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PubMed1982

Synthesis of chick brain GABA receptors by frog oocytes.

Poly(A)-mRNA, extracted from the optic lobe of chick embryos, directs the synthesis of gamma-aminobutyric acid (GABA) receptors in Xenopus laevis oocytes. The receptors are inserted into the oocyte membrane, where they form receptor--channel complexes. When activated by GABA, and related agonists, the chick brain receptors open membrane channels that are permeable to chloride ions. Thus, Xenopus oocytes provide a novel and useful approach to the study of brain receptors.

Animals↗
PubMed1999

A cystic fibrosis tracheal gland cell line, CF-KM4. Correction by adenovirus-mediated CFTR gene transfer.

Human tracheal gland serous (HTGS) cells are now considered one principal pulmonary target for the gene therapy of cystic fibrosis (CF). We developed a CF tracheal gland serous cell line, CF-KM4, obtained by the transformation of primary cultures of CF tracheal gland serous cells homozygous for the DeltaF508 mutation by using the wild-type SV40 virus. This cell line retained epithelial and secretory features of the native CF-HTGS cells in primary culture, namely, presence of cytokeratin, constitutive secretion of secretory leukocyte proteinase inhibitor, absence of responsiveness to carbachol and isoproterenol, and defective cyclic adenosine monophosphate-dependent chloride channel activity. Adenovirus-mediated CF transmembrane conductance regulator (CFTR) gene transfer into CF-KM4 cells corrected the defective chloride channel activity as well as the responsiveness to adrenergic and cholinergic agonists. In contrast, control transfection using adenovirus-mediated beta-galactosidase gene transfer was totally ineffective. In conclusion, these results present a stable CF tracheal gland cell line that has retained its epithelial and CF-specific defective secretory characteristics which are corrected after CFTR gene transfer. This cell line therefore appears to be a useful tool for large-scale molecular and cellular pharmacologic investigations designed to test potential therapies of the disease CF.

Animals↗
PubMed2001

Ethanol potentiation of glycine-induced responses in dissociated neurons of rat ventral tegmental area.

The potentiation of glycine-induced responses by ethanol (EtOH) was studied in neurons freshly dissociated from the ventral tegmental area (VTA) of 5- to 14-day-old postnatal rats using whole-cell and gramicidin-perforated patch-clamp techniques. Under current-clamp conditions, EtOH increased glycine-induced membrane depolarization and action potential firing. Under voltage-clamp conditions, EtOH (0. 1-40 mM) alone did not elicit a current. When coapplied with glycine, EtOH enhanced the glycine-induced current in 35% (180 of 474) of the neurons. The EtOH-induced enhancement of glycine current was independent of membrane potential (between -60 and +60 mV); the reversal potential was not changed. Concentration-response analysis showed that in the presence of EtOH (10 mM), the EC(50) for glycine decreased from 25 +/- 4 to 14 +/- 3 microM; the Hill coefficient increased from 1.5 +/- 0.2 to 1.9 +/- 0.3. Kinetic analysis of glycine currents indicated that EtOH decreased the time constant of activation and increased the time constant of deactivation of glycine-gated chloride channels. EtOH may accelerate glycine association with its receptor at the agonist binding site and increase the apparent agonist affinity. Our observations suggest that, at pharmacologically relevant concentrations, EtOH alters the function of glycine receptors and thus the excitability of neonatal VTA neurons. This action of EtOH may contribute to the neurobehavioral disturbances associated with fetal alcohol syndrome.

Algorithms↗
PubMedDate not supplied

[The reaction of the N-cholinoreceptors of the isolated neuron of the mollusk Planorbarius corneus to polymethylene-bis-trimethylammonium compounds].

Experiments have been made on isolated giant neurones of the mollusc Planorbarius corneus using clamp technique at temperatures 10 and 20 degrees C. The effect of polymethylene-bis-trimethylammonium compounds with 7-18 methylene groups in the molecule (C7...C18) on N-cholinoreceptors with chloride ionic channels was investigated. All these drugs were found to be agonists. Their cholinomimetic activity depends on the number of methylene groups (up to a certain extent) in their structure. This finding stands true also for skeletal muscles of frog and chick, as it had been shown in our earlier experiments. Analysis of membrane current fluctuations showed that the elementary current, the channel opened time, temperature coefficient (Q10) of the neuronal response to application of an agonist and the calculated Q10 of the reaction rate of the agonist with cholinoreceptor did not significantly differ for C8...C18 from the reaction rate of the agonist with cholinoreceptor. As compared with C8, C12...C18 exhibited 30 ... 40 times higher cholinomimetic activity, all other parameters in them being similar. Presumably, this difference is explained by concentrating capacity of C12...C18 at the membrane site because of their higher hydrophobic properties.

Animals↗
PubMed1995

Interaction of beta-carboline inverse agonists for the benzodiazepine site with another site on GABAA receptors.

1. We examined the effects of methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM), a beta-carboline inverse agonist for the benzodiazepine site, on gamma-aminobutyric acid (GABA)-induced Cl-currents in several cloned rat GABAA receptor subtypes expressed in human embryonic kidney cells. The Cl- currents were measured in the whole cell configuration of patch clamp techniques. 2. DMCM at low concentrations (< 0.5 microM) occupying only the benzodiazepine site decreased GABA-induced Cl currents in the alpha 1 beta 2 gamma 2 and alpha 3 beta 2 gamma 2 subtypes as expected from an inverse agonist, but produced no change in the alpha 6 beta 2 gamma 2 subtype (perhaps a neutral antagonist). The drug at higher concentrations (> 0.5 microM) enhanced Cl- currents in all the subtypes with a half maximal concentration of 6 to 20 microM, depending on the alpha isoform. In the alpha 1 beta 2 subtype, which is without the benzodiazepine site, DMCM monophasically increased Cl- currents with a half maximal concentration of 1.9 microM. 3. Ro 15-1788 (a classical benzodiazepine antagonist) had no effect on Cl- current enhancement by DMCM and, in fact, increased the current level through blocking current inhibition by DMCM via the benzodiazepine site. Also, Cl- current enhancement by pentobarbitone or by 3 alpha, 21-dihydroxy-5 alpha-pregnan-20-one was additive to that by DMCM at saturating doses. It appears that the agonist site for DMCM is distinct from those for benzodiazepines, barbiturates and neurosteroids. 4. Among beta-carboline analogues, methyl-beta-carboline-3-carboxylate and propyl-beta-carboline-3-carboxylate markedly enhanced GABA-induced Cl currents in the alpha 1 beta 2 gamma 2 subtype, while N-methyl-beta-carboline-3-carboxamide and 1-methyl-7-methoxy-3,4-dihydro-beta-carboline did not. It appears that the 3-carboxyl ester moiety is necessary for beta-carbolines to interact with a novel site on GABAA receptors as agonists.

Barbiturates↗
PubMed1997

Flumazenil reverses the decrease in the hypnotic activity of pentobarbital by social isolation stress: are endogenous benzodiazepine receptor ligands involved?

Long-term social isolation stress has been shown to cause a decrease in pentobarbital (PB)-induced sleeping time in mice. In the present study, to clarify whether the GABAA/benzodiazepine (BZD) receptor system is involved in the decrease in the hypnotic activity of PB by social isolation stress, we examined the effects of BZD receptor ligands on the PB-induced sleep in group-housed and socially isolated mice. Moreover, we also tested whether social isolation stress affects the ability of GABA to stimulate 36Cl- uptake or the modulatory effect of diazepam and PB and GABA-induced stimulation of 36Cl- uptake into synaptoneurosomes prepared from mouse brain. Social isolation stress significantly decreased the PB-induced sleeping time in mice. The BZD receptor diazepam (0.1-0.8 mg/kg, i.p.) dose-dependently prolonged PB sleep in group-housed and isolated mice, but the effect was weaker in isolated mice. In contrast, FG7142 (5-10 mg/kg, i.p.), a BZD receptor inverse agonist, shortened the sleep in group-housed but not in isolated mice. Flumazenil (16.5-33 nmol, i.c.v.), a selective BZD receptor antagonist, caused PB sleep in isolated mice to return to the level of group-housed mice, at the dose that antagonized the effects of diazepam and FG7142 on PB sleep in group-housed mice. However, this antagonist alone produced no effect on PB sleep in group-housed mice. Social isolation stress decreased the ability of GABA (0.6-200 microM) to stimulate 36Cl- uptake into synaptoneurosomes but this stress had no effect on PB- and diazepam-induced enhancement of GABA-stimulated 36Cl- uptake. These results suggest that endogenous substance(s) with an inverse BZD receptor agonist-like property and the changes in the ability of GABA to stimulate chloride ion channels are involved in the decrease in the hypnotic activity of pentobarbital following social isolation stress.

Animals↗
PubMed2000

Toxic effects of the fungicide benomyl on cell membranes.

This paper examines the toxicity of the fungicide benomyl towards cell membranes. Approaches to this aim were the study of its acute effects on the stimulatory response of a frog neuroepithelial synapse and on membrane models. The latter consisted of large unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and phospholipid multilayers built-up of DMPC and dimyristoylphosphatidylethanolamine (DMPE). Results showed that benomyl at concentrations as low as 10 microM decreased the stimulatory response of the potential difference (PD) and the short-circuit current (SCC) of the frog sympathetic junction. It is concluded that benomyl caused a dose-dependent reduction in the response of a sympathetic junction of the frog to stimulation leading to Cl(-) channel perturbation. This finding might be explained from those obtained from fluorescence spectroscopy and X-ray diffraction studies on membrane models. In fact, similar (0.01-1.0 mM) concentrations induced structural perturbations in DMPC large unilamellar vesicles and multilayers, respectively. Although it is still premature to define the precise molecular mechanism of benomyl toxicity, the experimental results confirm the important role played by the phospholipid bilayers in the interaction of the pesticide with cell membranes.

Adrenergic alpha-Agonists↗
PubMed1998

Functional changes in rat nigral GABA(A) receptors induced by degeneration of the striatonigral GABAergic pathway: an electrophysiological study of receptors incorporated into Xenopus oocytes.

Expression of rat brain gamma-aminobutyric acid type A (GABA(A)) receptors in Xenopus laevis oocytes can be achieved by injection of the oocytes with synaptosomes. This approach has now been applied to evaluate changes in the function of nigral GABA(A) receptors after degeneration of the striatonigral GABAergic pathway induced by the unilateral infusion of kainic acid into the rat striatum. Ten days after striatal injection, synaptosomal membranes were prepared from the substantia nigra and introduced into oocytes. Nigral GABA(A) receptors incorporated into the oocyte cell membrane were then characterized electrophysiologically under voltage-clamp conditions. The maximal amplitude of GABA-induced Cl- currents in oocytes injected with synaptosomes from denervated substantia nigra was twice that observed in oocytes injected with synaptosomes from control substantia nigra. The concentration of GABA required for the half-maximal response did not differ between the two groups of oocytes. In addition, the potentiation of GABA-induced currents by the benzodiazepine diazepam (1 microM) and the steroid derivative allopregnanolone (3 microM) was increased by approximately 65 and 60%, respectively, in oocytes injected with synaptosomes from denervated substantia nigra compared with those injected with control synaptosomes. The concentrations of diazepam and allopregnanolone giving half-maximal responses were not affected by denervation. In contrast, the inhibitory effects of the benzodiazepine receptor inverse agonists FG 7142 (10 microM) and 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid ethyl ester (1 microM) were reduced by 48 and 38%, respectively, after denervation. These results indicate that the up-regulation of nigral GABA(A) receptors induced by degeneration of the striatonigral GABAergic pathway is associated with an increased efficacy of positive allosteric modulators, such as benzodiazepines and steroids, and with a reduced efficacy of negative allosteric modulators such as beta-carbolines.

Animals↗
PubMed2001

Modulation of glycine responses by dihydropyridines and verapamil in rat spinal neurons.

Although glycine receptors (GlyRs) are responsible for the main spinal inhibitory responses in adult vertebrates, in the embryo they have been reported to mediate depolarizing responses, which can sometimes activate dihydropyridine-sensitive L-type calcium channels. However, these channels are not the only targets of dihydropyridines (DHPs), and we questioned whether GlyRs might be directly modulated by DHPs. By whole-cell recording of cultured spinal neurons, we investigated modulation of glycine responses by the calcium channel antagonists, nifedipine, nitrendipine, nicardipine and (R)-Bay K 8644, and by the calcium channel, agonist (S)-Bay K 8644. At concentrations between 1 and 10 microM, all these DHPs could block glycine responses, even in the absence of extracellular Ca2+. The block was stronger at higher glycine concentrations, and increased with time during each glycine application. Nicardipine blocked GABAA responses from the same neurons in a similar manner. In addition to their blocking effects, nitrendipine and nicardipine potentiated the peak responses to low glycine concentrations. Both effects of extracellular nitrendipine on glycine responses persisted when the drug was present in the intracellular solution. Thus, these modulations are related neither to calcium channel modulation nor to possible intracellular effects of DHPs. Another type of calcium antagonist, verapamil (10-50 microM), also blocked glycine responses. Our results suggest that some of the effects of calcium antagonists, including the neuroprotective and anticonvulsant effects of DHPs, might result partly from their interactions with ligand-gated chloride channels.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed2001

Increase in expression of the GABA(A) receptor alpha(4) subunit gene induced by withdrawal of, but not by long-term treatment with, benzodiazepine full or partial agonists.

The effects of long-term exposure to, and subsequent withdrawal of, diazepam or imidazenil (full and partial agonists of the benzodiazepine receptor, respectively) on the abundance of GABA(A) receptor subunit mRNAs and peptides were investigated in rat cerebellar granule cells in culture. Exposure of cells to 10 microM diazepam for 5 days significantly reduced the amounts of alpha(1) and gamma(2) subunit mRNAs, and had no effect on the amount of alpha(4) mRNA. These effects were accompanied by a decrease in the levels of alpha(1) and gamma(2) protein and by a reduction in the efficacy of diazepam with regard to potentiation of GABA-evoked Cl- current. Similar long-term treatment with 10 microM imidazenil significantly reduced the abundance of only the gamma(2)S subunit mRNA and had no effect on GABA(A) receptor function. Withdrawal of diazepam or imidazenil induced a marked increase in the amount of alpha(4) mRNA; withdrawal of imidazenil also reduced the amounts of alpha(1) and gamma(2) mRNAs. In addition, withdrawal of diazepam or imidazenil was associated with a reduced ability of diazepam to potentiate GABA action. These data give new insights into the different molecular events related to GABA(A) receptor gene expression and function produced by chronic treatment and withdrawal of benzodiazepines with full or partial agonist properties.

Animals↗
PubMed1994

Improved technique for studying ion channels expressed in Xenopus oocytes, including fast superfusion.

The study of whole-cell currents from ion channels expressed in Xenopus oocytes with conventional two-electrode voltage clamp has two major limitations. First, the large diameter and spherical geometry of oocytes prevent extremely fast solution changes. Second, the internal medium is not controlled, which limits the experimental versatility of the oocyte expression system. For example, because the internal medium is not controlled, endogenous calcium-activated chloride conductances can contaminate currents measured with channels that are permeable to calcium. We describe a new technique that combines vaseline-gap voltage clamp for oocytes with a fast superfusion system. The vaseline-gap procedure is simplified by having the micropipette that monitors voltage serve a dual role as a perfusion micropipette that controls the internal solution. In addition, the technique provides fast external solution changes that are complete in 30-50 ms. We applied the approach to measure the calcium permeability of a muscle and a neuronal nicotinic acetylcholine receptor. Very fast agonist induced currents were measured without contamination by the secondary activation of calcium-dependent chloride channels.

Acetylcholine↗
PubMed1999

Effects of Cl(-) substitution on electrophysiological properties, Ca(2+) influx and prolactin secretion of rat lactotropes in vitro.

In this study, we compared the effects of different chloride (Cl(-)) substitutes - methane sulfonate (CH(3)SO(-)(3)), bromide (Br(-)), nitrate (NO(-)(3)), thiocyanate (SCN(-)) and perchlorate (ClO(-)(4)) - on the secretory activity and calcium current activation of rat lactotropes in primary culture. We observed that CH(3)SO(-)(3) decreased basal prolactin (PRL) secretion. Br(-) had no effect, whereas the more lyotropic anions, such as NO(-3), SCN(-) and C1O(-4), increased basal PRL secretion. The latter three substitutes induced a significant shift in the voltage dependence of T-type calcium channel activation towards hyperpolarized values. However, this shift alone cannot explain the increase in secretion. Anion permeability studies also demonstrated that the organic anion CH(3)SO(-3) was less permeant than Cl(-), whereas monovalent inorganic anions were more permeant, with the following anion permeability sequence: SCN(-) > ClO(-4) > NO(-3) > Br(-). In conclusion, deprivation of Cl(-) ions has converse consequences on basal and induced secretion; permeating anions result in a transient increase in intracellular Ca(2+) ions. This process involves voltage-dependent Ca(2+) channels. We propose that an alteration in intracellular anion concentrations may influence the activation of internal effectors such as G proteins or channel proteins and, therefore, interfere with exocytosis. These effects are correlated with an external action of lyotropic anions, particularly NO(-3), ClO(-4) and SCN(-), on the gating properties of T-type calcium channels, probably through changes in cell surface charges. The results demonstrate the modulatory effect of anions on the secretory activity of rat lactotropes and underline the specific role played by chloride in stimulus-secretion coupling.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1996

Inhibition of GABA-gated chloride channels in brain by the arachidonic acid metabolite, thromboxane A2.

Previously, we showed that arachidonic acid and prostaglandin metabolites inhibited GABAA responses in rat cerebral cortex. Thromboxane A2 (TXA2), a metabolite of arachidonic acid, has potent actions on blood vessels and platelets, but its actions on neurons are not known. Here, we examined the effects of several TXA2 analogs on the functional and binding characteristics of GABAA receptors in rat brain. The stable analogs of TXA2, pinane and carbocyclic TXA2, and the TXA2 agonist, U-46619, inhibited muscimol-induced 36Cl- uptake in cerebral cortical synaptoneurosomes. Carbocyclic TXA2 decreased the maximal response to muscimol, consistent with a non-competitive interaction. The TXA2 antagonist, SQ 25,548, did not block the effects of either arachidonic acid or carbocyclic TXA2. Neither the biologically inactive metabolite of TXA2, TXB2, nor carbacyclin, a stable analog of prostacyclin (prostaglandin I2) had an effect on GABAA responses. Thus the pharmacology differs from that in vascular smooth muscle and platelets. To determine if GABAA receptors were sensitive to the thromboxanes, the effect of pinane TXA2 on the binding of [35S]t-butylbicyclophosphorothionate ([35S]TBPS) to GABA-gated Cl- channels was measured using receptor autoradiography. Pinane TXA2 inhibited [35S]TBPS binding in a regionally selective and non-competitive manner; the greatest inhibition was in the cerebral cortex, hippocampus and striatum, areas which are selectively vulnerable to cerebral ischemia. We conclude that TXA2 can interact with neuronal membranes to inhibit GABA receptor function, independent of its actions on the cerebrovasculature and on glial cells. This may be important during pathologic states such as ischemia, when TXA2 accumulates in extracellular spaces.

Animals↗
PubMed1994

Facilitation of GABAergic signaling in the retina by receptors stimulating adenylate cyclase.

The gamma-aminobutyric acid type A (GABAA) receptor is the predominant Cl(-)-channel protein mediating inhibition in the retina and elsewhere in the mammalian brain. We have observed a time-dependent increase of GABA-induced whole-cell currents when dopamine was applied externally to rat retinal amacrine cells. After 20 min, the peak current was increased to 208% +/- 10% of its initial value. A comparable effect was observed with the dopamine D1 receptor agonist (+)-1-phenyl-2,3,4,5-tetrahydro(1H)-3-benzazepine-7,8-diol hydrochloride (SKF-38393) but not with the D2 agonist bromocryptine. The action of dopamine involved phosphorylation of GABAA receptors by protein kinase A, as evident from intracellular application of protein kinase A, cAMP, and forskolin. Both guanosine 5'-[gamma-thio]triphosphate and cholera toxin augmented the GABA response, indicating a role for the guanosine 5'-triphosphate-binding protein Gs in the transduction cascade. Phosphorylation of GABAA receptors shifted the half-maximally effective GABA concentration from 71 microM to 47 microM without affecting the maximal response amplitude. The elevated binding affinity for GABA was caused by an increase of the open probability of the channels from 0.09 to 0.33 (2 microM GABA); conductance and mean open time did not change. Several other receptor agonists such as adenosine, histamine, somatostatin, enkephalin, and vasoactive intestinal peptide were found to couple to the same intracellular phosphorylation pathway. Since some of these cotransmitters colocalize with GABA in amacrine cells, they may fine-tune GABAergic inhibition in the retina.

Adenylyl Cyclases↗
PubMed1996

Regulation of GABAA receptor structure and function by chronic drug treatments in vivo and with stably transfected cells.

In this article, we review the use of stably transfected cells to study the regulation of receptor structure and function by chronic drug treatments and compare results from these cells to results obtained from other systems, including neuronal cultures and intact animals. We focus on the gamma-aminobutyric acid type A (GABAA) receptor complex. Sedative/hypnotic drugs such as benzodiazepines, barbiturates and alcohol that potentiate GABAA receptor function produce tolerance and dependence. Chronic treatment of GABAA receptor preparations from brain and neuronal cultures with GABAA agonists, as well as these other three classes of drugs, results in regulation of several properties of the receptor. Drug treatments may regulate levels of binding sites, allosteric binding interactions, receptor function, levels of receptor subunit mRNA and levels of receptor subunit protein. Some or all of these effects may comprise the molecular mechanisms of tolerance to these GABAA-modulatory drugs. The use of cells stably transfected with neurotransmitter receptors provides a homogeneous population that can be cultured under controlled conditions. As most preparations contain mixed populations of GABAA receptor subunits, stably transfected cells offer the advantage of the expression of receptors with a defined subunit composition. We conclude that chronic drug treatments regulate allosteric coupling and function of GABAA receptors in stably transfected cells. This regulation does not appear to be due to decreases in the expression of alpha 1- or beta 1-receptor subunits or to expression of subunits other than alpha 1, beta 1, gamma 2L. Therefore, it is unlikely to be due to changes in receptor subunit composition and probably represents post-translational changes. The rapid regulation of allosteric coupling and function by drug treatment of the stably transfected cells should provide insights to the mechanisms of coupling between GABAA and benzodiazepine receptors as well as tolerance and dependence of benzodiazepines and ethanol.

Animals↗
PubMed1995

Differential effects of 4'-chlorodiazepam on expressed human GABAA receptors.

The interactions of the atypical benzodiazepine 4'-chlorodiazepam (Ro 5-4864) with functionally expressed human GABAA receptor cDNAs were determined. Cotransfection of human alpha 2, beta 1, and gamma 2 subunits was capable of reconstituting a 4'-chlorodiazepam recognition site as revealed by a dose-dependent potentiation of t-[35S]butylbicyclophosphorothionate ([35S]-TBPS) binding to the GABA-activated chloride channel. This site is found on GABAA receptor complexes containing sites for GABA agonist-like benzodiazepines and neuroactive steroids. The importance of the alpha subunit was further demonstrated as substitution of either alpha 1 or alpha 3 for the alpha 2 subunit did not reconstitute a 4'-chlorodiazepam recognition site that was capable of modulating [35S]TBPS binding under the same experimental conditions. The 4'-chlorodiazepam modulatory site was shown to be distinct from the benzodiazepine site, but the phenylquinolines PK 8165 and PK 9084 produced effects similar to 4'-chlorodiazepam, consistent with the previous analysis of the 4'-chlorodiazepam site in brain homogenates. Further analysis of the subunit requirements revealed that coexpression of alpha 2 and beta 1 alone reconstituted a 4'-chlorodiazepam recognition site. It is interesting, however, that the 4'-chlorodiazepam site was found to inhibit [35S]TBPS binding to the GABA-activated chloride channel. Thus, the 4'-chlorodiazepam site may be reconstituted with only the alpha and beta polypeptides.

Benzodiazepinones↗
PubMed1998

Nitrous oxide-induced enhancement of gamma-aminobutyric acidA-mediated chloride currents in acutely dissociated hippocampal neurons.

BACKGROUND: Because the synaptic inhibition in the human brain is largely mediated by gamma-aminobutyric acid (GABA), the GABA receptor is of primary interest for the study of the working mechanism of general anesthetics. This article examines the interaction between this type of ion channel and nitrous oxide (N2O). METHODS: Patch clamp recording techniques were applied to investigate the effects of N2O on GABA(A) receptor channels in a whole-cell configuration at room temperature. Acutely dissociated rat hippocampal cells from the CA1 region were used. Rapid application of the agonist muscimol and anesthetics (N2O, pentobarbital, and ethanol) was accomplished using a Y tube application system. Peak chloride (Cl-) currents were measured. RESULTS: Short-term application of muscimol (5-30 microM) with dissolved N2O (80%, approximately 22.5 mM) increased the Cl- current (approximately 140%) compared with muscimol alone. This effect is comparable with results the authors obtained with ethanol (800 mM) and pentobarbital (100 microM). Prolonged exposure (9 min) to N2O further increased Cl- currents by an additional 50%. Concentrations of N2O lower than 12 mM did not show an enhancement of this current, whereas application of N2O alone did not result in any Cl- conductance. CONCLUSIONS: These results indicate that N2O can enhance GABA(A) channel-mediated Cl- currents by modulating the effect of the specific GABA(A) agonist; it is not active by itself.

Anesthetics, Inhalation↗
PubMed1996

Proton modulation of functionally distinct GABAA receptors in acutely isolated pyramidal neurons of rat hippocampus.

We have studied the effect of extracellular pH (pHo) on the GABAA receptor-mediated chloride conductance in acutely isolated pyramidal neurons from area CA1 of the rat hippocampus under whole-cell voltage clamp in bicarbonate-free solutions. The conductance evoked by saturating or near-saturating concentrations (200-1000 microM) of GABA showed a marked sensitivity to variations of pHo around 7.4. A decrease in pHo between 8.4 and 6.4 increased the GABAA receptor-mediated chloride conductance by about two-fold per pH unit. In contrast, when evoked by a low agonist concentration (1-10 microM) the conductance showed an equally marked decrease upon a decrease in pHo. The half-time for desensitization of the conductance induced by 500 microM GABA was around 900 ms at pHo 6.4 and 7.4, but decreased to 650 ms at pHo 8.4. A fall in pHo decreased the amount of desensitization of the conductance evoked by a 5 s application of 5 microM, but not of 500 microM, GABA. The concentration-response relationship of the GABA-induced conductance showed a local plateau between 50 and 100 microM of GABA, which was particularly evident at high pHo. Assuming two receptor populations with a high and a low affinity for GABA, the effect of H+ on the GABAA receptors could be explained as an increase in the EC50 of the high affinity receptor, and an apparently non-competitive potentiation of both the high and the low affinity receptors. The GABAA receptor-mediated conductance was markedly inhibited by 20-50 microM Zn2+. In addition, Zn2+ reverted the down-modulation by H+ observed at low GABA concentrations to up-modulation. Diazepam (1-10 microM) had only a marginal effect on the GABA-gated conductance. Taken together, the results suggest the coexistence in individual hippocampal neurons of two distinct GABAA receptor populations having differential sensitivities to H+. In the light of the inhibitory action of Zn2+ and the virtual absence of an effect of diazepam it is probable that a significant fraction of the GABAA receptors lack the gamma 2 subunit. The observation that an elevated pH has a strong suppressing effect on the conductance evoked by high concentrations of GABA may at least partly explain why an extracellular alkalosis leads to neuronal hyperexcitability.

Animals↗