Identification of gamma-aminobutyric acid and its binding sites in the rat ovary

Gamma-aminobutyric acid (GABA), GABA synthesizing enzyme and GABA binding sites were measured in rat ovaries. The concentration of GABA in the ovary (0.56 μg/mg protein) was less than that in the brain (1.2–3.4 μg/mg protein), but was six-fold higher than any other non-neuronal tissue examined. Glutamate decarboxylase, the GABA synthesizing enzyme was also found in high concentrations in whole ovarian homogenate but not in enriched ovarian granulosa cells, testis, anterior pituitary or muscles. Furthermore, high affinity (Kd = 15–21 nM), specific GABA binding sites were identified in the ovaries by specific [³H]muscimol binding and the majority of GABA binding sites were associated with the granulosa cells. These data suggest a possible role of GABA in the regulation of ovarian functions.     

Immunohistochemical localization of gamma-aminobutyric acid (GABA) in the rat pancreas

Summary The localization of gamma-aminobutyric acid (GABA) in rat pancreas was investigated using antiserum raised against GABA conjugated to bovine serum albumin with glutaraldehyde. Immunoreactive cells were only found in the center of the pancreatic islets, and these cells were surrounded by nonimmunoreactive cells. When two serial sections of rat pancreas were consecutively stained with GABA antiserum and with antibodies against insulin, both antisera stained the same population of endocrine cells within the islets. In rats pretreated with streptozotocin, a B-cell toxin, we observed a marked decrease in the number of cells exhibiting GABA-like immunoreactivity. These observations indicate that GABA is present in the B cells of rat pancreatic islets.This work was supported by the grants from the Ministry of Education, Science, and Culture, Japan     

Immunohistochemical localization of gamma-aminobutyric acid (GABA) in the rat pancreas

The localization of gamma-aminobutyric acid (GABA) in rat pancreas was investigated using antiserum raised against GABA conjugated to bovine serum albumin with glutaraldehyde. Immunoreactive cells were only found in the center of the pancreatic islets, and these cells were surrounded by nonimmunoreactive cells. When two serial sections of rat pancreas were consecutively stained with GABA antiserum and with antibodies against insulin, both antisera stained the same population of endocrine cells within the islets. In rats pretreated with streptozotocin, a B-cell toxin, we observed a marked decrease in the number of cells exhibiting GABA-like immunoreactivity. These observations indicate that GABA is present in the B cells of rat pancreatic islets.     

Control of rat gastric somatostatin release by gamma-aminobutyric acid (GABA)

The influence of gamma-aminobutyric acid (GABA) on gastric somatostatin and gastrin release was studied using an isolated perfused rat stomach preparation. GABA dose-dependently inhibited somatostatin release (maximal inhibition of 44% at 10(-5)M GABA), whereas gastrin secretion was not affected. The GABA agonist muscimol led to a decrease in somatostatin release of similar magnitude. The GABA-induced changes were partially reversed by 10(-5)M atropine. Gastrin secretion was not influenced by either protocol. It is concluded that GABA as a putative neurotransmitter in the enteric nervous system is inhibitory to rat gastric somatostatin release in vitro via cholinergic pathways.     

Molecular and physiological evidence for functional gamma-aminobutyric acid (GABA)-C receptors in growth hormone-secreting cells

The neurotransmitter gamma-aminobutyric acid (GABA), released by hypothalamic neurons as well as by growth hormone- (GH) and adrenocorticotropin-producing cells, is a regulator of pituitary endocrine functions. Different classes of GABA receptors may be involved. In this study, we report that GH cells, isolated by laser microdissection from rat pituitary slices, possess the GABA-C receptor subunit rho2. We also demonstrate that in the GH adenoma cell line, GH3, GABA-C receptor subunits are not only expressed but also form functional channels. GABA-induced Cl- currents were recorded using the whole cell patch clamp technique; these currents were insensitive to bicuculline (a GABA-A antagonist) but could be induced by the GABA-C agonist cis-4-aminocrotonic acid. In contrast to typical GABA-C mediated currents in neurons, they quickly desensitized. Ca2+i recordings were also performed on GH3 cells. The application of either GABA or cis-4-aminocrotonic acid led to Ca2+ transients of similar amplitude, indicating that the activation of GABA-C receptors in GH3 cells may cause membrane depolarization, opening of voltage-gated Ca2+ channels, and a subsequent Ca2+ influx. Our results point at a role for GABA in pituitary GH cells and disclose an additional pathway to the one known via GABA-B receptors.     

Autoradiographic studies of the gamma-aminobutyric acid (GABA) system in the rat pancreas

The beta-cells of the pancreatic islets have been shown to contain gamma-aminobutyric acid (GABA) together with insulin. Autoradiographic analysis indicated that high affinity GABA binding sites (GABA receptors) are not present in the pancreas. High affinity GABA uptake sites are present, not in beta-cells, but in a few cells on the periphery of the islets. These observations cast doubt on the suggestion that GABA has a paracrine role in the pancreas.     

Identification of gamma-aminobutyric acid and its binding sites in Caenorhabditis elegans

Gamma-aminobutyric acid (GABA), glutamate decarboxylase and GABA-transaminase were identified in the nematode Caenorhabditis elegans. The concentration of GABA in C. elegans (0.14 micrograms/mg protein) is approximately 10-fold lower than the concentration of GABA in rat brain. Glutamate decarboxylase and GABA-transaminase, the GABA anabolic and catabolic enzymes, are also present in C. elegans. Crude membrane fractions were prepared from C. elegans and used to study specific [3H] GABA binding sites. GABA binds to C. elegans membranes with high affinity (37 nM) and low capacity (Bmax = 2.25 pmol/mg protein). Muscimol is a competitive inhibitor of specific GABA binding with a KI value of 120 nM. None of the other GABA agonists or antagonists inhibited greater than 40% of the specific GABA binding at concentrations up to 10(-4)M. Thirteen spider venoms were examined as possible GABA agonists or antagonists, the venom from Calilena agelenidae inhibits specific GABA binding with a KI value of 6 nl/ml. These results suggest that GABA has a physiological role as a neurotransmitter in C. elegans.     

Neuropharmacology of supraoptic nucleus neurons: norepinephrine and gamma-aminobutyric acid receptors

The neurosecretory neurons in the mammalian hypothalamic supraoptic nucleus receive prominent GABAergic and noradrenergic projections arising from local interneurons and the A1 cells in the ventrolateral medulla, respectively. Intracellular recordings in in vitro perfused hypothalamic explants reveal an abundance of spontaneous inhibitory postsynaptic potentials (IPSPs) and a compound IPSP after electrical stimulation in the diagonal band of Broca area. The sensitivity of both spontaneous and evoked IPSPs to intracellular chloride injection, bicuculline, and pentobarbital is consistent with a GABA-activated, chloride-mediated inhibitory synaptic input. Parallel changes in membrane voltage and conductance are present during applications of GABA and muscimol, with similar sensitivity to ionic manipulation, bicuculline, and pentobarbital. These observations contrast with the consistently excitatory effects that follow either the stimulation of A1 cells or the application of norepinephrine and alpha 1-adrenergic agonists. Norepinephrine induces membrane depolarizations and bursting activity patterns that are blocked by the selective alpha 1 antagonist prazosin. Membrane response to norepinephrine is voltage dependent and is associated with little change in conductance. GABA and norepinephrine are proposed as transmitters in the final central pathways that mediate information to supraoptic vasopressinergic neurons from peripheral baroreceptors and chemoreceptors, respectively.     

Desensitization of gamma-aminobutyric acid receptor from rat brain: two distinguishable receptors on the same membrane

Transmembrane chloride flux mediated by gamma-aminobutyric acid (GABA) receptor can be measured with a mammalian brain homogenate preparation containing sealed membrane vesicles. The preparation can be mixed rapidly with solutions of defined composition. Influx of 36Cl- tracer initiated by mixing with GABA was rapidly terminated by mixing with bicuculline methiodide. The decrease in the isotope influx measurement due to prior incubation of the vesicle preparation with GABA, which increased with preincubation time and GABA concentration, was attributed to desensitization of the GABA receptor. By varying the time of preincubation with GABA between 10 ms and 50 s with quench-flow technique, the desensitization rates could be measured over their whole time course independently of the chloride ion flux rate. Most of the receptor activity decreased in a fast phase of desensitization complete in 200 ms (t 1/2 = 32 ms) at saturation with GABA. Remaining activity was desensitized in a few seconds (t 1/2 = 533 ms). These two phases of desensitization were each kinetically first order and were shown to correspond with two distinguishable GABA receptors on the same membrane. The receptor activities could be estimated, and the faster desensitizing receptor was the predominant one, giving on average ca. 80% of the total activity. The half-response concentrations were similar, 150 and 114 microM for the major and minor receptors, respectively. The dependence on GABA concentration indicated that desensitization is mediated by two GABA binding sites. The fast desensitization rate was approximately 20-fold faster than previously reported rates while the slower desensitization rate was slightly faster than previously reported rates.     

Detection of acetylcholinesterase activity and gamma-aminobutyric acid binding sites in Dicrocoelium dendriticum

In the present study we report the presence of acetylcholinesterase activity and gamma-aminobutyric acid binding sites in crude extracts of Dicrocoelium dendriticum. This indirectly demonstrates the presence of acetylcholine and GABA. The presence of these neurotransmitters could indicate the existence of two systems implicated in the neurotransmission of the Digenea.     

Chronic blockade of N-methyl-D-aspartate receptors alters gamma-aminobutyric acid type A receptor peptide expression and function in the rat

Chronic in vivo or in vitro application of GABA(A) receptor agonists alters GABA(A) receptor peptide expression and function. Furthermore, chronic in vitro application of N-methyl-D-aspartate (NMDA) agonists and antagonists alters GABA(A) receptor function and mRNA expression. However, it is unknown if chronic in vivo blockade of NMDA receptors alters GABA(A) receptor function and peptide expression in brain. Male Sprague-Dawley rats were chronically administered the noncompetitive NMDA receptor antagonist MK-801 (0.40 mg/kg, twice daily) for 14 days. Chronic blockade of NMDA receptors significantly increased hippocampal GABA(A) receptor alpha4 and gamma2 subunit expression while significantly decreasing hippocampal GABA(A) receptor alpha2 and beta2/3 subunit expression. Hippocampal GABA(A) receptor alpha1 subunit peptide expression was not altered. In contrast, no significant alterations in GABA(A) receptor subunit expression were found in cerebral cortex. Chronic MK-801 administration also significantly decreased GABA(A) receptor-mediated hippocampal Cl- uptake, whereas no change was found in GABA(A) receptor-mediated cerebral cortical Cl- uptake. Finally, chronic MK-801 administration did not alter NMDA receptor NR1, NR2A, or NR2B subunit peptide expression in either the cerebral cortex or the hippocampus. These data demonstrate heterogeneous regulation of GABA(A) receptors by glutamatergic activity in rat hippocampus but not cerebral cortex, suggesting a new mechanism of GABA(A) receptor regulation in brain.     

Measurement of gamma-aminobutyric acid (GABA) in blood.

Blood GABA levels can be readily determined using a radioreceptor assay or gas chromatography-mass spectrometry. After withdrawal of blood, GABA levels remain stable with 25–50% of the GABA in whole blood found in the plasma fraction. Whole blood GABA concentrations range from 500 pmoles/ml to 1200 pmoles/ml in 8 mammalian species with human values being about 900 pmoles/ml. $\text{in vivo}$ administration of aminooxyacetic acid increases both blood and brain GABA levels to a similar extent.     

Degradation of gamma-aminobutyric acid (GABA) by marine microorganisms

Abstract By degrading the settlement inducer gamma-aminobutyric acid (GABA), bacteria may affect the larval settlement of sedentary marine invertebrates. Nearly one third of bacterial isolates from surfaces suitable for abalone ( Haliotis ) settlement were able to grow on GABA as sole carbon source. Compared with similar compounds, GABA was a good source of carbon, nitrogen and energy, and it was utilized concomitantly with glucose. GABA-metabolizing enzymes were constitutive in Pseudomonas fluorescens and inducible in Aeromonas hydrophila . High-affinity ( K _m: 20–50 μ M) and low-affinity ( K _m: 7–9 mM) uptake systems were produced in response to low and high GABA concentrations, respectively, in the growth medium. Within the ecologically significant temperature range (12–24°C), specific GABA degradation rates varied 2.5-fold in young cells of P. fluorescens . This organism los its ability to degrade GABA during the stationary phase. The results suggest that marine bacteria have the potential to affect invertebrate larval settlement by removing GABA from the settlement habitat.     

Antagonist actions of bicuculline methiodide and picrotoxin on extrasynaptic gamma-aminobutyric acid receptors

The effects of picrotoxin and bicuculline methiodide to block depolarizing responses of extrasynaptic receptors for gamma-aminobutyric acid (GABA) are compared using excitability testing of myelinated axons in amphibian peripheral nerve. The actions of the antagonists appear both complex and dissimilar. Picrotoxin (10-1000 microM) produces large reversible depressions of the maximal response to GABA (0.01-10mM) and increases the EC50 from 0.33 to 12.6 mM. With high concentrations of agonist and antagonist an insensitive component is apparent. The action of picrotoxin is not classically noncompetitive: it may represent a mixed antagonism (competitive and noncompetitive) or a noncompetitive one, masked by the presence of receptor reserve and (or) secondary depolarizing influences (e.g., GABA-evoked [K+] o accumulation). Bicuculline methiodide (10-200 microM) shifts the GABA concentration-response curve to the right; maximal responses persist and are even enhanced. The impression that bicuculline methiodide has a competitive action is supported by analysis of its inhibition of responses to low concentrations of the agonist. It is suggested that the enhancement of GABA responses by bicuculline methiodide and their apparent resistance to block by picrotoxin may be due to a common secondary effect of the antagonists such as a decrease in membrane conductance to K+ and (or) block of transmitter uptake.