Stimulation of TM3 Leydig cell proliferation via GABA<Subscript>A</Subscript> receptors: A new role for testicular GABA

The neurotransmitter gamma-aminobutyric acid (GABA) and subtypes of GABA receptors were recently identified in adult testes. Since adult Leydig cells possess both the GABA biosynthetic enzyme glutamate decarboxylase (GAD), as well as GABA_A and GABA_B receptors, it is possible that GABA may act as auto-/paracrine molecule to regulate Leydig cell function. The present study was aimed to examine effects of GABA, which may include trophic action. This assumption is based on reports pinpointing GABA as regulator of proliferation and differentiation of developing neurons via GABA_A receptors. Assuming such a role for the developing testis, we studied whether GABA synthesis and GABA receptors are already present in the postnatal testis, where fetal Leydig cells and, to a much greater extend, cells of the adult Leydig cell lineage proliferate. Immunohistochemistry, RT-PCR, Western blotting and a radioactive enzymatic GAD assay evidenced that fetal Leydig cells of five-six days old rats possess active GAD protein, and that both fetal Leydig cells and cells of the adult Leydig cell lineage possess GABA_A receptor subunits. TM3 cells, a proliferating mouse Leydig cell line, which we showed to possess GABA_A receptor subunits by RT-PCR, served to study effects of GABA on proliferation. Using a colorimetric proliferation assay and Western Blotting for proliferating cell nuclear antigen (PCNA) we demonstrated that GABA or the GABA_A agonist isoguvacine significantly increased TM3 cell number and PCNA content in TM3 cells. These effects were blocked by the GABA_A antagonist bicuculline, implying a role for GABA_A receptors. In conclusion, GABA increases proliferation of TM3 Leydig cells via GABA_A receptor activation and proliferating Leydig cells in the postnatal rodent testis bear a GABAergic system. Thus testicular GABA may play an as yet unrecognized role in the development of Leydig cells during the differentiation of the testicular interstitial compartment.     

Modulation of Tonic GABA Currents by Anion Channel and Connexin Hemichannel Antagonists

Anion channels and connexin hemichannels are permeable to amino acid neurotransmitters. It is hypothesized that these conductive pathways release GABA, thereby influencing ambient GABA levels and tonic GABAergic inhibition. To investigate this, we measured the effects of anion channel/hemichannel antagonists on tonic GABA currents of rat hippocampal neurons. In contrast to predictions, blockade of anion channels and hemichannels with NPPB potentiated tonic GABA currents of neurons in culture and acute hippocampal slices. In contrast, the anion channel/hemichannel antagonist carbenoxolone (CBX) inhibited tonic currents. These findings could result from alterations of ambient GABA concentration or direct effects on GABA_A receptors. To test for effects on GABA_A receptors, we measured currents evoked by exogenous GABA. Coapplication of NPPB with GABA potentiated GABA-evoked currents. CBX dose-dependently inhibited GABA-evoked currents. These results are consistent with direct effects of NPPB and CBX on GABA_A receptors. GABA release from hippocampal cell cultures was directly measured using HPLC. Inhibition of anion channels with NPPB or CBX did not affect GABA release from cultured hippocampal neurons. NPPB reduced GABA release from pure astrocytic cultures by 21%, but the total GABA release from astrocytes was small compared to that of mixed cultures. These data indicate that drugs commonly used to antagonize anion channels and connexin hemichannels may affect tonic currents via direct effects on GABA_A receptors and have negligible effects on ambient GABA concentrations. Interpretation of experiments using NPPB or CBX should include consideration of their effects on tonic GABA currents.     

GABA Induces Functionally Active Low-Affinity GABA Receptors on Cultured Cerebellar Granule Cells

The effect of γ-aminobutyric acid (GABA) and its agonists muscimol and 4,5,6,7-tetrahydroisoxazolo[5-4-c]pyridin-3-ol (THIP) on the development of GABA receptors on cerebellar granule cells was studied by cultivation of the cells in media containing these substances. It was found that the presence of 50 μM GABA in the culture media led to the induction of low-affinity GABA receptors (K_D 546 ± 117 nM) in addition to the high-affinity receptors (K_D 7 ± 0.5 nM) which were present regardless of the presence of GABA in the culture media. The functional activity of the GABA receptors was tested by investigating the ability of GABA to modulate evoked glutamate release from the cells. It was found that GABA could inhibit evoked glutamate release (ED₅₀ 10 ± 3 (μM) only when the cells had been cultured in the presence of 50 νM GABA, 50 μM muscimol, or 150 μM THIP, i.e., under conditions where low-affinity GABA receptors were present on the cells. This inhibitory effect of GABA could be blocked by 120 μM bicuculline and mimicked by 50 μM muscimol or 150 μM THIP whereas 150 μM (-)-baclofen had no effect. It is concluded that GABA acting extracellularly induces formation of low-affinity receptors on cerebellar granule cells and that these receptors are necessary for mediating an inhibitory effect of GABA on evoked glutamate release. The pharmacological properties of these GABA receptors indicate that they belong to the so-called GABA_A receptors.     

Glutamic Acid and γ-Aminobutyric Acid Modulate Each Other's Release Through Heterocarriers Sited on the Axon Terminals of Rat Brain

Abstract: The effects of γ-aminobutyric acid (GABA) on the spontaneous release of endogenous glutamic acid (Glu) or aspartic acid (Asp) and the effects of Glu on the release of endogenous GABA or [³H]GABA were studied in superfused rat cerebral cortex synaptosomes. GABA increased the outflow of Glu (EC₅₀17.2 μM) and Asp (EC₅₀ 18.4 μM). GABA was not antagonized by bicuculline or picrotoxin. Neither muscimol nor (-)-baclofen mimicked GABA. The effects of GABA were prevented by GABA uptake inhibitors and were Na⁺ dependent. Glu enhanced the release of [³H]GABA (EC₅₀ 11.5 μM) from cortical synaptosomes. Glu was not mimicked by the glutamate receptor agonists N-methyl-d -aspartic, kainic, or quisqualic acid. The Glu effect was decreased by the Glu uptake inhibitor D-threo-hydroxyaspartic acid (THA) and it was Na⁺ sensitive. Similarly to Glu, D-Asp increased [³H]GABA release (EC₅₀ 9.9 μM), an effect blocked by THA. Glu also increased the release of endogenous GABA from cortex synaptosomes. In this case the effect was in part blocked by the (RS)-α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor antagonist 6-cyano-7-nitroquinoxaiine-2, 3-dione, whereas the 6-cyano-7-nitroquinoxaline- 2, 3-dione-insensitive portion of the effect was prevented by THA. GABA increased the [³H]D-Asp outflow (EC₅₀ 13.7 μM) from hippocampal synaptosomes in a muscimol-, (-)- baclofen-, bicuculline-, and picrotoxin-insensitive manner. The GABA effect was abolished by blocking GABA uptake and was Na⁺ dependent. Glu increased the release of [³H]- GABA from hippocampal synaptosomes (EC₅₀ 7.1 μM) in an N-methyl-d -aspartic acid-, kainic acid-, or quisqualic acid-insensitive way. The effect of Glu was prevented by THA and was Na⁺ dependent. As in the cortex, the effect of Glu was mimicked by D-Asp in a THA-sensitive manner. It is proposed that high-affinity GABA or Glu heterocarriers are sited respectively on glutamatergic or GA- BAergic nerve terminals in rat cerebral cortex and hippocampus. The uptake of GABA may modulate Glu and Asp release, whereas the uptake of Glu may modulate the release of GABA. The existence of these heterocarriers is in keeping with the reported colocalization of GABA and Glu in some cortical and hippocampal neurons. Preliminary data suggest that these mechanisms may also be present in rat cerebellum and spinal cord.     

Sensitivities of Achatina giant neurones to putative amino acid neurotransmitters

1. GABA receptors in Achatina identifiable giant neurones were classified into the muscimol I, muscimol II and baclofen types. Muscimol I and II type GABA receptors were sensitive to GABA and muscimol but insensitive to baclofen, whereas baclofen type receptors were sensitive to GABA and baclofen but insensitive to muscimol. Muscimol I and baclofen types were associated with the inhibition caused by GABA, while muscimol II type with the GABA excitation.2. GABA, muscimol and TACA produced a transient outward current (I_out) with an increase in membrane conductance (g) of an Achatina neurone, TAN, having the muscimol I type GABA receptors. Their relative potency values (RPV) at GABA ed₅₀ (approximately 10⁻⁴ M) were: GABA: muscimol: TACA = 1:0.6:0.3. The GABA effects were potentiated by pentobarbitone, antagonized competitively by pitrazepin and non-competitively by picrotoxin and diazepam, and unaffected by bicuculline. The ionic mechanism of effects of GABA and its two analogues was the increase in membrane Cl⁻ conductance (g_Cl).3. GABA and (±)-baclofen produced a slow I_out with a g increase of another Achatina neurone, RPeNLN, having the baclofen type GABA receptors. The two compounds were almost equipotent (ed₅₀: approximately 3 × 10⁻⁴ M). The ionic mechanism of their effects was the increase in g_k. The two compounds hardly affected the voltage-gated and slowly inactivating calcium current. I_out produced by GABA and (±)-baclofen were reduced by TEA, but unaffected by 4-AP, bicuculline, pitrazepin and picrotoxin.4. β-hydroxy-l-glutamic acid (l-BHGA) showed the marked effects on the Achatina giant neurones; the two neurones were excited by the compound, whereas the three inhibited. D-BHGA, l-Glu, d-Glu and NMDA were less effective than l-BHGA or almost ineffective. Erythro-l-BHGA was more or less effective than threo-l-BHGA according to the neurones tested.5. α-Kainic acid and domoic acid excited the two neurones, which were excited by l-BHGA. l-Quisqualic acid showed the similar effects to l-BHGA, which were mostly much stronger than l-BHGA. Erythro-l-tricholomic acid and dl-ibotenic acid showed the effects similar to l-BHGA selectively on some neurones.6. It was pointed out that the pharmacological features of GABA on the Achatina neurones are simpler than those of l-BHGA, due to the simpler structure of the former compound having less binding sites than the latter.     

GABA Concentration Sets the Conductance of Delayed GABAA Channels in Outside-out Patches from Rat Hippocampal Neurons

GABA_A channels were activated by GABA in outside-out patches from rat cultured hippocampal neurons. They were blocked by bicuculline and potentiated by diazepam. In 109 of 190 outside-out patches, no channels were active before exposure to GABA (silent patches). The other 81 patches showed spontaneous channel activity. In patches containing spontaneous channel activity, rapid application of GABA rapidly activated channels. In 93 of the silent patches, channels could be activated by GABA but only after a delay that was sometimes as long as 10 minutes. The maximum channel conductance of the channels activated after a delay increased with GABA concentration from less than 10 pS (0.5 μm GABA) to more than 100 pS (10 mm GABA). Fitting the data with a Hill-type equation gave an EC ₅₀ value of 33 μm and a Hill coefficient of 0.6. The channels showed outward rectification and were chloride selective. In the presence of 1 μm diazepam, the GABA EC ₅₀ decreased to 0.2 μm but the maximum conductance was unchanged. Diazepam decreased the average latency for channel opening. Bicuculline, a GABA antagonist, caused a concentration-dependent decrease in channel conductance. In channels activated with 100 μm GABA the bicuculline IC ₅₀ was 19 μm. The effect of GABA on channel conductance shows that the role of the ligand in GABA_A receptor channel function is more complex than previously thought. Received: 23 October 2000/Revised: 27 February 2001     

Function of taurine transporter (Slc6a6/TauT) as a GABA transporting protein and its relevance to GABA transport in rat retinal capillary endothelial cells

The purpose of this study was to identify the uptake mechanism of γ-aminobutyric acid (GABA) via taurine transporter (Slc6a6/TauT) and its relationship with GABA transport at the inner BRB. Rat Slc6a6/TauT-transfected HeLa cells exhibited Na⁺-, Cl⁻-, and concentration-dependent [³H]GABA uptake with a K_m of 1.5 mM. Taurine, β-alanine, and GABA markedly inhibited Slc6a6/TauT-mediated uptake of [³H]GABA. The uptake of [³H]GABA by a conditionally immortalized rat retinal capillary endothelial cell line (TR-iBRB2) was Na⁺-, Cl⁻-, and concentration-dependent with a K_m of 2.0 mM. This process was more potently inhibited by substrates of Slc6a6/TauT, taurine and β-alanine, than those of GABA transporters, GABA and betaine. In the presence of taurine, there was competitive inhibition with a K_i of 74 μM. [³H]Taurine also exhibited competitive inhibition with a K_i of 1.8 mM in the presence of GABA. In conclusion, rat Slc6a6/TauT has the ability to use GABA as a substrate and Slc6a6/TauT-mediated GABA transport appears to be present at the inner BRB.     

Pretreatment of Rat Brain Synaptosomes with GABA Increases Subsequent GABA Uptake Via GABAB Receptor Activation

Pretreatment with 100 M GABA of synaptosomes purified from rat brain results in an increased uptake of the labelled neurotransmitter in subsequent incubations. The effect is blocked by a GABA_B receptor antagonist, 2-hydroxy-saclofen. The effect is mimicked by baclofen and the baclofen effect is blocked by saclofen too. Lower GABA concentrations (up to 50 M) do not result in an increase of subsequent GABA uptake. Treatment of synaptosomes with 8-Br-cAMP results in a decreased GABA uptake. Since the uptake incubations were run with saturating concentrations of labelled GABA, the data indicates that GABA_B receptor activation in brain synaptosomes up-regulates their GABA uptake capacity by an increase in V_max. This mechanism appears of physiological relevance under conditions of sustained GABA release and substantial increase of its extracellular concentration.     

Decreased GABA<Subscript>A</Subscript> Receptor Function in the Brain Stem during Pancreatic Regeneration in Rats

Gamma amino butyric acid is a major inhibitory neurotransmitter in the central nervous system. In the present study we have investigated the alteration of GABA receptors in the brain stem of rats during pancreatic regeneration. Three groups of rats were used for the study: sham operated, 72 h and 7 days partially pancreatectomised. GABA was quantified by [³H]GABA receptor displacement method. GABA receptor kinetic parameters were studied by using the binding of [³H]GABA as ligand to the Triton X-100 treated membranes and displacement with unlabelled GABA. GABA_A receptor activity was studied by using the [³H]bicuculline and displacement with unlabelled bicuculline. GABA content significantly decreased (P < 0.001) in the brain stem during the regeneration of pancreas. The high affinity GABA receptor binding showed a significant decrease in B _max (P < 0.01) and K _d (P < 0.05) in 72 h and 7 days after partial pancreatectomy. [³H]bicuculline binding showed a significant decrease in B _max and K _d (P < 0.001) in 72 h pancreatectomised rats when compared with sham where as B _max and K _d reversed to near sham after 7 days of pancreatectomy. The results suggest that GABA through GABA receptors in brain stem has a regulatory role during active regeneration of pancreas which will have immense clinical significance in the treatment of diabetes.     

EFFECT OF ELECTRICAL STIMULATION AND CHLORPROMAZINE ON THE UPTAKE AND RELEASE OF TAURINE, γ-AMINOBUTYRIC ACID AND GLUTAMIC ACID IN MOUSE BRAIN SYNAPTOSOMES

—The concentrations of taurine and GABA were determined in isolated mouse brain synaptosomes incubated in Krebs-Ringer phosphate medium (pH 7·4). The concentration of GABA gradually decreased during incubation, but that of taurine remained approximately unchanged. In the presence of chlorpromazine the amount of GABA in the synaptosomes increased, but the efflux and influx of GABA were slightly reduced. The content and efflux of both taurine and GABA increased in electrically stimulated synaptosomes, and the influx of taurine, GABA and glutamate into the synaptosomes similarly increased. All three amino acids are taken up by the synaptosomes through at least two mechanisms: low-affinity and high-affinity. In the high-affinity system the K_m values were 33 μm for taurine, 24 μm for GABA and 68 μm for glutamate, and in the low-affinity one 1·1 mil, 0·9 mm and 1·2mm , respectively. The influx capacity (V_max) was highest for glutamate, second highest for GABA and lowest for taurine.     

mtDNA m.3635G>A may be classified as a common primary mutation for Leber hereditary optic neuropathy in the Chinese population

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian central nervous system and exerts its actions via ionotropic (GABA_A and GABA_C) and metabotropic (GABA_B) receptors. The GABA_B receptor is a dimer composed of R1 and R2 components. In addition to their location on neurons, GABA and functional GABA_B receptors also have been detected in some peripheral tissues. In the present study, we combined immunohistochemistry, immunoblot and tension recording to determine if the human fallopian tube express glutamic acid decarboxylase (GAD65/67), two isoforms for synthesis of GABA and functional GABA_B receptors. Immunoblots showed that the human fallopian tube tissue contained GABA_BR1 protein which was localized in the epithelial cells and smooth muscle cells by immunohistochemistry. In addition, epithelial cells also expressed GAD65/67. Tension recording found that both GABA and baclofen, a GABA_B receptor agonist increased the spontaneous activity of human fallopian tube. The expressions of GABA_BR and GAD65/67 were significantly upregulated in the ectopic pregnancy group than in the intrauterine pregnancy group. We conclude that the human fallopian tube is capable of synthesizing GABA and expresses functionally active GABA_B receptors. An upregulation of GABA synthesis and corresponding GABA_B receptors may involve in ectopic pregnancy.     

Unsaturated Analogues of the Neurotransmitter GABA: <Emphasis Type="Italic">trans</Emphasis>-4-Aminocrotonic, <Emphasis Type="Italic">cis</Emphasis>-4-Aminocrotonic and 4-Aminotetrolic Acids

Analogues of the neurotransmitter GABA containing unsaturated bonds are restricted in the conformations they can attain. This review traces three such analogues from their synthesis to their use as neurochemicals. trans-4-Aminocrotonic acid was the first conformationally restricted analogue to be extensively studied. It acts like GABA across a range of macromolecules from receptors to transporters. It acts similarly to GABA on ionotropic receptors. cis-4-Aminocrotonic acid selectively activates bicuculline-insensitive GABA_C receptors. 4-Aminotetrolic acid, containing a triple bond, activates bicuculline-sensitive GABA_A receptors. These findings indicate that GABA activates GABA_A receptors in extended conformations and GABA_C receptors in folded conformations. These and related analogues are important for the molecular modelling of ionotropic GABA receptors and to the development of new agents acting selectively on these receptors.     

Nitrate uptake and utilization is modulated by exogenous γ-aminobutyric acid in Arabidopsis thaliana seedlings

Exogenously applied GABA modulates root growth by inhibition of root elongation when seedlings were grown in vitro on full-strength Murashige and Skoog (MS) salts, but root elongation was stimulated when seedlings were grown on 1/8 strength MS salts. When the concentration of single ions in MS salts was individually varied, the control of growth between inhibition and stimulation was found to be related to the level of nitrate (NO₃^?) in the growth medium. At NO₃^? concentrations below 40 mM (full-strength MS salts level), root growth was stimulated by the addition of GABA to the growth medium; whereas at concentrations above 40 mM NO₃^?, the addition of GABA to the growth medium inhibited root elongation. GABA promoted NO₃^? uptake at low NO₃^?, while GABA inhibited NO₃^? uptake at high NO₃^?. Activities of several enzymes involved in nitrogen and carbon metabolism including nitrate reductase (NR), glutamine synthetase (GS), glutamate synthase (NADH-GOGAT), NADP-dependent isocitrate dehydrogenase (NADP-ICDH), and phosphoenol pyruvate carboxylase (PEPCase) were regulated by GABA in the growth medium. Supplementing 1/8 strength MS medium with 50 mM GABA enhanced the activities of all of the above enzymes except ICDH activities in root tissues. However, at full-strength MS, GABA showed no inhibitory effect on the activities of these enzymes, except on GS in both root and shoot tissues, and PEPCase activity in shoot tissues. Exogenous GABA increased the amount of NR protein rather than its activation status in the tissues. This study shows that GABA affects the growth of Arabidopsis, possibly by acting as a signaling molecule, modulating the activity of enzymes involved in primary nitrogen metabolism and nitrate uptake.     

Substrate Specificity of [3H] Muscimol Binding to a Particulate Fraction of a Neuron-enriched Culture of Embryonic Rat Brain

Abstract: The effects of some GABA analogues and some drugs on the binding of [³H]muscimol (3.08 nM) to thoroughly washed subcellular particles prepared from a neuron-enriched culture of embryonic rat brain were examined using Na+-free Tris-citrate medium and a centrifugation method. Competition for [³H]muscimol binding sites by excess(10^?5 M) unlabelled GABA provided estimates of “specific” binding. In accord with in vivo neuropharmacological studies on GABA receptors and with in vitro studies on cerebral membrane preparations, [³H]muscimol binding was potently inhibited by muscimol itself (IC₅₀, 2.5 nM), GABA (1C₅₀, 43 nM), isoguvacine (IC₅₀, 61 nM), and 3-aminopropanesulphonic acid (IC₅₀, 160 nM), and less potently inhibited by the GABA antagonist bicuculline methobromide (IC₅₀, 800 nM). δ- Aminovaleric acid (IC₅₀, 2.6 μM), the glycinelp-alanine antagonist strychnine (IC₅₀, 6.6 μM), and the predominantly glial GABA uptake inhibitors β-alanine (IC₅₀, 23 μM) and p-proline (IC₅₀, 66 μM) also inhibited [³H]muscimol binding. Other inhibitors of Na+-dependent GABA uptake, (±)-nipecotic acid, L- 2,4-diaminobutyric acid, and guvacine, as well as picrotoxinin, were relatively inactive as inhibitors of [³H]muscimol binding (IC₅₀≥ 1 mM). In addition to revealing that GABA receptors are present on neuronal membranes before the formation of most synapses, this binding of [³H]muscimol that occurs to neuronal, but not to glial, membranes might be useful as a “neuronal marker” and for the further characterization and isolation of GABA receptors.     

GABA regulates the buccal motor output of Helisoma by two pharmacologically distinct actions

GABA was tested for its effects on patterned motor activity (PMA) underlying feeding. Using buccal motoneuron B19 to monitor PMA through intracellular recordings, GABA was found to exert effects at two levels. First, GABA stimulated rhythmic patterned activity resembling fictive feeding, which is under the control of the buccal CPG. In addition, GABA produced a direct inhibition of neuron B19. Both effects were observed when the buccal ganglia were studied in isolation from the rest of the central nervous system, suggesting local interactions with GABA receptors of buccal neurons. Furthermore, these two actions of GABA were found to be pharmacologically distinguishable. The direct hyperpolarization of neuron B19 was mimicked by muscimol, but not baclofen, and involved an increased chloride conductance, which was blocked by picrotoxin.Baclofen duplicated CPG activation by GABA. Picrotoxin had no effect on GABA- or baclofen-induced PMA.These results demonstrate that the Helisoma buccal ganglia have two GABA receptor types which resemble, pharmacologically, mammalian GABA_A and GABA_B receptors, and that GABA plays a key role in feeding patterned motor activity in Helisoma.Abbreviations CPG central pattern generator - GABA gammaamino butyric acid - HPLC high performance liquid chromatography - IPSP inhibitory postsynaptic potential - PMA patterned motor activity - SLRT supralateral radular tensor muscle     

Molecular dynamics simulations of GABA binding to the GABAC receptor: the role of Arg104

GABA is the major inhibitory neurotransmitter in the nervous system and acts at a variety of receptors including GABA_C receptors, which are a subclass of GABA_A receptors. Here we have used molecular dynamics simulations of GABA docked into the extracellular domain of the GABA_C receptor to explain the molecular interactions of the neurotransmitter with the residues that contribute to the binding site; in particular, we have explored the interaction of GABA with Arg¹⁰⁴. The simulations suggest that the amine group of GABA forms cation-π interactions with Tyr¹⁰² and Tyr¹⁹⁸, and hydrogen-bonds with Gln⁸³, Glu²²⁰, Ser²⁴³, and Ser¹⁶⁸, and, most prominently, with Arg¹⁰⁴. Substituting Arg¹⁰⁴ with Ala, Glu, or Lys, which experimentally disrupt GABA_C receptor function, and repeating the simulation revealed fewer and different bonding patterns with GABA, or the rapid exit of GABA from the binding pocket. The simulations therefore unveil interactions of GABA within the binding pocket, and explain experimental data, which indicate that Arg¹⁰⁴ is critical for the efficient functioning of the receptor.     

High γ-aminobutyric acid content, a novel component associated with resistance to abamectin in Tetranychus cinnabarinus (Boisduval)

An abamectin-resistant strain of Tetranychus cinnabarinus (Boisduval) (Rf = 25.3) was selected in laboratory. We compared the γ-aminobutyric acid (GABA) content in abamectin-susceptible T. cinnabarinus individuals with that in resistant individuals and investigated its relationship to abamectin resistance. High performance liquid chromatography (HPLC) was used to ascertain GABA content in abamectin-susceptible (SS) and resistant (AR) strains of T. cinnabarinus. The results indicate that GABA content in the AR was significantly higher than that in the SS (1.39-fold). AR individuals treated with a sublethal dose of abamectin did not show significant differences in GABA levels compared with AR individuals that were not treated with abamectin. However in the SS, abamectin treated individuals had a significantly higher GABA content than those that were untreated (1.52-fold). Individuals in the SS that survived from selection with LC₉₅ of abamectin (SS-AR) showed significantly higher GABA levels compared to SS (1.41-fold). Similarly, progenies of the SS-AR parental generation (SS-ARF₁) also showed increased GABA levels (1.51-fold) compared to SS. In addition, behavioral observations have shown that all individuals from the AR, SS-AR and SS-ARF₁, which had more GABA content than the SS, demonstrated a significant decrease in crawling speed compared with SS individuals. This observation is consistent with excessive GABA levels had inhibitory effect on the central nervous system. Thus, we postulate that increasing GABA content in T. cinnabarinus is associated with resistance against abamectin.     

GABA Enhances Thermotolerance of Seeds Germination by Attenuating the ROS Damage in Arabidopsis

Seeds germination is strictly controlled by environment factor such as high temperature (HT) through altering the balance between gibberellin acid (GA) and abscisic acid (ABA). Gama-aminobutyric acid (GABA) is a small molecule with four-carbon amino acid, which plays a crucial role during plant physiological process associated with pollination, wounding or abiotic stress, but its role in seeds germination under HT remains elusive. In this study we found that HT induced the overaccumulation of ROS, mainly H₂O₂ and O₂^- , to suppress seeds germination, meanwhile, HT also activated the enzyme activity of GAD for the rapid accumulation of GABA, hinting the regulatory function of GABA in controlling seeds germination against HT stress. Applying GABA directly attenuated HT-induced ROS accumulation, upregulated GA biosynthesis and downregulated ABA biosynthesis, ultimately enhanced seeds germination. Consistently, genetic analysis using the gad1/2 mutant defective in GABA biosynthesis, or pop2-5 mutant with high endogenous GABA content supported the potential function of GABA in improving seeds germination tolerance to HT through scavenging ROS overaccumulation. Based on these data, we propose that GABA acts as a novel signal to enhance thermotolerance of seeds germination through alleviating the ROS damage to seeds viability.     

GABA exists as a negative regulator of cell proliferation in spermaogonial stem cells

γ-amino butyric acid (GABA) is the main inhibitory neurotransmitter in the mammalian central nervous system. GABA is also found in many peripheral tissues, where it has important functions during development. Here, we identified the existence of the GABA system in spermatogonial stem cells (SSCs) and found that GABA negatively regulates SSC proliferation. First, we demonstrated that GABA and its synthesizing enzymes were abundant in the testes 6 days postpartum (dpp), suggesting that GABA signaling regulates SSCs function in vivo. In order to directly examine the effect of GABA on SSC proliferation, we then established an in vitro culture system for long-term expansion of SSCs. We showed that GABA_A receptor subunits, including α1, α5, β1, β2, β3 and γ3, the synthesizing enzyme GAD67, and the transporter GAT-1, are expressed in SSCs. Using phosphorylated histone H3 (pH3) staining, we demonstrated that GABA or the GABA_AR-specific agonist muscimol reduced the proliferation of SSCs. This GABA regulation of SSC proliferation was shown to be independent of apoptosis using the TUNEL assay. These results suggest that GABA acts as a negative regulator of SSC proliferation to maintain the homeostasis of spermatogenesis in the testes.