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Sample records for 1-adrenergic receptor-mediated phosphoinositide

  1. Alpha-1 adrenergic receptor: Binding and phosphoinositide breakdown in human myometrium

    SciTech Connect

    Breuiller-Fouche, M.; Doualla-Bell Kotto Maka, F.; Geny, B.; Ferre, F. )

    1991-07-01

    Alpha-1 adrenergic receptors were examined in both inner and outer layers of human pregnant myometrium using radioligand binding of (3H)prazosin. (3H)prazosin bound rapidly and reversibly to a single class of high affinity binding sites in myometrial membrane preparations. Scatchard analysis gave similar values of equilibrium dissociation constants in both myometrial layers. In contrast, more alpha-1 adrenergic receptors were detected in the outer layer than in the inner layer. Antagonist inhibited (3H)prazosin binding with an order of potency of prazosin greater than phentolamine greater than idazoxan. Competition experiments have also revealed that a stable guanine nucleotide decreases the apparent affinity of norepinephrine for myometrial (3H)prazosin binding sites. The functional status of these alpha-1 adrenergic receptors was also assessed by measuring the norepinephrine-induced accumulation of inositol phosphates in myometrial tissue. Norepinephrine produced a concentration-dependent accumulation of inositol phosphates in both myometrial layers. However, norepinephrine-induced increases in inositol 1,4,5-triphosphate were only observed in the outer layer. These results indicate that alpha-1 adrenergic receptors in human myometrium at the end of pregnancy are linked to phosphoinositide hydrolysis and that this response occurs mainly in the outer layer.

  2. Effect of aging on alpha-1 adrenergic stimulation of phosphoinositide hydrolysis in various regions of rat brain

    SciTech Connect

    Burnett, D.M.; Bowyer, J.F.; Masserano, J.M.; Zahniser, N.R. )

    1990-12-01

    The effects of aging were examined on the ability of alpha-1 adrenergic receptor agonists to stimulate phosphoinositide hydrolysis in three brain regions. Tissue minces of thalamus, cerebral cortex and hippocampus from 3-, 18- and 28-month-old male Fischer 344 rats were prelabeled with ({sup 3}H)myoinositol. Exposure of these prelabeled minces to phenylephrine and (-)-norepinephrine revealed that accumulation of ({sup 3}H)inositol phosphates was selectively reduced by 20 to 30% in the thalamus and cerebral cortex of the oldest age group. Analysis of concentration-response and competition binding curves indicated that this decrease was due to diminished agonist efficacy rather than diminished receptor affinity. The reduction in responsiveness to phenylephrine and (-)-norepinephrine in the cerebral cortex and the lack of any changes in the hippocampus parallel previously reported changes in the density of alpha-1 adrenergic receptors with aging. These data indicate that the ability of alpha-1 adrenergic receptor agonists to stimulate phosphoinositide hydrolysis is reduced in some, but not all, brain regions of aged Fischer 344 rats.

  3. Role of a guanine nucleotide-binding protein in. cap alpha. /sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells

    SciTech Connect

    Cornett, L.E.; Norris, J.S.

    1987-11-01

    In this study the mechanisms involved in ..cap alpha../sub 1/-adrenergic receptor-mediated Ca/sup 2 +/ mobilization at the level of the plasma membrane were investigated. Stimulation of /sup 45/Ca/sup 2 +/ efflux from saponin-permeabilized DDT/sub 1/ MF-2 cells was observed with the addition of either the ..cap alpha../sub 1/-adrenergic agonist phenylephrine and guanosine-5'-triphosphate or the nonhydrolyzable guanine nucleotide guanylyl-imidodiphosphate. In the presence of (/sup 32/P) NAD, pertussis toxin was found to catalyze ADP-ribosylation of a M/sub r/ = 40,500 (n = 8) peptide in membranes prepared from DDT/sub 1/, MF-2 cells, possibly the ..cap alpha..-subunit of N/sub i/. However, stimulation of unidirectional /sup 45/Ca/sup 2 +/ efflux by phenylephrine was not affected by previous treatment of cells with 100 ng/ml pertussis toxin. These data suggest that the putative guanine nucleotide-binding protein which couples the ..cap alpha../sub 1/-adrenergic receptor to Ca/sup 2 +/ mobilization in DDT/sub 1/ MF-2 cells is not a pertussis toxin substrate and may possibly be an additional member of guanine nucleotide binding protein family.

  4. AGE-RELATED CHANGES IN RECEPTOR-MEDIATED PHOSPHOINOSITIDE HYDROLYSIS IN VARIOUS REGIONS OF RAT BRAIN

    EPA Science Inventory

    The effects of age on cholinergic markers and receptor-stimulated phosphoinositide hydrolysis was dined in the frontal cortex and striatum of male Fischer-344 rats. holine acetyltransferase activity was decreased 27% in the striatum of aged (24 month) rats cared to young (3 month...

  5. Leukotriene D4 receptor-mediated hydrolysis of phosphoinositide and mobilization of calcium in sheep tracheal smooth muscle cells

    SciTech Connect

    Mong, S.; Miller, J.; Wu, H.L.; Crooke, S.T.

    1988-02-01

    A sheep tracheal smooth muscle primary culture cell system was developed to characterize leukotriene D4 (LTD4) receptor-mediated biochemical and pharmacological effects. (/sup 3/H)LTD4 binding to the enriched plasma membrane receptor was specific, stereoselective and saturable. LTE4 and high affinity receptor antagonists bound to the receptors with a rank-order potency that was expected from previous smooth muscle contraction studies. In the (/sup 3/H)myoinositol labeled cells, LTD4 and LTE4 induced phosphoinositide hydrolysis. The biosynthesis of (/sup 3/H)inositol-trisphosphate was rapid and the induction of biosynthesis of (/sup 3/H)inositol-monophosphate by LTs was stereoselective and specific and was inhibited specifically by a receptor antagonist, SKF 104353. In the fura-2 loaded smooth muscle cells, LTD4 and LTE4 induced transient intracellular Ca++ mobilization. The fura-2/Ca++ transient was stereoselective and specific and was inhibited by receptor antagonist, SKF 104353. These results suggest that the cultured sheep tracheal smooth muscle cells have plasma membrane receptors for LTD4. These receptors were coupled to a phospholipase C that, when activated by agonists, induced hydrolysis of inositol containing phospholipids. The hydrolysis products, e.g. diacylglycerol and inositol-trisphosphate, may serve as intracellular messengers that trigger or contribute to the contractile effect in sheep tracheal smooth muscle.

  6. Phosphorylation of Src by phosphoinositide 3-kinase regulates beta-adrenergic receptor-mediated EGFR transactivation.

    PubMed

    Watson, Lewis J; Alexander, Kevin M; Mohan, Maradumane L; Bowman, Amber L; Mangmool, Supachoke; Xiao, Kunhong; Naga Prasad, Sathyamangla V; Rockman, Howard A

    2016-10-01

    β2-Adrenergic receptors (β2AR) transactivate epidermal growth factor receptors (EGFR) through formation of a β2AR-EGFR complex that requires activation of Src to mediate signaling. Here, we show that both lipid and protein kinase activities of the bifunctional phosphoinositide 3-kinase (PI3K) enzyme are required for β2AR-stimulated EGFR transactivation. Mechanistically, the generation of phosphatidylinositol (3,4,5)-tris-phosphate (PIP3) by the lipid kinase function stabilizes β2AR-EGFR complexes while the protein kinase activity of PI3K regulates Src activation by direct phosphorylation. The protein kinase activity of PI3K phosphorylates serine residue 70 on Src to enhance its activity and induce EGFR transactivation following βAR stimulation. This newly identified function for PI3K, whereby Src is a substrate for the protein kinase activity of PI3K, is of importance since Src plays a key role in pathological and physiological signaling. PMID:27169346

  7. Inhibition by islet-activating protein, pertussis toxin, of P2-purinergic receptor-mediated iodide efflux and phosphoinositide turnover in FRTL-5 cells

    SciTech Connect

    Okajima, F.; Sho, K.; Kondo, Y.

    1988-08-01

    Exposure of FRTL-5 thyroid cells to ATP (1 microM to 1 mM) resulted in the stimulation of I- efflux in association with the induction of inositol trisphosphate production and intracellular Ca2+ mobilization. Nonhydrolyzable ATP derivatives, ADP and GTP, were also as effective in magnitude as ATP, whereas neither AMP nor adenosine exerted significant effect on I- efflux, suggesting a P2-purinergic receptor-mediated activation of I- efflux. Treatment of the cells with the islet-activating protein (IAP) pertussis toxin, which ADP-ribosylated a 41,000 mol wt membrane protein, effectively suppressed the phosphoinositide response to ATP in addition to ATP-dependent I- efflux at agonist concentrations below 10 microM. In contrast, the I- efflux stimulated by TSH, A23187, or phorbol myristate acetate was insusceptible to IAP. The IAP substrate, probably GTP-binding protein, is hence proposed to mediate the activation of P2-purinergic receptor-linked phospholipase-C in FRTL-5 cells. However, the responses to ATP, its nonhydrolyzable derivatives, or ADP at the higher agonist concentrations, especially above 100 microM, were only partially inhibited by IAP, even though the IAP substrate was totally ADP ribosylated by the toxin. The responses to GTP in the whole concentration range tested were not influenced by IAP treatment. Thus, signals arising from the P2-receptor might be transduced to phospholipase-C by two different pathways, i.e. IAP-sensitive and insensitive ones, and result in the stimulation of I- efflux.

  8. Intratesticular alpha1-adrenergic receptors mediate stress-disturbed transcription of steroidogenic stimulator NUR77 as well as steroidogenic repressors DAX1 and ARR19 in Leydig cells of adult rats.

    PubMed

    Stojkov-Mimic, Natasa J; Bjelic, Maja M; Radovic, Sava M; Mihajlovic, Aleksandar I; Sokanovic, Srdjan J; Baburski, Aleksandar Z; Janjic, Marija M; Kostic, Tatjana S; Andric, Silvana A

    2015-09-01

    The aim of the present study was to define the role of testicular α1-adrenergic receptors (α1-ADRs) in stress-triggered adaptation of testosterone-producing Leydig cells of adult rats. Results showed that in vivo blockade of testicular α1-ADRs prevented partial recovery of circulating androgen levels registered after 10× repeated immobilization stress (10 × IMO). Moreover, α1-ADR-blockade diminished 10 × IMO-triggered recovery of Leydig cell androgen production, and abolished mitochondrial membrane potential recovery. In the same cells, 10 × IMO-induced increase in Star transcript was abolished, Lhcgr transcript decreased, while transcription of other steroidogenic proteins was not changed. α1-ADR-blockade recovered stress-induced decrease of Nur77, one of the main steroidogenic stimulator, while significantly reduced 10 × IMO-increased in the transcription of the main steroidogenic repressors, Arr19 and Dax1. In vitro experiments revealed an adrenaline-induced α1-ADR-mediated decrease in Nur77 transcription in Leydig cells. Adrenaline-induced increase of repressor Dax1 also involves ADRs in Leydig cells. Accordingly, α1-ADRs participate in some of the stress-triggered effects on the steroidogenic machinery of Leydig cells. PMID:26003139

  9. Alpha-1-Adrenergic Receptors: Targets for Agonist Drugs to Treat Heart Failure

    PubMed Central

    Jensen, Brian C.; O'Connell, Timothy D.; Simpson, Paul C.

    2011-01-01

    Evidence from cell, animal, and human studies demonstrates that α1-adrenergic receptors mediate adaptive and protective effects in the heart. These effects may be particularly important in chronic heart failure, when catecholamine levels are elevated and β-adrenergic receptors are down regulated and dysfunctional. This review summarizes these data and proposes that selectively activating α1-adrenergic receptors in the heart may represent a novel and effective way to treat heart failure. PMID:21118696

  10. Phosphoinositides regulate ion channels

    PubMed Central

    Hille, Bertil; Dickson, Eamonn J.; Kruse, Martin; Vivas, Oscar; Suh, Byung-Chang

    2014-01-01

    Phosphoinositides serve as signature motifs for different cellular membranes and often are required for the function of membrane proteins. Here, we summarize clear evidence supporting the concept that many ion channels are regulated by membrane phosphoinositides. We describe tools used to test their dependence on phosphoinositides, especially phosphatidylinositol 4,5-bisphosphate, and consider mechanisms and biological meanings of phosphoinositide regulation of ion channels. This lipid regulation can underlie changes of channel activity and electrical excitability in response to receptors. Since different intracellular membranes have different lipid compositions, the activity of ion channels still in transit towards their final destination membrane may be suppressed until they reach an optimal lipid environment. PMID:25241941

  11. Cardiac and neuroprotection regulated by α1-adrenergic receptor subtypes

    PubMed Central

    Perez, Dianne M.; Doze, Van A.

    2013-01-01

    Sympathetic nervous system regulation by the α1-adrenergic receptor (AR) subtypes (α1A, α1B, α1D) is complex, whereby chronic activity can be either detrimental or protective for both heart and brain function. This review will summarize the evidence that this dual regulation can be mediated through the different α1-AR subtypes in the context of cardiac hypertrophy, heart failure, apoptosis, ischemic preconditioning, neurogenesis, locomotion, neurodegeneration, cognition, neuroplasticity, depression, anxiety, epilepsy, and mental illness. PMID:21338248

  12. Mechanisms of alpha 1-adrenergic vascular desensitization in conscious dogs

    NASA Technical Reports Server (NTRS)

    Kiuchi, K.; Vatner, D. E.; Uemura, N.; Bigaud, M.; Hasebe, N.; Hempel, D. M.; Graham, R. M.; Vatner, S. F.

    1992-01-01

    To investigate the mechanisms of alpha 1-adrenergic vascular desensitization, osmotic minipumps containing either saline (n = 9) or amidephrine mesylate (AMD) (n = 9), a selective alpha 1-adrenergic receptor agonist, were implanted subcutaneously in dogs with chronically implanted arterial and right atrial pressure catheters and aortic flow probes. After chronic alpha 1-adrenergic receptor stimulation, significant physiological desensitization to acute AMD challenges was observed, i.e., pressor and vasoconstrictor responses to the alpha 1-adrenergic agonist were significantly depressed (p < 0.01) compared with responses in the same dogs studied in the conscious state before pump implantation. However, physiological desensitization to acute challenges of the neurotransmitter norepinephrine (NE) (0.1 micrograms/kg per minute) in the presence of beta-adrenergic receptor blockade was not observed for either mean arterial pressure (MAP) (30 +/- 7 versus 28 +/- 5 mm Hg) or total peripheral resistance (TPR) (29.8 +/- 4.9 versus 28.9 +/- 7.3 mm Hg/l per minute). In the presence of beta-adrenergic receptor plus ganglionic blockade after AMD pump implantation, physiological desensitization to NE was unmasked since the control responses to NE (0.1 micrograms/kg per minute) before the AMD pumps were now greater (p < 0.01) than after chronic AMD administration for both MAP (66 +/- 5 versus 32 +/- 2 mm Hg) and TPR (42.6 +/- 10.3 versus 23.9 +/- 4.4 mm Hg/l per minute). In the presence of beta-adrenergic receptor, ganglionic, plus NE-uptake blockade after AMD pump implantation, desensitization was even more apparent, since NE (0.1 micrograms/kg per minute) induced even greater differences in MAP (33 +/- 5 versus 109 +/- 6 mm Hg) and TPR (28.1 +/- 1.8 versus 111.8 +/- 14.7 mm Hg/l per minute). The maximal force of contraction induced by NE in the presence or absence of endothelium was significantly decreased (p < 0.05) in vitro in mesenteric artery rings from AMD pump dogs

  13. Regulation and ontogeny of subtypes of muscarinic receptors and muscarinic receptor-mediated

    SciTech Connect

    Lee, W.

    1989-01-01

    The densities of total and M1 muscarinic receptors were measured using the muscarinic receptor antagonists {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine, respectively. Thus, the difference between the density of {sup 3}H-quinuclidinyl benzilate and {sup 3}H-pirenzepine binding sites represents the density of M2 sites. In addition, there is no observable change in either acetylcholine-stimulated phosphoinositide breakdown (suggested to be an M1 receptor-mediated response) or in carbachol-mediated inhibition of cyclic AMP accumulation (suggested to be an M2 receptor-mediated response) in slices of cortex+dorsal hippocampus following chronic atropine administration. In other experiments, it has been shown that the M1 and M2 receptors in rat cortex have different ontogenetic profiles. The M2 receptor is present at adult levels at birth, while the M1 receptor develops slowly from low levels at postnatal week 1 to adult levels at postnatal week 3. The expression of acetylcholine-stimulated phosphoinositide breakdown parallels the development of M1 receptors, while the development of carbachol-mediated inhibition of cyclic AMP accumulation occurs abruptly between weeks 2 and 3 postnatally.

  14. Antagonism of Lateral Amygdala Alpha1-Adrenergic Receptors Facilitates Fear Conditioning and Long-Term Potentiation

    ERIC Educational Resources Information Center

    Lazzaro, Stephanie C.; Hou, Mian; Cunha, Catarina; LeDoux, Joseph E.; Cain, Christopher K.

    2010-01-01

    Norepinephrine receptors have been studied in emotion, memory, and attention. However, the role of alpha1-adrenergic receptors in fear conditioning, a major model of emotional learning, is poorly understood. We examined the effect of terazosin, an alpha1-adrenergic receptor antagonist, on cued fear conditioning. Systemic or intra-lateral amygdala…

  15. Dynamics of Phosphoinositide-Dependent Signaling in Sympathetic Neurons

    PubMed Central

    Kruse, Martin; Vivas, Oscar; Traynor-Kaplan, Alexis

    2016-01-01

    In neurons, loss of plasma membrane phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] leads to a decrease in exocytosis and changes in electrical excitability. Restoration of PI(4,5)P2 levels after phospholipase C activation is therefore essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We measured dynamic changes of PI(4,5)P2, phosphatidylinositol 4-phosphate, diacylglycerol, inositol 1,4,5-trisphosphate, and Ca2+ upon muscarinic stimulation in sympathetic neurons from adult male Sprague-Dawley rats with electrophysiological and optical approaches. We used this kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show and explain faster synthesis of PI(4,5)P2 in sympathetic neurons than in electrically nonexcitable tsA201 cells. They can be used to understand dynamic effects of receptor-mediated phospholipase C activation on excitability and other PI(4,5)P2-dependent processes in neurons. SIGNIFICANCE STATEMENT Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] is a minor phospholipid in the cytoplasmic leaflet of the plasma membrane. Depletion of PI(4,5)P2 via phospholipase C-mediated hydrolysis leads to a decrease in exocytosis and alters electrical excitability in neurons. Restoration of PI(4,5)P2 is essential for a return to basal neuronal activity. However, the dynamics of phosphoinositide metabolism have not been analyzed in neurons. We studied the dynamics of phosphoinositide metabolism in sympathetic neurons upon muscarinic stimulation and used the kinetic information to develop a quantitative description of neuronal phosphoinositide metabolism. The measurements and analysis show a several-fold faster synthesis of PI(4,5)P2 in sympathetic neurons than in an electrically nonexcitable cell line, and provide a framework for future studies of PI(4,5)P2-dependent processes in neurons. PMID:26818524

  16. Cholesterol stabilizes fluid phosphoinositide domains

    PubMed Central

    Jiang, Zhiping; Redfern, Roberta E.; Isler, Yasmin; Ross, Alonzo H.

    2014-01-01

    Local accumulation of phosphoinositides (PIPs) is an important factor for a broad range of cellular events including membrane trafficking and cell signaling. The negatively charged phosphoinositide headgroups can interact with cations or cationic proteins and this electrostatic interaction has been identified as the main phosphoinositide clustering mechanism. However, an increasing number of reports show that phosphoinositide-mediated signaling events are at least in some cases cholesterol dependent, suggesting other possible contributors to the segregation of phosphoinositides. Using fluorescence microscopy on giant unilamellar vesicles and monolayers at the air/water interface, we present data showing that cholesterol stabilizes fluid phosphoinositide-enriched phases. The interaction with cholesterol is observed for all investigated phosphoinositides (PI(4)P, PI(3,4)P2, PI(3,5)P2, PI(4,5)P2 and PI(3,4,5)P3) as well as phosphatidylinositol. We find that cholesterol is present in the phosphoinositide-enriched phase and that the resulting phase is fluid. Cholesterol derivatives modified at the hydroxyl group (cholestenone, cholesteryl ethyl ether) do not promote formation of phosphoinositide domains, suggesting an instrumental role of the cholesterol hydroxyl group in the observed cholesterol/phosphoinositide interaction. This leads to the hypothesis that cholesterol participates in an intermolecular hydrogen bond network formed among the phosphoinositide lipids. We had previously reported that the intra- and intermolecular hydrogen bond network between the phosphoinositide lipids leads to a reduction of the charge density at the phosphoinositide phosphomonoester groups (Kooijman et al. Biochemistry 48, (2009) 9360). We believe that cholesterol acts as a spacer between the phosphoinositide lipids, thereby reducing the electrostatic repulsion, while participating in the hydrogen bond network, leading to its further stabilization. To illustrate the effect of

  17. Norepinephrine-Induced Adrenergic Activation Strikingly Increased the Atrial Fibrillation Duration through β1- and α1-Adrenergic Receptor-Mediated Signaling in Mice

    PubMed Central

    Hasegawa, Nozomi; Cai, Wenqian; Jin, Huiling; Hidaka, Yuko; Prajapati, Rajesh; Umemura, Masanari; Yokoyama, Utako; Sato, Motohiko; Okumura, Satoshi; Ishikawa, Yoshihiro

    2015-01-01

    Background Atrial fibrillation (AF) is the most common arrhythmias among old people. It causes serious long-term health problems affecting the quality of life. It has been suggested that the autonomic nervous system is involved in the onset and maintenance of AF in human. However, investigation of its pathogenesis and potential treatment has been hampered by the lack of suitable AF models in experimental animals. Objectives Our aim was to establish a long-lasting AF model in mice. We also investigated the role of adrenergic receptor (AR) subtypes, which may be involved in the onset and duration of AF. Methods and Results Trans-esophageal atrial burst pacing in mice could induce AF, as previously shown, but with only a short duration (29.0±8.1 sec). We found that adrenergic activation by intraperitoneal norepinephrine (NE) injection strikingly increased the AF duration. It increased the duration to more than 10 minutes, i.e., by more than 20-fold (656.2±104.8 sec; P<0.001). In this model, a prior injection of a specific β1-AR blocker metoprolol and an α1-AR blocker prazosin both significantly attenuated NE-induced elongation of AF. To further explore the mechanisms underlying these receptors’ effects on AF, we assessed the SR Ca2+ leak, a major trigger of AF, and consequent spontaneous SR Ca2+ release (SCR) in atrial myocytes. Consistent with the results of our in-vivo experiments, both metoprolol and prazosin significantly inhibited the NE-induced SR Ca2+ leak and SCR. These findings suggest that both β1-AR and α1-AR may play important roles in the development of AF. Conclusions We have established a long-lasting AF model in mice induced by adrenergic activation, which will be valuable in future AF study using experimental animals, such as transgenic mice. We also revealed the important role of β1- and α1-AR-mediated signaling in the development of AF through in-vivo and in-vitro experiments. PMID:26203906

  18. A nuclear pathway for alpha 1-adrenergic receptor signaling in cardiac cells.

    PubMed Central

    Ardati, A; Nemer, M

    1993-01-01

    alpha 1-Adrenergic agonists and antagonists constitute an important class of therapeutic agents commonly used for the treatment of various cardiovascular diseases like hypertension, congestive heart failure and supraventricular tachycardia. At the heart level, activation of alpha 1-adrenergic receptors is associated with marked morphological and genetic changes. These include enhancement of contractility, myocardial growth (hypertrophy) and release of the heart major secretory product, atrial natriuretic factor (ANF). However, the signal transduction pathways which link extracellular activation of the receptors to cellular and genetic changes are not well understood. Using primary cardiocyte cultures from neonate rat hearts, an alpha 1-adrenergic regulatory sequence has been identified in the 5' flanking region of the ANF gene. This sequence, which is necessary and sufficient for transcriptional activation in response to the alpha 1-specific agonist phenylephrine, interacts with novel zinc-dependent proteins which are induced by alpha 1-adrenergic stimulation. Consistent with a conserved regulatory mechanism, the alpha 1 response element is highly conserved between rodent, bovine and human ANF genes, and is also present in the promoter region of other alpha 1-responsive cardiac genes. The identification of a nuclear pathway for alpha 1-receptor signaling will be useful for elucidating the intracellular effectors of alpha 1-adrenergic receptors. Images PMID:8262057

  19. The alpha 1-adrenergic transduction system in hamster brown adipocytes. Release of arachidonic acid accompanies activation of phospholipase C.

    PubMed Central

    Schimmel, R J

    1988-01-01

    Previous studies of brown adipocytes identified an increased breakdown of phosphoinositides after selective alpha 1-adrenergic-receptor activation. The present paper reports that this response, elicited with phenylephrine in the presence of propranolol and measured as the accumulation of [3H]inositol phosphates, is accompanied by increased release of [3H]arachidonic acid from cells prelabelled with [3H]arachidonic acid. Differences between stimulated arachidonic acid release and formation of inositol phosphates included a requirement for extracellular Ca2+ for stimulated release of arachidonic acid but not for the formation of inositol phosphates and the preferential inhibition of inositol phosphate formation by phorbol 12-myristate 13-acetate. The release of arachidonic acid in response to phenylephrine was associated with an accumulation of [3H]arachidonic acid-labelled diacylglycerol, and this response was not dependent on extracellular Ca2+ but was partially prevented by treatment with the phorbol ester. The release of arachidonic acid was also stimulated by melittin, which increases the activity of phospholipase A2, by ionophore A23187, by lipolytic stimulation with forskolin and by exogenous phospholipase C. The arachidonic acid response to phospholipase C was completely blocked by RHC 80267, an inhibitor of diacylglycerol lipase, but this inhibitor had no effect on release stimulated with melittin or A23187 and inhibited phenylephrine-stimulated release by only 40%. The arachidonate response to forskolin was additive with the responses to either phenylephrine or exogenous phospholipase C. These data indicate that brown adipocytes are capable of releasing arachidonic acid from neutral lipids via triacylglycerol lipolysis, and from phospholipids via phospholipase A2 or by the sequential activities of phospholipase C and diacylglycerol lipase. Our findings also suggest that the action of phenylephrine to promote the liberation of arachidonic acid utilizes both

  20. Phosphoinositide Phosphatases in Cell Biology and Disease

    PubMed Central

    Liu, Yang; Bankaitis, Vytas A.

    2010-01-01

    Phosphoinositides are essential signaling molecules linked to a diverse array of cellular processes in eukaryotic cells. The metabolic interconversions of these phospholipids are subject to exquisite spatial and temporal regulation executed by arrays of phosphatidylinositol (PtdIns) and phosphoinositide-metabolizing enzymes. These include PtdIns- and phosphoinositide-kinases that drive phosphoinositide synthesis, and phospholipases and phosphatases that regulate phosphoinositide degradation. In the past decade, phosphoinositide phosphatases have emerged as topics of particular interest. This interest is driven by the recent appreciation that these enzymes represent primary mechanisms for phosphoinositide degradation, and because of their ever-increasing connections with human diseases. Herein, we review the biochemical properties of six major phosphoinositide phosphatases, the functional involvements of these enzymes in regulating phosphoinositide metabolism, the pathologies that arise from functional derangements of individual phosphatases, and recent ideas concerning the involvements of phosphoinositide phosphatases in membrane traffic control. PMID:20043944

  1. BLOCKAGE OF A-1 ADRENERGIC RECEPTOR INHIBOTS HEPATIC DNA SYNTHESIS STIMULATED BY TUMOR PROMOTERS

    EPA Science Inventory

    Studies with regenerating liver and hepatocyte cultures have shown that the a-1 adrenergic receptor (A1AR) is involved in the early events which transmit a mitogenic signal to hepatocytes after 2/3 partial hepatectomy. n this study, we investigated the role of A1AR in DNA synthes...

  2. The functional role of the alpha-1 adrenergic receptors in cerebral blood flow regulation

    PubMed Central

    Purkayastha, Sushmita; Raven, Peter B.

    2011-01-01

    Cerebral vasculature is richly innervated by the α-1 adrenergic receptors similar to that of the peripheral vasculature. However, the functional role of the α-1adrenergic receptors in cerebral blood flow (CBF) regulation is yet to be established. The traditional thinking being that during normotension and normocapnia sympathetic neural activity does not play a significant role in CBF regulation. Reports in the past have stated that catecholamines do not penetrate the blood brain barrier (BBB) and therefore only influence cerebral vessels from outside the BBB and hence, have a limited role in CBF regulation. However, with the advent of dynamic measurement techniques, beat-to-beat CBF assessment can be done during dynamic changes in arterial blood pressure. Several studies in the recent years have reported a functional role of the α-1adrenergic receptors in CBF regulation. This review focuses on the recent developments on the role of the sympathetic nervous system, specifically that of the α-1 adrenergic receptors in CBF regulation. PMID:22021989

  3. Phosphoinositide signaling in somatosensory neurons.

    PubMed

    Rohacs, Tibor

    2016-05-01

    Somatosensory neurons of the dorsal root ganglia (DRG) and trigeminal ganglia (TG) are responsible for detecting thermal and tactile stimuli. They are also the primary neurons mediating pain and itch. A large number of cell surface receptors in these neurons couple to phospholipase C (PLC) enzymes leading to the hydrolysis of phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] and the generation of downstream signaling molecules. These neurons also express many different ion channels, several of which are regulated by phosphoinositides. This review will summarize the knowledge on phosphoinositide signaling in DRG neurons, with special focus on effects on sensory and other ion channels. PMID:26724974

  4. Altered hepatic vasopressin and alpha 1-adrenergic receptors after chronic endotoxin infusion

    SciTech Connect

    Roth, B.L.; Spitzer, J.A.

    1987-05-01

    Sepsis and septic shock are complicated by a number of hemodynamic and metabolic aberrations. These include catecholamine refractoriness and altered glucose metabolism. Recently, a nonshock rat model of continuous endotoxin infusion via an implanted osmotic pump was developed that reproduces some of the metabolic and cardiovascular findings of human sepsis. By using this model, we have found a decreased number of hepatic plasma membrane alpha 1-adrenergic and (Arg8)vasopressin receptors in rats continuously infused with endotoxin. There was a significant decrease in (/sup 3/H)prazosin (35 +/- 7%) and (/sup 3/H) (Arg8)vasopressin (43 +/- 8%) receptors after 30 h of continuous endotoxin infusion with no change in affinity. The ability of norepinephrine to form the high-affinity complex with alpha 1-adrenergic receptors was not altered after chronic endotoxin infusion. The results are consistent with the concept that alterations in receptor number might underlie certain of the metabolic consequences of chronic sepsis.

  5. A role for α1-adrenergic receptors in extinction of conditioned fear and cocaine conditioned preference

    PubMed Central

    Bernardi, Rick E.; Lattal, K. Matthew

    2010-01-01

    Previous work has demonstrated an important role for adrenergic receptors in memory processes in fear and drug conditioning paradigms. Recent studies have also demonstrated alterations in extinction in these paradigms using drug treatments targeting β- and α2-adrenergic receptors, but little is known about the role of α1-adrenergic receptors in extinction. The current study examined whether antagonism of α1-adrenergic receptors would impair the consolidation of extinction in fear and cocaine conditioned place preference (CPP) paradigms. After contextual fear conditioning, injections of prazosin (1.0 or 3.0 mg/kg) following nonreinforced context exposures slowed the loss of conditioned freezing over the course of five extinction sessions (Experiment 1). After cocaine place conditioning, prazosin had no effect on the rate of extinction over eight nonreinforced test sessions. Following post-extinction reconditioning, however, prazosin-treated mice showed a robust place preference, but vehicle-treated mice did not, suggesting that prazosin reduced the persistent effects of extinction (Experiment 2). These results confirm the involvement of the α1-adrenergic receptor in extinction processes in both appetitive and aversive preparations. PMID:20364880

  6. Effect of alpha 1-adrenergic blockade on myocardial blood flow during exercise after myocardial infarction.

    PubMed

    Herzog, C A; Dai, X Z; Bache, R J

    1991-08-01

    The effect of alpha 1-adrenergic blockade with prazosin on myocardial blood flow at rest and during two levels of treadmill exercise was assessed in 16 chronically instrumented dogs 9-14 days after myocardial infarction had been produced by occlusion of the left circumflex coronary artery. During resting conditions prazosin did not alter mean myocardial blood flow or the subendocardial-to-subepicardial flow ratio in either normally perfused or collateral-dependent myocardium. However, during exercise at comparable external work loads and comparable rate-pressure products, prazosin significantly increased blood flow to normally perfused (27% increase at the second level of exercise, P less than 0.001) and collateral-dependent myocardium (35% increase at the second level of exercise, P less than 0.001) compared with control. In addition, prazosin caused a small but significant decrease in the subendocardial-to-subepicardial flow ratio in both normal (1.27 +/- 0.04 to 1.19 +/- 0.04; P less than 0.01) and collateral-dependent myocardium (0.57 +/- 0.11 to 0.52 +/- 0.11; P less than 0.01) compared with control, reflecting a disproportionally greater increase in subepicardial flow in response to alpha 1-adrenergic blockade. These data demonstrate that alpha 1-adrenergic vasoconstriction inhibits coronary vasodilation during exercise, even in areas of collateral-dependent myocardium relatively early after coronary artery occlusion. PMID:1678929

  7. Muscarinic receptor-mediated inositol tetrakisphosphate response in bovine adrenal chromaffin cells

    SciTech Connect

    Sanborn, B.B.; Schneider, A.S. )

    1990-01-01

    Inositol trisphosphate (IP{sub 3}), a product of the phosphoinositide cycle, mobilizes intracellular Ca{sup 2+} in many cell types. New evidence suggests that inositol tetrakisphosphate (IP{sub 4}), an IP{sub 3} derivative, may act as another second messenger to further alter calcium homeostasis. However, the function and mechanism of action of IP{sub 4} are presently unresolved. We now report evidence of muscarinic receptor-mediated accumulation of IP{sub 4} in bovine adrenal chromaffin cells, a classic neurosecretory system in which calcium movements have been well studied. Muscarine stimulated an increase in ({sup 3}H)IP{sub 4} and ({sup 3}H)IP{sub 3} accumulation in chromaffin cells and this effect was completely blocked by atropine. ({sup 3}H)IP{sub 4} accumulation was detectable within 15 sec, increased to a maximum by 30 sec and thereafter declined. 2,3-diphosphoglycerate, an inhibitor of IP{sub 3} and IP{sub 4} hydrolysis, enhanced accumulation of these inositol polyphosphates. The results provide the first evidence of a rapid inositol tetrakisphosphate response in adrenal chromaffin cells, which should facilitate the future resolution of the relationship between IP{sub 4} and calcium homeostasis.

  8. Targeted gene delivery via N-acetylglucosamine receptor mediated endocytosis.

    PubMed

    Singh, Bijay; Maharjan, Sushila; Kim, You-Kyoung; Jiang, Tai; Islam, Mohammad Ariful; Kang, Sang-Kee; Cho, Myung-Haing; Choi, Yun-Jaie; Cho, Chong-Su

    2014-11-01

    Receptor-mediated endocytosis is a promising approach of gene delivery into the target cells via receptor-ligand interaction. Vimentins at the cell surface are recently known to bind N-acetylglucosamine (GlcNAc) residue, therefore, the cell surfaces of vimentin-expressing cells could be targeted by using the GlcNAc residue as a specific ligand for receptor-mediated gene delivery. Here, we have developed polymeric gene delivery vectors, based on poly(ethylene oxide)(PEO) and poly(aspartamide), namely poly[(aspartamide)(diethylenetriamine)]-b-[PEO-(GlcNAc)] (PADPG) and poly[(aspartamide)(diethylenetriamine)]-b-[PEO] (PADP) to elucidate the efficiency of GlcNAc ligand for gene delivery through receptor mediated endocytosis. To determine the efficiency of these polymeric vectors for specific gene delivery, the DNA condensation ability of PADPG and PADP and the subsequent formation of polymeric nanoparticles were confirmed by gel retardation assay and transmission electron microscopy respectively. Both PADPG and PADP had lower cytotoxicity than polyethylenimine 25 K (PEI 25 K). However, their transfection efficiency was comparatively lower than PEI 25 K due to hydrophilic property of PEO in the vectors. To observe the stability of polymeric nanoparticles, the transfection of PADPG and PADP was carried out in the presence of serum. Favorably, the interfering effect of serum on the transfection efficiency of PADPG and PADP was also very low. Finally, when the cell specificity of these polymeric vectors was investigated, PADPG had high gene transfection in vimentin-expressing cells than vimentin-deficiency cells. The high transfection efficiency of PADPG was attributed to the GlcNAc in the polymeric vector which interact specifically with vimentin in the cells for the receptor-mediated endocytosis. The competitive inhibition assay further proved the receptor-mediated endocytosis of PADPG. Thus, this study demonstrates that conjugation of GlcNAc is an effective and rational

  9. Identification and characterization of alpha 1 adrenergic receptors in the canine prostate using (/sup 125/I)-Heat

    SciTech Connect

    Lepor, H.; Baumann, M.; Shapiro, E.

    1987-11-01

    We have recently utilized radioligand receptor binding methods to characterize muscarinic cholinergic and alpha adrenergic receptors in human prostate adenomas. The primary advantages of radioligand receptor binding methods are that neurotransmitter receptor density is quantitated, the affinity of unlabelled drugs for receptor sites is determined, and receptors can be localized using autoradiography on slide-mounted tissue sections. Recently, (/sup 125/I)-Heat, a selective and high affinity ligand with high specific activity (2200 Ci/mmole) has been used to characterize alpha 1 adrenergic receptors in the brain. In this study alpha 1 adrenergic receptors in the dog prostate were characterized using (/sup 125/I)-Heat. The Scatchard plots were linear indicating homogeneity of (/sup 125/I)-Heat binding sites. The mean alpha 1 adrenergic receptor density determined from these Scatchard plots was 0.61 +/- 0.07 fmol/mg. wet wt. +/- S.E.M. The binding of (/sup 125/I)-Heat to canine prostate alpha 1 adrenergic binding sites was of high affinity (Kd = 86 +/- 19 pM). Steady state conditions were reached following an incubation interval of 30 minutes and specific binding and tissue concentration were linear within the range of tissue concentrations assayed. The specificity of (/sup 125/I)-Heat for alpha 1 adrenergic binding sites was confirmed by competitive displacement assays using unlabelled clonidine and prazosin. Retrospective analysis of the saturation experiments demonstrated that Bmax can be accurately calculated by determining specific (/sup 125/I)-Heat binding at a single ligand concentration. (/sup 125/I)-Heat is an ideal ligand for studying alpha 1 adrenergic receptors in the prostate and its favorable properties should facilitate the autoradiographic localization of alpha 1 adrenergic receptors in the prostate.

  10. Cocaine Increases Dopaminergic Neuron and Motor Activity via Midbrain α1 Adrenergic Signaling

    PubMed Central

    Goertz, Richard Brandon; Wanat, Matthew J; Gomez, Jorge A; Brown, Zeliene J; Phillips, Paul EM; Paladini, Carlos A

    2015-01-01

    Cocaine reinforcement is mediated by increased extracellular dopamine levels in the forebrain. This neurochemical effect was thought to require inhibition of dopamine reuptake, but cocaine is still reinforcing even in the absence of the dopamine transporter. Here, we demonstrate that the rapid elevation in dopamine levels and motor activity elicited by cocaine involves α1 receptor activation within the ventral midbrain. Activation of α1 receptors increases dopaminergic neuron burst firing by decreasing the calcium-activated potassium channel current (SK), as well as elevates dopaminergic neuron pacemaker firing through modulation of both SK and the hyperpolarization-activated cation currents (Ih). Furthermore, we found that cocaine increases both the pacemaker and burst-firing frequency of rat ventral-midbrain dopaminergic neurons through an α1 adrenergic receptor-dependent mechanism within the ventral tegmental area and substantia nigra pars compacta. These results demonstrate the mechanism underlying the critical role of α1 adrenergic receptors in the regulation of dopamine neurotransmission and behavior by cocaine. PMID:25374094

  11. Comparative analyses of lysophosphatidic acid receptor-mediated signaling.

    PubMed

    Fukushima, Nobuyuki; Ishii, Shoichi; Tsujiuchi, Toshifumi; Kagawa, Nao; Katoh, Kazutaka

    2015-06-01

    Lysophosphatidic acid (LPA) is a bioactive lipid mediator that activates G protein-coupled LPA receptors to exert fundamental cellular functions. Six LPA receptor genes have been identified in vertebrates and are classified into two subfamilies, the endothelial differentiation genes (edg) and the non-edg family. Studies using genetically engineered mice, frogs, and zebrafish have demonstrated that LPA receptor-mediated signaling has biological, developmental, and pathophysiological functions. Computational analyses have also identified several amino acids (aa) critical for LPA recognition by human LPA receptors. This review focuses on the evolutionary aspects of LPA receptor-mediated signaling by comparing the aa sequences of vertebrate LPA receptors and LPA-producing enzymes; it also summarizes the LPA receptor-dependent effects commonly observed in mouse, frog, and fish. PMID:25732591

  12. Multiscale Modeling of Virus Entry via Receptor-Mediated Endocytosis

    NASA Astrophysics Data System (ADS)

    Liu, Jin

    2012-11-01

    Virus infections are ubiquitous and remain major threats to human health worldwide. Viruses are intracellular parasites and must enter host cells to initiate infection. Receptor-mediated endocytosis is the most common entry pathway taken by viruses, the whole process is highly complex and dictated by various events, such as virus motions, membrane deformations, receptor diffusion and ligand-receptor reactions, occurring at multiple length and time scales. We develop a multiscale model for virus entry through receptor-mediated endocytosis. The binding of virus to cell surface is based on a mesoscale three dimensional stochastic adhesion model, the internalization (endocytosis) of virus and cellular membrane deformation is based on the discretization of Helfrich Hamiltonian in a curvilinear space using Monte Carlo method. The multiscale model is based on the combination of these two models. We will implement this model to study the herpes simplex virus entry into B78 cells and compare the model predictions with experimental measurements.

  13. Glucocorticoids down-regulate beta 1-adrenergic-receptor expression by suppressing transcription of the receptor gene.

    PubMed Central

    Kiely, J; Hadcock, J R; Bahouth, S W; Malbon, C C

    1994-01-01

    The expression of beta 2-adrenergic receptors is up-regulated by glucocorticoids. In contrast, beta 1-adrenergic receptors display glucocorticoid-induced down-regulation. In rat C6 glioma cells, which express both of these subtypes of beta-adrenergic receptors, the synthetic glucocorticoid dexamethasone stimulates no change in the total beta-adrenergic receptor content, but rather shifts the beta 1:beta 2 ratio from 80:20 to 50:50. Radioligand binding and immunoblotting demonstrate a sharp decline in beta 1-adrenergic receptor expression. Metabolic labelling of cells with [35S]-methionine in tandem with immunoprecipitation by beta 1-adrenergic-receptor-specific antibodies reveals a sharp decline in the synthesis of the receptor within 48 h for cells challenged with glucocorticoid. Steady-state levels of beta 1-adrenergic-receptor mRNA declined from 0.47 to 0.26 amol/microgram of total cellular RNA within 2 h of dexamethasone challenge, as measured by DNA-excess solution hybridization. The stability of receptor mRNA was not influenced by glucocorticoid; the half-lives of the beta 1- and beta 2-subtype mRNAs were 1.7 and 1.5 h respectively. Nuclear run-on assays revealed the basis for the down-regulation of receptor expression, i.e. a sharp decline in the relative rate of transcription for the beta 1-adrenergic-receptor gene in nuclei from dexamethasone-treated as compared with vehicle-treated cells. These data demonstrate transcriptional suppression as a molecular explanation for glucocorticoid-induced down-regulation of beta 1-adrenergic receptors. Images Figure 1 Figure 2 Figure 6 PMID:8092990

  14. Prazosin, an alpha 1-adrenergic receptor antagonist, suppresses experimental autoimmune encephalomyelitis in the Lewis rat.

    PubMed Central

    Brosnan, C F; Goldmuntz, E A; Cammer, W; Factor, S M; Bloom, B R; Norton, W T

    1985-01-01

    Prazosin, an antagonist of alpha 1-adrenergic receptors, has been found to suppress the clinical and histological expression of experimental autoimmune encephalomyelitis (EAE) in the Lewis rat. Suppression was more significant in females than in males and was a dose-dependent phenomenon. Analysis of the effect of other adrenergic receptor antagonists supports the conclusion that the suppressive effect of prazosin is a consequence of blockade of the alpha 1-receptor since treatment with either the alpha 2-antagonist yohimbine or the beta-antagonist propranolol exacerbated the disease, whereas treatment with the long-acting mixed alpha 1/alpha 2-antagonist phenoxybenzamine had some suppressive activity. Treatment with prazosin was also able to suppress clinical and histological signs of EAE in animals sensitized by adoptive transfer with activated spleen or lymph node cells. Whether prazosin acts through altering vascular permeability or the immune response, or both, remains to be determined. Images PMID:2994053

  15. Regulation of Golgi function via phosphoinositide lipids.

    PubMed

    Mayinger, Peter

    2009-09-01

    Phosphoinositides play important roles in Golgi traffic and structural integrity. Specific lipid kinases and phosphatases associate with the Golgi complex and regulate the multiplicity of trafficking routes from this organelle. Work in different model systems showed that the basic elements that regulate lipid signaling at the Golgi are conserved from yeast to humans. Many of the enzymes involved in Golgi phosphoinositide metabolism are essential for viability or cause severe human disease when malfunctioning. Phosphoinositide effectors at the Golgi control both non-vesicular transfer of lipids and sorting of secretory and membrane proteins. In addition, Golgi phosphoinositides were recently implicated in the metabolic and cell growth-dependent regulation of the secretory pathway. PMID:19508852

  16. Regulation of Golgi function via phosphoinositide lipids

    PubMed Central

    Mayinger, Peter

    2009-01-01

    Phosphoinositides play important roles in Golgi traffic and structural integrity. Specific lipid kinases and phosphatases associate with the Golgi complex and regulate the multiplicity of trafficking routes from this organelle. Work in different model systems showed that the basic elements that regulate lipid signaling at the Golgi are conserved form yeast to humans. Many of the enzymes involved in Golgi phosphoinositide metabolism are essential for viability or cause severe human disease when malfunctioning. Phosphoinositide effectors at the Golgi control both non-vesicular transfer of lipids and sorting of secretory and membrane proteins. In addition, Golgi phosphoinositides were recently implicated in the metabolic and cell growth-dependent regulation of the secretory pathway. PMID:19508852

  17. Phosphoinositides: Key modulators of energy metabolism☆

    PubMed Central

    Bridges, Dave; Saltiel, Alan R.

    2014-01-01

    Phosphoinositides are key players in many trafficking and signaling pathways. Recent advances regarding the synthesis, location and functions of these lipids have dramatically improved our understanding of how and when these lipids are generated and what their roles are in animal physiology. In particular, phosphoinositides play a central role in insulin signaling, and manipulation of PtdIns(3,4,5)P3 levels in particular, may be an important potential therapeutic target for the alleviation of insulin resistance associated with obesity and the metabolic syndrome. In this article we review the metabolism, regulation and functional roles of phosphoinositides in insulin signaling and the regulation of energy metabolism. This article is part of a Special Issue entitled Phosphoinositides. PMID:25463477

  18. Receptor-mediated mitophagy in yeast and mammalian systems.

    PubMed

    Liu, Lei; Sakakibara, Kaori; Chen, Quan; Okamoto, Koji

    2014-07-01

    Mitophagy, or mitochondria autophagy, plays a critical role in selective removal of damaged or unwanted mitochondria. Several protein receptors, including Atg32 in yeast, NIX/BNIP3L, BNIP3 and FUNDC1 in mammalian systems, directly act in mitophagy. Atg32 interacts with Atg8 and Atg11 on the surface of mitochondria, promoting core Atg protein assembly for mitophagy. NIX/BNIP3L, BNIP3 and FUNDC1 also have a classic motif to directly bind LC3 (Atg8 homolog in mammals) for activation of mitophagy. Recent studies have shown that receptor-mediated mitophagy is regulated by reversible protein phosphorylation. Casein kinase 2 (CK2) phosphorylates Atg32 and activates mitophagy in yeast. In contrast, in mammalian cells Src kinase and CK2 phosphorylate FUNDC1 to prevent mitophagy. Notably, in response to hypoxia and FCCP treatment, the mitochondrial phosphatase PGAM5 dephosphorylates FUNDC1 to activate mitophagy. Here, we mainly focus on recent advances in our understanding of the molecular mechanisms underlying the activation of receptor-mediated mitophagy and the implications of this catabolic process in health and disease. PMID:24903109

  19. Comprehensive Behavioral Phenotyping of Ts65Dn Mouse Model of Down Syndrome: Activation of β1-Adrenergic Receptor by Xamoterol as a Potential Cognitive Enhancer

    PubMed Central

    Faizi, Mehrdad; Bader, Patrick L.; Tun, Christine; Encarnacion, Angelo; Kleschevnikov, Alexander; Belichenko, Pavel; Saw, Nay; Priestley, Matthew; Tsien, Richard W; Mobley, William C; Shamloo, Mehrdad

    2012-01-01

    Down Syndrome (DS) is the most prevalent form of mental retardation caused by genetic abnormalities in humans. This has been successfully modeled in mice to generate the Ts65Dn mouse, a genetic model of DS. This transgenic mouse model shares a number of physical and functional abnormalities with people with DS, including changes in the structure and function of neuronal circuits. Significant abnormalities in noradrenergic (NE-ergic) afferents from the locus coeruleus to the hippocampus, as well as deficits in NE-ergic neurotransmission are detected in these animals. In the current study we characterized in detail the behavioral phenotype of Ts65Dn mice, in addition to using pharmacological tools for identification of target receptors mediating the learning and memory deficits observed in this model of DS. We undertook a comprehensive approach to mouse phenotyping using a battery of standard and novel tests encompassing: i) locomotion (Activity Chamber, PhenoTyper, and CatWalk), ii) learning and memory (spontaneous alternation, delayed matching-to-place water maze, fear conditioning, and Intellicage), and iii) social behavior. Ts65Dn mice showed increased locomotor activity in novel and home cage environments. There were significant and reproducible deficits in learning and memory tests including spontaneous alternation, delayed matching-to-place water maze, Intellicage place avoidance and contextual fear conditioning. Although Ts65Dn mice showed no deficit in sociability in the 3-chamber test, a marked impairment in social memory was detected. Xamoterol, a β1-adrenergic receptor (β1-ADR) agonist, effectively restored the memory deficit in contextual fear conditioning, spontaneous alternation and novel object recognition. These behavioral improvements were reversed by betaxolol, a selective β1-ADR antagonist. In conclusion, our results demonstrate that this mouse model of Down Syndrome display cognitive deficits which is mediated by imbalance in noradrenergic

  20. Anti-β1-adrenergic receptor autoantibodies in patients with chronic Chagas heart disease

    PubMed Central

    Labovsky, V; Smulski, C R; Gómez, K; Levy, G; Levin, M J

    2007-01-01

    Chronic Chagas heart disease (cChHD), a chronic manifestation of the Trypanosoma cruzi infection, is characterized by high antibody levels against the C-terminal region of the ribosomal P proteins (i.e. peptide R13, EEEDDDMGFGLFD) which bears similarity with the second extracellular loop of β1-adrenergic receptor (β1-AR, peptide H26R HWWRAESDEARRCYNDPKCCDFVTNR). Because it has not been demonstrated clearly that IgGs from cChHD patients bind to native human β1-AR, the aim of this study was to investigate further the physical interaction between cChHD IgGs and the human β1-AR. Immunofluorescence assays demonstrated the binding of these antibodies to the receptor expressed on stably transfected cells, together with a β1-AR agonist-like effect. In addition, immunoadsorption of the serum samples from cChHD patients with a commercially available matrix, containing peptides representing the first and the second extracellular loop of the β1-AR, completely abolished reactivity against the H26R peptide and the physiological response to the receptor. The follow-up of this specificity after in vitro immunoadsorption procedures suggests that this treatment might be used to diminish significantly the serum levels of anti-β1-AR antibodies in patients with Chagas heart disease. PMID:17419712

  1. Modeling the interactions between alpha(1)-adrenergic receptors and their antagonists.

    PubMed

    Du, Lupei; Li, Minyong

    2010-09-01

    As crucial members of the G-protein coupled receptor (GPCR) superfamily, alpha (1)-adrenergic receptors (alpha(1)-ARs) are recognized to intervene the actions of endogenous catecholamines such as norepinephrine and epinephrine. So far three distinct alpha(1)-AR subtypes, alpha(1A), alpha(1B) and alpha(1D), have been characterized by functional analysis, radio-ligand binding and molecular biology studies. The alpha(1)-ARs are of therapeutic interest because of their distinct and critical roles in many physiological processes, containing hypertension, benign prostatic hyperplasia, smooth muscle contraction, myocardial inotropy and chronotropy, and hepatic glucose metabolism. Accordingly, designing subtype-selective antagonists for each of the three alpha(1)-AR subtypes has been an enthusiastic region of medicinal research. Even though a large number of studies on GPCRs have been conducted, understanding of how known antagonists bind to alpha(1)-ARs still remains sketchy and has been a serious impediment to search for potent and subtype-selective alpha(1)-AR antagonists because of the lack of detailed experimental structural knowledge. This review deliberates the simulation of alpha(1)-ARs and their interactions with antagonists by using ligand-based (pharmacophore identification and QSAR modeling) and structure-based (comparative modeling and molecular docking) approaches. Combined with experimental data, these computational attempts could improve our understanding of the structural basis of antagonist binding and the molecular basis of receptor activation, thus offering a more reasonable approach in the design of drugs targeting alpha(1)-ARs. PMID:20412040

  2. β(1) Adrenergic receptor is key to cold- and diet-induced thermogenesis in mice.

    PubMed

    Ueta, Cintia B; Fernandes, Gustavo W; Capelo, Luciane P; Fonseca, Tatiane L; Maculan, Flávia D'Angelo; Gouveia, Cecilia H A; Brum, Patrícia C; Christoffolete, Marcelo A; Aoki, Marcelo S; Lancellotti, Carmen L; Kim, Brian; Bianco, Antonio C; Ribeiro, Miriam O

    2012-09-01

    Brown adipose tissue (BAT) is predominantly regulated by the sympathetic nervous system (SNS) and the adrenergic receptor signaling pathway. Knowing that a mouse with triple β-receptor knockout (KO) is cold intolerant and obese, we evaluated the independent role played by the β(1) isoform in energy homeostasis. First, the 30  min i.v. infusion of norepinephrine (NE) or the β(1) selective agonist dobutamine (DB) resulted in similar interscapular BAT (iBAT) thermal response in WT mice. Secondly, mice with targeted disruption of the β(1) gene (KO of β(1) adrenergic receptor (β(1)KO)) developed hypothermia during cold exposure and exhibited decreased iBAT thermal response to NE or DB infusion. Thirdly, when placed on a high-fat diet (HFD; 40% fat) for 5 weeks, β(1)KO mice were more susceptible to obesity than WT controls and failed to develop diet-induced thermogenesis as assessed by BAT Ucp1 mRNA levels and oxygen consumption. Furthermore, β(1)KO mice exhibited fasting hyperglycemia and more intense glucose intolerance, hypercholesterolemia, and hypertriglyceridemia when placed on the HFD, developing marked non-alcoholic steatohepatitis. In conclusion, the β(1) signaling pathway mediates most of the SNS stimulation of adaptive thermogenesis. PMID:22728333

  3. beta(1)-adrenergic antagonists improve sleep and behavioural disturbances in a circadian disorder, Smith-Magenis syndrome.

    PubMed

    De Leersnyder, H; de Blois, M C; Vekemans, M; Sidi, D; Villain, E; Kindermans, C; Munnich, A

    2001-09-01

    Smith-Magenis syndrome (SMS) is a clinically recognisable contiguous gene syndrome ascribed to interstitial deletions of chromosome 17p11.2. Patients have a phase shift of their circadian rhythm of melatonin with a paradoxical diurnal secretion of the hormone. Serum melatonin levels and day-night behaviour were studied in nine SMS children (aged 4 to 17 years) given acebutolol, a selective beta(1)-adrenergic antagonist (10 mg/kg early in the morning). Cardiac examination, serum melatonin, motor activity recordings, and sleep diaries were monitored before and after drug administration. The present study shows that a single morning dose of acebutolol suppressed the inappropriate secretion of melatonin in SMS. A significant improvement of inappropriate behaviour with increased concentration, delayed sleep onset, increased hours of sleep, and delayed waking were also noted. These results suggest that beta(1)-adrenergic antagonists help to manage hyperactivity, enhance cognitive performance, and reduce sleep disorders in SMS. PMID:11546826

  4. Channelopathies linked to plasma membrane phosphoinositides

    PubMed Central

    Logothetis, Diomedes E.; Petrou, Vasileios I.; Adney, Scott K.; Mahajan, Rahul

    2014-01-01

    The plasma membrane phosphoinositide phosphatidylinositol 4,5-bisphosphate (PIP2) controls the activity of most ion channels tested thus far through direct electrostatic interactions. Mutations in channel proteins that change their apparent affinity to PIP2 can lead to channelopathies. Given the fundamental role that membrane phosphoinositides play in regulating channel activity, it is surprising that only a small number of channelopathies have been linked to phosphoinositides. This review proposes that for channels whose activity is PIP2-dependent and for which mutations can lead to channelopathies, the possibility that the mutations alter channel-PIP2 interactions ought to be tested. Similarly, diseases that are linked to disorders of the phosphoinositide pathway result in altered PIP2 levels. In such cases, it is proposed that the possibility for a concomitant dysregulation of channel activity also ought to be tested. The ever-growing list of ion channels whose activity depends on interactions with PIP2 promises to provide a mechanism by which defects on either the channel protein or the phosphoinositide levels can lead to disease. PMID:20396900

  5. The use of alpha-1 adrenergic blockers in children with distal ureterolithiasis: a systematic review and meta-analysis

    PubMed Central

    Glina, F.P.; Castro, P.M.V.; Monteiro, G.G.R.; Guerra, G.C. Del; Glina, S.; Mazzurana, M.; Bernardo, W.M.

    2015-01-01

    ABSTRACT Introduction: Urinary lithiasis is the main urologic cause of emergency treatment in adult patient. In the past years, the incidence in children population has increased. However, literature about the use of alpha-1 adrenergic blockers in pediatric population with distal ureterolithiasis is still scarce. The drug acts by decreasing ureter contractions, especially in the distal portion, facilitating calculus expulsion. Objective: This review has the objective to evaluate the use of alpha-1 adrenergic blockers as medical expulsive treatment in children with distal ureterolithiasis. Evidence Acquisition: An electronic literature search was performed using the MEDLINE, COCHRANE, and LILACS databases. We further searched manually the references of the primary studies. Searches were concluded on October 4th, 2014. Articles were selected, independently and in pairs, by the respective titles and summaries. Any divergence was resolved by consensus. Evidence Synthesis: Alpha-1 adrenergic antagonists increased the probability of calculus expulsion by 27% (NNT=4). Calculi smaller than 5mm, increased by 33% (NNT=3). Larger than 5mm, increased by 34% (NNT=3). Conclusion: Alpha-1 adrenergic blocker use is related with a greater incidence of expulsion of ureteral calculi, smaller or greater than 5mm, and fewer episodes of pain when compared to ibuprofen. However it is necessary larger samples to enhance the power analysis of the expulsion of ureteral calculi larger than 5mm and the episodes of pain. Patient Summary: This review analyzed the outcome of alpha adrenergic antagonist in children with ureteral calculi. We conclude that it is the best medicine for use, since it helps the expulsion of the stone. PMID:26717117

  6. Nonlinear relationship between alpha 1-adrenergic receptor occupancy and norepinephrine-stimulated calcium flux in cultured vascular smooth muscle cells

    SciTech Connect

    Colucci, W.S.; Brock, T.A.; Gimbrone, M.A. Jr.; Alexander, R.W.

    1985-05-01

    To determine the relationship between vascular alpha 1-adrenergic receptor occupancy and receptor-coupled calcium flux, the authors have studied (/sup 3/H)prazosin binding and l-norepinephrine-induced /sup 45/Ca efflux in cultured vascular smooth muscle cells isolated from the rabbit aorta. In a crude cellular homogenate, (/sup 3/H)prazosin bound to a single high affinity site, whereas l-norepinephrine (NE) binding was best described by a two-site model. NE-stimulated /sup 45/Ca efflux was concentration-dependent (EC/sup 50/ = 108 nM) and potently inhibited by prazosin (IC/sup 50/ = 0.15 nM). For the total receptor pool identified by (/sup 3/H)prazosin binding, the relationship between receptor occupancy by NE and NE-stimulated /sup 45/Ca efflux was markedly nonlinear, such that 50% of maximum NE-stimulated efflux occurred with occupancy of only approximately 7% of receptors. These two experimental approaches provide direct evidence for the presence in cultured rabbit aortic smooth muscle cells of a sizable pool of alpha 1-adrenergic receptors in excess of those needed for maximum NE-stimulated /sup 45/Ca efflux. This evidence of ''spare'' receptors, together with the finding of two affinity states of agonist binding, raises the possibility of functional heterogeneity of alpha 1-adrenergic receptors in this system.

  7. Modeling the Effects of β1-Adrenergic Receptor Blockers and Polymorphisms on Cardiac Myocyte Ca2+ Handling

    PubMed Central

    Amanfu, Robert K.

    2014-01-01

    β-Adrenergic receptor blockers (β-blockers) are commonly used to treat heart failure, but the biologic mechanisms governing their efficacy are still poorly understood. The complexity of β-adrenergic signaling coupled with the influence of receptor polymorphisms makes it difficult to intuit the effect of β-blockers on cardiac physiology. While some studies indicate that β-blockers are efficacious by inhibiting β-adrenergic signaling, other studies suggest that they work by maintaining β-adrenergic responsiveness. Here, we use a systems pharmacology approach to test the hypothesis that in ventricular myocytes, these two apparently conflicting mechanisms for β-blocker efficacy can occur concurrently. We extended a computational model of the β1-adrenergic pathway and excitation-contraction coupling to include detailed receptor interactions for 19 ligands. Model predictions, validated with Ca2+ and Förster resonance energy transfer imaging of adult rat ventricular myocytes, surprisingly suggest that β-blockers can both inhibit and maintain signaling depending on the magnitude of receptor stimulation. The balance of inhibition and maintenance of β1-adrenergic signaling is predicted to depend on the specific β-blocker (with greater responsiveness for metoprolol than carvedilol) and β1-adrenergic receptor Arg389Gly polymorphisms. PMID:24867460

  8. Pharmacological Analysis and Structure Determination of 7-Methylcyanopindolol-Bound β1-Adrenergic Receptor.

    PubMed

    Sato, Tomomi; Baker, Jillian; Warne, Tony; Brown, Giles A; Leslie, Andrew G W; Congreve, Miles; Tate, Christopher G

    2015-12-01

    Comparisons between structures of the β1-adrenergic receptor (AR) bound to either agonists, partial agonists, or weak partial agonists led to the proposal that rotamer changes of Ser(5.46), coupled to a contraction of the binding pocket, are sufficient to increase the probability of receptor activation. (RS)-4-[3-(tert-butylamino)-2-hydroxypropoxy]-1H-indole-2-carbonitrile (cyanopindolol) is a weak partial agonist of β1AR and, based on the hypothesis above, we predicted that the addition of a methyl group to form 4-[(2S)-3-(tert-butylamino)-2-hydroxypropoxy]-7-methyl-1H-indole-2-carbonitrile (7-methylcyanopindolol) would dramatically reduce its efficacy. An eight-step synthesis of 7-methylcyanopindolol was developed and its pharmacology was analyzed. 7-Methylcyanopindolol bound with similar affinity to cyanopindolol to both β1AR and β2AR. As predicted, the efficacy of 7-methylcyanopindolol was reduced significantly compared with cyanopindolol, acting as a very weak partial agonist of turkey β1AR and an inverse agonist of human β2AR. The structure of 7-methylcyanopindolol-bound β1AR was determined to 2.4-Å resolution and found to be virtually identical to the structure of cyanopindolol-bound β1AR. The major differences in the orthosteric binding pocket are that it has expanded by 0.3 Å in 7-methylcyanopindolol-bound β1AR and the hydroxyl group of Ser(5.46) is positioned 0.8 Å further from the ligand, with respect to the position of the Ser(5.46) side chain in cyanopindolol-bound β1AR. Thus, the molecular basis for the reduction in efficacy of 7-methylcyanopindolol compared with cyanopindolol may be regarded as the opposite of the mechanism proposed for the increase in efficacy of agonists compared with antagonists. PMID:26385885

  9. Pharmacological Analysis and Structure Determination of 7-Methylcyanopindolol–Bound β1-Adrenergic Receptor

    PubMed Central

    Sato, Tomomi; Baker, Jillian; Warne, Tony; Brown, Giles A.; Leslie, Andrew G.W.; Congreve, Miles

    2015-01-01

    Comparisons between structures of the β1-adrenergic receptor (AR) bound to either agonists, partial agonists, or weak partial agonists led to the proposal that rotamer changes of Ser5.46, coupled to a contraction of the binding pocket, are sufficient to increase the probability of receptor activation. (RS)-4-[3-(tert-butylamino)-2-hydroxypropoxy]-1H-indole-2-carbonitrile (cyanopindolol) is a weak partial agonist of β1AR and, based on the hypothesis above, we predicted that the addition of a methyl group to form 4-[(2S)-3-(tert-butylamino)-2-hydroxypropoxy]-7-methyl-1H-indole-2-carbonitrile (7-methylcyanopindolol) would dramatically reduce its efficacy. An eight-step synthesis of 7-methylcyanopindolol was developed and its pharmacology was analyzed. 7-Methylcyanopindolol bound with similar affinity to cyanopindolol to both β1AR and β2AR. As predicted, the efficacy of 7-methylcyanopindolol was reduced significantly compared with cyanopindolol, acting as a very weak partial agonist of turkey β1AR and an inverse agonist of human β2AR. The structure of 7-methylcyanopindolol–bound β1AR was determined to 2.4-Å resolution and found to be virtually identical to the structure of cyanopindolol-bound β1AR. The major differences in the orthosteric binding pocket are that it has expanded by 0.3 Å in 7-methylcyanopindolol–bound β1AR and the hydroxyl group of Ser5.46 is positioned 0.8 Å further from the ligand, with respect to the position of the Ser5.46 side chain in cyanopindolol-bound β1AR. Thus, the molecular basis for the reduction in efficacy of 7-methylcyanopindolol compared with cyanopindolol may be regarded as the opposite of the mechanism proposed for the increase in efficacy of agonists compared with antagonists. PMID:26385885

  10. Estrogen alters the diurnal rhythm of alpha 1-adrenergic receptor densities in selected brain regions

    SciTech Connect

    Weiland, N.G.; Wise, P.M.

    1987-11-01

    Norepinephrine regulates the proestrous and estradiol-induced LH surge by binding to alpha 1-adrenergic receptors. The density of alpha 1-receptors may be regulated by estradiol, photoperiod, and noradrenergic neuronal activity. We wished to determine whether alpha 1-receptors exhibit a diurnal rhythm in ovariectomized and/or estradiol-treated female rats, whether estradiol regulates alpha 1-receptors in those areas of brain involved with LH secretion and/or sexual behavior, and whether the concentrations of alpha-receptors vary inversely relative to previously reported norepinephrine turnover patterns. Young female rats, maintained on a 14:10 light-dark cycle were ovariectomized. One week later, half of them were outfitted sc with Silastic capsules containing estradiol. Groups of animals were decapitated 2 days later at 0300, 1000, 1300, 1500, 1800, and 2300 h. Brains were removed, frozen, and sectioned at 20 micron. Sections were incubated with (/sup 3/H)prazosin in Tris-HCl buffer, washed, dried, and exposed to LKB Ultrofilm. The densities of alpha 1-receptors were quantitated using a computerized image analysis system. In ovariectomized rats, the density of alpha 1-receptors exhibited a diurnal rhythm in the suprachiasmatic nucleus (SCN), medial preoptic nucleus (MPN), and pineal gland. In SCN and MPN, receptor concentrations were lowest during the middle of the day and rose to peak levels at 1800 h. In the pineal gland, the density of alpha 1-receptors was lowest at middark phase, rose to peak levels before lights on, and remained elevated during the day. Estradiol suppressed the density of alpha 1 binding sites in the SCN, MPN, median eminence, ventromedial nucleus, and the pineal gland but had no effect on the lateral septum. Estrogen treatment altered the rhythm of receptor densities in MPN, median eminence, and the pineal gland.

  11. Backbone NMR reveals allosteric signal transduction networks in the β1-adrenergic receptor.

    PubMed

    Isogai, Shin; Deupi, Xavier; Opitz, Christian; Heydenreich, Franziska M; Tsai, Ching-Ju; Brueckner, Florian; Schertler, Gebhard F X; Veprintsev, Dmitry B; Grzesiek, Stephan

    2016-02-11

    G protein-coupled receptors (GPCRs) are physiologically important transmembrane signalling proteins that trigger intracellular responses upon binding of extracellular ligands. Despite recent breakthroughs in GPCR crystallography, the details of ligand-induced signal transduction are not well understood owing to missing dynamical information. In principle, such information can be provided by NMR, but so far only limited data of functional relevance on few side-chain sites of eukaryotic GPCRs have been obtained. Here we show that receptor motions can be followed at virtually any backbone site in a thermostabilized mutant of the turkey β1-adrenergic receptor (β1AR). Labelling with [(15)N]valine in a eukaryotic expression system provides over twenty resolved resonances that report on structure and dynamics in six ligand complexes and the apo form. The response to the various ligands is heterogeneous in the vicinity of the binding pocket, but gets transformed into a homogeneous readout at the intracellular side of helix 5 (TM5), which correlates linearly with ligand efficacy for the G protein pathway. The effect of several pertinent, thermostabilizing point mutations was assessed by reverting them to the native sequence. Whereas the response to ligands remains largely unchanged, binding of the G protein mimetic nanobody NB80 and G protein activation are only observed when two conserved tyrosines (Y227 and Y343) are restored. Binding of NB80 leads to very strong spectral changes throughout the receptor, including the extracellular ligand entrance pocket. This indicates that even the fully thermostabilized receptor undergoes activating motions in TM5, but that the fully active state is only reached in presence of Y227 and Y343 by stabilization with a G protein-like partner. The combined analysis of chemical shift changes from the point mutations and ligand responses identifies crucial connections in the allosteric activation pathway, and presents a general experimental

  12. Relationship between alpha 1-adrenergic receptor occupancy and response in BC3H-1 muscle cells

    SciTech Connect

    Brown, R.D.; Berger, K.D.; Taylor, P.

    1987-07-01

    The relationship between alpha 1-adrenergic receptor occupancy by agonists or antagonists and the regulation of intracellular Ca/sup 2 +/ was examined. Receptor occupancy was measured using the antagonist (/sup 3/H)prazosin and correlated with agonist-elicited /sup 45/Ca/sup 2 +/ fluxes. The agonists epinephrine (E), norepinephrine (NE), and phenylephrine (PE) coordinately activated Ca/sup 2 +/ efflux, reflecting a substantial mobilization of intracellular Ca/sup 2 +/, as well as a smaller /sup 45/Ca/sup 2 +/ influx. The agonist concentration dependences for influx and efflux were similar, with the order of potency expected for alpha 1 receptors (E greater than or equal to NE greater than PE). To determine the relationship between receptor occupancy and response, the slowly dissociating antagonist prazosin was used to inactivate specified fractions of the receptor population. A linear relationship was observed between the remaining activatable receptors and residual /sup 45/Ca/sup 2 +/ efflux elicited by E or NE, except at saturating agonist concentrations where some curvature was observed. Moreover, the concentration dependence for agonist-elicited /sup 45/Ca/sup 2 +/ efflux was shifted toward slightly higher concentrations of E or NE following prazosin inactivation. These results suggest the presence of a modest receptor reserve which is revealed by E or NE, but not by PE. Agonist occupation was measured over the same interval as receptor activation by competition with the initial rate of (/sup 3/H)prazosin association. All three agonists exhibited the major fraction of receptor occupation over the same concentration ranges required for the functional response. Exposure of receptors to specified agonist concentrations for 30 min had little effect on the number of receptors or their ligand affinities, whereas a 2.5-hr exposure to agonist decreased apparent agonist affinity as well as the number of receptors recognized by (/sup 3/H)prazosin.

  13. Alpha-1-Adrenergic Receptor Subtypes in Non-Failing and Failing Human Myocardium

    PubMed Central

    Jensen, Brian C.; Swigart, Philip M; DeMarco, Teresa; Hoopes, Charles; Simpson, Paul C.

    2009-01-01

    Background Alpha-1-adrenergic receptors (α1-ARs) play adaptive roles in the heart and protect against the development of heart failure (HF). The three α1-AR subtypes,α1A, α1B, and α1D, have distinct physiological roles in mouse heart, but very little is known about α1-subtypes in human heart. Here we test the hypothesis that the α1A and α1B subtypes are present in human myocardium, similar to the mouse, and are not down-regulated in heart failure. Methods and Results Hearts from transplant recipients and unused donors were failing (n = 12; mean EF 24%) or non-failing (n = 9; mean EF 59%), and similar in age (~44 years) and sex (~70% male). We measured the α1-AR subtypes in multiple regions of both ventricles by quantitative real-time reverse transcription PCR and radioligand binding. All three α1-AR subtype mRNAs were present, and α1A mRNA was most abundant (~65% of total α1-AR mRNA). However, only α1A and α1B binding were present, and the α1B was most abundant (60% of total). In failing hearts, α1A and α1B binding were not down-regulated, in contrast with β1-ARs. Conclusions Our data show for the first time that the α1A and α1B subtypes are both present in human myocardium, but α1D binding is not, and that the α1-subtypes are not down-regulated in HF. Since α1-subtypes in the human heart are similar to mouse, where adaptive and protective effects of α1-subtypes are most convincing, it might become feasible to treat HF with a drug targeting the α1A and/or α1B. PMID:19919991

  14. Solubilization and purification of the alpha 1-adrenergic receptor using a novel affinity resin.

    PubMed Central

    Graham, R M; Hess, H J; Homcy, C J

    1982-01-01

    The highly selective alpha 1-adrenergic receptor antagonist prazosin was used to identify binding sites having alpha-adrenergic specificity in rat hepatic plasma membranes. Solubilization of the membrane-bound receptors was achieved by incubation with the nonionic detergent digitonin, and binding activity was assayed by using [3H]prazosin and a polyethylene glycol precipitation technique. Only 20-30% of the total receptor pool was released by the solubilization procedure. However, binding of [3H]prazosin was saturable [maximal value, 206 +/- 8 fmol/mg of protein (membrane) vs. 74 +/- 4 fmol/mg of protein (soluble)] and of high affinity [Kd, 0.6 +/- 0.2 nM (membrane) vs. 0.8 +/- 0.2 nM (soluble)]. To aid in purification of the receptors, an affinity resin was developed using an analog of prazosin, 2-(4-succinoylpiperazin-1-yl)-4-amino-6,7-dimethoxyquinazoline (CP 57,609; Kd 2.7 X 10(-7) M) immobilized via an amide linkage to agarose. The resulting resin demonstrated high affinity (Kd 3.2 X 10(-7) M) for the solubilized receptors, as determined by competitive inhibition assay. The degree of substitution to the resin was determined by a direct radioimmunoassay using antibodies against albumin-complexed CP 57,609 and found to be 0.1 to 0.2 mumol/ml of agarose. Affinity chromatography using the resin resulted in 513-fold purification in a single step. Moreover, the specificity of the purified binding sites was similar to that of membrane-bound receptors. This novel affinity resin should thus provide a powerful tool for isolating the receptor protein in quantities sufficient for detailed biochemical characterization. PMID:6285370

  15. Role of phosphoinositides at the neuronal synapse

    PubMed Central

    Frere, Samuel G.; Chang-Ileto, Belle; Di Paolo, Gilbert

    2013-01-01

    Synaptic transmission is amongst the most sophisticated and tightly controlled biological phenomena in higher eukaryotes. In the past few decades, tremendous progress has been made in our understanding of the molecular mechanisms underlying multiple facets of neurotransmission, both pre- and postsynaptically. Brought under the spotlight by pioneer studies in the areas of secretion and signal transduction, phosphoinositides and their metabolizing enzymes have been increasingly recognized as key protagonists in fundamental aspects of neurotransmission. Not surprisingly, dysregulation of phosphoinositide metabolism has also been implicated in synaptic malfunction associated with a variety of brain disorders. In the present chapter, we summarize current knowledge on the role of phosphoinositides at the neuronal synapse and highlight some of the outstanding questions in this research field. PMID:22374090

  16. Phosphoinositide Control of Membrane Protein Function

    PubMed Central

    Logothetis, Diomedes E.; Petrou, Vasileios I.; Zhang, Miao; Mahajan, Rahul; Meng, Xuan-Yu; Adney, Scott K.; Cui, Meng; Baki, Lia

    2015-01-01

    Anionic phospholipids are critical constituents of the inner leaflet of the plasma membrane, ensuring appropriate membrane topology of transmembrane proteins. Additionally, in eukaryotes, the negatively charged phosphoinositides serve as key signals not only through their hydrolysis products but also through direct control of transmembrane protein function. Direct phosphoinositide control of the activity of ion channels and transporters has been the most convincing case of the critical importance of phospholipid-protein interactions in the functional control of membrane proteins. Furthermore, second messengers, such as [Ca2+]i, or posttranslational modifications, such as phosphorylation, can directly or allosterically fine-tune phospholipid-protein interactions and modulate activity. Recent advances in structure determination of membrane proteins have allowed investigators to obtain complexes of ion channels with phosphoinositides and to use computational and experimental approaches to probe the dynamic mechanisms by which lipid-protein interactions control active and inactive protein states. PMID:25293526

  17. Cell Permeable Ratiometric Fluorescent Sensors for Imaging Phosphoinositides.

    PubMed

    Mondal, Samsuzzoha; Rakshit, Ananya; Pal, Suranjana; Datta, Ankona

    2016-07-15

    Phosphoinositides are critical cell-signal mediators present on the plasma membrane. The dynamic change of phosphoinositide concentrations on the membrane including clustering and declustering mediates signal transduction. The importance of phosphoinositides is scored by the fact that they participate in almost all cell-signaling events, and a defect in phosphoinositide metabolism is linked to multiple diseases including cancer, bipolar disorder, and type-2 diabetes. Optical sensors for visualizing phosphoinositide distribution can provide information on phosphoinositide dynamics. This exercise will ultimately afford a handle into understanding and manipulating cell-signaling processes. The major requirement in phosphoinositide sensor development is a selective, cell permeable probe that can quantify phosphoinositides. To address this requirement, we have developed short peptide-based ratiometric fluorescent sensors for imaging phosphoinositides. The sensors afford a selective response toward two crucial signaling phosphoinositides, phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) and phosphatidylinositol-4-phosphate (PI4P), over other anionic membrane phospholipids and soluble inositol phosphates. Dissociation constant values indicate up to 4 times higher probe affinity toward PI(4,5)P2 when compared to PI4P. Significantly, the sensors are readily cell-permeable and enter cells within 15 min of incubation as indicated by multiphoton excitation confocal microscopy. Furthermore, the sensors light up signaling phosphoinositides present both on the cell membrane and on organelle membranes near the perinuclear space, opening avenues for quantifying and monitoring phosphoinositide signaling. PMID:27082310

  18. Rat beta 1-adrenergic receptor regulatory region containing consensus AP-2 elements recognizes novel transactivator proteins.

    PubMed

    Kirigiti, P; Yang, Y F; Li, X; Li, B; Midson, C N; Machida, C A

    2000-03-01

    beta 1-Adrenergic receptors (beta1-ARs) serve as important regulators of central nervous system (CNS)-mediated behavior and several neural functions, including mood, memory, neuroendocrine control, and stimulation of autonomic function. Using beta 1-AR-luciferase reporter recombinants, we have previously determined that important beta 1-AR genetic elements controlling expression within the C6 glioma cell line are contained within the region -396 to -299, relative to the translational start site. By conducting progressive internal deletions of the rat beta 1-AR 5' flanking region and with the use of beta 1-AR-luciferase recombinants, we have verified that this region contains the primary beta 1-AR promoter and/or major regulatory elements. To begin the identification of protein factors involved in beta 1-AR transcriptional activity conferred by this beta 1-AR region and flanking sequences, we conducted electrophoretic mobility shift assays using defined beta 1-AR DNA subregion probes. One probe (GS-1), encompassing the region -396 to -367, was found to produce two major and two minor mobility shift complexes when bound to nuclear extracts from the beta 1-AR expresser C6 cell line. UV-crosslinking of DNA-protein complexes, coupled with DNase I digestion, indicated that this beta 1-AR region interacts with one major protein of approximately 117 kDa molecular weight and additional minor proteins. GS-1 DNA-protein complexes were observed using beta 1-AR expresser tissues in the CNS, including cortex, hippocampus, and olfactory bulb. No DNA-protein complexes were observed when using nuclear extracts from beta 1-AR nonexpresser tissues; in some cases, using L6 cells, previously characterized to express little or no beta1-ARs, a reduction in intensities of the DNA-protein complexes was observed. Competition experiments indicate that nuclear protein binds to one of two subregions within the GS-1 sequence that contain AP-2-like consensus elements. Recombinant AP-2 protein

  19. Prostaglandin F/sub 2. cap alpha. activates phosphoinositide hydrolysis in rat aorta

    SciTech Connect

    Not Available

    1986-03-01

    The authors have previously demonstrated that norepinephrine (NE) and serotonin (5HT) activate a phosphoinositide-(PI) specific phospholipase C in rat aorta by interaction with ..cap alpha../sub 1/-adrenergic receptors and 5HT/sub 2/ receptor, respectively. They have subsequently noted that angiotensin II and vasopressin as well activate PI hydrolysis in the tissue. The most active agent they have thus far investigated is prostaglandin F/sub 2..cap alpha../ (PGF/sub 2..cap alpha../). Rat aortic rings were pre-labelled with (/sup 3/H)-inositol and then, in the presence of 10 mM LiCl, exposed to various doses of PGF/sub 2..cap alpha../. (/sup 3/H)-inositol monophosphate was the quantified by anion-exchange chromatography. After a 60 min incubation, PGF/sub 2..cap alpha../ caused a 10-15 fold increase over basal at maximal concentrations (0.1-1.0 mM). An EC/sub 50/ for PI hydrolysis was between 0.1-1.0 ..mu..M. PGF/sub 2..cap alpha../ caused maximal aortic contraction at 10 ..mu..M. PGF/sub 2..cap alpha../-induced PI hydrolysis, was inhibited by phorbol esters. These results suggest that PGF/sub 2..cap alpha../, similar to 5HT, NE, vasopressin and angiotensin II, causes vasoconstriction by activation of PI hydrolysis.

  20. Physical Foundations of PTEN/Phosphoinositide Interaction

    NASA Astrophysics Data System (ADS)

    Gericke, Arne; Jiang, Zhiping; Redfern, Roberta E.; Kooijman, Edgar E.; Ross, Alonzo H.

    2009-03-01

    Phosphoinositides act as signaling molecules by recruiting critical effectors to specific subcellular membranes to regulate cell proliferation, apoptosis and cytoskeletal reorganization, which requires a tight regulation of phosphoinositide generation and turnover as well as a high degree of compartmentalization. PTEN is a phosphatase specific for the 3 position of the phosophoinositide ring that is deleted or mutated in many different disease states. PTEN association with membranes requires the interaction of its C2 domain with phosphatidylserine and the interaction of its N-terminal end with phosphatidylinositol-4,5-bisphophate (PI(4,5)P2). We have investigated PTEN/PI(4,5)P2 interaction and found that Lys13 is crucial for the observed binding. We also found that the presence of cholesterol enhances PTEN binding to mixed PI(4,5)P2/POPC vesicles. Fluorescence microscopy experiments utilizing GUVs yielded results consistent with enhanced phosphoinositide domain formation in the presence of cholesterol. These experiments were accompanied by zeta potential measurements and solid state MAS ^31P-NMR experiments aimed at investigating the ionization behavior of phosphoinositides.

  1. Cerebellar vermis H₂ receptors mediate fear memory consolidation in mice.

    PubMed

    Gianlorenço, A C L; Riboldi, A M; Silva-Marques, B; Mattioli, R

    2015-02-01

    Histaminergic fibers are present in the molecular and granular layers of the cerebellum and have a high density in the vermis and flocullus. Evidence supports that the cerebellar histaminergic system is involved in memory consolidation. Our recent study showed that histamine injections facilitate the retention of an inhibitory avoidance task, which was abolished by pretreatment with an H2 receptor antagonist. In the present study, we investigated the effects of intracerebellar post training injections of H1 and H2 receptor antagonists as well as the selective H2 receptor agonist on fear memory consolidation. The cerebellar vermi of male mice were implanted with guide cannulae, and after three days of recovery, the inhibitory avoidance test was performed. Immediately after a training session, animals received a microinjection of the following histaminergic drugs: experiment 1, saline or chlorpheniramine (0.016, 0.052 or 0.16 nmol); experiment 2, saline or ranitidine (0.57, 2.85 or 5.07 nmol); and experiment 3, saline or dimaprit (1, 2 or 4 nmol). Twenty-four hours later, a retention test was performed. The data were analyzed using one-way analysis of variance (ANOVA) and Duncan's tests. Animals microinjected with chlorpheniramine did not show any behavioral effects at the doses that we used. Intra-cerebellar injection of the H2 receptor antagonist ranitidine inhibited, while the selective H2 receptor agonist dimaprit facilitated, memory consolidation, suggesting that H2 receptors mediate memory consolidation in the inhibitory avoidance task in mice. PMID:25524412

  2. Hemoglobin Uptake by Paracoccidioides spp. Is Receptor-Mediated

    PubMed Central

    Bailão, Elisa Flávia Luiz Cardoso; Parente, Juliana Alves; Pigosso, Laurine Lacerda; de Castro, Kelly Pacheco; Fonseca, Fernanda Lopes; Silva-Bailão, Mirelle Garcia; Báo, Sônia Nair; Bailão, Alexandre Melo; Rodrigues, Marcio L.; Hernandez, Orville; McEwen, Juan G.; Soares, Célia Maria de Almeida

    2014-01-01

    Iron is essential for the proliferation of fungal pathogens during infection. The availability of iron is limited due to its association with host proteins. Fungal pathogens have evolved different mechanisms to acquire iron from host; however, little is known regarding how Paracoccidioides species incorporate and metabolize this ion. In this work, host iron sources that are used by Paracoccidioides spp. were investigated. Robust fungal growth in the presence of the iron-containing molecules hemin and hemoglobin was observed. Paracoccidioides spp. present hemolytic activity and have the ability to internalize a protoporphyrin ring. Using real-time PCR and nanoUPLC-MSE proteomic approaches, fungal growth in the presence of hemoglobin was shown to result in the positive regulation of transcripts that encode putative hemoglobin receptors, in addition to the induction of proteins that are required for amino acid metabolism and vacuolar protein degradation. In fact, one hemoglobin receptor ortholog, Rbt5, was identified as a surface GPI-anchored protein that recognized hemin, protoporphyrin and hemoglobin in vitro. Antisense RNA technology and Agrobacterium tumefaciens-mediated transformation were used to generate mitotically stable Pbrbt5 mutants. The knockdown strain had a lower survival inside macrophages and in mouse spleen when compared with the parental strain, which suggested that Rbt5 could act as a virulence factor. In summary, our data indicate that Paracoccidioides spp. can use hemoglobin as an iron source most likely through receptor-mediated pathways that might be relevant for pathogenic mechanisms. PMID:24831516

  3. Receptor-mediated endocytosis and brain delivery of therapeutic biologics.

    PubMed

    Xiao, Guangqing; Gan, Liang-Shang

    2013-01-01

    Transport of macromolecules across the blood-brain-barrier (BBB) requires both specific and nonspecific interactions between macromolecules and proteins/receptors expressed on the luminal and/or the abluminal surfaces of the brain capillary endothelial cells. Endocytosis and transcytosis play important roles in the distribution of macromolecules. Due to the tight junction of BBB, brain delivery of traditional therapeutic proteins with large molecular weight is generally not possible. There are multiple pathways through which macromolecules can be taken up into cells through both specific and nonspecific interactions with proteins/receptors on the cell surface. This review is focused on the current knowledge of receptor-mediated endocytosis/transcytosis and brain delivery using the Angiopep-2-conjugated system and the molecular Trojan horses. In addition, the role of neonatal Fc receptor (FcRn) in regulating the efflux of Immunoglobulin G (IgG) from brain to blood, and approaches to improve the pharmacokinetics of therapeutic biologics by generating Fc fusion proteins, and increasing the pH dependent binding affinity between Fc and FcRn, are discussed. PMID:23840214

  4. H-Ras regulation of TRAIL death receptor mediated apoptosis

    PubMed Central

    Chen, Jun-Jie; Bozza, William P.; Di, Xu; Zhang, Yaqin; Hallett, William; Zhang, Baolin

    2014-01-01

    TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis through the death receptors (DRs) 4 and/or 5 expressed on the cell surface. Multiple clinical trials are underway to evaluate the antitumor activity of recombinant human TRAIL and agonistic antibodies to DR4 or DR5. However, their therapeutic potential is limited by the high frequency of cancer resistance. Here we provide evidence demonstrating the role of H-Ras in TRAIL receptor mediated apoptosis. By analyzing the genome wide mRNA expression data of the NCI60 cancer cell lines, we found that H-Ras expression was consistently upregulated in TRAIL-resistant cell lines. By contrast, no correlation was found between TRAIL sensitivity and K-Ras expression levels or their mutational profiles. Notably, H-Ras upregulation associated with a surface deficiency of TRAIL death receptors. Selective inhibition of H-Ras activity in TRAIL-resistant cells restored the surface expression of both DR4 and DR5 without changing their total protein levels. The resulting cells became highly susceptible to both TRAIL and agonistic DR5 antibody, whereas K-Ras inhibition had little or no effect on TRAIL-induced apoptosis, indicating H-Ras plays a distinct role in the regulation of TRAIL death receptors. Further studies are warranted to determine the therapeutic potential of H-Ras-specific inhibitors in combination with TRAIL receptor agonists. PMID:25026275

  5. [Glutamate receptor-mediated retinal neuronal injury in experimental glaucoma].

    PubMed

    Wang, Zhong-Feng; Yang, Xiong-Li

    2016-08-25

    Glaucoma, the second leading cause of blindness, is a neurodegenerative disease characterized by optic nerve degeneration related to apoptotic death of retinal ganglion cells (RGCs). In the pathogenesis of RGC death following the onset of glaucoma, functional changes of glutamate receptors are commonly regarded as important risk factors. During the past several years, we have explored the mechanisms underlying RGC apoptosis and retinal Müller cell reactivation (gliosis) in a rat chronic ocular hypertension (COH) model. We demonstrated that elevated intraocular pressure in COH rats may induce changes of various signaling pathways, which are involved in RGC apoptosis by modulating glutamate NMDA and AMPA receptors. Moreover, we also demonstrated that over-activation of group I metabotropic glutamate receptors (mGluR I) by excessive extracellular glutamate in COH rats could contribute to Müller cell gliosis by suppressing Kir4.1 channels. In this review, incorporating our results, we discuss glutamate receptor- mediated RGC apoptosis and Müller cell gliosis in experimental glaucoma. PMID:27546508

  6. Receptor-Mediated Endocytosis and Brain Delivery of Therapeutic Biologics

    PubMed Central

    Xiao, Guangqing

    2013-01-01

    Transport of macromolecules across the blood-brain-barrier (BBB) requires both specific and nonspecific interactions between macromolecules and proteins/receptors expressed on the luminal and/or the abluminal surfaces of the brain capillary endothelial cells. Endocytosis and transcytosis play important roles in the distribution of macromolecules. Due to the tight junction of BBB, brain delivery of traditional therapeutic proteins with large molecular weight is generally not possible. There are multiple pathways through which macromolecules can be taken up into cells through both specific and nonspecific interactions with proteins/receptors on the cell surface. This review is focused on the current knowledge of receptor-mediated endocytosis/transcytosis and brain delivery using the Angiopep-2-conjugated system and the molecular Trojan horses. In addition, the role of neonatal Fc receptor (FcRn) in regulating the efflux of Immunoglobulin G (IgG) from brain to blood, and approaches to improve the pharmacokinetics of therapeutic biologics by generating Fc fusion proteins, and increasing the pH dependent binding affinity between Fc and FcRn, are discussed. PMID:23840214

  7. Targeting receptor-mediated endocytotic pathways with nanoparticles: rationale and advances

    PubMed Central

    Xu, Shi; Olenyuk, Bogdan Z.; Okamoto, Curtis T.; Hamm-Alvarez, Sarah F.

    2012-01-01

    Targeting of drugs and their carrier systems by using receptor-mediated endocytotic pathways was in its nascent stages 25 years ago. In the intervening years, an explosion of knowledge focused on design and synthesis of nanoparticulate delivery systems as well as elucidation of the cellular complexity of what was previously-termed receptor-mediated endocytosis has now created a situation when it has become possible to design and test the feasibility of delivery of highly specific nanoparticle drug carriers to specific cells and tissue. This review outlines the mechanisms governing the major modes of receptor-mediated endocytosis used in drug delivery and highlights recent approaches using these as targets for in vivo drug delivery of nanoparticles. The review also discusses some of the inherent complexity associated with the simple shift from a ligand-drug conjugate versus a ligand-nanoparticle conjugate, in terms of ligand valency and its relationship to the mode of receptor-mediated internalization. PMID:23026636

  8. Bombesin receptor-mediated imaging and cytotoxicity: review and current status

    PubMed Central

    Sancho, Veronica; Di Florio, Alessia; Moody, Terry W.; Jensen, Robert T.

    2010-01-01

    The three mammalian bombesin (Bn) receptors (gastrin-releasing peptide [GRP] receptor, neuromedin B [NMB] receptor, BRS-3) are one of the classes of G protein-coupled receptors that are most frequently over-express/ectopically expressed by common, important malignancies. Because of the clinical success of somatostatin receptor-mediated imaging and cytotoxicity with neuroendocrine tumors, there is now increasing interest in pursuing a similar approach with Bn receptors. In the last few years then have been more than 200 studies in this area. In the present paper, the in vitro and in vivo results, as well as results of human studies from many of these studies are reviewed and the current state of Bn receptor-mediated imaging or cytotoxicity is discussed. Both Bn receptor-mediated imaging studies as well as Bn receptor-mediated tumoral cytotoxic studies using radioactive and non-radioactive Bn-based ligands are covered. PMID:21034419

  9. Exercise training normalizes renal blood flow responses to acute hypoxia in experimental heart failure: role of the α1-adrenergic receptor.

    PubMed

    Pügge, Carolin; Mediratta, Jai; Marcus, Noah J; Schultz, Harold D; Schiller, Alicia M; Zucker, Irving H

    2016-02-01

    Recent data suggest that exercise training (ExT) is beneficial in chronic heart failure (CHF) because it improves autonomic and peripheral vascular function. In this study, we hypothesized that ExT in the CHF state ameliorates the renal vasoconstrictor responses to hypoxia and that this beneficial effect is mediated by changes in α1-adrenergic receptor activation. CHF was induced in rabbits. Renal blood flow (RBF) and renal vascular conductance (RVC) responses to 6 min of 5% isocapnic hypoxia were assessed in the conscious state in sedentary (SED) and ExT rabbits with CHF with and without α1-adrenergic blockade. α1-adrenergic receptor expression in the kidney cortex was also evaluated. A significant decline in baseline RBF and RVC and an exaggerated renal vasoconstriction during acute hypoxia occurred in CHF-SED rabbits compared with the prepaced state (P < 0.05). ExT diminished the decline in baseline RBF and RVC and restored changes during hypoxia to those of the prepaced state. α1-adrenergic blockade partially prevented the decline in RBF and RVC in CHF-SED rabbits and eliminated the differences in hypoxia responses between SED and ExT animals. Unilateral renal denervation (DnX) blocked the hypoxia-induced renal vasoconstriction in CHF-SED rabbits. α1-adrenergic protein in the renal cortex of animals with CHF was increased in SED animals and normalized after ExT. These data provide evidence that the acute decline in RBF during hypoxia is caused entirely by the renal nerves but is only partially mediated by α1-adrenergic receptors. Nonetheless, α1-adrenergic receptors play an important role in the beneficial effects of ExT in the kidney. PMID:26607245

  10. Dopamine receptor-mediated regulation of neuronal "clock" gene expression.

    PubMed

    Imbesi, M; Yildiz, S; Dirim Arslan, A; Sharma, R; Manev, H; Uz, T

    2009-01-23

    Using a transgenic mice model (i.e. "clock" knockouts), clock transcription factors have been suggested as critical regulators of dopaminergic behaviors induced by drugs of abuse. Moreover, it has been shown that systemic administration of psychostimulants, such as cocaine and methamphetamine regulates the striatal expression of clock genes. However, it is not known whether dopamine receptors mediate these regulatory effects of psychostimulants at the cellular level. Primary striatal neurons in culture express dopamine receptors as well as clock genes and have been successfully used in studying dopamine receptor functioning. Therefore, we investigated the role of dopamine receptors on neuronal clock gene expression in this model using specific receptor agonists. We found an inhibitory effect on the expression of mClock and mPer1 genes with the D2-class (i.e. D2/D3) receptor agonist quinpirole. We also found a generalized stimulatory effect on the expression of clock genes mPer1, mClock, mNPAS2 (neuronal PAS domain protein 2), and mBmal1 with the D1-class (i.e. D1) receptor agonist SKF38393. Further, we tested whether systemic administration of dopamine receptor agonists causes similar changes in striatal clock gene expression in vivo. We found quinpirole-induced alterations in mPER1 protein levels in the mouse striatum (i.e. rhythm shift). Collectively, our results indicate that the dopamine receptor system may mediate psychostimulant-induced changes in clock gene expression. Using striatal neurons in culture as a model, further research is needed to better understand how dopamine signaling modulates the expression dynamics of clock genes (i.e. intracellular signaling pathways) and thereby influences neuronal gene expression, neuronal transmission, and brain functioning. PMID:19017537

  11. Association between β1 adrenergic receptor gene Arg389Gly polymorphism and risk of heart failure: a meta-analysis.

    PubMed

    Ma, S T; Zhao, W; Liu, B; Jia, R Y; Zhao, C J; Cui, L Q

    2015-01-01

    Numerous studies have evaluated the association between Arg389Gly polymorphism in the β1 adrenergic receptor gene and heart failure risk. However, the specific association is still controversial. We performed a meta-analysis of all case-control studies that evaluated the association between Arg389Gly polymorphism and heart failure in humans. Studies were identified in the PubMed, Embase, and China National Knowledge Infrastructure databases. Two reviewers independently assessed the studies. Six case-control studies with a total of 1736 participants were included in the meta-analysis, including 882 cases with heart failure and 854 controls, and our results showed no association between the Arg389Gly polymorphism and heart failure [ArgArg vs GlyGly: odds ratio (OR) = 0.84, 95% confidence interval (CI) 0.59-1.20; ArgArg vs ArgGly: OR = 0.95, 95%CI 0.78-1.16; dominant model: OR = 1.08, 95%CI 0.89-1.31; recessive model: OR = 0.96, 95%CI 0.69-1.35]. No publication bias was found in the present study (all P values > 0.05). In conclusion, the β1 adrenergic receptor gene Arg389Gly polymorphism might not be associated with heart failure risk. Further large and well-designed studies are needed to confirm this conclusion. PMID:26125791

  12. The α1 Adrenergic Receptor Antagonist Prazosin Reduces Heroin Self-Administration in Rats with Extended Access to Heroin Administration

    PubMed Central

    Greenwell, Thomas N.; Walker, Brendan M.; Cottone, Pietro; Zorrilla, Eric P.; Koob, George F.

    2009-01-01

    Previous studies have reported that noradrenergic antagonists alleviate some of the symptoms of opiate withdrawal and dependence. Clinical studies also have shown that modification of the noradrenergic system may help protect patients from relapse. The present study tested the hypothesis that a dysregulated noradrenergic system has motivational significance in heroin self-administration in dependent rats. Prazosin, an α1-adrenergic antagonist (0.5, 1.0, 1.5 and 2.0 mg/kg, i.p.), was administered to adult male Wistar rats with a history of limited (1 h/day; short access) or extended (12 h/day; long access) access to intravenous heroin self-administration. Prazosin dose-dependently reduced heroin self-administration in long-access rats but not short-access rats, with 2 mg/kg of systemic prazosin significantly decreasing 1 h and 2 h heroin intake. Prazosin also reversed some changes in meal pattern associated with extended heroin access, including the taking of smaller and briefer meals (at 3 h), while also increasing total food intake and slowing the eating rate within meals (both 3 h and 12 h). The data show that the α1-adrenergic system may contribute to mechanisms that promote dependence in rats with extended drug access, while also stimulating their food intake by restoring meals to the normal size and duration. PMID:18703080

  13. Antagonism of Nav channels and α1-adrenergic receptors contributes to vascular smooth muscle effects of ranolazine.

    PubMed

    Virsolvy, Anne; Farah, Charlotte; Pertuit, Nolwenn; Kong, Lingyan; Lacampagne, Alain; Reboul, Cyril; Aimond, Franck; Richard, Sylvain

    2015-01-01

    Ranolazine is a recently developed drug used for the treatment of patients with chronic stable angina. It is a selective inhibitor of the persistent cardiac Na(+) current (INa), and is known to reduce the Na(+)-dependent Ca(2+) overload that occurs in cardiomyocytes during ischemia. Vascular effects of ranolazine, such as vasorelaxation,have been reported and may involve multiple pathways. As voltage-gated Na(+) channels (Nav) present in arteries play a role in contraction, we hypothesized that ranolazine could target these channels. We studied the effects of ranolazine in vitro on cultured aortic smooth muscle cells (SMC) and ex vivo on rat aortas in conditions known to specifically activate or promote INa. We observed that in the presence of the Nav channel agonist veratridine, ranolazine inhibited INa and intracellular Ca(2+) calcium increase in SMC, and arterial vasoconstriction. In arterial SMC, ranolazine inhibited the activity of tetrodotoxin-sensitive voltage-gated Nav channels and thus antagonized contraction promoted by low KCl depolarization. Furthermore, the vasorelaxant effects of ranolazine, also observed in human arteries and independent of the endothelium, involved antagonization of the α1-adrenergic receptor. Combined α1-adrenergic antagonization and inhibition of SMCs Nav channels could be involved in the vascular effects of ranolazine. PMID:26655634

  14. Antagonism of Nav channels and α1-adrenergic receptors contributes to vascular smooth muscle effects of ranolazine

    PubMed Central

    Virsolvy, Anne; Farah, Charlotte; Pertuit, Nolwenn; Kong, Lingyan; Lacampagne, Alain; Reboul, Cyril; Aimond, Franck; Richard, Sylvain

    2015-01-01

    Ranolazine is a recently developed drug used for the treatment of patients with chronic stable angina. It is a selective inhibitor of the persistent cardiac Na+ current (INa), and is known to reduce the Na+-dependent Ca2+ overload that occurs in cardiomyocytes during ischemia. Vascular effects of ranolazine, such as vasorelaxation,have been reported and may involve multiple pathways. As voltage-gated Na+ channels (Nav) present in arteries play a role in contraction, we hypothesized that ranolazine could target these channels. We studied the effects of ranolazine in vitro on cultured aortic smooth muscle cells (SMC) and ex vivo on rat aortas in conditions known to specifically activate or promote INa. We observed that in the presence of the Nav channel agonist veratridine, ranolazine inhibited INa and intracellular Ca2+ calcium increase in SMC, and arterial vasoconstriction. In arterial SMC, ranolazine inhibited the activity of tetrodotoxin-sensitive voltage-gated Nav channels and thus antagonized contraction promoted by low KCl depolarization. Furthermore, the vasorelaxant effects of ranolazine, also observed in human arteries and independent of the endothelium, involved antagonization of the α1-adrenergic receptor. Combined α1-adrenergic antagonization and inhibition of SMCs Nav channels could be involved in the vascular effects of ranolazine. PMID:26655634

  15. Receptor-mediated inhibition of adenylate cyclase and stimulation of arachidonic acid release in 3T3 fibroblasts. Selective susceptibility to islet-activating protein, pertussis toxin

    SciTech Connect

    Murayama, T.; Ui, M.

    1985-06-25

    Thrombin exhibited diverse effects on mouse 3T3 fibroblasts. It (a) decreased cAMP in the cell suspension, (b) inhibited adenylate cyclase in the Lubrol-permeabilized cell suspension in a GTP-dependent manner, increased releases of (c) arachidonic acid and (d) inositol from the cell monolayer prelabeled with these labeled compounds, (e) increased /sup 45/Ca/sup 2 +/ uptake into the cell monolayer, and (f) increased /sup 86/Rb/sup +/ uptake into the cell monolayer in a ouabain-sensitive manner. Most of the effects were reproduced by bradykinin, platelet-activating factor, and angiotensin II. The receptors for these agonists are thus likely to be linked to three separate effector systems: the adenylate cyclase inhibition, the phosphoinositide breakdown leading to Ca/sup 2 +/ mobilization and phospholipase A2 activation, and the Na,K-ATPase activation. Among the effects of these agonists, (a), (b), (c), and (e) were abolished, but (d) and (f) were not, by prior treatment of the cells with islet-activating protein (IAP), pertussis toxin, which ADP-ribosylates the Mr = 41,000 protein, the alpha-subunit of the inhibitory guanine nucleotide regulatory protein (Ni), thereby abolishing receptor-mediated inhibition of adenylate cyclase. The effects (a), (c), (d), and (e) of thrombin, but not (b), were mimicked by A23187, a calcium ionophore. The effects of A23187, in contrast to those of receptor agonists, were not affected by the treatment of cells with IAP. Thus, the IAP substrate, the alpha-subunit of Ni, or the protein alike, may play an additional role in signal transduction arising from the Ca/sup 2 +/-mobilizing receptors, probably mediating process(es) distal to phosphoinositide breakdown and proximal to Ca/sup 2 +/ gating.

  16. NMDA receptors mediate calcium accumulation in myelin during chemical ischaemia.

    PubMed

    Micu, I; Jiang, Q; Coderre, E; Ridsdale, A; Zhang, L; Woulfe, J; Yin, X; Trapp, B D; McRory, J E; Rehak, R; Zamponi, G W; Wang, W; Stys, P K

    2006-02-23

    Central nervous system myelin is a specialized structure produced by oligodendrocytes that ensheaths axons, allowing rapid and efficient saltatory conduction of action potentials. Many disorders promote damage to and eventual loss of the myelin sheath, which often results in significant neurological morbidity. However, little is known about the fundamental mechanisms that initiate myelin damage, with the assumption being that its fate follows that of the parent oligodendrocyte. Here we show that NMDA (N-methyl-d-aspartate) glutamate receptors mediate Ca2+ accumulation in central myelin in response to chemical ischaemia in vitro. Using two-photon microscopy, we imaged fluorescence of the Ca2+ indicator X-rhod-1 loaded into oligodendrocytes and the cytoplasmic compartment of the myelin sheath in adult rat optic nerves. The AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid)/kainate receptor antagonist NBQX completely blocked the ischaemic Ca2+ increase in oligodendroglial cell bodies, but only modestly reduced the Ca2+ increase in myelin. In contrast, the Ca2+ increase in myelin was abolished by broad-spectrum NMDA receptor antagonists (MK-801, 7-chlorokynurenic acid, d-AP5), but not by more selective blockers of NR2A and NR2B subunit-containing receptors (NVP-AAM077 and ifenprodil). In vitro ischaemia causes ultrastructural damage to both axon cylinders and myelin. NMDA receptor antagonism greatly reduced the damage to myelin. NR1, NR2 and NR3 subunits were detected in myelin by immunohistochemistry and immunoprecipitation, indicating that all necessary subunits are present for the formation of functional NMDA receptors. Our data show that the mature myelin sheath can respond independently to injurious stimuli. Given that axons are known to release glutamate, our finding that the Ca2+ increase was mediated in large part by activation of myelinic NMDA receptors suggests a new mechanism of axo-myelinic signalling. Such a mechanism may represent a

  17. Developmental changes in the role of a pertussis toxin sensitive guanine nucleotide binding protein in the rat cardiac alpha sub 1 -adrenergic system

    SciTech Connect

    Han, H.M.

    1989-01-01

    During development, the cardiac alpha{sub 1}-adrenergic chronotropic response changes from positive in the neonate to negative in the adult. This thesis examined the possibility of a developmental change in coupling of a PT-sensitive G-protein to the alpha{sub 1}-adrenergic receptor. Radioligand binding experiments performed with the iodinated alpha{sub 1}-selective radioligand ({sup 125}I)-I-2-({beta}-(4-hydroxphenyl)ethylaminomethyl)tetralone (({sup 125}I)-IBE 2254) demonstrated that the alpha{sub 1}-adrenergic receptor is coupled to a G-protein in both neonatal and adult rat hearts. However, in the neonate the alpha{sub 1}-adrenergic receptor is coupled to a PT-insensitive G-protein, whereas in the adult the alpha{sub 1}-adrenergic receptor is coupled to both a PT-insensitive and a PT-sensitive G-protein. Consistent with the results from binding experiments, PT did not have any effect on the alpha{sub 1}-mediated positive chronotropic response in the neonate, whereas in the adult the alpha{sub 1}-mediated negative chronotropic response was completely converted to a positive one after PT-treatment. This thesis also examined the possibility of an alteration in coupling of the alpha{sub 1}-adrenergic receptor to its effector under certain circumstances such as high potassium (K{sup +}) depolarization in nerve-muscle (NM) co-cultures, a system which has been previously shown to be a convenient in vitro model to study the mature inhibitory alpha{sub 1}-response.

  18. Nonenzymatic domains of Kalirin7 contribute to spine morphogenesis through interactions with phosphoinositides and Abl

    PubMed Central

    Ma, Xin-Ming; Miller, Megan B.; Vishwanatha, K. S.; Gross, Maegan J.; Wang, Yanping; Abbott, Thomas; Lam, TuKiet T.; Mains, Richard E.; Eipper, Betty A.

    2014-01-01

    Like several Rho GDP/GTP exchange factors (GEFs), Kalirin7 (Kal7) contains an N-terminal Sec14 domain and multiple spectrin repeats. A natural splice variant of Kalrn lacking the Sec14 domain and four spectrin repeats is unable to increase spine formation; our goal was to understand the function of the Sec14 and spectrin repeat domains. Kal7 lacking its Sec14 domain still increased spine formation, but the spines were short. Strikingly, Kal7 truncation mutants containing only the Sec14 domain and several spectrin repeats increased spine formation. The Sec14 domain bound phosphoinositides, a minor but crucial component of cellular membranes, and binding was increased by a phosphomimetic mutation. Expression of KalSec14-GFP in nonneuronal cells impaired receptor-mediated endocytosis, linking Kal7 to membrane trafficking. Consistent with genetic studies placing Abl, a non–receptor tyrosine kinase, and the Drosophila orthologue of Kalrn into the same signaling pathway, Abl1 phosphorylated two sites in the fourth spectrin repeat of Kalirin, increasing its sensitivity to calpain-mediated degradation. Treating cortical neurons of the wild-type mouse, but not the Kal7KO mouse, with an Abl inhibitor caused an increase in linear spine density. Phosphorylation of multiple sites in the N-terminal Sec14/spectrin region of Kal7 may allow coordination of the many signaling pathways contributing to spine morphogenesis. PMID:24600045

  19. Co-translational formation and pharmacological characterization of beta1-adrenergic receptor/nanodisc complexes with different lipid environments.

    PubMed

    Rues, Ralf-Bernhardt; Dötsch, Volker; Bernhard, Frank

    2016-06-01

    G protein-coupled receptors are of key significance for biomedical research. Streamlined approaches for their efficient recombinant production are of pivotal interest in order to explore their intrinsic conformational dynamics and complex ligand binding behavior. We have systematically optimized the co-translational association and folding of G protein-coupled receptors with defined membranes of nanodiscs by cell-free expression approaches. Each optimization step was quantified and the ligand binding active fraction of the receptor samples could drastically be improved. The strategy was exemplified with a stabilized and a non-stabilized derivative of the turkey beta1-adrenergic receptor. Systematic lipid screens with preformed nanodiscs revealed that generation of ligand binding active conformations of the analyzed beta1-adrenergic receptors strongly depends on lipid charge, flexibility and chain length. The lipid composition of the nanodisc membranes modulates the affinities to a variety of ligands of both receptor derivatives. In addition, the thermostabilization procedure had a significant impact on specific ligand affinities of the receptor and abolished or reduced the binding of certain antagonists. Both receptors were highly stable after purification with optimized nanodisc membranes. The procedure avoids any detergent contact of the receptors and sample production takes less than two days. Moreover, even non-stabilized receptors can be analyzed and their prior purification is not necessary for the formation of nanodisc complexes. The established process appears therefore to be suitable as a new platform for the functional or even structural characterization of recombinant G protein-coupled receptors associated with defined lipid environments. PMID:26922884

  20. Rat hepatic. beta. /sub 2/-adrenergic receptor: structural similarities to the rat fat cell. beta. /sub 1/-adrenergic receptor

    SciTech Connect

    Graziano, M.P.

    1984-01-01

    The mammalian ..beta../sub 2/-adrenergic receptor from rat liver has been purified by sequential cycles of affinity chromatography followed by steric-exclusion high performance liquid chromatography. Electrophoresis of highly purified receptor preparations on polyacrylamide gels in the presence of sodium dodecyl sulfate under reducing conditions reveals a single peptide M/sub r/ = 67,000, as judged by silver staining. Purified ..beta../sub 2/-adrenergic receptor migrates on steric-exclusion high performance liquid chromatography in two peaks, with M/sub r/ = 140,000 and 67,000. Specific binding of the high affinity, ..beta..-adrenergic receptor antagonists (-)(/sup 3/H)dihydroalprenolol and (-)(/sup 125/I)iodocyanopindolol to purified rat liver ..beta..-adrenergic receptor preparations displays stereoselectivity for (-)isomers of agonists and a rank order of potencies for agonists characteristics of a ..beta../sub 2/-adrenergic receptor. Radioiodinated, ..beta../sub 1/-adrenergic receptors from rat fat cells and ..beta../sub 2/-adrenergic receptors from rat liver purified in the presence of protease inhibitors comigrate in electrophoretic separations on polyacrylamide gels in the presence of sodium dodecyl sulfate as 67,000-M/sub r/ peptides. Autoradiograms of two dimensional partial proteolytic digests of the purified, radioiodinated rat liver ..beta../sub 2/-adrenergic receptor, generated with ..cap alpha..-chymotrypsin, S. aureus V8 protease and elastase reveal a pattern of peptide fragments essentially identical to those generated by partial proteolytic digests of the purified, radioiodinated ..beta../sub 1/-adrenergic receptor from rat fat cells, by these same proteases. These data indicate that a high degree of homology exists between these two pharmacologically distinct mammalian ..beta..-adrenergic receptor proteins.

  1. Regulation of coronary vascular tone via redox modulation in the alpha1-adrenergic-angiotensin-endothelin axis of the myocardium.

    PubMed

    Yamaguchi, Osamu; Kaneshiro, Takashi; Saitoh, Shu-ichi; Ishibashi, Toshiyuki; Maruyama, Yukio; Takeishi, Yasuchika

    2009-01-01

    We hypothesized that alpha(1)-adrenoceptor stimulation of cardiac myocytes results in the production of an endothelin (ET)-releasing factor that stimulates the coronary vasculature to release ET and, by manipulating the redox state of cardiac and vascular cells, may influence the extent of alpha(1)-adrenergic-ET-1 vasoconstriction. Dihydroethidium (DHE) and dichlorodihydrofluorescein (DCF) intensities were increased by phenylephrine stimulation in isolated rat cardiac myocytes, which were enhanced by the mitochondrial electron transport chain complex I inhibitor rotenone (DHE: 20.4 +/- 1.2-fold and DCF: 25.2 +/- 0.9-fold, n = 8, P < 0.01, respectively) but not by the NADPH oxidase inhibitor apocynin. Olmesartan, an angiotensin II type 1 receptor antagonist, and enalaprilate did not change DHE and DCF intensities by phenylephrine. Next, we measured the vasoconstriction of isolated, pressurized rat coronary arterioles (diameter: 74 +/- 8 microm) in response to supernatant collected from isolated cardiac myocytes. The addition of supernatant from phenylephrine-stimulated myocytes to a 2-ml vessel bath (n = 8 each) caused volume-dependent vasoconstriction (500 microl: -14.8 +/- 2.2%). Olmesartan and TA0201, an ET type A receptor antagonist, converted vasoconstriction into vasodilation (8.5 +/- 1.2% and 10.5 +/- 0.5%, P < 0.01, respectively) in response to supernatant from phenylephrine-stimulated myocytes, which was eliminated with catalase. Vasoconstriction was weakened using supernatant from phenylephrine with rotenone-treated myocytes. Treatment of arterioles with apocynin to myocyte supernatant converted vasoconstriction into vasodilation (7.8 +/- 0.8%, P < 0.01). These results suggest that alpha(1)-adrenergic stimulation in cardiac myocytes produces angiotensin I and H(2)O(2) and that angiotensin releases ET-1 through NADPH oxidase in coronary arterioles. Thus, coronary vasoconstriction via the alpha-adrenergic-angiotensin-ET axis appears to require redox

  2. Different affinity states of alpha-1 adrenergic receptors defined by agonists and antagonists in bovine aorta plasma membranes

    SciTech Connect

    Jagadeesh, G.; Deth, R.C.

    1987-11-01

    Evidence for a nonlinear relationship between alpha-1 adrenergic receptor occupancy and tissue responses, together with the finding of different affinity states for agonist binding, has raised the possibility of functional heterogeneity of alpha-1 adrenergic receptors. We have conducted studies to examine: 1) binding characteristics of (/sup 3/H)prazosin, 2) competition of antagonists at these sites and 3) different affinity states of the receptor for agonists and modulation of these states by 5'-guanylylimidodiphosphate (Gpp(NH)p). A plasma membrane-enriched vesicular fraction (F2; 15%/33% sucrose interphase) was prepared from the muscular medial layer of bovine thoracic aorta. (/sup 3/H)Prazosin binding was characterized by a monophasic saturation isotherm (KD = 0.116 nM, Bmax = 112 fmol/mg of protein). Antagonist displacement studies yielded a relative potency order of prazosin greater than or equal to WB4104 much greater than phentolamine greater than corynanthine greater than yohimbine greater than or equal to idazoxan greater than rauwolscine. Competition curves for unlabeled prazosin, WB4101 (2-(2,6-dimethoxyphenoxyethyl)-aminomethyl-1,4 benzodioxane) and phentolamine were shallow and were best modeled to two binding sites with picomolar and nanomolar KD values. Gpp(NH)p was without effect on antagonist affinity. Agonist (epinephrine, norepinephrine and phenylephrine) competition with (/sup 3/H)prazosin binding was biphasic with pseudo-Hill slopes less than 1.0. Binding was best described by a two-site model in which the average contribution of high affinity sites was 23% of total binding. KD values for the high affinity site ranged from 2.9 to 18 nM, and 3.9 to 5.0 microM for the low affinity site.

  3. β1-Adrenergic and M2 Muscarinic Autoantibodies and Thyroid Hormone Facilitate Induction of Atrial Fibrillation in Male Rabbits.

    PubMed

    Li, Hongliang; Murphy, Taylor; Zhang, Ling; Huang, Bing; Veitla, Vineet; Scherlag, Benjamin J; Kem, David C; Yu, Xichun

    2016-01-01

    Activating autoantibodies to the β1-adrenergic and M2 muscarinic receptors are present in a very high percentage of patients with Graves' disease and atrial fibrillation (AF). The objective of this study was to develop a reproducible animal model and thereby to examine the impact of these endocrine-like autoantibodies alone and with thyroid hormone on induction of thyroid-associated atrial tachyarrhythmias. Five New Zealand white rabbits were coimmunized with peptides from the second extracellular loops of the β1-adrenergic and M2 muscarinic receptors to produce both sympathomimetic and parasympathomimetic antibodies. A catheter-based electrophysiological study was performed on anesthetized rabbits before and after immunization and subsequent treatment with thyroid hormone. Antibody expression facilitated the induction of sustained sinus, junctional and atrial tachycardias, but not AF. Addition of excessive thyroid hormone resulted in induced sustained AF in all animals. AF induction was blocked acutely by the neutralization of these antibodies with immunogenic peptides despite continued hyperthyroidism. The measured atrial effective refractory period as one parameter of AF propensity shortened significantly after immunization and was acutely reversed by peptide neutralization. No further decrease in the effective refractory period was observed after the addition of thyroid hormone, suggesting other cardiac effects of thyroid hormone may contribute to its role in AF induction. This study demonstrates autonomic autoantibodies and thyroid hormone potentiate the vulnerability of the heart to AF, which can be reversed by decoy peptide therapy. These data help fulfill Witebsky's postulates for an increased autoimmune/endocrine basis for Graves' hyperthyroidism and AF. PMID:26517045

  4. Photoaffinity labeling of mammalian. cap alpha. /sub 1/-adrenergic receptors: identification of the ligand binding subunit with a high affinity radioiodinated probe. [Rats, guinea pigs, rabbits

    SciTech Connect

    Leeb-Lundberg, L.M.F.; Dickinson, K.E.J.; Heald, S.L.; Wikberg, J.E.S.; Hagen, P.O.; DeBernardis, J.F.; Winn, M.; Arendsen, D.L.; Lefkowitz, R.J.; Caron, M.G.

    1984-02-01

    A description is given of the synthesised and characterization of a novel high affinity radioiodinated ..cap alpha../sub 1/-adrenergic receptor photoaffinity probe, 4-amino-6,7-dimethoxy-2-(4-(5-(4-azido-3-(/sup 125/I)iodophenyl)pentanoyl)-1-piperazinyl) quinazoline. In the absence of light, this ligand binds with high affinity (K/sub d/ = 130 pm) in a reverisble and saturable manner to sites in rat hepatic plasma membranes. The binding is stereoselective and competitively inhibited by adrenergic agonists and antagonists with an ..cap alpha../sub 1/-adrenergic specificity. Upon photolysis, this ligand incorporates irreversibly into plasma membranes prepared from several mammalian tissues including rat liver, rat, guinea pig, and rabbit spleen, rabbit lung, and rabbit aorta vascular smooth muscle cells, also with typical ..cap alpha../sub 1/-adrenergic specificity. Autoradiograms of such membrane samples subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis reveal a major specifically labeled polypeptide at M/sub 4/ = 78,000-85,000, depending on the tissue used, in addition to some lower molecular weight peptides. Protease inhibitors, in particular EDTA, a metalloprotease inhibitor, dramatically increases the predominance of the M/sub r/ = 78,000-85,000 polypeptide while attenuating the labeling of the lower molecular weight bands. This new high affinity radioiodinated photoaffinity probe should be of great value for the molecular characterization of the ..cap alpha../sub 1/-adrenergic receptor.

  5. Photoreceptor phagocytosis is mediated by phosphoinositide signaling

    PubMed Central

    Mustafi, Debarshi; Kevany, Brian M.; Genoud, Christel; Bai, Xiaodong; Palczewski, Krzysztof

    2013-01-01

    Circadian oscillations in peripheral tissues, such as the retinal compartment of the eye, are critical to anticipating changing metabolic demands. Circadian shedding of retinal photoreceptor cell discs with subsequent phagocytosis by the neighboring retinal pigmented epithelium (RPE) is essential for removal of toxic metabolites and lifelong survival of these postmitotic neurons. Defects in photoreceptor phagocytosis can lead to severe retinal pathology, but the biochemical mechanisms remain poorly defined. By first documenting a 2.8-fold burst of photoreceptor phagocytosis events in the mouse eye in the morning compared with the afternoon by serial block face imaging, we established time points to assess transcriptional readouts by RNA sequencing (RNA-Seq). We identified 365 oscillating protein-coding transcripts that implicated the phosphoinositide lipid signaling network mediating the discrete steps of photoreceptor phagocytosis. Moreover, examination of overlapping cistromic sites by core clock transcription factors and promoter elements of these effector genes provided a functional basis for the circadian cycling of these transcripts. RNA-Seq also revealed oscillating expression of 16 long intergenic noncoding RNAs and key histone modifying enzymes critical for circadian gene expression. Our phenotypic and genotypic characterization reveals a complex global landscape of overlapping and temporally controlled networks driving the essential circadian process in the eye.—Mustafi, D., Kevany, B. M., Genoud, C., Bai, X., Palczewski, K. Photoreceptor phagocytosis is mediated by phosphoinositide signaling. PMID:23913857

  6. The roles of phosphoinositides in mammalian autophagy.

    PubMed

    Jang, Deok-Jin; Lee, Jin-A

    2016-08-01

    Autophagy is an evolutionarily conserved cellular process for lysosomal degradation, which is involved in various physiological processes within cells. Its dysfunction is associated with many human diseases, such as cancer, liver diseases, heart diseases, and infectious diseases, including neurodegenerative diseases. Autophagy involves the formation of a double-membrane bound autophagosome and the degradation of cytosolic components via its fusion and maturation with the lysosome. One of the most important steps in the process of autophagy is membrane biogenesis during autophagosome formation/maturation from different membrane sources within cells. However, there is limited knowledge regarding: (1) how the core autophagy machinery is recruited to the initial site to initiate the formation of the isolation membrane and (2) how the autophagosome matures into the functional autolysosome. Lipid supply for nucleation/elongation of the autophagosome has been proposed as one possible mechanism. Accumulating evidence suggests the important role of phosphoinositides as phospholipids, which represent key membrane-localized signals in the regulation of fundamental cellular processes, in autophagosome formation and maturation. This review focuses on how phosphoinositides influence autophagy induction or autophagosome biogenesis/maturation, because the way they are altered by autophagy might contribute to the pathogenesis of human diseases. PMID:27350551

  7. Selective α1-adrenergic blockade disturbs the regional distribution of cerebral blood flow during static handgrip exercise.

    PubMed

    Fernandes, Igor A; Mattos, João D; Campos, Monique O; Machado, Alessandro C; Rocha, Marcos P; Rocha, Natalia G; Vianna, Lauro C; Nobrega, Antonio C L

    2016-06-01

    Handgrip-induced increases in blood flow through the contralateral artery that supplies the cortical representation of the arm have been hypothesized as a consequence of neurovascular coupling and a resultant metabolic attenuation of sympathetic cerebral vasoconstriction. In contrast, sympathetic restraint, in theory, inhibits changes in perfusion of the cerebral ipsilateral blood vessels. To confirm whether sympathetic nerve activity modulates cerebral blood flow distribution during static handgrip (SHG) exercise, beat-to-beat contra- and ipsilateral internal carotid artery blood flow (ICA; Doppler) and mean arterial pressure (MAP; Finometer) were simultaneously assessed in nine healthy men (27 ± 5 yr), both at rest and during a 2-min SHG bout (30% maximal voluntary contraction), under two experimental conditions: 1) control and 2) α1-adrenergic receptor blockade. End-tidal carbon dioxide (rebreathing system) was clamped throughout the study. SHG induced increases in MAP (+31.4 ± 10.7 mmHg, P < 0.05) and contralateral ICA blood flow (+80.9 ± 62.5 ml/min, P < 0.05), while no changes were observed in the ipsilateral vessel (-9.8 ± 39.3 ml/min, P > 0.05). The reduction in ipsilateral ICA vascular conductance (VC) was greater compared with contralateral ICA (contralateral: -0.8 ± 0.8 vs. ipsilateral: -2.6 ± 1.3 ml·min(-1)·mmHg(-1), P < 0.05). Prazosin was effective to induce α1-blockade since phenylephrine-induced increases in MAP were greatly reduced (P < 0.05). Under α1-adrenergic receptor blockade, SHG evoked smaller MAP responses (+19.4 ± 9.2, P < 0.05) but similar increases in ICAs blood flow (contralateral: +58.4 ± 21.5 vs. ipsilateral: +54.3 ± 46.2 ml/min, P > 0.05) and decreases in VC (contralateral: -0.4 ± 0.7 vs. ipsilateral: -0.4 ± 1.0 ml·min(-1)·mmHg(-1), P > 0.05). These findings indicate a role of sympathetic nerve activity in the regulation of cerebral blood flow distribution during SHG. PMID:27016578

  8. Phosphoinositides and the regulation of tubular-based endosomal sorting.

    PubMed

    Cullen, Peter J

    2011-08-01

    From the pioneering work of Mabel and Lowell Hokin in the 1950s, the biology of this specific isomer of hexahydroxycyclohexane and its phosphorylated derivatives, in the form of inositol phosphates and phosphoinositides, has expanded to fill virtually every corner of cell biology, whole-organism physiology and development. In the present paper, I give a personal view of the role played by phosphoinositides in regulating the function of the endosomal network, and, in so doing, highlight some of the basic properties through which phosphoinositides regulate cell function. PMID:21787311

  9. Phosphoinositides: Important lipids in the coordination of cell dynamics.

    PubMed

    Viaud, Julien; Mansour, Rana; Antkowiak, Adrien; Mujalli, Abdulrahman; Valet, Colin; Chicanne, Gaëtan; Xuereb, Jean-Marie; Terrisse, Anne-Dominique; Séverin, Sonia; Gratacap, Marie-Pierre; Gaits-Iacovoni, Frédérique; Payrastre, Bernard

    2016-06-01

    By interacting specifically with proteins, phosphoinositides organize the spatiotemporal formation of protein complexes involved in the control of intracellular signaling, vesicular trafficking and cytoskeleton dynamics. A set of specific kinases and phosphatases ensures the production, degradation and inter-conversion of phosphoinositides to achieve a high level of precision in the regulation of cellular dynamics coordinated by these lipids. The direct involvement of these enzymes in cancer, genetic or infectious diseases, and the recent arrival of inhibitors targeting specific phosphoinositide kinases in clinic, emphasize the importance of these lipids and their metabolism in the biomedical field. PMID:26391221

  10. Plant phosphoinositide signaling - dynamics on demand.

    PubMed

    Heilmann, Ingo

    2016-09-01

    Eukaryotic membranes contain small amounts of lipids with regulatory roles. An important class of such regulatory lipids are phosphoinositides (PIs). Within membranes, PIs serve as recruitment signals, as regulators of membrane protein function or as precursors for second messenger production, thereby influencing a multitude of cellular processes with key importance for plant function and development. Plant PIs occur locally and transiently within membrane microdomains, and their abundance is strictly controlled. To understand the functions of the plant PI-network it is important to understand not only downstream PI-effects, but also to identify and characterize factors contributing to dynamic PI formation. This article is part of a Special Issue entitled: Plant Lipid Biology edited by Kent D. Chapman and Ivo Feussner. PMID:26924252

  11. Phosphoinositide 3-kinases-a historical perspective.

    PubMed

    Toker, Alex

    2012-01-01

    The phosphoinositide 3-kinase (PI 3-K) signal relay pathway represents arguably one of the most intensely studied mechanisms by which extracellular signals elicit cellular responses through the generation of second messengers that are associated with cell growth and transformation. This chapter reviews the many landmark discoveries in the PI 3-K signaling pathway in biology and disease, from the identification of a novel phosphoinositide kinase activity associated with transforming oncogenes in the 1980s, to the identification of oncogenic mutations in the catalytic subunit of PI 3-K in the mid 2000s. Two and a half decades of intense research have provided clear evidence that the PI 3-K pathway controls virtually all aspects of normal cellular physiology, and that deregulation of one or more proteins that regulate or transduce the PI 3-K signal ultimately leads to human pathology. The most recent efforts have focused on the development of specific PI 3-K inhibitors that are currently being evaluated in clinical trials for a range of disease states.This chapter is devoted to a historical review of the landmark findings in the PI 3-K from its relatively humble beginnings in the early to mid 1980s up until the present day. When considering the key findings in the history of PI 3-K, it is essential to recognize the landmark studies by Lowell and Mabel Hokin in the 1950s who were the first to describe that extracellular agonists such as acetylcholine could stimulate the incorporation of radiolabeled phosphate into phospholipids (Hokin and Hokin 1953). Their work initiated an entirely new field of lipid signaling, and subsequent studies in the 1970s by Michell and Lapetina who linked phosphoinositide turnover to membrane-associated receptors that initiate intracellular calcium mobilization (Lapetina and Michell 1973). Later studies revealed that the phospholipase-mediated breakdown of the same minor membrane phospholipids such as PtdIns-4,5-P(2) (phosphatidylinositol-4

  12. Alpha-1-Adrenergic Receptors in Heart Failure: The Adaptive Arm of the Cardiac Response to Chronic Catecholamine Stimulation

    PubMed Central

    Jensen, Brian C.; O'Connell, Timothy D.; Simpson, Paul C.

    2013-01-01

    Alpha-1-adrenergic receptors are G-protein coupled receptors (GPCRs) activated by catecholamines. The alpha-1A and alpha-1B subtypes are expressed in mouse and human myocardium, whereas the alpha-1D protein is found only in coronary arteries. There are far fewer alpha-1-ARs than beta-ARs in the non-failing heart, but their abundance is maintained or increased in the setting of heart failure, which is characterized by pronounced chronic elevation of catecholamines and b□eta-AR dysfunction. Decades of evidence from gain- and loss-of-function studies in isolated cardiac myocytes and numerous animal models demonstrate important adaptive functions for cardiac alpha-1-ARs, to include physiological hypertrophy, positive inotropy, ischemic preconditioning, and protection from cell death. Clinical trial data indicate that blocking alpha-1-ARs is associated with incident heart failure in patients with hypertension. Collectively, these findings suggest that alpha-1-AR activation might mitigate the well-recognized toxic effects of beta-ARs in the hyperadrenergic setting of chronic heart failure. Thus, exogenous cardioselective activation of alpha-1-ARs might represent a novel and viable approach to the treatment of heart failure. PMID:24145181

  13. Alpha-1-adrenergic receptors in heart failure: the adaptive arm of the cardiac response to chronic catecholamine stimulation.

    PubMed

    Jensen, Brian C; OʼConnell, Timothy D; Simpson, Paul C

    2014-04-01

    Alpha-1-adrenergic receptors (ARs) are G protein-coupled receptors activated by catecholamines. The alpha-1A and alpha-1B subtypes are expressed in mouse and human myocardium, whereas the alpha-1D protein is found only in coronary arteries. There are far fewer alpha-1-ARs than beta-ARs in the nonfailing heart, but their abundance is maintained or increased in the setting of heart failure, which is characterized by pronounced chronic elevation of catecholamines and beta-AR dysfunction. Decades of evidence from gain and loss-of-function studies in isolated cardiac myocytes and numerous animal models demonstrate important adaptive functions for cardiac alpha-1-ARs to include physiological hypertrophy, positive inotropy, ischemic preconditioning, and protection from cell death. Clinical trial data indicate that blocking alpha-1-ARs is associated with incident heart failure in patients with hypertension. Collectively, these findings suggest that alpha-1-AR activation might mitigate the well-recognized toxic effects of beta-ARs in the hyperadrenergic setting of chronic heart failure. Thus, exogenous cardioselective activation of alpha-1-ARs might represent a novel and viable approach to the treatment of heart failure. PMID:24145181

  14. Phosphoinositides in the mammalian endo-lysosomal network

    PubMed Central

    Cullen, Peter J.; Carlton, Jeremy G.

    2014-01-01

    The endo-lysosomal system is an interconnected tubulo-vesicular network that acts as a sorting station to process and distribute internalised cargo. This network accepts cargoes from both the plasma membrane and the biosynthetic pathway, and directs these cargos either towards the lysosome for degradation, the peri-nuclear recycling endosome for return to the cell surface, or to the trans-Golgi network. These intracellular membranes are variously enriched in different phosphoinositides that help to shape compartmental identity. These lipids act to localise a number of phosphoinositide-binding proteins that function as sorting machineries to regulate endosomal cargo sorting. Herein we discuss regulation of these machineries by phosphoinositides and explore how phosphoinositide-switching contributes toward sorting decisions made at this platform. PMID:22374088

  15. Autoradiographic imaging of phosphoinositide turnover in the brain

    SciTech Connect

    Hwang, P.M.; Bredt, D.S.; Snyder, S.H. )

    1990-08-17

    With ({sup 3}H)cytidine as a precursor, phosphoinositide turnover can be localized in brain slices by selective autoradiography of the product ({sup 3}H)cytidine diphosphate diacylglycerol, which is membrane-bound. In the cerebellum, glutamatergic stimulation elicits an increase of phosphoinositide turnover only in Purkinje cells and the molecular layer. In the hippocampus, both glutamatergic and muscarinic cholinergic stimulation increase phosphoinositide turnover, but with distinct localizations. Cholinergic stimulation affects CA1, CA3, CA4, and subiculum, whereas glutamatergic effects are restricted to the subiculum and CA3. Imaging phosphoinositide turnover in brain slices, which are amenable to electrophysiologic studies, will permit a dynamic localized analysis of regulation of this second messenger in response to synaptic stimulation of specific neuronal pathways.

  16. Regional distribution of rat brain alpha/sub 1/-adrenergic receptors: correlation between (/sup 125/I)-heat membrane binding and in vitro autoradiography

    SciTech Connect

    Jones, L.S.; Miller, G.; Gauger, L.L.; Davis, J.N.

    1985-01-07

    (/sup 125/I)-HEAT has proven useful for in vitro autoradiography as a specific alpha/sub 1/-adrenergic radioligand. We compared the binding of (/sup 125/I)-HEAT to membranes from ten brain regions with the densitometric readings of these regions in autoradiographs. There was an excellent correlation between receptor numbers from membrane binding and relative optical densities from the autoradiography. The affinity of HEAT for binding to membranes from various regions was similar. The results of this direct comparison are further evidence that HEAT binds to alpha/sub 1/-adrenergic receptors in lightly fixed tissue sections. A further interesting observation is that in regions with a heterogeneous distribution of binding sites, membrane binding may not reflect the presence of a dense local population of receptors. 19 references, 3 figures, 1 table.

  17. The potential of metabolomic analysis techniques for the characterisation of α1-adrenergic receptors in cultured N1E-115 mouse neuroblastoma cells.

    PubMed

    Wenner, Maria I; Maker, Garth L; Dawson, Linda F; Drummond, Peter D; Mullaney, Ian

    2016-08-01

    Several studies of neuropathic pain have linked abnormal adrenergic signalling to the development and maintenance of pain, although the mechanisms underlying this are not yet fully understood. Metabolomic analysis is a technique that can be used to give a snapshot of biochemical status, and can aid in the identification of the mechanisms behind pathological changes identified in cells, tissues and biological fluids. This study aimed to use gas chromatography-mass spectrometry-based metabolomic profiling in combination with reverse transcriptase-polymerase chain reaction and immunocytochemistry to identify functional α1-adrenergic receptors on cultured N1E-115 mouse neuroblastoma cells. The study was able to confirm the presence of mRNA for the α1D subtype, as well as protein expression of the α1-adrenergic receptor. Furthermore, metabolomic data revealed changes to the metabolite profile of cells when exposed to adrenergic pharmacological intervention. Agonist treatment with phenylephrine hydrochloride (10 µM) resulted in altered levels of several metabolites including myo-inositol, glucose, fructose, alanine, leucine, phenylalanine, valine, and n-acetylglutamic acid. Many of the changes observed in N1E-115 cells by agonist treatment were modulated by additional antagonist treatment (prazosin hydrochloride, 100 µM). A number of these changes reflected what is known about the biochemistry of α1-adrenergic receptor activation. This preliminary study therefore demonstrates the potential of metabolomic profiling to confirm the presence of functional receptors on cultured cells. PMID:26408527

  18. β1-Adrenergic blocker bisoprolol reverses down-regulated ion channels in sinoatrial node of heart failure rats.

    PubMed

    Du, Yuan; Zhang, Junbo; Xi, Yutao; Wu, Geru; Han, Ke; Huang, Xin; Ma, Aiqun; Wang, Tingzhong

    2016-06-01

    Bisoprolol, an antagonist of β1-adrenergic receptors, is effective in reducing the morbidity and mortality in patients with heart failure (HF). It has been found that HF is accompanied with dysfunction of the sinoatrial node (SAN). However, whether bisoprolol reverses the decreased SAN function in HF and how the relevant ion channels in SAN change were relatively less studied. SAN function and messenger RNA (mRNA) expression of sodium channels and hyperpolarization-activated cyclic nucleotide-gated (HCN) channel subunits were assessed in sham-operated rats, abdominal arterio-venous shunt (volume overload)-induced HF rats, and bisoprolol- treated HF rats. SAN cells of rats were isolated by laser capture microdissection. Quantitative real-time PCR analysis was used to quantify mRNA expression of sodium channels and HCN channel subunits in SAN. Intrinsic heart rate declined and sinus node recovery time prolonged in HF rats, indicating the suppressed SAN function, which could be improved by bisoprolol treatment. Nav1.1, Nav1.6, and HCN4 mRNA expressions were reduced in SAN in HF rats compared with that in control rats. Treatment with bisoprolol could reverse both the SAN function and the Nav1.1, Nav1.6, and HCN4 mRNA expression partially. These data indicated that bisoprolol is effective in HF treatment partially due to improved SAN function by reversing the down-regulation of sodium channels (Nav1.1 and Nav1.6) and HCN channel (HCN4) subunits in SAN in failing hearts. PMID:26995749

  19. A Long Lasting β1 Adrenergic Receptor Stimulation of cAMP/Protein Kinase A (PKA) Signal in Cardiac Myocytes*

    PubMed Central

    Fu, Qin; Kim, Sungjin; Soto, Dagoberto; De Arcangelis, Vania; DiPilato, Lisa; Liu, Shubai; Xu, Bing; Shi, Qian; Zhang, Jin; Xiang, Yang K.

    2014-01-01

    Small-molecule, ligand-activated G protein-coupled receptors are generally thought to be rapidly desensitized within a period of minutes through receptor phosphorylation and internalization after repeated or prolonged stimulation. This transient G protein-coupled receptor activation remains at odds with many observed long-lasting cellular and physiological responses. Here, using live cell imaging of cAMP with a FRET-based biosensor and myocyte contraction assay, we show that the catecholamine-activated β1 adrenergic receptor (β1AR) continuously stimulates second messenger cAMP synthesis in primary cardiac myocytes and neurons, which lasts for more than 8 h (a decay t½ of 3.9 h) in cardiac myocytes. However, the β1AR-induced cAMP signal is counterbalanced and masked by the receptor-bound phosphodiesterase (PDE) 4D8-dependent cAMP hydrolysis. Inhibition of PDE4 activity recovers the receptor-induced cAMP signal and promotes contractile response in mouse hearts during extended periods of agonist stimulation. β1AR associates with PDE4D8 through the receptor C-terminal PDZ motif-dependent binding to synaptic-associated protein 97 (SAP97). Knockdown of SAP97 or mutation of the β1AR PDZ motif disrupts the complex and promotes sustained agonist-induced cAMP activity, PKA phosphorylation, and cardiac myocyte contraction response. Together, these findings unveil a long lasting adrenergic signal in neurons and myocytes under prolonged stimulation and an underappreciated role of PDE that is essential in classic receptor signaling desensitization and in maintaining a long lasting cAMP equilibrium for ligand-induced physiological response. PMID:24713698

  20. Agonist-promoted desensitization and phosphorylation of. cap alpha. /sub 1/-adrenergic receptors coupled to stimulation of phosphatidylinositol metabolism

    SciTech Connect

    Leeb-Lundberg, L.M.F.; Cotecchia, S.; Caron, M.G.; Lefkowitz, R.J.

    1986-03-05

    In the DDT/sub 1/ MF-2 hamster vas deferens smooth muscle cell line the ..cap alpha../sub 1/-adrenergic receptor (..cap alpha../sub 1/-AR) agonist norepinephrine (NE) promotes rapid attenuation of ..cap alpha../sub 1/-AR-mediated phosphatidylinositol (PI) metabolism which is paralleled by rapid phosphorylation of the ..cap alpha../sub 1/-AR. Cells were labeled by incubation with /sup 32/P/sub i/. Coincubation with NE (100 ..mu..M) significantly increases the rate of /sup 32/P-labeling of both PI and phosphatidic acid. Pretreatment of cells with 100 ..mu..M NE (in the presence of 1 ..mu..M propranolol to prevent ..beta..-AR interactions) results in a drastic attenuation of the NE response on PI metabolism. ..cap alpha../sub 1/-AR from labeled cells can be solubilized and purified by affinity chromatography on Affigel-A55414 and wheat germ agglutinin agarose chromatography. SDS-PAGE of purified ..cap alpha../sub 1/-AR shows a NE-promoted increase in phosphorylation of the M/sub r/ 80K ligand binding peptide. Stoichiometry of phosphorylation increases from approx. 1 mol phosphate/mol ..cap alpha../sub 1/-AR in the basal condition to approx. 2.5 after NE treatment. Both desensitization and phosphorylation are rapid being maximal within 10-20 min of agonist exposure. These results together with previous findings that phorbol esters promote rapid ..cap alpha../sub 1/-AR uncoupling and phosphorylation suggest that receptor phosphorylation is an important mechanism of regulation of ..cap alpha../sub 1/-AR receptor responsiveness.

  1. Phosphoregulation of Cardiac Inotropy via Myosin Binding Protein-C During Increased Pacing Frequency or β1-Adrenergic Stimulation

    PubMed Central

    Tong, Carl W.; Wu, Xin; Liu, Yang; Rosas, Paola C.; Sadayappan, Sakthivel; Hudmon, Andy; Muthuchamy, Mariappan; Powers, Patricia A.; Valdivia, Héctor H.; Moss, Richard L.

    2015-01-01

    Background Mammalian hearts exhibit positive inotropic responses to β-adrenergic stimulation as a consequence of protein kinase A (PKA)-mediated phosphorylation or as a result of increased beat frequency (the Bowditch effect). Several membrane and myofibrillar proteins are phosphorylated under these conditions, but the relative contributions of these to increased contractility are not known. Phosphorylation of cardiac myosin binding protein-C (cMyBP-C) by PKA accelerates the kinetics of force development in permeabilized heart muscle, but its role in vivo is unknown. Such understanding is important, since adrenergic responsiveness of the heart and the Bowditch effect are both depressed in heart failure. Methods and Results The roles of cMyBP-C phosphorylation were studied using mice in which either WT or nonphosphorylatable forms of cMyBP-C [ser273ala, ser282ala, ser302ala: cMyBP-C(t3SA)] were expressed at similar levels on a cMyBP-C null background. Force and [Ca2+]in measurements in isolated papillary muscles showed that the increased force and twitch kinetics due to increased pacing or β1-adrenergic stimulation were nearly absent in cMyBP-C(t3SA) myocardium, even though [Ca2+]intransients under each condition were similar to WT. Biochemical measurements confirmed that PKA phosphorylated ser273, ser282 and ser302 in WT cMyBP-C. In contrast, CaMKIIδ, which is activated by increased pacing, phosphorylated ser302 principally, ser282 to a lesser degree, and ser273 not at all. Conclusions Phosphorylation of cMyBP-C increases the force and kinetics of twitches in living cardiac muscle. Further, cMyBP-C is a principal mediator of increased contractility observed with β-adrenergic stimulation or increased pacing, due to PKA and CaMKIIδ phosphorylations of cMyB-C. PMID:25740838

  2. The Alpha-1D Is the Predominant Alpha-1-Adrenergic Receptor Subtype in Human Epicardial Coronary Arteries

    PubMed Central

    Jensen, Brian C.; Swigart, Philip M.; Laden, Marie-Eve; DeMarco, Teresa; Hoopes, Charles; Simpson, Paul C.

    2009-01-01

    Objectives The goal was to identify alpha-1-adrenergic receptor (α1-AR) subtypes in human coronary arteries. Background The α1-ARs regulate human coronary blood flow. α1-ARs exist as three molecular subtypes, α1A, α1B, and α1D, and the α1D subtype mediates coronary vasoconstriction in the mouse. However, the α1A is thought to be the only subtype in human coronary arteries. Methods We obtained human epicardial coronary arteries and left ventricular (LV) myocardium from 19 transplant recipients and 6 unused donors (age 19–70 years; 68% male; 32% with coronary artery disease). We cultured coronary rings and human coronary smooth muscle cells. We assayed α1- and β-AR subtype mRNAs by quantitative real-time reverse transcription PCR; and subtype proteins, by radioligand binding and ERK activation. Results The α1D subtype was 85% of total coronary α1-AR mRNA and 75% of total α1-AR protein, and α1D stimulation activated ERK. In contrast, the α1D was low in LV myocardium. Total coronary α1-AR levels were one-third of β-ARs, which were 99% the β2 subtype. Conclusions The α1D subtype is predominant and functional in human epicardial coronary arteries, whereas the α1A and α1B are present at very low levels. This distribution is similar to the mouse, where myocardial α1A and α1B-ARs mediate beneficial functional responses, and coronary α1Ds mediate vasoconstriction. Thus, α1D-selective antagonists might mediate coronary vasodilation, without the negative cardiac effects of non-selective α1-AR antagonists in current use. Furthermore, it could be possible to selectively activate beneficial myocardial α1A and/or α1B-AR signaling without causing coronary vasoconstriction. PMID:19761933

  3. α1-adrenergic receptors positively regulate Toll-like receptor cytokine production from human monocytes and macrophages.

    PubMed

    Grisanti, Laurel A; Woster, Andrew P; Dahlman, Julie; Sauter, Edward R; Combs, Colin K; Porter, James E

    2011-08-01

    Catecholamines released from the sympathetic nervous system in response to stress or injury affect expression of inflammatory cytokines generated by immune cells. α(1)-Adrenergic receptors (ARs) are expressed on innate immune cell populations, but their subtype expression patterns and signaling characteristics are not well characterized. Primary human monocytes, a human monocytic cell line, and monocyte-derived macrophage cells were used to measure expression of the proinflammatory mediator interleukin (IL)-1β responding to lipopolysaccharide (LPS) in the presence or absence of α(1)-AR activation. Based on our previous findings, we hypothesized that α(1)-AR stimulation on innate immune cells positively regulates LPS-initiated IL-1β production. IL-1β production in response to LPS was synergistically higher for both monocytes and macrophages in the presence of the selective α(1)-AR agonist (R)-(-)-phenylephrine hydrochloride (PE). This synergistic IL-1β response could be blocked with a selective α(1)-AR antagonist as well as inhibitors of protein kinase C (PKC). Radioligand binding studies characterized a homogenous α(1B)-AR subtype population on monocytes, which changed to a heterogeneous receptor subtype expression pattern when differentiated to macrophages. Furthermore, increased p38 mitogen-activated protein kinase (MAPK) activation was observed only with concurrent PE and LPS stimulation, peaking after 120 and 30 min in monocytes and macrophages, respectively. Blocking the PKC/p38 MAPK signaling pathway in both innate immune cell types inhibited the synergistic IL-1β increase observed with concurrent PE and LPS treatments. This study characterizes α(1)-AR subtype expression on both human monocyte and macrophage cells and illustrates a mechanism by which increased IL-1β production can be modulated by α(1)-AR input. PMID:21571945

  4. Mechanisms of postspaceflight orthostatic hypotension: low alpha1-adrenergic receptor responses before flight and central autonomic dysregulation postflight.

    PubMed

    Meck, Janice V; Waters, Wendy W; Ziegler, Michael G; deBlock, Heidi F; Mills, Paul J; Robertson, David; Huang, Paul L

    2004-04-01

    Although all astronauts experience symptoms of orthostatic intolerance after short-duration spaceflight, only approximately 20% actually experience presyncope during upright posture on landing day. The presyncopal group is characterized by low vascular resistance before and after flight and low norepinephrine release during orthostatic stress on landing day. Our purpose was to determine the mechanisms of the differences between presyncopal and nonpresyncopal groups. We studied 23 astronauts 10 days before launch, on landing day, and 3 days after landing. We measured pressor responses to phenylephrine injections; norepinephrine release with tyramine injections; plasma volumes; resting plasma levels of chromogranin A (a marker of sympathetic nerve terminal release), endothelin, dihydroxyphenylglycol (DHPG, an intracellular metabolite of norepinephrine); and lymphocyte beta(2)-adrenergic receptors. We then measured hemodynamic and neurohumoral responses to upright tilt. Astronauts were separated into two groups according to their ability to complete 10 min of upright tilt on landing day. Compared with astronauts who were not presyncopal on landing day, presyncopal astronauts had 1). significantly smaller pressor responses to phenylephrine both before and after flight; 2). significantly smaller baseline norepinephrine, but significantly greater DHPG levels, on landing day; 3). significantly greater norepinephrine release with tyramine on landing day; and 4). significantly smaller norepinephrine release, but significantly greater epinephrine and arginine vasopressin release, with upright tilt on landing day. These data suggest that the etiology of orthostatic hypotension and presyncope after spaceflight includes low alpha(1)-adrenergic receptor responsiveness before flight and a remodeling of the central nervous system during spaceflight such that sympathetic responses to baroreceptor input become impaired. PMID:14670816

  5. Mechanisms of postspaceflight orthostatic hypotension: low alpha1-adrenergic receptor responses before flight and central autonomic dysregulation postflight

    NASA Technical Reports Server (NTRS)

    Meck, Janice V.; Waters, Wendy W.; Ziegler, Michael G.; deBlock, Heidi F.; Mills, Paul J.; Robertson, David; Huang, Paul L.

    2004-01-01

    Although all astronauts experience symptoms of orthostatic intolerance after short-duration spaceflight, only approximately 20% actually experience presyncope during upright posture on landing day. The presyncopal group is characterized by low vascular resistance before and after flight and low norepinephrine release during orthostatic stress on landing day. Our purpose was to determine the mechanisms of the differences between presyncopal and nonpresyncopal groups. We studied 23 astronauts 10 days before launch, on landing day, and 3 days after landing. We measured pressor responses to phenylephrine injections; norepinephrine release with tyramine injections; plasma volumes; resting plasma levels of chromogranin A (a marker of sympathetic nerve terminal release), endothelin, dihydroxyphenylglycol (DHPG, an intracellular metabolite of norepinephrine); and lymphocyte beta(2)-adrenergic receptors. We then measured hemodynamic and neurohumoral responses to upright tilt. Astronauts were separated into two groups according to their ability to complete 10 min of upright tilt on landing day. Compared with astronauts who were not presyncopal on landing day, presyncopal astronauts had 1). significantly smaller pressor responses to phenylephrine both before and after flight; 2). significantly smaller baseline norepinephrine, but significantly greater DHPG levels, on landing day; 3). significantly greater norepinephrine release with tyramine on landing day; and 4). significantly smaller norepinephrine release, but significantly greater epinephrine and arginine vasopressin release, with upright tilt on landing day. These data suggest that the etiology of orthostatic hypotension and presyncope after spaceflight includes low alpha(1)-adrenergic receptor responsiveness before flight and a remodeling of the central nervous system during spaceflight such that sympathetic responses to baroreceptor input become impaired.

  6. Chemokine (C-X-C Motif) Receptor 4 and Atypical Chemokine Receptor 3 Regulate Vascular α1-Adrenergic Receptor Function

    PubMed Central

    Bach, Harold H; Wong, Yee M; Tripathi, Abhishek; Nevins, Amanda M; Gamelli, Richard L; Volkman, Brian F; Byron, Kenneth L; Majetschak, Matthias

    2014-01-01

    Chemokine (C-X-C motif) receptor (CXCR) 4 and atypical chemokine receptor (ACKR) 3 ligands have been reported to modulate cardiovascular function in various disease models. The underlying mechanisms, however, remain unknown. Thus, it was the aim of the present study to determine how pharmacological modulation of CXCR4 and ACKR3 regulate cardiovascular function. In vivo administration of TC14012, a CXCR4 antagonist and ACKR3 agonist, caused cardiovascular collapse in normal animals. During the cardiovascular stress response to hemorrhagic shock, ubiquitin, a CXCR4 agonist, stabilized blood pressure, whereas coactivation of CXCR4 and ACKR3 with CXC chemokine ligand 12 (CXCL12), or blockade of CXCR4 with AMD3100 showed opposite effects. While CXCR4 and ACKR3 ligands did not affect myocardial function, they selectively altered vascular reactivity upon α1-adrenergic receptor (AR) activation in pressure myography experiments. CXCR4 activation with ubiquitin enhanced α1-AR-mediated vasoconstriction, whereas ACKR3 activation with various natural and synthetic ligands antagonized α1-AR-mediated vasoconstriction. The opposing effects of CXCR4 and ACKR3 activation by CXCL12 could be dissected pharmacologically. CXCR4 and ACKR3 ligands did not affect vasoconstriction upon activation of voltage-operated Ca2+ channels or endothelin receptors. Effects of CXCR4 and ACKR3 agonists on vascular α1-AR responsiveness were independent of the endothelium. These findings suggest that CXCR4 and ACKR3 modulate α1-AR reactivity in vascular smooth muscle and regulate hemodynamics in normal and pathological conditions. Our observations point toward CXCR4 and ACKR3 as new pharmacological targets to control vasoreactivity and blood pressure. PMID:25032954

  7. A Trypanosoma cruzi Phosphatidylinositol 3-Kinase (TcVps34) Is Involved in Osmoregulation and Receptor-mediated Endocytosis*S⃞

    PubMed Central

    Schoijet, Alejandra C.; Miranda, Kildare; Girard-Dias, Wendell; de Souza, Wanderley; Flawiá, Mirtha M.; Torres, Héctor N.; Docampo, Roberto; Alonso, Guillermo D.

    2008-01-01

    Trypanosoma cruzi, the etiological agent of Chagas disease, has the ability to respond to a variety of environmental changes during its life cycle both in the insect vector and in the vertebrate host. Because regulation of transcription initiation seems to be nonfunctional in this parasite, it is important to investigate other regulatory mechanisms of adaptation. Regulatory mechanisms at the level of signal transduction pathways involving phosphoinositides are good candidates for this purpose. Here we report the identification of the first phosphatidylinositol 3-kinase (PI3K) in T. cruzi, with similarity with its yeast counterpart, Vps34p. TcVps34 specifically phosphorylates phosphatidylinositol to produce phosphatidylinositol 3-phosphate, thus confirming that it belongs to class III PI3K family. Overexpression of TcVps34 resulted in morphological and functional alterations related to vesicular trafficking. Although inhibition of TcVps34 with specific PI3K inhibitors, such as wortmannin and LY294,000, resulted in reduced regulatory volume decrease after hyposmotic stress, cells overexpressing this enzyme were resistant to these inhibitors. Furthermore, these cells were able to recover their original volume faster than wild type cells when they were submitted to severe hyposmotic stress. In addition, in TcVps34-overexpressing cells, the activities of vacuolar-H+-ATPase and vacuolar H+-pyrophosphatase were altered, suggesting defects in the acidification of intracellular compartments. Furthermore, receptor-mediated endocytosis was partially blocked although fluid phase endocytosis was not affected, confirming a function for TcVps34 in membrane trafficking. Taken together, these results strongly support that TcVps34 plays a prominent role in vital processes for T. cruzi survival such as osmoregulation, acidification, and vesicular trafficking. PMID:18801733

  8. Phosphoinositide metabolism and adrenergic receptors in astrocytes

    SciTech Connect

    Noble, E.P.; Ritchie, T.; de Vellis, J.

    1986-03-01

    Agonist-induced phosphoinositide (PI) breakdown functions as a signal generating system. Diacylglycerol, one breakdown product of phosphotidylinositol-4,5-diphosphate hydrolysis, can stimulate protein kinase C, whereas inositol triphosphate, the other product, has been proposed to be a second messenger for Ca/sup + +/ mobilization. Using purified astrocyte cultures from neonatal rat brain, the effects of adrenergic agonists and antagonists at 10/sup -5/ M were measured on PI breakdown. Astrocytes grown in culture were prelabeled with (/sup 3/H)inositol, and basal (/sup 3/H) inositol phosphate (IP/sub 1/) accumulation was measured in the presence of Li/sup +/. Epinephrine > norepinephrine (NE) were the most active stimulants of IP/sub 1/ production. The ..cap alpha../sub 1/ adrenoreceptor blockers, phentolamine and phenoxybenzamine, added alone had no effect on IP/sub 1/ production was reduced below basal levels. Propranolol partially blocked the effects of NE. Clonidine and isoproterenol, separately added, reduced IP/sub 1/ below basal levels and when added together diminished IP/sub 1/ accumulation even further. The role of adrenergic stimulation in the production of c-AMP.

  9. Chromosomal Instability and Phosphoinositide Pathway Gene Signatures in Glioblastoma Multiforme.

    PubMed

    Waugh, Mark G

    2016-01-01

    Structural rearrangements of chromosome 10 are frequently observed in glioblastoma multiforme and over 80 % of tumour samples archived in the catalogue of somatic mutations in cancer database had gene copy number loss for PI4K2A which encodes phosphatidylinositol 4-kinase type IIalpha. PI4K2A loss of heterozygosity mirrored that of PTEN, another enzyme that regulates phosphoinositide levels and also PIK3AP1, MINPP1, INPP5A and INPP5F. These results indicated a reduction in copy number for a set of phosphoinositide signalling genes that co-localise to chromosome 10q. This analysis was extended to a panel of phosphoinositide pathway genes on other chromosomes and revealed a number of previously unreported associations with glioblastoma multiforme. Of particular note were highly penetrant copy number losses for a group of X-linked phosphoinositide phosphatase genes OCRL, MTM1 and MTMR8; copy number amplifications for the chromosome 19 genes PIP5K1C, AKT2 and PIK3R2, and also for the phospholipase C genes PLCB1, PLCB4 and PLCG1 on chromosome 20. These mutations are likely to affect signalling and trafficking functions dependent on the PI(4,5)P2, PI(3,4,5)P3 and PI(3,5)P2 lipids as well as the inositol phosphates IP3, IP5 and IP6. Analysis of flanking genes with functionally unrelated products indicated that chromosomal instability as opposed to a phosphoinositide-specific process underlay this pattern of copy number variation. This in silico study suggests that in glioblastoma multiforme, karyotypic changes have the potential to cause multiple abnormalities in sets of genes involved in phosphoinositide metabolism and this may be important for understanding drug resistance and phosphoinositide pathway redundancy in the advanced disease state. PMID:25502460

  10. Phosphoinositide and Inositol Phosphate Analysis in Lymphocyte Activation

    PubMed Central

    Sauer, Karsten; Huang, Yina Hsing; Lin, Hongying; Sandberg, Mark; Mayr, Georg W.

    2015-01-01

    Lymphocyte antigen receptor engagement profoundly changes the cellular content of phosphoinositide lipids and soluble inositol phosphates. Among these, the phosphoinositides phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylinositol 3,4,5-trisphosphate (PIP3) play key signaling roles by acting as pleckstrin homology (PH) domain ligands that recruit signaling proteins to the plasma membrane. Moreover, PIP2 acts as a precursor for the second messenger molecules diacylglycerol and soluble inositol 1,4,5-trisphosphate (IP3), essential mediators of PKC, Ras/Erk, and Ca2+ signaling in lymphocytes. IP3 phosphorylation by IP3 3-kinases generates inositol 1,3,4,5-tetrakisphosphate (IP4), an essential soluble regulator of PH domain binding to PIP3 in developing T cells. Besides PIP2, PIP3, IP3, and IP4, lymphocytes produce multiple other phosphoinositides and soluble inositol phosphates that could have important physiological functions. To aid their analysis, detailed protocols that allow one to simultaneously measure the levels of multiple different phosphoinositide or inositol phosphate isomers in lymphocytes are provided here. They are based on thin layer, conventional and high-performance liquid chromatographic separation methods followed by radiolabeling or non-radioactive metal-dye detection. Finally, less broadly applicable nonchromatographic methods for detection of specific phosphoinositide or inositol phosphate isomers are discussed. Support protocols describe how to obtain pure unstimulated CD4+CD8+ thymocyte populations for analyses of inositol phosphate turnover during positive and negative selection, key steps in T cell development. PMID:19918943

  11. Phosphoinositides Regulate Ciliary Protein Trafficking to Modulate Hedgehog Signaling

    PubMed Central

    Roberson, Elle C.; Garcia, Galo; Abedin, Monika; Schurmans, Stéphane; Inoue, Takanari; Reiter, Jeremy F.

    2015-01-01

    SUMMARY Primary cilia interpret vertebrate Hedgehog (Hh) signals. Why cilia are essential for signaling is unclear. One possibility is that some forms of signaling require a distinct membrane lipid composition, found at cilia. We found that the ciliary membrane contains a particular phosphoinositide, PI(4)P, whereas a different phosphoinositide, PI(4,5)P2, is restricted to the membrane of the ciliary base. This distribution is created by Inpp5e, a ciliary phosphoinositide 5-phosphatase. Without Inpp5e, ciliary PI(4,5)P2 levels are elevated and Hh signaling is disrupted. Inpp5e limits the ciliary levels of inhibitors of Hh signaling, including Gpr161 and the PI(4,5)P2-binding protein Tulp3. Increasing ciliary PI(4,5)P2 levels or conferring the ability to bind PI(4)P on Tulp3 increases the ciliary localization of Tulp3. Lowering Tulp3 in cells lacking Inpp5e reduces ciliary Gpr161 levels and restores Hh signaling. Therefore, Inpp5e regulates ciliary membrane phosphoinositide composition, and Tulp3 reads out ciliary phosphoinositides to control ciliary protein localization, enabling Hh signaling. PMID:26305592

  12. Expression of α(1)-adrenergic receptors in rat prefrontal cortex: cellular co-localization with 5-HT(2A) receptors.

    PubMed

    Santana, Noemí; Mengod, Guadalupe; Artigas, Francesc

    2013-06-01

    The prefrontal cortex (PFC) is involved in behavioural control and cognitive processes that are altered in schizophrenia. The brainstem monoaminergic systems control PFC function, yet the cells/networks involved are not fully known. Serotonin (5-HT) and norepinephrine (NE) increase PFC neuronal activity through the activation of α(1)-adrenergic receptors (α(1)ARs) and 5-HT(2A) receptors (5-HT(2A)Rs), respectively. Neurochemical and behavioural interactions between these receptors have been reported. Further, classical and atypical antipsychotic drugs share nm in vitro affinity for α(1)ARs while having preferential affinity for D(2) and 5-HT(2A)Rs, respectively. Using double in situ hybridization we examined the cellular expression of α(1)ARs in pyramidal (vGluT1-positive) and GABAergic (GAD(65/67)-positive) neurons in rat PFC and their co-localization with 5-HT(2A)Rs. α(1)ARs are expressed by a high proportion of pyramidal (59-85%) and GABAergic (52-79%) neurons. The expression in pyramidal neurons exhibited a dorsoventral gradient, with a lower percentage of α(1)AR-positive neurons in infralimbic cortex compared to anterior cingulate and prelimbic cortex. The expression of α(1A), α(1B) and α(1D) adrenergic receptors was segregated in different layers and subdivisions. In all them there is a high co-expression with 5-HT(2A)Rs (∼80%). These observations indicate that NE controls the activity of most PFC pyramidal neurons via α(1)ARs, either directly or indirectly, via GABAergic interneurons. Antipsychotic drugs can thus modulate the activity of PFC via α(1)AR blockade. The high co-expression with 5-HT(2A)Rs indicates a convergence of excitatory serotonergic and noradrenergic inputs onto the same neuronal populations. Moreover, atypical antipsychotics may exert a more powerful control of PFC function through the simultaneous blockade of α(1)ARs and 5-HT(2A)Rs. PMID:23195622

  13. Beta-1 adrenergic agonist treatment mitigates negative changes in cancellous bone microarchitecture and inhibits osteocyte apoptosis during disuse.

    PubMed

    Swift, Joshua M; Swift, Sibyl N; Allen, Matthew R; Bloomfield, Susan A

    2014-01-01

    The sympathetic nervous system (SNS) plays an important role in mediating bone remodeling. However, the exact role that beta-1 adrenergic receptors (beta1AR) have in this process has not been elucidated. We have previously demonstrated the ability of dobutamine (DOB), primarily a beta1AR agonist, to inhibit reductions in cancellous bone formation and mitigate disuse-induced loss of bone mass. The purpose of this study was to characterize the independent and combined effects of DOB and hindlimb unloading (HU) on cancellous bone microarchitecture, tissue-level bone cell activity, and osteocyte apoptosis. Male Sprague-Dawley rats, aged 6-mos, were assigned to either normal cage activity (CC) or HU (n = 18/group) for 28 days. Animals were administered either daily DOB (4 mg/kg BW/d) or an equal volume of saline (VEH) (n = 9/gp). Unloading resulted in significantly lower distal femur cancellous BV/TV (-33%), Tb.Th (-11%), and Tb.N (-25%) compared to ambulatory controls (CC-VEH). DOB treatment during HU attenuated these changes in cancellous bone microarchitecture, resulting in greater BV/TV (+29%), Tb.Th (+7%), and Tb.N (+21%) vs. HU-VEH. Distal femur cancellous vBMD (+11%) and total BMC (+8%) were significantly greater in DOB- vs. VEH-treated unloaded rats. Administration of DOB during HU resulted in significantly greater osteoid surface (+158%) and osteoblast surface (+110%) vs. HU-VEH group. Furthermore, Oc.S/BS was significantly greater in HU-DOB (+55%) vs. CC-DOB group. DOB treatment during unloading fully restored bone formation, resulting in significantly greater bone formation rate (+200%) than in HU-VEH rats. HU resulted in an increased percentage of apoptotic cancellous osteocytes (+85%), reduced osteocyte number (-16%), lower percentage of occupied osteocytic lacunae (-30%) as compared to CC-VEH, these parameters were all normalized with DOB treatment. Altogether, these data indicate that beta1AR agonist treatment during disuse mitigates negative

  14. Ion channel regulation by phosphoinositides analyzed with VSPs—PI(4,5)P2 affinity, phosphoinositide selectivity, and PI(4,5)P2 pool accessibility

    PubMed Central

    Rjasanow, Alexandra; Leitner, Michael G.; Thallmair, Veronika; Halaszovich, Christian R.; Oliver, Dominik

    2015-01-01

    The activity of many proteins depends on the phosphoinositide (PI) content of the membrane. E.g., dynamic changes of the concentration of PI(4,5)P2 are cellular signals that regulate ion channels. The susceptibility of a channel to such dynamics depends on its affinity for PI(4,5)P2. Yet, measuring affinities for endogenous PIs has not been possible directly, but has relied largely on the response to soluble analogs, which may not quantitatively reflect binding to native lipids. Voltage-sensitive phosphatases (VSPs) turn over PI(4,5)P2 to PI(4)P when activated by depolarization. In combination with voltage-clamp electrophysiology VSPs are useful tools for rapid and reversible depletion of PI(4,5)P2. Because cellular PI(4,5)P2 is resynthesized rapidly, steady state PI(4,5)P2 changes with the degree of VSP activation and thus depends on membrane potential. Here we show that titration of endogenous PI(4,5)P2 with Ci-VSP allows for the quantification of relative PI(4,5)P2 affinities of ion channels. The sensitivity of inward rectifier and voltage-gated K+ channels to Ci-VSP allowed for comparison of PI(4,5)P2 affinities within and across channel subfamilies and detected changes of affinity in mutant channels. The results also reveal that VSPs are useful only for PI effectors with high binding specificity among PI isoforms, because PI(4,5)P2 depletion occurs at constant overall PI level. Thus, Kir6.2, a channel activated by PI(4,5)P2 and PI(4)P was insensitive to VSP. Surprisingly, despite comparable PI(4,5)P2 affinity as determined by Ci-VSP, the Kv7 and Kir channel families strongly differed in their sensitivity to receptor-mediated depletion of PI(4,5)P2. While Kv7 members were highly sensitive to activation of PLC by Gq-coupled receptors, Kir channels were insensitive even when PI(4,5)P2 affinity was lowered by mutation. We hypothesize that different channels may be associated with distinct pools of PI(4,5)P2 that differ in their accessibility to PLC and VSPs. PMID

  15. The Golgi apparatus is a functionally distinct Ca2+ store regulated by PKA and Epac branches of the β1-adrenergic signaling pathway

    PubMed Central

    Yang, Zhaokang.; Kirton, Hannah M.; MacDougall, David A.; Boyle, John P.; Deuchars, James; Frater, Brenda; Ponnambalam, Sreenivasan; Hardy, Matthew E.; White, Edward; Calaghan, Sarah C.; Peers, Chris; Steele, Derek S.

    2016-01-01

    Ca2+ release from the Golgi apparatus regulates key functions of the organelle, including vesicle trafficking. However, the signaling pathways that control this form of Ca2+ release are poorly understood and evidence of discrete Golgi Ca2+ release events is lacking. Here, we identified the Golgi apparatus as the source of prolonged Ca2+ release events that originate from the nuclear ‘poles’ of primary cardiac cells. Once initiated, Golgi Ca2+ release was unaffected by global depletion of sarcoplasmic reticulum Ca2+, and disruption of the Golgi apparatus abolished Golgi Ca2+ release without affecting sarcoplasmic reticulum function, suggesting functional and anatomical independence of Golgi and sarcoplasmic reticulum Ca2+ stores. Maximal activation of β1-adrenoceptors had only a small stimulating effect on Golgi Ca2+ release. However, inhibition of phosphodiesterase (PDE) 3 or 4, or downregulation of PDE 3 and 4 in heart failure markedly potentiated β1-adrenergic stimulation of Golgi Ca2+ release, consistent with compartmentalization of cAMP signaling within the Golgi apparatus microenvironment. β1-adrenergic stimulation of Golgi Ca2+ release involved activation of both Epac and PKA signaling pathways and CaMKII. Interventions that stimulated Golgi Ca2+ release induced trafficking of vascular growth factor receptor-1 (VEGFR-1) from the Golgi apparatus to the surface membrane. These data establish the Golgi apparatus as a juxtanuclear focal point for Ca2+ and β1-adrenergic signaling, which functions independently from the sarcoplasmic reticulum and the global Ca2+ transients that underlie the primary contractile function of the cell. PMID:26462734

  16. Analyzing Protein-Phosphoinositide Interactions with Liposome Flotation Assays.

    PubMed

    Busse, Ricarda A; Scacioc, Andreea; Schalk, Amanda M; Krick, Roswitha; Thumm, Michael; Kühnel, Karin

    2016-01-01

    Liposome flotation assays are a convenient tool to study protein-phosphoinositide interactions. Working with liposomes resembles physiological conditions more than protein-lipid overlay assays, which makes this method less prone to detect false positive interactions. However, liposome lipid composition must be well-considered in order to prevent nonspecific binding of the protein through electrostatic interactions with negatively charged lipids like phosphatidylserine. In this protocol we use the PROPPIN Hsv2 (homologous with swollen vacuole phenotype 2) as an example to demonstrate the influence of liposome lipid composition on binding and show how phosphoinositide binding specificities of a protein can be characterized with this method. PMID:26552682

  17. Tc-99m-galactosyl-neoglycoalbumin: in vivo characterization of receptor-mediated binding to hepatocyctes

    SciTech Connect

    Vera, D.R.; Krohn, K.A.; Stadalnik, R.C.; Scheibe, P.O.

    1984-04-01

    The biodistribution and kinetics of a receptor-binding hepatic radiopharmaceutical, Tc-99m-galactosyl-neoglycoalbumin (Tc-NGA), were investigated using mammalian and avian models. The radiopharmaceutical exhibited four significant features associated with receptor-mediated binding at the hepatocyte membrane in mammals: (a) high tissue specificity, (b) high molecular specificity, (c) affinity-dependent uptake, and (d) dose-dependent uptake. Diminished hepatic uptake by the avian model illustrated low nonspecific binding. The kinetic sensitivity to ligand-receptor affinity and stoichiometry illustrated the principal feature of receptor-binding radiopharmaceuticals, namely, quantitative assessment of tissue function based upon the biochemical interaction of a ligand and its specific receptor.

  18. Receptor-Mediated Drug Delivery to Macrophages in Chemotherapy of Leishmaniasis

    NASA Astrophysics Data System (ADS)

    Mukhopadhyay, Amitabha; Chaudhuri, Gautam; Arora, Sunil K.; Sehgal, Shobha; Basu, Sandip K.

    1989-05-01

    Methotrexate coupled to maleylated bovine serum albumin was taken up efficiently through the ``scavenger'' receptors present on macrophages and led to selective killing of intracellular Leishmania mexicana amazonensis amastigotes in cultured hamster peritoneal macrophages. The drug conjugate was nearly 100 times as effective as free methotrexate in eliminating the intracellular parasites. Furthermore, in a model of experimental cutaneous leishmaniasis in hamsters, the drug conjugate brought about more than 90% reduction in the size of footpad lesions within 11 days. In contrast, the free drug at a similar concentration did not significantly affect lesion size. These studies demonstrate the potential of receptor-mediated drug delivery in the therapy of macrophage-associated diseases.

  19. [Molecular physiology of receptor mediated endocytosis and its role in overcoming multidrug resistance].

    PubMed

    Severin, E S; Posypanova, G A

    2011-06-01

    Receptor-mediated endocytosis plays important role in the selective uptake of proteins at the plasma membrane of eukaryotic cells. Endocytosis regulates many processes of cell signalling by controlling the number of functional receptors on the cell surface. The article reviews the mechanism of clathrin-dependent endocytosis and the possibility of using this phenomenon for the targeted delivery of drugs. Use of certain proteins as targeting component of drug delivery systems can significantly improve the selectivity of this drug, as well as to overcome the multidrug resistance of cells resulting from the activity of the ABC-transporters. PMID:21874867

  20. The emerging role of phosphoinositide clustering in intracellular trafficking and signal transduction

    PubMed Central

    Picas, Laura; Gaits-Iacovoni, Frederique; Goud, Bruno

    2016-01-01

    Phosphoinositides are master regulators of multiple cellular processes: from vesicular trafficking to signaling, cytoskeleton dynamics, and cell growth. They are synthesized by the spatiotemporal regulated activity of phosphoinositide-metabolizing enzymes. The recent observation that some protein modules are able to cluster phosphoinositides suggests that alternative or complementary mechanisms might operate to stabilize the different phosphoinositide pools within cellular compartments. Herein, we discuss the different known and potential molecular players that are prone to engage phosphoinositide clustering and elaborate on how such a mechanism might take part in the regulation of intracellular trafficking and signal transduction. PMID:27092250

  1. Understanding magnetic nanoparticle osteoblast receptor-mediated endocytosis using experiments and modeling

    NASA Astrophysics Data System (ADS)

    Tran, Nhiem; Webster, Thomas J.

    2013-05-01

    Iron oxide nanoparticles are promising candidates for controlling drug delivery through an external magnetic force to treat a wide range of diseases, including osteoporosis. Previous studies have demonstrated that in the presence of hydroxyapatite coated magnetite (Fe3O4) nanoparticles, osteoblast (or bone forming cell) proliferation and long-term functions (such as calcium deposition) were significantly enhanced. Hydroxyapatite is the major inorganic component of bone. As a further attempt to understand why, in the current study, the uptake of such nanoparticles into osteoblasts was experimentally investigated and mathematically modeled. Magnetite nanoparticles were synthesized using a co-precipitation method and were coated with hydroxyapatite. A cellular uptake experiment at low temperatures indicated that receptor-mediated endocytosis contributed to the internalization of the magnetic nanoparticles into osteoblasts. A model was further developed to explain the uptake of magnetic nanoparticles into osteoblasts using receptor-mediated endocytosis. This model may explain the internalization of hydroxyapatite into osteoblasts to elevate intracellular calcium levels necessary to promote osteoblast functions to treat a wide range of orthopedic problems, including osteoporosis.

  2. Heterogeneity and probabilistic binding contributions to receptor-mediated cell detachment kinetics.

    PubMed Central

    Saterbak, A; Kuo, S C; Lauffenburger, D A

    1993-01-01

    Biospecific cell adhesion is mediated by receptor-ligand bonds. Early theoretical work presented a deterministic analysis of receptor-mediated cell attachment and detachment for a homogeneous cell population. However, initial comparison of a deterministic framework to experimental detachment profiles of model "cells" (antibody-coated latex beads) did not show qualitative or quantitative agreement (Cozens-Roberts, C., D.A. Lauffenburger, and J.A. Quinn. 1990. Biophys. J. 58:857-872). Hence, we determine the contributions of population heterogeneity and probabilistic binding to the detachment behavior of this experimental system which was designed to minimize experimental and theoretical complications. This work also corrects an error in the numerical solution of the probabilistic model of receptor-mediated cell attachment and detachment developed previously (Cozens-Roberts, C., D.A. Lauffenburger, and J.A. Quinn. 1990. Biophys J. 58:841-856). Measurement of the population distribution of the number of receptors per bead has enabled us to explicitly consider the effect of receptor number heterogeneity within the cell-surface contact area. A deterministic framework that incorporates receptor number heterogeneity qualitatively and quantitatively accounts in large part for the model cell detachment data. Using measured and estimated parameter values for the model cell system, we estimate that about 90% of the observed kinetic detachment behavior originates from heterogeneity effects, while about 10% is due to probabilistic binding effects. In general, these relative contributions may differ for other systems. PMID:8396454

  3. Adaptation in sound localization: from GABA(B) receptor-mediated synaptic modulation to perception.

    PubMed

    Stange, Annette; Myoga, Michael H; Lingner, Andrea; Ford, Marc C; Alexandrova, Olga; Felmy, Felix; Pecka, Michael; Siveke, Ida; Grothe, Benedikt

    2013-12-01

    Across all sensory modalities, the effect of context-dependent neural adaptation can be observed at every level, from receptors to perception. Nonetheless, it has long been assumed that the processing of interaural time differences, which is the primary cue for sound localization, is nonadaptive, as its outputs are mapped directly onto a hard-wired representation of space. Here we present evidence derived from in vitro and in vivo experiments in gerbils indicating that the coincidence-detector neurons in the medial superior olive modulate their sensitivity to interaural time differences through a rapid, GABA(B) receptor-mediated feedback mechanism. We show that this mechanism provides a gain control in the form of output normalization, which influences the neuronal population code of auditory space. Furthermore, psychophysical tests showed that the paradigm used to evoke neuronal GABA(B) receptor-mediated adaptation causes the perceptual shift in sound localization in humans that was expected on the basis of our physiological results in gerbils. PMID:24141311

  4. Receptor-mediated binding and uptake of GnRH agonist and antagonist by pituitary cells

    SciTech Connect

    Jennes, L.; Stumpf, W.E.; Conn, P.M.

    1984-01-01

    The intracellular pathway of an enzyme resistant GnRH agonist (D- Lys6 -GnRH) conjugated to ferritin or to colloidal gold was followed in cultured pituitary cells. After an initial uniform distribution over the cell surface of gonadotropes, the electrondense marker was internalized, either individually or in small groups. After longer incubation times, the marker appeared in the lysosomal compartment and the Golgi apparatus, where it could be found in the vesicular as well as cisternal portion. In addition, the receptor-mediated endocytosis of the GnRH antagonist D-p-Glu1-D-Phe2-D-Trp3-D- Lys6 -GnRH was studied by light and electron microscopic autoradiography after 30 and 60 min of incubation to ensure uptake. At both time points, in in vitro as well as in vivo studies, silver grains were localized over cytoplasmic organelles of castration cells, including dilated endoplasmic reticulum, lysosomes, and clear vesicles. No consistent association with cell nuclei, mitochondria, or secretory vesicles could be observed. The results suggest that both agonist and antagonist are binding selectively to the plasma membrane of gonadotropes and subsequently are taken up via receptor-mediated endocytosis for degradation or possible action on synthetic processes.

  5. Target shape dependence in a simple model of receptor-mediated endocytosis and phagocytosis

    PubMed Central

    Richards, David M.; Endres, Robert G.

    2016-01-01

    Phagocytosis and receptor-mediated endocytosis are vitally important particle uptake mechanisms in many cell types, ranging from single-cell organisms to immune cells. In both processes, engulfment by the cell depends critically on both particle shape and orientation. However, most previous theoretical work has focused only on spherical particles and hence disregards the wide-ranging particle shapes occurring in nature, such as those of bacteria. Here, by implementing a simple model in one and two dimensions, we compare and contrast receptor-mediated endocytosis and phagocytosis for a range of biologically relevant shapes, including spheres, ellipsoids, capped cylinders, and hourglasses. We find a whole range of different engulfment behaviors with some ellipsoids engulfing faster than spheres, and that phagocytosis is able to engulf a greater range of target shapes than other types of endocytosis. Further, the 2D model can explain why some nonspherical particles engulf fastest (not at all) when presented to the membrane tip-first (lying flat). Our work reveals how some bacteria may avoid being internalized simply because of their shape, and suggests shapes for optimal drug delivery. PMID:27185939

  6. Receptor-mediated tumor targeting with radiopeptides. Part 1. General principles and methods.

    PubMed

    Eberle, Alex N; Mild, Gabriele

    2009-01-01

    Radiolabeled peptides have become important tools for preclinical cancer research, and in nuclear oncology they serve as diagnostic and more recently also as therapeutic agents. In the latter application, radiolabeled peptides represent a distinct sector of the molecular targeting approach, which in many areas of therapy implements the old "magic bullet" concept by specifically directing the therapeutic agent to the site of action. Although in the past few years the development of receptor-mediated targeting for therapy has been confined to some radiolabeled antibodies and to somatostatin/SRIF, research into an increasing number of radiolabeled peptides and their receptors expressed by different tumors will soon lead to a wider use of peptide radiopharmaceuticals. In a consecutive series of six reviews we present a comprehensive overview of the literature on receptor-mediated tumor targeting with the different radiopeptides currently studied. Part 1 summarizes the concepts and methods of radiopeptide targeting, the selection of radioisotopes, chelators, the criteria of peptide ligand development and some general aspects of diagnostic and therapeutic application of peptide radiopharmaceuticals. PMID:19519167

  7. Interleukin 1 amplifies receptor-mediated activation of phospholipase A2 in 3T3 fibroblasts.

    PubMed Central

    Burch, R M; Connor, J R; Axelrod, J

    1988-01-01

    Human recombinant interleukin 1 alpha (IL-1 alpha) and IL-1 beta stimulated prostaglandin E2 synthesis in 3T3 fibroblasts in a time- and concentration-dependent manner. Enhanced prostaglandin E2 synthesis after IL-1 treatment was apparent by 1 hr and continued to increase for at least 2 days. Half-maximal stimulation occurred at 0.5 pM IL-1 alpha or IL-1 beta, and both interleukins were equally effective, with maximal stimulation occurring in response to 5-10 pM IL-1. In contrast to IL-1, bradykinin stimulation of prostaglandin E2 synthesis is rapid; its effect is maximal by 5 min. In cells that had been pretreated with IL-1 for 24 hr, prostaglandin E2 synthesis in response to bradykinin was amplified more than 10-fold. IL-1 also amplified the receptor-mediated formation of prostaglandin E2 by bombesin and thrombin. The lymphokine did not affect bradykinin receptor number or affinity. IL-1 treatment induced phospholipase A2 and cyclooxygenase but not phospholipase C or prostaglandin E isomerase. It also enhanced bradykinin-stimulated GTPase activity, suggesting possible induction of the GTP-binding regulatory protein coupled to the bradykinin receptor. Thus, IL-1 enhanced receptor-mediated release of prostaglandin E2 in response to bradykinin, bombesin, and thrombin by increasing the cellular levels of phospholipase A2, cyclooxygenase, and GTP-binding regulatory protein(s). PMID:2901097

  8. Opioid receptors mediate direct predictive fear learning: Evidence from one-trial blocking

    PubMed Central

    Cole, Sindy; McNally, Gavan P.

    2007-01-01

    Pavlovian fear learning depends on predictive error, so that fear learning occurs when the actual outcome of a conditioning trial exceeds the expected outcome. Previous research has shown that opioid receptors, including μ-opioid receptors in the ventrolateral quadrant of the midbrain periaqueductal gray (vlPAG), mediate such predictive fear learning. Four experiments reported here used a within-subject one-trial blocking design to study whether opioid receptors mediate a direct or indirect action of predictive error on Pavlovian association formation. In Stage I, rats were trained to fear conditioned stimulus (CS) A by pairing it with shock. In Stage II, CSA and CSB were co-presented once and co-terminated with shock. Two novel stimuli, CSC and CSD, were also co-presented once and co-terminated with shock in Stage II. The results showed one-trial blocking of fear learning (Experiment 1) as well as one-trial unblocking of fear learning when Stage II training employed a higher intensity footshock than was used in Stage I (Experiment 2). Systemic administrations of the opioid receptor antagonist naloxone (Experiment 3) or intra-vlPAG administrations of the selective μ-opioid receptor antagonist CTAP (Experiment 4) prior to Stage II training prevented one-trial blocking. These results show that opioid receptors mediate the direct actions of predictive error on Pavlovian association formation. PMID:17404385

  9. Arabidopsis AtPLC2 Is a Primary Phosphoinositide-Specific Phospholipase C in Phosphoinositide Metabolism and the Endoplasmic Reticulum Stress Response.

    PubMed

    Kanehara, Kazue; Yu, Chao-Yuan; Cho, Yueh; Cheong, Wei-Fun; Torta, Federico; Shui, Guanghou; Wenk, Markus R; Nakamura, Yuki

    2015-09-01

    Phosphoinositides represent important lipid signals in the plant development and stress response. However, multiple isoforms of the phosphoinositide biosynthetic genes hamper our understanding of the pivotal enzymes in each step of the pathway as well as their roles in plant growth and development. Here, we report that phosphoinositide-specific phospholipase C2 (AtPLC2) is the primary phospholipase in phosphoinositide metabolism and is involved in seedling growth and the endoplasmic reticulum (ER) stress responses in Arabidopsis thaliana. Lipidomic profiling of multiple plc mutants showed that the plc2-1 mutant increased levels of its substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, suggesting that the major phosphoinositide metabolic pathway is impaired. AtPLC2 displayed a distinct tissue expression pattern and localized at the plasma membrane in different cell types, where phosphoinositide signaling occurs. The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth. Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles. Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response. PMID:26401841

  10. Arabidopsis AtPLC2 Is a Primary Phosphoinositide-Specific Phospholipase C in Phosphoinositide Metabolism and the Endoplasmic Reticulum Stress Response

    PubMed Central

    Kanehara, Kazue; Yu, Chao-Yuan; Cho, Yueh; Cheong, Wei-Fun; Torta, Federico; Shui, Guanghou; Wenk, Markus R; Nakamura, Yuki

    2015-01-01

    Abstract Phosphoinositides represent important lipid signals in the plant development and stress response. However, multiple isoforms of the phosphoinositide biosynthetic genes hamper our understanding of the pivotal enzymes in each step of the pathway as well as their roles in plant growth and development. Here, we report that phosphoinositide-specific phospholipase C2 (AtPLC2) is the primary phospholipase in phosphoinositide metabolism and is involved in seedling growth and the endoplasmic reticulum (ER) stress responses in Arabidopsis thaliana. Lipidomic profiling of multiple plc mutants showed that the plc2-1 mutant increased levels of its substrates phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate, suggesting that the major phosphoinositide metabolic pathway is impaired. AtPLC2 displayed a distinct tissue expression pattern and localized at the plasma membrane in different cell types, where phosphoinositide signaling occurs. The seedlings of plc2-1 mutant showed growth defect that was complemented by heterologous expression of AtPLC2, suggesting that phosphoinositide-specific phospholipase C activity borne by AtPLC2 is required for seedling growth. Moreover, the plc2-1 mutant showed hypersensitive response to ER stress as evidenced by changes in relevant phenotypes and gene expression profiles. Our results revealed the primary enzyme in phosphoinositide metabolism, its involvement in seedling growth and an emerging link between phosphoinositide and the ER stress response. PMID:26401841

  11. Synthesis and Function of Membrane Phosphoinositides in Budding Yeast, Saccharomyces cerevisiae

    PubMed Central

    Strahl, Thomas; Thorner, Jeremy

    2007-01-01

    It is now well appreciated that derivatives of phosphatidylinositol (PtdIns) are key regulators of many cellular processes in eukaryotes. Of particular interest are phosphoinositides (mono- and polyphosphorylated adducts to the inositol ring in PtdIns), which are located at the cytoplasmic face of cellular membranes. Phosphoinositides serve both a structural and a signaling role via their recruitment of proteins that contain phosphoinositide-binding domains. Phosphoinositides also have a role as precursors of several types of second messengers for certain intracellular signaling pathways. Realization of the importance of phosphoinositides has brought increased attention to characterization of the enzymes that regulate their synthesis, interconversion, and turnover. Here we review the current state of our knowledge about the properties and regulation of the ATP-dependent lipid kinases responsible for synthesis of phosphoinositides and also the additional temporal and spatial controls exerted by the phosphatases and a phospholipase that act on phosphoinositides in yeast. PMID:17382260

  12. Presynaptic adenosine A2A receptors dampen cannabinoid CB1 receptor-mediated inhibition of corticostriatal glutamatergic transmission

    PubMed Central

    Ferreira, S G; Gonçalves, F Q; Marques, J M; Tomé, Â R; Rodrigues, R J; Nunes-Correia, I; Ledent, C; Harkany, T; Venance, L; Cunha, R A; Köfalvi, A

    2015-01-01

    Background and Purpose Both cannabinoid CB1 and adenosine A2A receptors (CB1 receptors and A2A receptors) control synaptic transmission at corticostriatal synapses, with great therapeutic importance for neurological and psychiatric disorders. A postsynaptic CB1−A2A receptor interaction has already been elucidated, but the presynaptic A2A receptor-mediated control of presynaptic neuromodulation by CB1 receptors remains to be defined. Because the corticostriatal terminals provide the major input to the basal ganglia, understanding the interactive nature of converging neuromodulation on them will provide us with novel powerful tools to understand the physiology of corticostriatal synaptic transmission and interpret changes associated with pathological conditions. Experimental Approach Pharmacological manipulation of CB1 and A2A receptors was carried out in brain nerve terminals isolated from rats and mice, using flow synaptometry, immunoprecipitation, radioligand binding, ATP and glutamate release measurement. Whole-cell patch-clamp recordings were made in horizontal corticostriatal slices. Key Results Flow synaptometry showed that A2A receptors were extensively co-localized with CB1 receptor-immunopositive corticostriatal terminals and A2A receptors co-immunoprecipitated CB1 receptors in these purified terminals. A2A receptor activation decreased CB1 receptor radioligand binding and decreased the CB1 receptor-mediated inhibition of high-K+-evoked glutamate release in corticostriatal terminals. Accordingly, A2A receptor activation prevented CB1 receptor-mediated paired-pulse facilitation and attenuated the CB1 receptor-mediated inhibition of synaptic transmission in glutamatergic synapses of corticostriatal slices. Conclusions and Implications Activation of presynaptic A2A receptors dampened CB1 receptor-mediated inhibition of corticostriatal terminals. This constitutes a thus far unrecognized mechanism to modulate the potent CB1 receptor-mediated presynaptic

  13. EP4 prostanoid receptor-mediated vasodilatation of human middle cerebral arteries

    PubMed Central

    Davis, Richard J; Murdoch, Colin E; Ali, Mozam; Purbrick, Stuart; Ravid, Rivka; Baxter, Gordon S; Tilford, Nick; Sheldrick, Robert L G; Clark, Kenneth L; Coleman, Robert A

    2004-01-01

    Dilatation of the cerebral vasculature is recognised to be involved in the pathophysiology of migraine. Furthermore, elevated levels of prostaglandin E2 (PGE2) occur in the blood, plasma and saliva of migraineurs during an attack, suggestive of a contributory role. In the present study, we have characterised the prostanoid receptors involved in the relaxation and contraction of human middle cerebral arteries in vitro. In the presence of indomethacin (3 μM) and the TP receptor antagonist GR32191 (1 μM), PGE2 was found to relax phenylephrine precontracted cerebral arterial rings in a concentration-dependent manner (mean pEC50 8.0±0.1, n=5). Establishment of a rank order of potency using the EP4>EP2 agonist 11-deoxy PGE1, and the EP2>EP4 agonist PGE1-OH (mean pEC50 of 7.6±0.1 (n=6) and 6.4±0.1 (n=4), respectively), suggested the presence of functional EP4 receptors. Furthermore, the selective EP2 receptor agonist butaprost at concentrations <1 μM failed to relax the tissues. Blockade of EP4 receptors with the EP4 receptor antagonists AH23848 and EP4A caused significant rightward displacements in PGE2 concentration–response curves, exhibiting pA2 and pKB values of 5.7±0.1, n=3, and 8.4, n=3, respectively. The IP receptor agonists iloprost and cicaprost relaxed phenylephrine precontracted cerebral arterial rings (mean pEC50 values 8.3±0.1 (n=4) and 8.1±0.1 (n=9), respectively). In contrast, the DP and FP receptor agonists PGD2 and PGF2α failed to cause appreciable relaxation or contraction at concentrations of up to 30 μM. In the absence of phenylephrine contraction and GR32191, the TP receptor agonist U46619 caused concentration-dependent contraction of cerebral artery (mean pEC50 7.4±0.3, n=3). These data demonstrate the presence of prostanoid EP4 receptors mediating PGE2 vasodilatation of human middle cerebral artery. IP receptors mediating relaxation and TP receptors mediating contraction were also functionally demonstrated. PMID:14744815

  14. Separation of fluorescently labeled phosphoinositides and sphingolipids by capillary electrophoresis

    PubMed Central

    Wang, Kelong; Jiang, Dechen; Sims, Christopher E.; Allbritton, Nancy L.

    2012-01-01

    Phosphoinositides (PIs) and sphingolipids regulate many aspects of cell behavior and are often involved in disease processes such as oncogenesis. Capillary electrophoresis with laser induced fluorescence detection (CE-LIF) is emerging as an important tool for enzymatic assays of the metabolism of these lipids, particularly in cell-based formats. Previous separations of phosphoinositide lipids by CE required a complex buffer with polymer additives which had the disadvantages of high cost and/or short shelf life. Further a simultaneous separation of these classes of lipids has not been demonstrated in a robust buffer system. In the current work, a simple separation buffer based on NaH2PO4 and 1-propanol was optimized to separate two sphingolipids and multiple phosphoinositides by CE. The NaH2PO4 concentration, pH, 1-propanol fraction, and a surfactant additive to the buffer were individually optimized to achieve simultaneous separation of the sphingolipids and phosphoinositides. Fluorescein-labeled sphingosine (SFL) and sphingosine 1-phosphate (S1PFL), fluorescein-labeled phosphatidyl-inositol 4,5-bisphosphate (PIP2) and phosphatidyl-inositol 3,4,5-trisphosphate (PIP3), and bodipy-fluorescein (BFL)-labeled PIP2 and PIP3 were separated pairwise and in combination to demonstrate the generalizability of the method. Theoretical plate numbers achieved were as high as 2×105 in separating fluorophore-labeled PIP2 and PIP3. Detection limits for the 6 analytes were in the range of 10−18 to 10−20 mol. The method also showed high reproducibility, as the relative standard deviation of the normalized migration time for each analyte in the simultaneous separation of all 6 compounds was less than 1%. The separation of a mixture composed of diacylglycerol (DAG) and multiple phosphoinositides was also demonstrated. As a final test, fluorescent lipid metabolites formed within cells loaded with BFLPIP2 were separated from a cell lysate as well as a single cell. This simple and

  15. Enzyme induction and histopathology elucidate aryl hydrocarbon receptor-mediated versus non-aryl hydrocarbon receptor-mediated effects of Aroclor 1268 in American mink (Neovison vison).

    PubMed

    Folland, William R; Newsted, John L; Fitzgerald, Scott D; Fuchsman, Phyllis C; Bradley, Patrick W; Kern, John; Kannan, Kurunthachalam; Zwiernik, Matthew J

    2016-03-01

    Polychlorinated biphenyl (PCB) concentrations reported in preferred prey and blubber of bottlenose dolphins from the Turtle-Brunswick River estuary (Georgia, USA) suggest the potential for adverse effects. However, PCBs in Turtle-Brunswick River estuary dolphins are primarily derived from Aroclor 1268, and predicting toxic effects of Aroclor 1268 is uncertain because of the mixture's unique composition and associated physiochemical characteristics. These differences suggest that toxicity benchmarks for other PCB mixtures may not be relevant to dolphins exposed to Aroclor 1268. American mink (Neovison vison) were used as a surrogate model for cetaceans to characterize mechanisms of action associated with Aroclor 1268 exposure. Mink share similarities in phylogeny and life history with cetaceans and are characteristically sensitive to PCBs, making them an attractive surrogate species for marine mammals in ecotoxicity studies. Adult female mink and a subsequent F1 generation were exposed to Aroclor 1268 through diet, and effects on enzyme induction, histopathology, thyroid hormone regulation, hematology, organ weights, and body condition index were compared to a negative control and a 3,3',4,4',5-pentachlorobiphenyl (PCB 126)-positive control. Aroclor 1268 dietary exposure concentrations ranged from 1.8 µg/g wet weight to 29 µg/g wet weight. Anemia, hypothyroidism, and hepatomegaly were observed in mink exposed to Aroclor 1268 beyond various dietary thresholds. Cytochrome P450 induction and squamous epithelial proliferation jaw lesions were low in Aroclor 1268 treatments relative to the positive control. Differences in enzyme induction and the development of squamous epithelial proliferation jaw lesions between Aroclor 1268 treatments and the positive control, coupled with effects observed in Aroclor 1268 treatments not observed in the positive control, indicate that mechanisms additional to the aryl hydrocarbon receptor-mediated pathway are associated with

  16. Receptor-mediated entry of diphtheria toxin into monkey kidney (Vero) cells: electron microscopic evaluation.

    PubMed Central

    Morris, R E; Gerstein, A S; Bonventre, P F; Saelinger, C B

    1985-01-01

    To express toxicity in living cells, diphtheria toxin (DT) must cross a membrane barrier and reach its target in the cytosol. Here we examine the entry of DT into the toxin-sensitive monkey kidney (Vero) cells. Using electron microscopy we directly demonstrated for the first time that DT is internalized by receptor-mediated endocytosis, i.e., via clathrin-coated pits, and enters the endosomal system. Methylamine, which is known to protect cells from DT, stopped the movement of toxin to coated areas of the cell membrane. In the presence of amine, prebound biotinyl-DT was internalized, but toxicity was inhibited. Biochemical evidence revealed that methylamine maintained toxin molecules at a site accessible to neutralization by antitoxin. The data suggest that DT entering Vero cells in the presence of methylamine is sequestered within the cell and does not express toxicity. Images PMID:4066029

  17. Administration of pyrene lipids by receptor-mediated endocytosis and their degradation in skin fibroblasts

    SciTech Connect

    Agmon, V.; Dinur, T.; Cherbu, S.; Dagan, A.; Gatt, S. )

    1991-10-01

    Sphingomyelin and seven glycosphingolipids were labeled with the fluorescent probe pyrene and administered into cultured fibroblasts by receptor-mediated endocytosis. For this purpose pyrene sphingomyelin or mixtures of pyrene glycolipid and unlabeled sphingomyelin were dispersed as small, unilamellar liposomes. Apolipoprotein E was then added and the receptor for this ligand on the cell surface was utilized for uptake of the liposomes and their transport to the lysosomes, where the respective pyrene lipids were degraded. Following incubation with each of the respective pyrene lipids, only the administered compound and the pyrene ceramide were present; intermediate hydrolysis products were not detected. This indicated that, in skin fibroblasts, the lysosomal ceramidase was limiting and controlled the rate of total degradation of the pyrene sphingolipids.

  18. Transferrin protein nanospheres: a nanoplatform for receptor-mediated cancer cell labeling and gene delivery

    NASA Astrophysics Data System (ADS)

    McDonald, Michael A.; Spurlin, Tighe A.; Tona, Alessandro; Elliott, John T.; Halter, Michael; Plant, Anne L.

    2010-02-01

    This paper presents preliminary results on the use of transferrin protein nanospheres (TfpNS) for targeting cancer cells in vitro. Protein nanospheres represent an easily prepared and modifiable nanoplatform for receptor-specific targeting, molecular imaging and gene delivery. Rhodamine B isothiocyanate conjugated TfpNS (RBITC-TfpNS) show significantly enhanced uptake in vitro in SK-MEL-28 human malignant melanoma cells known to overexpress transferrin receptors compared to controls. RBITCTfpNS labeling of the cancer cells is due to transferrin receptor-mediated uptake, as demonstrated by competitive inhibition with native transferrin. Initial fluorescence microscopy studies indicate GFP plasmid can be transfected into melanoma cells via GFP plasmid encapsulated by TfpNS.

  19. Targeting receptor-mediated transport for delivery of biologics across the blood-brain barrier.

    PubMed

    Lajoie, Jason M; Shusta, Eric V

    2015-01-01

    Biologics are an emerging class of medicines with substantial promise to treat neurological disorders such as Alzheimer's disease, stroke, and multiple sclerosis. However, the blood-brain barrier (BBB) presents a formidable obstacle that appreciably limits brain uptake and hence the therapeutic potential of biologics following intravenous administration. One promising strategy for overcoming the BBB to deliver biologics is the targeting of endogenous receptor-mediated transport (RMT) systems that employ vesicular trafficking to transport ligands across the BBB endothelium. If a biologic is modified with an appropriate targeting ligand, it can gain improved access to the brain via RMT. Various RMT-targeting strategies have been developed over the past 20 years, and this review explores exciting recent advances, emphasizing studies that show brain targeting in vivo. PMID:25340933

  20. Receptor-mediated uptake of low density lipoprotein stimulates bile acid synthesis by cultured rat hepatocytes

    SciTech Connect

    Junker, L.H.; Davis, R.A. )

    1989-12-01

    The cellular mechanisms responsible for the lipoprotein-mediated stimulation of bile acid synthesis in cultured rat hepatocytes were investigated. Adding 280 micrograms/ml of cholesterol in the form of human or rat low density lipoprotein (LDL) to the culture medium increased bile acid synthesis by 1.8- and 1.6-fold, respectively. As a result of the uptake of LDL, the synthesis of (14C)cholesterol from (2-14C)acetate was decreased and cellular cholesteryl ester mass was increased. Further studies demonstrated that rat apoE-free LDL and apoE-rich high density lipoprotein (HDL) both stimulated bile acid synthesis 1.5-fold, as well as inhibited the formation of (14C)cholesterol from (2-14C)acetate. Reductive methylation of LDL blocked the inhibition of cholesterol synthesis, as well as the stimulation of bile acid synthesis, suggesting that these processes require receptor-mediated uptake. To identify the receptors responsible, competitive binding studies using 125I-labeled apoE-free LDL and 125I-labeled apoE-rich HDL were performed. Both apoE-free LDL and apoE-rich HDL displayed an equal ability to compete for binding of the other, suggesting that a receptor or a group of receptors that recognizes both apolipoproteins is involved. Additional studies show that hepatocytes from cholestyramine-treated rats displayed 2.2- and 3.4-fold increases in the binding of apoE-free LDL and apoE-rich HDL, respectively. These data show for the first time that receptor-mediated uptake of LDL by the liver is intimately linked to processes activating bile acid synthesis.

  1. Potentiation of NMDA receptor-mediated transmission in striatal cholinergic interneurons.

    PubMed

    Oswald, Manfred J; Schulz, Jan M; Kelsch, Wolfgang; Oorschot, Dorothy E; Reynolds, John N J

    2015-01-01

    Pauses in the tonic firing of striatal cholinergic interneurons (CINs) emerge during reward-related learning in response to conditioning of a neutral cue. We have previously reported that augmenting the postsynaptic response to cortical afferents in CINs is coupled to the emergence of a cell-intrinsic afterhyperpolarization (AHP) underlying pauses in tonic activity. Here we investigated in a bihemispheric rat-brain slice preparation the mechanisms of synaptic plasticity of excitatory afferents to CINs and the association with changes in the AHP. We found that high frequency stimulation (HFS) of commissural corticostriatal afferents from the contralateral hemisphere induced a robust long-term depression (LTD) of postsynaptic potentials (PSP) in CINs. Depression of the PSP of smaller magnitude and duration was observed in response to HFS of the ipsilateral white matter or cerebral cortex. In Mg(2+)-free solution HFS induced NMDA receptor-dependent potentiation of the PSP, evident in both the maximal slope and amplitude of the PSP. The increase in maximal slope corroborates previous findings, and was blocked by antagonism of either D1-like dopamine receptors with SCH23390 or D2-like dopamine receptors with sulpiride during HFS in Mg(2+)-free solution. Potentiation of the slower PSP amplitude component was due to augmentation of the NMDA receptor-mediated potential as this was completely reversed on subsequent application of the NMDA receptor antagonist AP5. HFS similarly potentiated NMDA receptor currents isolated by blockade of AMPA/kainate receptors with CNQX. The plasticity-induced increase in the slow PSP component was directly associated with an increase in the subsequent AHP. Thus plasticity of cortical afferent synapses is ideally suited to influence the cue-induced firing dynamics of CINs, particularly through potentiation of NMDA receptor-mediated synaptic transmission. PMID:25914618

  2. Effect of Alpha-1-Adrenergic Agonist, Midodrine for the Management of Long-Standing Neurogenic Shock in Patient with Cervical Spinal Cord Injury: A Case Report

    PubMed Central

    Kim, Taikwan

    2015-01-01

    We report a rare case of a 71-year-old male patient who had suffered from long-lasting neurogenic shock for 13 weeks after cervical spinal cord injury (SCI) caused by a bicycle accident. The neurogenic shock was resolved dramatically 2 weeks after the administration of alpha-1-adrenergic agonist, midodrine hydrochloride. In usual cases, neurogenic shock tends to improve between 2 and 6 weeks after SCI; however, in a few cases, the shock lasts for several months. In our case, spinal shock lasted for 13 weeks and exhibited very sensitive decline of blood pressure for even a slight decrease of dopamine despite recovered bulbospongiosus reflex. Three days after midodrine hydrochloride was added, hypotension improved dramatically. We discuss our rare case with pertinent literatures. PMID:27169082

  3. Effect of Alpha-1-Adrenergic Agonist, Midodrine for the Management of Long-Standing Neurogenic Shock in Patient with Cervical Spinal Cord Injury: A Case Report.

    PubMed

    Kim, Taikwan; Jwa, Cheol Su

    2015-10-01

    We report a rare case of a 71-year-old male patient who had suffered from long-lasting neurogenic shock for 13 weeks after cervical spinal cord injury (SCI) caused by a bicycle accident. The neurogenic shock was resolved dramatically 2 weeks after the administration of alpha-1-adrenergic agonist, midodrine hydrochloride. In usual cases, neurogenic shock tends to improve between 2 and 6 weeks after SCI; however, in a few cases, the shock lasts for several months. In our case, spinal shock lasted for 13 weeks and exhibited very sensitive decline of blood pressure for even a slight decrease of dopamine despite recovered bulbospongiosus reflex. Three days after midodrine hydrochloride was added, hypotension improved dramatically. We discuss our rare case with pertinent literatures. PMID:27169082

  4. Biophysical Fragment Screening of the β1-Adrenergic Receptor: Identification of High Affinity Arylpiperazine Leads Using Structure-Based Drug Design

    PubMed Central

    2013-01-01

    Biophysical fragment screening of a thermostabilized β1-adrenergic receptor (β1AR) using surface plasmon resonance (SPR) enabled the identification of moderate affinity, high ligand efficiency (LE) arylpiperazine hits 7 and 8. Subsequent hit to lead follow-up confirmed the activity of the chemotype, and a structure-based design approach using protein–ligand crystal structures of the β1AR resulted in the identification of several fragments that bound with higher affinity, including indole 19 and quinoline 20. In the first example of GPCR crystallography with ligands derived from fragment screening, structures of the stabilized β1AR complexed with 19 and 20 were determined at resolutions of 2.8 and 2.7 Å, respectively. PMID:23517028

  5. Muscarinic and alpha(1)-adrenergic mechanisms contribute to the spinal mediation of stimulation-induced antinociception from the pedunculopontine tegmental nucleus in the rat.

    PubMed

    Dias, Quintino M; Crespilho, Simone F; Silveira, João Walter S; Prado, Wiliam A

    2009-05-01

    The effects of intraperitoneal (i.p.) or intrathecal (i.t.) injection of antagonists of acetylcholine, noradrenaline, serotonin, dopamine, opioids and GABA on stimulation-produced antinociception (SPA) from the pedunculopontine tegmental nucleus (PPTg) of rats were studied using the tail-flick test. The electrical stimulation of the PPTg produced a strong and long-lasting increase in tail-flick latency. The intensity and duration of the effect were significantly reduced in rats pretreated with i.p. or i.t. atropine (a non-selective muscarinic cholinergic antagonist), or i.t. phenoxybenzamine or WB 4101 (non-selective and selective alpha(1)-adrenergic antagonists, respectively). Intraperitoneal phenoxybenzamine, i.p. or i.t. methysergide or naloxone (non-selective serotonin and opioid antagonists, respectively), or i.t. idazoxan (a selective alpha(2)-adrenergic antagonist) only reduced the duration of the effect. The duration of SPA from the PPTg was increased by i.t. phaclofen (a GABA(B) antagonist). The effect from the nucleus was not altered following i.t. bicuculline (a GABA(A) antagonist), or i.p. or i.t. mecamylamine, propranolol or haloperidol (non-selective nicotinic cholinergic, beta-adrenergic and dopaminergic antagonists, respectively). Thus, SPA from the PPTg involves the spinal activation of muscarinic and alpha(1)-adrenergic but not nicotinic cholinergic, beta-adrenergic and dopaminergic mechanisms. Serotonergic, endogenous opioid and alpha(2)-adrenergic mechanisms are involved in the duration but not in the intensity of the effect. PMID:19463264

  6. The Golgi-associated PDZ Domain Protein PIST/GOPC Stabilizes the β1-Adrenergic Receptor in Intracellular Compartments after Internalization*

    PubMed Central

    Koliwer, Judith; Park, Minjong; Bauch, Carola; von Zastrow, Mark; Kreienkamp, Hans-Jürgen

    2015-01-01

    Many G-protein-coupled receptors carry C-terminal ligand motifs for PSD-95/discs large/ZO-1 (PDZ) domains; via interaction with PDZ domain-containing scaffold proteins, this allows for integration of receptors into signaling complexes. However, the presence of PDZ domain proteins attached to intracellular membranes suggests that PDZ-type interactions may also contribute to subcellular sorting of receptors. The protein interacting specifically with Tc10 (PIST; also known as GOPC) is a trans-Golgi-associated protein that interacts through its single PDZ domain with a variety of cell surface receptors. Here we show that PIST controls trafficking of the interacting β1-adrenergic receptor both in the anterograde, biosynthetic pathway and during postendocytic recycling. Overexpression and knockdown experiments show that PIST leads to retention of the receptor in the trans-Golgi network (TGN), to the effect that overexpressed PIST reduces activation of the MAPK pathway by β1-adrenergic receptor (β1AR) agonists. Receptors can be released from retention in the TGN by coexpression of the plasma membrane-associated scaffold PSD-95, which allows for transport of receptors to the plasma membrane. Stimulation of β1 receptors and activation of the cAMP pathway lead to relocation of PIST from the TGN to an endosome-like compartment. Here PIST colocalizes with SNX1 and the internalized β1AR and protects endocytosed receptors from lysosomal degradation. In agreement, β1AR levels are decreased in hippocampi of PIST-deficient mice. Our data suggest that PIST contributes to the fine-tuning of β1AR sorting both during biosynthetic and postendocytic trafficking. PMID:25614626

  7. Norepinephrine-induced alteration in the coupling of. cap alpha. /sub 1/-adrenergic receptor occupancy to calcium efflux in rabbit aortic smooth muscle cells

    SciTech Connect

    Colucci, W.S.; Alexander, R.W.

    1986-03-01

    To determine whether ..cap alpha..-adrenergic desensitization of vascular smooth muscle is due to an alteration in ..cap alpha../sub 1/-adrenergic receptor coupling, the authors determined the relationship between receptor occupancy and maximal receptor-coupled Ca/sup 2 +/ efflux in cultured rabbit aortic smooth muscle cells (i) under basal conditions as defined by receptor inactivation with phenoxybenzamine and (ii) after 48 hr of exposure to several concentrations of 1-norepinephrine (NE). Neither phenoxybenzamine nor NE exposure caused a change in binding affinity for (/sup 3/H)prazosin or NE. Maximal (/sup 3/H)prazosin binding capacity and maximal NE-stimulated /sup 45/Ca/sup 2 +/ efflux decreased progressively with exposure of incubated cells to increasing concentrations of phenoxybenzamine or NE. An approximately 80% decrease in maximal (/sup 3/H)prazosin binding capacity caused by either phenoxybenzamine or NE resulted in complete loss of NE-stimulated /sup 45/Ca/sup 2 +/ efflux, indicating that under these conditions approximately 20% of ..cap alpha../sub 1/-adrenergic receptors are not coupled to the Ca/sup 2 +/ efflux. Under basal conditions, the relationship between maximal (/sup 3/H)prazosin binding capacity and maximal NE-stimulated /sup 45/Ca/sup 2 +/ efflux was markedly nonlinear, so that a near maximal response could be elicited by occupancy of only approximately 40% of the receptors. Thus, an alteration in occupancy-response coupling at a step proximal to Ca/sup 2 +/ mobilization and/or influx, rather than a reduction in receptor number, is of primary importance in the process of agonist-induced ..cap alpha..-adrenergic receptor desensitization of vascular smooth muscle cells.

  8. Beneficial effects of desipramine on cognitive function of chronically stressed rats are mediated by alpha1-adrenergic receptors in medial prefrontal cortex

    PubMed Central

    Bondi, Corina O.; Jett, Julianne D.; Morilak, David A.

    2010-01-01

    Chronic stress is a risk factor for many psychopathological conditions, including depression and anxiety disorders. Cognitive impairments associated with prefrontal cortical dysfunction are a major component of such illnesses. Using an attentional set shifting test (AST), we have previously shown that elevating noradrenergic activity in rat medial prefrontal cortex (mPFC) can facilitate cognitive set-shifting, and that chronic unpredictable stress (CUS) caused set-shifting deficits. It is not known, however, if noradrenergic modulatory function is compromised by chronic stress, perhaps contributing to the stress-induced cognitive deficit. Thus, the first study investigated whether acutely elevating noradrenergic activity in mPFC still enhances cognitive function after chronic stress. As previously demonstrated, CUS impaired cognitive set-shifting on the AST. This deficit was abolished by acute systemic administration of the α2-adrenergic autoreceptor antagonist, atipamezole. Microdialysis revealed no differences in extracellular norepinephrine (NE) levels in mPFC of CUS-exposed and unstressed control rats at baseline or during behavioral testing, and comparable increases after atipamezole. In the second experiment, rats were treated chronically with the selective NE reuptake blocker, desipramine, during the CUS treatment through behavioral testing. Again, CUS impaired cognitive set-shifting in vehicle-treated rats, and chronic desipramine treatment prevented such deficits. Acute blockade of post-synaptic α1-adrenergic receptors in mPFC prior to testing blocked the beneficial effect of desipramine on cognitive set-shifting. These results suggest that desipramine restores cognitive set-shifting capability that has been compromised by chronic stress by activating α1-adrenergic receptors in the mPFC. Thus, noradrenergic modulatory capability in mPFC remains intact after CUS, and this represents one possible substrate by which antidepressants may exert their

  9. Studies on the characterization and regulation of alpha-1 adrenergic receptors and (/sup 3/H)WB4101 binding sites in the central nervous system

    SciTech Connect

    Morrow, A.L.

    1985-01-01

    The purpose of these studies has been to resolve the anomalous binding characteristics of two alpha adrenergic receptor ligands, (/sup 3/H)WB4101 and (/sup 3/H)prazosin and to study the regulation of the receptors labeled by these compounds after surgical denervation and chronic drug treatments. Preliminary studies indicated that (/sup 3/H)WB4101 binding sites, which were believed to represent alpha-1 adrenergic receptors, were increased in number following removal of the fimbrial afferents to the hippocampus. This increase was not due to removal of the adrenergic input into this structure since destruction of the locus coeruleus or the dorsal noradrenergic bundle did not produce the up-regulation. Characterization of alpha-1 adrenergic receptors using (/sup 3/H)prazosin and (/sup 3/H)WB4101 revealed evidence for subtypes of alpha-1 receptors designated alpha-1A and alpha-1B. The nanomolar affinity component of (/sup 3/H)WB4101 binding is not adrenergic but serotonergic. The serotonergic agonists, serotonin and 8-hydroxy-dipropylaminotetraline have affinities of 1.5 and 3.0 nM for this site, when studied in the presence of a 30 nM prazosin mask of the alpha-1 component of (/sup 3/H)WB4101 binding. Fimbria transection or 5,7 dihydroxytryptamine injections produced increases in the Bmax of the nanomolar affinity component of (/sup 3/H)WB4101 binding in the presence of a prazosin mask. The up-regulated site showed identical serotonergic pharmacology compared to control tissue. Thus, the author concluded that serotonergic denervation of the hippocampus produces the increase in serotonergic binding sites labeled by (/sup 3/H)WB4101.

  10. Phosphoinositides: Tiny Lipids With Giant Impact on Cell Regulation

    PubMed Central

    2013-01-01

    Phosphoinositides (PIs) make up only a small fraction of cellular phospholipids, yet they control almost all aspects of a cell's life and death. These lipids gained tremendous research interest as plasma membrane signaling molecules when discovered in the 1970s and 1980s. Research in the last 15 years has added a wide range of biological processes regulated by PIs, turning these lipids into one of the most universal signaling entities in eukaryotic cells. PIs control organelle biology by regulating vesicular trafficking, but they also modulate lipid distribution and metabolism via their close relationship with lipid transfer proteins. PIs regulate ion channels, pumps, and transporters and control both endocytic and exocytic processes. The nuclear phosphoinositides have grown from being an epiphenomenon to a research area of its own. As expected from such pleiotropic regulators, derangements of phosphoinositide metabolism are responsible for a number of human diseases ranging from rare genetic disorders to the most common ones such as cancer, obesity, and diabetes. Moreover, it is increasingly evident that a number of infectious agents hijack the PI regulatory systems of host cells for their intracellular movements, replication, and assembly. As a result, PI converting enzymes began to be noticed by pharmaceutical companies as potential therapeutic targets. This review is an attempt to give an overview of this enormous research field focusing on major developments in diverse areas of basic science linked to cellular physiology and disease. PMID:23899561

  11. Frequency and amplitude control of cortical oscillations by phosphoinositide waves.

    PubMed

    Xiong, Ding; Xiao, Shengping; Guo, Su; Lin, Qingsong; Nakatsu, Fubito; Wu, Min

    2016-03-01

    Rhythmicity is prevalent in the cortical dynamics of diverse single and multicellular systems. Current models of cortical oscillations focus primarily on cytoskeleton-based feedbacks, but information on signals upstream of the actin cytoskeleton is limited. In addition, inhibitory mechanisms--especially local inhibitory mechanisms, which ensure proper spatial and kinetic controls of activation--are not well understood. Here, we identified two phosphoinositide phosphatases, synaptojanin 2 and SHIP1, that function in periodic traveling waves of rat basophilic leukemia (RBL) mast cells. The local, phase-shifted activation of lipid phosphatases generates sequential waves of phosphoinositides. By acutely perturbing phosphoinositide composition using optogenetic methods, we showed that pulses of PtdIns(4,5)P2 regulate the amplitude of cyclic membrane waves while PtdIns(3,4)P2 sets the frequency. Collectively, these data suggest that the spatiotemporal dynamics of lipid metabolism have a key role in governing cortical oscillations and reveal how phosphatidylinositol 3-kinases (PI3K) activity could be frequency-encoded by a phosphatase-dependent inhibitory reaction. PMID:26751515

  12. Cooperation of phosphoinositides and BAR domain proteins in endosomal tubulation.

    PubMed

    Shinozaki-Narikawa, Naeko; Kodama, Tatsuhiko; Shibasaki, Yoshikazu

    2006-11-01

    Phosphorylated derivatives of phosphatidylinositol (PtdIns) regulate many intracellular events, including vesicular trafficking and actin remodeling, by recruiting proteins to their sites of function. PtdIns(4,5)-bisphosphate [PI(4,5)P2] and related phosphoinositides are mainly synthesized by type I PtdIns-4-phosphate 5-kinases (PIP5Ks). We found that PIP5K induces endosomal tubules in COS-7 cells. ADP-ribosylation factor (ARF) 6 has been shown to act upstream of PIP5K and regulate endocytic transport and tubulation. ARF GAP with coiled-coil, ankyrin repeat, and pleckstrin homology domains 1 (ACAP1) has guanosine triphosphatase-activating protein (GAP) activity for ARF6. While there were few tubules induced by the expression of ACAP1 alone, numerous endosomal tubules were induced by coexpression of PIP5K and ACAP1. ACAP1 has a pleckstrin homology (PH) domain known to bind phosphoinositide and a Bin/amphiphysin/Rvs (BAR) domain that has been reported to detect membrane curvature. Truncated and point mutations in the ACAP1 BAR and PH domains revealed that both BAR and PH domains are required for tubulation. These results suggest that two ARF6 downstream molecules, PIP5K and ACAP1, function together in endosomal tubulation and that phosphoinositide levels may regulate endosomal dynamics. PMID:17010122

  13. Phosphoinositide 3-kinase enhancer (PIKE) in the brain: is it simply a phosphoinositide 3-kinase/Akt enhancer?

    PubMed Central

    Chan, Chi Bun; Ye, Keqiang

    2013-01-01

    Since its discovery in 2000, phosphoinositide 3-kinase enhancer (PIKE) has been recognized as a class of GTPase that controls the enzymatic activities of phosphoinositide 3-kinase (PI3K) and Akt in the central nervous system (CNS). However, recent studies suggest that PIKEs are not only enhancers to PI3K/Akt but also modulators to other kinases including insulin receptor tyrosine kinase and focal adhesion kinases. Moreover, they regulate transcription factors such as signal transducer and activator of transcription and nuclear factor κB. Indeed, PIKE proteins participate in multiple cellular processes including control of cell survival, brain development, memory formation, gene transcription, and metabolism. In this review, we have summarized the functions of PIKE proteins in CNS and discussed their potential implications in various neurological disorders. PMID:22499674

  14. Nicotine impairs cyclooxygenase-2-dependent kinin-receptor-mediated murine airway relaxations

    SciTech Connect

    Xu, Yuan Cardell, Lars-Olaf

    2014-02-15

    Introduction: Cigarette smoke induces local inflammation and airway hyperreactivity. In asthmatics, it worsens the symptoms and increases the risk for exacerbation. The present study investigates the effects of nicotine on airway relaxations in isolated murine tracheal segments. Methods: Segments were cultured for 24 h in the presence of vehicle, nicotine (10 μM) and/or dexamethasone (1 μM). Airway relaxations were assessed in myographs after pre-contraction with carbachol (1 μM). Kinin receptors, cyclooxygenase (COX) and inflammatory mediator expressions were assessed by real-time PCR and confocal-microscopy-based immunohistochemistry. Results: The organ culture procedure markedly increased bradykinin- (selective B{sub 2} receptor agonist) and des-Arg{sup 9}-bradykinin- (selective B{sub 1} receptor agonist) induced relaxations, and slightly increased relaxation induced by isoprenaline, but not that induced by PGE{sub 2}. The kinin receptor mediated relaxations were epithelium-, COX-2- and EP2-receptor-dependent and accompanied by drastically enhanced mRNA levels of kinin receptors, as well as inflammatory mediators MCP-1 and iNOS. Increase in COX-2 and mPGES-1 was verified both at mRNA and protein levels. Nicotine selectively suppressed the organ-culture-enhanced relaxations induced by des-Arg{sup 9}-bradykinin and bradykinin, at the same time reducing mPGES-1 mRNA and protein expressions. α7-nicotinic acetylcholine receptor inhibitors α-bungarotoxin and MG624 both blocked the nicotine effects on kinin B{sub 2} receptors, but not those on B{sub 1}. Dexamethasone completely abolished kinin-induced relaxations. Conclusion: It is tempting to conclude that a local inflammatory process per se could have a bronchoprotective component by increasing COX-2 mediated airway relaxations and that nicotine could impede this safety mechanism. Dexamethasone further reduced airway inflammation together with relaxations. This might contribute to the steroid resistance seen in

  15. The phosphoinositide 3-kinase signaling pathway is involved in the control of modified low-density lipoprotein uptake by human macrophages.

    PubMed

    Michael, Daryn R; Davies, Thomas S; Laubertová, Lucia; Gallagher, Hayley; Ramji, Dipak P

    2015-03-01

    The transformation of macrophages into lipid-loaded foam cells is a critical early event in the pathogenesis of atherosclerosis. Both receptor-mediated uptake of modified LDL, mediated primarily by scavenger receptors-A (SR-A) and CD36 along with other proteins such as lipoprotein lipase (LPL), and macropinocytosis contribute to macrophage foam cell formation. The signaling pathways that are involved in the control of foam cell formation are not fully understood. In this study, we have investigated the role of phosphoinositide 3-kinase (PI3K) in relation to foam cell formation in human macrophages. The pan PI3K inhibitor LY294002 attenuated the uptake of modified LDL and macropinocytosis, as measured by Lucifer Yellow uptake, by human macrophages. In addition, the expression of SR-A, CD36 and LPL was attenuated by LY294002. The use of isoform-selective PI3K inhibitors showed that PI3K-β, -γ and -δ were all required for the expression of SR-A and CD36 whereas only PI3K-γ was necessary in the case of LPL. These studies reveal a pivotal role of PI3K in the control of macrophage foam cell formation and provide further evidence for their potential as therapeutic target against atherosclerosis. PMID:25663263

  16. Evidence that 5-HT1D receptors mediate inhibition of sympathetic ganglionic transmission in anaesthetized cats.

    PubMed Central

    Jones, J. F.; Martin, G. R.; Ramage, A. G.

    1995-01-01

    In anaesthetized cats, 5-carboxamidotryptamine (5-CT) or 5-hydroxytryptamine (5-HT) (0.3-300 micrograms kg-1,i.v.) inhibited the postganglionic compound action potential evoked by preganglionic electrical stimulation (0.5 Hz) with a similar potency in the stellate and splanchnic ganglia. In the 5-HT experiments transmission thorough the inferior mesenteric ganglia was also recorded. The maximal inhibitory effect of 5-HT was greater on the stellate and splanchnic ganglia (60 +/- 4 and 52 +/- 5%) than on the inferior mesenteric (15 +/- 2%). The effects of 5-HT were unaffected by pretreatment with antagonists (1 mg kg-1;i.v.) for 5-HT2 (BW501C67), 5-HT1A (WAY-100635) and 5-HT3 receptors (ondansetron). However, responses to both 5-HT and 5-CT were attenuated significantly by GR127935 (1 mg kg-1) except the responses to 5-HT at the inferior mesenteric ganglia. These results are consistent with the involvement of 5-HT1D receptors mediating inhibition of sympathetic ganglionic transmission in vivo. PMID:8528548

  17. Hormone stimulation of androgen receptor mediates dynamic changes in DNA methylation patterns at regulatory elements

    PubMed Central

    Dhiman, Vineet K.; Attwood, Kristopher; Campbell, Moray J.; Smiraglia, Dominic J.

    2015-01-01

    DNA methylation is an epigenetic modification that contributes to stable gene silencing by interfering with the ability of transcriptional regulators to bind to DNA. Recent findings have revealed that hormone stimulation of certain nuclear receptors induces rapid, dynamic changes in DNA methylation patterns alongside transcriptional responses at a subset of target loci, over time. However, the ability of androgen receptor (AR) to dynamically regulate gene transcription is relatively under-studied and its role in the regulation of DNA methylation patterns remains to be elucidated. Here we demonstrate in normal prostate cells that hormone stimulated AR activity results in dynamic changes in the transcription rate and DNA methylation patterns at the AR target genes, TIPARP and SGK1. Time-resolved chromatin immunoprecipitation experiments on the SGK1 locus reveals dynamic recruitment of AR and RNA Polymerase II, as well as the recruitment of proteins involved in the DNA demethylation process, TET1 and TDG. Furthermore, the presence of DNA methylation at dynamic regions inhibits protein binding and transcriptional activity of SGK1. These findings establish AR activity as a contributing factor to the dynamic regulation of DNA methylation patterns at target genes in prostate biology and infer further complexity involved in nuclear receptor mediation of transcriptional regulation. PMID:26646795

  18. S-nitrosylated SHP-2 contributes to NMDA receptor-mediated excitotoxicity in acute ischemic stroke

    PubMed Central

    Shi, Zhong-Qing; Sunico, Carmen R.; McKercher, Scott R.; Cui, Jiankun; Feng, Gen-Sheng; Nakamura, Tomohiro; Lipton, Stuart A.

    2013-01-01

    Overproduction of nitric oxide (NO) can cause neuronal damage, contributing to the pathogenesis of several neurodegenerative diseases and stroke (i.e., focal cerebral ischemia). NO can mediate neurotoxic effects at least in part via protein S-nitrosylation, a reaction that covalently attaches NO to a cysteine thiol (or thiolate anion) to form an S-nitrosothiol. Recently, the tyrosine phosphatase Src homology region 2-containing protein tyrosine phosphatase-2 (SHP-2) and its downstream pathways have emerged as important mediators of cell survival. Here we report that in neurons and brain tissue NO can S-nitrosylate SHP-2 at its active site cysteine, forming S-nitrosylated SHP-2 (SNO–SHP-2). We found that NMDA exposure in vitro and transient focal cerebral ischemia in vivo resulted in increased levels of SNO–SHP-2. S-Nitrosylation of SHP-2 inhibited its phosphatase activity, blocking downstream activation of the neuroprotective physiological ERK1/2 pathway, thus increasing susceptibility to NMDA receptor-mediated excitotoxicity. These findings suggest that formation of SNO–SHP-2 represents a key chemical reaction contributing to excitotoxic damage in stroke and potentially other neurological disorders. PMID:23382182

  19. Receptor-mediated endocytosis of lysozyme in renal proximal tubules of the frog Rana temporaria.

    PubMed

    Seliverstova, E V; Prutskova, N P

    2015-01-01

    The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endosomes), and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intracellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals. PMID:26150156

  20. Co-receptors are dispensable for tethering receptor-mediated phagocytosis of apoptotic cells.

    PubMed

    Park, B; Lee, J; Moon, H; Lee, G; Lee, D-H; Cho, J Hoon; Park, D

    2015-01-01

    During efferocytosis, phagocytic cells recognize dying cells by receptors binding to ligands specifically exposed on apoptotic cells. Multiple phagocytic receptors and some of their signaling pathways have been identified. However, the downstream pathways of tethering receptors that secure apoptotic cells remain elusive. It is generally assumed that tethering receptors induce signaling to mediate engulfment via interacting with co-receptors or other engulfment receptors located nearby. However, it is poorly understood whether co-receptors for tethering receptors exist during efferocytosis, and, if they do, whether they are indispensable for this process. Here, we address this issue using glycophosphatidylinositol (GPI)-anchored annexin A5 (Anxa5-GPI), an artificial tethering receptor without a putative co-receptor. Phagocytes expressing Anxa5-GPI exhibited enhanced binding of apoptotic cells, resulting in promoted ingestion of apoptotic cells in a phosphatidylserine-dependent manner. Anxa5-GPI-induced phagocytosis of apoptotic cells relied on the known cytoskeletal engulfment machinery but partially depended on the Elmo-Dock-Rac module or the integrin pathway. In addition, Anxa5-GPI-mediated efferocytosis provoked anti-inflammatory responses. Taken together, our work suggests that co-receptors are dispensable for tethering receptor-induced efferocytosis and that tethering receptors mediate the engulfment of apoptotic cells through multiple engulfment signaling pathways. PMID:26018733

  1. Evidence for 5-HT7 receptors mediating relaxation of human colonic circular smooth muscle

    PubMed Central

    Prins, Nicolaas H; Briejer, Michel R; Van Bergen, Patrick J E; Akkermans, Louis M A; Schuurkes, Jan A J

    1999-01-01

    5-HT4 receptors mediate relaxation of human colon circular muscle. However, after 5-HT4 receptor blockade (SB 204070 10 nM), 5-HT still induced a relaxation (pEC50 6.3). 5-HT4 receptors were sufficiently blocked, as the curves to 5-HT obtained in the presence of 10 and 100 nM SB 204070 were indistinguishable. This 5-HT-induced relaxation was tetrodotoxin-insensitive, indicative of a smooth muscle relaxant 5-HT receptor. This, and the rank order of potency (5-CT=5-MeOT=5-HT) suggested involvement of 5-HT1 or 5-HT7 receptors. Mesulergine, a 5-HT7 receptor antagonist at nanomolar concentrations, and a 5-HT1 receptor antagonist at micromolar concentrations, competitively antagonized the 5-HT-induced relaxation (pKB 8.3) and antagonized the relaxation to 5-CT. Methysergide antagonized the 5-HT-induced relaxation (pA2 7.6). It is concluded that the profile of the smooth muscle inhibitory 5-HT receptor resembles that of the 5-HT7 receptor. These data provide the first evidence for functional human 5-HT7 receptors. PMID:10556917

  2. The effect of vanadate on receptor-mediated endocytosis of asialoorosomucoid in rat liver parenchymal cells

    SciTech Connect

    Kindberg, G.M.; Gudmundsen, O.; Berg, T. )

    1990-06-05

    Vanadate is a phosphate analogue that inhibits enzymes involved in phosphate release and transfer reactions. Since such reactions may play important roles in endocytosis, we studied the effects of vanadate on various steps in receptor-mediated endocytosis of asialoorosomucoid labeled with 125I-tyramine-cellobiose (125I-TC-AOM). The labeled degradation products formed from 125I-TC-AOM are trapped in the lysosomes and may therefore serve as lysosomal markers in subcellular fractionation studies. Vanadate reduced the amount of active surface asialoglycoprotein receptors approximately 70%, but had no effect on the rate of internalization and retroendocytosis of ligand. The amount of surface asialoglycoprotein receptors can be reduced by lowering the incubation temperature gradually from 37 to 15 degrees C; vanadate affected only the temperature--sensitive receptors. Vanadate inhibited degradation of 125I-TC-AOM 70-80%. Degradation was much more sensitive to vanadate than binding; half-maximal effects were seen at approximately 1 mM vanadate for binding and approximately 0.1 mM vanadate for degradation. By subcellular fractionation in sucrose and Nycodenz gradients, it was shown that vanadate completely prevented the transfer of 125I-TC-AOM from endosomes to lysosomes. Therefore, the inhibition of degradation by vanadate was indirect; in the presence of vanadate, ligand did not gain access to the lysosomes. The limited degradation in the presence of vanadate took place in a prelysosomal compartment. Vanadate did not affect cell viability and ATP content.

  3. Effects of chronic ethanol administration on receptor mediated endocytosis of asialoorosomucoid (ASOR) in isolated rat hepatocytes

    SciTech Connect

    Casey, C.A.; Kragskow, S.L.; Sorrell, M.F.; Tuma, D.J.

    1986-05-01

    The authors have previously shown that acute and chronic ethanol administration decreases hepatic glycoprotein secretion and membrane biogenesis. The present study was undertaken to determine the effects of chronic ethanol feeding on receptor-mediated endocytosis using the endocytosis of ASOR as a model system. Rats were fed either rat chow ad lib or pair-fed with Lieber-DeCarli diet (ethanol or isocaloric glucose as 36% of total calories) for 5 to 7 weeks. Binding of /sup 125/I ASOR to isolated hepatocytes was studied at 0-4/sup 0/C. Internalization (cell-associated acid precipitable radioactivity) and degradation (acid soluble radioactivity) were determined at 37/sup 0/C for periods up to 240 min. Results were expressed as pmoles ASOR bound, degraded or internalized/10/sup 6/ cells. In ethanol-fed rats the number of pmoles ASOR bound/10/sup 6/ cells was decreased by 40-50% (p< 0.01) as compared to pair-fed and chow-fed animals. Rates of degradation and internalization of the ligand were also 50-70% lower (p< 0.01) in chronic ethanol-treated animals. No significant differences were observed for either binding or internalization of ASOR between chow-fed and pair-fed animals. These results indicate that chronic ethanol feeding decreases internalization and degradation of ASOR in rat hepatocytes.

  4. Progesterone stimulates respiration through a central nervous system steroid receptor-mediated mechanism in cat.

    PubMed Central

    Bayliss, D A; Millhorn, D E; Gallman, E A; Cidlowski, J A

    1987-01-01

    We have examined the effect on respiration of the steroid hormone progesterone, administered either intravenously or directly into the medulla oblongata in anesthetized and paralyzed male and female cats. The carotid sinus and vagus nerves were cut, and end-tidal PCO2 and temperature were kept constant with servo-controllers. Phrenic nerve activity was used to quantitate central respiratory activity. Repeated doses of progesterone (from 0.1 to 2.0 micrograms/kg, cumulative) caused a sustained (greater than 45 min) facilitation of phrenic nerve activity in female and male cats; however, the response was much more variable in females. Progesterone injected into the region of nucleus tractus solitarii, a respiratory-related area in the medulla oblongata, also caused a prolonged stimulation of respiration. Progesterone administration at high concentration by both routes also caused a substantial hypotension. Identical i.v. doses of other classes of steroid hormones (17 beta-estradiol, testosterone, and cortisol) did not elicit the same respiratory effect. Pretreatment with RU 486, a progesterone-receptor antagonist, blocked the facilitatory effect of progesterone. We conclude that progesterone acts centrally through a steroid receptor-mediated mechanism to facilitate respiration. PMID:3478727

  5. Plasma clearance of rat bikunin: evidence for receptor-mediated uptake.

    PubMed Central

    Sjöberg, E M; Blom, A; Larsson, B S; Alston-Smith, J; Sjöquist, M; Fries, E

    1995-01-01

    Bikunin is a chondroitin sulphate-containing protease inhibitor with a molecular mass of 25 kDa. It is secreted into the blood by hepatocytes, and recent observations indicate that it may have an extravascular function. Here we have studied the plasma clearance of bikunin in rats and mice. On intravenous injection, radiolabelled bikunin was found to have a half-life of 10 min; in rats with ligated renal arteries, the clearance time was twice as long, implying that the kidneys account for half the uptake. As judged by gel filtration, the size of bikunin is similar to that of albumin. Autoradiographic analysis of kidneys removed 2 min after the injection of radiolabelled bikunin indicated that, despite its size, bikunin is cleared by glomerular filtration. On ligation of the renal arteries, the plasma concentration of bikunin increased linearly to at least four times normal. This finding shows that the non-renal uptake system is saturated and therefore presumably receptor-mediated. Most of the non-renal uptake of injected bikunin was found to occur in non-visceral tissues such as the skin. Analysis of skin samples by autoradiography after injection of radiolabelled bikunin suggested that bikunin had been transferred from the plasma to the interstitial space. Images Figure 2 Figure 3 Figure 4 Figure 5 PMID:8948446

  6. Toll Receptor-Mediated Hippo Signaling Controls Innate Immunity in Drosophila.

    PubMed

    Liu, Bo; Zheng, Yonggang; Yin, Feng; Yu, Jianzhong; Silverman, Neal; Pan, Duojia

    2016-01-28

    The Hippo signaling pathway functions through Yorkie to control tissue growth and homeostasis. How this pathway regulates non-developmental processes remains largely unexplored. Here, we report an essential role for Hippo signaling in innate immunity whereby Yorkie directly regulates the transcription of the Drosophila IκB homolog, Cactus, in Toll receptor-mediated antimicrobial response. Loss of Hippo pathway tumor suppressors or activation of Yorkie in fat bodies, the Drosophila immune organ, leads to elevated cactus mRNA levels, decreased expression of antimicrobial peptides, and vulnerability to infection by Gram-positive bacteria. Furthermore, Gram-positive bacteria acutely activate Hippo-Yorkie signaling in fat bodies via the Toll-Myd88-Pelle cascade through Pelle-mediated phosphorylation and degradation of the Cka subunit of the Hippo-inhibitory STRIPAK PP2A complex. Our studies elucidate a Toll-mediated Hippo signaling pathway in antimicrobial response, highlight the importance of regulating IκB/Cactus transcription in innate immunity, and identify Gram-positive bacteria as extracellular stimuli of Hippo signaling under physiological settings. PMID:26824654

  7. Receptor-Mediated Entry of Pristine Octahedral DNA Nanocages in Mammalian Cells.

    PubMed

    Vindigni, Giulia; Raniolo, Sofia; Ottaviani, Alessio; Falconi, Mattia; Franch, Oskar; Knudsen, Birgitta R; Desideri, Alessandro; Biocca, Silvia

    2016-06-28

    DNA offers excellent programming properties for the generation of nanometer-scaled polyhedral structures with a broad variety of potential applications. Translation to biomedical applications requires improving stability in biological fluids, efficient and selective cell binding, and/or internalization of the assembled DNA nanostructures. Here, we report an investigation on the selective mechanism of cellular uptake of pristine DNA nanocages in cells expressing the receptor "oxidized low-density lipoprotein receptor-1" (LOX-1), a scavenger receptor associated with cardiovascular diseases and, more recently, identified as a tumor marker. For this purpose a truncated octahedral DNA nanocage functionalized with a single biotin molecule, which allows DNA cage detection through the biotin-streptavidin assays, was constructed. The results indicate that DNA nanocages are stable in biological fluids, including human serum, and are selectively bound and very efficiently internalized in vesicles only in LOX-1-expressing cells. The amount of internalized cages is 30 times higher in LOX-1-expressing cells than in normal fibroblasts, indicating that the receptor-mediated uptake of pristine DNA nanocages can be pursued for a selective cellular internalization. These results open the route for a therapeutic use of pristine DNA cages targeting LOX-1-overexpressing tumor cells. PMID:27214742

  8. Delivery of liposomes into cultured KB cells via folate receptor-mediated endocytosis.

    PubMed

    Lee, R J; Low, P S

    1994-02-01

    Folic acid was covalently conjugated to 66-nm liposomes via spacers of various lengths in an attempt to target the liposomes to KB cells expressing folate receptors. Spacers of short and intermediate lengths were unable to mediate association of folate-conjugated liposomes with receptor-bearing cells, however, use of a 250 A polyethyleneglycol spacer (PEG, M(r) approximately 3350) permitted avid uptake of the liposomes at approximately 2.5 x 10(5) sites/cell. The binding of folate-PEG liposomes to KB cells could be competitively inhibited by excess free folate or by antiserum against the folate receptor, demonstrating the interaction is mediated by the cell surface folate-binding protein. Following binding, cell-associated folate-PEG liposomes were internalized by folate-receptor-mediated endocytosis at 37 degrees C but not at 4 degrees C. These folate-PEG liposomes show potential for delivering large quantities of low molecular weight compounds nondestructively into folate receptor-bearing cells. PMID:8106354

  9. Killing of intracellular Mycobacterium tuberculosis by receptor-mediated drug delivery

    SciTech Connect

    Majumdar, S.; Basu, S.K. )

    1991-01-01

    p-Aminosalicylic acid (PAS) conjugated to maleylated bovine serum albumin (MBSA) was taken up efficiently through high-affinity MBSA-binding sites on macrophages. Binding of the radiolabeled conjugate to cultured mouse peritoneal macrophages at 4 degrees C was competed for by MBSA but not by PAS. At 37 degrees C, the radiolabeled conjugate was rapidly degraded by the macrophages, leading to release of acid-soluble degradation products in the medium. The drug conjugate was nearly 100 times as effective as free PAS in killing the intracellular mycobacteria in mouse peritoneal macrophages infected in culture with Mycobacterium tuberculosis. The killing of intracellular mycobacteria mediated by the drug conjugate was effectively prevented by simultaneous addition of excess MBSA (100 micrograms/ml) or chloroquine (3 microM) to the medium, whereas these agents did not affect the microbicidal action of free PAS. These results suggest that (i) uptake of the PAS-MBSA conjugate was mediated by cell surface receptors on macrophages which recognize MBSA and (ii) lysosomal hydrolysis of the internalized conjugate resulted in intracellular release of a pharmacologically active form of the drug, which led to selective killing of the M. tuberculosis harbored by mouse macrophages infected in culture. This receptor-mediated modality of delivering drugs to macrophages could contribute to greater therapeutic efficacy and minimization of toxic side effects in the management of tuberculosis and other intracellular mycobacterial infections.

  10. Progesterone stimulates respiration through a central nervous system steroid receptor-mediated mechanism in cat.

    PubMed

    Bayliss, D A; Millhorn, D E; Gallman, E A; Cidlowski, J A

    1987-11-01

    We have examined the effect on respiration of the steroid hormone progesterone, administered either intravenously or directly into the medulla oblongata in anesthetized and paralyzed male and female cats. The carotid sinus and vagus nerves were cut, and end-tidal PCO2 and temperature were kept constant with servo-controllers. Phrenic nerve activity was used to quantitate central respiratory activity. Repeated doses of progesterone (from 0.1 to 2.0 micrograms/kg, cumulative) caused a sustained (greater than 45 min) facilitation of phrenic nerve activity in female and male cats; however, the response was much more variable in females. Progesterone injected into the region of nucleus tractus solitarii, a respiratory-related area in the medulla oblongata, also caused a prolonged stimulation of respiration. Progesterone administration at high concentration by both routes also caused a substantial hypotension. Identical i.v. doses of other classes of steroid hormones (17 beta-estradiol, testosterone, and cortisol) did not elicit the same respiratory effect. Pretreatment with RU 486, a progesterone-receptor antagonist, blocked the facilitatory effect of progesterone. We conclude that progesterone acts centrally through a steroid receptor-mediated mechanism to facilitate respiration. PMID:3478727

  11. Receptor-Mediated Endocytosis of Lysozyme in Renal Proximal Tubules of the Frog Rana Temporaria

    PubMed Central

    Seliverstova, E.V.

    2015-01-01

    The mechanism of protein reabsorption in the kidney of lower vertebrates remains insufficiently investigated in spite of raising interest to the amphibian and fish kidneys as a useful model for physiological and pathophysiological examinations. In the present study, we examined the renal tubular uptake and the internalization rote of lysozyme after its intravenous injection in the wintering frog Rana temporaria using immunohisto- and immunocytochemistry and specific markers for some endocytic compartments. The distinct expression of megalin and cubilin in the proximal tubule cells of lysozyme-injected frogs was revealed whereas kidney tissue of control animals showed no positive immunoreactivity. Lysozyme was detected in the apical endocytic compartment of the tubular cells and colocalized with clathrin 10 min after injection. After 20 min, lysozyme was located in the subapical compartment negative to clathrin (endo-somes), and intracellular trafficking of lysozyme was coincided with the distribution of megalin and cubilin. However, internalized protein was retained in the endosomes and did not reach lysosomes within 30 min after treatment that may indicate the inhibition of intra-cellular trafficking in hibernating frogs. For the first time, we provided the evidence that lysozyme is filtered through the glomeruli and absorbed by receptor-mediated clathrin-dependent endocytosis in the frog proximal tubule cells. Thus, the protein uptake in the amphibian mesonephros is mediated by megalin and cubilin that confirms a critical role of endocytic receptors in the renal reabsorption of proteins in amphibians as in mammals. PMID:26150156

  12. Receptor-mediated adhesion phenomena. Model studies with the Radical-Flow Detachment Assay.

    PubMed Central

    Cozens-Roberts, C; Quinn, J A; Lauffenberger, D A

    1990-01-01

    Receptor-mediated cell adhesion phenomena play a vital role in many physiological and biotechnology-related processes. To investigate the physical and chemical factors that influence the cell/surface interaction, we have used a radial flow device, a so-called Radial-Flow Detachment Assay (RFDA). The RFDA allows us to make direct observations of the detachment process under specified experimental conditions. In results reported here, we have studied the detachment of receptor-coated latex beads (prototype cells) from ligand-coated glass surfaces. The receptors and ligands used in this work are complementary antibodies. The beads enable us to examine several aspects of the adhesion process with particles having uniform properties that can be varied systematically. Advantages of the RFDA are many, especially direct observation of cell detachment over a range of shear stresses with quantitative measurement of the adhesive force. We focus our studies on the effects of ligand and receptor densities, along with the influence of pH and ionic strength of the medium. These data are analyzed with a mathematical model based on the theoretical framework of Bell, G. I. (1978. Science [Wash. DC]. 200:618-627) and Hammer, D. A. and D. A. Lauffenburger (1987. Biophys. J. 52:475-487). We demonstrate experimental validation of a theoretical expression for the critical shear stress for particle detachment, and show that it is consistent with reasonable estimates for the receptor-ligand bond affinity. Images FIGURE 2 PMID:2166596

  13. LRP6 Protein Regulates Low Density Lipoprotein (LDL) Receptor-mediated LDL Uptake*

    PubMed Central

    Ye, Zhi-jia; Go, Gwang-Woong; Singh, Rajvir; Liu, Wenzhong; Keramati, Ali Reza; Mani, Arya

    2012-01-01

    Genetic variations in LRP6 gene are associated with high serum LDL cholesterol levels. We have previously shown that LDL clearance in peripheral B-lymphocytes of the LRP6R611C mutation carriers is significantly impaired. In this study we have examined the role of wild type LRP6 (LRP6WT) and LRP6R611C in LDL receptor (LDLR)-mediated LDL uptake. LDL binding and uptake were increased when LRP6WT was overexpressed and modestly reduced when it was knocked down in LDLR-deficient CHO (ldlA7) cells. These findings implicated LRP6 in LDLR-independent cellular LDL binding and uptake. However, LRP6 knockdown in wild type CHO cells resulted in a much greater decline in LDL binding and uptake compared with CHO-ldlA7 cells, suggesting impaired function of the LDLR. LDLR internalization was severely diminished when LRP6 was knocked down and was restored after LRP6 was reintroduced. Further analysis revealed that LRP6WT forms a complex with LDLR, clathrin, and ARH and undergoes a clathrin-mediated internalization after stimulation with LDL. LDLR and LRP6 internalizations as well as LDL uptake were all impaired in CHO-k1 cells expressing LRP6R611C. These studies identify LRP6 as a critical modulator of receptor-mediated LDL endocytosis and introduce a mechanism by which variation in LRP6 may contribute to high serum LDL levels. PMID:22128165

  14. Tonic GABAA Receptor-Mediated Inhibition in the Rat Dorsal Motor Nucleus of the Vagus

    PubMed Central

    Gao, Hong

    2010-01-01

    Type A γ-aminobutyric acid (GABAA) receptors expressed in the dorsal motor nucleus of vagus (DMV) critically regulate the activity of vagal motor neurons and, by inference, the gastrointestinal (GI) tract. Two types of GABAA receptor-mediated inhibition have been identified in the brain, represented by phasic (Iphasic) and tonic (Itonic) inhibitory currents. The hypothesis that Itonic regulates neuron activity was tested in the DMV using whole cell patch-clamp recordings in transverse brain stem slices from rats. An Itonic was present in a subset of DMV neurons, which was determined to be mediated by different receptors than those mediating fast, synaptic currents. Preapplication of tetrodotoxin significantly decreased the resting Itonic amplitude in DMV neurons, suggesting that most of the current was due to action potential (AP)–dependent GABA release. Blocking GABA transport enhanced Itonic and multiple GABA transporters cooperated to regulate Itonic. The Itonic was composed of both a gabazine-insensitive component that was nearly saturated under basal conditions and a gabazine-sensitive component that was activated when extracellular GABA concentration was elevated. Perfusion of THIP (10 μM) significantly increased Itonic amplitude without increasing Iphasic amplitude. The Itonic played a major role in determining the overall excitability of DMV neurons by contributing to resting membrane potential and AP frequency. Our results indicate that Itonic contributes to DMV neuron membrane potential and activity and is thus an important regulator of vagally mediated GI function. PMID:20018836

  15. Pharmacological characterization of the receptors mediating vasoactive intestinal peptide-induced vasodilation in rat aorta

    SciTech Connect

    Turner, J.T.; Bylund, D.B.

    1986-03-01

    Vasoactive intestinal peptide (VIP)-contain nerve fibers associated with blood vessels are widely distributed, both in the central nervous system and in the periphery. VIP has been shown to be a potent vasodilator in a variety of vascular preparations. The authors have evaluated VIP, the VIP fragment 10-28, and several related peptides including PHI-27, PHM-27 and secretin in terms of their potencies in (1) stimulating the synthesis of cyclic AMP, using the method of Shimizu, in aortic rings; and (2) reversing norepinephrine induced contraction in aortic rings. The authors results indicate that VIP is the most potent of the peptides in both experimental protocols and that the rank order potencies of the various peptides are consistent between the two parameters measured. The authors are currently conducting radioligand binding studies with (/sup 125/I)VIP to further characterize the receptors involved. Additionally, the authors experiments in rat aorta indicate that the presence of the endothelial layer is not required for VIP receptor mediated effects to occur. A potential role for synthetic compounds with high specificity for the VIP receptor in treating hypertension is suggested.

  16. Coupling of mitochondrial import and export translocases by receptor-mediated supercomplex formation.

    PubMed

    Qiu, Jian; Wenz, Lena-Sophie; Zerbes, Ralf M; Oeljeklaus, Silke; Bohnert, Maria; Stroud, David A; Wirth, Christophe; Ellenrieder, Lars; Thornton, Nicolas; Kutik, Stephan; Wiese, Sebastian; Schulze-Specking, Agnes; Zufall, Nicole; Chacinska, Agnieszka; Guiard, Bernard; Hunte, Carola; Warscheid, Bettina; van der Laan, Martin; Pfanner, Nikolaus; Wiedemann, Nils; Becker, Thomas

    2013-08-01

    The mitochondrial outer membrane harbors two protein translocases that are essential for cell viability: the translocase of the outer mitochondrial membrane (TOM) and the sorting and assembly machinery (SAM). The precursors of β-barrel proteins use both translocases-TOM for import to the intermembrane space and SAM for export into the outer membrane. It is unknown if the translocases cooperate and where the β-barrel of newly imported proteins is formed. We established a position-specific assay for monitoring β-barrel formation in vivo and in organello and demonstrated that the β-barrel was formed and membrane inserted while the precursor was bound to SAM. β-barrel formation was inhibited by SAM mutants and, unexpectedly, by mutants of the central import receptor, Tom22. We show that the cytosolic domain of Tom22 links TOM and SAM into a supercomplex, facilitating precursor transfer on the intermembrane space side. Our study reveals receptor-mediated coupling of import and export translocases as a means of precursor channeling. PMID:23911324

  17. Co-receptors are dispensable for tethering receptor-mediated phagocytosis of apoptotic cells

    PubMed Central

    Park, B; Lee, J; Moon, H; Lee, G; Lee, D-H; Hoon Cho, J; Park, D

    2015-01-01

    During efferocytosis, phagocytic cells recognize dying cells by receptors binding to ligands specifically exposed on apoptotic cells. Multiple phagocytic receptors and some of their signaling pathways have been identified. However, the downstream pathways of tethering receptors that secure apoptotic cells remain elusive. It is generally assumed that tethering receptors induce signaling to mediate engulfment via interacting with co-receptors or other engulfment receptors located nearby. However, it is poorly understood whether co-receptors for tethering receptors exist during efferocytosis, and, if they do, whether they are indispensable for this process. Here, we address this issue using glycophosphatidylinositol (GPI)-anchored annexin A5 (Anxa5-GPI), an artificial tethering receptor without a putative co-receptor. Phagocytes expressing Anxa5-GPI exhibited enhanced binding of apoptotic cells, resulting in promoted ingestion of apoptotic cells in a phosphatidylserine-dependent manner. Anxa5-GPI-induced phagocytosis of apoptotic cells relied on the known cytoskeletal engulfment machinery but partially depended on the Elmo-Dock-Rac module or the integrin pathway. In addition, Anxa5-GPI-mediated efferocytosis provoked anti-inflammatory responses. Taken together, our work suggests that co-receptors are dispensable for tethering receptor-induced efferocytosis and that tethering receptors mediate the engulfment of apoptotic cells through multiple engulfment signaling pathways. PMID:26018733

  18. Peptides in Receptor-Mediated Radiotherapy: From Design to the Clinical Application in Cancers

    PubMed Central

    Lozza, Catherine; Navarro-Teulon, Isabelle; Pèlegrin, André; Pouget, Jean-Pierre; Vivès, Eric

    2013-01-01

    Short peptides can show high affinity for specific receptors overexpressed on tumor cells. Some of these are already used in cancerology as diagnostic tools and others are in clinical trials for therapeutic applications. Therefore, peptides exhibit great potential as a diagnostic tool but also as an alternative or an additional antitumoral approach upon the covalent attachment of a therapeutic moiety such as a radionuclide or a cytotoxic drug. The chemistry offers flexibility to graft onto the targeting-peptide either fluorine or iodine directly, or metallic radionuclides through appropriate chelating agent. Since short peptides are straightforward to synthesize, there is an opportunity to further improve existing peptides or to design new ones for clinical applications. However, several considerations have to be taken into account to optimize the recognition properties of the targeting-peptide to its receptor, to improve its stability in the biological fluids and its residence in the body, or to increase its overall therapeutic effect. In this review, we highlight the different aspects which need to be considered for the development of an efficient peptide receptor-mediated radionuclide therapy in different neoplasms. PMID:24093086

  19. Contracting human skeletal muscle maintains the ability to blunt α1-adrenergic vasoconstriction during KIR channel and Na+/K+-ATPase inhibition

    PubMed Central

    Crecelius, Anne R; Kirby, Brett S; Hearon, Christopher M; Luckasen, Gary J; Larson, Dennis G; Dinenno, Frank A

    2015-01-01

    Sympathetic vasoconstriction in contracting skeletal muscle is blunted relative to that which occurs in resting tissue; however, the mechanisms underlying this ‘functional sympatholysis’ remain unclear in humans. We tested the hypothesis that α1-adrenergic vasoconstriction is augmented during exercise following inhibition of inwardly rectifying potassium (KIR) channels and Na+/K+-ATPase (BaCl2 + ouabain). In young healthy humans, we measured forearm blood flow (Doppler ultrasound) and calculated forearm vascular conductance (FVC) at rest, during steady-state stimulus conditions (pre-phenylephrine), and after 2 min of phenylephrine (PE; an α1-adrenoceptor agonist) infusion via brachial artery catheter in response to two different stimuli: moderate (15% maximal voluntary contraction) rhythmic handgrip exercise or adenosine infusion. In Protocol 1 (n = 11 subjects) a total of six trials were performed in three conditions: control (saline), combined enzymatic inhibition of nitric oxide (NO) and prostaglandin (PG) synthesis (l-NMMA + ketorolac) and combined inhibition of NO, PGs, KIR channels and Na+/K+-ATPase (l-NMMA + ketorolac + BaCl2 + ouabain). In Protocol 2 (n = 6) a total of four trials were performed in two conditions: control (saline), and combined KIR channel and Na+/K+-ATPase inhibition. All trials occurred after local β-adrenoceptor blockade (propranolol). PE-mediated vasoconstriction was calculated (%ΔFVC) in each condition. Contrary to our hypothesis, despite attenuated exercise hyperaemia of ∼30%, inhibition of KIR channels and Na+/K+-ATPase, combined with inhibition of NO and PGs (Protocol 1) or alone (Protocol 2) did not enhance α1-mediated vasoconstriction during exercise (Protocol 1: −27 ± 3%; P = 0.2 vs. control, P = 0.4 vs.l-NMMA + ketorolac; Protocol 2: −21 ± 7%; P = 0.9 vs. control). Thus, contracting human skeletal muscle maintains the ability to blunt α1-adrenergic vasoconstriction during

  20. Interaction of Ras with phosphoinositide 3-kinase gamma.

    PubMed Central

    Rubio, I; Rodriguez-Viciana, P; Downward, J; Wetzker, R

    1997-01-01

    Phosphoinositide 3-kinase gamma (PI3Kgamma) can be activated in vitro by both alpha and betagamma subunits of heterotrimeric G-proteins and does not interact with p85, the regulatory subunit of PI3Kalpha. Here we demonstrate the binding of Ras to PI3Kgamma in vitro. An N-terminal region of PI3Kgamma was identified as a binding site for Ras. After co-expression with PI3Kgamma in COS-7 cells, Ras induced only a modest increase in PI3K activity compared with the stimulation of PI3Kalpha by Ras in the same cells. PMID:9307042

  1. Silent NMDA receptor-mediated synapses are developmentally regulated in the dorsal horn of the rat spinal cord.

    PubMed

    Baba, H; Doubell, T P; Moore, K A; Woolf, C J

    2000-02-01

    In vitro whole cell patch-clamp recording techniques were utilized to study silent pure-N-methyl-D-aspartate (NMDA) receptor-mediated synaptic responses in lamina II (substantia gelatinosa, SG) and lamina III of the spinal dorsal horn. To clarify whether these synapses are present in the adult and contribute to neuropathic pain, transverse lumbar spinal cord slices were prepared from neonatal, naive adult and adult sciatic nerve transected rats. In neonatal rats, pure-NMDA receptor-mediated excitatory postsynaptic currents (EPSCs) were elicited in SG neurons either by focal intraspinal stimulation (n = 15 of 20 neurons) or focal stimulation of the dorsal root (n = 2 of 7 neurons). In contrast, in slices from naive adult rats, no silent pure-NMDA EPSCs were recorded in SG neurons following focal intraspinal stimulation (n = 27), and only one pure-NMDA EPSC was observed in lamina III (n = 23). Furthermore, in rats with chronic sciatic nerve transection, pure-NMDA EPSCs were elicited by focal intraspinal stimulation in only 2 of 45 SG neurons. Although a large increase in Abeta fiber evoked mixed alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) and NMDA receptor-mediated synapses was detected after sciatic nerve injury, Abeta fiber-mediated pure-NMDA EPSCs were not evoked in SG neurons by dorsal root stimulation. Pure-NMDA receptor-mediated EPSCs are therefore a transient, developmentally regulated phenomenon, and, although they may have a role in synaptic refinement in the immature dorsal horn, they are unlikely to be involved in receptive field plasticity in the adult. PMID:10669507

  2. The Orphan Nuclear Receptor ERRγ Regulates Hepatic CB1 Receptor-Mediated Fibroblast Growth Factor 21 Gene Expression

    PubMed Central

    Jung, Yoon Seok; Lee, Ji-Min; Kim, Don-Kyu; Lee, Yong-Soo; Kim, Ki-Sun; Kim, Yong-Hoon; Kim, Jina; Lee, Myung-Shik; Lee, In-Kyu; Kim, Seong Heon; Cho, Sung Jin; Jeong, Won-Il; Lee, Chul-Ho; Harris, Robert A.; Choi, Hueng-Sik

    2016-01-01

    Background Fibroblast growth factor 21 (FGF21), a stress inducible hepatokine, is synthesized in the liver and plays important roles in glucose and lipid metabolism. However, the mechanism of hepatic cannabinoid type 1 (CB1) receptor-mediated induction of FGF21 gene expression is largely unknown. Results Activation of the hepatic CB1 receptor by arachidonyl-2’-chloroethylamide (ACEA), a CB1 receptor selective agonist, significantly increased FGF21 gene expression. Overexpression of estrogen-related receptor (ERR) γ increased FGF21 gene expression and secretion both in hepatocytes and mice, whereas knockdown of ERRγ decreased ACEA-mediated FGF21 gene expression and secretion. Moreover, ERRγ, but not ERRα and ERRβ, induced FGF21 gene promoter activity. In addition, deletion and mutation analysis of the FGF21 promoter identified a putative ERRγ-binding motif (AGGTGC, a near-consensus response element). A chromatin immunoprecipitation assay revealed direct binding of ERRγ to the FGF21 gene promoter. Finally, GSK5182, an ERRγ inverse agonist, significantly inhibited hepatic CB1 receptor-mediated FGF21 gene expression and secretion. Conclusion Based on our data, we conclude that ERRγ plays a key role in hepatic CB1 receptor-mediated induction of FGF21 gene expression and secretion. PMID:27455076

  3. A pp32-retinoblastoma protein complex modulates androgen receptor-mediated transcription and associates with components of the splicing machinery

    SciTech Connect

    Adegbola, Onikepe; Pasternack, Gary R. . E-mail: gpastern@jhmi.edu

    2005-08-26

    We have previously shown pp32 and the retinoblastoma protein interact. pp32 and the retinoblastoma protein are nuclear receptor transcriptional coregulators: the retinoblastoma protein is a coactivator for androgen receptor, the major regulator of prostate cancer growth, while pp32, which is highly expressed in prostate cancer, is a corepressor of the estrogen receptor. We now show pp32 increases androgen receptor-mediated transcription and the retinoblastoma protein modulates this activity. Using affinity purification and mass spectrometry, we identify members of the pp32-retinoblastoma protein complex as PSF and nonO/p54nrb, proteins implicated in coordinate regulation of nuclear receptor-mediated transcription and splicing. We show that the pp32-retinoblastoma protein complex is modulated during TPA-induced K562 differentiation. Present evidence suggests that nuclear receptors assemble multiprotein complexes to coordinately regulate transcription and mRNA processing. Our results suggest that pp32 and the retinoblastoma protein may be part of a multiprotein complex that coordinately regulates nuclear receptor-mediated transcription and mRNA processing.

  4. The BCL2L1 and PGAM5 axis defines hypoxia-induced receptor-mediated mitophagy

    PubMed Central

    Wu, Hao; Xue, Danfeng; Chen, Guo; Han, Zhe; Huang, Li; Zhu, Chongzhuo; Wang, Xiaohui; Jin, Haijing; Wang, Jun; Zhu, Yushan; Liu, Lei; Chen, Quan

    2014-01-01

    Receptor-mediated mitophagy is one of the major mechanisms of mitochondrial quality control essential for cell survival. We previously have identified FUNDC1 as a mitophagy receptor for selectively removing damaged mitochondria in mammalian systems. A critical unanswered question is how receptor-mediated mitophagy is regulated in response to cellular and environmental cues. Here, we report the striking finding that BCL2L1/Bcl-xL, but not BCL2, suppresses mitophagy mediated by FUNDC1 through its BH3 domain. Mechanistically, we demonstrate that BCL2L1, but not BCL2, interacts with and inhibits PGAM5, a mitochondrially localized phosphatase, to prevent the dephosphorylation of FUNDC1 at serine 13 (Ser13), which activates hypoxia-induced mitophagy. Our results showed that the BCL2L1-PGAM5-FUNDC1 axis is critical for receptor-mediated mitophagy in response to hypoxia and that BCL2L1 possesses unique functions distinct from BCL2. PMID:25126723

  5. Electrophysiological actions of phenytoin on N-methyl-D-aspartate receptor-mediated responses in rat hippocampus in vitro.

    PubMed Central

    Laffling, A. J.; Scherr, P.; McGivern, J. G.; Patmore, L.; Sheridan, R. D.

    1995-01-01

    1. The effects of the anticonvulsant, phenytoin, have been examined on N-methyl-D-aspartate (NMDA) receptor-mediated population spikes in the CA1 region of the rat hippocampus in vitro. 2. The 'conventional' (AMPA receptor-mediated) CA1 population spike, evoked by electrical stimulation of the Schaffer collateral/commissural pathway, was abolished by 5 min treatment with 5 x 10(-6) M 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), after which superfusion with a nominally Mg(2+)-free Krebs solution (containing 5 x 10(-6) M CNQX) led to the appearance of an epileptiform population spike which was fully developed by 30-40 min. 3. The epileptiform population spike was abolished by the non-competitive NMDA antagonist, dizocilpine (1 x 10(-6) M, 20-30 min) and inhibited by the competitive NMDA receptor antagonist, D-CPP (IC50 for reducing the amplitude of the first spike in the train = 8.3 x 10(-7) M), demonstrating that the response was mediated by activation of NMDA receptors and validating its use as an assay for antagonists acting at the NMDA receptor/channel complex. 4. Phenytoin (0.1, 0.3 and 1 x 10(-4) M applied cumulatively for 30 min at each concentration) failed to inhibit the NMDA receptor-mediated epileptiform population response (n = 7 slices).(ABSTRACT TRUNCATED AT 250 WORDS) PMID:7647985

  6. [Changes in the phosphoinositide metabolism in the blood and tissues of benign and malignant uterine tumors].

    PubMed

    Damirov, M M; Sliusar', N N; Kulakov, V I; Bakuleva, I P; Matruk, T A

    1995-01-01

    Measurements of phosphoinositide levels in the blood, immunocompetent cells, and tumors of 105 patients with uterine myomas, 24 patients with cancer of the corpus uteri, and 17 ones with uterine sarcoma showed that the parameters of phosphoinositide metabolism in the blood of patients with tumors of the uterus reliably differed from those in healthy women. The content of phosphatidylinosites and other phosphoinositide fractions in patients with uterine myomas reliably differed from those in patients with malignant tumors of the uterus, this permitting the use of such measurements in the differential diagnosis. Phosphoinositide mechanism of development of tumors of the uterus is discussed, which is related to the "new" phosphoinositides and secondary messengers directly participating in transfer of cell growth signals. PMID:7785738

  7. 3D Structure Prediction of Human β1-Adrenergic Receptor via Threading-Based Homology Modeling for Implications in Structure-Based Drug Designing

    PubMed Central

    Ul-Haq, Zaheer; Saeed, Maria; Halim, Sobia Ahsan; Khan, Waqasuddin

    2015-01-01

    Dilated cardiomyopathy is a disease of left ventricular dysfunction accompanied by impairment of the β1-adrenergic receptor (β1-AR) signal cascade. The disturbed β1-AR function may be based on an elevated sympathetic tone observed in patients with heart failure. Prolonged adrenergic stimulation may induce metabolic and electrophysiological disturbances in the myocardium, resulting in tachyarrhythmia that leads to the development of heart failure in human and sudden death. Hence, β1-AR is considered as a promising drug target but attempts to develop effective and specific drug against this tempting pharmaceutical target is slowed down due to the lack of 3D structure of Homo sapiens β1-AR (hsβADR1). This study encompasses elucidation of 3D structural and physicochemical properties of hsβADR1 via threading-based homology modeling. Furthermore, the docking performance of several docking programs including Surflex-Dock, FRED, and GOLD were validated by re-docking and cross-docking experiments. GOLD and Surflex-Dock performed best in re-docking and cross docking experiments, respectively. Consequently, Surflex-Dock was used to predict the binding modes of four hsβADR1 agonists. This study provides clear understanding of hsβADR1 structure and its binding mechanism, thus help in providing the remedial solutions of cardiovascular, effective treatment of asthma and other diseases caused by malfunctioning of the target protein. PMID:25860348

  8. 3D structure prediction of human β1-adrenergic receptor via threading-based homology modeling for implications in structure-based drug designing.

    PubMed

    Ul-Haq, Zaheer; Saeed, Maria; Halim, Sobia Ahsan; Khan, Waqasuddin

    2015-01-01

    Dilated cardiomyopathy is a disease of left ventricular dysfunction accompanied by impairment of the β1-adrenergic receptor (β1-AR) signal cascade. The disturbed β1-AR function may be based on an elevated sympathetic tone observed in patients with heart failure. Prolonged adrenergic stimulation may induce metabolic and electrophysiological disturbances in the myocardium, resulting in tachyarrhythmia that leads to the development of heart failure in human and sudden death. Hence, β1-AR is considered as a promising drug target but attempts to develop effective and specific drug against this tempting pharmaceutical target is slowed down due to the lack of 3D structure of Homo sapiens β1-AR (hsβADR1). This study encompasses elucidation of 3D structural and physicochemical properties of hsβADR1 via threading-based homology modeling. Furthermore, the docking performance of several docking programs including Surflex-Dock, FRED, and GOLD were validated by re-docking and cross-docking experiments. GOLD and Surflex-Dock performed best in re-docking and cross docking experiments, respectively. Consequently, Surflex-Dock was used to predict the binding modes of four hsβADR1 agonists. This study provides clear understanding of hsβADR1 structure and its binding mechanism, thus help in providing the remedial solutions of cardiovascular, effective treatment of asthma and other diseases caused by malfunctioning of the target protein. PMID:25860348

  9. Discovery of the First Environment-Sensitive Near-Infrared (NIR) Fluorogenic Ligand for α1-Adrenergic Receptors Imaging in Vivo.

    PubMed

    Ma, Zhao; Lin, Yuxing; Cheng, Yanna; Wu, Wenxiao; Cai, Rong; Chen, Shouzhen; Shi, Benkang; Han, Bo; Shi, Xiaodong; Zhou, Yubin; Du, Lupei; Li, Minyong

    2016-03-10

    Fluorescent ligands are gaining popularity as tools to aid GPCR research. Nonetheless, in vivo application of such tools is hampered due to their short excitation wavelengths in the visible range and lack of fluorogenic switch. Here we report the discovery of fluorescent ligands (3a-f) for α1-adrenergic receptors (α1-ARs) by conjugating the environment-sensitive fluorophore cyane 5 (Cy5) with the quinazoline pharmacophore. Among them, the conjugated compound 3a, with acylated piperazine and the shortest carbon chain spacer, exhibited potent binding and remarkable changes in fluorescence (10-fold) upon binding to α1-AR. Furthermore, it could be employed to selectively and specifically label α1-ARs with no washing procedures in single cells, prostate tissue slices, intact tumor xenografts and organs in living mice. Especially, the slice imaging results gave direct and visual evidence that there is a close relationship between α1-ARs and pathological prostate. It is anticipated that our fluorescent tools will find broad applications in the study of α1-AR pharmacology and physiology to aid development of novel therapeutics. PMID:26821136

  10. Relationship between alpha/sub 1/-adrenergic receptor occupancy and regulation of intracellular Ca/sup + +/ in BC3H-1 muscle cells

    SciTech Connect

    Brown, R.D.; Berger, K.D.; Button, D.; Taylor, P.

    1986-05-01

    The relationship between ..cap alpha../sub 1/-adrenergic receptor occupancy by agonists or antagonists and functional response was examined. Receptor occupancy was measured using the antagonist (/sup 3/H)prazosin, and correlated with agonist-elicited unidirectional /sup 45/Ca/sup + +/ efflux. The agonists epinephrine (E), norepinephrine (NE), and phenylephrine (PE) activated /sup 45/Ca/sup + +/ efflux with the order of potency expected for ..cap alpha../sub 1/ receptors (E greater than or equal to NE > PE). A parabolic relationship suggesting the presence of a modest receptor reserve was observed between the number of activatable receptors after equilibration with specified (/sup 3/H)prazosin concentrations and residual /sup 45/Ca/sup + +/ efflux responses elicited by E or NE. A linear relationship was previously observed for PE. Agonist occupancy was independently measured by competition with the initial rate of (/sup 3/H)prazosin association. Both E and NE inhibited (/sup 3/H)prazosin binding over higher concentration ranges than those required to elicit /sup 45/Ca/sup + +/ efflux. Equilibration of cultures with agonist prior to measurement of (/sup 3/H)prazosin binding resulted in small decreases in apparent agonist affinities. These results indicate that BC3H-1 cells possess a small ..cap alpha../sub 1/-receptor reserve for agonist-elicited /sup 45/Ca/sup + +/ efflux which is reflected in the catecholamine agonists, and that exposure to agonist converts receptors to a state of reduced agonist affinity.

  11. Characterization of putative 5-HT7 receptors mediating tachycardia in the cat

    PubMed Central

    Villalón, Carlos M; Heiligers, Jan P C; Centurión, David; De Vries, Peter; Saxena, Pramod R

    1997-01-01

    , sumatriptan (30, 100 and 300 μg kg−1) and indorenate (300 and 1000 μg kg−1) or the 5-HT4 receptor (partial) agonist cisapride (300 and 1000 μg kg−1) were devoid of effects on feline heart rate per se and failed to modify significantly 5-HT-induced tachycardic responses. Based upon the above rank order of agonist potency, the failure of sumatriptan, indorenate or cisapride to produce cardioacceleration and the blockade by a series of drugs showing high affinity for the cloned 5-ht7 receptor, the present results indicate that the 5-HT receptor mediating tachycardia in the cat is operationally similar to other putative 5-HT7 receptors mediating vascular and non-vascular responses (e.g. relaxation of the rabbit femoral vein, canine external carotid and coronary arteries, rat systemic vasculature and guinea-pig ileum). Since these responses represent functional correlates of the 5-ht7 gene product, the 5-HT7 receptor appellation is reinforced. Therefore, the present experimental model, which is not complicated by the presence of other 5-HT receptors, can be utilized to characterize and develop new drugs with potential agonist and antagonist properties at functional 5-HT7 receptors. PMID:9249256

  12. Endothelin stimulates phosphoinositide hydrolysis and PAF synthesis in brain microvessels.

    PubMed

    Catalán, R E; Martínez, A M; Aragonés, M D; Martínez, A; Díaz, G

    1996-11-01

    Treatment of brain microvessels with the three endothelin (ET) isoforms resulted in an increase of phosphoinositide turnover by activation of phospholipase C in a dose- and time-dependent manner. Both ET-1 and ET-2 are maximally effective, whereas the effect evoked by ET-3 was smaller. Concomitantly, there was an enhanced production of a platelet-activating factor (PAF)-like material. This was identified by standard and biological probes in platelets, such as induction of aggregation, phosphatidic acid (PA) production, increase of endogenous protein phosphorylation, and reversal of these responses by a PAF antagonist. The effects evoked by endothelins on phosphoinositide metabolism and PAF production were, to a certain extent, dependent on the presence of extracellular Ca2+. In addition, ET induced changes in Ca2+ dynamics, evoking an initial and rapid intracellular mobilization and influx of Ca2+ and, later, a maintained Ca2+ influx. These findings contribute to the understanding of the pathophysiological role of ET in the blood-brain barrier (BBB). PMID:8898708

  13. Alcohol induced changes in phosphoinositide signaling system in rat brain

    SciTech Connect

    Pandey, S.; Piano, M.; Schwertz, D.; Davis, J.; Pandey, G. )

    1991-03-11

    Agonist-induced phosphoinositide break down functions as a signal generating system in a manner similar to the C-AMP system. In order to examine if the changes produced by chronic ethanol treatment on membrane lipid composition and metabolism effect the cellular functions of the neuron, the authors have examined the effect of chronic ethanol exposure on norepinephrine (NE) serotonin (5HT) and calcium ionophore (CI) stimulated phosphoinositide (PI) hydrolysis in rat cortical slices. Rats were maintained on liber-decarli diet alcohol and control liquid diet containing isocaloric sucrose substitute for two months. They were then sacrificed and brain was removed for determination of PI turnover. 5HT stimulated {sup 3}H- inositol monophosphate ({sup 3}H-IPI) formation was significantly lower in the cortex of alcohol treated rats as compared to control rats. However, neither CI nor NE stimulated IP1 formation was significantly different from control rats. The results thus indicate that chronic exposure to ethanol decreases 5HT induced PI breakdown in rat cortex. In order to examine if this decrease is related to a decrease in 5HT2 receptors, or decreased in coupling of receptor to the effector pathway, the authors are currently determining the number and affinity of 5HT2 receptors in alcohol treated rats.

  14. Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling.

    PubMed Central

    Shepherd, P R; Withers, D J; Siddle, K

    1998-01-01

    Insulin plays a key role in regulating a wide range of cellular processes. However, until recently little was known about the signalling pathways that are involved in linking the insulin receptor with downstream responses. It is now apparent that the activation of class 1a phosphoinositide 3-kinase (PI 3-kinase) is necessary and in some cases sufficient to elicit many of insulin's effects on glucose and lipid metabolism. The lipid products of PI 3-kinase act as both membrane anchors and allosteric regulators, serving to localize and activate downstream enzymes and their protein substrates. One of the major ways these lipid products of PI 3-kinase act in insulin signalling is by binding to pleckstrin homology (PH) domains of phosphoinositide-dependent protein kinase (PDK) and protein kinase B (PKB) and in the process regulating the phosphorylation of PKB by PDK. Using mechanisms such as this, PI 3-kinase is able to act as a molecular switch to regulate the activity of serine/threonine-specific kinase cascades important in mediating insulin's effects on endpoint responses. PMID:9677303

  15. Renal opiate receptor mediation of renin secretion to renal nerve stimulation in the dog.

    PubMed

    Koyama, S; Hosomi, H

    1986-06-01

    The present study was designed to evaluate renal opiate receptor mediation of the renin secretion response to electrical stimulation of the renal nerves in the pentobarbital sodium-anesthetized dog by use of the opiate agonist leucine-enkephalin (Leu-enk) and the opiate antagonist naloxone. In all animals studied, left kidneys were pump perfused at a constant renal blood flow. Renal perfusion pressure (RPP) and glomerular filtration rate (GFR) were unaltered at a stimulation frequency of 1.0 Hz; however, renin secretion rate (RSR) increased significantly in the nontreated group. High-frequency renal nerve stimulation (10 Hz) increased RPP and decreased GFR. RSR at the high-frequency stimulation was significantly augmented in the nontreated group. Renal arterial infusion of either Leu-enk (25 micrograms X kg-1 X min-1) or naloxone (7 micrograms X kg-1 X min-1) did not alter base-line levels of renal hemodynamics and RSR and did not produce significant changes in these variables even when renal nerves were stimulated at the low frequency; however, Leu-enk inhibited RPP and RSR responses to the high-frequency stimulation, and naloxone augmented these responses. Phentolamine (13 micrograms X kg-1 X min-1) prevented renal hemodynamic responses to the renal nerve stimulation, whereas RSR responses to the stimulation were unaffected. Propranolol (8 micrograms X kg-1 X min-1) resulted in decreases in RSR at the renal nerve stimulation despite the presence of changes in renal hemodynamics similar to the other groups. The results indicate that intrarenal opiate receptors may participate in inhibiting renal secretion of renin mediated by the renal nerves when renal vasoconstriction and reduction of GFR occurred at the high-frequency stimulation. PMID:3013030

  16. β2 Adrenergic receptors mediate important electrophysiological effects in human ventricular myocardium

    PubMed Central

    Lowe, M; Rowland, E; Brown, M; Grace, A

    2001-01-01

    OBJECTIVE—To define the effects of β2 adrenergic receptor stimulation on ventricular repolarisation in vivo.
DESIGN—Prospective study.
SETTING—Tertiary referral centre.
PATIENTS—85 patients with coronary artery disease and 22 normal controls.
INTERVENTIONS—Intravenous and intracoronary salbutamol (a β2 adrenergic receptor selective agonist; 10-30 µg/min and 1-10 µg/min), and intravenous isoprenaline (a mixed β1/β2 adrenergic receptor agonist; 1-5 µg/min), infused during fixed atrial pacing.
MAIN OUTCOME MEASURES—QT intervals, QT dispersion, monophasic action potential duration.
RESULTS—In patients with coronary artery disease, salbutamol decreased QTonset and QTpeak but increased QTend duration; QTonset-QTpeak and QTpeak-QTend intervals increased, resulting in T wave prolongation (mean (SEM): 201 (2) ms to 233 (2) ms; p < 0.01). There was a large increase in dispersion of QTonset, QTpeak, and QTend which was more pronounced in patients with coronary artery disease—for example, QTend dispersion: 50 (2) ms baseline v 98 (4) ms salbutamol (controls), and 70 (1) ms baseline v 108 (3) ms salbutamol (coronary artery disease); p < 0.001. Similar responses were obtained with isoprenaline. Monophasic action potential duration at 90% repolarisation shortened during intracoronary infusion of salbutamol, from 278 (4.1) ms to 257 (3.8) ms (p < 0.05).
CONCLUSIONS—β2 adrenergic receptors mediate important electrophysiological effects in human ventricular myocardium. The increase in dispersion of repolarisation provides a mechanism whereby catecholamines acting through this receptor subtype may trigger ventricular arrhythmias.


Keywords: β2 adrenergic receptors; ventricular repolarisation; QT dispersion; salbutamol; isoprenaline PMID:11410561

  17. Scavenger Receptors Mediate the Role of SUMO and Ftz-f1 in Drosophila Steroidogenesis

    PubMed Central

    Talamillo, Ana; Herboso, Leire; Pirone, Lucia; Pérez, Coralia; González, Monika; Sánchez, Jonatan; Mayor, Ugo; Lopitz-Otsoa, Fernando; Rodriguez, Manuel S.; Sutherland, James D.; Barrio, Rosa

    2013-01-01

    SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval–pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor), the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi–mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues. PMID:23637637

  18. Receptor-mediated cell attachment and detachment kinetics. I. Probabilistic model and analysis.

    PubMed Central

    Cozens-Roberts, C.; Lauffenburger, D. A.; Quinn, J. A.

    1990-01-01

    The kinetics of receptor-mediated cell adhesion to a ligand-coated surface play a key role in many physiological and biotechnology-related processes. We present a probabilistic model of receptor-ligand bond formation between a cell and surface to describe the probability of adhesion in a fluid shear field. Our model extends the deterministic model of Hammer and Lauffenburger (Hammer, D.A., and D.A. Lauffenburger. 1987. Biophys. J. 52:475-487) to a probabilistic framework, in which we calculate the probability that a certain number of bonds between a cell and surface exists at any given time. The probabilistic framework is used to account for deviations from ideal, deterministic behavior, inherent in chemical reactions involving relatively small numbers of reacting molecules. Two situations are investigated: first, cell attachment in the absence of fluid stress; and, second, cell detachment in the presence of fluid stress. In the attachment case, we examine the expected variance in bond formation as a function of attachment time; this also provides an initial condition for the detachment case. Focusing then on detachment, we predict transient behavior as a function of key system parameters, such as the distractive fluid force, the receptor-ligand bond affinity and rate constants, and the receptor and ligand densities. We compare the predictions of the probabilistic model with those of a deterministic model, and show how a deterministic approach can yield some inaccurate results; e.g., it cannot account for temporally continuous cell attach mentor detachment, it can underestimate the time needed for cell attachment, it can overestimate the time required for cell detachment for a given level of force, and it can overestimate the force necessary for cell detachment. PMID:2174271

  19. Direct muscarinic and nicotinic receptor-mediated excitation of rat medial vestibular nucleus neurons in vitro

    NASA Technical Reports Server (NTRS)

    Phelan, K. D.; Gallagher, J. P.

    1992-01-01

    We have utilized intracellular recording techniques to investigate the cholinoceptivity of rat medial vestibular nucleus (MVN) neurons in a submerged brain slice preparation. Exogenous application of the mixed cholinergic agonists, acetylcholine (ACh) or carbachol (CCh), produced predominantly membrane depolarization, induction of action potential firing, and decreased input resistance. Application of the selective muscarinic receptor agonist muscarine (MUSC), or the selective nicotinic receptor agonists nicotine (NIC) or 1,1-dimethyl-4-phenylpiperazinium (DMPP) also produced membrane depolarizations. The MUSC-induced depolarization was accompanied by decreased conductance, while an increase in conductance appeared to underlie the NIC- and DMPP-induced depolarizations. The muscarinic and nicotinic receptor mediated depolarizations persisted in tetrodotoxin and/or low Ca2+/high Mg2+ containing media, suggesting direct postsynaptic receptor activation. The MUSC-induced depolarization could be reversibly blocked by the selective muscarinic-receptor antagonist, atropine, while the DMPP-induced depolarization could be reversibly suppressed by the selective ganglionic nicotinic-receptor antagonist, mecamylamine. Some neurons exhibited a transient membrane hyperpolarization during the depolarizing response to CCh or MUSC application. This transient inhibition could be reversibly blocked by the gamma-aminobutyric acid (GABA) antagonist, bicuculline, suggesting that the underlying hyperpolarization results indirectly from the endogenous release of GABA acting at GABA receptors. This study confirms the cholinoceptivity of MVN neurons and establishes that individual MVN cells possess muscarinic as well as nicotinic receptors. The data provide support for a prominent role of cholinergic mechanisms in the direct and indirect regulation of the excitability of MVN neurons.

  20. Scavenger receptors mediate the role of SUMO and Ftz-f1 in Drosophila steroidogenesis.

    PubMed

    Talamillo, Ana; Herboso, Leire; Pirone, Lucia; Pérez, Coralia; González, Monika; Sánchez, Jonatan; Mayor, Ugo; Lopitz-Otsoa, Fernando; Rodriguez, Manuel S; Sutherland, James D; Barrio, Rosa

    2013-04-01

    SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval-pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor), the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi-mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues. PMID:23637637

  1. Reversal of endogenous dopamine receptor silencing in pituitary cells augments receptor-mediated apoptosis.

    PubMed

    Al-Azzawi, Haneen; Yacqub-Usman, Kiren; Richardson, Alan; Hofland, Leo J; Clayton, Richard N; Farrell, William E

    2011-02-01

    Dopamine (DA)-agonist targeting of the DA D(2) receptor (D2R) in prolactinomas is the first-line treatment choice for suppression of prolactin and induction of tumor shrinkage. Resistance to DA agonists seems to be related to receptor number. Using the MMQ and GH3 pituitary cell lines, that either do or do not express D2R, respectively, we explored the epigenetic profile associated with the presence or absence of D2R in these cells lines. These studies led us to explore pharmacological strategies designed to restore receptor expression and thereby potentially augment DA agonist-mediated apoptosis. We show in GH3 cells that the D2R harbors increased CpG island-associated methylation and enrichment for histone H3K27me3. Conversely, MMQ cells and normal pituitaries show enrichment for H3K9Ac and barely detectable H3K27me3. Coculture of GH3 cells with the demethylating agent zebularine and the histone deacetylase inhibitor trichostatin A was responsible for a decrease in CpG island methylation and enrichment for the histone H3K9Ac mark. In addition, challenge of GH3 cells with zebularine alone or coculture with both agents led to expression of endogenous D2R in these cells. Induced expression D2R in GH3 cells was associated with a significant increase in apoptosis indices to challenge with either DA or bromocriptine. Specificity of a receptor-mediated response was established in coincubations with specific D2R antagonist and siRNA approaches in GH3 cell and D2R expressing MMQ cell lines. These studies point to the potential efficacy of combined treatment with epigenetic drugs and DA agonists for the medical management of different pituitary tumor subtypes, resistant to conventional therapies. PMID:21177832

  2. Receptor-mediated mechanism for the transport of prolactin from blood to cerebrospinal fluid

    SciTech Connect

    Walsh, R.J.; Slaby, F.J.; Posner, B.I.

    1987-05-01

    Prolactin (PRL) interacts with areas of the central nervous system which reside behind the blood-brain barrier. While vascular PRL does not cross this barrier, it is readily accessible to the cerebrospinal fluid (CSF) from which it may gain access to the PRL-responsive areas of the brain. Studies were undertaken to characterize the mechanism responsible for the translocation of PRL from blood to CSF. Rats were given external jugular vein injections of (/sup 125/-I)iodo-PRL in the presence or absence of an excess of unlabeled ovine PRL (oPRL), human GH, bovine GH, or porcine insulin. CSF and choroid plexus were removed 60 min later. CSF samples were electrophoresed on sodium dodecyl sulfate-polyacrylamide slab gels and resultant autoradiographs were analyzed with quantitative microdensitometry. The data revealed that unlabeled lactogenic hormones, viz. oPRL and human GH, caused a statistically significant inhibition of (/sup 125/I)iodo-PRL transport from blood to CSF. In contrast, nonlactogenic hormones, viz bovine GH and insulin, had no effect on (/sup 125/I)iodo-PRL transport into the CSF. An identical pattern of competition was observed in the binding of hormone to the choroid plexus. Furthermore, vascular injections of (/sup 125/I)iodo-PRL administered with a range of concentrations of unlabeled oPRL revealed a dose-response inhibition in the transport of (/sup 125/I)iodo-PRL from blood to CSF. The study demonstrates that PRL enters the CSF by a specific, PRL receptor-mediated transport mechanism. The data is consistent with the hypothesis that the transport mechanism resides at the choroid plexus. The existence of this transport mechanism reflects the importance of the cerebroventricular system in PRL-brain interactions.

  3. Receptor-mediated uptake of labeled transferrin by embryonic chicken dorsal root ganglion neurons in culture.

    PubMed

    Markelonis, G J; Oh, T H; Park, L P; Azari, P; Max, S R

    1985-01-01

    Transferrin is a growth-promoting plasma protein which is known to occur within developing neurons. Since little information exists on the process by which transferrin is internalized by neurons, we studied this process using dissociated embryonic chicken dorsal root ganglion neurons in culture. Cultured dorsal root ganglion neurons were incubated in the presence of 3.75 nM (125)I-transferrin at 37°C, the cultures were extensively washed, the neurons were solubilized in a Triton-containing buffer and internalized (125)I-transferrin was quantified with a gamma counter. (125)I-transferrin was internalized in a linear fashion for at least 60 min, and this uptake was abolished by the presence of 1.25 μM unlabeled transferrin. No competition for the uptake of (125)I-transferrin was observed in the presence of 1.25 μM ovalbumin, cytochrome c, hemoglobin, insulin, horseradish peroxidase, aldolase or the carboxyl-terminal fragment ('half-site') of transferrin. By contrast, uptake was inhibited by approximately 50% in the presence of the ammo-terminal fragment ('half-site') of transferrin (1.25 μM) or in the presence of concanavalin A (1.25 μM). The binding of transferrin conjugated to fluorescein isothiocyanate to neurons at 4°C and its subsequent internalization at 37°C was demonstrated by fluorescence microscopy of unfixed cells following incubation of the neurons in the presence of the fluorescently labeled protein. Furthermore, the transferrin receptors were visualized immunocytochemically on the surface membranes of dorsal root ganglion neurons using rabbit antibodies directed against transferrin receptors from chicken reticulocytes. From these data, we conclude that transferrin is internalized by neurons via receptor-mediated endocytosis, and suggest that this protein may serve an important role in the development and survival of dorsal root ganglion neurons. PMID:24874753

  4. Regulation of muscarinic acetylcholine receptor-mediated synaptic responses by GABAB receptors in the rat hippocampus

    PubMed Central

    Morton, Robin A; Manuel, Nick A; Bulters, Diederick O; Cobb, Stuart R; Davies, Ceri H

    2001-01-01

    Both GABAB and muscarinic acetylcholine receptors (mAChRs) influence hippocampal-dependent mnemonic processing. Here the possibility of a direct interaction between GABAB receptors and mAChR-mediated synaptic responses has been studied using intracellular recording in rat hippocampal slices. The GABAB receptor agonist(−)-baclofen (5–10 μm) depressed an atropine-sensitive slow EPSP (EPSPM) and occluded the GABAB-receptor-mediated IPSP (IPSPB) which preceded it. These inhibitory effects were accompanied by postsynaptic hyperpolarization (9 ± 2 mV) and a reduction in cell input resistance (12 ± 3 %). The selective GABAB receptor antagonist CGP 55845A (1 μm) fully reversed the depressant effects of (−)-baclofen (5–10 μm) such that in the combined presence of (−)-baclofen and CGP 55845A the EPSPM was 134 ± 21 % of control. (−)-Baclofen (5–10 μm) caused a small (28 ± 11 %) inhibition of carbachol-induced (3.0 μm) postsynaptic depolarizations and increases in input resistance. CGP 55845A (1 μm) alone caused an increase in the amplitude of the EPSPM (253 ± 74 % of control) and blocked the IPSPB that preceded it. In contrast, the selective GABA uptake inhibitor NNC 05–0711 (10 μm) increased the amplitude of the IPSPB by 141 ± 38 % and depressed the amplitude of the EPSPM by 58 ± 10 %. This inhibition was abolished by CGP 55845A (1 μm). Taken together these data provide good evidence that synaptically released GABA activates GABAB receptors that inhibit mAChR-mediated EPSPs in hippocampal CA1 pyramidal neurones. The mechanism of inhibition may involve both pre- and postsynaptic elements. PMID:11559773

  5. Greater Beta-Adrenergic Receptor Mediated Vasodilation in Women Using Oral Contraceptives

    PubMed Central

    Limberg, Jacqueline K.; Peltonen, Garrett L.; Johansson, Rebecca E.; Harrell, John W.; Kellawan, Jeremy M.; Eldridge, Marlowe W.; Sebranek, Joshua J.; Walker, Benjamin J.; Schrage, William G.

    2016-01-01

    Background: β-adrenergic receptors play an important role in mitigating the pressor effects of sympathetic nervous system activity in young women. Based on recent data showing oral contraceptive use in women abolishes the relationship between muscle sympathetic nervous system activity and blood pressure, we hypothesized forearm blood flow responses to a β-adrenergic receptor agonist would be greater in young women currently using oral contraceptives (OC+, n = 13) when compared to those not using oral contraceptives (OC–, n = 10). Methods: Women (18–35 years) were studied during the early follicular phase of the menstrual cycle (days 1–5) or placebo phase of oral contraceptive use. Forearm blood flow (FBF, Doppler ultrasound) and mean arterial blood pressure (MAP, brachial arterial catheter) were measured at baseline and during graded brachial artery infusion of the β-adrenergic receptor agonist, Isoproterenol (ISO), as well as Acetylcholine (ACH, endothelium-dependent vasodilation) and Nitroprusside (NTP, endothelium-independent vasodilation). Forearm vascular conductance was calculated (FVC = FBF/MAP, ml/min/100 mmHg) and the rise in FVC from baseline during infusion quantified vasodilation (ΔFVC = FVCinfusion − FVCbaseline). Results: ISO increased FVC in both groups (p < 0.01) and ISO-mediated ΔFVC was greater in OC+ compared to OC– (Main effect of group, p = 0.02). Expressing data as FVC and FBF resulted in similar conclusions. FVC responses to both ACH and NTP were also greater in OC+ compared to OC–. Conclusions: These data are the first to demonstrate greater β-adrenergic receptor-mediated vasodilation in the forearm of women currently using oral contraceptives (placebo phase) when compared to those not using oral contraceptives (early follicular phase), and suggest oral contraceptive use influences neurovascular control. PMID:27375493

  6. Characterization of muscarinic receptors mediating relaxation and contraction in the rat iris dilator muscle.

    PubMed Central

    Masuda, Y; Yamahara, N S; Tanaka, M; Ryang, S; Kawai, T; Imaizumi, Y; Watanabe, M

    1995-01-01

    1. The characteristics of muscarinic receptors mediating relaxation and/or contraction in the rat iris dilator muscle were examined. 2. Relaxation was induced in a dilator muscle by application of acetylcholine (ACh) at low doses (3 microM or less) and contraction was induced by high doses. Methacholine and carbachol also showed biphasic effects similar to those of ACh; in contrast, bethanechol, arecoline, pilocarpine and McN-A-343 induced mainly relaxation but no substantial contraction. 3. After parasympathetic denervation by ciliary ganglionectomy, the relaxant response to muscarinic agonists disappeared upon nerve stimulation. Application of McN-A-343 and pilocarpine induced only small contractions in denervated dilator muscles, indicating that these are partial agonists for contraction. 4. pA2 values of pirenzepine, methoctramine, AF-DX 116, himbacine, and 4-DAMP for antagonism to pilocarpine-induced relaxation in normal dilator muscles and those for antagonism to ACh-induced contraction in denervated dilator muscles were determined. The pA2 values for antagonism to relaxation of all these antagonists were most similar to those for M3-type muscarinic receptors. 5. Although pA2 values for contraction of these antagonists, except for methoctramine, were very close to those for relaxation, contraction was not significantly antagonized by methoctramine. Contraction might be mediated by M3-like receptors which have a very low affinity for methoctramine. 6. In conclusion, ACh-induced biphasic responses in rat iris dilator muscles were clearly distinguished from each other by specific muscarinic agonists and parasympathetic denervation, whereas muscarinic receptors could not be subclassified according to the pA2 values of 5 specific antagonists only. PMID:7539696

  7. Evidence for 5-HT1-like receptor-mediated vasoconstriction in human pulmonary artery.

    PubMed Central

    MacLean, M. R.; Clayton, R. A.; Templeton, A. G.; Morecroft, I.

    1996-01-01

    1. The 5-hydroxytryptamine (5-HT) receptors mediating contraction of human isolated pulmonary artery rings were investigated. Responses to the agonists 5-carboximidotryptamine (5-CT, non-selective 5-HT1 agonist), sumatriptan (5-HT1D-like receptor agonist), 5-HT and 8-hydroxy-2-(di-n-propylamino)-tetralin (8-OH-DPAT, 5-HT1A receptor agonist) were studied. Responses to 5-HT and sumatriptan in the presence of the antagonists, methiothepin (non-selective 5-HT1+2-receptor antagonist), ketanserin (5-HT2A receptor antagonist) and the novel antagonist, GR55562 (5-HT1D receptor antagonist) were also studied. 2. All agonists contracted human pulmonary artery ring preparations in the following order of potency 5-CT > 5-HT = sumatriptan > 8-OH-DPAT. Maximum responses to 5-HT, 5-CT and sumatriptan were not significantly different. 3. Methiothepin 1 nM and 10 nM, but not 0.1 nM reduced the maximum contractile responses to 5-HT but did not alter tissue sensitivity to 5-HT. Methiothepin 0.1 nM, 1 nM and 10 nM had a similar effect on responses to sumatriptan. 4. The 5-HT2A receptor antagonist ketanserin (10 nM, 100 nM and 1 microM) also reduced the maximum contractile response to both 5-HT and sumatriptan without affecting tissue sensitivity to these agonists. 5. The novel 5-HT1D receptor antagonist, GR55562, inhibited responses to 5-HT and sumatriptan in a true competitive fashion. 6. The results suggest that the human pulmonary artery has a functional population of 5-HT1D-like receptors which are involved in the contractile response to 5-HT. PMID:8886409

  8. Self-Assembly into Nanoparticles Is Essential for Receptor Mediated Uptake of Therapeutic Antisense Oligonucleotides.

    PubMed

    Ezzat, Kariem; Aoki, Yoshitsugu; Koo, Taeyoung; McClorey, Graham; Benner, Leif; Coenen-Stass, Anna; O'Donovan, Liz; Lehto, Taavi; Garcia-Guerra, Antonio; Nordin, Joel; Saleh, Amer F; Behlke, Mark; Morris, John; Goyenvalle, Aurelie; Dugovic, Branislav; Leumann, Christian; Gordon, Siamon; Gait, Michael J; El-Andaloussi, Samir; Wood, Matthew J A

    2015-07-01

    Antisense oligonucleotides (ASOs) have the potential to revolutionize medicine due to their ability to manipulate gene function for therapeutic purposes. ASOs are chemically modified and/or incorporated within nanoparticles to enhance their stability and cellular uptake, however, a major challenge is the poor understanding of their uptake mechanisms, which would facilitate improved ASO designs with enhanced activity and reduced toxicity. Here, we study the uptake mechanism of three therapeutically relevant ASOs (peptide-conjugated phosphorodiamidate morpholino (PPMO), 2'Omethyl phosphorothioate (2'OMe), and phosphorothioated tricyclo DNA (tcDNA) that have been optimized to induce exon skipping in models of Duchenne muscular dystrophy (DMD). We show that PPMO and tcDNA have high propensity to spontaneously self-assemble into nanoparticles. PPMO forms micelles of defined size and their net charge (zeta potential) is dependent on the medium and concentration. In biomimetic conditions and at low concentrations, PPMO obtains net negative charge and its uptake is mediated by class A scavenger receptor subtypes (SCARAs) as shown by competitive inhibition and RNAi silencing experiments in vitro. In vivo, the activity of PPMO was significantly decreased in SCARA1 knockout mice compared to wild-type animals. Additionally, we show that SCARA1 is involved in the uptake of tcDNA and 2'OMe as shown by competitive inhibition and colocalization experiments. Surface plasmon resonance binding analysis to SCARA1 demonstrated that PPMO and tcDNA have higher binding profiles to the receptor compared to 2'OMe. These results demonstrate receptor-mediated uptake for a range of therapeutic ASO chemistries, a mechanism that is dependent on their self-assembly into nanoparticles. PMID:26042553

  9. Phosphoinositide-3-Kinase Is the Primary Mediator of Phosphoinositide-Dependent Inhibition in Mammalian Olfactory Receptor Neurons

    PubMed Central

    Ukhanov, Kirill; Corey, Elizabeth; Ache, Barry W.

    2016-01-01

    Odorants inhibit as well as excite primary olfactory receptor neurons (ORNs) in many animal species. Growing evidence suggests that inhibition of mammalian ORNs is mediated by phosphoinositide (PI) signaling through activation of phosphoinositide 3-kinase (PI3K), and that canonical adenylyl cyclase III signaling and PI3K signaling interact to provide the basis for ligand-induced selective signaling. As PI3K is known to act in concert with phospholipase C (PLC) in some cellular systems, the question arises as to whether they work together to mediate inhibitory transduction in mammalian ORNs. The present study is designed to test this hypothesis. While we establish that multiple PLC isoforms are expressed in the transduction zone of rat ORNs, that odorants can activate PLC in ORNs in situ, and that pharmacological blockade of PLC enhances the excitatory response to an odorant mixture in some ORNs in conjunction with PI3K blockade, we find that by itself PLC does not account for an inhibitory response. We conclude that PLC does not make a measurable independent contribution to odor-evoked inhibition, and that PI3K is the primary mediator of PI-dependent inhibition in mammalian ORNs. PMID:27147969

  10. VISUALIZIATION OF CELLULAR PHOSPHOINOSITIDE POOLS WITH GFP-FUSED PROTEIN-DOMAINS

    PubMed Central

    Balla, Tamas; Várnai, Péter

    2011-01-01

    This unit describes the method of following phosphoinositide dynamics in live cells. Inositol phospholipids have emerged as universal signaling molecules present in virtually every membrane of eukaryotic cells. Phosphoinositides are present only in tiny amounts compared to structural lipids but are metabolically very active as they are produced and degraded by the numerous inositide kinase and phosphatase enzymes. Phosphoinositides control the membrane-recruitment and activity of many protein signaling-complexes in specific membrane compartments and have been implicated in the regulation of a variety of signaling and trafficking pathways. It has been a challenge to develop methods that allow detection of phosphoinositides at the single cell level. The only available technique in live cell application is based on the use of the same protein domains selected by evolution to recognize cellular phosphoinositides. Some of these isolated protein modules when fused to fluorescent proteins can follow dynamic changes in phosphoinositides. While this technique can provide information on phosphoinositide dynamics in live cells with subcellular resolution and rapidly gained popularity, it also has several limitations that must be taken into account when interpreting the data. Here, we summarize the design and practical use of these constructs and also review important considerations for the interpretation of the data obtained by this technique. PMID:19283730

  11. Synthetic catecholamine triggers β1-adrenergic receptor activation and stimulates cardiotoxicity via oxidative stress mediated apoptotic cell death in rats: Abrogating action of thymol.

    PubMed

    Meeran, M F Nagoor; Jagadeesh, G S; Selvaraj, P

    2016-05-01

    Nowadays, there are considerable interests in the studies which are more connected with the impact of natural antioxidants against the free radical mediated damage in biological systems. Cardiotoxicity is one of the lethal manifestations of cardiovascular diseases (CVDs) which have been associated with the incidence of apoptotic cell death due to oxidative stress. We evaluated the impact of thymol, a dietary monoterpene phenol on isoproterenol (ISO), a synthetic catecholamine and a β1-adrenergic receptor agonist in rats. Thymol (7.5 mg/kg body weight) was pre and co-treated into male albino Wistar rats daily for a period of 7 days. Induction of cardiotoxicity was done by the subcutaneous administration of ISO (100 mg/kg body weight) into rats on 6th and 7th day. Cardiotoxicity in rats was confirmed by the increased levels/activity of serum troponin-T and creatine kinase in the serum alongwith decreased activity of creatine kinase in the heart. ISO induced cardiotoxic rats also showed a significant increase in the concentrations of lipid peroxidation products and a significant decrease in the activities/levels of antioxidants in the myocardium whereas Reverse Transcription Polymerase Chain Reaction study revealed an increased expression of caspase-8, caspase-9 and Fas genes along with a decreased expression of Bcl-xL gene in the myocardium. Thymol pre and co-treated ISO induced cardiotoxic rats showed considerable protective effects on all the biochemical parameters studied. Histopathological and in vitro findings are found in line with our biochemical findings. Thus, the present study revealed that thymol counters ISO induced cardiotoxicity by inhibiting oxidative stress and apoptotic cell death in rats by virtue of its potent antioxidant property. PMID:26996544

  12. Association of 14-3-3 Proteins to β1-Adrenergic Receptors Modulates Kv11.1 K+ Channel Activity in Recombinant Systems

    PubMed Central

    Tutor, Antonio S.; Delpón, Eva; Caballero, Ricardo; Gómez, Ricardo; Núñez, Lucía; Vaquero, Miguel; Tamargo, Juan; Penela, Petronila

    2006-01-01

    We identify a new mechanism for the β1-adrenergic receptor (β1AR)-mediated regulation of human ether-a-go-go–related gene (HERG) potassium channel (Kv11.1). We find that the previously reported modulatory interaction between Kv11.1 channels and 14-3-3ε proteins is competed by wild type β1AR by means of a novel interaction between this receptor and 14-3-3ε. The association between β1AR and 14-3-3ε is increased by agonist stimulation in both transfected cells and heart tissue and requires cAMP-dependent protein kinase (PKA) activity. The β1AR/14-3-3ε association is direct, since it can be recapitulated using purified 14-3-3ε and β1AR fusion proteins and is abolished in cells expressing β1AR phosphorylation–deficient mutants. Biochemical and electrophysiological studies of the effects of isoproterenol on Kv11.1 currents recorded using the whole-cell patch clamp demonstrated that β1AR phosphorylation–deficient mutants do not recruit 14-3-3ε away from Kv11.1 and display a markedly altered agonist-mediated modulation of Kv11.1 currents compared with wild-type β1AR, increasing instead of inhibiting current amplitudes. Interestingly, such differential modulation is not observed in the presence of 14-3-3 inhibitors. Our results suggest that the dynamic association of 14-3-3 proteins to both β1AR and Kv11.1 channels is involved in the adrenergic modulation of this critical regulator of cardiac repolarization and refractoriness. PMID:16914520

  13. S49G and R389G polymorphisms of the β1-adrenergic receptor influence signaling via the cAMP-PKA and ERK pathways

    PubMed Central

    Zhang, Fan

    2013-01-01

    Two functionally important β1-adrenergic receptor (β1AR) polymorphisms have been identified. The R389G polymorphism influences coupling to the Gs-cAMP pathway. R389-β1ARs display enhanced activation of cAMP/PKA; they provide short-term inotropic support but also cause a predisposition to cardiomyopathic decompensation. A second S49G polymorphism is implicated in the evolution of heart failure, but the mechanism remains uncertain. This study shows that position 49 and 389 polymorphisms function in a coordinate manner to influence agonist-dependent cAMP/PKA and ERK responses. cAMP/PKA and ERK responses are more robust in HEK293 cells that heterologously overexpress G49-β1ARs, compared with S49-β1ARs. However, this phenotype is most obvious on a G389-β1AR background; the more robust agonist-dependent cAMP/PKA and ERK responses in R389-β1AR cells effectively obscure the effect of the S49G polymorphism. We also show that isoproterenol (Iso) and carvedilol activate ERK via a similar EGFR-independent mechanism in cells expressing various β1AR haplotypes. However, Iso activates ERK via an Src-independent pathway, but carvedilol-dependent ERK activation requires Src. Since the S49G polymorphism has been linked to changes in β1AR trafficking, we examined whether β1AR polymorphisms influence partitioning to lipid raft membranes. Biochemical fractionation studies show that all four β1AR variants are recovered in buoyant flotillin-enriched membranes; the distinct signaling phenotypes of the different β1AR variants could not be attributed to any gross differences in basal compartmentalization to lipid raft membranes. The allele-specific differences in β1AR signaling phenotypes identified in this study could underlie interindividual differences in responsiveness to β-blocker therapy and clinical outcome in heart failure. PMID:24151242

  14. Analysis of hormone-induced changes of phosphoinositide metabolism in rat liver

    SciTech Connect

    Wallace, M.A.; Fain, J.N.

    1985-01-01

    The relationship between hormone-stimulated phosphoinositide turnover and Ca/sup 2 +/ flux can be investigated using radiolabelled hepatocytes and the subcellular fractions derived from them or from whole liver. Comparison of the results obtained using intact cells to those from subcellular fractions should ultimately lead to a reconstruction of the transmembrane signaling events through which hormone such as vasopressin, angiotensin, and catecholamines acutely activate liver glycogenolysis. The paper reviews hormone-stimulated phosphoinositide metabolism in intact hepatocytes as well as hepatic enzymes involved in phosphoinositide metabolism. Also discussed is the current status of studies on hormone action in broken cell preparations in liver.

  15. Pharmacological and biochemical characterization of the D-1 dopamine receptor mediating acetylcholine release in rabbit retina

    SciTech Connect

    Hensler, J.G.; Cotterell, D.J.; Dubocovich, M.L.

    1987-12-01

    Superfusion with dopamine (0.1 microM-10 mM) evokes calcium-dependent (/sup 3/H)acetylcholine release from rabbit retina labeled in vitro with (/sup 3/H)choline. This effect is antagonized by the D-1 dopamine receptor antagonist SCH 23390. Activation or blockade of D-2 dopamine, alpha-2 or beta receptors did not stimulate or attenuate the release of (/sup 3/H)acetylcholine from rabbit retina. Dopamine receptor agonists evoke the release of (/sup 3/H)acetylcholine with the following order of potency: apomorphine less than or equal to SKF(R)82526 < SKF 85174 < SKF(R)38393 less than or equal to pergolide less than or equal to dopamine (EC50 = 4.5 microM) < SKF(S)82526 less than or equal to SKF(S)38393. Dopamine receptor antagonists inhibited the dopamine-evoked release of (/sup 3/H)acetylcholine: SCH 23390 (IC50 = 1 nM) < (+)-butaclamol less than or equal to cis-flupenthixol < fluphenazine < perphenazine < trans-flupenthixol < R-sulpiride. The potencies of dopamine receptor agonists and antagonists at the dopamine receptor mediating (/sup 3/H)acetylcholine release is characteristic of the D-1 dopamine receptor. These potencies were correlated with the potencies of dopamine receptor agonists and antagonists at the D-1 dopamine receptor in rabbit retina as labeled by (/sup 3/H)SCH 23390, or as determined by adenylate cyclase activity. (/sup 3/H)SCH 23390 binding in rabbit retinal membranes was stable, saturable and reversible. Scatchard analysis of (/sup 3/H)SCH 23390 saturation data revealed a single high affinity binding site (Kd = 0.175 +/- 0.002 nM) with a maximum binding of 482 +/- 12 fmol/mg of protein. The potencies of dopamine receptor agonists to stimulate (/sup 3/H)acetylcholine release were correlated with their potencies to stimulate adenylate cyclase (r = 0.784, P less than .05, n = 7) and with their affinities at (/sup 3/H)SCH 23390 binding sites (r = 0.755, P < .05, n = 8).

  16. GABAA and GABAB receptor-mediated effects in guinea-pig ileum.

    PubMed

    Giotti, A; Luzzi, S; Spagnesi, S; Zilletti, L

    1983-03-01

    1 The effects of gamma-aminobutyric acid (GABA) and related substances were examined in guinea-pig ileum longitudinal muscle.2 GABA at doses ranging from 10(-7) M to 3 x 10(-6) M elicited a relaxation while at higher doses (3 x 10(-6) M - 10(-4) M), as previously described, it caused a contraction followed by relaxation.3 GABA-induced relaxation was bicuculline-insensitive, was mimicked by (-)-baclofen but not by homotaurine and muscimol. The effect of baclofen was stereospecific. GABA- and (-)-baclofen-induced relaxations were dose-dependent and their ED(50) values were similar. A specific cross-desensitization occurred between GABA and (-)-baclofen.4 The bicuculline-insensitive relaxation induced by GABA and (-)-baclofen was prevented by tetrodotoxin and hyoscine but not by phentolamine plus propranolol, naloxone or theophylline.5 In preparations in which the muscle tone was raised by histamine or prostaglandin F(2alpha), GABA and (-)-baclofen induced relaxation to the same extent as before increasing the tone. If the tone was raised by DMPP, a greater bicuculline-insensitive relaxation occurred.6 Contraction caused by GABA was bicuculline-sensitive and was mimicked by homotaurine and muscimol. Contraction was dose-dependent and muscimol was about three times more potent than GABA or homotaurine. A specific cross-desensitization occurred between the contractile effects of GABA and those of homotaurine or muscimol.7 Bicuculline competitively antagonized the contractile effects of GABA, homotaurine and muscimol and gave closely similar pA(2) values. The slope of the Schild plot for the above drugs was near 1, confirming the competitive nature of the antagonism.8 The bicuculline-sensitive contraction induced by GABA, homotaurine and muscimol was abolished by tetrodotoxin and was non-competitively antagonized by hyoscine, while it was unaffected by hexamethonium, mepyramine and methysergide.9 It is concluded that two receptors mediate the GABA effects in guinea

  17. Cannabinoid CB1 receptor mediates glucocorticoid effects on hormone secretion induced by volume and osmotic changes.

    PubMed

    Ruginsk, S G; Uchoa, E T; Elias, L L K; Antunes-Rodrigues, J

    2012-02-01

    The present study provides the first in vivo evidence that the cannabinoid CB(1) receptor mediates the effects of dexamethasone on hormone release induced by changes in circulating volume and osmolality. Male adult rats were administered with the CB(1) receptor antagonist rimonabant (10 mg/Kg, p.o.), followed or not in 1 hour by dexamethasone (1 mg/Kg, i.p.). Extracellular volume expansion (EVE, 2 mL/100 g of body weight, i.v.) was performed 2 hours after dexamethasone or vehicle treatment using either isotonic (I-EVE, 0.15 mol/L) or hypertonic (H-EVE, 0.30 mol/L) NaCl solution. Five minutes after EVE, animals were decapitated and trunk blood was collected for all plasma measurements. Rimonabant potentiated oxytocin (OT) secretion induced by H-EVE and completely reversed the inhibitory effects of dexamethasone in response to the same stimulus. These data suggest that glucocorticoid modulation of OT release is mediated by the CB(1) receptor. Although dexamethasone did not affect vasopressin (AVP) secretion induced by H-EVE, the administration of rimonabant potentiated AVP release in response to the same stimulus, supporting the hypothesis that the CB(1) receptor regulates AVP secretion independently of glucocorticoid-mediated signalling. Dexamethasone alone did not affect atrial natriuretic peptide (ANP) release stimulated by I-EVE or H-EVE. However, pretreatment with rimonabant potentiated ANP secretion induced by H-EVE, suggesting a possible role for the CB(1) receptor in the control of peripheral factors that modulate cardiovascular function. Rimonabant also reversed the inhibitory effects of dexamethasone on H-EVE-induced corticosterone secretion, reinforcing the hypothesis that the CB(1) receptor may be involved in the negative feedback exerted by glucocorticoids on the activity of the hypothalamic-pituitary-adrenal axis. Collectively, the results of the present study indicate that the CB(1) receptor modulates neurohypophyseal hormone secretion and

  18. The Impact of Hyperthermia on Receptor-Mediated Interleukin-6 Regulation in Mouse Skeletal Muscle

    PubMed Central

    Welc, Steven S.; Morse, Deborah A.; Mattingly, Alex J.; Laitano, Orlando; King, Michelle A.; Clanton, Thomas L.

    2016-01-01

    In inflammatory cells, hyperthermia inhibits lipopolysaccharide (LPS)-induced interleukin-6 (IL-6) gene expression and protein secretion. Since hyperthermia alone stimulates IL-6 in skeletal muscle, we hypothesized that it would amplify responses to other receptor-mediated stimuli. IL-6 regulation was tested in C2C12 myotubes and in soleus during treatment with epinephrine (EPI) or LPS. In EPI-treated myotubes (100 ng/ml), 1 h exposure at 40.5°C-42°C transiently increased IL-6 mRNA compared to EPI treatment alone at 37°C. In LPS-treated myotubes (1 μg/ml), exposure to 41°C-42°C also increased IL-6 mRNA. In isolated mouse soleus, similar amplifications of IL-6 gene expression were observed in 41°C, during both low (1 ng/ml) and high dose (100 ng/ml) EPI, but only in high dose LPS (1 μg/ml). In myotubes, heat increased IL-6 secretion during EPI exposure but had no effect or inhibited secretion with LPS. In soleus there were no effects of heat on IL-6 secretion during either EPI or LPS treatment. Mechanisms for the effects of heat on IL-6 mRNA were explored using a luciferase-reporter in C2C12 myotubes. Overexpression of heat shock factor-1 (HSF-1) had no impact on IL-6 promoter activity during EPI stimulation, but elevated IL-6 promoter activity during LPS stimulation. In contrast, when the activator protein-1 (AP-1) element was mutated, responses to both LPS and EPI were suppressed in heat. Using siRNA against activating transcription factor-3 (ATF-3), a heat-stress-induced inhibitor of IL-6, no ATF-3-dependent effects were observed. The results demonstrate that, unlike inflammatory cells, hyperthermia in muscle fibers amplifies IL-6 gene expression to EPI and LPS. The effect appears to reflect differential engagement of HSF-1 and AP-1 sensitive elements on the IL-6 gene, with no evidence for involvement of ATF-3. The functional significance of increased IL-6 mRNA expression during heat may serve to overcome the well-known suppression of protein synthetic

  19. Deciphering the roles of phosphoinositide lipids in phagolysosome biogenesis

    PubMed Central

    Jeschke, Andreas; Haas, Albert

    2016-01-01

    ABSTRACT Professional phagocytes engulf microbial invaders into plasma membrane-derived phagosomes. These mature into microbicidal phagolysosomes, leading to killing of the ingested microbe. Phagosome maturation involves sequential fusion of the phagosome with early endosomes, late endosomes, and the main degradative compartments in cells, lysosomes. Some bacterial pathogens manipulate the phosphoinositide (PIP) composition of phagosome membranes and are not delivered to phagolysosomes, pointing at a role of PIPs in phagosome maturation. This hypothesis is supported by comprehensive microscopic studies. Recently, cell-free reconstitution of fusion between phagosomes and endo(lyso)somes identified phosphatidylinositol 4-phosphate [PI(4)P] and phosphatidylinositol 3-phosphate [PI(3)P] as key regulators of phagolysosome biogenesis. Here, we describe the emerging roles of PIPs in phagosome maturation and we present tools to study PIP involvement in phagosome trafficking using intact cells or purified compartments. PMID:27489580

  20. Phosphoinositide kinase signaling controls ER-PM cross-talk

    PubMed Central

    Omnus, Deike J.; Manford, Andrew G.; Bader, Jakob M.; Emr, Scott D.; Stefan, Christopher J.

    2016-01-01

    Membrane lipid dynamics must be precisely regulated for normal cellular function, and disruptions in lipid homeostasis are linked to the progression of several diseases. However, little is known about the sensory mechanisms for detecting membrane composition and how lipid metabolism is regulated in response to membrane stress. We find that phosphoinositide (PI) kinase signaling controls a conserved PDK-TORC2-Akt signaling cascade as part of a homeostasis network that allows the endoplasmic reticulum (ER) to modulate essential responses, including Ca2+-regulated lipid biogenesis, upon plasma membrane (PM) stress. Furthermore, loss of ER-PM junctions impairs this protective response, leading to PM integrity defects upon heat stress. Thus PI kinase–mediated ER-PM cross-talk comprises a regulatory system that ensures cellular integrity under membrane stress conditions. PMID:26864629

  1. Lipoxin A4 inhibits phosphoinositide hydrolysis in human neutrophils.

    PubMed

    Grandordy, B M; Lacroix, H; Mavoungou, E; Krilis, S; Crea, A E; Spur, B W; Lee, T H

    1990-03-30

    Lipoxins (LX) are trihydroxytetraene metabolites derived from arachidonic acid via an interaction between the 5- and 15-lipoxygenases. Preincubation of [3H] myo-inositol labeled PMN with 10-7M and 10-5M LXA4 for 1 minute at 37 degrees C resulted in a concentration dependent inhibition of the generation of [3H] IP3 and [3H] IP in cells subsequently stimulated by increasing doses of LTB4 or FMLP for 1 minute at 37 degrees C. Preincubation of PMN with LXA4 did not inhibit specific binding of [3H] LTB4 to PMN. These results indicate that LXA4 inhibits chemotactic factor-induced phosphoinositide hydrolysis at a post-receptor level. PMID:2157419

  2. In vivo tracking of phosphoinositides in Drosophila photoreceptors

    PubMed Central

    Hardie, Roger C.; Liu, Che-Hsiung; Randall, Alexander S.; Sengupta, Sukanya

    2015-01-01

    ABSTRACT In order to monitor phosphoinositide turnover during phospholipase C (PLC)-mediated Drosophila phototransduction, fluorescently tagged lipid probes were expressed in photoreceptors and imaged both in dissociated cells, and in eyes of intact living flies. Of six probes tested, TbR332H (a mutant of the Tubby protein pleckstrin homology domain) was judged the best reporter for phosphatidylinositol (4,5)-bisphosphate [PtdIns(4,5)P2], and the P4M domain from Legionella SidM for phosphatidylinositol 4-phosphate (PtdIns4P). Using accurately calibrated illumination, we found that only ∼50% of PtdIns(4,5)P2 and very little PtdIns4P were depleted by full daylight intensities in wild-type flies, but both were severely depleted by ∼100-fold dimmer intensities in mutants lacking Ca2+-permeable transient receptor potential (TRP) channels or protein kinase C (PKC). Resynthesis of PtdIns4P (t½ ∼12 s) was faster than PtdIns(4,5)P2 (t½ ∼40 s), but both were greatly slowed in mutants of DAG kinase (rdgA) or PtdIns transfer protein (rdgB). The results indicate that Ca2+- and PKC-dependent inhibition of PLC is required for enabling photoreceptors to maintain phosphoinositide levels despite high rates of hydrolysis by PLC, and suggest that phosphorylation of PtdIns4P to PtdIns(4,5)P2 is the rate-limiting step of the cycle. PMID:26483384

  3. Enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses in the barrel cortex of Mecp2-null mice.

    PubMed

    Lo, Fu-Sun; Blue, Mary E; Erzurumlu, Reha S

    2016-03-01

    Rett syndrome (RTT) is a neurodevelopmental disorder that results from mutations in the X-linked gene for methyl-CpG-binding protein 2 (MECP2). The underlying cellular mechanism for the sensory deficits in patients with RTT is largely unknown. This study used the Bird mouse model of RTT to investigate sensory thalamocortical synaptic transmission in the barrel cortex of Mecp2-null mice. Electrophysiological results showed an excitation/inhibition imbalance, biased toward inhibition, due to an increase in efficacy of postsynaptic GABAA receptors rather than alterations in inhibitory network and presynaptic release properties. Enhanced inhibition impaired the transmission of tonic sensory signals from the thalamus to the somatosensory cortex. Previous morphological studies showed an upregulation of NMDA receptors in the neocortex of both RTT patients and Mecp2-null mice at early ages [Blue ME, Naidu S, Johnston MV. Ann Neurol 45: 541-545, 1999; Blue ME, Kaufmann WE, Bressler J, Eyring C, O'Driscoll C, Naidu S, Johnston MV. Anat Rec (Hoboken) 294: 1624-1634, 2011]. Although AMPA and NMDA receptor-mediated excitatory synaptic transmission was not altered in the barrel cortex of Mecp2-null mice, extrasynaptic NMDA receptor-mediated responses increased markedly. These responses were blocked by memantine, suggesting that extrasynaptic NMDA receptors play an important role in the pathogenesis of RTT. The results suggest that enhancement of postsynaptic GABAA and extrasynaptic NMDA receptor-mediated responses may underlie impaired somatosensation and that pharmacological blockade of extrasynaptic NMDA receptors may have therapeutic value for RTT. PMID:26683074

  4. Zonal differences in ethanol-induced impairments in receptor-mediated endocytosis of asialoglycoproteins in isolated rat hepatocytes

    SciTech Connect

    Casey, C.A.; Kragskow, S.L.; Sorrell, M.F.; Tuma, D.J. )

    1991-02-01

    We have shown previously that ethanol-induced defects in receptor-mediated endocytosis of asialoorosomucoid occurred as early as 1 wk after ethanol feeding. This study was undertaken as an initial attempt to establish a possible role of defective receptor-mediated endocytosis in liver injury by investigating whether differences exist in the effects of ethanol on receptor-mediated endocytosis in hepatocytes isolated from different regions of the liver. Perivenule cells, present in the distal half of the liver, are thought to be more susceptible to ethanol-induced liver injury than are the periportal cells located in the proximal half of the liver acini. For these studies, we fed male Sprague-Dawley rats for 7 days with liquid diets containing either ethanol (36% of calories) or isocaloric carbohydrate. Perivenule and periportal hepatocytes were then isolated using a digitonin-collagenase perfusion method. In control animals, cells isolated from the perivenule region bound significantly more ligand than did cells from the periportal region. Amounts of ligand internalized and degraded were also greater in perivenule than in periportal cells in these animals. After ethanol feeding, cells isolated from both the perivenule and periportal regions bound significantly less ligand than their respective controls. This impairment in surface and total binding was more pronounced in perivenule than in periportal cells. Internalization and degradation of the ligand were also more adversely affected in the centrilobular region as shown by decreases of greater than 60% in perivenule cells and by only 20% to 30% in periportal cells of ethanol-fed animals compared with controls.

  5. Wnt5a promotes cancer cell invasion and proliferation by receptor-mediated endocytosis-dependent and -independent mechanisms, respectively

    PubMed Central

    Shojima, Kensaku; Sato, Akira; Hanaki, Hideaki; Tsujimoto, Ikuko; Nakamura, Masahiro; Hattori, Kazunari; Sato, Yuji; Dohi, Keiji; Hirata, Michinari; Yamamoto, Hideki; Kikuchi, Akira

    2015-01-01

    Wnt5a activates the Wnt/β-catenin-independent pathway and its overexpression is associated with tumor aggressiveness enhancing invasive activity. For this action, Wnt5a-induced receptor endocytosis with clathrin is required. Wnt5a expression was previously believed to be associated with cancer cell motility but not proliferation. Recently, it was reported that Wnt5a is also implicated in cancer cell proliferation, but the mechanism was not clear. In this study, we generated a neutralizing anti-Wnt5a monoclonal antibody (mAb5A16) to investigate the mechanism by which Wnt5a regulates cancer cell proliferation. Wnt5a stimulated both invasion and proliferation of certain types of cancer cells, including HeLaS3 cervical cancer cells and A549 lung cancer cells although Wnt5a promoted invasion but not proliferation in other cancer cells such as KKLS gastric cancer cells. mAb5A16 did not affect the binding of Wnt5a to its receptor, but it suppressed Wnt5a-induced receptor-mediated endocytosis. mAb5A16 inhibited invasion but not proliferation of HeLaS3 and A549 cells. Wnt5a activated Src family kinases (SFKs) and Wnt5a-dependent cancer cell proliferation was dependent on SFKs, yet blockade of receptor-mediated endocytosis did not affect cancer cell proliferation and SFK activity. These results suggest that Wnt5a promotes invasion and proliferation of certain types of cancer cells through receptor-mediated endocytosis-dependent and -independent mechanisms, respectively. PMID:25622531

  6. Reactivation of apolipoprotein II gene transcription by cycloheximide reveals two steps in the deactivation of estrogen receptor-mediated transcription.

    PubMed

    Sensel, M G; Binder, R; Lazier, C B; Williams, D L

    1994-03-01

    In this report, we describe apolipoprotein II (apoII) gene expression in cell lines derived by stable expression of the chicken estrogen receptor in LMH chicken hepatoma cells. In cell lines expressing high levels of receptor (LMH/2A), apoII gene expression is increased by estrogen 300-fold compared with levels in the receptor-deficient parent LMH line. LMH/2A cells show apoII mRNA induction and turnover kinetics similar to those in chicken liver. Inhibition of protein synthesis with cycloheximide (CHX) or puromycin following estrogen withdrawal superinduces apoII mRNA without affecting apoII mRNA stability. Superinduction is due to an estrogen-independent reactivation of apoII gene transcription. The apoII gene can be reactivated by CHX for up to 24 h following hormone withdrawal, suggesting that the gene is in a repressed yet transcriptionally competent state. These results reveal two distinct events necessary for termination of estrogen receptor-mediated transcription. The first event, removal of hormone, is sufficient to stop transcription when translation is ongoing. The second event is revealed by the CHX-induced superinduction of apoII mRNA following hormone withdrawal. This superinduction suggests that deactivation of estrogen receptor-mediated transcription requires a labile protein. Furthermore, reactivation of apoII gene expression by CHX and estrogen is additive, suggesting that estrogen is unable to overcome repression completely. Thus, a labile protein may act to repress estrogen receptor-mediated transcription of the apoII gene. PMID:8114707

  7. Inhibitors of 3-hydroxy-3-methylglutaryl-CoA reductase reduce receptor-mediated endocytosis in opossum kidney cells.

    PubMed

    Sidaway, James E; Davidson, Robert G; McTaggart, Fergus; Orton, Terry C; Scott, Robert C; Smith, Graham J; Brunskill, Nigel J

    2004-09-01

    Renal proximal tubule cells are responsible for the reabsorption of proteins that are present in the tubular lumen. This occurs by receptor-mediated endocytosis, a process that has a requirement for some GTP-binding proteins. Statins are inhibitors of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase used for the therapeutic reduction of cholesterol-containing plasma lipoproteins. However, they can also reduce intracellular levels of isoprenoid pyrophosphates that are derived from the product of the enzyme, mevalonate, and are required for the prenylation and normal function of GTP-binding proteins. The hypothesis that inhibition of HMG-CoA reductase in renal proximal tubule cells could reduce receptor mediated-endocytosis was therefore tested. Five different statins inhibited the uptake of FITC-labeled albumin by the proximal tubule-derived opossum kidney cell line in a dose-dependent manner and in the absence of cytotoxicity. The reduction in albumin uptake was related to the degree of inhibition of HMG-CoA reductase. Simvastatin (e.g., statin) inhibited receptor-mediated endocytosis of both FITC-albumin and FITC-beta(2)-microglobulin to similar extents but without altering the binding of albumin to the cell surface. The effect on albumin endocytosis was prevented by mevalonate and by the isoprenoid geranylgeranyl pyrophosphate but not by cholesterol. Finally, evidence that the inhibitory effect of statins on endocytosis of proteins may be caused by reduced prenylation and thereby decreased function of one or more GTP-binding proteins is provided. These data establish the possibility in principle that inhibition of HMG-CoA reductase by statins in proximal tubule cells may reduce tubular protein reabsorption. PMID:15339975

  8. Effects of particle size and ligand density on the kinetics of receptor-mediated endocytosis of nanoparticles

    NASA Astrophysics Data System (ADS)

    Yuan, Hongyan; Zhang, Sulin

    2010-01-01

    We elucidate, from thermodynamic arguments, the governing factors of receptor-mediated endocytosis of nanoparticles (NPs). We show that the endocytic energetics specifies a minimal particle size and a minimal ligand density below which endocytosis is not possible. Due to the entropic penalty involved in ligand-receptor binding, endocytosis may occur with a large fraction of ligands unbound with receptors. Our analyses suggest that the endocytic time depends interrelatedly on the particle size and ligand density. There exists an optimal condition at which the endocytic time minimizes. These findings may provide valuable guidance to the rational designs of NP-based biomarkers and anticancer bioagents.

  9. Effects of rasagiline, its metabolite aminoindan and selegiline on glutamate receptor mediated signalling in the rat hippocampus slice in vitro

    PubMed Central

    2011-01-01

    Background Rasagiline, a new drug developed to treat Parkinson's disease, is known to inhibit monoamine oxidase B. However, its metabolite R-(-)-aminoindan does not show this kind of activity. The present series of in vitro experiments using the rat hippocampal slice preparation deals with effects of both compounds on the pyramidal cell response after electric stimulation of the Schaffer Collaterals in comparison to selegiline, another MAO B inhibitor. Method Stimulation of the Schaffer Collaterals by single stimuli (SS) or theta burst stimulation (TBS) resulted in stable responses of pyramidal cells measured as population spike amplitude (about 1 mV under control SS conditions or about 2 mV after TBS). Results During the first series, this response was attenuated in the presence of rasagiline and aminoindan-to a lesser degree of selegiline-in a concentration dependent manner (5-50 μM) after single stimuli as well as under TBS. During oxygen/glucose deprivation for 10 min the amplitude of the population spike breaks down by 75%. The presence of rasagiline and aminoindan, but rarely the presence of selegiline, prevented this break down. Following glutamate receptor mediated enhancements of neuronal transmission in a second series of experiments very clear differences could be observed in comparison to the action of selegiline: NMDA receptor, AMPA receptor as well as metabotropic glutamate receptor mediated increases of transmission were concentration dependently (0,3 - 2 μM) antagonized by rasagiline and aminoindan, but not by selegiline. On the opposite, only selegiline attenuated kainate receptor mediated increases of excitability. Thus, both monoamino oxidase (MAO) B inhibitors show attenuation of glutamatergic transmission in the hippocampus but interfere with different receptor mediated excitatory modulations at low concentrations. Conclusions Since aminoindan does not induce MAO B inhibition, these effects must be regarded as being independent from MAO B

  10. The Phox homology (PX) domain, a new player in phosphoinositide signalling.

    PubMed Central

    Xu, Y; Seet, L F; Hanson, B; Hong, W

    2001-01-01

    Phosphoinositides are key regulators of diverse cellular processes. The pleckstrin homology (PH) domain mediates the action of PtdIns(3,4)P(2), PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3), while the FYVE domain relays the pulse of PtdIns3P. The recent establishment that the Phox homology (PX) domain interacts with PtdIns3P and other phosphoinositides suggests another mechanism by which phosphoinositides can regulate/integrate multiple cellular events via a spectrum of PX domain-containing proteins. Together with the recent discovery that the epsin N-terminal homologue (ENTH) domain interacts with PtdIns(4,5)P(2), it is becoming clear that phosphoinositides regulate diverse cellular events through interactions with several distinct structural motifs present in many different proteins. PMID:11736640

  11. Inositol metabolism in WRK-1 cells. Relationship of hormone-sensitive to -insensitive pools of phosphoinositides

    SciTech Connect

    Monaco, M.E.

    1987-09-25

    Previous studies have indicated the existence of two separate pools of phosphoinositides in WRK-1 cells; one is labile and hormone-sensitive with respect to turnover, while the other is stable. Hormonal stimulation results in a rapid increase in /sup 32/Pi incorporation into the sensitive pool, while in the absence of hormone, incorporation of /sup 32/Pi into this pool is slow. Results are quite different when (/sup 3/H)inositol is the precursor utilized. Incorporation of (/sup 3/H)inositol into hormone-sensitive phosphoinositides is not stimulated in the presence of hormone, suggesting entry of this exogenous precursor into the cycle by a route other than the resynthetic phase of the cycle. Furthermore, failure of hormone to induce loss of (/sup 3/H)phosphoinositide in pulse-chase experiments in the absence of lithium suggests reutilization of the (/sup 3/H)inositol moiety generated by phosphodiesteratic cleavage of hormone-sensitive phosphoinositide. Time course studies indicate that the relative rates of incorporation of (/sup 3/H)inositol into sensitive and insensitive phosphoinositide remain constant from 2 to 24 h. Several factors are capable of increasing (/sup 3/H)inositol incorporation into hormone-insensitive phosphoinositide including vasopressin, calcium ionophores, and manganese. On the other hand, vasopressin treatment appears to decrease incorporation of (/sup 3/H)inositol into the hormone-sensitive pool, probably by shifting the equilibrium between phosphoinositides and inositol phosphates, since the decrease in radioactivity observed in the phosphoinositides is equaled by the increase observed in that in the inositol phosphates.

  12. Receptor-Mediated Endocytosis of Two-Dimensional Nanomaterials Undergoes Flat Vesiculation and Occurs by Revolution and Self-Rotation.

    PubMed

    Mao, Jian; Chen, Pengyu; Liang, Junshi; Guo, Ruohai; Yan, Li-Tang

    2016-01-26

    Two-dimensional nanomaterials, such as graphene and transitional metal dichalcogenide nanosheets, are promising materials for the development of antimicrobial surfaces and the nanocarriers for intracellular therapy. Understanding cell interaction with these emerging materials is an urgently important issue to promoting their wide applications. Experimental studies suggest that two-dimensional nanomaterials enter cells mainly through receptor-mediated endocytosis. However, the detailed molecular mechanisms and kinetic pathways of such processes remain unknown. Here, we combine computer simulations and theoretical derivation of the energy within the system to show that the receptor-mediated transport of two-dimensional nanomaterials, such as graphene nanosheet across model lipid membrane, experiences a flat vesiculation event governed by the receptor density and membrane tension. The graphene nanosheet is found to undergo revolution relative to the membrane and, particularly, unique self-rotation around its normal during membrane wrapping. We derive explicit expressions for the formation of the flat vesiculation, which reveals that the flat vesiculation event can be fundamentally dominated by a dimensionless parameter and a defined relationship determined by complicated energy contributions. The mechanism offers an essential understanding on the cellular internalization and cytotoxicity of the emerging two-dimensional nanomaterials. PMID:26741298

  13. Activation of innate antiviral immune response via double-stranded RNA-dependent RLR receptor-mediated necroptosis

    PubMed Central

    Wang, Wei; Wang, Wei-Hua; Azadzoi, Kazem M.; Su, Ning; Dai, Peng; Sun, Jianbin; Wang, Qin; Liang, Ping; Zhang, Wentao; Lei, Xiaoying; Yan, Zhen; Yang, Jing-Hua

    2016-01-01

    Viruses induce double-stranded RNA (dsRNA) in the host cells. The mammalian system has developed dsRNA-dependent recognition receptors such as RLRs that recognize the long stretches of dsRNA as PAMPs to activate interferon-mediated antiviral pathways and apoptosis in severe infection. Here we report an efficient antiviral immune response through dsRNA-dependent RLR receptor-mediated necroptosis against infections from different classes of viruses. We demonstrated that virus-infected A549 cells were efficiently killed in the presence of a chimeric RLR receptor, dsCARE. It measurably suppressed the interferon antiviral pathway but promoted IL-1β production. Canonical cell death analysis by morphologic assessment, phosphatidylserine exposure, caspase cleavage and chemical inhibition excluded the involvement of apoptosis and consistently suggested RLR receptor-mediated necroptosis as the underlying mechanism of infected cell death. The necroptotic pathway was augmented by the formation of RIP1-RIP3 necrosome, recruitment of MLKL protein and the activation of cathepsin D. Contributing roles of RIP1 and RIP3 were confirmed by gene knockdown. Furthermore, the necroptosis inhibitor necrostatin-1 but not the pan-caspase inhibitor zVAD impeded dsCARE-dependent infected cell death. Our data provides compelling evidence that the chimeric RLR receptor shifts the common interferon antiviral responses of infected cells to necroptosis and leads to rapid death of the virus-infected cells. This mechanism could be targeted as an efficient antiviral strategy. PMID:26935990

  14. Receptor-mediated endocytosis of albumin by kidney proximal tubule cells is regulated by phosphatidylinositide 3-kinase.

    PubMed

    Brunskill, N J; Stuart, J; Tobin, A B; Walls, J; Nahorski, S

    1998-05-15

    Receptor-mediated endocytosis of albumin is an important function of the kidney proximal tubule epithelium. We have measured endocytosis of [125I]-albumin in opossum kidney cells and examined the regulation of this process by phosphatidylinositide 3-kinase (PI 3-kinase). Albumin endocytosis was inhibited by both wortmannin (IC50 6.9 nM) and LY294002 (IC50 6.5 microM) at concentrations that suggested the involvement of PI 3-kinase in its regulation. Recycling rates were unaffected. We transfected OK cells with either a wild-type p85 subunit of PI 3-kinase, or a dominant negative form of the p85 subunit (Deltap85) using the LacSwitch expression system. Transfects were screened by immunoblotting with anti-PI 3-kinase antibodies. Under basal conditions, transfects demonstrated no expression of p85 or Deltap85, but expression was briskly induced by treatment of the cells with IPTG (EC50 13.7 microM). Inhibition of PI 3-kinase activity by Deltap85 was confirmed by in vitro kinase assay of anti-phosphotyrosine immunoprecipitates from transfected cells stimulated with insulin. Expression of Deltap85 resulted in marked inhibition of albumin endocytosis, predominantly as a result of reduction of the Vmax of the transport process. Expression of p85 had no significant effect on albumin uptake. The results demonstrate that PI 3-kinase regulates an early step in the receptor-mediated endocytosis of albumin by kidney proximal tubular cells. PMID:9593770

  15. Downregulation of Plk1 Expression By Receptor-Mediated Uptake of Antisense Oligonucleotide-Loaded Nanoparticles1

    PubMed Central

    Spänkuch, Birgit; Steinhauser, Isabel; Wartlick, Heidrun; Kurunci-Csacsko, Elisabeth; Strebhardt, Klaus I; Langer, Klaus

    2008-01-01

    Human serum albumin (HSA) nanoparticles represent a promising tool for targeted drug delivery to tumor cells. The coupling of the antibody trastuzumab to nanoparticles uses the capability of human epidermal growth factor receptor 2 (HER2)-positive cells to incorporate agents linked to HER2. In our present study, we developed targeted nanoparticles loaded with antisense oligonucleotides (ASOs) against polo-like kinase 1 (Plk1). We evaluated the receptor-mediated uptake into HER2-positive and -negative breast cancer and murine cell lines. We performed quantitative real-time PCR and Western blot analyses to monitor the impact on Plk1 expression in HER2-positive breast cancer cells. Antibody-conjugated nanoparticles showed a specific targeting to HER2-overexpressing cells with cellular uptake by receptor-mediated endocytosis and a release into HER2-positive BT-474 cells. We observed a significant reduction of Plk1 mRNA and protein expression and increased activation of Caspase 3/7. Thus, this is the first report about ASO-loaded HSA nanoparticles, where an impact on gene expression could be observed. The data provide the basis for the further development of carrier systems for Plk1-specific ASOs to reduce off-target effects evoked by systemically administered ASOs and to achieve a better penetration into primary and metastatic target cells. Treatment of tumors using trastuzumab-conjugated ASO-loaded HSA nanoparticles could be a promising approach to reach this goal. PMID:18320067

  16. Phosphoinositide kinases and the synthesis of polyphosphoinositides in higher plant cells

    NASA Technical Reports Server (NTRS)

    Drobak, B. K.; Dewey, R. E.; Boss, W. F.; Davies, E. (Principal Investigator)

    1999-01-01

    Phosphoinositides are a family of inositol-containing phospholipids which are present in all eukaryotic cells. Although in most cells these lipids, with the exception of phosphatidylinositol, constitute only a very minor proportion of total cellular lipids, they have received immense attention by researchers in the past 15-20 years. This is due to the discovery that these lipids, rather than just having structural functions, play key roles in a wide range of important cellular processes. Much less is known about the plant phosphoinositides than about their mammalian counterparts. However, it has been established that a functional phosphoinositide system exists in plant cells and it is becoming increasingly clear that inositol-containing lipids are likely to play many important roles throughout the life of a plant. It is not our intention to give an exhaustive overview of all aspects of the field, but rather we focus on the phosphoinositide kinases responsible for the synthesis of all phosphorylated forms of phosphatidylinositol. Also, we mention some of the aspects of current phosphoinositide research which, in our opinion, are most likely to provide a suitable starting point for further research into the role of phosphoinositides in plants.

  17. β-Adrenergic agonists mediate enhancement of β1-adrenergic receptor N-terminal cleavage and stabilization in vivo and in vitro.

    PubMed

    Hakalahti, Anna E; Khan, Hamayun; Vierimaa, Miia M; Pekkala, Emilia H; Lackman, Jarkko J; Ulvila, Johanna; Kerkelä, Risto; Petäjä-Repo, Ulla E

    2013-01-01

    The β(1)-adrenergic receptor (β(1)AR) is the predominant βAR in the heart and is the main target for β-adrenergic antagonists, widely used in the treatment of cardiovascular diseases. Previously, we have shown that the human (h) β(1)AR is cleaved in its N terminus by a metalloproteinase, both constitutively and in a receptor activation-dependent manner. In this study, we investigated the specific events involved in β(1)AR regulation, focusing on the effects of long-term treatment with β-adrenergic ligands on receptor processing in stably transfected human embryonic kidney 293(i) cells. The key findings were verified using the transiently transfected hβ(1)AR and the endogenously expressed receptor in neonatal rat cardiomyocytes. By using flow cytometry and Western blotting, we demonstrated that isoproterenol, S-propranolol, CGP-12177 [4-[3-[(1,1-dimethylethyl)amino]2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one], pindolol, and timolol, which displayed agonistic properties toward the β(1)AR in either the adenylyl cyclase or the mitogen-activated protein kinase signaling pathways, induced cleavage of the mature cell-surface receptor. In contrast, metoprolol, bisoprolol, and CGP-20712 [1-[2-((3-carbamoyl-4-hydroxy)phenoxy)ethylamino]-3-[4-(1-methyl-4-trifluoromethyl-2-imidazolyl)phenoxy]-2-propanol], which showed no agonistic activity, had only a marginal or no effect. Importantly, the agonists also stabilized intracellular receptor precursors, possibly via their pharmacological chaperone action, and they stabilized the receptor in vitro. The opposing effects on the two receptor forms thus led to an increase in the amount of cleaved receptor fragments at the plasma membrane. The results underscore the pluridimensionality of β-adrenergic ligands and extend this property from receptor activation and signaling to the regulation of β(1)AR levels. This phenomenon may contribute to the exceptional resistance of β(1)ARs to downregulation and tendency toward

  18. A Screen for Novel Phosphoinositide 3-kinase Effector Proteins*

    PubMed Central

    Dixon, Miles J.; Gray, Alexander; Boisvert, François-Michel; Agacan, Mark; Morrice, Nicholas A.; Gourlay, Robert; Leslie, Nicholas R.; Downes, C. Peter; Batty, Ian H.

    2011-01-01

    Class I phosphoinositide 3-kinases exert important cellular effects through their two primary lipid products, phosphatidylinositol 3,4,5-trisphosphate and phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). As few molecular targets for PtdIns(3,4)P2 have yet been identified, a screen for PI 3-kinase-responsive proteins that is selective for these is described. This features a tertiary approach incorporating a unique, primary recruitment of target proteins in intact cells to membranes selectively enriched in PtdIns(3,4)P2. A secondary purification of these proteins, optimized using tandem pleckstrin homology domain containing protein-1 (TAPP-1), an established PtdIns(3,4)P2 selective ligand, yields a fraction enriched in proteins of potentially similar lipid binding character that are identified by liquid chromatography-tandem MS. Thirdly, this approach is coupled to stable isotope labeling with amino acids in cell culture using differential isotope labeling of cells stimulated in the absence and presence of the PI 3-kinase inhibitor wortmannin. This provides a ratio-metric readout that distinguishes authentically responsive components from copurifying background proteins. Enriched fractions thus obtained from astrocytoma cells revealed a subset of proteins that exhibited ratios indicative of their initial, cellular responsiveness to PI 3-kinase activation. The inclusion among these of tandem pleckstrin homology domain containing protein-1, three isoforms of Akt, switch associated protein-70, early endosome antigen-1 and of additional proteins expressing recognized lipid binding domains demonstrates the utility of this strategy and lends credibility to the novel candidate proteins identified. The latter encompass a broad set of proteins that include the gene product of TBC1D2A, a putative Rab guanine nucleotide triphosphatase activating protein (GAP) and IQ motif containing GAP1, a potential tumor promoter. A sequence comparison of the former protein indicates

  19. Endogenous expression of histamine H1 receptors functionally coupled to phosphoinositide hydrolysis in C6 glioma cells: regulation by cyclic AMP.

    PubMed Central

    Peakman, M C; Hill, S J

    1994-01-01

    1. The effects of histamine receptor agonists and antagonists on phospholipid hydrolysis in rat-derived C6 glioma cells have been investigated. 2. Histamine H1 receptor-stimulation caused a concentration-dependent increase in the accumulation of total [3H]-inositol phosphates in cells prelabelled with [3H]-myo-inositol. The rank order of agonist potencies was histamine (EC50 = 24 microM) > N alpha-methylhistamine (EC50 = 31 microM) > 2-thiazolylethylamine (EC50 = 91 microM). 3. The response to 0.1 mM histamine was antagonized in a concentration-dependent manner by the H1-antagonists, mepyramine (apparent Kd = 1 nM) and (+)-chlorpheniramine (apparent Kd = 4 nM). In addition, (-)-chlorpheniramine was more than two orders of magnitude less potent than its (+)-stereoisomer. 4. Elevation of intracellular cyclic AMP accumulation with forskolin (10 microM, EC50 = 0.3 microM), isoprenaline (1 microM, EC50 = 4 nM) or rolipram (0.5 mM), significantly reduced the histamine-mediated (0.1 mM) inositol phosphate response by 37%, 43% and 26% respectively. In contrast, 1,9-dideoxyforskolin did not increase cyclic AMP accumulation and had no effect on the phosphoinositide response to histamine. 5. These data indicate the presence of functionally coupled, endogenous histamine H1 receptors in C6 glioma cells. Furthermore, the results also indicate that H1 receptor-mediated phospholipid hydrolysis is inhibited by the elevation of cyclic AMP levels in these cells. PMID:7889313

  20. Angiotensin II regulates phosphoinositide 3 kinase/Akt cascade via a negative crosstalk between AT1 and AT2 receptors in skin fibroblasts of human hypertrophic scars.

    PubMed

    Liu, Hong-Wei; Cheng, Biao; Yu, Wen-Lin; Sun, Rui-Xia; Zeng, Dong; Wang, Jie; Liao, Yuan-Xing; Fu, Xiao-Bing

    2006-06-27

    Angiotensin II (Ang II) stimulation has been shown to regulate proliferation of skin fibroblasts and production of extracellular matrix, which are very important process in skin wound healing and scarring; however, the signaling pathways involved in this process, especially in humans, are less explored. In the present study, we used skin fibroblasts of human hypertrophic scar, which expressed both AT1 and AT2 receptors, and observed that Ang II increased Akt phosphorylation and phosphoinositide 3 kinase (PI 3-K) activity. In addition, the Ang II-induced Akt phosphorylation was blocked by wortmannin, a PI 3-K inhibitor. This Ang II-activated PI 3-K/Akt cascade was markedly inhibited by valsartan, an AT(1) receptor-specific blocker, whereas it was enhanced by PD123319, an AT(2) receptor antagonist. On the other hand, the Ang II- or EGF-induced activation of PI 3-K/Akt was strongly attenuated by AG1478, an inhibitor of epidermal growth factor (EGF) receptor kinase. Moreover, Ang II stimulated tyrosine phosphorylation of EGF receptor and p85alpha subunit of PI 3-K accompanied by an increase in their association, which was inhibited by valsartan, and enhanced by PD123319. The Ang II-induced transactivation of EGF receptor resulted in activation of extracellular signal-regulated kinase (ERK) that was also inhibited by valsartan, and enhanced by PD123319. Taken together, our results showed that AT(1) receptor-mediated activation of PI 3-K/Akt cascades occurs at least partially via the transactivation of EGF receptor, which is under a negative control by AT(2) receptor in hypertrophic scar fibroblasts. These findings contribute to understanding the molecular mechanism of human hypertrophic scar formation. PMID:16522324

  1. Requirement of Phosphoinositides Containing Stearic Acid To Control Cell Polarity

    PubMed Central

    Laquel, Patricia; Testet, Eric; Tuphile, Karine; Fouillen, Laetitia; Bessoule, Jean-Jacques

    2015-01-01

    Phosphoinositides (PIPs) are present in very small amounts but are essential for cell signaling, morphogenesis, and polarity. By mass spectrometry, we demonstrated that some PIPs with stearic acyl chains were strongly disturbed in a psi1Δ Saccharomyces cerevisiae yeast strain deficient in the specific incorporation of a stearoyl chain at the sn-1 position of phosphatidylinositol. The absence of PIPs containing stearic acid induced disturbances in intracellular trafficking, although the total amount of PIPs was not diminished. Changes in PIPs also induced alterations in the budding pattern and defects in actin cytoskeleton organization (cables and patches). Moreover, when the PSI1 gene was impaired, a high proportion of cells with bipolar cortical actin patches that occurred concomitantly with the bipolar localization of Cdc42p was specifically found among diploid cells. This bipolar cortical actin phenotype, never previously described, was also detected in a bud9Δ/bud9Δ strain. Very interestingly, overexpression of PSI1 reversed this phenotype. PMID:26711260

  2. Identification and Structural Characterization of a Legionella Phosphoinositide Phosphatase*

    PubMed Central

    Toulabi, Leila; Wu, Xiaochun; Cheng, Yanshu; Mao, Yuxin

    2013-01-01

    Bacterial pathogen Legionella pneumophila is the causative agent of Legionnaires' disease, which is associated with intracellular replication of the bacteria in macrophages of human innate immune system. Recent studies indicate that pathogenic bacteria can subvert host cell phosphoinositide (PI) metabolism by translocated virulence effectors. However, in which manner Legionella actively exploits PI lipids to benefit its infection is not well characterized. Here we report that L. pneumophila encodes an effector protein, named SidP, that functions as a PI-3-phosphatase specifically hydrolyzing PI(3)P and PI(3,5)P2 in vitro. This activity of SidP rescues the growth phenotype of a yeast strain defective in PI(3)P phosphatase activity. Crystal structure of SidP orthologue from Legionella longbeachae reveals that this unique PI-3-phosphatase is composed of three distinct domains: a large catalytic domain, an appendage domain that is inserted into the N-terminal portion of the catalytic domain, and a C-terminal α-helical domain. SidP has a small catalytic pocket that presumably provides substrate specificity by limiting the accessibility of bulky PIs with multiple phosphate groups. Together, our identification of a unique family of Legionella PI phosphatases highlights a common scheme of exploiting host PI lipids in many intracellular bacterial pathogen infections. PMID:23843460

  3. Kappa opioid receptors stimulate phosphoinositide turnover in rat brain

    SciTech Connect

    Periyasamy, S.; Hoss, W. )

    1990-01-01

    The effects of various subtype-selective opioid agonists and antagonists on the phosphoinositide (PI) turnover response were investigated in the rat brain. The {kappa}-agonists U-50,488H and ketocyclazocine produced a concentration-dependent increase in the accumulation of IP's in hippocampal slices. The other {kappa}-agonists Dynorphin-A (1-13) amide, and its protected analog D(Ala){sup 2}-dynorphin-A (1-13) amide also produced a significant increase in the formation of ({sup 3}H)-IP's, whereas the {mu}-selective agonists (D-Ala{sup 2}-N-Me-Phe{sup 4}-Gly{sup 5}-ol)-enkephalin and morphine and the {delta}-selective agonist (D-Pen{sup 2,5})-enkephalin were ineffective. The increase in IP's formation elicited by U-50,488H was partially antagonized by naloxone and more completely antagonized by the {kappa}-selective antagonists nor-binaltorphimine and MR 2266. The formation of IP's induced by U-50,488H varies with the regions of the brain used, being highest in hippocampus and amygdala, and lowest in striatum and pons-medullar. The results indicate that brain {kappa}- but neither {mu}- nor {delta}- receptors are coupled to the PI turnover response.

  4. Phosphoinositide 3-kinase p85beta regulates invadopodium formation

    PubMed Central

    Cariaga-Martínez, Ariel E.; Cortés, Isabel; García, Esther; Pérez-García, Vicente; Pajares, María J.; Idoate, Miguel A.; Redondo-Muñóz, Javier; Antón, Inés M.; Carrera, Ana C.

    2014-01-01

    ABSTRACT The acquisition of invasiveness is characteristic of tumor progression. Numerous genetic changes are associated with metastasis, but the mechanism by which a cell becomes invasive remains unclear. Expression of p85β, a regulatory subunit of phosphoinositide-3-kinase, markedly increases in advanced carcinoma, but its mode of action is unknown. We postulated that p85β might facilitate cell invasion. We show that p85β localized at cell adhesions in complex with focal adhesion kinase and enhanced stability and maturation of cell adhesions. In addition, p85β induced development at cell adhesions of an F-actin core that extended several microns into the cell z-axis resembling the skeleton of invadopodia. p85β lead to F-actin polymerization at cell adhesions by recruiting active Cdc42/Rac at these structures. In accordance with p85β function in invadopodium-like formation, p85β levels increased in metastatic melanoma and p85β depletion reduced invadopodium formation and invasion. These results show that p85β enhances invasion by inducing cell adhesion development into invadopodia-like structures explaining the metastatic potential of tumors with increased p85β levels. PMID:25217619

  5. CELLULAR AND MOLECULAR INTERACTIONS OF PHOSPHOINOSITIDES AND PERIPHERAL PROTEINS

    PubMed Central

    Stahelin, Robert V.; Scott, Jordan L.; Frick, Cary T.

    2015-01-01

    Anionic lipids act as signals for the recruitment of proteins containing cationic clusters to biological membranes. A family of anionic lipids known as the phosphoinositides (PIPs) are low in abundance, yet play a critical role in recruitment of peripheral proteins to the membrane interface. PIPs are mono-, bis-, or trisphosphorylated derivatives of phosphatidylinositol (PI) yielding seven species with different structure and anionic charge. The differential spatial distribution and temporal appearance of PIPs is key to their role in communicating information to target proteins. Selective recognition of PIPs came into play with the discovery that the substrate of protein kinase C termed pleckstrin possessed the first PIP binding region termed the pleckstrin homology (PH) domain. Since the discovery of the PH domain, more than ten PIP binding domains have been identified including PH, ENTH, FYVE, PX, and C2 domains. Representative examples of each of these domains have been thoroughly characterized to understand how they coordinate PIP headgroups in membranes, translocate to specific membrane docking sites in the cell, and function to regulate the activity of their full-length proteins. In addition, a number of novel mechanisms of PIP-mediated membrane association have emerged, such as coincidence detection – specificity for two distinct lipid headgroups. Other PIP-binding domains may also harbor selectivity for a membrane physical property such as charge or membrane curvature. This review summarizes the current understanding of the cellular distribution of PIPs and their molecular interaction with peripheral proteins. PMID:24556335

  6. Classes of phosphoinositide 3-kinases at a glance

    PubMed Central

    Jean, Steve; Kiger, Amy A.

    2014-01-01

    ABSTRACT The phosphoinositide 3-kinase (PI3K) family is important to nearly all aspects of cell and tissue biology and central to human cancer, diabetes and aging. PI3Ks are spatially regulated and multifunctional, and together, act at nearly all membranes in the cell to regulate a wide range of signaling, membrane trafficking and metabolic processes. There is a broadening recognition of the importance of distinct roles for each of the three different PI3K classes (I, II and III), as well as for the different isoforms within each class. Ongoing issues include the need for a better understanding of the in vivo complexity of PI3K regulation and cellular functions. This Cell Science at a Glance article and the accompanying poster summarize the biochemical activities, cellular roles and functional requirements for the three classes of PI3Ks. In doing so, we aim to provide an overview of the parallels, the key differences and crucial interplays between the regulation and roles of the three PI3K classes. PMID:24587488

  7. Spatial Sensing in Fibroblasts Mediated by 3′ Phosphoinositides

    PubMed Central

    Haugh, Jason M.; Codazzi, Franca; Teruel, Mary; Meyer, Tobias

    2000-01-01

    The directed movement of fibroblasts towards locally released platelet-derived growth factor (PDGF) is a critical event in wound healing. Although recent studies have implicated polarized activation of phosphoinositide (PI) 3-kinase in G protein-mediated chemotaxis, the role of 3′ PI lipids in tyrosine kinase-triggered chemotaxis is not well understood. Using evanescent wave microscopy and green fluorescent protein–tagged Akt pleckstrin homology domain (GFP–AktPH) as a molecular sensor, we show that application of a shallow PDGF gradient triggers a markedly steeper gradient in 3′ PI lipids in the adhesion zone of fibroblasts. Polar GFP–AktPH gradients, as well as a new type of radial gradient, were measured from front to rear and from the periphery to the center of the adhesion zone, respectively. A strong spatial correlation between polarized 3′ PI production and rapid membrane spreading implicates 3′ PI lipids as a direct mediator of polarized migration. Analysis of the temporal changes of 3′ PI gradients in the adhesion zone revealed a fast diffusion coefficient (0.5 μm2/s) and short lifetime of 3′ PIs of <1 min. Together, this study suggests that the tyrosine kinase-coupled directional movement of fibroblasts and their radial membrane activity are controlled by local generation and rapid degradation of 3′ PI second messengers. PMID:11121441

  8. Structure, function, and control of phosphoinositide-specific phospholipase C.

    PubMed

    Rebecchi, M J; Pentyala, S N

    2000-10-01

    Phosphoinositide-specific phospholipase C (PLC) subtypes beta, gamma, and delta comprise a related group of multidomain phosphodiesterases that cleave the polar head groups from inositol lipids. Activated by all classes of cell surface receptor, these enzymes generate the ubiquitous second messengers inositol 1,4, 5-trisphosphate and diacylglycerol. The last 5 years have seen remarkable advances in our understanding of the molecular and biological facets of PLCs. New insights into their multidomain arrangement and catalytic mechanism have been gained from crystallographic studies of PLC-delta(1), while new modes of controlling PLC activity have been uncovered in cellular studies. Most notable is the realization that PLC-beta, -gamma, and -delta isoforms act in concert, each contributing to a specific aspect of the cellular response. Clues to their true biological roles were also obtained. Long assumed to function broadly in calcium-regulated processes, genetic studies in yeast, slime molds, plants, flies, and mammals point to specific and conditional roles for each PLC isoform in cell signaling and development. In this review we consider each subtype of PLC in organisms ranging from yeast to mammals and discuss their molecular regulation and biological function. PMID:11015615

  9. Mechanism-Based Tumor-Targeting Drug Delivery System. Validation of Efficient Vitamin Receptor-Mediated Endocytosis and Drug Release

    SciTech Connect

    Chen, S.; Wong, S.; Zhao, X.; Chen, J.; Chen, J.; Kuznetsova, L.; Ojima, I.

    2010-05-01

    An efficient mechanism-based tumor-targeting drug delivery system, based on tumor-specific vitamin-receptor mediated endocytosis, has been developed. The tumor-targeting drug delivery system is a conjugate of a tumor-targeting molecule (biotin: vitamin H or vitamin B-7), a mechanism-based self-immolative linker and a second-generation taxoid (SB-T-1214) as the cytotoxic agent. This conjugate (1) is designed to be (i) specific to the vitamin receptors overexpressed on tumor cell surface and (ii) internalized efficiently through receptor-mediated endocytosis, followed by smooth drug release via glutathione-triggered self-immolation of the linker. In order to monitor and validate the sequence of events hypothesized, i.e., receptor-mediated endocytosis of the conjugate, drug release, and drug-binding to the target protein (microtubules), three fluorescent/fluorogenic molecular probes (2, 3, and 4) were designed and synthesized. The actual occurrence of these processes was unambiguously confirmed by means of confocal fluorescence microscopy (CFM) and flow cytometry using L1210FR leukemia cells, overexpressing biotin receptors. The molecular probe 4, bearing the taxoid linked to fluorescein, was also used to examine the cell specificity (i.e., efficacy of receptor-based cell targeting) for three cell lines, L1210FR (biotin receptors overexpressed), L1210 (biotin receptors not overexpressed), and WI38 (normal human lung fibroblast, biotin receptor negative). As anticipated, the molecular probe 4 exhibited high specificity only to L1210FR. To confirm the direct correlation between the cell-specific drug delivery and anticancer activity of the probe 4, its cytotoxicity against these three cell lines was also examined. The results clearly showed a good correlation between the two methods. In the same manner, excellent cell-specific cytotoxicity of the conjugate 1 (without fluorescein attachment to the taxoid) against the same three cell lines was confirmed. This mechanism

  10. Increased levels of phosphoinositides cause neurodegeneration in a Drosophila model of amyotrophic lateral sclerosis

    PubMed Central

    Forrest, Stuart; Chai, Andrea; Sanhueza, Mario; Marescotti, Manuela; Parry, Katherine; Georgiev, Atanas; Sahota, Virender; Mendez-Castro, Raquel; Pennetta, Giuseppa

    2013-01-01

    The Vesicle-associated membrane protein (VAMP)-Associated Protein B (VAPB) is the causative gene of amyotrophic lateral sclerosis 8 (ALS8) in humans. Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterized by selective death of motor neurons leading to spasticity, muscle atrophy and paralysis. VAP proteins have been implicated in various cellular processes, including intercellular signalling, synaptic remodelling, lipid transport and membrane trafficking and yet, the molecular mechanisms underlying ALS8 pathogenesis remain poorly understood. We identified the conserved phosphoinositide phosphatase Sac1 as a Drosophila VAP (DVAP)-binding partner and showed that DVAP is required to maintain normal levels of phosphoinositides. Downregulating either Sac1 or DVAP disrupts axonal transport, synaptic growth, synaptic microtubule integrity and the localization of several postsynaptic components. Expression of the disease-causing allele (DVAP-P58S) in a fly model for ALS8 induces neurodegeneration, elicits synaptic defects similar to those of DVAP or Sac1 downregulation and increases phosphoinositide levels. Consistent with a role for Sac1-mediated increase of phosphoinositide levels in ALS8 pathogenesis, we found that Sac1 downregulation induces neurodegeneration in a dosage-dependent manner. In addition, we report that Sac1 is sequestered into the DVAP-P58S-induced aggregates and that reducing phosphoinositide levels rescues the neurodegeneration and suppresses the synaptic phenotypes associated with DVAP-P58S transgenic expression. These data underscore the importance of DVAP–Sac1 interaction in controlling phosphoinositide metabolism and provide mechanistic evidence for a crucial role of phosphoinositide levels in VAP-induced ALS. PMID:23492670