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GABA, the main inhibitory neurotransmitter, acts through [GABA.sub.A/C] and [GABA.sub.B] receptors ([GABA.sub.B]Rs); it is critical for gonadotropin regulation. We studied whether the lack of functional [GABA.sub.B]Rs in [GABA.sub.B1] knockout ([GABA.sub.B]KO) mice affected the gonadotropin axis physiology. Adult male and female [GABA.sub.B1]KO and wild-type (WT) mice were killed to collect blood and tissue samples. Gonadotropin-releasing hormone (GnRH) content in whole hypothalami (HT), olfactory bulbs (OB), and frontoparietal cortexes (CT) were determined (RIA). GnRH expression by quantitative real-time PCR (qRT-PCR) was evaluated in preoptic area-anterior hypothalamus (POA-AH), medial basal-posterior hypothalamus (MBH-PH), OB, and CT. Pulsatile GnRH secretion from hypothalamic explants was measured by RIA. GABA, glutamate, and taurine contents in HT and CT were determined by HPLC. Glutamic acid decarboxylase-67 (GAD-67) mRNA was measured by qRT-PCR in POA-AH, MBH-PH, and CT. Gonadotropin content, serum levels, and secretion from adenohypophyseal cell cultures (ACC) were measured by RIA. GnRH mRNA expression was increased in POA-AH of WT males compared with females; this pattern of expression was inversed in [GABA.sub.B1]KO mice. MBH-PH, OB, and CT did not follow this pattern. In [GABA.sub.B1]KO females, GnRH pulse frequency was increased and GABA and glutamate contents were augmented. POA-AH GAD-67 mRNA showed the same expression pattern as GnRH mRNA in this area. Gonadotropin pituitary contents and serum levels showed no differences between genotypes. Increased basal LH secretion and decreased GnRH-stimulated gonadotropin response were observed in [GABA.sub.B1]KO female ACCs. These results support the hypothesis that the absence of functional [GABA.sub.B]Rs alters GnRH physiology and critically affects sexual dimorphic expression of GnRH and GAD-67 in POA-AH. gonadotropin-releasing hormone pulsatility; gonadotropins; amino acid neurotransmitters doi:10.1152/ajpendo.00532.2009.

GABA is the major inhibitory neurotransmitter in the nervous system. It is also released by the insulin-producing [beta]-cells, providing them with a potential paracrine regulator. Because glucose was found to inhibit GABA release, we investigated whether extracellular GABA can serve as a marker for glucose-induced mitochondrial activity and thus for the functional state of [beta]-cells. GABA release by rat and human [beta]-cells was shown to reflect net GABA production, varying with the functional state of the cells. Net GABA production is the result of GABA formation through glutamate decarboxylase (GAD) and GABA catabolism involving a GABA-transferase (GABA-T)-mediated shunt to the TCA cycle. GABA-T exhibits [K.sub.m] values for GABA (1.25 mM) and for [alpha]-ketoglutarate ([alpha]-KG; 0.49 mM) that are, respectively, similar to and lower than those in brain. The GABA-T inhibitor [gamma]-vinyl GABA was used to assess the relative contribution of GABA formation and catabolism to net production and release. The nutrient status of the [beta]-cells was found to regulate both processes. Glutamine dose-dependently increased GAD-mediated formation of GABA, whereas glucose metabolism shunts part of this GABA to mitochondrial catabolism, involving [alpha]-KG-induced activation of GABA-T. In absence of extracellular glutamine, glucose also contributed to GABA formation through aminotransferase generation of glutamate from [alpha]-KG; this stimulatory effect increased GABA release only when GABA-T activity was suppressed. We conclude that GABA release from [beta]-cells is regulated by glutamine and glucose. Glucose inhibits glutaminedriven GABA formation and release through increasing GABA-T shunt activity by its cellular metabolism. Our data indicate that GABA release by [beta]-cells can be used to monitor their metabolic responsiveness to glucose irrespective of their insulin-secretory activity. glutamine; [gamma]-aminobutyric acid; [gamma]-aminobutyric acid transferase

The existence of endothelin binding sites on the catecholaminergic neurons of the hypothalamus suggests that endothelins (ETs) participate in the regulation of noradrenergic transmission modulating various hypothalamic-controlled processes such as blood pressure, cardiovascular activity, etc. The effects of ET-1 and ET-3 on the neuronal release of norepinephrine (NE) as well as the receptors and intracellular pathway involved were studied in the rat anterior hypothalamus. ET-1 (10 nM) and ET-3 (10 nM) diminished neuronal NE release and the effect blocked by the selective ET type B receptor antagonist BQ-788 (100 nM). [N.sup.[omega]]-nitro-L-arginine methyl ester (10 [micro]M), methylene blue (10 [micro]M), and KT5823 (2 [micro]M), inhibitors of nitric oxide synthase activity, guanylate cyclase, and protein kinase G, respectively, prevented the inhibitory effects of both ETs on neuronal NE release. In addition, both ETs increased nitric oxide synthase activity. Furthermore, 100 [micro]M picrotoxin, a GAB[A.sub.A]-receptor antagonist, inhibited ET-1 and ET-3 response. Our results show that ET-1 as well as ET-3 has an inhibitory neuromodulatory effect on NE release in the anterior hypothalamus mediated by the ET type B receptor and the involvement of a nitric oxide-dependent pathway and GAB[A.sub.A] receptors. ET-1 and ET-3 may thus diminish available NE in the synaptic gap leading to decreased noradrenergic activity. endothelin receptor; soluble guanylyl cyclase; BQ-610; BQ-788; GAB[A.sub.A] receptor, nitric oxide, norepinephrine

A single bout of exercise results in a postexercise hypotension (PEH) that is accompanied by a reduced baroreflex function. Based on the role of rostral ventrolateral medulla (RVLM) neurons in controlling sympathetic nerve activity (SNA) and blood pressure, the role of [gamma]-aminobutyric acid (GABA) in controlling RVLM neuronal activity, and the reduced baroreflex-SNA relationship during PEH, we determined whether: 1) RVLM neuronal activity is decreased during PEH, 2) GAB[A.sub.A]-receptor mechanisms mediate the decrease, and 3) baroreflex control of RVLM activity is reduced. Spontaneously hypertensive rats (SHR) were subjected to 40 min of treadmill or sham exercise (Sham PEH). PEH lasted 10 h in conscious and anesthetized SHR, indicating that the anesthetics did not affect the expression of PEH. Extracellular RVLM neuronal activity having a cardiac and sympathetic rhythm, lumbar SNA, and blood pressure were recorded at rest and during baroreflex function curves. Resting RVLM neuronal activity was lower and was increased to a greater extent by GAB[A.sub.A]-receptor antagonism in PEH versus Sham PEH (P < 0.05). Baroreflex control of RVLM neuronal activity operated with a reduced gain (P < 0.05). Thus increased GABA signaling at RVLM neurons may contribute to PEH. spontaneously hypertensive rats; central sympathetic network; extracellular recording

Research was conducted to examine the effects of microinjections of muscimol or baclofen into the lateral aspect of the commissural nucleus tractus solitarius (NTS) on the bradycardic and pressor responses to chemoreflex activation in awake rats. Results demonstrate that the activation of GABA(sub A) receptors in the NTS yields a substantial reduction in the receptors in the bradycardic response while the activation of GABA(sub B) receptors generate a sigificant reduction in the pressor response of the chemoreflex.

The mechanism of nitric oxide synthesis in intact longitudinal muscle-myenteric plexus preparations of the guinea pig ileum was investigated. To this end, the formation of ((super 3)H)citrulline during incubation with ((super 3)H)arginine was examined. Results show that the GABA receptor agonist muscimol and GABA reduced the electrically generated formation of ((super 3)H)citrulline, in contrast with baclofen which had no effect. The inhibitory effect of GABA, however, was antagonized by bicuculline.

The effects of microinjections of gamma-aminobutyric acid (GABA) into the phrenic nucleus are studied. The study also examined the effect of micropressure applications of GABA receptor agonists and antagonists on the extra-cellular action potentials of single phrenic neurons. Results of the study indicate the presence of GABA receptors on the neuros in the phrenic nucleus. Findings also indicate that their activation has a role in the decline of the phrenic nerve burst amplitude.

Researchers have sought to identify the impact of vitamin B12 (VB12) on the rat suprachiasmatic nucleus (SCN) by investigating the contents of upsilon-aminobutyric acid (GABA) and glutamate (Glu), its main neurotransmitters. The results indicated that VB12 produces a marked rise in GABA content in the SCN and cerebral cortex. It appears that GABA receptors may be involved in the regulation of the circadian clock system. GABA metabolism seems to be involved in the phase-resetting function as vigabatrin, an inhibitor of GABA catabolism, boosts light-induced phase delays.

An investigation of sympathetic nervous activity in rats has revealed the presence of gamma-aminobutyric acid (GABA) receptors in the bulbospinal neurons of the rostral ventrolateral medulla (RVLM). Tracer studies showed that the GABA receptors contribute to the regulation of firing frequency of RVLM vasomotor neurons. Upon activation, these receptors increases a potassium conductance which inhibits the discharge of RVLM neurons.

Male rat models were utilized to determine the effects of three doses of progesterone on the duration of vigilance states and sleep-state specific sleep encephalogram. Dose-specific administration of exogenous progestrone exhibits hypnotic properties that are similar to activity of gamma-aminobutyric acid A (GABAA) receptors. Dose-dependent elevations in the concentration of 5alpha- and 5beta-pregnanolone GABAA receptor agonistic metabolites were also detected during the administration of progesterone.

The application of gamma-aminobutyric acid (GABA), pentobarbital or pregnanolone on dissociated suprachiasmatic nucleus neurons of rat, induces inward currents. GABA(sub A) receptor is bicuculline sensitive, directly coupled with a Cl- channel, and potentiated by allosteric modulators like diazepam, pentobarbital and pregnanolone. Results show that the GABA response is mediated by the GABA(sub A) receptor. GABA(sub A) is also modulated by intracellular Ca+ and cAMP.

The mediation of changes in the cholinergic pathways in guinea pig ileum due to somatostatin by gamma-aminobutyric acid (GABA) neurons was examined. This involved the utilization of muscle tension studies and acetylcholine release experiments. Results indicate that the stimulation of GABA pathways is the means by which somatostatin exerts its effects on ileal cholinergic pathways.

[GABA.sub.B] receptors are inhibitory G protein-coupled receptors that are commonly associated with presynaptic inhibition of transmitter release in the central nervous system. In the brain-gastroesophageal axis, a role has recently been demonstrated for [GABA.sub.B] receptors on extrinsic afferent endings within the stomach and esophagus, where they reduce mechanosensitivity. This action is compounded by inhibition of communication centrally from these afferents in the brain stem and within central circuits. There is a final peripheral action on the motor pathway where [GABA.sub.B] receptors reduce output of acetylcholine from vagal preganglionic motoneurons. These potent, multiple actions of [GABA.sub.B] receptors may have therapeutic benefit by reducing the triggering of transient lower esophageal relaxations, which are the major cause of gastroesophageal reflux. An important clinical application is therefore emerging for this recent discovery. vagal afferents; mechanoreceptors; transient lower esophageal relaxations; gastroesophageal, reflux

Partosoedarso, Elita R., Richard L. Young, and L. Ashley Blackshaw. [GABA.sub.B] receptors on vagal afferent pathways: peripheral and central inhibition. Am J Physiol Gastrointest Liver Physiol 280: G658-G668, 2001.--To investigate [GABA.sub.B] receptors along vagal afferent pathways, we recorded from vagal afferents, medullary neurons, and vagal efferents in ferrets. Baclofen (7-14 [micro]mol/kg iv) reduced gastric tension receptor and nucleus tractus solitarii neuronal responses to gastric distension but not gastroduodenal mucosal receptor responses to cholecystokinin (CCK). [GABA.sub.B] antagonists CGP-35348 or CGP-62349 reversed effects of baclofen. Vagal efferents showed excitatory and inhibitory responses to distension and CCK. Baclofen (3 nmol icv or 7-14 [micro]mol/kg iv) reduced both distension response types but reduced only inhibitory responses to CCK. CGP-35348 (100 nmol icv or 100 [micro]mol/kg iv) reversed baclofen's effect on distension responses, but inhibitory responses to CCK remained attenuated. They were, however, reversed by CGP-62349 (0.4 nmol icv). In conclusion, [GABA.sub.B] receptors inhibit mechanosensitivity, not chemosensitivity, of vagal afferents peripherally. Mechanosensory input to brain stem neurons is also reduced centrally by [GABA.sub.B] receptors, but excitatory chemosensory input is unaffected. Inhibitory mechano-and chemosensory inputs to brain stem neurons (via inhibitory interneurons) are both reduced, but the pathway taken by chemosensory input involves GABAB receptors that are insensitive to CGP-35348. [Gamma]-aminobutyric acid B receptors; ferret; mechanoreceptors; vagal reflexes; cholecystokinin; nucleus tractus solitarii

Yao, Zhenhai, Bradley C. McPherson, Huiping Liu, Zuohui Shao, Changqing Li, Yimin Qin, Terry L. Vanden Hoek, Lance B. Becker, and Paul T. Schumacker. Signal transduction of flumazenil-induced preconditioning in myocytes. Am J Physiol Heart Circ Physiol 280: H1249-H1255, 2001.--The objective of this study was to examine the role of oxygen radicals, protein kinase C (PKC), and ATP-sensitive [K.sup.+] ([K.sub.ATP]) channels in mediating flumazenil-produced preconditioning. Chick cardiomyocyte death was quantified using propidium iodide, and oxygen radical generation was assessed using 2', 7'-dichlorofluorescin oxidation. Preconditioning was initiated with 10 min of ischemia followed by 10 min of reoxygenation. Alternatively, flumazenil was infused for 10 min and removed 10 min before ischemia. Flumazenil (10 [micro]M) and preconditioning increased oxygen radicals [1,693 [+ or -] 101 (n = 3) and 1,567 [+ or -] 98 (n = 3), respectively, vs. 345 [+ or -] 53 (n = 3) in control] and reduced cell death similarly [22 [+ or -] 3% (n = 5) and 18 [+ or -] 2% (n = 6), respectively, vs. controls 49 [+ or -] 5% (n = 8)]. Protection and increased oxygen radicals by flumazenil were abolished by pretreatment with the antioxidant thiol reductant 2-mercaptopropionyl glycine (800 [micro]M; 52 [+ or -] 10%, n = 6). Specific PKC inhibitors Go-6976 (0.1 [micro]M) and chelerythrine (2 [micro]M), given during ischemia and reoxygenation, blocked flumazenil-produced protection (47 [+ or -] 5%, n = 6). The PKC activator phorbol 12-myristate 13-acetate (0.2 [micro]M), given during ischemia and reoxygenation, reduced cell death similarly to that with flumazenil [17 [+ or -] 4% (n = 6) and 22 [+ or -] 3% (n = 5)]. Finally, 5-hydroxydecanoate (1 mM), a selective mitochondrial [K.sub.ATP] channel antagonist given during ischemia and reoxygenation, abolished the protection of flumazenil and phorbol 12-myristate 13-acetate. Thus flumazenil mimics preconditioning to reduce cell death in cardiomyocytes. Oxygen radicals activate mitochondrial [K.sub.ATP] channels via PKC during the process. mitochondria; ischemia; potassium channels; GABA receptors; signal transduction

Sha, L., S. M. Miller, and J. H. Szurszewski. Electrophysiological effects of GABA on cat pancreatic neurons. Am J Physiol Gastrointest Liver Physiol 280: G324-G331, 2001.--In mammalian peripheral sympathetic ganglia GABA acts presynaptically to facilitate cholinergic transmission and postsynaptically to depolarize membrane potential. The GABA effect on parasympathetic pancreatic ganglia is unknown. We aimed to determine the effect of locally applied GABA on cat pancreatic ganglion neurons. Ganglia with attached nerve trunks were isolated from cat pancreata. Conventional intracellular recording techniques were used to record electrical responses from ganglion neurons. GABA pressure microejection depolarized membrane potential with an amplitude of 17.4 [+ or -] 0.7 mV. Electrically evoked fast excitatory postsynaptic potentials were significantly inhibited (5.4 [+ or -] 0.3 to 2.9 [+ or -] 0.2 mV) after GABA application. GABA-evoked depolarizations were mimicked by the [GABA.sub.A] receptor agonist muscimol and abolished by the [GABA.sub.A] receptor antagonist bicuculline and the [Cl.sup.-] channel blocker picrotoxin. GABA was taken up and stored in ganglia during preincubation with 1 mM GABA; [Alpha]-aminobutyric acid application after GABA loading significantly (P [is less than] 0.05) increased depolarizing response to GABA (15.6 [+ or -] 1.0 vs. 7.8 [+ or -] 0.8 mV without GABA preincubation). Immunolabeling with antibodies to GABA, glial cell fibrillary acidic protein, protein gene product 9.5, and glutamic acid decarboxylase (GAD) immunoreactivity showed that GABA was present in glial cells, but not in neurons, and that glial cells did not contain GAD, whereas islet cells did. The data suggest that endogenous GABA released from ganglionic glial cells acts on pancreatic ganglion neurons through [GABA.sub.A] receptors. [Gamma]-Aminobutyric acid A receptors; glial cells; [Gamma]-aminobutyric acid release; electrophysiology; immunohistochemistry

Erlitzki, Ronit, Yuewen Gong, Manna Zhang, and Gerald Minuk. Identification of [Gamma]-aminobutyric acid receptor subunit types in human and rat liver. Am J Physiol Gastrointest Liver Physiol 279: G733-G739, 2000.--GABA is a potent inhibitory neurotransmitter that binds to heterooligomeric receptors in the mammalian brain. In a previous study, we documented specific GABA binding to isolated rat hepatocytes that resulted in inhibition of hepatocyte proliferation. The purpose of the present study was to define the nature of hepatic [GABA.sub.A] receptors and to document their expression during rapid liver growth (after partial hepatectomy). PCRs with gene-specific primers derived from published sequences were performed with Marathon-ready human and rat liver cDNA. Two [GABA.sub.A] receptor subunit types ([Beta]3 and [element of]) were expressed in human liver and one subunit type ([Beta]3) in rat liver. PCR amplification of the human [GABA.sub.A] receptor[Beta]3-subunit produced a single product (molecular mass 53-59 kDa). In the case of the [element of]-subunit, two PCR products were identified. After partial hepatectomy, [GABA.sub.A] receptor[Beta]3-subunit expression inversely correlated with regenerative activity (r = -0.527, P = 0.006). In conclusion, these results indicate that in the human liver [GABA.sub.A] receptors consist of the [Beta]3- and [element of]-subunit types, whereas in the rat liver only the [Beta]3-subunit type is expressed. The results also support the hypothesis that GABAergic activity serves to maintain hepatocytes in a quiescent state. gamma-aminobutyric acid A receptor; hepatocytes; receptors; neurotransmitters