Presence Of Gamma-aminobutyric Acid Research Articles

Gamma-Aminobutyric Acid Action on Membrane and Electrical Properties of Synaptosomes and Model Lipid Bilayers.

Dysfunction of the main inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is the underlying reason behind many neurological disorders including Alzheimer's and Huntington's diseases, autism spectrum disorders, anxiety, depression, hypertension, and cardiovascular diseases, among others. Here, we address neurotransmitter-induced alterations of synaptosomal and model membrane electrical properties for elucidating membrane-related biophysical mechanisms of neurological disorders. We focus on membrane surface characteristics of the pinched off nerve endings synaptosomes, which for decades have been a powerful tool in neurobiology. Microelectrophoretic measurements of GABA-treated negatively charged synaptosomes from rat cerebral cortex reveal lower negative zeta potential as a result of reduced electrical charge on the membrane surface at (1-4h) after isolation. Conversely, enhancement of the surface parameters of synaptosomes (17-22h) post isolation is obtained due to additional negatively exposed groups on the surface of the vesicles. The electrical properties of bilayer lipid membranes are probed by electrochemical impedance spectroscopy, reporting as light increase of the membrane electrical capacitance in the presence of GABA, likely related to membrane thinning and dielectric permittivity alterations. The neurotransmitter inhibits sodium-potassium as well as the total ATPase activity and slightly enhances magnesium-ATPase of native synaptic membranes. At low (pM) GABA concentrations the activity of acetylcholinesterase (AChE) in synaptic membranes increases. AChE inhibition is reported at higher GABA concentrations. The relation between the surface electrical properties of cells and the enzymatic activity of brain ATPases and AChE, as examined here, are expected to be helpful in the elucidation of membrane-mediated molecular mechanisms relevant to neurological disorders and conditions.

GABA Receptors and Kv7 Channels as Targets for GABAergic Regulation of Acetylcholine Release in Frog Neuromuscular Junction.

Effects of gamma-aminobutyric acid (GABA) and some selective GABAergic ligands on the quantal acetylcholine (ACh) release in the frog neuromuscular contacts were investigated using combination of microelectrode technique with fluorescent and immunohistochemical assays. Significant attenuation of ACh release was observed in the presence of GABA as well as selective GABAA and GABAB receptor agonists. Neither GABAA nor GABAB antagonists abolished to full extent this effect of GABA. Fluorescent assay allowed to detect the GABA-induced opening of K+ channels, which was inhibited by XE-991, a selective antagonist of Kv7 type. Electrophysiological recordings of endplate potentials in the presence of XE-991 confirmed the contribution of Kv7 type potassium channels to the effects of GABA on ACh release that was not associated with GABAA and GABAB receptors activation. Note that XE-991 effectively precluded the action of retigabine, neuronal Kv7 channel opener, on ACh release. Immunohistochemical assay revealed that frog mature skeletal muscle fibers contain a significant amount of GABA, and substantial amount of GABA can be released in the extracellular space at the muscle contractions induced by prolonged high-frequency nerve stimulation. Besides, some binding sites for exogenous GABA were detected on the plasma membranes. It is concluded that GABA, in addition to affecting GABAA and GABAB receptors, can directly activate Kv7 channels, thereby negatively modulating the evoked ACh release. Endogenous GABA may serve as a retrograde regulator of neurotransmitter exocytosis.

Gamma-aminobutyric acid modulates antioxidant and osmoprotective systems in seedlings of Triticum aestivum cultivars differing in drought tolerance

The stress-protective effects of plant neurotransmitters, including gamma-aminobutyric acid (GABA) have been intensively examined in recent years. However, studies on the GABA influence on stress protective systems in bread wheat cultivars with different drought adaptation strategies are still lacking. The aim of this work was to estimate the GABA effect on the state of antioxidant and osmoprotective systems in etiolated seedlings of two wheat cultivars differing significantly in drought tolerance, namely Doskonala (non-drought-resistant) and Tobak (drought-resistant) under model drought induced by PEG 6000. Two-day-old seedlings were transferred to 15% PEG 6000 and incubated for two days in the absence or presence of GABA. Treatment with 0.1 and 0.5 mM GABA significantly reduced the growth-inhibitory effect of PEG 6000 on the roots and shoots of both cultivars, but to a greater extent on those of non-resistant Doskonala. It was shown that GABA treatment reduced drought-induced accumulation of H2O2 and MDA, stabilized SOD and GPX activity, the level of sugars, anthocyanins and flavonoids in seedlings of both cultivars. Meanwhile GABA treatment enhanced the stress-induced increase in proline content in the Doskonala cultivar, but decreased it in the Tobak, completely prevented stress induced decrease in anthocyanins and flavonoids level in the Tobak and only partially in Doskonala cultivar. Thus, the stabilization of the stress-protective systems functioning in the wheat cultivars and variety-dependent differences in response to GABA were revealed. Keywords: anthocyanins, antioxidant system, drought resistance, gamma-aminobutyric acid, oxidative stress, proline, Triticum aestivum

The synergistic effects of gamma-aminobutyric acid and salinity during the enhancement of microalgal lipid production in photobioreactors

In the present work, combined application of gamma-aminobutyric acid (GABA) and salinity was used to stimulate biolipid production in the microalga Ankistrodesmus sp. EHY in a vertical tubular photobioreactor. Under 2.5 g L−1 NaCl combined with 50 μM GABA, the strain showed 1.36- and 1.27-fold increases in lipid content (59.42%) and lipid productivity (235.13 mg L−1 d−1), respectively, compared with the control. Wide-ranging analyses of the energy yield, biodiesel quality, cellular ultrastructure, signalling molecules, and lipogenesis genes were conducted to elucidate the role of GABA plus salinity on algal lipid accumulation. By using GABA and salinity treatments, the triacylglycerol (TAG) content and energy yield were increased, whereas the carbohydrate yield was decreased. The biodiesel quality of the algal biomass-derived lipids met the approved standards. The combinational strategy also increased the levels of endogenous reactive oxidative species (ROS), Ca2+, an autophagy-related protein (ATG8), glutathione (GSH) and proline, which might be associated with the increase in lipid synthesis and salt stress tolerance. Moreover, transcriptomic and metabolomic data indicated that the expression levels of TAG assembly, antioxidative system and autophagy-related genes, and the metabolite levels of the TCA cycle, GABA shunt and several stress hormones were consistent with the enhancements in lipid accumulation and stress tolerance. In the presence of GABA and salinity, exogenous proline (50 μM) and salicylic acid (SA; 12.5 mg L−1) further increased the lipid productivity to 280.48 mg L−1 d−1 and 263.58 mg L−1 d−1, respectively. Overall, the results demonstrate the significance, mechanism and applicability of using exogenous GABA combined with salinity to effectively stimulate bioenergy production by microalgae.

Interaction of agonists of a different subtype of the nAChR and carvacrol with GABA in Ascaris suum somatic muscle contractions.

Resistance of parasitic nematodes to anthelmintic drugs is a growing problem in human and veterinary medicine. The molecular mechanisms by which nematodes become resistant are different, but certainly one of the possible processes involves changing the drug binding site on the specific receptor. The significance of changes in individual subtypes of nicotinic acetylcholine receptors (nAChRs) for the development of resistance has not been clarified in detail. This study investigates the interaction of antinematodal drugs, agonist of different types of nAChRs and carvacrol with gamma aminobutyric acid (GABA) on the contractions of parasitic nematode A. suum. In our study, GABA (3 μM) produced significant increase of contractile EC50 value for pyrantel, and nonsignificant for bephenium and morantel, from 8.44 to 28.11 nM, 0.62 to 0.96 µM, and 3.72 to 5.69 nM, respectively. On the other hand, the maximal contractile effect (Rmax) did not change in the presence of GABA. However, when A. summ muscle flaps were incubated with GABA 3 μM and carvacrol 100 μM, the EC50 value of pyrantel, bephenium, and morantel was increased significantly to 44.62 nM, 1.40 μM, and nonsignificantly to 7.94 nM, respectively. Furthermore, Rmax decreased by 70, 60, and 65%. Presented results indicate that the combined use of GABA receptor agonists and nicotinic receptor antagonists can effectively inhibit the neuromuscular system of nematodes, even when one of the nicotinic receptor subtypes is dysfunctional, due to the potential development of resistance.

Gamma-aminobutyric acid production by Lactobacillus brevis A3: Optimization of production, antioxidant potential, cell toxicity, and antimicrobial activity.

In this study, whey powder was used as the basic compound for fermentation culture and the production of bioactive gamma‐aminobutyric acid (GABA) compound. GABA is a nonprotein four‐carbon amino acid that inhibits stress signals by preventing brain signals, reducing stress, and being effective in treating neurological disorders and decreasing the growth of cancer cells. Due to the side effects caused by the chemical type of GABA, the biological production of GABA has attracted. Three levels of whey powder (5%, 10%, and 15%), and monosodium glutamate (MSG) (1%, 3%, and 5%) were selected at temperatures (25, 30, and 37°C) and after fermentation, the presence of GABA in the culture medium was examined by thin‐layer chromatography. The optimal amount of GABA was measured by using high‐performance liquid chromatography. The results of the central composite design of the response surface methodology at a significant level of 95% showed that the optimal treatment was 14.96% whey powder, 4.95% MSG at temperature of 37°C and fermentation for 48 hr and under these conditions, GABA production was 553.5 ppm. The results of the fermented extract tests showed that the highest antimicrobial activity was on Escherichia coli and the highest free radical scavenging was 59.67%. The IC50 level in the Caco‐2 cancer cell cytotoxicity test was 39.5 mg/ml. According to the results, the combination of whey with MSG can be used as a cheap substrate to produce a valuable bioactive GABA product, and the cellular extract of this fermentation can also be used as an antimicrobial and antioxidant compound in food and pharmaceutical formulations.

Development and characterization of antioxidant and antimicrobial edible films based on chitosan and gamma-aminobutyric acid-rich fermented soy protein

In this study, biocomposite edible films of gamma-aminobutyric acid (GABA)-rich fermented soy protein (GABA-RFSP) with different chitosan concentrations (2, 2.5, and 3%) were prepared. The GABA-rich edible films based on 2.5 % chitosan had the highest a*, b*, browning index, and absorbance at 420 nm compared to other films, indicating the occurrence of Maillard reaction during film-formation period. The Maillard reaction fabricated 2.5 % chitosan-based edible films with high tensile strength and elongation at break, in conjugation with a more smooth, compact, and homogeneous surface with less pores or cracks, which was confirmed by Field emission scanning electron microscopy. The films also showed considerable antioxidant and antimicrobial activities. The covalent linkages between chitosan and GABA-RFSP, and the presence of GABA in the edible films were confirmed by Fourier transform infrared spectroscopy. The results suggested the potential use of GABA-RFSP and chitosan to fabricate novel bioactive-loaded edible films for food packaging purposes.

Spectroscopic investigation on alteration of dynamic properties of lipid membrane in presence of Gamma-Aminobutyric Acid (GABA)

The effect of neurotransmitters on the cell membrane is an elegant topic to be explored in the arena of chemistry and biology, particularly with respect to the understanding of various neurological disorders. In this present study, based on various spectroscopic and microscopic results, we find the alteration of membrane properties of model phospholipid vesicles due to interaction with the neurotransmitter Gamma-Aminobutyric Acid (GABA). The solvation dynamics and anisotropy measurements indicate the increased hydration and enhanced fluidity of lipid membrane in presence of GABA. An increased microheterogeneity of membrane system is reflected from fluorescence lifetime imaging microscopy (FLIM) study. Further, the zeta potential and 31PNMR spectroscopy confirm the mode and site of interaction of GABA to the membrane. The fluorescence correlation spectroscopy (FCS) measurement indicates that the dye molecules in the GABA-containing vesicles are diffusing at a slower rate due to the formation of large vesicular entity. Therefore, we establish the effect of interaction of elevated concentration of GABA with membrane, like, alteration of dynamic aspects, perturbation of membrane morphology, variation of rotational and translational diffusion properties of dye molecules in the vesicle, which would conspicuously research the status of the phospholipid membrane in presence of GABA under various neurological disorders.

Sex hormones and the nervous system, do they matter for physical performance?

There are well-known oscillations in circulating sex hormones that occur in both men and women on daily and monthly cycles, as well as across the lifespan. The non-reproductive effects of estrogen and progesterone, particularly in women, on physical performance have been an area of intense debate. This presentation will examine the evidence that sex hormones can modulate the nervous system from the cellular-level to a whole-body perspective and impact human physical performance. The cell-level and animal literature clearly indicates sex hormones have the capability to directly modulate the excitability of the nervous system through gamma-Aminobutyric acid (GABA) receptors. Pregnenolone, a first-order metabolite of progesterone, is able to directly activate GABA receptors creating either a neuro-excitatory or inhibitory effect depending on whether pregnenolone is sulfonated. Progesterone is able to enhance the GABA receptor response in the presence of GABA, increasing neural inhibition. Additionally, estrogen creates an excitatory effect on the nervous system by attenuating the release of GABA from neurons. Research from our work group has shown that the discharge of motor neurons are altered across the menstrual cycle 1 Tenan M.S. Peng Y.L. Hackney A.C. Griffin L. MSSE. 2013; 45: 2151-2157 Google Scholar and that quadriceps MVC may decrease by as much as 23% in the mid luteal phase of the menstrual cycle, when both estrogen and progesterone are high. 2 Tenan M.S. Hackney A.C. Griffin L. EJAP. 2016; 116: 153-160 Google Scholar In relative time to exhaustion, the mid luteal menstrual phase also has the least stable force production across the entire isometric task. At the cellular level, sex hormones clearly have the capability to modulate nervous system function. Our research indicates that this translates to measurable effects on human performance; however, this finding is not uniform across all studies. In conclusion, sex hormones have the theoretical ability to modify physical performance but the inconsistent findings at the clinical level may be a result of individual variability in the level of sex hormones both in circulation and uniquely within the central nervous system, making broad generalizations across a population of men or women challenging.

γ-Aminobutyric Acid Is Synthesized and Released by the Endothelium: Potential Implications.

Gamma aminobutyric acid (GABA), a neurotransmitter of the central nervous system, is found in the systemic circulation of humans at a concentration between 0.5 and 3 μmol/L. However, the potential source of circulating GABA and its significance on the vascular system remains unknown. We hypothesized that endothelial cells (ECs) may synthesize and release GABA to modulate some functions in the EC and after its release into the circulation. To assess whether GABA is synthesized and released by the EC and its potential functions. Utilizing the human umbilical vein ECs and aortic ECs, we demonstrated for the first time that ECs synthesize and release GABA from [1-(14)C]glutamate. Localization of GABA and the presence of the GABA-synthesizing enzyme, glutamic acid decarboxylase in EC were confirmed by immunostaining and immunoblot analysis, respectively. The presence of GABA was further confirmed by immunohistochemistry in the EC lining the human coronary vessel. EC-derived GABA regulated the key mechanisms of ATP synthesis, fatty acid, and pyruvate oxidation in EC. GABA protected EC by inhibiting the reactive oxygen species generation and prevented monocyte adhesion by attenuating vascular cell adhesion molecule -1 and monocyte chemoattractant protein-1 expressions. GABA had no relaxing effect on rat aortic rings. GABA exhibited a dose-dependent fall in blood pressure. However, the fall in BP was abolished after pretreatment with pentolinium. Our findings indicate novel potential functions of endothelium-derived GABA.

The Uptake of GABA in Trypanosoma cruzi.

Gamma aminobutyric acid (GABA) is widely known as a neurotransmitter and signal transduction molecule found in vertebrates, plants, and some protozoan organisms. However, the presence of GABA and its role in trypanosomatids is unknown. Here, we report the presence of intracellular GABA and the biochemical characterization of its uptake in Trypanosoma cruzi, the etiological agent of Chagas' disease. Kinetic parameters indicated that GABA is taken up by a single transport system in pathogenic and nonpathogenic forms. Temperature dependence assays showed a profile similar to glutamate transport, but the effect of extracellular cations Na(+) , K(+) , and H(+) on GABA uptake differed, suggesting a different uptake mechanism. In contrast to reports for other amino acid transporters in T. cruzi, GABA uptake was Na(+) dependent and increased with pH, with a maximum activity at pH 8.5. The sensitivity to oligomycin showed that GABA uptake is dependent on ATP synthesis. These data point to a secondary active Na(+) /GABA symporter energized by Na(+) -exporting ATPase. Finally, we show that GABA occurs in the parasite's cytoplasm under normal culture conditions, indicating that it is regularly taken up from the culture medium or synthesized through an still undescribed metabolic pathway.

Presence of Gamma-Aminobutyric Acid (Gaba) in the Pedal Mucus of the Critically Endangered Species Patella ferruginea.

Patella ferruginea is a giant patellid limpet endemic to the Mediterranean Sea. It presently is in danger of extinction, and many have called for developing conservation measures including the mass production of spats for re-introduction projects. However, so far all attempts have been relatively unsuccessful. Previous work analyzing the effects of gamma-aminobutyric acid (GABA) on the recruitment of patellid limpets has shown that larvae respond to the presence of this signaling molecule. This response could explain the gregarious distribution typical of this species. In the present study, we demonstrated that GABA is naturally secreted by P. ferruginea in the pedal mucus. GABA is preferentially secreted during the summer, coinciding with the reproductive resting period of the species. Further research should aim to analyze the effects of GABA on larval development and metamorphosis in order to assess its potential use to improve conservation efforts.

Fast detection of extrasynaptic GABA with a whole-cell sniffer.

Gamma-amino-butyric acid (GABA) is the main inhibitory transmitter of the brain. It operates by binding to specific receptors located both inside and outside synapses. The extrasynaptic receptors are activated by spillover from GABAergic synapses and by ambient GABA in the extracellular space. Ambient GABA is essential for adjusting the excitability of neurons. However, due to the lack of suitable methods, little is known about its dynamics. Here we describe a new technique that allows detection of GABA transients and measurement of the steady state GABA concentration with high spatial and temporal resolution. We used a human embryonic kidney (HEK) cell line that stably expresses GABAA receptors composed of α1, β2, and γ2 subunits. We recorded from such a HEK cell with the whole-cell patch-clamp technique. The presence of GABA near the HEK cell generated a measurable electric current whose magnitude increased with concentration. A fraction of the current did not inactivate during prolonged exposition to GABA. This technique, which we refer to as a “sniffer” allows the measurement of ambient GABA concentration inside nervous tissue with a resolution of few tens of nanomolars. In addition, the sniffer detects variations in the extrasynaptic GABA concentration with millisecond time resolution. Pilot experiments demonstrate that the sniffer is able to report spillover of GABA induced by synaptic activation in real time. This is the first report on a GABA sensor that combines the ability to detect fast transients and to measure steady concentrations.

The traditional antidiarrheal remedy, Garcinia buchananii stem bark extract, inhibits propulsive motility and fast synaptic potentials in the guinea pig distal colon.

Garcinia buchananii bark extract is a traditional African remedy for diarrhea, dysentery, abdominal discomfort, and pain. We investigated the mechanisms and efficacy of this extract using the guinea pig distal colon model of gastrointestinal motility. Stem bark was collected from G. buchananii trees in their natural habitat of Karagwe, Tanzania. Bark was sun dried and ground into fine powder, and suspended in Krebs to obtain an aqueous extract. Isolated guinea pig distal colon was used to determine the effect of the G. buchananii bark extract on fecal pellet propulsion. Intracellular recording was used to evaluate the extract action on evoked fast excitatory postsynaptic potentials (fEPSPs) in S-neurons of the myenteric plexus. Garcinia buchananii bark extract inhibited pellet propulsion in a concentration-dependent manner, with an optimal concentration of ∼10 mg powder per mL Krebs. Interestingly, washout of the extract resulted in an increase in pellet propulsion to a level above basal activity. The extract reversibly reduced the amplitude of evoked fEPSPs in myenteric neurons. The extract's inhibitory action on propulsive motility and fEPSPs was not affected by the opioid receptor antagonist, naloxone, or the alpha- 2 adrenoceptor antagonist, yohimbine. The extract inhibited pellet motility in the presence of gamma-aminobutyric acid (GABA), GABA(A) and GABA(B) receptor antagonists picrotoxin and phaclofen, respectively. However, phaclofen and picrotoxin inhibited recovery rebound of motility during washout. Garcinia buchananii extract has the potential to provide an effective, non-opiate antidiarrheal drug. Further studies are required to characterize bioactive components and elucidate the mechanisms of action, efficacy, and safety.

The presence of GABA in gastropod mucus and its role in inducing larval settlement

Chemical substances that induce larval settlement have been the focus of many gastropod studies due to the importance of wild stock recruitment and production within aquaculture facilities. Gamma-aminobutyric acid (GABA), GABA analogs, and GABA-mimetics associated with certain crustose coralline algae (CCA), are known to induce larval settlement in commercial abalone ( Haliotis) species, and other gastropods. Furthermore, mucus secreted from these gastropods has been shown to induce larval settlement, but the stimulatory components of mucus have not been thoroughly investigated. We now present data confirming that GABA is the settlement-inducing effector molecule contained within abalone mucus. To do this, we initially generated anti-GABA for use in immunoenzyme and immunofluorescent microscopy. Using these techniques GABA was identified in the nerves and epithelial cells of the foot, including mucus. Dried mucus samples subject to HPLC analysis revealed a mean concentration of 0.68 mM GABA after sample rehydration. The presence of GABA in these samples was confirmed by time-of-flight mass spectroscopy (TOF-MS). In addition, GABA was detected in the mucus of several abalone species and other gastropods by immunocytochemistry. Subsequent bioassays using both dry and fresh mucus strongly promoted induction of larval settlement.

Sexually Dimorphic Modulation of GABAA Receptor Currents by Melatonin in Rat Gonadotropin-Releasing Hormone Neurons

Gonadotropin-releasing hormone (GnRH) neurons represent the final output neurons in the central control of reproduction. gamma-Amino butyric acid (GABA), one of the major regulators of GnRH neurons, depolarizes GnRH neurons isolated from adult rats via GABA(A) receptors. The presence of GABA(A) receptors in GnRH neurons has also been demonstrated morphologically. Furthermore, the pineal hormone melatonin is involved in the regulation of reproductive function, including the timing of the luteinizing hormone surge. The suprachiasmatic nucleus and the GABAergic system in the medial preoptic area are considered as possible sites of the action of melatonin. Until now, however, a direct action of melatonin on GnRH neurons has not been reported. Therefore we examined the effect of melatonin on GABA(A) receptor currents in GnRH neurons isolated from GnRH-EGFP transgenic rats by means of perforated patch-clamp experiments. The GABA(A) receptor currents were modulated by melatonin in a sex-specific manner. In GnRH neurons from adult males, melatonin augmented these currents in 67% of the neurons examined, but attenuated the currents in only 19% of them. These modulations were blocked by the melatonin receptor antagonist luzindole, suggesting an involvement of melatonin receptors. The modulation by melatonin was not observed in GnRH neurons isolated from infantile rats. These findings indicate that GABA affects the excitability of GnRH neurons in adult rats through GABA(A) receptors, and that melatonin modifies this excitability via melatonin receptors in a sex-specific manner.

The GABAergic‐like system in the marine demosponge Chondrilla nucula

Gamma-amino butyric acid (GABA) is believed to be the principal inhibitory neurotransmitter in the mammalian central nervous system, a function that has been extended to a number of invertebrate systems. The presence of GABA in the marine demosponge Chondrilla nucula was verified using immunofluorescence detection and high-pressure liquid chromatography. A strong GABA-like immunoreactivity (IR) was found associated with choanocytes, exopinacocytes, endopinacocytes lining inhalant, and exhalant canals, as well as in archaeocytes scattered in the mesohyl. The capacity to synthesize GABA from glutamate and to transport it into the vesicles was confirmed by the presence in C. nucula of glutamate decarboxylase (GAD) and vesicular GABA transporters (vGATs), respectively. GAD-like and vGAT-like IR show the same distribution as GABA-like IR. Supporting the similarity between sponge and mammalian proteins, bands with an apparent molecular weight of about 65-67 kDa and 57 kDa were detected using antibodies raised against mammalian GAD and vGAT, respectively. A functional metabotropic GABA(B)-like receptor is also present in C. nucula. Indeed, both GABA(B) R1 and R2 isoforms were detected by immunoblot and immunofluorescence. Also in this case, IR was found in choanocytes, exopinacocytes, and endopinacocytes. The content of GABA in C. nucula amounts to 1225.75 +/- 79 pmol/mg proteins and GABA is released into the medium when sponge cells are depolarized. In conclusion, this study is the first indication of the existence of the GABA biosynthetic enzyme GAD and of the GABA transporter vGAT in sponges, as well as the first demonstration that the neurotransmitter GABA is released extracellularly.

Gamma-aminobutyric acid and related molecules in the sea fan Eunicella cavolini (Cnidaria: Octocorallia): a biochemical and immunohistochemical approach

The aim of this study has been the biochemical demonstration of the presence of gamma-aminobutyric acid (GABA) in the Mediterranean sea fan Eunicella cavolini by means of high-performance liquid chromatography, and the description of the distribution pattern of GABA and its related molecules, glutamic acid decarboxylase (GAD), vesicular GABA transporter (VGAT) and one of the GABA receptors (GABA(B) R) by immunohistochemical methods. The interrelationships of GABA, GAD and GABA receptor immunoreactivity have been established by using double-immunohistochemical methods and confocal microscopy. The immunodetection of monoclonal and/or polyclonal antibodies has revealed GABA immunoreactivity throughout the polyp tissue, both in neuronal and non-neuronal elements. GAD immunoreactivity has been mostly localized in the neuronal compartment, contacting epithelial and muscular elements. GABA(B) R immunoreactivity appears particularly intense in the nematocytes and in the oocyte envelope; its presence in GAD-immunoreactive neurons in the tentacles suggests an autocrine type of regulation. Western blot analysis has confirmed that a GABA(B) R, with a molecular weight of 142 kDa, similar to that of rat brain, is present in E. cavolini polyp tissue. The identification of the sites of the synthesis, vesicular transport, storage and reception of GABA strongly suggests the presence of an almost complete set of GABA-related molecules for the functioning of the GABAergic system in this simple nervous system. The distribution of these different immunoreactivities has allowed us to hypothesize GABA involvement in nematocyst discharge, in body wall and enteric muscular contraction, in neuronal integration and in male gametocyte differentiation.

Differential expression of γ-aminobutyric acid receptor A (GABAA) and effects of homocysteine

gamma-Aminobutyric acid (GABA) is a known inhibitory neurotransmitter in the mammalian central nervous system, and homocysteine (Hcy) behaves as an antagonist for GABA(A) receptor. Although the properties and functions of GABA(A) receptors are well studied in mouse neural tissue, its presence and significance in non-neural tissue remains obscure. The aim of the present study was to examine the expression of GABA(A) receptor and its subunits in non-neural tissue. The mice were analyzed. The presence of GABA(A) receptor and its subunits was evaluated using Western blot and reverse transcription polymerase chain reaction. We report that GABA(A) receptor protein is abundant in the renal medulla, cortex, heart, left ventricle, aorta and pancreas. Low levels of GABA(A) receptor protein were detected in the atria of the heart, right ventricle, lung and stomach. The mRNA protein expression of GABA(A) receptor subunit shows that alpha1, beta1, beta3 and gamma1 subunits are present only in brain. The mRNA protein expression levels of GABA(A) receptor alpha2, alpha6, beta2 and gamma3 subunits were highly expressed in brain compared to other tested tissue, while GABA(A) receptor gamma2 subunit was expressed only in brain and kidney. Treatment of microvascular endothelial cells with Hcy decreased GABA(A) receptor protein level, which was restored to its baseline level in the presence of GABA(A) receptor agonist, muscimol. The distribution of GABA(A) and GABA(B) receptors in wild type mice was determined and tissue-specific expression patterns were found showing that several receptor subtypes were also expressed in the central nervous system. Hcy, a GABA(A) agonist, was found to decrease GABA(A) expression levels. These data enlarge knowledge on distribution of GABA receptors and give novel ideas of the effects of Hcy on different organs.

Possible expression of a particular gamma-aminobutyric acid transporter isoform responsive to upregulation by hyperosmolarity in rat calvarial osteoblasts

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the brain, but widely distributed in different peripheral organs. We have previously shown the functional expression of GABA B receptors required for GABAergic signal input by cultured rat calvarial osteoblasts. This study focused on the possible functional expression of the machinery required for GABAergic signal termination such as GABA transporters. In rat calvarial osteoblasts cultured for 7 days, [ 3H]GABA accumulation was observed in a temperature-, sodium- and chloride-dependent manner, consisting of a single component with a K m value of 789.6 ± 9.0 μM and a V max value of 4.4 ± 0.1 nmol/min/mg protein, respectively. Both nipecotic and l-2,4-diaminobutyric acids significantly inhibited [ 3H]GABA accumulation in a concentration-dependent manner. Constitutive expression was seen with mRNA for the betaine/GABA transporter-1 (BGT-1) and taurine transporter (TauT), while hyperosmotic cultivation led to significant increases in both [ 3H]GABA accumulation and BGT-1 mRNA expression without affecting TauT mRNA expression. Highly immunoreactive cells were detected for the BGT-1 isoform at the surface of trabecular bone of neonatal rat tibias. Sustained exposure to GABA significantly inhibited alkaline phosphatase (ALP) activity, but not cellular viability, at concentrations above 0.1 mM in osteoblasts cultured for 3 to 28 days. Nipecotic acid not only decreased ALP activity alone, but also further decreased ALP activity in osteoblasts cultured in the presence of GABA. These results suggest that the BGT-1 isoform may be functionally expressed by rat calvarial osteoblasts to play a hitherto unidentified role in mechanisms underlying hyperosmotic regulation of osteoblastogenesis.