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Sample records for gamma-hydroxybutyrate receptors stimulation

  1. Experimental absence seizures: potential role of gamma-hydroxybutyric acid and GABAB receptors.

    PubMed

    Bernasconi, R; Lauber, J; Marescaux, C; Vergnes, M; Martin, P; Rubio, V; Leonhardt, T; Reymann, N; Bittiger, H

    1992-01-01

    We have investigated whether the pathogenesis of spontaneous generalized non-convulsive seizures in rats with genetic absence epilepsy is due to an increase in the brain levels of gamma-hydroxybutyric acid (GHB) or in the rate of its synthesis. Concentrations of GHB or of its precursor gamma-butyrolactone (GBL) were measured with a new GC/MS technique which allows the simultaneous assessment of GHB and GBL. The rate of GHB synthesis was estimated from the increase in GHB levels after inhibition of its catabolism with valproate. The results of this study do not indicate significant differences in GHB or GBL levels, or in their rates of synthesis in rats showing spike-and-wave discharges (SWD) as compared to rats without SWD. Binding data indicate that GHB, but not GBL, has a selective, although weak affinity for GABAB receptors (IC50 = 150 microM). Similar IC50 values were observed in membranes prepared from rats showing SWD and from control rats. The average GHB brain levels of 2.12 +/- 0.23 nmol/g measured in the cortex and of 4.28 +/- 0.90 nmol/g in the thalamus are much lower than the concentrations necessary to occupy a major part of the GABAB receptors. It is unlikely that local accumulations of GHB reach concentrations 30-70-fold higher than the average brain levels. After injection of 3.5 mmol/kg GBL, a dose sufficient to induce SWD, brain concentrations reach 240 +/- 31 nmol/g (Snead, 1991) and GHB could thus stimulate the GABAB receptor. Like the selective and potent GABAB receptor agonist R(-)-baclofen, GHB causes a dose-related decrease in cerebellar cGMP. This decrease and the increase in SWD caused by R(-)-baclofen were completely blocked by the selective and potent GABAB receptor antagonist CGP 35348, whereas only the increase in the duration of SWD induced by GHB was totally antagonized by CGP 35348. The decrease in cerebellar cGMP levels elicited by GHB was only partially antagonized by CGP 35348. These findings suggest that all effects of R

  2. Binding characteristics of gamma-hydroxybutyric acid as a weak but selective GABAB receptor agonist.

    PubMed

    Mathivet, P; Bernasconi, R; De Barry, J; Marescaux, C; Bittiger, H

    1997-02-19

    The aim of this study was to reexamine the concept that gamma-hydroxybutyric acid (GHB) is a weak but selective agonist at gamma-aminobutyric acidB (GABAB) receptors, using binding experiments with several radioligands. Ki values of GHB were similar (approximately equal to 100 microM) in three agonist radioligand assays for GABAB receptors, [3H]baclofen (beta-para-chlorophenyl-gamma-aminobutyric acid), [3H]CGP 27492 (3-aminopropyl-phosphinic acid) and [3H]GABA, in the presence of the GABAA receptor agonist isoguvacine with rat cortical, cerebellar and hippocampal membranes. In competition experiments between GHB and the GABAB receptor antagonist, [3H]CGP 54626 (3-N [1-{(S)-3,4-dichlorophenyl}-ethylamino]-2-(S)-hydroxypropyl cyclo-hexylmethyl phosphinic acid), the IC50 values were significantly increased with 300 microM of 5'-guanyl-imidodiphosphate (Gpp(NH)p), which suggested that guanine nucleotide binding proteins (G-proteins) modulate GHB binding on GABAB receptors. The inhibition by GHB of [3H]CGP 27492 binding in cortical membranes was not altered in the presence of 0.3 or 3 mM of the two GHB dehydrogenase inhibitors, valproate and ethosuximide. Thus, GHB is not reconverted into GABA by GHB dehydrogenase. Taken together, the results of this study demonstrated that GHB is an endogenous weak but selective agonist at GABAB receptors. PMID:9083788

  3. GABAB receptor-mediated activation of astrocytes by gamma-hydroxybutyric acid

    PubMed Central

    Gould, Timothy; Chen, Lixin; Emri, Zsuzsa; Pirttimaki, Tiina; Errington, Adam C.; Crunelli, Vincenzo; Parri, H. Rheinallt

    2014-01-01

    The gamma-aminobutyric acid (GABA) metabolite gamma-hydroxybutyric acid (GHB) shows a variety of behavioural effects when administered to animals and humans, including reward/addiction properties and absence seizures. At the cellular level, these actions of GHB are mediated by activation of neuronal GABAB receptors (GABABRs) where it acts as a weak agonist. Because astrocytes respond to endogenous and exogenously applied GABA by activation of both GABAA and GABABRs, here we investigated the action of GHB on astrocytes on the ventral tegmental area (VTA) and the ventrobasal (VB) thalamic nucleus, two brain areas involved in the reward and proepileptic action of GHB, respectively, and compared it with that of the potent GABABR agonist baclofen. We found that GHB and baclofen elicited dose-dependent (ED50: 1.6 mM and 1.3 µM, respectively) transient increases in intracellular Ca2+ in VTA and VB astrocytes of young mice and rats, which were accounted for by activation of their GABABRs and mediated by Ca2+ release from intracellular store release. In contrast, prolonged GHB and baclofen exposure caused a reduction in spontaneous astrocyte activity and glutamate release from VTA astrocytes. These findings have key (patho)physiological implications for our understanding of the addictive and proepileptic actions of GHB. PMID:25225100

  4. Gamma-hydroxybutyrate (GHB) induces cognitive deficits and affects GABAB receptors and IGF-1 receptors in male rats.

    PubMed

    Johansson, Jenny; Grönbladh, Alfhild; Hallberg, Mathias

    2014-08-01

    In recent years, the abuse of the club drug gamma-hydroxybutyrate (GHB) has become increasingly popular among adolescents. The drug induces euphoria but can also result in sedation, anaesthesia as well as short-term amnesia. In addition, the abuse of GHB causes cognitive impairments and the mechanism by which GHB induces these impairments is not clarified. The present study investigates the impact of GHB treatment on spatial learning and memory using a water maze (WM) test in rats. Furthermore, the behavioural data is combined with an autoradiographic analysis of the GABAB and the IGF-1 receptor systems. The results demonstrate that the animals administered with GHB display an impaired performance in the WM test as compared to controls. In addition, significant alterations in GABAB and IGF-1 receptor density as well as GABAB receptor functionality, were observed in several brain regions associated with cognitive functions e.g. hippocampus. To conclude, our findings suggest that GHB treatment can affect spatial learning and memory, and that this outcome at least to some extent is likely to involve both GABAB and IGF-1 receptors. PMID:24786330

  5. Gamma-hydroxybutyrate reduces mitogen-activated protein kinase phosphorylation via GABA B receptor activation in mouse frontal cortex and hippocampus.

    PubMed

    Ren, Xiuhai; Mody, Istvan

    2003-10-24

    gamma-Hydroxybutyrate (GHB) naturally occurs in the brain, but its exogenous administration induces profound effects on the central nervous system in animals and humans. The intracellular signaling mechanisms underlying its actions remain unclear. In the present study, the effects of GHB on the activation (phosphorylation) of mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase 1 and 2 (ERK1/2), were investigated. Acute administration of GHB (500 mg/kg, intraperitoneal) induced a fast and long lasting inhibition of MAP kinase phosphorylation in both frontal cortex and hippocampus. The reduced MAP kinase phosphorylation was observed in the CA1 and CA3 areas but not in the dentate gyrus. Pretreatment with the specific gamma-aminobutyric acid, type B (GABAB), receptor antagonist CGP56999A (20 mg/kg, intraperitoneal) prevented the action of GHB, and the effect of GHB was mimicked by baclofen, a selective GABAB receptor agonist, whereas the high affinity GHB receptor antagonist NCS-382 (200 mg/kg, intraperitoneal) had no effect on GHB-inhibited MAP kinase phosphorylation. Moreover, the GHB dehydrogenase inhibitor valproate (500 mg/kg, intraperitoneal), which inhibits the conversion of GHB into GABA, failed to block the effect of GHB on MAP kinase phosphorylation. Altogether, these data suggest that GHB, administered in vivo, reduces MAP kinase phosphorylation via a direct activation of GABAB receptors by GHB. In contrast, GHB (10 mm for 15 min) was found ineffective on MAP kinase phosphorylation in brain slices, indicating important differences in the conditions required for the second messenger activating action of GHB. PMID:12923192

  6. Characterization of the antiabsence effects of SCH 50911, a GABA-B receptor antagonist, in the lethargic mouse, gamma-hydroxybutyrate, and pentylenetetrazole models.

    PubMed

    Hosford, D A; Wang, Y; Liu, C C; Snead, O C

    1995-09-01

    Recent studies have shown that gamma-aminobutyric acidB (GABAB) receptor antagonists suppress absence seizures in animal models. (+)-5,5-Dimethyl-2-morpholineacetic acid, hydrochloride (SCH 50911) is a new GABAB antagonist that is structurally dissimilar to previously studied GABAB antagonists such as 3-aminopropyl-diethoxymethyl-phosphinic acid (CGP 35348), 3-aminopropyl-n-butyl-phosphinic acid (CGP 36742) or 3-aminopropyl-cyclohexylmethyl-phosphinic acid (CGP 46381). In this study we measured the antiabsence effects of SCH 50911 in three animal models: the lethargic (lh/lh) mutant mouse, which has spontaneous absence seizures; and two rat models in which absence seizures were induced by administration of either gamma-hydroxybutyrate or pentylenetetrazole. SCH 50911 abolished seizures in all three models in a dose-dependent fashion (ID100 = 8-170 mumol/kg). In each model SCH 50911 was more potent (ID50 = 2-22 mumol/kg) than the following antiabsence compounds: the GABAB antagonist CGP 35348 (ID50 = 210-890 mumol/kg); ethosuximide (ID50 < or = 142-1240 mumol/kg); trimethadione (ID50 = 520-1100 mumol/kg); and valproic acid (ID50 = 900-2360 mumol/kg). SCH 50911 was equipotent with the GABAB antagonist CGP 46381 (ID50 = 20 mumol/kg) in the lh/lh mouse model. These findings suggest that antiabsence activity may be a defining feature of GABAB receptor antagonists and provide a rationale for pursuing clinical trials of GABAB receptor antagonists in human patients with absence seizures. PMID:7562514

  7. Neurochemical and electrophysiological evidence for the existence of a functional gamma-hydroxybutyrate system in NCB-20 neurons.

    PubMed

    Kemmel, V; Taleb, O; Perard, A; Andriamampandry, C; Siffert, J C; Mark, J; Maitre, M

    1998-10-01

    Clonal neurohybridoma NCB-20 cells express a valproate-insensitive succinic semialdehyde reductase activity that transforms succinic semialdehyde into gamma-hydroxybutyrate. This activity (1.14+/-0.16 nmol/min/mg protein) was similar to the lowest activity existing in adult rat brain. [3H]gamma-Hydroxybutyrate labels a homogeneous population of sites on NCB-20 cell membranes (Kd=250+/-44.4nM, Bmax=180+/-16.2fmol/mg protein) that apparently represents specific gamma-hydroxybutyrate binding sites characterized previously on brain cell membranes. Finally, an Na+-dependent uptake of [3H]gamma-hydroxybutyrate was expressed in NCB-20 cells with a Km of 35+21.1 microM and a Vmax of 80+/-14.2 pmol/min/mg protein. A three-day treatment with 1 mM dibutyryl-cyclic-AMP induced a three-fold increase in the cellular succinic semialdehyde reductase activity. In parallel, a K+-evoked release of [3H]gamma-hydroxybutyrate occurred. This release was Ca2+ dependent and was not present in undifferentiated cells. Cyclic-AMP treatment induced a decrease of [3H]gamma-hydroxybutyrate binding sites, which could be due to spontaneous gamma-hydroxybutyrate release. Patch-clamp experiments carried out on differentiated NCB-20 cells revealed the presence of Ca2+ conductances which were partially inhibited by 50 microM gamma-hydroxybutyrate. This gamma-hydroxybutyrate-induced effect was blocked by the gamma-hydroxybutyrate receptor antagonist NCS-382, but not by the GABA(B) antagonist CGP-55845. These results demonstrate the presence of an active gamma-hydroxybutyratergic system in NCB-20 cells which possesses the ability to release gamma-hydroxybutyrate. These cells express specific gamma-hydroxybutyrate receptors which modulate Ca2+ currents independently of GABA(B) receptors. PMID:9692734

  8. Endogenous gamma-aminobutyric acid (GABA)(A) receptor active neurosteroids and the sedative/hypnotic action of gamma-hydroxybutyric acid (GHB): a study in GHB-S (sensitive) and GHB-R (resistant) rat lines.

    PubMed

    Barbaccia, Maria Luisa; Carai, Mauro A M; Colombo, Giancarlo; Lobina, Carla; Purdy, Robert H; Gessa, Gian Luigi

    2005-07-01

    In the rat brain, gamma-hydroxybutyric-acid (GHB) increases the concentrations of 3alpha-hydroxy,5alpha-pregnan-20-one (allopregnanolone, 3alpha,5alpha-THP) and 3alpha,21-dihydroxy,5alpha-pregnan-20-one (allotetrahydrodeoxycorticosterone/3alpha,5alphaTHDOC), two neurosteroids acting as positive allosteric modulators of gamma-aminobutyric acid (GABA)(A) receptors. This study was aimed at assessing whether neurosteroids play a role in GHB-induced loss of righting reflex (LORR). Basal and GHB-stimulated brain concentrations of endogenous 3alpha,5alpha-THP and 3alpha,5alpha-THDOC were analyzed in two rat lines, GHB-sensitive (GHB-S) and GHB-resistant (GHB-R), selectively bred for opposite sensitivity to GHB-induced sedation/hypnosis. Basal neurosteroid concentrations were similar in brain cortex of the two rat lines. However, in male GHB-S rats, administration of GHB (1000 mg/kg, i.p., 30 min) increased brain cortical concentrations of 3alpha,5alpha-THP and 3alpha,5alpha-THDOC 7- and 2.5-fold, respectively, whilst male GHB-R animals displayed only a 4- and 2-fold increase, respectively. In GHB-S rats this increase lasted up to 90 min and declined 180 min following GHB administration, a time course that matches LORR onset and duration. In contrast, in GHB-R rats, which failed to show GHB-induced LORR, brain cortical 3alpha,5alpha-THP and 3alpha,5alpha-THDOC had returned to control values within 90 min. At onset of LORR, a similar increase in brain cortical levels of 3alpha,5alpha-THP and 3alpha,5alpha-THDOC (2-3-fold) was observed in GHB-S female rats and in the few female GHB-R rats that lost the righting reflex after GHB administration, but not in female GHB-R rats failing to show LORR. Sub-hypnotic doses (7.5 and 12.5 mg/kg, i.p.) of pregnanolone, administered 10 min before GHB, dose-dependently facilitated the expression of GHB-induced LORR in GHB-R male rats. These results suggest that the GHB-induced increases of brain 3alpha,5alpha-THP and 3alpha,5alpha

  9. Central and Peripheral Metabolic Changes Induced by Gamma-Hydroxybutyrate

    PubMed Central

    Luca, Gianina; Vienne, Julie; Vaucher, Angélique; Jimenez, Sonia; Tafti, Mehdi

    2015-01-01

    Study Objectives: Gamma-hydroxybutyrate (GHB) was originally introduced as an anesthetic but was first abused by bodybuilders and then became a recreational or club drug.1 Sodium salt of GHB is currently used for the treatment of cataplexy in patients with narcolepsy. The mode of action and metabolism of GHB is not well understood. GHB stimulates growth hormone release in humans and induces weight loss in treated patients, suggesting an unexplored metabolic effect. In different experiments the effect of GHB administration on central (cerebral cortex) and peripheral (liver) biochemical processes involved in the metabolism of the drug, as well as the effects of the drug on metabolism, were evaluated in mice. Design: C57BL/6J, gamma-aminobutyric acid B (GABAB) knockout and obese (ob/ob) mice were acutely or chronically treated with GHB at 300 mg/kg. Measurements and Results: Respiratory ratio decreased under GHB treatment, independent of food intake, suggesting a shift in energy substrate from carbohydrates to lipids. GHB-treated C57BL/6J and GABAB null mice but not ob/ob mice gained less weight than matched controls. GHB dramatically increased the corticosterone level but did not affect growth hormone or prolactin. Metabolome profiling showed that an acute high dose of GHB did not increase the brain GABA level. In the brain and the liver, GHB was metabolized into succinic semialdehyde by hydroxyacid-oxoacid transhydrogenase. Chronic administration decreased glutamate, s-adenosylhomocysteine, and oxidized gluthathione, and increased omega-3 fatty acids. Conclusions: Our findings indicate large central and peripheral metabolic changes induced by gamma-hydroxybutyrate (GHB) with important relevance to its therapeutic use. Citation: Luca G, Vienne J, Vaucher A, Jimenez S, Tafti M. Central and peripheral metabolic changes induced by gamma-hydroxybutyrate. SLEEP 2015;38(2):305–313. PMID:25515097

  10. [The radioprotective effect of GABA-tropic substances, gamma-hydroxybutyrate and piracetam].

    PubMed

    Kulinskiĭ, V I; Klimova, A D

    1993-01-01

    From experiments in mice, it is shown that with a radiation dose of 8 Gy (LD96) the radioprotective effect was exerted by gamma-aminobutyric acid (GABA), substances that increase its concentration in tissues (progabide and valproate), and synthetic agonists of both receptor types, particularly baclofen, a GABA-receptor agonist. The radioprotective effect is also exerted by gamma-hydroxybutyrate, not piracetam. PMID:8469734

  11. Gamma hydroxybutyrate--a coma inducing recreational drug.

    PubMed Central

    Ryan, J M; Stell, I

    1997-01-01

    The effects of gamma hydroxybutyrate, a coma inducing recreational drug, are described and illustrated by case reports of five patients presenting to accident and emergency (A&E). All had depressed levels of consciousness. There was strong circumstantial evidence of gamma hydroxybutyrate ingestion in all cases, and laboratory evidence in two. All recovered and supportive treatment. gamma Hydroxybutyrate has become a fashionable recreational drug. The majority of people who have ingested it will recover spontaneously without long term sequelae but its toxic effects may be dramatic while they last, particularly when it is taken with other drugs or alcohol. Images Figure 3 Figure 1 PMID:9248920

  12. Baclofen and Gamma-Hydroxybutyrate Withdrawal

    PubMed Central

    LeTourneau, Jennifer L.; Hagg, Daniel S.; Smith, Stephen M.

    2008-01-01

    Introduction Benzodiazepine treatment of life-threatening gamma-hydroxybutyrate (GHB) withdrawal is frequently unsatisfactory. Animal studies suggest strongly that treatment with GABAB agonists, such as baclofen, will be a more effective strategy. Methods A case report from the medical intensive care unit (ICU) of the university tertiary care hospital. Results A 61-year-old woman was admitted to the medical ICU for severe withdrawal symptoms from chronic GHB use. This manifested as delirium, tremor, and seizures despite only small decreases in GHB dose and treatment with benzodiazepines. The addition of baclofen allowed the rapid sequential decreases in the GHB dose without seizure or delirium and resulted in long-term improvement of her tremor. Conclusions Baclofen, a GABAB agonist, may be a useful agent in the treatment of severe GHB withdrawal. PMID:18266111

  13. Gamma-hydroxybutyrate withdrawal syndrome: a case report

    PubMed Central

    2009-01-01

    Introduction To raise awareness among health care workers of the risk of withdrawal symptoms after longstanding and intense abuse of gamma-hydroxybutyric acid. Case presentation A 23 year old Caucasian woman presented with gamma-hydroxybutyric addiction and withdrawal syndrome. The symptoms of gamma-hydroxybutyric withdrawal in this patient initially went unrecognized, upon which her situation deteriorated in such a way that she needed to be admitted to the Intensive Care Unit for airway protection and mechanical ventilation. Treatment with high doses of benzodiazepines led to liberation of the ventilator and further recovery. Conclusion Withdrawal symptoms of gamma-hydroxybutyric addiction are often not well recognized and the responsible physicians at Emergency Department, Intensive Care Unit and the Psychiatry ward need better understanding of diagnose and treatment. Gamma-hydroxybutyric acid withdrawal is potentially life threatening and its management may require a multidisciplinary approach. Early recognition of gamma-hydroxybutyric acid withdrawal may lead to better management of these patients. PMID:20181164

  14. BEHAVIORAL EFFECTS OF GAMMA-HYDROXYBUTYRATE (GHB) IN HUMANS

    PubMed Central

    Oliveto, Alison; Gentry, W. Brooks; Pruzinsky, Rhonda; Gonsai, Kishorchandra; Kosten, Thomas R.; Martell, Bridget; Poling, James

    2010-01-01

    Despite the therapeutic use and abuse potential of gamma-hydroxybutyrate (GHB or Xyrem), relatively few studies have examined the behavioral effects of GHB in humans under controlled laboratory conditions. Thus, this eight-session study examined in 10 non substance-abusing volunteers the behavioral effects of GHB at each of the following doses: 0, 0.32, 0.56, 0.75, 1.0, 1.8, 2.4, 3.2 g/70 kg, p.o.. Order of dose testing was random, except that the first two participants received active doses in ascending order and 2.4 g/70 kg was always tested before 3.2 g/70 kg. Prior to drug administration and at several post-drug time points, self-report, observer-report, physiological, and psychomotor performance measures were obtained. Analyses based on area under the curve showed that GHB produced dose-related increases in subjective ratings of sedative-like, stimulant-like, positive mood, and dissociative effects, but no changes in psychomotor performance measures or blood pressure. Analyses based on peak effects generally showed dose-related increases in ratings indicating sedative-like, dissociative, and drug liking, although some measures showed U-shaped dose-related changes. These initial findings suggest that GHB at doses of 0.32–3.2 g/70 kg produces dissociative, sedating and some stimulant-like effects in humans without a history of sedative abuse. PMID:20526195

  15. [Qualitative and quantitative gamma-hydroxybutyrate analysis].

    PubMed

    Petek, Maja Jelena; Vrdoljak, Ana Lucić

    2006-12-01

    Gamma-hydroxybutyrate (GHB) is a naturally occurring compound present in the brain and peripheral tissues of mammals. It is a minor metabolite and precursor of gamma-aminobutyric acid (GABA). Just as GABA, GHB is believed to play a role in neurotransmission. GHB was first synthesized in vitro in 1960, when it revealed depressive and hypnotic effects on the central nervous system. In 1960s it was used as an anaesthetic and later as an alternative to anabolic steroids, in order to enhance muscle growth. However, after it was shown that it caused strong physical dependence and severe side effects, GHB was banned. For the last fifteen years, GHB has been abused for its intoxicating effects such as euphoria, reduced inhibitions and sedation. Illicitly it is available as white powder or as clear liquid. Paradoxically GHB can easily be manufactured from its precursor gamma-butyrolactone (GBL), which has not yet been banned. Because of many car accidents and criminal acts in which it is involved, GHB has become an important object of forensic laboratory analysis. This paper describes gas and liquid chromatography, infrared spectroscopy, microscopy, colourimetry and nuclear magnetic resonance as methods for detection and quantification of GHB in urine and illicit products. PMID:17265679

  16. Preference for Gamma-Hydroxybutyrate (GHB) in Current Users

    ERIC Educational Resources Information Center

    Roll, John M.; Newton, Thomas; Chudzynski, Joy; Cameron, Jennifer M.; McPherson, Sterling; Fong, Tim; Torrington, Matt

    2012-01-01

    Gamma-hydroxybutyrate (GHB) is a drug with significant abuse potential. The present study aimed to assess the relative value of escalating doses of GHB to current GHB users via the Multiple Choice Procedure (MCP), and to validate that the dose rated highest with the MCP would be self-administered at a greater rate than placebo. Participants were 5…

  17. [Life threatening symptoms of withdrawal of gamma-hydroxybutyrate].

    PubMed

    Veerman, S R T; Dijkstra, H N; Liefting-Kluft, I

    2010-01-01

    Acute psychosis and extreme agitation brought about by gamma-hydroxybutyrate GHB withdrawal can be life-threatening. In order to prevent states of excitement accompanied by aggression and somatic complications it is advisable to intervene by administering strong sedatives. It is argued that GHB should be tapered off as an alternative treatment for fixation and high doses of benzodiazepines. PMID:20544599

  18. A potential new metabolite of gamma-hydroxybutyrate: sulfonated gamma-hydroxybutyric acid.

    PubMed

    Hanisch, Stephanie; Stachel, Nicole; Skopp, Gisela

    2016-03-01

    Detection of gamma-hydroxybutyric acid (GHB) became crucial in many clinical and forensic settings due to its increasing use for recreational purposes and drug-facilitated sexual assault. Its narrow window of detection of about 3-12 h in urine represents a major problem. Analogous to ethyl glucuronide, the recently identified GHB-glucuronide exhibits a longer window of detection than the parent drug. It appeared reasonable that a sulfonated metabolite of GHB (GHB-SUL) will also be formed. Due to the lack of an appropriate standard, GHB was incubated with a human liver cytosolic fraction to produce GHB-SUL. Following development of a liquid chromatography/tandem mass spectrometry (LC-MS/MS) assay to measure GHB and GHB-SUL, authentic urine samples (n = 5) were tested for GHB-SUL. These investigations revealed detectable signals of both GHB and GHB-SUL, strongly indicating that GHB is not only glucuronidated but also sulfonated. Given that sulfonated metabolites generally have longer half-life times than the corresponding free drugs, GHB-SUL may serve as a biomarker of GHB misuse along with its glucuronide. PMID:26210636

  19. Gamma-hydroxybutyric acid as hypnotic. Clinical and pharmacokinetic evaluation of gamma-hydroxybutyric acid as hypnotic in man.

    PubMed

    Hoes, M J; Vree, T B; Guelen, P J

    1980-01-01

    Gamma-Hydroxybutyric acid (GOH) was administered to three groups of four patients in 50, 75 or 100 mg/kg dose respectively, and sleep effects were scored, by sleep observation and questionnaire; GOH plasma levels were determined at 75-100 mg/kg, saliva and urine excretion at 100 mg/kg. Sleep induction was rapid and irresistible, subjects awoke at plasma levels of 90 micrograms/ml; sleep scores were good for 75 mg/kg, excellent for 100 mg/kg, notably on mood. For 75 or 100 mg/kg GOH had virtually disappeared from the blood eight hours after intake; twelve hours after intake no more GOH was detectable in urine; no correlation between plasma- and saliva levels was found. Plasma levels for 100 mg/kg dose were not at an anaesthetic level. So, GOH is a safe and good hypnotic at 75 or 100 mg/kg in clinical use. PMID:7449723

  20. EXPERIENCES OF GAMMA HYDROXYBUTYRATE (GHB) INGESTION: A FOCUS GROUP STUDY

    PubMed Central

    Barker, Judith C.; Harris, Shana L.; Dyer, Jo E.

    2008-01-01

    GHB (gamma hydroxybutyrate) is a significant new drug of abuse added to the United States Controlled Substance Act in 2000. The majority of the published literature on GHB consists of clinical case reports, mainly from emergency departments, and a collection of laboratory-based studies, focused mainly on anesthesia. While comments about the various experiences and behaviors of human users are often included in such studies or reports, these aspects of GHB are only just beginning to be systematically investigated or detailed. Reported here are data from a qualitative study using focus group methods on the consumption habits, experiences, and beliefs of GHB users. A total of 51 people, 30 men and 21 women, mean age of 31.1±7.6 years (range 18 – 52 years), who report having used GHB for an average of 4.3±2.5 years (range 1–11 years), were interviewed in 10 separate groups held in 2004. This paper discusses broadly the general experience of the GHB ‘high,’ major perceived benefits including sexual responses to the drug, perceived risks and dangers of ingestion, co-ingestion, and various contexts of use. The paper concludes with a discussion of the implications drawn from this information for clinicians treating patients who use GHB. PMID:17703706

  1. Preference for gamma-hydroxybutyrate (GHB) in current users.

    PubMed

    Roll, John M; Newton, Thomas; Chudzynski, Joy; Cameron, Jennifer M; McPherson, Sterling; Fong, Timothy; Torrington, Matt

    2012-05-01

    Gamma-hydroxybutyrate (GHB) is a drug with significant abuse potential. The present study aimed to assess the relative value of escalating doses of GHB to current GHB users via the Multiple Choice Procedure (MCP), and to validate that the dose rated highest with the MCP would be self-administered at a greater rate than placebo. Participants were 5 current GHB users who were not currently trying to stop using GHB. To examine the value of escalating doses of GHB, the following doses of GHB were used: 0 (placebo), 12.5, 25, 37.5, and 50 mg/kg. Participants typically assigned higher doses of GHB had higher crossover points on the MCP. During choice sessions, participants made repeated choices between administering GHB, placebo or nothing. All participants selected GHB exclusively (5 out of 5 instances) except for one participant who selected GHB on 4 out of 5 instances, thus 96% (i.e., 24/25) of choices were for active GHB. Based on these data, GHB appears likely to function as a dose-dependent reinforcer for humans based on our sample. PMID:22693361

  2. Preference for Gamma-Hydroxybutyrate (GHB) in Current Users

    PubMed Central

    Roll, John M; Newton, Thomas; Chudzynski, Joy; Cameron, Jennifer M; McPherson, Sterling; Fong, Timothy; Torrington, Matt

    2012-01-01

    Gamma-hydroxybutyrate (GHB) is a drug with significant abuse potential. The present study aimed to assess the relative value of escalating doses of GHB to current GHB users via the Multiple Choice Procedure (MCP), and to validate that the dose rated highest with the MCP would be self-administered at a greater rate than placebo. Participants were 5 current GHB users who were not currently trying to stop using GHB. To examine the value of escalating doses of GHB, the following doses of GHB were used: 0 (placebo), 12.5, 25, 37.5, and 50 mg/kg. Participants typically assigned higher doses of GHB had higher crossover points on the MCP. During choice sessions, participants made repeated choices between administering GHB, placebo or nothing. All participants selected GHB exclusively (5 out of 5 instances) except for one participant who selected GHB on 4 out of 5 instances, thus 96% (i.e., 24/25) of choices were for active GHB. Based on these data, GHB appears likely to function as a dose-dependent reinforcer for humans based on our sample. PMID:22693361

  3. Acute poisoning from gamma-hydroxybutyrate in California.

    PubMed Central

    Chin, M. Y.; Kreutzer, R. A.; Dyer, J. E.

    1992-01-01

    We report a series of 5 representative patients in California who experienced adverse reactions from the illicitly marketed substance gamma-hydroxybutyrate (GHB). The drug is a putative neurotransmitter marketed as a growth hormone releaser for bodybuilders. The most commonly reported symptoms included abrupt drowsiness, dizziness, and a "high". Other effects were headache, nausea, vomiting, myoclonic jerking, and short-term coma. There have been no reported deaths. If product use is discontinued, full recovery with no long-term side effects is universal. No clear dose-response effect was observed; this may be attributable to differences in susceptibility, wide variations in doses taken by the same person, or the coingestion of other substances. Case interviews confirm that, despite being banned by the US Food and Drug Administration, GHB is still widely available in the underground drug market. Athletes and bodybuilders may take drugs for which there are claims of improved performance or body image. Physicians should be alert for signs of GHB poisoning in emergency department and clinic patients. PMID:1574880

  4. Gamma hydroxybutyrate: an ethnographic study of recreational use and abuse.

    PubMed

    Lee, Steven J; Levounis, Petros

    2008-09-01

    Gamma hydroxybutyrate (GHB) is a psychoactive substance with complex neurophysiological activity and significant potential for abuse, addiction, and dangerous toxicity. In this study, a semistructured interview was administered to 17 subjects to investigate GHB use, including: manner of use; setting; positive and negative consequences; other drug history; and sexual practices. Respondents were overwhelmingly male, but otherwise had a broad demographic background. Settings varied from nightclubs to private use at home. There was significant variability in the drug obtained, which subjects found problematic because of the narrow therapeutic window and ease of accidental overdose. Common positive experiences included increased sexual desire, decreased sexual inhibitions, and decreased anxiety. Common negative consequences included oversedation, loss of consciousness, motor incoordination, and mental confusion. Nine subjects reported that they would use GHB again, some despite severe negative consequences. Although most subjects reported negative experiences, only three felt their use was problematic, and none sought treatment for GHB abuse or addiction. Subjects were highly drug-experienced, most commonly using MDMA, ketamine, cocaine, alcohol, and methamphetamine. Some reported that GHB could cause poor decision making in sexual situations. This effect has significant ramifications for issues such as date rape and control of sexually transmitted diseases, such as HIV. PMID:19004416

  5. Effects of Gamma Hydroxybutyric Acid on Inhibition and Excitation in Rat Neocortex

    PubMed Central

    Li, Qiang; Kuhn, Cynthia M.; Wilson, Wilkie A.; Lewis, Darrell V.

    2008-01-01

    The mechanism by which the sedative and amnestic recreational drug gamma hydroxybutyric acid (GHB) acts is controversial. Some studies indicate that it acts at its unique receptor, while others demonstrate effects mediated through the GABAB receptor. We examined the effect of GHB on evoked GABAA receptor mediated mono- and polysynaptic IPSCs as well as on NMDA and AMPA mediated EPSCs in layers II/III pyramidal cells of the frontal cortex of rat brain. One millimolar (mM) GHB suppressed monosynaptic IPSCs by 20%, whereas polysynaptic IPSCs were reduced by 56%. GHB (1mM) also produced a significant suppression of NMDA-mediated EPSCs by 53% compared to 27% suppression of AMPA-mediated EPSCs. All effects of GHB on IPSCs and EPSCs were reversed by the specific GABAB antagonist CGP62349, but not by the GHB receptor antagonist NCS 382. Consistent with a presynaptic site of action, GHB reduced the frequency but not the amplitude of AMPA receptor mediated mEPSCs and had no effect on postsynaptic currents evoked by direct application of NMDA. Finally, even though GHB appeared to be acting at presynaptic GABAB receptors, GHB and the GABAB agonist baclofen appeared to have opposite potencies for depression of NMDA vs AMPA mediated EPSCs. GHB showed a preference for depressing NMDA responses while baclofen more potently suppressed AMPA responses. The suppression of NMDA more than AMPA responses by GHB at intoxicating doses may make it attractive as a recreational drug and may explain why GHB is abused and baclofen is not. PMID:17904295

  6. Safety and tolerability of gamma-hydroxybutyric acid in the treatment of alcohol-dependent patients.

    PubMed

    Beghè, F; Carpanini, M T

    2000-04-01

    Gamma-hydroxybutyric acid (GHB) has been in clinical use in Italy since 1991 for treatment of alcohol dependence. Results of phase III and phase IV studies have shown that the drug is effective and well tolerated in the treatment of alcohol withdrawal syndrome and in reducing alcohol consumption and alcohol craving. Pharmacosurveillance indicates that abuse of gamma-hydroxybutyric acid is a limited phenomenon in clinical settings when the drug is dispensed under strict medical surveillance and entrusted to a referring familiar member of the patient. PMID:10869863

  7. GC-MS Analysis of [gamma]-Hydroxybutyric Acid Analogs: A Forensic Chemistry Experiment

    ERIC Educational Resources Information Center

    Henck, Colin; Nally, Luke

    2007-01-01

    An upper-division forensic chemistry experiment is described. It involves using glycolic acid and sodium glycolate as analogs of [gamma]-hydroxybutyric acid and its sodium salt. The experiment shows the use of silylation in GC-MS analysis and gives students the opportunity to work with a commonly used silylating reagent,…

  8. gamma-Hydroxybutyrate conversion into GABA induces displacement of GABAB binding that is blocked by valproate and ethosuximide.

    PubMed

    Hechler, V; Ratomponirina, C; Maitre, M

    1997-05-01

    gamma-Hydroxybutyrate (GHB) has been reported to be a ligand for GABAB receptor(s), although with low or very low affinity (IC50 = 150-796 microM). In addition, several reports argue for a role of GHB via GABAB receptors in both in vivo and in vitro electro-physiological experiments. In the present study, we demonstrate that the inhibition of GHB's conversion into GABA by rat brain membranes blocks the ability of GHB to interfere with GABAB binding. In particular, the inhibition of GHB dehydrogenase by valproate or ethosuximide and the blockade of GABA-T by aminooxyacetic acid induce the disappearance of the GABA-like effect of GHB at GABAB, but also at GABAA, receptors. This finding could explain the misinterpretation of in vitro or in vivo experiments where GHB possesses a GABA-like effect. But in addition, it is postulated that the normal metabolism of GHB in brain induces GABAB mechanisms that could be blocked by the administration of valproate or ethosuximide. PMID:9152382

  9. Circadian rhythm in plasma concentrations of gamma-hydroxybutyric acid in alcoholics.

    PubMed

    Hoes, M J; Vree, T B; Guelen, P J

    1981-08-01

    Gamma-hydroxybutyric acid (GHB) was orally administered to six alcoholics at 09.00 and 23.00 h. The plasma concentrations of GHB show a clear circadian pattern, the area under the curve in the daytime experiments being 61% of that in the night experiments. The significance of alcohol dehydrogenase, the catabolic enzyme of GHB, for the difference is discussed. It is concluded that, although the activity of alcohol dehydrogenase in alcoholics is quantitatively disturbed, it remains subject to physiologic circadian activation. PMID:7341501

  10. [Variety of symptoms after drug use of gamma-hydroxybutyric acid (GHB)].

    PubMed

    Galldiks, N; Kadow, I; Bechdolf, A; Fink, G R; Klosterkötter, J; Kuhn, J

    2011-01-01

    Gamma-hydroxybutyric acid (GHB, "liquid ecstasy") and its legal prodrugs gamma-butyrolactone and 1,4-butanediol are gaining importance as recreational drugs in Germany. Because of the wide availability of GHB and its prodrugs physicians are increasingly being confronted with cases of intoxication. The effect of GHB intoxication is comparable with those of alcohol and/or benzodiazepines. Likewise, symptoms of withdrawal may occur. In this review, we summarise current data regarding the history, pharmacodynamics and pharmacokinetics of the drug as well as the relevant symptoms of intoxication or withdrawal as they pertain to neurology and psychiatry. PMID:21154180

  11. A proposed preventive role for Gamma-hydroxybutyrate (Xyrem(R)) in Alzheimer's disease.

    PubMed

    Maitre, Michel; Klein, Christian; Mensah-Nyagan, Ayikoe G

    2016-01-01

    Gamma-hydroxybutyrate (GHB or Xyrem(R)) is frequently used in humans for several clinical indications, including anesthesia, narcolepsy/cataplexy, and alcohol-withdrawal symptoms. Pharmacological effects induced in the brain by therapeutic doses of Xyrem(R) are generally GABAergic-dependent. These effects allow sedation, stress/anxiety reduction, deep sleep induction, decrease of neuroinflammation, and neuroprotection. Furthermore, Xyrem(R) promotes the expression of pivotal genes reducing toxic proteinopathies, as demonstrated in laboratory animal models. Altogether, these data represent additional evidence to suggest that Xyrem(R) may be tested during repeated short periods in populations at risk for Alzheimer's disease. PMID:27601032

  12. Effect of gamma-hydroxybutyrate on keratinocytes proliferation: A preliminary prospective controlled study in severe burn patients

    PubMed Central

    Rousseau, Anne-Françoise; Bargues, Laurent; Bever, Hervé Le; Vest, Philippe; Cavalier, Etienne; Ledoux, Didier; Piérard, Gérald E.; Damas, Pierre

    2014-01-01

    Background: Hypermetabolism and hyposomatotropism related to severe burns lead to impaired wound healing. Growth hormone (GH) boosts wound healing notably following stimulation of the production of insulin-like growth factor-1 (IGF1), a mitogen factor for keratinocytes. Gamma-hydroxybutyrate (GHB) stimulates endogenous GH secretion. Aim: To assess effects of GHB sedation on keratinocytes proliferation (based on immunohistochemical techniques). Design: Monocentric, prospective, controlled trial. Materials and Methods: Patients (aging 18-65 years, burn surface area >30%, expected to be sedated for at least one month) were alternately allocated, at the 5th day following injury, in three groups according to the intravenous GHB dose administered for 21 days: Evening bolus of 50 mg/kg (Group B), continuous infusion at the rate of 10 mg/kg/h (Group C), or absence of GHB (Group P). They all received local standard cares. Immunohistochemistry (Ki67/MIB-1, Ulex europaeus agglutinin-1 and Mac 387 antibodies) was performed at D21 on adjacent unburned skin sample for assessing any keratinocyte activation. Serum IGF1 levels were measured at initiation and completion of the protocol. Statistical Analysis: Categorical variables were compared with Chi-square test. Comparisons of medians were made using Kruskal-Wallis test. Post hoc analyses were performed using Mann-Whitney test with Bonferroni correction for multiple comparisons. A P < 0.05 was considered to be statistically significant. Results: A total of 14 patients completed the study (Group B: n = 5, Group C: n = 5, Group P: n = 4). Continuous administration of GHB was associated with a significant higher Ki67 immunolabeling at D21 (P = 0.049) and with a significant higher increase in the IGF1 concentrations at D21 (P = 0.024). No adverse effects were disclosed. Conclusions: Our preliminary data support a positive effect of GHB on keratinocyte proliferation and are encouraging enough to warrant large prospective studies. PMID

  13. Dilated cardiomyopathy and acute liver injury associated with combined use of ephedra, gamma-hydroxybutyrate, and anabolic steroids.

    PubMed

    Clark, Brychan M; Schofield, Richard S

    2005-05-01

    Anabolic-androgenic steroids are synthetic derivatives of testosterone that some athletes have used to enhance muscle mass and improve their athletic performance. Ephedrine is a potent sympathomimetic agent that can lead to cardiomyopathy similar to that seen with catecholamine excess. Adverse cardiovascular events attributed to anabolic steroid and ephedra use, such as arrhythmias, myocardial infarction, cardiomyopathy, and sudden death, are rarely reported. Bodybuilders have used gamma-hydroxybutyrate, a potent secretagogue of growth hormone, to promote muscle development. Although dilated cardiomyopathy is a known complication of excess growth hormone levels, it has not been associated with use of gamma-hydroxybutyrate. A healthy 40-year-old man was admitted to our hospital for new-onset congestive heart failure and severe acute hepatitis that developed several months after he began using anabolic-androgenic steroids, ephedra, and gamma-hydroxybutyrate supplements. Analysis with an objective causality assessment scale revealed a probable adverse drug reaction between the patient's use of anabolic steroids, ephedra, and gamma-hydroxybutyrate and the development of his cardiomyopathy and acute liver injury. PMID:15899737

  14. A web-based study of gamma hydroxybutyrate (GHB): patterns, experiences, and functions of use.

    PubMed

    Stein, L A R; Lebeau, Rebecca; Clair, Mary; Martin, Rosemarie; Bryant, Monte; Storti, Susan; Monti, Peter

    2011-01-01

    GHB (gamma hydroxybutyrate) was developed as a general anesthetic. Due to dosing difficulty and side effects, regular use was discontinued. Medical uses include treating sleep and alcohol disorders. In the 1990s, it was promoted as a supplement and taken to improve mood and sex. GHB and its analogs (gamma butyrolactone and butanediol) were widely available until federal regulations were put into effect with mounting evidence of adverse events. This survey (N = 61) study was conducted to assess patterns, experiences, and functions of use. Much of what is understood regarding GHB treatment is based on hospital case studies for overdose and withdrawal. Not enough is known about prevention, reducing use and associated problems, or relapse. We know little about specific drug effect expectancies, triggers, coping skills, and consequences of use (positive/negative). While the drug treatment literature has a wealth of information to draw upon, GHB-specific information may greatly assist relapse prevention.  PMID:21175918

  15. A Web-Based Study of Gamma Hydroxybutyrate (GHB): Patterns, Experiences, and Functions of Use

    PubMed Central

    Stein, LAR; Lebeau, Rebecca; Clair, Mary; Martin, Rosemarie; Bryant, Monte; Storti, Susan; Monti, Peter

    2011-01-01

    GHB (gamma hydroxybutyrate) was developed as a general anesthetic. Due to dosing difficulty and side effects, regular use was discontinued. Medical uses include treating sleep and alcohol disorders. In the 1990s, it was promoted as a supplement and taken to improve mood and sex. GHB and its analogs (gamma butyrolactone and butanediol) were widely available until federal regulations were put into effect with mounting evidence of adverse events. This survey (N = 61) study was conducted to assess patterns, experiences, and functions of use. Much of what is understood regarding GHB treatment is based on hospital case studies for overdose and withdrawal. Not enough is known about prevention, reducing use and associated problems, or relapse. We know little about specific drug effect expectancies, triggers, coping skills, and consequences of use (positive/negative). While the drug treatment literature has a wealth of information to draw upon, GHB-specific information may greatly assist relapse prevention. PMID:21175918

  16. [The knowledge about gamma-hydroxybutyric acid as by students of Physical Education Academy].

    PubMed

    Chwaluk, Paweł; Chwaluk, Agnieszka; Parnicki, Florian

    2009-01-01

    Gamma-hydroxybutyric acid is a substance stealthily used by criminals to facilitate sexual assaults. It is also known as doping agent in sports. Physical Education Academies should prepare their graduates to be educators for young people, their trainers, organizers of sports and recreational events. Second year students of two majors: physical education and tourism and recreation were surveyed by means of questionnaire on "date-rape drug". As much as 320 among 327 students surveyed had heard about "date-rape drug". However their knowledge on it was shallow and unsystematic. None of the surveyed knew that the substance of "date-rape drug" could also be used as a doping agent. Only 31% of respondents were aware of existence of the test to detect "date-rape drug" in drinks. Physical Education Academy students should be thoroughly and relevantly educated on the matter of pharmacologic doping agents and drugs endangerment. PMID:19788135

  17. Gamma-hydroxybutyrate (GHB): a scoping review of pharmacology, toxicology, motives for use, and user groups.

    PubMed

    Brennan, Rebekah; Van Hout, Marie Claire

    2014-01-01

    Gamma hydroxybutyrate (GHB) is a central nervous system depressant with euphoric and relaxant effects. Documentation of GHB prevalence and the underreporting of abuse remains problematic, given the availability of GHB and its precursors γ-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) and the ease of synthesis from kits available on the Internet. The continued abuse of and dependence on GHB, and associated fatalities, present an on-going public health problem. As the drug GHB remains an underresearched topic, a scoping review was chosen as a technique to map the available literature into a descriptive summarized account. PRISMA was used to assist in data retrieval, with subsequent data charting into three key themes (pharmacology and toxicology, outcomes, and user groups). Administered orally, GHB is dose-dependent and popular for certain uses (therapeutic, body enhancement, sexual assault) and amongst user sub groups (recreational party drug users, homosexual men). Despite the low prevalence of use in comparison to other club drugs, rising abuse of the drug is associated with dependence, withdrawal, acute toxicity, and fatal overdose. Clinical diagnosis and treatment is complicated by the co-ingestion of alcohol and other drugs. Limitations of the scoping review and potential for further research and harm reduction initiatives are discussed. PMID:25052883

  18. [Gamma-hydroxybutyrate (GHB) and its lactone (GBL) as psychoactive substances].

    PubMed

    Krajewska, Anna; Kwiecień-Obara, Ewelina; Szponar, Jarosław; Majewska, Magdalena; Kołodziej, Małgorzata

    2012-01-01

    Gammabutyrolactone is included in the solvent such as wheel cleaners, pesticides, cosmetics, drugs. After ingestion GBL is converted to gamma-hydroxybutyrate. Both substances are classified as so called "club drugs" and their action is characterized by euphoria, sedation, and induction of retrograde amnesia of events. These activities were basis for the use of GHB and its lactone as rape pill. Acute poisoning with these compounds causes confusion, agitation, ataxia, nausea, vomiting, nystagmus, dyskinesia, hallucinations, coma, irregular breathing, hypothermia, bradycardia, hypotension, convulsions, respiratory paralysis and thus respiratory arrest. These substances carry a risk of development of physical addiction of the hard proceeding of abstinence syndrome. In the USA there is a ban on the sale and promotion of these compounds. In Poland despite the fact that GHB is a controlled substance, there is no regulation of GBL trading. The aim of this paper is to summarize current knowledge regarding the pharmacology, impact on the human body, toxicity, and the effects of chronic abuse of these substances. PMID:23243924

  19. The involvement of gamma-hydroxybutyrate in reported sexual assaults: a systematic review.

    PubMed

    Németh, Zsófia; Kun, Bernadette; Demetrovics, Zsolt

    2010-09-01

    Over the past few years gamma-hydroxybutyrate (GHB) has generated widespread media interest as a possible 'date rape drug'. Our goal was to examine the extent to which GHB is associated with drug-facilitated sexual assaults. Literature was searched systematically and 11 studies, published between 1961 and June 30, 2009, were identified dealing specifically with the role of GHB in sexual assaults. GHB was detected in 0.2-4.4% of reported sexual assaults. The results demonstrate that a wide range of drugs may be present in cases of sexual assault, and many of them are much more frequent than GHB. Our results do not support the widespread labelling of GHB as a date rape drug as the prevalence of GHB is much lower than of other substances used in sexual assaults. On the other hand, however, the possible risk of GHB in this regard should not be neglected. Nevertheless, over-sensitive and sensation seeking media reports focusing on the association of sex crime and GHB might be counterproductive and misleading as they turn the attention away from other substances that are often used in sexual assaults. PMID:20488831

  20. Effects of gamma-hydroxybutyric acid and flunitrazepam on ethanol intake in male rats.

    PubMed

    Leonard, Stuart T; Gerak, Lisa R; Gurkovskaya, Olga; Moerschbaecher, Joseph M; Winsauer, Peter J

    2006-12-01

    Both gamma-hydroxybutyric acid (GHB) and flunitrazepam are often used illicitly in combination with ethanol. Nevertheless, the effects that these and other drugs of abuse have on the reinforcing effects of ethanol remain inconclusive. To test the effects of GHB and flunitrazepam on contingent ethanol intake, twelve male Long-Evans rats were trained to orally consume ethanol using a saccharin-fading procedure. After training, all animals preferentially consumed ethanol instead of water at each of five ethanol concentrations (0-32%) when tested with a two-bottle preference test in the homecage. Animals then received a noncontingent dose of ethanol (0.32, 0.56, 1, and 1.33 g/kg), flunitrazepam (0.032, 0.1, and 0.32 mg/kg), or GHB (100, 180, 320, and 560 mg/kg) prior to each subject's daily access to ethanol (18% v/v). Noncontingent doses of ethanol decreased ethanol intake, however, the subjects consumed enough ethanol to maintain a consistent total ethanol dose in g/kg. Flunitrazepam did not affect ethanol intake at any dose tested, whereas GHB only affected intake at the highest dose (560 mg/kg), a dose that also produced sedation. These data suggest that there are perceptible or qualitative differences between GHB, flunitrazepam, and ethanol in terms of their capacity for modulating oral ethanol intake in outbred rats. PMID:17208286

  1. Gamma-hydroxybutyric acid stability and formation in blood and urine.

    PubMed

    Beránková, Katerina; Mutnanská, Katerina; Balíková, Marie

    2006-09-12

    Gamma-hydroxybutyric acid (GHB) can cause problems in interpretation of toxicological findings due to its endogenous nature, significant production in tissues after death and potential formation in stored samples. Our study was designed to determine the influence of storage conditions on GHB levels and its possible in vitro formation in blood and urine in cases where no exogenous use of GHB or its precursors was suspected. The samples were prepared by validated method based on liquid-liquid reextraction with adipic acid internal standard and MSTFA derivatization and assayed on a GC-MS operating in EI SIM mode. The first part of the study was performed with pooled blood and urine samples obtained from living and deceased subjects stored with and without NaF (1% w/v) at 4 and -20 degrees C over 8 months. In ante-mortem samples (both blood and urine) no significant GHB production was found. After 4 months of storage, the substantial GHB rise up to 100 mg/Lwas observed in post-mortem blood stored at 4 degrees C without NaF with subsequent gradual decrease in following months. The inhibition of GHB production was apparent during storage in NaF treated frozen blood samples. In post-mortem urine only slight temporary GHB levels were ascertained (up to 8 mg/L). The second part of our study was aimed to analyse 20 individual post-mortem blood samples stored at 4 degrees C for 16-27 days between autopsy and analysis without preservation followed by storage at 4 degrees C with NaF for 4 months. The temporary GHB production with maximum of 28 mg/Lwas detected in some samples. PMID:16857333

  2. Intravenous self-administration of gamma-hydroxybutyrate (GHB) in baboons

    PubMed Central

    Goodwin, Amy K.; Kaminski, Barbara J.; Griffiths, Roland R.; Ator, Nancy A.; Weerts, Elise M.

    2010-01-01

    Background Abuse of gamma-hydroxybutyrate (GHB) poses a public health concern. In previous studies, intravenous (IV) self-administration of GHB doses up to 10 mg/kg was not maintained in non-human primates under limited-access conditions, which was inconsistent with the usual good correspondence between drugs abused by humans and those self-injected by laboratory animals. Methods Self-administration of GHB was studied in 10 baboons using procedures standard for our laboratory to assess drug abuse liability. Each self-injection depended on completion of 120 or 160 lever responses. Sessions ran continuously; a 3-h timeout limited the number of injections per 24 h to 8. Self-injection was established at 6–8 injections/day with cocaine (0.32 mg/kg/injection) prior to substitution of each GHB dose (3.2–178 mg/kg/injection) or vehicle for 15 days. Food pellets were available 24 h/day. Results GHB maintained significantly greater numbers of injections when compared to vehicle in 6 of the 9 baboons that completed GHB evaluations that included 32 mg/kg/injection or higher. The baboons that self-administered GHB at high rates were ones for which GHB was the first drug each had tested under the 24-hr/day cocaine baseline procedure. Self-injection of the highest doses of GHB decreased food-maintained responding. Conclusions High-dose GHB can function as a reinforcer in non-human primates under 24-h access, but self-administration history may be important. The findings are consistent with the demonstrated abuse liability of GHB in humans, and remove GHB as an exception to the typical good correspondence between those drugs abused by humans and those self-administered by nonhuman primates. PMID:21112162

  3. Symptoms and signs in interpreting Gamma-hydroxybutyrate (GHB) intoxication - an explorative study

    PubMed Central

    2014-01-01

    Background Acute poisoning with gamma-hydroxybutyrate (GHB) has been a serious medical and social problem in different parts of the world including Sweden. GHB is a drug of abuse which acts primarily as central nervous system (CNS) depressants. GHB has serious toxicity, although many young users do not recognise GHB as a dangerous drug. The aim of this pilot study was to explore how symptoms with risk of failure in vital functions would be valued among professionals that encounter GHB intoxication in the emergency phase. Methods A web-based survey focusing on the assessment of vital clinical signs for possible GHB intoxication using a numeric scale was carried out during April and May 2011. The participants, n 105, are all professionals who encounter GHB intoxicated in the emergency phase, but have different levels of training in GHB intoxication, mainly Registered Nurses (RNs) in southwest Sweden, employed in pre-hospital or emergency departments at somatic and most psychiatric health care facilities, as well as police officers who in their work come into contact with drug users. Responses in the survey were scored according to risk of GHB intoxication with serious failure of vital functions. The score value was then referred to a so-called evidence based priority (EBP) scale and analysed using descriptive statistics and Fisher's exact test. Results Cardiac arrest, coma, hypoxia, general convulsions, slow respiratory and heart rate and pale skin are symptoms with the highest risk of serious failure in vital physical functions and were predominantly recognised as such. Conclusion Despite the professionals' different levels of training in GHB intoxication, all of them were relatively well aware of and in accordance regarding the most risky symptoms. The interpretation score for the less risky symptoms and signs of GHB intoxication varied depending on their degree of training. The results should be viewed cautiously, as the size of the professional groups and their

  4. GHB (gamma-hydroxybutyrate) carrier-mediated transport across the blood-brain barrier.

    PubMed

    Bhattacharya, Indranil; Boje, Kathleen M K

    2004-10-01

    gamma-Hydroxybutyrate (sodium oxybate, GHB) is an approved therapeutic agent for cataplexy with narcolepsy. GHB is widely abused as an anabolic agent, euphoriant, and date rape drug. Recreational abuse or overdose of GHB (or its precursors gamma-butyrolactone or 1,4-butanediol) results in dose-dependent central nervous system (CNS) effects (respiratory depression, unconsciousness, coma, and death) as well as tolerance and withdrawal. An understanding of the CNS transport mechanisms of GHB may provide insight into overdose treatment approaches. The hypothesis that GHB undergoes carrier-mediated transport across the BBB was tested using a rat in situ brain perfusion technique. Various pharmacological agents were used to probe the pharmacological characteristics of the transporter. GHB exhibited carrier-mediated transport across the BBB consistent with a high-capacity, low-affinity transporter; averaged brain region parameters were V(max) = 709 +/- 214 nmol/min/g, K(m) = 11.0 +/- 3.56 mM, and CL(ns) = 0.019 +/- 0.003 cm(3)/min/g. Short-chain monocarboxylic acids (pyruvic, lactic, and beta-hydroxybutyric), medium-chain fatty acids (hexanoic and valproic), and organic anions (probenecid, benzoic, salicylic, and alpha-cyano-4-hydroxycinnamic acid) significantly inhibited GHB influx by 35 to 90%. Dicarboxylic acids (succinic and glutaric) and gamma-aminobutyric acid did not inhibit GHB BBB transport. Mutual inhibition was observed between GHB and benzoic acid, a well known substrate of the monocarboxylate transporter MCT1. These results are suggestive of GHB crossing the BBB via an MCT isoform. These novel findings of GHB BBB transport suggest potential therapeutic approaches in the treatment of GHB overdoses. We are currently conducting "proof-of-concept" studies involving the use of GHB brain transport inhibitors during GHB toxicity. PMID:15173314

  5. Fatal Combination with 3-Methylmethcathinone (3-MMC) and Gamma-Hydroxybutyric Acid (GHB).

    PubMed

    Jamey, Carole; Kintz, Pascal; Martrille, Laurent; Raul, Jean-Sébastien

    2016-09-01

    We reported the case of 69-year-old man who was discovered dead at a friend's home. 3-Methylmethcathinone (3-MMC) and poppers (alkyl nitrites) were found at the scene by the police. Autopsy specimens including peripheral and cardiac blood, urine, gastric content, bile and hair were sent to our laboratory to document a possible death involving abuse of drugs. Routine toxicological analysis was performed with gas chromatography with flame ionization detection (GC-FID), high performance liquid chromatography-diode array detection (HPLC-DAD), headspace gas chromatography-mass spectrometry (HS-GC-MS), gas chromatography-mass spectrometry (GC-MS) and liquid chromatography-mass spectrometry (LC-MS)-MS. After liquid-liquid extraction at alkaline pH, 3-MMC was identified with GC-MS (to allow the discrimination with 4-MMC) and quantified with ultra-performance liquid chromatography-mass spectrometry (UPLC-MS)-MS with the two following transitions: m/z 178.1 > 160 and 178.1 > 144.9. Gamma-hydroxybutyric acid (GHB) was analyzed by GC-MS for fluids and GC-MS-MS for hair. Toxicological analysis in peripheral blood revealed the presence of 3-MMC (0.33 mg/L), pseudoephedrine (0.03 mg/L) and GHB (576 mg/L). These molecules have also been found in other post-mortem fluids. Furthermore, testing for "poppers" by HS-GC-MS was negative. Hair analysis, without segmentation, demonstrated the presence of 3-MMC (206.7 ng/mg), pseudoephedrine (0.16 ng/mg) and GHB (96.3 ng/mg) and suggested a repeated consumption of these substances. However, one cannot exclude contamination by sweat during the agony period. Toxicological post-mortem results suggest a fatal combination of 3-MMC and GHB. Despite his age, the decedent was known to abuse drugs. PMID:27405362

  6. Flavonoids modulate monocarboxylate transporter-1-mediated transport of gamma-hydroxybutyrate in vitro and in vivo.

    PubMed

    Wang, Qi; Morris, Marilyn E

    2007-02-01

    The objective of this study was to determine the effects of flavonoids on the in vitro monocarboxylate transporter 1 (MCT1)-mediated transport and in vivo disposition of the drug of abuse, gamma-hydroxybutyrate (GHB). The uptake of GHB in rat MCT1 gene-transfected MDA-MB231 cells was significantly decreased in the presence of the flavonoids apigenin, biochanin A, chrysin, diosemin, fisetin, genistein, hesperitin, kaempferol, luteolin, morin, narigenin, phloretin, and quercetin, but was not affected by the flavonoid glycosides phloridzin and rutin. The IC(50) values for luteolin, morin, and phloretin were 0.41 +/- 0.14, 6.41 +/- 2.01, and 2.57 +/- 0.48 microM, with the inhibition mechanism for luteolin being competitive. [(3)H]Kaempferol and [(3)H]biochanin A did not exhibit MCT1-mediated uptake, suggesting that these flavonoids are not substrates for MCT1. The combination of luteolin and phloretin inhibited the uptake of GHB in a synergistic manner; however, the combination of luteolin and morin was antagonistic. GHB 1000 mg/kg was administered to rats by i.v. bolus, with or without the concomitant administration of luteolin 10 mg/kg i.v. After luteolin treatment, the renal and total clearances of GHB were significantly increased, probably because of inhibition of the MCT1-mediated renal reabsorption of GHB, and the sleep time significantly decreased (121 +/- 5 min versus 165 +/- 10 min) compared with control rats. Overall, the results of this study indicate that flavonoids from food or herbal products may significantly alter the pharmacokinetics and pharmacodynamics of MCT substrates. PMID:17108059

  7. Illicit gamma-hydroxybutyrate (GHB) and pharmaceutical sodium oxybate (Xyrem®): differences in characteristics and misuse

    PubMed Central

    Carter, Lawrence P.; Pardi, Daniel; Gorsline, Jane; Griffiths, Roland R.

    2009-01-01

    There are distinct differences in the accessibility, purity, dosing, and misuse associated with illicit gamma-hydroxybutyrate (GHB) compared to pharmaceutical sodium oxybate. Gamma-hydroxybutyrate sodium and sodium oxybate are the chemical and drug names, respectively, for the pharmaceutical product Xyrem® (sodium oxybate) oral solution. However, the acronym GHB is also used to refer to illicit formulations that are used for non-medical purposes. This review highlights important differences between illicit GHB and sodium oxybate with regard to their relative abuse liability, which includes the likelihood and consequences of abuse. Data are summarized from the scientific literature; from national surveillance systems in the U.S., Europe, and Australia (for illicit GHB); and from clinical trials and post-marketing surveillance with sodium oxybate (Xyrem). In the U.S., the prevalence of illicit GHB use, abuse, intoxication, and overdose has declined from 2000, the year that GHB was scheduled, to the present and is lower than that of most other licit and illicit drugs. Abuse and misuse of the pharmaceutical product, sodium oxybate, has been rare over the 5 years since its introduction to the market, which is likely due in part to the risk management program associated with this product. Differences in the accessibility, purity, dosing, and misuse of illicit GHB and sodium oxybate suggest that risks associated with illicit GHB are greater than those associated with the pharmaceutical product sodium oxybate. PMID:19493637

  8. [From alcohol to liquid ecstasy (GHB)--a survey of old and modern knockout agents. Part 3: gamma-hydroxybutyric acid ("liquid ecstasy")].

    PubMed

    Schütz, Harald; Jansen, Malin; Verhoff, Marcel A

    2011-01-01

    Currently, gamma-hydroxybutyric acid (GHB/"liquid ecstasy") is frequently abused as a knockout substance. Its detection and the interpretation of the results present numerous problems which are illustrated by case reports. In this context, hair analysis and the increasing significance of gamma-butyrolactone (GBL) are also discussed. PMID:22276366

  9. Determination of gamma-hydroxybutyric acid in human urine by capillary electrophoresis with indirect UV detection and confirmation with electrospray ionization ion-trap mass spectrometry.

    PubMed

    Baldacci, Andrea; Theurillat, Regula; Caslavska, Jitka; Pardubská, Helena; Brenneisen, Rudolf; Thormann, Wolfgang

    2003-03-21

    Gamma-hydroxybutyric acid (GHB), a minor metabolite or precursor of gamma-aminobutyric acid (GABA), acts as a neurotransmitter/neuromodulator via binding to GABA receptors and to specific presynaptic GHB receptors. Based upon the stimulatory effects, GHB is widely abused. Thus, there is great interest in monitoring GHB in body fluids and tissues. We have developed an assay for urinary GHB that is based upon liquid-liquid extraction and capillary zone electrophoresis (CZE) with indirect UV absorption detection. The background electrolyte is composed of 4 mM nicotinic acid (compound for indirect detection), 3 mM spermine (reversal of electroosmosis) and histidine (added to reach a pH of 6.2). Having a 50 microm I.D. capillary of 40 cm effective length, 1-octanesulfonic acid as internal standard, solute detection at 214 nm and a diluted urine with a conductivity of 2.4 mS/cm, GHB concentrations > or = 2 microg/ml can be detected. Limit of detection (LOD) and limit of quantitation (LOQ) were determined to be dependent on urine concentration and varied between 2-24 and 5-60 microg/ml, respectively. Data obtained suggest that LOD and LOQ (both in microg/ml) can be estimated with the relationships 0.83 kappa and 2.1 kappa, respectively, where kappa is the conductivity of the urine in mS/cm. The assay was successfully applied to urines collected after administration of 25 mg sodium GHB/kg body mass. Negative electrospray ionization ion-trap tandem mass spectrometry was used to confirm the presence of GHB in the urinary extract via selected reaction monitoring of the m/z 103.1-->m/z 85.1 precursor-product ion transition. Independent of urine concentration, this approach meets the urinary cut-off level of 10 microg/ml that is required for recognition of the presence of exogenous GHB. Furthermore, data obtained with injection of plain or diluted urine indicate that CZE could be used to rapidly recognize GHB amounts (in microg/ml) that are > or = 4 kappa. PMID:12685588

  10. Progesterone receptors and ventilatory stimulation by progestin.

    PubMed

    Brodeur, P; Mockus, M; McCullough, R; Moore, L G

    1986-02-01

    Progestin is thought to be a ventilatory stimulant but its effectiveness in raising ventilation is variable in humans and other species. We hypothesized that the level of progesterone receptors was an important determinant of the ventilatory response to progestin. Since estradiol induces progesterone receptor formation, we compared the ventilatory effect of the synthetic progestin medroxyprogesterone acetate (MPA) given in combination with estradiol with the effects of estradiol alone, MPA alone, or vehicle (saline) in ovariectomized rats. Animals receiving MPA alone had low numbers of progesterone receptors (2.43 pmol/g uterine wt) and had no change in ventilation, arterial Pco2, or Po2. MPA administration raised ventilation 23 +/- 5%, lowered arterial Pco2 3.2 +/- 0.9 Torr (both P less than 0.01) and tended to raise arterial Po2 when given in combination with estradiol to animals with increased numbers of progesterone receptors (4.85 pmol/g uterine wt). Estradiol alone produced the highest number of progesterone receptors (12.3 pmol/g uterine wt) but had no effect on ventilation or arterial Pco2 and decreased arterial Po2. Combined estradiol plus MPA treatment produced a greater fall in arterial Pco2 than did treatment with MPA alone, estradiol, or saline (all P less than 0.05). These results suggest that both an elevation in progestin levels and progesterone receptor numbers are required to stimulate ventilation. PMID:2936712

  11. Estimation of gamma-hydroxybutyrate (GHB) co-consumption in serum samples of drivers positive for amphetamine or ecstasy.

    PubMed

    Lott, S; Musshoff, F; Madea, B

    2012-09-10

    There is no toxicological analysis of gamma-hydroxybutyrate (GHB) applied routinely in cases of driving under influence (DUI); therefore the extent of consumption of this drug might be underestimated. Its consumption is described as occurring often concurrently with amphetamine or ecstasy. This study examines 196 serum samples which were collected by police during road side testing for GHB. The samples subject to this study have already been found to be positive for amphetamine, methamphetamine, 3,4-methylenedioxyamphetamine (MDA), 3,4-methylenedioxymethamphetamine (MDMA) and/or 3,4-methylenedioxyethamphetamine (MDEA). Analysis has been performed by LC/MS/MS in the multiple reaction monitoring (MRM) mode. Due to its polarity, chromatographic separation of GHB was achieved by a HILIC column. To differentiate endogenous and exogenous levels of GHB, a cut-off concentration of 4μg/ml was applied. Of the 196 samples, two have been found to be positive for GHB. Of these samples, one sample was also positive for amphetamine and one for MDMA. Whilst other amphetamine derivates were not detected in these samples, both samples were found to be positive for cannabinoids. These results suggest that co-consumption of GHB with amphetamine or ecstasy is relatively low (1%) for the collective of this study. PMID:22554869

  12. [Current knowledge on gamma-hydroxybutyric acid (GHB), gamma-butyrolactone (GBL) and 1 ,4-butanediol (1,4-BD)].

    PubMed

    Dematteis, Maurice; Pennel, Lucie; Mallaret, Michel

    2012-05-01

    Gamma-hydroxybutyric acid (GHB) is an old anaesthetic drug which was misused in the 80-90's as an anabolic agent (bodybuilding), recreational drug (drunkenness, euphoric, disinhibiting and aphrodisiac effects) and as a date rape drug (disinhibiting, hypnotic and amnesic effects). Its use in the general population is low, and mainly concerns gay population in nightclubs and young people in parties. The intoxications, above all with alcohol combination, can be severe, with coma and breathing depression, or even fatal. Chronic use leads to psychic and physical dependence; withdrawal syndrome can be severe, with agitation and delirium. In 1999, GHB classification as a narcotic resulted in the increased use of GHB prodrugs gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD), which were easily commercially available as solvent and cleaning products. Like GHB, they have a narrow window of use, and share similar toxicity. Their increased cases of recreational use and of severe drug intoxication, abuse and dependence, led the French Ministry of Health in 2011 to prohibit their sale and transfer to the public. PMID:22730800

  13. Determination of the illicit drug gamma-hydroxybutyrate (GHB) in human saliva and beverages by 1H NMR analysis.

    PubMed

    Grootveld, Martin; Algeo, Deborah; Silwood, Christopher J L; Blackburn, John C; Clark, Anthony D

    2006-01-01

    High resolution 1H NMR spectroscopy has been employed to investigate the detection and quantification of the illicit "date-rape" drug gamma-hydroxybutyrate (GHB) in both human saliva and a commonly-consumed low-alcohol beer product. Data acquired revealed that this multicomponent analytical technique provided unequivocal evidence for the detection of this agent by this technique in both of these matrices, i.e., all three of its resonances [those ascribable to the alpha-CH2 (t, delta=2.25 ppm), beta-CH2 (tt, delta=1.81 ppm) and gamma-CH2 (t, delta=3.61 ppm) group protons] were present in spectra acquired on human saliva, and two of these (the alpha- and beta-CH2 group signals) in the beverage product examined, the latter observation attributable to overlap of the gamma-CH2 1H resonance with those of carbohydrates. Since good linear calibration relationships between the intensities of each of the NMR-visible signals and added GHB concentration (the former normalised to that of an external 3-trimethylsilyl [2,2,3,3-2H4]- propionate standard present in a coaxial NMR tube insert) were observed, this illicit drug is also readily quantifiable in such multicomponent samples. Our data demonstrate the advantages offered by this technique when applied to the analysis of illicit drugs in multicomponent sample matrices such as human biofluids and beverage products. PMID:17012769

  14. Monitoring of the interconversion of gamma-butyrolactone (GBL) to gamma hydroxybutyric acid (GHB) by Raman spectroscopy.

    PubMed

    Munshi, Tasnim; Brewster, Victoria L; Edwards, Howell G M; Hargreaves, Michael D; Jilani, Shelina K; Scowen, Ian J

    2013-08-01

    Gamma-hydroxybutyric acid (GHB) is a drug-of-abuse that has recently become associated with drug-facilitated sexual assault, known as date rape. For this reason the drug is commonly found 'spiked' in alcoholic beverages. When GHB is in solution it may undergo conversion into the corresponding lactone, Gamma-butyrolactone (GBL). Studies have been carried out to determine the detection limits of GHB and GBL in various solutions by Raman spectroscopy and to monitor the interconversion of GHB and GBL in solution with different pH conditions and temperature. In this study, a portable Raman spectrometer was used to study the interconversion of GHB and GBL in water and ethanol solutions as a function of pH, time, and temperature. The aim of this was to determine the optimum pH range for conversion in order to relate this to the pH ranges that the drug is likely to be subjected to, first in spiked beverages and secondly after ingestion in the digestive system. The aim was also to identify a timescale for this conversion in relation to possible scenarios, for example if GHB takes a number of hours to convert to GBL, it is likely for the beverage to be ingested before esterification can take place. GHB and GBL were then spiked into a selection of beverages of known pH in order to study the stability of GHB and GBL in real systems. PMID:23225646

  15. The presence of gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL) in alcoholic and non-alcoholic beverages.

    PubMed

    Elliott, Simon; Burgess, Victoria

    2005-07-16

    Gamma-hydroxybutyric acid (GHB) and its precursor gamma-butyrolactone (GBL) are regularly implicated in instances of surreptitious drug administration, particularly in beverages (so-called "spiked drinks"). In order to assist in the interpretation of cases where analysis of the actual beverage is required, over 50 beverages purchased in the UK were analysed for the presence of GHB and GBL. It was found that naturally occurring GHB and GBL were detected in those beverages involving the fermentation of white and particularly red grapes. No GHB or GBL was detected in other drinks such as beer, juice, spirits or liqueurs. GHB/GBL was detected in red wine vermouth (8.2 mg/L), sherry (9.7 mg/L), port (GBL), red wine (4.1-21.4 mg/L) and white wine (<3-9.6 mg/L). The presence of GHB/GBL did not appear to be influenced by the alcohol content or the pH of the beverage. In addition, the concentration in wines did not appear to be related to the geographical origin of the grape type. This is believed to be the first published data concerning the endogenous presence of GHB and GBL in the beverages described. PMID:15939164

  16. The Impact of Gamma Hydroxybutyrate (GHB) Legal Restrictions on Patterns of Use: Results from an International Survey

    PubMed Central

    Anderson, IB; Kim-Katz, SY; Dyer, JE; Blanc, PD

    2009-01-01

    Aims To conduct an Internet-based survey of GHB use, identifying differences by respondent residence. Methods We recruited GHB-knowledgeable persons via “social networking Internet sites.” Individuals (n=314) or groups (n=66) were approached based on GHB-use testimonials. Data collected location, use, reason for cessation (if applicable). Findings We recruited 155 GHB users. U.S. respondents (53 of 70; 76%) compared to non-U.S. respondents (38 of 85; 45%) were older and more highly educated (p<0.05) but manifest a 3-fold greater adjusted odds of GHB cessation (Odds Ratio [OR] 3.1; 95% CI 1.4–6.9; p < 0.05). Of the 80 respondents stating reason for cessation, 36 (45%) cited legal risk, price, or access; 44 (55%) cited health or related concerns. U.S. compared to non-U.S. respondents more frequently invoked legal and related concerns (OR 2.5; 95% CI 0.99–6.3; p=0.05). In a nested analysis, narrowly stated legal (n=4/5 U.S.) versus health (n=6/18 U.S.) reasons differed by location (p=0.048, one-tailed). Conclusions In the U.S., where GHB has stricter legal penalties, GHB cessation is more likely, with legal and related reasons more commonly invoked for cessation. These findings support a link between declining U.S. GHB abuse and more stringent restrictions; although other un-assessed factors may also explain this association. The Impact of Gamma Hydroxybutyrate (GHB) Legal Restrictions on Patterns of Use: Results from an International Survey PMID:20953310

  17. Temporal differences in gamma-hydroxybutyrate overdoses involving injecting drug users versus recreational drug users in Helsinki: a retrospective study

    PubMed Central

    2012-01-01

    Background Gamma-hydroxybutyrate (GHB) and gamma-butyrolactone (GBL) have been profiled as 'party drugs' used mainly at dance parties and in nightclubs on weekend nights. The purpose of this study was to examine the frequency of injecting drug use among GHB/GBL overdose patients and whether there are temporal differences in the occurrence of GHB/GBL overdoses of injecting drug and recreational drug users. Methods In this retrospective study, the ambulance and hospital records of suspected GHB- and GBL overdose patients treated by the Helsinki Emergency Medical Service from January 1st 2006 to December 31st 2007 were reviewed. According to the temporal occurrence of the overdose, patients were divided in two groups. In group A, the overdose occurred on a Friday-Saturday or Saturday-Sunday night between 11 pm-6 am. Group B consisted of overdoses occurring on outside this time frame. Results Group A consisted of 39 patient contacts and the remaining 61 patient contacts were in group B. There were statistically significant differences between the two groups in (group A vs. B, respectively): history of injecting drug abuse (33% vs. 59%, p = 0.012), reported polydrug and ethanol use (80% vs. 62%, p = 0.028), the location where the patients were encountered (private or public indoors or outdoors, 10%, 41%, 41% vs. 25%, 18%, 53%, p = 0.019) and how the knowledge of GHB/GBL use was obtained (reported by patient/bystanders or clinical suspicion, 72%, 28% vs. 85%, 10%, p = 0.023). Practically all (99%) patients were transported to emergency department after prehospital care. Conclusion There appears to be at least two distinct groups of GHB/GBL users. Injecting drug users represent the majority of GHB/GBL overdose patients outside weekend nights. PMID:22296777

  18. Cloning of a rat brain succinic semialdehyde reductase involved in the synthesis of the neuromodulator gamma-hydroxybutyrate.

    PubMed Central

    Andriamampandry, C; Siffert, J C; Schmitt, M; Garnier, J M; Staub, A; Muller, C; Gobaille, S; Mark, J; Maitre, M

    1998-01-01

    The gamma-hydroxybutyrate biosynthetic enzyme succinic semialdehyde reductase (SSR) was purified to homogeneity from rat brain. Peptides were generated by tryptic cleavage and sequenced. PCR primers were designed from the amino acid sequences of two of the peptides showing a similarity (75-85%) to a mitochondrial aldehyde dehydrogenase. A PCR-amplified DNA fragment was generated from recombinant plasmids prepared by a mass excision procedure from a rat hippocampal cDNA library and used as a probe to screen this cDNA library. One cDNA of 1341 bp had an open reading frame encoding a protein of 447 residues with a deduced molecular mass of 47967 Da. The enzyme was expressed in Escherichia coli. Immunoblotting analysis revealed the existence of a protein with the same electrophoretic mobility as the SSR purified from rat brain and with an estimated molecular mass of 45 kDa. Northern blot experiments showed that this enzyme was not expressed in the kidney or in the liver. In the brain tissue, a single but rather broad band was labelled under high stringency conditions, suggesting the presence of more than one messenger species coding for SSR. Hybridization in situ performed on brain tissue slices showed specific labelling of the hippocampus, the upper cortex layer, the thalamus, the substantia nigra, the cerebellum, the pons medulla and the olfactory tract. The recombinant enzyme showed catalytic properties similar to those of the SSR purified from rat brain, particularly in regard to its substrate affinities and Ki for inhibition by phthalaldehydic acid. Valproic acid did not inhibit the cloned SSR. This enzyme had 20-35% identity in highly conserved regions involved in NADPH binding with four other proteins belonging to the aldo-oxo reductase family. PMID:9693100

  19. Cloning of a rat brain succinic semialdehyde reductase involved in the synthesis of the neuromodulator gamma-hydroxybutyrate.

    PubMed

    Andriamampandry, C; Siffert, J C; Schmitt, M; Garnier, J M; Staub, A; Muller, C; Gobaille, S; Mark, J; Maitre, M

    1998-08-15

    The gamma-hydroxybutyrate biosynthetic enzyme succinic semialdehyde reductase (SSR) was purified to homogeneity from rat brain. Peptides were generated by tryptic cleavage and sequenced. PCR primers were designed from the amino acid sequences of two of the peptides showing a similarity (75-85%) to a mitochondrial aldehyde dehydrogenase. A PCR-amplified DNA fragment was generated from recombinant plasmids prepared by a mass excision procedure from a rat hippocampal cDNA library and used as a probe to screen this cDNA library. One cDNA of 1341 bp had an open reading frame encoding a protein of 447 residues with a deduced molecular mass of 47967 Da. The enzyme was expressed in Escherichia coli. Immunoblotting analysis revealed the existence of a protein with the same electrophoretic mobility as the SSR purified from rat brain and with an estimated molecular mass of 45 kDa. Northern blot experiments showed that this enzyme was not expressed in the kidney or in the liver. In the brain tissue, a single but rather broad band was labelled under high stringency conditions, suggesting the presence of more than one messenger species coding for SSR. Hybridization in situ performed on brain tissue slices showed specific labelling of the hippocampus, the upper cortex layer, the thalamus, the substantia nigra, the cerebellum, the pons medulla and the olfactory tract. The recombinant enzyme showed catalytic properties similar to those of the SSR purified from rat brain, particularly in regard to its substrate affinities and Ki for inhibition by phthalaldehydic acid. Valproic acid did not inhibit the cloned SSR. This enzyme had 20-35% identity in highly conserved regions involved in NADPH binding with four other proteins belonging to the aldo-oxo reductase family. PMID:9693100

  20. Do capillary dried blood spot concentrations of gamma-hydroxybutyric acid mirror those in venous blood? A comparative study.

    PubMed

    Sadones, Nele; Archer, John R H; Ingels, Ann-Sofie M E; Dargan, Paul I; Wood, David M; Wood, Michelle; Neels, Hugo; Lambert, Willy E; Stove, Christophe P

    2015-04-01

    Gamma-hydroxybutyric acid (GHB) is a well-known illicit club and date-rape drug. Dried blood spot (DBS) sampling is a promising alternative for classical venous sampling in cases of (suspected) GHB intoxication since it allows rapid sampling, which is of interest for the extensively metabolized GHB. However, there is limited data if -and how- capillary DBS concentrations correlate with venous concentrations. We conducted a comparative study in 50 patients with suspected GHB intoxication, to determine and to correlate GHB concentrations in venous DBS (vDBS) and capillary DBS (cDBS). This is the first study that evaluates in a large cohort the correlation between capillary and venous concentrations of an illicit drug in real-life samples. Of the 50 paired samples, 7 were excluded: the vDBS concentration was below the LLOQ of 2 µg/mL in 3 cases and 4 samples were excluded after visual inspection of the DBS. Bland-Altman analysis revealed a mean % difference of -2.8% between cDBS and vDBS concentrations, with the zero value included in the 95% confidence interval of the mean difference in GHB concentration. A paired sample t-test confirmed this observation (p = 0.17). Also the requirement for incurred sample reproducibility was fulfilled: for more than two-thirds of the samples the concentrations obtained in cDBS and those in vDBS were within 20% of their mean. Since equivalent concentrations were observed in cDBS and vDBS, blood obtained by fingerprick can be considered a valid alternative for venous blood for GHB determination. PMID:25565078

  1. Coma in a 20-month-old child from an ingestion of a toy containing 1,4-butanediol, a precursor of gamma-hydroxybutyrate.

    PubMed

    Ortmann, Laura A; Jaeger, Matthew W; James, Laura P; Schexnayder, Steve M

    2009-11-01

    Ingestion of plastic toys is common in children and usually does not result in harm. We report a case of coma in a 20-month-old child after an ingestion of a toy containing 1,4-butanediol, an industrial solvent used to manufacture plastics. When ingested, 1,4-butanediol is metabolized to gamma-hydroxybutyrate, which can have significant systemic effects including death. Health care providers should suspect the possibility of a toxic component when a presumed nontoxic object causes unusual symptoms. PMID:19915428

  2. A Critical Evaluation of the Gamma-Hydroxybutyrate (GHB) Model of Absence Seizures

    PubMed Central

    Venzi, Marcello; Di Giovanni, Giuseppe; Crunelli, Vincenzo

    2015-01-01

    Typical absence seizures (ASs) are nonconvulsive epileptic events which are commonly observed in pediatric and juvenile epilepsies and may be present in adults suffering from other idiopathic generalized epilepsies. Our understanding of the pathophysiological mechanisms of ASs has been greatly advanced by the availability of genetic and pharmacological models, in particular the γ-hydroxybutyrate (GHB) model which, in recent years, has been extensively used in studies in transgenic mice. GHB is an endogenous brain molecule that upon administration to various species, including humans, induces not only ASs but also a state of sedation/hypnosis. Analysis of the available data clearly indicates that only in the rat does there exist a set of GHB-elicited behavioral and EEG events that can be confidently classified as ASs. Other GHB activities, particularly in mice, appear to be mostly of a sedative/hypnotic nature: thus, their relevance to ASs requires further investigation. At the molecular level, GHB acts as a weak GABA-B agonist, while the existence of a GHB receptor remains elusive. The pre- and postsynaptic actions underlying GHB-elicited ASs have been thoroughly elucidated in thalamus, but little is known about the cellular/network effects of GHB in neocortex, the other brain region involved in the generation of ASs. PMID:25403866

  3. A critical evaluation of the gamma-hydroxybutyrate (GHB) model of absence seizures.

    PubMed

    Venzi, Marcello; Di Giovanni, Giuseppe; Crunelli, Vincenzo

    2015-02-01

    Typical absence seizures (ASs) are nonconvulsive epileptic events which are commonly observed in pediatric and juvenile epilepsies and may be present in adults suffering from other idiopathic generalized epilepsies. Our understanding of the pathophysiological mechanisms of ASs has been greatly advanced by the availability of genetic and pharmacological models, in particular the γ-hydroxybutyrate (GHB) model which, in recent years, has been extensively used in studies in transgenic mice. GHB is an endogenous brain molecule that upon administration to various species, including humans, induces not only ASs but also a state of sedation/hypnosis. Analysis of the available data clearly indicates that only in the rat does there exist a set of GHB-elicited behavioral and EEG events that can be confidently classified as ASs. Other GHB activities, particularly in mice, appear to be mostly of a sedative/hypnotic nature: thus, their relevance to ASs requires further investigation. At the molecular level, GHB acts as a weak GABA-B agonist, while the existence of a GHB receptor remains elusive. The pre- and postsynaptic actions underlying GHB-elicited ASs have been thoroughly elucidated in thalamus, but little is known about the cellular/network effects of GHB in neocortex, the other brain region involved in the generation of ASs. PMID:25403866

  4. Quantitation of Gamma-Hydroxybutyric Acid in Dried Blood Spots: Feasibility Assessment for Newborn Screening of Succinic Semialdehyde Dehydrogenase (SSADH) Deficiency

    PubMed Central

    Forni, Sabrina; Pearl, Phillip L.; Gibson, K. Michael; Yu, Yuezhou; Sweetman, Lawrence

    2013-01-01

    Objective SSADH deficiency, the most prevalent autosomal recessive disorder of GABA degradation, is characterized by elevated gamma-hydroxybutyric acid (GHB). Neurological outcomes may be improved with early intervention and anticipatory guidance. Morbidity has been compounded by complications, e.g. hypotonia, in undiagnosed infants with otherwise routine childhood illnesses. We report pilot methodology on the feasibility of newborn screening for SSADH deficiency. Method Dried blood spot (DBS) cards from patients affected with SSADH deficiency were compared with 2831 archival DBS cards for gamma-hydroxybutyric acid content. Following extraction with methanol, GHB in DBS was separated and analyzed using ultra high-performance liquid chromatography tandem mass spectrometry. Results Methodology was validated to meet satisfactory accuracy and reproducibility criteria, including intra-day and inter-day validation. Archival refrigerated dried blood spots samples of babies, infants and children (N=2831) were screened for GHB, yielding a mean +/- S.D. of 8 ± 5 nM (99.9 %-tile 63 nM) (Min 0.0 Max 78 nM). The measured mean and median concentrations in blood spots derived from seven SSADH deficient patients were 1182 nM and 699 nM respectively (Min 124, Max 4851nM). Conclusions GHB concentration in all 2831 dried blood spot cards was well below the lowest concentration of affected children. These data provide proof-of-principle for screening methodology to detect SSADH deficiency with applicability to newborn screening and earlier diagnosis. PMID:23742746

  5. Report on the analysis of common beverages spiked with gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL) using NMR and the PURGE solvent-suppression technique.

    PubMed

    Lesar, Casey T; Decatur, John; Lukasiewicz, Elaan; Champeil, Elise

    2011-10-10

    In forensic evidence, the identification and quantitation of gamma-hydroxybutyric acid (GHB) in "spiked" beverages is challenging. In this report, we present the analysis of common alcoholic beverages found in clubs and bars spiked with gamma-hydroxybutyric acid (GHB) and gamma-butyrolactone (GBL). Our analysis of the spiked beverages consisted of using (1)H NMR with a water suppression method called Presaturation Utilizing Relaxation Gradients and Echoes (PURGE). The following beverages were analyzed: water, 10% ethanol in water, vodka-cranberry juice, rum and coke, gin and tonic, whisky and diet coke, white wine, red wine, and beer. The PURGE method allowed for the direct identification and quantitation of both compounds in all beverages except red and white wine where small interferences prevented accurate quantitation. The NMR method presented in this paper utilizes PURGE water suppression. Thanks to the use of a capillary internal standard, the method is fast, non-destructive, sensitive and requires no sample preparation which could disrupt the equilibrium between GHB and GBL. PMID:21775083

  6. Gamma-Hydroxybutyrate (GHB)

    MedlinePlus

    ... a muscle builder, a "party drug" and a "date-rape" drug. GHB is available in liquid form, powder form ... is why GHB is often used as a "date-rape" drug. What should I tell my children about GHB? ...

  7. Can Eph receptors stimulate the mind?

    PubMed

    Murai, Keith K; Pasquale, Elena B

    2002-01-17

    The Eph receptors are multitalented tyrosine kinases capable of performing many tasks. The receptors together with their ligands--the ephrins--are well known to play a critical role in the initial assembly of neuronal circuits in the embryo. However, the recently discovered function of these receptors in the adult brain is now receiving significant acclaim. Three new articles show that the Eph receptors continue to be important in modifying the strength of existing neuronal connections (synapses). They do so in close association with at least one family of ion channels, the NMDA receptors. PMID:11804564

  8. Gamma-hydroxybutyric acid endogenous production and post-mortem behaviour - the importance of different biological matrices, cut-off reference values, sample collection and storage conditions.

    PubMed

    Castro, André L; Dias, Mário; Reis, Flávio; Teixeira, Helena M

    2014-10-01

    Gamma-Hydroxybutyric Acid (GHB) is an endogenous compound with a story of clinical use, since the 1960's. However, due to its secondary effects, it has become a controlled substance, entering the illicit market for recreational and "dance club scene" use, muscle enhancement purposes and drug-facilitated sexual assaults. Its endogenous context can bring some difficulties when interpreting, in a forensic context, the analytical values achieved in biological samples. This manuscript reviewed several crucial aspects related to GHB forensic toxicology evaluation, such as its post-mortem behaviour in biological samples; endogenous production values, whether in in vivo and in post-mortem samples; sampling and storage conditions (including stability tests); and cut-off reference values evaluation for different biological samples, such as whole blood, plasma, serum, urine, saliva, bile, vitreous humour and hair. This revision highlights the need of specific sampling care, storage conditions, and cut-off reference values interpretation in different biological samples, essential for proper practical application in forensic toxicology. PMID:25287794

  9. Comparative profiles of sodium valproate and ethosuximide on electro-behavioural correlates in gamma-hydroxybutyrate and pentylenetetrazol induced absence seizures in rats.

    PubMed

    Kumaresan, S; David, J; Joseph, T

    2000-10-01

    Sodium valproate (VPA) and ethosuximide (ESM) were compared on behavioural and EEG changes in gamma-hydroxybutyrate (GHB) and pentylenetetrazole (PTZ) rat models of Absence Seizures (AS). Both GHB, 100 mg/kg i.p. and PTZ, 20 mg/kg i.p., produced repetitive episodes of staring and immobility with concomitant 6 to 9 Hz spike and wave discharges (SWDs) in the EEG. The parameters used for drug evaluation were the number and duration of SWDs/hour. Though the number of SWDs/hour produced by GHB and PTZ were not significantly different, the duration of SWDs was significantly longer in GHB treated rats (P < 0.001) VPA and ESM, at 200 mg/kg i.p., reduced SWD number and duration in GHB pretreated rats, whereas ESM, 50 mg/kg i.p., was four times more effective than VPA, 200 mg/kg i.p., in the PTZ model. Phenytoin (PHY) 20 and Carbamazepine (CBZ) 10 mg/kg i.p., worsened AS, a feature which has also been reported clinically. Both rat models of experimental AS can be used to defect potential anti-absence activity in new chemical entities. PMID:11214495

  10. An open randomized study of the treatment of escitalopram alone and combined with gamma-hydroxybutyric acid and naltrexone in alcoholic patients.

    PubMed

    Stella, Luigi; Addolorato, Giovanni; Rinaldi, Barbara; Capuano, Annalisa; Berrino, Liberato; Rossi, Francesca; Maione, Sabatino

    2008-04-01

    gamma-hydroxybutyric acid (GHB) and the selective serotonin reuptake inhibitor escitalopram are effective in inducing and maintaining abstinence in alcohol. Naltrexone (NTX), an opioid antagonist, may be effective in preventing relapse in alcohol-dependent subjects. To evaluate whether each drug and its combination help to maintain alcohol abstinence, we determined the relapse rate over 6 months in 3 groups of patients. Group 1 (11 patients) received escitalopram (20 mg/day) orally administered; group 2 (12 patients) received NTX (50 mg/day) and escitalopram (20 mg/day); group 3 (12 patients) received GHB (75 mg/kg body weight) and escitalopram (20 mg/day); and group 4 (12 patients) received NTX (50mg/day) plus GHB (75 mg/kg) and escitalopram (20 mg/day). All groups received psychological support and underwent urine tests for alcohol metabolites twice a week. In group 1 (escitalopram only), 6 patients relapsed within 3 months and 3 after 6 months; whereas 2 patients remained abstinent. In group 2 (SSRI+NTX), 5 patients relapsed after 3 months and 3 after 6 months; whereas 4 patients remained abstinent. In group 3 (GHB+SSRI), 3 patients relapsed after 3 months and 3 after 6 months; whereas 6 patients remained abstinent. Finally, in group 4 (NTX+GHB+SSRI), 1 patient relapsed after 3 months and 1 after 6 months, whereas 10 patients remain abstinent. In conclusion, the combination of NTX+GHB+SSRI was the most effective in preventing relapses. PMID:18434189

  11. Stimulation of the dopamine 1 receptor increases lung edema clearance.

    PubMed

    Barnard, M L; Ridge, K M; Saldias, F; Friedman, E; Gare, M; Guerrero, C; Lecuona, E; Bertorello, A M; Katz, A I; Sznajder, J I

    1999-09-01

    We previously reported that lung edema clearance was stimulated by dopamine (DA). The purpose of this study was to determine whether the DA-mediated stimulation of edema clearance occurs via an adrenergic or dopaminergic regulation of alveolar epithelial Na, K-ATPase. When isolated perfused rat lungs were coinstilled with DA and SCH 23390 (a specific D(1) receptor antagonist), there was a dose-dependent attenuation of the stimulatory effects of DA. Coinstillation with S-sulpiride (a specific D(2) receptor antagonist) or propranolol (a beta-adrenergic antagonist) did not alter DA-stimulated clearance. Similarly, the specific dopaminergic D(1) agonist fenoldopam increased lung edema clearance, but quinpirole (a specific dopaminergic D(2) agonist) did not. (125)I-SCH 23982 binding studies suggested that D(1) receptors are expressed on alveolar type II (ATII) cells with an apparent dissociation constant (K(d)) of 4.4 nM and binding maximum (Bmax) 9.8 pmol/mg. Consistent with these results, the D(1) receptor messenger RNA (mRNA) and protein were detected in ATII cells by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot analysis, respectively. These data demonstrate a novel mechanism involving the activation of dopaminergic D(1) receptors which mediates DA-stimulated edema removal from rat lungs. PMID:10471628

  12. Detection of gamma-hydroxybutyrate in hair: validation of GC-MS and LC-MS/MS methods and application to a real case.

    PubMed

    Bertol, Elisabetta; Argo, Antonina; Procaccianti, Paolo; Vaiano, Fabio; Di Milia, Maria Grazia; Furlanetto, Sandra; Mari, Francesco

    2012-11-01

    A gas chromatography-mass spectrometry (GC-MS) and a liquid chromatography tandem mass spectrometry (LC-MS/MS) method were validated for quantifying endogenous and exogenous hair concentrations of gamma-hydroxybutyrate (GHB). The GC-MS method is based on overnight extraction of 25 mg hair in NaOH at 56 °C, liquid/liquid extraction in ethylacetate and trimethylsylil derivatization; analysis is by electron ionization and single ion monitoring of three ions. The LC-MS/MS method entails a rapid digestion of 25 mg hair with NaOH at 75 °C for 40 min, liquid/liquid extraction in ethylacetate and reconstitution of the extract in the LC mobile phase; negative ion electrospray ionization and multiple reaction monitoring (MRM) analysis are employed for the LC-MS/MS detection. In both cases, GHB-d6 is used as an internal standard. The endogenous amount in "blank" hair are estimated by the standard addition method. Limits of detection are 0.4 and 0.5 ng/mg for GC-MS and LC-MS/MS respectively, while the limit of quantification (LOQ) is 0.6 ng/mg for both methods; the GC-MS method proved to be linear in the range 1-50 ng/mg whereas linearity was demonstrated from 0.6 to 50 ng/mg for the LC-MS/MS; imprecision and inaccuracy were always lower than 23% for quality controls samples. The two methods were applied to a real case of a man addicted to GHB; the drug concentration in segments from 17 cm hair strand well correlated with self-reported use of GHB in different periods of his life. Performances of the two methods were similar. PMID:22884787

  13. Behavioral effects and pharmacokinetics of gamma-hydroxybutyrate (GHB) precursors gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) in baboons

    PubMed Central

    Goodwin, A. K.; Brown, P. R.; Jansen, E. E. W.; Jakobs, C.; Gibson, K. M.; Weerts, E. M.

    2009-01-01

    Rationale Gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) are prodrugs for gamma-hydroxybutyrate (GHB). Like GHB, GBL and 1,4-BD are drugs of abuse, but their behavioral effects may differ from GHB under some conditions. Objectives The first study compared the behavioral effects of GBL (32−240 mg/kg) and 1,4-BD (32−240 mg/kg) with each other and to effects previously reported for GHB (32−420 mg/kg). A second study determined GHB pharmacokinetics following intragastric administration of GHB, GBL, and 1,4-BD. Methods Operant responding for food, observed behavioral effects, and a fine-motor task occurred at multiple time intervals after administration of drug or vehicle. In a separate pharmacokinetics study, blood samples were collected across multiple time points after administration of GHB, GBL, and 1,4-BD. Results Like GHB, GBL, and 1,4-BD impaired performance on the fine-motor task, but the onset of motor impairment differed across drugs. GBL and 1,4-BD dose dependently decreased the number of food pellets earned, but at lower doses than previously observed for GHB. Similar to GHB, both GBL and 1,4-BD produced sedation, muscle relaxation, gastrointestinal symptoms, and tremors/jerks. Administration of GBL and 1,4-BD produced higher maximum concentrations of GHB with shorter times to maximum concentrations of GHB in plasma when compared to GHB administration. Conclusions GBL and 1,4-BD produced behavioral effects similar to those previously reported with GHB and the time course of effects were related to blood levels of GHB. Given their higher potency and faster onset of effects, the abuse liability of GBL and 1,4-BD may be greater than GHB. PMID:19198808

  14. Self-administration of gamma-hydroxybutyric acid (GHB) precursors gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) in baboons

    PubMed Central

    Goodwin, Amy K.; Kaminski, Barbara J.; Weerts, Elise M.

    2012-01-01

    Rationale Gamma-butyrolactone (GBL) and 1,4-butanediol (1,4-BD) are gamma-hydroxybutyrate (GHB) pro-drugs and drugs of abuse. Objective Given the reports of abuse, and the ease at which GBL and 1,4-BD may be obtained, we investigated the reinforcing of GBL (n=5) and 1,4-BD (n=4) in baboons using IV self-administration procedures. Methods Sessions ran 24 h/day. Each injection was contingent upon completion of a fixed number (120 or 160) of lever responses. A 3-h timeout period followed each injection, limiting the total number of injections to 8/day. Self-administration was first established with cocaine (0.32 mg/kg/injection). GBL (10–130.0 mg/kg/injection), 1,4-BD (10–100 mg/kg/injection) or vehicle were substituted for cocaine at least 15 days. Food pellets were available ad libitum 24 h/day and were contingent upon completion of 10 lever responses. Results GBL (32–100 mg/kg/injection) maintained significantly greater numbers of injections when compared to vehicle in 4 of 5 baboons and mean rates of injection were high (>6 per day) in 3 baboons and moderate in the fourth baboon (4–6 per day). 1,4-BD (78–130 mg/kg/injection) maintained significantly greater numbers of injections when compared to vehicle in only 2 out of 4 baboons and rates were moderate to high in both baboons. Self-injection of these doses of GBL and 1,4-BD generally inhibited food-maintained responding. Conclusions GBL and 1,4-BD have abuse liability. Given that GBL and 1,4-BD are self-administered, are easier to obtain than GHB, and are detected in seized samples, additional legal control measures of these GHB pro-drugs may be needed. PMID:22945514

  15. Concentration-time profiles of gamma-hydroxybutyrate in blood after recreational doses are best described by zero-order rather than first-order kinetics.

    PubMed

    Jones, A W; Eklund, A; Kronstrand, R

    2009-01-01

    The recreational drug gamma-hydroxybutyrate (GHB) has a short plasma elimination half-life (t(1/2)) reported to be about 30-50 min. However, this represents a terminal half-life and therefore might not necessarily apply after large (abuse) doses are taken. Clinical studies with sodium oxybate (sodium salt of GHB) suggest that zero-order rather than first-order kinetics are more appropriate to describe post-peak concentration-time (C-T) profiles. We report the case of a 23-year-old male found unconscious by the police and a blood sample contained 100 mg/L GHB and 0.14 g% ethanol. On regaining consciousness the man admitted drinking alcohol about 6 h earlier but claimed that his drink must have been spiked with GHB. The police wanted to know how much GHB had been administered to account for the man's clinical condition. A back-calculation for 6 h, assuming a GHB half-life of 40 min, gives a very high concentration in blood of approximately 900 mg/L, which would probably have proven fatal. Back-calculating using zero-order kinetics and a proposed elimination rate of 18 mg/L per hour leads to a GHB concentration of 208 mg/L, which is much more realistic. Toxicologists should not arbitrarily apply the principles of first-order kinetics after abuse doses of drugs, when zero-order or saturation kinetics (Michaelis-Menten) are more appropriate. PMID:19653937

  16. Increased expression of the nicotinic acetylcholine receptor in stimulated muscle.

    PubMed

    O'Reilly, Clare; Pette, Dirk; Ohlendieck, Kay

    2003-01-10

    Chronic low-frequency stimulation has been used as a model for investigating responses of skeletal muscle fibres to enhanced neuromuscular activity under conditions of maximum activation. Fast-to-slow isoform shifting of markers of the sarcoplasmic reticulum and the contractile apparatus demonstrated successful fibre transitions prior to studying the effect of chronic electro-stimulation on the expression of the nicotinic acetylcholine receptor. Comparative immunoblotting revealed that the alpha- and delta-subunits of the receptor were increased in 10-78 day stimulated specimens, while an associated component of the surface utrophin-glycoprotein complex, beta-dystroglycan, was not drastically changed in stimulated fast skeletal muscle. Previous studies have shown that electro-stimulation induces degeneration of fast glycolytic fibres, trans-differentiation leading to fast-to-slow fibre transitions and activation of muscle precursor cells. In analogy, our results indicate a molecular modification of the central functional unit of the post-synaptic muscle surface within existing neuromuscular junctions and/or during remodelling of nerve-muscle contacts. PMID:12504123

  17. Manipulation of P2X Receptor Activities by Light Stimulation

    PubMed Central

    Kim, Sang Seong

    2016-01-01

    P2X receptors are involved in amplification of inflammatory responses in peripheral nociceptive fibers and in mediating pain-related signals to the CNS. Control of P2X activation has significant importance in managing unwanted hypersensitive neuron responses. To overcome the limitations of chemical ligand treatment, optical stimulation methods of optogenetics and photoswitching achieve efficient control of P2X activation while allowing specificity at the target site and convenient stimulation by light illumination. There are many potential applications for photosensitive elements, such as improved uncaging methods, photoisomerizable ligands, photoswitches, and gold nanoparticles. Each technique has both advantages and downsides, and techniques are selected according to the purpose of the application. Technical advances not only provide novel approaches to manage inflammation or pain mediated by P2X receptors but also suggest a similar approach for controlling other ion channels. PMID:26884649

  18. Gamma-hydroxybutyrate, acting through an anti-apoptotic mechanism, protects native and amyloid-precursor-protein-transfected neuroblastoma cells against oxidative stress-induced death.

    PubMed

    Wendt, G; Kemmel, V; Patte-Mensah, C; Uring-Lambert, B; Eckert, A; Schmitt, M J; Mensah-Nyagan, A G

    2014-03-28

    Clinical observations suggested that gamma-hydroxybutyrate (GHB) protects nerve cells against death but the direct proofs are missing. Here, we combined several approaches to investigate GHB capacity to protect human neuroblastoma SH-SY5Y cells against hydrogen peroxide (H2O2)-induced death. To increase the patho-physiological relevancy of our study, we used native SH-SY5Y cells and SH-SY5Y cells stably transfected with the wild-type amyloid-precursor-protein (APPwt) or control-vector-pCEP4. Trypan Blue exclusion and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide) assays combined with pharmacological analyses showed that H2O2 reduced native and genetically modified cell viability and APPwt-transfected cells were the most vulnerable. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) and activated caspase-3 staining assessed by flow cytometry revealed a basally elevated apoptotic signal in APPwt-transfected cells. Reverse-transcription, real-time quantitative polymerase chain reaction (qPCR) and Western blotting showed that mRNA and protein basal ratios of apoptotic modulators Bax/Bcl-2 were also high in APPwt-transfected cells. GHB efficiently and dose-dependently rescued native and genetically modified cells from H2O2-induced death. Interestingly, GHB, which strongly decreased elevated basal levels of TUNEL-staining, activated caspase 3-labeling and Bax/Bcl-2 in APPwt-transfected cells, also counteracted H2O2-evoked increased apoptotic markers in native and genetically modified SH-SY5Y cells. Since GHB did not promote cell proliferation, anti-apoptotic action through the down-regulation of Bax/Bcl-2 ratios and/or caspase 3 activity appears as a critical mechanism involved in GHB-induced protection of SH-SY5Y cells against APPwt-overexpression- or H2O2-evoked death. Altogether, these results, providing multi-parametric evidence for the existence of neuroprotective action of GHB, also open interesting perspectives for

  19. Endocannabinoids Stimulate Human Melanogenesis via Type-1 Cannabinoid Receptor*

    PubMed Central

    Pucci, Mariangela; Pasquariello, Nicoletta; Battista, Natalia; Di Tommaso, Monia; Rapino, Cinzia; Fezza, Filomena; Zuccolo, Michela; Jourdain, Roland; Finazzi Agrò, Alessandro; Breton, Lionel; Maccarrone, Mauro

    2012-01-01

    We show that a fully functional endocannabinoid system is present in primary human melanocytes (normal human epidermal melanocyte cells), including anandamide (AEA), 2-arachidonoylglycerol, the respective target receptors (CB1, CB2, and TRPV1), and their metabolic enzymes. We also show that at higher concentrations AEA induces normal human epidermal melanocyte apoptosis (∼3-fold over controls at 5 μm) through a TRPV1-mediated pathway that increases DNA fragmentation and p53 expression. However, at lower concentrations, AEA and other CB1-binding endocannabinoids dose-dependently stimulate melanin synthesis and enhance tyrosinase gene expression and activity (∼3- and ∼2-fold over controls at 1 μm). This CB1-dependent activity was fully abolished by the selective CB1 antagonist SR141716 or by RNA interference of the receptor. CB1 signaling engaged p38 and p42/44 mitogen-activated protein kinases, which in turn activated the cyclic AMP response element-binding protein and the microphthalmia-associated transcription factor. Silencing of tyrosinase or microphthalmia-associated transcription factor further demonstrated the involvement of these proteins in AEA-induced melanogenesis. In addition, CB1 activation did not engage the key regulator of skin pigmentation, cyclic AMP, showing a major difference compared with the regulation of melanogenesis by α-melanocyte-stimulating hormone through melanocortin 1 receptor. PMID:22431736

  20. Novel homodimeric and heterodimeric rat gamma-hydroxybutyrate synthases that associate with the Golgi apparatus define a distinct subclass of aldo-keto reductase 7 family proteins.

    PubMed Central

    Kelly, Vincent P; Sherratt, Philip J; Crouch, Dorothy H; Hayes, John D

    2002-01-01

    The aldo-keto reductase (AKR) 7 family is composed of the dimeric aflatoxin B(1) aldehyde reductase (AFAR) isoenzymes. In the rat, two AFAR subunits exist, designated rAFAR1 and rAFAR2. Herein, we report the molecular cloning of rAFAR2, showing that it shares 76% sequence identity with rAFAR1. By contrast with rAFAR1, which comprises 327 amino acids, rAFAR2 contains 367 amino acids. The 40 extra residues in rAFAR2 are located at the N-terminus of the polypeptide as an Arg-rich domain that may form an amphipathic alpha-helical structure. Protein purification and Western blotting have shown that the two AFAR subunits are found in rat liver extracts as both homodimers and as a heterodimer. Reductase activity in rat liver towards 2-carboxybenzaldehyde (CBA) was resolved by anion-exchange chromatography into three peaks containing rAFAR1-1, rAFAR1-2 and rAFAR2-2 dimers. These isoenzymes are functionally distinct; with NADPH as cofactor, rAFAR1-1 has a low K(m) and high activity with CBA, whereas rAFAR2-2 exhibits a low K(m) and high activity towards succinic semialdehyde. These data suggest that rAFAR1-1 is a detoxication enzyme, while rAFAR2-2 serves to synthesize the endogenous neuromodulator gamma-hydroxybutyrate (GHB). Subcellular fractionation of liver extracts showed that rAFAR1-1 was recovered in the cytosol whereas rAFAR2-2 was associated with the Golgi apparatus. The distinct subcellular localization of the rAFAR1 and rAFAR2 subunits was confirmed by immunocytochemistry in H4IIE cells. Association of rAFAR2-2 with the Golgi apparatus presumably facilitates secretion of GHB, and the novel N-terminal domain may either determine the targeting of the enzyme to the Golgi or regulate the secretory process. A murine AKR protein of 367 residues has been identified in expressed sequence tag databases that shares 91% sequence identity with rAFAR2 and contains the Arg-rich extended N-terminus of 40 amino acids. Further bioinformatic evidence is presented that full

  1. Stimulation by toll-like receptors inhibits osteoclast differentiation.

    PubMed

    Takami, Masamichi; Kim, Nacksung; Rho, Jaerang; Choi, Yongwon

    2002-08-01

    Osteoclasts, the cells capable of resorbing bone, are derived from hemopoietic precursor cells of monocyte-macrophage lineage. The same precursor cells can also give rise to macrophages and dendritic cells, which are essential for proper immune responses to various pathogens. Immune responses to microbial pathogens are often triggered because various microbial components induce the maturation and activation of immunoregulatory cells such as macrophages or dendritic cells by stimulating Toll-like receptors (TLRs). Since osteoclasts arise from the same precursors as macrophages, we tested whether TLRs play any role during osteoclast differentiation. We showed here that osteoclast precursors prepared from mouse bone marrow cells expressed all known murine TLRs (TLR1-TLR9). Moreover, various TLR ligands (e.g., peptidoglycan, poly(I:C) dsRNA, LPS, and CpG motif of unmethylated DNA, which act as ligands for TLR2, 3, 4, and 9, respectively) induced NF-kappa B activation and up-regulated TNF-alpha production in osteoclast precursor cells. Unexpectedly, however, TLR stimulation of osteoclast precursors by these microbial products strongly inhibited their differentiation into multinucleated, mature osteoclasts induced by TNF-related activation-induced cytokine. Rather, TLR stimulation maintained the phagocytic activity of osteoclast precursors in the presence of osteoclastogenic stimuli M-CSF and TNF-related activation-induced cytokine. Taken together, these results suggest that TLR stimulation of osteoclast precursors inhibits their differentiation into noninflammatory mature osteoclasts during microbial infection. This process favors immune responses and may be critical to prevent pathogenic effects of microbial invasion on bone. PMID:12133979

  2. Transferrin receptor expression by stimulated cells in mixed lymphocyte culture.

    PubMed Central

    Salmon, M; Bacon, P A; Symmons, D P; Walton, K W

    1985-01-01

    Transferrin receptor (TRFr) expression by cells in mixed lymphocyte culture increases steadily for the first 5 days, but then reaches a plateau. By the sixth day in culture, about 20% of viable cells express TRFr in two-way mixed lymphocyte reactions. This subpopulation of TRFr-positive cells represents the proliferating population; it is heterogeneous, containing T-cell blasts and smaller cells which are a mixture of T and non-T cells. A small group of non-T cells have phenotypic similarity to natural killer (NK) cells. T cells appear to divide earlier in the course of the response than non-T cells. The biphasic nature of this response and the slower non-T reactivity may be due to a secondary stimulation of non-T cells by factors released from activated T cells (such as interleukin-2). PMID:2982734

  3. Thrombin stimulates insulin secretion via protease-activated receptor-3

    PubMed Central

    Hänzelmann, Sonja; Wang, Jinling; Güney, Emre; Tang, Yunzhao; Zhang, Enming; Axelsson, Annika S; Nenonen, Hannah; Salehi, Albert S; Wollheim, Claes B; Zetterberg, Eva; Berntorp, Erik; Costa, Ivan G; Castelo, Robert; Rosengren, Anders H

    2015-01-01

    The disease mechanisms underlying type 2 diabetes (T2D) remain poorly defined. Here we aimed to explore the pathophysiology of T2D by analyzing gene co-expression networks in human islets. Using partial correlation networks we identified a group of co-expressed genes (‘module’) including F2RL2 that was associated with glycated hemoglobin. F2Rl2 is a G-protein-coupled receptor (GPCR) that encodes protease-activated receptor-3 (PAR3). PAR3 is cleaved by thrombin, which exposes a 6-amino acid sequence that acts as a ‘tethered ligand’ to regulate cellular signaling. We have characterized the effect of PAR3 activation on insulin secretion by static insulin secretion measurements, capacitance measurements, studies of diabetic animal models and patient samples. We demonstrate that thrombin stimulates insulin secretion, an effect that was prevented by an antibody that blocks the thrombin cleavage site of PAR3. Treatment with a peptide corresponding to the PAR3 tethered ligand stimulated islet insulin secretion and single β-cell exocytosis by a mechanism that involves activation of phospholipase C and Ca2+ release from intracellular stores. Moreover, we observed that the expression of tissue factor, which regulates thrombin generation, was increased in human islets from T2D donors and associated with enhanced β-cell exocytosis. Finally, we demonstrate that thrombin generation potential in patients with T2D was associated with increased fasting insulin and insulinogenic index. The findings provide a previously unrecognized link between hypercoagulability and hyperinsulinemia and suggest that reducing thrombin activity or blocking PAR3 cleavage could potentially counteract the exaggerated insulin secretion that drives insulin resistance and β-cell exhaustion in T2D. PMID:26742564

  4. Behavioral meaningful opioidergic stimulation activates kappa receptor gene expression

    PubMed Central

    Teodorov, E.; Ferrari, M.F.R.; Fior-Chadi, D.R.; Camarini, R.; Felício, L.F.

    2012-01-01

    The periaqueductal gray (PAG) has been reported to be a location for opioid regulation of pain and a potential site for behavioral selection in females. Opioid-mediated behavioral and physiological responses differ according to the activity of opioid receptor subtypes. The present study investigated the effects of the peripheral injection of the kappa-opioid receptor agonist U69593 into the dorsal subcutaneous region of animals on maternal behavior and on Oprk1 gene activity in the PAG of female rats. Female Wistar rats weighing 200-250 g at the beginning of the study were randomly divided into 2 groups for maternal behavior and gene expression experiments. On day 5, pups were removed at 7:00 am and placed in another home cage that was distant from their mother. Thirty minutes after removing the pups, the dams were treated with U69593 (0.15 mg/kg, sc) or 0.9% saline (up to 1 mL/kg) and after 30 min were evaluated in the maternal behavior test. Latencies in seconds for pup retrieval, grouping, crouching, and full maternal behavior were scored. The results showed that U69593 administration inhibited maternal behavior (P < 0.05) because a lower percentage of U69593 group dams showed retrieval of first pup, retrieving all pups, grouping, crouching and displaying full maternal behavior compared to the saline group. Opioid gene expression was evaluated using real-time reverse-transcription polymerase chain reaction (RT-PCR). A single injection of U69593 increased Oprk1 PAG expression in both virgin (P < 0.05) and lactating female rats (P < 0.01), with no significant effect on Oprm1 or Oprd1 gene activity. Thus, the expression of kappa-opioid receptors in the PAG may be modulated by single opioid receptor stimulation and behavioral meaningful opioidergic transmission in the adult female might occur simultaneously to specific changes in gene expression of kappa-opioid receptor subtype. This is yet another alert for the complex role of the opioid system in female

  5. In vivo stimulation of oestrogen receptor α increases insulin-stimulated skeletal muscle glucose uptake

    PubMed Central

    Gorres, Brittany K; Bomhoff, Gregory L; Morris, Jill K; Geiger, Paige C

    2011-01-01

    Abstract Previous studies suggest oestrogen receptor α (ERα) is involved in oestrogen-mediated regulation of glucose metabolism and is critical for maintenance of whole body insulin action. Despite this, the effect of direct ERα modulation in insulin-responsive tissues is unknown. The purpose of the current study was to determine the impact of ERα activation, using the ER subtype-selective ligand propylpyrazoletriyl (PPT), on skeletal muscle glucose uptake. Two-month-old female Sprague–Dawley rats, ovariectomized for 1 week, were given subcutaneous injections of PPT (10 mg kg−1), oestradiol benzoate (EB; 20 μg kg−1), the ERβ agonist diarylpropionitrile (DPN, 10 mg kg−1) or vehicle every 24 h for 3 days. On the fourth day, insulin-stimulated skeletal muscle glucose uptake was measured in vitro and insulin signalling intermediates were assessed via Western blotting. Activation of ERα with PPT resulted in increased insulin-stimulated glucose uptake into the slow-twitch soleus and fast-twitch extensor digitorum longus (EDL) muscles, activation of insulin signalling intermediates (as measured by phospho-Akt (pAkt) and pAkt substrate (PAS)) and phosphorylation of AMP-activated protein kinase (AMPK). GLUT4 protein was increased only in the EDL muscle. Rats treated with EB or DPN for 3 days did not show an increase in insulin-stimulated skeletal muscle glucose uptake compared to vehicle-treated animals. These new findings reveal that direct activation of ERα positively mediates glucose uptake and insulin action in skeletal muscle. Evidence that oestrogens and ERα stimulate glucose uptake has important implications for understanding mechanisms of glucose homeostasis, particularly in postmenopausal women. PMID:21486807

  6. Epidermal growth factor receptor-dependent stimulation of amphiregulin expression in androgen-stimulated human prostate cancer cells.

    PubMed Central

    Sehgal, I; Bailey, J; Hitzemann, K; Pittelkow, M R; Maihle, N J

    1994-01-01

    Amphiregulin is a heparin-binding epidermal growth factor (EGF)-related peptide that binds to the EGF receptor (EGF-R) with high affinity. In this study, we report a role for amphiregulin in androgen-stimulated regulation of prostate cancer cell growth. Androgen is known to enhance EGF-R expression in the androgen-sensitive LNCaP human prostate carcinoma cell line, and it has been suggested that androgenic stimuli may regulate proliferation, in part, through autocrine mechanisms involving the EGF-R. In this study, we demonstrate that LNCaP cells express amphiregulin mRNA and peptide and that this expression is elevated by androgenic stimulation. We also show that ligand-dependent EGF-R stimulation induces amphiregulin expression and that androgenic effects on amphiregulin synthesis are mediated through this EGF-R pathway. Parallel studies using the estrogen-responsive breast carcinoma cell line, MCF-7, suggest that regulation of amphiregulin by estrogen may also be mediated via an EGF-R pathway. In addition, heparin treatment of LNCaP cells inhibits androgen-stimulated cell growth further suggesting that amphiregulin can mediate androgen-stimulated LNCaP proliferation. Together, these results implicate an androgen-regulated autocrine loop composed of amphiregulin and its receptor in prostate cancer cell growth and suggest that the mechanism of steroid hormone regulation of amphiregulin synthesis may occur through androgen upregulation of the EGF-R and subsequent receptor-dependent pathways. Images PMID:8049525

  7. Comparative study of equimolar doses of gamma-hydroxybutyrate (GHB), 1,4-butanediol (1,4-BD) and gamma-butyrolactone (GBL) on catalepsy after acute and chronic administration.

    PubMed

    Towiwat, Pasarapa; Phattanarudee, Siripan; Maher, Timothy J

    2013-01-01

    Gamma-hydroxybutyrate (GHB), and its precursors 1,4-butanediol (1,4-BD) and gamma-butyrolactone (GBL) are known drugs of abuse. The ability of acute and chronic administration of equimolar doses of GHB (200mg/kg), 1,4-BD (174mg/kg) and GBL (166mg/kg) to produce catalepsy in male Swiss Webster mice was examined. GHB, 1,4-BD, GBL produced catalepsy when injected acutely. Drug treatment was then continued for 14days. Tolerance development was determined on days 6, 14, and challenged with a higher dose on day 15 in those chronically pretreated mice, and compared with naïve mice. Chronic GHB produced tolerance to catalepsy, as evidenced from area under the curve (AUC) of catalepsy versus time (min-sec) on days 6 (678±254), 14 (272±247), which were less than those on day 1 (1923±269). However, less tolerance was seen from GBL or 1,4-BD, as AUCs on days 6 and 14 were not significantly lower than that of day 1. In conclusion, although equimolar doses were used, expecting similar levels of GHB in the body, 1,4-BD and GBL shared only some of the in vivo effects of GHB. The rate of metabolic conversion of 1,4-BD and GBL into GHB might be responsible for the differences in the tolerance development to these drugs. PMID:23104245

  8. Calmodulin-stimulated phosphorylation of 17 beta-estradiol receptor on tyrosine.

    PubMed Central

    Migliaccio, A; Rotondi, A; Auricchio, F

    1984-01-01

    The calf uterine 17 beta-estradiol receptor is a phosphoprotein. Phosphorylation-dephosphorylation of the receptor is controlled by a cytosol receptor kinase that activates the hormone binding and by a nuclear phosphatase that inactivates this binding. This report concerns the nature of the 17 beta-estradiol receptor kinase. Highly purified calf uterus 17 beta-estradiol receptor preinactivated by the nuclear phosphatase was used as substrate of the purified receptor kinase. Ca2+ and calmodulin stimulate both the kinase-dependent activation of the hormone binding and 32P incorporation from [gamma-32P]-ATP into the receptor. Maximal stimulation of hormone binding activation requires 1 microM Ca2+ and 0.6 microM calmodulin. Fifteen micromolar trifluoperazine is the lowest concentration that will prevent completely Ca2+-calmodulin stimulation of the kinase. The receptor is phosphorylated by the receptor kinase exclusively on tyrosine. Phosphorylation of proteins on tyrosine is a rare event implicated in hormone-induced cell growth and cell transformation. Images PMID:6207535

  9. Decrease in the Sensitivity of Myocardium to M3 Muscarinic Receptor Stimulation during Postnatal Ontogenisis.

    PubMed

    Tapilina, S V; Abramochkin, D V

    2016-01-01

    Type 3 muscarinic receptors (M3 receptors) participate in the mediation of cholinergic effects in mammalian myocardium, along with M2 receptors. However, myocardium of adult mammals demonstrates only modest electrophysiological effects in response to selective stimulation of M3 receptors which are hardly comparable to the effects produced by M2 stimulation. In the present study, the effects of selective M3 stimulation induced by application of the muscarinic agonist pilocarpine (10 μM) in the presence of the selective M2 blocker methoctramine (100 nM) on the action potential (AP) waveform were investigated in isolated atrial and ventricular preparations from newborn and 3-week-old rats and compared to those in preparations from adult rats. In the atrial myocardium, stimulation of M3 receptors produced a comparable reduction of AP duration in newborn and adult rats, while in 3-week-old rats the effect was negligible. In ventricular myocardial preparations from newborn rats, the effect of M3 stimulation was more than 3 times stronger compared to that from adult rats, while preparations from 3-week old rats demonstrated no definite effect, similarly to atrial preparations. In all studied types of cardiac preparations, the effects of M3 stimulation were eliminated by the selective M3 antagonist 4-DAMP (10 nM). The results of RT-PCR show that the amount of product of the M3 receptor gene decreases with the maturation of animals both in atrial and ventricular myocardium. We concluded that the contribution of M3 receptors to the mediation of cardiac cholinergic responses decreases during postnatal ontogenesis. These age-related changes may be associated with downregulation of M3 receptor gene expression. PMID:27437147

  10. Decrease in the Sensitivity of Myocardium to M3 Muscarinic Receptor Stimulation during Postnatal Ontogenisis

    PubMed Central

    Tapilina, S.V.; Abramochkin, D.V.

    2016-01-01

    Type 3 muscarinic receptors (M3 receptors) participate in the mediation of cholinergic effects in mammalian myocardium, along with M2 receptors. However, myocardium of adult mammals demonstrates only modest electrophysiological effects in response to selective stimulation of M3 receptors which are hardly comparable to the effects produced by M2 stimulation. In the present study, the effects of selective M3 stimulation induced by application of the muscarinic agonist pilocarpine (10 μM) in the presence of the selective M2 blocker methoctramine (100 nM) on the action potential (AP) waveform were investigated in isolated atrial and ventricular preparations from newborn and 3-week-old rats and compared to those in preparations from adult rats. In the atrial myocardium, stimulation of M3 receptors produced a comparable reduction of AP duration in newborn and adult rats, while in 3-week-old rats the effect was negligible. In ventricular myocardial preparations from newborn rats, the effect of M3 stimulation was more than 3 times stronger compared to that from adult rats, while preparations from 3-week old rats demonstrated no definite effect, similarly to atrial preparations. In all studied types of cardiac preparations, the effects of M3 stimulation were eliminated by the selective M3 antagonist 4-DAMP (10 nM). The results of RT-PCR show that the amount of product of the M3 receptor gene decreases with the maturation of animals both in atrial and ventricular myocardium. We concluded that the contribution of M3 receptors to the mediation of cardiac cholinergic responses decreases during postnatal ontogenesis. These age-related changes may be associated with downregulation of M3 receptor gene expression. PMID:27437147

  11. Cyclin D1 stimulation of estrogen receptor transcriptional activity independent of cdk4.

    PubMed Central

    Neuman, E; Ladha, M H; Lin, N; Upton, T M; Miller, S J; DiRenzo, J; Pestell, R G; Hinds, P W; Dowdy, S F; Brown, M; Ewen, M E

    1997-01-01

    Cyclin D1 plays an important role in the development of breast cancer and is required for normal breast cell proliferation and differentiation associated with pregnancy. We show that ectopic expression of cyclin D1 can stimulate the transcriptional activity of the estrogen receptor in the absence of estradiol and that this activity can be inhibited by 4-hydroxytamoxifen and ICI 182,780. Cyclin D1 can form a specific complex with the estrogen receptor. Stimulation of the estrogen receptor by cyclin D1 is independent of cyclin-dependent kinase 4 activation. Cyclin D1 may manifest its oncogenic potential in breast cancer in part through binding to the estrogen receptor and activation of the transcriptional activity of the receptor. PMID:9271411

  12. Role of the Extracellular and Intracellular Loops of Follicle-Stimulating Hormone Receptor in Its Function

    PubMed Central

    Banerjee, Antara A.; Mahale, Smita D.

    2015-01-01

    Follicle-stimulating hormone receptor (FSHR) is a leucine-rich repeat containing class A G-protein coupled receptor belonging to the subfamily of glycoprotein hormone receptors (GPHRs), which includes luteinizing hormone/choriogonadotropin receptor (LH/CGR) and thyroid-stimulating hormone receptor. Its cognate ligand, follicle-stimulating hormone binds to, and activates FSHR expressed on the surface of granulosa cells of the ovary, in females, and Sertoli cells of the testis, in males, to bring about folliculogenesis and spermatogenesis, respectively. FSHR contains a large extracellular domain (ECD) consisting of leucine-rich repeats at the N-terminal end and a hinge region at the C-terminus that connects the ECD to the membrane spanning transmembrane domain (TMD). The TMD consists of seven α-helices that are connected to each other by means of three extracellular loops (ELs) and three intracellular loops (ILs) and ends in a short-cytoplasmic tail. It is well established that the ECD is the primary hormone binding domain, whereas the TMD is the signal transducing domain. However, several studies on the ELs and ILs employing site directed mutagenesis, generation of chimeric receptors and in vitro characterization of naturally occurring mutations have proven their indispensable role in FSHR function. Their role in every phase of the life cycle of the receptor like post translational modifications, cell surface trafficking, hormone binding, activation of downstream signaling, receptor phosphorylation, hormone–receptor internalization, and recycling of hormone–receptor complex have been documented. Mutations in the loops causing dysregulation of these processes lead to pathophysiological conditions. In other GPHRs as well, the loops have been convincingly shown to contribute to various aspects of receptor function. This review article attempts to summarize the extensive contributions of FSHR loops and C-terminal tail to its function. PMID:26236283

  13. Anandamide, an endogenous cannabimimetic eicosanoid, binds to the cloned human cannabinoid receptor and stimulates receptor-mediated signal transduction.

    PubMed Central

    Felder, C C; Briley, E M; Axelrod, J; Simpson, J T; Mackie, K; Devane, W A

    1993-01-01

    Arachidonylethanolamide (anandamide), a candidate endogenous cannabinoid ligand, has recently been isolated from porcine brain and displayed cannabinoid-like binding activity to synaptosomal membrane preparations and mimicked cannabinoid-induced inhibition of the twitch response in isolated murine vas deferens. In this study, anandamide and several congeners were evaluated as cannabinoid agonists by examining their ability to bind to the cloned cannabinoid receptor, inhibit forskolin-stimulated cAMP accumulation, inhibit N-type calcium channels, and stimulate one or more functional second messenger responses. Synthetic anandamide, and all but one congener, competed for [3H]CP55,940 binding to plasma membranes prepared from L cells expressing the rat cannabinoid receptor. The ability of anandamide to activate receptor-mediated signal transduction was evaluated in Chinese hamster ovary (CHO) cells expressing the human cannabinoid receptor (HCR, termed CHO-HCR cells) and compared to control CHO cells expressing the muscarinic m5 receptor (CHOm5 cells). Anandamide inhibited forskolin-stimulated cAMP accumulation in CHO-HCR cells, but not in CHOm5 cells, and this response was blocked with pertussis toxin. N-type calcium channels were inhibited by anandamide and several active congeners in N18 neuroblastoma cells. Anandamide stimulated arachidonic acid and intracellular calcium release in both CHOm5 and CHO-HCR cells and had no effect on the release of inositol phosphates or phosphatidylethanol, generated after activation of phospholipase C and D, respectively. Anandamide appears to exhibit the essential criteria required to be classified as a cannabinoid/anandamide receptor agonist and shares similar nonreceptor effects on arachidonic acid and intracellular calcium release as other cannabinoid agonists. PMID:8395053

  14. Serotonin-2C Receptor Agonists Decrease Potassium-Stimulated GABA Release In the Nucleus Accumbens

    PubMed Central

    Kasper, James M; Booth, Raymond G; Peris, Joanna

    2014-01-01

    The serotonin 5-HT2C receptor has shown promise in vivo as a pharmacotherapeutic target for alcoholism. For example, recently, a novel 4-phenyl-2-N,N-dimethylaminotetralin (PAT) drug candidate, that demonstrates 5-HT2C receptor agonist activity together with 5-HT2A/2B receptor inverse agonist activity, was shown to reduce operant responding for ethanol after peripheral administration to rats. Previous studies have shown that the 5-HT2C receptor is found throughout the mesoaccumbens pathway and that 5-HT2C receptor agonism causes activation of ventral tegmental area (VTA) GABA neurons. It is unknown what effect 5-HT2C receptor modulation has on GABA release in the nucleus accumbens core (NAcc). To this end, microdialysis coupled to capillary electrophoresis with laser-induced fluorescence was used to quantify extracellular neurotransmitter concentrations in the NAcc under basal and after potassium stimulation conditions, in response to PAT analogs and other 5-HT2C receptor modulators administered by reverse dialysis to rats. 5-HT2C receptor agonists specifically attenuated stimulated GABA release in the NAcc while 5-HT2C antagonists or inverse agonists had no effect. Agents with activity at 5-HT2A receptors had no effect on GABA release. Thus, in contrast to results reported for the VTA, current results suggest 5-HT2C receptor agonists decrease stimulated GABA release in the NAcc, and provide a possible mechanism of action for 5HT2C-mediated negative modulation of ethanol self-administration. PMID:25382408

  15. Dissociation of insulin receptor phosphorylation and stimulation of glucose transport in BC3H-1 myocytes

    SciTech Connect

    Mojsilovic, L.P.; Standaert, M.L.; Rosic, N.K.; Pollet, R.J.

    1986-05-01

    The authors have investigated insulin receptor phosphorylation in differentiated cultured BC3H-1 myocytes. As for other insulin-responsive cell systems in partially purified wheat germ agglutinin receptor preparations, insulin stimulates the phosphorylation of its own receptor (95K ..beta..-subunits) in a dose dependent manner (0-400 nM), as identified by immunoprecipitation with antiinsulin receptor antibodies and SDS-PAGE. In the same preparations they show that 12-0-tetradecanyl phorbol acetate (TPA), which in many respect ..beta..-subunits in the same dose dependent manner (0-5 ..mu..M). In addition, antiinsulin receptor antibodies (B-10) also induced phosphorylation of mimics insulin action, also induced phosphorylation of the insulin receptor and HPLC tryptic maps of the /sup 32/P-labeled ..beta..-subunit were identical to those for insulin-induced receptor phosphorylation. However, while insulin and TPA are potent stimulators of glucose transport in these muscle cells, the antireceptor antibodies alone failed to provoke glucose transport at any concentration. The specificity and activity of these antibodies were confirmed in their system by their ability to inhibit insulin binding and insulin-stimulated glucose transport in a concentration-dependent manner. Their results indicate that phosphorylation of insulin receptor is not a crucial event in mediating insulin action, at least with respect to glucose transport. While the effects of the B-10 antibody in the BC3H-1 myocyte differ from those in the adipocyte, their results provide independent confirmation of their essential conclusion that phosphorylation of the insulin receptor may not be necessary nor sufficient for its acute action in promoting glucose transport.

  16. Cross-adaptation to odor stimulation of olfactory receptor cells in the box turtle, Terrapene carolina.

    PubMed

    Tonosaki, K

    1993-01-01

    Electrical recording from small twigs of olfactory nerve and electro-olfactogram (EOG) from olfactory epithelium in a turtle shows that olfactory receptors in the nose are responsive to various odors. I have used the effects of cross-adaptation to odor stimulation on the olfactory receptors to investigate the stimulus-specific components of these responses and to provide information about the responsiveness of cells. The results of the cross-adaptation experiments strongly support the hypothesis that different categories of receptor cells exist in the olfactory epithelium. PMID:8386588

  17. Apical and basolateral ATP stimulates tracheal epithelial chloride secretion via multiple purinergic receptors.

    PubMed

    Hwang, T H; Schwiebert, E M; Guggino, W B

    1996-06-01

    Stimulation of Cl- secretion across the airway epithelium by ATP or UTP as agonists has therapeutic implications for cystic fibrosis. Our results demonstrate that ATP stimulates Cl- secretion in rat tracheal epithelial cell monolayers in primary culture from the apical or basolateral side of the monolayer. Multiple types of ATP-sensitive Cl- conductances in intact monolayers were elucidated through inhibition by Cl- channel-blocking drugs. Multiple Cl- conductances stimulated by ATP and adenosine 3',5'-cyclic monophosphate (cAMP) (tested for comparison) were also deciphered more specifically by nystatin permeabilization of the basolateral membrane, subsequent imposition of symmetrical Cl-, I-, or Br- solutions to test halide permselectivity, inhibition by Cl- channel-blocking drugs, and construction of current-voltage plots to study time and voltage dependence of the currents. Apical ATP stimulates Cl- secretion through P2U (or P2Y2) purinergic receptors via both intracellular Ca2+ (Ca(2+)i)-dependent and Cai(2+)-independent signaling pathways by opening outwardly rectifying Cl- channels (ORCCs), cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channels, and Cai(2+)-dependent Cl- channels. Basolateral ATP stimulates Cl- secretion via a combination of receptor subtypes (P2T and P2U) or a novel type of receptor (P2Y3), independent of Cai2+ or cAMP signaling by opening only CFTR channels. cAMP also stimulated multiple types of Cl- conductances, consistent with simultaneous activation of CFTR and ORCCs. Together, these results suggest that ATP as an agonist stimulates Cl- secretion via multiple purinergic receptors and multiple signal transduction pathways activated in different membrane domains of tracheal epithelia. PMID:8764143

  18. 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

  19. κ-Opioid Receptor Stimulation Improves Endothelial Function via Akt-stimulated NO Production in Hyperlipidemic Rats

    PubMed Central

    Tian, Fei; Zheng, Xu-Yang; Li, Juan; Zhang, Shu-Miao; Feng, Na; Guo, Hai-Tao; Jia, Min; Wang, Yue-Min; Fan, Rong; Pei, Jian-Ming

    2016-01-01

    This study was designed to investigate the effect of U50,488H (a selective κ-opioid receptor agonist) on endothelial function impaired by hyperlipidemia and to determine the role of Akt-stimulated NO production in it. Hyperlipidemic model was established by feeding rats with a high-fat diet for 14 weeks. U50,488H and nor-BNI (a selective κ-opioid receptor antagonist) were administered intraperitoneally. In vitro, the involvement of the PI3K/Akt/eNOS pathway in the effect of U50,488H was studied using cultured endothelial cells subjected to artificial hyperlipidemia. Serum total cholesterol and low-density lipoprotein cholesterol concentrations dramatically increased after high-fat diet feeding. Administration of U50,488H significantly alleviated endothelial ultrastructural destruction and endothelium-dependent vasorelaxation impairment caused by hyperlipidemia. U50,488H also increased Akt/eNOS phosphorylation and serum/medium NO level both in vivo and in vitro. U50,488H increased eNOS activity and suppressed iNOS activity in vivo. The effects of U50,488H were abolished in vitro by siRNAs targeting κ-opioid receptor and Akt or PI3K/Akt/eNOS inhibitors. All effects of U50,488H were blocked by nor-BNI. These results demonstrate that κ-opioid receptor stimulation normalizes endothelial ultrastructure and function under hyperlipidemic condition. Its mechanism is related to the preservation of eNOS phosphorylation through activation of the PI3K/Akt signaling pathway and downregulation of iNOS expression/activity. PMID:27226238

  20. κ-Opioid Receptor Stimulation Improves Endothelial Function via Akt-stimulated NO Production in Hyperlipidemic Rats.

    PubMed

    Tian, Fei; Zheng, Xu-Yang; Li, Juan; Zhang, Shu-Miao; Feng, Na; Guo, Hai-Tao; Jia, Min; Wang, Yue-Min; Fan, Rong; Pei, Jian-Ming

    2016-01-01

    This study was designed to investigate the effect of U50,488H (a selective κ-opioid receptor agonist) on endothelial function impaired by hyperlipidemia and to determine the role of Akt-stimulated NO production in it. Hyperlipidemic model was established by feeding rats with a high-fat diet for 14 weeks. U50,488H and nor-BNI (a selective κ-opioid receptor antagonist) were administered intraperitoneally. In vitro, the involvement of the PI3K/Akt/eNOS pathway in the effect of U50,488H was studied using cultured endothelial cells subjected to artificial hyperlipidemia. Serum total cholesterol and low-density lipoprotein cholesterol concentrations dramatically increased after high-fat diet feeding. Administration of U50,488H significantly alleviated endothelial ultrastructural destruction and endothelium-dependent vasorelaxation impairment caused by hyperlipidemia. U50,488H also increased Akt/eNOS phosphorylation and serum/medium NO level both in vivo and in vitro. U50,488H increased eNOS activity and suppressed iNOS activity in vivo. The effects of U50,488H were abolished in vitro by siRNAs targeting κ-opioid receptor and Akt or PI3K/Akt/eNOS inhibitors. All effects of U50,488H were blocked by nor-BNI. These results demonstrate that κ-opioid receptor stimulation normalizes endothelial ultrastructure and function under hyperlipidemic condition. Its mechanism is related to the preservation of eNOS phosphorylation through activation of the PI3K/Akt signaling pathway and downregulation of iNOS expression/activity. PMID:27226238

  1. Antinociceptive effects induced through the stimulation of spinal cannabinoid type 2 receptors in chronically inflamed mice.

    PubMed

    Curto-Reyes, Verdad; Boto, Tamara; Hidalgo, Agustín; Menéndez, Luis; Baamonde, Ana

    2011-10-01

    The stimulation of spinal cannabinoid type 2 (CB(2)) receptors is a suitable strategy for the alleviation of experimental pain symptoms. Several reports have described the up-regulation of spinal cannabinoid CB(2) receptors in neuropathic settings together with the analgesic effects derived from their activation. Besides, we have recently reported in two murine bone cancer models that the intrathecal administration of cannabinoid CB(2) receptor agonists completely abolishes hyperalgesia and allodynia, whereas spinal cannabinoid CB(2) receptor expression remains unaltered. The present experiments were designed to measure the expression of spinal cannabinoid CB(2) receptors as well as the analgesic efficacy derived from their stimulation in mice chronically inflamed by the intraplantar injection of complete Freund's adjuvant 1 week before. Both spinal cannabinoid CB(2) receptors mRNA measured by real-time PCR and cannabinoid CB(2) receptor protein levels measured by western blot remained unaltered in inflamed mice. Besides, the intrathecal (i.t.) administration of the cannabinoid CB(2) receptor agonists AM1241, (R,S)-3-(2-Iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole, (0.03-1 μg) and JWH 133, (6aR,10aR)-3-(1,1-Dimethylbutyl)-6a,7,10,10a-tetrahydro-6,6,9-trimethyl-6H-dibenzo[b,d]pyran, (3-30 μg) dose-dependently blocked inflammatory thermal hyperalgesia and mechanical allodynia. The analgesic effects induced by both agonists were counteracted by the coadministration of the selective cannabinoid CB(2) receptor antagonist SR144528, 5-(4-chloro-3-methylphenyl)-1-[(4-methylphenyl)methyl]-N-[(1S,2S,4R)-1,3,3-trimethylbicyclo[2.2.1]hept-2-yl]-1H-pyrazole-3-carboxamide, (5 μg) but not by the cannabinoid CB(1) receptor antagonist AM251, N-(Piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide, (10 μg). The effects induced by AM1241 were also inhibited by the coadministration of the opioid receptor antagonist, naloxone

  2. Excitation of type II anterior caudate neurons by stimulation of dopamine D3 receptors.

    PubMed

    Piercey, M F; Hyslop, D K; Hoffmann, W E

    1997-07-11

    Previous studies have demonstrated that both direct- and indirect-acting dopamine (DA) receptor agonists excite type II neurons in the anterior caudate (CN) by stimulation of DA receptors belonging to the D2 receptor subfamily (D2, D3, D4 receptor subtypes). In the present study, pramipexole, a D3-preferring DA agonist effective in treating Parkinson's disease, excited type II anterior CN neurons. As with other direct-acting agonists, excitation of the CN neurons occurred only at doses above those that silenced DA neurons in the substantia nigra pars compacta (SNPC). Although more potent than pramipexole in inhibiting SNPC cells, PNU-91356A, a D2-preferring agonist, did not excite type II CN cells. The D3-preferring antagonist (+)-AJ76 was weaker than haloperidol, a D2-preferring antagonist, in reversing the effects of amphetamine on firing rates in dopaminergic neurons in both the SNPC and the CN. However, in relationship to its potency in the SNPC, (+)-AJ76 was more potent than haloperidol in the CN. PNU-101387, a selective D4 antagonist, did not alter amphetamine-induced stimulation of type II CN neurons. We conclude that DA agonists may excite type II anterior CN neurons via D3 receptor activation. The stimulation of these neurons may contribute to the anti-parkinsonian effects of pramipexole. PMID:9262154

  3. Evidence for two different types of P2 receptors stimulating insulin secretion from pancreatic B cell.

    PubMed

    Petit, P; Hillaire-Buys, D; Manteghetti, M; Debrus, S; Chapal, J; Loubatières-Mariani, M M

    1998-11-01

    Adenine nucleotides have been shown to stimulate insulin secretion by acting on P2 receptors of the P2Y type. Since there have been some discrepancies in the insulin response of different analogues of ATP and ADP, we investigated whether two different types of P2 receptors exist on pancreatic B cells. The effects of alpha,beta-methylene ATP, which is more specific for the P2X subtype, were studied in vitro in pancreatic islets and isolated perfused pancreas from rats, in comparison with the potent P2Y receptor agonist ADPbetaS. In isolated islets, incubated with a slightly stimulating glucose concentration (8.3 mM), alpha,beta-me ATP (200 microM) and ADPbetaS (50 microM) similarly stimulated insulin secretion; by contrast, under a non stimulating glucose concentration (3 mM), alpha,beta-me ATP was still effective whereas ADPbetaS was not. In the same way, in islets perifused with 3 mM glucose, alpha,beta-me ATP but not ADPbetaS induced a partial but significant reduction in the peak 86Rb efflux induced by the ATP-dependent potassium channel opener diazoxide. In the isolated pancreas, perfused with a non stimulating glucose concentration (4.2 mM), ADPbetaS and alpha,beta-me ATP (5-50 microM), administered for 10 min, induced an immediate, transient and concentration-dependent increase in the insulin secretion; their relative potency was not significantly different. In contrast, with a slightly stimulating glucose concentration (8.3 mM), ADPbetaS was previously shown to be 100 fold more potent than alpha,beta-me ATP. Furthermore, at 4.2 mM glucose a second administration of alpha,beta-me ATP was ineffective. In the same way, ADPbetaS was also able to desensitize its own insulin response. At 3 mM glucose, alpha,beta-me ATP as well as ADPbetaS (50 microM) induced a transient stimulation of insulin secretion and down regulated the action of each other. These results give evidence that pancreatic B cells, in addition to P2Y receptors, which potentiate glucose

  4. Oestradiol stimulates tyrosine phosphorylation and hormone binding activity of its own receptor in a cell-free system.

    PubMed Central

    Auricchio, F; Migliaccio, A; Di Domenico, M; Nola, E

    1987-01-01

    Recent experiments have shown that calf uterus oestrogen receptor exists in a tyrosine-phosphorylated hormone binding form and in non-phosphorylated, non-hormone binding form. We report here that physiological concentrations of oestradiol in complex with the receptor stimulate the calf uterus receptor kinase that converts the non-hormone binding receptor into hormone binding receptor through phosphorylation of the receptor on tyrosine. The activity of this enzyme has been followed by reactivation of hormone binding sites and phosphorylation on tyrosine of calf uterus phosphatase-inactivated receptor. Phosphorylation of the receptor has been demonstrated by interaction of kinase 32P-phosphorylated proteins with anti-receptor antibody followed either by sucrose gradient centrifugation or SDS-PAGE of the immunoprecipitated proteins. Hormone stimulation of the kinase is inhibited by receptor occupancy of the anti-oestrogen tamoxifen. Oestradiol-receptor complex increases the affinity of the kinase for the dephosphorylated receptor. Findings of this report are consistent with the observation that several protein tyrosine kinases that are associated with peptide hormone receptors are stimulated by the binding of the hormone to the receptor. This is the first report on the activation of a tyrosine kinase by a steroid hormone. The finding that hormones can regulate their own receptor binding activity through a tyrosine kinase is also new. Images Fig. 2. Fig. 4. Fig. 5. PMID:3691476

  5. Epidermal growth factor receptor is required for estradiol-stimulated bovine satellite cell proliferation.

    PubMed

    Reiter, B C; Kamanga-Sollo, E; Pampusch, M S; White, M E; Dayton, W R

    2014-07-01

    The objective of this study was to assess the role of the epidermal growth factor receptor (EGFR) in estradiol-17β (E2)-stimulated proliferation of cultured bovine satellite cells (BSCs). Treatment of BSC cultures with AG1478 (a specific inhibitor of EGFR tyrosine kinase activity) suppresses E2-stimulated BSC proliferation (P < 0.05). In addition, E2-stimulated proliferation is completely suppressed (P < 0.05) in BSCs in which EGFR expression is silenced by treatment with EGFR small interfering RNA (siRNA). These results indicate that EGFR is required for E2 to stimulate proliferation in BSC cultures. Both AG1478 treatment and EGFR silencing also suppress proliferation stimulated by LR3-IGF-1 (an IGF1 analogue that binds normally to the insulin-like growth factor receptor (IGFR)-1 but has little or no affinity for IGF binding proteins) in cultured BSCs (P < 0.05). Even though EGFR siRNA treatment has no effect on IGFR-1β mRNA expression in cultured BSCs, IGFR-1β protein level is substantially reduced in BSCs treated with EGFR siRNA. These data suggest that EGFR silencing results in post-transcriptional modifications that result in decreased IGFR-1β protein levels. Although it is clear that functional EGFR is necessary for E2-stimulated proliferation of BSCs, the role of EGFR is not clear. Transactivation of EGFR may directly stimulate proliferation, or EGFR may function to maintain the level of IGFR-1β which is necessary for E2-stimulated proliferation. It also is possible that the role of EGFR in E2-stimulated BSC proliferation may involve both of these mechanisms. PMID:24906928

  6. Phorbol ester stimulates secretory activity while inhibiting receptor-activated aminopyrine uptake by gastric glands

    SciTech Connect

    Brown, M.R.; Chew, C.S.

    1986-03-05

    Both cyclic AMP-dependent and -independent secretagogues stimulate pepsinogen release, respiration and H/sup +/ secretory activity (AP uptake) in rabbit gastric glands. 12-O-tetradecanoylphorbol-13-acetate (T), a diacyglycerol analog, activates protein kinase C (PKC) and stimulates secretion in many systems. T stimulated respiration and pepsinogen release by glands and increased AP uptake by both glands and purified parietal cells. However, T reduced AP uptake by glands stimulated with carbachol (C) or histamine (H) with an apparent IC/sub 50/ of 1 nM. Preincubation with T for 30 min produced maximum inhibition which was not reversed by removal of T. T accelerated the decline of the transient C peak while the late steady state response to H was most inhibited. H-stimulated AP uptake was also inhibited by 50 ..mu..g/ml 1-oleoyl-2-acetyl-glycerol, a reported PKC activator, but not by the inactive phorbol, 4..cap alpha..-phorbol-12,13-didecanoate. In contrast, T potentiated AP uptake by glands stimulated with submaximal doses of dibutyryl cyclic AMP. These results suggest inhibition by T is a specific effect of PKC activators. The differing effects of T on secretion indicators may result from a dual action of T on receptor and post-receptor intracellular events.

  7. 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.

  8. Adrenaline Rush: The Role of Adrenergic Receptors in Stimulant-Induced Behaviors

    PubMed Central

    Schmidt, Karl T.

    2014-01-01

    Psychostimulants, such as cocaine and amphetamines, act primarily through the monoamine neurotransmitters dopamine (DA), norepinephrine, and serotonin. Although stimulant addiction research has largely focused on DA, medication development efforts targeting the dopaminergic system have thus far been unsuccessful, leading to alternative strategies aimed at abating stimulant abuse. Noradrenergic compounds have shown promise in altering the behavioral effects of stimulants in rodents, nonhuman primates, and humans. In this review, we discuss the contribution of each adrenergic receptor (AR) subtype (α1, α2, and β) to five stimulant-induced behaviors relevant to addiction: locomotor activity, conditioned place preference, anxiety, discrimination, and self-administration. AR manipulation has diverse effects on these behaviors; each subtype profoundly influences outcomes in some paradigms but is inconsequential in others. The functional neuroanatomy and intracellular signaling mechanisms underlying the impact of AR activation/blockade on these behaviors remain largely unknown, presenting a new frontier for research on psychostimulant–AR interactions. PMID:24499709

  9. Peripheral NMDA Receptors Mediate Antidromic Nerve Stimulation-Induced Tactile Hypersensitivity in the Rat

    PubMed Central

    Jang, Jun Ho; Nam, Taick Sang; Jun, Jaebeom; Jung, Se Jung; Kim, Dong-Wook; Leem, Joong Woo

    2015-01-01

    We investigated the role of peripheral NMDA receptors (NMDARs) in antidromic nerve stimulation-induced tactile hypersensitivity outside the skin area innervated by stimulated nerve. Tetanic electrical stimulation (ES) of the decentralized L5 spinal nerve, which induced enlargement of plasma extravasation, resulted in tactile hypersensitivity in the L4 plantar dermatome of the hind-paw. When intraplantar (i.pl.) injection was administered into the L4 dermatome before ES, NMDAR and group-I metabotropic Glu receptor (mGluR) antagonists and group-II mGluR agonist but not AMPA/kainate receptor antagonist prevented ES-induced hypersensitivity. I.pl. injection of PKA or PKC inhibitors also prevented ES-induced hypersensitivity. When the same injections were administered after establishment of ES-induced hypersensitivity, hypersensitivity was partially reduced by NMDAR antagonist only. In naïve animals, i.pl. Glu injection into the L4 dermatome induced tactile hypersensitivity, which was blocked by NMDAR antagonist and PKA and PKC inhibitors. These results suggest that the peripheral release of Glu, induced by antidromic nerve stimulation, leads to the expansion of tactile hypersensitive skin probably via nociceptor sensitization spread due to the diffusion of Glu into the skin near the release site. In addition, intracellular PKA- and PKC-dependent mechanisms mediated mainly by NMDAR activation are involved in Glu-induced nociceptor sensitization and subsequent hypersensitivity. PMID:26770021

  10. Muscarinic receptor agonists stimulate matrix metalloproteinase 1-dependent invasion of human colon cancer cells

    SciTech Connect

    Raufman, Jean-Pierre; Cheng, Kunrong; Saxena, Neeraj; Chahdi, Ahmed; Belo, Angelica; Khurana, Sandeep; Xie, Guofeng

    2011-11-18

    Highlights: Black-Right-Pointing-Pointer Muscarinic receptor agonists stimulated robust human colon cancer cell invasion. Black-Right-Pointing-Pointer Anti-matrix metalloproteinase1 antibody pre-treatment blocks cell invasion. Black-Right-Pointing-Pointer Bile acids stimulate MMP1 expression, cell migration and MMP1-dependent invasion. -- Abstract: Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers - this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre

  11. Muscarinic receptor agonists stimulate matrix metalloproteinase 1-dependent invasion of human colon cancer cells.

    PubMed

    Raufman, Jean-Pierre; Cheng, Kunrong; Saxena, Neeraj; Chahdi, Ahmed; Belo, Angelica; Khurana, Sandeep; Xie, Guofeng

    2011-11-18

    Mammalian matrix metalloproteinases (MMPs) which degrade extracellular matrix facilitate colon cancer cell invasion into the bloodstream and extra-colonic tissues; in particular, MMP1 expression correlates strongly with advanced colon cancer stage, hematogenous metastasis and poor prognosis. Likewise, muscarinic receptor signaling plays an important role in colon cancer; muscarinic receptors are over-expressed in colon cancer compared to normal colon epithelial cells. Muscarinic receptor activation stimulates proliferation, migration and invasion of human colon cancer cells. In mouse intestinal neoplasia models genetic ablation of muscarinic receptors attenuates carcinogenesis. In the present work, we sought to link these observations by showing that MMP1 expression and activation plays a mechanistic role in muscarinic receptor agonist-induced colon cancer cell invasion. We show that acetylcholine, which robustly increases MMP1 expression, stimulates invasion of HT29 and H508 human colon cancer cells into human umbilical vein endothelial cell monolayers - this was abolished by pre-incubation with atropine, a non-selective muscarinic receptor inhibitor, and by pre-incubation with anti-MMP1 neutralizing antibody. Similar results were obtained using a Matrigel chamber assay and deoxycholyltaurine (DCT), an amidated dihydroxy bile acid associated with colon neoplasia in animal models and humans, and previously shown to interact functionally with muscarinic receptors. DCT treatment of human colon cancer cells resulted in time-dependent, 10-fold increased MMP1 expression, and DCT-induced cell invasion was also blocked by pre-treatment with anti-MMP1 antibody. This study contributes to understanding mechanisms underlying muscarinic receptor agonist-induced promotion of colon cancer and, more importantly, indicates that blocking MMP1 expression and activation has therapeutic promise to stop or retard colon cancer invasion and dissemination. PMID:22027145

  12. Artificial Sweeteners Stimulate Adipogenesis and Suppress Lipolysis Independently of Sweet Taste Receptors*

    PubMed Central

    Simon, Becky R.; Parlee, Sebastian D.; Learman, Brian S.; Mori, Hiroyuki; Scheller, Erica L.; Cawthorn, William P.; Ning, Xiaomin; Gallagher, Katherine; Tyrberg, Björn; Assadi-Porter, Fariba M.; Evans, Charles R.; MacDougald, Ormond A.

    2013-01-01

    G protein-coupled receptors mediate responses to a myriad of ligands, some of which regulate adipocyte differentiation and metabolism. The sweet taste receptors T1R2 and T1R3 are G protein-coupled receptors that function as carbohydrate sensors in taste buds, gut, and pancreas. Here we report that sweet taste receptors T1R2 and T1R3 are expressed throughout adipogenesis and in adipose tissues. Treatment of mouse and human precursor cells with artificial sweeteners, saccharin and acesulfame potassium, enhanced adipogenesis. Saccharin treatment of 3T3-L1 cells and primary mesenchymal stem cells rapidly stimulated phosphorylation of Akt and downstream targets with functions in adipogenesis such as cAMP-response element-binding protein and FOXO1; however, increased expression of peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α was not observed until relatively late in differentiation. Saccharin-stimulated Akt phosphorylation at Thr-308 occurred within 5 min, was phosphatidylinositol 3-kinase-dependent, and occurred in the presence of high concentrations of insulin and dexamethasone; phosphorylation of Ser-473 occurred more gradually. Surprisingly, neither saccharin-stimulated adipogenesis nor Thr-308 phosphorylation was dependent on expression of T1R2 and/or T1R3, although Ser-473 phosphorylation was impaired in T1R2/T1R3 double knock-out precursors. In mature adipocytes, artificial sweetener treatment suppressed lipolysis even in the presence of forskolin, and lipolytic responses were correlated with phosphorylation of hormone-sensitive lipase. Suppression of lipolysis by saccharin in adipocytes was also independent of T1R2 and T1R3. These results suggest that some artificial sweeteners have previously uncharacterized metabolic effects on adipocyte differentiation and metabolism and that effects of artificial sweeteners on adipose tissue biology may be largely independent of the classical sweet taste receptors, T1R2 and T1R3. PMID

  13. Artificial sweeteners stimulate adipogenesis and suppress lipolysis independently of sweet taste receptors.

    PubMed

    Simon, Becky R; Parlee, Sebastian D; Learman, Brian S; Mori, Hiroyuki; Scheller, Erica L; Cawthorn, William P; Ning, Xiaomin; Gallagher, Katherine; Tyrberg, Björn; Assadi-Porter, Fariba M; Evans, Charles R; MacDougald, Ormond A

    2013-11-01

    G protein-coupled receptors mediate responses to a myriad of ligands, some of which regulate adipocyte differentiation and metabolism. The sweet taste receptors T1R2 and T1R3 are G protein-coupled receptors that function as carbohydrate sensors in taste buds, gut, and pancreas. Here we report that sweet taste receptors T1R2 and T1R3 are expressed throughout adipogenesis and in adipose tissues. Treatment of mouse and human precursor cells with artificial sweeteners, saccharin and acesulfame potassium, enhanced adipogenesis. Saccharin treatment of 3T3-L1 cells and primary mesenchymal stem cells rapidly stimulated phosphorylation of Akt and downstream targets with functions in adipogenesis such as cAMP-response element-binding protein and FOXO1; however, increased expression of peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α was not observed until relatively late in differentiation. Saccharin-stimulated Akt phosphorylation at Thr-308 occurred within 5 min, was phosphatidylinositol 3-kinase-dependent, and occurred in the presence of high concentrations of insulin and dexamethasone; phosphorylation of Ser-473 occurred more gradually. Surprisingly, neither saccharin-stimulated adipogenesis nor Thr-308 phosphorylation was dependent on expression of T1R2 and/or T1R3, although Ser-473 phosphorylation was impaired in T1R2/T1R3 double knock-out precursors. In mature adipocytes, artificial sweetener treatment suppressed lipolysis even in the presence of forskolin, and lipolytic responses were correlated with phosphorylation of hormone-sensitive lipase. Suppression of lipolysis by saccharin in adipocytes was also independent of T1R2 and T1R3. These results suggest that some artificial sweeteners have previously uncharacterized metabolic effects on adipocyte differentiation and metabolism and that effects of artificial sweeteners on adipose tissue biology may be largely independent of the classical sweet taste receptors, T1R2 and T1R3. PMID

  14. Extracellular matrix hyaluronan signals via its CD44 receptor in the increased responsiveness to mechanical stimulation.

    PubMed

    Ferrari, L F; Araldi, D; Bogen, O; Levine, J D

    2016-06-01

    We propose that the extracellular matrix (ECM) signals CD44, a hyaluronan receptor, to increase the responsiveness to mechanical stimulation in the rat hind paw. We report that intradermal injection of hyaluronidase induces mechanical hyperalgesia, that is inhibited by co-administration of a CD44 receptor antagonist, A5G27. The intradermal injection of low (LMWH) but not high (HMWH) molecular weight hyaluronan also induces mechanical hyperalgesia, an effect that was attenuated by pretreatment with HMWH or A5G27. Pretreatment with HMWH also attenuated the hyperalgesia induced by hyaluronidase. Similarly, intradermal injection of A6, a CD44 receptor agonist, produced hyperalgesia that was inhibited by HMWH and A5G27. Inhibitors of protein kinase A (PKA) and Src, but not protein kinase C (PKC), significantly attenuated the hyperalgesia induced by both A6 and LMWH. Finally, to determine if CD44 receptor signaling is involved in a preclinical model of inflammatory pain, we evaluated the effect of A5G27 and HMWH on the mechanical hyperalgesia associated with the inflammation induced by carrageenan. Both A5G27 and HMWH attenuated carrageenan-induced mechanical hyperalgesia. Thus, while LMWH acts at its cognate receptor, CD44, to induce mechanical hyperalgesia, HMWH acts at the same receptor as an antagonist. That the local administration of HMWH or A5G27 inhibits carrageenan-induced hyperalgesia supports the suggestion that carrageenan produces changes in the ECM that contributes to inflammatory pain. These studies define a clinically relevant role for signaling by the hyaluronan receptor, CD44, in increased responsiveness to mechanical stimulation. PMID:26996509

  15. Skeletal muscle beta-receptors and isoproterenol-stimulated vasodilation in canine heart failure

    SciTech Connect

    Frey, M.J.; Lanoce, V.; Molinoff, P.B.; Wilson, J.R. )

    1989-11-01

    To investigate whether heart failure alters beta-adrenergic receptors on skeletal muscle and its associated vasculature, the density of beta-adrenergic receptors, isoproterenol-stimulated adenylate cyclase activity, and coupling of the guanine nucleotide-binding regulatory protein were compared in 18 control dogs and 16 dogs with heart failure induced by 5-8 wk of ventricular pacing at 260 beats/min. Hindlimb vascular responses to isoproterenol were compared in eight controls and eight of the dogs with heart failure. In dogs with heart failure, the density of beta-receptors on skeletal muscle was reduced in both gastrocnemius (control: 50 +/- 5; heart failure: 33 +/- 8 fmol/mg of protein) and semitendinosus muscle (control: 43 +/- 9; heart failure: 27 +/- 9 fmol/mg of protein, both P less than 0.05). Receptor coupling to the ternary complex, as determined by isoproterenol competition curves with and without guanosine 5'-triphosphate (GTP), was unchanged. Isoproterenol-stimulated adenylate cyclase activity was significantly decreased in semitendinosus muscle (control: 52.4 +/- 4.6; heart failure: 36.5 +/- 9.5 pmol.mg-1.min-1; P less than 0.05) and tended to be decreased in gastrocnemius muscle (control: 40.1 +/- 8.5; heart failure: 33.5 +/- 4.5 pmol.mg-1.min-1; P = NS). Isoproterenol-induced hindlimb vasodilation was not significantly different in controls and in dogs with heart failure. These findings suggest that heart failure causes downregulation of skeletal muscle beta-adrenergic receptors, probably due to receptor exposure to elevated catecholamine levels, but does not reduce beta-receptor-mediated vasodilation in muscle.

  16. 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.

  17. Inhibitory effect of PYY on vagally stimulated acid secretion is mediated predominantly by Y1 receptors.

    PubMed

    Lloyd, K C; Grandt, D; Aurang, K; Eysselein, V E; Schimiczek, M; Reeve, J R

    1996-01-01

    Two molecular forms of peptide YY (PYY), PYY-(1--36) and PYY-(3--36), are abundant in rabbit intestine and blood. We have previously shown that PYY-(1--36) (PYYI) activates equipotently Y1 and Y2 receptors and PYY-(3--36) (PYY II) is a highly selective agonist for Y2 receptors. In the present study, we examined the effect of exogenous infusion of PYY on vagally stimulated gastric acid secretion in awake rabbits with chronic gastric fistula. To determine the specific PYY receptor(s) that mediates this effect, we used a highly selective Y1 agonist, Pro34-PYY, a synthetic PYY, and a Y2-selective agonist, PYY II. Vagal stimulation of acid secretion was elicited by an intravenous bolus injection of insulin (0.125 U/kg) 30 min after beginning a 180-min intravenous infusion of either PYY I, PYY II, or [Pro34]-PYY after a 50 micrograms/kg i.v. bolus of atropine followed immediately by a 500 micrograms/kg sc injection. During infusion of 200 pmol.kg 1.h-1 PYY I, acid output was significantly inhibited to 45 +/- 13% of maximum acid output 60 min after injection of insulin. Similarly, acid output during infusion of 200 pmol.kg-1.h-1 [Pro34]-PYY was significantly inhibited to 52 +/- 12% of maximum. In contrast, acid output during infusion of 200 pmol.kg-1.h-1 of PYY II was not significantly inhibited (101 +/- 18% of maximum). Infusion of double the dose (400 pmol.kg-1.h-1) of PYY II resulted in acid inhibition (51 = 15% of maximum), whereas infusion of the same dose did not significantly enhance acid inhibition by infusion of either PYY I or [Pro34]-PYY (28 +/- 11 and 42 +/- 15% of maximum). These results indicate that PYY, acting predominantly at Y1 receptors, is a potent inhibitor of vagally stimulated acid secretion in adult rabbits. PMID:8772509

  18. Synaptic NMDA receptor stimulation activates PP1 by inhibiting its phosphorylation by Cdk5

    PubMed Central

    Hou, Hailong; Sun, Lu; Siddoway, Benjamin A.; Petralia, Ronald S.; Yang, Hongtian; Gu, Hua; Nairn, Angus C.

    2013-01-01

    The serine/threonine protein phosphatase protein phosphatase 1 (PP1) is known to play an important role in learning and memory by mediating local and downstream aspects of synaptic signaling, but how PP1 activity is controlled in different forms of synaptic plasticity remains unknown. We find that synaptic N-methyl-d-aspartate (NMDA) receptor stimulation in neurons leads to activation of PP1 through a mechanism involving inhibitory phosphorylation at Thr320 by Cdk5. Synaptic stimulation led to proteasome-dependent degradation of the Cdk5 regulator p35, inactivation of Cdk5, and increased auto-dephosphorylation of Thr320 of PP1. We also found that neither inhibitor-1 nor calcineurin were involved in the control of PP1 activity in response to synaptic NMDA receptor stimulation. Rather, the PP1 regulatory protein, inhibitor-2, formed a complex with PP1 that was controlled by synaptic stimulation. Finally, we found that inhibitor-2 was critical for the induction of long-term depression in primary neurons. Our work fills a major gap regarding the regulation of PP1 in synaptic plasticity. PMID:24189275

  19. Gastrointestinal hormones stimulate growth of Foregut Neuroendocrine Tumors by transactivating the EGF receptor

    PubMed Central

    Di Florio, Alessia; Sancho, Veronica; Moreno, Paola; Fave, Gianfranco Delle; Jensen, Robert T.

    2012-01-01

    Foregut Neuroendocrine Tumors[NETs] usually pursuit a benign course, but some show aggressive behavior. The treatment of patients with advanced NETs is marginally effective and new approaches are needed. In other tumors, transactivation of the EGF receptor(EGFR) by growth factors, gastrointestinal(GI) hormones and lipids can stimulate growth, which has led to new treatments. Recent studies show a direct correlation between NET malignancy and EGFR expression, EGFR inhibition decreases basal NET growth and an autocrine growth effect exerted by GI hormones, for some NETs. To determine if GI hormones can stimulate NET growth by inducing transactivation of EGFR, we examined the ability of EGF, TGFα and various GI hormones to stimulate growth of the human foregut carcinoid, BON, the somatostatinoma QGP-1 and the rat islet tumor, Rin-14B-cell lines. The EGFR tyrosine-kinase inhibitor, AG1478 strongly inhibited EGF and the GI hormones stimulated cell growth, both in BON and QGP-1 cells. In all the three neuroendocrine cell lines studied, we found EGF, TGFα and the other growth-stimulating GI hormones increased Tyr1068 EGFR phosphorylation. In BON cells, both the GI hormones neurotensin and a bombesin analogue caused a time- and dose-dependent increase in EGFR phosphorylation, which was strongly inhibited by AG1478. Moreover, we found this stimulated phosphorylation was dependent on Src kinases, PKCs, matrix metalloproteinase activation and the generation of reactive oxygen species. These results raise the possibility that disruption of this signaling cascade by either EGFR inhibition alone or combined with receptor antagonists may be a novel therapeutic approach for treatment of foregut NETs/PETs. PMID:23220008

  20. A neomutation of the thyroid-stimulating hormone receptor in a severe neonatal hyperthyroidism.

    PubMed

    de Roux, N; Polak, M; Couet, J; Leger, J; Czernichow, P; Milgrom, E; Misrahi, M

    1996-06-01

    Until recently, neonatal hyperthyroidism has been considered to be related to the transplacental passage of thyroid-stimulating Ig present in the serum of the mother. We report here the case of a newborn who presented with severe hyperthyroidism, diffuse goiter, and important ocular signs (eyelid retraction and possibly proptosis). However, the absence of thyroid pathology in the parents and the lack of antithyroid antibodies in the mother and in the patient led us to suspect a nonimmune aetiology. Direct genomic sequencing of the last exon of the TSH receptor in the patient revealed a T-->C transversion yielding to a Met453-->Thr heterozygous substitution in the second transmembrane domain of the receptor. The mutation was absent in both parents. Eukaryotic expression analysis in COS-7 cells yielded a mutated receptor that produced constitutive activation of adenylate cyclase without enhancement of phospholipase C activity. PMID:8964822

  1. Properties of follicle-stimulating-hormone receptor in cell membranes of bovine testis.

    PubMed Central

    Cheng, K W

    1975-01-01

    A simple method for preparing plasma membranes from bovine testes is described. Bovine testicular receptor has a high affinity and specificity for 125I-labelled human FSH (follicle-stimulating hormone). The specific binding of 125I-labelled human FSH to the plasma membranes is a saturable process with respect to the amounts of receptor protein and FSH added. The association and dissociation of 125I-labelled human FSH are time- and temperature-dependent, and the binding of labelled human FSH to bovine testicular receptor is strong and not readily reversible. Scatchard [Ann. N.Y. Acad. Sci. (1949) 51, 660-672] analysis indicates a dissociation constant, Kd, of 9.8 X10(-11)M, and 5.9 X 10(-14)mol of binding sites/mg of membrane protein. The testicular membrane receptor is heat-labile. Preheating at 40 degrees C for 15 min destroyed 30% of the binding activity. Specific binding is pH-dependent, with an optimum between pH 7.0 and 7.5. Brief exposure to extremes of pH caused irreversible damage to the receptors. The ionic strength of the incubation medium markedly affects the association of 125I-labelled human FSH with its testicular receptor. Various cations at concentrations of 0.1M inhibit almost completely the binding of 125I-labelled human FSH. Nuclectides and steroid hormones at concentrations of 1mM and 5mu/ml respectively have no effect on the binding of FSH to its receptor. Incubation of membranes with and chymotrypsin resulted in an almost complete loss of binding activity, suggesting that protein moieties are essential for the binding of 125I-labelled human FSH. Binding of 125I-labelled human FSH to bovine testicular receptor does not result in destruction or degradation of the hormone. PMID:242318

  2. Direct angiotensin II type 2 receptor stimulation decreases dopamine synthesis in the rat striatum.

    PubMed

    Mertens, Birgit; Vanderheyden, Patrick; Michotte, Yvette; Sarre, Sophie

    2010-06-01

    A relationship between the central renin angiotensin system and the dopaminergic system has been described in the striatum. However, the role of the angiotensin II type 2 (AT(2)) receptor in this interaction has not yet been established. The present study examined the outcome of direct AT(2) receptor stimulation on dopamine (DA) release and synthesis by means of the recently developed nonpeptide AT(2) receptor agonist, compound 21 (C21). The effects of AT(2) receptor agonism on the release of DA and its major metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) and on the activity of tyrosine hydroxylase (TH), the rate-limiting enzyme in the catecholamine biosynthesis, were investigated using in vivo microdialysis. Local administration of C21 (0.1 and 1 microM) resulted in a decrease of the extracellular DOPAC levels, whereas extracellular DA concentrations remained unaltered, suggesting a reduced synthesis of DA. This effect was mediated by the AT(2) receptor since it could be blocked by the AT(2) receptor antagonist PD123319 (1 microM). A similar effect was observed after local striatal (10 nM) as well as systemic (0.3 and 3 mg/kg i.p.) administration of the AT(1) receptor antagonist, candesartan. TH activity as assessed by accumulation of extracellular levels of L-DOPA after inhibition of amino acid decarboxylase with NSD1015, was also reduced after local administration of C21 (0.1 and 1 microM) and candesartan (10 nM). Together, these data suggest that AT(1) and AT(2) receptors in the striatum exert an opposite effect on the modulation of DA synthesis rather than DA release. PMID:20097214

  3. Activation of EP4 receptors contributes to prostaglandin E2-mediated stimulation of renal sensory nerves.

    PubMed

    Kopp, Ulla C; Cicha, Michael Z; Nakamura, Kazuhiro; Nüsing, Rolf M; Smith, Lori A; Hökfelt, Tomas

    2004-12-01

    Induction of cyclooxygenase-2 (COX-2) in the renal pelvic wall increases prostaglandin E(2) (PGE(2)) leading to stimulation of cAMP production, which results in substance P (SP) release and activation of renal mechanosensory nerves. The subtype of PGE receptors involved, EP2 and/or EP4, was studied by immunohistochemistry and renal pelvic administration of agonists and antagonists of EP2 and EP4 receptors. EP4 receptor-like immunoreactivity (LI) was colocalized with calcitonin gene-related peptide (CGRP)-LI in dorsal root ganglia (DRGs) at Th(9)-L(1) and in nerve terminals in the renal pelvic wall. Th(9)-L(1) DRG neurons also contained EP3 receptor-LI and COX-2-LI, each of which was colocalized with CGRP-LI in some neurons. No renal pelvic nerves contained EP3 receptor-LI and only very few nerves COX-2-LI. The EP1/EP2 receptor antagonist AH-6809 (20 microM) had no effect on SP release produced by PGE(2) (0.14 microM) from an isolated rat renal pelvic wall preparation. However, the EP4 receptor antagonist L-161,982 (10 microM) blocked the SP release produced by the EP2/EP4 receptor agonist butaprost (10 microM) 12 +/- 2 vs. 2 +/- 1 and PGE(2), 9 +/- 1 vs. 1 +/- 0 pg/min. The SP release by butaprost and PGE(2) was similarly blocked by the EP4 receptor antagonist AH-23848 (30 microM). In anesthetized rats, the afferent renal nerve activity (ARNA) responses to butaprost 700 +/- 100 and PGE(2).780 +/- 100%.s (area under the curve of ARNA vs. time) were unaffected by renal pelvic perfusion with AH-6809. However, 1 microM L-161,982 and 10 microM AH-23848 blocked the ARNA responses to butaprost by 94 +/- 5 and 78 +/- 10%, respectively, and to PGE(2) by 74 +/- 16 and 74 +/- 11%, respectively. L-161,982 also blocked the ARNA response to increasing renal pelvic pressure 10 mmHg, 85 +/- 5%. In conclusion, PGE(2) increases renal pelvic release of SP and ARNA by activating EP4 receptors on renal sensory nerve fibers. PMID:15292051

  4. Hypotonicity stimulates renal epithelial sodium transport by activating JNK via receptor tyrosine kinases.

    PubMed

    Taruno, Akiyuki; Niisato, Naomi; Marunaka, Yoshinori

    2007-07-01

    We previously reported that hypotonic stress stimulated transepithelial Na(+) transport via a pathway dependent on protein tyrosine kinase (PTK; Niisato N, Van Driessche W, Liu M, Marunaka Y. J Membr Biol 175: 63-77, 2000). However, it is still unknown what type of PTK mediates this stimulation. In the present study, we investigated the role of receptor tyrosine kinase (RTK) in the hypotonic stimulation of Na(+) transport. In renal epithelial A6 cells, we observed inhibitory effects of AG1478 [an inhibitor of the EGF receptor (EGFR)] and AG1296 [an inhibitor of the PDGF receptor (PDGFR)] on both the hypotonic stress-induced stimulation of Na(+) transport and the hypotonic stress-induced ligand-independent activation of EGFR. We further studied whether hypotonic stress activates members of the MAP kinase family, ERK1/2, p38 MAPK, and JNK/SAPK, via an RTK-dependent pathway. The present study indicates that hypotonic stress induced phosphorylation of ERK1/2 and JNK/SAPK, but not p38 MAPK, that the hypotonic stress-induced phosphorylation of ERK1/2 and JNK/SAPK was diminished by coapplication of AG1478 and AG1296, and that only JNK/SAPK was involved in the hypotonic stimulation of Na(+) transport. A further study using cyclohexamide (a protein synthesis inhibitor) suggests that both RTK and JNK/SAPK contributed to the protein synthesis-independent early phase in hypotonic stress-induced Na(+) transport, but not to the protein synthesis-dependent late phase. The present study also suggests involvement of phosphatidylinositol 3-kinase (PI3-kinase) in RTK-JNK/SAPK cascade-mediated Na(+) transport. These observations indicate that 1) hypotonic stress activates JNK/SAPK via RTKs in a ligand-independent pathway, 2) the RTK-JNK/SAPK cascade acts as a mediator of hypotonic stress for stimulation of Na(+) transport, and 3) PI3-kinase is involved in the RTK-JNK/SAPK cascade for the hypotonic stress-induced stimulation of Na(+) transport. PMID:17344192

  5. Angiotensin AT2-receptor stimulation improves survival and neurological outcome after experimental stroke in mice.

    PubMed

    Schwengel, Katja; Namsolleck, Pawel; Lucht, Kristin; Clausen, Bettina H; Lambertsen, Kate L; Valero-Esquitino, Veronica; Thöne-Reineke, Christa; Müller, Susanne; Widdop, Robert E; Denton, Kate M; Horiuchi, Masatsugu; Iwai, Masaru; Boato, Francesco; Dahlöf, Björn; Hallberg, Anders; Unger, Thomas; Steckelings, U Muscha

    2016-08-01

    This study investigated the effect of post-stroke, direct AT2-receptor (AT2R) stimulation with the non-peptide AT2R-agonist compound 21 (C21) on infarct size, survival and neurological outcome after middle cerebral artery occlusion (MCAO) in mice and looked for potential underlying mechanisms. C57/BL6J or AT2R-knockout mice (AT2-KO) underwent MCAO for 30 min followed by reperfusion. Starting 45 min after MCAO, mice were treated once daily for 4 days with either vehicle or C21 (0.03 mg/kg ip). Neurological deficits were scored daily. Infarct volumes were measured 96 h post-stroke by MRI. C21 significantly improved survival after MCAO when compared to vehicle-treated mice. C21 treatment had no impact on infarct size, but significantly attenuated neurological deficits. Expression of brain-derived neurotrophic factor (BDNF), tyrosine kinase receptor B (TrkB) (receptor for BDNF) and growth-associated protein 43 (GAP-43) were significantly increased in the peri-infarct cortex of C21-treated mice when compared to vehicle-treated mice. Furthermore, the number of apoptotic neurons was significantly decreased in the peri-infarct cortex in mice treated with C21 compared to controls. There were no effects of C21 on neurological outcome, infarct size and expression of BDNF or GAP-43 in AT2-KO mice. From these data, it can be concluded that AT2R stimulation attenuates early mortality and neurological deficits after experimental stroke through neuroprotective mechanisms in an AT2R-specific way. Key message • AT2R stimulation after MCAO in mice reduces mortality and neurological deficits.• AT2R stimulation increases BDNF synthesis and protects neurons from apoptosis.• The AT2R-agonist C21 acts protectively when applied post-stroke and peripherally. PMID:26983606

  6. Interaction of urokinase with specific receptors stimulates mobilization of bovine adrenal capillary endothelial cells

    SciTech Connect

    Fibbi, G.; Ziche, M.; Morbidelli, L. ); Magnelli, L.; Del Rosso, M. )

    1988-12-01

    On the basis of {sup 125}I-labeled plasminogen activator binding analysis the authors have found that bovine adrenal capillary endothelial cells have specific receptors for human urinary-type plasminogen activator on the cell membrane. Each cell exposes about 37,000 free receptors with a K{sub d} of 0.8958{times}10{sup {minus}12} M. A monoclonal antibody against the 17,500 proteolytic fragment of the A chain of the plasminogen activator, not containing the catalytic site of the enzyme, impaired the specific binding, thus suggesting the involvement of a sequence present on the A chain in the interaction with the receptor, as previously shown in other cell model systems. Both the native molecule and the A chain are able to stimulate endothelial cell motility in the Boyden chamber, when used at nanomolar concentrations. The use of the same monoclonal antibody that can inhibit ligand-receptor interaction can impair the plasminogen activator and A-chain-induced endothelial cell motility, suggesting that under the conditions used in this in vitro model system, the motility of bovine adrenal capillary endothelial cells depends on the specific interaction of the ligand with free receptors on the surface of endothelial cells.

  7. Dissociation between neural and vascular responses to sympathetic stimulation : contribution of local adrenergic receptor function

    NASA Technical Reports Server (NTRS)

    Jacob, G.; Costa, F.; Shannon, J.; Robertson, D.; Biaggioni, I.

    2000-01-01

    Sympathetic activation produced by various stimuli, eg, mental stress or handgrip, evokes regional vascular responses that are often nonhomogeneous. This phenomenon is believed to be the consequence of the recruitment of differential central neural pathways or of a sympathetically mediated vasodilation. The purpose of this study was to determine whether a similar heterogeneous response occurs with cold pressor stimulation and to test the hypothesis that local differences in adrenergic receptor function could be in part responsible for this diversity. In 8 healthy subjects, local norepinephrine spillover and blood flow were measured in arms and legs at baseline and during sympathetic stimulation induced by baroreflex mechanisms (nitroprusside infusion) or cold pressor stimulation. At baseline, legs had higher vascular resistance (27+/-5 versus 17+/-2 U, P=0.05) despite lower norepinephrine spillover (0.28+/-0.04 versus 0.4+/-0.05 mg. min(-1). dL(-1), P=0.03). Norepinephrine spillover increased similarly in both arms and legs during nitroprusside infusion and cold pressor stimulation. On the other hand, during cold stimulation, vascular resistance increased in arms but not in legs (20+/-9% versus -7+/-4%, P=0.03). Increasing doses of isoproterenol and phenylephrine were infused intra-arterially in arms and legs to estimate beta-mediated vasodilation and alpha-induced vasoconstriction, respectively. beta-Mediated vasodilation was significantly lower in legs compared with arms. Thus, we report a dissociation between norepinephrine spillover and vascular responses to cold stress in lower limbs characterized by a paradoxical decrease in local resistance despite increases in sympathetic activity. The differences observed in adrenergic receptor responses cannot explain this phenomenon.

  8. Stimulation of pulmonary rapidly adapting receptors by inhaled wood smoke in rats.

    PubMed

    Lai, C J; Kou, Y R

    1998-04-15

    1. The stimulation of pulmonary rapidly adapting receptors (RARs) by wood smoke was investigated. Impulses from seventy RARs were recorded in fifty-nine anaesthetized, open-chest and artificially ventilated rats; responses to delivery of 6 ml of wood smoke into the lungs were studied in sixty-one receptors whereas responses to histamine (10 or 100 microg kg-1, i.v.) were studied in the other nine. 2. Delivery of wood smoke stimulated fifty-two of the sixty-one RARs studied. When stimulated, an intense burst of discharge was evoked within 1 or 2 s of smoke delivery. This increased activity quickly peaked in 1-3 s (Delta = 15.8 +/- 1.6 impulses s-1; n = 61; mean +/- s.e.m.), then declined and yet remained at a level higher than the baseline activity. The mean duration of the stimulation was 25.1 +/- 2.7 s. In contrast, smoke delivery did not affect tracheal pressure. 3. Peak responses of RARs to wood smoke were partially reduced by removal of smoke particulates and were largely attenuated by pretreatment with dimethylthiourea (DMTU, a hydroxyl radical scavenger), indomethacin (Indo, a cyclo-oxygenase inhibitor), or both DMTU and Indo (DMTU + Indo). Conversely, the peak responses of RARs were not significantly affected by pretreatment with isoprenaline (a bronchodilator) or vehicle for these chemicals. Additionally, pretreatment with DMTU, Indo, or DMTU + Indo did not significantly alter the RAR sensitivity to mechanical stimulation (constant-pressure lung inflation; 20 cmH2O). 4. Of the nine RARs tested, six were stimulated by histamine and their sensitivity to this chemical irritant was not altered by pretreatment with DMTU + Indo. 5. The results suggest that both the particulates and gas phases are responsible for, and both the hydroxyl radical and cyclo-oxygenase products are involved in, the stimulation of RARs by wood smoke. Furthermore, changes in lung mechanics following smoke delivery are not the cause of this afferent stimulation. PMID:9508820

  9. The therapeutic potential of erythropoiesis-stimulating agents for tissue protection: a tale of two receptors.

    PubMed

    Brines, Michael

    2010-01-01

    Erythropoietin (EPO) is a well-known therapeutic protein employed widely in the treatment of anemia. Over the past decade, abundant evidence has shown that in addition to its systemic role in the regulation of plasma pO(2) by modulating erythrocyte numbers, EPO is also a cytoprotective molecule made locally in response to injury or metabolic stress. Many studies have shown beneficial effects of EPO administration in reducing damage caused by ischemia-reperfusion, trauma, cytotoxicity, infection and inflammation in a variety of organs and tissues. Notably, the receptor mediating the nonerythropoietic effects of EPO differs from the one responsible for hematopoiesis. The tissue-protective receptor exhibits a lower affinity for EPO and is a heteromer consisting of EPO receptor monomers in association with the common receptor that is also employed by granulocyte macrophage colony-stimulating factor, interleukin 3, and interleukin 5. This heteromeric receptor is expressed immediately following injury, whereas EPO production is delayed. Thus, early administration of EPO can dramatically reduce the deleterious components of the local inflammatory cascade. However, a high dose of EPO is required and this also stimulates the bone marrow to produce highly reactive platelets and activates the vascular endothelium into a prothrombotic state. To circumvent these undesirable effects, the EPO molecule has been successfully altered to selectively eliminate erythropoietic and prothrombotic potencies, while preserving tissue-protective activities. Very recently, small peptide mimetics have been developed that recapitulate the tissue-protective activities of EPO. Nonerythropoietic tissue-protective molecules hold high promise in a wide variety of acute and chronic diseases. PMID:20093809

  10. Studies on the structure of the follicle-stimulating hormone receptor using photoaffinity labeling procedures

    SciTech Connect

    Smith, R.A.

    1985-01-01

    The general objective of this project was to study the structure of the follicle stimulating hormone (FSH) receptor using affinity labeling methods. A low density fraction derived from homogenates of bovine testis was found to contain high affinity and low capacity receptors specific for FSH. Electron microscopic examination of the fraction revealed structure resembling multilamellar membranous vesicles (MV). For photoaffinity labeling of the FSH receptors in MV, an azidobenzoyl-/sup 125/I-analog of human FSH was prepared (/sup 125/I-AB-hFSH) and binding of specific FSH receptors was studied. /sup 125/I-AB-hFSH binding of receptors was inhibited in a dose dependent manner by unlabeled hFSH, and binding was not prevented by structurally-related human chorionic gonadotropin (hCG). The formation of photocrosslinked protein of relative molecular mass (M/sub r/) 54,000, 64,000, 76,000, 84,000, 97,000 and 116,000 was found to be inhibited by unlabeled hFSH in a dose related manner, and to be dependent on photoactivation of the FSH derivative. The interpretation of the photoaffinity labeling experiments was that three proteins associated with the FSH receptor were photoaffinity labeled. Analysis by indirect means suggested that the three proteins were assembled to form oligomeric complexes, and based on the intensities and composition of the oligomeric species, spatial relationships of the polypeptides with respect to each other on the membrane surface were deduced. The results of photoaffinity labeling suggest the FSH receptor is composed of three subunits of M/sub r/ 38,000, 48,000, and 81,000 and exists in the membrane in part as a M/sub r/ 330,000 dimer.

  11. Preselection Thymocytes Are More Sensitive to T Cell Receptor Stimulation Than Mature T Cells

    PubMed Central

    Davey, Gayle M.; Schober, Sonya L.; Endrizzi, Bart T.; Dutcher, Angela K.; Jameson, Stephen C.; Hogquist, Kristin A.

    1998-01-01

    During T cell development, thymocytes which are tolerant to self-peptides but reactive to foreign peptides are selected. The current model for thymocyte selection proposes that self-peptide–major histocompatibility complex (MHC) complexes that bind the T cell receptor with low affinity will promote positive selection while those with high affinity will result in negative selection. Upon thymocyte maturation, such low affinity self-peptide–MHC ligands no longer provoke a response, but foreign peptides can incidentally be high affinity ligands and can therefore stimulate T cells. For this model to work, thymocytes must be more sensitive to ligand than mature T cells. Contrary to this expectation, several groups have shown that thymocytes are less responsive than mature T cells to anti-T cell receptor for antigen (TCR)/CD3 mAb stimulation. Additionally, the lower TCR levels on thymocytes, compared with T cells, would potentially correlate with decreased thymocyte sensitivity. Here we compared preselection thymocytes and mature T cells for early activation events in response to peptide–MHC ligands. Remarkably, the preselection thymocytes were more responsive than mature T cells when stimulated with low affinity peptide variants, while both populations responded equally well to the antigenic peptide. This directly demonstrates the increased sensitivity of thymocytes compared with T cells for TCR engagement by peptide–MHC complexes. PMID:9815264

  12. Thrombin stimulates fibroblast procollagen production via proteolytic activation of protease-activated receptor 1.

    PubMed Central

    Chambers, R C; Dabbagh, K; McAnulty, R J; Gray, A J; Blanc-Brude, O P; Laurent, G J

    1998-01-01

    Thrombin is a multifunctional serine protease that has a crucial role in blood coagulation. It is also a potent mesenchymal cell mitogen and chemoattractant and might therefore have an important role in the recruitment and local proliferation of mesenchymal cells at sites of tissue injury. We hypothesized that thrombin might also affect the deposition of connective tissue proteins at these sites by directly stimulating fibroblast procollagen production. To address this hypothesis, the effect of thrombin on procollagen production and gene expression by human foetal lung fibroblasts was assessed over 48 h. Thrombin stimulated procollagen production at concentrations of 1 nM and above, with maximal increases of between 60% and 117% at 10 nM thrombin. These effects of thrombin were, at least in part, due to increased steady-state levels of alpha1(I) procollagen mRNA. They could furthermore be reproduced with thrombin receptor-activating peptides for the protease-activated receptor 1 (PAR-1) and were completely abolished when thrombin was rendered proteolytically inactive with the specific inhibitors d-Phe-Pro-ArgCH2Cl and hirudin, indicating that thrombin is mediating these effects via the proteolytic activation of PAR-1. These results suggest that thrombin might influence the deposition of connective tissue proteins during normal wound healing and the development of tissue fibrosis by stimulating fibroblast procollagen production. PMID:9639571

  13. 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.

  14. 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

  15. Wedelolactone induces growth of breast cancer cells by stimulation of estrogen receptor signalling.

    PubMed

    Nehybova, Tereza; Smarda, Jan; Daniel, Lukas; Brezovsky, Jan; Benes, Petr

    2015-08-01

    Wedelolactone, a plant coumestan, was shown to act as anti-cancer agent for breast and prostate carcinomas in vitro and in vivo targeting multiple cellular proteins including androgen receptors, 5-lipoxygenase and topoisomerase IIα. It is cytotoxic to breast, prostate, pituitary and myeloma cancer cell lines in vitro at μM concentrations. In this study, however, a novel biological activity of nM dose of wedelolactone was demonstrated. Wedelolactone acts as agonist of estrogen receptors (ER) α and β as demonstrated by transactivation of estrogen response element (ERE) in cells transiently expressing either ERα or ERβ and by molecular docking of this coumestan into ligand binding pocket of both ERα and ERβ. In breast cancer cells, wedelolactone stimulates growth of estrogen receptor-positive cells, expression of estrogen-responsive genes and activates rapid non-genomic estrogen signalling. All these effects can be inhibited by pretreatment with pure ER antagonist ICI 182,780 and they are not observed in ER-negative breast cancer cells. We conclude that wedelolactone acts as phytoestrogen in breast cancer cells by stimulating ER genomic and non-genomic signalling pathways. PMID:25934092

  16. A model for the stimulation of taste receptor cells by salt.

    PubMed Central

    DeSimone, J A; Price, S

    1976-01-01

    A taste cell mucosal surface is regarded as a planar region containing bound anionic sites and openings to ionic channels. It is assumed that the bulk aqueous properties of the exterior phase are not continuous with the surface but terminate at a plane near the surface. The region between the (Stern) plane and the membrane is regarded as having a lower dielectric constant than bulk water. This fact admits the possibility of ion pair formation between fixed sites and mobile cations. Mobile ion pairs entering the region may also bind to a fixed anionic site. Thus, it is assumed that mobile cations and ion pairs are potential determining species at the surface. Binding cations neutralizes surface charges, whereas binding mobile ion pairs does not. This competition accounts for the observed anion effect on stimulation of tast receptors by sodium salts. The potential profile is constructed by superimposing the phase boundary potentials with an ionic diffusion potential across the membrane. The model accounts for the anion effect on receptor potential, pH effects, the reversal of polarity when cells are treated with FeCl3, and the so-called "water reponse," depolarization of the taste cell upon dilution of the stimulant solution below a critical lower limit. The proposed model does not require both bound cationic and anionic receptors, and further suggests that limited access to a Stern-like region continuous with membrane channels may generally serve to control transport of ions. PMID:938727

  17. Immunoglobulin-like domain containing receptor 1 mediates fat-stimulated cholecystokinin secretion.

    PubMed

    Chandra, Rashmi; Wang, Yu; Shahid, Rafiq A; Vigna, Steven R; Freedman, Neil J; Liddle, Rodger A

    2013-08-01

    Cholecystokinin (CCK) is a satiety hormone produced by discrete enteroendocrine cells scattered among absorptive cells of the small intestine. CCK is released into blood following a meal; however, the mechanisms inducing hormone secretion are largely unknown. Ingested fat is the major stimulant of CCK secretion. We recently identified a novel member of the lipoprotein remnant receptor family known as immunoglobulin-like domain containing receptor 1 (ILDR1) in intestinal CCK cells and postulated that this receptor conveyed the signal for fat-stimulated CCK secretion. In the intestine, ILDR1 is expressed exclusively in CCK cells. Orogastric administration of fatty acids elevated blood levels of CCK in wild-type mice but not Ildr1-deficient mice, although the CCK secretory response to trypsin inhibitor was retained. The uptake of fluorescently labeled lipoproteins in ILDR1-transfected CHO cells and release of CCK from isolated intestinal cells required a unique combination of fatty acid plus HDL. CCK secretion secondary to ILDR1 activation was associated with increased [Ca2+]i, consistent with regulated hormone release. These findings demonstrate that ILDR1 regulates CCK release through a mechanism dependent on fatty acids and lipoproteins and that absorbed fatty acids regulate gastrointestinal hormone secretion. PMID:23863714

  18. Interaction of the Clostridium difficile Binary Toxin CDT and Its Host Cell Receptor, Lipolysis-stimulated Lipoprotein Receptor (LSR)*

    PubMed Central

    Hemmasi, Sarah; Czulkies, Bernd A.; Schorch, Björn; Veit, Antonia; Aktories, Klaus; Papatheodorou, Panagiotis

    2015-01-01

    CDT (Clostridium difficile transferase) is a binary, actin ADP-ribosylating toxin frequently associated with hypervirulent strains of the human enteric pathogen C. difficile, the most serious cause of antibiotic-associated diarrhea and pseudomembranous colitis. CDT leads to the collapse of the actin cytoskeleton and, eventually, to cell death. Low doses of CDT result in the formation of microtubule-based protrusions on the cell surface that increase the adherence and colonization of C. difficile. The lipolysis-stimulated lipoprotein receptor (LSR) is the host cell receptor for CDT, and our aim was to gain a deeper insight into the interplay between both proteins. We show that CDT interacts with the extracellular, Ig-like domain of LSR with an affinity in the nanomolar range. We identified LSR splice variants in the colon carcinoma cell line HCT116 and disrupted the LSR gene in these cells by applying the CRISPR-Cas9 technology. LSR truncations ectopically expressed in LSR knock-out cells indicated that intracellular parts of LSR are not essential for plasma membrane targeting of the receptor and cellular uptake of CDT. By generating a series of N- and C-terminal truncations of the binding component of CDT (CDTb), we found that amino acids 757–866 of CDTb are sufficient for binding to LSR. With a transposon-based, random mutagenesis approach, we identified potential LSR-interacting epitopes in CDTb. This study increases our understanding about the interaction between CDT and its receptor LSR, which is key to the development of anti-toxin strategies for preventing cell entry of the toxin. PMID:25882847

  19. Vasopressin receptors in the brain, liver and kidney of rats following osmotic stimulation.

    PubMed

    Landgraf, R; Szot, P; Dorsa, D M

    1991-03-29

    The binding site concentration (Bmax) and equilibrium dissociation constant (Kd) for [3H]-arginine vasopressin (AVP) binding sites were measured in limbic brain areas (septum, dorsal hippocampus, amygdala) and liver and kidney of control and osmotically stimulated male Wistar rats. Membrane binding was performed in these five areas 30, 60 and 180 min following intraperitoneal injection of hypertonic saline. This paradigm resulted in no significant change in binding characteristics in the septum, dorsal hippocampus, amygdala and liver from control treated rats. In contrast, the kidney Bmax was significantly reduced 60 min following osmotic stimulation, with no effect on affinity. These results also suggest that AVP receptors in the CNS are relatively resistant to regulatory effects of an acute AVP exposure. PMID:1828184

  20. Leptospiral lipopolysaccharide stimulates the expression of toll-like receptor 2 and cytokines in pig fibroblasts.

    PubMed

    Guo, Yijie; Fukuda, Tomokazu; Donai, Kenichiro; Kuroda, Kengo; Masuda, Mizuki; Nakamura, Shuichi; Yoneyama, Hiroshi; Isogai, Emiko

    2015-02-01

    Pigs throughout the world are afflicted with leptospirosis, causing serious economic losses and potential hazards to human health. Although it has been known that leptospiral lipopolysaccharide (L-LPS) is involved in an immunological reaction between an antigen and a host cell, little is known about how the immune system of pigs can respond to L-LPS. Here, we stimulated pig fibroblasts by L-LPS and then quantitatively measured gene and protein expression levels of two toll-like receptors (TLRs), TLR2 and TLR4, by real-time PCR and Western blotting. As a result, expression of TLR2 was found to be significantly up-regulated within 24 h after L-LPS stimulation whereas induction of TLR4 expression was relatively weak. We also revealed that of myeloid differentiation primary response gene 88 (MyD88), interleukin 6 (IL-6) and IL-8 gene expressions were markedly up-regulated by L-LPS stimulation. These results may suggest that the pig cell can activate TLR2 rather than TLR4 by L-LPS stimulation, thereby inducing expression of cytokines. PMID:25039909

  1. Caloric restriction stimulates autophagy in rat cortical neurons through neuropeptide Y and ghrelin receptors activation

    PubMed Central

    Carmo-Silva, Sara; Botelho, Mariana; de Almeida, Luís Pereira; Cavadas, Cláudia

    2016-01-01

    Caloric restriction is an anti-aging intervention known to extend lifespan in several experimental models, at least in part, by stimulating autophagy. Caloric restriction increases neuropeptide Y (NPY) in the hypothalamus and plasma ghrelin, a peripheral gut hormone that acts in hypothalamus to modulate energy homeostasis. NPY and ghrelin have been shown to be neuroprotective in different brain areas and to induce several physiological modifications similar to those induced by caloric restriction. However, the effect of NPY and ghrelin in autophagy in cortical neurons is currently not known. Using a cell culture of rat cortical neurons we investigate the involvement of NPY and ghrelin in caloric restriction-induced autophagy. We observed that a caloric restriction mimetic cell culture medium stimulates autophagy in rat cortical neurons and NPY or ghrelin receptor antagonists blocked this effect. On the other hand, exogenous NPY or ghrelin stimulate autophagy in rat cortical neurons. Moreover, NPY mediates the stimulatory effect of ghrelin on autophagy in rat cortical neurons. Since autophagy impairment occurs in aging and age-related neurodegenerative diseases, NPY and ghrelin synergistic effect on autophagy stimulation may suggest a new strategy to delay aging process. PMID:27441412

  2. Caloric restriction stimulates autophagy in rat cortical neurons through neuropeptide Y and ghrelin receptors activation.

    PubMed

    Ferreira-Marques, Marisa; Aveleira, Célia A; Carmo-Silva, Sara; Botelho, Mariana; Pereira de Almeida, Luís; Cavadas, Cláudia

    2016-07-01

    Caloric restriction is an anti-aging intervention known to extend lifespan in several experimental models, at least in part, by stimulating autophagy. Caloric restriction increases neuropeptide Y (NPY) in the hypothalamus and plasma ghrelin, a peripheral gut hormone that acts in hypothalamus to modulate energy homeostasis. NPY and ghrelin have been shown to be neuroprotective in different brain areas and to induce several physiological modifications similar to those induced by caloric restriction. However, the effect of NPY and ghrelin in autophagy in cortical neurons is currently not known. Using a cell culture of rat cortical neurons we investigate the involvement of NPY and ghrelin in caloric restriction-induced autophagy. We observed that a caloric restriction mimetic cell culture medium stimulates autophagy in rat cortical neurons and NPY or ghrelin receptor antagonists blocked this effect. On the other hand, exogenous NPY or ghrelin stimulate autophagy in rat cortical neurons. Moreover, NPY mediates the stimulatory effect of ghrelin on autophagy in rat cortical neurons. Since autophagy impairment occurs in aging and age-related neurodegenerative diseases, NPY and ghrelin synergistic effect on autophagy stimulation may suggest a new strategy to delay aging process. PMID:27441412

  3. Functional interaction between mutations in the granulocyte colony-stimulating factor receptor in severe congenital neutropenia.

    PubMed

    Ward, Alister C; Gits, Judith; Majeed, Fidel; Aprikyan, Andrew A; Lewis, Rowena S; O'Sullivan, Lynda A; Freedman, Melvin; Shigdar, Sarah; Touw, Ivo P; Dale, David C; Dror, Yigal

    2008-08-01

    Most severe congenital neutropenia (SCN) cases possess constitutive neutrophil elastase mutations; a smaller cohort has acquired mutations truncating the granulocyte colony-stimulating factor receptor (G-CSF-R). We have described a case with constitutive extracellular G-CSF-R mutation hyporesponsive to ligand. Here we report two independent acquired G-CSF-R truncation mutations and a novel constitutive neutrophil elastase mutation in this patient. Co-expression of a truncated receptor chain restored STAT5 signalling responses of the extracellular G-CSF-R mutant, while constitutively-active STAT5 enhanced its proliferative capacity. These data add to our knowledge of SCN and further highlight the importance of STAT5 in mediating proliferative responses to G-CSF. PMID:18513286

  4. Stimulation of peripheral cholinergic nerves by glutamate indicates a new peripheral glutamate receptor.

    PubMed

    Aas, P; Tansø, R; Fonnum, F

    1989-05-01

    The bronchial smooth muscle of the rat was examined for contractile responses to excitatory amino acids. The nerve-mediated contraction induced by electrical field stimulation was enhanced by exogenous L-glutamate (L-Glu). The apparent affinity (ED50) of L-Glu was 3.5 +/- 0.1 mM. Both tetrodotoxin and hemicholinium-3 completely abolished the electrical field-induced contraction and therefore the potentiation by L-Glu, which indicates that L-Glu has a prejunctional effect. Concentrations of L-Glu higher than 22 mM inhibited the electrical field-induced contractions and enhanced the tonus of the smooth muscle by postjunctional stimulation. The ED50 of exogenous ACh was not altered by L-Glu. High concentrations (62 mM) of L-Glu increased the intrinsic activity (alpha) of ACh, indicating a postjunctional potentiation of ACh-induced contractions. L-Glu did not inhibit the activity of acetylcholinesterase, therefore the postjunctional potentiation was not due to ACh accumulation. Inhibition of the electrical field-induced contraction was seen with high concentrations of D-Glu, L-aspartate (L-Asp), L-alpha-amino adipate and ibotenate. Neither glutamate diethyl ester nor 2-amino-5-phosphonovalerate had any inhibitory effects on the L-Glu- and L-Asp-induced alterations of the electrical field-stimulated contraction or on the L-Glu-enhanced tonus of the bronchial smooth muscle. Kainate, N-methyl-D-aspartate, quisqualate and N-acetyl-aspartyl-glutamate had only minor transient potentiating effects on the electrical field-induced contraction. The results provide evidence for a L-Glu receptor in rat bronchi that has a different specificity for glutamate agonists and antagonists than the L-Glu receptor described in the CNS. The receptor seems to be located prejunctionally and enhances nerve-mediated responses and thereby stimulates the bronchial smooth muscle to contract. The possible involvement of this type of receptor in the 'Chinese restaurant syndrome' is discussed. PMID

  5. The prolactin receptor mediates HOXA1-stimulated oncogenicity in mammary carcinoma cells.

    PubMed

    Hou, Lin; Xu, Bing; Mohankumar, Kumarasamypet M; Goffin, Vincent; Perry, Jo K; Lobie, Peter E; Liu, Dong-Xu

    2012-12-01

    The HOX genes are a highly conserved subgroup of homeodomain-containing transcription factors that are crucial to normal development. Forced expression of HOXA1 results in oncogenic transformation of immortalized human mammary cells with aggressive tumour formation in vivo. Microarray analysis identified that the prolactin receptor (PRLR) was significantly upregulated by forced expression of HOXA1 in mammary carcinoma cells. To determine prolactin (PRL) involvement in HOXA1‑induced oncogenicity in mammary carcinoma cells (MCF-7), we examined the effect of human prolactin (hPRL)-initiated PRLR signal transduction on changes in cellular behaviour mediated by HOXA1. Forced expression of HOXA1 in MCF-7 cells increased PRLR mRNA and protein expression. Forced expression of HOXA1 also enhanced hPRL-stimulated phosphorylation of both STAT5A/B and p44/42 MAPK, and increased subsequent transcriptional activity of STAT5A and STAT5B, and Elk-1 and Sap1a, respectively. Moreover, forced expression of HOXA1 in MCF-7 cells enhanced the hPRL‑stimulated increase in total cell number as a consequence of enhanced cell proliferation and cell survival, and also enhanced hPRL-stimulated anchorage-independent growth in soft agar. Increased anchorage-independent growth was attenuated by the PRLR antagonist ∆1-9-G129R‑hPRL. In conclusion, we have demonstrated that HOXA1 increases expression of the cell surface receptor PRLR and enhances PRLR-mediated signal transduction. Thus, the PRLR is one mediator of HOXA1‑stimulated oncogenicity in mammary carcinoma cells. PMID:23064471

  6. A selective TSH receptor antagonist inhibits stimulation of thyroid function in female mice.

    PubMed

    Neumann, Susanne; Nir, Eshel A; Eliseeva, Elena; Huang, Wenwei; Marugan, Juan; Xiao, Jingbo; Dulcey, Andrés E; Gershengorn, Marvin C

    2014-01-01

    Because the TSH receptor (TSHR) plays an important role in the pathogenesis of thyroid disease, a TSHR antagonist could be a novel treatment. We attempted to develop a small molecule, drug-like antagonist of TSHR signaling that is selective and active in vivo. We synthesized NCGC00242364 (ANTAG3) by chemical modification of a previously reported TSHR antagonist. We tested its potency, efficacy, and selectivity in a model cell system in vitro by measuring its activity to inhibit stimulation of cAMP production stimulated by TSH, LH, or FSH. We tested the in vivo activity of ANTAG3 by measuring its effects to lower serum free T4 and thyroid gene expression in female BALB/c mice continuously treated with ANTAG3 for 3 days and given low doses of TRH continuously or stimulated by a single administration of a monoclonal thyroid-stimulating antibody M22. ANTAG3 was selective for TSHR inhibition; half-maximal inhibitory doses were 2.1 μM for TSHR and greater than 30 μM for LH and FSH receptors. In mice treated with TRH, ANTAG3 lowered serum free T4 by 44% and lowered mRNAs for sodium-iodide cotransporter and thyroperoxidase by 75% and 83%, respectively. In mice given M22, ANTAG3 lowered serum free T4 by 38% and lowered mRNAs for sodium-iodide cotransporter and thyroperoxidase by 73% and 40%, respectively. In conclusion, we developed a selective TSHR antagonist that is effective in vivo in mice. This is the first report of a small-molecule TSHR antagonist active in vivo and may lead to a drug to treat Graves' disease. PMID:24169564

  7. Extracellular Nucleotides Inhibit Insulin Receptor Signaling, Stimulate Autophagy and Control Lipoprotein Secretion

    PubMed Central

    Chatterjee, Cynthia; Sparks, Daniel L.

    2012-01-01

    Hyperglycemia is associated with abnormal plasma lipoprotein metabolism and with an elevation in circulating nucleotide levels. We evaluated how extracellular nucleotides may act to perturb hepatic lipoprotein secretion. Adenosine diphosphate (ADP) (>10 µM) acts like a proteasomal inhibitor to stimulate apoB100 secretion and inhibit apoA-I secretion from human liver cells at 4 h and 24 h. ADP blocks apoA-I secretion by stimulating autophagy. The nucleotide increases cellular levels of the autophagosome marker, LC3-II, and increases co-localization of LC3 with apoA-I in punctate autophagosomes. ADP affects autophagy and apoA-I secretion through P2Y13. Overexpression of P2Y13 increases cellular LC3-II levels by ∼50% and blocks induction of apoA-I secretion. Conversely, a siRNA-induced reduction in P2Y13 protein expression of 50% causes a similar reduction in cellular LC3-II levels and a 3-fold stimulation in apoA-I secretion. P2Y13 gene silencing blocks the effects of ADP on autophagy and apoA-I secretion. A reduction in P2Y13 expression suppresses ERK1/2 phosphorylation, increases the phosphorylation of IR-β and protein kinase B (Akt) >3-fold, and blocks the inhibition of Akt phosphorylation by TNFα and ADP. Conversely, increasing P2Y13 expression significantly inhibits insulin-induced phosphorylation of insulin receptor (IR-β) and Akt, similar to that observed after treatment with ADP. Nucleotides therefore act through P2Y13, ERK1/2 and insulin receptor signaling to stimulate autophagy and affect hepatic lipoprotein secretion. PMID:22590634

  8. Misfolding Ectodomain Mutations of the Lutropin Receptor Increase Efficacy of Hormone Stimulation.

    PubMed

    Charmandari, E; Guan, R; Zhang, M; Silveira, L G; Fan, Q R; Chrousos, G P; Sertedaki, A C; Latronico, A C; Segaloff, D L

    2016-01-01

    We demonstrate 2 novel mutations of the LHCGR, each homozygous, in a 46,XY patient with severe Leydig cell hypoplasia. One is a mutation in the signal peptide (p.Gln18_Leu19ins9; referred to here as SP) that results in an alteration of the coding sequence of the N terminus of the mature mutant receptor. The other mutation (p.G71R) is also within the ectodomain. Similar to many other inactivating mutations, the cell surface expression of recombinant human LHR(SP,G71R) is greatly reduced due to intracellular retention. However, we made the unusual discovery that the intrinsic efficacy for agonist-stimulated cAMP in the reduced numbers of receptors on the cell surface was greatly increased relative to the same low number of cell surface wild-type receptor. Remarkably, this appears to be a general attribute of misfolding mutations in the ectodomains, but not serpentine domains, of the gonadotropin receptors. These findings suggest that there must be a common, shared mechanism by which disparate mutations in the ectodomain that cause misfolding and therefore reduced cell surface expression concomitantly confer increased agonist efficacy to those receptor mutants on the cell surface. Our data further suggest that, due to their increased agonist efficacy, extremely small changes in cell surface expression of misfolded ectodomain mutants cause larger than expected alterations in the cellular response to agonist. Therefore, for inactivating LHCGR mutations causing ectodomain misfolding, the numbers of cell surface mutant receptors on fetal Leydig cells of 46,XY individuals exert a more exquisite effect on the relative severity of the clinical phenotypes than already appreciated. PMID:26554443

  9. PGE2 Signaling Through the EP4 Receptor on Fibroblasts Upregulates RANKL and Stimulates Osteolysis

    PubMed Central

    Tsutsumi, Ryosuke; Xie, Chao; Wei, Xiaochao; Zhang, Minjie; Zhang, Xinping; Flick, Lisa M.; Schwarz, Edward M.; O'Keefe, Regis J.

    2009-01-01

    Periprosthetic osteolysis is the most common cause of aseptic loosening in total joint arthroplasty. The role of inflammatory mediators such as prostaglandin E2 (PGE2) and osteoclast promoting factors including RANKL in the pathogenesis of osteolysis has been well characterized. However, the PGE2 receptor (EP1, EP2, or EP4), and cell type in which it is expressed, which is responsible for PGE2 induction of RANKL during wear debris–induced osteolysis, has yet to be elucidated. To address this, we used mice genetically deficient in these EP receptors to assess PGE2 and wear debris responses in vitro and in vivo. Wear debris–induced osteolysis and RANKL expression were observed at similar levels in WT, EP1−/−, and EP2−/− mice, indicating that these receptors do not mediate PGE2 signals in this process. A conditional knockout approach was used to eliminate EP4 expression in FSP1+ fibroblasts that are the predominant source of RANKL. In the absence of EP4, fibroblasts do not express RANKL after stimulation with particles or PGE2, nor do they exhibit high levels of osteoclasts and osteolysis. These results show that periprosthetic fibroblasts are important mediators of osteolysis through the expression of RANKL, which is induced after PGE2 signaling through the EP4 receptor. PMID:19419302

  10. Effects of serotonin 2A/1A receptor stimulation on social exclusion processing.

    PubMed

    Preller, Katrin H; Pokorny, Thomas; Hock, Andreas; Kraehenmann, Rainer; Stämpfli, Philipp; Seifritz, Erich; Scheidegger, Milan; Vollenweider, Franz X

    2016-05-01

    Social ties are crucial for physical and mental health. However, psychiatric patients frequently encounter social rejection. Moreover, an increased reactivity to social exclusion influences the development, progression, and treatment of various psychiatric disorders. Nevertheless, the neuromodulatory substrates of rejection experiences are largely unknown. The preferential serotonin (5-HT) 2A/1A receptor agonist, psilocybin (Psi), reduces the processing of negative stimuli, but whether 5-HT2A/1A receptor stimulation modulates the processing of negative social interactions remains unclear. Therefore, this double-blind, randomized, counterbalanced, cross-over study assessed the neural response to social exclusion after the acute administration of Psi (0.215 mg/kg) or placebo (Pla) in 21 healthy volunteers by using functional magnetic resonance imaging (fMRI) and resting-state magnetic resonance spectroscopy (MRS). Participants reported a reduced feeling of social exclusion after Psi vs. Pla administration, and the neural response to social exclusion was decreased in the dorsal anterior cingulate cortex (dACC) and the middle frontal gyrus, key regions for social pain processing. The reduced neural response in the dACC was significantly correlated with Psi-induced changes in self-processing and decreased aspartate (Asp) content. In conclusion, 5-HT2A/1A receptor stimulation with psilocybin seems to reduce social pain processing in association with changes in self-experience. These findings may be relevant to the normalization of negative social interaction processing in psychiatric disorders characterized by increased rejection sensitivity. The current results also emphasize the importance of 5-HT2A/1A receptor subtypes and the Asp system in the control of social functioning, and as prospective targets in the treatment of sociocognitive impairments in psychiatric illnesses. PMID:27091970

  11. Protection against ventricular fibrillation via cholinergic receptor stimulation and the generation of nitric oxide

    PubMed Central

    Kalla, Manish; Chotalia, Minesh; Coughlan, Charles; Hao, Guoliang; Crabtree, Mark J.; Tomek, Jakub; Bub, Gil; Paterson, David J.

    2016-01-01

    Key points Animal studies suggest an anti‐fibrillatory action of the vagus nerve on the ventricle, although the exact mechanism is controversial.Using a Langendorff perfused rat heart, we show that the acetylcholine analogue carbamylcholine raises ventricular fibrillation threshold (VFT) and flattens the electrical restitution curve.The anti‐fibrillatory action of carbamylcholine was prevented by the nicotinic receptor antagonist mecamylamine, inhibitors of neuronal nitric oxide synthase (nNOS) and soluble guanylyl cyclase (sGC), and can be mimicked by the nitric oxide (NO) donor sodium nitroprusside.Carbamylcholine increased NO metabolite content in the coronary effluent and this was prevented by mecamylamine.The anti‐fibrillatory action of both carbamylcholine and sodium nitroprusside was ultimately dependent on muscarinic receptor stimulation as all effects were blocked by atropine.These data demonstrate a protective effect of carbamylcholine on VFT that depends upon both muscarinic and nicotinic receptor stimulation, where the generation of NO is likely to be via a neuronal nNOS–sGC dependent pathway. Abstract Implantable cardiac vagal nerve stimulators are a promising treatment for ventricular arrhythmia in patients with heart failure. Animal studies suggest the anti‐fibrillatory effect may be nitric oxide (NO) dependent, although the exact site of action is controversial. We investigated whether a stable analogue of acetylcholine could raise ventricular fibrillation threshold (VFT), and whether this was dependent on NO generation and/or muscarinic/nicotinic receptor stimulation. VFT was determined in Langendorff perfused rat hearts by burst pacing until sustained VF was induced. Carbamylcholine (CCh, 200 nmol l–1, n = 9) significantly (P < 0.05) reduced heart rate from 292 ± 8 to 224 ± 6 b.p.m. Independent of this heart rate change, CCh caused a significant increase in VFT (control 1.5 ± 0.3 mA, CCh 2.4 ± 0.4 mA, wash 1.1

  12. The human D2 dopamine receptor synergizes with the A2A adenosine receptor to stimulate adenylyl cyclase in PC12 cells.

    PubMed

    Kudlacek, Oliver; Just, Herwig; Korkhov, Vladimir M; Vartian, Nina; Klinger, Markus; Pankevych, Halyna; Yang, Qiong; Nanoff, Christian; Freissmuth, Michael; Boehm, Stefan

    2003-07-01

    The adenosine A(2A) receptor and the dopamine D(2) receptor are prototypically coupled to G(s) and G(i)/G(o), respectively. In striatal intermediate spiny neurons, these receptors are colocalized in dendritic spines and act as mutual antagonists. This antagonism has been proposed to occur at the level of the receptors or of receptor-G protein coupling. We tested this model in PC12 cells which endogenously express A(2A) receptors. The human D(2) receptor was introduced into PC12 cells by stable transfection. A(2A)-agonist-mediated inhibition of D(2) agonist binding was absent in PC12 cell membranes but present in HEK293 cells transfected as a control. However, in the resulting PC12 cell lines, the action of the D(2) agonist quinpirole depended on the expression level of the D(2) receptor: at low and high receptor levels, the A(2A)-agonist-induced elevation of cAMP was enhanced and inhibited, respectively. Forskolin-stimulated cAMP formation was invariably inhibited by quinpirole. The effects of quinpirole were abolished by pretreatment with pertussis toxin. A(2A)-receptor-mediated cAMP formation was inhibited by other G(i)/G(o)-coupled receptors that were either endogenously present (P(2y12)-like receptor for ADP) or stably expressed after transfection (A(1) adenosine, metabotropic glutamate receptor-7A). Similarly, voltage activated Ca(2+) channels were inhibited by the endogenous P(2Y) receptor and by the heterologously expressed A(1) receptor but not by the D(2) receptor. These data indicate functional segregation of signaling components. Our observations are thus compatible with the proposed model that D(2) and A(2A) receptors are closely associated, but they highlight the fact that this interaction can also support synergism. PMID:12784121

  13. Androgen receptor and miRNA-126* axis controls follicle-stimulating hormone receptor expression in porcine ovarian granulosa cells.

    PubMed

    Du, Xing; Li, Qiqi; Pan, Zengxiang; Li, Qifa

    2016-08-01

    Androgen, which acts via the androgen receptor (AR), plays crucial roles in mammalian ovarian function. Recent studies showed that androgen/AR signaling regulates follicle-stimulating hormone receptor (FSHR) expression in follicles; however, the detailed mechanism underlying this regulation remained unknown. Here, we demonstrate that AR and miR-126* cooperate to inhibit FSHR expression and function in pig follicular granulosa cells (pGCs). In pGCs, overexpression of AR decreased, whereas knockdown increased, FSHR mRNA and protein expression; however, neither manipulation affected FSHR promoter activity. Using a dual-luciferase reporter assay, we found that the FSHR gene is a direct target of miR-126*, which inhibits FSHR expression and increases the rate of AR-induced apoptosis in pGCs. Collectively, our data show for the first time that the AR/miR-126* axis exerts synergetic effects in the regulation of FSHR expression and apoptosis in pGCs. Our findings thus define a novel pathway, AR/miR-126*/FSHR, that regulates mammalian GC functions. PMID:27222597

  14. Stimulation of Sigma-1 Receptor Ameliorates Depressive-like Behaviors in CaMKIV Null Mice.

    PubMed

    Moriguchi, Shigeki; Sakagami, Hiroyuki; Yabuki, Yasushi; Sasaki, Yuzuru; Izumi, Hisanao; Zhang, Chen; Han, Feng; Fukunaga, Kohji

    2015-12-01

    Sigma-1 receptor (Sig-1R) is a molecular chaperone regulating calcium efflux from the neuronal endoplasmic reticulum to the mitochondria. Calcium/calmodulin-dependent protein kinase IV (CaMKIV) null mice exhibit depressive-like behaviors and impaired neurogenesis as assessed by bromodeoxyuridine (BrdU) incorporation into newborn cells of the hippocampal dentate gyrus (DG). Here, we demonstrate that chronic stimulation of Sig-1R by treatment with the agonist SA4503 or the SSRI fluvoxamine for 14 days improves depressive-like behaviors in CaMKIV null mice. By contrast, treatment with paroxetine, which lacks affinity for Sig-1R, did not alter these behaviors. Reduced numbers of BrdU-positive cells and decreased brain-derived neurotrophic factor (BDNF) mRNA expression and protein kinase B (Akt; Ser-473) phosphorylation seen in the DG of CaMKIV null mice were significantly rescued by chronic Sig-1R stimulation. Interestingly, reduced ATP production observed in the DG of CaMKIV null mice was improved by chronic Sig-1R stimulation. Such stimulation also improved hippocampal long-term potentiation (LTP) induction and maintenance, which are impaired in the DG of CaMKIV null mice. LTP rescue was closely associated with both increases in calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation and GluA1 (Ser-831) phosphorylation. Taken together, Sig-1R stimulation by SA4503 or fluvoxamine treatment increased hippocampal neurogenesis, which is closely associated with amelioration of depressive-like behaviors in CaMKIV null mice. PMID:25316382

  15. Profiles of Basal and stimulated receptor signaling networks predict drug response in breast cancer lines.

    PubMed

    Niepel, Mario; Hafner, Marc; Pace, Emily A; Chung, Mirra; Chai, Diana H; Zhou, Lili; Schoeberl, Birgit; Sorger, Peter K

    2013-09-24

    Identifying factors responsible for variation in drug response is essential for the effective use of targeted therapeutics. We profiled signaling pathway activity in a collection of breast cancer cell lines before and after stimulation with physiologically relevant ligands, which revealed the variability in network activity among cells of known genotype and molecular subtype. Despite the receptor-based classification of breast cancer subtypes, we found that the abundance and activity of signaling proteins in unstimulated cells (basal profile), as well as the activity of proteins in stimulated cells (signaling profile), varied within each subtype. Using a partial least-squares regression approach, we constructed models that significantly predicted sensitivity to 23 targeted therapeutics. For example, one model showed that the response to the growth factor receptor ligand heregulin effectively predicted the sensitivity of cells to drugs targeting the cell survival pathway mediated by PI3K (phosphoinositide 3-kinase) and Akt, whereas the abundance of Akt or the mutational status of the enzymes in the pathway did not. Thus, basal and signaling protein profiles may yield new biomarkers of drug sensitivity and enable the identification of appropriate therapies in cancers characterized by similar functional dysregulation of signaling networks. PMID:24065145

  16. Stimulation of olfactory receptors alters regulation of [Cai] in olfactory neurons of the catfish (Ictalurus punctatus).

    PubMed

    Restrepo, D; Boyle, A G

    1991-03-01

    Intracellular calcium was measured in single olfactory neurons from the channel catfish (Ictalurus punctatus) using the fluorescent Ca2+ indicator fura 2. In 5% of the cells, olfactory stimuli (amino acids) elicited an influx of calcium through the plasma membrane which led to a rapid transient increase in intracellular calcium concentration. Amino acids did not induce release of calcium from internal stores in these cells. Some cells responded specifically to one stimulus (L-alanine, L-arginine, L-norleucine and L-glutamate) while one cell responded to all stimuli. An increase in intracellular calcium could also be elicited in 50% of the cells by direct G-protein stimulation using aluminum fluoride. Because the fraction of cells which respond to direct G-protein stimulation is substantially larger than the fraction of cells responding to amino acids, we tested for possible damage of receptor proteins due to exposure of the olfactory neurons to papain during cell isolation. We find that pretreatment with papain does not alter specific binding of L-alanine and L-arginine to olfactory receptor sites in isolated olfactory cilia. The results are discussed in terms of their relevance to olfactory transduction. PMID:2051471

  17. Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats.

    PubMed

    Gowen, M; Stroup, G B; Dodds, R A; James, I E; Votta, B J; Smith, B R; Bhatnagar, P K; Lago, A M; Callahan, J F; DelMar, E G; Miller, M A; Nemeth, E F; Fox, J

    2000-06-01

    Parathyroid hormone (PTH) is an effective bone anabolic agent, but it must be administered parenterally. An orally active anabolic agent would provide a valuable alternative for treating osteoporosis. NPS 2143 is a novel, selective antagonist (a "calcilytic") of the parathyroid cell Ca(2+) receptor. Daily oral administration of NPS 2143 to osteopenic ovariectomized (OVX) rats caused a sustained increase in plasma PTH levels, provoking a dramatic increase in bone turnover but no net change in bone mineral density. Concurrent oral administration of NPS 2143 and subcutaneous infusion of 17beta-estradiol also resulted in increased bone turnover. However, the antiresorptive action of estrogen decreased the extent of bone resorption stimulated by the elevated PTH levels, leading to an increase in bone mass compared with OVX controls or to either treatment alone. Despite the sustained stimulation to the parathyroid gland, parathyroid cells did not undergo hyperplasia. These data demonstrate that an increase in endogenous PTH secretion, induced by antagonism of the parathyroid cell Ca(2+) receptor with a small molecule, leads to a dramatic increase in bone turnover, and they suggest a novel approach to the treatment of osteoporosis. PMID:10841518

  18. Antagonizing the parathyroid calcium receptor stimulates parathyroid hormone secretion and bone formation in osteopenic rats

    PubMed Central

    Gowen, Maxine; Stroup, George B.; Dodds, Robert A.; James, Ian E.; Votta, Bart J.; Smith, Brian R.; Bhatnagar, Pradip K.; Lago, Amparo M.; Callahan, James F.; DelMar, Eric G.; Miller, Michael A.; Nemeth, Edward F.; Fox, John

    2000-01-01

    Parathyroid hormone (PTH) is an effective bone anabolic agent, but it must be administered parenterally. An orally active anabolic agent would provide a valuable alternative for treating osteoporosis. NPS 2143 is a novel, selective antagonist (a “calcilytic”) of the parathyroid cell Ca2+ receptor. Daily oral administration of NPS 2143 to osteopenic ovariectomized (OVX) rats caused a sustained increase in plasma PTH levels, provoking a dramatic increase in bone turnover but no net change in bone mineral density. Concurrent oral administration of NPS 2143 and subcutaneous infusion of 17β-estradiol also resulted in increased bone turnover. However, the antiresorptive action of estrogen decreased the extent of bone resorption stimulated by the elevated PTH levels, leading to an increase in bone mass compared with OVX controls or to either treatment alone. Despite the sustained stimulation to the parathyroid gland, parathyroid cells did not undergo hyperplasia. These data demonstrate that an increase in endogenous PTH secretion, induced by antagonism of the parathyroid cell Ca2+ receptor with a small molecule, leads to a dramatic increase in bone turnover, and they suggest a novel approach to the treatment of osteoporosis. PMID:10841518

  19. Beta2-adrenergic receptor stimulation inhibits nitric oxide generation by Mycobacterium avium infected macrophages.

    PubMed

    Boomershine, C S; Lafuse, W P; Zwilling, B S

    1999-11-01

    Catecholamine regulation of nitric oxide (NO) production by IFNgamma-primed macrophages infected with Mycobacterium avium was investigated. Epinephrine treatment of IFNgamma-primed macrophages at the time of M. avium infection inhibited the anti-mycobacterial activity of the cells. The anti-mycobacterial activity of macrophages correlated with NO production. Using specific adrenergic receptor agonists, the abrogation of mycobacterial killing and decreased NO production by catecholamines was shown to be mediated via the beta2-adrenergic receptor. Elevation of intracellular cAMP levels mimicked the catecholamine-mediated inhibition of NO in both M. avium infected and LPS stimulated macrophages. Specific inhibitors of both adenylate cyclase and protein kinase A prevented the beta2-adrenoceptor-mediated inhibition of nitric oxide production. Beta2-adrenoreceptor stimulation at the time of M. avium infection of IFNgamma-primed macrophages also inhibited expression of iNOS mRNA. These observations show that catecholamine hormones can affect the outcome of macrophage-pathogen interactions and suggest that one result of sympathetic nervous system activation is the suppression of the capacity of macrophages to produce anti-microbial effector molecules. PMID:10580815

  20. Acute inhalation of ozone stimulates bronchial C-fibers and rapidly adapting receptors in dogs

    SciTech Connect

    Coleridge, J.C.G.; Coleridge, H.M.; Schelegle, E.S.; Green, J.F. Univ. of California, San Francisco )

    1993-05-01

    To identify the afferents responsible for initiating the vagally mediated respiratory changes evoked by acute exposure to ozone, the authors recorded vagal impulses in anesthetized, open-chest, artificially ventilated dogs and examined the pulmonary afferent response to ozone (2--3 ppM in air) delivered to the lower trachea for 20--60 min. Bronchial C-fibers (BrCs) were the lung afferents most susceptible to ozone, the activity of 10 of 11 BrCs increasing from 0.2 [+-] 0.2 to 4.6 [+-] 1.3 impulses/s within 1--7 min of ozone exposure. Ten of 15 rapidly adapting receptors (RARs) were stimulated by ozone, their activity increasing from 1.5 [+-] 0.4 to 4.7 [+-] 0.7 impulses/s. Stimulation of RARs (but not of BrCs) appeared secondary to the ozone-induced reduction of lung compliance because it was abolished by hyperinflation of the lungs. Ozone had little effect on pulmonary C-fibers or slowly adapting pulmonary stretch receptors. The authors' results suggest that both BrCs and RARs contribute to the tachypnea and bronchoconstriction evoked by acute exposure to ozone when vagal conduction is intact and that BrCs alone are responsible for the vagally mediated tachypnea that survives vagal cooling to 7[degrees]C. 23 refs., 5 figs.

  1. Rac-mediated Stimulation of Phospholipase Cγ2 Amplifies B Cell Receptor-induced Calcium Signaling.

    PubMed

    Walliser, Claudia; Tron, Kyrylo; Clauss, Karen; Gutman, Orit; Kobitski, Andrei Yu; Retlich, Michael; Schade, Anja; Röcker, Carlheinz; Henis, Yoav I; Nienhaus, G Ulrich; Gierschik, Peter

    2015-07-10

    The Rho GTPase Rac is crucially involved in controlling multiple B cell functions, including those regulated by the B cell receptor (BCR) through increased cytosolic Ca(2+). The underlying molecular mechanisms and their relevance to the functions of intact B cells have thus far remained unknown. We have previously shown that the activity of phospholipase Cγ2 (PLCγ2), a key constituent of the BCR signalosome, is stimulated by activated Rac through direct protein-protein interaction. Here, we use a Rac-resistant mutant of PLCγ2 to functionally reconstitute cultured PLCγ2-deficient DT40 B cells and to examine the effects of the Rac-PLCγ2 interaction on BCR-mediated changes of intracellular Ca(2+) and regulation of Ca(2+)-regulated and nuclear-factor-of-activated-T-cell-regulated gene transcription at the level of single, intact B cells. The results show that the functional Rac-PLCγ2 interaction causes marked increases in the following: (i) sensitivity of B cells to BCR ligation; (ii) BCR-mediated Ca(2+) release from intracellular stores; (iii) Ca(2+) entry from the extracellular compartment; and (iv) nuclear translocation of the Ca(2+)-regulated nuclear factor of activated T cells. Hence, Rac-mediated stimulation of PLCγ2 activity serves to amplify B cell receptor-induced Ca(2+) signaling. PMID:25903139

  2. Phencyclidine-induced social withdrawal results from deficient stimulation of cannabinoid CB₁ receptors: implications for schizophrenia.

    PubMed

    Seillier, Alexandre; Martinez, Alex A; Giuffrida, Andrea

    2013-08-01

    The neuronal mechanisms underlying social withdrawal, one of the core negative symptoms of schizophrenia, are not well understood. Recent studies suggest an involvement of the endocannabinoid system in the pathophysiology of schizophrenia and, in particular, of negative symptoms. We used biochemical, pharmacological, and behavioral approaches to investigate the role played by the endocannabinoid system in social withdrawal induced by sub-chronic administration of phencyclidine (PCP). Pharmacological enhancement of endocannabinoid levels via systemic administration of URB597, an inhibitor of endocannabinoid degradation, reversed social withdrawal in PCP-treated rats via stimulation of CB1 receptors, but reduced social interaction in control animals through activation of a cannabinoid/vanilloid-sensitive receptor. In addition, the potent CB agonist CP55,940 reversed PCP-induced social withdrawal in a CB₁-dependent manner, whereas pharmacological blockade of CB₁ receptors by either AM251 or SR141716 reduced the time spent in social interaction in control animals. PCP-induced social withdrawal was accompanied by a decrease of anandamide (AEA) levels in the amygdala and prefrontal cortex, and these deficits were reversed by URB597. As CB₁ receptors are predominantly expressed on GABAergic interneurons containing the anxiogenic peptide cholecystokinin (CCK), we also examined whether the PCP-induced social withdrawal resulted from deficient CB₁-mediated modulation of CCK transmission. The selective CCK2 antagonist LY225910 blocked both PCP- and AM251-induced social withdrawal, but not URB597 effect in control rats. Taken together, these findings indicate that AEA-mediated activation of CB₁ receptors is crucial for social interaction, and that PCP-induced social withdrawal results from deficient endocannabinoid transmission. PMID:23563893

  3. Association of follicle stimulating hormone receptor promoter with ovarian response in IVF-ET patients

    PubMed Central

    Dan, Wang; Jing, Gao; Liangbin, Xia; Ting, Zhang; Ying, Zeng

    2015-01-01

    Background: Poor ovarian response phenomenon has been observed in some of the in vitro fertilization-embryo transfer patients. Some investigations found that follicle stimulating hormone receptor (FSHR) gene plays a role in the process, but no direct evidence shows the correlation between genotypes of FSHR and ovarian response. Objective: Exploring the molecular mechanism behind the mutation of FSHR promoter association with ovarian granulosa cells and poor ovarian response. Materials and Methods: This cross sectional study was performed using 158 women undergoing the controlled short program ovarian stimulation for IVF treatment. The 263 bp DNA fragments before the follicle stimulating hormone (FSH) receptor 5' initiation site were sequenced in the patients under IVF cycle, 70 of which had poor ovarian response and 88 showed normal ovarian responses. Results: With a mutation rate of 40%, 63 in 158 cases showed a 29th site G→A point mutation; among the mutated cases, the mutation rate of the poor ovarian responders was significantly higher than the normal group (60% versus 23.9%; χ2=21.450, p<0.001). Besides, the variability was also obvious in antral follicle count, and ovum pick-ups. The estradiol peak values and the number of mature eggs between the two groups had significant difference. However, there was no obvious variability (t=0.457, p=0.324) in the basic FSH values between the two groups (normal group, 7.2±2.3 U/L; mutation group, 7.1±2.0 U/L). Conclusion: The activity of FSHR promoter is significantly affected by the 29th site G→A mutation that will weaken promoter activity and result in poor response to FSH. PMID:26730247

  4. Molecular characterization and quantification of the follicle-stimulating hormone receptor in turbot (Scophthalmus maximus).

    PubMed

    Jia, Yudong; Sun, Ai; Meng, Zhen; Liu, Baoliang; Lei, Jilin

    2016-02-01

    Molecular cloning, characterization, and functional analysis of follicle-stimulating hormone receptor (FSHR) in female turbot (Scophthalmus maximus) were evaluated. Results showed that the full-length FSHR cDNA was 3824 bp long and contained a 2202 bp open reading frame that encoded a mature protein of 733 amino acids (aa) and a signal peptide of 18 aa. Multiple sequence analyses showed that turbot FSHR has high homology with the corresponding genes of other teleosts and significant homology with that of Hippoglossus hippoglossus. Turbot FSHR has the typical structural architecture of glycoprotein hormone receptors consisting of a large N-terminal extracellular domain, seven transmembrane domains and short C-terminal intracellular domain. FSHR mRNA was found to be abundant in the ovaries, but deficient in eyes, intestine, brain, muscle, gills, spleen, stomach, heart and kidney. Furthermore, FSHR mRNA was found to increase gradually from pre-vitellogenesis to migratory nucleus stages, with the highest values observed during the late vitellogenesis stage of the reproductive cycle. However, FSHR mRNA was found to decrease dramatically during the atresia stage. Meanwhile, functional analysis with HEK293T cells continual expressing FSHR demonstrated that FSHR was specifically stimulated by ovine FSH, but not ovine LH. These results indicate that turbot FSHR is mainly involved in the stimulation of vitellogenesis, regulation of oocyte maturation as well as promotion of ovarian development via specific ligand binding. These findings open doors to further investigation of physiological functions of FSHR, which will be valuable for fish reproduction and broodstock management. PMID:26358315

  5. Activation of brain somatostatin2 receptors stimulates feeding in mice: analysis of food intake microstructure

    PubMed Central

    Stengel, Andreas; Goebel, Miriam; Wang, Lixin; Rivier, Jean; Kobelt, Peter; Mönnikes, Hubert; Taché, Yvette

    2010-01-01

    We recently reported that the oligosomatostatin receptor agonist, ODT8-SST increases food intake in rats via the somatostatin2 receptor (sst2). We characterized ingestive behavior following intracerebroventricular (icv) injection of a selective sst2 agonist in freely fed mice during the light phase. The sst2 agonist (0.01, 0.03, 0.1, 0.3 or 1µg/mouse) injected icv under short inhalation anesthesia dose-dependently increased cumulative light phase food intake over 4h compared to vehicle with a 3.1-times increase at 1µg/mouse (p<0.05). Likewise, the sst2,3,5 agonist octreotide (0.3 or 1µg/mouse) dose-dependently increased 4-h food intake, whereas selective sst1 or sst4 agonists at 1µg/mouse did not. In vehicle-treated mice, high fat diet increased caloric intake/4h by 2.8-times compared to regular diet (p<0.05) and values were further increased 1.4-times/4h by the sst2 agonist. Automated continuous assessment of food intake established a 6.6-times higher food intake during the dark phase due to increased number of meals, meal size, meal duration and rate of ingestion compared to non-treated mice during the light phase. During the first 4h post icv sst2 agonist injection, mice had a 57% increase in number of meals with a 60% higher rate of ingestion, and a 61% reduction in inter-meal intervals, whereas meal sizes were not altered compared to vehicle. These data indicate that activation of brain sst2 receptors potently stimulates ingestive behavior under basal or high fat diet-stimulated conditions in mice. The shortened inter-meal interval suggests an inhibitory effect of the sst2 agonist on “satiety”, whereas “satiation” is not altered as indicated by normal meal size. PMID:20851136

  6. Magnesium Sulfate Protects Against the Bioenergetic Consequences of Chronic Glutamate Receptor Stimulation

    PubMed Central

    Clerc, Pascaline; Young, Christina A.; Bordt, Evan A.; Grigore, Alina M.; Fiskum, Gary; Polster, Brian M.

    2013-01-01

    Extracellular glutamate is elevated following brain ischemia or trauma and contributes to neuronal injury. We tested the hypothesis that magnesium sulfate (MgSO4, 3 mM) protects against metabolic failure caused by excitotoxic glutamate exposure. Rat cortical neuron preparations treated in medium already containing a physiological concentration of Mg2+ (1 mM) could be segregated based on their response to glutamate (100 µM). Type I preparations responded with a decrease or small transient increase in oxygen consumption rate (OCR). Type II neurons responded with >50% stimulation in OCR, indicating a robust response to increased energy demand without immediate toxicity. Pre-treatment with MgSO4 improved the initial bioenergetic response to glutamate and ameliorated subsequent loss of spare respiratory capacity, measured following addition of the uncoupler FCCP, in Type I but not Type II neurons. Spare respiratory capacity in Type I neurons was also improved by incubation with MgSO4 or NMDA receptor antagonist MK801 in the absence of glutamate treatment. This finding indicates that the major difference between Type I and Type II preparations is the amount of endogenous glutamate receptor activity. Incubation of Type II neurons with 5 µM glutamate prior to excitotoxic (100 µM) glutamate exposure recapitulated a Type I phenotype. MgSO4 protected against an excitotoxic glutamate-induced drop in neuronal ATP both with and without prior 5 µM glutamate exposure. Results indicate that MgSO4 protects against chronic moderate glutamate receptor stimulation and preserves cellular ATP following treatment with excitotoxic glutamate. PMID:24236167

  7. Methylphenidate and μ opioid receptor interactions: A pharmacological target for prevention of stimulant abuse

    PubMed Central

    Zhu, Jinmin; Spencer, Thomas J.; Kachroo, Anil; Liu-Chen, Lee-Yuan; Biederman, Joseph; Bhide, Pradeep G.

    2011-01-01

    Methylphenidate (MPH) is one of the most commonly used and highly effective treatments for attention deficit hyperactivity disorder (ADHD) in children and adults. As the therapeutic use of MPH has increased, so has its abuse and illicit street-use. Yet, the mechanisms associated with development of MPH-associated abuse and dependence are not well understood making it difficult to develop methods to help its mitigation. As a result, many ADHD patients especially children and youth, that could benefit from MPH treatment do not receive it and risk life-long disabilities associated with untreated ADHD. Therefore, understanding the mechanisms associated with development of MPH addiction and designing methods to prevent it assume high public health significance. Using a mouse model we show that supra-therapeutic doses of MPH produce rewarding effects (surrogate measure for addiction in humans) in a conditioned place preference paradigm and upregulate μ opioid receptor (MOPR) activity in the striatum and nucleus accumbens, brain regions associated with reward circuitry. Co-administration of naltrexone, a non-selective opioid receptor antagonist, prevents MPH-induced MOPR activation and the rewarding effects. The MPH-induced MOPR activation and rewarding effect require activation of the dopamine D1 but not the D2 receptor. These findings identify the MOPR as a potential target for attenuating rewarding effects of MPH and suggest that a formulation that combines naltrexone with MPH could be a useful pharmaceutical approach to alleviate abuse potential of MPH and other stimulants. PMID:21545805

  8. Extranuclear Actions of the Androgen Receptor Enhance Glucose-Stimulated Insulin Secretion in the Male.

    PubMed

    Navarro, Guadalupe; Xu, Weiwei; Jacobson, David A; Wicksteed, Barton; Allard, Camille; Zhang, Guanyi; De Gendt, Karel; Kim, Sung Hoon; Wu, Hongju; Zhang, Haitao; Verhoeven, Guido; Katzenellenbogen, John A; Mauvais-Jarvis, Franck

    2016-05-10

    Although men with testosterone deficiency are at increased risk for type 2 diabetes (T2D), previous studies have ignored the role of testosterone and the androgen receptor (AR) in pancreatic β cells. We show that male mice lacking AR in β cells (βARKO) exhibit decreased glucose-stimulated insulin secretion (GSIS), leading to glucose intolerance. The AR agonist dihydrotestosterone (DHT) enhances GSIS in cultured male islets, an effect that is abolished in βARKO(-/y) islets and human islets treated with an AR antagonist. In β cells, DHT-activated AR is predominantly extranuclear and enhances GSIS by increasing islet cAMP and activating the protein kinase A. In mouse and human islets, the insulinotropic effect of DHT depends on activation of the glucagon-like peptide-1 (GLP-1) receptor, and accordingly, DHT amplifies the incretin effect of GLP-1. This study identifies AR as a novel receptor that enhances β cell function, a finding with implications for the prevention of T2D in aging men. PMID:27133133

  9. Prolactin stimulates cell proliferation through a long form of prolactin receptor and K+ channel activation.

    PubMed Central

    Van Coppenolle, Fabien; Skryma, Roman; Ouadid-Ahidouch, Halima; Slomianny, Christian; Roudbaraki, Morad; Delcourt, Philippe; Dewailly, Etienne; Humez, Sandrine; Crépin, Alexandre; Gourdou, Isabelle; Djiane, Jean; Bonnal, Jean-Louis; Mauroy, Brigitte; Prevarskaya, Natalia

    2004-01-01

    PRL (prolactin) has been implicated in the proliferation and differentiation of numerous tissues, including the prostate gland. However, the PRL-R (PRL receptor) signal transduction pathway, leading to the stimulation of cell proliferation, remains unclear and has yet to be mapped. The present study was undertaken to develop a clear understanding of the mechanisms involved in this pathway and, in particular, to determine the role of K(+) channels. We used androgen-sensitive prostate cancer (LNCaP) cells whose proliferation is known to be stimulated by PRL. Reverse transcriptase PCR analysis showed that LNCaP cells express a long form of PRL-R, but do not produce its intermediate isoform. Patch-clamp techniques showed that the application of 5 nM PRL increased both the macroscopic K(+) current amplitude and the single K(+)-channel open probability. This single-channel activity increase was reduced by the tyrosine kinase inhibitors genistein, herbimycin A and lavandustine A, thereby indicating that tyrosine kinase phosphorylation is required in PRL-induced K(+) channel stimulation. PRL enhances p59( fyn ) phosphorylation by a factor of 2 after a 10 min application in culture. In addition, where an antip59( fyn ) antibody is present in the patch pipette, PRL no longer increases K(+) current amplitude. Furthermore, the PRL-stimulated proliferation is inhibited by the K(+) channel inhibitors alpha-dendrotoxin and tetraethylammonium. Thus, as K(+) channels are known to be involved in LNCaP cell proliferation, we suggest that K(+) channel modulation by PRL, via p59( fyn ) pathway, is the primary ionic event in PRL signal transduction, triggering cell proliferation. PMID:14565846

  10. Central oxytocin receptor stimulation attenuates the orexigenic effects of butorphanol tartrate.

    PubMed

    Olszewski, Pawel K; Klockars, Oscar A; Klockars, Anica; Levine, Allen S

    2016-09-28

    Butorphanol tartrate (BT), a mixed µ/κ/δ opioid receptor agonist, is one of the most potent orexigens known to date. The central mechanisms through which BT causes hyperphagia are largely unknown. Interestingly, BT suppresses meal-end activation of neurons synthesizing anorexigenic neuropeptide, oxytocin (OT), which suggests that BT promotes hyperphagia by silencing OT-derived satiety signaling. As OT terminates consumption by acting by distinct hindbrain and forebrain circuits, we investigated whether stimulation of the OT receptor in the forebrain or the hindbrain [through lateral ventricular (LV) and fourth ventricular (4V) OT injections] leads to termination of food intake induced by BT. We established effective doses of BT on chow intake in ad-libitum-fed and overnight-deprived rats as well as effective doses of LV and 4V OT in deprived animals. Then, we determined doses of LV and 4V OT that reduce hyperphagia produced by BT in sated and deprived rats. Finally, we assessed whether OT's effects on BT-induced feeding can be suppressed by an OT receptor antagonist. 4 mg/kg BT increased intake in ad-libitum-fed and overnight-deprived rats, whereas LV and 4V OT at 1 μg caused a decrease in deprived rats. BT-induced chow intake in hungry and sated animals was suppressed by a very low, 0.1 μg dose of 4V OT, whereas 1 μg OT was effective LV. The effect of OT was attenuated by OT receptor antagonist, L-368 899. Reduced activity of the OT circuit, especially its hindbrain component, is a critical factor in shaping the magnitude of consumption in response to BT treatment. PMID:27471903

  11. Nitric oxide and hypoxia stimulate erythropoietin receptor via MAPK kinase in endothelial cells.

    PubMed

    Cokic, Bojana B Beleslin; Cokic, Vladan P; Suresh, Sukanya; Wirt, Stacey; Noguchi, Constance Tom

    2014-03-01

    Erythropoietin receptor (EPOR) expression level determines the extent of erythropoietin (EPO) response. Previously we showed that EPOR expression in endothelial cells is increased at low oxygen tension and that EPO stimulation of endothelial cells during hypoxia can increase endothelial nitric oxide (NO) synthase (eNOS) expression and activation as well as NO production. We now observe that while EPO can stimulate NO production, NO in turn can regulate EPOR expression. Human umbilical vein endothelial cells (HUVEC) treated with 10-50 μM of NO donor diethylenetriamine NONOate (DETANO) for 24h showed significant induction of EPOR gene expression at 5% and 2% of oxygen. Also human bone marrow microvascular endothelial cell line (TrHBMEC) cultured at 21 and 2% oxygen with 50 μM DETANO demonstrated a time and oxygen dependent induction of EPOR mRNA expression after 24 and 48 h, particularly at low oxygen tension. EPOR protein was also induced by DETANO at 2% oxygen in TrHBMEC and HUVEC. The activation of signaling pathways by NO donor stimulation appeared to be distinct from EPO stimulation. In reporter gene assays, DETANO treatment of HeLa cells at 2% oxygen increased EPOR promoter activity indicated by a 48% increase in luciferase activity with a 2 kb EPOR promoter fragment and a 71% increase in activity with a minimal EPOR promoter fragment containing 0.2 kb 5'. We found that DETANO activated MAPK kinase in TrHBMEC both in normoxia and hypoxia, while MAPK kinase inhibition showed significant reduction of EPOR mRNA gene expression at low oxygen tension, suggesting MAPK involvement in NO mediated induction of EPOR. Furthermore, DETANO stimulated Akt anti-apoptotic activity after 30 min in normoxia, whereas it inhibited Akt phosphorylation in hypoxia. In contrast, EPO did not significantly increase MAPK activity while EPO stimulated Akt phosphorylation in TrHBMEC in normoxia and hypoxia. These observations provide a new effect of NO on EPOR expression to enhance EPO

  12. Stimulation of rat hepatic low density lipoprotein receptors by glucagon. Evidence of a novel regulatory mechanism in vivo.

    PubMed Central

    Rudling, M; Angelin, B

    1993-01-01

    We studied the influence of glucagon on hepatic LDL receptors and plasma lipoproteins in rats. A dose-dependent (maximum, threefold) increase in LDL-receptor binding was evident already at a dose of 2 x 4 micrograms, and detectable 3 h after injection; concomitantly, cholesterol and apolipoprotein (apo) B and apoE within LDL and large HDL decreased in plasma. LDL receptor mRNA levels were however unaltered or reduced. Hepatic microsomal cholesterol was increased and the enzymatic activities of 3-hydroxy-3-methylglutaryl coenzyme A reductase and cholesterol 7 alpha-hydroxylase in hepatic microsomes were reduced. Insulin alone increased receptor binding and receptor mRNA levels twofold, but plasma cholesterol was unchanged and plasma apoE and apoB increased. Administration of insulin to glucagon-treated animals reduced the LDL-receptor binding to control levels and apoB appeared in LDL particles. Estrogen treatment increased LDL-receptor binding and mRNA levels five- and eightfold, respectively. Combined treatment with glucagon and estrogen reduced the stimulation of LDL-receptor mRNA levels by 80% although LDL-receptor binding was unchanged. Immunoblot analysis showed that glucagon increased the number of hepatic LDL receptors. We conclude that glucagon induces the number of hepatic LDL receptors by a mechanism not related to increased mRNA levels, suggesting the presence of a posttranscriptional regulatory mechanism present in the liver in vivo. Images PMID:8514887

  13. Positive AMPA receptor modulation rapidly stimulates BDNF release and increases dendritic mRNA translation.

    PubMed

    Jourdi, Hussam; Hsu, Yu-Tien; Zhou, Miou; Qin, Qingyu; Bi, Xiaoning; Baudry, Michel

    2009-07-01

    Brain-derived neurotrophic factor (BDNF) stimulates local dendritic mRNA translation and is involved in formation and consolidation of memory. 2H,3H,6aH-pyrrolidino[2'',1''-3',2']1,3-oxazino[6',5'-5,4]-benzo[e]1,4-dioxan-10-one (CX614), one of the best-studied positive AMPA receptor modulators (also known as ampakines), increases BDNF mRNA and protein and facilitates long-term potentiation (LTP) induction. Several other ampakines also improve performance in various behavioral and learning tasks. Since local dendritic protein synthesis has been implicated in LTP stabilization and in memory consolidation, this study investigated whether CX614 could influence synaptic plasticity by upregulating dendritic protein translation. CX614 treatment of primary neuronal cultures and acute hippocampal slices rapidly activated the translation machinery and increased local dendritic protein synthesis. CX614-induced activation of translation was blocked by K252a [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-hydroxy-9-methyl-1-oxo-9,12-epoxy-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester], CNQX, APV, and TTX, and was inhibited in the presence of an extracellular BDNF scavenger, TrkB-Fc. The acute effect of CX614 on translation was mediated by increased BDNF release as demonstrated with a BDNF scavenging assay using TrkB-Fc during CX614 treatment of cultured primary neurons and was blocked by nifedipine, ryanodine, and lack of extracellular Ca(2+) in acute hippocampal slices. Finally, CX614, like BDNF, rapidly increased dendritic translation of an exogenous translation reporter. Together, our results demonstrate that positive modulation of AMPA receptors rapidly stimulates dendritic translation, an effect mediated by BDNF secretion and TrkB receptor activation. They also suggest that increased BDNF secretion and stimulation of local protein synthesis contribute to the effects of ampakines on synaptic plasticity. PMID:19587275

  14. What is the effect of nicotinic acetylcholine receptor stimulation on osteoarthritis in a rodent animal model?

    PubMed Central

    Bock, Kilian; Plaass, Christian; Coger, Vincent; Peck, Claas-Tido; Reimers, Kerstin; Stukenborg-Colsman, Christina; Claassen, Leif

    2016-01-01

    Objectives: Despite the rising number of patients with osteoarthritis, no sufficient chondroprotective and prophylactic therapy for osteoarthritis has been established yet. The purpose of this study was to verify whether stimulation of the nicotinic acetylcholine receptor via nicotine has a beneficial effect on cartilage degeneration in the development of osteoarthritis and is capable of reducing the expression of proinflammatory cytokines and cartilage degrading enzymes in synovial membranes after osteoarthritis induction. Methods: Experimental osteoarthritis was induced in Lewis rats using a standardized osteoarthritis model with monoiodoacetate. A total of 16 Lewis rats were randomized into four groups: control, sham + nicotine application, osteoarthritis, and osteoarthritis + nicotine application. Nicotine (0.625 mg/kg twice daily) was administered intraperitoneally for 42 days. We analyzed histological sections, radiological images and the expression of the proinflammatory cytokines, such as interleukin-1β, tumor necrosis factor-α and interleukin-6, and of matrix metalloproteases 3, 9 and 13 and tissue inhibitors of metalloprotease-1 in synovial membranes via quantitative polymerase chain reaction. Results: Histological and x-ray examination revealed cartilage degeneration in the osteoarthritis group compared to control or sham + nicotine groups (histological control vs osteoarthritis: p = 0.002 and x-ray control vs osteoarthritis: p = 0.004). Nicotine treatment reduced the cartilage degeneration without significant differences. Osteoarthritis induction led to a higher expression of proinflammatory cytokines and matrix metalloproteases as compared to control groups. This effect was attenuated after nicotine administration. The differences of proinflammatory cytokines and matrix metalloproteases did not reach statistical significance. Conclusion: With the present small-scale study, we could not prove a positive effect of nicotinic

  15. Caffeine-induced behavioral stimulation is dose-dependent and associated with A1 adenosine receptor occupancy.

    PubMed

    Kaplan, G B; Greenblatt, D J; Kent, M A; Cotreau, M M; Arcelin, G; Shader, R I

    1992-05-01

    Caffeine's psychomotor stimulant effects may relate to its blockade of central adenosine receptors. We examined acute caffeine effects on motor activity, adenosine receptor occupancy in vivo, and receptor affinity and density ex vivo. Acute doses of caffeine-sodium benzoate (0, 20, 40, and 60 mg/kg, intraperitoneally [0, 0.10, 0.21, 0.31 mu mol/kg]) were given to CD-1 mice and their activity was measured in an animal activity monitor over a 1-hour period. Adenosine receptor occupancy in vivo was quantified in mice 1 hour postdosage, using the high-affinity, A1 receptor selective adenosine antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine. Adenosine receptor binding affinities and densities were determined from analyses of binding studies in cortical, hippocampal, and brainstem membranes from treated mice (0 and 40 mg/kg caffeine). Caffeine doses of 20 and 40 mg/kg, corresponding to mean brain concentrations of 5 and 17 micrograms/g, increased all horizontal and vertical motor activity measures and stereotypy counts, as compared to doses of 0 and 60 mg/kg. Additionally, all acute caffeine doses significantly altered specific A1 binding in vivo (decreasing binding between 55% and 73% versus vehicle), presumably as it occupied A1 receptors. Therefore, at doses of 20 and 40 mg/kg, caffeine stimulated motor activity as it occupied A1 receptors; at a dose of 60 mg/kg (mean brain concentration of 26 micrograms/g) caffeine had no stimulant effect even though it appeared to occupy A1 receptors. Acute caffeine dosage did not alter ex vivo adenosine receptor binding affinity or density in any brain regions.(ABSTRACT TRUNCATED AT 250 WORDS) PMID:1599605

  16. Electrical Stimulation Decreases Coupling Efficiency Between Beta-Adrenergic Receptors and Cyclic AMP Production in Cultured Muscle Cells

    NASA Technical Reports Server (NTRS)

    Young, R. B.; Bridge, K. Y.

    1999-01-01

    Electrical stimulation of skeletal muscle cells in culture is an effective way to simulate the effects of muscle contraction and its effects on gene expression in muscle cells. Expression of the beta-adrenergic receptor and its coupling to cyclic AMP synthesis are important components of the signaling system that controls muscle atrophy and hypertrophy, and the goal of this project was to determine if electrical stimulation altered the beta-adrenergic response in muscle cells. Chicken skeletal muscle cells that had been grown for seven days in culture were subjected to electrical stimulation for an additional two days at a pulse frequency of 0.5 pulses/sec and a pulse duration of 200 msec. At the end of this two-day stimulation period, beta-adrenergic receptor population was measured by the binding of tritium-labeled CGP-12177 to muscle cells, and coupling to cAMP synthesis was measured by Radioimmunoassay (RIA) after treating the cells for 10 min with the potent (beta)AR agonist, isoproterenol. The number of beta adrenergic receptors and the basal levels of intracellular cyclic AMP were not affected by electrical stimulation. However, the ability of these cells to synthesize cyclic AMP was reduced by approximately 50%. Thus, an enhanced level of contraction reduces the coupling efficiency of beta-adrenergic receptors for cyclic AMP production.

  17. Basal and adenosine receptor-stimulated levels of cAMP are reduced in lymphocytes from alcoholic patients

    SciTech Connect

    Diamond, I.; Wrubel, B.; Estrin, W.; Gordon, A.

    1987-03-01

    Alcoholism causes serious neurologic disease that may be due, in part, to the ability of ethanol to interact with neural cell membranes and change neuronal function. Adenosine receptors are membrane-bound proteins that appear to mediate some of the effects of ethanol in the brain. Human lymphocytes also have adenosine receptors, and their activation causes increases in cAMP levels. To test the hypothesis that basal and adenosine receptor-stimulated cAMP levels in lymphocytes might be abnormal in alcoholism, the authors studied lymphocytes from 10 alcoholic subjects, 10 age- and sex-matched normal individuals, and 10 patients with nonalcoholic liver disease. Basal and adenosine receptor-stimulated cAMP levels were reduced 75% in lymphocytes from alcoholic subjects. Also, there was a 76% reduction in ethanol stimulation of cAMP accumulation in lymphocytes from alcoholics. Similar results were demonstrable in isolated T cells. Unlike other laboratory tests examined, these measurements appeared to distinguish alcoholics from normal subjects and from patients with nonalcoholic liver disease. Reduced basal and adenosine receptor-stimulated levels of cAMP in lymphocytes from alcoholics may reflect a change in cell membranes due either to chronic alcohol abuse or to a genetic predisposition unique to alcoholic subjects.

  18. [Leu]enkephalin stimulates carbohydrate metabolism in isolated hepatocytes and kidney tubule fragments by interaction with angiotensin II receptors.

    PubMed Central

    Hothi, S K; Randall, D P; Titheradge, M A

    1989-01-01

    The possibility that the effects of [Leu]enkephalin in vitro on hepatic carbohydrate metabolism are mediated by interaction with angiotensin II receptors has been examined. Preincubation of hepatocytes with either the angiotensin II receptor antagonist [Sar1,Ile8]angiotensin II or 10 mM-dithiothreitol abolished the ability of both angiotensin II and [Leu]enkephalin to increase phosphorylase a in hepatocytes prepared from fed rats. Dithiothreitol had no effect on the stimulation of phosphorylase in the presence of glucagon or phenylephrine, although it also inhibited the response to vasopressin. [Leu]enkephalin displaced specifically bound 125I-labelled angiotensin II from hepatic plasma membranes over a concentration range of 10(-7)-10(-5) M. This correlated with the dose-response required to stimulate phosphorylase activity in intact hepatocytes and suggests that the effects of the opioid peptides on carbohydrate metabolism in liver are the result of cross-reactivity of the peptides with angiotensin II receptors. Addition of 10(-5) M-[Leu]enkephalin to isolated kidney tubule fragments stimulated gluconeogenesis from 5 mM-pyruvate, the magnitude of stimulation being comparable to that by either angiotensin II or adrenaline. This effect of the opioid peptide was also abolished by pretreatment of the tubules with [Sar1,Ile8]angiotensin II, suggesting that the ability of [Leu]enkephalin to interact with angiotensin II receptors is not restricted to the liver, but may occur in other tissues where both receptors occur together. PMID:2930480

  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. Enhancement of peripheral nerve regeneration due to treadmill training and electrical stimulation is dependent on androgen receptor signaling.

    PubMed

    Thompson, Nicholas J; Sengelaub, Dale R; English, Arthur W

    2014-05-01

    Moderate exercise in the form of treadmill training and brief electrical nerve stimulation both enhance axon regeneration after peripheral nerve injury. Different regimens of exercise are required to enhance axon regeneration in male and female mice (Wood et al.: Dev Neurobiol 72 (2012) 688-698), and androgens are suspected to be involved. We treated mice with the androgen receptor blocker, flutamide, during either exercise or electrical stimulation, to evaluate the role of androgen receptor signaling in these activity-based methods of enhancing axon regeneration. The common fibular (CF) and tibial (TIB) nerves of thy-1-YFP-H mice, in which axons in peripheral nerves are marked by yellow fluorescent protein (YFP), were transected and repaired using CF and TIB nerve grafts harvested from non-fluorescent donor mice. Silastic capsules filled with flutamide were implanted subcutaneously to release the drug continuously. Exercised mice were treadmill trained 5 days/week for 2 weeks, starting on the third day post-transection. For electrical stimulation, the sciatic nerve was stimulated continuously for 1 h prior to nerve transection. After 2 weeks, lengths of YFP+ profiles of regenerating axons were measured from harvested nerves. Both exercise and electrical stimulation enhanced axon regeneration, but this enhancement was blocked completely by flutamide treatments. Signaling through androgen receptors is necessary for the enhancing effects of treadmill exercise or electrical stimulation on axon regeneration in cut peripheral nerves. PMID:24293191

  1. Targeting the thyroid-stimulating hormone receptor with small molecule ligands and antibodies

    PubMed Central

    Davies, Terry F; Latif, Rauf

    2015-01-01

    Introduction The thyroid-stimulating hormone receptor (TSHR) is the essential molecule for thyroid growth and thyroid hormone production. Since it is also a key autoantigen in Graves’ disease and is involved in thyroid cancer pathophysiology, the targeting of the TSHR offers a logical model for disease control. Areas covered We review the structure and function of the TSHR and the progress in both small molecule ligands and TSHR antibodies for their therapeutic potential. Expert opinion Stabilization of a preferential conformation for the TSHR by allosteric ligands and TSHR antibodies with selective modulation of the signaling pathways is now possible. These tools may be the next generation of therapeutics for controlling the pathophysiological consequences mediated by the effects of the TSHR in the thyroid and other extrathyroidal tissues. PMID:25768836

  2. Graves' Disease Mechanisms: The Role of Stimulating, Blocking, and Cleavage Region TSH Receptor Antibodies.

    PubMed

    Morshed, S A; Davies, T F

    2015-09-01

    The immunologic processes involved in Graves' disease (GD) have one unique characteristic--the autoantibodies to the TSH receptor (TSHR)--which have both linear and conformational epitopes. Three types of TSHR antibodies (stimulating, blocking, and cleavage) with different functional capabilities have been described in GD patients, which induce different signaling effects varying from thyroid cell proliferation to thyroid cell death. The establishment of animal models of GD by TSHR antibody transfer or by immunization with TSHR antigen has confirmed its pathogenic role and, therefore, GD is the result of a breakdown in TSHR tolerance. Here we review some of the characteristics of TSHR antibodies with a special emphasis on new developments in our understanding of what were previously called "neutral" antibodies and which we now characterize as autoantibodies to the "cleavage" region of the TSHR ectodomain. PMID:26361259

  3. Activating Parabrachial Cannabinoid CB1 Receptors Selectively Stimulates Feeding of Palatable Foods in Rats

    PubMed Central

    DiPatrizio, Nicholas V.; Simansky, Kenny J.

    2009-01-01

    The endocannabinoid system is emerging as an integral component in central and peripheral regulation of feeding and energy balance. Our investigation analyzed behavioral roles for cannabinoid mechanisms of the pontine parabrachial nucleus (PBN) in modulating intake of presumably palatable foods containing fat and/or sugar. The PBN serves to gate neurotransmission associated with, but not limited to, the gustatory properties of food. Immunofluorescence and in vitro [35S]GTPγS autoradiography of rat tissue sections containing the PBN revealed the presence of cannabinoid receptors and their functional capability to couple to their G-proteins following incubation with the endocannabinoid, 2-arachidonoyl glycerol (2-AG). The selective cannabinoid 1 receptor (CB1R) antagonist, AM251, prevented the response, demonstrating CB1R mediation of 2-AG induced coupling. Microinfusions of 2-AG into the PBN in behaving rats robustly stimulated feeding of pellets high in content of fat and sucrose (HFS), pure sucrose and pure fat (Crisco®), during the first 30min following infusion. In contrast, 2-AG failed to increase consumption of standard chow, even when the feeding regimen was manipulated to match baseline intakes of HFS. Orexigenic responses to 2-AG were attenuated by AM251, again indicating CB1R mediation of 2-AG actions. Furthermore, responses were regionally specific, as 2-AG failed to alter intake when infused into sites ~500µm caudal to infusions that successfully stimulated feeding. Our data suggest that hedonically-positive sensory properties of food enable endocannabinoids at PBN CB1Rs to initiate increases in eating and more generally, these pathways may serve a larger role in brain functions controlling behavioral responses for natural reward. PMID:18815256

  4. Trypanosoma cruzi and Its Soluble Antigens Induce NET Release by Stimulating Toll-Like Receptors

    PubMed Central

    Diniz, Larissa Figueiredo Alves; Souza, Priscila Silva Sampaio; Pinge-Filho, Phileno; Toledo, Karina Alves

    2015-01-01

    Neutrophils release fibrous traps of DNA, histones, and granule proteins known as neutrophil extracellular traps (NETs), which contribute to microbicidal killing and have been implicated in autoimmunity. The role of NET formation in the host response to nonbacterial pathogens is not well-understood. In this study, we investigated the release of NETs by human neutrophils upon their interaction with Trypanosoma cruzi (Y strain) parasites. Our results showed that human neutrophils stimulated by T. cruzi generate NETs composed of DNA, histones, and elastase. The release occurred in a dose-, time-, and reactive oxygen species-dependent manner to decrease trypomastigote and increase amastigote numbers of the parasites without affecting their viability. NET release was decreased upon blocking with antibodies against Toll-like receptors 2 and 4. In addition, living parasites were not mandatory in the release of NETs induced by T. cruzi, as the same results were obtained when molecules from its soluble extract were tested. Our results increase the understanding of the stimulation of NETs by parasites, particularly T. cruzi. We suggest that contact of T. cruzi with NETs during Chagas’s disease can limit infection by affecting the infectivity/pathogenicity of the parasite. PMID:26431537

  5. The angiotensin II-AT1 receptor stimulates reactive oxygen species within the cell nucleus

    SciTech Connect

    Pendergrass, Karl D.; Gwathmey, TanYa M.; Michalek, Ryan D.; Grayson, Jason M.; Chappell, Mark C.

    2009-06-26

    We and others have reported significant expression of the Ang II Type 1 receptor (AT1R) on renal nuclei; thus, the present study assessed the functional pathways and distribution of the intracellular AT1R on isolated nuclei. Ang II (1 nM) stimulated DCF fluorescence, an intranuclear indicator of reactive oxygen species (ROS), while the AT1R antagonist losartan or the NADPH oxidase (NOX) inhibitor DPI abolished the increase in ROS. Dual labeling of nuclei with antibodies against nucleoporin 62 (Nup62) and AT1R or the NADPH oxidase isoform NOX4 revealed complete overlap of the Nup62 and AT1R (99%) by flow cytometry, while NOX4 was present on 65% of nuclei. Treatment of nuclei with a PKC agonist increased ROS while the PKC inhibitor GF109203X or PI3 kinase inhibitor LY294002 abolished Ang II stimulation of ROS. We conclude that the Ang II-AT1R-PKC axis may directly influence nuclear function within the kidney through a redox sensitive pathway.

  6. Promotion of adipogenesis by an EP2 receptor agonist via stimulation of angiogenesis in pulmonary emphysema.

    PubMed

    Tsuji, Takao; Yamaguchi, Kazuhiro; Kikuchi, Ryota; Itoh, Masayuki; Nakamura, Hiroyuki; Nagai, Atsushi; Aoshiba, Kazutetsu

    2014-08-01

    Body weight loss is a common manifestation in patients with chronic obstructive pulmonary disease (COPD), particularly those with severe emphysema. Adipose angiogenesis is a key mediator of adipogenesis and use of pro-angiogenic agents may serve as a therapeutic option for lean COPD patients. Since angiogenesis is stimulated by PGE2, we examined whether ONO-AE1-259, a selective E-prostanoid (EP) 2 receptor agonist, might promote adipose angiogenesis and adipogenesis in a murine model of elastase-induced pulmonary emphysema (EIE mice). Mice were intratracheally instilled with elastase or saline, followed after 4 weeks by intraperitoneal administration of ONO-AE1-259 for 4 weeks. The subcutaneous adipose tissue (SAT) weight decreased in the EIE mice, whereas in the EIE mice treated with ONO-AE1-259, the SAT weight was largely restored, which was associated with significant increases in SAT adipogenesis, angiogenesis, and VEGF protein production. In contrast, ONO-AE1-259 administration induced no alteration in the weight of the visceral adipose tissue. These results suggest that in EIE mice, ONO-AE1-259 stimulated adipose angiogenesis possibly via VEGF production, and thence, adipogenesis. Our data pave the way for the development of therapeutic interventions for weight loss in emphysema patients, e.g., use of pro-angiogenic agents targeting the adipose tissue vascular component. PMID:24911647

  7. Enhanced emotional empathy after mineralocorticoid receptor stimulation in women with borderline personality disorder and healthy women.

    PubMed

    Wingenfeld, Katja; Kuehl, Linn K; Janke, Katrin; Hinkelmann, Kim; Dziobek, Isabel; Fleischer, Juliane; Otte, Christian; Roepke, Stefan

    2014-07-01

    The mineralocorticoid receptor (MR) is highly expressed in the hippocampus and prefrontal cortex. MR have an important role in appraisal processes and in modulating stress-associated emotional reactions but it is not known whether the MR affects empathy. Borderline personality disorder (BPD) is characterized by disturbed emotion regulation and alterations in empathy. In the current study, we examined whether stimulation of the MR enhances empathy in patients with BPD and healthy individuals. In a placebo-controlled study, we randomized 38 women with BPD and without psychotropic medication, and 35 healthy women to either placebo or 0.4 mg fludrocortisone, an MR agonist. Subsequently, all participants underwent two tests of social cognition, the Multifaceted Empathy Test (MET) and the Movie for the Assessment of Social Cognition (MASC), measuring cognitive and emotional facets of empathy. Eighteen BPD patients and 18 healthy women received placebo, whereas 20 BPD patients and 17 healthy women received fludrocortisone. In the MET, fludrocortisone enhanced emotional empathy across groups, whereas cognitive empathy was not affected. In the MASC, no effect of fludrocortisone could be revealed. In both tests, BPD patients and healthy women did not differ significantly in cognitive and emotional empathy and in their response to fludrocortisone. Stimulation of MR enhanced emotional empathy in healthy women and in BPD patients. Whether fludrocortisone might have a therapeutic role in psychotherapeutic processes, remains to be elucidated. PMID:24535100

  8. Impaired Cognition after Stimulation of P2Y1 Receptors in the Rat Medial Prefrontal Cortex

    PubMed Central

    Koch, Holger; Bespalov, Anton; Drescher, Karla; Franke, Heike; Krügel, Ute

    2015-01-01

    We hypothesize that cortical ATP and ADP accumulating in the extracellular space, eg during prolonged network activity, contribute to a decline in cognitive performance in particular via stimulation of the G protein-coupled P2Y1 receptor (P2Y1R) subtype. Here, we report first evidence on P2Y1R-mediated control of cognitive functioning in rats using bilateral microinfusions of the selective agonist MRS2365 into medial prefrontal cortex (mPFC). MRS2365 attenuated prepulse inhibition of the acoustic startle reflex while having no impact on startle amplitude. Stimulation of P2Y1Rs deteriorated performance accuracy in the delayed non-matching to position task in a delay dependent manner and increased the rate of magazine entries consistent with both working memory disturbances and impaired impulse control. Further, MRS2365 significantly impaired performance in the reversal learning task. These effects might be related to MRS2365-evoked increase of dopamine observed by microdialysis to be short-lasting in mPFC and long-lasting in the nucleus accumbens. P2Y1Rs were identified on pyramidal cells and parvalbumin-positive interneurons, but not on tyrosine hydroxylase-positive fibers, which argues for an indirect activation of dopaminergic afferents in the cortex by MRS2365. Collectively, these results suggest that activation of P2Y1Rs in the mPFC impairs inhibitory control and behavioral flexibility mediated by increased mesocorticolimbic activity and local disinhibition. PMID:25027332

  9. Statins stimulate the production of a soluble form of the receptor for advanced glycation end products

    PubMed Central

    Quade-Lyssy, Patricia; Kanarek, Anna Maria; Baiersdörfer, Markus; Postina, Rolf; Kojro, Elzbieta

    2013-01-01

    The beneficial effects of statin therapy in the reduction of cardiovascular pathogenesis, atherosclerosis, and diabetic complications are well known. The receptor for advanced glycation end products (RAGE) plays an important role in the progression of these diseases. In contrast, soluble forms of RAGE act as decoys for RAGE ligands and may prevent the development of RAGE-mediated disorders. Soluble forms of RAGE are either produced by alternative splicing [endogenous secretory RAGE (esRAGE)] or by proteolytic shedding mediated by metalloproteinases [shed RAGE (sRAGE)]. Therefore we analyzed whether statins influence the production of soluble RAGE. Lovastatin treatment of either mouse alveolar epithelial cells endogenously expressing RAGE or HEK cells overexpressing RAGE caused induction of RAGE shedding, but did not influence secretion of esRAGE from HEK cells overexpressing esRAGE. Lovastatin-induced secretion of sRAGE was also evident after restoration of the isoprenylation pathway, demonstrating a correlation of sterol biosynthesis and activation of RAGE shedding. Lovastatin-stimulated induction of RAGE shedding was completely abolished by a metalloproteinase ADAM10 inhibitor. We also demonstrate that statins stimulate RAGE shedding at low physiologically relevant concentrations. Our results show that statins, due to their cholesterol-lowering effects, increase the soluble RAGE level by inducing RAGE shedding, and by doing this, might prevent the development of RAGE-mediated pathogenesis. PMID:23966666

  10. Muscarinic acetylcholine receptor-mediated stimulation of retinal ganglion cell photoreceptors.

    PubMed

    Sodhi, Puneet; Hartwick, Andrew T E

    2016-09-01

    Melanopsin-dependent phototransduction in intrinsically photosensitive retinal ganglion cells (ipRGCs) involves a Gq-coupled phospholipase C (PLC) signaling cascade. Acetylcholine, released in the mammalian retina by starburst amacrine cells, can also activate Gq-PLC pathways through certain muscarinic acetylcholine receptors (mAChRs). Using multielectrode array recordings of rat retinas, we demonstrate that robust spiking responses can be evoked in neonatal and adult ipRGCs after bath application of the muscarinic agonist carbachol. The stimulatory action of carbachol on ipRGCs was a direct effect, as confirmed through calcium imaging experiments on isolated ipRGCs in purified cultures. Using flickering (6 Hz) yellow light stimuli at irradiances below the threshold for melanopsin activation, spiking responses could be elicited in ipRGCs that were suppressed by mAChR antagonism. Therefore, this work identified a novel melanopsin-independent pathway for stimulating sustained spiking in ganglion cell photoreceptors. This mAChR-mediated pathway could enhance ipRGC spiking responses in conditions known to evoke retinal acetylcholine release, such as those involving flickering or moving visual stimuli. Furthermore, this work identifies a pharmacological approach for light-independent ipRGC stimulation that could be targeted by mAChR agonists. PMID:27055770

  11. Tamoxifen stimulates in vivo growth of drug-resistant estrogen receptor-negative breast cancer.

    PubMed

    Maenpaa, J; Wiebe, V; Koester, S; Wurz, G; Emshoff, V; Seymour, R; Sipila, P; DeGregorio, M

    1993-01-01

    An estrogen receptor-negative, multidrug-resistant MDA-MB-A1 human breast cancer cell line was grown in culture with and without a noninhibitory concentration (0.5 microM) of tamoxifen for 122 days. Tamoxifen-treated and control cells were inoculated into opposite flanks of nine nude mice, where they produced measurable tumors in every case. Six of the animals were treated with tamoxifen at 500 micrograms/day for 22 days. Although no inhibitory nor stimulatory effect of tamoxifen was seen in vitro, tamoxifen had a clear tumor-growth-stimulating effect in mice. The most pronounced stimulatory effects were observed in the cells that had been cultured with tamoxifen. Within 3 weeks of the start of tamoxifen therapy, the cells grown in the presence of tamoxifen produced tumors with a mean size of 380 mm2, whereas the cells not pretreated with tamoxifen had tumors of 220 mm2. In contrast, in mice not receiving tamoxifen, the sizes of the tumors were 190 and 140 mm2, respectively. These preliminary results suggest that prolonged in vitro tamoxifen exposure induces cellular changes that result in tumors that are stimulated to grow faster in mice following tamoxifen treatment. PMID:8339392

  12. Iron Mediates N-Methyl-d-aspartate Receptor-dependent Stimulation of Calcium-induced Pathways and Hippocampal Synaptic Plasticity*

    PubMed Central

    Muñoz, Pablo; Humeres, Alexis; Elgueta, Claudio; Kirkwood, Alfredo; Hidalgo, Cecilia; Núñez, Marco T.

    2011-01-01

    Iron deficiency hinders hippocampus-dependent learning processes and impairs cognitive performance, but current knowledge on the molecular mechanisms underlying the unique role of iron in neuronal function is sparse. Here, we investigated the participation of iron on calcium signal generation and ERK1/2 stimulation induced by the glutamate agonist N-methyl-d-aspartate (NMDA), and the effects of iron addition/chelation on hippocampal basal synaptic transmission and long-term potentiation (LTP). Addition of NMDA to primary hippocampal cultures elicited persistent calcium signals that required functional NMDA receptors and were independent of calcium influx through L-type calcium channels or α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors; NMDA also promoted ERK1/2 phosphorylation and nuclear translocation. Iron chelation with desferrioxamine or inhibition of ryanodine receptor (RyR)-mediated calcium release with ryanodine-reduced calcium signal duration and prevented NMDA-induced ERK1/2 activation. Iron addition to hippocampal neurons readily increased the intracellular labile iron pool and stimulated reactive oxygen species production; the antioxidant N-acetylcysteine or the hydroxyl radical trapper MCI-186 prevented these responses. Iron addition to primary hippocampal cultures kept in calcium-free medium elicited calcium signals and stimulated ERK1/2 phosphorylation; RyR inhibition abolished these effects. Iron chelation decreased basal synaptic transmission in hippocampal slices, inhibited iron-induced synaptic stimulation, and impaired sustained LTP in hippocampal CA1 neurons induced by strong stimulation. In contrast, iron addition facilitated sustained LTP induction after suboptimal tetanic stimulation. Together, these results suggest that hippocampal neurons require iron to generate RyR-mediated calcium signals after NMDA receptor stimulation, which in turn promotes ERK1/2 activation, an essential step of sustained LTP. PMID:21296883

  13. Development of Follicle-Stimulating Hormone Receptor Binding Probes to Image Ovarian Xenografts

    PubMed Central

    Lee, Chung-Wein; Guo, Lili; Matei, Daniela; Stantz, Keith

    2015-01-01

    The Follicle-Stimulating Hormone Receptor (FSHR) is used as an imaging biomarker for the detection of ovarian cancer (OC). FSHR is highly expressed on ovarian tumors and involved with cancer development and metastatic signaling pathways. A decapeptide specific to the FSHR extracellular domain is synthesized and conjugated to fluorescent dyes to image OC cells in vitro and tumors xenograft model in vivo. The in vitro binding curve and the average number of FSHR per cell are obtained for OVCAR-3 cells by a high resolution flow cytometer. For the decapeptide, the measured EC50 was 160 μM and the average number of receptors per cell was 1.7 × 107. The decapeptide molecular imaging probe reached a maximum tumor to muscle ratio five hours after intravenous injection and a dose-dependent plateau after 24–48 hours. These results indicate the potential application of a small molecular weight imaging probe specific to ovarian cancer through binding to FSHR. Based on these results, multimeric constructs are being developed to optimize binding to ovarian cells and tumors. PMID:26779384

  14. 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

  15. The Therapeutic Potential of Toll-like Receptor 7 Stimulation in Asthma

    PubMed Central

    Drake, Matthew G.; Kaufman, Elad H.; Fryer, Allison D.; Jacoby, David B.

    2012-01-01

    Asthma is an inflammatory disorder of the airways frequently characterized by an excessive Th2 adaptive immune response. Activation of Toll-like receptor (TLR)-7, a single-stranded viral RNA receptor that is highly expressed in the airways, triggers a rapid innate immune response and favors a subsequent Th1 response. Because of this role in pulmonary immunoregulation, TLR7 has gained considerable interest as a therapeutic target in asthma. Synthetic TLR7 ligands, including the imidazoquinolines imiquimod (R837) and resiquimod (R848), and 8-hydroxyadenine derivatives have been developed for other clinical indications. TLR7 activation prevents ovalbumin-induced airway hyperreactivity, eosinophilic inflammation, goblet cell hyperplasia and airway remodeling in murine models of asthma. TLR7 activation also inhibits viral replication in the lung and prevents virus-induced airway hyperreactivity. Furthermore, it has recently been shown that stimulating TLR7 rapidly relaxes airway smooth muscle, dilating the airways. This bronchodilating effect, which occurs in seconds to minutes and depends on rapid production of nitric oxide, indicates that TLR7 can signal via previously unrecognized pathways. The effects of decreasing the allergic Th2 response, acting as an immediate bronchodilator, and promoting an antiviral immune environment, make TLR7 an attractive drug target. We examine the current understanding of TLR7 as a therapeutic target and its translation to asthma treatment in humans. PMID:23078048

  16. Monte Carlo loop refinement and virtual screening of the thyroid-stimulating hormone receptor transmembrane domain

    PubMed Central

    Ali, M. Rejwan; Latif, Rauf; Davies, Terry F.; Mezei, Mihaly

    2015-01-01

    Metropolis Monte Carlo (MMC) loop refinement has been performed on the three extracellular loops (ECLs) of rhodopsin and opsin-based homology models of the thyroid-stimulating hormone receptor transmembrane domain, a class A type G protein-coupled receptor. The Monte Carlo sampling technique, employing torsion angles of amino acid side chains and local moves for the six consecutive backbone torsion angles, has previously reproduced the conformation of several loops with known crystal structures with accuracy consistently less than 2 Å. A grid-based potential map, which includes van der Waals, electrostatics, hydrophobic as well as hydrogen-bond potentials for bulk protein environment and the solvation effect, has been used to significantly reduce the computational cost of energy evaluation. A modified sigmoidal distance-dependent dielectric function has been implemented in conjunction with the desolvation and hydrogen-bonding terms. A long high-temperature simulation with 2 kcal/mol repulsion potential resulted in extensive sampling of the conformational space. The slow annealing leading to the low-energy structures predicted secondary structure by the MMC technique. Molecular docking with the reported agonist reproduced the binding site within 1.5 Å. Virtual screening performed on the three lowest structures showed that the ligand-binding mode in the inter-helical region is dependent on the ECL conformations. PMID:25012978

  17. 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

  18. Activation of macrophages stimulated by the bengkoang fiber extract through toll-like receptor 4.

    PubMed

    Kumalasari, Ika Dyah; Nishi, Kosuke; Putra, Agus Budiawan Naro; Sugahara, Takuya

    2014-07-25

    Bengkoang (Pachyrhizus erosus (L.) Urban) is an edible root tuber containing fairly large amounts of carbohydrates and crude fibers. Our previous studies showed that the bengkoang fiber extract (BFE) stimulates activation of macrophages, leading to induction of phagocytotic activity and cytokine production. In the present study we investigated the mechanism underlying activation of murine macrophages by BFE. BFE increased production of TNF-α, IL-6, and nitric oxide by J774.1 cells. In addition BFE also facilitated the gene expression levels of inducible nitric oxide synthase. We examined the effect of a TLR4 inhibitor on cytokine production to investigate the membrane receptor of macrophage activation by BFE. Treatment of J774.1 cells with the TLR4 inhibitor significantly inhibited production of IL-6 and TNF-α, suggesting that TLR4 is the target membrane receptor for BFE. The main signal molecules located downstream of TLR4 such as JNK, p38, ERK, and NF-κB were activated by BFE treatment. The immunostimulatory effect of BFE was cancelled by the pectinase treatment, suggesting that the active ingredient in BFE is pectin-like molecules. Overall results suggested that BFE activates J774.1 cells via the MAPK and NF-κB signaling pathways. PMID:24770453

  19. Monte Carlo loop refinement and virtual screening of the thyroid-stimulating hormone receptor transmembrane domain.

    PubMed

    Ali, M Rejwan; Latif, Rauf; Davies, Terry F; Mezei, Mihaly

    2015-01-01

    Metropolis Monte Carlo (MMC) loop refinement has been performed on the three extracellular loops (ECLs) of rhodopsin and opsin-based homology models of the thyroid-stimulating hormone receptor transmembrane domain, a class A type G protein-coupled receptor. The Monte Carlo sampling technique, employing torsion angles of amino acid side chains and local moves for the six consecutive backbone torsion angles, has previously reproduced the conformation of several loops with known crystal structures with accuracy consistently less than 2 Å. A grid-based potential map, which includes van der Waals, electrostatics, hydrophobic as well as hydrogen-bond potentials for bulk protein environment and the solvation effect, has been used to significantly reduce the computational cost of energy evaluation. A modified sigmoidal distance-dependent dielectric function has been implemented in conjunction with the desolvation and hydrogen-bonding terms. A long high-temperature simulation with 2 kcal/mol repulsion potential resulted in extensive sampling of the conformational space. The slow annealing leading to the low-energy structures predicted secondary structure by the MMC technique. Molecular docking with the reported agonist reproduced the binding site within 1.5 Å. Virtual screening performed on the three lowest structures showed that the ligand-binding mode in the inter-helical region is dependent on the ECL conformations. PMID:25012978

  20. Alpha-melanocyte-stimulating hormone down-regulates CXC receptors through activation of neutrophil elastase.

    PubMed

    Manna, Sunil K; Sarkar, Abira; Sreenivasan, Yashin

    2006-03-01

    Considering the role of interleukin-8 (IL-8) in a large number of acute and chronic inflammatory diseases, the regulation of IL-8-mediated biological responses is important. Alpha-melanocyte-stimulating hormone (alpha-MSH), a tridecapeptide, inhibits most forms of inflammation by an unknown mechanism. In the present study, we have found that alpha-MSH interacts predominantly with melanocortin-1 receptors and inhibits several IL-8-induced biological responses in macrophages and neutrophils. It down-regulated receptors for IL-8 but not for TNF, IL-4, IL-13 or TNF-related apoptosis-inducing ligand (TRAIL) in neutrophils. It down-regulated CXCR type 1 and 2 but not mRNA levels. alpha-MSH did not inhibit IL-8 binding in purified cell membrane or affinity-purified CXCR. IL-8 or anti-CXCR Ab protected against alpha-MSH-mediated inhibition of IL-8 binding. The level of neutrophil elastase, a specific serine protease, but not cathepsin G or proteinase 3 increased in alpha-MSH-treated cells, and restoration of CXCR by specific neutrophil elastase or serine protease inhibitors indicates the involvement of elastase in alpha-MSH-induced down-regulation of CXCR. These studies suggest that alpha-MSH inhibits IL-8-mediated biological responses by down-regulating CXCR through induction of serine protease and that alpha-MSH acts as a potent immunomodulator in neutrophil-driven inflammatory distress. PMID:16479540

  1. δ-Opioid receptors stimulate the metabolic sensor AMP-activated protein kinase through coincident signaling with G(q/11)-coupled receptors.

    PubMed

    Olianas, Maria C; Dedoni, Simona; Olianas, Alessandra; Onali, Pierluigi

    2012-02-01

    AMP-activated protein kinase (AMPK) and δ-opioid receptors (DORs) are both involved in controlling cell survival, energy metabolism, and food intake, but little is known on the interaction between these two signaling molecules. Here we show that activation of human DORs stably expressed in Chinese hamster ovary (CHO) cells increased AMPK activity and AMPK phosphorylation on Thr172. DOR-induced AMPK phosphorylation was prevented by pertussis toxin, reduced by protein kinase A (PKA) activators, and unaffected by PKA, transforming growth factor-β-activated kinase 1, mitogen-activated protein kinase, and protein kinase C inhibitors. Conversely, the DOR effect was reduced by Ca(2+)/calmodulin-dependent protein kinase kinase (CaMKK) inhibition, apyrase treatment, G(q/11) antagonism, and blockade of P2 purinergic receptors. Apyrase treatment also depressed DOR stimulation of intracellular Ca(2+) concentration, whereas P2 receptor antagonism blocked DOR stimulation of inositol phosphate accumulation. In SH-SY5Y neuroblastoma cells and primary olfactory bulb neurons, DOR activation failed to affect AMPK phosphorylation per se but potentiated the stimulation by either muscarinic agonists or 2-methyl-thio-ADP. Sequestration of G protein βγ subunits (Gβγ) blocked the DOR potentiation of AMPK phosphorylation induced by oxotremorine-M. In CHO cells, the AMPK activator 5-aminoimidazole-4-carboxamide1-β-D-ribonucleoside stimulated AMPK phosphorylation and glucose uptake, whereas pharmacological inhibition of AMPK, expression of a dominant-negative mutant of AMPKα1, and P2Y receptor blockade reduced DOR-stimulated glucose uptake. The data indicate that in different cell systems, DOR activation up-regulates AMPK through a Gβγ-dependent synergistic interaction with G(q/11)-coupled receptors, potentiating Ca(2+) release and CaMKKβ-dependent AMPK phosphorylation. In CHO cells, this coincident signaling mechanism is involved in DOR-induced glucose uptake. PMID:22031472

  2. Vasopressin/V2 receptor stimulates renin synthesis in the collecting duct.

    PubMed

    Gonzalez, Alexis A; Cifuentes-Araneda, Flavia; Ibaceta-Gonzalez, Cristobal; Gonzalez-Vergara, Alex; Zamora, Leonardo; Henriquez, Ricardo; Rosales, Carla B; Navar, L Gabriel; Prieto, Minolfa C

    2016-02-15

    Renin is synthesized in the principal cells of the collecting duct (CD), and its production is increased via cAMP in angiotensin (ANG) II-dependent hypertension, despite suppression of juxtaglomerular (JG) renin. Vasopressin, one of the effector hormones of the renin-angiotensin system (RAS) via the type 2-receptor (V2R), activates the cAMP/PKA/cAMP response element-binding protein (CREB) pathway and aquaporin-2 expression in principal cells of the CD. Accordingly, we hypothesized that activation of V2R increases renin synthesis via PKA/CREB, independently of ANG II type 1 (AT1) receptor activation in CD cells. Desmopressin (DDAVP; 10(-6) M), a selective V2R agonist, increased renin mRNA (∼3-fold), prorenin (∼1.5-fold), and renin (∼2-fold) in cell lysates and cell culture media in the M-1 CD cell line. Cotreatment with DDAVP+H89 (PKA inhibitor) or CREB short hairpin (sh) RNA prevented this response. H89 also blunted DDAVP-induced CREB phosphorylation and nuclear localization. In 48-h water-deprived (WD) mice, prorenin-renin protein levels were increased in the renal inner medulla (∼1.4- and 1.8-fold). In WD mice treated with an ACE inhibitor plus AT1 receptor blockade, renin mRNA and prorenin protein levels were still higher than controls, while renin protein content was not changed. In M-1 cells, ANG II or DDAVP increased prorenin-renin protein levels; however, there were no further increases by combined treatment. These results indicate that in the CD the activation of the V2R stimulates renin synthesis via the PKA/CREB pathway independently of RAS, suggesting a critical role for vasopressin in the regulation of renin in the CD. PMID:26608789

  3. Homeobox A7 stimulates breast cancer cell proliferation by up-regulating estrogen receptor-alpha

    SciTech Connect

    Zhang, Yu; Cheng, Jung-Chien; Huang, He-Feng; Leung, Peter C.K.

    2013-11-01

    Highlights: •HOXA7 regulates MCF7 cell proliferation. •HOXA7 up-regulates ERα expression. •HOXA7 mediates estrogen-induced MCF7 cell proliferation. -- Abstract: Breast cancer is the most common hormone-dependent malignancy in women. Homeobox (HOX) transcription factors regulate many cellular functions, including cell migration, proliferation and differentiation. The aberrant expression of HOX genes has been reported to be associated with human reproductive cancers. Estradiol (E2) and its nuclear receptors, estrogen receptor (ER)-alpha and ER-beta, are known to play critical roles in the regulation of breast cancer cell growth. However, an understanding of the potential relationship between HOXA7 and ER in breast cancer cells is limited. In this study, our results demonstrate that knockdown of HOXA7 in MCF7 cells significantly decreased cell proliferation and ERα expression. In addition, HOXA7 knockdown attenuated E2-induced cell proliferation as well as progesterone receptor (PR) expression. The stimulatory effects of E2 on cell proliferation and PR expression were abolished by co-treatment with ICI 182780, a selective ERα antagonist. In contrast, overexpression of HOXA7 significantly stimulated cell proliferation and ERα expression. Moreover, E2-induced cell proliferation, as well as PR expression, was enhanced by the overexpression of HOXA7. Neither knockdown nor overexpression of HOXA7 affected the ER-beta levels. Our results demonstrate a novel mechanistic role for HOXA7 in modulating breast cancer cell proliferation via regulation of ERα expression. This finding contributes to our understanding of the role HOXA7 plays in regulating the proliferation of ER-positive cancer cells.

  4. Icariin Stimulates Differentiation and Suppresses Adipocytic Transdifferentiation of Primary Osteoblasts Through Estrogen Receptor-Mediated Pathway.

    PubMed

    Zhang, Dawei; Fong, Chichun; Jia, Zhenbin; Cui, Liao; Yao, Xinsheng; Yang, Mengsu

    2016-08-01

    Icariin, the main constituent of Herba Epimedii, appears to be a promising alternative to classic drugs used to treat osteoporosis. However, the detailed molecular mechanisms of its action and the role of icariin in the cross-talk between osteoblasts and adipocytes remain unclear. The present study was designed to investigate the gene expression profile of primary osteoblasts in the presence of icariin, and the effects of icariin on the differentiation and adipogenic transdifferentiation of osteoblasts. Cellular and molecular markers expressed during osteoblastic differentiation were assessed by cytochemical analysis, real-time quantitative PCR, Western blotting, and cDNA microarray analysis. Results indicated that icariin up-regulated the expression of runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2 (Bmp2), and collagen type 1 (Col1) genes, and down-regulated the expression of the peroxisome proliferator-activated receptor γ (Pparg) and CCAAT/enhancer-binding protein β (Cebpb) genes. These effects were blocked by ICI 182,780, suggesting that icariin may be acting via the estrogen receptor (ER). Results also demonstrated that the ratio of osteoprotegerin (Opg)/receptor activator of nuclear factor kappa B ligand (Rankl) expression was up-regulated following treatment with icariin. In total, osteoblastic gene expression profile analysis suggested that 33 genes were affected by icariin; these could be sub-divided into nine functional categories. It appears that icariin could stimulate the differentiation and mineralization of osteoblasts, regulate the differentiation of osteoclasts, and inhibit the adipogenic transdifferentiation of osteoblasts, therefore increasing the number of osteoblasts undergoing differentiation to mature osteoblasts, via an ER-mediated pathway. In summary, icariin may exhibit beneficial effects on bone health, especially for patients with osteoporosis and obesity. PMID:27061090

  5. Ceramide stimulates ABCA12 expression via peroxisome proliferator-activated receptor {delta} in human keratinocytes.

    PubMed

    Jiang, Yan J; Uchida, Yoshikazu; Lu, Biao; Kim, Peggy; Mao, Cungui; Akiyama, Masashi; Elias, Peter M; Holleran, Walter M; Grunfeld, Carl; Feingold, Kenneth R

    2009-07-10

    ABCA12 (ATP binding cassette transporter, family 12) is a cellular membrane transporter that facilitates the delivery of glucosylceramides to epidermal lamellar bodies in keratinocytes, a process that is critical for permeability barrier formation. Following secretion of lamellar bodies into the stratum corneum, glucosylceramides are metabolized to ceramides, which comprise approximately 50% of the lipid in stratum corneum. Gene mutations of ABCA12 underlie harlequin ichthyosis, a devastating skin disorder characterized by abnormal lamellar bodies and a severe barrier abnormality. Recently we reported that peroxisome proliferator-activated receptor (PPAR) and liver X receptor activators increase ABCA12 expression in human keratinocytes. Here we demonstrate that ceramide (C(2)-Cer and C(6)-Cer), but not C(8)-glucosylceramides, sphingosine, or ceramide 1-phosphate, increases ABCA12 mRNA expression in a dose- and time-dependent manner. Inhibitors of glucosylceramide synthase, sphingomyelin synthase, and ceramidase and small interfering RNA knockdown of human alkaline ceramidase, which all increase endogenous ceramide levels, also increased ABCA12 mRNA levels. Moreover, simultaneous treatment with C(6)-Cer and each of these same inhibitors additively increased ABCA12 expression, indicating that ceramide is an important inducer of ABCA12 expression and that the conversion of ceramide to other sphingolipids or metabolites is not required. Finally, both exogenous and endogenous ceramides preferentially stimulate PPARdelta expression (but not other PPARs or liver X receptors), whereas PPARdelta knockdown by siRNA transfection specifically diminished the ceramide-induced increase in ABCA12 mRNA levels, indicating that PPARdelta is a mediator of the ceramide effect. Together, these results show that ceramide, an important lipid component of epidermis, up-regulates ABCA12 expression via the PPARdelta-mediated signaling pathway, providing a substrate-driven, feed

  6. Nitric oxide stimulates the proliferation of neural stem cells bypassing the epidermal growth factor receptor.

    PubMed

    Carreira, Bruno Pereira; Morte, Maria Inês; Inácio, Angela; Costa, Gabriel; Rosmaninho-Salgado, Joana; Agasse, Fabienne; Carmo, Anália; Couceiro, Patrícia; Brundin, Patrik; Ambrósio, António Francisco; Carvalho, Caetana Monteiro; Araújo, Inês Maria

    2010-07-01

    Nitric oxide (NO) was described to inhibit the proliferation of neural stem cells. Some evidence suggests that NO, under certain conditions, can also promote cell proliferation, although the mechanisms responsible for a potential proliferative effect of NO in neural stem cells have remained unaddressed. In this work, we investigated and characterized the proliferative effect of NO in cell cultures obtained from the mouse subventricular zone. We found that the NO donor NOC-18 (10 microM) increased cell proliferation, whereas higher concentrations (100 microM) inhibited cell proliferation. Increased cell proliferation was detected rapidly following exposure to NO and was prevented by blocking the mitogen-activated kinase (MAPK) pathway, independently of the epidermal growth factor (EGF) receptor. Downstream of the EGF receptor, NO activated p21Ras and the MAPK pathway, resulting in a decrease in the nuclear presence of the cyclin-dependent kinase inhibitor 1, p27(KIP1), allowing for cell cycle progression. Furthermore, in a mouse model that shows increased proliferation of neural stem cells in the hippocampus following seizure injury, we observed that the absence of inducible nitric oxide synthase (iNOS(-/-) mice) prevented the increase in cell proliferation observed following seizures in wild-type mice, showing that NO from iNOS origin is important for increased cell proliferation following a brain insult. Overall, we show that NO is able to stimulate the proliferation of neural stem cells bypassing the EGF receptor and promoting cell division. Moreover, under pathophysiological conditions in vivo, NO from iNOS origin also promotes proliferation in the hippocampus. PMID:20506358

  7. Insulin Receptor Substrate 1, the Hub Linking Follicle-stimulating Hormone to Phosphatidylinositol 3-Kinase Activation.

    PubMed

    Law, Nathan C; Hunzicker-Dunn, Mary E

    2016-02-26

    The ubiquitous phosphatidylinositol 3-kinase (PI3K) signaling pathway regulates many cellular functions. However, the mechanism by which G protein-coupled receptors (GPCRs) signal to activate PI3K is poorly understood. We have used ovarian granulosa cells as a model to investigate this pathway, based on evidence that the GPCR agonist follicle-stimulating hormone (FSH) promotes the protein kinase A (PKA)-dependent phosphorylation of insulin receptor substrate 1 (IRS1) on tyrosine residues that activate PI3K. We report that in the absence of FSH, granulosa cells secrete a subthreshold concentration of insulin-like growth factor-1 (IGF-1) that primes the IGF-1 receptor (IGF-1R) but fails to promote tyrosine phosphorylation of IRS1. FSH via PKA acts to sensitize IRS1 to the tyrosine kinase activity of the IGF-1R by activating protein phosphatase 1 (PP1) to promote dephosphorylation of inhibitory Ser/Thr residues on IRS1, including Ser(789). Knockdown of PP1β blocks the ability of FSH to activate PI3K in the presence of endogenous IGF-1. Activation of PI3K thus requires both PKA-mediated relief of IRS1 inhibition and IGF-1R-dependent tyrosine phosphorylation of IRS1. Treatment with FSH and increasing concentrations of exogenous IGF-1 triggers synergistic IRS1 tyrosine phosphorylation at PI3K-activating residues that persists downstream through protein kinase B (AKT) and FOXO1 (forkhead box protein O1) to drive synergistic expression of genes that underlies follicle maturation. Based on the ability of GPCR agonists to synergize with IGFs to enhance gene expression in other cell types, PP1 activation to relieve IRS1 inhibition may be a more general mechanism by which GPCRs act with the IGF-1R to activate PI3K/AKT. PMID:26702053

  8. Role of CRF receptor 1 in central CRF-induced stimulation of colonic propulsion in rats.

    PubMed

    Martínez, V; Taché, Y

    2001-03-01

    The CRF receptor subtype mediating the colonic and gastric motor responses to central CRF was investigated in conscious rats. CRF (0.6 microg/rat) injected intracerebroventicularly (i.c.v.) or 1 h water avoidance stress stimulated defecation (pellet/60 min: 4.1+/-1.0 and 8.7+/-0.7 respectively vs. 0.3+/-0.3 in i.c.v. vehicle/no stress). The CRF receptor 1 (CRF-R1) antagonist, NBI-27914 (50-100 microg/rat) injected i.c.v., abolished the colonic response to i.c.v. CRF and dose-dependently reduced that induced by water avoidance stress. NBI-27914 (100 microg/rat) injected peripherally did not influence the defecatory response to stress. The peptide CRF-R1/R2 antagonist, astressin (10 microg/rat, i.c.v.) inhibited the colonic motor response to i.c.v. CRF and stress similarly as NBI-27914 injected i.c.v. at 100 microg/rat. Intracisternal (i.c.) injection of astressin (10 microg/rat) also completely prevented CRF (0.6 g, i.c.)-induced delayed gastric emptying while i.c. NBI-27914 (50 or 100 microg) had no effect. These results indicate a differential role of central CRF receptor subtypes in the colonic stimulatory and gastric inhibitory motor responses to central CRF and that the CRF component of stress-related activation of colonic expulsion is primarily mediated by CRF-R1. PMID:11222989

  9. Regulation of surface expression of the granulocyte/macrophage colony-stimulating factor receptor in normal human myeloid cells

    SciTech Connect

    Cannistra, S.A.; Groshek, P.; Griffin, J.D. ); Garlick, R.; Miller, J. )

    1990-01-01

    Recombinant human granulocyte/macrophage colony-stimulating factor (GM-CSF) exerts stimulatory effects on hematopoietic cells through binding to specific, high-affinity receptors. By using radiolabeled GM-CSF with high specific activity, the authors have investigated the factors and mechanisms that regulate GM-CSF receptor expression in normal human neutrophils, monocytes, and partially purified bone marrow myeloid progenitor cells. The neutrophil GM-CSF receptor was found to rapidly internalize in the presence of ligand through a mechanism that required endocytosis. Out of a large panel of naturally occurring humoral factors tested, only GM-CSF itself, tumor necrosis factor, and formyl-Met-Leu-Phe were found to down-regulate neutrophil GM-CSF receptor expression after a 2-hr exposure at biologically active concentrations. Since formyl-Met-Leu-Phe is known to stimulate neutrophil protein kinase C activity, they also tested the ability of protein kinase C agonists to modulate GM-CSF receptor expression. Phorbol 12-myristate 13-acetate, bryostatin-1, and 1,2-dioctanoylglycerol were found to induce rapid down-regulation of the GM-CSF receptor in neutrophils, monocytes, and partially purified myeloid progenitor cells, suggesting that this effect may be at least partially mediated by protein kinase C. These data suggest that certain activators of neutrophil function may negatively regulate their biological effects by inducing down-regulation of the GM-CSF receptor.

  10. Effects of left atrial receptor stimulation on carotid chemoreceptor-induced renal responses in dogs.

    PubMed

    Karim, F; al-Obaidi, M

    1992-10-01

    1. Dogs were anaesthetized with thiopentone sodium and alpha-chloralose and artificially ventilated. The carotid sinus regions were vascularly isolated and perfused with arterial or venous blood to stimulate the chemoreceptors. Left atrial receptors were stimulated by distending four balloons, three in the left pulmonary vein-atrial junctions and one in the left atrial appendage. Mean aortic pressure was held constant by means of a pressure control device. Atenolol and atropine (2.0 and 0.5 mg kg-1, respectively), and gallamine triethiodide (3.0 mg kg-1 h-1) were given I.V. Renal blood flow was measured by an electromagnetic flowmeter, glomerular filtration rate by creatinine clearance, urinary sodium by flame photometry and solute excretion by osmometry. 2. In fifteen tests in eight dogs (in one dog responses of both left and right kidneys were determined), at a constant aortic pressure (AoP) of 92.0 +/- 3.2 mmHg, and carotid sinus pressure (CSP) of 95.0 +/- 2.0 mmHg, stimulation of left atrial receptors with balloon inflation resulted in significant increases in renal blood flow (RBF) by 8.3 +/- 0.9 from 255.0 +/- 14.6 ml min-1 (100 g kidney weight)-1 (n = 9), in glomerular filtration rate (GFR) by 4.1 +/- 0.6 from 21.2 +/- 1.9 ml min-1 (100 g)-1, in filtration fraction (FF) by 0.04 +/- 0.003 from 0.20 +/- 0.01, in urine flow rate (V) by 0.08 +/- 0.02 from 0.33 +/- 0.05 ml min-1 (100 g)-1, in sodium excretion (UNaV) by 4.4 +/- 0.9 from 27.7 +/- 4.2 mumol min-1 (100 g)-1, in osmolar excretion (UosmV) by 62.0 +/- 5.6 from 303.0 +/- 28.3 mu osmol min-1 (100 g)-1, and in a decrease in free water clearance (CH2O) by 0.13 +/- 0.03 from -0.63 +/- 0.04 ml min-1 (100 g)-1. Left atrial pressure (LAP) and heart rate (HR) did not change significantly from 6.9 +/- 0.3 cmH2O, and 133.0 +/- 3.4 beats min-1 respectively.(ABSTRACT TRUNCATED AT 400 WORDS) PMID:1293287

  11. Activation of transmembrane bile acid receptor TGR5 stimulates insulin secretion in pancreatic {beta} cells

    SciTech Connect

    Kumar, Divya P.; Rajagopal, Senthilkumar; Mahavadi, Sunila; Mirshahi, Faridoddin; Grider, John R.; Murthy, Karnam S.; Sanyal, Arun J.

    2012-10-26

    Highlights: Black-Right-Pointing-Pointer G protein coupled receptor TGR5 is expressed in mouse and human islets. Black-Right-Pointing-Pointer TGR5 is coupled to activation of Gs and Ca{sup 2+} release via cAMP/Epac/PLC-{epsilon} pathway. Black-Right-Pointing-Pointer Activation of TGR5 by bile salts and selective ligands causes insulin secretion. Black-Right-Pointing-Pointer TGR5 could be a potential therapeutic target to treat diabetes. -- Abstract: Bile acids act as signaling molecules and stimulate the G protein coupled receptor, TGR5, in addition to nuclear farnesoid X receptor to regulate lipid, glucose and energy metabolism. Bile acid induced activation of TGR5 in the enteroendocrine cells promotes glucagon like peptide-1 (GLP-1) release, which has insulinotropic effect in the pancreatic {beta} cells. In the present study, we have identified the expression of TGR5 in pancreatic {beta} cell line MIN6 and also in mouse and human pancreatic islets. TGR5 selective ligands, oleanolic acid (OA) and INT-777 selectively activated G{alpha}{sub s} and caused an increase in intracellular cAMP and Ca{sup 2+}. OA and INT-777 also increased phosphoinositide (PI) hydrolysis and the increase was blocked by NF449 (a selective G{alpha}{sub s} inhibitor) or (U73122) (PI hydrolysis inhibitor). OA, INT-777 and lithocholic acid increased insulin release in MIN6 and human islets and the increase was inhibited by treatment with NF449, (U73122) or BAPTA-AM (chelator of calcium), but not with myristoylated PKI (PKA inhibitor), suggesting that the release is dependent on G{sub s}/cAMP/Ca{sup 2+} pathway. 8-pCPT-2 Prime -O-Me-cAMP, a cAMP analog, which activates Epac, but not PKA also stimulated PI hydrolysis. In conclusion, our study demonstrates that the TGR5 expressed in the pancreatic {beta} cells regulates insulin secretion and highlights the importance of ongoing therapeutic strategies targeting TGR5 in the control of glucose homeostasis.

  12. Stimulation of serotonin-1A receptors in mammals to alleviate motion sickness and emesis induced by chemical agents

    NASA Technical Reports Server (NTRS)

    Lucot, James B. (Inventor); Crampton, George H. (Inventor)

    1990-01-01

    A method for the alleviation of both motion sickness and chemically-induced emesis is provided which includes the administration of a nontoxic, therapeutically effective amount of a composition which stimulates serotonin-1A receptors in a mammal in need of such treatment. The preferred compounds for use are buspirone and 8-hydroxy-2(di-n-propylamino)-tetralin (8-OH-DPAT).

  13. Serum amyloid A stimulates macrophage foam cell formation via lectin-like oxidized low-density lipoprotein receptor 1 upregulation

    SciTech Connect

    Lee, Ha Young; Kim, Sang Doo; Baek, Suk-Hwan; Choi, Joon Hyuk; Cho, Kyung-Hyun; Zabel, Brian A.; Bae, Yoe-Sik

    2013-03-29

    Highlights: ► SAA induced macrophage foam cell formation. ► SAA stimulated upregulation of lectin-like oxidized low-density lipoprotein receptor 1 (LOX1). ► SAA-induced LOX1 expression and foam cell formation is mediated by JNK/NF-κB signaling. ► HDL-conjugated SAA also stimulates foam cell formation via LOX1 upregulation. ► The finding reveals a novel mechanism of action of SAA in the pathogenesis of atherosclerosis. -- Abstract: Elevated levels of serum amyloid A (SAA) is a risk factor for cardiovascular diseases, however, the role of SAA in the pathophysiology of atherosclerosis remains unclear. Here we show that SAA induced macrophage foam cell formation. SAA-stimulated foam cell formation was mediated by c-jun N-terminal kinase (JNK) signaling. Moreover, both SAA and SAA-conjugated high density lipoprotein stimulated the expression of the important scavenger receptor lectin-like oxidized low-density lipoprotein receptor 1 (LOX1) via nuclear factor-κB (NF-κB). A LOX1 antagonist carrageenan significantly blocked SAA-induced foam cell formation, indicating that SAA promotes foam cell formation via LOX1 expression. Our findings therefore suggest that SAA stimulates foam cell formation via LOX1 induction, and thus likely contributes to atherogenesis.

  14. Dopamine D1 receptor stimulation modulates the formation and retrieval of novel object recognition memory: Role of the prelimbic cortex

    PubMed Central

    Pezze, Marie A.; Marshall, Hayley J.; Fone, Kevin C.F.; Cassaday, Helen J.

    2015-01-01

    Previous studies have shown that dopamine D1 receptor antagonists impair novel object recognition memory but the effects of dopamine D1 receptor stimulation remain to be determined. This study investigated the effects of the selective dopamine D1 receptor agonist SKF81297 on acquisition and retrieval in the novel object recognition task in male Wistar rats. SKF81297 (0.4 and 0.8 mg/kg s.c.) given 15 min before the sampling phase impaired novel object recognition evaluated 10 min or 24 h later. The same treatments also reduced novel object recognition memory tested 24 h after the sampling phase and when given 15 min before the choice session. These data indicate that D1 receptor stimulation modulates both the encoding and retrieval of object recognition memory. Microinfusion of SKF81297 (0.025 or 0.05 μg/side) into the prelimbic sub-region of the medial prefrontal cortex (mPFC) in this case 10 min before the sampling phase also impaired novel object recognition memory, suggesting that the mPFC is one important site mediating the effects of D1 receptor stimulation on visual recognition memory. PMID:26277743

  15. Endothelin receptor B protects granulocyte macrophage colony-stimulating factor mRNA from degradation.

    PubMed

    Jungck, David; Knobloch, Jürgen; Körber, Sandra; Lin, Yingfeng; Konradi, Jürgen; Yanik, Sarah; Stoelben, Erich; Koch, Andrea

    2015-06-01

    Evidence is lacking on the differential effects of the two therapeutic concepts of endothelin receptor antagonists (ERAs): the blockade of only the endothelin receptor A (ETAR; selective antagonism) versus both ETAR and endothelin receptor B (ETBR; dual blockade). Ambrisentan, a selective ERA, and bosentan, a dual blocker, are both available for therapy. We hypothesized that there are differences in the potential of ERAs to ameliorate inflammatory processes in human airway smooth muscle cells (HASMCs) and aimed to unravel underlying mechanisms. We used HASMC culture, enzyme-linked immunosorbent assay, and quantitative reverse-transcription polymerase chain reaction. Tumor necrosis factor α (TNFα) induced transcription and expression of chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand 3 (CXCL3), granulocyte macrophage colony-stimulating factor (GM-CSF), and matrix metalloproteinase 12 (MMP12) in HASMCs. In concentration-response experiments, bosentan led to a significantly greater reduction of GM-CSF and MMP12 protein release than ambrisentan, whereas there was no significant difference in their effect on GM-CSF and MMP12 mRNA. Both ERAs reduced CXCL3 protein and mRNA equally but had no effect on CXCL2. Blocking mitogen-activated protein kinases revealed that both ETAR and ETBR signal through p38 mitogen-activated protein kinase, but ETBR also signals through extracellular signal-regulated kinase (ERK) 1/2 to induce GM-CSF expression. In the presence of the transcription inhibitor actinomycin D, bosentan, but not ambrisentan, reduced GM-CSF but not MMP12 or CXCL3 mRNA. In conclusion, blockade of each endothelin receptor subtype reduces GM-CSF transcription, but blocking ETBR additionally protects GM-CSF mRNA from degradation via ERK-1/2. Accordingly, blocking both ETAR and ETBR leads to a stronger reduction of TNFα-induced GM-CSF protein expression. This mechanism might be specific to GM-CSF. Our data stress the anti-inflammatory potential

  16. The NOP (ORL1) receptor antagonist Compound B stimulates mesolimbic dopamine release and is rewarding in mice by a non-NOP-receptor-mediated mechanism.

    PubMed

    Koizumi, Miwako; Sakoori, Kazuto; Midorikawa, Naoko; Murphy, Niall P

    2004-09-01

    1. Compound B (1-[(3R, 4R)-1-cyclooctylmethyl-3-hydroxymethyl-4-piperidyl]-3-ethyl-1,3-dihydro-2H-benzimidazol-2-one, CompB) is a nociceptin/orphanin FQ (N/OFQ) antagonist showing high selectivity for the NOP (ORL1) receptor over classical opioid receptors. We studied the effect of subcutaneous CompB administration on the release of mesolimbic dopamine (DA) and the expression of hedonia in mice. 2. CompB (0.3-30 mg kg(-1)) dose dependently stimulated mesolimbic DA release as measured by in vivo freely moving microdialysis, without any change in locomotor activity. However, intracerebroventricular administered N/OFQ (endogenous agonist of the NOP receptor, 6 nmol) did not influence CompB- (10 mg kg(-1)) induced DA release, despite clearly suppressing release when administered alone. 3. Studies using NOP receptor knockout mice and no-net-flux microdialysis revealed mildly, but not statistically significantly higher endogenous DA levels in mice lacking the NOP receptor compared to wild-type mice. Administration of CompB (10 mg kg(-1)) induced identical increases in mesolimbic DA release in wild-type and NOP receptor knockout mice. 4. CompB was rewarding in approximately the same dose range in which CompB induced major increases in mesolimbic DA release when assayed using a conditioned place preference paradigm. The rewarding effect of CompB (30 mg kg(-1)) was maintained in NOP receptor knockout mice. 5. These results show that CompB stimulates mesolimbic DA release and is rewarding by an action independent of the NOP receptor, the precise site of which is unclear. Consequently, caution should be exercised when interpreting the results of studies using this drug, particularly when administered by a peripheral route. PMID:15289286

  17. Thin-fibre receptors responding to mechanical, chemical, and thermal stimulation in the skeletal muscle of the dog

    PubMed Central

    Kumazawa, T.; Mizumura, K.

    1977-01-01

    1. Unitary activities of muscular thin fibre afferents, which were not sensitive to muscle stretching, were recorded from the nerve of the medial gastrocnemius muscle of the dog. Responses to mechanical stimulation, intra-arterial injection and local application of chemical solutions, and thermal stimulation of the surface of the muscle were studied. It was observed that polymodal receptors which responded to all types of stimulation existed in the thin fibre afferents of the muscle. 2. The receptive area of these units tested by mechanical stimulation was spot-like and appeared to be located not only on the surface but in the midst of the muscle. 3. The mechanical response varied among these units with respect to the threshold and the pattern of discharges. 4. In these units, NaCl, KCl, and bradykinin consistently evoked responses, with differences in the latencies and discharge patterns, while solutions of histamine, acetylcholine and sodium citrate caused responses less consistently and less effectively. In the stretch receptors, chemical stimulation applied in the same way as tested in the thin fibre afferents produced quite different features in their responses. 5. Heating the receptive area of the muscle surface caused responses in twenty-five out of thirty-six units, which were sensitive both to mechanical and to chemical stimulations. The threshold varied from 38·0 to 48·3 °C, with a mean of 43·1 °C for C fibre units and 41 °C for A-δ fibre units. 6. The responses to heating were consistently obtained in the units responding to the surface application of chemical solutions. However, the above response was never obtained in the units which did not respond to surface chemical stimulation but responded to intra-arterial injection. These results suggest a large population of polymodal receptors in the muscular thin fibre afferents. PMID:599419

  18. An Antagonistic Vascular Endothelial Growth Factor (VEGF) Variant Inhibits VEGF-Stimulated Receptor Autophosphorylation and Proliferation of Human Endothelial Cells

    NASA Astrophysics Data System (ADS)

    Siemeister, Gerhard; Schirner, Michael; Reusch, Petra; Barleon, Bernhard; Marme, Dieter; Martiny-Baron, Georg

    1998-04-01

    Vascular endothelial growth factor (VEGF) is a potent mitogen with a unique specificity for endothelial cells and a key mediator of aberrant endothelial cell proliferation and vascular permeability in a variety of human pathological situations, such as tumor angiogenesis, diabetic retinopathy, rheumatoid arthritis, or psoriasis. VEGF is a symmetric homodimeric molecule with two receptor binding interfaces lying on each pole of the molecule. Herein we report on the construction and recombinant expression of an asymmetric heterodimeric VEGF variant with an intact receptor binding interface at one pole and a mutant receptor binding interface at the second pole of the dimer. This VEGF variant binds to VEGF receptors but fails to induce receptor activation. In competition experiments, the heterodimeric VEGF variant antagonizes VEGF-stimulated receptor autophosphorylation and proliferation of endothelial cells. A 15-fold excess of the heterodimer was sufficient to inhibit VEGF-stimulated endothelial cell proliferation by 50%, and a 100-fold excess resulted in an almost complete inhibition. By using a rational approach that is based on the structure of VEGF, we have shown the feasibility to construct a VEGF variant that acts as an VEGF antagonist.

  19. NGF stimulation of erk phosphorylation is impaired by a point mutation in the transmembrane domain of trkA receptor.

    PubMed

    Monshipouri, M; Jiang, H; Lazarovici, P

    2000-01-01

    The nerve growth factor (NGF) trkA receptor is a transmembrane glycoprotein composed of a large extracellular ligand-binding region connected to the cytoplasmic tyrosine kinase region by a single transmembrane domain (TMD). To explore the role of TMD in the process of receptor activation, we substituted the hydrophobic amino-acid residue valine 432 with the charged amino-acid glutamic acid (designated V432E mutant) by utilizing in vitro site-directed mutagenesis. NIH 3T3 cells lacking endogenous NGF receptors were stably transfected with a pRc/CMV vector carrying either wild-type (trkA) or mutated (V432E) receptors. Stable transfectants were shown, using 125I-NGF binding and Western-blot analysis, to express the trkA recombinant receptors. Scatchard analysis revealed similar affinity for NGF in wild-type and V432E receptors. Although the level of basal trkA receptor tyrosine phosphorylation was higher in the mutant than in the wild-type, NGF stimulation of WT 11 and V432E transfectants resulted in a rapid increase in receptor tyrosine phosphorylation and of its intracellular adaptor protein SHC. In contrast to WT 11, V432E mutants showed very low levels of NGF-, and moderate levels of FGF-induced erks phosphorylation, respectively. Collectively, these findings suggest that a single substitution (V432E) in the trkA TMD results in a selective impairment of trkA-mediated erks signaling pathway. PMID:10854038

  20. Expression of transferrin receptors on mitogen-stimulated human peripheral blood lymphocytes: relation to cellular activation and related metabolic events.

    PubMed Central

    Galbraith, R M; Galbraith, G M

    1981-01-01

    Mitogen-activated normal human peripheral blood lymphocytes bind transferrin to specific membrane receptors. In this study, lymphocytes stimulated with phytohaemagglutinin for 0-66 hr were examined to determine the relation of this phenomenon to cellular activation and related metabolic events. Transferrin receptors were first detected at 20-24 hr. This event was consistently preceded by RNA and protein turnover which commenced during the first 6 hr of culture, whereas initiation of DNA synthesis was detected concurrently with the appearance of receptors or slightly later (24-30 hr). Exposure of cells to inhibitors of RNA and protein synthesis early during culture (at 0 or 24 hr) prevented the expression of transferrin receptors, but also caused generalized metabolic failure, and abrogated cellular activation. In contrast, later addition of these agents at 48 hr did not interfere significantly with the process of activation, but did suppress the terminal increase in receptor-bearing cells observed during the final 18 hr in control cultures lacking inhibitor. After deliberate thermal stripping of receptors from activated cells, the reappearance of membrance binding sites which normally occurred within 30 min, was also blocked by cycloheximide, puromycin and actinomycin D. However, similar inhibition of DNA which was induced by hydroxyurea had much less effect upon both the initial appearance of receptors and their reappearance after ligand-induced depletion. These results demonstrate that the appearance of transferrin receptors upon human lymphocytes is dependent upon cellular activation and requires synthesis of protein and RNA. PMID:6172372

  1. Stimulation of glutamate receptors in the ventral tegmental area is necessary for serotonin-2 receptor-induced increases in mesocortical dopamine release.

    PubMed

    Pehek, E A; Hernan, A E

    2015-04-01

    Modulation of dopamine (DA) released by serotonin-2 (5-HT2) receptors has been implicated in the mechanism of action of antipsychotic drugs. The mesocortical DA system has been implicated particularly in the cognitive deficits observed in schizophrenia. Agonism at 5-HT2A receptors in the prefrontal cortex (PFC) is associated with increases in cortical DA release. Evidence indicates that 5-HT2A receptors in the cortex regulate mesocortical DA release through stimulation of a "long-loop" feedback system from the PFC to the ventral tegmental area (VTA) and back. However, a causal role for VTA glutamate in the 5-HT2-induced increases in PFC DA has not been established. The present study does so by measuring 5-HT2 agonist-induced DA release in the cortex after infusions of glutamate antagonists into the VTA of the rat. Infusions of a combination of a N-methyl-d-aspartic acid (NMDA) (AP-5: 2-amino-5-phosphopentanoic acid) and an AMPA/kainate (CNQX: 6-cyano-7-nitroquinoxaline-2,3-dione) receptor antagonist into the VTA blocked the increases in cortical DA produced by administration of the 5-HT2 agonist DOI [(±)-2,5-dimethoxy-4-iodoamphetamine] (2.5mg/kg s.c.). These results demonstrate that stimulation of glutamate receptors in the VTA is necessary for 5-HT2 agonist-induced increases in cortical DA. PMID:25637799

  2. Angiotensin AT2 Receptor Stimulation Inhibits Early Renal Inflammation in Renovascular Hypertension

    PubMed Central

    Matavelli, Luis C.; Huang, Jiqian; Siragy, Helmy M.

    2011-01-01

    Angiotensin II type 2 receptor (AT2R) counteracts most effects of angiotensin II type 1 receptor (AT1R). We hypothesized that direct AT2R stimulation reduces renal production of the inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and transforming growth factor-β1 (TGF-β1) and enhances the production of nitric oxide (NO) and cyclic guanosine 3′,5′-monophosphate (cGMP) in the clipped kidney of 2-kidney, 1-clip (2K1C) hypertension rat model. We used Sprague-Dawley rats to evaluate changes in renal interstitial fluid recovery levels of TNF-α, IL-6, NO, and cGMP; renal expression of AT1R, AT2R, TGF-β1, TNF-α, and IL-6 in sham and 2K1C rats treated for 4 days with vehicle, AT2R agonist compound 21 (C21), or AT2R antagonist PD123319 (PD), alone and combined (n=6, each group). Systolic blood pressure increased significantly in 2K1C and was not influenced by any treatment. Clipped kidneys showed significant increases in renal expression of AT1R, AT2R, TNF-α, IL-6, TGF-β1 and decreases in NO and cGMP levels. These factors were not influenced by PD treatment. In contrast, C21 caused significant decrease in renal TNF-α, IL-6, TGF-β1 and an increase in NO and cGMP levels. Combined C21 and PD treatment partially reversed the observed C21 effects. Compared to sham, there were no significant changes in TNF-α, IL-6, TGF-β1, NO, or cGMP in the nonclipped kidneys of 2K1C animals. We conclude that direct AT2R stimulation reduces early renal inflammatory responses and improves production of NO and cGMP in renovascular hypertension independent of blood pressure reduction. PMID:21189405

  3. Hypoxia stimulates urokinase receptor expression through a heme protein-dependent pathway.

    PubMed

    Graham, C H; Fitzpatrick, T E; McCrae, K R

    1998-05-01

    Hypoxia underlies a number of biologic processes in which cellular migration and invasion occur. Because earlier studies have shown that the receptor for urokinase-type plasminogen activator (uPAR) may facilitate such events, we studied the effect of hypoxia on the expression of uPAR by first trimester human trophoblasts (HTR-8/SVneo) and human umbilical vein endothelial cells (HUVEC). Compared with control cells cultured under standard conditions (20% O2), HTR-8/SVneo cells and HUVEC cultured in 1% O2 expressed more uPAR, as determined by flow cytometric and [125I]-prourokinase ligand binding analyses. Increased uPAR expression paralleled increases in uPAR mRNA. The involvement of a heme protein in the hypoxia-induced expression of uPAR was suggested by the observations that culture of cells with cobalt chloride, or sodium 4, 5-dihydroxybenzene-1,3-disulfonate (Tiron), an iron-chelating agent, also stimulated uPAR expression, and that the hypoxia-induced uPAR expression was inhibited by adding carbon monoxide to the hypoxic atmosphere. Culture of HTR-8/SVneo cells with vascular endothelial growth factor (VEGF) did not increase uPAR mRNA levels, suggesting that the hypoxia-mediated effect on uPAR expression by these cells did not occur through a VEGF-dependent mechanism. The functional importance of these findings is suggested by the fact that HTR-8/SVneo cells cultured under hypoxia displayed higher levels of cell surface plasminogen activator activity and greater invasion through a reconstituted basement membrane. These results suggest that hypoxia may promote cellular invasion by stimulating the expression of uPAR through a heme protein-dependent pathway. PMID:9558386

  4. Role of urokinase and its receptor in basal and stimulated colonic epithelial cell migration in vitro

    PubMed Central

    Wilson, A; Gibson, P

    2000-01-01

    BACKGROUND—Migration of colonic epithelial cells is important for mucosal repair following injury. The urokinase (u-PA) system regulates migration in other cell types.
AIM—To examine the role of u-PA and its receptor (u-PAR) in colonic epithelial cell migration.
METHODS—Migration was assessed over 24 hours in circular wounds made in confluent monolayers of LIM1215 and Caco-2 human colon cancer cells. The function of u-PA and u-PAR was ablated with antisense oligonucleotides to block expression, with synthetic u-PA peptides to block interaction, and with aprotinin to block u-PA mediated proteolysis.
RESULTS—Migration was stimulated two to threefold by exogenous u-PA, an effect dependent on u-PAR binding but independent of u-PA mediated mitogenesis and proteolysis. Expression of u-PA and u-PAR was inhibited by 80% by the appropriate antisense oligonucleotide. Basal migration and the motogenic effects of butyrate, epidermal growth factor, and phorbol-12-myristate-13-acetate were suppressed by the u-PAR antisense oligonucleotide (40-60%) but were at best minimally affected following inhibition of u-PA expression and binding. 
CONCLUSIONS—In an in vitro model of wounded colonic epithelium, u-PAR promotes cell migration through mechanisms that are not exclusively dependent on u-PA binding. Therefore, u-PA and u-PAR may contribute to colonic mucosal repair in vivo.


Keywords: colon; migration; urokinase; urokinase receptor; epidermal growth factor; butyrate; protein kinase C PMID:10861271

  5. Differential Roles of GABAA Receptor Subtypes in Benzodiazepine-Induced Enhancement of Brain-Stimulation Reward

    PubMed Central

    Reynolds, Lauren M; Engin, Elif; Tantillo, Gabriella; Lau, Hew Mun; Muschamp, John W; Carlezon, William A; Rudolph, Uwe

    2012-01-01

    Benzodiazepines such as diazepam are widely prescribed as anxiolytics and sleep aids. Continued use of benzodiazepines, however, can lead to addiction in vulnerable individuals. Here, we investigate the neural mechanisms of the behavioral effects of benzodiazepines using the intracranial self-stimulation (ICSS) test, a procedure with which the reward-enhancing effects of these drugs can be measured. Benzodiazepines bind nonselectively to several different GABAA receptor subtypes. To elucidate the α subunit(s) responsible for the reward-enhancing effects of benzodiazepines, we examined mice carrying a histidine-to-arginine point mutation in the α1, α2, or α3 subunit, which renders the targeted subunit nonresponsive to diazepam, other benzodiazepines and zolpidem. In wild-type and α1-point-mutated mice, diazepam caused a dose-dependent reduction in ICSS thresholds (reflecting a reward-enhancing effect) that is comparable to the reduction observed following cocaine administration. This effect was abolished in α2- and α3-point-mutant mice, suggesting that these subunits are necessary for the reward-enhancing action of diazepam. α2 Subunits appear to be particularly important, since diazepam increased ICSS thresholds (reflecting an aversive-like effect) in α2-point-mutant animals. Zolpidem, an α1-preferring benzodiazepine-site agonist, had no reward-enhancing effects in any genotype. Our findings implicate α2 and α3 subunit containing GABAA receptors as key mediators of the reward-related effects of benzodiazepines. This finding has important implications for the development of new medications that retain the therapeutic effects of benzodiazepines but lack abuse liability. PMID:22763624

  6. Stimulation of the mineralocorticoid receptor improves memory in young and elderly healthy individuals.

    PubMed

    Hinkelmann, Kim; Wingenfeld, Katja; Kuehl, Linn K; Fleischer, Juliane; Heuser, Isabella; Wiedemann, Klaus; Otte, Christian

    2015-02-01

    Glucocorticoids play an important role in cognitive function and act on glucocorticoid receptors and mineralocorticoid receptors (MRs) in the brain. Previously, the blockade of the MR has been shown to impair visuospatial and working memory in healthy young men. Here, we investigated the effects of the MR agonist fludrocortisone on memory in young and elderly healthy individuals. Thirty-one young (mean age 25.4 ± 4.6 years) and 22 elderly (mean age 63.2 ± 8.2 years) healthy participants received the MR agonist fludrocortisone (0.4 mg) or placebo at least 3 days apart in a randomized, double-blind within-subject cross-over design. We measured verbal memory (auditory verbal learning test), nonverbal memory (Rey/Taylor complex figure test), and working memory (digit-span task). As expected, young participants performed significantly better than elderly individuals in visuospatial memory (effect of group: F = 42.7, p < 0.01), verbal memory (F = 33.1, p < 0.01), and working memory (digit-span backward: F = 4.5, p = 0.04). For visuospatial memory (F = 5.0, p = 0.03) and short-term and working memory (digit-span forward: F = 4.2, p = 0.05), we found a significant treatment effect indicating better memory performance after fludrocortisone compared with placebo across groups. In concert with the previous studies, our data suggest a role of the MR in memory function. A cognitive enhancing effect by MR stimulation warrants future studies. PMID:25442112

  7. Calcitonin Receptor-Zonula Occludens-1 Interaction Is Critical for Calcitonin-Stimulated Prostate Cancer Metastasis

    PubMed Central

    Aljameeli, Ahmed; Thakkar, Arvind; Thomas, Shibu; Lakshmikanthan, Vijaybasker; Iczkowski, Kenneth A.; Shah, Girish V.

    2016-01-01

    The role of neuroendocrine peptide calcitonin (CT) and its receptor (CTR) in epithelial cancer progression is an emerging concept with great clinical potential. Expression of CT and CTR is frequently elevated in prostate cancers (PCs) and activation of CT–CTR axis in non-invasive PC cells induces an invasive phenotype. Here we show by yeast-two hybrid screens that CTR associates with the tight junction protein Zonula Occludens-1 (ZO-1) via the interaction between the type 1 PDZ motif at the carboxy-terminus of CTR and the PDZ3 domain of ZO-1. Mutation of either the CTR C-PDZ-binding motif or the ZO-1-PDZ3 domain did not affect binding of CTR with its ligand or G-protein-mediated signaling but abrogated destabilizing actions of CT on tight junctions and formation of distant metastases by orthotopically implanted PC cells in nude mice, indicating that these PDZ domain interactions were pathologically relevant. Further, we observed CTR-ZO-1 interactions in PC specimens by proximity ligation immunohistochemistry, and identified that the number of interactions in metastatic PC specimens was several-fold larger than in non-metastatic PC. Our results for the first time demonstrate a mechanism by which PDZ-mediated interaction between CTR and ZO1 is required for CT-stimulated metastasis of prostate cancer. Since many receptors contain PDZ-binding motifs, this would suggest that PDZ-binding motif-adaptor protein interactions constitute a common mechanism for cancer metastasis. PMID:26934365

  8. The frequency of follicle stimulating hormone receptor gene polymorphisms in Iranian infertile men with azoospermia

    PubMed Central

    Gharesi-Fard, Behrouz; Ghasemi, Zahra; Shakeri, Saeed; Behdin, Shabnam; Aghaei, Fatemeh; Malek-Hosseini, Zahra

    2015-01-01

    Background: Azoospermia is the medical condition of a man not having any measurable level of sperm in his semen. Follicle stimulating hormone (FSH) is a member of the glycoprotein hormone family that plays an important role in human reproduction because of its essential role in normal spermatogenesis. Various Single Nucleotide Polymorphisms (SNPs) have been reported within FSH receptor (FSHR) gene that may affect the receptor function. Objective: The present study aimed to investigate the correlation between two FSHR SNPs at positions A919G, A2039G, and susceptibility to azoospermia in a group of Iranian azoospermic men. The association between FSH levels within the sera and A919G and A2039G alleles and genotypes were also investigated. Materials and Methods: This case control study was performed on 212 men with azoospermia (126 non-obstructive and 86 obstructive) and 200 healthy Iranian men. Two FSHR gene SNPs were genotyped using PCR-RFLP method. The relationship between FSH levels within the sera and A919G and A2039G alleles and genotypes were also investigated. Results: Statistical analysis indicated that at A919G position, AA genotype and A allele were more frequent in obstructive azoospermia cases compared to non-obstructive or normal men (p=0.001). Regarding A2039G polymorphisms, no significant difference was observed between both azoospermia groups and the controls. The mean level of serum FSH was higher in the non-obstructive men compared to the obstructive patients (23.8 versus 13.8, respectively, p= 0.04). Conclusion: The results of the present study indicated that the genetic polymorphisms in the FSHR gene might increase the susceptibility to azoospermia in Iranian men. PMID:26730241

  9. Serotonin stimulates lateral habenula via activation of the post-synaptic serotonin 2/3 receptors and transient receptor potential channels.

    PubMed

    Zuo, Wanhong; Zhang, Yong; Xie, Guiqin; Gregor, Danielle; Bekker, Alex; Ye, Jiang-Hong

    2016-02-01

    There is growing interest on the role of the lateral habenula (LHb) in depression, because it closely and bilaterally connects with the serotoninergic raphe nuclei. The LHb sends glutamate efferents to the raphe nuclei, while it receives serotoninergic afferents, and expresses a high density of serotonin (5-HT) receptors. Recent studies suggest that 5-HT receptors exist both in the presynaptic and postsynaptic sites of LHb neurons, and activation of these receptors may have different effects on the activity of LHb neurons. The current study focused on the effect of 5-HT on the postsynaptic membrane. We found that 5-HT initiated a depolarizing inward current (I((5-HTi))) and accelerated spontaneous firing in ∼80% of LHb neurons in rat brain slices. I((5-HTi)) was also induced by the 5-HT uptake blocker citalopram, indicating activity of endogenous 5-HT. I((5-HTi)) was diminished by 5-HT(2/3) receptor antagonists (ritanserin, SB-200646 or ondansetron), and activated by the selective 5-HT(2/3) agonists 1-(3-Chlorophenyl) piperazine hydrochloride or 1-(3-Chlorophenyl) biguanide hydrochloride. Furthermore, I((5-HTi)) was attenuated by 2-Aminoethyl diphenylborinate, a blocker of transient receptor potential channels, and an IP3 receptor inhibitor, indicating the involvement of transient receptor potential channels. These results demonstrate that the reciprocal connection between the LHb and the 5-HT system highlights a key role for 5-HT stimulation of LHb neurons that may be important in the pathogenesis of depression. PMID:26471419

  10. Epidermal growth factor–stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis

    PubMed Central

    Garay, Camilo; Judge, Gurjeet; Lucarelli, Stefanie; Bautista, Stephen; Pandey, Rohan; Singh, Tanveer; Antonescu, Costin N.

    2015-01-01

    Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits—membrane structures containing clathrin and other proteins—eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation. PMID:26246598

  11. Roles of mitogen activated protein kinases and EGF receptor in arsenite-stimulated matrix metalloproteinase-9 production

    SciTech Connect

    Cooper, Karen L.; Myers, Terrance Alix; Rosenberg, Martina; Chavez, Miquella; Hudson, Laurie G. . E-mail: lghudson@unm.edu

    2004-11-01

    The dermatotoxicity of arsenic is well established and epidemiological studies identify an increased incidence of keratinocytic tumors (basal cell and squamous cell carcinoma) associated with arsenic exposure. Little is known about the underlying mechanisms of arsenic-mediated skin carcinogenesis, but activation of mitogen-activated protein (MAP) kinases and subsequent regulation of downstream target genes may contribute to tumor promotion and progression. In this study, we investigated activation of the extracellular signal regulated kinase (ERK) and the stress-associated kinase p38 by arsenite in HaCat cells, a spontaneously immortalized human keratinocyte cell line. Arsenite concentrations {>=}100 {mu}M stimulate rapid activation of p38 and ERK MAP kinases. However, upon extended exposure (24 h), persistent stimulation of p38 and ERK MAP kinases was detected at low micromolar concentrations of arsenite. Although ERK and p38 were activated with similar time and concentration dependence, the mechanism of activation differed for these two MAP kinases. ERK activation by arsenite was fully dependent on the catalytic activity of the epidermal growth factor (EGF) receptor and partially dependent on Src-family kinase activity. In contrast, p38 activation was independent of EGF receptor or Src-family kinase activity. Arsenite-stimulated MAP kinase signal transduction resulted in increased production of matrix metalloproteinase (MMP)-9, an AP-1 regulated gene product. MMP-9 induction by arsenite was prevented when EGF receptor or MAP kinase signaling was inhibited. These studies indicate that EGF receptor activation is a component of arsenite-mediated signal transduction and gene expression in keratinocytes and that low micromolar concentrations of arsenite stimulate key signaling pathways upon extended exposure. Stimulation of MAP kinase cascades by arsenic and subsequent regulation of genes including c-fos, c-jun, and the matrix degrading proteases may play an important

  12. Eicosanoid receptor subtype-mediated opposing regulation of TLR-stimulated expression of astrocyte glial-derived neurotrophic factor

    PubMed Central

    Li, Xianwu; Cudaback, Eiron; Breyer, Richard M.; Montine, Kathleen S.; Keene, C. Dirk; Montine, Thomas J.

    2012-01-01

    A major therapeutic target for Parkinson's disease (PD) is providing increased glial-derived neurotrophic factor (GDNF) to dopaminergic neurons. We tested the hypothesis that innate immune activation increases astrocyte GDNF production and that this is regulated by specific eicosanoid receptors. Innate immune-activated primary murine astrocytes were assayed for GDNF expression and secretion. Controls were agent vehicle exposure and wild-type mice. Rank order for up to 10-fold selectively increased GDNF expression was activators of TLR3 > TLR2 or TLR4 > TLR9. TLR3 activator-stimulated GDNF expression was selectively JNK-dependent, followed cyclooxygenase (COX)-2, was coincident with membranous PGE2 synthase, and was not significantly altered by a nonspecific COX- or a COX-2-selective inhibitor. Specific eicosanoid receptors had opposing effects on TLR3 activator-induced GDNF expression: ∼60% enhancement by blocking or ablating of PGE2 receptor subtype 1 (EP1), ∼30% enhancement by activating PGF2α receptor or thromboxane receptor, or ∼15% enhancement by activating EP4. These results demonstrate functionally antagonistic eicosanoid receptor subtype regulation of innate immunity-induced astrocyte GDNF expression and suggest that selective inhibition of EP1 signaling might be a means to augment astrocyte GDNF secretion in the context of innate immune activation in diseased regions of brain in PD.—Li, X., Cudaback, E., Breyer, R. M., Montine, K. S., Keene, C. D., Montine, T. J. Eicosanoid receptor subtype-mediated opposing regulation of Toll-like receptor-stimulated expression of astrocyte glial-derived neurotrophic factor. PMID:22499581

  13. A Gα-Stimulated RapGEF Is a Receptor-Proximal Regulator of Dictyostelium Chemotaxis.

    PubMed

    Liu, Youtao; Lacal, Jesus; Veltman, Douwe M; Fusetti, Fabrizia; van Haastert, Peter J M; Firtel, Richard A; Kortholt, Arjan

    2016-06-01

    Chemotaxis, or directional movement toward extracellular chemical gradients, is an important property of cells that is mediated through G-protein-coupled receptors (GPCRs). Although many chemotaxis pathways downstream of Gβγ have been identified, few Gα effectors are known. Gα effectors are of particular importance because they allow the cell to distinguish signals downstream of distinct chemoattractant GPCRs. Here we identify GflB, a Gα2 binding partner that directly couples the Dictyostelium cyclic AMP GPCR to Rap1. GflB localizes to the leading edge and functions as a Gα-stimulated, Rap1-specific guanine nucleotide exchange factor required to balance Ras and Rap signaling. The kinetics of GflB translocation are fine-tuned by GSK-3 phosphorylation. Cells lacking GflB display impaired Rap1/Ras signaling and actin and myosin dynamics, resulting in defective chemotaxis. Our observations demonstrate that GflB is an essential upstream regulator of chemoattractant-mediated cell polarity and cytoskeletal reorganization functioning to directly link Gα activation to monomeric G-protein signaling. PMID:27237792

  14. Notoginsenoside R1 stimulates osteogenic function in primary osteoblasts via estrogen receptor signaling.

    PubMed

    Wang, Ting; Wan, Daqian; Shao, Lei; Dai, Jiezhi; Jiang, Chaoyin

    2015-10-16

    Notoginsenoside R1 (NGR1), a novel phytoestrogen isolated from Panax notoginseng, has been widely used in the treatment of microcirculatory diseases in Asian countries. Here we investigated the effect of NGR1 on osteoblast differentiation and mineralization process. Furthermore, we also evaluated NGR1's estrogenic properties, especially its effects on estrogen receptors (ERs). NGR1 activated the transcriptional activity of phosphorylated estrogen response element (pERE)-luciferase (Luc) and induced ERα phosphorylation in hBMSC. In addition, ER activation correlated with induction and was associated with osteoblast differentiation biomarkers including alkaline phosphatase activity and transcription of osteoblastic genes, e.g., type I collagen (COL1), osteonectin, osteocalcin (OC), runt related protein 2 (Runx2), and osterix. NGR1 also promoted the mineralization process of osteoblasts. The NGR1-induced effects were confirmed to be mediated by the ER by the observation that pretreatment of the osteoblasts with the ER antagonist, ICI 182,780 fully blocked the effects. Our results showed that NGR1 stimulates osteogenic differentiation of cultured osteoblasts by activating ER signaling and in turn might be a potential therapeutic alternative for the prevention and treatment of osteoporosis. PMID:26362186

  15. The non-benzodiazepine anxiolytic drug etifoxine causes a rapid, receptor-independent stimulation of neurosteroid biosynthesis.

    PubMed

    do Rego, Jean Luc; Vaudry, David; Vaudry, Hubert

    2015-01-01

    Neurosteroids can modulate the activity of the GABAA receptors, and thus affect anxiety-like behaviors. The non-benzodiazepine anxiolytic compound etifoxine has been shown to increase neurosteroid concentrations in brain tissue but the mode of action of etifoxine on neurosteroid formation has not yet been elucidated. In the present study, we have thus investigated the effect and the mechanism of action of etifoxine on neurosteroid biosynthesis using the frog hypothalamus as an experimental model. Exposure of frog hypothalamic explants to graded concentrations of etifoxine produced a dose-dependent increase in the biosynthesis of 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone and tetrahydroprogesterone, associated with a decrease in the production of dihydroprogesterone. Time-course experiments revealed that a 15-min incubation of hypothalamic explants with etifoxine was sufficient to induce a robust increase in neurosteroid synthesis, suggesting that etifoxine activates steroidogenic enzymes at a post-translational level. Etifoxine-evoked neurosteroid biosynthesis was not affected by the central-type benzodiazepine (CBR) receptor antagonist flumazenil, the translocator protein (TSPO) antagonist PK11195 or the GABAA receptor antagonist bicuculline. In addition, the stimulatory effects of etifoxine and the triakontatetraneuropeptide TTN, a TSPO agonist, were additive, indicating that these two compounds act through distinct mechanisms. Etifoxine also induced a rapid stimulation of neurosteroid biosynthesis from frog hypothalamus homogenates, a preparation in which membrane receptor signalling is disrupted. In conclusion, the present study demonstrates that etifoxine stimulates neurosteroid production through a membrane receptor-independent mechanism. PMID:25785994

  16. The Non-Benzodiazepine Anxiolytic Drug Etifoxine Causes a Rapid, Receptor-Independent Stimulation of Neurosteroid Biosynthesis

    PubMed Central

    do Rego, Jean Luc; Vaudry, David; Vaudry, Hubert

    2015-01-01

    Neurosteroids can modulate the activity of the GABAA receptors, and thus affect anxiety-like behaviors. The non-benzodiazepine anxiolytic compound etifoxine has been shown to increase neurosteroid concentrations in brain tissue but the mode of action of etifoxine on neurosteroid formation has not yet been elucidated. In the present study, we have thus investigated the effect and the mechanism of action of etifoxine on neurosteroid biosynthesis using the frog hypothalamus as an experimental model. Exposure of frog hypothalamic explants to graded concentrations of etifoxine produced a dose-dependent increase in the biosynthesis of 17-hydroxypregnenolone, dehydroepiandrosterone, progesterone and tetrahydroprogesterone, associated with a decrease in the production of dihydroprogesterone. Time-course experiments revealed that a 15-min incubation of hypothalamic explants with etifoxine was sufficient to induce a robust increase in neurosteroid synthesis, suggesting that etifoxine activates steroidogenic enzymes at a post-translational level. Etifoxine-evoked neurosteroid biosynthesis was not affected by the central-type benzodiazepine (CBR) receptor antagonist flumazenil, the translocator protein (TSPO) antagonist PK11195 or the GABAA receptor antagonist bicuculline. In addition, the stimulatory effects of etifoxine and the triakontatetraneuropeptide TTN, a TSPO agonist, were additive, indicating that these two compounds act through distinct mechanisms. Etifoxine also induced a rapid stimulation of neurosteroid biosynthesis from frog hypothalamus homogenates, a preparation in which membrane receptor signalling is disrupted. In conclusion, the present study demonstrates that etifoxine stimulates neurosteroid production through a membrane receptor-independent mechanism. PMID:25785994

  17. Mitochondrial respiratory chain is involved in insulin-stimulated hydrogen peroxide production and plays an integral role in insulin receptor autophosphorylation in neurons

    PubMed Central

    Storozhevykh, Tatiana P; Senilova, Yana E; Persiyantseva, Nadezhda A; Pinelis, Vsevolod G; Pomytkin, Igor A

    2007-01-01

    Background Accumulated evidence suggests that hydrogen peroxide (H2O2) generated in cells during insulin stimulation plays an integral role in insulin receptor signal transduction. The role of insulin-induced H2O2 in neuronal insulin receptor activation and the origin of insulin-induced H2O2 in neurons remain unclear. The aim of the present study is to test the following hypotheses (1) whether insulin-induced H2O2 is required for insulin receptor autophosphorylation in neurons, and (2) whether mitochondrial respiratory chain is involved in insulin-stimulated H2O2 production, thus playing an integral role in insulin receptor autophosphorylation in neurons. Results Insulin stimulation elicited rapid insulin receptor autophosphorylation accompanied by an increase in H2O2 release from cultured cerebellar granule neurons (CGN). N-acetylcysteine (NAC), a H2O2 scavenger, inhibited both insulin-stimulated H2O2 release and insulin-stimulated autophosphorylation of insulin receptor. Inhibitors of respiratory chain-mediated H2O2 production, malonate and carbonyl cyanide-4-(trifluoromethoxy)-phenylhydrazone (FCCP), inhibited both insulin-stimulated H2O2 release from neurons and insulin-stimulated autophosphorylation of insulin receptor. Dicholine salt of succinic acid, a respiratory substrate, significantly enhanced the effect of suboptimal insulin concentration on the insulin receptor autophosphorylation in CGN. Conclusion Results of the present study suggest that insulin-induced H2O2 is required for the enhancement of insulin receptor autophosphorylation in neurons. The mitochondrial respiratory chain is involved in insulin-stimulated H2O2 production, thus playing an integral role in the insulin receptor autophosphorylation in neurons. PMID:17919343

  18. mu-Opioid receptor stimulation in the nucleus accumbens elevates fatty tastant intake by increasing palatability and suppressing satiety signals.

    PubMed

    Katsuura, Yoshihiro; Heckmann, Jennifer A; Taha, Sharif A

    2011-07-01

    Infusion of a μ-opioid receptor (MOR) agonist into the nucleus accumbens (NAcc) drives voracious food intake, an effect hypothesized to occur through increased tastant palatability. While intake of many palatable foods is elevated by MOR stimulation, this manipulation has a preferential effect on fatty food ingestion. Consumption of high-fat foods is increased by NAcc MOR stimulation even in rats that prefer a carbohydrate-rich alternative under baseline conditions. This suggests that NAcc MOR stimulation may not simply potentiate palatability signals and raises the possibility that mechanisms mediating fat intake may be distinct from those underlying intake of other tastants. The present study was conducted to investigate the physiological mechanisms underlying the effects of NAcc MOR stimulation on fatty food intake. In experiment 1, we analyzed lick microstructure in rats ingesting Intralipid to identify the changes underlying feeding induced by infusion of a MOR-specific agonist into the NAcc. MOR stimulation in the NAcc core, but not shell, increased burst duration and first-minute licks, while simultaneously increasing the rate and duration of Intralipid ingestion. These results suggest that MOR activation in the core increases Intralipid palatability and attenuates inhibitory postingestive feedback. In experiment 2, we measured the effects of MOR stimulation in the NAcc core on consumption of nonnutritive olestra. A MOR-specific agonist dose dependently increased olestra intake, demonstrating that caloric signaling is not required for hyperphagia induced by NAcc MOR stimulation. Feeding induced by drug infusion in both experiments 1 and 2 was blocked by a MOR antagonist. In experiment 3, we determined whether MOR activation in the NAcc core could attenuate satiety-related signaling caused by infusion of the melanocortin agonist MTII into the third ventricle. Suppression of intake caused by MTII was reversed by MOR stimulation. Together, our results suggest

  19. Activation of Distinct P2Y Receptor Subtypes Stimulates Insulin Secretion in MIN6 Mouse Pancreatic β Cells

    PubMed Central

    Balasubramanian, Ramachandran; de Azua, Inigo Ruiz; Wess, Jürgen; Jacobson, Kenneth A.

    2010-01-01

    Extracellular nucleotides and their receptor antagonists have therapeutic potential in disorders such as inflammation, brain disorders, and cardiovascular diseases. Pancreatic β cells express several purinergic receptors, and reported nucleotide effects on insulin secretion are contradictory. We studied the effect of P2Y receptors on insulin secretion and cell death in MIN6, mouse pancreatic β cells. Expression of P2Y1 and P2Y6 receptors was revealed by total mRNA analysis using RT-PCR. MIN6 cells were stimulated in the presence of 16.7 mM glucose with or without P2Y1 and P2Y6 agonists, 2-MeSADP and Up3U, respectively. Both the agonists increased insulin secretion with EC50 values of 44.6±7.0 nM and 30.7±12.7 nM respectively. The insulin secretion by P2Y1 and P2Y6 agonists was blocked by their selective antagonists MRS2179 and MRS2578, respectively. Binding of the selective P2Y1 receptor antagonist radioligand [125I]MRS2500 in MIN6 cell membranes was saturable (KD 4.74±0.47 nM), and known P2Y1 ligands competed with high affinities. Inflammation and glucose toxicity leads to pancreatic β cell death in diabetes. Flow cytometric analysis revealed that Up3U but not 2-MeSADP protected MIN6 cells against TNF-α induced apoptosis. Overall, the results demonstrate that selective stimulation of P2Y1 and P2Y6 receptors increases insulin secretion that accompanies intracellular calcium release, suggesting potential application of P2Y receptor ligands in the treatment of diabetes. PMID:20067775

  20. Stimulation of LDL receptor activity in Hep-G2 cells by a serum factor(s)

    SciTech Connect

    Ellsworth, J.L.; Brown, C.; Cooper, A.D.

    1988-05-01

    The regulation of low-density lipoprotein (LDL) receptor activity in the human hepatoma cell line Hep-G2 by serum components was examined. Incubation of dense monolayers of Hep-G2 cells with fresh medium containing 10% fetal calf serum (FM) produced a time-dependent increase in LDL receptor activity. Uptake and degradation of 125I-LDL was stimulated two- to four-fold, as compared with that of Hep-G2 cells cultured in the same media in which they had been grown to confluence (CM); the maximal 125I-LDL uptake plus degradation increased from 0.2 microgram/mg cell protein/4 h to 0.8 microgram/mg cell protein/4 h. In addition, a two-fold increase in cell surface binding of 125I-LDL to Hep-G2 cells was observed when binding was measured at 4 degrees C. There was no change in the apparent Kd. The stimulation of LDL receptor activity was suppressed in a concentration-dependent manner by the addition of cholesterol, as LDL, to the cell medium. In contrast to the stimulation of LDL receptor activity, FM did not affect the uptake or degradation of 125I-asialoorosomucoid. Addition of FM increased the protein content per dish, and DNA synthesis was stimulated approximately five-fold, as measured by (3H)thymidine incorporation into DNA; however, the cell number did not change. Cellular cholesterol biosynthesis was also stimulated by FM; (14C)acetate incorporation into unesterified and esterified cholesterol was increased approximately five-fold. Incubation of Hep-G2 cells with high-density lipoproteins (200 micrograms protein/ml) or albumin (8.0 mg/ml) in the absence of the serum factor did not significantly increase the total processed 125I-LDL. Stimulation of LDL receptor activity was dependent on a heat-stable, nondialyzable serum component that eluted in the inclusion volume of a Sephadex G-75 column.

  1. Stimulation of renin release by prostaglandin E2 is mediated by EP2 and EP4 receptors in mouse kidneys.

    PubMed

    Schweda, Frank; Klar, Jürgen; Narumiya, Shuh; Nüsing, Rolf M; Kurtz, Armin

    2004-09-01

    PGE(2) is a potent stimulator of renin release. So far, the contribution of each of the four PGE(2) receptor subtypes (EP(1)-EP(4)) in the regulation of renin release has not been characterized. Therefore, we investigated the effects PGE(2) on renin secretion rates (RSR) from isolated, perfused kidneys of EP(1)-/-, EP(2)-/-, EP(3)-/-, EP(4)-/-, and wild-type mice. PGE(2) concentration dependently stimulated RSR from kidneys of all four knockout strains with a threshold concentration of 1 nM in EP(1)-/-, EP(2)-/-, EP(3)-/-, and wild-type mice, whereas the threshold concentration was shifted to 10 nM in EP(4)-/- mice. Moreover, the maximum stimulation of RSR by PGE(2) at 1 microM was significantly reduced in EP(4)-/- (12.8-fold of control) and EP(2)-/- (15.9-fold) compared with wild-type (20.7-fold), EP(1)-/- (23.8-fold), and EP(3)-/- (20.1-fold). In contrast, stimulation of RSR by either the loop diuretic bumetanide or the beta-adrenoceptor agonist isoproterenol was similar in all strains. PGE(2) exerted a dual effect on renal vascular tone, inducing vasodilatation at low concentrations (1 nmol/) and vasoconstriction at higher concentrations (100 nmol/) in kidneys of wild-type mice. In kidneys of EP(2)-/- as well as EP(4)-/- mice, vasodilatation at low PGE(2) concentrations was prevented, whereas vasoconstriction at higher concentrations was augmented. In contrast, the vasodilatory component was pronounced in kidneys of EP(1) and EP(3) knockout mice, whereas in both genotypes the vasoconstriction at higher PGE(2) concentrations was markedly blunted. Our data provide evidence that PGE(2) stimulates renin release via activation of EP(2) and EP(4) receptors, whereas EP(1) and EP(3) receptors appear to be without functional relevance in juxtaglomerular cells. In contrast, all four receptor subtypes are involved in the control of renal vascular tone, EP(1) and EP(3) receptors increasing, and EP(2) as well as EP(4) receptors, decreasing it. PMID:15113745

  2. Lipoic acid stimulates cAMP production via G protein-coupled receptor-dependent and -independent mechanisms.

    PubMed

    Salinthone, Sonemany; Schillace, Robynn V; Tsang, Catherine; Regan, John W; Bourdette, Dennis N; Carr, Daniel W

    2011-07-01

    Lipoic acid (LA) is a naturally occurring fatty acid that exhibits anti-oxidant and anti-inflammatory properties and is being pursued as a therapeutic for many diseases including multiple sclerosis, diabetic polyneuropathy and Alzheimer's disease. We previously reported on the novel finding that racemic LA (50:50 mixture of R-LA and S-LA) stimulates cAMP production, activates prostanoid EP2 and EP4 receptors and adenylyl cyclases (AC), and suppresses activation and cytotoxicity in NK cells. In this study, we present evidence that furthers our understanding of the mechanisms of action of LA. Using various LA derivatives, such as dihydrolipoic acid (DHLA), S,S-dimethyl lipoic acid (DMLA) and lipoamide (LPM), we discovered that only LA is capable of stimulating cAMP production in NK cells. Furthermore, there is no difference in cAMP production after stimulation with either R-LA, S-LA or racemic LA. Competition and synergistic studies indicate that LA may also activate AC independent of the EP2 and EP4 receptors. Pretreatment of PBMCs with KH7 (a specific peptide inhibitor of soluble AC) and the calcium inhibitor (Bapta) prior to LA treatment resulted in reduced cAMP levels, suggesting that soluble AC and calcium signaling mediate LA stimulation of cAMP production. In addition, pharmacological inhibitor studies demonstrate that LA also activates other G protein-coupled receptors, including histamine and adenosine but not the β-adrenergic receptors. These novel findings provide information to better understand the mechanisms of action of LA, which can help facilitate the use of LA as a therapeutic for various diseases. PMID:21036588

  3. Stimulation of 5-HT1B receptors enhances cocaine reinforcement yet reduces cocaine-seeking behavior

    PubMed Central

    Pentkowski, Nathan S.; Acosta, Jazmin I.; Browning, Jenny R.; Hamilton, Elizabeth C.; Neisewander, Janet L.

    2010-01-01

    Paradoxically, stimulation of 5-HT1B receptors (5-HT1BRs) enhances sensitivity to the reinforcing effects of cocaine but attenuates incentive motivation for cocaine as measured using the extinction/reinstatement model. We revisited this issue by examining the effects of a 5-HT1BR agonist, CP94253, on cocaine reinforcement and cocaine-primed reinstatement, predicting that CP94253would enhance cocaine-seeking behavior reinstated by a low priming dose, similar to its effect on cocaine reinforcement. Rats were trained to self-administer cocaine (0.75 mg/kg, i.v.) paired with light and tone cues. For reinstatement experiments, they then underwent daily extinction training to reduce cocaine-seeking behavior (operant responses without cocaine reinforcement). Next, they were pre-treated with CP94253 (3–10 mg/kg, s.c.) and either tested for cocaine-primed (10 or 2.5 mg/kg, i.p.) or cue-elicited reinstatement of extinguished cocaine-seeking behavior. For reinforcement, effects of CP94253 (5.6 mg/kg) across a range of self-administered cocaine doses (0–1.5 mg/kg, i.v.) were examined. Cocaine dose-dependently reinstated cocaine-seeking behavior, but contrary to our prediction, CP94253 reduced reinstatement with both priming doses. Similarly, CP94253 reduced cue-elicited reinstatement. In contrast, CP94253 shifted the self-administration dose-effect curve leftward, consistent with enhanced cocaine reinforcement. When saline was substituted for cocaine, CP94253 reduced response rates (i.e. cocaine-seeking behavior). In subsequent control experiments, CP94253 decreased open-arm exploration in an elevated plus-maze suggesting an anxiogenic effect, but had no effect on locomotion or sucrose reinforcement. These results provide strong evidence that stimulation of 5-HT1BRs produces opposite effects on cocaine reinforcement and cocaine-seeking behavior, and further suggest that 5-HT1BRs may be a novel target for developing medications for cocaine dependence. PMID:19650818

  4. Stimulation of 5-HT(1B) receptors enhances cocaine reinforcement yet reduces cocaine-seeking behavior.

    PubMed

    Pentkowski, Nathan S; Acosta, Jazmin I; Browning, Jenny R; Hamilton, Elizabeth C; Neisewander, Janet L

    2009-09-01

    Paradoxically, stimulation of 5-HT(1B) receptors (5-HT(1B)Rs) enhances sensitivity to the reinforcing effects of cocaine but attenuates incentive motivation for cocaine as measured using the extinction/reinstatement model. We revisited this issue by examining the effects of a 5-HT(1B)R agonist, CP94253, on cocaine reinforcement and cocaine-primed reinstatement, predicting that CP94253 would enhance cocaine-seeking behavior reinstated by a low priming dose, similar to its effect on cocaine reinforcement. Rats were trained to self-administer cocaine (0.75 mg/kg, i.v.) paired with light and tone cues. For reinstatement experiments, they then underwent daily extinction training to reduce cocaine-seeking behavior (operant responses without cocaine reinforcement). Next, they were pre-treated with CP94253 (3-10 mg/kg, s.c.) and either tested for cocaine-primed (10 or 2.5 mg/kg, i.p.) or cue-elicited reinstatement of extinguished cocaine-seeking behavior. For reinforcement, effects of CP94253 (5.6 mg/kg) across a range of self-administered cocaine doses (0-1.5 mg/kg, i.v.) were examined. Cocaine dose-dependently reinstated cocaine-seeking behavior, but contrary to our prediction, CP94253 reduced reinstatement with both priming doses. Similarly, CP94253 reduced cue-elicited reinstatement. In contrast, CP94253 shifted the self-administration dose-effect curve leftward, consistent with enhanced cocaine reinforcement. When saline was substituted for cocaine, CP94253 reduced response rates (i.e. cocaine-seeking behavior). In subsequent control experiments, CP94253 decreased open-arm exploration in an elevated plus-maze suggesting an anxiogenic effect, but had no effect on locomotion or sucrose reinforcement. These results provide strong evidence that stimulation of 5-HT(1B)Rs produces opposite effects on cocaine reinforcement and cocaine-seeking behavior, and further suggest that 5-HT(1B)Rs may be a novel target for developing medications for cocaine dependence. PMID:19650818

  5. Enhanced type 1alpha metabotropic glutamate receptor-stimulated phosphoinositide signaling after pertussis toxin treatment.

    PubMed

    Carruthers, A M; Challiss, R A; Mistry, R; Saunders, R; Thomsen, C; Nahorski, S R

    1997-09-01

    The regulation of phosphoinositide hydrolysis by the type 1alpha metabotropic glutamate receptor (mGluR1alpha) was investigated in stably transfected baby hamster kidney (BHK) cells. Incubation of the cells with L-glutamate, quisqualate, and 1-aminocyclopentane-1S, 3R-dicarboxylic acid resulted in a marked accumulation of [3H]inositol monophosphate (InsP1) and inositol-1,4,5-trisphosphate [Ins(1,4,5)P3] mass in a time- and concentration-dependent manner. Pretreatment of BHK-mGluR1alpha cells with pertussis toxin [ 100 ng/ml, 24 hr] led to a dramatic 12-16-fold increase in the accumulation of [3H]InsP1 and a 2-fold increase in Ins(1,4,5)P3 in the absence of added agonist. Although only very low levels (/=75%, and the EC50 shifted leftward by 65-fold [-log EC50 values (molar), 7.26 +/- 0.23 versus 5.45 +/- 0.07; n = 4) in PTX-treated compared with control cells. In contrast, antagonist effects on agonist-stimulated [3H]InsP1 responses were similar in control and PTX-treated BHK-mGluR1alpha cells. These changes in the concentration-effect curves for mGluR agonists are consistent with a model in which the receptor associates with PTX-sensitive inhibitory (Gi/o) and PTX-insensitive stimulatory (Gq/11) G proteins that can each influence PIC activity. The present observations are consistent with a dual regulation of mGluR1alpha-mediated PIC activity that could be fundamental in

  6. Endothelin-stimulated secretion of natriuretic peptides by rat atrial myocytes is mediated by endothelin A receptors.

    PubMed

    Thibault, G; Doubell, A F; Garcia, R; Larivière, R; Schiffrin, E L

    1994-03-01

    Endothelin (ET), a potent vasoconstrictor peptide, is known to enhance the secretion of atrial natriuretic factor (ANF) by the heart. In the present study, we investigated the potency of ET isopeptides to stimulate ANF and brain natriuretic peptide (BNP) secretion in primary cultures of neonatal atrial myocytes, and we characterized the receptor mediating these effects. All ET isopeptides caused a twofold increase of ANF and BNP secretion with the following order of potency: ET-1 approximately ET-2 > sarafotoxin 6b > ET-3. Secretion of the natriuretic peptides was blocked by BQ-123, an ETA-receptor antagonist, but was not affected by either IRL-1620 or [Ala1,3,11,15]ET-1, two ETB-receptor agonists. ET receptors were localized by autoradiography on the surface of atrial myocytes, indicating that contaminating cells were not responsible for 125I-ET-1 binding. Competition binding analyses were then used to assess the ET-receptor subtype on atrial myocyte membrane preparations. A high-affinity (100 pmol/L) binding site with high density (approximately 1500 fmol/mg) was found to preferentially bind the ET isopeptides in the following order: ET-1 > or = ET-2 > or = sarafotoxin 6b > ET-3. Binding was totally displaced by BQ-123 but not by IRL-1620. The ET binding site therefore had the characteristics of an ETA-like receptor. Analysis by cross-linking and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that it possessed a molecular mass of approximately 50 kD. Northern blot analysis of both ETA- and ETB-receptor mRNAs allowed only the detection of the former, indicating that the ETB receptor may be expressed in very small amounts. These results demonstrate that ANF and BNP secretion by atrial myocytes is enhanced by ET via binding to an ETA-like receptor. PMID:8118954

  7. Modulation of the vagal bradycardia evoked by stimulation of upper airway receptors by central 5-HT1 receptors in anaesthetized rabbits

    PubMed Central

    Dando, Simon B; Skinner, Matthew R; Jordan, David; Ramage, Andrew G

    1998-01-01

    The effects of central application of 5-HT1A and 5-HT1B/1D receptor ligands on the reflex bradycardia, apnoea, renal sympathoexcitation and pressor response evoked by stimulating upper airway receptors with smoke in atenolol-pretreated anaesthetized rabbits were studied.Intracisternal administration of the 5-HT1A receptor antagonists WAY-100635 (100 μg kg−1) and (−)pindolol (100 μg kg−1) significantly reduced the smoke-induced bradycardia, attenuated the pressor response and in the case of (−)pindolol, sympathetic nerve activity. The same dose of WAY-100635 i.v. was without effect.Buspirone (200 μg kg−1, i.c.) potentiated the reflex bradycardia. This action was prevented if the animals were pretreated with WAY-100635 (100 μg kg−1, i.v.)(+)8-OH-DPAT (25 μg kg−1, i.c.) attenuated the evoked bradycardia, pressor response, apnoea and renal sympathoexcitation. The attenuation of the apnoea and renal sympathoexcitation, but not the bradycardia or pressor response was prevented in animals pretreated with WAY-100635 (100 μg kg−1, i.v.). The attenuation of the reflex bradycardia and the reduction in the renal sympathoexcitation were reduced by pretreatment with the 5-HT1B/1D receptor antagonist GR127935 (100 μg kg−1, i.v.).In WAY-100635 (100 μg kg−1, i.v.) pretreated animals, sumatriptan (a 5-HT1B/1D receptor agonist) reduced the reflex bradycardia and the pressor response. The 5-HT1B/1D receptor antagonist GR127935 (20 μg kg−1, i.c. or 100 μg kg−1, i.v.) had no effect on the reflex responses.In conclusion, the present data are consistent with the hypothesis that activation of central 5-HT1A receptors potentiate whilst activation of 5-HT1B/1D receptors attenuate the reflex activation of cardiac preganglionic vagal motoneurones evoked by stimulation of upper airway receptors with smoke in rabbits. PMID:9786516

  8. Antimicrobial peptide scolopendrasin VII, derived from the centipede Scolopendra subspinipes mutilans, stimulates macrophage chemotaxis via formyl peptide receptor 1.

    PubMed

    Park, Yoo Jung; Lee, Ha Young; Jung, Young Su; Park, Joon Seong; Hwang, Jae Sam; Bae, Yoe-Sik

    2015-08-01

    In this study, we report that one of the antimicrobial peptides scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates actin polymerization and the subsequent chemotactic migration of macrophages through the activation of ERK and protein kinase B (Akt) activity. The scolopendrasin VII-induced chemotactic migration of macrophages is inhibited by the formyl peptide receptor 1 (FPR1) antagonist cyclosporine H. We also found that scolopendrasin VII stimulate the chemotactic migration of FPR1-transfected RBL-2H3 cells, but not that of vector-transfected cells; moreover, scolopendrasin VII directly binds to FPR1. Our findings therefore suggest that the antimicrobial peptide scolopendrasin VII, derived from Scolopendra subspinipes mutilans, stimulates macrophages, resulting in chemotactic migration via FPR1 signaling, and the peptide can be useful in the study of FPR1-related biological responses. PMID:26129676

  9. Insulin-stimulated Na/sup +/ transport in a model renal epithelium: protein synthesis dependence and receptor interactions

    SciTech Connect

    Blazer-Yost, B.L.; Cox, M.

    1987-05-01

    The urinary bladder of the toad, Bufo marinus, is a well characterized model of the mammalian distal nephron. Porcine insulin (approx. 0.5-5.0 ..mu..M) stimulates net mucosal to serosal Na/sup +/ flux within 10 minutes of hormone addition. The response is maintained for at least 5 hr and is completely abolished by low doses (10..mu..M) of the epithelial Na/sup +/ channel blocker amiloride. Insulin-stimulated Na/sup +/ transport does not require new protein synthesis since it is actinomycin-D (10..mu..g/ml) insensitive. Also in 3 separate experiments in which epithelial cell proteins were examined by /sup 35/S-methionine labeling, 2-dimensional polyacrylamide gel electrophoresis/autoradiography, no insulin induced proteins were observed. Equimolar concentrations of purified porcine proinsulin and insulin (0.64..mu..M) stimulate Na/sup +/ transport to the same extent. Thus, the putative toad insulin receptor may have different affinity characteristics than those demonstrated for insulin and proinsulin in mammalian tissues. Alternatively, the natriferic action of insulin in toad urinary bladders may be mediated by occupancy of another receptor. Preliminary experiments indicating that nanomolar concentrations of IGF/sub 1/ stimulate Na/sup +/ transport in this tissue support the latter contention.

  10. RNA editing of the human serotonin 5-HT(2C) receptor delays agonist-stimulated calcium release.

    PubMed

    Price, R D; Sanders-Bush, E

    2000-10-01

    RNA encoding the human 5-HT(2C) receptor undergoes adenosine-to-inosine RNA editing events at five positions in the putative second intracellular loop, with a corresponding reduction in receptor/G-protein coupling. Agonist-stimulated calcium release was examined in NIH-3T3 fibroblasts stably expressing the nonedited human INI (hINI) or the edited hVSV or hVGV variants. We hypothesized that different receptor isoforms would show altered dynamics of agonist-induced calcium release. The three isoforms showed a rightward shift in agonist concentration-response curves for eliciting calcium release (EC(50) values: hINI, 2.2 nM; hVSV, 15 nM; hVGV, 49 nM). Additionally, the hVGV receptor showed a blunted and delayed [Ca(2+)](i) peak compared with the hINI or hVSV receptor isoforms. These distinctions in agonist-induced [Ca(2+)](i) release imply that edited 5-HT(2C) receptors may produce distinct physiological responses within the central nervous system. PMID:10999958