidney (HEK) cell line that stably expresses GABAA receptors composed of α1, β2, and γ2 subunits. We recorded from such a HEK cell with the whole-cell patch-clamp technique. The presence of GABA near the HEK cell generated a measurable electric current whose magnitude increased with concentration. A fraction of the current did not inactivate during prolonged exposition to GABA. This technique, which we refer to as a "sniffer" allows the measurement of ambient GABA concentration inside nervous tissue with a resolution of few tens of nanomolars. In addition, the sniffer detects variations in the extrasynaptic GABA concentration with millisecond time resolution. Pilot experiments demonstrate that the sniffer is able to report spillover of GABA induced by synaptic activation in real time. This is the first report on a GABA sensor that combines the ability to detect fast transients and to measure steady concentrations.

  4. Rho/ROCK-dependent inhibition of 3T3-L1 adipogenesis by G-protein-deamidating dermonecrotic toxins: differential regulation of Notch1, Pref1/Dlk1, and β-catenin signaling

    PubMed Central

    Bannai, Yuka; Aminova, Leila R.; Faulkner, Melinda J.; Ho, Mengfei; Wilson, Brenda A.

    2012-01-01

    The dermonecrotic toxins from Pasteurella multocida (PMT), Bordetella (DNT), Escherichia coli (CNF1-3), and Yersinia (CNFY) modulate their G-protein targets through deamidation and/or transglutamination of an active site Gln residue, which results in activation of the G protein and its cognate downstream signaling pathways. Whereas DNT and the CNFs act on small Rho GTPases, PMT acts on the α subunit of heterotrimeric Gq, Gi, and G12/13 proteins. We previously demonstrated that PMT potently blocks adipogenesis and adipocyte differentiation in a calcineurin-independent manner through downregulation of Notch1 and stabilization of β-catenin and Pref1/Dlk1, key proteins in signaling pathways strongly linked to cell fate decisions, including fat and bone development. Here, we report that similar to PMT, DNT, and CNF1 completely block adipogenesis and adipocyte differentiation by preventing upregulation of adipocyte markers, PPARγ and C/EBPα, while stabilizing the expression of Pref1/Dlk1 and β-catenin. We show that the Rho/ROCK inhibitor Y-27632 prevented or reversed these toxin-mediated effects, strongly supporting a role for Rho/ROCK signaling in dermonecrotic toxin-mediated inhibition of adipogenesis and adipocyte differentiation. Toxin treatment was also accompanied by downregulation of Notch1 expression, although this inhibition was independent of Rho/ROCK signaling. We further show that PMT-mediated downregulation of Notch1 expression occurs primarily through G12/13 signaling. Our results reveal new details of the pathways involved in dermonecrotic toxin action on adipocyte differentiation, and the role of Rho/ROCK signaling in mediating toxin effects on Wnt/β-catenin and Notch1 signaling, and in particular the role of Gq and G12/13 in mediating PMT effects on Rho/ROCK and Notch1 signaling. PMID:22919671

  5. Calcium/calmodulin-dependent kinase II phosphorylation of the GABAA receptor alpha1 subunit modulates benzodiazepine binding.

    PubMed

    Churn, Severn B; Rana, Aniruddha; Lee, Kangmin; Parsons, J Travis; De Blas, Angel; Delorenzo, Robert J

    2002-09-01

    gamma-Aminobutyric acid (GABA) is the primary neurotransmitter that is responsible for the fast inhibitory synaptic transmission in the central nervous system. A major post-translational mechanism that can rapidly regulate GABAAR function is receptor phosphorylation. This study was designed to test the effect of endogenous calcium and calmodulin-dependent kinase II (CaM kinase II) activation on both allosteric modulator binding and GABAA receptor subunit phosphorylation. Endogenous CaM kinase II activity was stimulated, and GABAA receptors were subsequently analyzed for bothallosteric modulator binding properties and immunoprecipitated and analyzed for subunit phosphorylation levels. A significant increase in allosteric-modulator binding of the GABAAR was observed under conditions maximal for CaM kinase II activation. In addition, CaM kinase II activation resulted in a direct increase in phosphorylation of the GABAA receptor alpha1 subunit. The data suggest that the CaM kinase II-dependent phosphorylation of the GABAA receptor alpha1 subunit modulated allosteric modulator binding to the GABAA receptor.

  6. RHGF-2 Is an Essential Rho-1 Specific RhoGEF that binds to the Multi-PDZ Domain Scaffold Protein MPZ-1 in Caenorhabditis elegans

    PubMed Central

    Lin, Li; Tran, Thuy; Hu, Shuang; Cramer, Todd; Komuniecki, Richard; Steven, Robert M.

    2012-01-01

    RhoGEF proteins activate the Rho family of small GTPases and thus play a key role in regulating fundamental cellular processes such as cell morphology and polarity, cell cycle progression and gene transcription. We identified a Caenorhabditis elegans RhoGEF protein, RHGF-2, as a binding partner of the C. elegans multi-PDZ domain scaffold protein MPZ-1 (MUPP1 in mammals). RHGF-2 exhibits significant identity to the mammalian RhoGEFs PLEKHG5/Tech/Syx and contains a class I C-terminal PDZ binding motif (SDV) that interacts most strongly to MPZ-1 PDZ domain eight. RHGF-2 RhoGEF activity is specific to the C. elegans RhoA homolog RHO-1 as determined by direct binding, GDP/GTP exchange and serum response element-driven reporter activity. rhgf-2 is an essential gene since rhgf-2 deletion mutants do not elongate during embryogenesis and hatch as short immobile animals that arrest development. Interestingly, the expression of a functional rhgf-2::gfp transgene appears to be exclusively neuronal and rhgf-2 overexpression results in loopy movement with exaggerated body bends. Transient expression of RHGF-2 in N1E-115 neuroblastoma cells prevents neurite outgrowth similar to constitutive RhoA activation in these cells. Together, these observations indicate neuronally expressed RHGF-2 is an essential RHO-1 specific RhoGEF that binds most strongly to MPZ-1 PDZ domain eight and is required for wild-type C. elegans morphology and growth. PMID:22363657

  7. Decreased GABA receptor in the cerebral cortex of epileptic rats: effect of Bacopa monnieri and Bacoside-A

    PubMed Central

    2012-01-01

    Abstact Background Gamma amino butyric acid (GABA), the principal inhibitory neurotransmitter in the cerebral cortex, maintains the inhibitory tones that counter balances neuronal excitation. When this balance is perturbed, seizures may ensue. Methods In the present study, alterations of the general GABA, GABAA and GABAB receptors in the cerebral cortex of the epileptic rat and the therapeutic application of Bacopa monnieri were investigated. Results Scatchard analysis of [3H]GABA, [3H]bicuculline and [3H]baclofen in the cerebral cortex of the epileptic rat showed significant decrease in Bmax (P < 0.001) compared to control. Real Time PCR amplification of GABA receptor subunits such as GABAAά1, GABAAγ, GABAAδ, GABAB and GAD where down regulated (P < 0.001) in epileptic rats. GABAAά5 subunit and Cyclic AMP responsible element binding protein were up regulated. Confocal imaging study confirmed the decreased GABA receptors in epileptic rats. Epileptic rats have deficit in radial arm and Y maze performance. Conclusions Bacopa monnieri and Bacoside-A treatment reverses epilepsy associated changes to near control suggesting that decreased GABA receptors in the cerebral cortex have an important role in epileptic occurrence; Bacopa monnieri and Bacoside-A have therapeutic application in epilepsy management. PMID:22364254

  8. Activated RhoA Is a Positive Feedback Regulator of the Lbc Family of Rho Guanine Nucleotide Exchange Factor Proteins*

    PubMed Central

    Medina, Frank; Carter, Angela M.; Dada, Olugbenga; Gutowski, Stephen; Hadas, Jana; Chen, Zhe; Sternweis, Paul C.

    2013-01-01

    The monomeric Rho GTPases are essential for cellular regulation including cell architecture and movement. A direct mechanism for hormonal regulation of the RhoA-type GTPases is their modulation by the G12 and G13 proteins via RH (RGS homology) containing RhoGEFs. In addition to the interaction of the G protein α subunits with the RH domain, activated RhoA also binds to the pleckstrin homology (PH) domain of PDZRhoGEF. The latter interaction is now extended to all seven members of the homologous Lbc family of RhoGEFs which includes the RH-RhoGEFs. This is evinced by direct measurements of binding or through effects on selected signaling pathways in cells. Overexpression of these PH domains alone can block RhoA-dependent signaling in cells to various extents. Whereas activated RhoA does not modulate the intrinsic activity of the RhoGEFs, activated RhoA associated with phospholipid vesicles can facilitate increased activity of soluble RhoGEFs on vesicle-delimited substrate (RhoA-GDP). This demonstrates feasibility of the hypothesis that binding of activated RhoA to the PH domains acts as a positive feedback mechanism. This is supported by cellular studies in which mutation of this binding site on PH strongly attenuates the stimulation of RhoA observed by overexpression of five of the RhoGEF DH-PH domains. This mutation is even more dramatic in the context of full-length p115RhoGEF. The utilization of this mechanism by multiple RhoGEFs suggests that this regulatory paradigm may be a common feature in the broader family of RhoGEFs. PMID:23493395

  9. The RhoGEF Net1 Is Required for Normal Mammary Gland Development

    PubMed Central

    Zuo, Yan; Berdeaux, Rebecca

    2014-01-01

    Neuroepithelial transforming gene 1 (Net1) is a RhoA subfamily-specific guanine nucleotide exchange factor that is overexpressed in human breast cancer and is required for breast cancer cell migration and invasion. However, the role of Net1 in normal mammary gland development or function has never been assessed. To understand the role of Net1 in the mammary gland, we have created a conditional Net1 knockout mouse model. Whole-body deletion of Net1 results in delayed mammary gland development during puberty characterized by slowed of ductal extension and reduced ductal branching. Epithelial cells within the developing ducts show reduced proliferation that is accompanied by diminished estrogen receptor-α expression and activity. Net1-deficient mammary glands also exhibit reduced phosphorylation of regulatory subunits of myosin light chain and myosin light-chain phosphatase, indicating that RhoA-dependent actomyosin contraction is compromised. Net1 deficiency also leads to disorganization of myoepithelial and ductal epithelial cells and increased periductal collagen deposition. Mammary epithelial cell transplantation experiments indicate that reduced ductal branching and disorganization are cell autonomous. These data identify for the first time a role for NET1 in vivo and indicate that NET1 expression is essential for the proliferation and differentiation of mammary epithelial cells in the developing mammary gland. PMID:25321414

  10. Fast detection of extrasynaptic GABA with a whole-cell sniffer

    PubMed Central

    Christensen, Rasmus K.; Petersen, Anders V.; Schmitt, Nicole; Perrier, Jean-François

    2014-01-01

    Gamma-amino-butyric acid (GABA) is the main inhibitory transmitter of the brain. It operates by binding to specific receptors located both inside and outside synapses. The extrasynaptic receptors are activated by spillover from GABAergic synapses and by ambient GABA in the extracellular space. Ambient GABA is essential for adjusting the excitability of neurons. However, due to the lack of suitable methods, little is known about its dynamics. Here we describe a new technique that allows detection of GABA transients and measurement of the steady state GABA concentration with high spatial and temporal resolution. We used a human embryonic kidney (HEK) cell line that stably expresses GABAA receptors composed of α1, β2, and γ2 subunits. We recorded from such a HEK cell with the whole-cell patch-clamp technique. The presence of GABA near the HEK cell generated a measurable electric current whose magnitude increased with concentration. A fraction of the current did not inactivate during prolonged exposition to GABA. This technique, which we refer to as a “sniffer” allows the measurement of ambient GABA concentration inside nervous tissue with a resolution of few tens of nanomolars. In addition, the sniffer detects variations in the extrasynaptic GABA concentration with millisecond time resolution. Pilot experiments demonstrate that the sniffer is able to report spillover of GABA induced by synaptic activation in real time. This is the first report on a GABA sensor that combines the ability to detect fast transients and to measure steady concentrations. PMID:24860433

  11. Inhibitory effect of rhynchophylline on contraction of cerebral arterioles to endothelin 1: role of rho kinase.

    PubMed

    Hao, Hui-Feng; Liu, Li-Mei; Liu, Yu-Ying; Liu, Juan; Yan, Li; Pan, Chun-Shui; Wang, Ming-Xia; Wang, Chuan-She; Fan, Jing-Yu; Gao, Yuan-Sheng; Han, Jing-Yan

    2014-08-08

    Rhynchophylline (Rhy) is a major ingredient of Uncaria rhynchophylla (UR) used to reduce blood pressure and ameliorate brain ailments. This study was to examine the role of Rho kinase (ROCK) in the inhibition of Rhy on contraction of cerebral arterioles caused by endothelin 1 (ET-1). Cerebral arterioles of male Wistar rats were constricted with ET-1 for 10 min followed by perfusion of Rhy for 20 min. Changes in the diameters of the arterioles were recorded. The effects of Rhy on contraction of middle cerebral arteries (MCAs) were determined by a Multi-Myograph. Western blotting and immunofluorescent staining were used to examine the effects of Rhy on RhoA translocation and myosin phosphatase target subunit 1 (MYPT1) phosphorylation. In vivo, Rhy (30-300 µM) relaxed cerebral arterioles constricted with ET-1 dose-dependently. In vitro, Rhy at lower concentrations (1-100 µM) caused relaxation of rat MCAs constricted with KCl and Bay-K8644 (an agonist of L-type voltage-dependent calcium channels (L-VDCCs)). Rhy at higher concentrations (>100 µM) caused relaxation of rat MCAs constricted with ET-1, which was inhibited by Y27632, a ROCK׳s inhibitor. Western blotting of rat aortas showed that Rhy inhibited RhoA translocation and MYPT1 phosphorylation. Immunofluorescent staining of MCAs confirmed that phosphorylation of MYPT1 caused by ET-1 was inhibited by Rhy. These results demonstrate that Rhy is a potent inhibitor of contraction of cerebral arteries caused by ET-1 in vivo and in vitro. The effect of Rhy was in part mediated by inhibiting RhoA-ROCK signaling. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  12. GABA metabolism pathway genes, UGA1 and GAD1, regulate replicative lifespan in Saccharomycescerevisiae

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Kamei, Yuka; Tamura, Takayuki; Yoshida, Ryo

    2011-04-01

    Highlights: {yields}We demonstrate that two genes in the yeast GABA metabolism pathway affect aging. {yields} Deletion of the UGA1 or GAD1 genes extends replicative lifespan. {yields} Addition of GABA to wild-type cultures has no effect on lifespan. {yields} Intracellular GABA levels do not differ in longevity mutants and wild-type cells. {yields} Levels of tricarboxylic acid cycle intermediates positively correlate with lifespan. -- Abstract: Many of the genes involved in aging have been identified in organisms ranging from yeast to human. Our previous study showed that deletion of the UGA3 gene-which encodes a zinc-finger transcription factor necessary for {gamma}-aminobutyric acid (GABA)-dependentmore » induction of the UGA1 (GABA aminotransferase), UGA2 (succinate semialdehyde dehydrogenase), and UGA4 (GABA permease) genes-extends replicative lifespan in the budding yeast Saccharomycescerevisiae. Here, we found that deletion of UGA1 lengthened the lifespan, as did deletion of UGA3; in contrast, strains with UGA2 or UGA4 deletions exhibited no lifespan extension. The {Delta}uga1 strain cannot deaminate GABA to succinate semialdehyde. Deletion of GAD1, which encodes the glutamate decarboxylase that converts glutamate into GABA, also increased lifespan. Therefore, two genes in the GABA metabolism pathway, UGA1 and GAD1, were identified as aging genes. Unexpectedly, intracellular GABA levels in mutant cells (except for {Delta}uga2 cells) did not differ from those in wild-type cells. Addition of GABA to culture media, which induces transcription of the UGA structural genes, had no effect on replicative lifespan of wild-type cells. Multivariate analysis of {sup 1}H nuclear magnetic resonance spectra for the whole-cell metabolite levels demonstrated a separation between long-lived and normal-lived strains. Gas chromatography-mass spectrometry analysis of identified metabolites showed that levels of tricarboxylic acid cycle intermediates positively correlated with

  13. RhoG regulates anoikis through a phosphatidylinositol 3-kinase-dependent mechanism

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Yamaki, Nao; Negishi, Manabu; Katoh, Hironori

    2007-08-01

    In normal epithelial cells, cell-matrix interaction is required for cell survival and proliferation, whereas disruption of this interaction causes epithelial cells to undergo apoptosis called anoikis. Here we show that the small GTPase RhoG plays an important role in the regulation of anoikis. HeLa cells are capable of anchorage-independent cell growth and acquire resistance to anoikis. We found that RNA interference-mediated knockdown of RhoG promoted anoikis in HeLa cells. Previous studies have shown that RhoG activates Rac1 and induces several cellular functions including promotion of cell migration through its effector ELMO and the ELMO-binding protein Dock180 that function as amore » Rac-specific guanine nucleotide exchange factor. However, RhoG-induced suppression of anoikis was independent of the ELMO- and Dock180-mediated activation of Rac1. On the other hand, the regulation of anoikis by RhoG required phosphatidylinositol 3-kinase (PI3K) activity, and constitutively active RhoG bound to the PI3K regulatory subunit p85{alpha} and induced the PI3K-dependent phosphorylation of Akt. Taken together, these results suggest that RhoG protects cells from apoptosis caused by the loss of anchorage through a PI3K-dependent mechanism, independent of its activation of Rac1.« less

  14. GABA-A receptor beta3 and alpha5 subunit gene cluster on chromosome 15q11-q13 and bipolar disorder: a genetic association study.

    PubMed

    Papadimitriou, G N; Dikeos, D G; Karadima, G; Avramopoulos, D; Daskalopoulou, E G; Stefanis, C N

    2001-05-08

    There is accumulated evidence that the genes coding for the receptor of gamma aminobutyric acid (GABA), the most important inhibitory neurotransmitter in the CNS, may be involved in the pathogenesis of affective disorders. In a previous study, we have found a genetic association between the GABA-A receptor alpha5 subunit gene locus (GABRA5) on chromosome 15q11-of 13 and bipolar affective disorder. The aim of the present study was to examine the same subjects to see if there exists a genetic association between bipolar affective disorder and the GABA receptor beta3 subunit gene (GABRB3), which is located within 100 kb from GABRA5. The sample consisted of 48 bipolar patients compared to 44 controls (blood donors). All subjects were Greek, unrelated, and personally interviewed. Diagnosis was based on DSM-IV and ICD-10 criteria. The marker used was a dinucleotide (CA) repeat polymorphism with 12 alleles 179 to 201 bp long; genotyping was successful in all patients and 43 controls. The distribution of GABRB3 genotypes among the controls did not deviate significantly from the Hardy-Weinberg equilibrium. No differences in allelic frequencies between bipolar patients and controls were found for GABRB3, while this locus and GABRA5 did not seem to be in significant linkage disequilibrium. In conclusion, the GABRB3 CA-repeat polymorphism we investigated does not present the observed association between bipolar affective illness and GABRA5. This could be due to higher mutation rate in the GABRB3 CA-repeat polymorphism, but it might also signify that GABRA5 is the gene actually associated with the disease. Copyright 2001 Wiley-Liss, Inc.

  15. Control of cortical neuronal migration by glutamate and GABA

    PubMed Central

    Luhmann, Heiko J.; Fukuda, A.; Kilb, W.

    2015-01-01

    Neuronal migration in the cortex is controlled by the paracrine action of the classical neurotransmitters glutamate and GABA. Glutamate controls radial migration of pyramidal neurons by acting primarily on NMDA receptors and regulates tangential migration of inhibitory interneurons by activating non-NMDA and NMDA receptors. GABA, acting on ionotropic GABAA-rho and GABAA receptors, has a dichotomic action on radially migrating neurons by acting as a GO signal in lower layers and as a STOP signal in upper cortical plate (CP), respectively. Metabotropic GABAB receptors promote radial migration into the CP and tangential migration of interneurons. Besides GABA, the endogenous GABAergic agonist taurine is a relevant agonist controlling radial migration. To a smaller extent glycine receptor activation can also influence radial and tangential migration. Activation of glutamate and GABA receptors causes increases in intracellular Ca2+ transients, which promote neuronal migration by acting on the cytoskeleton. Pharmacological or genetic manipulation of glutamate or GABA receptors during early corticogenesis induce heterotopic cell clusters in upper layers and loss of cortical lamination, i.e., neuronal migration disorders which can be associated with neurological or neuropsychiatric diseases. The pivotal role of NMDA and ionotropic GABA receptors in cortical neuronal migration is of major clinical relevance, since a number of drugs acting on these receptors (e.g., anti-epileptics, anesthetics, alcohol) may disturb the normal migration pattern when present during early corticogenesis. PMID:25688185

  16. Parafascicular thalamic nucleus deep brain stimulation decreases NMDA receptor GluN1 subunit gene expression in the prefrontal cortex.

    PubMed

    Fernández-Cabrera, Mónica R; Selvas, Abraham; Miguéns, Miguel; Higuera-Matas, Alejandro; Vale-Martínez, Anna; Ambrosio, Emilio; Martí-Nicolovius, Margarita; Guillazo-Blanch, Gemma

    2017-04-21

    The rodent parafascicular nucleus (PFn) or the centromedian-parafascicular complex of primates is a posterior intralaminar nucleus of the thalamus related to cortical activation and maintenance of states of consciousness underlying attention, learning and memory. Deep brain stimulation (DBS) of the PFn has been proved to restore arousal and consciousness in humans and to enhance performance in learning and memory tasks in rats. The primary expected effect of PFn DBS is to induce plastic changes in target neurons of brain areas associated with cognitive function. In this study, Wistar rats were stimulated for 20mins in the PFn following a DBS protocol that had previously facilitated memory in rats. NMDA and GABA B receptor binding, and gene expression of the GluN1subunit of the NMDA receptor (NMDAR) were assessed in regions related to cognitive functions, such as the prefrontal cortex and hippocampus. The results showed that PFn DBS induced a decrease in NMDAR GluN1 subunit gene expression in the cingulate and prelimbic cortices, but no significant statistical differences were found in the density of NMDA or GABA B receptors in any of the analyzed regions. Taken together, our findings suggest a possible role for the NMDAR GluN1 subunit in the prefrontal cortex in the procognitive actions of the PFn DBS. Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.

  17. Aldehyde dehydrogenase 1a1 mediates a GABA synthesis pathway in midbrain dopaminergic neurons.

    PubMed

    Kim, Jae-Ick; Ganesan, Subhashree; Luo, Sarah X; Wu, Yu-Wei; Park, Esther; Huang, Eric J; Chen, Lu; Ding, Jun B

    2015-10-02

    Midbrain dopamine neurons are an essential component of the basal ganglia circuitry, playing key roles in the control of fine movement and reward. Recently, it has been demonstrated that γ-aminobutyric acid (GABA), the chief inhibitory neurotransmitter, is co-released by dopamine neurons. Here, we show that GABA co-release in dopamine neurons does not use the conventional GABA-synthesizing enzymes, glutamate decarboxylases GAD65 and GAD67. Our experiments reveal an evolutionarily conserved GABA synthesis pathway mediated by aldehyde dehydrogenase 1a1 (ALDH1a1). Moreover, GABA co-release is modulated by ethanol (EtOH) at concentrations seen in blood alcohol after binge drinking, and diminished ALDH1a1 leads to enhanced alcohol consumption and preference. These findings provide insights into the functional role of GABA co-release in midbrain dopamine neurons, which may be essential for reward-based behavior and addiction. Copyright © 2015, American Association for the Advancement of Science.

  18. Rapid Substrate-Induced Charge Movements of the GABA Transporter GAT1

    PubMed Central

    Bicho, Ana; Grewer, Christof

    2005-01-01

    The GABA transporter GAT1 removes the neurotransmitter GABA from the synaptic cleft by coupling of GABA uptake to the co-transport of two sodium ions and one chloride ion. The aim of this work was to investigate the individual reaction steps of GAT1 after a GABA concentration jump. GAT1 was transiently expressed in HEK293 cells and its pre-steady-state kinetics were studied by combining the patch-clamp technique with the laser-pulse photolysis of caged GABA, which allowed us to generate GABA concentration jumps within <100 μs. Recordings of transport currents generated by GAT1, both in forward and exchange transport modes, showed multiple charge movements that can be separated along the time axis. The individual reactions associated with these charge movements differ from the well-characterized electrogenic “sodium-occlusion” reaction by GAT1. One of the observed electrogenic reactions is shown to be associated with the GABA-translocating half-cycle of the transporter, in contradiction to previous studies that showed no charge movements associated with these reactions. Interestingly, reactions of the GABA-bound transporter were not affected by the absence of extracellular chloride, suggesting that Cl− may not be co-translocated with GABA. Based on the results, a new alternating access sequential-binding model is proposed for GAT1's transport cycle that describes the results presented here and those by others. PMID:15849242

  19. RhoA/Rho-Kinase in the Cardiovascular System.

    PubMed

    Shimokawa, Hiroaki; Sunamura, Shinichiro; Satoh, Kimio

    2016-01-22

    Twenty years ago, Rho-kinase was identified as an important downstream effector of the small GTP-binding protein, RhoA. Thereafter, a series of studies demonstrated the important roles of Rho-kinase in the cardiovascular system. The RhoA/Rho-kinase pathway is now widely known to play important roles in many cellular functions, including contraction, motility, proliferation, and apoptosis, and its excessive activity induces oxidative stress and promotes the development of cardiovascular diseases. Furthermore, the important role of Rho-kinase has been demonstrated in the pathogenesis of vasospasm, arteriosclerosis, ischemia/reperfusion injury, hypertension, pulmonary hypertension, and heart failure. Cyclophilin A is secreted by vascular smooth muscle cells and inflammatory cells and activated platelets in a Rho-kinase-dependent manner, playing important roles in a wide range of cardiovascular diseases. Thus, the RhoA/Rho-kinase pathway plays crucial roles under both physiological and pathological conditions and is an important therapeutic target in cardiovascular medicine. Recently, functional differences between ROCK1 and ROCK2 have been reported in vitro. ROCK1 is specifically cleaved by caspase-3, whereas granzyme B cleaves ROCK2. However, limited information is available on the functional differences and interactions between ROCK1 and ROCK2 in the cardiovascular system in vivo. Herein, we will review the recent advances about the importance of RhoA/Rho-kinase in the cardiovascular system. © 2016 American Heart Association, Inc.

  20. The adjustment of γ-aminobutyric acidA tonic subunits in Huntington's disease: from transcription to translation to synaptic levels into the neostriatum.

    PubMed

    Rosas-Arellano, Abraham; Estrada-Mondragón, Argel; Mantellero, Carola A; Tejeda-Guzmán, Carlos; Castro, Maite A

    2018-04-01

    γ-Aminobutyric acid (GABA), plays a key role in all stages of life, also is considered the main inhibitory neurotransmitter. GABA activates two kind of membrane receptors known as GABA A and GABA B , the first one is responsible to render tonic inhibition by pentameric receptors containing α4-6, β3, δ, or ρ1-3 subunits, they are located at perisynaptic and/or in extrasynaptic regions. The biophysical properties of GABA A tonic inhibition have been related with cellular protection against excitotoxic injury and cell death in presence of excessive excitation. On this basis, GABA A tonic inhibition has been proposed as a potential target for therapeutic intervention of Huntington's disease. Huntington's disease is a neurodegenerative disorder caused by a genetic mutation of the huntingtin protein. For experimental studies of Huntington's disease mouse models have been developed, such as R6/1, R6/2, HdhQ92, HdhQ150, as well as YAC128. In all of them, some key experimental reports are focused on neostriatum. The neostriatum is considered as the most important connection between cerebral cortex and basal ganglia structures, its cytology display two pathways called direct and indirect constituted by medium sized spiny neurons expressing dopamine D1 and D2 receptors respectively, they display strong expression of many types of GABA A receptors, including tonic subunits. The studies about of GABA A tonic subunits and Huntington's disease into the neostriatum are rising in recent years, suggesting interesting changes in their expression and localization which can be used as a strategy to delay the cellular damage caused by the imbalance between excitation and inhibition, a hallmark of Huntington's disease.

  1. RhoA and RhoC are involved in stromal cell-derived factor-1-induced cell migration by regulating F-actin redistribution and assembly.

    PubMed

    Luo, Jixian; Li, Dingyun; Wei, Dan; Wang, Xiaoguang; Wang, Lan; Zeng, Xianlu

    2017-12-01

    Stromal cell-derived factor-1 (SDF-1) signaling is important to the maintenance and progression of T-cell acute lymphoblastic leukemia by inducing chemotaxis migration. To identify the mechanism of SDF-1 signaling in the migration of T-ALL, Jurkat acute lymphoblastic leukemia cells were used. Results showed that SDF-1 induces Jurkat cell migration by F-actin redistribution and assembly, which is dependent on Rho activity. SDF-1 induced RhoA and RhoC activation, as well as reactive oxygen species (ROS) production, which was inhibited by Rho inhibitor. The Rho-dependent ROS production led to subsequent cytoskeleton redistribution and assembly in the process of migration. Additionally, RhoA and RhoC were involved in SDF-1-induced Jurkat cell migration. Taken together, we found a SDF-1/CXCR4-RhoA and RhoC-ROS-cytoskeleton pathway that regulates Jurkat cell migration in response to SDF-1. This work will contribute to a clearer insight into the migration mechanism of acute lymphoblastic leukemia.

  2. Increased GABA-A receptor binding and reduced connectivity at the motor cortex in children with hemiplegic cerebral palsy: a multimodal investigation using 18F-fluoroflumazenil PET, immunohistochemistry, and MR imaging.

    PubMed

    Park, Hae-Jeong; Kim, Chul Hoon; Park, Eun Sook; Park, Bumhee; Oh, So Ra; Oh, Maeng-Keun; Park, Chang Il; Lee, Jong Doo

    2013-08-01

    γ-aminobutyric acid (GABA)-A receptor-mediated neural transmission is important to promote practice-dependent plasticity after brain injury. This study investigated alterations in GABA-A receptor binding and functional and anatomic connectivity within the motor cortex in children with cerebral palsy (CP). We conducted (18)F-fluoroflumazenil PET on children with hemiplegic CP to investigate whether in vivo GABA-A receptor binding is altered in the ipsilateral or contralateral hemisphere of the lesion site. To evaluate changes in the GABA-A receptor subunit after prenatal brain injury, we performed GABA-A receptor immunohistochemistry using rat pups with a diffuse hypoxic ischemic insult. We also performed diffusion tensor MR imaging and resting-state functional MR imaging on the same children with hemiplegic CP to investigate alterations in anatomic and functional connectivity at the motor cortex with increased GABA-A receptor binding. In children with hemiplegic CP, the (18)F-fluoroflumazenil binding potential was increased within the ipsilateral motor cortex. GABA-A receptors with the α1 subunit were highly expressed exclusively within cortical layers III, IV, and VI of the motor cortex in rat pups. The motor cortex with increased GABA-A receptor binding in children with hemiplegic CP had reduced thalamocortical and corticocortical connectivity, which might be linked to increased GABA-A receptor distribution in cortical layers in rats. Increased expression of the GABA-A receptor α1 subunit within the ipsilateral motor cortex may be an important adaptive mechanism after prenatal brain injury in children with CP but may be associated with improper functional connectivity after birth and have adverse effects on the development of motor plasticity.

  3. Characteristics of concatemeric GABAA receptors containing α4/δ subunits expressed in Xenopus oocytes

    PubMed Central

    Shu, Hong-Jin; Bracamontes, John; Taylor, Amanda; Wu, Kyle; Eaton, Megan M; Akk, Gustav; Manion, Brad; Evers, Alex S; Krishnan, Kathiresan; Covey, Douglas F; Zorumski, Charles F; Steinbach, Joe Henry; Mennerick, Steven

    2012-01-01

    BACKGROUND AND PURPOSE GABAA receptors mediate both synaptic and extrasynaptic actions of GABA. In several neuronal populations, α4 and δ subunits are key components of extrasynaptic GABAA receptors that strongly influence neuronal excitability and could mediate the effects of neuroactive agents including neurosteroids and ethanol. However, these receptors can be difficult to study in native cells and recombinant δ subunits can be difficult to express in heterologous systems. EXPERIMENTAL APPROACH We engineered concatemeric (fused) subunits to ensure δ and α4 subunit expression. We tested the pharmacology of the concatemeric receptors, compared with a common synaptic-like receptor subunit combination (α1 +β2 +γ2L), and with free-subunit α4/δ receptors, expressed in Xenopus oocytes. KEY RESULTS δ-β2 −α4 +β2-α4 cRNA co-injected into Xenopus oocytes resulted in GABA-gated currents with the expected pharmacological properties of α4/δ-containing receptors. Criteria included sensitivity to agonists of different efficacy, sensitivity to the allosteric activator pentobarbital, and modulation of agonist responses by DS2 (4-chloro-N-[2-(2-thienyl)imidazo[1,2-a]pyridine-3-yl benzamide; a δ-selective positive modulator), furosemide, and Zn2+. We used the concatemers to examine neurosteroid sensitivity of extrasynaptic-like, δ-containing receptors. We found no qualitative differences between extrasynaptic-like receptors and synaptic-like receptors in the actions of either negative or positive neurosteroid modulators of receptor function. Quantitative differences were explained by the partial agonist effects of the natural agonist GABA and by a mildly increased sensitivity to low steroid concentrations. CONCLUSIONS AND IMPLICATIONS The neurosteroid structure-activity profile for α4/δ-containing extrasynaptic receptors is unlikely to differ from that of synaptic-like receptors such as α1/β2/γ2-containing receptors. PMID:21950777

  4. Behavioural endophenotypes in mice lacking the auxiliary GABAB receptor subunit KCTD16.

    PubMed

    Cathomas, Flurin; Sigrist, Hannes; Schmid, Luca; Seifritz, Erich; Gassmann, Martin; Bettler, Bernhard; Pryce, Christopher R

    2017-01-15

    Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the brain and is implicated in the pathophysiology of a number of neuropsychiatric disorders. The GABA B receptors are G-protein coupled receptors consisting of principle subunits and auxiliary potassium channel tetramerization domain (KCTD) subunits. The KCTD subunits 8, 12, 12b and 16 are cytosolic proteins that determine the kinetics of the GABA B receptor response. Previously, we demonstrated that Kctd12 null mutant mice (Kctd12 -/- ) exhibit increased auditory fear learning and that Kctd12 +/- mice show altered circadian activity, as well as increased intrinsic excitability in hippocampal pyramidal neurons. KCTD16 has been demonstrated to influence neuronal excitability by regulating GABA B receptor-mediated gating of postsynaptic ion channels. In the present study we investigated for behavioural endophenotypes in Kctd16 -/- and Kctd16 +/- mice. Compared with wild-type (WT) littermates, auditory and contextual fear conditioning were normal in both Kctd16 -/- and Kctd16 +/- mice. When fear memory was tested on the following day, Kctd16 -/- mice exhibited less extinction of auditory fear memory relative to WT and Kctd16 +/- mice, as well as more contextual fear memory relative to WT and, in particular, Kctd16 +/- mice. Relative to WT, both Kctd16 +/- and Kctd16 -/- mice exhibited normal circadian activity. This study adds to the evidence that auxillary KCTD subunits of GABA B receptors contribute to the regulation of behaviours that could constitute endophenotypes for hyper-reactivity to aversive stimuli in neuropsychiatric disorders. Copyright © 2016 Elsevier B.V. All rights reserved.

  5. The RhoU/Wrch1 Rho GTPase gene is a common transcriptional target of both the gp130/STAT3 and Wnt-1 pathways

    PubMed Central

    SCHIAVONE, Davide; DEWILDE, Sarah; VALLANIA, Francesco; TURKSON, James; CUNTO, Ferdinando DI; POLI, Valeria

    2010-01-01

    STAT3 (signal transducer and activator of transcription 3) is a transcription factor activated by cytokines, growth factors and oncogenes, whose activity is required for cell survival/proliferation of a wide variety of primary tumours and tumour cell lines. Prominent among its multiple effects on tumour cells is the stimulation of cell migration and metastasis, whose functional mechanisms are however not completely characterized. RhoU/Wrch1 (Wnt-responsive Cdc42 homologue) is an atypical Rho GTPase thought to be constitutively bound to GTP. RhoU was first identified as a Wnt-1-inducible mRNA and subsequently shown to act on the actin cytoskeleton by stimulating filopodia formation and stress fibre dissolution. It was in addition recently shown to localize to focal adhesions and to Src-induced podosomes and enhance cell migration. RhoU overexpression in mammary epithelial cells stimulates quiescent cells to re-enter the cell cycle and morphologically phenocopies Wnt-1-dependent transformation. In the present study we show that Wnt-1-mediated RhoU induction occurs at the transcriptional level. Moreover, we demonstrate that RhoU can also be induced by gp130 cytokines via STAT3, and we identify two functional STAT3-binding sites on the mouse RhoU promoter. RhoU induction by Wnt-1 is independent of β-catenin, but does not involve STAT3. Rather, it is mediated by the Wnt/planar cell polarity pathway through the activation of JNK (c-Jun N-terminal kinase). Both the so-called non-canonical Wnt pathway and STAT3 are therefore able to induce RhoU, which in turn may be involved in mediating their effects on cell migration. PMID:19397496

  6. Behavioral deficit and decreased GABA receptor functional regulation in the hippocampus of epileptic rats: effect of Bacopa monnieri.

    PubMed

    Mathew, Jobin; Gangadharan, Gireesh; Kuruvilla, Korah P; Paulose, C S

    2011-01-01

    In the present study, alterations of the General GABA and GABA(A) receptors in the hippocampus of pilocarpine-induced temporal lobe epileptic rats and the therapeutic application of Bacopa monnieri and its active component Bacoside-A were investigated. Bacopa monnieri (Linn.) is a herbaceous plant belonging to the family Scrophulariaceae. Hippocampus is the major region of the brain belonging to the limbic system and plays an important role in epileptogenesis, memory and learning. Scatchard analysis of [³H]GABA and [³H]bicuculline in the hippocampus of the epileptic rat showed significant decrease in B(max) (P < 0.001) compared to control. Real Time PCR amplification of GABA(A) receptor sub-units such as GABA(Aά₁), GABA(Aά₅) GABA(Aδ), and GAD were down regulated (P < 0.001) in the hippocampus of the epileptic rats compared to control. GABA(Aγ) subunit was up regulated. Epileptic rats have deficit in the radial arm and Y maze performance. Bacopa monnieri and Bacoside-A treatment reverses all these changes near to control. Our results suggest that decreased GABA receptors in the hippocampus have an important role in epilepsy associated behavioral deficit, Bacopa monnieri and Bacoside-A have clinical significance in the management of epilepsy.

  7. Carbachol-Induced Signaling through Thr696-Phosphorylation of Myosin Phosphatase Targeting Subunit 1 (MYPT1) in rat Bladder Smooth Muscle Cells

    PubMed Central

    Alwaal, Amjad; Wang, Guifang; Banie, Lia; Lin, Ching-Shwun; Lin, Guiting; Lue, Tom F.

    2016-01-01

    Purpose Lines of evidence suggest that Rho-associated protein kinase (ROCK) mediated myosin phosphatase targeting subunit 1 (MYPT1) phosphorylation play a central role in smooth muscle contraction. However, the physiological significance of MYPT1 phosphorylation at Thr696 catalyzed by ROCK in bladder smooth muscle remains controversial. We attempt to directly observe the quantitative protein expression of RhoA/ROCK and phosphorylation of MYPT1 at Thr696 after carbachol administration in rat bladder smooth muscle cells (RBMSCs). Materials and Methods Primary cultured smooth muscle cells were obtained from rat bladders. The effects of both concentration and time-course induced by the muscarinic agonist carbachol were investigated by assessing the expression of Rho A/ROCK and MYPT1 phosphorylation at Thr696 using Western blot. Results In the dose-course studies, carbachol showed significant increase of phosphorylation of MYPT1 at Thr696 (p-MYPT1) from concentrations of 15 μM to 100 μM based on Western blot results (p < 0.05, ANOVA test). In the time-course studies, treatment of cells with 15 μM of carbachol significantly enhanced the expression of p-MYPT1 from 3 to 15 hr (p < 0.05, ANOVA test) and induced the expression of Rho A from 10 to 120 min (p < 0.05, ANOVA test). Conclusions Carbachol can induce the expression of ROCK pathway, leading to MYPT1 phosphorylation at Thr696 and thereby sustained RBSMCs contraction. PMID:27118568

  8. Carbachol-induced signaling through Thr696-phosphorylation of myosin phosphatase-targeting subunit 1 (MYPT1) in rat bladder smooth muscle cells.

    PubMed

    Liu, Benchun; Lee, Yung-Chin; Alwaal, Amjad; Wang, Guifang; Banie, Lia; Lin, Ching-Shwun; Lin, Guiting; Lue, Tom F

    2016-08-01

    Lines of evidence suggest that Rho-associated protein kinase (ROCK)-mediated myosin phosphatase-targeting subunit 1 (MYPT1) phosphorylation plays a central role in smooth muscle contraction. However, the physiological significance of MYPT1 phosphorylation at Thr696 catalyzed by ROCK in bladder smooth muscle remains controversial. We attempt to directly observe the quantitative protein expression of Rho A/ROCK and phosphorylation of MYPT1 at Thr696 after carbachol administration in rat bladder smooth muscle cells (RBMSCs). Primary cultured smooth muscle cells were obtained from rat bladders. The effects of both concentration and time-course induced by the muscarinic agonist carbachol were investigated by assessing the expression of Rho A/ROCK and MYPT1 phosphorylation at Thr696 using Western blot. In the dose-course studies, carbachol showed significant increase in phosphorylation of MYPT1 at Thr696 (p-MYPT1) from concentrations of 15-100 μM based on Western blot results (p < 0.05, ANOVA test). In the time-course studies, treatment of cells with 15 μM of carbachol significantly enhanced the expression of p-MYPT1 from 3 to 15 h (p < 0.05, ANOVA test) and induced the expression of Rho A from 10 to 120 min (p < 0.05, ANOVA test). Carbachol can induce the expression of ROCK pathway, leading to MYPT1 phosphorylation at Thr696 and thereby sustained RBSMCs contraction.

  9. Neuropeptide-stimulated cell migration in prostate cancer cells is mediated by RhoA kinase signaling and inhibited by neutral endopeptidase.

    PubMed

    Zheng, R; Iwase, A; Shen, R; Goodman, O B; Sugimoto, N; Takuwa, Y; Lerner, D J; Nanus, D M

    2006-09-28

    The neuropeptides bombesin and endothelin-1 stimulate prostate cancer (PC) cell migration and invasion (J Clin Invest, 2000; 106: 1399-1407). The intracellular signaling pathways that direct this cell movement are not well delineated. The monomeric GTPase RhoA is required for migration in several cell types including neutrophils, monocytes and fibroblasts. We demonstrate that bombesin-stimulated PC cell migration occurs via the heterotrimeric G-protein-coupled receptors (G-protein) G alpha 13 subunit leading to activation of RhoA, and Rho-associated coiled-coil forming protein kinase (ROCK). Using siRNA to suppress expression of the three known G-protein alpha-subunit-associated RhoA guanine nucleotide exchange factors (GEFs), we also show that two of these RhoA GEFs, PDZ-RhoGEF and leukemia-associated RhoGEF (LARG), link bombesin receptors to RhoA in a non-redundant manner in PC cells. We next show that focal adhesion kinase, which activates PDZ-RhoGEF and LARG, is required for bombesin-stimulated RhoA activation. Neutral endopeptidase (NEP) is expressed on normal prostate epithelium whereas loss of NEP expression contributes to PC progression. We also demonstrate that NEP inhibits neuropeptide activation of RhoA. Together, these results establish a contiguous signaling pathway from the bombesin receptor to ROCK in PC cells, and they implicate NEP as a major regulator of neuropeptide-stimulated RhoA in these cells. This work also identifies members of this signaling pathway as potential targets for rational pharmacologic manipulation of neuropeptide-stimulated migration of PC cells.

  10. Loss of ethanol conditioned taste aversion and motor stimulation in knockin mice with ethanol-insensitive α2-containing GABA(A) receptors.

    PubMed

    Blednov, Y A; Borghese, C M; McCracken, M L; Benavidez, J M; Geil, C R; Osterndorff-Kahanek, E; Werner, D F; Iyer, S; Swihart, A; Harrison, N L; Homanics, G E; Harris, R A

    2011-01-01

    GABA type A receptors (GABA(A)-Rs) are potential targets of ethanol. However, there are multiple subtypes of this receptor, and, thus far, individual subunits have not been definitively linked with specific ethanol behavioral actions. Interestingly, though, a chromosomal cluster of four GABA(A)-R subunit genes, including α2 (Gabra2), was associated with human alcoholism (Am J Hum Genet 74:705-714, 2004; Pharmacol Biochem Behav 90:95-104, 2008; J Psychiatr Res 42:184-191, 2008). The goal of our study was to determine the role of receptors containing this subunit in alcohol action. We designed an α2 subunit with serine 270 to histidine and leucine 277 to alanine mutations that was insensitive to potentiation by ethanol yet retained normal GABA sensitivity in a recombinant expression system. Knockin mice containing this mutant subunit were tested in a range of ethanol behavioral tests. These mutant mice did not develop the typical conditioned taste aversion in response to ethanol and showed complete loss of the motor stimulant effects of ethanol. Conversely, they also demonstrated changes in ethanol intake and preference in multiple tests. The knockin mice showed increased ethanol-induced hypnosis but no difference in anxiolytic effects or recovery from acute ethanol-induced motor incoordination. Overall, these studies demonstrate that the effects of ethanol at GABAergic synapses containing the α2 subunit are important for specific behavioral effects of ethanol that may be relevant to the genetic linkage of this subunit with human alcoholism.

  11. Effects of chronic ethanol consumption on rat GABA(A) and strychnine-sensitive glycine receptors expressed by lateral/basolateral amygdala neurons.

    PubMed

    McCool, Brian A; Frye, Gerald D; Pulido, Marisa D; Botting, Shaleen K

    2003-02-14

    It is well known that the anxiolytic potential of ethanol is maintained during chronic exposure. We have confirmed this using a light-dark box paradigm following chronic ethanol ingestion via a liquid diet. However, cessation from chronic ethanol exposure is known to cause severe withdrawal anxiety. These opposing effects on anxiety likely result from neuro-adaptations of neurotransmitter systems within the brain regions regulating anxiety. Recent work highlights the importance of amygdala ligand-gated chloride channels in the expression of anxiety. We have therefore examined the effects of chronic ethanol exposure on GABA(A) and strychnine-sensitive glycine receptors expressed by acutely isolated adult rat lateral/basolateral amygdala neurons. Chronic ethanol exposure increased the functional expression of GABA(A) receptors in acutely isolated basolateral amygdala neurons without altering strychnine-sensitive glycine receptors. Neither the acute ethanol nor benzodiazepine sensitivity of either receptor system was affected. We explored the likelihood that subunit composition might influence each receptor's response to chronic ethanol. Importantly, when expressed in a mammalian heterologous system, GABA(A) receptors composed of unique alpha subunits were differentially sensitive to acute ethanol. Likewise, the presence of the beta subunit appeared to influence the acute ethanol sensitivity of glycine receptors containing the alpha(2) subunit. Our results suggest that the facilitation of GABA(A) receptors during chronic ethanol exposure may help explain the maintenance of ethanol's anti-anxiety effects during chronic ethanol exposure. Furthermore, the subunit composition of GABA(A) and strychnine-sensitive glycine receptors may ultimately influence the response of each system to chronic ethanol exposure.

  12. Ionotropic GABA and Glutamate Receptor Mutations and Human Neurologic Diseases.

    PubMed

    Yuan, Hongjie; Low, Chian-Ming; Moody, Olivia A; Jenkins, Andrew; Traynelis, Stephen F

    2015-07-01

    The advent of whole exome/genome sequencing and the technology-driven reduction in the cost of next-generation sequencing as well as the introduction of diagnostic-targeted sequencing chips have resulted in an unprecedented volume of data directly linking patient genomic variability to disorders of the brain. This information has the potential to transform our understanding of neurologic disorders by improving diagnoses, illuminating the molecular heterogeneity underlying diseases, and identifying new targets for therapeutic treatment. There is a strong history of mutations in GABA receptor genes being involved in neurologic diseases, particularly the epilepsies. In addition, a substantial number of variants and mutations have been found in GABA receptor genes in patients with autism, schizophrenia, and addiction, suggesting potential links between the GABA receptors and these conditions. A new and unexpected outcome from sequencing efforts has been the surprising number of mutations found in glutamate receptor subunits, with the GRIN2A gene encoding the GluN2A N-methyl-d-aspartate receptor subunit being most often affected. These mutations are associated with multiple neurologic conditions, for which seizure disorders comprise the largest group. The GluN2A subunit appears to be a locus for epilepsy, which holds important therapeutic implications. Virtually all α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mutations, most of which occur within GRIA3, are from patients with intellectual disabilities, suggesting a link to this condition. Similarly, the most common phenotype for kainate receptor variants is intellectual disability. Herein, we summarize the current understanding of disease-associated mutations in ionotropic GABA and glutamate receptor families, and discuss implications regarding the identification of human mutations and treatment of neurologic diseases. Copyright © 2015 by The American Society for Pharmacology and Experimental

  13. Ionotropic GABA and Glutamate Receptor Mutations and Human Neurologic Diseases

    PubMed Central

    Yuan, Hongjie; Low, Chian-Ming; Moody, Olivia A.; Jenkins, Andrew

    2015-01-01

    The advent of whole exome/genome sequencing and the technology-driven reduction in the cost of next-generation sequencing as well as the introduction of diagnostic-targeted sequencing chips have resulted in an unprecedented volume of data directly linking patient genomic variability to disorders of the brain. This information has the potential to transform our understanding of neurologic disorders by improving diagnoses, illuminating the molecular heterogeneity underlying diseases, and identifying new targets for therapeutic treatment. There is a strong history of mutations in GABA receptor genes being involved in neurologic diseases, particularly the epilepsies. In addition, a substantial number of variants and mutations have been found in GABA receptor genes in patients with autism, schizophrenia, and addiction, suggesting potential links between the GABA receptors and these conditions. A new and unexpected outcome from sequencing efforts has been the surprising number of mutations found in glutamate receptor subunits, with the GRIN2A gene encoding the GluN2A N-methyl-d-aspartate receptor subunit being most often affected. These mutations are associated with multiple neurologic conditions, for which seizure disorders comprise the largest group. The GluN2A subunit appears to be a locus for epilepsy, which holds important therapeutic implications. Virtually all α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor mutations, most of which occur within GRIA3, are from patients with intellectual disabilities, suggesting a link to this condition. Similarly, the most common phenotype for kainate receptor variants is intellectual disability. Herein, we summarize the current understanding of disease-associated mutations in ionotropic GABA and glutamate receptor families, and discuss implications regarding the identification of human mutations and treatment of neurologic diseases. PMID:25904555

  14. Cell type specificity of GABA(A) receptor mediated signaling in the hippocampus.

    PubMed

    Semyanov, A

    2003-08-01

    Inhibitory signaling mediated by ionotropic GABA(1) receptors generally acts as a major brake against excessive excitability in the brain. This is especially relevant in epilepsy-prone structures such as the hippocampus, in which GABA(A) receptor mediated inhibition is critical in suppressing epileptiform activity. Indeed, potentiating GABA(A) receptor mediated signaling is an important target for antiepileptic drug therapy. GABA(A) receptor mediated inhibition has different roles in the network dependent on the target neuron. Inhibiting principal cells will thus reduce network excitability, whilst inhibiting interneurons will increase network excitability; GABAergic therapeutic agents do not distinguish between these two alternatives, which may explain why, on occasion, GABAergic antiepileptic drugs can be proconvulsant. The importance of the target-cell for the effect of neuroactive drugs has emerged from a number of recent studies. Immunocytochemical data have suggested non-uniform distribution of GABA(A) receptor subunits among hippocampal interneurons and pyramidal cells. This has been confirmed by subsequent electropharmacological data. These have demonstrated that compounds which act on GABA(A) receptors or the extracellular GABA concentration can have distinct effects in different neuronal populations. Recently, it has also been discovered that presynaptic glutamate heteroreceptors can modulate GABA release in the hippocampus in a postsynaptic cell-specific manner. Since systemically administrated drugs may act on different neuronal subtypes, they can exhibit paradoxical effects. Distinguishing compounds that have target specific effects on GABAergic signaling may lead to novel and more effective treatments against epilepsy.

  15. RhoA/rho kinase signaling reduces connexin43 expression in high glucose-treated glomerular mesangial cells with zonula occludens-1 involvement

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xie, Xi; Department of Pharmaceutical Engineering, Ocean College, Hainan University, Haikou 570228; Chen, Cheng

    RhoA/Rho kinase (ROCK) signaling has been suggested to be involved in diabetic nephropathy (DN) pathogenesis. Altered expression of connexin43 (Cx43) has been found in kidneys of diabetic animals. Both of them have been found to regulate nuclear factor kappa-B (NF-κB) activation in high glucose-treated glomerular mesangial cells (GMCs). The aim of this study was to investigate the relationship between RhoA/ROCK signaling and Cx43 in the DN pathogenesis. We found that upregulation of Cx43 expression inhibited NF-κB p65 nuclear translocation induced by RhoA/ROCK signaling in GMCs. Inhibition of RhoA/ROCK signaling attenuated the high glucose-induced decrease in Cx43. F-actin accumulation and anmore » enhanced interaction between zonula occludens-1 (ZO-1) and Cx43 were observed in high glucose-treated GMCs. ZO-1 depletion or disruption of F-actin formation also inhibited the reduction in Cx43 protein levels induced by high glucose. In conclusion, activated RhoA/ROCK signaling induces Cx43 degradation in GMCs cultured in high glucose, depending on F-actin regulation. Increased F-actin induced by RhoA/ROCK signaling promotes the association between ZO-1 and Cx43, which possibly triggered Cx43 endocytosis, a mechanism of NF-κB activation in high glucose-treated GMCs. - Highlights: • RhoA/ROCK signaling induces Cx43 degradation in GMCs. • F-actin and ZO-1 have functions in the regulation of Cx43 by RhoA/ROCK signaling. • We reveal the relationship between RhoA/ROCK and Cx43 in the activation of NF-κB.« less

  16. Unique Structural and Nucleotide Exchange Features of the Rho1 GTPase of Entamoeba histolytica

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Bosch, Dustin E.; Wittchen, Erika S.; Qiu, Connie

    The single-celled human parasite Entamoeba histolytica possesses a dynamic actin cytoskeleton vital for its intestinal and systemic pathogenicity. The E. histolytica genome encodes several Rho family GTPases known to regulate cytoskeletal dynamics. EhRho1, the first family member identified, was reported to be insensitive to the Rho GTPase-specific Clostridium botulinum C3 exoenzyme, raising the possibility that it may be a misclassified Ras family member. Here, we report the crystal structures of EhRho1 in both active and inactive states. EhRho1 is activated by a conserved switch mechanism, but diverges from mammalian Rho GTPases in lacking a signature Rho insert helix. EhRho1 engagesmore » a homolog of mDia, EhFormin1, suggesting a role in mediating serum-stimulated actin reorganization and microtubule formation during mitosis. EhRho1, but not a constitutively active mutant, interacts with a newly identified EhRhoGDI in a prenylation-dependent manner. Furthermore, constitutively active EhRho1 induces actin stress fiber formation in mammalian fibroblasts, thereby identifying it as a functional Rho family GTPase. EhRho1 exhibits a fast rate of nucleotide exchange relative to mammalian Rho GTPases due to a distinctive switch one isoleucine residue reminiscent of the constitutively active F28L mutation in human Cdc42, which for the latter protein, is sufficient for cellular transformation. Nonconserved, nucleotide-interacting residues within EhRho1, revealed by the crystal structure models, were observed to contribute a moderating influence on fast spontaneous nucleotide exchange. Collectively, these observations indicate that EhRho1 is a bona fide member of the Rho GTPase family, albeit with unique structural and functional aspects compared with mammalian Rho GTPases.« less

  17. Three-dimensional T1rho-weighted MRI at 1.5 Tesla.

    PubMed

    Borthakur, Arijitt; Wheaton, Andrew; Charagundla, Sridhar R; Shapiro, Erik M; Regatte, Ravinder R; Akella, Sarma V S; Kneeland, J Bruce; Reddy, Ravinder

    2003-06-01

    To design and implement a magnetic resonance imaging (MRI) pulse sequence capable of performing three-dimensional T(1rho)-weighted MRI on a 1.5-T clinical scanner, and determine the optimal sequence parameters, both theoretically and experimentally, so that the energy deposition by the radiofrequency pulses in the sequence, measured as the specific absorption rate (SAR), does not exceed safety guidelines for imaging human subjects. A three-pulse cluster was pre-encoded to a three-dimensional gradient-echo imaging sequence to create a three-dimensional, T(1rho)-weighted MRI pulse sequence. Imaging experiments were performed on a GE clinical scanner with a custom-built knee-coil. We validated the performance of this sequence by imaging articular cartilage of a bovine patella and comparing T(1rho) values measured by this sequence to those obtained with a previously tested two-dimensional imaging sequence. Using a previously developed model for SAR calculation, the imaging parameters were adjusted such that the energy deposition by the radiofrequency pulses in the sequence did not exceed safety guidelines for imaging human subjects. The actual temperature increase due to the sequence was measured in a phantom by a MRI-based temperature mapping technique. Following these experiments, the performance of this sequence was demonstrated in vivo by obtaining T(1rho)-weighted images of the knee joint of a healthy individual. Calculated T(1rho) of articular cartilage in the specimen was similar for both and three-dimensional and two-dimensional methods (84 +/- 2 msec and 80 +/- 3 msec, respectively). The temperature increase in the phantom resulting from the sequence was 0.015 degrees C, which is well below the established safety guidelines. Images of the human knee joint in vivo demonstrate a clear delineation of cartilage from surrounding tissues. We developed and implemented a three-dimensional T(1rho)-weighted pulse sequence on a 1.5-T clinical scanner. Copyright 2003

  18. Further exploration of MRI techniques for liver T1rho quantification.

    PubMed

    Zhao, Feng; Yuan, Jing; Deng, Min; Lu, Pu-Xuan; Ahuja, Anil T; Wang, Yi-Xiang J

    2013-12-01

    With biliary duct ligation and CCl4 induced rat liver fibrosis models, recent studies showed that MR T1rho imaging is able to detect liver fibrosis, and the degree of fibrosis is correlated with the degree of elevation of the T1rho measurements, suggesting liver T1rho quantification may play an important role for liver fibrosis early detection and grading. It has also been reported it is feasible to obtain consistent liver T1rho measurement for human subjects at 3 Tesla (3 T), and preliminary clinical data suggest liver T1rho is increased in patients with cirrhosis. In these previous studies, T1rho imaging was used with the rotary-echo spin-lock pulse for T1rho preparation, and number of signal averaging (NSA) was 2. Due to the presence of inhomogeneous B0 field, artifacts may occur in the acquired T1rho-weighted images. The method described by Dixon et al. (Magn Reson Med 1996;36:90-4), which is a hard RF pulse with 135° flip angle and same RF phase as the spin-locking RF pulse is inserted right before and after the spin-locking RF pulse, has been proposed to reduce sensitivity to B0 field inhomogeneity in T1rho imaging. In this study, we compared the images scanned by rotary-echo spin-lock pulse method (sequence 1) and the pulse modified according to Dixon method (sequence 2). When the artifacts occurred in T1rho images, we repeated the same scan until satisfactory. We accepted images if artifact in liver was less than 10% of liver area by visual estimation. When NSA =2, the breath-holding duration for data acquisition of one slice scanning was 8 sec due to a delay time of 6,000 ms for magnetization restoration. If NSA =1, the duration was shortened to be 2 sec. In previous studies, manual region of interest (ROI) analysis of T1rho map was used. In this current study, histogram analysis was also applied to evaluate liver T1rho value on T1rho maps. MRI data acquisition was performed on a 3 T clinical scanner. There were 29 subjects with 61 examinations obtained

  19. Altered cortical GABA neurotransmission in schizophrenia: insights into novel therapeutic strategies.

    PubMed

    Stan, Ana D; Lewis, David A

    2012-06-01

    Altered markers of cortical GABA neurotransmission are among the most consistently observed abnormalities in postmortem studies of schizophrenia. The altered markers are particularly evident between the chandelier class of GABA neurons and their synaptic targets, the axon initial segment (AIS) of pyramidal neurons. For example, in the dorsolateral prefrontal cortex of subjects with schizophrenia immunoreactivity for the GABA membrane transporter is decreased in presynaptic chandelier neuron axon terminals, whereas immunoreactivity for the GABAA receptor α2 subunit is increased in postsynaptic AIS. Both of these molecular changes appear to be compensatory responses to a presynaptic deficit in GABA synthesis, and thus could represent targets for novel therapeutic strategies intended to augment the brain's own compensatory mechanisms. Recent findings that GABA inputs from neocortical chandelier neurons can be powerfully excitatory provide new ideas about the role of these neurons in the pathophysiology of cortical dysfunction in schizophrenia, and consequently in the design of pharmacological interventions.

  20. Valerian Inhibits Rat Hepatocarcinogenesis by Activating GABA(A) Receptor-Mediated Signaling

    PubMed Central

    Kakehashi, Anna; Kato, Ayumi; Ishii, Naomi; Wei, Min; Morimura, Keiichirou; Fukushima, Shoji; Wanibuchi, Hideki

    2014-01-01

    Valerian is widely used as a traditional medicine to improve the quality of sleep due to interaction of several active components with the γ-aminobutyric acid (GABA) A receptor (GABA(A)R) system. Recently, activation of GABA signaling in stem cells has been reported to suppress cell cycle progression in vivo. Furthermore, possible inhibitory effects of GABA(A)R agonists on hepatocarcinogenesis have been reported. The present study was performed to investigate modulating effects of Valerian on hepatocarcinogenesis using a medium-term rat liver bioassay. Male F344 rats were treated with one of the most powerful Valerian species (Valeriana sitchensis) at doses of 0, 50, 500 and 5000 ppm in their drinking water after initiation of hepatocarcinogenesis with diethylnitrosamine (DEN). Formation of glutathione S-transferase placental form positive (GST-P+) foci was significantly inhibited by Valerian at all applied doses compared with DEN initiation control rats. Generation of 8-hydroxy-2′-deoxyguanosine in the rat liver was significantly suppressed by all doses of Valerian, likely due to suppression of Nrf2, CYP7A1 and induction of catalase expression. Cell proliferation was significantly inhibited, while apoptosis was induced in areas of GST-P+ foci of Valerian groups associated with suppression of c-myc, Mafb, cyclin D1 and induction of p21Waf1/Cip1, p53 and Bax mRNA expression. Interestingly, expression of the GABA(A)R alpha 1 subunit was observed in GST-P+ foci of DEN control rats, with significant elevation associated with Valerian treatment. These results indicate that Valerian exhibits inhibitory effects on rat hepatocarcinogenesis by inhibiting oxidative DNA damage, suppressing cell proliferation and inducing apoptosis in GST-P+ foci by activating GABA(A)R-mediated signaling. PMID:25419570

  1. Adolescent female rats exhibiting activity-based anorexia express elevated levels of GABA(A) receptor α4 and δ subunits at the plasma membrane of hippocampal CA1 spines.

    PubMed

    Aoki, Chiye; Sabaliauskas, Nicole; Chowdhury, Tara; Min, Jung-Yun; Colacino, Anna Rita; Laurino, Kevin; Barbarich-Marsteller, Nicole C

    2012-05-01

    Activity-based anorexia (ABA) is an animal model for anorexia nervosa that has revealed genetic links to anxiety traits and neurochemical characteristics within the hypothalamus. However, few studies have used this animal model to investigate the biological basis for vulnerability of pubertal and adolescent females to ABA, even though the great majority of the anorexia nervosa cases are females exhibiting the first symptoms during puberty. GABAergic inhibition of the hippocampus strongly regulates anxiety as well as plasticity throughout life. We recently showed that the hippocampal CA1 of female mice undergo a dramatic change at puberty onset--from expressing virtually none of the nonsynaptic α4βδ GABA(A) receptors (GABARs) prepubertally to expressing these GABARs at ~7% of the CA1 dendritic spine membranes at puberty onset. Furthermore, we showed that this change underlies the enhanced modulation of anxiety, neuronal excitability, and NMDA receptor-dependent synaptic plasticity in the hippocampus by the stress neurosteroid, THP (3α-OH-5α[β]-pregnan-20-one or [allo]pregnanolone). Here, we used quantitative electron microscopy to determine whether ABA induction in female rats during adolescence also elevates the expression of α4 and δ subunits of α4βδ GABARs, as was observed at puberty onset for mice. Our analysis revealed that rats also exhibit a rise of α4 and δ subunits of α4βδ GABARs at puberty onset, in that these subunits are detectable at ~6% of the dendritic spine membranes of CA1 pyramidal cells at puberty onset (postnatal day 32-36; P32-36) but this drops to about 2% by P40-P44. The levels of α4 and δ subunits at the CA1 spines remained low following exposure of females to either of the two environmental factors needed to generate ABA--food restriction and access to a running wheel for 4 days--from P40 to P44. This pattern contrasted greatly from those of ABA animals, for which the two environmental factors were combined. Within the

  2. A Residue in Loop 9 of the β2-Subunit Stabilizes the Closed State of the GABAA Receptor*

    PubMed Central

    Williams, Carrie A.; Bell, Shannon V.; Jenkins, Andrew

    2010-01-01

    In γ-aminobutyric acid type A (GABAA) receptors, the structural elements that couple ligand binding to channel opening remain poorly defined. Here, site-directed mutagenesis was used to determine if Loop 9 on the non-GABA binding site interface of the β2-subunit may be involved in GABAA receptor activation. Specifically, residues Gly170-Gln185 of the β2-subunit were mutated to alanine, co-expressed with wild-type α1- and γ2S-subunits in human embryonic kidney (HEK) 293 cells and assayed for their activation by GABA, the intravenous anesthetic propofol and the endogenous neurosteroid pregnanolone using whole cell macroscopic recordings. Three mutants, G170A, V175A, and G177A, produced 2.5-, 6.7-, and 5.6-fold increases in GABA EC50 whereas one mutant, Q185A, produced a 5.2-fold decrease in GABA EC50. None of the mutations affected the ability of propofol or pregnanolone to potentiate a submaximal GABA response, but the Q185A mutant exhibited 8.3- and 3.5-fold increases in the percent direct activation by propofol and pregnanolone, respectively. Mutant Q185A receptors also had an increased leak current that was sensitive to picrotoxin, indicating an increased gating efficiency. Further Q185E, Q185L, and Q185W substitutions revealed a strong correlation between the hydropathy of the amino acid at this position and the GABA EC50. Taken together, these results indicate that β2 Loop 9 is involved in receptor activation by GABA, propofol, and pregnanolone and that β2(Q185) participates in hydrophilic interactions that are important for stabilizing the closed state of the GABAA receptor. PMID:20007704

  3. Nonvesicular inhibitory neurotransmission via reversal of the GABA transporter GAT-1

    PubMed Central

    Wu, Yuanming; Wang, Wengang; Díez-Sampedro, Ana; Richerson, George B.

    2007-01-01

    SUMMARY GABA transporters play an important but poorly understood role in neuronal inhibition. They can reverse, but this is widely thought to occur only under pathological conditions. Here we use a heterologous expression system to show that the reversal potential of GAT-1 under physiologically relevant conditions is near the normal resting potential of neurons, and that reversal can occur rapidly enough to release GABA during simulated action potentials. We then use paired recordings from cultured hippocampal neurons, and show that GABAergic transmission is not prevented by four methods widely used to block vesicular release. This nonvesicular neurotransmission was potently blocked by GAT-1 antagonists, and was enhanced as predicted by agents that increase cytosolic [GABA] or [Na+]. The results indicate that GAT-1 regulates tonic inhibition by clamping ambient [GABA] at a level high enough to activate high affinity GABAA receptors, and that transporter-mediated GABA release can contribute to phasic inhibition. PMID:18054861

  4. Enhanced phasic GABA inhibition during the repair phase of stroke: a novel therapeutic target

    PubMed Central

    Paz, Jeanne T.; Wang, Eric Hou Jen; Badgely, Corrine; Olson, Andrew; Micheva, Kristina D.; Wang, Gordon; Lemmens, Robin; Tran, Kevin V.; Nishiyama, Yasuhiro; Liang, Xibin; Hamilton, Scott A.; O’Rourke, Nancy; Smith, Stephen J.; Huguenard, John R.; Bliss, Tonya M.

    2016-01-01

    Abstract Ischaemic stroke is the leading cause of severe long-term disability yet lacks drug therapies that promote the repair phase of recovery. This repair phase of stroke occurs days to months after stroke onset and involves brain remapping and plasticity within the peri-infarct zone. Elucidating mechanisms that promote this plasticity is critical for the development of new therapeutics with a broad treatment window. Inhibiting tonic (extrasynaptic) GABA signalling during the repair phase was reported to enhance functional recovery in mice suggesting that GABA plays an important function in modulating brain repair. While tonic GABA appears to suppress brain repair after stroke, less is known about the role of phasic (synaptic) GABA during the repair phase. We observed an increase in postsynaptic phasic GABA signalling in mice within the peri-infarct cortex specific to layer 5; we found increased numbers of α1 receptor subunit-containing GABAergic synapses detected using array tomography, and an associated increased efficacy of spontaneous and miniature inhibitory postsynaptic currents in pyramidal neurons. Furthermore, we demonstrate that enhancing phasic GABA signalling using zolpidem, a Food and Drug Administration (FDA)-approved GABA-positive allosteric modulator, during the repair phase improved behavioural recovery. These data identify potentiation of phasic GABA signalling as a novel therapeutic strategy, indicate zolpidem’s potential to improve recovery, and underscore the necessity to distinguish the role of tonic and phasic GABA signalling in stroke recovery. PMID:26685158

  5. Enhanced phasic GABA inhibition during the repair phase of stroke: a novel therapeutic target.

    PubMed

    Hiu, Takeshi; Farzampour, Zoya; Paz, Jeanne T; Wang, Eric Hou Jen; Badgely, Corrine; Olson, Andrew; Micheva, Kristina D; Wang, Gordon; Lemmens, Robin; Tran, Kevin V; Nishiyama, Yasuhiro; Liang, Xibin; Hamilton, Scott A; O'Rourke, Nancy; Smith, Stephen J; Huguenard, John R; Bliss, Tonya M; Steinberg, Gary K

    2016-02-01

    Ischaemic stroke is the leading cause of severe long-term disability yet lacks drug therapies that promote the repair phase of recovery. This repair phase of stroke occurs days to months after stroke onset and involves brain remapping and plasticity within the peri-infarct zone. Elucidating mechanisms that promote this plasticity is critical for the development of new therapeutics with a broad treatment window. Inhibiting tonic (extrasynaptic) GABA signalling during the repair phase was reported to enhance functional recovery in mice suggesting that GABA plays an important function in modulating brain repair. While tonic GABA appears to suppress brain repair after stroke, less is known about the role of phasic (synaptic) GABA during the repair phase. We observed an increase in postsynaptic phasic GABA signalling in mice within the peri-infarct cortex specific to layer 5; we found increased numbers of α1 receptor subunit-containing GABAergic synapses detected using array tomography, and an associated increased efficacy of spontaneous and miniature inhibitory postsynaptic currents in pyramidal neurons. Furthermore, we demonstrate that enhancing phasic GABA signalling using zolpidem, a Food and Drug Administration (FDA)-approved GABA-positive allosteric modulator, during the repair phase improved behavioural recovery. These data identify potentiation of phasic GABA signalling as a novel therapeutic strategy, indicate zolpidem's potential to improve recovery, and underscore the necessity to distinguish the role of tonic and phasic GABA signalling in stroke recovery. © The Author (2015). Published by Oxford University Press on behalf of the Guarantors of Brain.

  6. p120Ras-GAP binds the DLC1 Rho-GAP tumor suppressor protein and inhibits its RhoA GTPase and growth-suppressing activities.

    PubMed

    Yang, X-Y; Guan, M; Vigil, D; Der, C J; Lowy, D R; Popescu, N C

    2009-03-19

    DLC1 (deleted in liver cancer 1), which encodes a Rho GTPase-activating protein (Rho-GAP), is a potent tumor suppressor gene that is frequently inactivated in several human cancers. DLC1 is a multidomain protein that has been shown previously to bind members of the tensin gene family. Here we show that p120Ras-GAP (Ras-GAP; also known as RASA1) interacts and extensively colocalizes with DLC1 in focal adhesions. The binding was mapped to the SH3 domain located in the N terminus of Ras-GAP and to the Rho-GAP catalytic domain located in the C terminus of the DLC1. In vitro analyses with purified proteins determined that the isolated Ras-GAP SH3 domain inhibits DLC1 Rho-GAP activity, suggesting that Ras-GAP is a negative regulator of DLC1 Rho-GAP activity. Consistent with this possibility, we found that ectopic overexpression of Ras-GAP in a Ras-GAP-insensitive tumor line impaired the growth-suppressing activity of DLC1 and increased RhoA activity in vivo. Our observations expand the complexity of proteins that regulate DLC1 function and define a novel mechanism of the cross talk between Ras and Rho GTPases.1R01CA129610

  7. Rho-kinase signaling controls nucleocytoplasmic shuttling of class IIa Histone Deacetylase (HDAC7) and transcriptional activation of orphan nuclear receptor NR4A1

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Compagnucci, Claudia; Barresi, Sabina; Petrini, Stefania

    2015-04-03

    Rho-kinase (ROCK) has been well documented to play a key role in RhoA-induced actin remodeling. ROCK activation results in myosin light chain (MLC) phosphorylation either by direct action on MLC kinase (MLCK) or by inhibition of MLC phosphatase (MLCP), modulating actin–myosin contraction. We found that inhibition of the ROCK pathway in induced pluripotent stem cells, leads to nuclear export of HDAC7 and transcriptional activation of the orphan nuclear receptor NR4A1 while in cells with constitutive ROCK hyperactivity due to loss of function of the RhoGTPase activating protein Oligophrenin-1 (OPHN1), the orphan nuclear receptor NR4A1 is downregulated. Our study identify amore » new target of ROCK signaling via myosin phosphatase subunit (MYPT1) and Histone Deacetylase (HDAC7) at the nuclear level and provide new insights in the cellular functions of ROCK. - Highlights: • ROCK regulates nucleocytoplasmic shuttling of HDAC7 via phosphorylation of MYPT1. • Nuclear export of HDAC7 and upregulation of NR4A1 occurs with low ROCK activity. • High levels of ROCK activity due to OPHN1 loss of function downregulate NR4A1.« less

  8. Overexpressing wild-type γ2 subunits rescued the seizure phenotype in Gabrg2+/Q390X Dravet syndrome mice.

    PubMed

    Huang, Xuan; Zhou, Chengwen; Tian, Mengnan; Kang, Jing-Qiong; Shen, Wangzhen; Verdier, Kelienne; Pimenta, Aurea; MacDonald, Robert L

    2017-08-01

    The mutant γ-aminobutyric acid type A (GABA A ) receptor γ2(Q390X) subunit (Q351X in the mature peptide) has been associated with the epileptic encephalopathy, Dravet syndrome, and the epilepsy syndrome genetic epilepsy with febrile seizures plus (GEFS+). The mutation generates a premature stop codon that results in translation of a stable truncated and misfolded γ2 subunit that accumulates in neurons, forms intracellular aggregates, disrupts incorporation of γ2 subunits into GABA A receptors, and affects trafficking of partnering α and β subunits. Heterozygous Gabrg2 +/Q390X knock-in (KI) mice had reduced cortical inhibition, spike wave discharges on electroencephalography (EEG), a lower seizure threshold to the convulsant drug pentylenetetrazol (PTZ), and spontaneous generalized tonic-clonic seizures. In this proof-of-principal study, we attempted to rescue these deficits in KI mice using a γ2 subunit gene (GABRG2) replacement therapy. We introduced the GABRG2 allele by crossing Gabrg2 +/Q390X KI mice with bacterial artificial chromosome (BAC) transgenic mice overexpressing HA (hemagglutinin)-tagged human γ2 HA subunits, and compared GABA A receptor subunit expression by Western blot and immunohistochemical staining, seizure threshold by monitoring mouse behavior after PTZ-injection, and thalamocortical inhibition and network oscillation by slice recording. Compared to KI mice, adult mice carrying both mutant allele and transgene had increased wild-type γ2 and partnering α1 and β2/3 subunits, increased miniature inhibitory postsynaptic current (mIPSC) amplitudes recorded from layer VI cortical neurons, reduced thalamocortical network oscillations, and higher PTZ seizure threshold. Based on these results we suggest that seizures in a genetic epilepsy syndrome caused by epilepsy mutant γ2(Q390X) subunits with dominant negative effects could be rescued potentially by overexpression of wild-type γ2 subunits. Wiley Periodicals, Inc. © 2017 International

  9. RHON1 mediates a Rho-like activity for transcription termination in plastids of Arabidopsis thaliana.

    PubMed

    Chi, Wei; He, Baoye; Manavski, Nikolay; Mao, Juan; Ji, Daili; Lu, Congming; Rochaix, Jean David; Meurer, Jörg; Zhang, Lixin

    2014-12-01

    Although transcription termination is essential to generate functional RNAs, its underlying molecular mechanisms are still poorly understood in plastids of vascular plants. Here, we show that the RNA binding protein RHON1 participates in transcriptional termination of rbcL (encoding large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase) in Arabidopsis thaliana. Inactivation of RHON1 leads to enhanced rbcL read-through transcription and to aberrant accD (encoding β-subunit of the acetyl-CoA carboxylase) transcriptional initiation, which may result from inefficient transcription termination of rbcL. RHON1 can bind to the mRNA as well as to single-stranded DNA of rbcL, displays an RNA-dependent ATPase activity, and terminates transcription of rbcL in vitro. These results suggest that RHON1 terminates rbcL transcription using an ATP-driven mechanism similar to that of Rho of Escherichia coli. This RHON1-dependent transcription termination occurs in Arabidopsis but not in rice (Oryza sativa) and appears to reflect a fundamental difference between plastomes of dicotyledonous and monocotyledonous plants. Our results point to the importance and significance of plastid transcription termination and provide insights into its machinery in an evolutionary context. © 2014 American Society of Plant Biologists. All rights reserved.

  10. Extrasynaptic αβ subunit GABAA receptors on rat hippocampal pyramidal neurons

    PubMed Central

    Mortensen, Martin; Smart, Trevor G

    2006-01-01

    Extrasynaptic GABAA receptors that are tonically activated by ambient GABA are important for controlling neuronal excitability. In hippocampal pyramidal neurons, the subunit composition of these extrasynaptic receptors may include α5βγ and/or α4βδ subunits. Our present studies reveal that a component of the tonic current in the hippocampus is highly sensitive to inhibition by Zn2+. This component is probably not mediated by either α5βγ or α4βδ receptors, but might be explained by the presence of αβ isoforms. Using patch-clamp recording from pyramidal neurons, a small tonic current measured in the absence of exogenous GABA exhibited both high and low sensitivity to Zn2+ inhibition (IC50 values, 1.89 and 223 μm, respectively). Using low nanomolar and micromolar GABA concentrations to replicate tonic currents, we identified two components that are mediated by benzodiazepine-sensitive and -insensitive receptors. The latter indicated that extrasynaptic GABAA receptors exist that are devoid of γ2 subunits. To distinguish whether the benzodiazepine-insensitive receptors were αβ or αβδ isoforms, we used single-channel recording. Expressing recombinant α1β3γ2, α5β3γ2, α4β3δ and α1β3 receptors in human embryonic kidney (HEK) or mouse fibroblast (Ltk) cells, revealed similar openings with high main conductances (∼25–28 pS) for γ2 or δ subunit-containing receptors whereas αβ receptors were characterized by a lower main conductance state (∼11 pS). Recording from pyramidal cell somata revealed a similar range of channel conductances, indicative of a mixture of GABAA receptors in the extrasynaptic membrane. The lowest conductance state (∼11 pS) was the most sensitive to Zn2+ inhibition in accord with the presence of αβ receptors. This receptor type is estimated to account for up to 10% of all extrasynaptic GABAA receptors on hippocampal pyramidal neurons. PMID:17023503

  11. Decreased GABA receptor in the striatum and spatial recognition memory deficit in epileptic rats: effect of Bacopa monnieri and bacoside-A.

    PubMed

    Mathew, Jobin; Soman, Smijin; Sadanandan, Jayanarayanan; Paulose, Cheramadathikudyil Skaria

    2010-07-20

    Gamma-aminobutyric acid A receptors are the principal mediators of synaptic inhibition in striatal neurons and play an important role in preventing the spreading of seizures through the striatum. In the present study, effect of Bacopa monnieri (L.) Pennel and its active component bacoside-A on spatial recognition memory deficit and alterations of GABA receptor in the striatum of epileptic rats were investigated. Total GABA and GABA(A) receptor numbers in the control and epileptic rats were evaluated using [(3)H]GABA and [(3)H]bicuculline binding. GABA(Aalpha1,) GABA(Aalpha5,) GABA(Agamma3) and GABA(Adelta) gene expressions were studied. Behavioral performance was assed using Y-maze. Scatchard analysis of [(3)H]GABA and [(3)H]bicuculline in the striatum of epileptic rats showed significant decrease in B(max) compared to control. Real-Time PCR amplification of GABA(A) receptor subunits such as GABA(Aalpha1,) GABA(Aalpha5) and GABA(Adelta), were down regulated (p<0.001) in the striatum of epileptic rats compared to control. Epileptic rats have deficit in Y-maze performance. Bacopa monnieri and bacoside-A treatment reversed these changes to near control. Our results suggest that decreased GABA receptors in the striatum have an important role in epilepsy associated motor learning deficits and Bacopa monnieri and bacoside-A has a beneficial effect in the management of epilepsy. Copyright 2010 Elsevier Ireland Ltd. All rights reserved.

  12. Acutely increasing δGABA(A) receptor activity impairs memory and inhibits synaptic plasticity in the hippocampus.

    PubMed

    Whissell, Paul D; Eng, Dave; Lecker, Irene; Martin, Loren J; Wang, Dian-Shi; Orser, Beverley A

    2013-01-01

    Extrasynaptic γ-aminobutyric acid type A (GABA(A)) receptors that contain the δ subunitGABA(A) receptors) are expressed in several brain regions including the dentate gyrus (DG) and CA1 subfields of the hippocampus. Drugs that increase δGABA(A) receptor activity have been proposed as treatments for a variety of disorders including insomnia, epilepsy and chronic pain. Also, long-term pretreatment with the δGABA(A) receptor-preferring agonist 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP) enhances discrimination memory and increases neurogenesis in the DG. Despite the potential therapeutic benefits of such treatments, the effects of acutely increasing δGABA(A) receptor activity on memory behaviors remain unknown. Here, we studied the effects of THIP (4 mg/kg, i.p.) on memory performance in wild-type (WT) and δGABA(A) receptor null mutant (Gabrd(-/-)) mice. Additionally, the effects of THIP on long-term potentiation (LTP), a molecular correlate of memory, were studied within the DG and CA1 subfields of the hippocampus using electrophysiological recordings of field potentials in hippocampal slices. The results showed that THIP impaired performance in the Morris water maze, contextual fear conditioning and object recognition tasks in WT mice but not Gabrd(-/-) mice. Furthermore, THIP inhibited LTP in hippocampal slices from WT but not Gabrd(-/-) mice, an effect that was blocked by GABA(A) receptor antagonist bicuculline. Thus, acutely increasing δGABA(A) receptor activity impairs memory behaviors and inhibits synaptic plasticity. These results have important implications for the development of therapies aimed at increasing δGABA(A) receptor activity.

  13. γ-Aminobutyric Acid Type A α4, β2, and δ Subunits Assemble to Produce More Than One Functionally Distinct Receptor Type

    PubMed Central

    Eaton, Megan M.; Bracamontes, John; Shu, Hong-Jin; Li, Ping; Mennerick, Steven; Steinbach, Joe Henry

    2014-01-01

    Native γ-aminobutyric acid (GABA)A receptors consisting of α4, β1–3, and δ subunits mediate responses to the low, tonic concentration of GABA present in the extracellular milieu. Previous studies on heterologously expressed α4βδ receptors have shown a large degree of variability in functional properties, including sensitivity to the transmitter. We studied properties of α4β2δ receptors employing free subunits and concatemeric constructs, expressed in Xenopus oocytes, HEK 293 cells, and cultured hippocampal neurons. The expression system had a strong effect on the properties of receptors containing free subunits. The midpoint of GABA activation curve was 10 nM for receptors in oocytes versus 2300 nM in HEK cells. Receptors activated by the steroid alfaxalone had an estimated maximal open probability of 0.6 in oocytes and 0.01 in HEK cells. Irrespective of the expression system, receptors resulting from combining the tandem construct β2-δ and a free α4 subunit exhibited large steroid responses. We propose that free α4, β2, and δ subunits assemble in different configurations with distinct properties in oocytes and HEK cells, and that subunit linkage can overcome the expression system-dependent preferential assembly of free subunits. Hippocampal neurons transfected with α4 and the picrotoxin-resistant δ(T269Y) subunit showed large responses to alfaxalone in the presence of picrotoxin, suggesting that α4βδ receptors may assemble in a similar configuration in neurons and oocytes. PMID:25238745

  14. Regulation of hepatic Na+/K+-ATPase in obese female and male rats: involvement of ERK1/2, AMPK, and Rho/ROCK.

    PubMed

    Stanimirovic, Julijana; Obradovic, Milan; Panic, Anastasija; Petrovic, Voin; Alavantic, Dragan; Melih, Irena; Isenovic, Esma R

    2018-03-01

    In this study, we assessed whether the disturbed regulation of sodium/potassium-adenosine-triphosphatase (Na + /K + -ATPase) occurs as a consequence of obesity-induced IR in sex-specific manner. We also assessed whether alterations of IRS/PI3K/Akt, ERK1/2, AMPKα, and RhoA/ROCK signaling cascades have an important role in this pathology. Female and male Wistar rats (150-200 g, 8 weeks old) were fed a standard laboratory diet or a high-fat (HF) diet (42% fat) for 10 weeks. The activity of hepatic Na + /K + -ATPase and Rho, and the association of IRS-1/p85 were assessed in liver. Furthermore, the protein level of α 1 Na + /K + -ATPase in plasma membrane fractions, and protein levels of IRS-1, PI3K-p85, -p110, RhoA, ROCK1, ROCK2, ERK1/2, AMPKα, ERα, and ERβ in liver lysates were assessed. The expression of hepatic α 1 Na + /K + -ATPase mRNA was also analyzed by qRT-PCR. The results show that HF-fed female rats exhibited an increase in hepatic ERK1/2 (p < 0.05) and AMPKα (p < 0.05) phosphorylation levels, unchanged level of Na + /K + -ATPase α 1 mRNA, decreased level of Na + /K + -ATPase activity (p < 0.05), and decreased α 1 Na + /K + -ATPase protein expression (p < 0.01). In liver of HF-fed male rats, results show decreased levels of Na + /K + -ATPase activity (p < 0.01), both protein and mRNA of α 1 subunit (p < 0.05), but significant increase in Rho activity (p < 0.05). Our results indicate significant sex differences in α 1 Na + /K + -ATPase mRNA expression and activation of ERK1/2, AMPKα, and Rho in the liver. Exploring the sex-specific factors and pathways that promote obesity-related diseases may lead to a better understanding of pathogenesis and discovering new therapeutic targets.

  15. Mechanisms of the gabapentinoids and α 2 δ-1 calcium channel subunit in neuropathic pain.

    PubMed

    Patel, Ryan; Dickenson, Anthony H

    2016-04-01

    The gabapentinoid drugs gabapentin and pregabalin are key front-line therapies for various neuropathies of peripheral and central origin. Originally designed as analogs of GABA, the gabapentinoids bind to the α 2 δ-1 and α 2 δ-2 auxiliary subunits of calcium channels, though only the former has been implicated in the development of neuropathy in animal models. Transgenic approaches also identify α 2 δ-1 as key in mediating the analgesic effects of gabapentinoids, however the precise molecular mechanisms remain unclear. Here we review the current understanding of the pathophysiological role of the α 2 δ-1 subunit, the mechanisms of analgesic action of gabapentinoid drugs and implications for efficacy in the clinic. Despite widespread use, the number needed to treat for gabapentin and pregabalin averages from 3 to 8 across neuropathies. The failure to treat large numbers of patients adequately necessitates a novel approach to treatment selection. Stratifying patients by sensory profiles may imply common underlying mechanisms, and a greater understanding of these mechanisms could lead to more direct targeting of gabapentinoids.

  16. Membrane depolarization-induced RhoA/Rho-associated kinase activation and sustained contraction of rat caudal arterial smooth muscle involves genistein-sensitive tyrosine phosphorylation

    PubMed Central

    Mita, Mitsuo; Tanaka, Hitoshi; Yanagihara, Hayato; Nakagawa, Jun-ichi; Hishinuma, Shigeru; Sutherland, Cindy; Walsh, Michael P.; Shoji, Masaru

    2013-01-01

    Rho-associated kinase (ROK) activation plays an important role in K+-induced contraction of rat caudal arterial smooth muscle (Mita et al., Biochem J. 2002; 364: 431–40). The present study investigated a potential role for tyrosine kinase activity in K+-induced RhoA activation and contraction. The non-selective tyrosine kinase inhibitor genistein, but not the src family tyrosine kinase inhibitor PP2, inhibited K+-induced sustained contraction (IC50 = 11.3 ± 2.4 µM). Genistein (10 µM) inhibited the K+-induced increase in myosin light chain (LC20) phosphorylation without affecting the Ca2+ transient. The tyrosine phosphatase inhibitor vanadate induced contraction that was reversed by genistein (IC50 = 6.5 ± 2.3 µM) and the ROK inhibitor Y-27632 (IC50 = 0.27 ± 0.04 µM). Vanadate also increased LC20 phosphorylation in a genistein- and Y-27632-dependent manner. K+ stimulation induced translocation of RhoA to the membrane, which was inhibited by genistein. Phosphorylation of MYPT1 (myosin-targeting subunit of myosin light chain phosphatase) was significantly increased at Thr855 and Thr697 by K+ stimulation in a genistein- and Y-27632-sensitive manner. Finally, K+ stimulation induced genistein-sensitive tyrosine phosphorylation of proteins of ∼55, 70 and 113 kDa. We conclude that a genistein-sensitive tyrosine kinase, activated by the membrane depolarization-induced increase in [Ca2+]i, is involved in the RhoA/ROK activation and sustained contraction induced by K+. Ca2+ sensitization, myosin light chain phosphatase, RhoA, Rho-associated kinase, tyrosine kinase PMID:24133693

  17. Imbalance between Glutamate and GABA in Fmr1 Knockout Astrocytes Influences Neuronal Development

    PubMed Central

    Wang, Lu; Wang, Yan; Zhou, Shimeng; Yang, Liukun; Shi, Qixin; Li, Yujiao; Zhang, Kun; Yang, Le; Zhao, Minggao; Yang, Qi

    2016-01-01

    Fragile X syndrome (FXS) is a form of inherited mental retardation that results from the absence of the fragile X mental retardation protein (FMRP), the product of the Fmr1 gene. Numerous studies have shown that FMRP expression in astrocytes is important in the development of FXS. Although astrocytes affect neuronal dendrite development in Fmr1 knockout (KO) mice, the factors released by astrocytes are still unclear. We cultured wild type (WT) cortical neurons in astrocyte-conditioned medium (ACM) from WT or Fmr1 KO mice. Immunocytochemistry and Western blotting were performed to detect the dendritic growth of both WT and KO neurons. We determined glutamate and γ-aminobutyric acid (GABA) levels using high-performance liquid chromatography (HPLC). The total neuronal dendritic length was reduced when cultured in the Fmr1 KO ACM. This neurotoxicity was triggered by an imbalanced release of glutamate and GABA from Fmr1 KO astrocytes. We found increased glutaminase and GABA transaminase (GABA-T) expression and decreased monoamine oxidase B expression in Fmr1 KO astrocytes. The elevated levels of glutamate contributed to oxidative stress in the cultured neurons. Vigabatrin (VGB), a GABA-T inhibitor, reversed the changes caused by glutamate and GABA release in Fmr1 KO astrocytes and the abnormal behaviors in Fmr1 KO mice. Our results indicate that the imbalance in the astrocytic glutamate and GABA release may be involved in the neuropathology and the underlying symptoms of FXS, and provides a therapeutic target for treatment. PMID:27517961

  18. Enhancement of GABA release through endogenous activation of axonal GABA(A) receptors in juvenile cerebellum.

    PubMed

    Trigo, Federico F; Chat, Mireille; Marty, Alain

    2007-11-14

    Recent evidence indicates the presence of presynaptic GABA(A) receptors (GABA(A)Rs) in the axon domain of several classes of central neurons, including cerebellar basket and stellate cells. Here, we investigate the possibility that these receptors could be activated in the absence of electrical or chemical stimulation. We find that low concentrations of GABA increase the frequency of miniature GABAergic synaptic currents. Submaximal concentrations of a GABA(A)R blocker, gabazine, decrease both the miniature current frequency and the probability of evoked GABA release. Zolpidem, an agonist of the benzodiazepine binding site, and NO-711 (1-[2-[[(diphenylmethylene)imino]oxy]ethyl]-1,2,5,6-tetrahydro-3-pyridinecarboxylic acid hydrochloride), a blocker of GABA uptake, both increase the frequency of miniature currents. These effects occur up to postnatal day 14, but not later. Immunohistochemistry indicates the presence of alpha1-containing GABA(A)Rs in interneuron presynaptic terminals with a similar age dependence. We conclude that, under resting conditions, axonal GABA(A)Rs are significantly activated, that this activation results in enhanced GABA release, and that it can be augmented by increasing the affinity of GABA(A)Rs or reducing GABA uptake. Our findings suggest the existence of a positive-feedback mechanism involving presynaptic GABA(A)Rs that maintains a high release rate and a high local GABA concentration in the immature cerebellar network.

  19. RhoB controls endothelial barrier recovery by inhibiting Rac1 trafficking to the cell border

    PubMed Central

    Marcos-Ramiro, Beatriz; García-Weber, Diego; Barroso, Susana; Feito, Jorge; Ortega, María C.; Cernuda-Morollón, Eva; Reglero-Real, Natalia; Fernández-Martín, Laura; Durán, Maria C.; Alonso, Miguel A.; Correas, Isabel; Cox, Susan; Ridley, Anne J.

    2016-01-01

    Endothelial barrier dysfunction underlies chronic inflammatory diseases. In searching for new proteins essential to the human endothelial inflammatory response, we have found that the endosomal GTPase RhoB is up-regulated in response to inflammatory cytokines and expressed in the endothelium of some chronically inflamed tissues. We show that although RhoB and the related RhoA and RhoC play additive and redundant roles in various aspects of endothelial barrier function, RhoB specifically inhibits barrier restoration after acute cell contraction by preventing plasma membrane extension. During barrier restoration, RhoB trafficking is induced between vesicles containing RhoB nanoclusters and plasma membrane protrusions. The Rho GTPase Rac1 controls membrane spreading and stabilizes endothelial barriers. We show that RhoB colocalizes with Rac1 in endosomes and inhibits Rac1 activity and trafficking to the cell border during barrier recovery. Inhibition of endosomal trafficking impairs barrier reformation, whereas induction of Rac1 translocation to the plasma membrane accelerates it. Therefore, RhoB-specific regulation of Rac1 trafficking controls endothelial barrier integrity during inflammation. PMID:27138256

  20. GABAA receptors: Various stoichiometrics of subunit arrangement in α1β3 and α1β3ε receptors.

    PubMed

    Has, Ahmad Tarmizi Che; Chebib, Mary

    2018-05-15

    GABAA receptors (GABAARs) are members of the Cys-loop ligand-gated ion channel (LGIC) superfamily, which includes nicotinic acetylcholine, glycine, and serotonin (5HT3) receptors [1,2,3,4]. LGICs typically mediate fast synaptic transmission via the movement of ions through channels gated by neurotransmitters, such as acetylcholine for nicotinic receptors and GABA for GABAARs [5]. The term Cys-loop receptors originates from the presence of a conserved disulphide bond (or bridge) which holds together two cysteine amino acids of the loop that forms from the structure of polypeptides in the extracellular domain of the receptor's subunit [6]. GABAARs are pentameric transmembrane protein complexes consisting of five subunits from a variety of polypeptide subunits [7,8]. All of these subunits are pseudo-symmetrically organized in the plane of the membrane, with a Cl--selective channel in the middle of the complex. To date, nineteen GABAAR subunits have been identified and categorized into eight classes, α1-6, β1-3, γ1-3, δ, ε, θ, π and ρ1-3, but their variety is further broadened by the existence of several splice forms for certain subunits (e.g., α6, β2 and γ2) [9,10,11,12]. The subunits within each class have an amino acid sequence homology of 70% or more, whereas those with a sequence homology of 30% or less are grouped into different classes [13,14]. A subunit consists of four transmembrane domains (TM1-TM4), each forming an α-helix; a large extracellular N-terminal domain that incorporates part of the orthosteric agonist/antagonist binding site; a large intracellular loop between the TM3 and TM4; a small intracellular loop between TM1 and TM2; a small extracellular loop between TM2 and TM3; and a C-terminal extracellular domain [15,16]. Each subunit is arranged in such a way as to create principal and complementary interfaces with the other subunits, and in a position such that the TM2 of each subunit forms the wall of the channel pore [17,18,19]. The

  1. Mechanism of Inactivation of GABA Aminotransferase by (E)- and (Z)-(1S,3S)-3-Amino-4-fluoromethylenyl-1-cyclopentanoic Acid

    PubMed Central

    Lee, Hyunbeom; Le, Hoang V.; Wu, Rui; Doud, Emma; Sanishvili, Ruslan; Kellie, John F.; Compton, Phillip D.; Pachaiyappan, Boobalan; Liu, Dali; Kelleher, Neil L.

    2015-01-01

    When γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian central nervous system, falls below a threshold level, seizures occur. One approach to raise GABA concentrations is to inhibit GABA aminotransferase (GABA-AT), a pyridoxal 5’-phosphate-dependent enzyme that degrades GABA. We have previously developed (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115), which is 186 times more efficient in inactivating GABA-AT than vigabatrin, the only FDA-approved inactivator of GABA-AT. We also developed (E)- and (Z)-(1S,3S)-3-amino-4-fluoromethylenyl-1-cyclopentanoic acid (1 and 2, respectively), monofluorinated analogs of CPP-115, which are comparable to vigabatrin in inactivating GABA-AT. Here we report the mechanism of inactivation of GABA-AT by 1 and 2. Both produce a metabolite that induces disruption of the Glu270-Arg445 salt bridge to accommodate interaction between the metabolite formyl group and Arg445. This is the second time that Arg445 has interacted with a ligand and is involved in GABA-AT inactivation, thereby confirming the importance of Arg445 in future inactivator design. PMID:26110556

  2. Mechanism of Inactivation of GABA Aminotransferase by (E)- and (Z)-(1S,3S)-3-Amino-4-fluoromethylenyl-1-cyclopentanoic Acid.

    PubMed

    Lee, Hyunbeom; Le, Hoang V; Wu, Rui; Doud, Emma; Sanishvili, Ruslan; Kellie, John F; Compton, Phillip D; Pachaiyappan, Boobalan; Liu, Dali; Kelleher, Neil L; Silverman, Richard B

    2015-09-18

    When γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian central nervous system, falls below a threshold level, seizures occur. One approach to raise GABA concentrations is to inhibit GABA aminotransferase (GABA-AT), a pyridoxal 5'-phosphate-dependent enzyme that degrades GABA. We have previously developed (1S,3S)-3-amino-4-difluoromethylene-1-cyclopentanoic acid (CPP-115), which is 186 times more efficient in inactivating GABA-AT than vigabatrin, the only FDA-approved inactivator of GABA-AT. We also developed (E)- and (Z)-(1S,3S)-3-amino-4-fluoromethylenyl-1-cyclopentanoic acid (1 and 2, respectively), monofluorinated analogs of CPP-115, which are comparable to vigabatrin in inactivating GABA-AT. Here, we report the mechanism of inactivation of GABA-AT by 1 and 2. Both produce a metabolite that induces disruption of the Glu270-Arg445 salt bridge to accommodate interaction between the metabolite formyl group and Arg445. This is the second time that Arg445 has interacted with a ligand and is involved in GABA-AT inactivation, thereby confirming the importance of Arg445 in future inactivator design.

  3. Early continuous white noise exposure alters l-alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor subunit glutamate receptor 2 and gamma-aminobutyric acid type a receptor subunit beta3 protein expression in rat auditory cortex.

    PubMed

    Xu, Jinghong; Yu, Liping; Zhang, Jiping; Cai, Rui; Sun, Xinde

    2010-02-15

    Auditory experience during the postnatal critical period is essential for the normal maturation of auditory function. Previous studies have shown that rearing infant rat pups under conditions of continuous moderate-level noise delayed the emergence of adult-like topographic representational order and the refinement of response selectivity in the primary auditory cortex (A1) beyond normal developmental benchmarks and indefinitely blocked the closure of a brief, critical-period window. To gain insight into the molecular mechanisms of these physiological changes after noise rearing, we studied expression of the AMPA receptor subunit GluR2 and GABA(A) receptor subunit beta3 in the auditory cortex after noise rearing. Our results show that continuous moderate-level noise rearing during the early stages of development decreases the expression levels of GluR2 and GABA(A)beta3. Furthermore, noise rearing also induced a significant decrease in the level of GABA(A) receptors relative to AMPA receptors. However, in adult rats, noise rearing did not have significant effects on GluR2 and GABA(A)beta3 expression or the ratio between the two units. These changes could have a role in the cellular mechanisms involved in the delayed maturation of auditory receptive field structure and topographic organization of A1 after noise rearing. Copyright 2009 Wiley-Liss, Inc.

  4. RhoA/ROCK may involve in cardiac hypertrophy induced by experimental hyperthyroidism.

    PubMed

    Na, Wang; Peng, Guan; Jianping, Zhang; Yanzhong, Chang; Shengjiang, Guan; Li, Chu

    2012-10-01

    In this study, the role of the RhoA/Rho-kinase (RhoA/ROCK)-signaling pathway in cardiovascular dysfunction associated with hyperthyroidism was examined with the use of fasudil, a Rho-kinase inhibitor. Male Spraque-Dawley rats were treated with l-thyroxine (T(4)) alone, T(4) + low-dose fasudil (2 mg/kg/day) or T(4) + high-dose fasudil (10 mg/kg/day) and compared with control animals. Rats in the T(4) group showed an increase in the ratio of heart weight to body weight, which was ameliorated by fasudil at both low and high doses. Morphometric and hemodynamic parameters were also evaluated and confirmed that fasudil attenuated the cardiac hypertrophy induced by T(4). The extent of phosphorylation of the myosin phosphatase targeting subunit was quantified by Western blotting to evaluate the activity of Rho-kinase in the heart tissue. Both Western blotting and reverse transcriptase-polymerase chain reaction analyses revealed enhancement of Rho-kinase and activator protein 1 activity and reduction of c-FLIP(L) expression in the T(4) group, and this response was inhibited by fasudil in a dose-dependent manner. Furthermore, fasudil inhibited apoptosis induced by T(4) as evidenced by the detection of terminal deoxynucleotidyl transferase dUTP nick end labeling-positive cells and the expressions of bax and bcl-2. These results suggested that the RhoA/ROCK pathway is involved in the cardiac hypertrophy induced by experimental hyperthyroidism. The antagonism of this pathway may thus be useful as an alternative target in the treatment of hyperthyroid heart disease.

  5. Superoxide constricts rat pulmonary arteries via Rho-kinase-mediated Ca2+ sensitization

    PubMed Central

    Shaifta, Yasin; Connolly, Michelle; Drndarski, Svetlana; Noah, Anthony; Pourmahram, Ghazaleh E.; Becker, Silke; Aaronson, Philip I.; Ward, Jeremy P.T.

    2018-01-01

    Reactive oxygen species play a key role in vascular disease, pulmonary hypertension, and hypoxic pulmonary vasoconstriction. We investigated contractile responses, intracellular Ca2+ ([Ca2+]i), Rho-kinase translocation, and phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light chain (MLC20) in response to LY83583, a generator of superoxide anion, in small intrapulmonary arteries (IPA) of rat. LY83583 caused concentration-dependent constrictions in IPA and greatly enhanced submaximal PGF2α-mediated preconstriction. In small femoral or mesenteric arteries of rat, LY83583 alone was without effect, but it relaxed a PGF2α-mediated preconstriction. Constrictions in IPA were inhibited by superoxide dismutase and tempol, but not catalase, and were endothelium and guanylate cyclase independent. Constrictions were also inhibited by the Rho-kinase inhibitor Y27632 and the Src-family kinase inhibitor SU6656. LY83583 did not raise [Ca2+]i, but caused a Y27632-sensitive constriction in α-toxin-permeabilized IPA. LY83583 triggered translocation of Rho-kinase from the nucleus to the cytosol in pulmonary artery smooth muscle cells and enhanced phosphorylation of MYPT-1 at Thr-855 and of MLC20 at Ser-19 in IPA. This enhancement was inhibited by superoxide dismutase and abolished by Y27632. Hydrogen peroxide did not activate Rho-kinase. We conclude that in rat small pulmonary artery, superoxide triggers Rho-kinase-mediated Ca2+ sensitization and vasoconstriction independent of hydrogen peroxide. PMID:19103285

  6. RhoA/Rho Kinase Mediates Neuronal Death Through Regulating cPLA2 Activation.

    PubMed

    Wu, Xiangbing; Walker, Chandler L; Lu, Qingbo; Wu, Wei; Eddelman, Daniel B; Parish, Jonathan M; Xu, Xiao-Ming

    2017-11-01

    Activation of RhoA/Rho kinase leads to growth cone collapse and neurite retraction. Although RhoA/Rho kinase inhibition has been shown to improve axon regeneration, remyelination and functional recovery, its role in neuronal cell death remains unclear. To determine whether RhoA/Rho kinase played a role in neuronal death after injury, we investigated the relationship between RhoA/Rho kinase and cytosolic phospholipase A 2 (cPLA 2 ), a lipase that mediates inflammation and cell death, using an in vitro neuronal death model and an in vivo contusive spinal cord injury model performed at the 10th thoracic (T10) vertebral level. We found that co-administration of TNF-α and glutamate induced spinal neuron death, and activation of RhoA, Rho kinase and cPLA 2 . Inhibition of RhoA, Rho kinase and cPLA 2 significantly reduced TNF-α/glutamate-induced cell death by 33, 52 and 43 %, respectively (p < 0.001). Inhibition of RhoA and Rho kinase also significantly downregulated cPLA 2 activation by 66 and 60 %, respectively (p < 0.01). Furthermore, inhibition of RhoA and Rho kinase reduced the release of arachidonic acid, a downstream substrate of cPLA 2 . The immunofluorescence staining showed that ROCK 1 or ROCK 2 , two isoforms of Rho kinase, was co-localized with cPLA 2 in neuronal cytoplasm. Interestingly, co-immunoprecipitation (Co-IP) assay showed that ROCK 1 or ROCK 2 bonded directly with cPLA 2 and phospho-cPLA 2 . When the Rho kinase inhibitor Y27632 was applied in mice with T10 contusion injury, it significantly decreased cPLA 2 activation and expression and reduced injury-induced apoptosis at and close to the lesion site. Taken together, our results reveal a novel mechanism of RhoA/Rho kinase-mediated neuronal death through regulating cPLA 2 activation.

  7. Targeted deletion of the GABRA2 gene encoding alpha2-subunits of GABA(A) receptors facilitates performance of a conditioned emotional response, and abolishes anxiolytic effects of benzodiazepines and barbiturates.

    PubMed

    Dixon, C I; Rosahl, T W; Stephens, D N

    2008-07-01

    Mice with point-mutated alpha2 GABA(A) receptor subunits (rendering them diazepam insensitive) are resistant to the anxiolytic-like effects of benzodiazepines (BZs) in the conditioned emotional response (CER) test, but show normal anxiolytic effects of a barbiturate. We investigated the consequence of deleting the alpha2-subunit on acquisition of the CER with increasing intensity of footshock, and on the anxiolytic efficacy of a benzodiazepine, diazepam, and a barbiturate, pentobarbital. alpha2 knockout (KO) and wildtype (WT) mice were trained in a conditioned emotional response (CER) task, in which lever pressing for food on a variable interval (VI) schedule was suppressed during the presentation of a compound light/tone conditioned stimulus (CS+) that predicted footshock. The ability of diazepam and of pentobarbital to reduce suppression during the CS+ was interpreted as an anxiolytic response. There were no differences between the genotypes in shock sensitivity, as assessed by their flinch responses to increasing levels of shock. However, alpha2 KO mice showed a greater suppression of lever pressing than WT littermates in the presence of a compound cue signalling footshock. Diazepam (0, 0.5, 1 and 2 mg/kg) induced a dose-dependent anxiolytic-like effect in WT mice but no such effect was seen in KO mice. Similarly, although pentobarbital (20 mg/kg) reduced the ability of the CS+ to reduce lever pressing rates in WT mice, this effect was not seen in the KO. These findings suggest that alpha2-containing GABA(A) receptors mediate the anxiolytic effects of barbiturates, as well as benzodiazepines, and that they may be involved in neuronal circuits underlying conditioned anxiety.

  8. Association between GABA-A receptor alpha 5 subunit gene locus and schizophrenia of a later age of onset.

    PubMed

    Papadimitriou, G; Dikeos, D; Daskalopoulou, E; Karadima, G; Avramopoulos, D; Contis, C; Stefanis, C

    2001-01-01

    Heritability is considered to be a major etiologic factor for schizophrenia. Among the genes considered as candidates for the disease, are those related to GABAergic neurotransmission. Our aim was to test for a genetic association between GABA-A receptor alpha 5 subunit gene locus (GABRA(5)) and schizophrenia. Genotyping of the GABRA(5) locus was performed by the use of a dinucleotide (CA) repeat marker in 46 schizophrenic patients and 50 healthy individuals, all unrelated Greeks. Eight alleles were identified, 276-290 bp long. A nonsignificant excess of the 282-bp allele, which was found in a previous study in a Greek population to be associated with bipolar affective disorder, was observed in schizophrenic patients (33.8 vs. 23.9% in the controls). The frequency of this allele was 43.3% among patients with a later age of onset (over 25 years), differing at a statistically significant level from the controls (p < 0.05). These results suggest that common pathophysiological mechanisms may possibly underlie affective disorders and schizophrenia, at least in a subgroup of patients. Copyright 2001 S. Karger AG, Basel

  9. Molecular characterization of a novel RhoGAP, RRC-1 of the nematode Caenorhabditis elegans

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Delawary, Mina; Nakazawa, Takanobu; Tezuka, Tohru

    2007-06-01

    The GTPase-activating proteins for Rho family GTPases (RhoGAP) transduce diverse intracellular signals by negatively regulating Rho family GTPase-mediated pathways. In this study, we have cloned and characterized a novel RhoGAP for Rac1 and Cdc42, termed RRC-1, from Caenorhabditis elegans. RRC-1 was highly homologous to mammalian p250GAP and promoted GTP hydrolysis of Rac1 and Cdc42 in cells. The rrc-1 mRNA was expressed in all life stages. Using an RRC-1::GFP fusion protein, we found that RRC-1 was localized to the coelomocytes, excretory cell, GLR cells, and uterine-seam cell in adult worms. These data contribute toward understanding the roles of Rho family GTPasesmore » in C. elegans.« less

  10. Dose-dependent EEG effects of zolpidem provide evidence for GABA(A) receptor subtype selectivity in vivo.

    PubMed

    Visser, S A G; Wolters, F L C; van der Graaf, P H; Peletier, L A; Danhof, M

    2003-03-01

    Zolpidem is a nonbenzodiazepine GABA(A) receptor modulator that binds in vitro with high affinity to GABA(A) receptors expressing alpha(1) subunits but with relatively low affinity to receptors expressing alpha(2), alpha(3), and alpha(5) subunits. In the present study, it was investigated whether this subtype selectivity could be detected and quantified in vivo. Three doses (1.25, 5, and 25 mg) of zolpidem were administered to rats in an intravenous infusion over 5 min. The time course of the plasma concentrations was determined in conjunction with the change in the beta-frequency range of the EEG as pharmacodynamic endpoint. The concentration-effect relationship of the three doses showed a dose-dependent maximum effect and a dose-dependent potency. The data were analyzed for one- or two-site binding using two pharmacodynamic models based on 1) the descriptive model and 2) a novel mechanism-based pharmacokinetic/pharmacodynamic (PK/PD) model for GABA(A) receptor modulators that aims to separates drug- and system-specific properties, thereby allowing the estimation of in vivo affinity and efficacy. The application of two-site models significantly improved the fits compared with one-site models. Furthermore, in contrast to the descriptive model, the mechanism-based PK/PD model yielded dose-independent estimates for affinity (97 +/- 40 and 33,100 +/- 14,800 ng x ml(-1)). In conclusion, the mechanism-based PK/PD model is able to describe and explain the observed dose-dependent EEG effects of zolpidem and suggests the subtype selectivity of zolpidem in vivo.

  11. Attenuated sensitivity to neuroactive steroids in γ-aminobutyrate type A receptor delta subunit knockout mice

    PubMed Central

    Mihalek, Robert M.; Banerjee, Pradeep K.; Korpi, Esa R.; Quinlan, Joseph J.; Firestone, Leonard L.; Mi, Zhi-Ping; Lagenaur, Carl; Tretter, Verena; Sieghart, Werner; Anagnostaras, Stephan G.; Sage, Jennifer R.; Fanselow, Michael S.; Guidotti, Alessandro; Spigelman, Igor; Li, Zhiwei; DeLorey, Timothy M.; Olsen, Richard W.; Homanics, Gregg E.

    1999-01-01

    γ-Aminobutyric acid (GABA) type A receptors mediate fast inhibitory synaptic transmission and have been implicated in responses to sedative/hypnotic agents (including neuroactive steroids), anxiety, and learning and memory. Using gene targeting technology, we generated a strain of mice deficient in the δ subunit of the GABA type A receptors. In vivo testing of various behavioral responses revealed a strikingly selective attenuation of responses to neuroactive steroids, but not to other modulatory drugs. Electrophysiological recordings from hippocampal slices revealed a significantly faster miniature inhibitory postsynaptic current decay time in null mice, with no change in miniature inhibitory postsynaptic current amplitude or frequency. Learning and memory assessed with fear conditioning were normal. These results begin to illuminate the novel contributions of the δ subunit to GABA pharmacology and sedative/hypnotic responses and behavior and provide insights into the physiology of neurosteroids. PMID:10536021

  12. Behavioral deficit and decreased GABA receptor functional regulation in the cerebellum of epileptic rats: effect of Bacopa monnieri and bacoside A.

    PubMed

    Mathew, Jobin; Peeyush Kumar, T; Khan, Reas S; Paulose, C S

    2010-04-01

    In the present study, the effects of Bacopa monnieri and its active component, bacoside A, on motor deficit and alterations of GABA receptor functional regulation in the cerebellum of epileptic rats were investigated. Scatchard analysis of [(3)H]GABA and [(3)H]bicuculline in the cerebellum of epileptic rats revealed a significant decrease in B(max) compared with control. Real-time polymerase chain reaction amplification of GABA(A) receptor subunits-GABA(Aalpha1), GABA(Aalpha5,) and GABA(Adelta)-was downregulated (P<0.001) in the cerebellum of epileptic rats compared with control rats. Epileptic rats exhibit deficits in radial arm and Y-maze performance. Treatment with B. monnieri and bacoside A reversed these changes to near-control levels. Our results suggest that changes in GABAergic activity, motor learning, and memory deficit are induced by the occurrence of repetitive seizures. Treatment with B. monnieri and bacoside A prevents the occurrence of seizures thereby reducing the impairment of GABAergic activity, motor learning, and memory deficit. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  13. Functional role of ambient GABA in refining neuronal circuits early in postnatal development

    PubMed Central

    Cellot, Giada; Cherubini, Enrico

    2013-01-01

    Early in development, γ-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the mature brain, depolarizes and excites targeted neurons by an outwardly directed flux of chloride, resulting from the peculiar balance between the cation-chloride importer NKCC1 and the extruder KCC2. The low expression of KCC2 at birth leads to accumulation of chloride inside the cell and to the equilibrium potential for chloride positive respect to the resting membrane potential. GABA exerts its action via synaptic and extrasynaptic GABAA receptors mediating phasic and tonic inhibition, respectively. Here, recent data on the contribution of “ambient” GABA to the refinement of neuronal circuits in the immature brain have been reviewed. In particular, we focus on the hippocampus, where, prior to the formation of conventional synapses, GABA released from growth cones and astrocytes in a calcium- and SNARE (soluble N-ethylmaleimide-sensitive-factor attachment protein receptor)-independent way, diffuses away to activate in a paracrine fashion extrasynaptic receptors localized on distal neurons. The transient increase in intracellular calcium following the depolarizing action of GABA leads to inhibition of DNA synthesis and cell proliferation. Tonic GABA exerts also a chemotropic action on cell migration. Later on, when synapses are formed, GABA spilled out from neighboring synapses, acting mainly on extrasynaptic α5, β2, β3, and γ containing GABAA receptor subunits, provides the membrane depolarization necessary for principal cells to reach the window where intrinsic bursts are generated. These are instrumental in triggering calcium transients associated with network-driven giant depolarizing potentials which act as coincident detector signals to enhance synaptic efficacy at emerging GABAergic and glutamatergic synapses. PMID:23964205

  14. Activation of G protein-coupled estrogen receptor 1 induces coronary artery relaxation via Epac/Rap1-mediated inhibition of RhoA/Rho kinase pathway in parallel with PKA.

    PubMed

    Yu, Xuan; Zhang, Qiao; Zhao, Yan; Schwarz, Benjamin J; Stallone, John N; Heaps, Cristine L; Han, Guichun

    2017-01-01

    Previously, we reported that cAMP/PKA signaling is involved in GPER-mediated coronary relaxation by activating MLCP via inhibition of RhoA pathway. In the current study, we tested the hypothesis that activation of GPER induces coronary artery relaxation via inhibition of RhoA/Rho kinase pathway by cAMP downstream targets, exchange proteins directly activated by cAMP (Epac) as well as PKA. Our results show that Epac inhibitors, brefeldin A (BFA, 50 μM), or ESI-09 (20 μM), or CE3F4 (100 μM), all partially inhibited porcine coronary artery relaxation response to the selective GPER agonist, G-1 (0.3-3 μM); while concurrent administration of BFA and PKI (5 μM), a PKA inhibitor, almost completely blocked the relaxation effect of G-1. The Epac specific agonist, 8-CPT-2Me-cAMP (007, 1-100 μM), induced a concentration-dependent relaxation response. Furthermore, the activity of Ras-related protein 1 (Rap1) was up regulated by G-1 (1 μM) treatment of porcine coronary artery smooth muscle cells (CASMCs). Phosphorylation of vasodilator-stimulated phosphoprotein (p-VASP) was elevated by G-1 (1 μM) treatment, but not by 007 (50 μM); and the effect of G-1 on p-VASP was blocked by PKI, but not by ESI-09, an Epac antagonist. RhoA activity was similarly down regulated by G-1 and 007, whereas ESI-09 restored most of the reduced RhoA activity by G-1 treatment. Furthermore, G-1 decreased PGF2α-induced p-MYPT1, which was partially reversed with either ESI-09 or PKI; whereas, concurrent administration of ESI-09 and PKI totally prevented the inhibitory effect of G-1. The inhibitory effects of G-1 on p- MLC levels in CASMCs were mostly restored by either ESI-09 or PKI. These results demonstrate that activation of GPER induces coronary artery relaxation via concurrent inhibition of RhoA/Rho kinase by Epac/Rap1 and PKA. GPER could be a potential drug target for preventing and treating cardiovascular diseases.

  15. Expression of GABA receptor subunits in the hippocampus and thalamus after experimental traumatic brain injury

    PubMed Central

    Drexel, Meinrad; Puhakka, Noora; Kirchmair, Elke; Hörtnagl, Heide; Pitkänen, Asla; Sperk, Günther

    2015-01-01

    Traumatic brain injury is a major cause of death and disability worldwide and often associated with post-traumatic epilepsy. We recently demonstrated that TBI induces acquired GABAA receptors channelopathy that associates with hyperexcitability in granule cell layer (GCL). We now assessed the expression of GABAA and GABAB receptor subunit mRNAs between 6 h and 6 months post-TBI in the hippocampus and thalamus. The expression of major GABAA receptor subunit mRNAs (α1, α2, α5, β2, β3, γ2 and δ) was, often bilaterally, down-regulated in the GCL and in the CA3 pyramidal cells. Instead, expression of α4 (GCL, CA3, CA1), α5 (CA1) and γ2 (GCL, CA3, CA1) mRNA was up-regulated after 10 d and/or 4 months. Many of these changes were reversible. In the thalamus, we found decreases in α1, α4, β2, γ2 and δ mRNAs in the laterodorsal thalamus and in the area combining the posterior thalamic nuclear group, ventroposterolateral and ventroposteromedial complex at 6 h to 4 months post-TBI. Unlike in the hippocampus, thalamic subunit down-regulations were irreversible and limited to the ipsilateral side. However, contralaterally there was up-regulation of the subunits δ and α4 6 h and 4 months after TBI, respectively. PCR array analysis suggested a mild long-lasting GABAA receptor channelopathy in the GCL and thalamus after TBI. Whereas TBI induces transient changes in the expression of GABAA receptor subunits in the hippocampus (presumably representing compensatory mechanisms), alterations of GABAA receptor subunit mRNAs in the thalamus are long-lasting and related to degeneration of receptor-containing neurons in thalamo-cortical relay nuclei. This article is part of the Special Issue entitled ‘GABAergic Signaling in Health and Disease’. PMID:25229716

  16. Expression of GABA receptor subunits in the hippocampus and thalamus after experimental traumatic brain injury.

    PubMed

    Drexel, Meinrad; Puhakka, Noora; Kirchmair, Elke; Hörtnagl, Heide; Pitkänen, Asla; Sperk, Günther

    2015-01-01

    Traumatic brain injury is a major cause of death and disability worldwide and often associated with post-traumatic epilepsy. We recently demonstrated that TBI induces acquired GABAA receptors channelopathy that associates with hyperexcitability in granule cell layer (GCL). We now assessed the expression of GABAA and GABAB receptor subunit mRNAs between 6 h and 6 months post-TBI in the hippocampus and thalamus. The expression of major GABAA receptor subunit mRNAs (α1, α2, α5, β2, β3, γ2 and δ) was, often bilaterally, down-regulated in the GCL and in the CA3 pyramidal cells. Instead, expression of α4 (GCL, CA3, CA1), α5 (CA1) and γ2 (GCL, CA3, CA1) mRNA was up-regulated after 10 d and/or 4 months. Many of these changes were reversible. In the thalamus, we found decreases in α1, α4, β2, γ2 and δ mRNAs in the laterodorsal thalamus and in the area combining the posterior thalamic nuclear group, ventroposterolateral and ventroposteromedial complex at 6 h to 4 months post-TBI. Unlike in the hippocampus, thalamic subunit down-regulations were irreversible and limited to the ipsilateral side. However, contralaterally there was up-regulation of the subunits δ and α4 6 h and 4 months after TBI, respectively. PCR array analysis suggested a mild long-lasting GABAA receptor channelopathy in the GCL and thalamus after TBI. Whereas TBI induces transient changes in the expression of GABAA receptor subunits in the hippocampus (presumably representing compensatory mechanisms), alterations of GABAA receptor subunit mRNAs in the thalamus are long-lasting and related to degeneration of receptor-containing neurons in thalamo-cortical relay nuclei. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  17. A novel GABA(A) alpha 5 receptor inhibitor with therapeutic potential.

    PubMed

    Ling, István; Mihalik, Balázs; Etherington, Lori-An; Kapus, Gábor; Pálvölgyi, Adrienn; Gigler, Gábor; Kertész, Szabolcs; Gaál, Attila; Pallagi, Katalin; Kiricsi, Péter; Szabó, Éva; Szénási, Gábor; Papp, Lilla; Hársing, László G; Lévay, György; Spedding, Michael; Lambert, Jeremy J; Belelli, Delia; Barkóczy, József; Volk, Balázs; Simig, Gyula; Gacsályi, István; Antoni, Ferenc A

    2015-10-05

    Novel 2,3-benzodiazepine and related isoquinoline derivatives, substituted at position 1 with a 2-benzothiophenyl moiety, were synthesized to produce compounds that potently inhibited the action of GABA on heterologously expressed GABAA receptors containing the alpha 5 subunit (GABAA α5), with no apparent affinity for the benzodiazepine site. Substitutions of the benzothiophene moiety at position 4 led to compounds with drug-like properties that were putative inhibitors of extra-synaptic GABAA α5 receptors and had substantial blood-brain barrier permeability. Initial characterization in vivo showed that 8-methyl-5-[4-(trifluoromethyl)-1-benzothiophen-2-yl]-1,9-dihydro-2H-[1,3]oxazolo[4,5-h][2,3]benzodiazepin-2-one was devoid of sedative, pro-convulsive or motor side-effects, and enhanced the performance of rats in the object recognition test. In summary, we have discovered a first-in-class GABA-site inhibitor of extra-synaptic GABAA α5 receptors that has promising drug-like properties and warrants further development. Copyright © 2015 Elsevier B.V. All rights reserved.

  18. Deregulation of HEF1 Impairs M-Phase Progression by Disrupting the RhoA Activation Cycle

    PubMed Central

    Dadke, Disha; Jarnik, Michael; Pugacheva, Elena N.; Singh, Mahendra K.; Golemis, Erica A.

    2006-01-01

    The focal adhesion-associated signaling protein HEF1 undergoes a striking relocalization to the spindle at mitosis, but a function for HEF1 in mitotic signaling has not been demonstrated. We here report that overexpression of HEF1 leads to failure of cells to progress through cytokinesis, whereas depletion of HEF1 by small interfering RNA (siRNA) leads to defects earlier in M phase before cleavage furrow formation. These defects can be explained mechanistically by our determination that HEF1 regulates the activation cycle of RhoA. Inactivation of RhoA has long been known to be required for cytokinesis, whereas it has recently been determined that activation of RhoA at the entry to M phase is required for cellular rounding. We find that increased HEF1 sustains RhoA activation, whereas depleted HEF1 by siRNA reduces RhoA activation. Furthermore, we demonstrate that chemical inhibition of RhoA is sufficient to reverse HEF1-dependent cellular arrest at cytokinesis. Finally, we demonstrate that HEF1 associates with the RhoA-GTP exchange factor ECT2, an orthologue of the Drosophila cytokinetic regulator Pebble, providing a direct means for HEF1 control of RhoA. We conclude that HEF1 is a novel component of the cell division control machinery and that HEF1 activity impacts division as well as cell attachment signaling events. PMID:16394104

  19. A Single Amino Acid Residue at Transmembrane Domain 4 of the α Subunit Influences Carisoprodol Direct Gating Efficacy at GABAA Receptors.

    PubMed

    Kumar, Manoj; Kumar, Manish; Freund, John M; Dillon, Glenn H

    2017-09-01

    The muscle relaxant carisoprodol has recently been controlled at the federal level as a Schedule IV drug due to its high abuse potential and consequences of misuse, such as withdrawal syndrome, delusions, seizures, and even death. Recent work has shown that carisoprodol can directly gate and allosterically modulate the type A GABA (GABA A ) receptor. These actions are subunit-dependent; compared with other GABA A receptors, carisoprodol has nominal direct gating effects in α 3 β 2 γ 2 receptors. Here, using site-directed mutagenesis and whole-cell patch-clamp electrophysiology in transiently transfected human embryonic kidney 293 cells, we examined the role of GABA A receptor α subunit transmembrane domain 4 (TM4) amino acids in direct gating and allosteric modulatory actions of carisoprodol. Mutation of α 3 valine at position 440 to leucine (present in the equivalent position in the α 1 subunit) significantly increased the direct gating effects of carisoprodol without affecting its allosteric modulatory effects. The corresponding reverse mutation, α 1(L415V), decreased carisoprodol direct gating potency and efficacy. Analysis of a series of amino acid mutations at the 415 position demonstrated that amino acid volume correlated positively with carisoprodol efficacy, whereas polarity inversely correlated with carisoprodol efficacy. We conclude that α 1(415) of TM4 is involved in the direct gating, but not allosteric modulatory, actions of carisoprodol. In addition, the orientation of alkyl or hydroxyl groups at this position influences direct gating effects. These findings support the likelihood that the direct gating and allosteric modulatory effects of carisoprodol are mediated via distinct binding sites. Copyright © 2017 by The American Society for Pharmacology and Experimental Therapeutics.

  20. Measurement of branching fractions and charge asymmetries in B+/--->rho+/-pi0 and B+/--->rho0pi+/- decays, and search for B0-->rho0pi0.

    PubMed

    Aubert, B; Barate, R; Boutigny, D; Couderc, F; Gaillard, J-M; Hicheur, A; Karyotakis, Y; Lees, J P; Robbe, P; Tisserand, V; Zghiche, A; Palano, A; Pompili, A; Chen, J C; Qi, N D; Rong, G; Wang, P; Zhu, Y S; Eigen, G; Ofte, I; Stugu, B; Abrams, G S; Borgland, A W; Breon, A B; Brown, D N; Button-Shafer, J; Cahn, R N; Charles, E; Day, C T; Gill, M S; Gritsan, A V; Groysman, Y; Jacobsen, R G; Kadel, R W; Kadyk, J; Kerth, L T; Kolomensky, Yu G; Kukartsev, G; LeClerc, C; Levi, M E; Lynch, G; Mir, L M; Oddone, P J; Orimoto, T J; Pripstein, M; Roe, N A; Romosan, A; Ronan, M T; Shelkov, V G; Telnov, A V; Wenzel, W A; Ford, K; Harrison, T J; Hawkes, C M; Knowles, D J; Morgan, S E; Penny, R C; Watson, A T; Watson, N K; Goetzen, K; Held, T; Koch, H; Lewandowski, B; Pelizaeus, M; Peters, K; Schmuecker, H; Steinke, M; Boyd, J T; Chevalier, N; Cottingham, W N; Kelly, M P; Latham, T E; Mackay, C; Wilson, F F; Abe, K; Cuhadar-Donszelmann, T; Hearty, C; Mattison, T S; McKenna, J A; Thiessen, D; Kyberd, P; McKemey, A K; Teodorescu, L; Blinov, V E; Bukin, A D; Golubev, V B; Ivanchenko, V N; Kravchenko, E A; Onuchin, A P; Serednyakov, S I; Skovpen, Yu I; Solodov, E P; Yushkov, A N; Best, D; Bruinsma, M; Chao, M; Kirkby, D; Lankford, A J; Mandelkern, M; Mommsen, R K; Roethel, W; Stoker, D P; Buchanan, C; Hartfiel, B L; Gary, J W; Layter, J; Shen, B C; Wang, K; del Re, D; Hadavand, H K; Hill, E J; MacFarlane, D B; Paar, H P; Rahatlou, Sh; Sharma, V; Berryhill, J W; Campagnari, C; Dahmes, B; Levy, S L; Long, O; Lu, A; Mazur, M A; Richman, J D; Verkerke, W; Beck, T W; Beringer, J; Eisner, A M; Heusch, C A; Lockman, W S; Schalk, T; Schmitz, R E; Schumm, B A; Seiden, A; Spradlin, P; Turri, M; Walkowiak, W; Williams, D C; Wilson, M G; Albert, J; Chen, E; Dubois-Felsmann, G P; Dvoretskii, A; Erwin, R J; Hitlin, D G; Narsky, I; Piatenko, T; Porter, F C; Ryd, A; Samuel, A; Yang, S; Jayatilleke, S; Mancinelli, G; Meadows, B T; Sokoloff, M D; Abe, T; Blanc, F; Bloom, P; Chen, S; Clark, P J; Ford, W T; Nauenberg, U; Olivas, A; Rankin, P; Roy, J; Smith, J G; van Hoek, W C; Zhang, L; Harton, J L; Hu, T; Soffer, A; Toki, W H; Wilson, R J; Zhang, J; Altenburg, D; Brandt, T; Brose, J; Colberg, T; Dickopp, M; Dubitzky, R S; Hauke, A; Lacker, H M; Maly, E; Müller-Pfefferkorn, R; Nogowski, R; Otto, S; Schubert, J; Schubert, K R; Schwierz, R; Spaan, B; Wilden, L; Bernard, D; Bonneaud, G R; Brochard, F; Cohen-Tanugi, J; Grenier, P; Thiebaux, Ch; Vasileiadis, G; Verderi, M; Khan, A; Lavin, D; Muheim, F; Playfer, S; Swain, J E; Andreotti, M; Azzolini, V; Bettoni, D; Bozzi, C; Calabrese, R; Cibinetto, G; Luppi, E; Negrini, M; Piemontese, L; Sarti, A; Treadwell, E; Baldini-Ferroli, R; Calcaterra, A; de Sangro, R; Falciai, D; Finocchiaro, G; Patteri, P; Piccolo, M; Zallo, A; Buzzo, A; Capra, R; Contri, R; Crosetti, G; Lo Vetere, M; Macri, M; Monge, M R; Passaggio, S; Patrignani, C; Robutti, E; Santroni, A; Tosi, S; Bailey, S; Morii, M; Won, E; Bhimji, W; Bowerman, D A; Dauncey, P D; Egede, U; Eschrich, I; Gaillard, J R; Morton, G W; Nash, J A; Taylor, G P; Grenier, G J; Lee, S-J; Mallik, U; Cochran, J; Crawley, H B; Lamsa, J; Meyer, W T; Prell, S; Rosenberg, E I; Yi, J; Davier, M; Grosdidier, G; Höcker, A; Laplace, S; Le Diberder, F; Lepeltier, V; Lutz, A M; Petersen, T C; Plaszczynski, S; Schune, M H; Tantot, L; Wormser, G; Brigljević, V; Cheng, C H; Lange, D J; Simani, M C; Wright, D M; Bevan, A J; Coleman, J P; Fry, J R; Gabathuler, E; Gamet, R; Kay, M; Parry, R J; Payne, D J; Sloane, R J; Touramanis, C; Back, J J; Harrison, P F; Shorthouse, H W; Vidal, P B; Brown, C L; Cowan, G; Flack, R L; Flaecher, H U; George, S; Green, M G; Kurup, A; Marker, C E; McMahon, T R; Ricciardi, S; Salvatore, F; Vaitsas, G; Winter, M A; Brown, D; Davis, C L; Allison, J; Barlow, N R; Barlow, R J; Hart, P A; Hodgkinson, M C; Jackson, F; Lafferty, G D; Lyon, A J; Weatherall, J H; Williams, J C; Farbin, A; Jawahery, A; Kovalskyi, D; Lae, C K; Lillard, V; Roberts, D A; Blaylock, G; Dallapiccola, C; Flood, K T; Hertzbach, S S; Kofler, R; Koptchev, V B; Moore, T B; Saremi, S; Staengle, H; Willocq, S; Cowan, R; Sciolla, G; Taylor, F; Yamamoto, R K; Mangeol, D J J; Patel, P M; Robertson, S H; Lazzaro, A; Palombo, F; Bauer, J M; Cremaldi, L; Eschenburg, V; Godang, R; Kroeger, R; Reidy, J; Sanders, D A; Summers, D J; Zhao, H W; Brunet, S; Cote-Ahern, D; Taras, P; Nicholson, H; Cartaro, C; Cavallo, N; De Nardo, G; Fabozzi, F; Gatto, C; Lista, L; Paolucci, P; Piccolo, D; Sciacca, C; Baak, M A; Raven, G; LoSecco, J M; Gabriel, T A; Brau, B; Gan, K K; Honscheid, K; Hufnagel, D; Kagan, H; Kass, R; Pulliam, T; Wong, Q K; Brau, J; Frey, R; Igonkina, O; Potter, C T; Sinev, N B; Strom, D; Torrence, E; Colecchia, F; Dorigo, A; Galeazzi, F; Margoni, M; Morandin, M; Posocco, M; Rotondo, M; Simonetto, F; Stroili, R; Tiozzo, G; Voci, C; Benayoun, M; Briand, H; Chauveau, J; David, P; de la Vaissière, Ch; Del Buono, L; Hamon, O; John, M J J; Leruste, Ph; Ocariz, J; Pivk, M; Roos, L; Stark, J; T'Jampens, S; Therin, G; Manfredi, P F; Re, V; Behera, P K; Gladney, L; Guo, Q H; Panetta, J; Anulli, F; Biasini, M; Peruzzi, I M; Pioppi, M; Angelini, C; Batignani, G; Bettarini, S; Bondioli, M; Bucci, F; Calderini, G; Carpinelli, M; Del Gamba, V; Forti, F; Giorgi, M A; Lusiani, A; Marchiori, G; Martinez-Vidal, F; Morganti, M; Neri, N; Paoloni, E; Rama, M; Rizzo, G; Sandrelli, F; Walsh, J; Haire, M; Judd, D; Paick, K; Wagoner, D E; Danielson, N; Elmer, P; Lu, C; Miftakov, V; Olsen, J; Smith, A J S; Tanaka, H A; Varnes, E W; Bellini, F; Cavoto, G; Faccini, R; Ferrarotto, F; Ferroni, F; Gaspero, M; Mazzoni, M A; Morganti, S; Pierini, M; Piredda, G; Safai Tehrani, F; Voena, C; Christ, S; Wagner, G; Waldi, R; Adye, T; De Groot, N; Franek, B; Geddes, N I; Gopal, G P; Olaiya, E O; Xella, S M; Aleksan, R; Emery, S; Gaidot, A; Ganzhur, S F; Giraud, P-F; Hamel de Monchenault, G; Kozanecki, W; Langer, M; Legendre, M; London, G W; Mayer, B; Schott, G; Vasseur, G; Yeche, Ch; Zito, M; Purohit, M V; Weidemann, A W; Yumiceva, F X; Aston, D; Bartoldus, R; Berger, N; Boyarski, A M; Buchmueller, O L; Convery, M R; Cristinziani, M; Dong, D; Dorfan, J; Dujmic, D; Dunwoodie, W; Elsen, E E; Field, R C; Glanzman, T; Gowdy, S J; Grauges-Pous, E; Hadig, T; Halyo, V; Hast, C; Hryn'ova, T; Innes, W R; Jessop, C P; Kelsey, M H; Kim, P; Kocian, M L; Langenegger, U; Leith, D W G S; Libby, J; Luitz, S; Luth, V; Lynch, H L; Marsiske, H; Messner, R; Muller, D R; O'Grady, C P; Ozcan, V E; Perazzo, A; Perl, M; Petrak, S; Ratcliff, B N; Roodman, A; Salnikov, A A; Schindler, R H; Schwiening, J; Simi, G; Snyder, A; Soha, A; Stelzer, J; Su, D; Sullivan, M K; Va'vra, J; Wagner, S R; Weaver, M; Weinstein, A J R; Wisniewski, W J; Wright, D H; Young, C C; Burchat, P R; Edwards, A J; Meyer, T I; Petersen, B A; Roat, C; Ahmed, M; Ahmed, S; Alam, M S; Ernst, J A; Saeed, M A; Saleem, M; Wappler, F R; Bugg, W; Krishnamurthy, M; Spanier, S M; Eckmann, R; Kim, H; Ritchie, J L; Schwitters, R F; Izen, J M; Kitayama, I; Lou, X C; Ye, S; Bianchi, F; Bona, M; Gallo, F; Gamba, D; Borean, C; Bosisio, L; Della Ricca, G; Dittongo, S; Grancagnolo, S; Lanceri, L; Poropat, P; Vitale, L; Vuagnin, G; Panvini, R S; Banerjee, Sw; Brown, C M; Fortin, D; Jackson, P D; Kowalewski, R; Roney, J M; Band, H R; Dasu, S; Datta, M; Eichenbaum, A M; Johnson, J R; Kutter, P E; Li, H; Liu, R; Di Lodovico, F; Mihalyi, A; Mohapatra, A K; Pan, Y; Prepost, R; Sekula, S J; von Wimmersperg-Toeller, J H; Wu, J; Wu, S L; Yu, Z; Neal, H

    2004-07-30

    We present measurements of branching fractions and charge asymmetries in B-meson decays to rho(+)pi(0), rho(0)pi(+), and rho(0)pi(0). The data sample comprises 89x10(6) Upsilon(4S)-->BBmacr; decays collected with the BABAR detector at the PEP-II asymmetric-energy B Factory at SLAC. We find the charge-averaged branching fractions B(B+-->rho(+)pi(0))=[10.9+/-1.9(stat)+/-1.9(syst)]x10(-6) and B(B+-->rho(0)pi(+))=(9.5+/-1.1+/-0.9)x10(-6), and we set a 90% confidence-level upper limit B(B0-->rho(0)pi(0))<2.9x10(-6). We measure the charge asymmetries ACP(pi(0))(rho(+))=0.24+/-0.16+/-0.06 and ACP(pi(+))(rho(0))=-0.19+/-0.11+/-0.02.

  1. Is GABA neurotransmission enhanced in auditory thalamus relative to inferior colliculus?

    PubMed Central

    Cai, Rui; Kalappa, Bopanna I.; Brozoski, Thomas J.; Ling, Lynne L.

    2013-01-01

    Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central auditory system. Sensory thalamic structures show high levels of non-desensitizing extrasynaptic GABAA receptors (GABAARs) and a reduction in the redundancy of coded information. The present study compared the inhibitory potency of GABA acting at GABAARs between the inferior colliculus (IC) and the medial geniculate body (MGB) using quantitative in vivo, in vitro, and ex vivo experimental approaches. In vivo single unit studies compared the ability of half maximal inhibitory concentrations of GABA to inhibit sound-evoked temporal responses, and found that GABA was two to three times (P < 0.01) more potent at suppressing MGB single unit responses than IC unit responses. In vitro whole cell patch-clamp slice recordings were used to demonstrate that gaboxadol, a δ-subunit selective GABAAR agonist, was significantly more potent at evoking tonic inhibitory currents from MGB neurons than IC neurons (P < 0.01). These electrophysiological findings were supported by an in vitro receptor binding assay which used the picrotoxin analog [3H]TBOB to assess binding in the GABAAR chloride channel. MGB GABAARs had significantly greater total open chloride channel capacity relative to GABAARs in IC (P < 0.05) as shown by increased total [3H]TBOB binding. Finally, a comparative ex vivo measurement compared endogenous GABA levels and suggested a trend towards higher GABA concentrations in MGB than in IC. Collectively, these studies suggest that, per unit GABA, high affinity extrasynaptic and synaptic GABAARs confer a significant inhibitory GABAAR advantage to MGB neurons relative to IC neurons. This increased GABA sensitivity likely underpins the vital filtering role of auditory thalamus. PMID:24155003

  2. The atypical structure and function of newborn arterial endothelium is mediated by Rho/Rho kinase signaling.

    PubMed

    Flavahan, Sheila; Flavahan, Nicholas A

    2014-08-15

    Endothelium of fetal or newborn arteries is atypical, displaying actin stress fibers and reduced nitric oxide (NO)-mediated dilatation. This study tested the hypothesis that Rho/Rho kinase signaling, which promotes endothelial stress fibers and inhibits endothelial dilatation, contributed to this phenotype. Carotid arteries were isolated from newborn [postnatal day 1 (P1)], P7, and P21 mice. Endothelial dilatation to acetylcholine (pressure myograph) was minimal at P1, increased at P7, and further increased at P21. Inhibition of Rho (C3 transferase) or Rho kinase (Y27632, fasudil) significantly increased dilatation to acetylcholine in P1 arteries but had no effect in P7 or P21 arteries. After inhibition of NO synthase (N(G)-nitro-l-arginine methyl ester), Rho kinase inhibition no longer increased acetylcholine responses in P1 arteries. Rho kinase inhibition did not affect dilatation to the NO donor DEA-NONOate. The endothelial actin cytoskeleton was labeled with phalloidin and visualized by laser-scanning microscopy. In P1 arteries, the endothelium had prominent transcytoplasmic stress fibers, whereas in P7 and P21 arteries, the actin fibers had a significantly reduced intensity and were restricted to cell borders. Phosphorylation of myosin light chains, a Rho kinase substrate, was highest in P1 endothelium and significantly reduced in P7 and P21 endothelium (laser-scanning microscopy). In P1 arteries, inhibition of Rho (C3 transferase) or Rho kinase (Y27632) significantly reduced the intensity of actin fibers, which were restricted to cell borders. Similarly, in P1 arteries, Rho inhibition significantly reduced endothelial levels of phosphorylated myosin light chains. These results indicate that the atypical function and morphology of newborn endothelium is mediated by Rho/Rho kinase signaling. Copyright © 2014 the American Physiological Society.

  3. Abnormal relationship between GABA, neurophysiology and impulsive behavior in neurofibromatosis type 1.

    PubMed

    Ribeiro, Maria J; Violante, Inês R; Bernardino, Inês; Edden, Richard A E; Castelo-Branco, Miguel

    2015-03-01

    Neurofibromatosis type 1 (NF1) is a neurodevelopmental disorder characterized by a broad spectrum of cognitive deficits. In particular, executive dysfunction is recognized as a core deficit of NF1, including impairments in executive attention and inhibitory control. Yet, the neural mechanisms behind these important deficits are still unknown. Here, we studied inhibitory control in a visual go/no-go task in children and adolescents with NF1 and age- and gender-matched controls (n = 16 per group). We applied a multimodal approach using high-density electroencephalography (EEG), to study the evoked brain responses, and magnetic resonance spectroscopy (MRS) to measure the levels of GABA and glutamate + glutamine in the medial frontal cortex, a brain region that plays a pivotal role in inhibitory control, and also in a control region, the occipital cortex. Finally, we run correlation analyses to identify the relationship between inhibitory control, levels of neurotransmitters, and EEG markers of neural function. Individuals with NF1 showed impaired impulse control and reduced EEG correlates of early visual processing (parieto-occipital P1) and inhibitory control (frontal P3). MRS data revealed a reduction in medial frontal GABA+/tCr (total Creatine) levels in the NF1 group, in parallel with the already reported reduced occipital GABA levels. In contrast, glutamate + glutamine/tCr levels were normal, suggesting the existence of abnormal inhibition/excitation balance in this disorder. Notably, medial frontal but not occipital GABA levels correlated with general intellectual abilities (IQ) in NF1, and inhibitory control in both groups. Surprisingly, the relationship between inhibitory control and medial frontal GABA was reversed in NF1: higher GABA was associated with a faster response style whereas in controls it was related to a cautious strategy. Abnormal GABAergic physiology appears, thus, as an important factor underlying impaired cognition in NF1, in a level and

  4. In vivo electroretinographic studies of the role of GABA C receptors in retinal signal processing

    DOE PAGES

    Wang, Jing; Mojumder, Deb Kumar; Yan, Jun; ...

    2015-07-08

    The retina expresses all three classes of receptors for the inhibitory neurotransmitter GABA (GABAR). Our study investigated roles of GABAR, especially GABA(C)R (GABA(A)-rho), in retinal signaling in vivo by studying effects on the mouse electroretinogram (ERG) of genetic deletion of GABA(C)R versus pharmacological blockade using receptor antagonists. Brief full-field flash ERGs were recorded from anesthetized GABA(C)R(-/-) mice, and WT C57BL/6 (B6) mice, before and after intravitreal injection of GABA(C)R antagonists, TPMPA, 3-APMPA, or the more recently developed 2-AEMP; GABA(A)R antagonist, SR95531; GABA(B)R antagonist, CGP, and agonist, baclofen. Intravitreal injections of TPMPA and SR95531 were also made in Brown Norway rats.more » The effect of 2-AEMP on GABA-induced current was tested directly in isolated rat rod bipolar cells, and 2-AEMP was found to preferentially block GABA(C)R in those cells. Maximum amplitudes of dark (DA) and light-adapted (LA) ERG b-waves were reduced in GABA(C)R(-/-) mice, compared to B6 mice, by 30-60%; a-waves were unaltered and oscillatory potential amplitudes were increased. In B6 mice, after injection of TPMPA (also in rats), 3-APMPA or 2-AEMP, ERGs became similar to ERGs of GABA(C)R(-/-) mice. Blockade of GABA(A)Rs and GABA(B)Rs, or agonism of GABA(B)Rs did not alter B6 DA b-wave amplitude. Furthermore, the negative scotopic threshold response (nSTR) was slightly less sensitive in GABA(C)R(-/-) than in B6 mice, and unaltered by 2-AEMP. However, amplitudes of nSTR and photopic negative response (PhNR), both of which originate from inner retina, were enhanced by TPMPA and 3-APMPA, each of which has GABA(B) agonist properties, and further increased by baclofen. The finding that genetic deletion of GABA(C)R, the GABA(C)R antagonist 2-AEMP, and other antagonists all reduced ERG b-wave amplitude, supports a role for CABA(C)R in determining the maximum response amplitude of bipolar cells contributing to the b-wave. GABA

  5. Effect of chronic treatment with the GABA transaminase inhibitors gamma-vinyl GABA and ethanolamine O-sulphate on the in vitro GABA release from rat hippocampus.

    PubMed

    Qume, M; Fowler, L J

    1997-10-01

    1. The effects of 2, 8 and 21 day oral treatment with the specific gamma-aminobutyric acid transaminase (GABA-T) inhibitors gamma-vinyl GABA (GVG) and ethanolamine O-sulphate (EOS) on brain GABA levels, GABA-T activity, and basal and stimulated GABA release from rat cross-chopped brain hippocampal slices was investigated. 2. Treatment with GABA-T inhibitors lead to a reduction in brain GABA-T activity by 65-80% compared with control values, with a concomitant increase in brain GABA content of 40-100%. 3. Basal hippocampal GABA release was increased to 250-450% of control levels following inhibition of GABA-T activity. No Ca2+ dependence was observed in either control or treated tissues. 4. GVG and EOS administration led to a significant elevation in the potassium stimulated release of GABA from cross-chopped hippocampal slices compared with that of controls. Although stimulated GABA release from control tissues was decreased in the presence of a low Ca2+ medium, GVG and EOS treatment abolished this Ca2+ dependency. 5. GABA compartmentalization, Na+ and Cl- coupled GABA uptake carriers and glial release may provide explanations for the loss of the Ca2+ dependency of stimulated GABA release observed following GVG and EOS treatment. 6. Administration of GABA-T inhibitors led to increases in both basal and stimulated hippocampal GABA release. However, it is not clear which is the most important factor in the anticonvulsant activity of these drugs, the increased GABA content 'leaking' out of neurones and glia leading to widespread inhibition, or the increase in stimulated GABA release which may occur following depolarization caused by an epileptic discharge.

  6. Metabotropic GABAB receptors mediate GABA inhibition of acetylcholine release in the rat neuromuscular junction.

    PubMed

    Malomouzh, Artem I; Petrov, Konstantin A; Nurullin, Leniz F; Nikolsky, Evgeny E

    2015-12-01

    Gamma-aminobutyric acid (GABA) is an amino acid which acts as a neurotransmitter in the central nervous system. Here, we studied the effects of GABA on non-quantal, spontaneous, and evoked quantal acetylcholine (ACh) release from motor nerve endings. We found that while the application of 10 μM of GABA had no effect on spontaneous quantal ACh release, as detected by the frequency of miniature endplate potentials, GABA reduced the non-quantal ACh release by 57%, as determined by the H-effect value. Finally, the evoked quantal ACh release, estimated by calculating the quantal content of full-sized endplate potentials (EPPs), was reduced by 34%. GABA's inhibitory effect remained unchanged after pre-incubation with picrotoxin, an ionotropic GABAA receptor blocker, but was attenuated following application of the GABAB receptor blocker CGP 55845, which itself had no effect on ACh release. An inhibitor of phospholipase C, U73122, completely prevented the GABA-induced decrease in ACh release. Immunofluorescence demonstrated the presence of both subunits of the GABAB receptor (GABAB R1 and GABAB R2) in the neuromuscular junction. These findings suggest that metabotropic GABAB receptors are expressed in the mammalian neuromuscular synapse and their activation results in a phospholipase C-mediated reduction in the intensity of non-quantal and evoked quantal ACh release. We investigated the effect of gamma-aminobutyric acid (GABA) on neuromuscular transmission. GABA reduced the non-quantal and evoked quantal release of acetylcholine. These effects are mediated by GABAB receptors and are implemented via phospholipase C (PLC) activation. Our findings suggest that in the mammalian neuromuscular synapse, metabotropic GABAB receptors are expressed and their activation results in a reduction in the intensity of acetylcholine release. © 2015 International Society for Neurochemistry.

  7. Gamma-vinyl GABA increases nonvesicular release of GABA and glutamate in the nucleus accumbens in rats via action on anion channels and GABA transporters

    PubMed Central

    Peng, Xiao-Qing; Gardner, Eliot L.

    2013-01-01

    Rationale γ-Amino butyric acid (GABA) is a well-characterized inhibitory neurotransmitter in the central nervous system, which may also stimulate nonvesicular release of other neurotransmitters under certain conditions. We have recently reported that γ-vinyl GABA (GVG), an irreversible GABA transaminase inhibitor, elevates extracellular GABA but fails to alter dopamine release in the nucleus accumbens (NAc). Objectives Here, we investigated the mechanism(s) by which GVG elevates extracellular GABA levels and whether GVG also alters glutamate release in the NAc. Materials and methods In vivo microdialysis was used to simultaneously measure extracellular NAc GABA and glutamate before and after GVG administration in freely moving rats. Results Systemic administration of GVG or intra-NAc local perfusion of GVG significantly increased extracellular NAc GABA and glutamate. GVG-enhanced GABA was completely blocked by intra-NAc local perfusion of 5-nitro-2, 3-(phenylpropylamino)-benzoic acid (NPPB), a selective anion channel blocker and partially blocked by SKF89976A, a type 1 GABA transporter inhibitor. GVG-enhanced glutamate was completely blocked by NPPB or SKF89976A. Tetrodotoxin, a voltage-dependent Na+-channel blocker, failed to alter GVG-enhanced GABA and glutamate. Conclusions These data suggest that GVG-enhanced extracellular GABA and glutamate are mediated predominantly by the opening of anion channels and partially by the reversal of GABA transporters. Enhanced extracellular glutamate may functionally attenuate the pharmacological action of GABA and prevent enhanced GABA-induced excess inhibition. PMID:20033132

  8. Altered cortical expression of GABA-related genes in schizophrenia: illness progression vs developmental disturbance.

    PubMed

    Hoftman, Gil D; Volk, David W; Bazmi, H Holly; Li, Siyu; Sampson, Allan R; Lewis, David A

    2015-01-01

    Schizophrenia is a neurodevelopmental disorder with altered expression of GABA-related genes in the prefrontal cortex (PFC). However, whether these gene expression abnormalities reflect disturbances in postnatal developmental processes before clinical onset or arise as a consequence of clinical illness remains unclear. Expression levels for 7 GABA-related transcripts (vesicular GABA transporter [vGAT], GABA membrane transporter [GAT1], GABAA receptor subunit α1 [GABRA1] [novel in human and monkey cohorts], glutamic acid decarboxylase 67 [GAD67], parvalbumin, calretinin, and somatostatin [previously reported in human cohort, but not in monkey cohort]) were quantified in the PFC from 42 matched pairs of schizophrenia and comparison subjects and from 49 rhesus monkeys ranging in age from 1 week postnatal to adulthood. Levels of vGAT and GABRA1, but not of GAT1, messenger RNAs (mRNAs) were lower in the PFC of the schizophrenia subjects. As previously reported, levels of GAD67, parvalbumin, and somatostatin, but not of calretinin, mRNAs were also lower in these subjects. Neither illness duration nor age accounted for the levels of the transcripts with altered expression in schizophrenia. In monkey PFC, developmental changes in expression levels of many of these transcripts were in the opposite direction of the changes observed in schizophrenia. For example, mRNA levels for vGAT, GABRA1, GAD67, and parvalbumin all increased with age. Together with published reports, these findings support the interpretation that the altered expression of GABA-related transcripts in schizophrenia reflects a blunting of normal postnatal development changes, but they cannot exclude a decline during the early stages of clinical illness. © The Author 2013. Published by Oxford University Press on behalf of the Maryland Psychiatric Research Center. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  9. Paracrine GABA and insulin regulate pancreatic alpha cell proliferation in a mouse model of type 1 diabetes.

    PubMed

    Feng, Allen L; Xiang, Yun-Yan; Gui, Le; Kaltsidis, Gesthika; Feng, Qingping; Lu, Wei-Yang

    2017-06-01

    This study aimed to elucidate the mechanism of increased proliferation of alpha cells in recent-onset type 1 diabetes. Pancreatic beta cells express GAD and produce γ-aminobutyric acid (GABA), which inhibits alpha cell secretion of glucagon. We explored the roles of GABA in alpha cell proliferation in conditions corresponding to type 1 diabetes in a mouse model and in vitro. Type 1 diabetes was induced by injecting the mice with streptozotocin (STZ). Some of the STZ-injected mice were treated with GABA (10 mg/kg daily) for 12 days. Isolated pancreatic islets were treated with STZ or STZ together with GABA for 2 days. The effects of GABA treatment on STZ-induced alpha cell proliferation in vivo and in vitro were assessed. The effect of muscimol, a GABA receptor agonist, on αTC1-6 cell proliferation was also examined. STZ injection substantially decreased levels of GAD, GABA and insulin in pancreatic beta cells 12 h after injection; this was followed by an upsurge of phosphorylated mechanistic target of rapamycin (p-mTOR) in the alpha cells at day 1, and a significant increase in alpha cell mass at day 3. Treating STZ-injected mice with GABA largely restored the immunodetectable levels of insulin and GAD in the beta cells and significantly decreased the number of aldehyde dehydrogenase 1 family, member A3 (ALDH1a3)-positive cells, alpha cell mass and hyperglucagonaemia. STZ treatment also increased alpha cell proliferation in isolated islets, which was reversed by co-treatment with GABA. Muscimol, together with insulin, significantly lowered the level of cytosolic Ca 2+ and p-mTOR, and decreased the proliferation rate of αTC1-6 cells. GABA signalling critically controls the alpha cell population in pancreatic islets. Low intraislet GABA may contribute to alpha cell hyperplasia in early type 1 diabetes.

  10. Sodium and T1rho MRI for molecular and diagnostic imaging of articular cartilage.

    PubMed

    Borthakur, Arijitt; Mellon, Eric; Niyogi, Sampreet; Witschey, Walter; Kneeland, J Bruce; Reddy, Ravinder

    2006-11-01

    In this article, both sodium magnetic resonance (MR) and T1rho relaxation mapping aimed at measuring molecular changes in cartilage for the diagnostic imaging of osteoarthritis are reviewed. First, an introduction to structure of cartilage, its degeneration in osteoarthritis (OA) and an outline of diagnostic imaging methods in quantifying molecular changes and early diagnostic aspects of cartilage degeneration are described. The sodium MRI section begins with a brief overview of the theory of sodium NMR of biological tissues and is followed by a section on multiple quantum filters that can be used to quantify both bi-exponential relaxation and residual quadrupolar interaction. Specifically, (i) the rationale behind the use of sodium MRI in quantifying proteoglycan (PG) changes, (ii) validation studies using biochemical assays, (iii) studies on human OA specimens, (iv) results on animal models and (v) clinical imaging protocols are reviewed. Results demonstrating the feasibility of quantifying PG in OA patients and comparison with that in healthy subjects are also presented. The section concludes with the discussion of advantages and potential issues with sodium MRI and the impact of new technological advancements (e.g. ultra-high field scanners and parallel imaging methods). In the theory section on T1rho, a brief description of (i) principles of measuring T1rho relaxation, (ii) pulse sequences for computing T1rho relaxation maps, (iii) issues regarding radio frequency power deposition, (iv) mechanisms that contribute to T1rho in biological tissues and (v) effects of exchange and dipolar interaction on T1rho dispersion are discussed. Correlation of T1rho relaxation rate with macromolecular content and biomechanical properties in cartilage specimens subjected to trypsin and cytokine-induced glycosaminoglycan depletion and validation against biochemical assay and histopathology are presented. Experimental T1rho data from osteoarthritic specimens, animal models

  11. Aluminum-Activated Malate Transporters Can Facilitate GABA Transport.

    PubMed

    Ramesh, Sunita A; Kamran, Muhammad; Sullivan, Wendy; Chirkova, Larissa; Okamoto, Mamoru; Degryse, Fien; McLaughlin, Michael; Gilliham, Matthew; Tyerman, Stephen D

    2018-05-01

    Plant aluminum-activated malate transporters (ALMTs) are currently classified as anion channels; they are also known to be regulated by diverse signals, leading to a range of physiological responses. Gamma-aminobutyric acid (GABA) regulation of anion flux through ALMT proteins requires a specific amino acid motif in ALMTs that shares similarity with a GABA binding site in mammalian GABA A receptors. Here, we explore why TaALMT1 activation leads to a negative correlation between malate efflux and endogenous GABA concentrations ([GABA] i ) in both wheat ( Triticum aestivum ) root tips and in heterologous expression systems. We show that TaALMT1 activation reduces [GABA] i because TaALMT1 facilitates GABA efflux but GABA does not complex Al 3+ TaALMT1 also leads to GABA transport into cells, demonstrated by a yeast complementation assay and via 14 C-GABA uptake into TaALMT1 -expressing Xenopus laevis oocytes; this was found to be a general feature of all ALMTs we examined. Mutation of the GABA motif (TaALMT1 F213C ) prevented both GABA influx and efflux, and resulted in no correlation between malate efflux and [GABA] i We conclude that ALMTs are likely to act as both GABA and anion transporters in planta. GABA and malate appear to interact with ALMTs in a complex manner to regulate each other's transport, suggestive of a role for ALMTs in communicating metabolic status. © 2018 American Society of Plant Biologists. All rights reserved.

  12. Immunocytochemical localization of the NMDA-R2A receptor subunit in the cat retina.

    PubMed

    Goebel, D J; Aurelia, J L; Tai, Q; Jojich, L; Poosch, M S

    1998-10-19

    Immunocytochemical studies were performed to determine the distribution and cellular localization of the NMDA-R2A receptor subunit (R2A) in the cat retina. R2A-immunoreactivity (R2A-IR) was noted in all layers of the retina, with specific localizations in the outer segments of red/green and blue cone photoreceptors, B-type horizontal cells, several types of amacrine cells, Müller cells and the majority of cells in the ganglion cell layer. In the inner nuclear layer, 48% of all cells residing in the amacrine cell layer were R2A-IR including a cell resembling the GABAergic A17 amacrine cell. Interestingly, the AII rod amacrine cell was devoid of R2A-IR. Although the localization of the R2A subunit was anticipated in ganglion cells, amacrines and Müller cells, the presence of this receptor subunit to the cells in the outer retina was not expected. Here, both the R2A and the R2B subunits were found to be present in the outer segments of cone photoreceptors and to the tips of rod outer segments. Although the function of these receptor subunits in rod and cone photoreceptors remains to be determined, the fact that both R2A and R2B receptor subunits are localized to cone outer segments suggests a possible alternative pathway for calcium entry into a region where this cation plays such a crucial role in the process of phototransduction. To further classify the cells that display NR2A-IR, we performed dual labeling experiments showing the relationship between R2A-labeled cells with GABA. Results showed that all GABAergic-amacrines and displaced amacrines express the R2A-subunit protein. In addition, approximately 11% of the NR2A-labeled amacrines, did not stain for GABA. These findings support pharmacological data showing that NMDA directly facilitates GABA release in retina and retinal cultures [I.L. Ferreira, C.B. Duarte, P.F. Santos, C.M. Carvalho, A.P. Carvalho, Release of [3H]GABA evoked by glutamate receptor agonist in cultured chick retinal cells: effect of Ca2

  13. Hap1 and GABA: thinking about food intake.

    PubMed

    Woods, Stephen C; Seeley, Randy J

    2006-06-01

    GABA stimulation of hypothalamic GABAA receptors increases food intake and body weight. Huntingtin-associated protein-1 (Hap1), is highly expressed in the hypothalamus and increases activity at GABAA receptors; mice lacking Hap1 are hypophagic. A recent paper (Sheng et al.,2006) further explores the role of Hap1 in the control of food intake.

  14. GABA Levels Are Decreased After Stroke and GABA Changes During Rehabilitation Correlate With Motor Improvement

    PubMed Central

    Blicher, Jakob Udby; Near, Jamie; Næss-Schmidt, Erhard; Stagg, Charlotte J.; Johansen-Berg, Heidi; Nielsen, Jørgen Feldbæk; Østergaard, Leif; Ho, Yi-Ching Lynn

    2017-01-01

    Background and Objective γ-Aminobutyric acid (GABA) is the dominant inhibitory neurotransmitter in the brain and is important in motor learning. We aimed to measure GABA content in primary motor cortex poststroke (using GABA-edited magnetic resonance spectroscopy [MRS]) and in relation to motor recovery during 2 weeks of constraint-induced movement therapy (CIMT). Methods Twenty-one patients (3-12 months poststroke) and 20 healthy subjects were recruited. Magnetic resonance imaging structural T1 and GABA-edited MRS were performed at baseline and after CIMT, and once in healthy subjects. GABA:creatine (GABA:Cr) ratio was measured by GABA-edited MRS. Motor function was measured using Wolf Motor Function Test (WMFT). Results Baseline comparison between stroke patients (n = 19) and healthy subjects showed a significantly lower GABA:Cr ratio in stroke patients (P < .001) even after correcting for gray matter content in the voxel (P < .01) and when expressing GABA relative to N-acetylaspartic acid (NAA; P = .03). After 2 weeks of CIMT patients improved significantly on WMFT, but no consistent change across the group was observed for the GABA:Cr ratio (n = 17). However, the extent of improvement on WMFT correlated significantly with the magnitude of GABA:Cr changes (P < .01), with decreases in GABA:Cr ratio being associated with better improvements in motor function. Conclusions In patients 3 to 12 months poststroke, GABA levels are lower in the primary motor cortex than in healthy subjects. The observed association between GABA and recovery warrants further studies on the potential use of GABA MRS as a biomarker in poststroke recovery. PMID:25055837

  15. Conserved regional patterns of GABA-related transcript expression in the neocortex of subjects with schizophrenia.

    PubMed

    Hashimoto, Takanori; Bazmi, H Holly; Mirnics, Karoly; Wu, Qiang; Sampson, Allan R; Lewis, David A

    2008-04-01

    Individuals with schizophrenia exhibit disturbances in a number of cognitive, affective, sensory, and motor functions that depend on the circuitry of different cortical areas. The cognitive deficits associated with dysfunction of the dorsolateral prefrontal cortex result, at least in part, from abnormalities in GABA neurotransmission, as reflected in a specific pattern of altered expression of GABA-related genes. Consequently, the authors sought to determine whether this pattern of altered gene expression is restricted to the dorsolateral prefrontal cortex or could also contribute to the dysfunction of other cortical areas in subjects with schizophrenia. Real-time quantitative polymerase chain reaction was used to assess the levels of eight GABA-related transcripts in four cortical areas (dorsolateral prefrontal cortex, anterior cingulate cortex, and primary motor and primary visual cortices) of subjects (N=12) with schizophrenia and matched normal comparison subjects. Expression levels of seven transcripts were lower in subjects with schizophrenia, with the magnitude of reduction for each transcript comparable across the four areas. The largest reductions were detected for mRNA encoding somatostatin and parvalbumin, followed by moderate decreases in mRNA expression for the 67-kilodalton isoform of glutamic acid decarboxylase, the GABA membrane transporter GAT-1, and the alpha 1 and delta subunits of GABA(A) receptors. In contrast, the expression of calretinin mRNA did not differ between the subject groups in any of the four areas. Because the areas examined represent the major functional domains (e.g., association, limbic, motor, and sensory) of the cerebral cortex, our findings suggest that a conserved set of molecular alterations affecting GABA neurotransmission contribute to the pathophysiology of different clinical features of schizophrenia.

  16. Expression of GABA signaling molecules KCC2, NKCC1, and GAD1 in cortical development and schizophrenia.

    PubMed

    Hyde, Thomas M; Lipska, Barbara K; Ali, Towhid; Mathew, Shiny V; Law, Amanda J; Metitiri, Ochuko E; Straub, Richard E; Ye, Tianzhang; Colantuoni, Carlo; Herman, Mary M; Bigelow, Llewellyn B; Weinberger, Daniel R; Kleinman, Joel E

    2011-07-27

    GABA signaling molecules are critical for both human brain development and the pathophysiology of schizophrenia. We examined the expression of transcripts derived from three genes related to GABA signaling [GAD1 (GAD67 and GAD25), SLC12A2 (NKCC1), and SLC12A5 (KCC2)] in the prefrontal cortex (PFC) and hippocampal formation of a large cohort of nonpsychiatric control human brains (n = 240) across the lifespan (from fetal week 14 to 80 years) and in patients with schizophrenia (n = 30-31), using quantitative RT-PCR. We also examined whether a schizophrenia risk-associated promoter SNP in GAD1 (rs3749034) is related to expression of these transcripts. Our studies revealed that development and maturation of both the PFC and hippocampal formation are characterized by progressive switches in expression from GAD25 to GAD67 and from NKCC1 to KCC2. Previous studies have demonstrated that the former leads to GABA synthesis, and the latter leads to switching from excitatory to inhibitory neurotransmission. In the hippocampal formation, GAD25/GAD67 and NKCC1/KCC2 ratios are increased in patients with schizophrenia, reflecting a potentially immature GABA physiology. Remarkably, GAD25/GAD67 and NKCC1/KCC2 expression ratios are associated with rs3749034 genotype, with risk alleles again predicting a relatively less mature pattern. These findings suggest that abnormalities in GABA signaling critical to brain development contribute to genetic risk for schizophrenia.

  17. Drug-induced GABA transporter currents enhance GABA release to induce opioid withdrawal behaviors.

    PubMed

    Bagley, Elena E; Hacker, Jennifer; Chefer, Vladimir I; Mallet, Christophe; McNally, Gavan P; Chieng, Billy C H; Perroud, Julie; Shippenberg, Toni S; Christie, MacDonald J

    2011-10-30

    Neurotransmitter transporters can affect neuronal excitability indirectly via modulation of neurotransmitter concentrations or directly via transporter currents. A physiological or pathophysiological role for transporter currents has not been described. We found that GABA transporter 1 (GAT-1) cation currents directly increased GABAergic neuronal excitability and synaptic GABA release in the periaqueductal gray (PAG) during opioid withdrawal in rodents. In contrast, GAT-1 did not indirectly alter GABA receptor responses via modulation of extracellular GABA concentrations. Notably, we found that GAT-1-induced increases in GABAergic activity contributed to many PAG-mediated signs of opioid withdrawal. Together, these data support the hypothesis that GAT-1 activity directly produces opioid withdrawal signs through direct hyperexcitation of GABAergic PAG neurons and nerve terminals, which presumably enhances GABAergic inhibition of PAG output neurons. These data provide, to the best of our knowledge, the first evidence that dysregulation of a neurotransmitter transporter current is important for the maladaptive plasticity that underlies opiate withdrawal.

  18. The RNA-mediated, asymmetric ring regulatory mechanism of the transcription termination Rho helicase decrypted by time-resolved nucleotide analog interference probing (trNAIP).

    PubMed

    Soares, Emilie; Schwartz, Annie; Nollmann, Marcello; Margeat, Emmanuel; Boudvillain, Marc

    2014-08-01

    Rho is a ring-shaped, ATP-dependent RNA helicase/translocase that dissociates transcriptional complexes in bacteria. How RNA recognition is coupled to ATP hydrolysis and translocation in Rho is unclear. Here, we develop and use a new combinatorial approach, called time-resolved Nucleotide Analog Interference Probing (trNAIP), to unmask RNA molecular determinants of catalytic Rho function. We identify a regulatory step in the translocation cycle involving recruitment of the 2'-hydroxyl group of the incoming 3'-RNA nucleotide by a Rho subunit. We propose that this step arises from the intrinsic weakness of one of the subunit interfaces caused by asymmetric, split-ring arrangement of primary RNA tethers around the Rho hexamer. Translocation is at highest stake every seventh nucleotide when the weak interface engages the incoming 3'-RNA nucleotide or breaks, depending on RNA threading constraints in the Rho pore. This substrate-governed, 'test to run' iterative mechanism offers a new perspective on how a ring-translocase may function or be regulated. It also illustrates the interest and versatility of the new trNAIP methodology to unveil the molecular mechanisms of complex RNA-based systems. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

  19. Rho'ing in and out of cells: viral interactions with Rho GTPase signaling.

    PubMed

    Van den Broeke, Céline; Jacob, Thary; Favoreel, Herman W

    2014-01-01

    Rho GTPases are key regulators of actin and microtubule dynamics and organization. Increasing evidence shows that many viruses have evolved diverse interactions with Rho GTPase signaling and manipulate them for their own benefit. In this review, we discuss how Rho GTPase signaling interferes with many steps in the viral replication cycle, especially entry, replication, and spread. Seen the diversity between viruses, it is not surprising that there is considerable variability in viral interactions with Rho GTPase signaling. However, several largely common effects on Rho GTPases and actin architecture and microtubule dynamics have been reported. For some of these processes, the molecular signaling and biological consequences are well documented while for others we just begin to understand them. A better knowledge and identification of common threads in the different viral interactions with Rho GTPase signaling and their ultimate consequences for virus and host may pave the way toward the development of new antiviral drugs that may target different viruses.

  20. Cell and receptor type-specific alterations in markers of GABA neurotransmission in the prefrontal cortex of subjects with schizophrenia.

    PubMed

    Lewis, David A; Hashimoto, Takanori; Morris, Harvey M

    2008-10-01

    Impairments in cognitive control, such as those involved in working memory, are associated with dysfunction of the dorsolateral prefrontal cortex (DLPFC) in individuals with schizophrenia. This dysfunction appears to result, at least in part, from abnormalities in GABA-mediated neurotransmission. In this paper, we review recent findings indicating that the altered DLPFC circuitry in subjects with schizophrenia reflects changes in the expression of genes that encode selective presynaptic and postsynaptic components of GABA neurotransmission. Specifically, using a combination of methods, we found that subjects with schizophrenia exhibited expression deficits in GABA-related transcripts encoding presynaptic regulators of GABA neurotransmission, neuropeptide markers of specific subpopulations of GABA neurons, and certain subunits of the GABA(A) receptor. In particular, alterations in the expression of the neuropeptide somatostatin suggested that GABA neurotransmission is impaired in the Martinotti subset of GABA neurons that target the dendrites of pyramidal cells. In contrast, none of the GABA-related transcripts assessed to date were altered in the DLPFC of monkeys chronically exposed to antipsychotic medications, suggesting that the effects observed in the human studies reflect the disease process and not its treatment. In concert with previous findings, these data suggest that working memory dysfunction in schizophrenia may be attributable to altered GABA neurotransmission in specific DLPFC microcircuits.

  1. IκB Kinase γ/Nuclear Factor-κB-Essential Modulator (IKKγ/NEMO) Facilitates RhoA GTPase Activation, which, in Turn, Activates Rho-associated Kinase (ROCK) to Phosphorylate IKKβ in Response to Transforming Growth Factor (TGF)-β1*

    PubMed Central

    Kim, Hee-Jun; Kim, Jae-Gyu; Moon, Mi-Young; Park, Seol-Hye; Park, Jae-Bong

    2014-01-01

    Transforming growth factor (TGF)-β1 plays several roles in a variety of cellular functions. TGF-β1 transmits its signal through Smad transcription factor-dependent and -independent pathways. It was reported that TGF-β1 activates NF-κB and RhoA, and RhoA activates NF-κB in several kinds of cells in a Smad-independent pathway. However, the activation molecular mechanism of NF-κB by RhoA upon TGF-β1 has not been clearly elucidated. We observed that RhoA-GTP level was increased by TGF-β1 in RAW264.7 cells. RhoA-GDP and RhoGDI were bound to N- and C-terminal domains of IKKγ, respectively. Purified IKKγ facilitated GTP binding to RhoA complexed with RhoGDI. Furthermore, Dbs, a guanine nucletotide exchange factor of RhoA much more enhanced GTP binding to RhoA complexed with RhoGDI in the presence of IKKγ. Indeed, si-IKKγ abolished RhoA activation in response to TGF-β1 in cells. However, TGF-β1 stimulated the release of RhoA-GTP from IKKγ and Rho-associated kinase (ROCK), an active RhoA effector protein, directly phosphorylated IKKβ in vitro, whereas TGF-β1-activated kinase 1 activated RhoA upon TGF-β1 stimulation. Taken together, our data indicate that IKKγ facilitates RhoA activation via a guanine nucletotide exchange factor, which in turn activates ROCK to phosphorylate IKKβ, leading to NF-κB activation that induced the chemokine expression and cell migration upon TGF-β1. PMID:24240172

  2. Gamma-aminobutyric acid (GABA) receptors genes polymorphisms and risk for restless legs syndrome.

    PubMed

    Jiménez-Jiménez, Félix Javier; Esguevillas, Gara; Alonso-Navarro, Hortensia; Zurdo, Martín; Turpín-Fenoll, Laura; Millán-Pascual, Jorge; Adeva-Bartolomé, Teresa; Cubo, Esther; Navacerrada, Francisco; Amo, Gemma; Rojo-Sebastián, Ana; Rubio, Lluisa; Díez-Fairén, Mónica; Pastor, Pau; Calleja, Marisol; Plaza-Nieto, José Francisco; Pilo-de-la-Fuente, Belén; Arroyo-Solera, Margarita; García-Albea, Esteban; Agúndez, José A G; García-Martín, Elena

    2018-05-03

    The possible role of gammaaminobutyric acid (GABA) in the pathophysiology of restless legs syndrome (RLS) is suggested by the symptomatic improvement achieved with GABAergic drugs. Thalamic GABA levels have shown positive correlation with periodic limb movements indices and with RLS severity. We tried to investigate the possible association between the most common single nucleotide polymorphisms (SNPs) in the GABA receptors (GABR) genes rho1, 2, and 3 (GABRR1, GABRR2, GABRR3), alpha4 (GABRA4), epsilon (GABRE), and theta (GABRQ) with the risk of developing RLS. We studied the genotype and allelic variant frequencies of the most common SNPs in the GABRR1(rs12200969, rs1186902), GABRR2(rs282129), GABRR3(rs832032), GABRA4(rs2229940), GABRE(rs1139916), and GABRQ(rs3810651) genes in 205 RLS patients and 230 age- and gender-matched healthy controls using specific TaqMan assays. The frequencies of the GABRR3 rs832032TT genotype and the allelic variant GABRR3 rs832032T were significantly higher in RLS patients than in controls (odds ratio [95% confidence intervals] 7.08[1.48-46.44] and 1.66[1.16-2.37], respectively), although only the higher frequency of the rs832032T allele remained as significant after multiple comparison analysis, both in the whole series and in the female gender. The frequencies of the other genotypes of allelic variants did not differ significantly between RLS patients and controls. RLS patients carrying the GABRA4 rs2229940TT genotype showed a significantly younger age at onset of RLS symptoms than those with the other two genotypes. These results suggest association between GABRR3rs832032 polymorphism and the risk for RLS, and a modifier effect of GABRA4 rs2229940 on the age of onset of RLS.

  3. Synthesis and biological evaluation of novel 2,3-disubstituted benzofuran analogues of GABA as neurotropic agents.

    PubMed

    Coaviche-Yoval, Arturo; Luna, Hector; Tovar-Miranda, Ricardo; Soriano-Ursua, Marvin Antonio; Trujillo-Ferrara, Jose G

    2018-05-23

    Benzofurans are heterocyclic compounds with neurotropic activity. Some have been developed for the treatment of acute and degenerative neuronal injuries. To evaluate the in silico binding of some promising benzofurans on the GABA receptors, and the in vivo neurotropic activity of benzofuran analogues (BZF 6-10) of gamma-aminobutyric acid (GABA) on a seizure model. The ligands with the best physicochemical attributes were docked on two GABA receptors (the alpha-1 subunit of GABAA-R and GBR1 subunit of GABAB-R). Selected benzofuran derivatives were synthesized by a multistep procedure and characterized. To examine the neurotropic effects, mice were pretreated with different concentrations of the compounds prior to PTZ- or 4-AP-induced seizures. We assessed acute toxicity, motor behavior, and the effects on seizures. The tested ligands that complied with Lipinski's rule of five were tested in silico with GABAA-R (ΔG = -5.51 to -5.84 kcal/mol) at the allosteric site for benzodiazepines. They bound to a similar cluster of residues as the reference compound (gaboxadol, ΔG = -5.51 kcal/mol). Synthesis was achieved with good overall yields (42-9.7%). Two compounds were selected for biological tests (BZF-7 and rac-BZF-10) on a mouse model of seizures, induced by pentylenetetrazol (PTZ) or 4-aminopyridine (4-AP). PTZ-induced seizures are associated with GABA receptors, and those 4-AP-induced with the blockage of the delayed rectifier-type potassium channel, which promotes the release of the NMDA-sensitive glutamatergic ionotropic receptor and other neurotransmitters. The biological assays demonstrated that BZF-7 and rac-BZF-10 do not protect against seizures. Indeed, BZF-7 increased the number of PTZ-induced seizures and decreased latency time. The 4-AP model apparently showed a potentiation of seizure effects after administration of the BZF-analogues, evidenced by the incidence and severity of the seizures and reduced latency time. The results suggest that the test

  4. Rho A and the Rho kinase pathway regulate fibroblast contraction: Enhanced contraction in constitutively active Rho A fibroblast cells

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nobe, Koji, E-mail: kojinobe@pharm.showa-u.ac.jp; Nobe, Hiromi; Department of Physical Therapy, Bunkyo-Gakuin University

    Research highlights: {yields} Mechanisms of fibroblast cell contraction in collagen matrix. {yields} Assessed an isometric force development using 3D-reconstituted-fibroblast fiber. {yields} Constitutively active Rho A induced the over-contraction of fibroblast cells. {yields} Rho A and Rho kinase pathway has a central role in fibroblast cell contraction. -- Abstract: Fibroblast cells play a central role in the proliferation phase of wound healing processes, contributing to force development. The intracellular signaling pathways regulating this non-muscle contraction are only partially understood. To study the relations between Rho A and contractile responses, constitutively active Rho A (CA-Rho A) fibroblast cells were reconstituted into fibersmore » and the effects of calf serum (CS) on isometric force were studied. CS-induced force in CA-Rho A fibroblast fibers was twice as large as that in wild type (NIH 3T3) fibroblast fibers. During this response, the translocation of Rho A from the cytosol to the membrane was detected by Rho A activity assays and Western blot analysis. Pre-treatment with a Rho specific inhibitor (C3-exoenzyme) suppressed translocation as well as contraction. These results indicate that Rho A activation is essential for fibroblast contraction. The Rho kinase inhibitor ( (Y27632)) inhibited both NIH 3T3 and CA-Rho A fibroblast fiber contractions. Activation of Rho A is thus directly coupled with Rho kinase activity. We conclude that the translocation of Rho A from the cytosol to the membrane and the Rho kinase pathway can regulate wound healing processes mediated by fibroblast contraction.« less

  5. A structural study of the complex between neuroepithelial cell transforming gene 1 (Net1) and RhoA reveals a potential anticancer drug hot spot.

    PubMed

    Petit, Alain-Pierre; Garcia-Petit, Christel; Bueren-Calabuig, Juan A; Vuillard, Laurent M; Ferry, Gilles; Boutin, Jean A

    2018-06-08

    The GTPase RhoA is a major player in many different regulatory pathways. RhoA catalyzes GTP hydrolysis, and its catalysis is accelerated when RhoA forms heterodimers with proteins of the guanine nucleotide exchange factor (GEF) family. Neuroepithelial cell transforming gene 1 (Net1) is a RhoA-interacting GEF implicated in cancer, but the structural features supporting the RhoA/Net1 interaction are unknown. Taking advantage of a simple production and purification process, here we solved the structure of a RhoA/Net1 heterodimer with X-ray crystallography at 2-Å resolution. Using a panel of several techniques, including molecular dynamics simulations, we characterized the RhoA/Net1 interface. Moreover, deploying an extremely simple peptide-based scanning approach, we found that short peptides (penta- to nonapeptides) derived from the protein/protein interaction region of RhoA could disrupt the RhoA/Net1 interaction and thereby diminish the rate of nucleotide exchange. The most inhibitory peptide, EVKHF, spanning residues 102-106 in the RhoA sequence, displayed an IC 50 of ∼100 μm without further modifications. The peptides identified here could be useful in further investigations of the RhoA/Net1 interaction region. We propose that our structural and functional insights might inform chemical approaches for transforming the pentapeptide into an optimized pseudopeptide that antagonizes Net1-mediated RhoA activation with therapeutic anticancer potential. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Association of N-cadherin levels and downstream effectors of Rho GTPases with dendritic spine loss induced by chronic stress in rat hippocampal neurons.

    PubMed

    Castañeda, Patricia; Muñoz, Mauricio; García-Rojo, Gonzalo; Ulloa, José L; Bravo, Javier A; Márquez, Ruth; García-Pérez, M Alexandra; Arancibia, Damaris; Araneda, Karina; Rojas, Paulina S; Mondaca-Ruff, David; Díaz-Véliz, Gabriela; Mora, Sergio; Aliaga, Esteban; Fiedler, Jenny L

    2015-10-01

    Chronic stress promotes cognitive impairment and dendritic spine loss in hippocampal neurons. In this animal model of depression, spine loss probably involves a weakening of the interaction between pre- and postsynaptic cell adhesion molecules, such as N-cadherin, followed by disruption of the cytoskeleton. N-cadherin, in concert with catenin, stabilizes the cytoskeleton through Rho-family GTPases. Via their effector LIM kinase (LIMK), RhoA and ras-related C3 botulinum toxin substrate 1 (RAC) GTPases phosphorylate and inhibit cofilin, an actin-depolymerizing molecule, favoring spine growth. Additionally, RhoA, through Rho kinase (ROCK), inactivates myosin phosphatase through phosphorylation of the myosin-binding subunit (MYPT1), producing actomyosin contraction and probable spine loss. Some micro-RNAs negatively control the translation of specific mRNAs involved in Rho GTPase signaling. For example, miR-138 indirectly activates RhoA, and miR-134 reduces LIMK1 levels, resulting in spine shrinkage; in contrast, miR-132 activates RAC1, promoting spine formation. We evaluated whether N-cadherin/β-catenin and Rho signaling is sensitive to chronic restraint stress. Stressed rats exhibit anhedonia, impaired associative learning, and immobility in the forced swim test and reduction in N-cadherin levels but not β-catenin in the hippocampus. We observed a reduction in spine number in the apical dendrites of CA1 pyramidal neurons, with no effect on the levels of miR-132 or miR-134. Although the stress did not modify the RAC-LIMK-cofilin signaling pathway, we observed increased phospho-MYPT1 levels, probably mediated by RhoA-ROCK activation. Furthermore, chronic stress raises the levels of miR-138 in accordance with the observed activation of the RhoA-ROCK pathway. Our findings suggest that a dysregulation of RhoA-ROCK activity by chronic stress could potentially underlie spine loss in hippocampal neurons. © 2015 Wiley Periodicals, Inc.

  7. RhoGAP18B Isoforms Act on Distinct Rho-Family GTPases and Regulate Behavioral Responses to Alcohol via Cofilin

    PubMed Central

    Kalahasti, Geetha; Rodan, Aylin R.; Rothenfluh, Adrian

    2015-01-01

    Responses to the effects of ethanol are highly conserved across organisms, with reduced responses to the sedating effects of ethanol being predictive of increased risk for human alcohol dependence. Previously, we described that regulators of actin dynamics, such as the Rho-family GTPases Rac1, Rho1, and Cdc42, alter Drosophila’s sensitivity to ethanol-induced sedation. The GTPase activating protein RhoGAP18B also affects sensitivity to ethanol. To better understand how different RhoGAP18B isoforms affect ethanol sedation, we examined them for their effects on cell shape, GTP-loading of Rho-family GTPase, activation of the actin-severing cofilin, and actin filamentation. Our results suggest that the RhoGAP18B-PA isoform acts on Cdc42, while PC and PD act via Rac1 and Rho1 to activate cofilin. In vivo, a loss-of-function mutation in the cofilin-encoding gene twinstar leads to reduced ethanol-sensitivity and acts in concert with RhoGAP18B. Different RhoGAP18B isoforms, therefore, act on distinct subsets of Rho-family GTPases to modulate cofilin activity, actin dynamics, and ethanol-induced behaviors. PMID:26366560

  8. Involvement of PI3K, Akt, and RhoA in oestradiol regulation of cardiac iNOS expression.

    PubMed

    Zafirovic, Sonja; Sudar-Milovanovic, Emina; Obradovic, Milan; Djordjevic, Jelena; Jasnic, Nebojsa; Borovic, Milica Labudovic; Isenovic, Esma R

    2018-02-12

    Oestradiol is an important regulatory factor with several positive effects on the cardiovascular (CV) system. We evaluated the molecular mechanism of the in vivo effects of oestradiol on the regulation of cardiac inducible nitric oxide (NO) synthase (iNOS) expression and activity. Male Wistar rats were treated with oestradiol (40 mg/kg, intraperitoneally) and after 24 h the animals were sacrificed. The concentrations of NO and L-Arginine (L-Arg) were determined spectrophotometrically. For protein expressions of iNOS, p65 subunit of nuclear factor-κB (NFκB-p65), Ras homolog gene family-member A (RhoA), angiotensin II receptor type 1 (AT1R), insulin receptor substrate 1 (IRS-1), p85, p110 and protein kinase B (Akt), Western blot method was used. Co-immunoprecipitation was used for measuring the association of IRS-1 with the p85 subunit of phosphatidylinositol-3-kinase (PI3K). The expression of iNOS messenger ribonucleic acid (mRNA) was measured with the quantitative real-time polymerase chain reaction (qRT-PCR). Immunohistochemical analysis of the tissue was used to detect localization and expression of iNOS in heart tissue. Oestradiol treatment reduced L-Arg concentration (p<0.01), iNOS mRNA (p<0.01) and protein (p<0.001) expression, level of RhoA (p<0.05) and AT1R (p<0.001) protein. In contrast, plasma NO (p<0.05), Akt phosphorylation at Thr308 (p<0.05) and protein level of p85 (p<0.001) increased after oestradiol treatment. Our results suggest that oestradiol in vivo regulates cardiac iNOS expression via the PI3K/Akt signaling pathway, through attenuation of RhoA and AT1R. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  9. Rho Associated Coiled-Coil Kinase-1 Regulates Collagen-Induced Phosphatidylserine Exposure in Platelets

    PubMed Central

    Dasgupta, Swapan K.; Le, Anhquyen; Haudek, Sandra B.; Entman, Mark L.; Rumbaut, Rolando E.; Thiagarajan, Perumal

    2013-01-01

    Background The transbilayer movement of phosphatidylserine mediates the platelet procoagulant activity during collagen stimulation. The Rho-associated coiled-coil kinase (ROCK) inhibitor Y-27632 inhibits senescence induced but not activation induced phosphatidylserine exposure. To investigate further the specific mechanisms, we now utilized mice with genetic deletion of the ROCK1 isoform. Methods and Results ROCK1-deficient mouse platelets expose significantly more phosphatidylserine and generate more thrombin upon activation with collagen compared to wild-type platelets. There were no significant defects in platelet shape change, aggregation, or calcium response compared to wild-type platelets. Collagen-stimulated ROCK1-deficient platelets also displayed decreased phosphorylation levels of Lim Kinase-1 and cofilin-1. However, there was no reduction in phosphorylation levels of myosin phosphatase subunit-1 (MYPT1) or myosin light chain (MLC). In an in vivo light/dye-induced endothelial injury/thrombosis model, ROCK1-deficient mice presented a shorter occlusion time in cremasteric venules when compared to wild-type littermates (3.16 ± 1.33 min versus 6.6 ± 2.6 min; p = 0.01). Conclusions These studies define ROCK1 as a new regulator for collagen-induced phosphatidylserine exposure in platelets with functional consequences on thrombosis. This effect was downstream of calcium signaling and was mediated by Lim Kinase-1 / cofilin-1-induced cytoskeletal changes. PMID:24358370

  10. The amino acid motif L/IIxxFE defines a novel actin-binding sequence in PDZ-RhoGEF

    PubMed Central

    Banerjee, Jayashree; Fischer, Christopher C.; Wedegaertner, Philip B.

    2009-01-01

    PDZ-RhoGEF is a member of the regulator of G protein signaling (RGS) domain-containing RhoGEFs (RGS-RhoGEFs) that link activated heterotrimeric G protein α subunits of the G12 family to activation of the small GTPase RhoA. Unique among the RGS-RhoGEFs, PDZ-RhoGEF contains a short sequence that localizes the protein to the actin cytoskeleton. In this report, we demonstrate that the actin-binding domain, located between amino acids 561–585, directly binds to F-actin in vitro. Extensive mutagenesis identifies isoleucine 568, isoleucine 569, phenylalanine 572, and glutamic acid 573 as necessary for binding to actin and for co-localization with the actin cytoskeleton in cells. These results define a novel actin-binding sequence in PDZ-RhoGEF with a critical amino acid motif of IIxxFE. Moreover, sequence analysis identifies a similar actin-binding motif in the N-terminus of the RhoGEF frabin, and, as with PDZ-RhoGEF, mutagenesis and actin interaction experiments demonstrate a motif of LIxxFE, consisting of the key amino acids leucine 23, isoleucine 24, phenylalanine 27, and glutamic acid 28. Taken together, results with PDZ-RhoGEF and frabin identify a novel actin binding sequence. Lastly, inducible dimerization of the actin-binding region of PDZ-RhoGEF revealed a dimerization-dependent actin bundling activity in vitro. PDZ-RhoGEF exists in cells as a dimer, raising the possibility that PDZ-RhoGEF could influence actin structure independent of its ability to activate RhoA. PMID:19618964

  11. Hypoglycemia induced behavioural deficit and decreased GABA receptor, CREB expression in the cerebellum of streptozoticin induced diabetic rats.

    PubMed

    Sherin, A; Peeyush, K T; Naijil, G; Chinthu, R; Paulose, C S

    2010-11-20

    Intensive glycemic control during diabetes is associated with an increased incidence of hypoglycemia, which is the major barrier in blood glucose homeostasis during diabetes therapy. The CNS neurotransmitters play an important role in the regulation of glucose homeostasis. In the present study, we showed the effects of hypoglycemia in diabetic and non- diabetic rats on motor functions and alterations of GABA receptor and CREB expression in the cerebellum. Cerebellar dysfunction is associated with seizure generation, motor deficits and memory impairment. Scatchard analysis of [(3)H]GABA binding in the cerebellum of diabetic hypoglycemic and control hypoglycemic rats showed significant (P<0.01) decrease in B(max) and K(d) compared to diabetic and control rats. Real-time PCR amplification of GABA receptor subunit GABA(Aα1) and GAD showed significant (P<0.001) down-regulation in the cerebellum of hypoglycemic rats compared to diabetic and control rats. Confocal imaging study confirmed the decreased GABA receptors in hypoglycemic rats. CREB mRNA expression was down-regulated during recurrent hypoglycemia. Both diabetic and non-diabetic hypoglycemic rats showed impaired performance in grid walk test compared to diabetic and control. Impaired GABA receptor and CREB expression along with motor function deficit were more prominent in hypoglycemic rats than hyperglycemic which showed that hypoglycemia is causing more neuronal damage at molecular level. These molecular changes observed during hypo/hyperglycemia contribute to motor and learning deficits which has clinical significance in diabetes treatment. 2010 Elsevier Inc. All rights reserved.

  12. Enhanced p122RhoGAP/DLC-1 Expression Can Be a Cause of Coronary Spasm

    PubMed Central

    Kinjo, Takahiko; Tanaka, Makoto; Osanai, Tomohiro; Shibutani, Shuji; Narita, Ikuyo; Tanno, Tomohiro; Nishizaki, Kimitaka; Ichikawa, Hiroaki; Kimura, Yoshihiro; Ishida, Yuji; Yokota, Takashi; Shimada, Michiko; Homma, Yoshimi; Tomita, Hirofumi; Okumura, Ken

    2015-01-01

    Background We previously showed that phospholipase C (PLC)-δ1 activity was enhanced by 3-fold in patients with coronary spastic angina (CSA). We also reported that p122Rho GTPase-activating protein/deleted in liver cancer-1 (p122RhoGAP/DLC-1) protein, which was discovered as a PLC-δ1 stimulator, was upregulated in CSA patients. We tested the hypothesis that p122RhoGAP/DLC-1 overexpression causes coronary spasm. Methods and Results We generated transgenic (TG) mice with vascular smooth muscle (VSM)-specific overexpression of p122RhoGAP/DLC-1. The gene and protein expressions of p122RhoGAP/DLC-1 were markedly increased in the aorta of homozygous TG mice. Stronger staining with anti-p122RhoGAP/DLC-1 in the coronary artery was found in TG than in WT mice. PLC activities in the plasma membrane fraction and the whole cell were enhanced by 1.43 and 2.38 times, respectively, in cultured aortic vascular smooth muscle cells from homozygous TG compared with those from WT mice. Immediately after ergometrine injection, ST-segment elevation was observed in 1 of 7 WT (14%), 6 of 7 heterozygous TG (84%), and 7 of 7 homozygous TG mice (100%) (p<0.05, WT versus TGs). In the isolated Langendorff hearts, coronary perfusion pressure was increased after ergometrine in TG, but not in WT mice, despite of the similar response to prostaglandin F2α between TG and WT mice (n = 5). Focal narrowing of the coronary artery after ergometrine was documented only in TG mice. Conclusions VSM-specific overexpression of p122RhoGAP/DLC-1 enhanced coronary vasomotility after ergometrine injection in mice, which is relevant to human CSA. PMID:26624289

  13. Human α1β3γ2L gamma-aminobutyric acid type A receptors: High-level production and purification in a functional state.

    PubMed

    Dostalova, Zuzana; Zhou, Xiaojuan; Liu, Aiping; Zhang, Xi; Zhang, Yinghui; Desai, Rooma; Forman, Stuart A; Miller, Keith W

    2014-02-01

    Gamma-aminobutyric acid type A receptors (GABA(A)Rs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA(A)Rs determine their function and pharmacological profile. GABAA Rs are heteropentamers of subunits, and (α1)2 (β3)2 (γ2L)1 is a common subtype. Biochemical and biophysical studies of GABA(A)Rs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high-level production of active human α1β3 GABA(A)R using tetracycline-inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline-inducible HEK293-TetR cell line expressing human (N)-FLAG-α1β3γ2L-(C)-(GGS)3 GK-1D4 GABA(A)R. These cells achieved expression levels of 70-90 pmol [(3)H]muscimol binding sites/15-cm plate at a specific activity of 15-30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [(3)H]flunitrazepam to [(3)H]muscimol binding sites and sensitivity of GABA-induced currents to benzodiazepines and zinc. The α1β3γ2L GABA(A)Rs were solubilized in dodecyl-D-maltoside, purified by anti-FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ∼ 30%. Typical purifications yielded 1.0-1.5 nmoles of [(3)H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [(3)H]muscimol binding were maintained in the purified state. © 2013 The Protein Society.

  14. Interplay between DISC1 and GABA signaling regulates neurogenesis in mice and risk for schizophrenia.

    PubMed

    Kim, Ju Young; Liu, Cindy Y; Zhang, Fengyu; Duan, Xin; Wen, Zhexing; Song, Juan; Feighery, Emer; Lu, Bai; Rujescu, Dan; St Clair, David; Christian, Kimberly; Callicott, Joseph H; Weinberger, Daniel R; Song, Hongjun; Ming, Guo-li

    2012-03-02

    How extrinsic stimuli and intrinsic factors interact to regulate continuous neurogenesis in the postnatal mammalian brain is unknown. Here we show that regulation of dendritic development of newborn neurons by Disrupted-in-Schizophrenia 1 (DISC1) during adult hippocampal neurogenesis requires neurotransmitter GABA-induced, NKCC1-dependent depolarization through a convergence onto the AKT-mTOR pathway. In contrast, DISC1 fails to modulate early-postnatal hippocampal neurogenesis when conversion of GABA-induced depolarization to hyperpolarization is accelerated. Extending the period of GABA-induced depolarization or maternal deprivation stress restores DISC1-dependent dendritic regulation through mTOR pathway during early-postnatal hippocampal neurogenesis. Furthermore, DISC1 and NKCC1 interact epistatically to affect risk for schizophrenia in two independent case control studies. Our study uncovers an interplay between intrinsic DISC1 and extrinsic GABA signaling, two schizophrenia susceptibility pathways, in controlling neurogenesis and suggests critical roles of developmental tempo and experience in manifesting the impact of susceptibility genes on neuronal development and risk for mental disorders. Copyright © 2012 Elsevier Inc. All rights reserved.

  15. A new chromanone derivative isolated from Hypericum lissophloeus (Hypericaceae) potentiates GABAA receptor currents in a subunit specific fashion.

    PubMed

    Crockett, Sara; Baur, Roland; Kunert, Olaf; Belaj, Ferdinand; Sigel, Erwin

    2016-02-15

    A phytochemical investigation of the lipophilic extract of Hypericum lissophloeus (smoothbark St. John's wort, Hypericaceae) was conducted, resulting in the isolation and identification of a new chromanone derivative: 5,7-dihydroxy-2,3-dimethyl-6-(3-methyl-but-2-enyl)-chroman-4-one (1). This compound was demonstrated to act as a potent stimulator of currents elicited by GABA in recombinant α1β2γ2 GABAA receptors, with a half-maximal potentiation observed at a concentration of about 4μM and a maximal potentiation of >4000%. Significant potentiation was already evident at a concentration as low as 0.1μM. Extent of potentiation strongly depends on the type of α subunit, the type of β subunit and the presence of the γ subunit. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. mRNA and Protein Levels for GABA[subscript A][alpha]4, [alpha]5, [beta]1 and GABA[subscript B]R1 Receptors are Altered in Brains from Subjects with Autism

    ERIC Educational Resources Information Center

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Rooney, Robert J.; Patel, Diven H.; Thuras, Paul D.

    2010-01-01

    We have shown altered expression of gamma-aminobutyric acid A (GABA[subscript A]) and gamma-aminobutyric acid B (GABA[subscript B]) receptors in the brains of subjects with autism. In the current study, we sought to verify our western blotting data for GABBR1 via qRT-PCR and to expand our previous work to measure mRNA and protein levels of 3…

  17. The postnatal 5-HT1A receptor regulates adult anxiety and depression differently via multiple molecules.

    PubMed

    Ishikawa, Chihiro; Shiga, Takashi

    2017-08-01

    Serotonin (5-HT) and the 5-HT 1A receptor during development are known to modulate anxiety and depression in later life. However, the brain mechanisms linking the postnatal 5-HT system and adult behavior remain unknown. Here, we examined the effects of pharmacological 5-HT 1A receptor activation during the postnatal period on anxiety and depression-like behavior in adult BALB/c male mice. To elucidate the underlying mechanisms, we measured mRNA expression of the 5-HT 1A receptor, brain-derived neurotrophic factor (BDNF), GABA A receptor subunits, and AMPA receptor subunits in the medial prefrontal cortex (mPFC), amygdala, and hippocampus. Treatment with the selective 5-HT reuptake inhibitor (fluoxetine) and 5-HT 1A receptor agonist (8-OH-DPAT) during the postnatal period decreased anxiety-like behavior in adulthood, whereas only 8-OH-DPAT treatment increased depression-like behavior. Concomitantly with the behavioral effects, postnatal treatment with fluoxetine and 8-OH-DPAT decreased the mRNA expression of the GABA A receptor α3 subunit in the mPFC and ventral hippocampus in adulthood, while 8-OH-DPAT, but not fluoxetine, decreased the mRNA expression of the 5-HT 1A receptor and BDNF in the mPFC and the GABA A receptor α2 subunit in the mPFC and ventral hippocampus. On the basis of the correlative changes between behavior and mRNA expression, these results suggest that the GABA A receptor α3 subunit in the mPFC and ventral hippocampus may regulate anxiety-like behavior. In contrast, depression-like behavior may be regulated by the 5-HT 1A receptor and BDNF in the mPFC and by the GABA A receptor α2 subunit in the mPFC and ventral hippocampus. In summary, activation of the 5-HT 1A receptor during the postnatal period may reduce anxiety levels, but increase depression levels during adulthood via different multiple molecules in the mPFC and ventral hippocampus. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. Laser photolysis of DPNI-GABA, a tool for investigating the properties and distribution of GABA receptors and for silencing neurons in situ.

    PubMed

    Trigo, Federico F; Papageorgiou, George; Corrie, John E T; Ogden, David

    2009-07-30

    Laser photolysis to release GABA at precisely defined times and locations permits investigation of the distribution of functional GABA(A) receptors in neuronal compartments, the activation kinetics and pharmacology of GABA(A) receptors in situ, and the role of individual neurons in neural circuits by selective silencing with low GABA concentrations. We describe the experimental evaluation and applications of a new nitroindoline-caged GABA, DPNI-GABA, modified to minimize the pharmacological interference commonly found with caged GABA reagents, but retaining the advantages of nitroindoline cages. Unlike the 5-methoxycarbonylmethyl-7-nitroindolinyl-GABA tested previously, DPNI-GABA inhibited GABA(A) receptors with much lower affinity, reducing peak GABA-evoked responses with an IC(50) of approximately 0.5 mM. Most importantly, the kinetics of receptor activation, determined as 10-90% rise-times, were comparable to synaptic events and were little affected by DPNI-GABA present at 1mM concentration, permitting photolysis of DPNI-GABA to mimic synaptic activation of GABA(A) receptors. With a laser spot of 1 microm applied to cerebellar molecular layer interneurons, the spatial resolution of uncaging DPNI-GABA in dendrites was estimated as 2 microm laterally and 7.5 microm focally. Finally, at low DPNI-GABA concentration, photorelease restricted to the area of the soma suppressed spiking in single Purkinje neurons or molecular layer interneurons for periods controlled by the flash intensity and duration. DPNI-GABA has properties better adapted for fast kinetic studies with laser photolysis at GABA(A) receptors than previously reported caged GABA reagents, and can be used in experiments where spatial resolution is determined by the dimensions of the laser light spot.

  19. Increased Rho kinase activity in congestive heart failure

    PubMed Central

    Dong, Ming; Liao, James K.; Fang, Fang; Lee, Alex Pui-Wai; Yan, Bryan Ping-Yen; Liu, Ming; Yu, Cheuk-Man

    2012-01-01

    Aims Rho kinases (ROCKs) are the best characterized effectors of the small G-protein RhoA, and play a role in enhanced vasoconstriction in animal models of congestive heart failure (CHF). This study examined if ROCK activity is increased in CHF and how it is associated with the outcome in CHF. Methods and results Patients admitted with CHF (n =178), disease controls (n =31), and normal subjects (n =30) were studied. Baseline ROCK activity was measured by phosphorylation of themyosin-binding subunit in peripheral leucocytes. The patients were followed up for 14.4 ± 7.2 months (range 0.5–26 months) or until the occurrence of cardiac death. The ROCK activity in CHF patients (2.93 ± 0.87) was significantly higher than that of the disease control (2.06 ± 0.38, P < 0.001) and normal control (1.57 ± 0.43, P < 0.001) groups. Similarly, protein levels of ROCK1 and ROCK2 as well as the activity of RhoA in CHF were significantly higher than in disease controls and normal controls (all P < 0.05). Dyspnoea at rest (β =0.338, P < 0.001), low left ventricular ejection fraction (β = –0.277, P < 0.001), and high creatinine (β =0.202, P =0.006) were independent predictors of the baseline ROCK activity in CHF. Forty-five patients died within 2 years follow-up (25.3%). Combining ROCK activity and N-terminal pro brain natriuretic peptide (NT-proBNP) had an incremental value (log rank χ2 =11.62) in predicting long-term mortality when compared with only NT-proBNP (log rank χ2 =5.16, P < 0.05). Conclusion ROCK activity is increased in CHF and it might be associated with the mortality in CHF. ROCK activity might be a complementary biomarker to CHF risk stratification. PMID:22588320

  20. GIRK Channels Modulate Opioid-Induced Motor Activity in a Cell Type- and Subunit-Dependent Manner

    PubMed Central

    Kotecki, Lydia; Hearing, Matthew; McCall, Nora M.; Marron Fernandez de Velasco, Ezequiel; Pravetoni, Marco; Arora, Devinder; Victoria, Nicole C.; Munoz, Michaelanne B.; Xia, Zhilian; Slesinger, Paul A.; Weaver, C. David

    2015-01-01

    G-protein-gated inwardly rectifying K+ (GIRK/Kir3) channel activation underlies key physiological effects of opioids, including analgesia and dependence. GIRK channel activation has also been implicated in the opioid-induced inhibition of midbrain GABA neurons and consequent disinhibition of dopamine (DA) neurons in the ventral tegmental area (VTA). Drug-induced disinhibition of VTA DA neurons has been linked to reward-related behaviors and underlies opioid-induced motor activation. Here, we demonstrate that mouse VTA GABA neurons express a GIRK channel formed by GIRK1 and GIRK2 subunits. Nevertheless, neither constitutive genetic ablation of Girk1 or Girk2, nor the selective ablation of GIRK channels in GABA neurons, diminished morphine-induced motor activity in mice. Moreover, direct activation of GIRK channels in midbrain GABA neurons did not enhance motor activity. In contrast, genetic manipulations that selectively enhanced or suppressed GIRK channel function in midbrain DA neurons correlated with decreased and increased sensitivity, respectively, to the motor-stimulatory effect of systemic morphine. Collectively, these data support the contention that the unique GIRK channel subtype in VTA DA neurons, the GIRK2/GIRK3 heteromer, regulates the sensitivity of the mouse mesolimbic DA system to drugs with addictive potential. PMID:25948263

  1. Gad1 mRNA as a reliable indicator of altered GABA release from orexigenic neurons in the hypothalamus

    PubMed Central

    Dicken, Matthew S.; Hughes, Alexander R.; Hentges, Shane T.

    2016-01-01

    The strength of γ-aminobutyric acid (GABA)-mediated inhibitory synaptic input is a principle determinant of neuronal activity. However, because of differences in the number of GABA afferent inputs and the sites of synapses, it is difficult to directly assay for altered GABA transmission between specific cells. The present study tested the hypothesis that the level of mRNA for the GABA synthetic enzyme glutamate decarboxylase (GAD) can provide a reliable proxy for GABA release. This was tested in a mouse hypothalamic circuit important in the regulation of energy balance. Fluorescent in situ hybridization results show that the expression of Gad1 mRNA (encoding the GAD67 enzyme) was increased in hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons after an overnight fast, consistent with the ability of GABA from these neurons to stimulate food intake. Optogenetic studies confirmed that the observed increase in Gad1 mRNA correlated with an increase in the probability of GABA release from NPY/AgRP neurons onto downstream proopiomelanocortin neurons. Likewise, there was an increase in the readily releasable pool of GABA in NPY/AgRP neurons. Selective inhibition of GAD activity in NPY/AgRP neurons decreased GABA release, indicating that GAD67 activity, which is largely dictated by expression level, is a key determinant of GABA release. Altogether, it appears that Gad expression may be a reliable proxy of altered GABAergic transmission. Examining changes in Gad mRNA as a proxy for GABA release may be particularly helpful when the downstream targets are not known or when limited tools exist for detecting GABA release at a particular synapse. PMID:26370162

  2. Impact of exogenous GABA treatments on endogenous GABA metabolism in anthurium cut flowers in response to postharvest chilling temperature.

    PubMed

    Aghdam, Morteza Soleimani; Naderi, Roohangiz; Jannatizadeh, Abbasali; Babalar, Mesbah; Sarcheshmeh, Mohammad Ali Askari; Faradonbe, Mojtaba Zamani

    2016-09-01

    Anthurium flowers are susceptible to chilling injury, and the optimum storage temperature is 12.5-20 °C. The γ-aminobutyric acid (GABA) shunt pathway may alleviate chilling stress in horticultural commodities by providing energy (ATP), reducing molecules (NADH), and minimizing accumulation of reactive oxygen species (ROS). In this experiment, the impact of a preharvest spray treatment with 1 mM GABA and postharvest treatment of 5 mM GABA stem-end dipping on GABA shunt pathway activity of anthurium cut flowers (cv. Sirion) in response to cold storage (4 °C for 21 days) was investigated. GABA treatments resulted in lower glutamate decarboxylase (GAD) and higher GABA transaminase (GABA-T) activities in flowers during cold storage, which was associated with lower GABA content and coincided with higher ATP content. GABA treatments also enhanced accumulation of endogenous glycine betaine (GB) in flowers during cold storage, as well as higher spathe relative water content (RWC). These findings suggest that GABA treatments may alleviate chilling injury of anthurium cut flowers by enhancing GABA shunt pathway activity leading to provide sufficient ATP and promoting endogenous GB accumulation. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

  3. An activating mutant of Rac1 that fails to interact with Rho GDP-dissociation inhibitor stimulates membrane ruffling in mammalian cells.

    PubMed Central

    Gandhi, Payal N; Gibson, Richard M; Tong, Xiaofeng; Miyoshi, Jun; Takai, Yoshimi; Konieczkowski, Martha; Sedor, John R; Wilson-Delfosse, Amy L

    2004-01-01

    Rac1, a member of the Rho family of small GTP-binding proteins, is involved in the regulation of the actin cytoskeleton via activation of lamellipodia and membrane ruffle formation. RhoGDI (Rho-family-specific GDP-dissociation inhibitor) forms a complex with Rho proteins in the cytosol of mammalian cells. It not only regulates guanine nucleotide binding to Rho proteins, but may also function as a molecular shuttle to carry Rho proteins from an inactive cytosolic pool to the membrane for activation. These studies tested if RhoGDI is necessary for the translocation of Rac1 from the cytosol to the plasma membrane for the formation of membrane ruffles. We describe a novel mutant of Rac1, R66E (Arg66-->Glu), that fails to bind RhoGDI. This RhoGDI-binding-defective mutation is combined with a Rac1-activating mutation G12V, resulting in a double-mutant [Rac1(G12V/R66E)] that fails to interact with RhoGDI in COS-7 cells, but remains constitutively activated. This double mutant stimulates membrane ruffling to a similar extent as that observed after epidermal growth factor treatment of non-transfected cells. To confirm that Rac1 can signal ruffle formation in the absence of interaction with RhoGDI, Rac1(G12V) was overexpressed in cultured mesangial cells derived from a RhoGDI knockout mouse. Rac1-mediated membrane ruffling was indistinguishable between the RhoGDI(-/-) and RhoGDI(+/+) cell lines. In both the COS-7 and cultured mesangial cells, Rac1(G12V) and Rac1(G12V/R66E) co-localize with membrane ruffles. These findings suggest that interaction with RhoGDI is not essential in the mechanism by which Rac1 translocates to the plasma membrane to stimulate ruffle formation. PMID:14629200

  4. Rho1- and Pkc1-dependent phosphorylation of the F-BAR protein Syp1 contributes to septin ring assembly

    PubMed Central

    Merlini, Laura; Bolognesi, Alessio; Juanes, Maria Angeles; Vandermoere, Franck; Courtellemont, Thibault; Pascolutti, Roberta; Séveno, Martial; Barral, Yves; Piatti, Simonetta

    2015-01-01

    In many cell types, septins assemble into filaments and rings at the neck of cellular appendages and/or at the cleavage furrow to help compartmentalize the plasma membrane and support cytokinesis. How septin ring assembly is coordinated with membrane remodeling and controlled by mechanical stress at these sites is unclear. Through a genetic screen, we uncovered an unanticipated link between the conserved Rho1 GTPase and its effector protein kinase C (Pkc1) with septin ring stability in yeast. Both Rho1 and Pkc1 stabilize the septin ring, at least partly through phosphorylation of the membrane-associated F-BAR protein Syp1, which colocalizes asymmetrically with the septin ring at the bud neck. Syp1 is displaced from the bud neck upon Pkc1-dependent phosphorylation at two serines, thereby affecting the rigidity of the new-forming septin ring. We propose that Rho1 and Pkc1 coordinate septin ring assembly with membrane and cell wall remodeling partly by controlling Syp1 residence at the bud neck. PMID:26179915

  5. Gad1 mRNA as a reliable indicator of altered GABA release from orexigenic neurons in the hypothalamus.

    PubMed

    Dicken, Matthew S; Hughes, Alexander R; Hentges, Shane T

    2015-11-01

    The strength of γ-aminobutyric acid (GABA)-mediated inhibitory synaptic input is a principle determinant of neuronal activity. However, because of differences in the number of GABA afferent inputs and the sites of synapses, it is difficult to directly assay for altered GABA transmission between specific cells. The present study tested the hypothesis that the level of mRNA for the GABA synthetic enzyme glutamate decarboxylase (GAD) can provide a reliable proxy for GABA release. This was tested in a mouse hypothalamic circuit important in the regulation of energy balance. Fluorescent in situ hybridization results show that the expression of Gad1 mRNA (encoding the GAD67 enzyme) was increased in hypothalamic neuropeptide Y/agouti-related peptide (NPY/AgRP) neurons after an overnight fast, consistent with the ability of GABA from these neurons to stimulate food intake. Optogenetic studies confirmed that the observed increase in Gad1 mRNA correlated with an increase in the probability of GABA release from NPY/AgRP neurons onto downstream proopiomelanocortin neurons. Likewise, there was an increase in the readily releasable pool of GABA in NPY/AgRP neurons. Selective inhibition of GAD activity in NPY/AgRP neurons decreased GABA release, indicating that GAD67 activity, which is largely dictated by expression level, is a key determinant of GABA release. Altogether, it appears that Gad expression may be a reliable proxy of altered GABAergic transmission. Examining changes in Gad mRNA as a proxy for GABA release may be particularly helpful when the downstream targets are not known or when limited tools exist for detecting GABA release at a particular synapse. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  6. Effect of chronic treatment with the GABA transaminase inhibitors γ-vinyl GABA and ethanolamine O-sulphate on the in vitro GABA release from rat hippocampus

    PubMed Central

    Qume, M; Fowler, L J

    1997-01-01

    The effects of 2, 8 and 21 day oral treatment with the specific γ-aminobutyric acid transaminase (GABA-T) inhibitors γ-vinyl GABA (GVG) and ethanolamine O-sulphate (EOS) on brain GABA levels, GABA-T activity, and basal and stimulated GABA release from rat cross-chopped brain hippocampal slices was investigated. Treatment with GABA-T inhibitors lead to a reduction in brain GABA-T activity by 65–80% compared with control values, with a concomitant increase in brain GABA content of 40–100%. Basal hippocampal GABA release was increased to 250–450% of control levels following inhibition of GABA-T activity. No Ca2+ dependence was observed in either control or treated tissues. GVG and EOS administration led to a significant elevation in the potassium stimulated release of GABA from cross-chopped hippocampal slices compared with that of controls. Although stimulated GABA release from control tissues was decreased in the presence of a low Ca2+ medium, GVG and EOS treatment abolished this Ca2+ dependency. GABA compartmentalization, Na+ and Cl− coupled GABA uptake carriers and glial release may provide explanations for the loss of the Ca2+ dependency of stimulated GABA release observed following GVG and EOS treatment. Administration of GABA-T inhibitors led to increases in both basal and stimulated hippocampal GABA release. However, it is not clear which is the most important factor in the anticonvulsant activity of these drugs, the increased GABA content ‘leaking' out of neurones and glia leading to widespread inhibition, or the increase in stimulated GABA release which may occur following depolarization caused by an epileptic discharge. PMID:9351512

  7. Rho-associated coiled-coil containing kinases (ROCK)

    PubMed Central

    Julian, Linda; Olson, Michael F

    2014-01-01

    Rho-associated coiled-coil containing kinases (ROCK) were originally identified as effectors of the RhoA small GTPase.1–5 They belong to the AGC family of serine/threonine kinases6 and play vital roles in facilitating actomyosin cytoskeleton contractility downstream of RhoA and RhoC activation. Since their discovery, ROCK kinases have been extensively studied, unveiling their manifold functions in processes including cell contraction, migration, apoptosis, survival, and proliferation. Two mammalian ROCK homologs have been identified, ROCK1 (also called ROCK I, ROKβ, Rho-kinase β, or p160ROCK) and ROCK2 (also known as ROCK II, ROKα, or Rho kinase), hereafter collectively referred to as ROCK. In this review, we will focus on the structure, regulation, and functions of ROCK. PMID:25010901

  8. Mechanisms of anabolic androgenic steroid inhibition of mammalian ɛ-subunit-containing GABAA receptors

    PubMed Central

    Jones, Brian L; Whiting, Paul J; Henderson, Leslie P

    2006-01-01

    GABAergic transmission regulates the activity of gonadotrophin-releasing hormone (GnRH) neurons in the preoptic area/hypothalamus that control the onset of puberty and the expression of reproductive behaviours. One of the hallmarks of illicit use of anabolic androgenic steroids (AAS) is disruption of behaviours under neuroendocrine control. GnRH neurons are among a limited population of cells that express high levels of the ɛ-subunit of the GABAA receptor. To better understand the actions of AAS on neuroendocrine mechanisms, we have characterized modulation of GABAA receptor-mediated currents in mouse native GnRH neurons and in heterologous cells expressing recombinant α2β3ɛ-receptors. GnRH neurons exhibited robust currents in response to millimolar concentrations of GABA and a picrotoxin (PTX)-sensitive, bicuculline-insensitive current that probably arises from spontaneous openings of GABAA receptors. The AAS 17α-methyltestosterone (17α-MeT) inhibited spontaneous and GABA-evoked currents in GnRH neurons. For recombinant α2β3ɛ-receptors, 17α-MeT inhibited phasic and tonic GABA-elicited responses, accelerated desensitization and slowed paired pulse response recovery. Single channel analysis indicated that GABA-evoked events could be described by three open dwell components and that 17α-MeT enhanced residence in the intermediate dwell state. This AAS also inhibited a PTX-sensitive, spontaneous current (open probability, ∼0.15–0.2) in a concentration-dependent fashion (IC50 ≈ 9 μm). Kinetic modelling indicated that the inhibition induced by 17α-MeT occurs by an allosteric block in which the AAS interacts preferentially with a closed state and promotes accumulation in that state. Finally, studies with a G302S mutant ɛ-subunit suggest that this residue within the transmembrane domain TM2 plays a role in mediating AAS binding and modulation. In sum, our results indicate that inclusion of the ɛ-subunit significantly alters the profile of AAS

  9. Association of GABA(B)R1 receptor gene polymorphism with obstructive sleep apnea syndrome.

    PubMed

    Bayazit, Yildirim A; Yilmaz, Metin; Kokturk, Oguz; Erdal, M Emin; Ciftci, Tansu; Gokdogan, Tuba; Kemaloglu, Yusuf; Ileri, Fikret

    2007-01-01

    GABA(B)R (gamma-amino butyric acid B receptor)-mediated neurotransmission has been implicated in the pathophysiology of a variety of neuropsychiatric disorders. GABA(B)R1 gene variants were identified by single-strand conformation analysis. The nucleotide exchanges cause a substitution of alanine to valine in exon 1a1 (Ala20Val), a substitution of glycine to serine in exon 7 (Gly489Ser) and a silent C to G nucleotide exchange encoding the amino acid phenylalanine in exon 11 (Phe658Phe). The significance of GABA(B)R1a gene polymorphism in obstructive sleep apnea syndrome (OSAS) as well as the association of these polymorphisms with the polysomnography findings in OSAS patients are not known. In this study, we aimed to assess the significance of 3 different GABA(B)R1 gene polymorphisms (Ala20Val, Gly489Ser and Phe658Phe) in OSAS. Seventy-five patients (23 female and 52 male) with OSAS and 99 healthy volunteers (51 female, 48 male) were included in the study to assess Ala20Val, Gly489Ser and Phe658Phe polymorphisms of the GABA(B)R1 gene. For the Ala20Val variants, there was no significant difference between the genotypes and allele frequencies of the patients and controls, nor between both genders (p > 0.05). For Phe658Phe polymorphism, there was no significant difference between genotypes and allele frequencies of the patients and controls (p > 0.05). However, the C/C genotype was overrepresented and the T/C genotype was less frequent in male than female patients (p = 0.03). The C/C genotype was overrepresented and the T/C genotype was less frequent in male patients than male controls (p = 0.01). For GABA(B)R1-Gly489Ser polymorphism, all of the patients and controls had G/G genotype. The apnea arousal index scores of the male patients with C/C genotype were significantly higher than in the patients with C/T genotype (p = 0.01). The percent total sleep time in non-REM 1 scores of the male patients with T/T genotype were significantly higher than in the patients with T

  10. Fine structure of OXI1, the mitochondrial gene coding for subunit II of yeast cytochrome c oxidase.

    PubMed

    Weiss-Brummer, B; Guba, R; Haid, A; Schweyen, R J

    1979-12-01

    Genetic and biochemical studies have been performed with 110 mutants which are defective in cytochrome a·a3 and map in the regions on mit DNA previously designated OXI1 and OXI2. With 88 mutations allocated to OXI1 fine structure mapping was achieved by the analysis of rho (-) deletions. The order of six groups of mutational sites (A 1, A2, B 1, B2, C 1, C2) thus determined was confirmed by oxi i x oxi j recombination analysis.Analysis of mitochondrially translated polypeptides of oxil mutants by SDS-polyacrylamide electrophoresis reveals three classes of mutant patterns: i) similar to wild-tpye (19 mutants); ii) lacking SU II of cytochrome c oxidase (53 mutants); iii) lacking this subunit and exhibiting a single new polypeptide of lower Mr (16 mutants). Mutations of each of these classes are scattered over the OXI1 region without any detectable clustering; this is consistent with the assumption that all oxil mutations studied are within the same gene.New polypeptides observed in oxil mutants of class iii) vary in Mr in the range from 10,500 to 33,000. Those of Mr 17,000 to 33,000 are shown to be antigenically related to subunit II of cytochrome c oxidase. Colinearity is established between the series of new polypeptides of Mr values increasing from 10,500 to 31,500 and the order of the respective mutational sites on the map, e.g. mutations mapping in A 1 generate the smallest and mutations mapping in C2 the largest mutant fragments.From these data we conclude that i) all mutations allocated to the OXI1 region are in the same gene; ii) this gene codes for subunit II of cytochrome c oxidase; iii) the direction of translation is from CAP to 0X12. Out of 19 mutants allocated to OXI2 three exhibit a new polypeptide; these and all the other oxi2 mutants lack subunit III of cytochrome oxidase. This result provides preliminary evidence that the OXI2 region harbours the structural gene for this subunit III.

  11. Mutations in the GABA Transporter SLC6A1 Cause Epilepsy with Myoclonic-Atonic Seizures

    PubMed Central

    Carvill, Gemma L.; McMahon, Jacinta M.; Schneider, Amy; Zemel, Matthew; Myers, Candace T.; Saykally, Julia; Nguyen, John; Robbiano, Angela; Zara, Federico; Specchio, Nicola; Mecarelli, Oriano; Smith, Robert L.; Leventer, Richard J.; Møller, Rikke S.; Nikanorova, Marina; Dimova, Petia; Jordanova, Albena; Petrou, Steven; Helbig, Ingo; Striano, Pasquale; Weckhuysen, Sarah; Berkovic, Samuel F.; Scheffer, Ingrid E.; Mefford, Heather C.

    2015-01-01

    GAT-1, encoded by SLC6A1, is one of the major gamma-aminobutyric acid (GABA) transporters in the brain and is responsible for re-uptake of GABA from the synapse. In this study, targeted resequencing of 644 individuals with epileptic encephalopathies led to the identification of six SLC6A1 mutations in seven individuals, all of whom have epilepsy with myoclonic-atonic seizures (MAE). We describe two truncations and four missense alterations, all of which most likely lead to loss of function of GAT-1 and thus reduced GABA re-uptake from the synapse. These individuals share many of the electrophysiological properties of Gat1-deficient mice, including spontaneous spike-wave discharges. Overall, pathogenic mutations occurred in 6/160 individuals with MAE, accounting for ∼4% of unsolved MAE cases. PMID:25865495

  12. Identification of a Novel, Putative Rho-specific GDP/GTP Exchange Factor and a RhoA-binding Protein: Control of Neuronal Morphology

    PubMed Central

    Gebbink, Martijn F.B.G.; Kranenburg, Onno; Poland, Mieke; van Horck, Francis P.G.; Houssa, Brahim; Moolenaar, Wouter H.

    1997-01-01

    The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein–coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116Rip, that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116Rip stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116Rip fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein–coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116Rip inhibits RhoA-stimulated contractility and promotes neurite outgrowth. PMID:9199174

  13. A tip-localized RhoGAP controls cell polarity by globally inhibiting Rho GTPase at the cell apex.

    PubMed

    Hwang, Jae-Ung; Vernoud, Vanessa; Szumlanski, Amy; Nielsen, Erik; Yang, Zhenbiao

    2008-12-23

    Highly elongated eukaryotic cells (e.g., neuronal axons, fungal hyphae, and pollen tubes) are generated through continuous apically restricted growth (tip growth), which universally requires tip-localized Rho GTPases. We used the oscillating pollen tube as a model system to determine the function and regulation of Rho GTPases in tip growth. Our previous work showed that the spatiotemporal dynamics of the apical cap of the activated Rho-like GTPase from Plant 1 (ROP1) are critical for tip growth in pollen tubes. However, the underlying mechanism for the generation and maintenance of this dynamic apical cap is poorly understood. A screen for mutations that enhance ROP1-overexpression-induced depolarization of pollen-tube growth identified REN1 (ROP1 enhancer 1) in Arabidopsis, whose null mutations turn elongated pollen tubes into bulbous cells. REN1 encodes a novel Rho GTPase-activating protein (RhoGAP) required for restricting the ROP1 activity to the pollen-tube tip. REN1 was localized to exocytic vesicles accumulated in the pollen-tube apex, as well as to the apical plasma membrane at the site of ROP1 activation. The apical localization of REN1 and its function in controlling growth polarity was compromised by disruption of ROP1-dependent F-actin and vesicular trafficking, which indicates that REN1 targeting and function is regulated by ROP1 downstream signaling. Our findings suggest that the REN1 RhoGAP controls a negative-feedback-based global inhibition of ROP1. This function provides a critical self-organizing mechanism, by which ROP signaling is spatially limited to the growth site and temporally oscillates during continuous tip growth. Similar spatiotemporal control of Rho GTPase signaling may also play an important role in cell-polarity control in other systems, including tip growth in fungi and cell movement in animals.

  14. The effects of agonists of ionotropic GABA(A) and metabotropic GABA(B) receptors on learning.

    PubMed

    Zyablitseva, Evgeniya A; Kositsyn, Nikolay S; Shul'gina, Galina I

    2009-05-01

    The research described here investigates the role played by inhibitory processes in the discriminations made by the nervous system of humans and animals between familiar and unfamiliar and significant and nonsignificant events. This research compared the effects of two inhibitory mediators of gamma-aminobutyric acid (GABA): 1) phenibut, a nonselective agonist of ionotropic GABA(A) and metabotropic GABA(B) receptors and 2) gaboxadol a selective agonist of ionotropic GABA(A) receptors on the process of developing active defensive and inhibitory conditioned reflexes in alert non-immobilized rabbits. It was found that phenibut, but not gaboxadol, accelerates the development of defensive reflexes at an early stage of conditioning. Both phenibut and gaboxadol facilitate the development of conditioned inhibition, but the effect of gaboxadol occurs at later stages of conditioning and is less stable than that of phenibut. The earlier and more stable effects of phenibut, as compared to gaboxadol, on storage in memory of the inhibitory significance of a stimulus may occur because GABA(B) receptors play the dominant role in the development of internal inhibition during an early stage of conditioning. On the other hand this may occur because the participation of both GABA(A) and GABA(B) receptors are essential to the process. We discuss the polyfunctionality of GABA receptors as a function of their structure and the positions of the relevant neurons in the brain as this factor can affect regulation of various types of psychological processes.

  15. RhoB controls coordination of adult angiogenesis and lymphangiogenesis following injury by regulating VEZF1-mediated transcription

    NASA Astrophysics Data System (ADS)

    Gerald, Damien; Adini, Irit; Shechter, Sharon; Perruzzi, Carole; Varnau, Joseph; Hopkins, Benjamin; Kazerounian, Shiva; Kurschat, Peter; Blachon, Stephanie; Khedkar, Santosh; Bagchi, Mandrita; Sherris, David; Prendergast, George C.; Klagsbrun, Michael; Stuhlmann, Heidi; Rigby, Alan C.; Nagy, Janice A.; Benjamin, Laura E.

    2013-11-01

    Mechanisms governing the distinct temporal dynamics that characterize post-natal angiogenesis and lymphangiogenesis elicited by cutaneous wounds and inflammation remain unclear. RhoB, a stress-induced small GTPase, modulates cellular responses to growth factors, genotoxic stress and neoplastic transformation. Here we show, using RhoB null mice, that loss of RhoB decreases pathological angiogenesis in the ischaemic retina and reduces angiogenesis in response to cutaneous wounding, but enhances lymphangiogenesis following both dermal wounding and inflammatory challenge. We link these unique and opposing roles of RhoB in blood versus lymphatic vasculatures to the RhoB-mediated differential regulation of sprouting and proliferation in primary human blood versus lymphatic endothelial cells. We demonstrate that nuclear RhoB-GTP controls expression of distinct gene sets in each endothelial lineage by regulating VEZF1-mediated transcription. Finally, we identify a small-molecule inhibitor of VEZF1-DNA interaction that recapitulates RhoB loss in ischaemic retinopathy. Our findings establish the first intra-endothelial molecular pathway governing the phased response of angiogenesis and lymphangiogenesis following injury.

  16. Somatostatin, acting at receptor subtype 1, inhibits Rho activity, the assembly of actin stress fibers, and cell migration.

    PubMed

    Buchan, Alison M J; Lin, Chin-Yu; Choi, Jimmy; Barber, Diane L

    2002-08-09

    Somatostatin regulates multiple biological functions by acting through a family of five G protein-coupled receptors, somatostatin receptors (SSTRs) 1-5. Although all five receptor subtypes inhibit adenylate cyclase activity and decrease intracellular cAMP levels, specific receptor subtypes also couple to additional signaling pathways. In CCL39 fibroblasts expressing either human SSTR1 or SSTR2, we demonstrate that activation of SSTR1 (but not SSTR2) attenuated both thrombin- and integrin-stimulated Rho-GTP complex formation. The reduction in Rho-GTP formation in the presence of somatostatin was associated with decreased translocation of Rho and LIM kinase to the plasma membrane and fewer focal contacts. Activation of Rho resulted in the formation of intracellular actin stress fibers and cell migration. In CCL39-R1 cells, somatostatin treatment prevented actin stress fiber assembly and attenuated thrombin-stimulated cell migration through Transwell membranes to basal levels. To show that native SSTR1 shares the ability to inhibit Rho activation, we demonstrated that somatostatin treatment of human umbilical vein endothelial cells attenuated thrombin-stimulated Rho-GTP accumulation. These data show for the first time that a G protein-coupled receptor, SSTR1, inhibits the activation of Rho, the assembly of focal adhesions and actin stress fibers, and cell migration.

  17. The RhoGAP activity of CYK-4/MgcRacGAP functions non-canonically by promoting RhoA activation during cytokinesis

    PubMed Central

    Zhang, Donglei; Glotzer, Michael

    2015-01-01

    Cytokinesis requires activation of the GTPase RhoA. ECT-2, the exchange factor responsible for RhoA activation, is regulated to ensure spatiotemporal control of contractile ring assembly. Centralspindlin, composed of the Rho family GTPase-activating protein (RhoGAP) MgcRacGAP/CYK-4 and the kinesin MKLP1/ZEN-4, is known to activate ECT-2, but the underlying mechanism is not understood. We report that ECT-2-mediated RhoA activation depends on the ability of CYK-4 to localize to the plasma membrane, bind RhoA, and promote GTP hydrolysis by RhoA. Defects resulting from loss of CYK-4 RhoGAP activity can be rescued by activating mutations in ECT-2 or depletion of RGA-3/4, which functions as a conventional RhoGAP for RhoA. Consistent with CYK-4 RhoGAP activity contributing to GEF activation, the catalytic domains of CYK-4 and ECT-2 directly interact. Thus, counterintuitively, CYK-4 RhoGAP activity promotes RhoA activation. We propose that the most active form of the cytokinetic RhoGEF involves complex formation between ECT-2, centralspindlin and RhoA. DOI: http://dx.doi.org/10.7554/eLife.08898.001 PMID:26252513

  18. Reduced Brain GABA in Primary Insomnia: Preliminary Data from 4T Proton Magnetic Resonance Spectroscopy (1H-MRS)

    PubMed Central

    Winkelman, John W.; Buxton, Orfeu M.; Jensen, J. Eric; Benson, Kathleen L.; O'Connor, Shawn P.; Wang, Wei; Renshaw, Perry F.

    2008-01-01

    Study Objectives: Both basic and clinical data suggest a potential significant role for GABA in the etiology and maintenance of primary insomnia (PI). Proton magnetic resonance spectroscopy (1H-MRS) can non-invasively determine GABA levels in human brain. Our objective was to assess GABA levels in unmedicated individuals with PI, using 1H-MRS. Design and Setting: Matched-groups, cross-sectional study conducted at two university-based hospitals. Participants: Sixteen non-medicated individuals (8 women) with PI (mean age = 37.3 +/− 8.1) and 16 (7 women) well-screened normal sleepers (mean age = 37.6 +/− 4.5). Methods and Measurements: PI was established with an unstructured clinical interview, a Structured Clinical Interview for DSM-IV (SCID), sleep diary, actigraphy and polysomnography (PSG). 1H-MRS data were collected on a Varian 4 Tesla magnetic resonance imaging/spectroscopy scanner. Global brain GABA levels were averaged from samples in the basal ganglia, thalamus, and temporal, parietal, and occipital white-matter and cortex. Results: Average brain GABA levels were nearly 30% lower in patients with PI (.18 +/− .06) compared to controls (.25 +/− .11). GABA levels were negatively correlated with wake after sleep onset (WASO) on two independent PSGs (r = −0.71, p = 0.0024 and −0.70, p = 0.0048). Conclusions: Our preliminary finding of a global reduction in GABA in non-medicated individuals with PI is the first demonstration of a neurochemical difference in the brains of those with PI compared to normal sleeping controls. 1H-MRS is a valuable tool to assess GABA in vivo, and may provide a means to shed further light on the neurobiology of insomnia. Citation: Winkelman JW; Buxton OM; Jensen JE; Benson KL; O'Connor SP; Wang W; Renshaw PF. Reduced brain GABA in primary insomnia: preliminary data from 4T proton magnetic resonance spectroscopy (1H-MRS). SLEEP 2008;31(11):1499–1506. PMID:19014069

  19. [ERK activation effects on GABA secretion inhibition induced by SDF-1 in hippocampal neurons of rats].

    PubMed

    Zhang, Zi-juan; Guo, Mei-xia; Xing, Ying

    2015-09-01

    To investigate the effect of extracellular regulating kinase (ERK) signaling pathway on the secretion of gamma-aminobutyric acid (GABA) in cultured rat hippocampal neurons induced by stromal cell derived factor-1 (SDF-1). The hippocampal neurons of newborn SD rats were cultured and identified in vitro; the phosphorylation level of ERK1/2 was examined by Western blot; ELISA was used to detect the effect of PD98059, a ERK1/2 specific blocker on GABA secretion of cultured hippocampal neurons and Western blot were adopted to measure the protein expression levels of glutamate decarboxylase (GAD65/67) and gamma aminobutyric acid transporter (GAT); after blocking ERK1/2 signaling pathway with PD98059; RT-PCR was used to detect the mRNA expression levels of GAT-1 and GAD65 after treated with PD98059. The levels of ERKl/2 phosphorylation were increased significantly by SDF1 acting on hippocampal neurons, and CX-CR4 receptor blocker AMD3100, could inhibit SDF-1 induced ERK1/2 activation; SDF-1 could inhibit the secretion of GABA in cultured hippocampal neurons, and ERK1/2 specific inhibitor PD98059, could partly reverse the inhibition of GABA secretion by SDF-1. The effects of SDF-1 on cultured hippocampal neurons was to decrease the mRNA genesis of glutamic acid decarboxylase GAD65 and GABA transporter GAT-1, besides, ERK inhibitor PD98059 could effectively flip the effect of SDF-1. The results of Western blot showed that SDF-1 could inhibit the protein expression of GAT-1 and GAD65/67 in hippocampal neurons and the inhibition of GAT-1 and GAD65/67 protein expression could be partially restored by ERK1/2 blocker. SDF-1 acts on the CXCR4 of hippocampal neurons in vitro, and inhibits the expression of GAD by activating the ERK1/2 signaling pathway, and this may represent one possible pathway of GABA secretion inhibition.

  20. Embryonic GABA(B) receptor blockade alters cell migration, adult hypothalamic structure, and anxiety- and depression-like behaviors sex specifically in mice.

    PubMed

    Stratton, Matthew S; Staros, Michelle; Budefeld, Tomaz; Searcy, Brian T; Nash, Connor; Eitel, Chad; Carbone, David; Handa, Robert J; Majdic, Gregor; Tobet, Stuart A

    2014-01-01

    Neurons of the paraventricular nucleus of the hypothalamus (PVN) regulate the hypothalamic- pituitary-adrenal (HPA) axis and the autonomic nervous system. Females lacking functional GABA(B) receptors because of a genetic disruption of the R1 subunit have altered cellular characteristics in and around the PVN at birth. The genetic disruption precluded appropriate assessments of physiology or behavior in adulthood. The current study was conducted to test the long term impact of a temporally restricting pharmacological blockade of the GABA(B) receptor to a 7-day critical period (E11-E17) during embryonic development. Experiments tested the role of GABA(B) receptor signaling in fetal development of the PVN and later adult capacities for adult stress related behaviors and physiology. In organotypic slices containing fetal PVN, there was a female specific, 52% increase in cell movement speeds with GABA(B) receptor antagonist treatment that was consistent with a sex-dependent lateral displacement of cells in vivo following 7 days of fetal exposure to GABA(B) receptor antagonist. Anxiety-like and depression-like behaviors, open-field activity, and HPA mediated responses to restraint stress were measured in adult offspring of mothers treated with GABA(B) receptor antagonist. Embryonic exposure to GABA(B) receptor antagonist resulted in reduced HPA axis activation following restraint stress and reduced depression-like behaviors. There was also increased anxiety-like behavior selectively in females and hyperactivity in males. A sex dependent response to disruptions of GABA(B) receptor signaling was identified for PVN formation and key aspects of physiology and behavior. These changes correspond to sex specific prevalence in similar human disorders, namely anxiety disorders and hyperactivity.

  1. RhoA influences the nuclear localization of extracellular signal-regulated kinases to modulate p21Waf/Cip1 expression.

    PubMed

    Zuckerbraun, Brian S; Shapiro, Richard A; Billiar, Timothy R; Tzeng, Edith

    2003-08-19

    The 42/44-kD mitogen-activated protein kinases (extracellular signal-regulated kinases, ERKs) regulate smooth muscle cell (SMC) cell-cycle progression and can either promote or inhibit proliferation depending on the activation status of the small GTPase RhoA. RhoA is involved in the regulation of the actin cytoskeleton and converges on multiple signaling pathways. However, the mechanism by which RhoA modulates ERK signaling is not well defined. The purpose of this investigation was to examine whether RhoA regulates ERK downstream signaling and cellular proliferation through its effects on the cytoskeleton and the nuclear localization of ERK. Treatment of SMCs with Clostridia botulinum C3 exoenzyme, which inhibits RhoA activation, decreased SMC proliferation to 24+/-7% of that of controls and increased p21Waf1/Cip1 transcription and protein levels. These effects of RhoA were reversed by inhibition of ERK phosphorylation. However, inactivation of RhoA did not alter levels of ERK phosphorylation but did increase nuclear localization of phosphorylated ERK. In addition, immunostaining demonstrated that phosphorylated ERK associated with the actin cytoskeleton, which was disrupted by C3 exoenzyme. Leptomycin B, an inhibitor of Crm1 that results in ERK nuclear accumulation, similarly increased p21Waf1/Cip1. RhoA inhibition increased levels of phosphorylated ERK in the cell nucleus. Inhibition of RhoA or pharmacological inhibition of nuclear export resulted in increased p21Waf1/Cip1 expression and decreased SMC proliferation, effects that were partially dependent on ERK. RhoA regulation of the actin cytoskeleton may determine ERK subcellular localization and its subsequent effects on SMC proliferation.

  2. Salmon lice (Lepeophtheirus salmonis) showing varying emamectin benzoate susceptibilities differ in neuronal acetylcholine receptor and GABA-gated chloride channel mRNA expression.

    PubMed

    Carmichael, Stephen N; Bron, James E; Taggart, John B; Ireland, Jacqueline H; Bekaert, Michaël; Burgess, Stewart Tg; Skuce, Philip J; Nisbet, Alasdair J; Gharbi, Karim; Sturm, Armin

    2013-06-18

    Caligid copepods, also called sea lice, are fish ectoparasites, some species of which cause significant problems in the mariculture of salmon, where the annual cost of infection is in excess of €300 million globally. At present, caligid control on farms is mainly achieved using medicinal treatments. However, the continued use of a restricted number of medicine actives potentially favours the development of drug resistance. Here, we report transcriptional changes in a laboratory strain of the caligid Lepeophtheirus salmonis (Krøyer, 1837) that is moderately (~7-fold) resistant to the avermectin compound emamectin benzoate (EMB), a component of the anti-salmon louse agent SLICE® (Merck Animal Health). Suppression subtractive hybridisation (SSH) was used to enrich transcripts differentially expressed between EMB-resistant (PT) and drug-susceptible (S) laboratory strains of L. salmonis. SSH libraries were subjected to 454 sequencing. Further L. salmonis transcript sequences were available as expressed sequence tags (EST) from GenBank. Contiguous sequences were generated from both SSH and EST sequences and annotated. Transcriptional responses in PT and S salmon lice were investigated using custom 15 K oligonucleotide microarrays designed using the above sequence resources. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains, these genes being enriched for functions such as calcium ion binding, chitin metabolism and muscle structure. γ-aminobutyric acid (GABA)-gated chloride channel (GABA-Cl) and neuronal acetylcholine receptor (nAChR) subunits showed significantly lower transcript levels in PT lice compared to S lice. Using RT-qPCR, the decrease in mRNA levels was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR. Salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 μg L-1 of EMB, a drug concentration tolerated by PT lice, but toxic for S lice

  3. Gamma-aminobutyric acid (GABA)-B receptor 1 in cerebellar cortex of essential tremor.

    PubMed

    Luo, C; Rajput, A H; Robinson, C A; Rajput, A

    2012-06-01

    Some reports suggest cerebellar dysfunction as the basis of essential tremor (ET). Several drugs with the action of gamma-aminobutyric acid (GABA) are known to improve ET. Autopsy studies were performed on brains from nine former patients followed at the Movement Disorders Clinic Saskatchewan, Canada, and compared with five normal control brains. We aimed to measure the concentration of GABA B receptor 1 (GBR1) in the brains of patients who had had ET and to compare them to the GABA concentration in brains of controls. Western blot was used to determine the expression of GBR1 in cerebellar cortex tissue. We found that compared to the controls, the ET brains had three different patterns of GBR1 protein concentration--two with high, four comparable, and three with marginally low levels. There was no association between the age of onset, severity or duration of tremor, the response to alcohol or other drugs and GBR1 level. Thus, we conclude that our study does not support that GBR1 is involved in ET. Further studies are needed to verify these results. Copyright © 2011 Elsevier Ltd. All rights reserved.

  4. GABA as a rising gliotransmitter

    PubMed Central

    Yoon, Bo-Eun; Lee, C. Justin

    2014-01-01

    Gamma-amino butyric acid (GABA) is the major inhibitory neurotransmitter that is known to be synthesized and released from GABAergic neurons in the brain. However, recent studies have shown that not only neurons but also astrocytes contain a considerable amount of GABA that can be released and activate GABA receptors in neighboring neurons. These exciting new findings for glial GABA raise further interesting questions about the source of GABA, its mechanism of release and regulation and the functional role of glial GABA. In this review, we highlight recent studies that identify the presence and release of GABA in glial cells, we show several proposed potential pathways for accumulation and modulation of glial intracellular and extracellular GABA content, and finally we discuss functional roles for glial GABA in the brain. PMID:25565970

  5. Nicotinic receptors and functional regulation of GABA cell microcircuitry in bipolar disorder and schizophrenia.

    PubMed

    Benes, Francine M

    2012-01-01

    Studies of the hippocampus in postmortem brains from patients with schizophrenia and bipolar disorder have provided evidence for a defect of GABAergic interneurons. Significant decreases in the expression of GAD67, a marker for GABA cell function, have been found repeatedly in several different brain regions that include the hippocampus. In this region, nicotinic receptors are thought to play an important role in modulating the activity of GABAergic interneurons by influences of excitatory cholinergic afferents on their activity. In bipolar disorder, this influence appears to be particularly prominent in the stratum oriens of sectors CA3/2 and CA1, two sites where these cells constitute the exclusive neuronal cell type. In sector CA3/2, this layer receives a robust excitatory projection from the basolateral amygdala (BLA) and this is thought to play a central role in regulating GABA cells at this locus. Using laser microdissection, recent studies have focused selectively on these two layers and their associated GABA cells using microarray technology. The results have provided support for the idea that nicotinic cholinergic receptors play a particularly important role in regulating the activity of GABA neurons at these loci by regulating the progression of cell cycle and the repair of damaged DNA. In bipolar disorder, there is a prominent reduction in the expression of mRNAs for several different nicotinic subunit isoforms. These decreases could reflect a diminished influence of this receptor system on these GABA cells, particularly in sector CA3/2 where a preponderance of abnormalities have been observed in postmortem studies. In patients with bipolar disorder, excitatory nicotinic cholinergic fibers from the medial septum may converge with glutamatergic fibers from the BLA on GABAergic interneurons in the stratum oriens of CA3/2 and result in disturbances of their genomic and functional integrity, ones that may induce disruptions of the integration of

  6. RhoA/Rho-kinase triggers epithelial-mesenchymal transition in mesothelial cells and contributes to the pathogenesis of dialysis-related peritoneal fibrosis

    PubMed Central

    Wang, Qinglian; Yang, Xiaowei; Xu, Ying; Shen, Zhenwei; Cheng, Hongxia; Cheng, Fajuan; Liu, Xiang; Wang, Rong

    2018-01-01

    Peritoneal fibrosis (PF) with associated peritoneal dysfunction is almost invariably observed in long-term peritoneal dialysis (PD) patients. Advanced glycation end products (AGEs) are pro-oxidant compounds produced in excess during the metabolism of glucose and are present in high levels in standard PD solutions. The GTPase RhoA has been implicated in PF, but its specific role remains poorly understood. Here, we studied the effects of RhoA/Rho-kinase signaling in AGEs-induced epithelial-mesenchymal transition (EMT) in human peritoneal mesothelial cells (HPMCs), and evaluated morphological and molecular changes in a rat model of PD-related PF. Activation of RhoA/Rho-kinase and activating protein-1 (AP-1) was assessed in HPMCs using pull-down and electrophoretic mobility shift assays, respectively, while expression of transforming growth factor-β, fibronectin, α-smooth muscle actin, vimentin, N-cadherin, and E-cadherin expression was assessed using immunohistochemistry and western blot. AGEs exposure activated Rho/Rho-kinase in HPMCs and upregulated EMT-related genes via AP-1. These changes were prevented by the Rho-kinase inhibitors fasudil and Y-27632, and by the AP-1 inhibitor curcumin. Importantly, fasudil normalized histopathological and molecular alterations and preserved peritoneal function in rats. These data support the therapeutic potential of Rho-kinase inhibitors in PD-related PF. PMID:29581852

  7. Inhibition of GABA-gated chloride channels by 12,14-dichlorodehydroabietic acid in mammalian brain.

    PubMed

    Nicholson, R A; Lees, G; Zheng, J; Verdon, B

    1999-03-01

    1. 12,14-dichlorodehydroabietic acid (12,14-Cl2DHA) reduced GABA-stimulated uptake of 36Cl- into mouse brain synaptoneurosomes suggesting inhibition of mammalian GABA(A) receptor function. 2. 12,14-Cl2DHA did not affect the binding of [3H]-muscimol to brain membranes but displaced specifically bound [3H]-EBOB. The inhibitory effect on [3H]-EBOB binding was not reversible. 12,14-Cl2DHA reduced the availability of [3H]-EBOB binding sites (Bmax) without changing the KD of the radioligand for remaining sites. 12,14-Cl2DHA did not affect the rate of association of [3H]-EBOB with its chloride channel receptor, but increased the initial rate of [3H]-EBOB dissociation. 3. 12,14-Cl2DHA enhanced the incidence of EPSCs when rapidly applied to cultured rat cortical neurones. Longer exposures produced block of IPSCs with marked increases in the frequency of EPSCs and min EPSCs. 12,14-Cl2DHA also irreversibly suppressed chloride currents evoked by pulses of exogenous GABA in these cells. 4. Ultimately, 12,14-Cl2DHA inhibited all synaptic traffic and action currents in current clamped cells indicating that, in contrast to picrotoxinin (which causes paroxysmal bursting), it is not fully selective for the GABA(A) receptor-chloride channel complex. 5. The depolarizing block seen with 12,14-Cl2DHA in amphotericin-perforated preparations implicates loss of Ca2+ buffering in the polarity change and this may account for inhibition of spontaneous action potentials. 6. Our investigation demonstrates that 12,14-Cl2DHA blocks GABA-dependent chloride entry in mammalian brain and operates as a non-competitive insurmountable GABA(A) antagonist. The mechanism likely involves either irreversible binding of 12,14-Cl2DHA to the trioxabicyclooctane recognition site or a site that is allosterically coupled to it. We cannot exclude, however, the possibility that 12,14-Cl2DHA causes localized proteolysis or more extensive conformational change within a critical subunit of the chloride channel.

  8. Rapid PCR-mediated synthesis of competitor molecules for accurate quantification of beta(2) GABA(A) receptor subunit mRNA.

    PubMed

    Vela, J; Vitorica, J; Ruano, D

    2001-12-01

    We describe a fast and easy method for the synthesis of competitor molecules based on non-specific conditions of PCR. RT-competitive PCR is a sensitive technique that allows quantification of very low quantities of mRNA molecules in small tissue samples. This technique is based on the competition established between the native and standard templates for nucleotides, primers or other factors during PCR. Thus, the most critical parameter is the use of good internal standards to generate a standard curve from which the amount of native sequences can be properly estimated. At the present time different types of internal standards and methods for their synthesis have been described. Normally, most of these methods are time-consuming and require the use of different sets of primers, different rounds of PCR or specific modifications, such as site-directed mutagenesis, that need subsequent analysis of the PCR products. Using our method, we obtained in a single round of PCR and with the same primer pair, competitor molecules that were successfully used in RT-competitive PCR experiments. The principal advantage of this method is high versatility and economy. Theoretically it is possible to synthesize a specific competitor molecule for each primer pair used. Finally, using this method we have been able to quantify the increase in the expression of the beta(2) GABA(A) receptor subunit mRNA that occurs during rat hippocampus development.

  9. Merkel Cell Polyomavirus Small T Antigen Drives Cell Motility via Rho-GTPase-Induced Filopodium Formation.

    PubMed

    Stakaitytė, Gabrielė; Nwogu, Nnenna; Dobson, Samuel J; Knight, Laura M; Wasson, Christopher W; Salguero, Francisco J; Blackbourn, David J; Blair, G Eric; Mankouri, Jamel; Macdonald, Andrew; Whitehouse, Adrian

    2018-01-15

    Cell motility and migration is a complex, multistep, and multicomponent process intrinsic to progression and metastasis. Motility is dependent on the activities of integrin receptors and Rho family GTPases, resulting in the remodeling of the actin cytoskeleton and formation of various motile actin-based protrusions. Merkel cell carcinoma (MCC) is an aggressive skin cancer with a high likelihood of recurrence and metastasis. Merkel cell polyomavirus (MCPyV) is associated with the majority of MCC cases, and MCPyV-induced tumorigenesis largely depends on the expression of the small tumor antigen (ST). Since the discovery of MCPyV, a number of mechanisms have been suggested to account for replication and tumorigenesis, but to date, little is known about potential links between MCPyV T antigen expression and the metastatic nature of MCC. Previously, we described the action of MCPyV ST on the microtubule network and how it impacts cell motility and migration. Here, we demonstrate that MCPyV ST affects the actin cytoskeleton to promote the formation of filopodia through a mechanism involving the catalytic subunit of protein phosphatase 4 (PP4C). We also show that MCPyV ST-induced cell motility is dependent upon the activities of the Rho family GTPases Cdc42 and RhoA. In addition, our results indicate that the MCPyV ST-PP4C interaction results in the dephosphorylation of β 1 integrin, likely driving the cell motility pathway. These findings describe a novel mechanism by which a tumor virus induces cell motility, which may ultimately lead to cancer metastasis, and provides opportunities and strategies for targeted interventions for disseminated MCC. IMPORTANCE Merkel cell polyomavirus (MCPyV) is the most recently discovered human tumor virus. It causes the majority of cases of Merkel cell carcinoma (MCC), an aggressive skin cancer. However, the molecular mechanisms implicating MCPyV-encoded proteins in cancer development are yet to be fully elucidated. This study builds

  10. Rac1/RhoA antagonism defines cell-to-cell heterogeneity during epidermal morphogenesis in nematodes

    PubMed Central

    Ouellette, Marie-Hélène

    2016-01-01

    The antagonism between the GTPases Rac1 and RhoA controls cell-to-cell heterogeneity in isogenic populations of cells in vitro and epithelial morphogenesis in vivo. Its involvement in the regulation of cell-to-cell heterogeneity during epidermal morphogenesis has, however, never been addressed. We used a quantitative cell imaging approach to characterize epidermal morphogenesis at a single-cell level during early elongation of Caenorhabditis elegans embryos. This study reveals that a Rac1-like pathway, involving the Rac/Cdc42 guanine-exchange factor β-PIX/PIX-1 and effector PAK1/PAK-1, and a RhoA-like pathway, involving ROCK/LET-502, control the remodeling of apical junctions and the formation of basolateral protrusions in distinct subsets of hypodermal cells. In these contexts, protrusions adopt lamellipodia or an amoeboid morphology. We propose that lamella formation may reduce tension building at cell–cell junctions during morphogenesis. Cell-autonomous antagonism between these pathways enables cells to switch between Rac1- and RhoA-like morphogenetic programs. This study identifies the first case of cell-to-cell heterogeneity controlled by Rac1/RhoA antagonism during epidermal morphogenesis. PMID:27821782

  11. Wash functions downstream of Rho1 GTPase in a subset of Drosophila immune cell developmental migrations

    PubMed Central

    Verboon, Jeffrey M.; Rahe, Travis K.; Rodriguez-Mesa, Evelyn; Parkhurst, Susan M.

    2015-01-01

    Drosophila immune cells, the hemocytes, undergo four stereotypical developmental migrations to populate the embryo, where they provide immune reconnoitering, as well as a number of non–immune-related functions necessary for proper embryogenesis. Here, we describe a role for Rho1 in one of these developmental migrations in which posteriorly located hemocytes migrate toward the head. This migration requires the interaction of Rho1 with its downstream effector Wash, a Wiskott–Aldrich syndrome family protein. Both Wash knockdown and a Rho1 transgene harboring a mutation that prevents Wash binding exhibit the same developmental migratory defect as Rho1 knockdown. Wash activates the Arp2/3 complex, whose activity is needed for this migration, whereas members of the WASH regulatory complex (SWIP, Strumpellin, and CCDC53) are not. Our results suggest a WASH complex–independent signaling pathway to regulate the cytoskeleton during a subset of hemocyte developmental migrations. PMID:25739458

  12. Low doses of alcohol potentiate GABA sub B inhibition of spontaneous activity of hippocampal CA1 neurons in vivo

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Criado, J.R.; Thies, R.

    1991-03-11

    Low doses of alcohol facilitate firing of hippocampal neurons. Such doses also enhance the inhibitory actions of GABA. Alcohol is known to potentiate inhibition via GABA{sub A} receptors. However, the effects of alcohol on GABA{sub B} receptor function are not understood. Spontaneous activity of single units was recorded from CA1 neurons of male rats anesthetized with 1.0% halothane. Electrical recordings and local application of drugs were done with multi-barrel pipettes. CA1 pyramidal neurons fired spontaneous bursts of action potentials. Acute alcohol decreased the interval between bursts, a mild excitatory action. Alcohol also more than doubled the period of complete inhibitionmore » produced by local application of both GABA and baclofen. These data suggest that GABA{sub B}-mediated inhibition is also potentiated by low doses of alcohol.« less

  13. Effects of new fluorinated analogues of GABA, pregabalin bioisosters, on the ambient level and exocytotic release of [3H]GABA from rat brain nerve terminals.

    PubMed

    Borisova, T; Pozdnyakova, N; Shaitanova, E; Gerus, I; Dudarenko, M; Haufe, G; Kukhar, V

    2017-01-15

    Recently, we have shown that new fluorinated analogues of γ-aminobutyric acid (GABA), bioisosters of pregabalin (β-i-Bu-GABA), i.e. β-polyfluoroalkyl-GABAs (FGABAs), with substituents: β-CF 3 -β-OH (1), β-CF 3 (2); β-CF 2 CF 2 H (3), are able to increase the initial rate of [ 3 H]GABA uptake by isolated rat brain nerve terminals (synaptosomes), and this effect is higher than that of pregabalin. So, synthesized FGABAs are structural but not functional analogues of GABA. Herein, we assessed the effects of synthesized FGABAs (100μM) on the ambient level and exocytotic release of [ 3 H]GABA in nerve terminals and compared with those of pregabalin (100μM). It was shown that FGABAs 1-3 did not influence the ambient level of [ 3 H]GABA in the synaptosomal preparations, and this parameter was also not altered by pregabalin. During blockage of GABA transporters GAT1 by specific inhibitor NO-711, FGABAs and pregabalin also did not change ambient [ 3 H]GABA in synaptosomal preparations. Exocytotic release of [ 3 H]GABA from synaptosomes decreased in the presence of FGABAs 1-3 and pregabalin, and the effects of FGABAs 1 &3 were more significant than those of FGABAs 2 and pregabalin. FGABAs 1-3/pregabalin-induced decrease in exocytotic release of [ 3 H]GABA from synaptosomes was not a result of changes in the potential of the plasma membrane. Therefore, new synthesized FGABAs 1 &3 were able to decrease exocytotic release of [ 3 H]GABA from nerve terminals more effectively in comparison to pregabalin. Absence of unspecific side effects of FGABAs 1 &3 on the membrane potential makes these compounds perspective for medical application. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Rho/Rho-dependent kinase affects locomotion and actin-myosin II activity of Amoeba proteus.

    PubMed

    Kłopocka, W; Redowicz, M J

    2004-10-01

    The highly motile free-living unicellular organism Amoeba proteus has been widely used as a model to study cell motility. However, the molecular mechanisms underlying its unique locomotion are still scarcely known. Recently, we have shown that blocking the amoebae's endogenous Rac- and Rho-like proteins led to distinct and irreversible changes in the appearance of these large migrating cells as well as to a significant inhibition of their locomotion. In order to elucidate the mechanism of the Rho pathway, we tested the effects of blocking the endogenous Rho-dependent kinase (ROCK) by anti-ROCK antibodies and Y-27632, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl)cyclohexanecarboxamide dihydrochloride, a specific inhibitor of ROCK, on migrating amoebae and the effect of the Rho and ROCK inhibition on the actin-activated Mg-ATPase of the cytosolic fraction of the amoebae. Amoebae microinjected with anti-ROCK inhibitors remained contracted and strongly attached to the glass surface and exhibited an atypical locomotion. Despite protruding many pseudopodia that were advancing in various directions, the amoebae could not effectively move. Immunofluorescence studies showed that ROCK-like protein was dispersed throughout the cytoplasm and was also found in the regions of actin-myosin II interaction during both isotonic and isometric contraction. The Mg-ATPase activity was about two- to threefold enhanced, indicating that blocking the Rho/Rho-dependent kinase activated myosin. It is possible then that in contrast to the vertebrate cells, the inactivation of Rho/Rho-dependent kinase in amoebae leads to the activation of myosin II and to the observed hypercontracted cells which cannot exert effective locomotion.

  15. RhoA, Rac1, and Cdc42 differentially regulate αSMA and collagen I expression in mesenchymal stem cells.

    PubMed

    Ge, Jianfeng; Burnier, Laurent; Adamopoulou, Maria; Kwa, Mei Qi; Schaks, Matthias; Rottner, Klemens; Brakebusch, Cord

    2018-06-15

    Mesenchymal stem cells (MSC) are suggested to be important progenitors of myofibroblasts in fibrosis. To understand the role of Rho GTPase signaling in TGFβ-induced myofibroblast differentiation of MSC, we generated a novel MSC line and its descendants lacking functional Rho GTPases and Rho GTPase signaling components. Unexpectedly, our data revealed that Rho GTPase signaling is required for TGFβ-induced expression of α-smooth muscle actin (αSMA) but not of collagen I α1 ( col1a1 ). Whereas loss of RhoA and Cdc42 reduced αSMA expression, ablation of the Rac1 gene had the opposite effect. Although actin polymerization and MRTFa were crucial for TGFβ-induced αSMA expression, neither Arp2/3-dependent actin polymerization nor cofilin-dependent severing and depolymerization of F-actin were required. Instead, F-actin levels were dependent on cell contraction, and TGFβ-induced actin polymerization correlated with increased cell contraction mediated by RhoA and Cdc42. Finally, we observed impaired collagen I secretion in MSC lacking RhoA or Cdc42. These data give novel molecular insights into the role of Rho GTPases in TGFβ signaling and have implications for our understanding of MSC function in fibrosis. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

  16. Role of phosphatidylserine in the activation of Rho1-related Pkc1 signaling in Saccharomyces cerevisiae.

    PubMed

    Nomura, Wataru; Ito, Yusuke; Inoue, Yoshiharu

    2017-02-01

    Protein kinase C (PKC) belongs to a family of serine/threonine kinases and is evolutionary conserved among eukaryotes. It contains several functional domains, with the C1 domain being identified as a membrane-targeting module. Diacylglycerol (DAG) and phorbol esters bind to the C1 domain to enhance its kinase activity. The C1 domain is conserved in PKC (Pkc1) in the budding yeast Saccharomyces cerevisiae; however, its kinase activity does not respond to DAG. Although the C1 domain of Pkc1 physically interacts with the small GTPase Rho1, the interaction between C1 domain and lipids has not yet been characterized. We herein provide evidence to show the physical interaction between the C1 domain of Pkc1 and phosphatidylserine (PS), but not DAG. The stress-induced activation of Pkc1 signaling was abolished in a cho1 mutant, which was defective in PS synthase. The deletion of CHO1 perturbed the appropriate localization of Pkc1 at the bud tip, and impaired the physical interaction between Pkc1 and GTP-bound Rho1 in vivo. Our results suggest that PS is necessary for Pkc1 signaling due to its role in regulating the localization of Pkc1 as well as the physical interaction between Rho1 and Pkc1. Copyright © 2017 Elsevier Inc. All rights reserved.

  17. [Effect of acupuncture at different acupoints on expression of hypothalamic GABA and GABA(A) receptor proteins in insomnia rats].

    PubMed

    Zhou, Yan-Li; Gao, Xi-Yan; Wang, Pei-Yu; Ren, Shan

    2012-08-01

    To observe the effect of acupuncture of "Shenmai" (BL 62) and "Zhaohai" (KI 6), "Shenmen" (HT 7), etc. on the expression of hypothalamic gamma-aminobutyric acid (GABA) and GABA(A) receptor (GABA(A)R) proteins in experimental insomnia rats so as to explore its mechanism underlying improving sleeping. Seventy Wistar rats were randomly divided into normal control, model, "Sanyinjiao" (SP6), "Neiguan" (PC 6), "Zusanli" (ST 36), "Shenmen" (HT7), and "Shenmai" (BL 62)-Zhaohai (KI 6, BL 62-KI 6) groups, with 10 rats in each group. Insomnia model was established by intraperitoneal injection of chlorophenylalanine solution (PCPA, 1 mL/100 g). An acupuncture needle was inserted into each of the bilateral HT 7, PC 6, SP 6, ST 36 and BL 62-KI 6 respectively and manipulated for about 1 min, once daily for 7 days. Hypothamic GABA and GABA(A)R protein expressions were detected by immunohistochemistry. The animals' physical ability was evaluated by using pole-climbing test in a water tank. In comparison with the normal control group, the numbers of hypothalamic GABA immunoreaction (IR)- and GABA(A)R IR-positive neurons and the pole-climbing time were reduced significantly in the model group (P < 0.05). While in comparison with the model group, the numbers of hypothalamic GABA IR-positive neurons and those of hypothalamic GABA(A)R IR-positive neurons in the HT 7, PC 6, SP 6, ST 36 and BL 62-KI 6 groups, as well as the pole-climbing duration in the SP 6, ST 36 and BL 62-KI 6 groups were increased considerably (P < 0.05, P < 0.01). The effects of HT 7 and BL 62-KI 6 groups were significantly superior to those of PC 6, ST 36 and SP 6 groups in up-regulating GABA and GABA(A)R expression, and the effect of BL 62-KI 6 group was remarkably better than those of HT 7, PC 6, SP 6 and ST 36 groups in lengthening the pole-climbing time (P < 0.05). Acupuncture can effectively suppress insomnia induced down-regulation of hypothalamic GABA and GABA(A)R in rats and lengthen pole-climbing time

  18. RhoA-Mediated Functions in C3H10T1/2 Osteoprogenitors Are Substrate Topography Dependent.

    PubMed

    Ogino, Yoichiro; Liang, Ruiwei; Mendonça, Daniela B S; Mendonça, Gustavo; Nagasawa, Masako; Koyano, Kiyoshi; Cooper, Lyndon F

    2016-03-01

    Surface topography broadly influences cellular responses. Adherent cell activities are regulated, in part, by RhoA, a member of the Rho-family of GTPases. In this study, we evaluated the influence of surface topography on RhoA activity and associated cellular functions. The murine mesenchymal stem cell line C3H10T1/2 cells (osteoprogenitor cells) were cultured on titanium substrates with smooth topography (S), microtopography (M), and nanotopography (N) to evaluate the effect of surface topography on RhoA-mediated functions (cell spreading, adhesion, migration, and osteogenic differentiation). The influence of RhoA activity in the context of surface topography was also elucidated using RhoA pharmacologic inhibitor. Following adhesion, M and N adherent cells developed multiple projections, while S adherent cells had flattened and widespread morphology. RhoA inhibitor induced remarkable longer and thinner cytoplasmic projections on all surfaces. Cell adhesion and osteogenic differentiation was topography dependent with S < M and N surfaces. RhoA inhibition increased adhesion on S and M surfaces, but not N surfaces. Cell migration in a wound healing assay was greater on S versus M versus N surfaces and RhoA inhibitor increased S adherent cell migration, but not N adherent cell migration. RhoA inhibitor enhanced osteogenic differentiation in S adherent cells, but not M or N adherent cells. RhoA activity was surface topography roughness dependent (S < M, N). RhoA activity and -mediated functions are influenced by surface topography. Smooth surface adherent cells appear highly sensitive to RhoA function, while nano-scale topography adherent cell may utilize alternative cellular signaling pathway(s) to influence adherent cellular functions regardless of RhoA activity. © 2015 Wiley Periodicals, Inc.

  19. Comparison Of The Direct Costs, Length Of Recovery, And Incidence Of Post Operative Anti Emetic Use After Anesthesia Induction With Propofol Or A 1:1 Mixture Of Thiopental And Propofol

    DTIC Science & Technology

    1999-10-01

    1.2% purified egg phosphatide as a stabilizer (Doyle, 1998; Searle & Sahab, 1993). Propofol is rapidly metabolized with less than 20% recovered...affect the neuro transmitter gamma- aminobutyric acid A (GABA A ) receptor sites present in the central nervous system. A GABA A receptor is an...Gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, binds to alpha or beta sub-units on the receptor

  20. Brain gamma-aminobutyric acid (GABA) abnormalities in bipolar disorder

    PubMed Central

    Brady, Roscoe O; McCarthy, Julie M; Prescot, Andrew P; Jensen, J Eric; Cooper, Alissa J; Cohen, Bruce M; Renshaw, Perry F; Ongür, Dost

    2017-01-01

    Objectives Gamma-aminobutyric acid (GABA) abnormalities have been implicated in bipolar disorder. However, due to discrepant studies measuring postmortem, cerebrospinal fluid, plasma, and in vivo brain levels of GABA, the nature of these abnormalities is unclear. Using proton magnetic resonance spectroscopy, we investigated tissue levels of GABA in the anterior cingulate cortex and parieto-occipital cortex of participants with bipolar disorder and healthy controls. Methods Fourteen stably medicated euthymic outpatients with bipolar disorder type I (mean age 32.6 years, eight male) and 14 healthy control participants (mean age 36.9 years, 10 male) completed a proton magnetic resonance spectroscopy scan at 4-Tesla after providing informed consent. We collected data from two 16.7-mL voxels using MEGAPRESS, and they were analyzed using LCModel. Results GABA/creatine ratios were elevated in bipolar disorder participants compared to healthy controls [F(1,21) = 4.4, p = 0.048] in the anterior cingulate cortex (25.1% elevation) and the parieto-occipital cortex (14.6% elevation). Bipolar disorder participants not taking GABA-modulating medications demonstrated greater GABA/creatine elevations than patients taking GABA-modulating medications. Conclusions We found higher GABA/creatine levels in euthymic bipolar disorder outpatients compared to healthy controls, and the extent of this elevation may be affected by the use of GABA-modulating medications. Our findings suggest that elevated brain GABA levels in bipolar disorder may be associated with GABAergic dysfunction and that GABA-modulating medications reduce GABA levels in this condition. PMID:23634979

  1. REVISITING {rho}{sup 1} CANCRI e: A NEW MASS DETERMINATION OF THE TRANSITING SUPER-EARTH

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Endl, Michael; Cochran, William D.; MacQueen, Phillip J.

    2012-11-01

    We present a mass determination for the transiting super-Earth {rho}{sup 1} Cancri e based on nearly 700 precise radial velocity (RV) measurements. This extensive RV data set consists of data collected by the McDonald Observatory planet search and published data from Lick and Keck observatories. We obtained 212 RV measurements with the Tull Coude Spectrograph at the Harlan J. Smith 2.7 m Telescope and combined them with a new Doppler reduction of the 131 spectra that we have taken in 2003-2004 with the High-Resolution Spectrograph (HRS) at the Hobby-Eberly Telescope for the original discovery of {rho}{sup 1} Cancri e. Usingmore » this large data set we obtain a five-planet Keplerian orbital solution for the system and measure an RV semi-amplitude of K = 6.29 {+-} 0.21 m s{sup -1} for {rho}{sup 1} Cnc e and determine a mass of 8.37 {+-} 0.38 M {sub Circled-Plus }. The uncertainty in mass is thus less than 5%. This planet was previously found to transit its parent star, which allowed them to estimate its radius. Combined with the latest radius estimate from Gillon et al., we obtain a mean density of {rho} = 4.50 {+-} 0.20 g cm{sup -3}. The location of {rho}{sup 1} Cnc e in the mass-radius diagram suggests that the planet contains a significant amount of volatiles, possibly a water-rich envelope surrounding a rocky core.« less

  2. The first transmembrane domain (TM1) of β2-subunit binds to the transmembrane domain S1 of α-subunit in BK potassium channels

    PubMed Central

    Morera, Francisco J.; Alioua, Abderrahmane; Kundu, Pallob; Salazar, Marcelo; Gonzalez, Carlos; Martinez, Agustin D.; Stefani, Enrico; Toro, Ligia; Latorre, Ramon

    2012-01-01

    The BK channel is one of the most broadly expressed ion channels in mammals. In many tissues, the BK channel pore-forming α-subunit is associated to an auxiliary β-subunit that modulates the voltage- and Ca2+-dependent activation of the channel. Structural components present in β-subunits that are important for the physical association with the α-subunit are yet unknown. Here, we show through co-immunoprecipitation that the intracellular C-terminus, the second transmembrane domain (TM2) and the extracellular loop of the β2-subunit are dispensable for association with the α-subunit pointing transmembrane domain 1 (TM1) as responsible for the interaction. Indeed, the TOXCAT assay for transmembrane protein–protein interactions demonstrated for the first time that TM1 of the β2-subunit physically binds to the transmembrane S1 domain of the α-subunit. PMID:22710124

  3. Development of tolerance to the effects of vigabatrin (gamma-vinyl-GABA) on GABA release from rat cerebral cortex, spinal cord and retina.

    PubMed Central

    Neal, M. J.; Shah, M. A.

    1990-01-01

    1. The effects of acute and chronic vigabatrin (gamma-vinyl-GABA) (GVG) administration on gamma-aminobutyric acid (GABA) levels and release in rat cortical slices, spinal cord slices and retinas were studied. 2. GVG (250 mgkg-1 i.p.) administered to rats 18 h before death (acute administration) produced an almost 3 fold increase in GABA levels of the cortex and spinal cord and a 6 fold increase in retinal GABA. The levels of glutamate, aspartate, glycine and taurine were unaffected. 3. When GVG (250 mgkg-1 i.p.) was administered daily for 17 days (chronic administration) a similar (almost 3 fold) increase in cortical GABA occurred but the increases in spinal and retinal GABA were reduced by approximately 40%. 4. Acute administration of GVG strikingly increased the potassium-evoked release (KCl 50 mM) of GABA from all three tissues. This enhanced evoked release was reduced by about 50% in tissues taken from rats that had been chronically treated with GVG. 5. Acute administration of GVG reduced GABA-transaminase (GABA-T) activity by approximately 80% in cortex and cord and by 98% in the retina. Following the chronic administration of GVG, there was a trend for GABA-T activities to recover (significant only in cortex). Acute administration of GVG had no effect on glutamic acid decarboxylase (GAD) activity in cortex or spinal cord. However, chronic treatment resulted in significant decreases in GAD activity in both the cortex and cord (35% and 50% reduction respectively).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2379037

  4. Signaling efficiency of Gαq through its effectors p63RhoGEF and GEFT depends on their subcellular location.

    PubMed

    Goedhart, Joachim; van Unen, Jakobus; Adjobo-Hermans, Merel J W; Gadella, Theodorus W J

    2013-01-01

    The p63RhoGEF and GEFT proteins are encoded by the same gene and both members of the Dbl family of guanine nucleotide exchange factors. These proteins can be activated by the heterotrimeric G-protein subunit Gαq. We show that p63RhoGEF is located at the plasma membrane, whereas GEFT is confined to the cytoplasm. Live-cell imaging studies yielded quantitative information on diffusion coefficients, association rates and encounter times of GEFT and p63RhoGEF. Calcium signaling was examined as a measure of the signal transmission, revealing more efficient signaling through the membrane-associated p63RhoGEF. A rapamycin dependent recruitment system was used to dynamically alter the subcellular location and concentration of GEFT, showing efficient signaling through GEFT only upon membrane recruitment. Together, our results show efficient signal transmission through membrane located effectors, and highlight a role for increased concentration rather than increased encounter times due to membrane localization in the Gαq mediated pathways to p63RhoGEF and PLCβ.

  5. GABBR1 and SLC6A1, two genes involved in modulation of GABA synaptic transmission influence risk for alcoholism; results from three ethnically diverse populations

    PubMed Central

    Enoch, Mary-Anne; Hodgkinson, Colin A.; Shen, Pei-Hong; Gorodetsky, Elena; Marietta, Cheryl A.; Roy, Alex; Goldman, David

    2015-01-01

    Background Animal and human studies indicate that GABBR1, encoding the GABAB1 receptor subunit, and SLC6A1, encoding the neuronal GABA transporter GAT1, play a role in addiction by modulating synaptic GABA. Therefore variants in these genes might predict risk/resilience for alcoholism. Methods This study included three populations that differed by ethnicity and alcoholism phenotype: African American (AA) men: 401 treatment-seeking inpatients with single/comorbid diagnoses of alcohol and drug dependence, 193 controls; Finnish Caucasian men: 159 incarcerated alcoholics, half with comorbid ASPD, 181 controls; a community sample of Plains Indian (PI) men and women: 239 alcoholics, 178 controls. Seven GABBR1 tag SNPs were genotyped in the AA and Finnish samples; rs29220 was genotyped in the PI for replication. Also, a uniquely African, functional SLC6A1 insertion promoter polymorphism (IND) was genotyped in the AAs. Results We found a significant and congruent association between GABBR1 rs29220 and alcoholism in all three populations. The major genotype (heterozygotes in AAs, Finns) and the major allele in PIs were significantly more common in alcoholics. Moreover, SLC6A1 IND was more abundant in controls, i.e. the major genotype predicted alcoholism. An analysis of combined GABBR1 rs29220 and SLC6A1 IND genotypes showed that rs29220 heterozygotes, irrespective of their IND status, had an increased risk for alcoholism whereas carriers of the IND allele and either rs29220 homozygote were more resilient. Conclusions Our results show that with both GABBR1 and SLC6A1, the minor genotypes/alleles were protective against risk for alcoholism. Finally, GABBR1 rs29220 might predict treatment response/adverse effects for baclofen, a GABAB receptor agonist. PMID:26727527

  6. Acetylation of the RhoA GEF Net1A controls its subcellular localization and activity

    PubMed Central

    Song, Eun Hyeon; Oh, Wonkyung; Ulu, Arzu; Carr, Heather S.; Zuo, Yan; Frost, Jeffrey A.

    2015-01-01

    ABSTRACT Net1 isoform A (Net1A) is a RhoA GEF that is required for cell motility and invasion in multiple cancers. Nuclear localization of Net1A negatively regulates its activity, and we have recently shown that Rac1 stimulates Net1A relocalization to the plasma membrane to promote RhoA activation and cytoskeletal reorganization. However, mechanisms controlling the subcellular localization of Net1A are not well understood. Here, we show that Net1A contains two nuclear localization signal (NLS) sequences within its N-terminus and that residues surrounding the second NLS sequence are acetylated. Treatment of cells with deacetylase inhibitors or expression of active Rac1 promotes Net1A acetylation. Deacetylase inhibition is sufficient for Net1A relocalization outside the nucleus, and replacement of the N-terminal acetylation sites with arginine residues prevents cytoplasmic accumulation of Net1A caused by deacetylase inhibition or EGF stimulation. By contrast, replacement of these sites with glutamine residues is sufficient for Net1A relocalization, RhoA activation and downstream signaling. Moreover, the N-terminal acetylation sites are required for rescue of F-actin accumulation and focal adhesion maturation in Net1 knockout MEFs. These data indicate that Net1A acetylation regulates its subcellular localization to impact on RhoA activity and actin cytoskeletal organization. PMID:25588829

  7. Cytoplasmic p21(Cip1/WAF1) regulates neurite remodeling by inhibiting Rho-kinase activity.

    PubMed

    Tanaka, Hiroyuki; Yamashita, Toshihide; Asada, Minoru; Mizutani, Shuki; Yoshikawa, Hideki; Tohyama, Masaya

    2002-07-22

    p21(Cip1/WAF1) has cell cycle inhibitory activity by binding to and inhibiting both cyclin/Cdk kinases and proliferating cell nuclear antigen. Here we show that p21(Cip1/WAF1) is induced in the cytoplasm during the course of differentiation of chick retinal precursor cells and N1E-115 cells. Ectopic expression of p21(Cip1/WAF1) lacking the nuclear localization signal in N1E-115 cells and NIH3T3 cells affects the formation of actin structures, characteristic of inactivation of Rho. p21(Cip1/WAF1) forms a complex with Rho-kinase and inhibits its activity in vitro and in vivo. Neurite outgrowth and branching from the hippocampal neurons are promoted if p21(Cip1/WAF1) is expressed abundantly in the cytoplasm. These results suggest that cytoplasmic p21(Cip1/WAF1) may contribute to the developmental process of the newborn neurons that extend axons and dendrites into target regions.

  8. GABA receptors, alcohol dependence and criminal behavior.

    PubMed

    Terranova, Claudio; Tucci, Marianna; Sartore, Daniela; Cavarzeran, Fabiano; Di Pietra, Laura; Barzon, Luisa; Palù, Giorgio; Ferrara, Santo D

    2013-09-01

    The aim of this study was to analyze the connection between alcohol dependence and criminal behavior by an integrated genetic-environmental approach. The research, structured as a case-control study, examined 186 alcohol-dependent males; group 1 (N = 47 convicted subjects) was compared with group 2 (N = 139 no previous criminal records). Genetic results were innovative, highlighting differences in genotype distribution (p = 0.0067) in group 1 for single-nucleotide polymorphism rs 3780428, located in the intronic region of subunit 2 of the GABA B receptor gene (GABBR2). Some environmental factors (e.g., grade repetition) were associated with criminal behavior; others (e.g., attendance at Alcoholics Anonymous) were inversely related to convictions. The concomitant presence of the genetic and environmental factors found to be associated with the condition of alcohol-dependent inmate showed a 4-fold increase in the risk of antisocial behavior. The results need to be replicated on a larger population to develop new preventive and therapeutic proposals. © 2013 American Academy of Forensic Sciences.

  9. PAS Kinase Promotes Cell Survival and Growth Through Activation of Rho1

    PubMed Central

    Cardon, Caleb M.; Beck, Thomas; Hall, Michael N.; Rutter, Jared

    2014-01-01

    In Saccharomyces cerevisiae, phosphorylation of Ugp1 by either of the yeast PASK family protein kinases (yPASK), Psk1 or Psk2, directs this metabolic enzyme to deliver glucose to the periphery for synthesis of the cell wall. However, we isolated PSK1 and PSK2 in a high-copy suppressor screen of a temperature-sensitive mutant of target of rapamycin 2 (TOR2). Posttranslational activation of yPASK, either by cell integrity stress or by growth on nonfermentative carbon sources, also suppressed the growth defect resulting from tor2 mutation. Although suppression of the tor2 mutant growth phenotype by activation of the kinase activity of yPASK required phosphorylation of the metabolic enzyme Ugp1 on serine 11, this resulted in the formation of a complex that induced Rho1 activation, rather than required the glucose partitioning function of Ugp1. In addition to phosphorylated Ugp1, this complex contained Rom2, a Rho1 guanine nucleotide exchange factor, and Ssd1, an mRNA-binding protein. Activation of yPASK-dependent Ugp1 phosphorylation, therefore, enables two processes that are required for cell growth and stress resistance: synthesis of the cell wall through partitioning glucose to the periphery and the formation of the signaling complex with Rom2 and Ssd1 to promote Rho1-dependent polarized cell growth. This complex may integrate metabolic and signaling responses required for cell growth and survival in suboptimal conditions. PMID:22296835

  10. LncRNA PCGEM1 Induces Ovarian Carcinoma Tumorigenesis and Progression Through RhoA Pathway.

    PubMed

    Chen, Shuo; Wang, Li-Li; Sun, Kai-Xuan; Liu, Yao; Guan, Xue; Zong, Zhi-Hong; Zhao, Yang

    2018-06-27

    Prostate cancer gene expression marker 1 (PCGEM1) is a long noncoding RNA (lncRNA) and is well known as a promoter in prostate cancer and osteoarthritis synoviocytes. However, the role PCGEM1 plays in epithelial ovarian cancer is unknown. PCGEM1 expression was examined in epithelial ovarian cancer and normal ovarian tissues using reverse transcription-PCR. Ovarian cancer cell phenotypes and genotypes were examined after PCGEM1 overexpression or downregulation in vitro; besides, the effects of PCGEM1 overexpression was also examined in vivo. PCGEM1 expression level was higher in epithelial ovarian cancer tissues than in normal ovarian tissues and was positively associated with differentiation (Well vs. Mod/Poor). Upregulation of PCGEM1 induced cancer cell proliferation, migration, and invasion, but decreased cell apoptosis through upregulating RhoA, YAP (Yes-associated protein), MMP2 (matrix metalloproteinase 2), Bcl-xL, and P70S6K expression; while PCGEM1 downregulation had the opposite effect. The nude mouse xenograft assay demonstrated that PCGEM1 overexpression promoted tumor growth. Furthermore, silencing RhoA expression reversed the effect of PCGEM1 and significantly inhibited RhoA, YAP, MMP2, Bcl-xL, and P70S6K protein expression. In conclusion, we suggest that PCGEM1 may be an inducer in epithelial ovarian cancer tumorigenesis and progression by upregulating RhoA and the subsequent expression of YAP, P70S6K, MMP2, and Bcl-xL. © 2018 The Author(s). Published by S. Karger AG, Basel.

  11. Gα12 facilitates shortening in human airway smooth muscle by modulating phosphoinositide 3-kinase-mediated activation in a RhoA-dependent manner.

    PubMed

    Yoo, Edwin J; Cao, Gaoyuan; Koziol-White, Cynthia J; Ojiaku, Christie A; Sunder, Krishna; Jude, Joseph A; Michael, James V; Lam, Hong; Pushkarsky, Ivan; Damoiseaux, Robert; Di Carlo, Dino; Ahn, Kwangmi; An, Steven S; Penn, Raymond B; Panettieri, Reynold A

    2017-12-01

    PI3K-dependent activation of Rho kinase (ROCK) is necessary for agonist-induced human airway smooth muscle cell (HASMC) contraction, and inhibition of PI3K promotes bronchodilation of human small airways. The mechanisms driving agonist-mediated PI3K/ROCK axis activation, however, remain unclear. Given that G 12 family proteins activate ROCK pathways in other cell types, their role in M 3 muscarinic acetylcholine receptor-stimulated PI3K/ROCK activation and contraction was examined. Gα 12 coupling was evaluated using co-immunoprecipitation and serum response element (SRE)-luciferase reporter assays. siRNA and pharmacological approaches, as well as overexpression of a regulator of G-protein signaling (RGS) proteins were applied in HASMCs. Phosphorylation levels of Akt, myosin phosphatase targeting subunit-1 (MYPT1), and myosin light chain-20 (MLC) were measured. Contraction and shortening were evaluated using magnetic twisting cytometry (MTC) and micro-pattern deformation, respectively. Human precision-cut lung slices (hPCLS) were utilized to evaluate bronchoconstriction. Knockdown of M 3 receptors or Gα 12 attenuated activation of Akt, MYPT1, and MLC phosphorylation. Gα 12 coimmunoprecipitated with M 3 receptors, and p115RhoGEF-RGS overexpression inhibited carbachol-mediated induction of SRE-luciferase reporter. p115RhoGEF-RGS overexpression inhibited carbachol-induced activation of Akt, HASMC contraction, and shortening. Moreover, inhibition of RhoA blunted activation of PI3K. Lastly, RhoA inhibitors induced dilation of hPCLS. Gα 12 plays a crucial role in HASMC contraction via RhoA-dependent activation of the PI3K/ROCK axis. Inhibition of RhoA activation induces bronchodilation in hPCLS, and targeting Gα 12 signaling may elucidate novel therapeutic targets in asthma. These findings provide alternative approaches to the clinical management of airway obstruction in asthma. © 2017 The British Pharmacological Society.

  12. Interaction between src family kinases and rho-kinase in agonist-induced Ca2+-sensitization of rat pulmonary artery.

    PubMed

    Knock, Greg A; Shaifta, Yasin; Snetkov, Vladimir A; Vowles, Benjamin; Drndarski, Svetlana; Ward, Jeremy P T; Aaronson, Philip I

    2008-02-01

    We investigated the role of src family kinases (srcFK) in agonist-mediated Ca2+-sensitization in pulmonary artery and whether this involves interaction with the rho/rho-kinase pathway. Intra-pulmonary arteries (IPAs) and cultured pulmonary artery smooth muscle cells (PASMC) were obtained from rat. Expression of srcFK was determined at the mRNA and protein levels. Ca2+-sensitization was induced by prostaglandin F(2 alpha) (PGF(2 alpha)) in alpha-toxin-permeabilized IPAs. Phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light-chain-20 (MLC20) and translocation of rho-kinase in response to PGF(2 alpha) were also determined. Nine srcFK were expressed at the mRNA level, including src, fyn, and yes, and PGF(2 alpha) enhanced phosphorylation of three srcFK proteins at tyr-416. In alpha-toxin-permeabilized IPAs, PGF(2 alpha) enhanced the Ca2+-induced contraction (pCa 6.9) approximately three-fold. This enhancement was inhibited by the srcFK blockers SU6656 and PP2 and by the rho-kinase inhibitor Y27632. Y27632, but not SU6656 or PP2, also inhibited the underlying pCa 6.9 contraction. PGF(2 alpha) enhanced phosphorylation of MYPT-1 at thr-697 and thr-855 and of MLC20 at ser-19. This enhancement, but not the underlying basal phosphorylation, was inhibited by SU6656. Y27632 suppressed both basal and PGF(2 alpha)-mediated phosphorylation. The effects of SU6656 and Y27632, on both contraction and MYPT-1 and MLC20 phosphorylation, were not additive. PGF(2 alpha) triggered translocation of rho-kinase in PASMC, and this was inhibited by SU6656. srcFK are activated by PGF(2 alpha) in the rat pulmonary artery and may contribute to Ca2+-sensitization and contraction via rho-kinase translocation and phosphorylation of MYPT-1.

  13. Interaction between src family kinases and rho-kinase in agonist-induced Ca2+-sensitization of rat pulmonary artery

    PubMed Central

    Knock, Greg A.; Shaifta, Yasin; Snetkov, Vladimir A.; Vowles, Benjamin; Drndarski, Svetlana; Ward, Jeremy P.T.; Aaronson, Philip I.

    2008-01-01

    Abstract Aims We investigated the role of src family kinases (srcFK) in agonist-mediated Ca2+-sensitization in pulmonary artery and whether this involves interaction with the rho/rho-kinase pathway. Methods and results Intra-pulmonary arteries (IPAs) and cultured pulmonary artery smooth muscle cells (PASMC) were obtained from rat. Expression of srcFK was determined at the mRNA and protein levels. Ca2+-sensitization was induced by prostaglandin F2α (PGF2α) in α-toxin-permeabilized IPAs. Phosphorylation of the regulatory subunit of myosin phosphatase (MYPT-1) and of myosin light-chain-20 (MLC20) and translocation of rho-kinase in response to PGF2α were also determined. Nine srcFK were expressed at the mRNA level, including src, fyn, and yes, and PGF2α enhanced phosphorylation of three srcFK proteins at tyr-416. In α-toxin-permeabilized IPAs, PGF2α enhanced the Ca2+-induced contraction (pCa 6.9) approximately three-fold. This enhancement was inhibited by the srcFK blockers SU6656 and PP2 and by the rho-kinase inhibitor Y27632. Y27632, but not SU6656 or PP2, also inhibited the underlying pCa 6.9 contraction. PGF2α enhanced phosphorylation of MYPT-1 at thr-697 and thr-855 and of MLC20 at ser-19. This enhancement, but not the underlying basal phosphorylation, was inhibited by SU6656. Y27632 suppressed both basal and PGF2α-mediated phosphorylation. The effects of SU6656 and Y27632, on both contraction and MYPT-1 and MLC20 phosphorylation, were not additive. PGF2α triggered translocation of rho-kinase in PASMC, and this was inhibited by SU6656. Conclusions srcFK are activated by PGF2α in the rat pulmonary artery and may contribute to Ca2+-sensitization and contraction via rho-kinase translocation and phosphorylation of MYPT-1. PMID:18032393

  14. Exogenous Application of GABA Improves PEG-Induced Drought Tolerance Positively Associated with GABA-Shunt, Polyamines, and Proline Metabolism in White Clover.

    PubMed

    Yong, Bin; Xie, Huan; Li, Zhou; Li, Ya-Ping; Zhang, Yan; Nie, Gang; Zhang, Xin-Quan; Ma, Xiao; Huang, Lin-Kai; Yan, Yan-Hong; Peng, Yan

    2017-01-01

    In order to investigate the physiological effects of exogenous γ-aminobutyric acid (GABA) on drought tolerance in white clover (Trifolium repens), GABA shunt, polyamines (PAs), and proline (Pro) metabolism were examined after plants pretreated with or without GABA (8 mM) and then exposed to water or 15% PEG-induced drought stress in growth chamber. In this study, exogenous application of GABA effectively alleviated drought-induced damage in leaves, as reflected by significantly higher relative water content, lower electrolyte leakage, lipid peroxidation, and leaf wilt. Exogenous GABA further promoted drought-induced increases in GABA transaminase and alpha ketone glutarate dehydrogenase activities, but inhibited glutamate decarboxylase activity under both control and drought conditions, resulting in an increase in endogenous glutamate (Glu) and GABA content. Besides, exogenous GABA could well accelerated PAs synthesis and suppressed PAs catabolism, which lead to the extremely enhanced different types of PAs content (free Put and Spd, insoluble bound Spd and Spm, soluble conjugated Spd and Spm, and total Put, Spd and Spm) under drought stress. In addition, exogenous GABA application further activated drought-induced Δ 1 -pyrroline-5-carboxylate synthetase and proline dehydrogenase activities, but suppressed drought-facilitated ornithine -δ-amino transferase activities, leading to a higher Pro accumulation and metabolism in GABA-pretreated plants in the middle and last period of drought. The results suggested that increased endogenous GABA by exogenous GABA treatment could improve drought tolerance of white clover associated with a positive regulation in the GABA-shunt, PAs and Pro metabolism.

  15. Exogenous Application of GABA Improves PEG-Induced Drought Tolerance Positively Associated with GABA-Shunt, Polyamines, and Proline Metabolism in White Clover

    PubMed Central

    Yong, Bin; Xie, Huan; Li, Zhou; Li, Ya-Ping; Zhang, Yan; Nie, Gang; Zhang, Xin-Quan; Ma, Xiao; Huang, Lin-Kai; Yan, Yan-Hong; Peng, Yan

    2017-01-01

    In order to investigate the physiological effects of exogenous γ-aminobutyric acid (GABA) on drought tolerance in white clover (Trifolium repens), GABA shunt, polyamines (PAs), and proline (Pro) metabolism were examined after plants pretreated with or without GABA (8 mM) and then exposed to water or 15% PEG-induced drought stress in growth chamber. In this study, exogenous application of GABA effectively alleviated drought-induced damage in leaves, as reflected by significantly higher relative water content, lower electrolyte leakage, lipid peroxidation, and leaf wilt. Exogenous GABA further promoted drought-induced increases in GABA transaminase and alpha ketone glutarate dehydrogenase activities, but inhibited glutamate decarboxylase activity under both control and drought conditions, resulting in an increase in endogenous glutamate (Glu) and GABA content. Besides, exogenous GABA could well accelerated PAs synthesis and suppressed PAs catabolism, which lead to the extremely enhanced different types of PAs content (free Put and Spd, insoluble bound Spd and Spm, soluble conjugated Spd and Spm, and total Put, Spd and Spm) under drought stress. In addition, exogenous GABA application further activated drought-induced Δ1-pyrroline-5-carboxylate synthetase and proline dehydrogenase activities, but suppressed drought-facilitated ornithine -δ-amino transferase activities, leading to a higher Pro accumulation and metabolism in GABA-pretreated plants in the middle and last period of drought. The results suggested that increased endogenous GABA by exogenous GABA treatment could improve drought tolerance of white clover associated with a positive regulation in the GABA-shunt, PAs and Pro metabolism. PMID:29312009

  16. The Role of the Rho/ROCK Pathway in Ang II and TGF-β1-Induced Atrial Remodeling

    PubMed Central

    Lu, Gui-Hua; Xu, Cheng-Gui; Xu, Zhe; Tang, Kai; Cheng, Yun-Jiu; Gao, Xiu-Ren; Wu, Su-Hua

    2016-01-01

    Objectives To study the role of the Rho/ROCK pathway in Ang II and TGF-β1-induced atrial remodeling. Methods and Results A canine atrial fibrillation (AF) model was established by rapid atrial pacing (RAP) of the left atrium. The roles of TGF-β1, the RhoA/ROCK signaling pathway and connective tissue growth factor (CTGF) in atrial remodeling were studied via both in vitro and in vivo experiments. Each of the dogs that received RAP developed persistent AF within 4 weeks. The mRNA expression levels of TGF-β1 (1.32±0.38), Collagen-I(1.33±0.91), CTGF(5.83±3.71), RhoA(1.23±0.57) and ROCK-1 (1.02±0.27) in the left atrium were significantly increased following 4 weeks of RAP. Angiotensin II (Ang II) induced the proliferation of atrial fibroblasts and up-regulated the expression of both CTGF and ROCK-1 in a dose-dependent manner. Simvastatin and Y27632 reversed Ang II-induced CFs proliferation, as well as ROCK-1(0.89±0.05 and 1.27±0.03, respectively) and CTGF (0.87±0.04 and 0.91±0.02, respectively) expression. The expression mRNA of ROCK-1(1.74±0.13) and CTGF (2.28±0.11) can upregulated by TGF-β1, and down-regulated by Simvastatin (1.22±0.03 vs 2.27±0.11), Y27632 (1.01±0.04 vs 1.64±0.03), Los (1.04±0.11 vs 1.26±0.05), respectively. Losartan and Simvastatin attenuated the effects of TGF-β1, inhibited RhoA activity as opposed to RhoA protein expression. Y27632 had no effect on either the expression or the activity of RhoA. Conclusions The increased expression of profibrotic factors (CTGF, ROCK1 and Smad2/3) played an important role in our RAP-induced AF model. Increased atrial profibrotic factors involve the activation of either the TGF-β1/RhoA/ROCK-1 or the TGF-β1/Smad2/3 signaling pathway. PMID:27611832

  17. Delta Subunit-Containing Gamma-Aminobutyric Acid A Receptor Disinhibits Lateral Amygdala and Facilitates Fear Expression in Mice.

    PubMed

    Liu, Zhi-Peng; He, Qing-Hai; Pan, Han-Qing; Xu, Xiao-Bin; Chen, Wen-Bing; He, Ye; Zhou, Jin; Zhang, Wen-Hua; Zhang, Jun-Yu; Ying, Xiao-Ping; Han, Ren-Wen; Li, Bao-Ming; Gao, Tian-Ming; Pan, Bing-Xing

    2017-06-15

    Maintaining gamma-aminobutyric acidergic (GABAergic) inhibition in the amygdala within a physiological range is critical for the appropriate expression of emotions such as fear and anxiety. The synaptic GABA type A receptor (GABA A R) is generally known to mediate the primary component of amygdala inhibition and prevent inappropriate expression of fear. However, little is known about the contribution of the extrasynaptic GABA A R to amygdala inhibition and fear. By using mice expressing green fluorescent protein in interneurons (INs) and lacking the δ subunit-containing GABA A R (GABA A (δ)R), which is exclusively situated in the extrasynaptic membrane, we systematically investigated the role of GABA A (δ)R in regulating inhibition in the lateral amygdala (LA) and fear learning using the combined approaches of immunohistochemistry, electrophysiology, and behavior. In sharp contrast to the established role of synaptic GABA A R in mediating LA inhibition, we found that either pharmacological or physiological recruitment of GABA A (δ)R resulted in the weakening of GABAergic transmission onto projection neurons in LA while leaving the glutamatergic transmission unaltered, suggesting disinhibition by GABA A (δ)R. The disinhibition arose from IN-specific expression of GABA A (δ)R with its activation decreasing the input resistance of local INs and suppressing their activation. Genetic deletion of GABA A (δ)R attenuated its role in suppressing LA INs and disinhibiting LA. Importantly, the GABA A (δ)R facilitated long-term potentiation in sensory afferents to LA and permitted the expression of learned fear. Our findings suggest that GABA A (δ)R serves as a brake rather than a mediator of GABAergic inhibition in LA. The disinhibition by GABA A (δ)R may help to prevent excessive suppression of amygdala activity and thus ensure the expression of emotion. Copyright © 2016 Society of Biological Psychiatry. Published by Elsevier Inc. All rights reserved.

  18. GABA predicts visual intelligence.

    PubMed

    Cook, Emily; Hammett, Stephen T; Larsson, Jonas

    2016-10-06

    Early psychological researchers proposed a link between intelligence and low-level perceptual performance. It was recently suggested that this link is driven by individual variations in the ability to suppress irrelevant information, evidenced by the observation of strong correlations between perceptual surround suppression and cognitive performance. However, the neural mechanisms underlying such a link remain unclear. A candidate mechanism is neural inhibition by gamma-aminobutyric acid (GABA), but direct experimental support for GABA-mediated inhibition underlying suppression is inconsistent. Here we report evidence consistent with a global suppressive mechanism involving GABA underlying the link between sensory performance and intelligence. We measured visual cortical GABA concentration, visuo-spatial intelligence and visual surround suppression in a group of healthy adults. Levels of GABA were strongly predictive of both intelligence and surround suppression, with higher levels of intelligence associated with higher levels of GABA and stronger surround suppression. These results indicate that GABA-mediated neural inhibition may be a key factor determining cognitive performance and suggests a physiological mechanism linking surround suppression and intelligence. Copyright © 2016 The Authors. Published by Elsevier Ireland Ltd.. All rights reserved.

  19. Na+, K+-ATPase β1 subunit associates with α1 subunit modulating a "higher-NKA-in-hyposmotic media" response in gills of euryhaline milkfish, Chanos chanos.

    PubMed

    Hu, Yau-Chung; Chu, Keng-Fu; Yang, Wen-Kai; Lee, Tsung-Han

    2017-10-01

    The euryhaline milkfish (Chanos chanos) is a popular aquaculture species that can be cultured in fresh water, brackish water, or seawater in Southeast Asia. In gills of the milkfish, Na + , K + -ATPase (i.e., NKA; sodium pump) responds to salinity challenges including changes in mRNA abundance, protein amount, and activity. The functional pump is composed of a heterodimeric protein complex composed of α- and β-subunits. Among the NKA genes, α11 isozyme comprises the major form of NKA subunits in mammalian osmoregulatory organs; however, most studies on fish gills have focused on the α1 subunit and did not verify the α11 isozyme. Based on the sequenced milkfish transcriptome, an NKA β1 subunit gene was identified that had the highest amino acid homology to β233, a NKA β1 subunit paralog originally identified in the eel. Despite this high level of homology to β233, phylogenetic analysis and the fact that only a single NKA β1 subunit gene exists in the milkfish suggest that the milkfish gene should be referred to as the NKA β1 subunit gene. The results of accurate domain prediction of the β1 subunit, co-localization of α1 and β1 subunits in epithelial ionocytes, and co-immunoprecipitation of α1 and β1 subunits, indicated the formation of a α11 complex in milkfish gills. Moreover, when transferred to hyposmotic media (fresh water) from seawater, parallel increases in branchial mRNA and protein expression of NKA α1 and β1 subunits suggested their roles in hypo-osmoregulation of euryhaline milkfish. This study molecularly characterized the NKA β1 subunit and provided the first evidence for an NKA α11 association in gill ionocytes of euryhaline teleosts.

  20. Pathophysiological effects of RhoA and Rho-associated kinase on cardiovascular system.

    PubMed

    Cai, Anping; Li, Liwen; Zhou, Yingling

    2016-01-01

    In past decades, growing evidence from basic and clinical researches reveal that small guanosine triphosphate binding protein ras homolog gene family, member A (RhoA) and its main effector Rho-associated kinase (ROCK) play central and complex roles in cardiovascular systems, and increasing RhoA and ROCK activity is associated with a broad range of cardiovascular diseases such as congestive heart failure, atherosclerosis, and hypertension. Favorable outcomes have been observed with ROCK inhibitors treatment. In this review, we briefly summarize the pathophysiological roles of RhoA/ROCK signaling pathway on cardiovascular system, displaying the potential benefits in the cardiovascular system with controlling RhoA/ROCK signaling pathway.

  1. Raf-1/CK2 and RhoA/ROCK signaling promote TNF-α-mediated endothelial apoptosis via regulating vimentin cytoskeleton.

    PubMed

    Yang, Lifeng; Tang, Lian; Dai, Fan; Meng, Guoliang; Yin, Runting; Xu, Xiaole; Yao, Wenjuan

    2017-08-15

    Both RhoA/ROCK and Raf-1/CK2 pathway play essential roles in cell proliferation, apoptosis, differentiation, and multiple other common cellular functions. We previously reported that vimentin is responsible for TNF-α-induced cell apoptosis. Herein, we investigated the regulation of RhoA/ROCK and Raf-1/CK2 signaling on vimentin filaments and endothelial apoptosis mediated by TNF-α. Treatment with TNF-α significantly induced the activation of RhoA and ROCK, and the expression of ROCK1. RhoA deficiency could obviously inhibit ROCK activation and ROCK1 expression induced by TNF-α. Both RhoA deficiency and ROCK activity inhibition (Y-27632) greatly inhibited endothelial apoptosis and preserved cell viability in TNF-α-induced human umbilical vein endothelial cells (HUVECs). Also vimentin phosphorylation and the remodeling of vimentin or phospho-vimentin induced by TNF-α were obviously attenuated by RhoA suppression and ROCK inhibition. TNF-α-mediated vimentin cleavage was significantly inhibited by RhoA suppression and ROCK inhibition through decreasing the activation of caspase3 and 8. Furthermore, TNF-α treatment greatly enhanced the activation of Raf-1. Suppression of Raf-1 or CK2 by its inhibitor (GW5074 or TBB) blocked vimentin phosphorylation, remodeling and endothelial apoptosis, and preserved cell viability in TNF-α-induced HUVECs. However, Raf-1 inhibition showed no significant effect on TNF-α-induced ROCK expression and activation, suggesting that the regulation of Raf-1/CK2 signaling on vimentin was independent of ROCK. Taken together, these results indicate that both RhoA/ROCK and Raf-1/CK2 pathway are responsible for TNF-α-mediated endothelial cytotoxicity via regulating vimentin cytoskeleton. Copyright © 2017 Elsevier B.V. All rights reserved.

  2. Abnormal Concentration of GABA and Glutamate in The Prefrontal Cortex in Schizophrenia.-An in Vivo 1H-MRS Study.

    PubMed

    Chen, Tianyi; Wang, Yingchan; Zhang, Jianye; Wang, Zuowei; Xu, Jiale; Li, Yao; Yang, Zhilei; Liu, Dengtang

    2017-10-25

    The etiology and pathomechanism of schizophrenia are unknown. The traditional dopamine (DA) hypothesis is unable to fully explain its pathology and therapeutics. The glutamate (Glu) and γ-aminobutyric acid (GABA) hypotheses suggest Glu or GABA concentrations are abnormal in the brains of patients with schizophrenia. Magnetic resonance spectroscopy (MRS) show glutamate level increases in the ventromedial prefrontal cortex (vmPFC) including the anterior cingulated cortex (ACC) in those with schizophrenia. To investigate the function of the glutamate system (glutamate and γ-aminobutyric acid) in the etiology and pathomechanism of schizophrenia. 24 drug naïve patients with schizophrenia and 24 healthy volunteers were matched by gender, age, and educational level. The Siemens 3T MRI system was used to collect the magnetic resonance spectroscopy (MRS) data of the subjects. The regions of interest included the left dorsolateral prefrontal cortex (IDLPFC), ventromedial prefrontal cortex (vmPFC), and anterior cingulate cortex (ACC). LCModel software was used to analyze the concentrations of γ-aminobutyric acid (GABA), glutamate (Glu), glutamine (Gln), N-acetylaspartate (NAA), and N-acetylaspartylglutamate (NAAG) in the region of interest. Meanwhile, the Positive and Negative Syndrome Scale (PANSS) and the Clinical Global Impression Scale (CGI) were used to assess the mental symptoms and severity of the disease. The median GABA concentrations in the anterior cingulate cortex of the schizophrenia group and the healthy control group were 1.90 (Q1=1.55, Q3=2.09) and 2.16 (Q1=1.87, Q3=2.59) respectively; the mean (sd) Glu concentrations were 6.07 (2.48) and 6.54 (1.99); the median Gln concentrations were 0.36 (Q1=0.00, Q3=0.74) and 0.29 (Q1=0.00, Q3=0.59); the between-group difference of the GABA concentrations was statistically significant ( Z =-2.95, p =0.003); the between-group difference of the GABA/(NAA+NAAG) was statistically significant ( Z =-2.72, p =0.012); the

  3. Rho1- and Pkc1-dependent phosphorylation of the F-BAR protein Syp1 contributes to septin ring assembly.

    PubMed

    Merlini, Laura; Bolognesi, Alessio; Juanes, Maria Angeles; Vandermoere, Franck; Courtellemont, Thibault; Pascolutti, Roberta; Séveno, Martial; Barral, Yves; Piatti, Simonetta

    2015-09-15

    In many cell types, septins assemble into filaments and rings at the neck of cellular appendages and/or at the cleavage furrow to help compartmentalize the plasma membrane and support cytokinesis. How septin ring assembly is coordinated with membrane remodeling and controlled by mechanical stress at these sites is unclear. Through a genetic screen, we uncovered an unanticipated link between the conserved Rho1 GTPase and its effector protein kinase C (Pkc1) with septin ring stability in yeast. Both Rho1 and Pkc1 stabilize the septin ring, at least partly through phosphorylation of the membrane-associated F-BAR protein Syp1, which colocalizes asymmetrically with the septin ring at the bud neck. Syp1 is displaced from the bud neck upon Pkc1-dependent phosphorylation at two serines, thereby affecting the rigidity of the new-forming septin ring. We propose that Rho1 and Pkc1 coordinate septin ring assembly with membrane and cell wall remodeling partly by controlling Syp1 residence at the bud neck. © 2015 Merlini et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

  4. Comparison of T2, T1rho, and diffusion metrics in assessment of liver fibrosis in rats.

    PubMed

    Zhang, Hui; Yang, Qihua; Yu, Taihui; Chen, Xiaodong; Huang, Jingwen; Tan, Cui; Liang, Biling; Guo, Hua

    2017-03-01

    To evaluate the value of T 2 , T 1 rho, and diffusion metrics in assessment of liver fibrosis in rats. Liver fibrosis in a rat model (n = 72) was induced by injection of carbon tetrachloride (CCl 4 ) at 3T. T 2 , T 1 rho, and diffusion parameters (apparent diffusion coefficient (ADC), D true ) via spin echo (SE) diffusion-weighted imaging (DWI) and stimulated echo acquisition mode (STEAM) DWI with three diffusion times (DT: 80, 106, 186 msec) were obtained in surviving rats with hepatic fibrosis (n = 52) and controls (n = 8). Liver fibrosis stage (F0-F6) was identified based on pathological results using the traditional liver fibrosis staging method for rodents. Nonparametric statistical methods and receiver operating characteristic (ROC) curve analysis were employed to determine the diagnostic accuracy. Mean T 2 , T 1 rho, ADC, and D true with DT = 186 msec correlated with the severity of fibrosis with r = 0.73, 0.83, -0.83, and -0.85 (all P < 0.001), respectively. The average areas under the ROC curve at different stages for T 1 rho and diffusion parameters (DT = 186 msec) were larger than those of T 2 and SE DWI (0.92, 0.92, and 0.92 vs. 0.86, 0.82, and 0.83). The corresponding average sensitivity and specificity for T 1 rho and diffusion parameters with a long DT were larger (89.35 and 88.90, 88.36 and 89.97, 90.16 and 87.13) than T 2 and SE DWI (90.28 and 79.93, 85.30 and 77.64, 78.21 and 82.41). The performances of T 1 rho and D true (DT = 186 msec) were comparable (average AUC: 0.92 and 0.92). Among the evaluated sequences, T 1 rho and STEAM DWI with a long DT may serve as superior imaging biomarkers for assessing liver fibrosis and monitoring disease severity. 1 J. Magn. Reson. Imaging 2017;45:741-750. © 2016 International Society for Magnetic Resonance in Medicine.

  5. Protein Tyrosine Phosphatase-PEST and β8 Integrin Regulate Spatiotemporal Patterns of RhoGDI1 Activation in Migrating Cells

    PubMed Central

    Lee, Hye Shin; Cheerathodi, Mujeeburahiman; Chaki, Sankar P.; Reyes, Steve B.; Zheng, Yanhua; Lu, Zhimin; Paidassi, Helena; DerMardirossian, Celine; Lacy-Hulbert, Adam; Rivera, Gonzalo M.

    2015-01-01

    Directional cell motility is essential for normal development and physiology, although how motile cells spatiotemporally activate signaling events remains largely unknown. Here, we have characterized an adhesion and signaling unit comprised of protein tyrosine phosphatase (PTP)-PEST and the extracellular matrix (ECM) adhesion receptor β8 integrin that plays essential roles in directional cell motility. β8 integrin and PTP-PEST form protein complexes at the leading edge of migrating cells and balance patterns of Rac1 and Cdc42 signaling by controlling the subcellular localization and phosphorylation status of Rho GDP dissociation inhibitor 1 (RhoGDI1). Translocation of Src-phosphorylated RhoGDI1 to the cell's leading edge promotes local activation of Rac1 and Cdc42, whereas dephosphorylation of RhoGDI1 by integrin-bound PTP-PEST promotes RhoGDI1 release from the membrane and sequestration of inactive Rac1/Cdc42 in the cytoplasm. Collectively, these data reveal a finely tuned regulatory mechanism for controlling signaling events at the leading edge of directionally migrating cells. PMID:25666508

  6. Zinc ion enhances GABA tea-mediated oxidative DNA damage.

    PubMed

    Chuang, Show-Mei; Wang, Hsueh-Fang; Hsiao, Ching-Chuan; Cherng, Shur-Hueih

    2012-02-15

    GABA tea is a tea product that contains a high level of γ-aminobutyric acid (GABA). Previous study has demonstrated a synergistic effect of GABA tea and copper ions on DNA breakage. This study further explored whether zinc (Zn), a nonredox metal, modulated DNA cleavage induced by GABA tea extract. In a cell-free system, Zn(2+) significantly enhanced GABA tea extract and (-)-epigallocatechin-3-gallate (EGCG)- or H(2)O(2)-induced DNA damage at 24 h of incubation. Additionally, low dosages of GABA tea extract (1-10 μg/mL) possessed pro-oxidant activity to increase H(2)O(2)/Zn(2+)-induced DNA cleavage in a dose-dependent profile. By use of various reactive oxygen scavengers, it was observed that glutathione, catalase, and potassium iodide effectively inhibited DNA degradation caused by the GABA tea extract/H(2)O(2)/Zn(2+) system. Moreover, the data showed that the GABA tea extract itself (0.5-5 mg/mL) could induce DNA cleavage in a long-term exposure (48 h). EGCG, but not the GABA tea extract, enhanced H(2)O(2)-induced DNA cleavage. In contrast, GABA decreased H(2)O(2)- and EGCG-induced DNA cleavage, suggesting that GABA might contribute the major effect on the antioxidant activity of GABA tea extract. Furthermore, a comet assay revealed that GABA tea extract (0.25 mg/mL) and GABA had antioxidant activity on H(2)O(2)-induced DNA breakage in human peripheral lymphocytes. Taken together, these findings indicate that GABA tea has the potential of both pro-oxidant and antioxidant. It is proposed that a balance between EGCG-induced pro-oxidation and GABA-mediated antioxidation may occur in a complex mixture of GABA tea extract.

  7. Role of a gamma-aminobutryic acid (GABA) receptor mutation in the evolution and spread of Diabrotica virgifera virgifera resistance to cyclodiene insecticides

    USDA-ARS?s Scientific Manuscript database

    An alanine to serine amino acid substitution within the Rdl subunit of the gamma-aminobutyric acid (GABA) receptor confers resistance to cyclodiene insecticides in many species. The corn rootworm, Diabrotica virgifera virgifera, is a damaging pest of cultivated corn that was partially controlled by ...

  8. Grb2 mediates semaphorin-4D-dependent RhoA inactivation.

    PubMed

    Sun, Tianliang; Krishnan, Rameshkumar; Swiercz, Jakub M

    2012-08-01

    Signaling through the semaphorin 4D (Sema4D) receptor plexin-B1 is modulated by its interaction with tyrosine kinases ErbB-2 and Met. In cells expressing the plexin-B1-ErbB-2 receptor complex, ligand stimulation results in the activation of small GTPase RhoA and stimulation of cellular migration. By contrast, in cells expressing plexin-B1 and Met, ligand stimulation results in an association with the RhoGTPase-activating protein p190 RhoGAP and subsequent RhoA inactivation--a process that involves the tyrosine phosphorylation of plexin-B1 by Met. Inactivation of RhoA is necessary for Sema4D-mediated inhibition of cellular migration. It is, however, unknown how plexin-B1 phosphorylation regulates RhoGAP interaction and activity. Here we show that the activation of plexin-B1 by Sema4D and its subsequent tyrosine phosphorylation by Met creates a docking site for the SH2 domain of growth factor receptor bound-2 (Grb2). Grb2 is thereby recruited into the plexin-B1 receptor complex and, through its SH3 domain, interacts with p190 RhoGAP and mediates RhoA deactivation. Phosphorylation of plexin-B1 by Met and the recruitment of Grb2 have no effect on the R-RasGAP activity of plexin-B1, but are required for Sema4D-induced, RhoA-dependent antimigratory effects of Sema4D on breast cancer cells. These data show Grb2 as a direct link between plexin and p190-RhoGAP-mediated downstream signaling.

  9. Relationship among Glutamine, γ-Aminobutyric Acid, and Social Cognition in Autism Spectrum Disorders

    PubMed Central

    Sikoglu, Elif M.; Hodge, Steven M.; Edden, Richard A.E.; Foley, Ann; Kennedy, David N.; Moore, Constance M.; Frazier, Jean A.

    2015-01-01

    Abstract Objective: An imbalance of excitatory and inhibitory neurotransmission in autism spectrum disorder (ASD) has been proposed. We compared glutamate (Glu), glutamine (Gln), and γ-aminobutyric acid (GABA) levels in the anterior cingulate cortex (ACC) of 13 males with ASD and 14 typically developing (TD) males (ages 13–17), and correlated these levels with intelligence quotient (IQ) and measures of social cognition. Methods: Social cognition was evaluated by administration of the Social Responsiveness Scale (SRS) and the Reading the Mind in the Eyes Test (RMET). We acquired proton magnetic resonance spectroscopy (1H-MRS) data from the bilateral ACC using the single voxel point resolved spectroscopy sequence (PRESS) to quantify Glu and Gln, and Mescher–Garwood point-resolved spectroscopy sequence (MEGA-PRESS) to quantify GABA levels referenced to creatine (Cr). Results: There were higher Gln levels (p=0.04), and lower GABA/Cre levels (p=0.09) in the ASD group than in the TD group. There was no difference in Glu levels between groups. Gln was negatively correlated with RMET score (rho=−0.62, p=0.001) and IQ (rho=−0.56, p=0.003), and positively correlated with SRS scores (rho=0.53, p=0.007). GABA/Cre levels were positively correlated with RMET score (rho=0.34, p=0.09) and IQ (rho=0.36, p=0.07), and negatively correlated with SRS score (rho=−0.34, p=0.09). Conclusions: These data suggest an imbalance between glutamatergic neurotransmission and GABA-ergic neurotransmission in ASD. Higher Gln levels and lower GABA/Cre levels were associated with lower IQ and greater impairments in social cognition across groups. PMID:25919578

  10. Inherent conformational flexibility of F1-ATPase α-subunit.

    PubMed

    Hahn-Herrera, Otto; Salcedo, Guillermo; Barril, Xavier; García-Hernández, Enrique

    2016-09-01

    The core of F1-ATPase consists of three catalytic (β) and three noncatalytic (α) subunits, forming a hexameric ring in alternating positions. A wealth of experimental and theoretical data has provided a detailed picture of the complex role played by catalytic subunits. Although major conformational changes have only been seen in β-subunits, it is clear that α-subunits have to respond to these changes in order to be able to transmit information during the rotary mechanism. However, the conformational behavior of α-subunits has not been explored in detail. Here, we have combined unbiased molecular dynamics (MD) simulations and calorimetrically measured thermodynamic signatures to investigate the conformational flexibility of isolated α-subunits, as a step toward deepening our understanding of its function inside the α3β3 ring. The simulations indicate that the open-to-closed conformational transition of the α-subunit is essentially barrierless, which is ideal to accompany and transmit the movement of the catalytic subunits. Calorimetric measurements of the recombinant α-subunit from Geobacillus kaustophilus indicate that the isolated subunit undergoes no significant conformational changes upon nucleotide binding. Simulations confirm that the nucleotide-free and nucleotide-bound subunits show average conformations similar to that observed in the F1 crystal structure, but they reveal an increased conformational flexibility of the isolated α-subunit upon MgATP binding, which might explain the evolutionary conserved capacity of α-subunits to recognize nucleotides with considerable strength. Furthermore, we elucidate the different dependencies that α- and β-subunits show on Mg(II) for recognizing ATP. Copyright © 2016 Elsevier B.V. All rights reserved.

  11. Perisylvian GABA levels in schizophrenia and bipolar disorder.

    PubMed

    Atagün, Murat İlhan; Şıkoğlu, Elif Muazzez; Soykan, Çağlar; Serdar Süleyman, Can; Ulusoy-Kaymak, Semra; Çayköylü, Ali; Algın, Oktay; Phillips, Mary Louise; Öngür, Dost; Moore, Constance Mary

    2017-01-10

    The aim of this study is to measure GABA levels of perisylvian cortices in schizophrenia and bipolar disorder patients, using proton magnetic resonance spectroscopy ( 1 H-MRS). Patients with schizophrenia (n=25), bipolar I disorder (BD-I; n=28) and bipolar II disorder (BD-II; n=20) were compared with healthy controls (n=30). 1 H-MRS data was acquired using a Siemens 3T whole body scanner to quantify right and left perisylvian structures' (including superior temporal lobes) GABA levels. Right perisylvian GABA values differed significantly between groups [χ 2 =9.62, df: 3, p=0.022]. GABA levels were significantly higher in the schizophrenia group compared with the healthy control group (p=0.002). Furthermore, Chlorpromazine equivalent doses of antipsychotics correlated with right hemisphere GABA levels (r 2 =0.68, p=0.006, n=33). GABA levels are elevated in the right hemisphere in patients with schizophrenia in comparison to bipolar disorder and healthy controls. The balance between excitatory and inhibitory controls over the cortical circuits may have direct relationship with GABAergic functions in auditory cortices. In addition, GABA levels may be altered by brain regions of interest, psychotropic medications, and clinical stage in schizophrenia and bipolar disorder. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  12. Perisylvian GABA levels in schizophrenia and bipolar disorder

    PubMed Central

    ATAGÜN, Murat İlhan; ŞIKOĞLU, Elif Muazzez; SOYKAN, Çağlar; CAN, Serdar Süleyman; ULUSOY-KAYMAK, Semra; ÇAYKÖYLÜ, Ali; ALGIN, Oktay; PHILLIPS, Mary Louise; ÖNGÜR, Dost; MOORE, Constance Mary

    2016-01-01

    The aim of this study is to measure GABA levels of perisylvian cortices in schizophrenia and bipolar disorder patients, using proton magnetic resonance spectroscopy (1H-MRS). Patients with schizophrenia (n=25), bipolar I disorder (BD-I; n=28) and bipolar II disorder (BD-II; n=20) were compared with healthy controls (n=30). 1H-MRS data was acquired using a Siemens 3 Tesla whole body scanner to quantify right and left perisylvian structures’ (including superior temporal lobes) GABA levels. Right perisylvian GABA values differed significantly between groups [χ2=9.62, df: 3, p = 0.022]. GABA levels were significantly higher in the schizophrenia group compared with the healthy control group (p=0.002). Furthermore, Chlorpromazine equivalent doses of antipsychotics correlated with right hemisphere GABA levels (r2=0.68, p=0.006, n=33). GABA levels are elevated in the right hemisphere in patients with schizophrenia in comparison to bipolar disorder and healthy controls. The balance between excitatory and inhibitory controls over the cortical circuits may have direct relationship with GABAergic functions in auditory cortices. In addition, GABA levels may be altered by brain regions of interest, psychotropic medications, and clinical stage in schizophrenia and bipolar disorder. PMID:27890741

  13. Salmon lice (Lepeophtheirus salmonis) showing varying emamectin benzoate susceptibilities differ in neuronal acetylcholine receptor and GABA-gated chloride channel mRNA expression

    PubMed Central

    2013-01-01

    Background Caligid copepods, also called sea lice, are fish ectoparasites, some species of which cause significant problems in the mariculture of salmon, where the annual cost of infection is in excess of €300 million globally. At present, caligid control on farms is mainly achieved using medicinal treatments. However, the continued use of a restricted number of medicine actives potentially favours the development of drug resistance. Here, we report transcriptional changes in a laboratory strain of the caligid Lepeophtheirus salmonis (Krøyer, 1837) that is moderately (~7-fold) resistant to the avermectin compound emamectin benzoate (EMB), a component of the anti-salmon louse agent SLICE® (Merck Animal Health). Results Suppression subtractive hybridisation (SSH) was used to enrich transcripts differentially expressed between EMB-resistant (PT) and drug-susceptible (S) laboratory strains of L. salmonis. SSH libraries were subjected to 454 sequencing. Further L. salmonis transcript sequences were available as expressed sequence tags (EST) from GenBank. Contiguous sequences were generated from both SSH and EST sequences and annotated. Transcriptional responses in PT and S salmon lice were investigated using custom 15 K oligonucleotide microarrays designed using the above sequence resources. In the absence of EMB exposure, 359 targets differed in transcript abundance between the two strains, these genes being enriched for functions such as calcium ion binding, chitin metabolism and muscle structure. γ-aminobutyric acid (GABA)-gated chloride channel (GABA-Cl) and neuronal acetylcholine receptor (nAChR) subunits showed significantly lower transcript levels in PT lice compared to S lice. Using RT-qPCR, the decrease in mRNA levels was estimated at ~1.4-fold for GABA-Cl and ~2.8-fold for nAChR. Salmon lice from the PT strain showed few transcriptional responses following acute exposure (1 or 3 h) to 200 μg L-1 of EMB, a drug concentration tolerated by PT lice, but

  14. LINKING GABAA RECEPTOR SUBUNITS TO ALCOHOL-INDUCED CONDITIONED TASTE AVERSION AND RECOVERY FROM ACUTE ALCOHOL INTOXICATION

    PubMed Central

    Blednov, Y.A.; Benavidez, J.M.; Black, M.; Chandra, D.; Homanics, G.E.; Rudolph, U.; Harris, R.A.

    2012-01-01

    GABA type A receptors (GABAA-R) are important for ethanol actions and it is of interest to link individual subunits with specific ethanol behaviors. We studied null mutant mice for six different GABAA-R subunits1, α2, α3, α4, α5 and δ). Only mice lacking the α2 subunit showed reduction of conditioned taste aversion (CTA) to ethanol. These results are in agreement with data from knock-in mice with mutation of the ethanol-sensitive site in the α2-subunit (Blednov et al., 2011) and indicate this aversive property of ethanol is dependent on ethanol action on α2-containing GABAA-R. Deletion of the α2-subunit led to faster recovery whereas absence of the α3-subunit slowed recovery from ethanol-induced incoordination (rotarod). Deletion of the other four subunits did not affect this behavior. Similar changes in this behavior for the α2 and α3 null mutants were found for flurazepam motor-incoordination. However, no differences in recovery were found in motor-incoordinating effects of an α1-selective modulator (zolpidem) or an α4-selective agonist (gaboxadol). Therefore, recovery of rotarod incoordination is under control of two GABAA-R subunits: α2 and α3. For motor activity, α3 null mice demonstrated higher activation by ethanol (1 g/kg) whereas both α2 and α3 (-/-) knockout mice were less sensitive to ethanol-induced reduction of motor activity (1.5 g/kg). These studies demonstrate that the effects of ethanol at GABAergic synapses containing α2 subunit are important for specific behavioral effects of ethanol which may be relevant to the genetic linkage of the α2 subunit with human alcoholism. PMID:23147414

  15. Demonstration of elevation and localization of Rho-kinase activity in the brain of a rat model of cerebral infarction.

    PubMed

    Yano, Kazuo; Kawasaki, Koh; Hattori, Tsuyoshi; Tawara, Shunsuke; Toshima, Yoshinori; Ikegaki, Ichiro; Sasaki, Yasuo; Satoh, Shin-ichi; Asano, Toshio; Seto, Minoru

    2008-10-10

    Evidence that Rho-kinase is involved in cerebral infarction has accumulated. However, it is uncertain whether Rho-kinase is activated in the brain parenchyma in cerebral infarction. To answer this question, we measured Rho-kinase activity in the brain in a rat cerebral infarction model. Sodium laurate was injected into the left internal carotid artery, inducing cerebral infarction in the ipsilateral hemisphere. At 6 h after injection, increase of activating transcription factor 3 (ATF3) and c-Fos was found in the ipsilateral hemisphere, suggesting that neuronal damage occurs. At 0.5, 3, and 6 h after injection of laurate, Rho-kinase activity in extracts of the cerebral hemispheres was measured by an ELISA method. Rho-kinase activity in extracts of the ipsilateral hemisphere was significantly increased compared with that in extracts of the contralateral hemisphere at 3 and 6 h but not 0.5 h after injection of laurate. Next, localization of Rho-kinase activity was evaluated by immunohistochemical analysis in sections of cortex and hippocampus including infarct area 6 h after injection of laurate. Staining for phosphorylation of myosin-binding subunit (phospho-MBS) and myosin light chain (phospho-MLC), substrates of Rho-kinase, was elevated in neuron and blood vessel, respectively, in ipsilateral cerebral sections, compared with those in contralateral cerebral sections. These findings indicate that Rho-kinase is activated in neuronal and vascular cells in a rat cerebral infarction model, and suggest that Rho-kinase could be an important target in the treatment of cerebral infarction.

  16. GABA transaminases from Saccharomyces cerevisiae and Arabidopsis thaliana complement function in cytosol and mitochondria.

    PubMed

    Cao, Juxiang; Barbosa, Jose M; Singh, Narendra; Locy, Robert D

    2013-07-01

    GABA transaminase (GABA-T) catalyses the conversion of GABA to succinate semialdehyde (SSA) in the GABA shunt pathway. The GABA-T from Saccharomyces cerevisiae (ScGABA-TKG) is an α-ketoglutarate-dependent enzyme encoded by the UGA1 gene, while higher plant GABA-T is a pyruvate/glyoxylate-dependent enzyme encoded by POP2 in Arabidopsis thaliana (AtGABA-T). The GABA-T from A. thaliana is localized in mitochondria and mediated by an 18-amino acid N-terminal mitochondrial targeting peptide predicated by both web-based utilities TargetP 1.1 and PSORT. Yeast UGA1 appears to lack a mitochondrial targeting peptide and is localized in the cytosol. To verify this bioinformatic analysis and examine the significance of ScGABA-TKG and AtGABA-T compartmentation and substrate specificity on physiological function, expression vectors were constructed to modify both ScGABA-TKG and AtGABA-T, so that they express in yeast mitochondria and cytosol. Physiological function was evaluated by complementing yeast ScGABA-TKG deletion mutant Δuga1 with AtGABA-T or ScGABA-TKG targeted to the cytosol or mitochondria for the phenotypes of GABA growth defect, thermosensitivity and heat-induced production of reactive oxygen species (ROS). This study demonstrates that AtGABA-T is functionally interchangeable with ScGABA-TKG for GABA growth, thermotolerance and limiting production of ROS, regardless of location in mitochondria or cytosol of yeast cells, but AtGABA-T is about half as efficient in doing so as ScGABA-TKG. These results are consistent with the hypothesis that pyruvate/glyoxylate-limited production of NADPH mediates the effect of the GABA shunt in moderating heat stress in Saccharomyces. Copyright © 2013 John Wiley & Sons, Ltd.

  17. The Putative Exchange Factor Gef3p Interacts with Rho3p GTPase and the Septin Ring during Cytokinesis in Fission Yeast*

    PubMed Central

    Muñoz, Sofía; Manjón, Elvira; Sánchez, Yolanda

    2014-01-01

    The small GTP-binding proteins of the Rho family and its regulatory proteins play a central role in cytokinetic actomyosin ring assembly and cytokinesis. Here we show that the fission yeast guanine nucleotide exchange factor Gef3p interacts with Rho3p at the division site. Gef3p contains a putative DH homology domain and a BAR/IMD-like domain. The protein localized to the division site late in mitosis, where it formed a ring that did not constrict with actomyosin ring (cytokinetic actomyosin ring) invagination; instead, it split into a double ring that resembled the septin ring. Gef3p co-localized with septins and Mid2p and required septins and Mid2p for its localization. Gef3p interacts physically with the GTP-bound form of Rho3p. Although Gef3p is not essential for cell separation, the simultaneous disruption of gef3+ and Rho3p-interacting proteins, such as Sec8p, an exocyst component, Apm1p, a subunit of the clathrin adaptor complex or For3p, an actin-polymerizing protein, yielded cells with strong defects in septation and polarity respectively. Our results suggest that interactions between septins and Rho-GEFs provide a new targeting mechanism for GTPases in cytokinesis, in this case probably contributing to Rho3p function in vesicle tethering and vesicle trafficking in the later steps of cell separation. PMID:24947517

  18. Critical functions of RhoB in support of glioblastoma tumorigenesis

    PubMed Central

    Ma, Yufang; Gong, Yuanying; Cheng, Zhixiang; Loganathan, Sudan; Kao, Crystal; Sarkaria, Jann N.; Abel, Ty W.; Wang, Jialiang

    2015-01-01

    Background RhoB is a member of the Rho small GTPase family that regulates cytoskeletal dynamics and vesicle trafficking. The RhoB homologs, RhoA and RhoC, have been shown to promote cancer progression and metastasis. In contrast, the functions of RhoB in human cancers are context dependent. Although expression of RhoB inversely correlates with disease progression in several epithelial cancers, recent data suggest that RhoB may support malignant phenotypes in certain cancer types. Methods We assessed RhoB protein levels in glioma surgical specimens and patient-derived xenografts. The roles of RhoB in glioblastoma were determined by loss-of-function and gain-of-function assays in vitro and in vivo. The impact on p53 and STAT3 signaling was investigated. Results RhoB expression was similar in tumor specimens compared with normal neural tissues obtained from epilepsy surgery. RhoB was expressed in the vast majority of xenograft tumors and spheroid cultures. Knockdown of RhoB induced cell-cycle arrest and apoptosis and compromised in vivo tumorigenic potential. However, overexpression of wild-type RhoB or a constitutively active mutant (RhoB-V14) did not significantly affect cell growth, which suggests that RhoB is not a rate-limiting oncogenic factor and is consistent with the scarcity of RhoB mutations in human cancer. Knockdown of RhoB reduced basal STAT3 activity and impaired cytokine-induced STAT3 activation. In glioblastoma tumors retaining wild-type p53, depletion of RhoB also activated p53 and induced expression of p21CIP1/WAF1. Conclusions Our data suggest that RhoB belongs to an emerging class of “nononcogene addiction” factors that are essential for maintenance of malignant phenotypes in human cancers. PMID:25216671

  19. RhoGTPase Regulators Orchestrate Distinct Stages of Synaptic Development

    PubMed Central

    Martin-Vilchez, Samuel; Whitmore, Leanna; Asmussen, Hannelore; Zareno, Jessica; Horwitz, Rick; Newell-Litwa, Karen

    2017-01-01

    Small RhoGTPases regulate changes in post-synaptic spine morphology and density that support learning and memory. They are also major targets of synaptic disorders, including Autism. Here we sought to determine whether upstream RhoGTPase regulators, including GEFs, GAPs, and GDIs, sculpt specific stages of synaptic development. The majority of examined molecules uniquely regulate either early spine precursor formation or later maturation. Specifically, an activator of actin polymerization, the Rac1 GEF β-PIX, drives spine precursor formation, whereas both FRABIN, a Cdc42 GEF, and OLIGOPHRENIN-1, a RhoA GAP, regulate spine precursor elongation. However, in later development, a novel Rac1 GAP, ARHGAP23, and RhoGDIs inactivate actomyosin dynamics to stabilize mature synapses. Our observations demonstrate that specific combinations of RhoGTPase regulatory proteins temporally balance RhoGTPase activity during post-synaptic spine development. PMID:28114311

  20. Cytoplasmic p21Cip1/WAF1 regulates neurite remodeling by inhibiting Rho-kinase activity

    PubMed Central

    Tanaka, Hiroyuki; Yamashita, Toshihide; Asada, Minoru; Mizutani, Shuki; Yoshikawa, Hideki; Tohyama, Masaya

    2002-01-01

    p21Cip1/WAF1 has cell cycle inhibitory activity by binding to and inhibiting both cyclin/Cdk kinases and proliferating cell nuclear antigen. Here we show that p21Cip1/WAF1 is induced in the cytoplasm during the course of differentiation of chick retinal precursor cells and N1E-115 cells. Ectopic expression of p21Cip1/WAF1 lacking the nuclear localization signal in N1E-115 cells and NIH3T3 cells affects the formation of actin structures, characteristic of inactivation of Rho. p21Cip1/WAF1 forms a complex with Rho-kinase and inhibits its activity in vitro and in vivo. Neurite outgrowth and branching from the hippocampal neurons are promoted if p21Cip1/WAF1 is expressed abundantly in the cytoplasm. These results suggest that cytoplasmic p21Cip1/WAF1 may contribute to the developmental process of the newborn neurons that extend axons and dendrites into target regions. PMID:12119358

  1. Glial GABA, synthesized by monoamine oxidase B, mediates tonic inhibition

    PubMed Central

    Yoon, Bo-Eun; Woo, Junsung; Chun, Ye-Eun; Chun, Heejung; Jo, Seonmi; Bae, Jin Young; An, Heeyoung; Min, Joo Ok; Oh, Soo-Jin; Han, Kyung-Seok; Kim, Hye Yun; Kim, Taekeun; Kim, Young Soo; Bae, Yong Chul; Lee, C Justin

    2014-01-01

    GABA is the major inhibitory transmitter in the brain and is released not only from a subset of neurons but also from glia. Although neuronal GABA is well known to be synthesized by glutamic acid decarboxylase (GAD), the source of glial GABA is unknown. After estimating the concentration of GABA in Bergmann glia to be around 5–10 mm by immunogold electron microscopy, we demonstrate that GABA production in glia requires MAOB, a key enzyme in the putrescine degradation pathway. In cultured cerebellar glia, both Ca2+-induced and tonic GABA release are significantly reduced by both gene silencing of MAOB and the MAOB inhibitor selegiline. In the cerebellum and striatum of adult mice, general gene silencing, knock out of MAOB or selegiline treatment resulted in elimination of tonic GABA currents recorded from granule neurons and medium spiny neurons. Glial-specific rescue of MAOB resulted in complete rescue of tonic GABA currents. Our results identify MAOB as a key synthesizing enzyme of glial GABA, which is released via bestrophin 1 (Best1) channel to mediate tonic inhibition in the brain. PMID:25239459

  2. Different roles of the small GTPases Rac1, Cdc42, and RhoG in CALEB/NGC-induced dendritic tree complexity.

    PubMed

    Schulz, Jana; Franke, Kristin; Frick, Manfred; Schumacher, Stefan

    2016-10-01

    Rho GTPases play prominent roles in the regulation of cytoskeletal reorganization. Many aspects have been elaborated concerning the individual functions of Rho GTPases in distinct signaling pathways leading to cytoskeletal rearrangements. However, major questions have yet to be answered regarding the integration and the signaling hierarchy of different Rho GTPases in regulating the cytoskeleton in fundamental physiological events like neuronal process differentiation. Here, we investigate the roles of the small GTPases Rac1, Cdc42, and RhoG in defining dendritic tree complexity stimulated by the transmembrane epidermal growth factor family member CALEB/NGC. Combining gain-of-function and loss-of-function analysis in primary hippocampal neurons, we find that Rac1 is essential for CALEB/NGC-mediated dendritic branching. Cdc42 reduces the complexity of dendritic trees. Interestingly, we identify the palmitoylated isoform of Cdc42 to adversely affect dendritic outgrowth and dendritic branching, whereas the prenylated Cdc42 isoform does not. In contrast to Rac1, CALEB/NGC and Cdc42 are not directly interconnected in regulating dendritic tree complexity. Unlike Rac1, the Rac1-related GTPase RhoG reduces the complexity of dendritic trees by acting upstream of CALEB/NGC. Mechanistically, CALEB/NGC activates Rac1, and RhoG reduces the amount of CALEB/NGC that is located at the right site for Rac1 activation at the cell membrane. Thus, Rac1, Cdc42, and RhoG perform very specific and non-redundant functions at different levels of hierarchy in regulating dendritic tree complexity induced by CALEB/NGC. Rho GTPases play a prominent role in dendritic branching. CALEB/NGC is a transmembrane member of the epidermal growth factor (EGF) family that mediates dendritic branching, dependent on Rac1. CALEB/NGC stimulates Rac1 activity. RhoG inhibits CALEB/NGC-mediated dendritic branching by decreasing the amount of CALEB/NGC at the plasma membrane. Palmitoylated, but not prenylated form

  3. CB1-Dependent Long-Term Depression in Ventral Tegmental Area GABA Neurons: A Novel Target for Marijuana

    PubMed Central

    Friend, Lindsey; Sandoval, Philip; Nufer, Teresa; Ostlund, Isaac

    2017-01-01

    The VTA is necessary for reward behavior with dopamine cells critically involved in reward signaling. Dopamine cells in turn are innervated and regulated by neighboring inhibitory GABA cells. Using whole-cell electrophysiology in juvenile-adolescent GAD67-GFP male mice, we examined excitatory plasticity in fluorescent VTA GABA cells. A novel CB1-dependent LTD was induced in GABA cells that was dependent on metabotropic glutamate receptor 5, and cannabinoid receptor 1 (CB1). LTD was absent in CB1 knock-out mice but preserved in heterozygous littermates. Bath applied Δ9-tetrahydrocannabinol depressed GABA cell activity, therefore downstream dopamine cells will be disinhibited; and thus, this could potentially result in increased reward. Chronic injections of Δ9-tetrahydrocannabinol occluded LTD compared with vehicle injections; however, a single exposure was insufficient to do so. As synaptic modifications by drugs of abuse are often tied to addiction, these data suggest a possible mechanism for the addictive effects of Δ9-tetrahydrocannabinol in juvenile-adolescents, by potentially altering reward behavioral outcomes. SIGNIFICANCE STATEMENT The present study identifies a novel form of glutamatergic synaptic plasticity in VTA GABA neurons, a currently understudied cell type that is critical for the brain's reward circuit, and how Δ9-tetrahydrocannabinol occludes this plasticity. This study specifically addresses a potential unifying mechanism whereby marijuana could exert rewarding and addictive/withdrawal effects. Marijuana use and legalization are a pressing issue for many states in the United States. Although marijuana is the most commonly abused illicit drug, the implications of legalized, widespread, or continued usage are speculative. This study in juvenile-adolescent aged mice identifies a novel form of synaptic plasticity in VTA GABA cells, and the synaptic remodeling that can occur after Δ9-tetrahydrocannabinol use. PMID:29038246

  4. CB1-Dependent Long-Term Depression in Ventral Tegmental Area GABA Neurons: A Novel Target for Marijuana.

    PubMed

    Friend, Lindsey; Weed, Jared; Sandoval, Philip; Nufer, Teresa; Ostlund, Isaac; Edwards, Jeffrey G

    2017-11-08

    The VTA is necessary for reward behavior with dopamine cells critically involved in reward signaling. Dopamine cells in turn are innervated and regulated by neighboring inhibitory GABA cells. Using whole-cell electrophysiology in juvenile-adolescent GAD67-GFP male mice, we examined excitatory plasticity in fluorescent VTA GABA cells. A novel CB1-dependent LTD was induced in GABA cells that was dependent on metabotropic glutamate receptor 5, and cannabinoid receptor 1 (CB1). LTD was absent in CB1 knock-out mice but preserved in heterozygous littermates. Bath applied Δ 9 -tetrahydrocannabinol depressed GABA cell activity, therefore downstream dopamine cells will be disinhibited; and thus, this could potentially result in increased reward. Chronic injections of Δ 9 -tetrahydrocannabinol occluded LTD compared with vehicle injections; however, a single exposure was insufficient to do so. As synaptic modifications by drugs of abuse are often tied to addiction, these data suggest a possible mechanism for the addictive effects of Δ 9 -tetrahydrocannabinol in juvenile-adolescents, by potentially altering reward behavioral outcomes. SIGNIFICANCE STATEMENT The present study identifies a novel form of glutamatergic synaptic plasticity in VTA GABA neurons, a currently understudied cell type that is critical for the brain's reward circuit, and how Δ 9 -tetrahydrocannabinol occludes this plasticity. This study specifically addresses a potential unifying mechanism whereby marijuana could exert rewarding and addictive/withdrawal effects. Marijuana use and legalization are a pressing issue for many states in the United States. Although marijuana is the most commonly abused illicit drug, the implications of legalized, widespread, or continued usage are speculative. This study in juvenile-adolescent aged mice identifies a novel form of synaptic plasticity in VTA GABA cells, and the synaptic remodeling that can occur after Δ 9 -tetrahydrocannabinol use. Copyright © 2017 the

  5. GABAA receptors involved in sleep and anaesthesia: β1- versus β3-containing assemblies.

    PubMed

    Yanovsky, Yevgenij; Schubring, Stephan; Fleischer, Wiebke; Gisselmann, Günter; Zhu, Xin-Ran; Lübbert, Hermann; Hatt, Hanns; Rudolph, Uwe; Haas, Helmut L; Sergeeva, Olga A

    2012-01-01

    The histaminergic neurons of the posterior hypothalamus (tuberomamillary nucleus-TMN) control wakefulness, and their silencing through activation of GABA(A) receptors (GABA(A)R) induces sleep and is thought to mediate sedation under propofol anaesthesia. We have previously shown that the β1 subunit preferring fragrant dioxane derivatives (FDD) are highly potent modulators of GABA(A)R in TMN neurons. In recombinant receptors containing the β3N265M subunit, FDD action is abolished and GABA potency is reduced. Using rat, wild-type and β3N265M mice, FDD and propofol, we explored the relative contributions of β1- and β3-containing GABA(A)R to synaptic transmission from the GABAergic sleep-on ventrolateral preoptic area neurons to TMN. In β3N265M mice, GABA potency remained unchanged in TMN neurons, but it was decreased in cultured posterior hypothalamic neurons with impaired modulation of GABA(A)R by propofol. Spontaneous and evoked GABAergic synaptic currents (IPSC) showed β1-type pharmacology, with the same effects achieved by 3 μM propofol and 10 μM PI24513. Propofol and the FDD PI24513 suppressed neuronal firing in the majority of neurons at 5 and 100 μM, and in all cells at 10 and 250 μM, respectively. FDD given systemically in mice induced sedation but not anaesthesia. Propofol-induced currents were abolished (1-6 μM) or significantly reduced (12 μM) in β3N265M mice, whereas gating and modulation of GABA(A)R by PI24513 as well as modulation by propofol were unchanged. In conclusion, β1-containing (FDD-sensitive) GABA(A)R represent the major receptor pool in TMN neurons responding to GABA, while β3-containing (FDD-insensitive) receptors are gated by low micromolar doses of propofol. Thus, sleep and anaesthesia depend on different GABA(A)R types.

  6. Role of glutamic acid decarboxylase 67 in regulating cortical parvalbumin and GABA membrane transporter 1 expression: implications for schizophrenia.

    PubMed

    Curley, Allison A; Eggan, Stephen M; Lazarus, Matt S; Huang, Z Josh; Volk, David W; Lewis, David A

    2013-02-01

    Markers of GABA neurotransmission are altered in multiple regions of the neocortex in individuals with schizophrenia. Lower levels of glutamic acid decarboxylase 67 (GAD67) mRNA and protein, which is responsible for most cortical GABA synthesis, are accompanied by lower levels of GABA membrane transporter 1 (GAT1) mRNA. These alterations are thought to be most prominent in the parvalbumin (PV)-containing subclass of interneurons, which also contain lower levels of PV mRNA. Since GAT1 and PV each reduce the availability of GABA at postsynaptic receptors, lower levels of GAT1 and PV mRNAs have been hypothesized to represent compensatory responses to an upstream reduction in cortical GABA synthesis in schizophrenia. However, such cause-and-effect hypotheses cannot be directly tested in a human illness. Consequently, we used two mouse models with reduced GAD67 expression specifically in PV neurons (PV(GAD67+/-)) or in all interneurons (GABA(GAD67+/-)) and quantified GAD67, GAT1 and PV mRNA levels using methods identical to those employed in studies of schizophrenia. Cortical levels of PV or GAT1 mRNAs were not altered in PV(GAD67+/-) mice during postnatal development or in adulthood. Furthermore, cellular analyses confirmed the predicted reduction in GAD67 mRNA, but failed to show a deficit in PV mRNA in these animals. Levels of PV and GAT1 mRNAs were also unaltered in GABA(GAD67+/-) mice. Thus, mouse lines with cortical reductions in GAD67 mRNA that match or exceed those present in schizophrenia, and that differ in the developmental timing and cell type-specificity of the GAD67 deficit, failed to provide proof-of-concept evidence that lower PV and GAT1 expression in schizophrenia are a consequence of lower GAD67 expression. Together, these findings suggest that the correlated decrements in cortical GAD67, PV and GAT1 mRNAs in schizophrenia may be a common consequence of some other upstream factor. Copyright © 2012 Elsevier Inc. All rights reserved.

  7. Brain GABA levels across psychiatric disorders: A systematic literature review and meta-analysis of (1) H-MRS studies.

    PubMed

    Schür, Remmelt R; Draisma, Luc W R; Wijnen, Jannie P; Boks, Marco P; Koevoets, Martijn G J C; Joëls, Marian; Klomp, Dennis W; Kahn, René S; Vinkers, Christiaan H

    2016-09-01

    The inhibitory gamma-aminobutyric acid (GABA) system is involved in the etiology of most psychiatric disorders, including schizophrenia, autism spectrum disorder (ASD) and major depressive disorder (MDD). It is therefore not surprising that proton magnetic resonance spectroscopy ((1) H-MRS) is increasingly used to investigate in vivo brain GABA levels. However, integration of the evidence for altered in vivo GABA levels across psychiatric disorders is lacking. We therefore systematically searched the clinical (1) H-MRS literature and performed a meta-analysis. A total of 40 studies (N = 1,591) in seven different psychiatric disorders were included in the meta-analysis: MDD (N = 437), schizophrenia (N = 517), ASD (N = 150), bipolar disorder (N = 129), panic disorder (N = 81), posttraumatic stress disorder (PTSD) (N = 104), and attention deficit/hyperactivity disorder (ADHD) (N = 173). Brain GABA levels were lower in ASD (standardized mean difference [SMD] = -0.74, P = 0.001) and in depressed MDD patients (SMD = -0.52, P = 0.005), but not in remitted MDD patients (SMD = -0.24, P = 0.310) compared with controls. In schizophrenia this finding did not reach statistical significance (SMD = -0.23, P = 0.089). No significant differences in GABA levels were found in bipolar disorder, panic disorder, PTSD, and ADHD compared with controls. In conclusion, this meta-analysis provided evidence for lower brain GABA levels in ASD and in depressed (but not remitted) MDD patients compared with healthy controls. Findings in schizophrenia were more equivocal. Even though future (1) H-MRS studies could greatly benefit from a longitudinal design and consensus on the preferred analytical approach, it is apparent that (1) H-MRS studies have great potential in advancing our understanding of the role of the GABA system in the pathogenesis of psychiatric disorders. Hum Brain Mapp 37:3337-3352, 2016. © 2016 Wiley Periodicals

  8. Targeting RhoA/Rho kinase and p21-activated kinase signaling to prevent cancer development and progression.

    PubMed

    Chang, Yu-Wen E; Bean, Ronald R; Jakobi, Rolf

    2009-06-01

    Elevated RhoA/Rho kinase and p21-activated kinase signaling have been shown to promote cancer development and metastasis and have drawn much attention as potential targets of anti-cancer therapy. Elevated RhoA and Rho kinase activity promote cancer cell invasion and eventually lead to metastasis by disrupting E-cadherin-mediated adherens junctions and degradation of the extracellular matrix. Elevated p21-activated kinase activity promotes invasion by stimulating cell motility but also promotes cancer cell survival and growth. In this review we describe normal functions of RhoA/Rho kinase and p21-activated kinase signaling, mechanisms that lead to constitutive activation of RhoA/Rho kinase and p21-activated kinase pathways, and processes by which constitutive RhoA/Rho kinase and p21-activated kinase activity promote cancer development and progression to more aggressive and metastatic phenotypes. In addition, we summarize relevant patents on RhoA/Rho kinase and p21-activated kinase as targets of anti-cancer therapy and discuss the clinical potential of different approaches to modulate RhoA/Rho kinase and p21-activated kinase signaling.

  9. DNA topoisomerase IIβ stimulates neurite outgrowth in neural differentiated human mesenchymal stem cells through regulation of Rho-GTPases (RhoA/Rock2 pathway) and Nurr1 expression.

    PubMed

    Zaim, Merve; Isik, Sevim

    2018-04-25

    DNA topoisomerase IIβ (topo IIβ) is known to regulate neural differentiation by inducing the neuronal genes responsible for critical neural differentiation events such as neurite outgrowth and axon guidance. However, the pathways of axon growth controlled by topo IIβ have not been clarified yet. Microarray results of our previous study have shown that topo IIβ silencing in neural differentiated primary human mesenchymal stem cells (hMSCs) significantly alters the expression pattern of genes involved in neural polarity, axonal growth, and guidance, including Rho-GTPases. This study aims to further analyze the regulatory role of topo IIβ on the process of axon growth via regulation of Rho-GTPases. For this purpose, topo IIβ was silenced in neurally differentiated hMSCs. Cells lost their morphology because of topo IIβ deficiency, becoming enlarged and flattened. Additionally, a reduction in both neural differentiation efficiency and neurite length, upregulation in RhoA and Rock2, downregulation in Cdc42 gene expression were detected. On the other hand, cells were transfected with topo IIβ gene to elucidate the possible neuroprotective effect of topo IIβ overexpression on neural-induced hMSCs. Topo IIβ overexpression prompted all the cells to exhibit neural cell morphology as characterized by longer neurites. RhoA and Rock2 expressions were downregulated, whereas Cdc42 expression was upregulated. Nurr1 expression level correlated with topo IIβ in both topo IIβ-overexpressed and -silenced cells. Furthermore, differential translocation of Rho-GTPases was detected by immunostaining in response to topo IIβ. Our results suggest that topo IIβ deficiency could give rise to neurodegeneration through dysregulation of Rho-GTPases. However, further in-vivo research is needed to demonstrate if re-regulation of Rho GTPases by topo IIβ overexpression could be a neuroprotective treatment in the case of neurodegenerative diseases.

  10. Multiple roles of the Rho GEF ephexin1 in synapse remodeling

    PubMed Central

    Shi, Lei; Fu, Amy KY

    2010-01-01

    Synapse remodeling, which involves changes in the synaptic structure and their molecular composition, is required for the maturation and refinement of neural circuits. Although synapse remodeling is known to be tightly dependent on the assembly of local actin cytoskeleton, how actin directs the structural changes of synapse and targeting of synaptic proteins are not fully understood. Recently, we identified ephexin1, a Rho guanine nucleotide exchange factor (GEF) that regulates actin dynamics, to play an essential role in the maturation and functioning of the mammalian neuromuscular junction (NMJ). We showed that ephexin1 regulates the synaptic organization of the neurotransmitter receptor acetylcholine receptor (AChR) clusters through RhoA-dependent actin reorganization. Interestingly, ephexin1 has been implicated in the regulation of postsynaptic structure as well as the presynaptic vesicle release at various types of synapses. Our findings thus establish a novel function of ephexin1 in synapse remodeling through regulating the synaptic targeting of neurotransmitter receptors, revealing a versatile role of ephexin1 at synapses. PMID:21331259

  11. Manganese exposure alters extracellular GABA, GABA receptor and transporter protein and mRNA levels in the developing rat brain

    PubMed Central

    Anderson, Joel G.; Fordahl, Steve C.; Cooney, Paula T.; Weaver, Tara L.; Colyer, Christa L.; Erikson, Keith M.

    2011-01-01

    Unlike other essential trace elements (e.g., zinc and iron) it is the toxicity of manganese (Mn) that is more common in human populations than its deficiency. Data suggest alterations in dopamine biology may drive the effects associated with Mn neurotoxicity, though recently γ-aminobutyric acid (GABA) has been implicated. In addition, iron deficiency (ID), a common nutritional problem, may cause disturbances in neurochemistry by facilitating accumulation of Mn in the brain. Previous data from our lab have shown decreased brain tissue levels of GABA as well as decreased 3H-GABA uptake in synaptosomes as a result of Mn exposure and ID. These results indicate a possible increase in the concentration of extracellular GABA due to alterations in expression of GABA transport and receptor proteins. In this study weanling-male Sprague-Dawley rats were randomly placed into one of four dietary treatment groups: control (CN; 35 mg Fe/kg diet), iron-deficient (ID; 6 mg Fe/kg diet), CN with Mn supplementation (via the drinking water; 1 g Mn/L) (CNMn), and ID with Mn supplementation (IDMn). Using in vivo microdialysis, an increase in extracellular GABA concentrations in the striatum was observed in response to Mn exposure and ID although correlational analysis reveals that extracellular GABA is related more to extracellular iron levels and not Mn. A diverse effect of Mn exposure and ID was observed in the regions examined via Western blot and RT-PCR analysis, with effects on mRNA and protein expression of GAT-1, GABAA, and GABAB differing between and within the regions examined. For example, Mn exposure reduced GAT-1 protein expression by approximately 50% in the substantia nigra, while increasing mRNA expression approximately four-fold, while in the caudate putamen mRNA expression was decreased with no effect on protein expression. These data suggest that Mn exposure results in an increase in extracellular GABA concentrations via altered expression of transport and receptor

  12. Rotation of Subunits During Catalysis by Escherichia coli F_1-ATPase

    NASA Astrophysics Data System (ADS)

    Duncan, Thomas M.; Bulygin, Vladimir V.; Zhou, Yuantai; Hutcheon, Marcus L.; Cross, Richard L.

    1995-11-01

    During oxidative and photo-phosphorylation, F_0F_1-ATP synthases couple the movement of protons down an electrochemical gradient to the synthesis of ATP. One proposed mechanistic feature that has remained speculative is that this coupling process requires the rotation of subunits within F_0F_1. Guided by a recent, high-resolution structure for bovine F_1 [Abrahams, J. P., Leslie, A. G., Lutter, R. & Walker, J. E. (1994) Nature (London) 370, 621-628], we have developed a critical test for rotation of the central γ subunit relative to the three catalytic β subunits in soluble F_1 from Escherichia coli. In the bovine F_1 structure, a specific point of contact between the γ subunit and one of the three catalytic β subunits includes positioning of the homolog of E. coli γ-subunit C87 (γC87) close to the β-subunit 380DELSEED386 sequence. A βD380C mutation allowed us to induce formation of a specific disulfide bond between β and γC87 in soluble E. coli F_1. Formation of the crosslink inactivated βD380C-F_1, and reduction restored full activity. Using a dissociation/reassembly approach with crosslinked βD380C-F_1, we incorporated radiolabeled β subunits into the two noncrosslinked β-subunit positions of F_1. After reduction of the initial nonradio-active β-γ crosslink, only exposure to conditions for catalytic turnover results in similar reactivities of unlabeled and radiolabeled β subunits with γC87 upon reoxidation. The results demonstrate that γ subunit rotates relative to the β subunits during catalysis.

  13. The Guanine Nucleotide Exchange Factor Tiam1 Affects Neuronal Morphology; Opposing Roles for the Small GTPases Rac and Rho

    PubMed Central

    van Leeuwen, Frank N.; Kain, Hendrie E.T.; van der Kammen, Rob A.; Michiels, Frits; Kranenburg, Onno W.; Collard, John G.

    1997-01-01

    The invasion-inducing T-lymphoma invasion and metastasis 1 (Tiam1) protein functions as a guanine nucleotide exchange factor (GEF) for the small GTPase Rac1. Differentiation-dependent expression of Tiam1 in the developing brain suggests a role for this GEF and its effector Rac1 in the control of neuronal morphology. Here we show that overexpression of Tiam1 induces cell spreading and affects neurite outgrowth in N1E-115 neuroblastoma cells. These effects are Rac-dependent and strongly promoted by laminin. Overexpression of Tiam1 recruits the α6β1 integrin, a laminin receptor, to specific adhesive contacts at the cell periphery, which are different from focal contacts. Cells overexpressing Tiam1 no longer respond to lysophosphatidic acid– induced neurite retraction and cell rounding, processes mediated by Rho, suggesting that Tiam1-induced activation of Rac antagonizes Rho signaling. This inhibition can be overcome by coexpression of constitutively active RhoA, which may indicate that regulation occurs at the level of Rho or upstream. Conversely, neurite formation induced by Tiam1 or Rac1 is further promoted by inactivating Rho. These results demonstrate that Rac- and Rho-mediated pathways oppose each other during neurite formation and that a balance between these pathways determines neuronal morphology. Furthermore, our data underscore the potential role of Tiam1 as a specific regulator of Rac during neurite formation and illustrate the importance of reciprocal interactions between the cytoskeleton and the extracellular matrix during this process. PMID:9348295

  14. Immunoreactivity for GABA, GAD65, GAD67 and Bestrophin-1 in the meninges and the choroid plexus: implications for non-neuronal sources for GABA in the developing mouse brain.

    PubMed

    Tochitani, Shiro; Kondo, Shigeaki

    2013-01-01

    Neural progenitors in the developing neocortex, neuroepithelial cells and radial glial cells, have a bipolar shape with a basal process contacting the basal membrane of the meninge and an apical plasma membrane facing the lateral ventricle, which the cerebrospinal fluid is filled with. Recent studies revealed that the meninges and the cerebrospinal fluid have certain roles to regulate brain development. γ-aminobutyric acid (GABA) is a neurotransmitter which appears first during development and works as a diffusible factor to regulate the properties of neural progenitors. In this study, we examined whether GABA can be released from the meninges and the choroid plexus in the developing mouse brain. Immunohistochemical analyses showed that glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67), both of which are GABA-synthesizing enzymes, are expressed in the meninges. The epithelial cells in the choroid plexus express GAD65. GABA immunoreactivity could be observed beneath the basal membrane of the meninge and in the epithelial cells of the choroid plexus. Expression analyses on Bestrophin-1, which is known as a GABA-permeable channel in differentiated glial cells, suggested that the cells in the meninges and the epithelial cells in the choroid plexus have the channels able to permeate non-synaptic GABA into the extracellular space. Further studies showed that GAD65/67-expressing meningeal cells appear in a manner with rostral to caudal and lateral to dorsal gradient to cover the entire neocortex by E14.5 during development, while the cells in the choroid plexus in the lateral ventricle start to express GAD65 on E11-E12, the time when the choroid plexus starts to develop in the developing brain. These results totally suggest that the meninges and the choroid plexus can work as non-neuronal sources for ambient GABA which can modulate the properties of neural progenitors during neocortical development.

  15. Ester to amide substitution improves selectivity, efficacy and kinetic behavior of a benzodiazepine positive modulator of GABAA receptors containing the α5 subunit.

    PubMed

    Stamenić, Tamara Timić; Poe, Michael M; Rehman, Sabah; Santrač, Anja; Divović, Branka; Scholze, Petra; Ernst, Margot; Cook, James M; Savić, Miroslav M

    2016-11-15

    We have synthesized and characterized MP-III-022 ((R)-8-ethynyl-6-(2-fluorophenyl)-N,4-dimethyl-4H-benzo[f]imidazo[1,5-a][1,4]diazepine-3-carboxamide) in vitro and in vivo as a binding- and efficacy-selective positive allosteric modulator of GABA A receptors containing the α5 subunit (α5GABA A Rs). By approximation of the electrophysiological responses which the estimated free rat brain concentrations can induce, we demonstrated that convenient systemic administration of MP-III-022 in the dose range 1-10mg/kg may result in a selective potentiation, over a wide range from mild to moderate to strong, of α5βγ2 GABA A receptors. For eliciting a comparable range of potentiation, the widely studied parent ligand SH-053-2'F-R-CH3 containing an ester moiety needs to be administered over a much wider dose range (10-200mg/kg), but at the price of activating non-α5 GABA A Rs as well as the desired α5GABA A Rs at the highest dose. At the dose of 10mg/kg, which elicits a strong positive modulation of α5GABA A Rs, MP-III-022 caused mild, but significant muscle relaxation, while at doses 1-10mg/kg was devoid of ataxia, sedation or an influence on the anxiety level, characteristic for non-selective benzodiazepines. As an amide compound with improved stability and kinetic properties, MP-III-022 may represent an optimized tool to study the influence of α5GABA A Rs on the neuronal pathways related to CNS disorders such as schizophrenia, Alzheimer's disease, Down syndrome or autism. Copyright © 2016 Elsevier B.V. All rights reserved.

  16. Novel GABA receptor pesticide targets.

    PubMed

    Casida, John E; Durkin, Kathleen A

    2015-06-01

    The γ-aminobutyric acid (GABA) receptor has four distinct but overlapping and coupled targets of pesticide action importantly associated with little or no cross-resistance. The target sites are differentiated by binding assays with specific radioligands, resistant strains, site-directed mutagenesis and molecular modeling. Three of the targets are for non-competitive antagonists (NCAs) or channel blockers of widely varied chemotypes. The target of the first generation (20th century) NCAs differs between the larger or elongated compounds (NCA-IA) including many important insecticides of the past (cyclodienes and polychlorocycloalkanes) or present (fiproles) and the smaller or compact compounds (NCA-IB) highly toxic to mammals and known as cage convulsants, rodenticides or chemical threat agents. The target of greatest current interest is designated NCA-II for the second generation (21st century) of NCAs consisting for now of isoxazolines and meta-diamides. This new and uniquely different NCA-II site apparently differs enough between insects and mammals to confer selective toxicity. The fourth target is the avermectin site (AVE) for allosteric modulators of the chloride channel. NCA pesticides vary in molecular surface area and solvent accessible volume relative to avermectin with NCA-IBs at 20-22%, NCA-IAs at 40-45% and NCA-IIs at 57-60%. The same type of relationship relative to ligand-docked length is 27-43% for NCA-IBs, 63-71% for NCA-IAs and 85-105% for NCA-IIs. The four targets are compared by molecular modeling for the Drosophila melanogaster GABA-R. The principal sites of interaction are proposed to be: pore V1' and A2' for NCA-IB compounds; pore A2', L6' and T9' for NCA-IA compounds; pore T9' to S15' in proximity to M1/M3 subunit interface (or alternatively an interstitial site) for NCA-II compounds; and M1/M3, M2 interfaces for AVE. Understanding the relationships of these four binding sites is important in resistance management and in the discovery and use

  17. RCP-driven α5β1 recycling suppresses Rac and promotes RhoA activity via the RacGAP1-IQGAP1 complex.

    PubMed

    Jacquemet, Guillaume; Green, David M; Bridgewater, Rebecca E; von Kriegsheim, Alexander; Humphries, Martin J; Norman, Jim C; Caswell, Patrick T

    2013-09-16

    Inhibition of αvβ3 or expression of mutant p53 promotes invasion into fibronectin (FN)-containing extracellular matrix (ECM) by enhancing Rab-coupling protein (RCP)-dependent recycling of α5β1 integrin. RCP and α5β1 cooperatively recruit receptor tyrosine kinases, including EGFR1, to regulate their trafficking and downstream signaling via protein kinase B (PKB)/Akt, which, in turn, promotes invasive migration. In this paper, we identify a novel PKB/Akt substrate, RacGAP1, which is phosphorylated as a consequence of RCP-dependent α5β1 trafficking. Phosphorylation of RacGAP1 promotes its recruitment to IQGAP1 at the tips of invasive pseudopods, and RacGAP1 then locally suppresses the activity of the cytoskeletal regulator Rac and promotes the activity of RhoA in this subcellular region. This Rac to RhoA switch promotes the extension of pseudopodial processes and invasive migration into FN-containing matrices, in a RhoA-dependent manner. Thus, the localized endocytic trafficking of α5β1 within the tips of invasive pseudopods elicits signals that promote the reorganization of the actin cytoskeleton, protrusion, and invasion into FN-rich ECM.

  18. Tetramethylpyrazine Protects Against Oxygen-Glucose Deprivation-Induced Brain Microvascular Endothelial Cells Injury via Rho/Rho-kinase Signaling Pathway.

    PubMed

    Yang, Guang; Qian, Chen; Wang, Ning; Lin, Chenyu; Wang, Yan; Wang, Guangyun; Piao, Xinxin

    2017-05-01

    Tetramethylpyrazine (TMP, also known as Ligustrazine), which is isolated from Chinese Herb Medicine Ligustium wollichii Franchat (Chuan Xiong), has been widely used in China for the treatment of ischemic stroke by Chinese herbalists. Brain microvascular endothelial cells (BMECs) are the integral parts of the blood-brain barrier (BBB), protecting BMECs against oxygen-glucose deprivation (OGD) which is important for the treatment of ischemic stroke. Here, we investigated the protective mechanisms of TMP, focusing on OGD-injured BMECs and the Rho/Rho-kinase (Rho-associated kinases, ROCK) signaling pathway. The model of OGD-injured BMECs was established in this study. BMECs were identified by von Willebrand factor III staining and exposed to fasudil, or TMP at different concentrations (14.3, 28.6, 57.3 µM) for 2 h before 24 h of OGD injury. The effect of each treatment was examined by cell viability assays, measurement of intracellular reactive oxygen species (ROS), and transendothelial electric resistance and western blot analysis (caspase-3, endothelial nitric oxide synthase (eNOS), RhoA, Rac1). Our results show that TMP significantly attenuated apoptosis and the permeability of BMECs induced by OGD. In addition, TMP could notably down-regulate the characteristic proteins in Rho/ROCK signaling pathway such as RhoA and Rac1, which triggered abnormal changes of eNOS and ROS, respectively. Altogether, our results show that TMP has a strong protective effect against OGD-induced BMECs injury and suggest that the mechanism might be related to the inhibition of the Rho/ROCK signaling pathway.

  19. Evidence for B{sup 0}{yields}{rho}{sup 0}{rho}{sup 0} Decays and Implications for the Cabibbo-Kobayashi-Maskawa Angle {alpha}

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Aubert, B.; Bona, M.; Boutigny, D.

    2007-03-16

    We search for the decays B{sup 0}{yields}{rho}{sup 0}{rho}{sup 0}, B{sup 0}{yields}{rho}{sup 0}f{sub 0}(980), and B{sup 0}{yields}f{sub 0}(980)f{sub 0}(980) in a sample of about 384x10{sup 6} {upsilon}(4S){yields}BB decays collected with the BABAR detector at the PEP-II asymmetric-energy e{sup +}e{sup -} collider at Stanford Linear Accelerator Center. We find evidence for B{sup 0}{yields}{rho}{sup 0}{rho}{sup 0} with 3.5{sigma} significance and measure the branching fraction B=(1.07{+-}0.33{+-}0.19)x10{sup -6} and longitudinal polarization fraction f{sub L}=0.87{+-}0.13{+-}0.04, where the first uncertainty is statistical, and the second is systematic. The uncertainty on the Cabibbo-Kobayashi-Maskawa quark-mixing matrix unitarity angle {alpha} due to penguin contributions in B{yields}{rho}{rho} decays is 18 deg.more » at the 1{sigma} level. We also set upper limits on the B{sup 0}{yields}{rho}{sup 0}f{sub 0}(980) and B{sup 0}{yields}f{sub 0}(980)f{sub 0}(980) decay rates.« less

  20. P311 Accelerates Skin Wound Reepithelialization by Promoting Epidermal Stem Cell Migration Through RhoA and Rac1 Activation.

    PubMed

    Yao, Zhihui; Li, Haisheng; He, Weifeng; Yang, Sisi; Zhang, Xiaorong; Zhan, Rixing; Xu, Rui; Tan, Jianglin; Zhou, Junyi; Wu, Jun; Luo, Gaoxing

    2017-03-15

    P311 is a newly discovered functional gene, and it has been proved to play a key role in blood pressure homeostasis, glioblastoma invasion, renal fibrosis, hypertrophic scar formation, and others. In this study, for the first time, we found that P311 could enhance reepithelialization during wound healing via promoting epidermal stem cell (EpSC) migration through Rho GTPases. P311 expression was highly increased in neo-epidermal cells during human and mouse skin wound healing, and P311was co-localized with 5-bromo-2'-deoxyuridine positive label-retaining cells in a mouse superficial second-degree burn wound model. Furthermore, transfection of human EpSCs with adenovirus encoding P311 significantly accelerated the cell migration in vitro. Moreover, highly expressed P311 could enhance the activities of the Rho GTPases (RhoA, Rac1, and Cdc42) in cultured human EpSCs. P311-knockout mouse EpSCs showed dramatically decreased cell migration and activities of Rho GTPases (RhoA, Rac1, and Cdc42). Besides, both the RhoA-specific inhibitor and the Rac1 inhibitor, not the Cdc42 inhibitor, could significantly suppress P311-induced human EpSC migration. In vivo, the reepithelialization was markedly impaired during wound healing after P311 was knocked out. Together, our results suggested that P311 could accelerate skin wound reepithelialization by promoting the migration of EpSCs through RhoA and Rac1 activation. P311 could serve as a novel target for regulation of EpSC migration during cutaneous wound healing.

  1. Elevating Endogenous GABA Levels with GAT-1 Blockade Modulates Evoked but Not Induced Responses in Human Visual Cortex

    PubMed Central

    Muthukumaraswamy, Suresh D; Myers, Jim F M; Wilson, Sue J; Nutt, David J; Hamandi, Khalid; Lingford-Hughes, Anne; Singh, Krish D

    2013-01-01

    The electroencephalographic/magnetoencephalographic (EEG/MEG) signal is generated primarily by the summation of the postsynaptic currents of cortical principal cells. At a microcircuit level, these glutamatergic principal cells are reciprocally connected to GABAergic interneurons. Here we investigated the relative sensitivity of visual evoked and induced responses to altered levels of endogenous GABAergic inhibition. To do this, we pharmacologically manipulated the GABA system using tiagabine, which blocks the synaptic GABA transporter 1, and so increases endogenous GABA levels. In a single-blinded and placebo-controlled crossover study of 15 healthy participants, we administered either 15 mg of tiagabine or a placebo. We recorded whole-head MEG, while participants viewed a visual grating stimulus, before, 1, 3 and 5 h post tiagabine ingestion. Using beamformer source localization, we reconstructed responses from early visual cortices. Our results showed no change in either stimulus-induced gamma-band amplitude increases or stimulus-induced alpha amplitude decreases. However, the same data showed a 45% reduction in the evoked response component at ∼80 ms. These data demonstrate that, in early visual cortex the evoked response shows a greater sensitivity compared with induced oscillations to pharmacologically increased endogenous GABA levels. We suggest that previous studies correlating GABA concentrations as measured by magnetic resonance spectroscopy to gamma oscillation frequency may reflect underlying variations such as interneuron/inhibitory synapse density rather than functional synaptic GABA concentrations. PMID:23361120

  2. Regulation of Local Ambient GABA Levels via Transporter-Mediated GABA Import and Export for Subliminal Learning.

    PubMed

    Hoshino, Osamu

    2015-06-01

    Perception of supraliminal stimuli might in general be reflected in bursts of action potentials (spikes), and their memory traces could be formed through spike-timing-dependent plasticity (STDP). Memory traces for subliminal stimuli might be formed in a different manner, because subliminal stimulation evokes a fraction (but not a burst) of spikes. Simulations of a cortical neural network model showed that a subliminal stimulus that was too brief (10 msec) to perceive transiently (more than about 500 msec) depolarized stimulus-relevant principal cells and hyperpolarized stimulus-irrelevant principal cells in a subthreshold manner. This led to a small increase or decrease in ongoing-spontaneous spiking activity frequency (less than 1 Hz). Synaptic modification based on STDP during this period effectively enhanced relevant synaptic weights, by which subliminal learning was improved. GABA transporters on GABAergic interneurons modulated local levels of ambient GABA. Ambient GABA molecules acted on extrasynaptic receptors, provided principal cells with tonic inhibitory currents, and contributed to achieving the subthreshold neuronal state. We suggest that ongoing-spontaneous synaptic alteration through STDP following subliminal stimulation may be a possible neuronal mechanism for leaving its memory trace in cortical circuitry. Regulation of local ambient GABA levels by transporter-mediated GABA import and export may be crucial for subliminal learning.

  3. Rho/Rho kinase and phosphoinositide 3-kinase are parallel pathways in the development of spontaneous arterial tone in deoxycorticosterone acetate-salt hypertension.

    PubMed

    Wehrwein, Erica A; Northcott, Carrie A; Loberg, Robert D; Watts, Stephanie W

    2004-06-01

    Hypertension is characterized by abnormal vascular contractility and function. Arteries from deoxycorticosterone acetate (DOCA)-salt hypertensive rats develop spontaneous tone that is not observed in arteries from normotensive rats. Inhibition of phosphoinositide 3-kinase (PI3-kinase) by 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002) reduces spontaneous tone development. The Rho/Rho-kinase pathway has been suggested to play a role in hypertension and may be dependent on PI3-kinase activity. We hypothesized that Rhokinase is involved in spontaneous tone development and that Rho/Rho-kinase is a downstream effector of PI3-kinase. Using endothelium-denuded aortic strips in isolated tissue bath, we demonstrated that (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) (Y27632) (1 microM), a Rho-kinase inhibitor, significantly reduced spontaneous tone in the DOCA aorta but that it did not affect sham aorta basal tone (DOCA 63.5 +/- 15.9 versus sham 1.2 +/- 0.4 total change in percentage of phenylephrine contraction). We examined the interaction between the PI3-kinase and Rho pathways by observing the effects of LY294002 on a Rhokinase effector, myosin phosphatase (MYPT), and Y27632 on a PI3-kinase effector, Akt, using Western blot analysis. Inhibition of PI3-kinase reduced spontaneous tone, but it had no effect on the phosphorylation status of MYPT, indicating that PI3-kinase is not a downstream effector of Rho/Rho-kinase. These data indicate that there is little interaction between the Rho/Rhokinase and PI3-kinase pathways in the DOCA-salt aorta, and the two pathways seem to operate in parallel in supporting spontaneous arterial tone. These data reflect spontaneous tone only and do not rule out the possibility of interaction between these pathways in agonist-stimulated tone.

  4. Up-regulation of the RhoA/Rho-kinase signaling pathway in corpus cavernosum from endothelial nitric-oxide synthase (NOS), but not neuronal NOS, null mice.

    PubMed

    Priviero, Fernanda B M; Jin, Li-Ming; Ying, Zhekang; Teixeira, Cleber E; Webb, R Clinton

    2010-04-01

    We tested the hypothesis that the basal release of nitric oxide (NO) from endothelial cells modulates contractile activity in the corpus cavernosum (CC) via inhibition of the RhoA/Rho-kinase signaling pathway. Cavernosal strips from wild-type (WT), endothelial nitric-oxide synthase knockout [eNOS(-/-)], and neuronal nitric-oxide synthase knockout [nNOS(-/-)] mice were mounted in myographs, and isometric force was recorded. mRNA and protein expression of key molecules in the RhoA/Rho-kinase pathway were analyzed by real-time polymerase chain reaction and Western blot, respectively. The cGMP levels were determined. The Rho-kinase inhibitors (R)-(+)-trans-N-(4-pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide (Y-27632) and (S)-(+)-2-methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl] homopiperazine (H-1152) reduced cavernosal contractions evoked by phenylephrine or electrical field stimulation (EFS) in a concentration-dependent manner, although this inhibition was less effective in tissues from eNOS(-/-) mice. Y-27632 enhanced relaxations induced by sodium nitroprusside, EFS, and NO (administered as acidified NaNO2) without affecting the cGMP content of the cavernosal strips. This enhancement was less prominent in CC from eNOS(-/-). The protein expression of RhoA, Rho-guanine dissociation inhibitor, and Rho-kinase beta did not differ among the strains. However, in eNOS(-/-) CC, the protein expression of Rho-kinase alpha and both mRNA and protein expression of p115-Rho-associated guanine exchange factor (RhoGEF), PDZ-RhoGEF, and leukemia-associated RhoGEF were up-regulated. Phosphorylation of MYPT1 at Thr696 was higher in tissues from eNOS(-/-) mice. A high concentration of Y-27632 significantly enhanced NO release in CC stimulated by EFS. These results suggest a basal release of NO from endothelial cells, which inhibits contractions mediated by the RhoA/Rho-kinase pathway and modulates the expression of proteins related to this pathway in mouse CC. It indicates that

  5. GABBR1 and SLC6A1, Two Genes Involved in Modulation of GABA Synaptic Transmission, Influence Risk for Alcoholism: Results from Three Ethnically Diverse Populations.

    PubMed

    Enoch, Mary-Anne; Hodgkinson, Colin A; Shen, Pei-Hong; Gorodetsky, Elena; Marietta, Cheryl A; Roy, Alec; Goldman, David

    2016-01-01

    Animal and human studies indicate that GABBR1, encoding the GABAB1 receptor subunit, and SLC6A1, encoding the neuronal gamma-aminobutyric acid (GABA) transporter GAT1, play a role in addiction by modulating synaptic GABA. Therefore, variants in these genes might predict risk/resilience for alcoholism. This study included 3 populations that differed by ethnicity and alcoholism phenotype: African American (AA) men: 401 treatment-seeking inpatients with single/comorbid diagnoses of alcohol and drug dependence, 193 controls; Finnish Caucasian men: 159 incarcerated alcoholics, half with comorbid antisocial personality disorder, 181 controls; and a community sample of Plains Indian (PI) men and women: 239 alcoholics, 178 controls. Seven GABBR1 tag single nucleotide polymorphisms were genotyped in the AA and Finnish samples; rs29220 was genotyped in the PI for replication. Also, a uniquely African, functional SLC6A1 insertion promoter polymorphism (IND) was genotyped in the AAs. We found a significant and congruent association between GABBR1 rs29220 and alcoholism in all 3 populations. The major genotype (heterozygotes in AAs, Finns) and the major allele in PIs were significantly more common in alcoholics. Moreover, SLC6A1 IND was more abundant in controls, that is, the major genotype predicted alcoholism. An analysis of combined GABBR1 rs29220 and SLC6A1 IND genotypes showed that rs29220 heterozygotes, irrespective of their IND status, had an increased risk for alcoholism, whereas carriers of the IND allele and either rs29220 homozygote were more resilient. Our results show that with both GABBR1 and SLC6A1, the minor genotypes/alleles were protective against risk for alcoholism. Finally, GABBR1 rs29220 might predict treatment response/adverse effects for baclofen, a GABAB receptor agonist. Copyright © 2016 by the Research Society on Alcoholism. This article has been contributed to by US Government employees and their work is in the public domain in the USA.

  6. Hydrostatic pressure promotes the proliferation and osteogenic/chondrogenic differentiation of mesenchymal stem cells: The roles of RhoA and Rac1.

    PubMed

    Zhao, Yin-Hua; Lv, Xin; Liu, Yan-Li; Zhao, Ying; Li, Qiang; Chen, Yong-Jin; Zhang, Min

    2015-05-01

    Our previous studies have shown that hydrostatic pressure can serve as an active regulator for bone marrow mesenchymal stem cells (BMSCs). The current work further investigates the roles of cytoskeletal regulatory proteins Ras homolog gene family member A (RhoA) and Ras-related C3 botulinum toxin substrate 1 (Rac1) in hydrostatic pressure-related effects on BMSCs. Flow cytometry assays showed that the hydrostatic pressure promoted cell cycle initiation in a RhoA- and Rac1-dependent manner. Furthermore, fluorescence assays confirmed that RhoA played a positive and Rac1 displayed a negative role in the hydrostatic pressure-induced F-actin stress fiber assembly. Western blots suggested that RhoA and Rac1 play central roles in the pressure-inhibited ERK phosphorylation, and Rac1 but not RhoA was involved in the pressure-promoted JNK phosphorylation. Finally, real-time polymerase chain reaction (PCR) experiments showed that pressure promoted the expression of osteogenic marker genes in BMSCs at an early stage of osteogenic differentiation through the up-regulation of RhoA activity. Additionally, the PCR results showed that pressure enhanced the expression of chondrogenic marker genes in BMSCs during chondrogenic differentiation via the up-regulation of Rac1 activity. Collectively, our results suggested that RhoA and Rac1 are critical to the pressure-induced proliferation and differentiation, the stress fiber assembly, and MAPK activation in BMSCs. Copyright © 2015. Published by Elsevier B.V.

  7. NET1 and HFI1 genes of yeast mediate both chromosome maintenance and mitochondrial rho(-) mutagenesis.

    PubMed

    Koltovaya, N A; Guerasimova, A S; Tchekhouta, I A; Devin, A B

    2003-08-01

    An increase in the mitochondrial rho(-) mutagenesis is a well-known response of yeast cells to mutations in numerous nuclear genes as well as to various kinds of stress. Despite extensive studies for several decades, the biological significance of this response is still not fully understood. The genetic approach to solving this enigma includes a study of genes that are required for the high incidence of spontaneous rho(-) mutants. We have obtained mutations of a few nuclear genes of that sort and found that mutations in certain genes, including CDC28, the central cell-cycle regulation gene, result in a decrease in spontaneous rho(-) mutability and simultaneously affect the maintenance of the yeast chromosomes and plasmids. Two more genes resembling CDC28 in this respect are identified in the present work as a result of the characterization of four new mutants. These two genes are NET1 and HFI1 which mediate important regulatory protein-protein interactions in the yeast cell. The effects of four mutations, including net1-srm and hfi1-srm, on the maintenance of the yeast mitochondrial genome, chromosomes and plasmids, as well as on the cell's sensitivity to ionizing radiation, are also described. The data presented suggest that the pleiotropic srm mutations determining coordinate changes in the fidelity of mitotic transmission of chromosomes, plasmids and mtDNA molecules identify genes that most probably operate high up in the hierarchy of the general genetic regulation of yeast. Copyright 2003 John Wiley & Sons, Ltd.

  8. Alteration of glutamate/GABA balance during acute alcohol intoxication in rats: effect of Xingnaojing injection.

    PubMed

    Wei, Jingjing; Yao, Limei; Yang, Lei; Zhao, Wei; Shi, Si; Cai, Qingyan; Chen, Dingsheng; Li, Weirong; Wang, Qi

    2015-05-26

    Xingnaojing Injection (XNJI) is a modern Chinese formula came from famous Chinese medicine An Gong Niu Huang Pill. XNJI has been used for treatment of cerebral diseases and stroke in China, and is approved by the State Food and Drug Administration of China for the treatment of acute alcohol intoxication (AAI). XNJI belongs to the ethnopharmacological family of medicines. In this study, we investigated the mechanisms of the XNJI effect on AAI. To investigate the effects of XNJI on glutamate, gamma-aminobutyric acid (GABA) and related receptor in lateral hypothalamic area (LHA) of AAI rat. Adult male Sprague-Dawley rats were implanted with microdialysis probes in LHA. Rats were randomly divided into control, model, 1.36mg/kg XNJI, 0.68mg/kg XNJI and 0.34mg/kg XNJI groups. During microdialysis, baseline samples were collected from 1h to 2.5h; thereafter, the rats were given an intraperitoneal injection of 52% ethanol, 5.2g/kg, or saline for control group. Twenty minutes later, three doses of XNJI was given by unilateral injection respectively, while saline for control and model groups, and samples were collected for the next 4h. The extracellular glutamate and GABA levels were measured in the LHA by a high performance liquid chromatography coupled with fluorescence detector (HPLC-FLU). The expression levels of related receptors N-methyl-d-aspartate receptor (NR) subunit NR2A, NR2B and GABAA were analyzed by reverse transcription polymerase chain reaction (RT-PCR). Ethanol (5.2g/kg) significantly decreased the extracellular levels of glutamate and increased extracellular GABA in LHA. On the other hand ethanol significantly decreased NR2A and NR2B mRNAs expression, and increase GABAA mRNA expression. XNJI could increase the extracellular level of glutamate and decrease that of GABA; moreover, induced an increase in NR2A and NR2B mRNA expression, and a decrease in GABAA mRNA expression in LHA. The current changes in glutamate, GABA and mRNA expressions of related

  9. GABA pharmacology: the search for analgesics.

    PubMed

    McCarson, Kenneth E; Enna, S J

    2014-10-01

    Decades of research have been devoted to defining the role of GABAergic transmission in nociceptive processing. Much of this work was performed using rigid, orthosteric GABA analogs created by Povl Krogsgaard-Larsen and his associates. A relationship between GABA and pain is suggested by the anatomical distribution of GABA receptors and the ability of some GABA agonists to alter nociceptive responsiveness. Outlined in this report are data supporting this proposition, with particular emphasis on the anatomical localization and function of GABA-containing neurons and the molecular and pharmacological properties of GABAA and GABAB receptor subtypes. Reference is made to changes in overall GABAergic tone, GABA receptor expression and activity as a function of the duration and intensity of a painful stimulus or exposure to GABAergic agents. Evidence is presented that the plasticity of this receptor system may be responsible for the variability in the antinociceptive effectiveness of compounds that influence GABA transmission. These findings demonstrate that at least some types of persistent pain are associated with a regionally selective decline in GABAergic tone, highlighting the need for agents that enhance GABA activity in the affected regions without compromising GABA function over the long-term. As subtype selective positive allosteric modulators may accomplish these goals, such compounds might represent a new class of analgesic drugs.

  10. Assembly of the stator in Escherichia coli ATP synthase. Complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha

    PubMed Central

    Senior, Alan E.; Muharemagi, Alma; Wilke-Mounts, Susan

    2008-01-01

    Alpha subunit of Escherichia coli ATP synthase was expressed with a C-terminal 6-His tag and purified. Pure alpha was monomeric, competent in nucleotide binding, and had normal N-terminal sequence. In F1-subunit dissociation/reassociation experiments it supported full reconstitution of ATPase, and reassociated complexes were able to bind to F1-depleted membranes with restoration of ATP-driven proton pumping. Therefore interaction between the stator delta subunit and the N-terminal residue 1-22 region of alpha occurred normally when pure alpha was complexed with other F1 subunits. On the other hand, three different types of experiment showed that no interaction occurred between pure delta and isolated alpha subunit. Unlike in F1, the N-terminal region of isolated alpha was not susceptible to trypsin cleavage. Therefore, during assembly of ATP synthase, complexation of alpha subunit with other F1 subunits is prerequisite for delta subunit binding to the N-terminal region of alpha. We suggest that the N-terminal 1-22 residues of alpha are sequestered in isolated alpha until released by binding of beta to alpha subunit. This prevents 1/1 delta/alpha complexes from forming, and provides a satisfactory explanation of the stoichiometry of one delta per three alpha seen in the F1 sector of ATP synthase, assuming that steric hindrance prevents binding of more than one delta to the alpha3/beta3 hexagon. The cytoplasmic fragment of the b subunit (bsol) did not bind to isolated alpha. It might also be that complexation of alpha with beta subunits is prerequisite for direct binding of stator b subunit to the F1-sector. PMID:17176112

  11. MEDU-05. THE ROLE OF GABA METABOLISM IN MEDULLOBLASTOMA

    PubMed Central

    Martirosian, Vahan; Deshpande, Krutika; Shackelford, Gregory; Julian, Alex; Lin, Michelle; Erdreich-Epstein, Anat; Chen, Thomas; Neman, Josh

    2017-01-01

    Abstract BACKGROUND: Brain tumors are the most common cause of childhood oncological death, and medulloblastoma (originating in the cerebellum) is the most common malignant pediatric brain tumor. In the microenvironment of the brain, especially the cerebellum, variables related to GABA, the major inhibitory neurotransmitter in the nervous system, are particularly prominent. Abnormal GABAergic Receptor activation has been described in in aggressive MYC-driven Group 3 medulloblastoma. However these studies did not look at the metabolic contribution of GABA for the development of medulloblastomas. In addition to its role in neurotransmission through GABA receptor, GABA can act as a trophic factor during nervous system development to influence cellular events including proliferation, migration, differentiation, synapse maturation, and cell death. Under conditions that inhibit the tricarboxylic acid cycle (TCA), impair respiration, and enhance the accumulation of reactive oxygen intermediates, GABA can be used as an NADH energy source for growth through the GABA-shunt pathway regulators (ABAT, SSADH, GAT-1, GAT-3). Therefore, we hypothesize that blocking GABA-metabolic-shunt will lead to growth suppression and invasiveness of medulloblastoma in the cerebellar GABA-rich microenvironment. RESULTS: Our results show RNA microarray from patient medulloblastoma tissue have high expression of GABA-shunt regulators with ~3-fold increase in the expression of ABAT in MYC amplified versus non-amplified MYC tumors. When medulloblastomas were supplemented with GABA, there was a significant fold change in expression of GABA-shunt mediators and induction of large and stable tumor spheres with Epithelial-Mesenchymal Transition gene expression signature. We next investigated whether a novel perrilyl alcohol-based small molecule NEO216 targeted the GABA-shunt metabolic pathway. NEO216 administration significantly reduced GABA-mediated NADH levels, reversed EMT-profiling, leading to loss

  12. The GABA Hypothesis in Essential Tremor: Lights and Shadows.

    PubMed

    Gironell, Alexandre

    2014-01-01

    The gamma-aminobutyric acid (GABA) hypothesis in essential tremor (ET) implies a disturbance of the GABAergic system, especially involving the cerebellum. This review examines the evidence of the GABA hypothesis. The review is based on published data about GABA dysfunction in ET, taking into account studies on cerebrospinal fluid, pathology, electrophysiology, genetics, neuroimaging, experimental animal models, and human drug therapies. Findings from several studies support the GABA hypothesis in ET. The hypothesis follows four steps: 1) cerebellar neurodegeneration with Purkinje cell loss; 2) a decrease in GABA system activity in deep cerebellar neurons; 3) disinhibition in output deep cerebellar neurons with pacemaker activity; and 4) an increase in rhythmic activity of the thalamus and thalamo-cortical circuit, contributing to the generation of tremor. Doubts have been cast on this hypothesis, however, by the fact that it is based on relatively few works, controversial post-mortem findings, and negative genetic studies on the GABA system. Furthermore, GABAergic drug efficacy is low and some GABAergic drugs do not have antitremoric efficacy. The GABA hypothesis continues to be the most robust pathophysiological hypothesis to explain ET. There is light in all GABA hypothesis steps, but a number of shadows cannot be overlooked. We need more studies to clarify the neurodegenerative nature of the disease, to confirm the decrease of GABA activity in the cerebellum, and to test more therapies that enhance the GABA transmission specifically in the cerebellum area.

  13. Stiffness of γ subunit of F(1)-ATPase.

    PubMed

    Okuno, Daichi; Iino, Ryota; Noji, Hiroyuki

    2010-11-01

    F(1)-ATPase is a molecular motor in which the γ subunit rotates inside the α(3)β(3) ring upon adenosine triphosphate (ATP) hydrolysis. Recent works on single-molecule manipulation of F(1)-ATPase have shown that kinetic parameters such as the on-rate of ATP and the off-rate of adenosine diphosphate (ADP) strongly depend on the rotary angle of the γ subunit (Hirono-Hara et al. 2005; Iko et al. 2009). These findings provide important insight into how individual reaction steps release energy to power F(1) and also have implications regarding ATP synthesis and how reaction steps are reversed upon reverse rotation. An important issue regarding the angular dependence of kinetic parameters is that the angular position of a magnetic bead rotation probe could be larger than the actual position of the γ subunit due to the torsional elasticity of the system. In the present study, we assessed the stiffness of two different portions of F(1) from thermophilic Bacillus PS3: the internal part of the γ subunit embedded in the α(3)β(3) ring, and the complex of the external part of the γ subunit and the α(3)β(3) ring (and streptavidin and magnetic bead), by comparing rotational fluctuations before and after crosslinkage between the rotor and stator. The torsional stiffnesses of the internal and remaining parts were determined to be around 223 and 73 pNnm/radian, respectively. Based on these values, it was estimated that the actual angular position of the internal part of the γ subunit is one-fourth of the magnetic bead position upon stalling using an external magnetic field. The estimated elasticity also partially explains the accommodation of the intrinsic step size mismatch between F(o) and F(1)-ATPase.

  14. Glucose, Lactate, β-Hydroxybutyrate, Acetate, GABA, and Succinate as Substrates for Synthesis of Glutamate and GABA in the Glutamine-Glutamate/GABA Cycle.

    PubMed

    Hertz, Leif; Rothman, Douglas L

    2016-01-01

    The glutamine-glutamate/GABA cycle is an astrocytic-neuronal pathway transferring precursors for transmitter glutamate and GABA from astrocytes to neurons. In addition, the cycle carries released transmitter back to astrocytes, where a minor fraction (~25 %) is degraded (requiring a similar amount of resynthesis) and the remainder returned to the neurons for reuse. The flux in the cycle is intense, amounting to the same value as neuronal glucose utilization rate or 75-80 % of total cortical glucose consumption. This glucose:glutamate ratio is reduced when high amounts of β-hydroxybutyrate are present, but β-hydroxybutyrate can at most replace 60 % of glucose during awake brain function. The cycle is initiated by α-ketoglutarate production in astrocytes and its conversion via glutamate to glutamine which is released. A crucial reaction in the cycle is metabolism of glutamine after its accumulation in neurons. In glutamatergic neurons all generated glutamate enters the mitochondria and its exit to the cytosol occurs in a process resembling the malate-aspartate shuttle and therefore requiring concomitant pyruvate metabolism. In GABAergic neurons one half enters the mitochondria, whereas the other one half is released directly from the cytosol. A revised concept is proposed for the synthesis and metabolism of vesicular and nonvesicular GABA. It includes the well-established neuronal GABA reuptake, its metabolism, and use for resynthesis of vesicular GABA. In contrast, mitochondrial glutamate is by transamination to α-ketoglutarate and subsequent retransamination to releasable glutamate essential for the transaminations occurring during metabolism of accumulated GABA and subsequent resynthesis of vesicular GABA.

  15. K+ channel TASK-1 knockout mice show enhanced sensitivities to ataxic and hypnotic effects of GABA(A) receptor ligands.

    PubMed

    Linden, Anni-Maija; Aller, M Isabel; Leppä, Elli; Rosenberg, Per H; Wisden, William; Korpi, Esa R

    2008-10-01

    TASK two-pore-domain leak K(+) channels occur throughout the brain. However, TASK-1 and TASK-3 knockout (KO) mice have few neurological impairments and only mildly reduced sensitivities to inhalational anesthetics, contrasting with the anticipated functions and importance of these channels. TASK-1/-3 channel expression can compensate for the absence of GABA(A) receptors in GABA(A) alpha6 KO mice. To investigate the converse, we analyzed the behavior of TASK-1 and -3 KO mice after administering drugs with preferential efficacies at GABA(A) receptor subtypes: benzodiazepines (diazepam and flurazepam, active at alpha1betagamma2, alpha2betagamma2, alpha3betagamma2, and alpha5betagamma2 subtypes), zolpidem (alpha1betagamma2 subtype), propofol (beta2-3-containing receptors), gaboxadol (alpha4betadelta and alpha6betadelta subtypes), pregnanolone, and pentobarbital (many subtypes). TASK-1 KO mice showed increased motor impairment in rotarod and beam-walking tests after diazepam and flurazepam administration but not after zolpidem. They also showed prolonged loss of righting reflex induced by propofol and pentobarbital. Autoradiography indicated no change in GABA(A) receptor ligand binding levels. These altered behavioral responses to GABAergic drugs suggest functional up-regulation of alpha2beta2/3gamma2 and alpha3beta2/3gamma2 receptor subtypes in TASK-1 KO mice. In addition, female, but not male, TASK-1 KO mice were more sensitive to gaboxadol, suggesting an increased influence of alpha4betadelta or alpha6betadelta subtypes. The benzodiazepine sensitivity of TASK-3 KO mice was marginally increased. Our results underline that TASK-1 channels perform such key functions in the brain that compensation is needed for their absence. Furthermore, because inhalation anesthetics act partially through GABA(A) receptors, the up-regulation of GABA(A) receptor function in TASK-1 KO mice might mask TASK-1 channel's significance as a target for inhalation anesthetics.

  16. Decreasing the Expression of GABAA α5 Subunit-Containing Receptors Partially Improves Cognitive, Electrophysiological, and Morphological Hippocampal Defects in the Ts65Dn Model of Down Syndrome.

    PubMed

    Vidal, Verónica; García-Cerro, Susana; Martínez, Paula; Corrales, Andrea; Lantigua, Sara; Vidal, Rebeca; Rueda, Noemí; Ozmen, Laurence; Hernández, Maria-Clemencia; Martínez-Cué, Carmen

    2018-06-01

    Trisomy 21 or Down syndrome (DS) is the most common cause of intellectual disability of a genetic origin. The Ts65Dn (TS) mouse, which is the most commonly used and best-characterized mouse model of DS, displays many of the cognitive, neuromorphological, and biochemical anomalies that are found in the human condition. One of the mechanisms that have been proposed to be responsible for the cognitive deficits in this mouse model is impaired GABA-mediated inhibition. Because of the well-known modulatory role of GABA A α5 subunit-containing receptors in cognitive processes, these receptors are considered to be potential targets for improving the intellectual disability in DS. The chronic administration of GABA A α5-negative allosteric modulators has been shown to be procognitive without anxiogenic or proconvulsant side effects. In the present study, we use a genetic approach to evaluate the contribution of GABA A α5 subunit-containing receptors to the cognitive, electrophysiological, and neuromorphological deficits in TS mice. We show that reducing the expression of GABA A α5 receptors by deleting one or two copies of the Gabra5 gene in TS mice partially ameliorated the cognitive impairments, improved long-term potentiation, enhanced neural differentiation and maturation, and normalized the density of the GABAergic synapse markers. Reducing the gene dosage of Gabra5 in TS mice did not induce motor alterations and anxiety or affect the viability of the mice. Our results provide further evidence of the role of GABA A α5 receptor-mediated inhibition in cognitive impairment in the TS mouse model of DS.

  17. Interaction of p190A RhoGAP with eIF3A and Other Translation Preinitiation Factors Suggests a Role in Protein Biosynthesis.

    PubMed

    Parasuraman, Prasanna; Mulligan, Peter; Walker, James A; Li, Bihua; Boukhali, Myriam; Haas, Wilhelm; Bernards, Andre

    2017-02-17

    The negative regulator of Rho family GTPases, p190A RhoGAP, is one of six mammalian proteins harboring so-called FF motifs. To explore the function of these and other p190A segments, we identified interacting proteins by tandem mass spectrometry. Here we report that endogenous human p190A, but not its 50% identical p190B paralog, associates with all 13 eIF3 subunits and several other translational preinitiation factors. The interaction involves the first FF motif of p190A and the winged helix/PCI domain of eIF3A, is enhanced by serum stimulation and reduced by phosphatase treatment. The p190A/eIF3A interaction is unaffected by mutating phosphorylated p190A-Tyr 308 , but disrupted by a S296A mutation, targeting the only other known phosphorylated residue in the first FF domain. The p190A-eIF3 complex is distinct from eIF3 complexes containing S6K1 or mammalian target of rapamycin (mTOR), and appears to represent an incomplete preinitiation complex lacking several subunits. Based on these findings we propose that p190A may affect protein translation by controlling the assembly of functional preinitiation complexes. Whether such a role helps to explain why, unique among the large family of RhoGAPs, p190A exhibits a significantly increased mutation rate in cancer remains to be determined. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Immunoreactivity for GABA, GAD65, GAD67 and Bestrophin-1 in the Meninges and the Choroid Plexus: Implications for Non-Neuronal Sources for GABA in the Developing Mouse Brain

    PubMed Central

    Tochitani, Shiro; Kondo, Shigeaki

    2013-01-01

    Neural progenitors in the developing neocortex, neuroepithelial cells and radial glial cells, have a bipolar shape with a basal process contacting the basal membrane of the meninge and an apical plasma membrane facing the lateral ventricle, which the cerebrospinal fluid is filled with. Recent studies revealed that the meninges and the cerebrospinal fluid have certain roles to regulate brain development. γ-aminobutyric acid (GABA) is a neurotransmitter which appears first during development and works as a diffusible factor to regulate the properties of neural progenitors. In this study, we examined whether GABA can be released from the meninges and the choroid plexus in the developing mouse brain. Immunohistochemical analyses showed that glutamic acid decarboxylase 65 and 67 (GAD65 and GAD67), both of which are GABA-synthesizing enzymes, are expressed in the meninges. The epithelial cells in the choroid plexus express GAD65. GABA immunoreactivity could be observed beneath the basal membrane of the meninge and in the epithelial cells of the choroid plexus. Expression analyses on Bestrophin-1, which is known as a GABA-permeable channel in differentiated glial cells, suggested that the cells in the meninges and the epithelial cells in the choroid plexus have the channels able to permeate non-synaptic GABA into the extracellular space. Further studies showed that GAD65/67-expressing meningeal cells appear in a manner with rostral to caudal and lateral to dorsal gradient to cover the entire neocortex by E14.5 during development, while the cells in the choroid plexus in the lateral ventricle start to express GAD65 on E11–E12, the time when the choroid plexus starts to develop in the developing brain. These results totally suggest that the meninges and the choroid plexus can work as non-neuronal sources for ambient GABA which can modulate the properties of neural progenitors during neocortical development. PMID:23437266

  19. RCP-driven α5β1 recycling suppresses Rac and promotes RhoA activity via the RacGAP1–IQGAP1 complex

    PubMed Central

    Jacquemet, Guillaume; Green, David M.; Bridgewater, Rebecca E.; von Kriegsheim, Alexander; Humphries, Martin J.; Norman, Jim C.

    2013-01-01

    Inhibition of αvβ3 or expression of mutant p53 promotes invasion into fibronectin (FN)-containing extracellular matrix (ECM) by enhancing Rab-coupling protein (RCP)–dependent recycling of α5β1 integrin. RCP and α5β1 cooperatively recruit receptor tyrosine kinases, including EGFR1, to regulate their trafficking and downstream signaling via protein kinase B (PKB)/Akt, which, in turn, promotes invasive migration. In this paper, we identify a novel PKB/Akt substrate, RacGAP1, which is phosphorylated as a consequence of RCP-dependent α5β1 trafficking. Phosphorylation of RacGAP1 promotes its recruitment to IQGAP1 at the tips of invasive pseudopods, and RacGAP1 then locally suppresses the activity of the cytoskeletal regulator Rac and promotes the activity of RhoA in this subcellular region. This Rac to RhoA switch promotes the extension of pseudopodial processes and invasive migration into FN-containing matrices, in a RhoA-dependent manner. Thus, the localized endocytic trafficking of α5β1 within the tips of invasive pseudopods elicits signals that promote the reorganization of the actin cytoskeleton, protrusion, and invasion into FN-rich ECM. PMID:24019536

  20. Fine tuning of Rac1 and RhoA alters cuspal shapes by remolding the cellular geometry

    PubMed Central

    Li, Liwen; Tang, Qinghuang; Nakamura, Takashi; Suh, Jun-Gyo; Ohshima, Hayato; Jung, Han-Sung

    2016-01-01

    The anatomic and functional combinations of cusps and lophs (ridges) define the tooth shape of rodent molars, which distinguishes species. The species-specific cusp patterns result from the spatiotemporal induction of enamel knots (EKs), which require precisely controlled cellular behavior to control the epithelial invagination. Despite the well-defined roles of EK in cusp patterning, the determinants of the ultimate cuspal shapes and involvement of epithelial cellular geometry are unknown. Using two typical tooth patterns, the lophodont in gerbils and the bunodont in mice, we showed that the cuspal shape is determined by the dental epithelium at the cap stage, whereas the cellular geometry in the inner dental epithelium (IDE) is correlated with the cuspal shape. Intriguingly, fine tuning Rac1 and RhoA interconvert cuspal shapes between two species by remolding the cellular geometry. Either inhibition of Rac1 or ectopic expression of RhoA could region-distinctively change the columnar shape of IDE cells in gerbils to drive invagination to produce cusps. Conversely, RhoA reduction in mice inhibited invagination and developed lophs. Furthermore, we found that Rac1 and RhoA modulate the choices of cuspal shape by coordinating adhesion junctions, actin distribution, and fibronectin localization to drive IDE invagination. PMID:27892530

  1. Genetic manipulation of the γ-aminobutyric acid (GABA) shunt in rice: overexpression of truncated glutamate decarboxylase (GAD2) and knockdown of γ-aminobutyric acid transaminase (GABA-T) lead to sustained and high levels of GABA accumulation in rice kernels.

    PubMed

    Shimajiri, Yasuka; Oonishi, Takayuki; Ozaki, Kae; Kainou, Kumiko; Akama, Kazuhito

    2013-06-01

    Gamma-aminobutyric acid (GABA) is a non-protein amino acid commonly present in all organisms. Because cellular levels of GABA in plants are mainly regulated by synthesis (glutamate decarboxylase, GAD) and catabolism (GABA-transaminase, GABA-T), we attempted seed-specific manipulation of the GABA shunt to achieve stable GABA accumulation in rice. A truncated GAD2 sequence, one of five GAD genes, controlled by the glutelin (GluB-1) or rice embryo globulin promoters (REG) and GABA-T-based trigger sequences in RNA interference (RNAi) cassettes controlled by one of these promoters as well, was introduced into rice (cv. Koshihikari) to establish stable transgenic lines under herbicide selection using pyriminobac. T₁ and T₂ generations of rice lines displayed high GABA concentrations (2-100 mg/100 g grain). In analyses of two selected lines from the T₃ generation, there was a strong correlation between GABA level and the expression of truncated GAD2, whereas the inhibitory effect of GABA-T expression was relatively weak. In these two lines both with two T-DNA copies, their starch, amylose, and protein levels were slightly lower than non-transformed cv. Koshihikari. Free amino acid analysis of mature kernels of these lines demonstrated elevated levels of GABA (75-350 mg/100 g polished rice) and also high levels of several amino acids, such as Ala, Ser, and Val. Because these lines of seeds could sustain their GABA content after harvest (up to 6 months), the strategy in this study could lead to the accumulation GABA and for these to be sustained in the edible parts. © 2013 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

  2. Distribution of 3H-GABA uptake sites in the nematode Ascaris

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Guastella, J.; Stretton, A.O.

    1991-05-22

    The distribution of uptake sites for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA) in the nematode Ascaris suum was examined by autoradiography of 3H-GABA uptake. Single neural processes in both the ventral and dorsal nerve cords were labeled with 3H-GABA. Serial section analysis identified the cells of origin of these processes as the RMEV-like and RMED-like neurons. These cells belong to a set of four neurons in the nerve ring, all of which are labeled by 3H-GABA. 3H-GABA labeling of at least two other sets of cephalic neurons was seen. One of these pairs consists of medium-sized lateral ganglia neurons, locatedmore » at the level of the amphid commissure bundle. A second pair is located in the lateral ganglia at the level of the deirid commissure bundle. The position and size of these lateral ganglia cells suggest that they are the GABA-immunoreactive lateral ganglia cells frequently seen in whole-mount immunocytochemical preparations. Four neuronal cell bodies located in the retrovesicular ganglion were also labeled with 3H-GABA. These cells, which are probably cholinergic excitatory motor neurons, do not contain detectable GABA-like immunoreactivity. Heavy labeling of muscle cells was also observed. The ventral and dorsal nerve cord inhibitory motor neurons, which are known to contain GABA-like immunoreactivity, were not labeled above background with 3H-GABA. Together with the experiments reported previously, these results define three classes of GABA-associated neurons in Ascaris: (1) neurons that contain endogenous GABA and possess a GABA uptake system; (2) neurons that contain endogenous GABA, but that either lack a GABA uptake system or possess a GABA uptake system of low activity; (3) neurons that possess a GABA uptake system, but that lack endogenous GABA.« less

  3. The atypical Rho GTPase RhoD is a regulator of actin cytoskeleton dynamics and directed cell migration

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Blom, Magdalena; Reis, Katarina; Heldin, Johan

    RhoD belongs to the Rho GTPases, a protein family responsible for the regulation and organization of the actin cytoskeleton, and, consequently, many cellular processes like cell migration, cell division and vesicle trafficking. Here, we demonstrate that the actin cytoskeleton is dynamically regulated by increased or decreased protein levels of RhoD. Ectopic expression of RhoD has previously been shown to give an intertwined weave of actin filaments. We show that this RhoD-dependent effect is detected in several cell types and results in a less dynamic actin filament system. In contrast, RhoD depletion leads to increased actin filament-containing structures, such as corticalmore » actin, stress fibers and edge ruffles. Moreover, vital cellular functions such as cell migration and proliferation are defective when RhoD is silenced. Taken together, we present data suggesting that RhoD is an important component in the control of actin dynamics and directed cell migration. - Highlights: • Increased RhoD expression leads to loss of actin structures, e.g. stress fibers and gives rise to decreased actin dynamics. • RhoD knockdown induces various actin-containing structures such as edge ruffles, stress fibers and cortical actin, in a cell-type specific manner. • RhoD induces specific actin rearrangements depending on its subcellular localization. • RhoD knockdown has effects on cellular processes, such as directed cell migration and proliferation.« less

  4. RhoA Regulation of Cardiomyocyte Differentiation

    PubMed Central

    Kaarbø, Mari; Crane, Denis I.; Murrell, Wayne G.

    2013-01-01

    Earlier findings from our laboratory implicated RhoA in heart developmental processes. To investigate factors that potentially regulate RhoA expression, RhoA gene organisation and promoter activity were analysed. Comparative analysis indicated strict conservation of both gene organisation and coding sequence of the chick, mouse, and human RhoA genes. Bioinformatics analysis of the derived promoter region of mouse RhoA identified putative consensus sequence binding sites for several transcription factors involved in heart formation and organogenesis generally. Using luciferase reporter assays, RhoA promoter activity was shown to increase in mouse-derived P19CL6 cells that were induced to differentiate into cardiomyocytes. Overexpression of a dominant negative mutant of mouse RhoA (mRhoAN19) blocked this cardiomyocyte differentiation of P19CL6 cells and led to the accumulation of the cardiac transcription factors SRF and GATA4 and the early cardiac marker cardiac α-actin. Taken together, these findings indicate a fundamental role for RhoA in the differentiation of cardiomyocytes. PMID:23935420

  5. [Influence of exogenous gamma-aminobutyric acid (GABA) on GABA metabolism and amino acid contents in roots of melon seedling under hypoxia stress].

    PubMed

    Wang, Chun-Yan; Li, Jing-Rui; Xia, Qing-Ping; Wu, Xiao-Lei; Gao, Hong-Bo

    2014-07-01

    This paper investigated the influence of gamma-aminobutyric acid (GABA) on GABA metabolism and amino acid content under hypoxia stress by accurately controlling the level of dissolved oxygen in hydroponics, using the roots of melon 'Xiyu 1' seedlings as the test material. The results showed that compared with the control, the growth of roots was inhibited seriously under hypoxia stress. Meanwhile, the hypoxia-treated roots had significantly higher activities of glutamate decarboxylase (GAD), glutamate dehydrogenase (GDH), glutamate synthase (GOGAT), glutamine synthetase (GS), alanine aminotransferase (ALT), aspartate aminotransferase (AST) as well as the contents of GABA, pyruvic acid, alanine (Ala) and aspartic acid (Asp). But the contents of glutamic acid (Glu) and alpha-keto glutaric acid in roots under hypoxia stress was obviously lower than those of the control. Exogenous treatment with GABA alleviated the inhibition effect of hypoxia stress on root growth, which was accompanied by an increase in the contents of endogenous GABA, Glu, alpha-keto glutaric acid and Asp. Furthermore, under hypoxia stress, the activities of GAD, GDH, GOGAT, GS, ALT, AST as well as the contents of pyruvic acid and Ala significantly decreased in roots treated with GABA. However, adding GABA and viny-gamma-aminobutyric acid (VGB) reduced the alleviation effect of GABA on melon seedlings under hypoxia stress. The results suggested that absorption of GABA by roots could alleviate the injury of hypoxia stress to melon seedlings. This meant that GABA treatment allows the normal physiological metabolism under hypoxia by inhibiting the GAD activity through feedback and maintaining higher Glu content as well as the bal- ance of carbon and nitrogen.

  6. Multiple functions of GABA A and GABA B receptors during pattern processing in the zebrafish olfactory bulb.

    PubMed

    Tabor, Rico; Yaksi, Emre; Friedrich, Rainer W

    2008-07-01

    gamma-Aminobutyric acid (GABA)ergic synapses are thought to play pivotal roles in the processing of activity patterns in the olfactory bulb (OB), but their functions have been difficult to study during odor responses in the intact system. We pharmacologically manipulated GABA(A) and GABA(B) receptors in the OB of zebrafish and analysed the effects on odor responses of the output neurons, the mitral cells (MCs), by electrophysiological recordings and temporally deconvolved two-photon Ca2+ imaging. The blockade of GABA(B) receptors enhanced presynaptic Ca2+ influx into afferent axon terminals, and changed the amplitude and time course of a subset of MC responses, indicating that GABA(B) receptors have a modulatory influence on OB output activity. The blockade of GABA(A) receptors induced epileptiform firing, enhanced excitatory responses and abolished fast oscillations in the local field potential. Moreover, the topological reorganization and decorrelation of MC activity patterns during the initial phase of the response was perturbed. These results indicate that GABA(A) receptor-containing circuits participate in the balance of excitation and inhibition, the regulation of total OB output activity, the synchronization of odor-dependent neuronal ensembles, and the reorganization of odor-encoding activity patterns. GABA(A) and GABA(B) receptors are therefore differentially involved in multiple functions of neuronal circuits in the OB.

  7. GABA and Glutamate in Children with Primary Complex Motor Stereotypies: A 1H MRS Study at 7T

    PubMed Central

    Harris, A. D.; Singer, H. S.; Horska, A.; Kline, T.; Ryan, M.; Edden, R. A. E.; Mahone, E. Mark

    2015-01-01

    Background and Purpose Complex motor stereotypies (CMS) are rhythmic, repetitive, fixed, purposeful but purposeless movements that stop with distraction. They can occur in otherwise normal healthy children (primary stereotypies), as well in those with autism spectrum disorders (secondary stereotypies). The underlying neurobiological basis for these movements is unknown, but thought to involve cortical-striatal-thalamo-cortical pathways. In order to further clarify potential neurochemical alterations, GABA, glutamate (Glu), glutamine (Gln), N-acetyl aspartate (NAA) and choline (Cho) levels were measured in four frontostriatal regions, using 1H MRS at 7T. Materials and Methods A total of 18 children with primary CMS and 24 typically developing controls, ages 5-10 years completed MRS at 7T. Single voxel STEAM acquisitions from the anterior cingulate cortex (ACC), premotor cortex (PMC), dorsolateral prefrontal cortex (DLPFC) and striatum were obtained and metabolites were quantified with respect to creatine using LCModel. Results The 7T scan was well tolerated by all participants. Compared to controls, children with CMS had lower levels of GABA ACC (GABA/Cr, p=0.049; GABA/Glu: p=0.051) and striatum (GABA/Cr: p= 0.028; GABA/Glu: p=0.0037), but not the DLPFC or PMC. Glu, Gln, NAA, and Cho levels did not differ between groups in any of the aforementioned regions. Within the CMS group, reduced GABA/Cr in the ACC was significantly associated with greater severity of motor stereotypies (r=-0.59, p= 0.021). Conclusions These results suggest possible GABAergic dysfunction within corticostriatal pathways in children with primary CMS. PMID:26542237

  8. Up-regulation of Rho-associated kinase 1/2 by glucocorticoids promotes migration, invasion and metastasis of melanoma.

    PubMed

    Huang, Gao-Xiang; Wang, Yan; Su, Jie; Zhou, Peng; Li, Bo; Yin, Li-Juan; Lu, Jian

    2017-12-01

    Although glucocorticoids (GCs) regulate proliferation, differentiation and apoptosis of tumor cells, their influence on metastasis of tumor cells is poorly understood. Melanoma is a type of skin cancers with high metastasis. We investigated the effect of GCs on metastasis of melanoma cells and its mechanism. We found that GCs significantly promoted the adhesion, migration, invasion of melanoma cells in vitro and lung metastasis in experimental melanoma metastasis mice. Dexamethasone (Dex), a synthetic GC, did not change the RhoA, RhoB and RhoC signalings, but significantly increased the expression and activity of Rho-associated kinase 1/2 (ROCK1/2). The effect of Dex was to increase ROCK1/2 stability mediated by glucocorticoid receptor. Inhibiting ROCK1/2 activity with Y-27632, a ROCK1/2 inhibitor abrogated the pro-migration and pro-metastasis effects of GCs in vitro and in vivo, indicating that ROCK1/2 mediated the pro-metastasis effects of GCs. Activation of PI3K/AKT also contributed to the pro-migration and pro-invasion effects of Dex partially through up-regulating ROCK1/2 expression. Additionally, Dex also down-regulated the expression of tissue inhibitors of matrix metalloproteinase-2. Taken together, our findings provide new data to understand the possible promoting roles and mechanisms of GCs in melanoma metastasis. Copyright © 2017. Published by Elsevier B.V.

  9. 4,5-Substituted 3-Isoxazolols with Insecticidal Activity Act as Competitive Antagonists of Housefly GABA Receptors.

    PubMed

    Liu, Genyan; Ozoe, Fumiyo; Furuta, Kenjiro; Ozoe, Yoshihisa

    2015-07-22

    The insect GABA receptor (GABAR), which is composed of five RDL subunits, represents an important target for insecticides. A series of 4,5-disubstituted 3-isoxazolols, including muscimol analogues, were synthesized and examined for their activities against four splice variants (ac, ad, bc, and bd) of housefly GABARs expressed in Xenopus oocytes. Muscimol was a more potent agonist than GABA in all four splice variants, whereas synthesized analogues did not exhibit agonism but rather antagonism in housefly GABARs. The introduction of bicyclic aromatic groups at the 4-position of muscimol and the simultaneous replacement of the aminomethyl group with a carbamoyl group at the 5-position to afford six 4-aryl-5-carbamoyl-3-isoxazolols resulted in compounds that exhibited significantly enhanced antagonism with IC50 values in the low micromolar range in the ac variant. The inhibition of GABA-induced currents by 100 μM analogues was approximately 1.5-4-fold greater in the ac and bc variants than in the ad and bd variants. 4-(3-Biphenylyl)-5-carbamoyl-3-isoxazolol displayed competitive antagonism, with IC50 values of 30, 34, 107, and 96 μM in the ac, bc, ad, and bd variants, respectively, and exhibited moderate insecticidal activity against houseflies, with an LD50 value of 5.6 nmol/fly. These findings suggest that these 3-isoxazolol analogues are novel lead compounds for the design and development of insecticides that target the orthosteric site of housefly GABARs.

  10. Control of T lymphocyte morphology by the GTPase Rho

    NASA Technical Reports Server (NTRS)

    Woodside, Darren G.; Wooten, David K.; Teague, T. Kent; Miyamoto, Yuko J.; Caudell, Eva G.; Udagawa, Taturo; Andruss, Bernard F.; McIntyre, Bradley W.

    2003-01-01

    BACKGROUND: Rho family GTPase regulation of the actin cytoskeleton governs a variety of cell responses. In this report, we have analyzed the role of the GTPase Rho in maintenance of the T lymphocyte actin cytoskeleton. RESULTS: Inactivation of the GTPase Rho in the human T lymphocytic cell line HPB-ALL does not inhibit constitutively high adhesion to the integrin beta1 substrate fibronectin. It did however result in the aberrant extension of finger-like dendritic processes on the substrates VCAM-1, Fn, and mAb specific to beta1 integrins. Time-lapse video microscopy demonstrated that C3 induced extensions were primarily the result of an altered pseudopod elongation rather than retraction. Once the stellate pseudopodia extended, none retracted, and cells became completely immobile. Filipodial structures were absent and the dendritic-like processes in C3 treated cells were rich in filamentous actin. Immunolocalization of RhoA in untreated HPB-ALL cells spreading on fibronectin demonstrated a diffuse staining pattern within the pseudopodia. In C3 treated cells, clusters of RhoA were pronounced and localized within the altered extensions. CONCLUSIONS: GTPase Rho is actively involved in the regulation of T lymphocyte morphology and motility.

  11. Immunochemical analysis of Micrococcus lysodeikticus (luteus) F1-ATPase and its subunits.

    PubMed

    Urban, C; Salton, M R

    1983-08-31

    The F1-ATPase from Micrococcus lysodeikticus has been purified to 95% protein homogeneity in this laboratory and as all other bacterial F1S, possesses five distinct subunits with molecular weights ranging from 60 000 to 10 000 (Huberman, M. and Salton, M.R.J. (1979) Biochim. Biophys. Acta 547, 230-240). In this communication, we demonstrate the immunochemical reactivities of antibodies to native and SDS-dissociated subunits with the native and dissociated F1-ATPase and show that: (1) the antibodies generated to the native or SDS-dissociated subunits react with the native molecule; (2) all of the subunits comprising the F1 are antigenically unique as determined by crossed immunoelectrophoresis and the Ouchterlony double-diffusion techniques; (3) antibodies to the SDS-denatured individual delta- and epsilon-subunits can be used to destabilize the interaction of these specific subunits with the rest of the native F1; and (4) all subunit antibodies as well as anti-native F1 were found to inhibit ATPase activity to varying degrees, the strongest inhibition being seen with antibodies to the total F1 and anti-alpha- and anti-beta-subunit antibodies. The interaction of specific subunit antibodies may provide a new and novel way to study further and characterize the catalytic portions of F1-ATPases and in general may offer an additional method for the examination of multimeric proteins.

  12. Role of RhoA, mDia, and ROCK in cell shape-dependent control of the Skp2-p27kip1 pathway and the G1/S transition.

    PubMed

    Mammoto, Akiko; Huang, Sui; Moore, Kimberly; Oh, Philmo; Ingber, Donald E

    2004-06-18

    Cell shape-dependent control of cell-cycle progression underlies the spatial differentials of growth that drive tissue morphogenesis, yet little is known about how cell distortion impacts the biochemical signaling machinery that is responsible for growth control. Here we show that the Rho family GTPase, RhoA, conveys the "cell shape signal" to the cell-cycle machinery in human capillary endothelial cells. Cells accumulating p27(kip1) and arrested in mid G(1) phase when spreading were inhibited by restricted extracellular matrix adhesion, whereas constitutively active RhoA increased expression of the F-box protein Skp2 required for ubiquitination-dependent degradation of p27(kip1) and restored G(1) progression in these cells. Studies with dominant-negative and constitutively active forms of mDia1, a downstream effector of RhoA, and with a pharmacological inhibitor of ROCK, another RhoA target, revealed that RhoA promoted G(1) progression by altering the balance of activities between these two downstream effectors. These data indicate that signaling proteins such as mDia1 and ROCK, which are thought to be involved primarily in cytoskeletal remodeling, also mediate cell growth regulation by coupling cell shape to the cell-cycle machinery at the level of signal transduction.

  13. Early life stress is a risk factor for excessive alcohol drinking and impulsivity in adults and is mediated via a CRF/GABA(A) mechanism.

    PubMed

    Gondré-Lewis, Marjorie C; Warnock, Kaitlin T; Wang, Hong; June, Harry L; Bell, Kimberly A; Rabe, Holger; Tiruveedhula, Veera Venkata Naga Phani Babu; Cook, James; Lüddens, Hartmut; Aurelian, Laure; June, Harry L

    2016-01-01

    Childhood stress and trauma are associated with substance use disorders in adulthood, but the neurological changes that confer increased vulnerability are largely unknown. In this study, maternal separation (MS) stress, restricted to the pre-weaning period, was used as a model to study mechanisms of protracted effects of childhood stress/traumatic experiences on binge drinking and impulsivity. Using an operant self-administration model of binge drinking and a delay discounting assay to measure impulsive-like behavior, we report that early life stress due to MS facilitated acquisition of binge drinking and impulsivity during adulthood in rats. Previous studies have shown heightened levels of corticotropin releasing factor (CRF) after MS, and here, we add that MS increased expression levels of GABA(A) α2 subunit in central stress circuits. To investigate the precise role of these circuits in regulating impulsivity and binge drinking, the CRF1 receptor antagonist antalarmin and the novel GABA(A) α2 subunit ligand 3-PBC were infused into the central amygdala (CeA) and medial prefrontal cortex (mPFC). Antalarmin and 3-PBC at each site markedly reduced impulsivity and produced profound reductions on binge-motivated alcohol drinking, without altering responding for sucrose. Furthermore, whole-cell patch-clamp studies showed that low concentrations of 3-PBC directly reversed the effect of relatively high concentrations of ethanol on α2β3γ2 GABA(A) receptors, by a benzodiazepine site-independent mechanism. Together, our data provide strong evidence that maternal separation, i.e. early life stress, is a risk factor for binge drinking, and is linked to impulsivity, another key risk factor for excessive alcohol drinking. We further show that pharmacological manipulation of CRF and GABA receptor signaling is effective to reverse binge drinking and impulsive-like behavior in MS rats. These results provide novel insights into the role of the brain stress systems in the

  14. The effect of the Ras homolog gene family (Rho), member A/Rho associated coiled-coil forming protein kinase pathway in atrial fibrosis of type 2 diabetes in rats.

    PubMed

    Chen, Jinling; Li, Qingqing; Dong, Ruiqing; Gao, Huikuan; Peng, Hui; Wu, Yongquan

    2014-09-01

    Diabetes mellitus promotes atrial structural remodeling, thereby producing atrial arrhythmogenicity. Atrial arrhythmia can substantially increase the risk of premature death. The aim of this study was to investigate the role of Ras homolog gene family, member A (RhoA)/Rho associated coiled-coil forming protein kinase (ROCK) in atrial fibrosis in diabetic hearts, and the effects of fasudil hydrochloride hydrate on atrial fibrosis. An eight-week-old male Sprague-Dawley rat model of type 2 diabetes was established using a high-fat diet combined with streptozotocin [30 mg/kg, once, intraperitoneal (i.p.)]. Animals were randomly divided into three groups: Control rats, untreated diabetic rats that received vehicle, and treated diabetic rats that received Rho kinase inhibitor fasudil hydrochloride hydrate (10 mg/kg/day, i.p., for 14 weeks). The morphological features of atrial fibrosis were observed using Masson staining. The mRNA expression levels of RhoA, ROCK1, ROCK2, type-I and type-III procollagen were assessed with quantitative polymerase chain reaction. The protein levels of RhoA, ROCK1 and ROCK2 were evaluated using western blot analysis. The atria of untreated diabetic rats showed evident atrial fibrosis as compared to the control rats; the mRNA expression levels of RhoA, ROCK1, ROCK2, type-I and type-III procollagen were upregulated; and the protein levels of RhoA, ROCK1 and ROCK2 were increased. The treatment with fasudil hydrochloride hydrate significantly reduced atrial fibrosis, mRNA levels of RhoA, ROCK1, ROCK2, type-I and type-III procollagen, and the protein levels of RhoA, ROCK1 and ROCK2. The results suggested that RhoA/ROCK was involved in atrial fibrosis, and that fasudil hydrochloride hydrate ameliorates atrial fibrosis through the RhoA/ROCK pathway in rats with type 2 diabetes.

  15. Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection

    PubMed Central

    Goffinet, Marine; Chinestra, Patrick; Lajoie-Mazenc, Isabelle; Medale-Giamarchi, Claire; Favre, Gilles; Faye, Jean-Charles

    2008-01-01

    Background The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv) that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings. Results After five rounds of phage selection using a constitutively activated mutant of RhoB (RhoBQ63L), three scFvs (A8, C1 and D11) were selected for subsequent analysis. Further biochemical characterization was pursued for the single clone, C1, exhibiting an scFv structure. C1 was selective for the GTP-bound form of RhoA, RhoB, as well as RhoC, and failed to recognize GTP-loaded Rac1 or Cdc42, two other members of the Rho family. To enhance its production, soluble C1 was expressed in fusion with the N-terminal domain of phage protein pIII (scFv C1-N1N2), it appeared specifically associated with GTP-loaded recombinant RhoA and RhoB via immunoprecipitation, and endogenous activated Rho in HeLa cells as determined by immunofluorescence. Conclusion We identified an antibody, C1-N1N2, specific for the GTP-bound form of RhoB from a phage library, and confirmed its specificity towards GTP-bound RhoA and RhoC, as well as RhoB. The success of C1-N1N2 in discriminating activated Rho in

  16. γ-Aminobutyric acid (GABA) signalling in plants.

    PubMed

    Ramesh, Sunita A; Tyerman, Stephen D; Gilliham, Matthew; Xu, Bo

    2017-05-01

    The role of γ-aminobutyric acid (GABA) as a signal in animals has been documented for over 60 years. In contrast, evidence that GABA is a signal in plants has only emerged in the last 15 years, and it was not until last year that a mechanism by which this could occur was identified-a plant 'GABA receptor' that inhibits anion passage through the aluminium-activated malate transporter family of proteins (ALMTs). ALMTs are multigenic, expressed in different organs and present on different membranes. We propose GABA regulation of ALMT activity could function as a signal that modulates plant growth, development, and stress response. In this review, we compare and contrast the plant 'GABA receptor' with mammalian GABA A receptors in terms of their molecular identity, predicted topology, mode of action, and signalling roles. We also explore the implications of the discovery that GABA modulates anion flux in plants, its role in signal transduction for the regulation of plant physiology, and predict the possibility that there are other GABA interaction sites in the N termini of ALMT proteins through in silico evolutionary coupling analysis; we also explore the potential interactions between GABA and other signalling molecules.

  17. Subunits of the Snf1 kinase heterotrimer show interdependence for association and activity.

    PubMed

    Elbing, Karin; Rubenstein, Eric M; McCartney, Rhonda R; Schmidt, Martin C

    2006-09-08

    The Snf1 kinase and its mammalian orthologue, the AMP-activated protein kinase (AMPK), function as heterotrimers composed of a catalytic alpha-subunit and two non-catalytic subunits, beta and gamma. The beta-subunit is thought to hold the complex together and control subcellular localization whereas the gamma-subunit plays a regulatory role by binding to and blocking the function of an auto-inhibitory domain (AID) present in the alpha-subunit. In addition, catalytic activity requires phosphorylation by a distinct upstream kinase. In yeast, any one of three Snf1-activating kinases, Sak1, Tos3, or Elm1, can fulfill this role. We have previously shown that Sak1 is the only Snf1-activating kinase that forms a stable complex with Snf1. Here we show that the formation of the Sak1.Snf1 complex requires the beta- and gamma-subunits in vivo. However, formation of the Sak1.Snf1 complex is not necessary for glucose-regulated phosphorylation of the Snf1 activation loop. Snf1 kinase purified from cells lacking the beta-subunits do not contain any gamma-subunit, indicating that the Snf1 kinase does not form a stable alphagamma dimer in vivo. In vitro kinase assays using purified full-length and truncated Snf1 proteins demonstrate that the kinase domain, which lacks the AID, is significantly more active than the full-length Snf1 protein. Addition of purified beta- and gamma-subunits could stimulate the kinase activity of the full-length alpha-subunit but only when all three subunits were present, suggesting an interdependence of all three subunits for assembly of a functional complex.

  18. Effects of Traumatic Stress Induced in the Juvenile Period on the Expression of Gamma-Aminobutyric Acid Receptor Type A Subunits in Adult Rat Brain

    PubMed Central

    Lu, Cui Yan; Liu, De Xiang; Jiang, Hong; Ho, Cyrus S. H.; Ho, Roger C. M.

    2017-01-01

    Studies have found that early traumatic experience significantly increases the risk of posttraumatic stress disorder (PTSD). Gamma-aminobutyric acid (GABA) deficits were proposed to be implicated in development of PTSD, but the alterations of GABA receptor A (GABAAR) subunits induced by early traumatic stress have not been fully elucidated. Furthermore, previous studies suggested that exercise could be more effective than medications in reducing severity of anxiety and depression but the mechanism is unclear. This study used inescapable foot-shock to induce PTSD in juvenile rats and examined their emotional changes using open-field test and elevated plus maze, memory changes using Morris water maze, and the expression of GABAAR subunits (γ2, α2, and α5) in subregions of the brain in the adulthood using western blotting and immunohistochemistry. We aimed to observe the role of GABAAR subunits changes induced by juvenile trauma in the pathogenesis of subsequent PTSD in adulthood. In addition, we investigated the protective effects of exercise for 6 weeks and benzodiazepine (clonazepam) for 2 weeks. This study found that juvenile traumatic stress induced chronic anxiety and spatial memory loss and reduced expression of GABAAR subunits in the adult rat brains. Furthermore, exercise led to significant improvement as compared to short-term BZ treatment. PMID:28352479

  19. Progression of Human Renal Cell Carcinoma via Inhibition of RhoA-ROCK Axis by PARG1.

    PubMed

    Miyazaki, Junichiro; Ito, Keiichi; Fujita, Tomonobu; Matsuzaki, Yuriko; Asano, Takako; Hayakawa, Masamichi; Asano, Tomohiko; Kawakami, Yutaka

    2017-04-01

    Renal cell carcinoma (RCC) is the most lethal urological malignancy with high risk of recurrence; thus, new prognostic biomarkers are needed. In this study, a new RCC antigen, PTPL1 associated RhoGAP1 (PARG1), was identified by using serological identification of recombinant cDNA expression cloning with sera from RCC patients. PARG1 protein was found to be differentially expressed in RCC cells among patients. High PARG1 expression is significantly correlated with various clinicopathological factors relating to cancer cell proliferation and invasion, including G3 percentage (P = .0046), Ki-67 score (p expression is also correlated with high recurrence of N0M0 patients (P = .0084) and poor prognosis in RCC patients (P = .0345). Multivariate analysis has revealed that high PARG1 expression is an independent factor for recurrence (P = .0149) of N0M0 RCC patients. In in vitro studies, depletion of PARG1by siRNA in human RCC cell lines inhibited their proliferation through inducing G1 cell cycle arrest via upregulation of p53 and subsequent p21 Cip1/Waf1 , which are mediated by increased RhoA-ROCK activities. Similarly, PARG1 depletion cells inhibited invasion ability via increasing RhoA-ROCK activities in the RCC cell lines. Conversely, overexpression of PARG1 on human embryonic kidney cell line HEK293T promotes its cell proliferation and invasion. These results indicate that PARG1 plays crucial roles in progression of human RCC in increasing cell proliferation and invasion ability via inhibition of the RhoA-ROCK axis, and PARG1 is a poor prognostic marker, particularly for high recurrence of N0M0 RCC patients. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  20. Engineering the intracellular metabolism of Escherichia coli to produce gamma-aminobutyric acid by co-localization of GABA shunt enzymes.

    PubMed

    Pham, Van Dung; Somasundaram, Sivachandiran; Lee, Seung Hwan; Park, Si Jae; Hong, Soon Ho

    2016-02-01

    To direct the carbon flux from Krebs cycle into the gamma-aminobutyric acid (GABA) shunt pathway for the production of GABA by protein scaffold introduction in Escherichia coli. Escherichia coli was engineered to produce GABA from glucose by the co-localization of enzymes succinate semialdehyde dehydrogenase (GadD), GABA aminotransferase (PuuE) and GABA transporter (GadC) by protein scaffold. 0.7 g GABA l(-1) was produced from 10 g glucose l(-1) while no GABA was produced in wild type E. coli. pH 6 and 30 °C were optimum for GABA production, and GABA concentration increased to 1.12 g GABA l(-1) when 20 g glucose l(-1) was used. When competing metabolic networks were inactivated, GABA increased by 24 % (0.87 g GABA l(-1)). The novel GABA production system was constructed by co-localization of GABA shunt enzymes.

  1. Genetic Screening in C. Elegans Identifies Rho-GTPAse Activating Protein 6 as Novel HERG Regulator

    PubMed Central

    Potet, Franck; Petersen, Christina I.; Boutaud, Olivier; Shuai, Wen; Stepanovic, Svetlana Z.; Balser, Jeffrey R.; Kupershmidt, Sabina

    2009-01-01

    The human ether-a-go-go related gene (HERG) constitutes the pore forming subunit of IKr, a K+ current involved in repolarization of the cardiac action potential. While mutations in HERG predispose patients to cardiac arrhythmias (Long QT syndrome; LQTS), altered function of HERG regulators are undoubtedly LQTS risk factors. We have combined RNA interference with behavioral screening in Caenorhabditis elegans to detect genes that influence function of the HERG homolog, UNC-103. One such gene encodes the worm ortholog of the rho-GTPase activating protein 6 (ARHGAP6). In addition to its GAP function, ARHGAP6 induces cytoskeletal rearrangements and activates phospholipase C (PLC). Here we show that IKr recorded in cells co-expressing HERG and ARHGAP6 was decreased by 43% compared to HERG alone. Biochemical measurements of cell-surface associated HERG revealed that ARHGAP6 reduced membrane expression of HERG by 35%, which correlates well with the reduction in current. In an atrial myocyte cell line, suppression of endogenous ARHGAP6 by virally transduced shRNA led to a 53 % enhancement of IKr. ARHGAP6 effects were maintained when we introduced a dominant negative rho-GTPase, or ARHGAP6 devoid of rhoGAP function, indicating ARHGAP6 regulation of HERG is independent of rho activation. However, ARHGAP6 lost effectiveness when PLC was inhibited. We further determined that ARHGAP6 effects are mediated by a consensus SH3 binding domain within the C-terminus of HERG, although stable ARHGAP6-HERG complexes were not observed. These data link a rhoGAP-activated PLC pathway to HERG membrane expression and implicate this family of proteins as candidate genes in disorders involving HERG. PMID:19038263

  2. Exploiting RhoA Mutations in Diffuse Gastric Adenocarcinoma and Targeting Intertwined RhoA and Yap1 Pathways for Therapeutic Advantage

    DTIC Science & Technology

    2017-10-01

    fluorescent marker mOrange into MIT’s Dr. Zhang’s pLenti- Crispr -v2, making transfection into mammalian cells easier and visible under fluorescent...microscope, it the same time, those cells under Crispr editing are also selectable with puromycin. We have successfully knocked-out RhoA expression in cell...15. SUBJECT TERMS RHOA, YAP1, mouse model, CRISPR -CAS9, plasmid, cell lines, diffuse gastric adenocarcinoma, mutations, gastric adenocarcinoma 16

  3. Effect of Rho family GTP-binding proteins on Amoeba proteus.

    PubMed

    Kłopocka, W; Redowicz, M J

    2003-03-01

    While there is a number of studies on the effects of Rho GTPases on the actin-based cytoskeleton in higher eukaryotes, studies in protozoans are rather limited. The problem seems to be intriguing since the structure of protozoan cytoskeletons is distinct from most vertebrate cells. By blocking endogenous Rho family proteins of highly motile Amoeba proteus with C3 transferase and antibodies against human RhoA and Rac1, we tried to assess the in vivo role of these proteins in amoebae. In migrating amoebae, both proteins are concentrated in the cortical layer and seem to colocalize with filamentous actin. Endogenous Rac1, but not RhoA, is accumulated in the perinuclear cytoskeleton. Blocking Rac- or Rho-like proteins caused distinct and irreversible changes in the locomotive shape of the examined amoebae and significant inhibition of their migration. Amoebae microinjected with anti-Rac1 antibodies were contracted, shortened, and developed only few wide pseudopodia. More pronounced changes were observed in cells treated with anti-RhoA antibodies. They exhibited an atypical locomotion not leading to their effective displacement. After treatment with 50 microg of C3 transferase per ml, cells rapidly contracted and almost completely rounded up, became refractile with the granules beaten into a dense mass, detached from the surface and died. Ten times lower concentration of the enzyme caused similar changes as the inhibition of endogenous RhoA-like protein. These results indicate that Rho family-based regulation plays a key role in amoebic migration.

  4. The adjustment of γ-aminobutyric acidA tonic subunits in Huntington's disease: from transcription to translation to synaptic levels into the neostriatum

    PubMed Central

    Rosas-Arellano, Abraham; Estrada-Mondragón, Argel; Mantellero, Carola A.; Tejeda-Guzmán, Carlos; Castro, Maite A.

    2018-01-01

    γ-Aminobutyric acid (GABA), plays a key role in all stages of life, also is considered the main inhibitory neurotransmitter. GABA activates two kind of membrane receptors known as GABAA and GABAB, the first one is responsible to render tonic inhibition by pentameric receptors containing α4−6, β3, δ, or ρ1−3 subunits, they are located at perisynaptic and/or in extrasynaptic regions. The biophysical properties of GABAA tonic inhibition have been related with cellular protection against excitotoxic injury and cell death in presence of excessive excitation. On this basis, GABAA tonic inhibition has been proposed as a potential target for therapeutic intervention of Huntington's disease. Huntington's disease is a neurodegenerative disorder caused by a genetic mutation of the huntingtin protein. For experimental studies of Huntington's disease mouse models have been developed, such as R6/1, R6/2, HdhQ92, HdhQ150, as well as YAC128. In all of them, some key experimental reports are focused on neostriatum. The neostriatum is considered as the most important connection between cerebral cortex and basal ganglia structures, its cytology display two pathways called direct and indirect constituted by medium sized spiny neurons expressing dopamine D1 and D2 receptors respectively, they display strong expression of many types of GABAA receptors, including tonic subunits. The studies about of GABAA tonic subunits and Huntington's disease into the neostriatum are rising in recent years, suggesting interesting changes in their expression and localization which can be used as a strategy to delay the cellular damage caused by the imbalance between excitation and inhibition, a hallmark of Huntington's disease. PMID:29722299

  5. Influence of GABA and GABA-producing Lactobacillus brevis DPC 6108 on the development of diabetes in a streptozotocin rat model.

    PubMed

    Marques, T M; Patterson, E; Wall, R; O'Sullivan, O; Fitzgerald, G F; Cotter, P D; Dinan, T G; Cryan, J F; Ross, R P; Stanton, C

    2016-06-01

    The aim of this study was to investigate if dietary administration of γ-aminobutyric acid (GABA)-producing Lactobacillus brevis DPC 6108 and pure GABA exert protective effects against the development of diabetes in streptozotocin (STZ)-induced diabetic Sprague Dawley rats. In a first experiment, healthy rats were divided in 3 groups (n=10/group) receiving placebo, 2.6 mg/kg body weight (bw) pure GABA or L. brevis DPC 6108 (~10(9)microorganisms). In a second experiment, rats (n=15/group) were randomised to five groups and four of these received an injection of STZ to induce type 1 diabetes. Diabetic and non-diabetic controls received placebo [4% (w/v) yeast extract in dH2O], while the other three diabetic groups received one of the following dietary supplements: 2.6 mg/kg bw GABA (low GABA), 200 mg/kg bw GABA (high GABA) or ~10(9) L. brevis DPC 6108. L. brevis DPC 6108 supplementation was associated with increased serum insulin levels (P<0.05), but did not alter other metabolic markers in healthy rats. Diabetes induced by STZ injection decreased body weight (P<0.05), increased intestinal length (P<0.05) and stimulated water and food intake. Insulin was decreased (P<0.05), whereas glucose was increased (P<0.001) in all diabetic groups, compared with non-diabetic controls. A decrease (P<0.01) in glucose levels was observed in diabetic rats receiving L. brevis DPC 6108, compared with diabetic-controls. Both the composition and diversity of the intestinal microbiota were affected by diabetes. Microbial diversity in diabetic rats supplemented with low GABA was not reduced (P>0.05), compared with non-diabetic controls while all other diabetic groups displayed reduced diversity (P<0.05). L. brevis DPC 6108 attenuated hyperglycaemia induced by diabetes but additional studies are needed to understand the mechanisms involved in this reduction.

  6. The Rho GTPase effector ROCK regulates cyclin A, cyclin D1, and p27Kip1 levels by distinct mechanisms.

    PubMed

    Croft, Daniel R; Olson, Michael F

    2006-06-01

    The members of the Rho GTPase family are well known for their regulation of actin cytoskeletal structures. In addition, they influence progression through the cell cycle. The RhoA and RhoC proteins regulate numerous effector proteins, with a central and vital signaling role mediated by the ROCK I and ROCK II serine/threonine kinases. The requirement for ROCK function in the proliferation of numerous cell types has been revealed by studies utilizing ROCK-selective inhibitors such as Y-27632. However, the mechanisms by which ROCK signaling promotes cell cycle progression have not been thoroughly characterized. Using a conditionally activated ROCK-estrogen receptor fusion protein, we found that ROCK activation is sufficient to stimulate G1/S cell cycle progression in NIH 3T3 mouse fibroblasts. Further analysis revealed that ROCK acts via independent pathways to alter the levels of cell cycle regulatory proteins: cyclin D1 and p21(Cip1) elevation via Ras and the mitogen-activated protein kinase pathway, increased cyclin A via LIM kinase 2, and reduction of p27(Kip1) protein levels. Therefore, the influence of ROCK on cell cycle regulatory proteins occurs by multiple independent mechanisms.

  7. Through its F-BAR and RhoGAP domains, Rgd1p acts in different polarized growth processes in budding yeast

    PubMed Central

    Lefebvre, Fabien; Prouzet-Mauléon, Valérie; Vieillemard, Aurélie; Thoraval, Didier; Crouzet, Marc

    2009-01-01

    Protein domain architecture can be used to construct supramolecular structures, to carry out specific functions and to mediate signaling in prokaryotic and eukaryotic cells. The Rgd1p protein of budding yeast contains two domains with different functions in the cell: the F-BAR and RhoGAP domains. The F-BAR domain has been shown to interact with membrane phospholipids and is thought to induce or sense membrane curvature. The RhoGAP domain activates the GTP hydrolysis of two Rho GTPases, thereby regulating different cellular pathways. Specific molecular interactions with the F-BAR and RhoGAP domains, cell signaling and interplay between these domains may allow the Rgd1p protein to act in several different biological processes, all of which are required for polarized growth in yeast. PMID:19704907

  8. Single channel analysis of the blocking actions of BIDN and fipronil on a Drosophila melanogaster GABA receptor (RDL) stably expressed in a Drosophila cell line

    PubMed Central

    Grolleau, Françoise; Sattelle, David B

    2000-01-01

    Single channel recordings were obtained from a Drosophila S2 cell line stably expressing the wild-type RDLac Drosophila melanogaster homomer-forming ionotropic GABA receptor subunit, a product of the resistance to dieldrin gene, Rdl. GABA (50 μM) was applied by pressure ejection to outside-out patches from S2-RDL cells at a holding potential of −60 mV. The resulting inward current was completely blocked by 100 μM picrotoxin (PTX). The unitary current-voltage relationship was linear at negative potentials but showed slight inward rectification at potentials more positive than 0 mV. The reversal potential of the current (EGABA=−1.4 mV) was close to the calculated chloride equilibrium potential. The single channel conductance elicited by GABA was 36 pS. A 71 pS conductance channel was also observed when the duration of the pulse, used to eject GABA, was longer than 80 ms. The mean open time distribution of the unitary events was fitted best by two exponential functions suggesting two open channel states. When either 1 μM fipronil or 1 μM BIDN was present in the external saline, the GABA-gated channels were completely blocked. When BIDN or fipronil was applied at a concentration close to the IC50 value for suppression of open probability (281 nM, BIDN; 240 nM, fipronil), the duration of channel openings was shortened. In addition, the blocking action of BIDN resulted in the appearance of a novel channel conductance (17 pS). The effects of co-application of BIDN and fipronil were examined. Co-application of BIDN (300 nM) with various concentrations (100–1000 nM) of fipronil resulted in an additional BIDN-induced dose-dependent reduction of the maximum Po value. Thus both BIDN and fipronil shorten the duration of wild-type RDLac GABA receptor channel openings but appear to act at distinct sites. PMID:10952672

  9. GABAA receptor subunit gene expression in human prefrontal cortex: comparison of schizophrenics and controls

    NASA Technical Reports Server (NTRS)

    Akbarian, S.; Huntsman, M. M.; Kim, J. J.; Tafazzoli, A.; Potkin, S. G.; Bunney, W. E. Jr; Jones, E. G.; Bloom, F. E. (Principal Investigator)

    1995-01-01

    The prefrontal cortex of schizophrenics is hypoactive and displays changes related to inhibitory, GABAergic neurons, and GABAergic synapses. These changes include decreased levels of glutamic acid decarboxylase (GAD), the enzyme for GABA synthesis, upregulation of muscimol binding, and downregulation of benzodiazepine binding to GABAA receptors. Studies in the visual cortex of nonhuman primates have demonstrated that gene expression for GAD and for several GABAA receptor subunit polypeptides is under control of neuronal activity, raising the possibility that similar mechanisms in the hypoactive prefrontal cortex of schizophrenics may explain the abnormalities in GAD and in GABAA receptor regulation. In the present study, which is the first of its type on human cerebral cortex, levels of mRNAs for six GABAA receptor subunits (alpha 1, alpha 2, alpha 5, beta 1, beta 2, gamma 2) and their laminar expression patterns were analyzed in the prefrontal cortex of schizophrenics and matched controls, using in situ hybridization histochemistry and densitometry. Three types of laminar expression pattern were observed: mRNAs for the alpha 1, beta 2, and gamma 2 subunits, which are the predominant receptor subunits expressed in the mature cortex, were expressed at comparatively high levels by cells of all six cortical layers, but most intensely by cells in lower layer III and layer IV. mRNAs for the alpha 2, alpha 5, and beta 1 subunits were expressed at lower levels; alpha 2 and beta 1 were expressed predominantly by cells in layers II, III, and IV; alpha 5 was expressed predominantly in layers IV, V, and VI. There were no significant changes in overall mRNA levels for any of the receptor subunits in the prefrontal cortex of schizophrenics, and the laminar expression pattern of all six receptor subunit mRNAs did not differ between schizophrenics and controls. Because gene expression for GABAA receptor subunits is not consistently altered in the prefrontal cortex of

  10. GPER inhibits diabetes-mediated RhoA activation to prevent vascular endothelial dysfunction.

    PubMed

    Li, Zilin; Cheng, Liang; Liang, Hongliang; Duan, Weixun; Hu, Jing; Zhi, Weiwei; Yang, Jinbao; Liu, Zhenhua; Zhao, Minggao; Liu, Jincheng

    2016-02-01

    The effect of estrogen receptors on diabetes-induced vascular dysfunction is critical, but ambiguous. Individuals with diabetic vascular disease may require estrogen receptor-specific targeted therapy in the future. The G protein-coupled estrogen receptor (GPER) has beneficial effects on vascular function. However, its fundamental mechanisms are unclear. The RhoA/Rho-kinase pathway contributes to diabetic vascular complications, whereas estrogen can suppress Rho-kinase function. Thus, we assumed that GPER inhibits diabetes-mediated RhoA activation to prevent vascular dysfunction. We further investigated the underlying mechanisms involved in this process. Vascular endothelial cells and ex vivo cultured ovariectomized (OVX) C57BL/6 mouse aortae were treated with high glucose (HG) alone or in combination with GPER agonist (G1). G1 treatment was also administered to OVX db/db mice for 8 weeks. An ex-vivo isovolumic myograph was used to analyze the endothelium-dependent vasodilation and endothelium-independent contraction of mouse aortae. Apoptosis, oxidative stress, and inflammation were attenuated in G1-pretreated vascular endothelial cells. G1 significantly decreased the phosphorylation of inhibitory endothelial nitric oxide (NO) synthase residue threonine 495 (eNOS Thr495), inhibited RhoA expression, and increased NO production. Additionally, G1 rescued the impaired endothelium-dependent relaxation and inhibited RhoA activation in the thoracic aorta of OVX db/db mice and ex-vivo cultured OVX C57BL/6 mouse aortae treated with HG. Estrogens acting via GPER could protect vascular endothelium, and GPER activation might elicit ERα-independent effect to inhibit RhoA/Rho-kinase pathway. Additionally, GPER activation might reduce vascular smooth muscle contraction by inhibiting RhoA activation. Thus, the results of the present study suggest a new therapeutic paradigm for end-stage vascular dysfunction by inhibiting RhoA/Rho-kinase pathway via GPER activation. Copyright

  11. Neuroprotection of ebselen against ischemia/reperfusion injury involves GABA shunt enzymes.

    PubMed

    Seo, Jeong Yeol; Lee, Choong Hyun; Cho, Jun Hwi; Choi, Jung Hoon; Yoo, Ki-Yeon; Kim, Dae Won; Park, Ok Kyu; Li, Hua; Choi, Soo Young; Hwang, In Koo; Won, Moo-Ho

    2009-10-15

    Seleno-organic compound, ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one), is a substrate with radical-scavenging activity. In this study, we observed the neuroprotective effects of ebselen against ischemic damage and on GABA shunt enzymes such as glutamic acid decarboxylase 67 (GAD67), GABA transaminse (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) in the hippocampal CA1 region after 5 min of transient forebrain ischemia in gerbils. For this, vehicle (physiological saline) or ebselen was administered 30 min before or after ischemia/reperfusion and sacrificed 4 days after ischemia/reperfusion. The administration of ebselen significantly reduced the neuronal death in the CA1 region induced by ischemia/reperfusion. In addition, treatment with ebselen markedly elevated GAD67, GABA-T and SSADH immunoreactivity and their protein levels compared to that in the vehicle-treated group, respectively. These results suggest that ebselen protects neurons from ischemic damage via control of the expressions of GABA shunt enzymes to enter the TCA cycle.

  12. Inhibition of RhoA/Rho kinase pathway and smooth muscle contraction by hydrogen sulfide.

    PubMed

    Nalli, Ancy D; Wang, Hongxia; Bhattacharya, Sayak; Blakeney, Bryan A; Murthy, Karnam S

    2017-10-01

    Hydrogen sulfide (H 2 S) plays an important role in smooth muscle relaxation. Here, we investigated the expression of enzymes in H 2 S synthesis and the mechanism regulating colonic smooth muscle function by H 2 S. Expression of cystathionine-γ-lyase (CSE), but not cystathionine-β-synthase (CBS), was identified in the colonic smooth muscle of rabbit, mouse, and human. Carbachol (CCh)-induced contraction in rabbit muscle strips and isolated muscle cells was inhibited by l-cysteine (substrate of CSE) and NaHS (an exogenous H 2 S donor) in a concentration-dependent fashion. H 2 S induced S-sulfhydration of RhoA that was associated with inhibition of RhoA activity. CCh-induced Rho kinase activity also was inhibited by l-cysteine and NaHS in a concentration-dependent fashion. Inhibition of CCh-induced contraction by l-cysteine was blocked by the CSE inhibitor, dl-propargylglycine (DL-PPG) in dispersed muscle cells. Inhibition of CCh-induced Rho kinase activity by l-cysteine was blocked by CSE siRNA in cultured cells and DL-PPG in dispersed muscle cells. Stimulation of Rho kinase activity and muscle contraction in response to CCh was also inhibited by l-cysteine or NaHS in colonic muscle cells from mouse and human. Collectively, our studies identified the expression of CSE in colonic smooth muscle and determined that sulfhydration of RhoA by H 2 S leads to inhibition of RhoA and Rho kinase activities and muscle contraction. The mechanism identified may provide novel therapeutic approaches to mitigate gastrointestinal motility disorders. © 2017 The Authors. Pharmacology Research & Perspectives published by John Wiley & Sons Ltd, British Pharmacological Society and American Society for Pharmacology and Experimental Therapeutics.

  13. GABA(B) receptor modulation of feedforward inhibition through hippocampal neurogliaform cells.

    PubMed

    Price, Christopher J; Scott, Ricardo; Rusakov, Dmitri A; Capogna, Marco

    2008-07-02

    Feedforward inhibition of neurons is a fundamental component of information flow control in the brain. We studied the roles played by neurogliaform cells (NGFCs) of stratum lacunosum moleculare of the hippocampus in providing feedforward inhibition to CA1 pyramidal cells. We recorded from synaptically coupled pairs of anatomically identified NGFCs and CA1 pyramidal cells and found that, strikingly, a single presynaptic action potential evoked a biphasic unitary IPSC (uIPSC), consisting of two distinct components mediated by GABA(A) and GABA(B) receptors. A GABA(B) receptor-mediated unitary response has not previously been observed in hippocampal excitatory neurons. The decay of the GABA(A) receptor-mediated response was slow (time constant = 50 ms), and was tightly regulated by presynaptic GABA(B) receptors. Surprisingly, the GABA(B) receptor ligands baclofen and (2S)-3-{[(1S)-1-(3,4-dichlorophenyl)ethyl]amino-2-hydroxypropyl}(phenylmethyl)phosphinic acid (CGP55845), while affecting the NGFC-mediated uIPSCs, had no effect on action potential-evoked presynaptic Ca2+ signals monitored in individual axonal boutons of NGFCs with two-photon microscopy. In contrast, baclofen clearly depressed presynaptic Ca2+ transients in non-NGF interneurons. Changes in extracellular Ca2+ concentration that mimicked the effects of baclofen or CGP55845 on uIPSCs significantly altered presynaptic Ca2+ transients. Electrophysiological data suggest that GABA(B) receptors expressed by NGFCs contribute to the dynamic control of the excitatory input to CA1 pyramidal neurons from the temporoammonic path. The NGFC-CA1 pyramidal cell connection therefore provides a unique and subtle mechanism to shape the integration time domain for signals arriving via a major excitatory input to CA1 pyramidal cells.

  14. Neuroimaging studies of GABA in schizophrenia: a systematic review with meta-analysis.

    PubMed

    Egerton, A; Modinos, G; Ferrera, D; McGuire, P

    2017-06-06

    Data from animal models and from postmortem studies suggest that schizophrenia is associated with brain GABAergic dysfunction. The extent to which this is reflected in data from in vivo studies of GABA function in schizophrenia is unclear. The Medline database was searched to identify articles published until 21 October 2016. The search terms included GABA, proton magnetic resonance spectroscopy ( 1 H-MRS), positron emission tomography (PET), single photon emission computed tomography (SPECT), schizophrenia and psychosis. Sixteen GABA 1 H-MRS studies (538 controls, 526 patients) and seven PET/SPECT studies of GABA A /benzodiazepine receptor (GABA A /BZR) availability (118 controls, 113 patients) were identified. Meta-analyses of 1 H-MRS GABA in the medial prefrontal cortex (mPFC), parietal/occipital cortex (POC) and striatum did not show significant group differences (mFC: g=-0.3, 409 patients, 495 controls, 95% confidence interval (CI): -0.6 to 0.1; POC: g=-0.3, 139 patients, 111 controls, 95% CI: -0.9 to 0.3; striatum: g=-0.004, 123 patients, 95 controls, 95% CI: -0.7 to 0.7). Heterogeneity across studies was high (I 2 >50%), and this was not explained by subsequent moderator or meta-regression analyses. There were insufficient PET/SPECT receptor availability studies for meta-analyses, but a systematic review did not suggest replicable group differences in regional GABA A /BZR availability. The current literature does not reveal consistent alterations in in vivo GABA neuroimaging measures in schizophrenia, as might be hypothesized from animal models and postmortem data. The analysis highlights the need for further GABA neuroimaging studies with improved methodology and addressing potential sources of heterogeneity.

  15. Ghrelin Causes a Decline in GABA Release by Reducing Fatty Acid Oxidation in Cortex.

    PubMed

    Mir, Joan Francesc; Zagmutt, Sebastián; Lichtenstein, Mathieu P; García-Villoria, Judit; Weber, Minéia; Gracia, Ana; Fabriàs, Gemma; Casas, Josefina; López, Miguel; Casals, Núria; Ribes, Antònia; Suñol, Cristina; Herrero, Laura; Serra, Dolors

    2018-02-02

    Lipid metabolism, specifically fatty acid oxidation (FAO) mediated by carnitine palmitoyltransferase (CPT) 1A, has been described to be an important actor of ghrelin action in hypothalamus. However, it is not known whether CPT1A and FAO mediate the effect of ghrelin on the cortex. Here, we show that ghrelin produces a differential effect on CPT1 activity and γ-aminobutyric acid (GABA) metabolism in the hypothalamus and cortex of mice. In the hypothalamus, ghrelin enhances CPT1A activity while GABA transaminase (GABAT) activity, a key enzyme in GABA shunt metabolism, is unaltered. However, in cortex CPT1A activity and GABAT activity are reduced after ghrelin treatment. Furthermore, in primary cortical neurons, ghrelin reduces GABA release through a CPT1A reduction. By using CPT1A floxed mice, we have observed that genetic ablation of CPT1A recapitulates the effect of ghrelin on GABA release in cortical neurons, inducing reductions in mitochondrial oxygen consumption, cell content of citrate and α-ketoglutarate, and GABA shunt enzyme activity. Taken together, these observations indicate that ghrelin-induced changes in CPT1A activity modulate mitochondrial function, yielding changes in GABA metabolism. This evidence suggests that the action of ghrelin on GABA release is region specific within the brain, providing a basis for differential effects of ghrelin in the central nervous system.

  16. The role of Rho-kinase and calcium ions in constriction triggered by ET-1.

    PubMed

    Wiciński, Michał; Szadujkis-Szadurska, Katarzyna; Węclewicz, Mateusz M; Malinowski, Bartosz; Matusiak, Grzegorz; Walczak, Maciej; Wódkiewicz, Eryk; Grześk, Grzegorz; Pawlak-Osińska, Katarzyna

    2018-05-05

    Endothelin-1 (ET-1) is one of the key factors regulating tension of smooth muscles in blood vessels. It is believed that ET-1 plays an important role in pathogenesis of hypertension, and cardiovascular diseases; therefore, research in order to limit ET-1-mediated action is still in progress. The main objective of this paper was to evaluate the role of Rho-kinase in the ET-1-induced constriction of arteries. The analysis also included significance of intra- and extracellular pool of calcium ions in constriction triggered by ET-1. The studies were performed on perfused Wistar rat tail arteries. Concentration response curve (CRC) was determined for ET-1 in the presence of increased concentrations of Rho-kinase inhibitor (Y-27632) and IP3-receptor antagonist (2APB), both in reference to constriction triggered by solely ET-1. Afterwards, the influence of calcium ions present in the perfusion fluid was evaluated in terms of the effect triggered by 2APB and occurring in arteries constricted by ET-1. ET-1, in concentration dependent manner, leads to increase in perfusion pressure. Y-27632 and 2APB lead to shift of the concentration response curve for ET-1 to the right with simultaneously lowered maximum effect. There was no difference in reaction of the artery constricted by ET-1 and treated with 2APB in solution containing calcium and in calcium-free solution. Vasoconstrictive action of endothelin is not significantly dependent on the inflow of extracellular calcium, but it is proportional to inflow of Ca 2+ related to activation of IP3 receptors and to Rho-kinase activity. Copyright © 2018. Published by Elsevier Inc.

  17. The endogenous GABA bioactivity of camel, bovine, goat and human milks.

    PubMed

    Limon, Agenor; Gallegos-Perez, Jose-Luis; Reyes-Ruiz, Jorge M; Aljohi, Mohammad A; Alshanqeeti, Ali S; Miledi, Ricardo

    2014-02-15

    GABA orally administered has several beneficial effects on health, including the regulation of hyperglycaemic states in humans. Those effects are similar to the effects reported for camel milk (CMk); however, it is not known whether compounds with GABAergic activity are present in milk from camels or other species. We determined CMk free-GABA concentration by LS/MS and its bioactivity on human GABA receptors. We found that camel and goat milks have significantly more bioavailable GABA than cow and human milks and are able to activate GABAρ receptors. The relationship between GABA and taurine concentrations suggests that whole camel milk may be more efficient to activate GABAρ1 receptors than goat milk. Because GABAρ receptors are normally found in enteroendocrine cells in the lumen of the digestive tract, these results suggest that GABA in camel and goat milk may participate in GABA-modulated functions of enteroendocrine cells in the GI lumen. Copyright © 2013 Elsevier Ltd. All rights reserved.

  18. Plasticity of rat central inhibitory synapses through GABA metabolism

    PubMed Central

    Engel, Dominique; Pahner, Ingrid; Schulze, Katrin; Frahm, Christiane; Jarry, Hubertus; Ahnert-Hilger, Gudrun; Draguhn, Andreas

    2001-01-01

    The production of the central inhibitory transmitter GABA (γ-aminobutyric acid) varies in response to different patterns of activity. It therefore seems possible that GABA metabolism can determine inhibitory synaptic strength and that presynaptic GABA content is a regulated parameter for synaptic plasticity. We altered presynaptic GABA metabolism in cultured rat hippocampal slices using pharmacological tools. Degradation of GABA by GABA-transaminase (GABA-T) was blocked by γ-vinyl-GABA (GVG) and synthesis of GABA through glutamate decarboxylase (GAD) was suppressed with 3-mercaptopropionic acid (MPA). We measured miniature GABAergic postsynaptic currents (mIPSCs) in CA3 pyramidal cells using the whole-cell patch clamp technique. Elevated intra-synaptic GABA levels after block of GABA-T resulted in increased mIPSC amplitude and frequency. In addition, tonic GABAergic background noise was enhanced by GVG. Electron micrographs from inhibitory synapses identified by immunogold staining for GABA confirmed the enhanced GABA content but revealed no further morphological alterations. The suppression of GABA synthesis by MPA had opposite functional consequences: mIPSC amplitude and frequency decreased and current noise was reduced compared with control. However, we were unable to demonstrate the decreased GABA content in biochemical analyses of whole slices or in electron micrographs. We conclude that the transmitter content of GABAergic vesicles is variable and that postsynaptic receptors are usually not saturated, leaving room for up-regulation of inhibitory synaptic strength. Our data reveal a new mechanism of plasticity at central inhibitory synapses and provide a rationale for the activity-dependent regulation of GABA synthesis in mammals. PMID:11533137

  19. Transglutaminase-dependent RhoA activation and depletion by serotonin in vascular smooth muscle cells.

    PubMed

    Guilluy, Christophe; Rolli-Derkinderen, Malvyne; Tharaux, Pierre-Louis; Melino, Gerry; Pacaud, Pierre; Loirand, Gervaise

    2007-02-02

    The small G protein RhoA plays a major role in several vascular processes and cardiovascular disorders. Here we analyze the mechanisms of RhoA regulation by serotonin (5-HT) in arterial smooth muscle. 5-HT (0.1-10 microM) induced activation of RhoA followed by RhoA depletion at 24-72 h. Inhibition of 5-HT1 receptors reduced the early phase of RhoA activation but had no effect on 5-HT-induced delayed RhoA activation and depletion, which were suppressed by the 5-HT transporter inhibitor fluoxetine and the transglutaminase inhibitor monodansylcadaverin and in type 2 transglutaminase-deficient smooth muscle cells. Coimmunoprecipitations demonstrated that 5-HT associated with RhoA both in vitro and in vivo. This association was calcium-dependent and inhibited by fluoxetine and monodansylcadaverin. 5-HT promotes the association of RhoA with the E3 ubiquitin ligase Smurf1, and 5-HT-induced RhoA depletion was inhibited by the proteasome inhibitor MG132 and the RhoA inhibitor Tat-C3. Simvastatin, the Rho kinase inhibitor Y-27632, small interfering RNA-mediated RhoA gene silencing, and long-term 5-HT stimulation induced Akt activation. In contrast, inhibition of 5-HT-mediated RhoA degradation by MG132 prevented 5-HT-induced Akt activation. Long-term 5-HT stimulation also led to the inhibition of the RhoA/Rho kinase component of arterial contraction. Our data provide evidence that 5-HT, internalized through the 5-HT transporter, is transamidated to RhoA by transglutaminase. Transamidation of RhoA leads to RhoA activation and enhanced proteasomal degradation, which in turn is responsible for Akt activation and contraction inhibition. The observation of transamidation of 5-HT to RhoA in pulmonary artery of hypoxic rats suggests that this process could participate in pulmonary artery remodeling and hypertension.

  20. Relative Neurotoxicity of Ivermectin and Moxidectin in Mdr1ab (−/−) Mice and Effects on Mammalian GABA(A) Channel Activity

    PubMed Central

    Ménez, Cécile; Sutra, Jean-François; Prichard, Roger; Lespine, Anne

    2012-01-01

    The anthelmintics ivermectin (IVM) and moxidectin (MOX) display differences in toxicity in several host species. Entrance into the brain is restricted by the P-glycoprotein (P-gp) efflux transporter, while toxicity is mediated through the brain GABA(A) receptors. This study compared the toxicity of IVM and MOX in vivo and their interaction with GABA(A) receptors in vitro. Drug toxicity was assessed in Mdr1ab(−/−) mice P-gp-deficient after subcutaneous administration of increasing doses (0.11–2.0 and 0.23–12.9 µmol/kg for IVM and MOX in P-gp-deficient mice and half lethal doses (LD50) in wild-type mice). Survival was evaluated over 14-days. In Mdr1ab(−/−) mice, LD50 was 0.46 and 2.3 µmol/kg for IVM and MOX, respectively, demonstrating that MOX was less toxic than IVM. In P-gp-deficient mice, MOX had a lower brain-to-plasma concentration ratio and entered into the brain more slowly than IVM. The brain sublethal drug concentrations determined after administration of doses close to LD50 were, in Mdr1ab(−/−) and wild-type mice, respectively, 270 and 210 pmol/g for IVM and 830 and 740–1380 pmol/g for MOX, indicating that higher brain concentrations are required for MOX toxicity than IVM. In rat α1β2γ2 GABA channels expressed in Xenopus oocytes, IVM and MOX were both allosteric activators of the GABA-induced response. The Hill coefficient was 1.52±0.45 for IVM and 0.34±0.56 for MOX (p<0.001), while the maximum potentiation caused by IVM and MOX relative to GABA alone was 413.7±66.1 and 257.4±40.6%, respectively (p<0.05), showing that IVM causes a greater potentiation of GABA action on this receptor. Differences in the accumulation of IVM and MOX in the brain and in the interaction of IVM and MOX with GABA(A) receptors account for differences in neurotoxicity seen in intact and Mdr1-deficient animals. These differences in neurotoxicity of IVM and MOX are important in considering their use in humans. PMID:23133688

  1. Expression and distribution of Kv4 potassium channel subunits and potassium channel interacting proteins in subpopulations of interneurons in the basolateral amygdala.

    PubMed

    Dabrowska, J; Rainnie, D G

    2010-12-15

    The Kv4 potassium channel α subunits, Kv4.1, Kv4.2, and Kv4.3, determine some of the fundamental physiological properties of neurons in the CNS. Kv4 subunits are associated with auxiliary β-subunits, such as the potassium channel interacting proteins (KChIP1 - 4), which are thought to regulate the trafficking and gating of native Kv4 potassium channels. Intriguingly, KChIP1 is thought to show cell type-selective expression in GABA-ergic inhibitory interneurons, while other β-subunits (KChIP2-4) are associated with principal glutamatergic neurons. However, nothing is known about the expression of Kv4 family α- and β-subunits in specific interneurons populations in the BLA. Here, we have used immunofluorescence, co-immunoprecipitation, and Western Blotting to determine the relative expression of KChIP1 in the different interneuron subtypes within the BLA, and its co-localization with one or more of the Kv4 α subunits. We show that all three α-subunits of Kv4 potassium channel are found in rat BLA neurons, and that the immunoreactivity of KChIP1 closely resembles that of Kv4.3. Indeed, Kv4.3 showed almost complete co-localization with KChIP1 in the soma and dendrites of a distinct subpopulation of BLA neurons. Dual-immunofluorescence studies revealed this to be in BLA interneurons immunoreactive for parvalbumin, cholecystokin-8, and somatostatin. Finally, co-immunoprecipitation studies showed that KChIP1 was associated with all three Kv4 α subunits. Together our results suggest that KChIP1 is selectively expressed in BLA interneurons where it may function to regulate the activity of A-type potassium channels. Hence, KChIP1 might be considered as a cell type-specific regulator of GABAergic inhibitory circuits in the BLA. Copyright © 2010 IBRO. Published by Elsevier Ltd. All rights reserved.

  2. Inhibition of Rho Is Required for cAMP-induced Melanoma Cell Differentiation

    PubMed Central

    Buscà, Roser; Bertolotto, Corine; Abbe, Patricia; Englaro, Walter; Ishizaki, Toshimasa; Narumiya, Shuh; Boquet, Patrice; Ortonne, Jean-Paul; Ballotti, Robert

    1998-01-01

    Up-regulation of the cAMP pathway by forskolin or α-melanocyte stimulating hormone induces melanocyte and melanoma cell differentiation characterized by stimulation of melanin synthesis and dendrite development. Here we show that forskolin-induced dendricity is associated to a disassembly of actin stress fibers. Since Rho controls actin organization, we studied the role of this guanosine triphosphate (GTP)-binding protein in cAMP-induced dendrite formation. Clostridium botulinum C3 exotransferase, which inhibits Rho, mimicked the effect of forskolin in promoting dendricity and stress fiber disruption, while the Escherichia coli toxin cytotoxic necrotizing factor-1 (CNF-1), which activates Rho and the expression of a constitutively active Rho mutant, blocked forskolin-induced dendrite outgrowth. In addition, overexpression of a constitutively active form of the Rho target p160 Rho-kinase (P160ROCK) prevented the dendritogenic effects of cAMP. Our results suggest that inhibition of Rho and of its target p160ROCK are required events for cAMP-induced dendrite outgrowth in B16 cells. Furthermore, we present evidence that Rho is involved in the regulation of melanogenesis. Indeed, Rho inactivation enhanced the cAMP stimulation of tyrosinase gene transcription and protein expression, while Rho constitutive activation impaired these cAMP-induced effects. This reveals that, in addition to controlling dendricity, Rho also participates in the regulation of melanin synthesis by cAMP. PMID:9614180

  3. Deciphering the function of the CNGB1b subunit in olfactory CNG channels.

    PubMed

    Nache, Vasilica; Wongsamitkul, Nisa; Kusch, Jana; Zimmer, Thomas; Schwede, Frank; Benndorf, Klaus

    2016-07-11

    Olfactory cyclic nucleotide-gated (CNG) ion channels are key players in the signal transduction cascade of olfactory sensory neurons. The second messengers cAMP and cGMP directly activate these channels, generating a depolarizing receptor potential. Olfactory CNG channels are composed of two CNGA2 subunits and two modulatory subunits, CNGA4, and CNGB1b. So far the exact role of the modulatory subunits for channel activation is not fully understood. By measuring ligand binding and channel activation simultaneously, we show that in functional heterotetrameric channels not only the CNGA2 subunits and the CNGA4 subunit but also the CNGB1b subunit binds cyclic nucleotides and, moreover, also alone translates this signal to open the pore. In addition, we show that the CNGB1b subunit is the most sensitive subunit in a heterotetrameric channel to cyclic nucleotides and that it accelerates deactivation to a similar extent as does the CNGA4 subunit. In conclusion, the CNGB1b subunit participates in ligand-gated activation of olfactory CNG channels and, particularly, contributes to rapid termination of odorant signal in an olfactory sensory neuron.

  4. Artifacts correction for T1rho imaging with constant amplitude spin-lock

    NASA Astrophysics Data System (ADS)

    Chen, Weitian

    2017-01-01

    T1rho imaging with constant amplitude spin-lock is prone to artifacts in the presence of B1 RF and B0 field inhomogeneity. Despite significant technological progress, improvements on the robustness of constant amplitude spin-lock are necessary in order to use it for routine clinical practice. This work proposes methods to simultaneously correct for B1 RF and B0 field inhomogeneity in constant amplitude spin-lock. By setting the maximum B1 amplitude of the excitation adiabatic pulses equal to the expected constant amplitude spin-lock frequency, the spins become aligned along the effective field throughout the spin-lock process. This results in T1rho-weighted images free of artifacts, despite the spatial variation of the effective field caused by B1 RF and B0 field inhomogeneity. When the pulse is long, the relaxation effect during the adiabatic half passage may result in a non-negligible error in the mono-exponential relaxation model. A two-acquisition approach is presented to solve this issue. Simulation, phantom, and in-vivo scans demonstrate the proposed methods achieve superior image quality compared to existing methods, and that the two-acquisition method is effective in resolving the relaxation effect during the adiabatic half passage.

  5. Reactivation of the chloroplast CF1-ATPase beta subunit by trace amounts of the CF1 alpha subunit suggests a chaperonin-like activity for CF1 alpha.

    PubMed

    Avni, A; Avital, S; Gromet-Elhanan, Z

    1991-04-25

    Incubation of tobacco and lettuce thylakoids with 2 M LiCl in the presence of MgATP removes the beta subunit from their CF1-ATPase (CF1 beta) together with varying amounts of the CF1 alpha subunit (CF1 alpha). These 2 M LiCl extracts, as with the one obtained from spinach thylakoids (Avital, S., and Gromet-Elhanan, Z. (1991) J. Biol. Chem. 266, 7067-7072), could form active hybrid ATPases when reconstituted into inactive beta-less Rhodospirillum rubrum chromatophores. Pure CF1 beta fractions that have been isolated from these extracts could not form such active hybrids by themselves, but could do so when supplemented with trace amounts (less than 5%) of CF1 alpha. A mitochondrial F1-ATPase alpha subunit was recently reported to be a heat-shock protein, having two amino acid sequences that show a highly conserved identity with sequences found in molecular chaperones (Luis, A. M., Alconada, A., and Cuezva, J. M. (1990) J. Biol. Chem. 265, 7713-7716). These sequences are also conserved in CF1 alpha isolated from various plants, but not in F1 beta subunits. The above described reactivation of CF1 beta by trace amounts of CF1 alpha could thus be due to a chaperonin-like function of CF1 alpha, which involves the correct, active folding of isolated pure CF1 beta.

  6. Assessment of Rho GTPase signaling during neurite outgrowth.

    PubMed

    Feltrin, Daniel; Pertz, Olivier

    2012-01-01

    Rho GTPases are key regulators of the cytoskeleton during the process of neurite outgrowth. Based on overexpression of dominant-positive and negative Rho GTPase constructs, the classic view is that Rac1 and Cdc42 are important for neurite elongation whereas RhoA regulates neurite retraction in response to collapsing agents. However, recent work has suggested a much finer control of spatiotemporal Rho GTPase signaling in this process. Understanding this complexity level necessitates a panel of more sensitive tools than previously used. Here, we discuss a novel assay that enables the biochemical fractionation of the neurite from the soma of differentiating N1E-115 neuronal-like cells. This allows for spatiotemporal characterization of a large number of protein components, interactions, and post-translational modifications using classic biochemical and also proteomics approaches. We also provide protocols for siRNA-mediated knockdown of genes and sensitive assays that allow quantitative analysis of the neurite outgrowth process.

  7. Activation of G protein-coupled bile acid receptor, TGR5, induces smooth muscle relaxation via both Epac- and PKA-mediated inhibition of RhoA/Rho kinase pathway.

    PubMed

    Rajagopal, Senthilkumar; Kumar, Divya P; Mahavadi, Sunila; Bhattacharya, Sayak; Zhou, Ruizhe; Corvera, Carlos U; Bunnett, Nigel W; Grider, John R; Murthy, Karnam S

    2013-03-01

    The present study characterized the TGR5 expression and the signaling pathways coupled to this receptor that mediates the relaxation of gastric smooth muscle. TGR5 was detected in gastric muscle cells by RT-PCR and Western blotting. Treatment of cells with the TGR5-selective ligand oleanolic acid (OA) activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3, and increased cAMP levels. OA did not elicit contraction, but caused relaxation of carbachol-induced contraction of gastric muscle cells from wild-type mice, but not tgr5(-/-) mice. OA, but not a selective exchange protein activated by cAMP (Epac) ligand (8-pCPT-2'-O-Me-cAMP), caused phosphorylation of RhoA and the phosphorylation was blocked by the PKA inhibitor, myristoylated PKI, and by the expression of phosphorylation-deficient mutant RhoA (S188A). Both OA and Epac ligand stimulated Ras-related protein 1 (Rap1) and inhibited carbachol (CCh)-induced Rho kinase activity. Expression of RhoA (S188A) or PKI partly reversed the inhibition of Rho kinase activity by OA but had no effect on inhibition by Epac ligand. However, suppression of Rap1 with siRNA blocked the inhibition of Rho kinase by Epac ligand, and partly reversed the inhibition by OA; the residual inhibition was blocked by PKI. Muscle relaxation in response to OA, but not Epac ligand, was partly reversed by PKI. We conclude that activation of TGR5 causes relaxation of gastric smooth muscle and the relaxation is mediated through inhibition of RhoA/Rho kinase pathway via both cAMP/Epac-dependent stimulation of Rap1 and cAMP/PKA-dependent phosphorylation of RhoA at Ser(188). TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids.

  8. Determination of γ-Aminobutyric Acid (GABA) in Rambutan Fruit cv. Rongrian by HPLC-ELSD and Separation of GABA from Rambutan Fruit Using Dowex 50W-X8 Column.

    PubMed

    Meeploy, Maneerat; Deewatthanawong, Rujira

    2016-03-01

    A high-performance liquid chromatography method coupled with an evaporative light scattering detector (ELSD) was validated for the determination of γ-aminobutyric acid (GABA) in rambutan fruit without any sample pretreatment or derivatization. In the concentration range of 0.05-1.0 mg/mL GABA, the ELSD response was linear with a correlation coefficient (r) >0.999. Limit of detection and limit of quantitation were found to be 0.7 and 2.0 µg/mL, respectively. The method enabled the complete separation of GABA in the aqueous extract of rambutan flesh from the impurity peaks at 45.7 min. The recoveries of sample added GABA were obtained in the range of 92.0-99.3%. Intraday and interday relative standard deviations were <5.3%. Repeatability of the extraction process showed the acceptable precision. From the analysis of GABA content in rambutan flesh, 0.71 ± 0.23 mg of GABA was found in 1 g fresh weight. The recovery of GABA after passing through the Dowex 50W-X8 column was 96.65%. The analytical methodology could be potentially applied to the detection and quantification of GABA in other fruits and complex matrices when a sufficient quantity is available. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  9. RhoC and ROCKs regulate cancer cell interactions with endothelial cells.

    PubMed

    Reymond, Nicolas; Im, Jae Hong; Garg, Ritu; Cox, Susan; Soyer, Magali; Riou, Philippe; Colomba, Audrey; Muschel, Ruth J; Ridley, Anne J

    2015-06-01

    RhoC is a member of the Rho GTPase family that is implicated in cancer progression by stimulating cancer cell invasiveness. Here we report that RhoC regulates the interaction of cancer cells with vascular endothelial cells (ECs), a crucial step in the metastatic process. RhoC depletion by RNAi reduces PC3 prostate cancer cell adhesion to ECs, intercalation between ECs as well as transendothelial migration in vitro. Depletion of the kinases ROCK1 and ROCK2, two known RhoC downstream effectors, similarly decreases cancer interaction with ECs. RhoC also regulates the extension of protrusions made by cancer cells on vascular ECs in vivo. Transient RhoC depletion is sufficient to reduce both early PC3 cell retention in the lungs and experimental metastasis formation in vivo. Our results indicate RhoC plays a central role in cancer cell interaction with vascular ECs, which is a critical event for cancer progression. Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.

  10. Involvement of rho-gtpases in fibroblast adhesion and fibronectine fibrillogenesis under stretch

    NASA Astrophysics Data System (ADS)

    Guignandon, A.; Lambert, C.; Rattner, A.; Servotte, S.; Lapiere, C.; Nusgens, B.; Vico, L.

    The Rho family small GTPases play a crucial role in mediating cellular adaptation to mechanical stimulation (MS), and possibly to microgravity (μg), through effects on the cytoskeleton and cell adhesion which is, in turn, mainly regulated by fibronectin fibrillogenesis (FnF). It remains unclear how mechanical stimulation is transduced to the Rho signaling pathways and how it impacts on fibronectin (fbn) fibrillogenesis (FnF). μg (2 days, mission STS-095) led to de-adhesion of fibroblasts and modification of the underlying extracellular matrix. To determine whether GTPases modulated FnF, we generated stable cell lines expressing high level of activated RhoA and Rac1 (QL) as compared to wild type (WI26-WT). After MS application [8% deformation, 1Hz, 15 min., 3 times/day for 1-2 days], we quantified focal adhesion (vinculin, paxillin, FAKY397), f-actin stress fibers (Sf) and FnF with home-developed softwares. We reported that after MS, Sf are more rapidly (30min) formed under the nucleus in Wi26-WT (+100%) and Rac1 (+200%) than in RhoA (+20%). Vinculin & paxillin were only restricted to the cell edge in static conditions and homogeneously distributed after MS in WT and Rac1. The relative area of contacts (vinculin & paxillin) was more dramatically enhanced by MS in Rac1 (+80%) than in WT (+40%) and RhoA (+25%) indicating that new focal contacts are formed under MS and supported the presence of Sf. MS Activation of FAK (FAKY397) was clear in WT and Rac1 and reduced in RhoA. FnF was restricted to cell-cell contacts zone without any change in the relative area of fbn after a 2-days MS. However we found more numerous spots of fbn at the cell center in Rac1 as compared with RhoA & WT suggesting that these fibrillar contacts will grow upon maturation and modulate FnF. The results indicate that MS induces formation of Sf and focal adhesions and enhances FF. RhoA has been shown to induce the formation of Sf and focal adhesions, and Rac1 activation decreases Rho activity in

  11. IKs channels open slowly because KCNE1 accessory subunits slow the movement of S4 voltage sensors in KCNQ1 pore-forming subunits

    PubMed Central

    Ruscic, Katarina J.; Miceli, Francesco; Villalba-Galea, Carlos A.; Dai, Hui; Mishina, Yukiko; Bezanilla, Francisco; Goldstein, Steve A. N.

    2013-01-01

    Human IKs channels activate slowly with the onset of cardiac action potentials to repolarize the myocardium. IKs channels are composed of KCNQ1 (Q1) pore-forming subunits that carry S4 voltage-sensor segments and KCNE1 (E1) accessory subunits. Together, Q1 and E1 subunits recapitulate the conductive and kinetic properties of IKs. How E1 modulates Q1 has been unclear. Investigators have variously posited that E1 slows the movement of S4 segments, slows opening and closing of the conduction pore, or modifies both aspects of electromechanical coupling. Here, we show that Q1 gating current can be resolved in the absence of E1, but not in its presence, consistent with slowed movement of the voltage sensor. E1 was directly demonstrated to slow S4 movement with a fluorescent probe on the Q1 voltage sensor. Direct correlation of the kinetics of S4 motion and ionic current indicated that slowing of sensor movement by E1 was both necessary and sufficient to determine the slow-activation time course of IKs. PMID:23359697

  12. Gα73B is a downstream effector of JAK/STAT signalling and a regulator of Rho1 in Drosophila haematopoiesis.

    PubMed

    Bausek, Nina; Zeidler, Martin P

    2014-01-01

    JAK/STAT signalling regulates many essential developmental processes including cell proliferation and haematopoiesis, whereas its inappropriate activation is associated with the majority of myeloproliferative neoplasias and numerous cancers. Furthermore, high levels of JAK/STAT pathway signalling have also been associated with enhanced metastatic invasion by cancerous cells. Strikingly, gain-of-function mutations in the single Drosophila JAK homologue, Hopscotch, result in haemocyte neoplasia, inappropriate differentiation and the formation of melanised haemocyte-derived 'tumour' masses; phenotypes that are partly orthologous to human gain-of-function JAK2-associated pathologies. Here we show that Gα73B, a novel JAK/STAT pathway target gene, is necessary for JAK/STAT-mediated tumour formation in flies. In addition, although Gα73B does not affect haemocyte differentiation, it does regulate haemocyte morphology and motility under non-pathological conditions. We show that Gα73B is required for constitutive, but not injury-induced, activation of Rho1 and for the localisation of Rho1 into filopodia upon haemocyte activation. Consistent with these results, we also show that Rho1 interacts genetically with JAK/STAT signalling, and that wild-type levels of Rho1 are necessary for tumour formation. Our findings link JAK/STAT transcriptional outputs, Gα73B activity and Rho1-dependent cytoskeletal rearrangements and cell motility, therefore connecting a pathway associated with cancer with a marker indicative of invasiveness. As such, we suggest a mechanism by which JAK/STAT pathway signalling may promote metastasis.

  13. Thermodynamic characterization of two homologous protein complexes: Associations of the semaphorin receptor plexin-B1 RhoGTPase binding domain with Rnd1 and active Rac1

    PubMed Central

    Hota, Prasanta K; Buck, Matthias

    2009-01-01

    Plexin receptors function in response to semaphorin guidance cues in a variety of developmental processes involving cell motility. Interactions with Rho, as well as Ras family small GTPases are critical events in the cell signaling mechanism. We have recently determined the structure of a cytoplasmic domain (RBD) of plexin-B1 and mapped its binding interface with several Rho-GTPases, Rac1, Rnd1, and RhoD. All three GTPases associate with a similar region of this plexin domain, but show different functional behavior in cells. To understand whether thermodynamic properties of the GTPase–RBD interaction contribute to such different behavior, we have examined the interaction at different temperatures, buffer, and pH conditions. Although the binding affinity of both Rnd1 and Rac1 with the plexin-B1 RBD is similar, the detailed thermodynamic properties of the interactions are considerably different. These data suggest that on Rac1 binding to the plexin-B1 RBD, the proteins become more rigid in the complex. By contrast, Rnd1 binding is consistent with unchanged or slightly increased flexibility in one or both proteins. Both GTPases show an appreciable reduction in affinity for the dimeric plexin-B1 RBD indicating that GTPase binding is not cooperative with dimer formation, but that a partial steric hindrance destabilizes the dimer. However, a reduced affinity binding mode to a disulphide stabilized model for the dimeric RBD is also possible. Consistent with cellular studies, the interaction thermodynamics imply that further levels of regulation involving additional binding partners and/or regions outside of the RhoGTPase binding domain are required for receptor activation. PMID:19388051

  14. Glutamate modulation of GABA transport in retinal horizontal cells of the skate

    PubMed Central

    Kreitzer, Matthew A; Andersen, Kristen A; Malchow, Robert Paul

    2003-01-01

    Transport of the amino acid GABA into neurons and glia plays a key role in regulating the effects of GABA in the vertebrate retina. We have examined the modulation of GABA-elicited transport currents of retinal horizontal cells by glutamate, the likely neurotransmitter of vertebrate photoreceptors. Enzymatically isolated external horizontal cells of skate were examined using whole-cell voltage-clamp techniques. GABA (1 mm) elicited an inward current that was completely suppressed by the GABA transport inhibitors tiagabine (10 μm) and SKF89976-A (100 μm), but was unaffected by 100 μm picrotoxin. Prior application of 100 μm glutamate significantly reduced the GABA-elicited current. Glutamate depressed the GABA dose-response curve without shifting the curve laterally or altering the voltage dependence of the current. The ionotropic glutamate receptor agonists kainate and AMPA also reduced the GABA-elicited current, and the effects of glutamate and kainate were abolished by the ionotropic glutamate receptor antagonist 6-cyano-7-nitroquinoxaline. NMDA neither elicited a current nor modified the GABA-induced current, and metabotropic glutamate analogues were also without effect. Inhibition of the GABA-elicited current by glutamate and kainate was reduced when extracellular calcium was removed and when recording pipettes contained high concentrations of the calcium chelator BAPTA. Caffeine (5 mm) and thapsigargin (2 nm), agents known to alter intracellular calcium levels, also reduced the GABA-elicited current, but increases in calcium induced by depolarization alone did not. Our data suggest that glutamate regulates GABA transport in retinal horizontal cells through a calcium-dependent process, and imply a close physical relationship between calcium-permeable glutamate receptors and GABA transporters in these cells. PMID:12562999

  15. Inhibition of RhoA GTPase and the subsequent activation of PTP1B protects cultured hippocampal neurons against amyloid β toxicity

    PubMed Central

    2011-01-01

    Background Amyloid beta (Aβ) is the main agent responsible for the advent and progression of Alzheimer's disease. This peptide can at least partially antagonize nerve growth factor (NGF) signalling in neurons, which may be responsible for some of the effects produced by Aβ. Accordingly, better understanding the NGF signalling pathway may provide clues as to how to protect neurons from the toxic effects of Aβ. Results We show here that Aβ activates the RhoA GTPase by binding to p75NTR, thereby preventing the NGF-induced activation of protein tyrosine phosphatase 1B (PTP1B) that is required for neuron survival. We also show that the inactivation of RhoA GTPase and the activation of PTP1B protect cultured hippocampal neurons against the noxious effects of Aβ. Indeed, either pharmacological inhibition of RhoA with C3 ADP ribosyl transferase or the transfection of cultured neurons with a dominant negative form of RhoA protects cultured hippocampal neurons from the effects of Aβ. In addition, over-expression of PTP1B also prevents the deleterious effects of Aβ on cultured hippocampal neurons. Conclusion Our findings indicate that potentiating the activity of NGF at the level of RhoA inactivation and PTP1B activation may represent a new means to combat the noxious effects of Aβ in Alzheimer's disease. PMID:21294893

  16. Activation of p115-RhoGEF Requires Direct Association of G[alpha subscript 13] and the Dbl Homology Domain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Chen, Zhe; Guo, Liang; Hadas, Jana

    2012-09-05

    RGS-containing RhoGEFs (RGS-RhoGEFs) represent a direct link between the G{sub 12} class of heterotrimeric G proteins and the monomeric GTPases. In addition to the canonical Dbl homology (DH) and pleckstrin homology domains that carry out the guanine nucleotide exchange factor (GEF) activity toward RhoA, these RhoGEFs also possess RGS homology (RH) domains that interact with activated {alpha} subunits of G{sub 12} and G{sub 13}. Although the GEF activity of p115-RhoGEF (p115), an RGS-RhoGEF, can be stimulated by G{alpha}{sub 13}, the exact mechanism of the stimulation has remained unclear. Using combined studies with small angle x-ray scattering, biochemistry, and mutagenesis, wemore » identify an additional binding site for activated G{alpha}{sub 13} in the DH domain of p115. Small angle x-ray scattering reveals that the helical domain of G{alpha}{sub 13} docks onto the DH domain, opposite to the surface of DH that binds RhoA. Mutation of a single tryptophan residue in the {alpha}3b helix of DH reduces binding to activated G{alpha}{sub 13} and ablates the stimulation of p115 by G{alpha}{sub 13}. Complementary mutations at the predicted DH-binding site in the {alpha}B-{alpha}C loop of the helical domain of G{alpha}{sub 13} also affect stimulation of p115 by G{alpha}{sub 13}. Although the GAP activity of p115 is not required for stimulation by G{alpha}{sub 13}, two hydrophobic motifs in RH outside of the consensus RGS box are critical for this process. Therefore, the binding of G{alpha}{sub 13} to the RH domain facilitates direct association of G{alpha}{sub 13} to the DH domain to regulate its exchange activity. This study provides new insight into the mechanism of regulation of the RGS-RhoGEF and broadens our understanding of G protein signaling.« less

  17. GABA from reactive astrocytes impairs memory in mouse models of Alzheimer's disease.

    PubMed

    Jo, Seonmi; Yarishkin, Oleg; Hwang, Yu Jin; Chun, Ye Eun; Park, Mijeong; Woo, Dong Ho; Bae, Jin Young; Kim, Taekeun; Lee, Jaekwang; Chun, Heejung; Park, Hyun Jung; Lee, Da Yong; Hong, Jinpyo; Kim, Hye Yun; Oh, Soo-Jin; Park, Seung Ju; Lee, Hyo; Yoon, Bo-Eun; Kim, YoungSoo; Jeong, Yong; Shim, Insop; Bae, Yong Chul; Cho, Jeiwon; Kowall, Neil W; Ryu, Hoon; Hwang, Eunmi; Kim, Daesoo; Lee, C Justin

    2014-08-01

    In Alzheimer's disease (AD), memory impairment is the most prominent feature that afflicts patients and their families. Although reactive astrocytes have been observed around amyloid plaques since the disease was first described, their role in memory impairment has been poorly understood. Here, we show that reactive astrocytes aberrantly and abundantly produce the inhibitory gliotransmitter GABA by monoamine oxidase-B (Maob) and abnormally release GABA through the bestrophin 1 channel. In the dentate gyrus of mouse models of AD, the released GABA reduces spike probability of granule cells by acting on presynaptic GABA receptors. Suppressing GABA production or release from reactive astrocytes fully restores the impaired spike probability, synaptic plasticity, and learning and memory in the mice. In the postmortem brain of individuals with AD, astrocytic GABA and MAOB are significantly upregulated. We propose that selective inhibition of astrocytic GABA synthesis or release may serve as an effective therapeutic strategy for treating memory impairment in AD.

  18. Partially hydrolyzed guar gum enhances colonic epithelial wound healing via activation of RhoA and ERK1/2.

    PubMed

    Horii, Yusuke; Uchiyama, Kazuhiko; Toyokawa, Yuki; Hotta, Yuma; Tanaka, Makoto; Yasukawa, Zenta; Tokunaga, Makoto; Okubo, Tsutomu; Mizushima, Katsura; Higashimura, Yasuki; Dohi, Osamu; Okayama, Tetsuya; Yoshida, Naohisa; Katada, Kazuhiro; Kamada, Kazuhiro; Handa, Osamu; Ishikawa, Takeshi; Takagi, Tomohisa; Konishi, Hideyuki; Naito, Yuji; Itoh, Yoshito

    2016-07-13

    Healing of the intestinal mucosal epithelium was found to be a critical factor in the treatment of inflammatory bowel disease (IBD). In this study, we provide further evidence that partially hydrolyzed dietary fiber (PHGG) enhances colonic epithelial cell wound healing, and partially characterize the mechanism that governs this process. Young adult mouse colonic (YAMC) epithelial cells were scraped with a 10 μl micro-pipette tip to denude a round of the monolayer and were incubated with PHGG. The area of cell migration was measured using Image J software. Meanwhile, Rho activation assays were utilized to monitor Rho activation levels. To assess in vivo effects, C57B6 mice were treated with DSS for 7 days and then provided food supplemented with PHGG for 8 days. YAMC cells treated with PHGG exhibited significantly enhanced wound healing compared to the control cells; however, this enhancement was inhibited by both Y-27632 (RhoA inhibitor) and U0126 (ERK1/2 inhibitor). Likewise, there was a PHGG-dependent increase in F-actin accumulation and Rho kinase activity that was blocked by U0126. Meanwhile, PHGG-dependent ERK1/2 activity was not inhibited by Y-27632. In the DSS-induced mouse colitis model, animals that received food supplemented with PHGG exhibited significant recovery of the colonic mucosa. In this study, we demonstrate that PHGG promotes colonic epithelial cell wound healing via activation of RhoA, which occurs downstream of ERK1/2 activation. These findings indicate that PHGG could be utilized as a therapeutic agent for patients with intestinal mucosal damage such as those with IBD.

  19. Interaction of 3beta, 5alpha-tetrahydrodeoxycorticosterone in rat and guinea-pig neurons: a comparison of Ca2+ - and GABA(A)-CI- -channel current modulation.

    PubMed

    ffrench-Mullen, J M

    1999-01-01

    A comparison of the interaction of 3beta, 5alpha-tetrahydrodeoxycorticosterone (TDOC) on voltage-gated Ca2+ -and the gamma-aminobutyric receptor (GABA(A)) gated-Cl- -channels was examined in freshly dissociated guinea-pig (GP) and rat hippocampal CA1 neurons and rat hypothalamic ventromedial nucleus (VMN) neurons. The steady-state inhibition of the peak Ca2+ channel current evoked by depolarized steps from -80 to -10 mV by TDOC increased in concentration-dependent manner with IC50 values of 1 and 6 pM for rat and GP CA1 neurons, respectively and 3 nM for rat VMN neurons. TDOC rapidly and reversibly inhibited a fraction (up to 26%) of the total Ca2+ channel current in all neurons. Intracellular dialysis with GDP-beta-S (500 microM) significantly diminished the TDOC inhibition of the Ca2+ channel current, suggesting a G-protein involvement. In neurons isolated from pertussis-toxin-treated animals by chronic intracerebroventricular (1000 ng/24/48 h) infusion, the TDOC inhibition was also significantly diminished, suggesting modulation by the Galphai and/or Galphao G-protein subunits. The peak GABA-gated inward Cl- current was enhanced in both species from 0.1 to 10 microM with the greatest increase (48% at 10 microM) seen in the VMN. There was no difference in the enhancement of the GABA current in the CA1 region of both species. The results show that in contrast to the 3a-series, the 3beta-series weakly enhance the GABA-evoked Cl- current but potently inhibit the Ca2+ channel current. In addition, these results also suggest a common mode of action and a lack of interspecies difference for this steroid.

  20. Interaction of KCNE subunits with the KCNQ1 K+ channel pore

    PubMed Central

    Panaghie, Gianina; Tai, Kwok-Keung; Abbott, Geoffrey W

    2006-01-01

    KCNQ1 α subunits form functionally distinct potassium channels by coassembling with KCNE ancillary subunits MinK and MiRP2. MinK-KCNQ1 channels generate the slowly activating, voltage-dependent cardiac IKs current. MiRP2-KCNQ1 channels form a constitutively active current in the colon. The structural basis for these contrasting channel properties, and the mechanisms of α subunit modulation by KCNE subunits, are not fully understood. Here, scanning mutagenesis located a tryptophan-tolerant region at positions 338–340 within the KCNQ1 pore-lining S6 domain, suggesting an exposed region possibly amenable to interaction with transmembrane ancillary subunits. This hypothesis was tested using concomitant mutagenesis in KCNQ1 and in the membrane-localized ‘activation triplet’ regions of MinK and MiRP2 to identify pairs of residues that interact to control KCNQ1 activation. Three pairs of mutations exerted dramatic effects, ablating channel function or either removing or restoring control of KCNQ1 activation. The results place KCNE subunits close to the KCNQ1 pore, indicating interaction of MiRP2-72 with KCNQ1-338; and MinK-59,58 with KCNQ1-339, 340. These data are consistent either with perturbation of the S6 domain by MinK or MiRP2, dissimilar positioning of MinK and MiRP2 within the channel complex, or both. Further, the results suggest specifically that two of the interactions, MiRP2-72/KCNQ1-338 and MinK-58/KCNQ1-340, are required for the contrasting gating effects of MinK and MiRP2. PMID:16308347

  1. β-Subunits of the SnRK1 Complexes Share a Common Ancestral Function Together with Expression and Function Specificities; Physical Interaction with Nitrate Reductase Specifically Occurs via AKINβ1-Subunit1[C][OA

    PubMed Central

    Polge, Cécile; Jossier, Mathieu; Crozet, Pierre; Gissot, Lionel; Thomas, Martine

    2008-01-01

    The SNF1/AMPK/SnRK1 kinases are evolutionary conserved kinases involved in yeast, mammals, and plants in the control of energy balance. These heterotrimeric enzymes are composed of one α-type catalytic subunit and two γ- and β-type regulatory subunits. In yeast it has been proposed that the β-type subunits regulate both the localization of the kinase complexes within the cell and the interaction of the kinases with their targets. In this work, we demonstrate that the three β-type subunits of Arabidopsis (Arabidopsis thaliana; AKINβ1, AKINβ2, and AKINβ3) restore the growth phenotype of the yeast sip1Δsip2Δgal83Δ triple mutant, thus suggesting the conservation of an ancestral function. Expression analyses, using AKINβ promoter∷β-glucuronidase transgenic lines, reveal different and specific patterns of expression for each subunit according to organs, developmental stages, and environmental conditions. Finally, our results show that the β-type subunits are involved in the specificity of interaction of the kinase with the cytosolic nitrate reductase. Together with previous cell-free phosphorylation data, they strongly support the proposal that nitrate reductase is a real target of SnRK1 in the physiological context. Altogether our data suggest the conservation of ancestral basic function(s) together with specialized functions for each β-type subunit in plants. PMID:18768910

  2. Activation of G protein-coupled bile acid receptor, TGR5, induces smooth muscle relaxation via both Epac- and PKA-mediated inhibition of RhoA/Rho kinase pathway

    PubMed Central

    Rajagopal, Senthilkumar; Kumar, Divya P.; Mahavadi, Sunila; Bhattacharya, Sayak; Zhou, Ruizhe; Corvera, Carlos U.; Bunnett, Nigel W.; Grider, John R.

    2013-01-01

    The present study characterized the TGR5 expression and the signaling pathways coupled to this receptor that mediates the relaxation of gastric smooth muscle. TGR5 was detected in gastric muscle cells by RT-PCR and Western blotting. Treatment of cells with the TGR5-selective ligand oleanolic acid (OA) activated Gαs, but not Gαq, Gαi1, Gαi2, or Gαi3, and increased cAMP levels. OA did not elicit contraction, but caused relaxation of carbachol-induced contraction of gastric muscle cells from wild-type mice, but not tgr5−/− mice. OA, but not a selective exchange protein activated by cAMP (Epac) ligand (8-pCPT-2′-O-Me-cAMP), caused phosphorylation of RhoA and the phosphorylation was blocked by the PKA inhibitor, myristoylated PKI, and by the expression of phosphorylation-deficient mutant RhoA (S188A). Both OA and Epac ligand stimulated Ras-related protein 1 (Rap1) and inhibited carbachol (CCh)-induced Rho kinase activity. Expression of RhoA (S188A) or PKI partly reversed the inhibition of Rho kinase activity by OA but had no effect on inhibition by Epac ligand. However, suppression of Rap1 with siRNA blocked the inhibition of Rho kinase by Epac ligand, and partly reversed the inhibition by OA; the residual inhibition was blocked by PKI. Muscle relaxation in response to OA, but not Epac ligand, was partly reversed by PKI. We conclude that activation of TGR5 causes relaxation of gastric smooth muscle and the relaxation is mediated through inhibition of RhoA/Rho kinase pathway via both cAMP/Epac-dependent stimulation of Rap1 and cAMP/PKA-dependent phosphorylation of RhoA at Ser188. TGR5 receptor activation on smooth muscle reveals a novel mechanism for the regulation of gut motility by bile acids. PMID:23275618

  3. Modulation of GABA-stimulated chloride influx into membrane vesicles from rat cerebral cortex by triazolobenzodiazepines

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Obata, T.; Yamamura, H.I.

    1988-01-01

    The effects of triazolobenzodiazepines of GABA-stimulated /sup 36/Cl/sup -/ uptake by membrane vesicles from rat cerebral cortex were examined. Triazolam and alprazolam showed a significant enhancement of GABA-stimulated /sup 36/Cl/sup -/ uptake at 0.01-10 uM. On the other hand, adinazolam showed a small enhancement at 0.1-1 uM followed by a significant inhibition of GABA-stimulated /sup 36/Cl/sup -/ uptake at 100 uM. The enhancement of GABA-stimulated /sup 36/Cl/sup -/ uptake by 1 uM alprazolam was antagonized by Ro15-1788, a benzodiazepine antagonist, but the inhibition of this response by 30 uM adinazolam was not antagonized by Ro15-1788. These results indicate that triazolobenzodiazepinesmore » enhanced GABA-stimulated /sup 36/Cl/sup -/ uptake through benzodiazepine receptors. High concentrations of adinazolam inhibit GABA-stimulated /sup 36/Cl/sup -/ uptake which may be due to the direct blockade of GABA-gated chloride channel. 23 references, 4 figures.« less

  4. Piezoelectric ceramic (PZT) modulates axonal guidance growth of rat cortical neurons via RhoA, Rac1, and Cdc42 pathways.

    PubMed

    Wen, Jianqiang; Liu, Meili

    2014-03-01

    Electrical stimulation is critical for axonal connection, which can stimulate axonal migration and deformation to promote axonal growth in the nervous system. Netrin-1, an axonal guidance cue, can also promote axonal guidance growth, but the molecular mechanism of axonal guidance growth under indirect electric stimulation is still unknown. We investigated the molecular mechanism of axonal guidance growth under piezoelectric ceramic lead zirconate titanate (PZT) stimulation in the primary cultured cortical neurons. PZT induced marked axonal elongation. Moreover, PZT activated the excitatory postsynaptic currents (EPSCs) by increasing the frequency and amplitude of EPSCs of the cortical neurons in patch clamp assay. PZT downregulated the expression of Netrin-1 and its receptor Deleted in Colorectal Cancer (DCC). Rho GTPase signaling is involved in interactions of Netrin-1 and DCC. PZT activated RhoA. Dramatic decrease of Cdc42 and Rac1 was also observed after PZT treatment. RhoA inhibitor Clostridium botulinum C3 exoenzyme (C3-Exo) prevented the PZT-induced downregulation of Netrin-1 and DCC. We suggest that PZT can promote axonal guidance growth by downregulation of Netrin-1 and DCC to mediate axonal repulsive responses via the Rho GTPase signaling pathway. Obviously, piezoelectric materials may provide a new approach for axonal recovery and be beneficial for clinical therapy in the future.

  5. Activation of VTA GABA neurons disrupts reward consumption

    PubMed Central

    van Zessen, Ruud; Phillips, Jana L.; Budygin, Evgeny A.; Stuber, Garret D.

    2012-01-01

    The activity of Ventral Tegmental Area (VTA) dopamine (DA) neurons promotes behavioral responses to rewards and environmental stimuli that predict them. VTA GABA inputs synapse directly onto DA neurons and may regulate DA neuronal activity to alter reward-related behaviors, however, the functional consequences of selective activation of VTA GABA neurons remains unknown. Here, we show that in vivo optogenetic activation of VTA GABA neurons disrupts reward consummatory behavior, but not conditioned anticipatory behavior in response to reward-predictive cues. In addition, direct activation of VTA GABA projections to the nucleus accumbens (NAc) resulted in detectable GABA release, but did not alter reward consumption. Furthermore, optogenetic stimulation of VTA GABA neurons directly suppressed the activity and excitability of neighboring DA neurons, as well as the release of DA in the NAc, suggesting that the dynamic interplay between VTA DA and GABA neurons can control the initiation and termination of reward-related behaviors. PMID:22445345

  6. The Small GTPase Rac1 Contributes to Extinction of Aversive Memories of Drug Withdrawal by Facilitating GABAA Receptor Endocytosis in the vmPFC.

    PubMed

    Wang, Weisheng; Ju, Yun-Yue; Zhou, Qi-Xin; Tang, Jian-Xin; Li, Meng; Zhang, Lei; Kang, Shuo; Chen, Zhong-Guo; Wang, Yu-Jun; Ji, Hui; Ding, Yu-Qiang; Xu, Lin; Liu, Jing-Gen

    2017-07-26

    Extinction of aversive memories has been a major concern in neuropsychiatric disorders, such as anxiety disorders and drug addiction. However, the mechanisms underlying extinction of aversive memories are not fully understood. Here, we report that extinction of conditioned place aversion (CPA) to naloxone-precipitated opiate withdrawal in male rats activates Rho GTPase Rac1 in the ventromedial prefrontal cortex (vmPFC) in a BDNF-dependent manner, which determines GABA A receptor (GABA A R) endocytosis via triggering synaptic translocation of activity-regulated cytoskeleton-associated protein (Arc) through facilitating actin polymerization. Active Rac1 is essential and sufficient for GABA A R endocytosis and CPA extinction. Knockdown of Rac1 expression within the vmPFC of rats using Rac1-shRNA suppressed GABA A R endocytosis and CPA extinction, whereas expression of a constitutively active form of Rac1 accelerated GABA A R endocytosis and CPA extinction. The crucial role of GABA A R endocytosis in the LTP induction and CPA extinction is evinced by the findings that blockade of GABA A R endocytosis by a dynamin function-blocking peptide (Myr-P4) abolishes LTP induction and CPA extinction. Thus, the present study provides first evidence that Rac1-dependent GABA A R endocytosis plays a crucial role in extinction of aversive memories and reveals the sequence of molecular events that contribute to learning experience modulation of synaptic GABA A R endocytosis. SIGNIFICANCE STATEMENT This study reveals that Rac1-dependent GABA A R endocytosis plays a crucial role in extinction of aversive memories associated with drug withdrawal and identifies Arc as a downstream effector of Rac1 regulations of synaptic plasticity as well as learning and memory, thereby suggesting therapeutic targets to promote extinction of the unwanted memories. Copyright © 2017 the authors 0270-6474/17/377096-15$15.00/0.

  7. Interaction between mDia1 and ROCK in Rho-induced migration and adhesion of human dental pulp cells.

    PubMed

    Cheng, L; Xu, J; Qian, Y Y; Pan, H Y; Yang, H; Shao, M Y; Cheng, R; Hu, T

    2017-01-01

    To investigate the effects of mammalian homologue of Drosophila diaphanous-1(mDia1) and Rho-associated coiled-coil-containing protein kinase (ROCK) on the migration and adhesion of dental pulp cells (DPCs). Lysophosphatidic acid (LPA) was used to activate Rho signalling. mDia1 and ROCK were inhibited by short interfering RNA and the specific inhibitor, Y-27632, respectively. The migration of DPCs was assessed using the transwell migration assay and scratch test. Formation of cytoskeleton and focal adhesions(FAs) was observed by confocal laser scanning microscopy. Cell adhesion and spreading assays were performed. Phosphorylation of focal adhesion kinase (FAK) and paxillin was detected by Western blotting, and the bands were analysed using Adobe Photoshop CS5 software. All experiments were performed at least three times, and data were analysed with one-way anova and a post hoc test. LPA-triggered activation of Rho and inhibition of ROCK significantly increased the cell migration rate. Cell migration was inhibited by silencing mDia1. mDia1 silencing and ROCK inhibition suppressed the LPA-induced formation of the cytoskeleton, FA and phosphorylation of FAK and paxillin. Inhibition of ROCK or mDia1 facilitated early cell adhesion and spreading; by contrast, the combined inhibition of ROCK and mDia1 neutralized these effects. mDia1 promoted RhoA-induced migration of DPCs, but ROCK had an opposite effect. Both mDia1 and ROCK participated in cytoskeleton formation and adhesion of DPCs. The interactions between mDia1 and ROCK might influence dental pulp repair by determining the migration and adhesion of DPCs. © 2015 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  8. Chemical crosslinking of the subunits of HIV-1 reverse transcriptase.

    PubMed Central

    Debyser, Z.; De Clercq, E.

    1996-01-01

    The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) is composed of two subunits of 66 and 51 kDa in a 1 to 1 ratio. Because dimerization is a prerequisite for enzymatic activity, interference with the dimerization process could constitute an alternative antiviral strategy for RT inhibition. Here we describe an in vitro assay for the study of the dimerization state of HIV-1 reverse transcriptase based on chemical crosslinking of the subunits with dimethylsuberimidate. Crosslinking results in the formation of covalent bonds between the subunits, so that the crosslinked species can be resolved by denaturing gel electrophoresis. Crosslinked RT species with molecular weight greater than that of the dimeric form accumulate during a 1-15-min time course. Initial evidence suggests that those high molecular weight species represent trimers and tetramers and may be the result of intramolecular crosslinking of the subunits of a higher-order RT oligomer. A peptide that corresponds to part of the tryptophan repeat motif in the connection domain of HIV-1 RT inhibits crosslink formation as well as enzymatic activity. The crosslinking assay thus allows the investigation of the effect of inhibitors on the dimerization of HIV-1 RT. PMID:8745406

  9. Structural and molecular characterization of the prefoldin beta subunit from Thermococcus strain KS-1.

    PubMed

    Kida, Hiroshi; Sugano, Yuri; Iizuka, Ryo; Fujihashi, Masahiro; Yohda, Masafumi; Miki, Kunio

    2008-11-14

    Prefoldin (PFD) is a heterohexameric molecular chaperone that is found in eukaryotic cytosol and archaea. PFD is composed of alpha and beta subunits and forms a "jellyfish-like" structure. PFD binds and stabilizes nascent polypeptide chains and transfers them to group II chaperonins for completion of their folding. Recently, the whole genome of Thermococcus kodakaraensis KOD1 was reported and shown to contain the genes of two alpha and two beta subunits of PFD. The genome of Thermococcus strain KS-1 also possesses two sets of alpha (alpha1 and alpha2) and beta subunits (beta1 and beta2) of PFD (TsPFD). However, the functions and roles of each of these PFD subunits have not been investigated in detail. Here, we report the crystal structure of the TsPFD beta1 subunit at 1.9 A resolution and its functional analysis. TsPFD beta1 subunits form a tetramer with four coiled-coil tentacles resembling the jellyfish-like structure of heterohexameric PFD. The beta hairpin linkers of beta1 subunits assemble to form a beta barrel "body" around a central fourfold axis. Size-exclusion chromatography and multi-angle light-scattering analyses show that the beta1 subunits form a tetramer at pH 8.0 and a dimer of tetramers at pH 6.8. The tetrameric beta1 subunits can protect against aggregation of relatively small proteins, insulin or lysozyme. The structural and biochemical analyses imply that PFD beta1 subunits act as molecular chaperones in living cells of some archaea.

  10. Rho-associated kinase is a therapeutic target in neuroblastoma.

    PubMed

    Dyberg, Cecilia; Fransson, Susanne; Andonova, Teodora; Sveinbjörnsson, Baldur; Lännerholm-Palm, Jessika; Olsen, Thale K; Forsberg, David; Herlenius, Eric; Martinsson, Tommy; Brodin, Bertha; Kogner, Per; Johnsen, John Inge; Wickström, Malin

    2017-08-08

    Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. Here we show that neuroblastoma harbors frequent mutations of genes controlling the Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. RhoA is activated in tumors from neuroblastoma patients, and elevated expression of Rho-associated kinase (ROCK)2 is associated with poor patient survival. Pharmacological or genetic inhibition of ROCK1 and 2, key molecules in Rho signaling, resulted in neuroblastoma cell differentiation and inhibition of neuroblastoma cell growth, migration, and invasion. Molecularly, ROCK inhibition induced glycogen synthase kinase 3β-dependent phosphorylation and degradation of MYCN protein. Small-molecule inhibition of ROCK suppressed MYCN -driven neuroblastoma growth in TH- MYCN homozygous transgenic mice and MYCN gene-amplified neuroblastoma xenograft growth in nude mice. Interference with Rho/Rac signaling might offer therapeutic perspectives for high-risk neuroblastoma.

  11. Rho-associated kinase is a therapeutic target in neuroblastoma

    PubMed Central

    Dyberg, Cecilia; Fransson, Susanne; Andonova, Teodora; Sveinbjörnsson, Baldur; Lännerholm-Palm, Jessika; Olsen, Thale K.; Martinsson, Tommy; Brodin, Bertha; Kogner, Per; Johnsen, John Inge

    2017-01-01

    Neuroblastoma is a peripheral neural system tumor that originates from the neural crest and is the most common and deadly tumor of infancy. Here we show that neuroblastoma harbors frequent mutations of genes controlling the Rac/Rho signaling cascade important for proper migration and differentiation of neural crest cells during neuritogenesis. RhoA is activated in tumors from neuroblastoma patients, and elevated expression of Rho-associated kinase (ROCK)2 is associated with poor patient survival. Pharmacological or genetic inhibition of ROCK1 and 2, key molecules in Rho signaling, resulted in neuroblastoma cell differentiation and inhibition of neuroblastoma cell growth, migration, and invasion. Molecularly, ROCK inhibition induced glycogen synthase kinase 3β-dependent phosphorylation and degradation of MYCN protein. Small-molecule inhibition of ROCK suppressed MYCN-driven neuroblastoma growth in TH-MYCN homozygous transgenic mice and MYCN gene-amplified neuroblastoma xenograft growth in nude mice. Interference with Rho/Rac signaling might offer therapeutic perspectives for high-risk neuroblastoma. PMID:28739902

  12. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development.

    PubMed

    Beier, Anna; Teichert, Ines; Krisp, Christoph; Wolters, Dirk A; Kück, Ulrich

    2016-06-21

    The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. The striatin-interacting phosphatase and kinase (STRIPAK) complex is highly conserved from yeasts to humans and is an important regulator of numerous eukaryotic developmental processes, such as cellular signaling and cell development. Although functional insights into the STRIPAK complex are accumulating, the detailed molecular mechanisms of single subunits are only partially understood

  13. Structural Basis of Membrane Targeting by the Dock180 Family of Rho Family Guanine Exchange Factors (Rho-GEFs)*

    PubMed Central

    Premkumar, Lakshmanane; Bobkov, Andrey A.; Patel, Manishha; Jaroszewski, Lukasz; Bankston, Laurie A.; Stec, Boguslaw; Vuori, Kristiina; Côté, Jean-Francois; Liddington, Robert C.

    2010-01-01

    The Dock180 family of atypical Rho family guanine nucleotide exchange factors (Rho-GEFs) regulate a variety of processes involving cellular or subcellular polarization, including cell migration and phagocytosis. Each contains a Dock homology region-1 (DHR-1) domain that is required to localize its GEF activity to a specific membrane compartment where levels of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) are up-regulated by the local activity of PtdIns 3-kinase. Here we define the structural and energetic bases of phosphoinositide specificity by the DHR-1 domain of Dock1 (a GEF for Rac1), and show that DHR-1 utilizes a C2 domain scaffold and surface loops to create a basic pocket on its upper surface for recognition of the PtdIns(3,4,5)P3 head group. The pocket has many of the characteristics of those observed in pleckstrin homology domains. We show that point mutations in the pocket that abolish phospholipid binding in vitro ablate the ability of Dock1 to induce cell polarization, and propose a model that brings together recent mechanistic and structural studies to rationalize the central role of DHR-1 in dynamic membrane targeting of the Rho-GEF activity of Dock180. PMID:20167601

  14. RhoA/ROCK downregulates FPR2-mediated NADPH oxidase activation in mouse bone marrow granulocytes.

    PubMed

    Filina, Julia V; Gabdoulkhakova, Aida G; Safronova, Valentina G

    2014-10-01

    Polymorphonuclear neutrophils (PMNs) express the high and low affinity receptors to formylated peptides (mFPR1 and mFPR2 in mice, accordingly). RhoA/ROCK (Rho activated kinase) pathway is crucial for cell motility and oxidase activity regulated via FPRs. There are contradictory data on RhoA-mediated regulation of NADPH oxidase activity in phagocytes. We have shown divergent Rho GTPases signaling via mFPR1 and mFPR2 to NADPH oxidase in PMNs from inflammatory site. The present study was aimed to find out the role of RhoA/ROCK in the respiratory burst activated via mFPR1 and mFPR2 in the bone marrow PMNs. Different kinetics of RhoA activation were detected with 0.1μM fMLF and 1μM WKYMVM operating via mFPR1 and mFPR2, accordingly. RhoA was translocated in fMLF-activated cells towards the cell center and juxtamembrane space versus uniform allocation in the resting cells. Specific inhibition of RhoA by CT04, Rho inhibitor I, weakly depressed the respiratory burst induced via mFPR1, but significantly increased the one induced via mFPR2. Inhibition of ROCK, the main effector of RhoA, by Y27632 led to the same effect on the respiratory burst. Regulation of mFPR2-induced respiratory response by ROCK was impossible under the cytoskeleton disruption by cytochalasin D, whereas it persisted in the case of mFPR1 activation. Thus we suggest RhoA to be one of the regulatory and signal transduction components in the respiratory burst through FPRs in the mouse bone marrow PMNs. Both mFPR1 and mFPR2 binding with a ligand trigger the activation of RhoA. FPR1 signaling through RhoA/ROCK increases NADPH-oxidase activity. But in FPR2 action RhoA/ROCK together with cytoskeleton-linked systems down-regulates NADPH-oxidase. This mechanism could restrain the reactive oxygen species dependent damage of own tissues during the chemotaxis of PMNs and in the resting cells. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. GABA-B receptor activation and conflict behavior

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ketelaars, C.E.J.; Bollen, E.L.; Rigter, H.

    1988-01-01

    Baclofen and oxazepam enhance extinction of conflict behavior in the Geller-Seifter test while baclofen and diazepam release punished behavior in Vogel's conflict test. In order to investigate the possibility that the effect of the selective GABA-B receptor agonist baclofen is mediated indirectly via the GABA-A/benzodiazepine receptor complex, the effect of pretreatment of rats with baclofen on (/sup 3/H)-diazepam binding to washed and unwashed cortical and cerebellar membranes of rats has been studied. Baclofen pretreatment increase Bmax in washed cerebellar membranes when bicuculline was present in the incubation mixture. No effect was seen in cortical membranes. The present results render itmore » unlikely that the effect of baclofen on extinction of conflict behavior and punished drinking is mediated via the GABA-A/benzodiazepine receptor complex. 50 references, 1 figure, 4 tables.« less

  16. Production of gaba (γ - Aminobutyric acid) by microorganisms: a review.

    PubMed

    Dhakal, Radhika; Bajpai, Vivek K; Baek, Kwang-Hyun

    2012-10-01

    GABA (γ-aminobutyric acid) is a four carbon non-protein amino acid that is widely distributed in plants, animals and microorganisms. As a metabolic product of plants and microorganisms produced by the decarboxylation of glutamic acid, GABA functions as an inhibitory neurotransmitter in the brain that directly affects the personality and the stress management. A wide range of traditional foods produced by microbial fermentation contain GABA, in which GABA is safe and eco-friendly, and also has the possibility of providing new health-benefited products enriched with GABA. Synthesis of GABA is catalyzed by glutamate decarboxylase, therefore, the optimal fermentation condition is mainly based on the biochemical properties of the enzyme. Major GABA producing microorganisms are lactic acid bacteria (LAB), which make food spoilage pathogens unable to grow and act as probiotics in the gastrointestinal tract. The major factors affecting the production of GABA by microbial fermentation are temperature, pH, fermentation time and different media additives, therefore, these factors are summarized to provide the most up-dated information for effective GABA synthesis. There has been a huge accumulation of knowledge on GABA application for human health accompanying with a demand on natural GABA supply. Only the GABA production by microorganisms can fulfill the demand with GABA-enriched health beneficial foods.

  17. Activation of Rho GTPases by Cytotoxic Necrotizing Factor 1 Induces Macropinocytosis and Scavenging Activity in Epithelial Cells

    PubMed Central

    Fiorentini, Carla; Falzano, Loredana; Fabbri, Alessia; Stringaro, Annarita; Logozzi, Mariaantonia; Travaglione, Sara; Contamin, Stéphanette; Arancia, Giuseppe; Malorni, Walter; Fais, Stefano

    2001-01-01

    Macropinocytosis, a ruffling-driven process that allows the capture of large material, is an essential aspect of normal cell function. It can be either constitutive, as in professional phagocytes where it ends with the digestion of captured material, or induced, as in epithelial cells stimulated by growth factors. In this case, the internalized material recycles back to the cell surface. We herein show that activation of Rho GTPases by a bacterial protein toxin, the Escherichia coli cytotoxic necrotizing factor 1 (CNF1), allowed epithelial cells to engulf and digest apoptotic cells in a manner similar to that of professional phagocytes. In particular, we have demonstrated that 1) the activation of all Rho, Rac, and Cdc42 by CNF1 was essential for the capture and internalization of apoptotic cells; and 2) such activation allowed the discharge of macropinosomal content into Rab7 and lysosomal associated membrane protein-1 acidic lysosomal vesicles where the ingested particles underwent degradation. Taken together, these findings indicate that CNF1-induced “switching on” of Rho GTPases may induce in epithelial cells a scavenging activity, comparable to that exerted by professional phagocytes. The activation of such activity in epithelial cells may be relevant, in mucosal tissues, in supporting or integrating the scavenging activity of resident macrophages. PMID:11452003

  18. P120-Catenin Protects Endplate Chondrocytes From Intermittent Cyclic Mechanical Tension Induced Degeneration by Inhibiting the Expression of RhoA/ROCK-1 Signaling Pathway.

    PubMed

    Xu, Hong-Guang; Ma, Ming-Ming; Zheng, Quan; Shen, Xiang; Wang, Hong; Zhang, Shu-Feng; Xu, Jia-Jia; Wang, Chuan-Dong; Zhang, Xiao-Ling

    2016-08-15

    The changes of endplate chondrocytes induced by intermittent cyclic mechanical tension (ICMT) were observed by realtime reverse transcription-polymerase chain reaction, immunofluorescence, and Western blot analysis. To investigate the role of RhoA/ROCK-1 signaling pathway and E-cadherin/P120-catenin complex in endplate chondrocytes degeneration induced by ICMT. ICMT can induce the endplate chondrocyte degeneration. However, the relationship between P120-catenin or RhoA/ROCK-1 signaling pathway and endplate chondrocytes degeneration induced by ICMT is not clear. ICMT (strain at 0.5 Hz sinusoidal curve at 8% elongation) was applied to rat endplate chondrocytes for 6 days, 16 hours a day. The cell viability and apoptosis were examined by the LIVE/DEAD assay and flow cytometry. Histological staining was used to examine the lumbar disc tissue morphology and extracellular matrix. To regulate RhoA/ROCK-1 signaling pathway and the expression of E-cadherin and P120-catenin, RhoA/ROCK-1 pathway-specific inhibitors, E-cadherin, and p120-catenin plasmid were applied. Coimmunoprecipitation was employed to examine the interaction between E-cadherin and P120-catenin, P120-catenin, and RhoA. The related gene expression and protein location was examined by realtime reverse transcription-polymerase chain reaction, Western blot, and immunofluorescence. There was no change of viability verified by LIVE/DEAD assay and flow cytometry after ICMT loading. ICMT loading led to RhoA/ROCK-1 signaling activation and the loss of the chondrogenic phenotype of endplate chondrocytes. Inhibition of RhoA/ROCK-1 signaling pathway significantly ameliorated the degeneration induced by ICMT. The expression of P120-catenin and E-cadherin were inhibited by ICMT. ICMT reduced the interaction between P120-catenin and E-cadherin. Furthermore, over-expression of P120-catenin and E-cadherin can suppress the expression of chondrogenic gene, over-expression of P120-catenin can suppress the RhoA/ROCK-1

  19. Escherichia coli cytotoxic necrotizing factor 1: evidence for induction of actin assembly by constitutive activation of the p21 Rho GTPase.

    PubMed Central

    Fiorentini, C; Donelli, G; Matarrese, P; Fabbri, A; Paradisi, S; Boquet, P

    1995-01-01

    Cytotoxic necrotizing factor type 1 (CNF1) induces in HEp-2 cells an increase in F-actin structures, which was detectable by fluorescence-activated cell sorter analysis 24 h after addition of this factor to the culture medium. Increase in F-actin was correlated with the augmentation of both the cell volume and the total cell actin content. Actin assembly-disassembly is controlled by small GTP-binding proteins of the Rho family, which have been reported recently to be modified by CNF1 treatment. Clostridium difficile toxin B and Clostridium botulinum exoenzyme C3, both known to act on the Rho GTPase, were used as biological tools to study the effect of CNF1 on this protein. CNF1 incubated before, during, or after exposure to the chimeric toxin C3B (which is the product of a genetic fusion between the DNA coding for C3 and the one coding for the B fragment of diphtheria toxin) protected HEp-2 cells from the disruption of F-actin structures caused by inactivation of the Rho GTPase through its ADP-ribosylation. On the other hand, C. difficile toxin B cytopathic effect was not observed upon preincubation of cells with CNF1. Toxins acting through a Rho-independent mechanism, such as cytochalasin D and Clostridium spiroforme iota-like toxin, could not be modified in their cellular activities by CNF1 treatment. All of our results suggest that CNF1 modifies the Rho molecule, thus probably protecting this GTPase from further bacterial toxin modification. PMID:7558302

  20. Escherichia coli cytotoxic necrotizing factor 1: evidence for induction of actin assembly by constitutive activation of the p21 Rho GTPase.

    PubMed

    Fiorentini, C; Donelli, G; Matarrese, P; Fabbri, A; Paradisi, S; Boquet, P

    1995-10-01

    Cytotoxic necrotizing factor type 1 (CNF1) induces in HEp-2 cells an increase in F-actin structures, which was detectable by fluorescence-activated cell sorter analysis 24 h after addition of this factor to the culture medium. Increase in F-actin was correlated with the augmentation of both the cell volume and the total cell actin content. Actin assembly-disassembly is controlled by small GTP-binding proteins of the Rho family, which have been reported recently to be modified by CNF1 treatment. Clostridium difficile toxin B and Clostridium botulinum exoenzyme C3, both known to act on the Rho GTPase, were used as biological tools to study the effect of CNF1 on this protein. CNF1 incubated before, during, or after exposure to the chimeric toxin C3B (which is the product of a genetic fusion between the DNA coding for C3 and the one coding for the B fragment of diphtheria toxin) protected HEp-2 cells from the disruption of F-actin structures caused by inactivation of the Rho GTPase through its ADP-ribosylation. On the other hand, C. difficile toxin B cytopathic effect was not observed upon preincubation of cells with CNF1. Toxins acting through a Rho-independent mechanism, such as cytochalasin D and Clostridium spiroforme iota-like toxin, could not be modified in their cellular activities by CNF1 treatment. All of our results suggest that CNF1 modifies the Rho molecule, thus probably protecting this GTPase from further bacterial toxin modification.

  1. Rho GTPases at the crossroad of signaling networks in mammals: impact of Rho-GTPases on microtubule organization and dynamics.

    PubMed

    Wojnacki, José; Quassollo, Gonzalo; Marzolo, María-Paz; Cáceres, Alfredo

    2014-01-01

    Microtubule (MT) organization and dynamics downstream of external cues is crucial for maintaining cellular architecture and the generation of cell asymmetries. In interphase cells RhoA, Rac, and Cdc42, conspicuous members of the family of small Rho GTPases, have major roles in modulating MT stability, and hence polarized cell behaviors. However, MTs are not mere targets of Rho GTPases, but also serve as signaling platforms coupling MT dynamics to Rho GTPase activation in a variety of cellular conditions. In this article, we review some of the key studies describing the reciprocal relationship between small Rho-GTPases and MTs during migration and polarization.

  2. Design and Mechanism of Tetrahydrothiophene-based GABA Aminotransferase Inactivators

    PubMed Central

    Le, Hoang V.; Hawker, Dustin D.; Wu, Rui; Doud, Emma; Widom, Julia; Sanishvili, Ruslan; Liu, Dali; Kelleher, Neil L.; Silverman, Richard B.

    2015-01-01

    Low levels of γ-aminobutyric acid (GABA), one of two major neurotransmitters that regulate brain neuronal activity, are associated with many neurological disorders, such as epilepsy, Parkinson’s disease, Alzheimer’s disease, Huntington’s disease, and cocaine addiction. One of the main methods to raise the GABA level in human brain is to use small molecules that cross the blood-brain barrier and inhibit the activity of γ-aminobutyric acid aminotransferase (GABA-AT), the enzyme that degrades GABA. We have designed a series of conformationally-restricted, tetrahydrothiophene-based GABA analogs with a properly-positioned leaving group that could facilitate a ring-opening mechanism, leading to inactivation of GABA-AT. One compound in the series is eight times more efficient an inactivator of GABA-AT than vigabatrin, the only FDA-approved inactivator of GABA-AT. Our mechanistic studies show that the compound inactivates GABA-AT by a new mechanism. The metabolite resulting from inactivation does not covalently bind to amino acid residues of GABA-AT but stays in the active site via H-bond interactions with Arg-192, a π-π interaction with Phe-189, and a weak nonbonded S···O=C interaction with Glu-270, thereby inactivating the enzyme. PMID:25781189

  3. Src family kinases mediate the inhibition of substance P release in the rat spinal cord by μ-opioid receptors and GABA(B) receptors, but not α2 adrenergic receptors.

    PubMed

    Zhang, Guohua; Chen, Wenling; Marvizón, Juan Carlos G

    2010-09-01

    GABA(B) , μ-opioid and adrenergic α(2) receptors inhibit substance P release from primary afferent terminals in the dorsal horn. Studies in cell expression systems suggest that μ-opioid and GABA(B) receptors inhibit transmitter release from primary afferents by activating Src family kinases (SFKs), which then phosphorylate and inhibit voltage-gated calcium channels. This study investigated whether SFKs mediate the inhibition of substance P release by these three receptors. Substance P release was measured as neurokinin 1 receptor (NK1R) internalization in spinal cord slices and in vivo. In slices, NK1R internalization induced by high-frequency dorsal root stimulation was inhibited by the μ-opioid agonist DAMGO and the GABA(B) agonist baclofen. This inhibition was reversed by the SFK inhibitor PP1. NK1R internalization induced by low-frequency stimulation was also inhibited by DAMGO, but PP1 did not reverse this effect. In vivo, NK1R internalization induced by noxious mechanical stimulation of the hind paw was inhibited by intrathecal DAMGO and baclofen. This inhibition was reversed by intrathecal PP1, but not by the inactive PP1 analog PP3. PP1 produced no effect by itself. The α(2) adrenergic agonists medetomidine and guanfacine produced a small but statistically significant inhibition of NK1R internalization induced by low-frequency dorsal root stimulation. PP1 did not reverse the inhibition by guanfacine. These results show that SFKs mediate the inhibition of substance P release by μ-opioid and GABA(B) receptors, but not by α(2) receptors, which is probably mediated by the binding of G protein βγ subunits to calcium channels. European Journal of Neuroscience © 2010 Federation of European Neuroscience Societies and Blackwell Publishing Ltd. No claim to original US government works.

  4. Big GABA: Edited MR spectroscopy at 24 research sites.

    PubMed

    Mikkelsen, Mark; Barker, Peter B; Bhattacharyya, Pallab K; Brix, Maiken K; Buur, Pieter F; Cecil, Kim M; Chan, Kimberly L; Chen, David Y-T; Craven, Alexander R; Cuypers, Koen; Dacko, Michael; Duncan, Niall W; Dydak, Ulrike; Edmondson, David A; Ende, Gabriele; Ersland, Lars; Gao, Fei; Greenhouse, Ian; Harris, Ashley D; He, Naying; Heba, Stefanie; Hoggard, Nigel; Hsu, Tun-Wei; Jansen, Jacobus F A; Kangarlu, Alayar; Lange, Thomas; Lebel, R Marc; Li, Yan; Lin, Chien-Yuan E; Liou, Jy-Kang; Lirng, Jiing-Feng; Liu, Feng; Ma, Ruoyun; Maes, Celine; Moreno-Ortega, Marta; Murray, Scott O; Noah, Sean; Noeske, Ralph; Noseworthy, Michael D; Oeltzschner, Georg; Prisciandaro, James J; Puts, Nicolaas A J; Roberts, Timothy P L; Sack, Markus; Sailasuta, Napapon; Saleh, Muhammad G; Schallmo, Michael-Paul; Simard, Nicholas; Swinnen, Stephan P; Tegenthoff, Martin; Truong, Peter; Wang, Guangbin; Wilkinson, Iain D; Wittsack, Hans-Jörg; Xu, Hongmin; Yan, Fuhua; Zhang, Chencheng; Zipunnikov, Vadim; Zöllner, Helge J; Edden, Richard A E

    2017-10-01

    Magnetic resonance spectroscopy (MRS) is the only biomedical imaging method that can noninvasively detect endogenous signals from the neurotransmitter γ-aminobutyric acid (GABA) in the human brain. Its increasing popularity has been aided by improvements in scanner hardware and acquisition methodology, as well as by broader access to pulse sequences that can selectively detect GABA, in particular J-difference spectral editing sequences. Nevertheless, implementations of GABA-edited MRS remain diverse across research sites, making comparisons between studies challenging. This large-scale multi-vendor, multi-site study seeks to better understand the factors that impact measurement outcomes of GABA-edited MRS. An international consortium of 24 research sites was formed. Data from 272 healthy adults were acquired on scanners from the three major MRI vendors and analyzed using the Gannet processing pipeline. MRS data were acquired in the medial parietal lobe with standard GABA+ and macromolecule- (MM-) suppressed GABA editing. The coefficient of variation across the entire cohort was 12% for GABA+ measurements and 28% for MM-suppressed GABA measurements. A multilevel analysis revealed that most of the variance (72%) in the GABA+ data was accounted for by differences between participants within-site, while site-level differences accounted for comparatively more variance (20%) than vendor-level differences (8%). For MM-suppressed GABA data, the variance was distributed equally between site- (50%) and participant-level (50%) differences. The findings show that GABA+ measurements exhibit strong agreement when implemented with a standard protocol. There is, however, increased variability for MM-suppressed GABA measurements that is attributed in part to differences in site-to-site data acquisition. This study's protocol establishes a framework for future methodological standardization of GABA-edited MRS, while the results provide valuable benchmarks for the MRS community

  5. The activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 to the distal CCAAT box of the RhoB promoter

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Ahn, Jiwon; Department of Microbiology, Chungnam National University, Daejeon 305-764; Choi, Jeong-Hae

    2011-06-03

    Highlights: {yields} Regulation of transcriptional activation of RhoB is still unclear. {yields} We examine the effect of p38 MAPK inhibition, and c-Jun and RhoB depletion on UV-induced RhoB expression and apoptosis. {yields} We identify the regions of RhoB promoter necessary to confer UV responsiveness using pRhoB-luciferase reporter assays. {yields} c-Jun, ATF2 and p300 are dominantly associated with NF-Y on the distal CCAAT box. {yields} The activation of p38 MAPK primarily contribute to UV-induced RhoB expression by recruiting the c-Jun and p300 proteins on distal CCAAT box of RhoB promoter. -- Abstract: The Ras-related small GTP-binding protein RhoB is rapidly inducedmore » in response to genotoxic stresses caused by ionizing radiation. It is known that UV-induced RhoB expression results from the binding of activating transcription factor 2 (ATF2) via NF-Y to the inverted CCAAT box (-23) of the RhoB promoter. Here, we show that the association of c-Jun with the distal CCAAT box (-72) is primarily involved in UV-induced RhoB expression and p38 MAPK regulated RhoB induction through the distal CCAAT box. UV-induced RhoB expression and apoptosis were markedly attenuated by pretreatment with the p38 MAPK inhibitor. siRNA knockdown of RhoB, ATF2 and c-Jun resulted in decreased RhoB expression and eventually restored the growth of UV-irradiated Jurkat cells. In the reporter assay using luciferase under the RhoB promoter, inhibition of RhoB promoter activity by the p38 inhibitor and knockdown of c-Jun using siRNA occurred through the distal CCAAT box. Immunoprecipitation and DNA affinity protein binding assays revealed the association of c-Jun and p300 via NF-YA and the dissociation of histone deacetylase 1 (HDAC1) via c-Jun recruitment to the CCAAT boxes of the RhoB promoter. These results suggest that the activation of p38 MAPK primarily contributes to UV-induced RhoB expression by recruiting the c-Jun and p300 proteins to the distal CCAAT box of the RhoB promoter

  6. Interactions between the bud emergence proteins Bem1p and Bem2p and Rho-type GTPases in yeast.

    PubMed

    Peterson, J; Zheng, Y; Bender, L; Myers, A; Cerione, R; Bender, A

    1994-12-01

    The SH3 domain-containing protein Bem1p is needed for normal bud emergence and mating projection formation, two processes that require asymmetric reorganizations of the cortical cytoskeleton in Saccharomyces cerevisiae. To identify proteins that functionally and/or physically interact with Bem1p, we screened for mutations that display synthetic lethality with a mutant allele of the BEM1 gene and for genes whose products display two-hybrid interactions with the Bem1 protein. CDC24, which is required for bud emergence and encodes a GEF (guanine-nucleotide exchange factor) for the essential Rho-type GTPase Cdc42p, was identified during both screens. The COOH-terminal 75 amino acids of Cdc24p, outside of the GEF domain, can interact with a portion of Bem1p that lacks both SH3 domains. Bacterially expressed Cdc24p and Bem1p bind to each other in vitro, indicating that no other yeast proteins are required for this interaction. The most frequently identified gene that arose from the bem1 synthetic-lethal screen was the bud-emergence gene BEM2 (Bender and Pringle. 1991. Mol. Cell Biol. 11:1295-1395), which is allelic with IPL2 (increase in ploidy; Chan and Botstein, 1993. Genetics. 135:677-691). Here we show that Bem2p contains a GAP (GTPase-activating protein) domain for Rho-type GTPases, and that this portion of Bem2p can stimulate in vitro the GTPase activity of Rho1p, a second essential yeast Rho-type GTPase. Cells deleted for BEM2 become large and multinucleate. These and other genetic, two-hybrid, biochemical, and phenotypic data suggest that multiple Rho-type GTPases control the reorganization of the cortical cytoskeleton in yeast and that the functions of these GTPases are tightly coupled. Also, these findings raise the possibility that Bem1p may regulate or be a target of action of one or more of these GTPases.

  7. Interactions between the bud emergence proteins Bem1p and Bem2p and Rho- type GTPases in yeast

    PubMed Central

    1994-01-01

    The SH3 domain-containing protein Bem1p is needed for normal bud emergence and mating projection formation, two processes that require asymmetric reorganizations of the cortical cytoskeleton in Saccharomyces cerevisiae. To identify proteins that functionally and/or physically interact with Bem1p, we screened for mutations that display synthetic lethality with a mutant allele of the BEM1 gene and for genes whose products display two-hybrid interactions with the Bem1 protein. CDC24, which is required for bud emergence and encodes a GEF (guanine- nucleotide exchange factor) for the essential Rho-type GTPase Cdc42p, was identified during both screens. The COOH-terminal 75 amino acids of Cdc24p, outside of the GEF domain, can interact with a portion of Bem1p that lacks both SH3 domains. Bacterially expressed Cdc24p and Bem1p bind to each other in vitro, indicating that no other yeast proteins are required for this interaction. The most frequently identified gene that arose from the bem1 synthetic-lethal screen was the bud-emergence gene BEM2 (Bender and Pringle. 1991. Mol. Cell Biol. 11:1295-1395), which is allelic with IPL2 (increase in ploidy; Chan and Botstein, 1993. Genetics. 135:677-691). Here we show that Bem2p contains a GAP (GTPase-activating protein) domain for Rho-type GTPases, and that this portion of Bem2p can stimulate in vitro the GTPase activity of Rho1p, a second essential yeast Rho-type GTPase. Cells deleted for BEM2 become large and multinucleate. These and other genetic, two-hybrid, biochemical, and phenotypic data suggest that multiple Rho-type GTPases control the reorganization of the cortical cytoskeleton in yeast and that the functions of these GTPases are tightly coupled. Also, these findings raise the possibility that Bem1p may regulate or be a target of action of one or more of these GTPases. PMID:7962098

  8. Wsc1 and Mid2 Are Cell Surface Sensors for Cell Wall Integrity Signaling That Act through Rom2, a Guanine Nucleotide Exchange Factor for Rho1

    PubMed Central

    Philip, Bevin; Levin, David E.

    2001-01-01

    Wsc1 and Mid2 are highly O-glycosylated cell surface proteins that reside in the plasma membrane of Saccharomyces cerevisiae. They have been proposed to function as mechanosensors of cell wall stress induced by wall remodeling during vegetative growth and pheromone-induced morphogenesis. These proteins are required for activation of the cell wall integrity signaling pathway that consists of the small G-protein Rho1, protein kinase C (Pkc1), and a mitogen-activated protein kinase cascade. We show here by two-hybrid experiments that the C-terminal cytoplasmic domains of Wsc1 and Mid2 interact with Rom2, a guanine nucleotide exchange factor (GEF) for Rho1. At least with regard to Wsc1, this interaction is mediated by the Rom2 N-terminal domain. This domain is distinct from the Rho1-interacting domain, suggesting that the GEF can interact simultaneously with a sensor and with Rho1. We also demonstrate that extracts from wsc1 and mid2 mutants are deficient in the ability to catalyze GTP loading of Rho1 in vitro, providing evidence that the function of the sensor-Rom2 interaction is to stimulate nucleotide exchange toward this G-protein. In a related line of investigation, we identified the PMT2 gene in a genetic screen for mutations that confer an additive cell lysis defect with a wsc1 null allele. Pmt2 is a member of a six-protein family in yeast that catalyzes the first step in O mannosylation of target proteins. We demonstrate that Mid2 is not mannosylated in a pmt2 mutant and that this modification is important for signaling by Mid2. PMID:11113201

  9. Genetics Home Reference: GABA-transaminase deficiency

    MedlinePlus

    ... Chiriboga CA, Ichikawa K, Osaka H, Tsuji M, Gibson KM, Bonnen PE, Pearl PL. Phenotype of GABA- ... Meirleir L, Jaeken J, Jakobs C, Nyhan WL, Gibson KM. 4-Aminobutyrate aminotransferase (GABA-transaminase) deficiency. J ...

  10. Ultra-low field T1 vs. T1rho at 3T and 7T: study of rotationally immobilized protein gels and animal brain tissues

    NASA Astrophysics Data System (ADS)

    Dong, Hui; Inglis, Ben; Barr, Ian; Clarke, John

    2015-03-01

    Clinical magnetic resonance imaging (MRI) machines operating in static fields of typically 1.5 T or 3 T can capture information on slow molecular dynamics utilizing the so-called T1rho technique. This technique, in which a radiofrequency (RF) spin-lock field is applied with microtesla amplitude, has been used, for example, to determine the onset time of stroke in studies on rats. The long RF pulse, however, may exceed the specific absorption rate (SAR) limit, putting subjects at risk. Ultra-low-field (ULF) MRI, based on Superconducting Quantum Interference Devices (SQUIDs), directly detects proton signals at a static magnetic field of typically 50-250 μT. Using our ULF MRI system with adjustable static field of typically 55 to 240 μT, we systematically measured the T1 and T2 dispersion profiles of rotationally immobilized protein gels (bovine serum albumin), ex vivo pig brains, and ex vivo rat brains with induced stroke. Comparing the ULF results with T1rho dispersion obtained at 3 T and 7 T, we find that the degree of protein immobilization determines the frequency-dependence of both T1 and T1rho. Furthermore, T1rho and ULF T1 show similar results for stroke, suggesting that ULF MRI may be used to image traumatic brain injury with negligible SAR. This research was supported by the Henry H. Wheeler, Jr. Brain Imaging Center and the Donaldson Trust.

  11. Flexibility of the zeolite RHO framework. In situ X-ray and neutron powder structural characterization of cation-exchanged BePO and BeAsORHO analogs

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Nenoff, T.M.; Parise, J.B.; Jones, G.A.

    This is an extensive study of the non-aluminosilicate analogs of the zeolite RHO. This molecular sieve is of great interest commercially because of its catalytic properties. In the absence of rigid supporting structural subunits (smaller cages or channels), the aluminosilicate RHO exhibits atypical framework flexibility with large displacivere arrangements. The beryllophosphate and beryalloarsenate analogs are easily synthesized under very mild reaction conditions and therefore maybe of interest for inexpensive and rapid commercial production. However,t hey have decreased thermal stability. In an effort to increase thermal stability and explore framework flexibility, we have synthesized and characterized a series of analogs ofmore » the non-aluminosilicate RHO framework. All materials crystallize in the space group I23, ranging from a = 13.584-(2) A for Li-BePO RHO to a = 14.224(4) A for Ba-RbBeAsO RHO for hydrated phases. The extra framework cations are distributed over the double 8-ring, single 8-ring, and two single 6-ring sites. Partially and fully dehydrated phases were also studied for changes in framework stability. Predictive trends based on the type of cation exchanged into the framework were determined by {sup 9}Be and {sup 31}P MAS NMR. 50 refs., 8 figs., 6 tabs.« less

  12. Coordination of microtubule and microfilament dynamics by Drosophila Rho1, Spire and Cappuccino.

    PubMed

    Rosales-Nieves, Alicia E; Johndrow, James E; Keller, Lani C; Magie, Craig R; Pinto-Santini, Delia M; Parkhurst, Susan M

    2006-04-01

    The actin-nucleation factors Spire and Cappuccino (Capu) regulate the onset of ooplasmic streaming in Drosophila melanogaster. Although this streaming event is microtubule-based, actin assembly is required for its timing. It is not understood how the interaction of microtubules and microfilaments is mediated in this context. Here, we demonstrate that Capu and Spire have microtubule and microfilament crosslinking activity. The spire locus encodes several distinct protein isoforms (SpireA, SpireC and SpireD). SpireD was recently shown to nucleate actin, but the activity of the other isoforms has not been addressed. We find that SpireD does not have crosslinking activity, whereas SpireC is a potent crosslinker. We show that SpireD binds to Capu and inhibits F-actin/microtubule crosslinking, and activated Rho1 abolishes this inhibition, establishing a mechanistic basis for the regulation of Capu and Spire activity. We propose that Rho1, cappuccino and spire are elements of a conserved developmental cassette that is capable of directly mediating crosstalk between microtubules and microfilaments.

  13. Temperature dependence and GABA modulation of (TH)triazolam binding in the rat brain

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Earle, M.E.; Concas, A.; Wamsley, J.K.

    1987-07-27

    The hypnotic triazolam (TZ), a triazolobenzodiazepine displays a short physiological half life and has been used for the treatment of insomnia related to anxiety states. The authors major objectives were the direct measurement of the temperature dependence and the gamma-aminobutyric acid (GABA) effect of (TH)TZ binding in the rat brain. Saturation studies showed a shift to lower affinity with increasing temperatures (K/sub d/ = 0.27 +/- 08 nM at 0C; K/sub d/ = 1.96 +/- 0.85 nM at 37C) while the B/sub max/ values remained unchanged (1220 +/- 176 fmoles/mg protein at 0C and 1160 +/- 383 fmoles/mg protein atmore » 37C). Saturation studies of (TH)TZ binding in the presence or absence of GABA (100 M) showed a GABA-shift. At 0C the K/sub d/ values were (K/sub d/ = 0.24 +/- 0.03 nM/-GABA; K/sub d/ = 0.16 +/- 0.04/+GABA) and at 37C the K/sub d/ values were (K/sub d/ = 1.84 +/- 0.44 nM/-GABA; K/sub d/ = 0.95 +/- 0.29 nM/+GABA). In contrast to reported literature, the authors findings show that TZ interacts with benzodiazepine receptors with a temperature dependence and GABA-shift consistent with predicted behavior for benzodiazepine agonists. 20 references, 3 tables.« less

  14. RhoA/Rho-kinase signaling: a therapeutic target in pulmonary hypertension.

    PubMed

    Barman, Scott A; Zhu, Shu; White, Richard E

    2009-01-01

    Pulmonary arterial hypertension (PAH) is a devastating disease characterized by progressive elevation of pulmonary arterial pressure and vascular resistance due to pulmonary vasoconstriction and vessel remodeling as well as inflammation. Rho-kinases (ROCKs) are one of the best-described effectors of the small G-protein RhoA, and ROCKs are involved in a variety of cellular functions including muscle cell contraction, proliferation and vascular inflammation through inhibition of myosin light chain phosphatase and activation of downstream mediators. A plethora of evidence in animal models suggests that heightened RhoA/ROCK signaling is important in the pathogenesis of pulmonary hypertension by causing enhanced constriction and remodeling of the pulmonary vasculature. Both animal and clinical studies suggest that ROCK inhibitors are effective for treatment of severe PAH with minimal risk, which supports the premise that ROCKs are important therapeutic targets in pulmonary hypertension and that ROCK inhibitors are a promising new class of drugs for this devastating disease.

  15. ε Subunit of Bacillus subtilis F1-ATPase Relieves MgADP Inhibition

    PubMed Central

    Mizumoto, Junya; Kikuchi, Yuka; Nakanishi, Yo-Hei; Mouri, Naoto; Cai, Anrong; Ohta, Tokushiro; Haruyama, Takamitsu; Kato-Yamada, Yasuyuki

    2013-01-01

    MgADP inhibition, which is considered as a part of the regulatory system of ATP synthase, is a well-known process common to all F1-ATPases, a soluble component of ATP synthase. The entrapment of inhibitory MgADP at catalytic sites terminates catalysis. Regulation by the ε subunit is a common mechanism among F1-ATPases from bacteria and plants. The relationship between these two forms of regulatory mechanisms is obscure because it is difficult to distinguish which is active at a particular moment. Here, using F1-ATPase from Bacillus subtilis (BF1), which is strongly affected by MgADP inhibition, we can distinguish MgADP inhibition from regulation by the ε subunit. The ε subunit did not inhibit but activated BF1. We conclude that the ε subunit relieves BF1 from MgADP inhibition. PMID:23967352

  16. Rotenone Activates Phagocyte NADPH Oxidase through Binding to Its Membrane Subunit gp91phox

    PubMed Central

    Zhou, Hui; Zhang, Feng; Chen, Shih-heng; Zhang, Dan; Wilson, Belinda; Hong, Jau-shyong; Gao, Hui-Ming

    2011-01-01

    Rotenone, a widely used pesticide, reproduces Parkinsonism in rodents and associates with increased risk for Parkinson’s disease. We previously reported rotenone increased superoxide production through stimulating microglial phagocyte NADPH oxidase (PHOX). The present study identified a novel mechanism by which rotenone activates PHOX. Ligand-binding assay revealed that rotenone directly bound to membrane gp91phox, the catalytic subunit of PHOX; such binding was inhibited by diphenyleneiodonium, a PHOX inhibitor with a binding site on gp91phox. Functional studies showed both membrane and cytosolic subunits were required for rotenone-induced superoxide production in cell-free systems, intact phagocytes, and COS7 cells transfected with membrane subunits (gp91phox/p22phox) and cytosolic subunits (p67phox and p47phox). Rotenone-elicited extracellular superoxide release in p47phox-deficient macrophages suggested rotenone enabled to activate PHOX through a p47phox-independent mechanism. Increased membrane translocation of p67phox, elevated binding of p67phox to rotenone-treated membrane fractions, and co-immunoprecipitation of p67phox and gp91phox in rotenone-treated wild-type and p47phox-deficient macrophages indicated p67phox played a critical role in rotenone-induced PHOX activation via its direct interaction with gp91phox. Rac1, a Rho-like small GTPase, enhanced p67phox-gp91phox interaction; Rac1 inhibition decreased rotenone-elicited superoxide release. In conclusion, rotenone directly interacted with gp91phox; such an interaction triggered membrane translocation of p67phox, leading to PHOX activation and superoxide production. PMID:22094225

  17. GABA+ levels in postmenopausal women with mild-to-moderate depression

    PubMed Central

    Wang, Zhensong; Zhang, Aiying; Zhao, Bin; Gan, Jie; Wang, Guangbin; Gao, Fei; Liu, Bo; Gong, Tao; Liu, Wen; Edden, Richard A.E.

    2016-01-01

    Abstract Background: It is increasingly being recognized that alterations of the GABAergic system are implicated in the pathophysiology of depression. This study aimed to explore in vivo gamma-aminobutyric acid (GABA) levels in the anterior cingulate cortex/medial prefrontal cortex (ACC/mPFC) and posterior-cingulate cortex (PCC) of postmenopausal women with depression using magnetic resonance spectroscopy (1H-MRS). Methods: Nineteen postmenopausal women with depression and thirteen healthy controls were enrolled in the study. All subjects underwent 1H-MRS of the ACC/mPFC and PCC using the “MEGA Point Resolved Spectroscopy Sequence” (MEGA-PRESS) technique. The severity of depression was assessed by 17-item Hamilton Depression Scale (HAMD). Quantification of MRS data was performed using Gannet program. Differences of GABA+ levels from patients and controls were tested using one-way analysis of variance. Spearman correlation coefficients were used to evaluate the linear associations between GABA+ levels and HAMD scores, as well as estrogen levels. Results: Significantly lower GABA+ levels were detected in the ACC/mPFC of postmenopausal women with depression compared to healthy controls (P = 0.002). No significant correlations were found between 17-HAMD/14-HAMA and GABA+ levels, either in ACC/mPFC (P = 0.486; r = 0.170/P = 0.814; r = −0.058) or PCC (P = 0.887; r = 0.035/ P = 0.987; r = −0.004) in the patients; there is also no significant correlation between GABA+ levels and estrogen levels in patients group (ACC/mPFC: P = 0.629, r = −0.018; PCC: P = 0.861, r = 0.043). Conclusion: Significantly lower GABA+ levels were found in the ACC/mPFC of postmenopausal women with depression, suggesting that the dysfunction of the GABAergic system may also be involved in the pathogenesis of depression in postmenopausal women. PMID:27684829

  18. Rho/Rock cross-talks with transforming growth factor-β/Smad pathway participates in lung fibroblast-myofibroblast differentiation.

    PubMed

    Ji, Hong; Tang, Haiying; Lin, Hongli; Mao, Jingwei; Gao, Lili; Liu, Jia; Wu, Taihua

    2014-11-01

    The differentiation of fibroblasts, which are promoted by transforming growth factor-β (TGF-β)/Smad, is involved in the process of pulmonary fibrosis. The Rho/Rho-associated coiled-coil-forming protein kinase (Rock) pathway may regulate the fibroblast differentiation and myofibroblast expression of α-smooth muscle actin (α-SMA), however, the mechanism is not clear. The aim of the present study was to evaluate the role of Rho/Rock and TGF-β/Smad in TGF-β1-induced lung fibroblasts differentiation. Human embryonic lung fibroblasts were stimulated by TGF-β1, Y-27632 (inhibitor of Rho/Rock signaling) and staurosporine (inhibitor of TGF-β/Smad signaling). The α-SMA expression, cell cycle progression, content of the extracellular matrix (ECM) in cell culture supernatants and the expression of RhoA, RhoC, Rock1 and Smad2 were detected. The results demonstrated that α-SMA-positive cells significantly increased following TGF-β1 stimulation. Rho/Rock and TGF-β/Smad inhibitors suppressed TGF-β1-induced lung fibroblast differentiation. The inhibitors increased G 0 /G 1 and decreased S and G 2 /M percentages. The concentrations of the ECM proteins in the supernatant were significantly increased by TGF-β1 stimulation, whereas they were decreased by inhibitor stimulation. RhoA, RhoC, Rock1, Smad2 and tissue inhibitor of metalloproteinase-1 were upregulated by TGF-β1 stimulation. The Rho/Rock inhibitor downregulated Smad2 expression and the TGF-β/Smad inhibitor downregulated RhoA, RhoC and Rock1 expression. Therefore, the Rho/Rock pathway and Smad signaling were involved in the process of lung fibroblasts transformation, induced by TGF-β1, to myofibroblasts. The two pathways may undergo cross-talk in the lung fibroblasts differentiation in vitro .

  19. Caspase-activated ROCK-1 allows erythroblast terminal maturation independently of cytokine-induced Rho signaling

    PubMed Central

    Gabet, A-S; Coulon, S; Fricot, A; Vandekerckhove, J; Chang, Y; Ribeil, J-A; Lordier, L; Zermati, Y; Asnafi, V; Belaid, Z; Debili, N; Vainchenker, W; Varet, B; Hermine, O; Courtois, G

    2011-01-01

    Stem cell factor (SCF) and erythropoietin are strictly required for preventing apoptosis and stimulating proliferation, allowing the differentiation of erythroid precursors from colony-forming unit-E to the polychromatophilic stage. In contrast, terminal maturation to generate reticulocytes occurs independently of cytokine signaling by a mechanism not fully understood. Terminal differentiation is characterized by a sequence of morphological changes including a progressive decrease in cell size, chromatin condensation in the nucleus and disappearance of organelles, which requires transient caspase activation. These events are followed by nucleus extrusion as a consequence of plasma membrane and cytoskeleton reorganization. Here, we show that in early step, SCF stimulates the Rho/ROCK pathway until the basophilic stage. Thereafter, ROCK-1 is activated independently of Rho signaling by caspase-3-mediated cleavage, allowing terminal maturation at least in part through phosphorylation of the light chain of myosin II. Therefore, in this differentiation system, final maturation occurs independently of SCF signaling through caspase-induced ROCK-1 kinase activation. PMID:21072057

  20. Increased GABA Levels in Medial Prefrontal Cortex of Young Adults with Narcolepsy

    PubMed Central

    Kim, Seog Ju; Lyoo, In Kyoon; Lee, Yujin S.; Sung, Young Hoon; Kim, Hengjun J.; Kim, Jihyun H.; Kim, Kye Hyun; Jeong, Do-Un

    2008-01-01

    Study Objectives: To explore absolute concentrations of brain metabolites including gamma amino-butyric acid (GABA) in the medial prefrontal cortex and basal ganglia of young adults with narcolepsy. Design: Proton magnetic resonance (MR) spectroscopy centered on the medial prefrontal cortex and the basal ganglia was acquired. The absolute concentrations of brain metabolites including GABA and glutamate were assessed and compared between narcoleptic patients and healthy comparison subjects. Setting: Sleep and Chronobiology Center at Seoul National University Hospital; A high strength 3.0 Tesla MR scanner in the Department of Radiology at Seoul National University Hospital. Patients or Participants: Seventeen young adults with a sole diagnosis of HLA DQB1 0602 positive narcolepsy with cataplexy (25.1 ± 4.6 years old) and 17 healthy comparison subjects (26.8 ± 4.8 years old). Interventions: N/A. Measurements and Results: Relative to comparison subjects, narcoleptic patients had higher GABA concentration in the medial prefrontal cortex (t = 4.10, P <0.001). Narcoleptic patients with nocturnal sleep disturbance had higher GABA concentration in the medial prefrontal cortex than those without nocturnal sleep disturbance (t = 2.45, P= 0.03), but had lower GABA concentration than comparison subjects (t = 2.30, P = 0.03). Conclusions: The current study reports that young adults with narcolepsy had a higher GABA concentration in the medial prefrontal cortex, which was more prominent in patients without nocturnal sleep disturbance. Our findings suggest that the medial prefrontal GABA level may be increased in narcolepsy, and the increased medial prefrontal GABA might be a compensatory mechanism to reduce nocturnal sleep disturbances in narcolepsy. Citation: Kim SJ; Lyoo IK; Lee YS; Sung YH; Kim HJ; Kim JH; Kim KH; Jeong DU. Increased GABA levels in medial prefrontal cortex of young adults with narcolepsy. SLEEP 2008;31(3):342-347. PMID:18363310

  1. Technological and safety properties of newly isolated GABA-producing Lactobacillus futsaii strains.

    PubMed

    Sanchart, C; Rattanaporn, O; Haltrich, D; Phukpattaranont, P; Maneerat, S

    2016-09-01

    To evaluate the technological and safety properties of Lactobacillus futsaii CS3 and CS5 isolated from Thai fermented shrimp products (Kung-Som) in order to develop a valuable gamma-aminobutyric acid (GABA)-producing starter culture. Both strains showed a high GABA-producing ability (>8 mg ml(-1) ) in MRS broth containing 20 mg ml(-1) monosodium glutamate (MSG) for 120 h. They also exhibited inhibitory activity against foodborne pathogens and spoilage bacteria. Cell surface hydrophobicity and proteolytic activity were observed in both strains. Strain CS3 survived better under simulated gastrointestinal tract conditions with only 1·5 log-units cell decrease over 8 h. Both strains showed the ability to deconjugate taurocholate and taurodeoxycholate acid. Neither virulence genes nor biogenic amine production was detected. Strain CS3 exhibited susceptibility to all tested antibiotics with the exception of vancomycin, while strain CS5 showed resistance to vancomycin, ampicillin and chloramphenicol. Based on the results obtained, Lact. futsaii CS3 is very promising as a GABA-producing and potentially probiotic starter culture strain for applications in functional fermented foods. This study focuses on the technological and safety characteristics of Lact. futsaii CS3 and CS5 including their high GABA-producing capacity for the first time. This provides a way of replacing chemical GABA by natural GABA using a GABA-producing starter culture candidate, at the same time offering the consumer new attractive food products. © 2016 The Society for Applied Microbiology.

  2. A multilevel prediction of physiological response to challenge: Interactions among child maltreatment, neighborhood crime, endothelial nitric oxide synthase gene (eNOS), and GABA(A) receptor subunit alpha-6 gene (GABRA6).

    PubMed

    Lynch, Michael; Manly, Jody Todd; Cicchetti, Dante

    2015-11-01

    Physiological response to stress has been linked to a variety of healthy and pathological conditions. The current study conducted a multilevel examination of interactions among environmental toxins (i.e., neighborhood crime and child maltreatment) and specific genetic polymorphisms of the endothelial nitric oxide synthase gene (eNOS) and GABA(A) receptor subunit alpha-6 gene (GABRA6). One hundred eighty-six children were recruited at age 4. The presence or absence of child maltreatment as well as the amount of crime that occurred in their neighborhood during the previous year were determined at that time. At age 9, the children were brought to the lab, where their physiological response to a cognitive challenge (i.e., change in the amplitude of the respiratory sinus arrhythmia) was assessed and DNA samples were collected for subsequent genotyping. The results confirmed that complex Gene × Gene, Environment × Environment, and Gene × Environment interactions were associated with different patterns of respiratory sinus arrhythmia reactivity. The implications for future research and evidence-based intervention are discussed.

  3. GABA concentration in schizophrenia patients and the effects of antipsychotic medication: a proton magnetic resonance spectroscopy study.

    PubMed

    Tayoshi, Shin'Ya; Nakataki, Masahito; Sumitani, Satsuki; Taniguchi, Kyoko; Shibuya-Tayoshi, Sumiko; Numata, Shusuke; Iga, Jun-ichi; Ueno, Shu-ichi; Harada, Masafumi; Ohmori, Tetsuro

    2010-03-01

    Gamma-amino butyric acid (GABA) is thought to play a role in the pathophysiology of schizophrenia. High magnetic field proton magnetic resonance spectroscopy ((1)H-MRS) provides a reliable measurement of GABA in specific regions of the brain. This study measured GABA concentration in the anterior cingulate cortex (ACC) and in the left basal ganglia (ltBG) in 38 patients with chronic schizophrenia and 29 healthy control subjects. There was no significant difference in GABA concentration between the schizophrenia patients and the healthy controls in either the ACC (1.36+/-0.45 mmol/l in schizophrenia patients and 1.52+/-0.54 mmol/l in control subjects) or the ltBG (1.13+/-0.26 mmol/l in schizophrenia patients and 1.18+/-0.20 mmol/l in control subjects). Among the right handed schizophrenia patients, the GABA concentration in the ltBG was significantly higher in patients taking typical antipsychotics (1.25+/-0.24 mmol/l) than in those taking atypical antipsychotics (1.03+/-0.24 mmol/l, p=0.026). In the ACC, the GABA concentration was negatively correlated with the dose of the antipsychotics (rs=-0.347, p=0.035). In the ltBG, the GABA concentration was positively correlated with the dose of the anticholinergics (rs=0.403, p=0.015). To the best of our knowledge, this is the first study to have directly measured GABA concentrations in schizophrenia patients using (1)H-MRS. Our results suggest that there are no differences in GABA concentrations in the ACC or the ltBG of schizophrenia patients compared to healthy controls. Antipsychotic medication may cause changes in GABA concentration, and atypical and typical antipsychotics may have differing effects. It is possible that medication effects conceal inherent differences in GABA concentrations between schizophrenia patients and healthy controls. (c) 2009 Elsevier B.V. All rights reserved.

  4. RhoA GTPase inhibition organizes contraction during epithelial morphogenesis

    PubMed Central

    Mason, Frank M.; Xie, Shicong; Vasquez, Claudia G.; Tworoger, Michael

    2016-01-01

    During morphogenesis, contraction of the actomyosin cytoskeleton within individual cells drives cell shape changes that fold tissues. Coordination of cytoskeletal contractility is mediated by regulating RhoA GTPase activity. Guanine nucleotide exchange factors (GEFs) activate and GTPase-activating proteins (GAPs) inhibit RhoA activity. Most studies of tissue folding, including apical constriction, have focused on how RhoA is activated by GEFs to promote cell contractility, with little investigation as to how GAPs may be important. Here, we identify a critical role for a RhoA GAP, Cumberland GAP (C-GAP), which coordinates with a RhoA GEF, RhoGEF2, to organize spatiotemporal contractility during Drosophila melanogaster apical constriction. C-GAP spatially restricts RhoA pathway activity to a central position in the apical cortex. RhoGEF2 pulses precede myosin, and C-GAP is required for pulsation, suggesting that contractile pulses result from RhoA activity cycling. Finally, C-GAP expression level influences the transition from reversible to irreversible cell shape change, which defines the onset of tissue shape change. Our data demonstrate that RhoA activity cycling and modulating the ratio of RhoGEF2 to C-GAP are required for tissue folding. PMID:27551058

  5. An Electrostatic Funnel in the GABA-Binding Pathway

    PubMed Central

    Lightstone, Felice C.

    2016-01-01

    The γ-aminobutyric acid type A receptor (GABAA-R) is a major inhibitory neuroreceptor that is activated by the binding of GABA. The structure of the GABAA-R is well characterized, and many of the binding site residues have been identified. However, most of these residues are obscured behind the C-loop that acts as a cover to the binding site. Thus, the mechanism by which the GABA molecule recognizes the binding site, and the pathway it takes to enter the binding site are both unclear. Through the completion and detailed analysis of 100 short, unbiased, independent molecular dynamics simulations, we have investigated this phenomenon of GABA entering the binding site. In each system, GABA was placed quasi-randomly near the binding site of a GABAA-R homology model, and atomistic simulations were carried out to observe the behavior of the GABA molecules. GABA fully entered the binding site in 19 of the 100 simulations. The pathway taken by these molecules was consistent and non-random; the GABA molecules approach the binding site from below, before passing up behind the C-loop and into the binding site. This binding pathway is driven by long-range electrostatic interactions, whereby the electrostatic field acts as a ‘funnel’ that sweeps the GABA molecules towards the binding site, at which point more specific atomic interactions take over. These findings define a nuanced mechanism whereby the GABAA-R uses the general zwitterionic features of the GABA molecule to identify a potential ligand some 2 nm away from the binding site. PMID:27119953

  6. A Conserved RhoGAP Limits M-phase Contractility and Coordinates with Microtubule Asters to Restrict Active RhoA to the Cell Equator During Cytokinesis

    PubMed Central

    Zanin, Esther; Desai, Arshad; Poser, Ina; Toyoda, Yusuke; Andree, Cordula; Moebius, Claudia; Bickle, Marc; Conradt, Barbara; Piekny, Alisa; Oegema, Karen

    2014-01-01

    SUMMARY During animal cell cytokinesis, the spindle directs contractile ring assembly by activating RhoA in a narrow equatorial zone. Rapid GTPase activating protein (GAP)-mediated inactivation (RhoA flux) is proposed to limit RhoA zone dimensions. Testing the significance of RhoA flux has been hampered by the fact that the GAP targeting RhoA is not known. Here, we identify M-phase GAP (MP-GAP) as the primary GAP targeting RhoA during mitosis/cytokinesis. MP-GAP inhibition caused excessive RhoA activation in M-phase leading to the uncontrolled formation of large cortical protrusions and late cytokinesis failure. RhoA zone width was broadened by attenuation of the centrosomal asters but was not affected by MP-GAP inhibition alone. Simultaneous aster attenuation and MP-GAP inhibition led to RhoA accumulation around the entire cell periphery. These results identify the major GAP restraining RhoA during cell division and delineate the relative contributions of RhoA flux and centrosomal asters in controlling RhoA zone dimensions. PMID:24012485

  7. Impact of Precooling and Controlled-Atmosphere Storage on γ-Aminobutyric Acid (GABA) Accumulation in Longan (Dimocarpus longan Lour.) Fruit.

    PubMed

    Zhou, Molin; Ndeurumio, Kessy H; Zhao, Lei; Hu, Zhuoyan

    2016-08-24

    Longan (Dimocarpus longan Lour.) fruit cultivars 'Chuliang' and 'Shixia' were analyzed for γ-aminobutyric acid (GABA) accumulation after precooling and in controlled-atmosphere storage. Fruit were exposed to 5% O2 plus 3%, 5%, or 10% CO2 at 4 °C, and GABA and associated enzymes, aril firmness, and pericarp color were measured. Aril softening and pericarp browning were delayed by 5% CO2 + 5% O2. GABA concentrations and glutamate decarboxylase (GAD; EC 4.1.1.15) activities declined during storage at the higher-CO2 treatments. However, GABA aminotransferase (GABA-T; EC 2.6.1.19) activities in elevated CO2-treated fruit fluctuated during storage. GABA concentrations increased after precooling treatments. GAD activity and GABA-T activity were different between cultivars after precooling. GABA concentrations in fruit increased after 3 days of 10% CO2 + 5% O2 treatment and then declined as storage time increased. GABA accumulation was associated with stimulation of GAD activity rather than inhibition of GABA-T activity.

  8. A study on the involvement of GABA-transaminase in MCT induced pulmonary hypertension.

    PubMed

    Lingeshwar, Poorella; Kaur, Gurpreet; Singh, Neetu; Singh, Seema; Mishra, Akanksha; Shukla, Shubha; Ramakrishna, Rachumallu; Laxman, Tulsankar Sachin; Bhatta, Rabi Sankar; Siddiqui, Hefazat H; Hanif, Kashif

    2016-02-01

    Increased sympathetic nervous system (SNS) activity is associated with cardiovascular diseases but its role has not been completely explored in pulmonary hypertension (PH). Increased SNS activity is distinguished by elevated level of norepinephrine (NE) and activity of γ-Amino butyric acid Transminase (GABA-T) which degrades GABA, an inhibitory neurotransmitter within the central and peripheral nervous system. Therefore, we hypothesized that GABA-T may contribute in pathophysiology of PH by modulating level of GABA and NE. The effect of daily oral administration of GABA-T inhibitor, Vigabatrin (GVG, 50 and 75 mg/kg/day, 35 days) was studied following a single subcutaneous administration of monocrotaline (MCT, 60 mg/kg) in male SD rats. The pressure and hypertrophy of right ventricle (RV), oxidative stress, inflammation, pulmonary vascular remodelling were assessed after 35 days in MCT treated rats. The expression of GABA-T and HIF-1α was studied in lung tissue. The levels of plasma NE (by High performance liquid chromatography coupled with electrochemical detector; HPLC-ECD) and lung GABA (by liquid chromatography-mass spectrometry) were also estimated. GVG at both doses significantly attenuated increased in pressure (35.82 ± 4.80 mm Hg, p < 0.001; 28.37 ± 3.32 mm Hg, p < 0.001 respectively) and hypertrophy of RV, pulmonary vascular remodelling, oxidative stress and inflammation in lungs of MCT exposed rats. GVG also reduced the expression of GABA-T and HIF-1α in MCT treated rats. Increased NE level and decreased GABA level was also reversed by GVG in MCT exposed rats. GABA-T plays an important role in PH by modulating SNS activity and may be considered as a therapeutic target in PH. Copyright © 2015 Elsevier Ltd. All rights reserved.

  9. RhoA-ROCK and p38MAPK-MSK1 mediate vitamin D effects on gene expression, phenotype, and Wnt pathway in colon cancer cells.

    PubMed

    Ordóñez-Morán, Paloma; Larriba, María Jesús; Pálmer, Héctor G; Valero, Ruth A; Barbáchano, Antonio; Duñach, Mireia; de Herreros, Antonio García; Villalobos, Carlos; Berciano, María Teresa; Lafarga, Miguel; Muñoz, Alberto

    2008-11-17

    The active vitamin D metabolite 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) inhibits proliferation and promotes differentiation of colon cancer cells through the activation of vitamin D receptor (VDR), a transcription factor of the nuclear receptor superfamily. Additionally, 1,25(OH)(2)D(3) has several nongenomic effects of uncertain relevance. We show that 1,25(OH)(2)D(3) induces a transcription-independent Ca(2+) influx and activation of RhoA-Rho-associated coiled kinase (ROCK). This requires VDR and is followed by activation of the p38 mitogen-activated protein kinase (p38MAPK) and mitogen- and stress-activated kinase 1 (MSK1). As shown by the use of chemical inhibitors, dominant-negative mutants and small interfering RNA, RhoA-ROCK, and p38MAPK-MSK1 activation is necessary for the induction of CDH1/E-cadherin, CYP24, and other genes and of an adhesive phenotype by 1,25(OH)(2)D(3). RhoA-ROCK and MSK1 are also required for the inhibition of Wnt-beta-catenin pathway and cell proliferation. Thus, the action of 1,25(OH)(2)D(3) on colon carcinoma cells depends on the dual action of VDR as a transcription factor and a nongenomic activator of RhoA-ROCK and p38MAPK-MSK1.

  10. Coupling constant for N*(1535)N{rho}

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Xie Jujun; Graduate University of Chinese Academy of Sciences, Beijing 100049; Wilkin, Colin

    2008-05-15

    The value of the N*(1535)N{rho} coupling constant g{sub N*N{rho}} derived from the N*(1535){yields}N{rho}{yields}N{pi}{pi} decay is compared with that deduced from the radiative decay N*(1535){yields}N{gamma} using the vector-meson-dominance model. On the basis of an effective Lagrangian approach, we show that the values of g{sub N*N{rho}} extracted from the available experimental data on the two decays are consistent, though the error bars are rather large.

  11. Negative modulation of the GABAA ρ1 receptor function by l-cysteine.

    PubMed

    Beltrán González, Andrea N; Vicentini, Florencia; Calvo, Daniel J

    2018-01-01

    l-Cysteine is an endogenous sulfur-containing amino acid with multiple and varied roles in the central nervous system, including neuroprotection and the maintenance of the redox balance. However, it was also suggested as an excitotoxic agent implicated in the pathogenesis of neurological disorders such as Parkinson's and Alzheimer's disease. l-Cysteine can modulate the activity of ionic channels, including voltage-gated calcium channels and glutamatergic NMDA receptors, whereas its effects on GABAergic neurotransmission had not been studied before. In the present work, we analyzed the effects of l-cysteine on responses mediated by homomeric GABA A ρ1 receptors, which are known for mediating tonic γ-aminobutyric acid (GABA) responses in retinal neurons. GABA A ρ1 receptors were expressed in Xenopus laevis oocytes and GABA-evoked chloride currents recorded by two-electrode voltage-clamp in the presence or absence of l-cysteine. l-Cysteine antagonized GABA A ρ1 receptor-mediated responses; inhibition was dose-dependent, reversible, voltage independent, and susceptible to GABA concentration. Concentration-response curves for GABA were shifted to the right in the presence of l-cysteine without a substantial change in the maximal response. l-Cysteine inhibition was insensitive to chemical protection of the sulfhydryl groups of the ρ1 subunits by the irreversible alkylating agent N-ethyl maleimide. Our results suggest that redox modulation is not involved during l-cysteine actions and that l-cysteine might be acting as a competitive antagonist of the GABA A ρ1 receptors. © 2017 International Society for Neurochemistry.

  12. Kv7 potassium channel subunits and M currents in cultured hippocampal interneurons.

    PubMed

    Grigorov, Alexej; Moskalyuk, Anastasia; Kravchenko, Mykola; Veselovsky, Nikolai; Verkhratsky, Alexei; Fedulova, Svetlana

    2014-09-01

    Potassium channels of the Kv7 family that mediate the non-inactivating M current regulate the excitability of many types of neurons in the central nervous system, including some in the hippocampus. We report here that individual interneurons from newborn rat hippocampi in long-term culture strongly express messenger RNA specific for Kv7.2 and Kv7.3 and, to a lesser extent, Kv7.5 channel subunits but not for the Kv7.4 subunit. An M-like current was electrophysiologically identified in two subpopulations of interneurons distinct in their spiking behaviour (regular or fast spiking). The M-channel enhancer retigabine reduced interneuronal excitability by constraining the number of action potentials generated during imposed depolarisations; this effect was inhibited by specific the M-channel blocking drugs. In paired synaptically connected interneuron-target cell recordings, anatomically localised applications of retigabine indicated that M channels were present in both the interneuron soma and its GABA-ergic inhibitory axon. We conclude that M-channel subunits and functional M channels are broadly expressed in hippocampal interneurons and their axons and are potentially capable of strongly regulating their firing properties.

  13. Quantification of γ-aminobutyric acid (GABA) in 1 H MRS volumes composed heterogeneously of grey and white matter.

    PubMed

    Mikkelsen, Mark; Singh, Krish D; Brealy, Jennifer A; Linden, David E J; Evans, C John

    2016-11-01

    The quantification of γ-aminobutyric acid (GABA) concentration using localised MRS suffers from partial volume effects related to differences in the intrinsic concentration of GABA in grey (GM) and white (WM) matter. These differences can be represented as a ratio between intrinsic GABA in GM and WM: r M . Individual differences in GM tissue volume can therefore potentially drive apparent concentration differences. Here, a quantification method that corrects for these effects is formulated and empirically validated. Quantification using tissue water as an internal concentration reference has been described previously. Partial volume effects attributed to r M can be accounted for by incorporating into this established method an additional multiplicative correction factor based on measured or literature values of r M weighted by the proportion of GM and WM within tissue-segmented MRS volumes. Simulations were performed to test the sensitivity of this correction using different assumptions of r M taken from previous studies. The tissue correction method was then validated by applying it to an independent dataset of in vivo GABA measurements using an empirically measured value of r M . It was shown that incorrect assumptions of r M can lead to overcorrection and inflation of GABA concentration measurements quantified in volumes composed predominantly of WM. For the independent dataset, GABA concentration was linearly related to GM tissue volume when only the water signal was corrected for partial volume effects. Performing a full correction that additionally accounts for partial volume effects ascribed to r M successfully removed this dependence. With an appropriate assumption of the ratio of intrinsic GABA concentration in GM and WM, GABA measurements can be corrected for partial volume effects, potentially leading to a reduction in between-participant variance, increased power in statistical tests and better discriminability of true effects. Copyright © 2016 John

  14. Determination of GABA and vigabatrin in human plasma by a rapid and simple HPLC method: correlation between clinical response to vigabatrin and increase in plasma GABA.

    PubMed

    Löscher, W; Fassbender, C P; Gram, L; Gramer, M; Hörstermann, D; Zahner, B; Stefan, H

    1993-03-01

    The novel antiepileptic drug vigabatrin (Sabril) acts by inhibiting degradation of the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), increasing the GABA concentrations in the brain. Because the GABA degrading enzyme GABA aminotransferase (GABA-T) is also present in peripheral tissues, including blood platelets, measurement of plasma GABA levels might be a useful indication of the pharmacological response to vigabatrin during therapeutic monitoring. However, because of the very low concentrations of GABA in plasma, the few methods available for plasma GABA analysis are time-consuming, difficult to perform and/or not selective enough because of potential interference with other plasma constituents. In the present study, a rapid, selective and sensitive amino acid analysis HPLC method has been developed for plasma GABA determination with fluorescence detection, using o-phthaldialdehyde as a precolumn derivatizing agent. By employing a 3 microns particle size reversed-phase column and a multi-step gradient system of two solvents, the very low endogenous concentration of GABA in human plasma could be reproducibly quantitated without interference of other endogenous compounds. Incubation of human plasma samples with GABA degrading enzyme(s) resulted in an almost total loss of the GABA peak, thus demonstrating the specificity of the method for GABA analysis. In addition to GABA and other endogenous amino acids, the HPLC method could be used to quantitate plasma levels of vigabatrin. Thus, this improved HPLC amino acid assay might be used to examine whether concomitant monitoring of plasma GABA and vigabatrin is useful for clinical purposes. This was examined in 20 epileptic patients undergoing chronic treatment with vigabatrin. The average plasma GABA level of these 20 patients did not differ significantly from non-epileptic controls. However, when epileptic patients were subdivided according to their clinical response to vigabatrin, vigabatrin responders

  15. Evidence of two populations of GABA(A) receptors in cerebellar granule cells in culture: different desensitization kinetics, pharmacology, serine/threonine kinase sensitivity, and localization.

    PubMed

    Robello, M; Amico, C; Cupello, A

    1999-12-20

    GABA(A) receptors of rat cerebellar granule cells in culture have been studied by the whole cell patch clamp technique. The biphasic desensitization kinetic observed could be due either to different desensitization mechanisms of a single receptor population or to different receptor populations. The overall data indicate that the latter hypothesis is most probably the correct one. In fact, the fast desensitizing component was selectively potentiated by a benzodiazepine agonist and preferentially down-regulated by activation of the protein serine/threonine kinases A and G, as a consequence of the latter characteristic that receptor population was preferentially down-regulated by previous activation of N-methyl-d-aspartate glutamate receptors, via production of nitric oxide and PKG activation, most probably in dendrites. The other population is benzodiazepine insensitive and not influenced by activation of PKA or PKG. This slowly desensitizing population may correspond to the extrasynaptic delta subunit containing GABA(A) receptors described by other authors. Instead, the rapidly desensitizing population appears to represent dendritic synaptic GABA(A) receptors. Copyright 1999 Academic Press.

  16. [Effect of Different Stimulating Strength of Electroacupuncture on Gastrointestinal Motility and RhoA/ROCK Signaling in Gastric Antral Smooth Muscle in Diabetic Gastroparesis Rats].

    PubMed

    Wu, Xue-Fen; Chen, Xiao-Li; Zheng, Xue-Na; Guo, Xin; Xie, Zhi-Qiang; Liu, Li; Wei, Xin-Ran; Yue, Zeng-Hui

    2018-03-25

    To observe the effect of different strength of electroacupuncture (EA) stimulation on gastrointestinal motility and Ras homolog gene family member (RhoA)/Rho associated coiled-coil forming protein kinase (ROCK) signaling in diabetic gastroparesis (DGP) rats, so as to reveal the underlying mechanisms of EA for improving DGP. Sixty SD rats were randomly and equally divided into blank control, DGP model, weak EA, medium EA, and strong EA groups ( n =12 rats in each). The DGP model was established by intraperitoneal injection of streptozotocin (STZ, 55 mmol/kg, 2%) and high-sugar and high-fat fodder feeding for 8 weeks. EA (0.12, 0.24, 0.36 mA, 20 Hz/100 Hz) was applied to "Zusanli" (ST 36), "Sanyinjiao" (SP 6) and "Liangmen" (ST 21) for 20 min, once daily for 15 successive days. Blood glucose levels were measured weekly with blood glucose meter and blood glucose test paper. Fecal phenol red excretion method was used to display gastric emptying and small intestinal propulsion function. The expression of RhoA protein in the gastric antral smooth muscle tissue was detected by immunohistochemistry and Western blot (WB), separately, and that of ROCK, myosin phosphatase target subunit 1 (MYPT 1) and phosphorylated (p)-MYPT 1 proteins in gastric antrum detected by WB. Compared with the blank control group, the gastric emptying rate and small intestine propulsion rate of the model group were significantly decreased ( P <0.05), and the blood glucose level was remarkably increased ( P <0.05). Moreover, the expression levels of RhoA, ROCK, MYPT 1 and p-MYPT 1 proteins in the gastric antrum were significantly down-regulated relevant to the control group ( P <0.05). After administration of EA, the decreased gastric emptying rate and intestinal propulsion rate, and the down-regulated expression of RhoA, ROCK, MYPT 1 and p-MYPT 1 proteins were significantly increased in the strong, medium and weak EA stimulation groups ( P <0.05). Comparison among the 3 EA groups showed that the

  17. Localization of a GABA transporter to glial cells in the developing and adult olfactory pathway of the moth Manduca sexta1

    PubMed Central

    Oland, Lynne A; Gibson, Nicholas J; Tolbert, Leslie P

    2010-01-01

    Glial cells have several critical roles in the developing and adult olfactory (antennal) lobe of the moth Manduca sexta. Early in development, glial cells occupy discrete regions of the developing olfactory pathway and processes of GABAergic neurons extend into some of these regions. Because GABA is known to have developmental effects in a variety of systems, we explored the possibility that the glial cells express a GABA transporter that could regulate GABA levels to which olfactory neurons and glial cells are exposed. Using an antibody raised against a characterized high-affinity M. sexta GABA transporter with high sequence homology to known mammalian GABA transporters (Mbungu et al., 1995; Umesh and Gill, 2002), we found that the GABA transporter is localized to subsets of centrally derived glial cells during metamorphic adult development. The transporter persists into adulthood in a subset of the neuropil-associated glial cells, but its distribution pattern as determined by light- and electron-microscopic-level immunocytochemistry indicates that it could not serve to regulate GABA concentration in the synaptic cleft. Rather its role is more likely to regulate extracellular GABA levels within the glomerular neuropil. Expression in the sorting zone glial cells disappears after the period of olfactory receptor axon ingrowth, but may be important during ingrowth if GABA regulates axon growth. Glial cells take up GABA, and that uptake can be blocked by DABA. This is the first molecular evidence that the central glial cell population in this pathway is heterogeneous. PMID:20058309

  18. ADP binding to TF1 and its subunits induces ultraviolet spectral changes.

    PubMed

    Hisabori, T; Yoshida, M; Sakurai, H

    1986-09-01

    Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF1) were investigated by UV spectroscopy and by equilibrium dialysis. When ADP was mixed with TF1 in the presence and in the absence of Mg2+, an UV absorbance change was induced (t1/2 approximately 1 min) with a peak at about 278 nm and a trough at about 250 nm. Similar spectral changes were induced by ADP with the isolated beta subunits in the presence and in the absence of Mg2+, and with the isolated alpha subunits in the presence of Mg2+ although the magnitudes of the changes were different. From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF1 in the presence of Mg2+, three high-affinity sites (Kd = 61 nM) and three low-affinity sites (Kd = 87 microM). In the absence of Mg2+, TF1 has one high-affinity site (Kd less than 10 nM) and five low-affinity sites (Kd = 100 microM). Moreover, we found a single Mg2+-dependent ADP binding site on the isolated alpha subunit and a single Mg2+-independent ADP binding site on the isolated beta subunit. From the above observations, we concluded that the three Mg2+-dependent high-affinity sites for ADP are located on the alpha subunit in TF1 and that the single high-affinity site is located on one of the beta subunits in TF1 in the absence of Mg2+.

  19. Production of gaba (γ – Aminobutyric acid) by microorganisms: a review

    PubMed Central

    Dhakal, Radhika; Bajpai, Vivek K.; Baek, Kwang-Hyun

    2012-01-01

    GABA (γ-aminobutyric acid) is a four carbon non-protein amino acid that is widely distributed in plants, animals and microorganisms. As a metabolic product of plants and microorganisms produced by the decarboxylation of glutamic acid, GABA functions as an inhibitory neurotransmitter in the brain that directly affects the personality and the stress management. A wide range of traditional foods produced by microbial fermentation contain GABA, in which GABA is safe and eco-friendly, and also has the possibility of providing new health-benefited products enriched with GABA. Synthesis of GABA is catalyzed by glutamate decarboxylase, therefore, the optimal fermentation condition is mainly based on the biochemical properties of the enzyme. Major GABA producing microorganisms are lactic acid bacteria (LAB), which make food spoilage pathogens unable to grow and act as probiotics in the gastrointestinal tract. The major factors affecting the production of GABA by microbial fermentation are temperature, pH, fermentation time and different media additives, therefore, these factors are summarized to provide the most up-dated information for effective GABA synthesis. There has been a huge accumulation of knowledge on GABA application for human health accompanying with a demand on natural GABA supply. Only the GABA production by microorganisms can fulfill the demand with GABA-enriched health beneficial foods. PMID:24031948

  20. A Transmembrane Amino Acid in the GABAA Receptor β2 Subunit Critical for the Actions of Alcohols and Anesthetics

    PubMed Central

    McCracken, Mandy L.; Borghese, Cecilia M.; Trudell, James R.

    2010-01-01

    Alcohols and inhaled anesthetics enhance the function of GABAA receptors containing α, β, and γ subunits. Molecular analysis has focused on the role of the α subunits; however, there is evidence that the β subunits may also be important. The goal of our study was to determine whether Asn265, which is homologous to the site implicated in the α subunit (Ser270), contributes to an alcohol and volatile anesthetic binding site in the GABAA receptor β2 subunit. We substituted cysteine for Asn265 and exposed the mutant to the sulfhydryl-specific reagent octyl methanethiosulfonate (OMTS). We used two-electrode voltage-clamp electrophysiology in Xenopus laevis oocytes and found that, after OMTS application, GABA-induced currents were irreversibly potentiated in mutant α1β2(N265C)γ2S receptors [but not α1β2(I264C)γ2S], presumably because of the covalent linking of octanethiol to the thiol group in the substituted cysteine. It is noteworthy that this effect was blocked when OMTS was applied in the presence of octanol. We found that potentiation by butanol, octanol, or isoflurane in the N265C mutant was nearly abolished after the application of OMTS, suggesting that an alcohol and volatile anesthetic binding site at position 265 of the β2 subunit was irreversibly occupied by octanethiol and consequently prevented butanol or isoflurane from binding and producing their effects. OMTS did not affect modulation or direct activation by pentobarbital, but there was a partial reduction of allosteric modulation by flunitrazepam and alphaxalone in mutant α1β2(N265C)γ2S receptors after OMTS was applied. Our findings provide evidence that Asn265 may contribute to an alcohol and anesthetic binding site. PMID:20826568

  1. Occipital GABA correlates with cognitive failures in daily life.

    PubMed

    Sandberg, Kristian; Blicher, Jakob Udby; Dong, Mia Yuan; Rees, Geraint; Near, Jamie; Kanai, Ryota

    2014-02-15

    The brain has limited capacity, and so selective attention enhances relevant incoming information while suppressing irrelevant information. This process is not always successful, and the frequency of such cognitive failures varies to a large extent between individuals. Here we hypothesised that individual differences in cognitive failures might be reflected in inhibitory processing in the sensory cortex. To test this hypothesis, we measured GABA in human visual cortex using MR spectroscopy and found a negative correlation between occipital GABA (GABA+/Cr ratio) and cognitive failures as measured by an established cognitive failures questionnaire (CFQ). For a second site in parietal cortex, no correlation between CFQ score and GABA+/Cr ratio was found, thus establishing the regional specificity of the link between occipital GABA and cognitive failures. We further found that grey matter volume in the left superior parietal lobule (SPL) correlated with cognitive failures independently from the impact of occipital GABA and together, occipital GABA and SPL grey matter volume statistically explained around 50% of the individual variability in daily cognitive failures. We speculate that the amount of GABA in sensory areas may reflect the potential capacity to selectively suppress irrelevant information already at the sensory level, or alternatively that GABA influences the specificity of neural representations in visual cortex thus improving the effectiveness of successful attentional modulation. © 2013. Published by Elsevier Inc. All rights reserved.

  2. Adaptive Mutations in RNA Polymerase and the Transcriptional Terminator Rho Have Similar Effects on Escherichia coli Gene Expression.

    PubMed

    González-González, Andrea; Hug, Shaun M; Rodríguez-Verdugo, Alejandra; Patel, Jagdish Suresh; Gaut, Brandon S

    2017-11-01

    Modifications to transcriptional regulators play a major role in adaptation. Here, we compared the effects of multiple beneficial mutations within and between Escherichia coli rpoB, the gene encoding the RNA polymerase β subunit, and rho, which encodes a transcriptional terminator. These two genes have harbored adaptive mutations in numerous E. coli evolution experiments but particularly in our previous large-scale thermal stress experiment, where the two genes characterized alternative adaptive pathways. To compare the effects of beneficial mutations, we engineered four advantageous mutations into each of the two genes and measured their effects on fitness, growth, gene expression and transcriptional termination at 42.2 °C. Among the eight mutations, two rho mutations had no detectable effect on relative fitness, suggesting they were beneficial only in the context of epistatic interactions. The remaining six mutations had an average relative fitness benefit of ∼20%. The rpoB mutations affected the expression of ∼1,700 genes; rho mutations affected the expression of fewer genes but most (83%) were a subset of those altered by rpoB mutants. Across the eight mutants, relative fitness correlated with the degree to which a mutation restored gene expression back to the unstressed, 37.0 °C state. The beneficial mutations in the two genes did not have identical effects on fitness, growth or gene expression, but they caused parallel phenotypic effects on gene expression and genome-wide transcriptional termination. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

  3. Meroterpenoid Chrodrimanins Are Selective and Potent Blockers of Insect GABA-Gated Chloride Channels

    PubMed Central

    Ihara, Makoto; Ling, Yun; Yang, Xinling; Kai, Kenji; Hayashi, Hideo; Matsuda, Kazuhiko

    2015-01-01

    Meroterpenoid chrodrimanins, produced from Talaromyces sp. YO-2, are known to paralyze silkworm (Bombyx mori) larvae, but their target is unknown. We have investigated the actions of chrodrimanin B on ligand-gated ion channels of silkworm larval neurons using patch-clamp electrophysiology. Chrodrimanin B had no effect on membrane currents when tested alone at 1 μM. However, it completely blocked the γ-aminobutyric acid (GABA)-induced current and showed less pronounced actions on acetylcholine- and L-glutamate-induced currents, when delivered at 1 μM for 1 min prior to co-application with transmitter GABA. Thus, chrodrimanins were also tested on a wild-type isoform of the B. mori GABA receptor (GABAR) RDL using two-electrode voltage-clamp electrophysiology. Chrodrimanin B attenuated the peak current amplitude of the GABA response of RDL with an IC50 of 1.66 nM. The order of the GABAR-blocking potency of chrodrimanins B > D > A was in accordance with their reported insecticidal potency. Chrodrimanin B had no open channel blocking action when tested at 3 nM on the GABA response of RDL. Co-application with 3 nM chrodrimanin B shifted the GABA concentration response curve to a higher concentration and further increase of chrodrimanin B concentration to10 nM; it reduced maximum current amplitude of the GABA response, pointing to a high-affinity competitive action and a lower affinity non-competitive action. The A282S;T286V double mutation of RDL, which impairs the actions of fipronil, hardly affected the blocking action of chrodrimanin B, indicating a binding site of chrodrimanin B distinct from that of fipronil. Chrodrimanin B showed approximately 1,000-fold lower blocking action on human α1β2γ2 GABAR compared to RDL and thus is a selective blocker of insect GABARs. PMID:25902139

  4. Relative positioning of classical benzodiazepines to the γ2-subunit of GABAA receptors.

    PubMed

    Middendorp, Simon J; Hurni, Evelyn; Schönberger, Matthias; Stein, Marco; Pangerl, Michael; Trauner, Dirk; Sigel, Erwin

    2014-08-15

    GABAA receptors are the major inhibitory neurotransmitter receptors in the brain. Benzodiazepine exert their action via a high affinity-binding site at the α/γ subunit interface on some of these receptors. Diazepam has sedative, hypnotic, anxiolytic, muscle relaxant, and anticonvulsant effects. It acts by potentiating the current evoked by the agonist GABA. Understanding specific interaction of benzodiazepines in the binding pocket of different GABAA receptor isoforms might help to separate these divergent effects. As a first step, we characterized the interaction between diazepam and the major GABAA receptor isoform α1β2γ2. We mutated several amino acid residues on the γ2-subunit assumed to be located near or in the benzodiazepine binding pocket individually to cysteine and studied the interaction with three ligands that are modified with a cysteine-reactive isothiocyanate group (-NCS). When the reactive NCS group is in apposition to the cysteine residue this leads to a covalent reaction. In this way, three amino acid residues, γ2Tyr58, γ2Asn60, and γ2Val190 were located relative to classical benzodiazepines in their binding pocket on GABAA receptors.

  5. In Vivo Measurements of Glutamate, GABA, and NAAG in Schizophrenia

    PubMed Central

    Rowland, Laura M.

    2013-01-01

    The major excitatory and inhibitory neurotransmitters, glutamate (Glu) and gamma-aminobutyric acid (GABA), respectively, are implicated in the pathophysiology of schizophrenia. N-acetyl-aspartyl-glutamate (NAAG), a neuropeptide that modulates the Glu system, may also be altered in schizophrenia. This study investigated GABA, Glu + glutamine (Glx), and NAAG levels in younger and older subjects with schizophrenia. Forty-one subjects, 21 with chronic schizophrenia and 20 healthy controls, participated in this study. Proton magnetic resonance spectroscopy (1H-MRS) was used to measure GABA, Glx, and NAAG levels in the anterior cingulate (AC) and centrum semiovale (CSO) regions. NAAG in the CSO was higher in younger schizophrenia subjects compared with younger control subjects. The opposite pattern was observed in the older groups. Glx was reduced in the schizophrenia group irrespective of age group and brain region. There was a trend for reduced AC GABA in older schizophrenia subjects compared with older control subjects. Poor attention performance was correlated to lower AC GABA levels in both groups. Higher levels of CSO NAAG were associated with greater negative symptom severity in schizophrenia. These results provide support for altered glutamatergic and GABAergic function associated with illness course and cognitive and negative symptoms in schizophrenia. The study also highlights the importance of studies that combine MRS measurements of NAAG, GABA, and Glu for a more comprehensive neurochemical characterization of schizophrenia. PMID:23081992

  6. In vivo measurements of glutamate, GABA, and NAAG in schizophrenia.

    PubMed

    Rowland, Laura M; Kontson, Kimberly; West, Jeffrey; Edden, Richard A; Zhu, He; Wijtenburg, S Andrea; Holcomb, Henry H; Barker, Peter B

    2013-09-01

    The major excitatory and inhibitory neurotransmitters, glutamate (Glu) and gamma-aminobutyric acid (GABA), respectively, are implicated in the pathophysiology of schizophrenia. N-acetyl-aspartyl-glutamate (NAAG), a neuropeptide that modulates the Glu system, may also be altered in schizophrenia. This study investigated GABA, Glu + glutamine (Glx), and NAAG levels in younger and older subjects with schizophrenia. Forty-one subjects, 21 with chronic schizophrenia and 20 healthy controls, participated in this study. Proton magnetic resonance spectroscopy ((1)H-MRS) was used to measure GABA, Glx, and NAAG levels in the anterior cingulate (AC) and centrum semiovale (CSO) regions. NAAG in the CSO was higher in younger schizophrenia subjects compared with younger control subjects. The opposite pattern was observed in the older groups. Glx was reduced in the schizophrenia group irrespective of age group and brain region. There was a trend for reduced AC GABA in older schizophrenia subjects compared with older control subjects. Poor attention performance was correlated to lower AC GABA levels in both groups. Higher levels of CSO NAAG were associated with greater negative symptom severity in schizophrenia. These results provide support for altered glutamatergic and GABAergic function associated with illness course and cognitive and negative symptoms in schizophrenia. The study also highlights the importance of studies that combine MRS measurements of NAAG, GABA, and Glu for a more comprehensive neurochemical characterization of schizophrenia.

  7. Low dose of kaempferol suppresses the migration and invasion of triple-negative breast cancer cells by downregulating the activities of RhoA and Rac1.

    PubMed

    Li, Shoushan; Yan, Ting; Deng, Rong; Jiang, Xuesong; Xiong, Huaping; Wang, Yuan; Yu, Qiao; Wang, Xiaohua; Chen, Cheng; Zhu, Yichao

    2017-01-01

    Triple-negative breast cancer (TNBC) is an especially aggressive and hard-to-treat disease. Although the anticancer role of kaempferol has been reported in breast cancer, the effect of kaempferol on TNBC remains unclear. This experiment investigated the migration-suppressive role of a low dose of kaempferol in TNBC cells. Wound-healing assays and cell invasion assays were used to confirm the migration and invasion of cells treated with kaempferol or transfected indicated constructs. We evaluated the activations of RhoA, Rac1 and Cdc42 in TNBC cells with a Rho activation assay. A panel of inhibitors of estrogen receptor/progesterone receptor/human epidermal growth factor receptor 2 (ER/PR/HER2) treated non-TNBC (SK-BR-3 and MCF-7) cells and blocked the ER/PR/HER2 activity. Wound-healing assays and Rho activation assays were employed to measure the effect of kaempferol and ER/PR/HER2 inhibitors on Rho activation and cell migration rates. A low dose of kaempferol (20 μmol/L) had a potent inhibitory effect on the migration and invasion of TNBC cells, but not on the migration of non-TNBC (SK-BR-3 and MCF-7) cells. The low dose of kaempferol downregulated the activations of RhoA and Rac1 in TNBC cells. Moreover, the low dose of kaempferol also inhibited the migration and RhoA activations of HER2-silence SK-BR-3 and ER/PR-silence MCF-7 cells. Overexpressed HER2 rescued the cell migration and RhoA and Rac1 activations of kaempferol-treated MDA-MB-231 cells. The low dose of kaempferol inhibits the migration and invasion of TNBC cells via blocking RhoA and Rac1 signaling pathway.

  8. Rho GTPases and their roles in cancer metabolism

    PubMed Central

    Wilson, Kristin F.; Erickson, Jon W.; Antonyak, Marc A.; Cerione, Richard A.

    2013-01-01

    Recently, the small molecule 968 was found to block the Rho GTPase-dependent growth of cancer cells in cell culture and mouse xenografts, and when the target of 968 was found to be mitochondrial enzyme glutaminase (GLS1) it revealed a surprising link between Rho GTPases and mitochondrial glutamine metabolism. Signal transduction via the Rho GTPases, together with NFκB, appears to elevate mitochondrial glutaminase activity in cancer cells, thereby helping cancer cells satisfy their altered metabolic demands. Here, we review what is known about the mechanism of glutaminase activation in cancer cells, as well as compare the properties of two distinct glutaminase inhibitors, and discuss recent findings that shed new light on how glutamine metabolism might affect cancer progression. PMID:23219172

  9. Subunit rotation of ATP synthase embedded in membranes: a or β subunit rotation relative to the c subunit ring

    PubMed Central

    Nishio, Kazuaki; Iwamoto-Kihara, Atsuko; Yamamoto, Akitsugu; Wada, Yoh; Futai, Masamitsu

    2002-01-01

    ATP synthase FoF1 (α3β3γδɛab2c10–14) couples an electrochemical proton gradient and a chemical reaction through the rotation of its subunit assembly. In this study, we engineered FoF1 to examine the rotation of the catalytic F1 β or membrane sector Fo a subunit when the Fo c subunit ring was immobilized; a biotin-tag was introduced onto the β or a subunit, and a His-tag onto the c subunit ring. Membrane fragments were obtained from Escherichia coli cells carrying the recombinant plasmid for the engineered FoF1 and were immobilized on a glass surface. An actin filament connected to the β or a subunit rotated counterclockwise on the addition of ATP, and generated essentially the same torque as one connected to the c ring of FoF1 immobilized through a His-tag linked to the α or β subunit. These results established that the γɛc10–14 and α3β3δab2 complexes are mechanical units of the membrane-embedded enzyme involved in rotational catalysis. PMID:12357031

  10. The GIRK1 subunit potentiates G protein activation of cardiac GIRK1/4 hetero-tetramers

    PubMed Central

    Touhara, Kouki K; Wang, Weiwei; MacKinnon, Roderick

    2016-01-01

    G protein gated inward rectifier potassium (GIRK) channels are gated by direct binding of G protein beta-gamma subunits (Gβγ), signaling lipids, and intracellular Na+. In cardiac pacemaker cells, hetero-tetramer GIRK1/4 channels and homo-tetramer GIRK4 channels play a central role in parasympathetic slowing of heart rate. It is known that the Na+ binding site of the GIRK1 subunit is defective, but the functional difference between GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers remains unclear. Here, using purified proteins and the lipid bilayer system, we characterize Gβγ and Na+ regulation of GIRK1/4 hetero-tetramers and GIRK4 homo-tetramers. We find in GIRK4 homo-tetramers that Na+ binding increases Gβγ affinity and thereby increases the GIRK4 responsiveness to G protein stimulation. GIRK1/4 hetero-tetramers are not activated by Na+, but rather are in a permanent state of high responsiveness to Gβγ, suggesting that the GIRK1 subunit functions like a GIRK4 subunit with Na+ permanently bound. DOI: http://dx.doi.org/10.7554/eLife.15750.001 PMID:27074664

  11. New Pharmacotherapy Targeting Cognitive Dysfunction of Schizophrenia via Modulation of GABA Neuronal Function

    PubMed Central

    Jeon, Won Je; Sumiyoshi, Tomiki; Kurachi, Masayoshi

    2015-01-01

    Schizophrenia is considered a neurodevelopmental and neurodegenerative disorder. Cognitive impairment is a core symptom in patients with the illness, and has been suggested a major predictor of functional outcomes. Reduction of parvalbumin (PV)-positive γ-aminobutyric acid (GABA) interneurons has been associated with the pathophysiology of schizophrenia, in view of the link between the abnormality of GABA neurons and cognitive impairments of the disease. It is assumed that an imbalance of excitatory and inhibitory (E-I) activity induced by low activity of glutamatergic projections and PV-positive GABA interneurons in the prefrontal cortex resulted in sustained neural firing and gamma oscillation, leading to impaired cognitive function. Therefore, it is important to develop novel pharmacotherapy targeting GABA neurons and their activities. Clinical evidence suggests serotonin (5-HT) 1A receptor agonist improves cognitive disturbances of schizophrenia, consistent with results from preclinical studies, through mechanism that corrects E-I imbalance via the suppression of GABA neural function. On the other hand, T-817MA, a novel neurotrophic agent, ameliorated loss of PV-positive GABA neurons in the medial prefrontal cortex and reduction of gamma-band activity, as well as cognitive dysfunction in animal model of schizophrenia. In conclusion, a pharmacotherapy to alleviate abnormalities in GABA neurons through 5-HT1A agonists and T-817MA is expected to prevent the onset and/or progression of schizophrenia. PMID:26630957

  12. New Pharmacotherapy Targeting Cognitive Dysfunction of Schizophrenia via Modulation of GABA Neuronal Function.

    PubMed

    Uehara, Takashi; Sumiyoshi, Tomiki; Kurachi, Masayoshi

    2015-01-01

    Schizophrenia is considered a neurodevelopmental and neurodegenerative disorder. Cognitive impairment is a core symptom in patients with the illness, and has been suggested a major predictor of functional outcomes. Reduction of parvalbumin (PV)-positive γ-aminobutyric acid (GABA) interneurons has been associated with the pathophysiology of schizophrenia, in view of the link between the abnormality of GABA neurons and cognitive impairments of the disease. It is assumed that an imbalance of excitatory and inhibitory (E-I) activity induced by low activity of glutamatergic projections and PV-positive GABA interneurons in the prefrontal cortex resulted in sustained neural firing and gamma oscillation, leading to impaired cognitive function. Therefore, it is important to develop novel pharmacotherapy targeting GABA neurons and their activities. Clinical evidence suggests serotonin (5-HT) 1A receptor agonist improves cognitive disturbances of schizophrenia, consistent with results from preclinical studies, through mechanism that corrects E-I imbalance via the suppression of GABA neural function. On the other hand, T-817MA, a novel neurotrophic agent, ameliorated loss of PV-positive GABA neurons in the medial prefrontal cortex and reduction of gamma-band activity, as well as cognitive dysfunction in animal model of schizophrenia. In conclusion, a pharmacotherapy to alleviate abnormalities in GABA neurons through 5-HT1A agonists and T-817MA is expected to prevent the onset and/or progression of schizophrenia.

  13. Brain GABA Detection in vivo with the J-editing 1H MRS Technique: A Comprehensive Methodological Evaluation of Sensitivity Enhancement, Macromolecule Contamination and Test-Retest Reliability

    PubMed Central

    Shungu, Dikoma C.; Mao, Xiangling; Gonzales, Robyn; Soones, Tacara N.; Dyke, Jonathan P.; van der Veen, Jan Willem; Kegeles, Lawrence S.

    2016-01-01

    Abnormalities in brain γ-aminobutyric acid (GABA) have been implicated in various neuropsychiatric and neurological disorders. However, in vivo GABA detection by proton magnetic resonance spectroscopy (1H MRS) presents significant challenges arising from low brain concentration, overlap by much stronger resonances, and contamination by mobile macromolecule (MM) signals. This study addresses these impediments to reliable brain GABA detection with the J-editing difference technique on a 3T MR system in healthy human subjects by (a) assessing the sensitivity gains attainable with an 8-channel phased-array head coil, (b) determining the magnitude and anatomic variation of the contamination of GABA by MM, and (c) estimating the test-retest reliability of measuring GABA with this method. Sensitivity gains and test-retest reliability were examined in the dorsolateral prefrontal cortex (DLPFC), while MM levels were compared across three cortical regions: the DLPFC, the medial prefrontal cortex (MPFC) and the occipital cortex (OCC). A 3-fold higher GABA detection sensitivity was attained with the 8-channel head coil compared to the standard single-channel head coil in DLPFC. Despite significant anatomic variation in GABA+MM and MM across the three brain regions (p < 0.05), the contribution of MM to GABA+MM was relatively stable across the three voxels, ranging from 41% to 49%, a non-significant regional variation (p = 0.58). The test-retest reliability of GABA measurement, expressed either as ratios to voxel tissue water (W) or total creatine, was found to be very high for both the single-channel coil and the 8-channel phased-array coil. For the 8-channel coil, for example, Pearson’s correlation coefficient of test vs. retest for GABA/W was 0.98 (R2 = 0.96, p = 0.0007), the percent coefficient of variation (CV) was 1.25%, and the intraclass correlation coefficient (ICC) was 0.98. Similar reliability was also found for the co-edited resonance of combined glutamate and

  14. Functional neuroanatomy of the ventral striopallidal GABA pathway. New sites of intervention in the treatment of schizophrenia.

    PubMed

    O'Connor, W T

    2001-08-15

    Microdialysis was employed to investigate the dopamine, cholecystokinin (CCK) and neurotensin receptor regulation of ventral striopallidal GABA transmission by intra-accumbens perfusion with selective receptor ligands and monitoring local or ipsilateral ventral pallidal GABA release. In the dual probe studies intra-accumbens perfusion with the dopamine D1 and D2 receptor agonists SKF28293 and pergolide had no effect on ventral pallidal GABA, while both the D1 and D2 receptor antagonists SCH23390 and raclopride increased ventral pallidal GABA release. In contrast, intra-accumbens CCK decreased ventral pallidal GABA release and this was reversed by local perfusion with the CCK2 receptor antagonist PD134308 but not the CCK1 receptor antagonist L-364,718. In a single probe study intra-accumbens neurotensin increased local GABA release, which was strongly potentiated when the peptidase inhibitor phosphodiepryl 08 was perfused together with neurotensin. In addition, the neurotensin receptor antagonist SR48692 counteracted this phosphodiepryl 08 induced potentiated increased in GABA release. Taken together, these findings indicate that mesolimbic dopamine and CCK exert a respective tonic and phasic inhibition of ventral pallidal GABA release while the antipsychotic activity associated with D1 and D2 receptor antagonists may be explained by their ability to increase ventral striopallidal GABA transmission. Furthermore, the findings suggest that CCK2 receptor antagonists and neurotensin endopeptidase inhibitors may be useful antipsychotics.

  15. The Andes Virus Nucleocapsid Protein Directs Basal Endothelial Cell Permeability by Activating RhoA

    PubMed Central

    Gorbunova, Elena E.; Simons, Matthew J.; Gavrilovskaya, Irina N.

    2016-01-01

    ABSTRACT Andes virus (ANDV) predominantly infects microvascular endothelial cells (MECs) and nonlytically causes an acute pulmonary edema termed hantavirus pulmonary syndrome (HPS). In HPS patients, virtually every pulmonary MEC is infected, MECs are enlarged, and infection results in vascular leakage and highly lethal pulmonary edema. We observed that MECs infected with the ANDV hantavirus or expressing the ANDV nucleocapsid (N) protein showed increased size and permeability by activating the Rheb and RhoA GTPases. Expression of ANDV N in MECs increased cell size by preventing tuberous sclerosis complex (TSC) repression of Rheb-mTOR-pS6K. N selectively bound the TSC2 N terminus (1 to 1403) within a complex containing TSC2/TSC1/TBC1D7, and endogenous TSC2 reciprocally coprecipitated N protein from ANDV-infected MECs. TSCs normally restrict RhoA-induced MEC permeability, and we found that ANDV infection or N protein expression constitutively activated RhoA. This suggests that the ANDV N protein alone is sufficient to activate signaling pathways that control MEC size and permeability. Further, RhoA small interfering RNA, dominant-negative RhoA(N19), and the RhoA/Rho kinase inhibitors fasudil and Y27632 dramatically reduced the permeability of ANDV-infected MECs by 80 to 90%. Fasudil also reduced the bradykinin-directed permeability of ANDV and Hantaan virus-infected MECs to control levels. These findings demonstrate that ANDV activation of RhoA causes MEC permeability and reveal a potential edemagenic mechanism for ANDV to constitutively inhibit the basal barrier integrity of infected MECs. The central importance of RhoA activation in MEC permeability further suggests therapeutically targeting RhoA, TSCs, and Rac1 as potential means of resolving capillary leakage during hantavirus infections. PMID:27795403

  16. GABA promotes elastin synthesis and elastin fiber formation in normal human dermal fibroblasts (HDFs).

    PubMed

    Uehara, Eriko; Hokazono, Hideki; Hida, Mariko; Sasaki, Takako; Yoshioka, Hidekatsu; Matsuo, Noritaka

    2017-06-01

    The multiple physiological effects of γ-aminobutyric acid (GABA) as a functional food component have been recently reported. We previously reported that GABA upregulated the expression of type I collagen in human dermal fibroblasts (HDFs), and that oral administration of GABA significantly increased skin elasticity. However, details of the regulatory mechanism still remain unknown. In this study, we further examined the effects of GABA on elastin synthesis and elastin fiber formation in HDFs. Real-time PCR indicated that GABA significantly increased the expression of tropoelastin transcript in a dose-dependent manner. Additionally, the expression of fibrillin-1, fibrillin-2, and fibulin-5/DANCE, but not lysyl oxidase and latent transforming factor-β-binding protein 4, were also significantly increased in HDFs. Finally, immunohistochemical analysis confirmed that treatment with GABA dramatically increased the formation of elastic fibers in HDFs. Taken together, our results showed that GABA improves skin elasticity in HDFs by upregulating elastin synthesis and elastin fiber formation.

  17. Antagonism of GABA-B but not GABA-A receptors in the VTA prevents stress- and intra-VTA CRF-induced reinstatement of extinguished cocaine seeking in rats

    PubMed Central

    Blacktop, Jordan M.; Vranjkovic, Oliver; Mayer, Matthieu; Van Hoof, Matthew; Baker, David A.; Mantsch, John R.

    2015-01-01

    Stress-induced reinstatement of cocaine seeking requires corticotropin releasing factor (CRF) actions in the ventral tegmental area (VTA). However the mechanisms through which CRF regulates VTA function to promote cocaine use are not fully understood. Here we examined the role of GABAergic neurotransmission in the VTA mediated by GABA-A or GABA-B receptors in the reinstatement of extinguished cocaine seeking by a stressor, uncontrollable intermittent footshock, or bilateral intra-VTA administration of CRF. Rats underwent repeated daily cocaine self-administration (1.0 mg/kg/ing; 14 × 6 hrs/day) and extinction and were tested for reinstatement in response to footshock (0.5 mA, 0.5” duration, average every 40 sec; range 10–70 sec) or intra-VTA CRF delivery (500 ng/side) following intra-VTA pretreatment with the GABA-A antagonist, bicuculline, the GABA-B antagonist, 2-hydroxysaclofen or vehicle. Intra-VTA bicuculline (1, 10 or 20 ng/side) failed to block footshock- or CRF-induced cocaine seeking at either dose tested. By contrast, 2-hydroxysaclofen (0.2 or 2 µg/side) prevented reinstatement by both footshock and intra-VTA CRF at a concentration that failed to attenuate food-reinforced lever pressing (45 mg sucrose-sweetened pellets; FR4 schedule) in a separate group of rats. These data suggest that GABA-B receptor-dependent CRF actions in the VTA mediate stress-induced cocaine seeking and that GABA-B receptor antagonists may have utility for the management of stress-induced relapse in cocaine addicts. PMID:26596556

  18. Biochemical magnetic resonance imaging of knee articular cartilage: T1rho and T2 mapping as cartilage degeneration biomarkers.

    PubMed

    Le, Jenna; Peng, Qi; Sperling, Karen

    2016-11-01

    Osteoarthritis (OA) is a disease whose hallmark is the degeneration of articular cartilage. There is a worsening epidemic of OA in the United States today, with considerable economic costs. In order to develop more effective treatments for OA, noninvasive biomarkers that permit early diagnosis and treatment monitoring are necessary. T1rho and T2 mapping are two magnetic resonance imaging techniques that have shown great promise as noninvasive biomarkers of cartilage degeneration. Each of the two techniques is endowed with advantages and disadvantages: T1rho can discern earlier biochemical changes of OA than T2 mapping, while T2 mapping is more widely available and can be incorporated into existing imaging protocols in a more time-efficient manner than T1rho. Both techniques have been applied in numerous instances to study how cartilage is affected by OA risk factors, such as age and exercise. Additionally, both techniques have been repeatedly applied to the study of posttraumatic OA in patients with torn anterior cruciate ligaments. © 2016 New York Academy of Sciences.

  19. The Role of Primary Motor Cortex (M1) Glutamate and GABA Signaling in l-DOPA-Induced Dyskinesia in Parkinsonian Rats

    PubMed Central

    Conti, Melissa M.; Ostock, Corinne Y.; George, Jessica A.; Goldenberg, Adam A.; Melikhov-Sosin, Mitchell; Nuss, Emily E.

    2016-01-01

    Long-term treatment of Parkinson's disease with l-DOPA almost always leads to the development of involuntary movements termed l-DOPA-induced dyskinesia. Whereas hyperdopaminergic signaling in the basal ganglia is thought to cause dyskinesia, alterations in primary motor cortex (M1) activity are also prominent during dyskinesia, suggesting that the cortex may represent a therapeutic target. The present study used the rat unilateral 6-hydroxydopamine lesion model of Parkinson's disease to characterize in vivo changes in GABA and glutamate neurotransmission within M1 and determine their contribution to behavioral output. 6-Hydroxydopamine lesion led to parkinsonian motor impairment that was partially reversed by l-DOPA. Among sham-lesioned rats, l-DOPA did not change glutamate or GABA efflux. Likewise, 6-hydroxydopamine lesion did not impact GABA or glutamate among rats chronically treated with saline. However, we observed an interaction of lesion and treatment whereby, among lesioned rats, l-DOPA given acutely (1 d) or chronically (14–16 d) reduced glutamate efflux and enhanced GABA efflux. Site-specific microinjections into M1 demonstrated that l-DOPA-induced dyskinesia was reduced by M1 infusion of a D1 antagonist, an AMPA antagonist, or a GABAA agonist. Overall, the present study demonstrates that l-DOPA-induced dyskinesia is associated with increased M1 inhibition and that exogenously enhancing M1 inhibition may attenuate dyskinesia, findings that are in agreement with functional imaging and transcranial magnetic stimulation studies in human Parkinson's disease patients. Together, our study suggests that increasing M1 inhibitory tone is an endogenous compensatory response designed to limit dyskinesia severity and that potentiating this response is a viable therapeutic strategy. SIGNIFICANCE STATEMENT Most Parkinson's disease patients will receive l-DOPA and eventually develop hyperkinetic involuntary movements termed dyskinesia. Such symptoms can be as

  20. The Role of Primary Motor Cortex (M1) Glutamate and GABA Signaling in l-DOPA-Induced Dyskinesia in Parkinsonian Rats.

    PubMed

    Lindenbach, David; Conti, Melissa M; Ostock, Corinne Y; George, Jessica A; Goldenberg, Adam A; Melikhov-Sosin, Mitchell; Nuss, Emily E; Bishop, Christopher

    2016-09-21

    Long-term treatment of Parkinson's disease with l-DOPA almost always leads to the development of involuntary movements termed l-DOPA-induced dyskinesia. Whereas hyperdopaminergic signaling in the basal ganglia is thought to cause dyskinesia, alterations in primary motor cortex (M1) activity are also prominent during dyskinesia, suggesting that the cortex may represent a therapeutic target. The present study used the rat unilateral 6-hydroxydopamine lesion model of Parkinson's disease to characterize in vivo changes in GABA and glutamate neurotransmission within M1 and determine their contribution to behavioral output. 6-Hydroxydopamine lesion led to parkinsonian motor impairment that was partially reversed by l-DOPA. Among sham-lesioned rats, l-DOPA did not change glutamate or GABA efflux. Likewise, 6-hydroxydopamine lesion did not impact GABA or glutamate among rats chronically treated with saline. However, we observed an interaction of lesion and treatment whereby, among lesioned rats, l-DOPA given acutely (1 d) or chronically (14-16 d) reduced glutamate efflux and enhanced GABA efflux. Site-specific microinjections into M1 demonstrated that l-DOPA-induced dyskinesia was reduced by M1 infusion of a D1 antagonist, an AMPA antagonist, or a GABAA agonist. Overall, the present study demonstrates that l-DOPA-induced dyskinesia is associated with increased M1 inhibition and that exogenously enhancing M1 inhibition may attenuate dyskinesia, findings that are in agreement with functional imaging and transcranial magnetic stimulation studies in human Parkinson's disease patients. Together, our study suggests that increasing M1 inhibitory tone is an endogenous compensatory response designed to limit dyskinesia severity and that potentiating this response is a viable therapeutic strategy. Most Parkinson's disease patients will receive l-DOPA and eventually develop hyperkinetic involuntary movements termed dyskinesia. Such symptoms can be as debilitating as the disease

  1. TGR5 suppresses high glucose-induced upregulation of fibronectin and transforming growth factor-β1 in rat glomerular mesangial cells by inhibiting RhoA/ROCK signaling.

    PubMed

    Xiong, Fengxiao; Li, Xuejuan; Yang, Zhiying; Wang, Yu; Gong, Wenyan; Huang, Junying; Chen, Cheng; Liu, Peiqing; Huang, Heqing

    2016-12-01

    RhoA/ROCK can cause renal inflammation and fibrosis in the context of diabetes by activating nuclear factor-κB (NF-κB). TGR5 is known for its role in maintaining metabolic homeostasis and anti-inflammation, which is closely related to NF-κB inhibition. Given that TGR5 is highly enriched in kidney, we aim to investigate the regulatory role of TGR5 on fibronectin (FN) and transforming growth factor-β1 (TGF-β1) in high glucose (HG)-treated rat glomerular mesangial cells (GMCs). Both the factors are closely related to renal inflammations and mediated by NF-κB. Moreover, our study determines whether such regulation is achieved by the inhibition of RhoA/ROCK and the subsequent NF-κB suppression. Polymerase chain reaction was taken to test the mRNA level of TGR5. Western blot was used to measure the protein expressions of TGR5, FN, TGF-β1, p65, IκBα, phospho-MYPT1 (Thr853), and MYPT1. Glutathione S-transferase-pull down and immunofluorescence were conducted to test the activation of RhoA, the distribution of TGR5, and p65, respectively. Electrophoretic mobility shift assay was adopted to measure the DNA binding activity of NF-κB. In GMCs, TGR5 activation or overexpression significantly suppressed FN and TGF-β1 protein expressions, NF-κB, and RhoA/ROCK activation induced by HG or transfection of constitutively active RhoA. By contrast, TGR5 RNA interference caused enhancement of FN, TGF-β1 protein expressions, increase of RhoA/ROCK activation. However, TGR5 cannot suppress RhoA/ROCK activation when a selective Protein kinase A (PKA) inhibitor was used. This study suggests that in HG-treated GMCs, TGR5 significantly suppresses the NF-κB-mediated upregulation of FN and TGF-β1, which are hallmarks of diabetic nephropathy. These functions are closely related to the suppression of RhoA/ROCK via PKA.

  2. Recombinant Production, Reconstruction in Lipid-Protein Nanodiscs, and Electron Microscopy of Full-Length α-Subunit of Human Potassium Channel Kv7.1.

    PubMed

    Shenkarev, Z O; Karlova, M G; Kulbatskii, D S; Kirpichnikov, M P; Lyukmanova, E N; Sokolova, O S

    2018-05-01

    Voltage-gated potassium channel Kv7.1 plays an important role in the excitability of cardiac muscle. The α-subunit of Kv7.1 (KCNQ1) is the main structural element of this channel. Tetramerization of KCNQ1 in the membrane results in formation of an ion channel, which comprises a pore and four voltage-sensing domains. Mutations in the human KCNQ1 gene are one of the major causes of inherited arrhythmias, long QT syndrome in particular. The construct encoding full-length human KCNQ1 protein was synthesized in this work, and an expression system in the Pichia pastoris yeast cells was developed. The membrane fraction of the yeast cells containing the recombinant protein (rKCNQ1) was solubilized with CHAPS detergent. To better mimic the lipid environment of the channel, lipid-protein nanodiscs were formed using solubilized membrane fraction and MSP2N2 protein. The rKCNQ1/nanodisc and rKCNQ1/CHAPS samples were purified using the Rho1D4 tag introduced at the C-terminus of the protein. Protein samples were examined using transmission electron microscopy with negative staining. In both cases, homogeneous rKCNQ1 samples were observed based on image analysis. Statistical analysis of the images of individual protein particles solubilized in the detergent revealed the presence of a tetrameric structure confirming intact subunit assembly. A three-dimensional channel structure reconstructed at 2.5-nm resolution represents a compact density with diameter of the membrane part of ~9 nm and height ~11 nm. Analysis of the images of rKCNQ1 in nanodiscs revealed additional electron density corresponding to the lipid bilayer fragment and the MSP2N2 protein. These results indicate that the nanodiscs facilitate protein isolation, purification, and stabilization in solution and can be used for further structural studies of human Kv7.1.

  3. Myc requires RhoA/SRF to reprogram glutamine metabolism.

    PubMed

    Haikala, Heidi M; Marques, Elsa; Turunen, Mikko; Klefström, Juha

    2018-05-04

    RhoA regulates actin cytoskeleton but recent evidence suggest a role for this conserved Rho GTPase also in other cellular processes, including transcriptional control of cell proliferation and survival. Interestingy, loss of RhoA is synthetic lethal with oncogenic Myc, a master transcription factor that turns on anabolic metabolism to promote cell growth in many cancers. We show evidence indicating that the synthetic lethal interaction between RhoA loss and Myc arises from deficiency in glutamine utilization, resulting from impaired co-regulation of glutaminase expression and anaplerosis by Myc and RhoA - serum response factor (SRF) pathway. The results suggest metabolic coordination between Myc and RhoA/SRF in sustaining cancer cell viability and indicate RhoA/SRF as a potential vulnerability in cancer cells for therapeutic targeting.

  4. E-Cadherin Antagonizes Transforming Growth Factor β1 Gene Induction in Hepatic Stellate Cells by Inhibiting RhoA–Dependent Smad3 Phosphorylation

    PubMed Central

    Cho, Il Je; Kim, Young Woo; Han, Chang Yeob; Kim, Eun Hyun; Anderson, Richard A.; Lee, Young Sok; Lee, Chang Ho; Hwang, Se Jin; Kim, Sang Geon

    2011-01-01

    Cadherins mediate cell-cell adhesion and catenin (ctn)-related signaling pathways. Liver fibrosis is accompanied by the loss of E-cadherin (ECAD), which promotes the process of epithelial-mesenchymal transition. Currently, no information is available about the inhibitory role of ECAD in hepatic stellate cell activation. Because of ECAD’s potential for inhibiting the induction of transforming growth factor β1 (TGFβ1), we investigated whether ECAD overexpression prevents TGFβ1 gene induction; we also examined what the molecular basis could be. Forced expression of ECAD decreased α-smooth muscle actin and vimentin levels and caused decreases in the constitutive and inducible expression of the TGFβ1 gene and its downstream genes. ECAD overexpression decreased Smad3 phosphorylation, weakly decreased Smad2 phosphorylation, and thus inhibited Smad reporter activity induced by either treatment with TGFβ1 or Smad3 overexpression. Overexpression of a dominant negative mutant of ras homolog gene family A (RhoA) diminished the ability of TGFβ1 to elicit its own gene induction. Consistently, transfection with a constitutively active mutant of RhoA reversed the inhibition of TGFβ1-inducible or Smad3-inducible reporter activity by ECAD. Studies using the mutant constructs of ECAD revealed that the p120-ctn binding domain of ECAD was responsible for TGFβ1 repression. Consistently, ECAD was capable of binding p120-ctn, which recruited RhoA; this prevented TGFβ1 from increasing RhoA-mediated Smad3 phosphorylation. In the liver samples of patients with mild or severe fibrosis, ECAD expression reciprocally correlated with the severity of fibrosis. Conclusion Our results demonstrate that ECAD inhibits Smad3/2 phosphorylation by recruiting RhoA to p120-ctn at the p120-ctn binding domain, whereas the loss of ECAD due to cadherin switching promotes the up-regulation of TGFβ1 and its target genes, and facilitates liver fibrosis. PMID:20890948

  5. Activity-dependent rapid local RhoA synthesis is required for hippocampal synaptic plasticity.

    PubMed

    Briz, Victor; Zhu, Guoqi; Wang, Yubin; Liu, Yan; Avetisyan, Mariam; Bi, Xiaoning; Baudry, Michel

    2015-02-04

    Dendritic protein synthesis and actin cytoskeleton reorganization are important events required for the consolidation of hippocampal LTP and memory. However, the temporal and spatial relationships between these two processes remain unclear. Here, we report that treatment of adult rat hippocampal slices with BDNF or with tetraethylammonium (TEA), which induces a chemical form of LTP, produces a rapid and transient increase in RhoA protein levels. Changes in RhoA were restricted to dendritic spines of CA3 and CA1 and require de novo protein synthesis regulated by mammalian target of rapamycin (mTOR). BDNF-mediated stimulation of RhoA activity, cofilin phosphorylation, and actin polymerization were completely suppressed by protein synthesis inhibitors. Furthermore, intrahippocampal injections of RhoA antisense oligodeoxynucleotides inhibited theta burst stimulation (TBS)-induced RhoA upregulation in dendritic spines and prevented LTP consolidation. Addition of calpain inhibitors after BDNF or TEA treatment maintained RhoA levels elevated and prolonged the effects of BDNF and TEA on actin polymerization. Finally, the use of isoform-selective calpain inhibitors revealed that calpain-2 was involved in RhoA synthesis, whereas calpain-1 mediated RhoA degradation. Overall, this mechanism provides a novel link between dendritic protein synthesis and reorganization of the actin cytoskeleton in hippocampal dendritic spines during LTP consolidation. Copyright © 2015 the authors 0270-6474/15/352269-14$15.00/0.

  6. RhoA mediates the expression of acidic extracellular pH-induced matrix metalloproteinase-9 mRNA through phospholipase D1 in mouse metastatic B16-BL6 melanoma cells.

    PubMed

    Maeda, Toyonobu; Yuzawa, Satoshi; Suzuki, Atsuko; Baba, Yuh; Nishimura, Yukio; Kato, Yasumasa

    2016-03-01

    Solid tumors are characterized by acidic extracellular pH (pHe). The present study examined the contribution of small GTP-binding proteins to phospholipase D (PLD) activation of acidic pHe-induced matrix metalloproteinase-9 (MMP-9) production. Acidic pHe-induced MMP-9 production was reduced by C3 exoenzyme, which inhibits the Rho family of GTPases; cytochalasin D, which inhibits actin reorganization; and simvastatin, which inhibits geranylgeranylation of Rho. Small interfering RNA (siRNA) against RhoA, but not against Rac1 or Cdc42, significantly inhibited acidic pHe induction of MMP-9. Pull-down assays showed that acidic pHe increased the activated form of RhoA. Forced expression of constitutively active RhoA induced MMP-9 production, even at neutral pHe. RhoA siRNA also reduced acidic pHe induced PLD activity. Specific inhibition of PLD1 and Pld1 gene knockout significantly reduced acidic pHe-induced MMP-9 expression. In contrast, PLD2 inhibition or knockout had no effect on MMP-9 expression. These findings suggested that RhoA-PLD1 signaling is involved in acidic pHe induction of MMP-9.

  7. Effect of GABA agonists and GABA-A receptor modulators on cocaine- and food-maintained responding and cocaine discrimination in rats.

    PubMed

    Barrett, Andrew C; Negus, S Stevens; Mello, Nancy K; Caine, S Barak

    2005-11-01

    Recent studies indicate that GABAergic ligands modulate abuse-related effects of cocaine. The goal of this study was to evaluate the effects of a mechanistically diverse group of GABAergic ligands on the discriminative stimulus and reinforcing effects of cocaine in rats. One group of rats was trained to discriminate 5.6 mg/kg cocaine from saline in a two-lever, food-reinforced, drug discrimination procedure. In two other groups, responding was maintained by cocaine (0-3.2 mg/kg/injection) or liquid food (0-100%) under a fixed ratio 5 schedule. Six GABA agonists were tested: the GABA-A receptor agonist muscimol, the GABA-B receptor agonist baclofen, the GABA transaminase inhibitor gamma-vinyl-GABA (GVG), and three GABA-A receptor modulators (the barbiturate pentobarbital, the high-efficacy benzodiazepine midazolam, and the low-efficacy benzodiazepine enazenil). When tested alone, none of the compounds substituted fully for the discriminative stimulus effects of cocaine. As acute pretreatments, select doses of midazolam and pentobarbital produced 2.2- to 3.6-fold rightward shifts in the cocaine dose-effect function. In contrast, muscimol, baclofen, GVG, and enazenil failed to alter the discriminative stimulus effects of cocaine. In assays of cocaine- and food-maintained responding, midazolam and pentobarbital decreased cocaine self-administration at doses 9.6- and 3.3-fold lower, respectively, than those that decreased food-maintained responding. In contrast, muscimol, baclofen, and GVG decreased cocaine self-administration at doses that also decreased food-maintained responding. Enazenil failed to alter cocaine self-administration. Together with previous studies, these data suggest that among mechanistically diverse GABA agonists, high-efficacy GABA-A modulators may be the most effective for modifying the abuse-related effects of cocaine.

  8. γ-amino butyric acid (GABA) level as an overall survival risk factor in breast cancer.

    PubMed

    Brzozowska, Anna; Burdan, Franciszek; Duma, Dariusz; Solski, Janusz; Mazurkiewicz, Maria

    2017-09-21

    The γ-amino butyric acid (GABA) plays important role in the proliferation and migration of cancer cells. The aim of the study was to evaluate the level of GABA in breast cancer, in relation to clinical and epidemiological data. The study was conducted on 89 patients with breast cancer in stage I-II. GABA level was assessed using spectrofluorometric method in tumour homogenates. Immunoexpression of E-cadherin was evaluated histologically on paraffin fixed specimens. Overall and disease-free survival was assessed for a 15-year interval period. Median overall survival was significantly longer (127.2 months) in patients with a high level of GABA (>89.3 μg/1), compared with a group with a low level of the amino acid (106.4 months). Disease-free survival was insignificantly different - 99 and 109 months, respectively. A significantly longer overall survival (131.2 months) was seen among patients with a high level of GABA and positive E-cadherin immunoexpression, compared with a group characterized by a low level of GABA and lack of E-cadherin immunorectivity (98.1 months). The co-existence of negative immunoexpression of E-cadherin and low GABA concentration resulted in a six-fold increase in the risk of death (HR=6.03). GABA has a significant prognostic value in breast cancer. Co-existence of a low level of GABA and loss of E-cadherin immune-expression seems to be a new, independent, and negative prognostic marker of the neoplasm.

  9. Binding and Translocation of Termination Factor Rho Studied at the Single-Molecule Level

    PubMed Central

    Koslover, Daniel J.; Fazal, Furqan M.; Mooney, Rachel A.; Landick, Robert; Block, Steven M.

    2012-01-01

    Rho termination factor is an essential hexameric helicase responsible for terminating 20–50% of all mRNA synthesis in E. coli. We used single- molecule force spectroscopy to investigate Rho-RNA binding interactions at the Rho- utilization (rut) site of the ? tR1 terminator. Our results are consistent with Rho complexes adopting two states, one that binds 57 ±2 nucleotides of RNA across all six of the Rho primary binding sites, and another that binds 85 ±2 nucleotides at the six primary sites plus a single secondary site situated at the center of the hexamer. The single-molecule data serve to establish that Rho translocates 5′-to-3′ towards RNA polymerase (RNAP) by a tethered-tracking mechanism, looping out the intervening RNA between the rut site and RNAP. These findings lead to a general model for Rho binding and translocation, and establish a novel experimental approach that should facilitate additional single- molecule studies of RNA-binding proteins. PMID:22885804

  10. Neuroimaging studies of GABA in schizophrenia: a systematic review with meta-analysis

    PubMed Central

    Egerton, A; Modinos, G; Ferrera, D; McGuire, P

    2017-01-01

    Data from animal models and from postmortem studies suggest that schizophrenia is associated with brain GABAergic dysfunction. The extent to which this is reflected in data from in vivo studies of GABA function in schizophrenia is unclear. The Medline database was searched to identify articles published until 21 October 2016. The search terms included GABA, proton magnetic resonance spectroscopy (1H-MRS), positron emission tomography (PET), single photon emission computed tomography (SPECT), schizophrenia and psychosis. Sixteen GABA 1H-MRS studies (538 controls, 526 patients) and seven PET/SPECT studies of GABAA/benzodiazepine receptor (GABAA/BZR) availability (118 controls, 113 patients) were identified. Meta-analyses of 1H-MRS GABA in the medial prefrontal cortex (mPFC), parietal/occipital cortex (POC) and striatum did not show significant group differences (mFC: g=−0.3, 409 patients, 495 controls, 95% confidence interval (CI): −0.6 to 0.1; POC: g=−0.3, 139 patients, 111 controls, 95% CI: −0.9 to 0.3; striatum: g=−0.004, 123 patients, 95 controls, 95% CI: −0.7 to 0.7). Heterogeneity across studies was high (I2>50%), and this was not explained by subsequent moderator or meta-regression analyses. There were insufficient PET/SPECT receptor availability studies for meta-analyses, but a systematic review did not suggest replicable group differences in regional GABAA/BZR availability. The current literature does not reveal consistent alterations in in vivo GABA neuroimaging measures in schizophrenia, as might be hypothesized from animal models and postmortem data. The analysis highlights the need for further GABA neuroimaging studies with improved methodology and addressing potential sources of heterogeneity. PMID:28585933

  11. Bombesin stimulates invasion and migration of Isreco1 colon carcinoma cells in a Rho-dependent manner.

    PubMed

    Saurin, Jean-Christophe; Fallavier, Marjorie; Sordat, Bernard; Gevrey, Jean-Claude; Chayvialle, Jean-Alain; Abello, Jacques

    2002-08-15

    The membrane receptor for the neuropeptide bombesin/gastrin-releasing peptide (GRP) is expressed by a large fraction of human colorectal carcinoma cells. We reported previously a stimulation of cell adhesion and lamellipodia formation by the neuropeptide bombesin in the human, bombesin/GRP receptor-expressing, Isreco1 colorectal cancer cell line (J. C. Saurin et al., Cancer Res., 59: 962-967, 1999). Using invasion and motility assays, we demonstrate in this report that bombesin can both enhance the invasive capacity of Isreco1 cells in a dose-dependent manner (maximal effect at 1 nM) and stimulate the closure of wounds performed on confluent Isreco1 cells. These effects were reversed fully by the specific bombesin/GRP receptor antagonist D-Phe(6)-Bn(6-13)OMe used at 1 micro M. MMP-9 and urokinase-type plasminogen activator were expressed by Isreco1 cells, and bombesin did not significantly alter their level of secretion. Interestingly, exoenzyme C3 (10 micro g/ml) decreased cell invasiveness induced by bombesin by 70% and completely inhibited the migration of Isreco1 cells. Similarly, the Rho-kinase inhibitor Y-27632 dose-dependently reduced the effect of bombesin on cell invasion. Moreover, pull-down assays for GTP-bound RhoA demonstrated that bombesin was able to activate the small G-protein in Isreco1 cells. These results show that the neuropeptide bombesin is able to modulate invasiveness of Isreco1 colorectal carcinoma cells in vitro through a Rho-dependent pathway, leading to an increase in cell locomotion without a significant effect on tumor-cell associated proteolytic activity. These findings indicate that bombesin/GRP receptor expression may contribute to the cellular events that are critical for invasion/migration of colorectal carcinoma cells.

  12. Fiat lux! Phylogeny and bioinformatics shed light on GABA functions in plants.

    PubMed

    Renault, Hugues

    2013-06-01

    The non-protein amino acid γ-aminobutyric acid (GABA) accumulates in plants in response to a wide variety of environmental cues. Recent data point toward an involvement of GABA in tricarboxylic acid (TCA) cycle activity and respiration, especially in stressed roots. To gain further insights into potential GABA functions in plants, phylogenetic and bioinformatic approaches were undertaken. Phylogenetic reconstruction of the GABA transaminase (GABA-T) protein family revealed the monophyletic nature of plant GABA-Ts. However, this analysis also pointed to the common origin of several plant aminotransferases families, which were found more similar to plant GABA-Ts than yeast and human GABA-Ts. A computational analysis of AtGABA-T co-expressed genes was performed in roots and in stress conditions. This second approach uncovered a strong connection between GABA metabolism and glyoxylate cycle during stress. Both in silico analyses open new perspectives and hypotheses for GABA metabolic functions in plants.

  13. GABA (γ-Aminobutyric Acid) Uptake Via the GABA Permease GabP Represses Virulence Gene Expression in Pseudomonas syringae pv. tomato DC3000.

    PubMed

    McCraw, S L; Park, D H; Jones, R; Bentley, M A; Rico, A; Ratcliffe, R G; Kruger, N J; Collmer, A; Preston, G M

    2016-12-01

    The nonprotein amino acid γ-aminobutyric acid (GABA) is the most abundant amino acid in the tomato (Solanum lycopersicum) leaf apoplast and is synthesized by Arabidopsis thaliana in response to infection by the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 (hereafter called DC3000). High levels of exogenous GABA have previously been shown to repress the expression of the type III secretion system (T3SS) in DC3000, resulting in reduced elicitation of the hypersensitive response (HR) in the nonhost plant tobacco (Nicotiana tabacum). This study demonstrates that the GABA permease GabP provides the primary mechanism for GABA uptake by DC3000 and that the gabP deletion mutant ΔgabP is insensitive to GABA-mediated repression of T3SS expression. ΔgabP displayed an enhanced ability to elicit the HR in young tobacco leaves and in tobacco plants engineered to produce increased levels of GABA, which supports the hypothesis that GABA uptake via GabP acts to regulate T3SS expression in planta. The observation that P. syringae can be rendered insensitive to GABA through loss of gabP but that gabP is retained by this bacterium suggests that GabP is important for DC3000 in a natural setting, either for nutrition or as a mechanism for regulating gene expression. [Formula: see text] Copyright © 2016 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .

  14. Physical and genetic interaction between ammonium transporters and the signaling protein Rho1 in the plant pathogen Ustilago maydis.

    PubMed

    Paul, Jinny A; Barati, Michelle T; Cooper, Michael; Perlin, Michael H

    2014-10-01

    Dimorphic transitions between yeast-like and filamentous forms occur in many fungi and are often associated with pathogenesis. One of the cues for such a dimorphic switch is the availability of nutrients. Under conditions of nitrogen limitation, fungal cells (such as those of Saccharomyces cerevisiae and Ustilago maydis) switch from budding to pseudohyphal or filamentous growth. Ammonium transporters (AMTs) are responsible for uptake and, in some cases, for sensing the availability of ammonium, a preferred nitrogen source. Homodimer and/or heterodimer formation may be required for regulating the activity of the AMTs. To investigate the potential interactions of Ump1 and Ump2, the AMTs of the maize pathogen U. maydis, we first used the split-ubiquitin system, followed by a modified split-YFP (yellow fluorescent protein) system, to validate the interactions in vivo. This analysis showed the formation of homo- and hetero-oligomers by Ump1 and Ump2. We also demonstrated the interaction of the high-affinity ammonium transporter, Ump2, with the Rho1 GTPase, a central protein in signaling, with roles in controlling polarized growth. This is the first demonstration in eukaryotes of the physical interaction in vivo of an ammonium transporter with the signaling protein Rho1. Moreover, the Ump proteins interact with Rho1 during the growth of cells in low ammonium concentrations, a condition required for the expression of the Umps. Based on these results and the genetic evidence for the interaction of Ump2 with both Rho1 and Rac1, another small GTPase, we propose a model for the role of these interactions in controlling filamentation, a fundamental aspect of development and pathogenesis in U. maydis. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  15. GABA-independent GABAA Receptor Openings Maintain Tonic Currents

    PubMed Central

    Wlodarczyk, Agnieszka I.; Sylantyev, Sergiy; Herd, Murray B.; Kersanté, Flavie; Lambert, Jeremy J.; Rusakov, Dmitri A.; Linthorst, Astrid C.E.; Semyanov, Alexey; Belelli, Delia; Pavlov, Ivan; Walker, Matthew C.

    2013-01-01

    Activation of GABAA receptors (GABAARs) produces two forms of inhibition: ‘phasic’ inhibition generated by the rapid, transient activation of synaptic GABAARs by presynaptic GABA release, and tonic inhibition generated by the persistent activation of peri- or extrasynaptic GABAARs which can detect extracellular GABA. Such tonic GABAAR-mediated currents are particularly evident in dentate granule cells in which they play a major role in regulating cell excitability. Here we show that in rat dentate granule cells in ex-vivo hippocampal slices, tonic currents are predominantly generated by GABA-independent GABAA receptor openings. This tonic GABAAR conductance is resistant to the competitive GABAAR antagonist SR95531, which at high concentrations acts as a partial agonist, but can be blocked by an open channel blocker picrotoxin. When slices are perfused with 200 nM GABA, a concentration that is comparable to cerebrospinal fluid concentrations but is twice that measured by us in the hippocampus in vivo using zero-net-flux microdialysis, negligible GABA is detected by dentate granule cells. Spontaneously opening GABAARs, therefore, maintain dentate granule cell tonic currents in the face of low extracellular GABA concentrations. PMID:23447601

  16. Antagonism of GABA-B but not GABA-A receptors in the VTA prevents stress- and intra-VTA CRF-induced reinstatement of extinguished cocaine seeking in rats.

    PubMed

    Blacktop, Jordan M; Vranjkovic, Oliver; Mayer, Matthieu; Van Hoof, Matthew; Baker, David A; Mantsch, John R

    2016-03-01

    Stress-induced reinstatement of cocaine seeking requires corticotropin releasing factor (CRF) actions in the ventral tegmental area (VTA). However the mechanisms through which CRF regulates VTA function to promote cocaine use are not fully understood. Here we examined the role of GABAergic neurotransmission in the VTA mediated by GABA-A or GABA-B receptors in the reinstatement of extinguished cocaine seeking by a stressor, uncontrollable intermittent footshock, or bilateral intra-VTA administration of CRF. Rats underwent repeated daily cocaine self-administration (1.0 mg/kg/ing; 14 × 6 h/day) and extinction and were tested for reinstatement in response to footshock (0.5 mA, 0.5" duration, average every 40 s; range 10-70 s) or intra-VTA CRF delivery (500 ng/side) following intra-VTA pretreatment with the GABA-A antagonist, bicuculline, the GABA-B antagonist, 2-hydroxysaclofen or vehicle. Intra-VTA bicuculline (1, 10 or 20 ng/side) failed to block footshock- or CRF-induced cocaine seeking at either dose tested. By contrast, 2-hydroxysaclofen (0.2 or 2 μg/side) prevented reinstatement by both footshock and intra-VTA CRF at a concentration that failed to attenuate food-reinforced lever pressing (45 mg sucrose-sweetened pellets; FR4 schedule) in a separate group of rats. These data suggest that GABA-B receptor-dependent CRF actions in the VTA mediate stress-induced cocaine seeking and that GABA-B receptor antagonists may have utility for the management of stress-induced relapse in cocaine addicts. Copyright © 2015 Elsevier Ltd. All rights reserved.

  17. Strain-specific programming of prenatal ethanol exposure across generations.

    PubMed

    Popoola, Daniel O; Nizhnikov, Michael E; Cameron, Nicole M

    2017-05-01

    Behavioral consequences of prenatal alcohol exposure (PAE) can be transmitted from in utero-exposed F1 generation to their F2 offspring. This type of transmission is modulated by genetic and epigenetic mechanisms. This study investigated the intergenerational consequences of prenatal exposure to a low ethanol dose (1 g/kg) during gestational days 17-20, on ethanol-induced hypnosis in adolescent male F1 and F2 generations, in two strains of rats. Adolescent Long-Evans and Sprague-Dawley male rats were tested for sensitivity to ethanol-induced hypnosis at a 3.5-g/kg or 4.5-g/kg ethanol dose using the loss of righting reflex (LORR) paradigm. We hypothesized that PAE would attenuate sensitivity to ethanol-induced hypnosis in the ethanol-exposed animals in these two strains and in both generations. Interestingly, we only found this effect in Sprague-Dawley rats. Lastly, we investigated PAE related changes in expression of GABA A receptor α1, α4, and δ subunits in the cerebral cortex of the PAE sensitive Sprague-Dawley strain. We hypothesized a reduction in the cerebral cortex GABA A receptor subunits' expression in the F1 and F2 PAE groups compared to control animals. GABA A receptor α1, α4, and δ subunits protein expressions were quantified in the cerebral cortex of F1 and F2 male adolescents by western blotting. PAE did not alter cerebral cortical GABA A receptor subunit expressions in the F1 generation, but it decreased GABA A receptor α4 and δ subunits' expressions in the F2 generation, and had a tendency to decrease α1 subunit expression. We also found correlations between some of the subunits in both generations. These strain-dependent vulnerabilities to ethanol sensitivity, and intergenerational PAE-mediated changes in sensitivity to alcohol indicate that genetic and epigenetic factors interact to determine the outcomes of PAE animals and their offspring. Copyright © 2017 Elsevier Inc. All rights reserved.

  18. In vivo measurement of GABA transmission in healthy subjects and schizophrenia patients.

    PubMed

    Frankle, W Gordon; Cho, Raymond Y; Prasad, Konasale M; Mason, N Scott; Paris, Jennifer; Himes, Michael L; Walker, Christopher; Lewis, David A; Narendran, Rajesh

    2015-11-01

    Postmortem studies in schizophrenia reveal alterations in gene products that regulate the release and extracellular persistence of GABA. However, results of in vivo studies of schizophrenia measuring total tissue GABA with magnetic resonance spectroscopy (MRS) have been inconsistent. Neither the postmortem nor the MRS studies directly address the physiological properties of GABA neurotransmission. The present study addresses this question through an innovative positron emission tomography (PET) paradigm. The binding of [(11)C]flumazenil, a benzodiazepine-specific PET radiotracer, was measured before and after administration of tiagabine (0.2 mg/kg of body weight), a GABA membrane transporter (GAT1) blocker, in 17 off-medication patients with schizophrenia and 22 healthy comparison subjects. Increased extracellular GABA, through GAT1 blockade, enhances the affinity of GABAA receptors for benzodiazepine ligands, detected as an increase in [(11)C]flumazenil tissue distribution volume (VT). [(11)C]Flumazenil VT was significantly increased across all cortical brain regions in the healthy comparison group but not in the schizophrenia group. This lack of effect was most prominent in the antipsychotic-naive schizophrenia group. In this subgroup, [(11)C]flumazenil ΔVT in the medial temporal lobe was correlated with positive symptoms, and baseline [(11)C]flumazenil VT in the medial temporal lobe was negatively correlated with visual learning. In the healthy comparison group but not the schizophrenia group, [(11)C]flumazenil ΔVT was positively associated with gamma-band oscillation power. This study demonstrates, for the first time, an in vivo impairment in GABA transmission in schizophrenia, most prominent in antipsychotic-naive individuals. The impairment in GABA transmission appears to be linked to clinical symptoms, disturbances in cortical oscillations, and cognition.

  19. The Improvement of Sleep by Oral Intake of GABA and Apocynum venetum Leaf Extract.

    PubMed

    Yamatsu, Atsushi; Yamashita, Yusuke; Maru, Isafumi; Yang, Jinwei; Tatsuzaki, Jin; Kim, Mujo

    2015-01-01

    The effects of two food materials, γ-aminobutyric acid (GABA) produced by natural fermentation and Apocynum venetum leaf extract (AVLE), on the improvement of sleep were investigated in humans. The electroencephalogram (EEG) test revealed that oral administration of GABA (100 mg) and AVLE (50 mg) had beneficial effects on sleep. GABA shortened sleep latency by 5.3 min and AVLE increased non-rapid eye movement (REM) sleep time by 7.6%. Simultaneous intake of GABA and AVLE shortened sleep latency by 4.3 min and increased non-REM sleep time by 5.1%. The result of questionnaires showed that GABA and AVLE enabled subjects to realize the effects on sleep. These results mean that GABA can help people to fall asleep quickly, AVLE induces deep sleep, and they function complementarily with simultaneous intake. Since both GABA and AVLE are materials of foods and have been ingested for a long time, they can be regarded as safe and appropriate for daily intake in order to improve the quality of sleep.

  20. An RNA motif advances transcription by preventing Rho-dependent termination

    PubMed Central

    Sevostyanova, Anastasia; Groisman, Eduardo A.

    2015-01-01

    The transcription termination factor Rho associates with most nascent bacterial RNAs as they emerge from RNA polymerase. However, pharmacological inhibition of Rho derepresses only a small fraction of these transcripts. What, then, determines the specificity of Rho-dependent transcription termination? We now report the identification of a Rho-antagonizing RNA element (RARE) that hinders Rho-dependent transcription termination. We establish that RARE traps Rho in an inactive complex but does not prevent Rho binding to its recruitment sites. Although translating ribosomes normally block Rho access to an mRNA, inefficient translation of an open reading frame in the leader region of the Salmonella mgtCBR operon actually enables transcription of its associated coding region by favoring an RNA conformation that sequesters RARE. The discovery of an RNA element that inactivates Rho signifies that the specificity of nucleic-acid binding proteins is defined not only by the sequences that recruit these proteins but also by sequences that antagonize their activity. PMID:26630006

  1. Yeast Inner-Subunit PA-NZ-1 Labeling Strategy for Accurate Subunit Identification in a Macromolecular Complex through Cryo-EM Analysis.

    PubMed

    Wang, Huping; Han, Wenyu; Takagi, Junichi; Cong, Yao

    2018-05-11

    Cryo-electron microscopy (cryo-EM) has been established as one of the central tools in the structural study of macromolecular complexes. Although intermediate- or low-resolution structural information through negative staining or cryo-EM analysis remains highly valuable, we lack general and efficient ways to achieve unambiguous subunit identification in these applications. Here, we took advantage of the extremely high affinity between a dodecapeptide "PA" tag and the NZ-1 antibody Fab fragment to develop an efficient "yeast inner-subunit PA-NZ-1 labeling" strategy that when combined with cryo-EM could precisely identify subunits in macromolecular complexes. Using this strategy combined with cryo-EM 3D reconstruction, we were able to visualize the characteristic NZ-1 Fab density attached to the PA tag inserted into a surface-exposed loop in the middle of the sequence of CCT6 subunit present in the Saccharomyces cerevisiae group II chaperonin TRiC/CCT. This procedure facilitated the unambiguous localization of CCT6 in the TRiC complex. The PA tag was designed to contain only 12 amino acids and a tight turn configuration; when inserted into a loop, it usually has a high chance of maintaining the epitope structure and low likelihood of perturbing the native structure and function of the target protein compared to other tagging systems. We also found that the association between PA and NZ-1 can sustain the cryo freezing conditions, resulting in very high occupancy of the Fab in the final cryo-EM images. Our study demonstrated the robustness of this strategy combined with cryo-EM in efficient and accurate subunit identification in challenging multi-component complexes. Copyright © 2018 Elsevier Ltd. All rights reserved.

  2. Role of RhoA and its effectors ROCK and mDia1 in the modulation of deformation-induced FAK, ERK, p38, and MLC motogenic signals in human Caco-2 intestinal epithelial cells

    PubMed Central

    Chaturvedi, Lakshmi S.; Marsh, Harold M.

    2011-01-01

    Repetitive deformation enhances intestinal epithelial migration across tissue fibronectin. We evaluated the contribution of RhoA and its effectors Rho-associated kinase (ROK/ROCK) and mammalian diaphanous formins (mDia1) to deformation-induced intestinal epithelial motility across fibronectin and the responsible focal adhesion kinase (FAK), extracellular signal-regulated kinase (ERK), p38, and myosin light chain (MLC) signaling. We reduced RhoA, ROCK1, ROCK2, and mDia1 by smart-pool double-stranded short-interfering RNAs (siRNA) and pharmacologically inhibited RhoA, ROCK, and FAK in human Caco-2 intestinal epithelial monolayers on fibronectin-coated membranes subjected to 10% repetitive deformation at 10 cycles/min. Migration was measured by wound closure. Stimulation of migration by deformation was prevented by exoenzyme C3, Y27632, or selective RhoA, ROCK1, and ROCK2 or mDia1 siRNAs. RhoA, ROCK inhibition, or RhoA, ROCK1, ROCK2, mDia1, and FAK reduction by siRNA blocked deformation-induced nuclear ERK phosphorylation without preventing ERK phosphorylation in the cytoplasmic protein fraction. Furthermore, RhoA, ROCK inhibition or RhoA, ROCK1, ROCK2, and mDia1 reduction by siRNA also blocked strain-induced FAK-Tyr925, p38, and MLC phosphorylation. These results suggest that RhoA, ROCK, mDia1, FAK, ERK, p38, and MLC all mediate the stimulation of intestinal epithelial migration by repetitive deformation. This pathway may be an important target for interventions to promote mechanotransduced mucosal healing during inflammation. PMID:21849669

  3. Distinct c-Met activation mechanisms induce cell rounding or invasion through pathways involving integrins, RhoA and HIP1.

    PubMed

    Mai, Anja; Muharram, Ghaffar; Barrow-McGee, Rachel; Baghirov, Habib; Rantala, Juha; Kermorgant, Stéphanie; Ivaska, Johanna

    2014-05-01

    Many carcinomas have acquired oncogenic mechanisms for activating c-Met, including c-Met overexpression and excessive autocrine or paracrine stimulation with hepatocyte growth factor (HGF). However, the biological outcome of c-Met activation through these distinct modes remains ambiguous. Here, we report that HGF-mediated c-Met stimulation triggers a mesenchymal-type collective cell invasion. By contrast, the overexpression of c-Met promotes cell rounding. Moreover, in a high-throughput siRNA screen that was performed using a library of siRNAs against putative regulators of integrin activity, we identified RhoA and the clathrin-adapter protein HIP1 as crucial c-Met effectors in these morphological changes. Transient RhoA activation was necessary for the HGF-induced invasion, whereas sustained RhoA activity regulated c-Met-induced cell rounding. In addition, c-Met-induced cell rounding correlated with the phosphorylation of filamin A and the downregulation of active cell-surface integrins. By contrast, a HIP1-mediated increase in β1-integrin turnover was required for the invasion triggered by HGF. Taken together, our results indicate that c-Met induces distinct cell morphology alterations depending on the stimulus that activates c-Met.

  4. Effect of Cavβ Subunits on Structural Organization of Cav1.2 Calcium Channels

    PubMed Central

    Duong, Son Q.; Thomas, Sam; Harry, Jo Beth; Patel, Chirag; Lao, Qi Zong; Soldatov, Nikolai M.

    2009-01-01

    Background Voltage-gated Cav1.2 calcium channels play a crucial role in Ca2+ signaling. The pore-forming α1C subunit is regulated by accessory Cavβ subunits, cytoplasmic proteins of various size encoded by four different genes (Cavβ1 - β4) and expressed in a tissue-specific manner. Methods and Results Here we investigated the effect of three major Cavβ types, β1b, β2d and β3, on the structure of Cav1.2 in the plasma membrane of live cells. Total internal reflection fluorescence microscopy showed that the tendency of Cav1.2 to form clusters depends on the type of the Cavβ subunit present. The highest density of Cav1.2 clusters in the plasma membrane and the smallest cluster size were observed with neuronal/cardiac β1b present. Cav1.2 channels containing β3, the predominant Cavβ subunit of vascular smooth muscle cells, were organized in a significantly smaller number of larger clusters. The inter- and intramolecular distances between α1C and Cavβ in the plasma membrane of live cells were measured by three-color FRET microscopy. The results confirm that the proximity of Cav1.2 channels in the plasma membrane depends on the Cavβ type. The presence of different Cavβ subunits does not result in significant differences in the intramolecular distance between the termini of α1C, but significantly affects the distance between the termini of neighbor α1C subunits, which varies from 67 Å with β1b to 79 Å with β3. Conclusions Thus, our results show that the structural organization of Cav1.2 channels in the plasma membrane depends on the type of Cavβ subunits present. PMID:19492014

  5. High-mobility group box 1 inhibits HCO3− absorption in the medullary thick ascending limb through RAGE-Rho-ROCK-mediated inhibition of basolateral Na+/H+ exchange

    PubMed Central

    Watts, Bruns A.; George, Thampi; Badalamenti, Andrew

    2016-01-01

    High-mobility group box 1 (HMGB1) is a nuclear protein released extracellularly in response to infection or injury, where it activates immune responses and contributes to the pathogenesis of kidney dysfunction in sepsis and sterile inflammatory disorders. Recently, we demonstrated that HMGB1 inhibits HCO3− absorption in perfused rat medullary thick ascending limbs (MTAL) through a basolateral receptor for advanced glycation end products (RAGE)-dependent pathway that is additive to Toll-like receptor 4 (TLR4)-ERK-mediated inhibition by LPS (Good DW, George T, Watts BA III. Am J Physiol Renal Physiol 309: F720–F730, 2015). Here, we examined signaling and transport mechanisms that mediate inhibition by HMGB1. Inhibition of HCO3− absorption by HMGB1 was eliminated by the Rho-associated kinase (ROCK) inhibitor Y27632 and by a specific inhibitor of Rho, the major upstream activator of ROCK. HMGB1 increased RhoA and ROCK1 activity. HMGB1-induced ROCK1 activation was eliminated by the RAGE antagonist FPS-ZM1 and by inhibition of Rho. The Rho and ROCK inhibitors had no effect on inhibition of HCO3− absorption by bath LPS. Inhibition of HCO3− absorption by HMGB1 was eliminated by bath amiloride, 0 Na+ bath, and the F-actin stabilizer jasplakinolide, three conditions that selectively prevent inhibition of MTAL HCO3− absorption mediated through NHE1. HMGB1 decreased basolateral Na+/H+ exchange activity through activation of ROCK. We conclude that HMGB1 inhibits HCO3− absorption in the MTAL through a RAGE-RhoA-ROCK1 signaling pathway coupled to inhibition of NHE1. The HMGB1-RAGE-RhoA-ROCK1 pathway thus represents a potential target to attenuate MTAL dysfunction during sepsis and other inflammatory disorders. HMGB1 and LPS inhibit HCO3− absorption through different receptor signaling and transport mechanisms, which enables these pathogenic mediators to act directly and independently to impair MTAL function. PMID:27358052

  6. Conopeptide Vt3.1 preferentially inhibits BK potassium channels containing β4 subunits via electrostatic interactions.

    PubMed

    Li, Min; Chang, Shan; Yang, Longjin; Shi, Jingyi; McFarland, Kelli; Yang, Xiao; Moller, Alyssa; Wang, Chunguang; Zou, Xiaoqin; Chi, Chengwu; Cui, Jianmin

    2014-02-21

    BK channel β subunits1-β4) modulate the function of channels formed by slo1 subunits to produce tissue-specific phenotypes. The molecular mechanism of how the homologous β subunits differentially alter BK channel functions and the role of different BK channel functions in various physiologic processes remain unclear. By studying channels expressed in Xenopus laevis oocytes, we show a novel disulfide-cross-linked dimer conopeptide, Vt3.1 that preferentially inhibits BK channels containing the β4 subunit, which is most abundantly expressed in brain and important for neuronal functions. Vt3.1 inhibits the currents by a maximum of 71%, shifts the G-V relation by 45 mV approximately half-saturation concentrations, and alters both open and closed time of single channel activities, indicating that the toxin alters voltage dependence of the channel. Vt3.1 contains basic residues and inhibits voltage-dependent activation by electrostatic interactions with acidic residues in the extracellular loops of the slo1 and β4 subunits. These results suggest a large interaction surface between the slo1 subunit of BK channels and the β4 subunit, providing structural insight into the molecular interactions between slo1 and β4 subunits. The results also suggest that Vt3.1 is an excellent tool for studying β subunit modulation of BK channels and for understanding the physiological roles of BK channels in neurophysiology.

  7. GABA and primary motor cortex inhibition in young and older adults: a multimodal reliability study.

    PubMed

    Mooney, Ronan A; Cirillo, John; Byblow, Winston D

    2017-07-01

    The effects of healthy aging on γ-aminobutyric acid (GABA) within primary motor cortex (M1) remain poorly understood. Studies have reported contrasting results, potentially due to limitations with the common assessment technique. The aim of the present study was to investigate the effect of healthy aging on M1 GABA concentration and neurotransmission using a multimodal approach. Fifteen young and sixteen older adults participated in this study. Magnetic resonance spectroscopy (MRS) was used to measure M1 GABA concentration. Single-pulse and threshold-tracking paired-pulse transcranial magnetic stimulation (TMS) protocols were used to examine cortical silent period duration, short- and long-interval intracortical inhibition (SICI and LICI), and late cortical disinhibition (LCD). The reliability of TMS measures was examined with intraclass correlation coefficient analyses. SICI at 1 ms was reduced in older adults (15.13 ± 2.59%) compared with young (25.66 ± 1.44%; P = 0.002). However, there was no age-related effect for cortical silent period duration, SICI at 3 ms, LICI, or LCD (all P > 0.66). The intersession reliability of threshold-tracking measures was good to excellent for both young (range 0.75-0.96) and older adults (range 0.88-0.93). Our findings indicate that extrasynaptic inhibition may be reduced with advancing age, whereas GABA concentration and synaptic inhibition are maintained. Furthermore, MRS and threshold-tracking TMS provide valid and reliable assessment of M1 GABA concentration and neurotransmission, respectively, in young and older adults. NEW & NOTEWORTHY γ-Aminobutyric acid (GABA) in primary motor cortex was assessed in young and older adults using magnetic resonance spectroscopy and threshold-tracking paired-pulse transcranial magnetic stimulation. Older adults exhibited reduced extrasynaptic inhibition (short-interval intracortical inhibition at 1 ms) compared with young, whereas GABA concentration and synaptic inhibition were

  8. Neurochemical correlates of. gamma. -aminobutyrate (GABA) inhibition in cat visual cortex

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Balcar, V.J.; Dreher, B.

    1990-01-01

    High affinity binding of ({sup 3}H){gamma}-aminobutyric acid (GABA) to neuronal membranes from different parts of cat visual cortex was tested for sensitivity to GABA{sub A} agonists isoguvacine and THIP, GABA{sub A} antagonist SR95531 and GABA{sub B} agonist baclofen. Some of the GABA{sub A}-binding sites were found to have a very low affinity for THIP, suggesting the presence and, possibly, uneven distribution of non-synaptic GABA{sub A} receptors in cat visual cortex. There were no differences in K{sub m} and V{sub max} values of high affinity uptake of GABA and in the potency of K{sup +}-stimulated release of GABA, between primary andmore » association cortices. Consequently, the present results indicate that despite the anatomical and physiological differences between the primary and association feline visual cortices the neurochemical characteristics of GABAergic inhibition are very similar in the two regions.« less

  9. Gamma-amino butyric acid (GABA) release in the ciliated protozoon Paramecium occurs by neuronal-like exocytosis.

    PubMed

    Ramoino, P; Milanese, M; Candiani, S; Diaspro, A; Fato, M; Usai, C; Bonanno, G

    2010-04-01

    Paramecium primaurelia expresses a significant amount of gamma-amino butyric acid (GABA). Paramecia possess both glutamate decarboxylase (GAD)-like and vesicular GABA transporter (vGAT)-like proteins, indicating the ability to synthesize GABA from glutamate and to transport GABA into vesicles. Using antibodies raised against mammalian GAD and vGAT, bands with an apparent molecular weight of about 67 kDa and 57 kDa were detected. The presence of these bands indicated a similarity between the proteins in Paramecium and in mammals. VAMP, syntaxin and SNAP, putative proteins of the release machinery that form the so-called SNARE complex, are present in Paramecium. Most VAMP, syntaxin and SNAP fluorescence is localized in spots that vary in size and density and are primarily distributed near the plasma membrane. Antibodies raised against mammal VAMP-3, sintaxin-1 or SNAP-25 revealed protein immunoblot bands having molecular weights consistent with those observed in mammals. Moreover, P. primaurelia spontaneously releases GABA into the environment, and this neurotransmitter release significantly increases after membrane depolarization. The depolarization-induced GABA release was strongly reduced not only in the absence of extracellular Ca(2+) but also by pre-incubation with bafilomycin A1 or with botulinum toxin C1 serotype. It can be concluded that GABA occurs in Paramecium, where it is probably stored in vesicles capable of fusion with the cell membrane; accordingly, GABA can be released from Paramecium by stimulus-induced, neuronal-like exocytotic mechanisms.

  10. Upregulated STAT3 and RhoA signaling in colorectal cancer (CRC) regulate the invasion and migration of CRC cells.

    PubMed

    Zhang, G-Y; Yang, W-H; Chen, Z

    2016-05-01

    We aimed to reveal the expression and activation of signal transducers and activators of transcription 3 (STAT3) and RhoA/Rho-associated coiled-coil forming kinase 1 (ROCK1) signaling in CRC tissues, and to investigate the regulatory role of STAT3 and RhoA signaling in the invasion and migration of colorectal cancer cells. We examined the expression of STAT3, RhoA and ROCK1 in CRC tissues with real-time PCR and Western blotting methods. And then we examined the interaction between STAT3 and RhoA/ROCK1 signaling in CRC HT-29 cells with gain-of-function and loss-of-function strategies. In addition, we determined the regulation by STAT3 and RhoA/ROCK1 on the invasion and migration of CRC HT-29 cells. Our study demonstrated a significant upregulation of RhoA and ROCK1 expression and STAT3-Y705 phosphorylation in 32 CRC specimens, compared to the 17 normal CRC tissues. Further study demonstrated there was a coordination between STAT3 and RhoA/Rock signaling in the HT-29 cells. Moreover, STAT3 knockdown or RhoA knockdown significantly repressed the migration and invasion in HT-29 cells and vice versa. STAT3 and RhoA signaling regulate the invasion and migration of CRC cells, implying the orchestrated and oncogenic roles of STAT3 and RhoA/ROCK1 signaling in CRC.

  11. Essential arginine in subunit a and aspartate in subunit c of FoF1 ATP synthase: effect of repositioning within helix 4 of subunit a and helix 2 of subunit c.

    PubMed

    Langemeyer, Lars; Engelbrecht, Siegfried

    2007-07-01

    FoF1 ATP synthase couples proton flow through the integral membrane portion Fo (ab2c10) to ATP-synthesis in the extrinsic F1-part ((alphabeta)3gammadeltaepsilon) (Escherichia coli nomenclature and stoichiometry). Coupling occurs by mechanical rotation of subunits c10gammaepsilon relative to (alphabeta)3deltaab2. Two residues were found to be essential for proton flow through ab2c10, namely Arg210 in subunit a (aR210) and Asp61 in subunits c (cD61). Their deletion abolishes proton flow, but "horizontal" repositioning, by anchoring them in adjacent transmembrane helices, restores function. Here, we investigated the effects of "vertical" repositioning aR210, cD61, or both by one helical turn towards the N- or C-termini of their original helices. Other than in the horizontal the vertical displacement changes the positions of the side chains within the depth of the membrane. Mutant aR210A/aN214R appeared to be short-circuited in that it supported proton conduction only through EF1-depleted EFo, but not in EFoEF1, nor ATP-driven proton pumping. Mutant cD61N/cM65D grew on succinate, retained the ability to synthesize ATP and supported passive proton conduction but apparently not ATP hydrolysis-driven proton pumping.

  12. GABA[subscript A] Receptor Downregulation in Brains of Subjects with Autism

    ERIC Educational Resources Information Center

    Fatemi, S. Hossein; Reutiman, Teri J.; Folsom, Timothy D.; Thuras, Paul D.

    2009-01-01

    Gamma-aminobutyric acid A (GABA[subscript A]) receptors are ligand-gated ion channels responsible for mediation of fast inhibitory action of GABA in the brain. Preliminary reports have demonstrated altered expression of GABA receptors in the brains of subjects with autism suggesting GABA/glutamate system dysregulation. We investigated the…

  13. The interdependence of the Rho GTPases and apicobasal cell polarity.

    PubMed

    Mack, Natalie Ann; Georgiou, Marios

    2014-01-01

    Signaling via the Rho GTPases provides crucial regulation of numerous cell polarization events, including apicobasal (AB) polarity, polarized cell migration, polarized cell division and neuronal polarity. Here we review the relationships between the Rho family GTPases and epithelial AB polarization events, focusing on the 3 best-characterized members: Rho, Rac and Cdc42. We discuss a multitude of processes that are important for AB polarization, including lumen formation, apical membrane specification, cell-cell junction assembly and maintenance, as well as tissue polarity. Our discussions aim to highlight the immensely complex regulatory mechanisms that encompass Rho GTPase signaling during AB polarization. More specifically, in this review we discuss several emerging common themes, that include: 1) the need for Rho GTPase activities to be carefully balanced in both a spatial and temporal manner through a multitude of mechanisms; 2) the existence of signaling feedback loops and crosstalk to create robust cellular responses; and 3) the frequent multifunctionality that exists among AB polarity regulators. Regarding this latter theme, we provide further discussion of the potential plasticity of the cell polarity machinery and as a result the possible implications for human disease.

  14. In Vivo Measurement of GABA Transmission in Healthy Subjects and Schizophrenia Patients

    PubMed Central

    Frankle, W. Gordon; Cho, Raymond Y.; Prasad, Konasale M.; Mason, N. Scott; Paris, Jennifer; Himes, Michael L.; Walker, Christopher; Lewis, David A.; Narendran, Rajesh

    2016-01-01

    Objective Postmortem studies in schizophrenia reveal alterations in gene products that regulate the release and extracellular persistence of GABA. However, results of in vivo studies of schizophrenia measuring total tissue GABA with magnetic resonance spectroscopy (MRS) have been inconsistent. Neither the postmortem nor the MRS studies directly address the physiological properties of GABA neurotransmission. The present study addresses this question through an innovative positron emission tomography (PET) paradigm. Method The binding of [11C]flumazenil, a benzodiazepine-specific PET radiotracer, was measured before and after administration of tiagabine (0.2 mg/kg of body weight), a GABA membrane transporter (GAT1) blocker, in 17 off-medication patients with schizophrenia and 22 healthy comparison subjects. Increased extracellular GABA, through GAT1 blockade, enhances the affinity of GABAA receptors for benzodiazepine ligands, detected as an increase in [11C]flumazenil tissue distribution volume (VT). Results [11C]Flumazenil VT was significantly increased across all cortical brain regions in the healthy comparison group but not in the schizophrenia group. This lack of effect was most prominent in the antipsychotic-naive schizophrenia group. In this subgroup, [11C]flumazenil ΔVT in the medial temporal lobe was correlated with positive symptoms, and baseline [11C] flumazenil VT in the medial temporal lobe was negatively correlated with visual learning. In the healthy comparison group but not the schizophrenia group, [11C]flumazenil ΔVT was positively associated with gamma-band oscillation power. Conclusions This study demonstrates, for the first time, an in vivo impairment in GABA transmission in schizophrenia, most prominent in antipsychotic-naive individuals. The impairment in GABA transmission appears to be linked to clinical symptoms, disturbances in cortical oscillations, and cognition. PMID:26133962

  15. Rho GTPases and their downstream effectors in megakaryocyte biology.

    PubMed

    Pleines, Irina; Cherpokova, Deya; Bender, Markus

    2018-06-18

    Megakaryocytes differentiate from hematopoietic stem cells in the bone marrow. The transition of megakaryocytes to platelets is a complex process. Thereby, megakaryocytes extend proplatelets into sinusoidal blood vessels, where the proplatelets undergo fission to release platelets. Defects in platelet production can lead to a low platelet count (thrombocytopenia) with increased bleeding risk. Rho GTPases comprise a family of small signaling G proteins that have been shown to be master regulators of the cytoskeleton controlling many aspects of intracellular processes. The generation of Pf4-Cre transgenic mice was a major breakthrough that enabled studies in megakaryocyte-/platelet-specific knockout mouse lines and provided new insights into the central regulatory role of Rho GTPases in megakaryocyte maturation and platelet production. In this review, we will summarize major findings on the role of Rho GTPases in megakaryocyte biology with a focus on mouse lines in which knockout strategies have been applied to study the function of the best-characterized members Rac1, Cdc42 and RhoA and their downstream effector proteins.

  16. Lipid Emulsion Inhibits Vasodilation Induced by a Toxic Dose of Bupivacaine via Attenuated Dephosphorylation of Myosin Phosphatase Target Subunit 1 in Isolated Rat Aorta

    PubMed Central

    Ok, Seong-Ho; Byon, Hyo-Jin; Kwon, Seong-Chun; Park, Jungchul; Lee, Youngju; Hwang, Yeran; Baik, Jiseok; Choi, Mun-Jeoung; Sohn, Ju-Tae

    2015-01-01

    Lipid emulsions are widely used for the treatment of systemic toxicity that arises from local anesthetics. The goal of this in vitro study was to examine the cellular mechanism associated with the lipid emulsion-mediated attenuation of vasodilation induced by a toxic dose of bupivacaine in isolated endothelium-denuded rat aorta. The effects of lipid emulsion on vasodilation induced by bupivacaine, mepivacaine, and verapamil were assessed in isolated aorta precontracted with phenylephrine, the Rho kinase stimulant NaF, and the protein kinase C activator phorbol 12,13-dibutyrate (PDBu). The effects of Rho kinase inhibitor Y-27632 on contraction induced by phenylephrine or NaF were assessed. The effects of bupivacaine on intracellular calcium concentrations ([Ca2+]i) and tension induced by NaF were simultaneously measured. The effects of bupivacaine alone and lipid emulsion plus bupivacaine on myosin phosphatase target subunit 1 (MYPT1) phosphorylation induced by NaF were examined in rat aortic vascular smooth muscle cells. In precontracted aorta, the lipid emulsion attenuated bupivacaine-induced vasodilation but had no effect on mepivacaine-induced vasodilation. Y-27632 attenuated contraction induced by either phenylephrine or NaF. The lipid emulsion attenuated verapamil-induced vasodilation. Compared with phenylephrine-induced precontracted aorta, bupivacaine-induced vasodilation was slightly attenuated in NaF-induced precontracted aorta. The magnitude of the bupivacaine-induced vasodilation was higher than that of a bupivacaine-induced decrease in [Ca2+]i. Bupivacaine attenuated NaF-induced MYPT1 phosphorylation, whereas lipid emulsion pretreatment attenuated the bupivacaine-induced inhibition of MYPT1 phosphorylation induced by NaF. Taken together, these results suggest that lipid emulsions attenuate bupivacaine-induced vasodilation via the attenuation of inhibition of MYPT1 phosphorylation evoked by NaF. PMID:26664257

  17. Prefoldin Subunits Are Protected from Ubiquitin-Proteasome System-mediated Degradation by Forming Complex with Other Constituent Subunits*

    PubMed Central

    Miyazawa, Makoto; Tashiro, Erika; Kitaura, Hirotake; Maita, Hiroshi; Suto, Hiroo; Iguchi-Ariga, Sanae M. M.; Ariga, Hiroyoshi

    2011-01-01

    The molecular chaperone prefoldin (PFD) is a complex comprised of six different subunits, PFD1-PFD6, and delivers newly synthesized unfolded proteins to cytosolic chaperonin TRiC/CCT to facilitate the folding of proteins. PFD subunits also have functions different from the function of the PFD complex. We previously identified MM-1α/PFD5 as a novel c-Myc-binding protein and found that MM-1α suppresses transformation activity of c-Myc. However, it remains unclear how cells regulate protein levels of individual subunits and what mechanisms alter the ratio of their activities between subunits and their complex. In this study, we found that knockdown of one subunit decreased protein levels of other subunits and that transfection of five subunits other than MM-1α into cells increased the level of endogenous MM-1α. We also found that treatment of cells with MG132, a proteasome inhibitor, increased the level of transfected/overexpressed MM-1α but not that of endogenous MM-1α, indicating that overexpressed MM-1α, but not endogenous MM-1α, was degraded by the ubiquitin proteasome system (UPS). Experiments using other PFD subunits showed that the UPS degraded a monomer of PFD subunits, though extents of degradation varied among subunits. Furthermore, the level of one subunit was increased after co-transfection with the respective subunit, indicating that there are specific combinations between subunits to be stabilized. These results suggest mutual regulation of protein levels among PFD subunits and show how individual subunits form the PFD complex without degradation. PMID:21478150

  18. E-cadherin antagonizes transforming growth factor β1 gene induction in hepatic stellate cells by inhibiting RhoA-dependent Smad3 phosphorylation.

    PubMed

    Cho, Il Je; Kim, Young Woo; Han, Chang Yeob; Kim, Eun Hyun; Anderson, Richard A; Lee, Young Sok; Lee, Chang Ho; Hwang, Se Jin; Kim, Sang Geon

    2010-12-01

    Cadherins mediate cell-cell adhesion and catenin (ctn)-related signaling pathways. Liver fibrosis is accompanied by the loss of E-cadherin (ECAD), which promotes the process of epithelial-mesenchymal transition. Currently, no information is available about the inhibitory role of ECAD in hepatic stellate cell activation. Because of ECAD's potential for inhibiting the induction of transforming growth factor β1 (TGFβ1), we investigated whether ECAD overexpression prevents TGFβ1 gene induction; we also examined what the molecular basis could be. Forced expression of ECAD decreased α-smooth muscle actin and vimentin levels and caused decreases in the constitutive and inducible expression of the TGFβ1 gene and its downstream genes. ECAD overexpression decreased Smad3 phosphorylation, weakly decreased Smad2 phosphorylation, and thus inhibited Smad reporter activity induced by either treatment with TGFβ1 or Smad3 overexpression. Overexpression of a dominant negative mutant of ras homolog gene family A (RhoA) diminished the ability of TGFβ1 to elicit its own gene induction. Consistently, transfection with a constitutively active mutant of RhoA reversed the inhibition of TGFβ1-inducible or Smad3-inducible reporter activity by ECAD. Studies using the mutant constructs of ECAD revealed that the p120-ctn binding domain of ECAD was responsible for TGFβ1 repression. Consistently, ECAD was capable of binding p120-ctn, which recruited RhoA; this prevented TGFβ1 from increasing RhoA-mediated Smad3 phosphorylation. In the liver samples of patients with mild or severe fibrosis, ECAD expression reciprocally correlated with the severity of fibrosis. Our results demonstrate that ECAD inhibits Smad3/2 phosphorylation by recruiting RhoA to p120-ctn at the p120-ctn binding domain, whereas the loss of ECAD due to cadherin switching promotes the up-regulation of TGFβ1 and its target genes, and facilitates liver fibrosis. Copyright © 2010 American Association for the Study of Liver

  19. Regulation of (/sup 3/H)GABA release from strips of guinea pig urinary bladder

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Shirakawa, J.; Taniyama, K.; Iwai, S.

    1988-12-01

    The presence of receptors that regulate the release of gamma-aminobutyric acid (GABA) was studied in strips of the guinea pig urinary bladder. GABA (10(-8)-10(-5) M) and muscimol (10(-8)-10(-5) M), but not baclofen (10(-5) M), reduced the Ca2+-dependent, tetrodotoxin-resistant release of (/sup 3/H)GABA evoked by high K+ from the urinary bladder strips preloaded with (/sup 3/H)GABA. The inhibitory effect of muscimol was antagonized by bicuculline and potentiated by diazepam, clonazepam, and pentobarbital sodium. The potentiating effect of clonazepam was antagonized by Ro 15-1788. Acetylcholine (ACh) inhibited the high K+-evoked release of (/sup 3/H)GABA. The inhibitory effect of ACh was antagonized bymore » atropine sulfate and pirenzepine but not by hexamethonium. Norepinephrine (NE) inhibited the evoked release of (/sup 3/H)GABA. The inhibitory effect of NE was mimicked by clonidine, but not by phenylephrine, and was antagonized by yohimbine but not by prazosin. These results provide evidence that the release of GABA from strips of guinea pig urinary bladder is regulated via the bicuculline-sensitive GABAA receptor, M1-muscarinic, and alpha 2-adrenergic receptors.« less

  20. GABA action in immature neocortical neurons directly depends on the availability of ketone bodies.

    PubMed

    Rheims, Sylvain; Holmgren, Carl D; Chazal, Genevieve; Mulder, Jan; Harkany, Tibor; Zilberter, Tanya; Zilberter, Yuri

    2009-08-01

    In the early postnatal period, energy metabolism in the suckling rodent brain relies to a large extent on metabolic pathways alternate to glucose such as the utilization of ketone bodies (KBs). However, how KBs affect neuronal excitability is not known. Using recordings of single NMDA and GABA-activated channels in neocortical pyramidal cells we studied the effects of KBs on the resting membrane potential (E(m)) and reversal potential of GABA-induced anionic currents (E(GABA)), respectively. We show that during postnatal development (P3-P19) if neocortical brain slices are adequately supplied with KBs, E(m) and E(GABA) are both maintained at negative levels of about -83 and -80 mV, respectively. Conversely, a KB deficiency causes a significant depolarization of both E(m) (>5 mV) and E(GABA) (>15 mV). The KB-mediated shift in E(GABA) is largely determined by the interaction of the NKCC1 cotransporter and Cl(-)/HCO3 transporter(s). Therefore, by inducing a hyperpolarizing shift in E(m) and modulating GABA signaling mode, KBs can efficiently control the excitability of neonatal cortical neurons.

  1. (±)-(1S,2R,5S)-5-Amino-2-fluorocyclohex-3-ene Carboxylic Acid. A Potent GABA Aminotransferase Inactivator that Irreversibly Inhibits through an Elimination-Aromatization Pathway†

    PubMed Central

    Wang, Zhiyong; Yuan, Hai; Nikolic, Dejan; Van Breemen, Richard B.; Silverman, Richard B.

    2008-01-01

    Inhibition of γ-aminobutyric acid aminotransferase (GABA-AT) raises the concentration of GABA, an inhibitory neurotransmitter in human brain, which could have therapeutic applications for a variety of neurological diseases including epilepsy. Based on studies of several previously synthesized conformationally-restricted GABA-AT inhibitors, (±)- (1S,2R,5S)-5-amino-2-fluorocyclohex-3-ene carboxylic acid (12) was designed as a mechanismbased inactivator. This compound was shown to irreversibly inhibit GABA-AT; substrate protects the enzyme from inactivation. Mechanistic experiments demonstrated the loss of one fluoride ion per active site during inactivation and the formation of N-m-carboxyphenylpyridoxamine 5′-phosphate (26), the same product generated by inactivation of GABA-AT by gabaculine (8). An elimination-aromatization mechanism is proposed to account for these results. PMID:17128990

  2. Blocking RhoA/ROCK inhibits the pathogenesis of pemphigus vulgaris by suppressing oxidative stress and apoptosis through TAK1/NOD2-mediated NF-κB pathway.

    PubMed

    Liang, Junqin; Zeng, Xuewen; Halifu, Yilinuer; Chen, Wenjing; Hu, Fengxia; Wang, Peng; Zhang, Huan; Kang, Xiaojing

    2017-12-01

    Oxidative stress and apoptosis play critical roles in pemphigus vulgaris (PV). The main aim of the present study was to investigate the effects of RhoA/ROCK signaling on UVB-induced oxidative damage, and to delineate the molecular mechanisms involved in the UVB-mediated inflammatory and apoptotic response. In HaCaT cells, we observed that blockage of RhoA/ROCK signaling with the inhibitor CT04 or Y27632 greatly inhibited the UVB-mediated increase in intracellular reactive oxygen species (ROS). Additionally, inhibition of RhoA/ROCK signaling reduced UVB-induced apoptosis, as exemplified by a reduction in DNA fragmentation, and also elevated anti-apoptotic Bcl-2 protein, concomitant with reduced levels of pro-apoptotic protein Bax, caspase-3 cleavage and decreased PARP-1 protein. The release of inflammatory mediators TNF-α, IL-1β, and IL-6 was also attenuated. Mechanically, we observed that blockage of RhoA/ROCK repressed the TAK1/NOD2-mediated NF-κB pathway in HaCaT cells exposed to UVB. Taken together, these data reveal that RhoA/ROCK signaling is one of the regulators contributing to oxidative damage and apoptosis in human keratinocytes, suggesting that RhoA/ROCK signaling has strong potential to be used as a useful therapeutic target in skin diseases including PV.

  3. The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence

    PubMed Central

    2014-01-01

    Background The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. Findings This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. Conclusions In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle. PMID:25379053

  4. The subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3: dynamics and interdependence.

    PubMed

    Uzunova, Sonya Dimitrova; Zarkov, Alexander Stefanov; Ivanova, Anna Marianova; Stoynov, Stoyno Stefanov; Nedelcheva-Veleva, Marina Nedelcheva

    2014-01-01

    The S-phase checkpoint aims to prevent cells from generation of extensive single-stranded DNA that predisposes to genome instability. The S. cerevisiae complex Tof1/Csm3/Mrc1 acts to restrain the replicative MCM helicase when DNA synthesis is prohibited. Keeping the replication machinery intact allows restart of the replication fork when the block is relieved. Although the subunits of the Tof1/Csm3/Mrc1 complex are well studied, the impact of every single subunit on the triple complex formation and function needs to be established. This work studies the cellular localization and the chromatin binding of GFP-tagged subunits when the complex is intact and when a subunit is missing. We demonstrate that the complex is formed in cell nucleus, not the cytoplasm, as Tof1, Csm3 and Mrc1 enter the nucleus independently from one another. Via in situ chromatin binding assay we show that a Tof1-Csm3 dimer formation and chromatin binding is required to ensure the attachment of Mrc1 to chromatin. Our study indicates that the translocation into the nucleus is not the process to regulate the timing of chromatin association of Mrc1. We also studied the nuclear behavior of Mrc1 subunit in the process of adaptation to the presence hydroxyurea. Our results indicate that after prolonged HU incubation, cells bypass the S-phase checkpoint and proceed throughout the cell cycle. This process is accompanied by Mrc1 chromatin detachment and Rad53 dephosphorylation. In S. cerevisiae the subunits of the S-phase checkpoint complex Mrc1/Tof1/Csm3 independently enter the cell nucleus, where a Tof1-Csm3 dimer is formed to ensure the chromatin binding of Mrc1 and favor DNA replication and S-phase checkpoint fork arrest. In the process of adaptation to the presence of hydroxyurea Mrc1 is detached from chromatin and Rad53 checkpoint activity is diminished in order to allow S-phase checkpoint escape and completion of the cell cycle.

  5. Regulation of α3-containing GABAA receptors in guinea-pig adrenal medullary cells by adrenal steroids.

    PubMed

    Inoue, M; Harada, K; Nakamura, J; Matsuoka, H

    2013-12-03

    GABA is thought to function as a paracrine factor in adrenal medullary (AM) cells. Thus, we electrophysiologically and immunologically examined the properties of GABAA receptors (GABAARs) in guinea-pig AM cells. Bath application of GABA produced an inward current at -60 mV in a dose-dependent manner with an EC50 of 32.3 μM. This GABA-induced current was enhanced by allopregnanolone at concentrations of 0.01 μM and more. A prior exposure to allopregnanolone resulted in a decrease in an EC50 for GABA in activating GABAARs. The GABA-induced current was suppressed by Zn(2+) in a dose-dependent manner with an IC50 of 18 μM, whereas it was enhanced by 100 μM La(3+). The benzodiazepine analog diazepam was three times more potent than zolpidem in enhancing the GABA current, and it was also augmented by L-838,417, which has no action on α1-containing GABAARs. The GABAAR α3, but not α1, and γ2 subunits were immunologically detected at the cell periphery. The expression of α3 subunits in PC12 cells was enhanced by glucocorticoid activity. The results indicated that GABAARs in guinea-pig AM cells mainly comprise α3, β, and γ2 subunits and are enhanced by allopreganalone and glucocorticoids may play a major role in the expression of α3 subunits. Copyright © 2013 IBRO. Published by Elsevier Ltd. All rights reserved.

  6. Cocaine Dysregulates Opioid Gating of GABA Neurotransmission in the Ventral Pallidum

    PubMed Central

    Scofield, Michael D.; Rice, Kenner C.; Cheng, Kejun; Roques, Bernard P.

    2014-01-01

    The ventral pallidum (VP) is a target of dense nucleus accumbens projections. Many of these projections coexpress GABA and the neuropeptide enkephalin, a δ and μ opioid receptor (MOR) ligand. Of these two, the MOR in the VP is known to be involved in reward-related behaviors, such as hedonic responses to palatable food, alcohol intake, and reinstatement of cocaine seeking. Stimulating MORs in the VP decreases extracellular GABA, indicating that the effects of MORs in the VP on cocaine seeking are via modulating GABA neurotransmission. Here, we use whole-cell patch-clamp on a rat model of withdrawal from cocaine self-administration to test the hypothesis that MORs presynaptically regulate GABA transmission in the VP and that cocaine withdrawal changes the interaction between MORs and GABA. We found that in cocaine-extinguished rats pharmacological activation of MORs no longer presynaptically inhibited GABA release, whereas blocking the MORs disinhibited GABA release. Moreover, MOR-dependent long-term depression of GABA neurotransmission in the VP was lost in cocaine-extinguished rats. Last, GABA neurotransmission was found to be tonically suppressed in cocaine-extinguished rats. These substantial synaptic changes indicated that cocaine was increasing tone on MOR receptors. Accordingly, increasing endogenous tone by blocking the enzymatic degradation of enkephalin inhibited GABA neurotransmission in yoked saline rats but not in cocaine-extinguished rats. In conclusion, our results indicate that following withdrawal from cocaine self-administration enkephalin levels in the VP are elevated and the opioid modulation of GABA neurotransmission is impaired. This may contribute to the difficulties withdrawn addicts experience when trying to resist relapse. PMID:24431463

  7. Depolarizing actions of GABA in immature neurons depend neither on ketone bodies nor on pyruvate.

    PubMed

    Tyzio, Roman; Allene, Camille; Nardou, Romain; Picardo, Michel A; Yamamoto, Sumii; Sivakumaran, Sudhir; Caiati, Maddalena D; Rheims, Sylvain; Minlebaev, Marat; Milh, Mathieu; Ferré, Pascal; Khazipov, Rustem; Romette, Jean-Louis; Lorquin, Jean; Cossart, Rosa; Khalilov, Ilgam; Nehlig, Astrid; Cherubini, Enrico; Ben-Ari, Yehezkel

    2011-01-05

    GABA depolarizes immature neurons because of a high [Cl(-)](i) and orchestrates giant depolarizing potential (GDP) generation. Zilberter and coworkers (Rheims et al., 2009; Holmgren et al., 2010) showed recently that the ketone body metabolite DL-3-hydroxybutyrate (DL-BHB) (4 mM), lactate (4 mM), or pyruvate (5 mM) shifted GABA actions to hyperpolarizing, suggesting that the depolarizing effects of GABA are attributable to inadequate energy supply when glucose is the sole energy source. We now report that, in rat pups (postnatal days 4-7), plasma D-BHB, lactate, and pyruvate levels are 0.9, 1.5, and 0.12 mM, respectively. Then, we show that DL-BHB (4 mM) and pyruvate (200 μM) do not affect (i) the driving force for GABA(A) receptor-mediated currents (DF(GABA)) in cell-attached single-channel recordings, (2) the resting membrane potential and reversal potential of synaptic GABA(A) receptor-mediated responses in perforated patch recordings, (3) the action potentials triggered by focal GABA applications, or (4) the GDPs determined with electrophysiological recordings and dynamic two-photon calcium imaging. Only very high nonphysiological concentrations of pyruvate (5 mM) reduced DF(GABA) and blocked GDPs. Therefore, DL-BHB does not alter GABA signals even at the high concentrations used by Zilberter and colleagues, whereas pyruvate requires exceedingly high nonphysiological concentrations to exert an effect. There is no need to alter conventional glucose enriched artificial CSF to investigate GABA signals in the developing brain.

  8. GABAB receptor attenuation of GABAA currents in neurons of the mammalian central nervous system.

    PubMed

    Shen, Wen; Nan, Changlong; Nelson, Peter T; Ripps, Harris; Slaughter, Malcolm M

    2017-03-01

    Ionotropic receptors are tightly regulated by second messenger systems and are often present along with their metabotropic counterparts on a neuron's plasma membrane. This leads to the hypothesis that the two receptor subtypes can interact, and indeed this has been observed in excitatory glutamate and inhibitory GABA receptors. In both systems the metabotropic pathway augments the ionotropic receptor response. However, we have found that the metabotropic GABA B receptor can suppress the ionotropic GABA A receptor current, in both the in vitro mouse retina and in human amygdala membrane fractions. Expression of amygdala membrane microdomains in Xenopus oocytes by microtransplantation produced functional ionotropic and metabotropic GABA receptors. Most GABA A receptors had properties of α -subunit containing receptors, with ~5% having ρ -subunit properties. Only GABA A receptors with α -subunit-like properties were regulated by GABA B receptors. In mouse retinal ganglion cells, where only α -subunit-containing GABA A receptors are expressed, GABA B receptors suppressed GABA A receptor currents. This suppression was blocked by GABA B receptor antagonists, G-protein inhibitors, and GABA B receptor antibodies. Based on the kinetic differences between metabotropic and ionotropic receptors, their interaction would suppress repeated, rapid GABAergic inhibition. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

  9. Direct multiplex imaging and optogenetics of Rho GTPases enabled by near-infrared FRET.

    PubMed

    Shcherbakova, Daria M; Cox Cammer, Natasha; Huisman, Tsipora M; Verkhusha, Vladislav V; Hodgson, Louis

    2018-06-01

    Direct visualization and light control of several cellular processes is a challenge, owing to the spectral overlap of available genetically encoded probes. Here we report the most red-shifted monomeric near-infrared (NIR) fluorescent protein, miRFP720, and the fully NIR Förster resonance energy transfer (FRET) pair miRFP670-miRFP720, which together enabled design of biosensors compatible with CFP-YFP imaging and blue-green optogenetic tools. We developed a NIR biosensor for Rac1 GTPase and demonstrated its use in multiplexed imaging and light control of Rho GTPase signaling pathways. Specifically, we combined the Rac1 biosensor with CFP-YFP FRET biosensors for RhoA and for Rac1-GDI binding, and concurrently used the LOV-TRAP tool for upstream Rac1 activation. We directly observed and quantified antagonism between RhoA and Rac1 dependent on the RhoA-downstream effector ROCK; showed that Rac1 activity and GDI binding closely depend on the spatiotemporal coordination between these two molecules; and simultaneously observed Rac1 activity during optogenetic manipulation of Rac1.

  10. Isochoric p-{rho}-T measurements on 1,1-difluoroethane (R152a) from 158 to 400 K and 1,1,1-trifluoroethane (R143a) from 166 to 400 K at pressures to 35 MPa

    DOE Office of Scientific and Technical Information (OSTI.GOV)

    Magee, J.W.

    1998-09-01

    The p-{rho}-T relationships have been measured for 1,1-difluoroethane (R152a) and 1,1,1-trifluoroethane (R143a) by an isochoric method with gravimetric determinations of the amount of substance. Temperatures ranged from 158 to 400K for R152a and from 166 to 400 K for R143a, while pressures were up to 35 MPa. Measurements were conducted on compressed liquid samples. Determinations of saturated liquid densities were made by extrapolating each isochore to the vapor pressure, and determining the temperature and density at the intersection. Published p-{rho}-T data are in good agreement with this study. For the p-{rho}-T apparatus, the uncertainty of the temperature is {+-}0.03 K,more » and for pressure it is {+-}0.01% at p > 3 MPa and {+-}0.05% at p > 3 MPa and {+-}0.05% at p < 3MPa. The principal source of uncertainty is the cell volume ({approximately}28.5 cm{sup 3}), which has a standard uncertainty of {+-}0.003 cm{sup 3}. When all components of experimental uncertainty are considered, the expanded relative uncertainty (with a coverage factor k = 2 and thus a two-standard deviation estimate) of the density measurement is estimated to be {+-}0.05%.« less

  11. Proline antagonizes GABA-induced quenching of quorum-sensing in Agrobacterium tumefaciens

    PubMed Central

    Haudecoeur, E.; Planamente, S.; Cirou, A.; Tannières, M.; Shelp, B. J.; Moréra, S.; Faure, D.

    2009-01-01

    Plants accumulate free L-proline (Pro) in response to abiotic stresses (drought and salinity) and presence of bacterial pathogens, including the tumor-inducing bacterium Agrobacterium tumefaciens. However, the function of Pro accumulation in host-pathogen interaction is still unclear. Here, we demonstrated that Pro antagonizes plant GABA-defense in the A. tumefaciens C58-induced tumor by interfering with the import of GABA and consequently the GABA-induced degradation of the bacterial quorum-sensing signal, 3-oxo-octanoylhomoserine lactone. We identified a bacterial receptor Atu2422, which is implicated in the uptake of GABA and Pro, suggesting that Pro acts as a natural antagonist of GABA-signaling. The Atu2422 amino acid sequence contains a Venus flytrap domain that is required for trapping GABA in human GABAB receptors. A constructed atu2422 mutant was more virulent than the wild type bacterium; moreover, transgenic plants with a low level of Pro exhibited less severe tumor symptoms than did their wild-type parents, revealing a crucial role for Venus flytrap GABA-receptor and relative abundance of GABA and Pro in host-pathogen interaction. PMID:19706545

  12. Regulation of MT1-MMP and MMP-2 by leptin in cardiac fibroblasts involves Rho/ROCK-dependent actin cytoskeletal reorganization and leads to enhanced cell migration.

    PubMed

    Schram, Kristin; Ganguly, Riya; No, Eun Kyung; Fang, Xiangping; Thong, Farah S L; Sweeney, Gary

    2011-05-01

    Altered leptin action has been implicated in the pathophysiology of heart failure in obesity, a hallmark of which is extracellular matrix remodeling. Here, we characterize the direct influence of leptin on matrix metalloproteinase (MMP) activity in primary adult rat cardiac fibroblasts and focus on elucidating the molecular mechanisms responsible. Leptin increased expression and cell surface localization of membrane type 1 (MT1)-MMP, measured by cell surface biotinylation assay and antibody-based colorimetric detection of an exofacial epitope in intact cells. Coimmunoprecipitation analysis showed that leptin also induced the formation of a cluster of differentiation 44/MT1-MMP complex. Qualitative analysis using rhodamine-conjugated phalloidin immunofluorescence indicated that leptin stimulated actin cytoskeletal reorganization and enhanced stress fiber formation. Hence, we analyzed activation of Ras homolog gene family (Rho), member A GTPase activity and found a rapid increase in response to leptin that corresponded with increased phosphorylation of cofilin. Quantitative analysis of cytoskeleton reorganization upon separation of globular and filamentous actin by differential centrifugation confirmed the significant increase in filamentous to globular actin ratio in response to leptin, which was prevented by pharmacological inhibition of Rho (C3 transferase) or its downstream effector kinase Rho-associated coiled-coil-forming protein kinase (ROCK) (Y-27632). Inhibition of Rho or ROCK also attenuated leptin-stimulated increases in cell surface MT1-MMP content. Pro-MMP-2 is a known MT1-MMP substrate, and we observed that enhanced cell surface MT1-MMP in response to leptin resulted in enhanced extracellular activation of pro-MMP-2 measured by gelatin zymography, which was again attenuated by inhibition of Rho or ROCK. Using wound scratch assays, we observed enhanced cell migration, but not proliferation, measured by 5-bromo2'-deoxy-uridine incorporation, in response

  13. l-Proline, GABA Synthesis and Gamma Oscillations in Schizophrenia.

    PubMed

    Volk, David W; Gonzalez-Burgos, Guillermo; Lewis, David A

    2016-12-01

    Altered inhibition from parvalbumin-containing GABA neurons is thought to contribute to impaired gamma frequency oscillations and cognitive deficits in schizophrenia. Crabtree and colleagues report that proline dehydrogenase deficits produce excessive cytosolic levels of the GABA-mimetic l-proline which impairs GABA synthesis and gamma oscillations in a manner that mimics schizophrenia. Copyright © 2016 Elsevier Ltd. All rights reserved.

  14. Verification of γ-Amino-Butyric Acid (GABA) Signaling System Components in Periodontal Ligament Cells In Vivo and In Vitro.

    PubMed

    Konermann, Anna; Kantarci, Alpdogan; Wilbert, Steven; Van Dyke, Thomas; Jäger, Andreas

    2016-11-01

    CNS key neurotransmitter γ-amino-butyric acid (GABA) and its signaling components are likewise detectable in non-neuronal tissues displaying inter alia immunomodulatory functions. This study aimed at identifying potential glutamate decarboxylase (GAD)65 and GABA receptor expression in periodontal ligament (PDL) cells in vivo and in vitro, with particular regard to inflammation and mechanical loading. Gene expression was analyzed in human PDL cells at rest or in response to IL-1ß (5 ng/ml) or TNFα (5 ng/ml) challenge via qRT-PCR. Western blot determined constitutive receptor expression, and confocal laser scanning fluorescence microscopy visualized expression changes induced by inflammation. ELISA quantified GAD65 release. Immunocytochemistry was performed for GABA component detection in vitro on mechanically loaded PDL cells, and in vivo on rat upper jaw biopsies with mechanically induced root resorptions. Statistical significance was set at p < 0.05. GABA B1 , GABA B2 , GABA A1 , and GABA A3 were ubiquitously expressed both on gene and protein level. GABA A2 and GAD65 were undetectable in resting cells, but induced by inflammation. GABA B1 exhibited the highest basal gene expression (6.97 % ± 0.16). IL-1ß markedly increased GABA B2 on a transcriptional (57.28-fold ± 12.40) and protein level seen via fluorescence microscopy. TNFα-stimulated PDL cells released GAD65 (3.68 pg/ml ± 0.17 after 24 h, 5.77 pg/ml ± 0.65 after 48 h). Immunocytochemistry revealed GAD65 expression in mechanically loaded PDL cells. In vivo, GABA components were varyingly expressed in an inflammatory periodontal environment. PDL cells differentially express GABA signaling components and secrete GAD65. Inflammation and mechanical loading regulate these neurotransmitter molecules, which are also detectable in vivo and are potentially involved in periodontal pathophysiology.

  15. A human anti-neuronal autoantibody against GABA B receptor induces experimental autoimmune agrypnia.

    PubMed

    Frisullo, Giovanni; Della Marca, Giacomo; Mirabella, Massimiliano; Caggiula, Marcella; Broccolini, Aldobrando; Rubino, Marco; Mennuni, Gioacchino; Tonali, Pietro Attilio; Batocchi, Anna Paola

    2007-04-01

    In the serum and cerebrospinal fluid of a patient with recurrent acute episodes of respiratory crises, autonomic symptoms and total insomnia (agrypnia), we identified a novel anti-neural complement fixing antibody directed against GABA(B) receptor (GABA(B)R). Patient purified IgG recognized a band of approximately 110 kDa on protein extracts of mouse cerebellum, cortex and brainstem and immunolabelled cultured Chinese hamster ovary (CHO) cells, transfected with human GABA(B)R1 and rat GABA(B)R2 receptors. Western blot analysis of transfected CHO homogenates showed the same band using both patient purified IgG and anti-GABA(B)R1 antibody. In order to verify the pathogenic role of these purified antibodies, we injected patient IgG intrathecally into cisterna magna of C57BL/6 mice pre-implanted with EEG electrodes and we observed severe ataxia followed by breathing depression and total suppression of slow wave sleep, as evidenced by EEG recording, in a dose-dependent manner. Immunohistochemistry on brain sections of mice injected with patient IgG showed the simultaneous presence of bound human IgG and C5b-9 deposits on Purkinje cells and cerebellar granular layer. After incubation with anti-GABA(B)R antibody, a marked reduction of receptor immunostaining was found with relative sparing of neuronal architecture. In conclusion we recognized an anti-neuronal autoantibody directed against GABA(B)R that is associated with autoimmune agrypnia and we showed that our patient purified IgG was able to induce in mice experimental autoimmune agrypnia characterized by a complex neurological syndrome affecting several CNS functions.

  16. Endogenous concentrations, pharmacokinetics, and selected pharmacodynamic effects of a single dose of exogenous GABA in horses.

    PubMed

    Knych, H K; Steinmetz, S J; McKemie, D S

    2015-04-01

    The anti-anxiety and calming effects following activation of the GABA receptor have been exploited in performance horses by administering products containing GABA. The primary goal of the study reported here was to describe endogenous concentrations of GABA in horses and the pharmacokinetics, selected pharmacodynamic effects, and CSF concentrations following administration of a GABA-containing product. The mean (±SD) endogenous GABA level was 36.4 ± 12.5 ng/mL (n = 147). Sixteen of these horses received a single intravenous and oral dose of GABA (1650 mg). Blood, urine, and cerebrospinal fluid (n = 2) samples were collected at time 0 and at various times for up to 48 h and analyzed using LC-MS. Plasma clearance and volume of distribution was 155.6 and 147.6 L/h and 0.154 and 7.39 L for the central and peripheral compartments, respectively. Terminal elimination half-life was 22.1 (intravenous) and 25.1 (oral) min. Oral bioavailability was 9.81%. Urine GABA concentrations peaked rapidly returning to baseline levels by 3 h. Horses appeared behaviorally unaffected following oral administration, while sedative-like changes following intravenous administration were transient. Heart rate was increased for 1 h postintravenous administration, and gastrointestinal sounds decreased for approximately 30 min following both intravenous and oral administration. Based on a limited number of horses and time points, exogenously administered GABA does not appear to enter the CSF to an appreciable extent. © 2014 John Wiley & Sons Ltd.

  17. Baclofen and phaclofen modulate GABA release from slices of rat cerebral cortex and spinal cord but not from retina.

    PubMed Central

    Neal, M. J.; Shah, M. A.

    1989-01-01

    1. The effects of (-)-baclofen, muscimol and phaclofen on endogenous gamma-aminobutyric acid (GABA) release from rat cortical slices, spinal cord slices and entire retinas were studied. 2. The spontaneous resting release of GABA from the three tissues was 3 to 6 pmol mg-1 wet wt 10 min-1. Depolarization of cortical slices with KCl (50 mM) (high-K) produced an 8 fold increase in GABA release but high-K did not evoke an increased release of GABA from spinal slices or retinas. 3. When rats were injected with gamma-vinyl-GABA (250 mg kg-1 i.p.) (GVG) 18 h before death, the tissue GABA stores were increased 3 to 6 fold and high-K then evoked striking Ca-dependent releases of GABA from all three tissues. Thus, in subsequent experiments, unless otherwise stated, the nervous tissues were taken from GVG-treated rats. 4. (-)-Baclofen (10 microM) significantly reduced the K-evoked release of GABA from cortical and spinal slices but retinal release was not affected, even at a concentration of (+/-)-baclofen of 1 mM. For cortical slices, the IC50 for baclofen was approximately 5.2 microM. The inhibitory effect of baclofen on GABA release from cortical slices also occurred in slices prepared from saline-injected rats, indicating that GVG treatment did not qualitatively affect the results. 5. The inhibitory effect of (-)-baclofen on the K-evoked release of GABA from cortical and spinal slices was antagonised by phaclofen (500 microM), confirming that baclofen was producing its effects by acting at the GABAB-receptor.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:2804540

  18. Rotation in USco and rho Oph with K2

    NASA Astrophysics Data System (ADS)

    Rebull, Luisa; Stauffer, John; K2 Clusters Team

    2018-01-01

    K2 observed Upper Scorpius and rho Oph as part of their Campaign 2 in 2014. At ~8 and ~1 Myr respectively, the stars in Upper Sco and rho Oph exhibit greater diversity of light curve shapes than are found in older clusters observed with K2 such as Pleiades or Praesepe. Nonetheless, we are able to derive rotation periods for 85% (971/1136) of the USco members and 80% (71/88) of the rho Oph members. About 25% of the periodic stars have evidence for multiple periods. These light curves sample smaller amplitudes to lower masses and with a far better cadence, than has even been probed before. We can compare USco with similar stars in Praesepe (~700 Myr) and the Pleiades (~125 Myr), all with K2 light curves.

  19. Macrophage differentiation induced by PMA is mediated by activation of RhoA/ROCK signaling.

    PubMed

    Yang, Lifeng; Dai, Fan; Tang, Lian; Le, Yulan; Yao, Wenjuan

    2017-01-01

    In order to investigate the effects of RhoA/ROCK signaling in macrophage differentiation, we used 100 ng/mL PMA to induce macrophage differentiation from U937 cells in vitro. The observation of cell morphology and the expression of CD68 and SR-A were performed to confirm the differentiation induced by PMA. Western blot analysis showed that the expression of ROCK1 and ROCK2 and the phosphorylation of MYPT1 were significantly increased after PMA treatment. Pulldown assay showed that the activation of RhoA was obviously enhanced when U937 cells were treated with PMA. In order to further demonstrate whether RhoA/ROCK signaling could mediate the macrophage differentiation induced by PMA, we successfully suppressed the expression of RhoA, ROCK1 and ROCK2 by performing siRNA technology in U937 cells, respectively. The macrophage differentiation and the expression of CD68 and SR-A were significantly inhibited by the suppression of RhoA, ROCK1 or ROCK2 in PMA-induced U937 cells, indicating that the macrophage differentiation induced by PMA is associated with RhoA/ROCK signaling pathway. In addition, we pretreated U937 cells with Y27632 (ROCK inhibitor, 20 μM) for 30 min and then observed the macrophage differentiation induced by PMA. The result illustrated that Y27632 pretreatment obviously inhibited PMA-induced differentiation and the expression of CD68 and SR-A. In conclusion, the activation of RhoA/ROCK signaling is responsible for the macrophage differentiation induced by PMA.

  20. PKCε Phosphorylates and Mediates the Cell Membrane Localization of RhoA

    PubMed Central

    Su, Tizhi; Bao, Liwei; Xie, Xiujie; Lehner, Caryn L.; Cavey, Greg S.; Teknos, Theodoros N.

    2013-01-01

    Protein kinase Cε (PKCε) signals through RhoA to modulate cell invasion and motility. In this study, the multifaceted interaction between PKCε and RhoA was defined. Phosphopeptide mapping revealed that PKCε phosphorylates RhoA at T127 and S188. Recombinant PKCε bound to recombinant RhoA in the absence of ATP indicating that the association between PKCε and RhoA does not require an active ATP-docked PKCε conformation. Activation of PKCε resulted in a dramatic coordinated translocation of PKCε and RhoA from the cytoplasm to the cell membrane using time-lapse fluorescence microscopy. Stoichiometric FRET analysis revealed that the molecular interaction between PKCε and RhoA is a biphasic event, an initial peak at the cytoplasm and a gradual prolonged increase at the cell membrane for the entire time-course (12.5 minutes). These results suggest that the PKCε-RhoA complex is assembled in the cytoplasm and subsequently recruited to the cell membrane. Kinase inactive (K437R) PKCε is able to recruit RhoA to the cell membrane indicating that the association between PKCε and RhoA is proximal to the active catalytic site and perhaps independent of a PKCε-RhoA phosphorylation event. This work demonstrates, for the first time, that PKCε phosphorylates and modulates the cell membrane translocation of RhoA. PMID:24191200