The agonistic action of pentobarbital on GABAA beta-subunit homomeric receptors

Murine gamma-aminobutyric acid type A (GABAA) receptor beta 1, beta 2, and beta 3 subunits were expressed in Xenopus oocytes and studied using the two electrode voltage clamp technique. Although all three beta-subunits were unresponsive to GABA when expressed as homomers, the intravenous general anaesthetics pentobarbital, etomidate and propofol induced currents in beta 2 and beta 3 homomers. The pentobarbital-induced currents in beta 3 homomers showed a dose dependence with an ED50 of 89 +/- 8.9 microM and a Hill coefficient of 0.94 +/- 0.08. Zinc (50 microM) blocked (61.1 +/- 5.6% of control) and 200 microM lanthanum potentiated (139 +/- 8.6% of control) the pentobarbital-induced current. This current was also blocked by picrotoxin but was insensitive to the GABAA receptor antagonist bicuculline. These observations indicate that the full expression of the agonistic action of GABA requires the presence of an alpha-subunit, in contrast to the agonistic action of intravenous general anesthetics, where the presence of a beta2 or beta 3-subunit is sufficient. The difference in the agonistic action of intravenous anaesthetics among these highly homologous beta-subunits suggests that the beta-subunit homomeric receptors may be useful to further define the molecular sites of action of intravenous general anaesthetics and other functional domains on GABAA receptors.     

Direct activation of GABAA receptors by loreclezole, an anticonvulsant drug with selectivity for the beta-subunit

Loreclezole, an anticonvulsant and antiepileptic compound, potentiates gamma-aminobutyric acid (GABA) type A receptor function, by interacting with a specific allosteric modulatory site on receptor beta-subunits. A similar selectivity for GABAA receptor beta-subunits is apparent for the direct activation of receptor-operated Cl- channels, by the general anesthetics propofol and pentobarbital. The ability of loreclezole to activate GABAA receptors directly has now been compared, biochemically and electrophysiologically, with that of propofol. In well-washed rat cortical membranes (devoid of endogenous GABA), loreclezole and propofol increased t-[35S]butylbicyclophosphorothionate ([35S]TBPS) binding by up to 28% (at 5 microM) and 80% (at 10 microM), respectively. Higher concentrations (50-100 microM) of both compounds inhibited [35S]TBPS binding with great efficacy, an effect mimicked by GABA. In contrast, the benzodiazepine diazepam increased [35S]TBPS binding, but failed to inhibit this parameter, even at high concentrations. At concentrations of 50-100 microM, loreclezole induced inward Cl- currents in the absence of GABA, in Xenopus oocytes expressing human recombinant GABAA receptors, comprised of alpha 1-, beta 2- and gamma 2S-subunits. At 100 microM, the current evoked by loreclezole was 26% of that induced by 5 microM GABA. The current evoked by 100 microM propofol was 98% of that induced by 5 microM GABA. Currents induced by loreclezole, like those evoked by propofol, were potentiated by diazepam in a flumazenil-sensitive manner and blocked by either bicuculline or picrotoxin. These data suggest that loreclezole shares, with propofol, an agonistic action at GABAA receptors containing the beta 2-subunit and that the different efficacies of the two compounds in this regard, may underlie the difference in their pharmacological profiles. The failure of loreclezole to activate GABAA receptors containing the beta 1-subunit may be responsible for its lack of hypnotic effect.     

Calcium released from intracellular stores inhibits GABAA-mediated currents in ganglion cells of the turtle retina

We studied spiking neurons isolated from turtle retina by the whole cell version of the patch clamp. The studied cells had perikaryal diameters > 15 microns and fired multiple spikes in response to depolarizing current steps, indicating they were ganglion cells. In symmetrical [Cl-], currents elicited by puffs of 100 microM gamma-aminobutyric acid (GABA) were inward at a holding potential of -80 mV. All of the GABA-evoked current was blocked by SR95331 (20 microM), indicating that it was mediated by a GABAA receptor. The GABA-evoked currents were unaltered by eliciting a transmembrane calcium current either just before or during the response to GABA. On the other hand caffeine (10 mM), which induces Ca2+ release from intracellular stores, inhibited the GABA-evoked current on average by 30%. The caffeine effect was blocked by introducing the calcium buffer bis-(o-aminophenoxy)-N,N,N',N'-tetraacetic acid (BAPTA) into the cell but was unaffected by replacing [Ca2+]o with equimolar cobalt. Thapsigargin (10 microM), an inhibitor of intracellular calcium pumps, and ryanodine (20 microM), which depletes intracellular calcium stores, both markedly reduced a caffeine-induced inhibition of the GABA-evoked current. Another activator of intracellular calcium release, inositol trisphosphate (IP3; 50 microM), also progressively reduced the GABA-induced current when introduced into the cell. Dibutyryl adenosine 3'5'-cyclic monophosphate (cAMP; 0.5 mM), a membrane-permeable analogue of cAMP, did not reduce GABA-evoked currents, suggesting that cAMP-dependent kinases are not involved in suppressing GABAA currents, whereas calmidazolium (30 microM) and cyclosporin A (20 microM), which inhibit Ca/calmodulin-dependent phosphatases, did reduce the caffeine-induced inhibition of the GABA-evoked current. Alkaline phosphatase (150 micrograms/ml) and calcineurin (300 micrograms/ml) had a similar action to caffeine or IP3. Antibodies directed against the ryanodine receptor or the IP3 receptor reacted with the great majority of neurons in the ganglion cell layer. We found that these two antibodies colocalized in large ganglion cells. In summary, intracellular calcium plays a role in reducing the currents elicited by GABA, acting through GABAA receptors. The modulatory action of calcium on GABA responses appears to work through one or more Ca-dependent phosphatases.     

Modulation of recombination human gamma-aminobutyric acidA receptors by isoflurane: influence of the delta subunit

BACKGROUND: The gamma-aminobutyric acid (GABA)A receptor/chloride channel has a broad-spectrum anesthetic sensitivity and is a key regulator of arousal. Each receptor/channel complex is an assembly of five protein subunits. Six subunit classes have been identified, each containing one to six members; many combinations are expressed throughout the brain. Benzodiazepines and intravenous anesthetic agents are clearly subunit dependent, but the literature to date suggests that volatile anesthetics are not. The physiological role of the delta subunit remains enigmatic, and it has not been examined as a determinant of anesthetic sensitivity. METHODS: Combinations of GABA(A) receptor subunit cDNAs were injected into Xenopus laevis oocytes: alpha1beta1, alpha1beta1gamma2L, alpha1beta1delta, and alpha1beta1gamma2Ldelta. Expression of functional ion channels with distinct signalling and pharmacologic properties was demonstrated within 1-4 days by established electrophysiological methods. RESULTS: Co-expression of the delta subunit produced changes in receptor affinity; current density; and the modulatory efficacy of diazepam, zinc, and lanthanum; it also produced subtle changes in the rate of desensitization in response to GABA. Isoflurane enhanced GABA-induced responses from all combinations: alphabeta delta (>10-fold) > alphabeta > alphabeta gamma > or = alphabeta gammadelta (approximately 5-fold). Dose-response plots were bell shaped. Compared with alphabeta gamma receptors (EC50 = 225 microM), both alphabeta delta (EC50 = 372 microM) and alphabeta gammadelta (EC50 = 399 microM) had a reduced affinity for isoflurane. Isoflurane (at a concentration close to the EC50 for each subunit) increased the affinity of GABA for its receptor but depressed the maximal response (alphabeta gamma and alphabeta gammadelta). In contrast, the small currents through alphabeta delta receptors were enhanced, even at saturating agonist concentrations. CONCLUSIONS: Delta subunit expression alters GABA(A) receptor function but is not an absolute determinant of anesthetic sensitivity.     

Modulation of GABAA receptor function by alcohols: effects of subunit composition and differential effects of ethanol

We sought to test the hypotheses that closely related alcohols would have effects on GABAA receptor function that were not predicted by differences in lipid solubility, and that the subunit structure of the GABAA receptor would significantly affect the actions of different alcohols. Cloned subunits of human GABAA receptors were expressed in Xenopus oocytes, and two-electrode voltage-clamp recording was used to quantify the membrane current response to GABA in the presence and absence of different alcohols. 1-Butanol and 2-butanol differentially potentiated the response to 20 microM GABA in oocytes expressing the alpha 1 beta 2 gamma 2L and alpha 2 beta 2 gamma 2L receptor isoforms. In the alpha 1 beta 2 gamma 2L receptor construct, 1-butanol was more potent than 2-butanol to potentiate GABAA receptor function, but 2-butanol had a greater efficacy. In the alpha 2 beta 2 gamma 2L receptor construct, 1-butanol and 2-butanol were equipotent, but 2-butanol again had a greater efficacy. In the alpha 2 beta 2 receptor construct, both 1-butanol and 2-butanol produced large potentiations of the current response to 3 microM GABA. The efficacy for butanol potentiation of GABA responses in the absence of a gamma 2L subunit was greater, but the potency was greatly reduced. Low concentrations (20 mM) of ethanol potentiated GABA responses in the alpha 1 beta 2 gamma 2L receptor construct. Ethanol potentiation of GABAA receptor function was completely blocked by the benzodiazepine receptor partial inverse agonist RO15-4513 at a concentration (0.5 microM) that did not alter the control GABA response. In contrast, RO15-4513 did not block potentiation of GABAA receptor activity induced by n-propanol, 1-butanol, 2-butanol, 1-heptanol, or propofol (2,6-diisopropylphenol). These results suggest that alcohols have specific interactions with GABAA receptors, and that ethanol may have unique effects not shared by other longer chain alcohols.     

Effects of dopamine and estrogen on the regulation of Pit-1 alpha, Pit-1 beta, and PL-II gene expression in the rat placenta

We investigated the effects of pregnenolone sulfate (PS) on the [Ca2+]i increase induced by gamma-aminobutyric acid (GABA) and N-methyl-D-aspartate (NMDA) using fluorescence imaging. PS inhibited the 50 microM GABA-induced increase in [Ca2+]i in a dose-dependent manner with an IC50 of 30 microM. The inhibitory effect of PS was apparent within 5 min and was in a non-competitive manner, suggesting that PS may act directly to the membrane level but indirectly to the GABA binding sites. Our previous study has already shown that the GABA-induced Ca2+ increase involves GABAA receptors and the similar pathway to a high K(+)-induced Ca2+ response (Takebayashi et al., 1996). Because 50 microM of PS could not inhibit a 25 mM K(+)-induced Ca2+ increase, it seems likely that the site of the inhibitory action of PS on the GABA-induced Ca2+ increase may be independent of the pathway of the high K(+)-induced Ca2+ response, but rather at GABAA receptor complex. In contrast, PS potentiated the 50 microM NMDA-induced increase in [Ca2+]i in a dose-dependent manner. The magnitude of the NMDA response was approximately doubled in the presence of 100 microM of PS. However, PS did not affect the acetylcholine(Ach)-induced increase in [Ca2+]i. Furthermore, corticosterone had little effect on the GABA- and NMDA-induced Ca2+ increases, indicating that the alteration of the Ca2+ response is specific for PS. In conclusion, it is suggested that PS modulates differentially [Ca2+]i increase induced by GABA and NMDA.     

Maintenance of recombinant type A gamma-aminobutyric acid receptor function: role of protein tyrosine phosphorylation and calcineurin

In the present study, rundown of gamma-aminobutyric acid (GABA)-activated Cl- channels was studied in recombinant GABAA receptors stably expressed in human embryonic kidney cells (HEK 293), with conventional whole-cell and amphotericin B-perforated patch recording. When [ATP]i was lowered to 1 mM and resting [Ca++]i was buffered to a relatively high level, the response of alpha 3 beta 2 gamma 2 GABAA receptors to relatively low [GABA] (up to 50 microM) did not show rundown in the whole-cell configuration. However, high [GABA] (greater than 200 microM) induced significant rundown, which was observed by decreases in both the maximum GABA-induced current and GABA EC50. Rundown was prevented completely with a solution containing 4 mM Mg(++)-ATP and low resting [Ca++]i, or during perforated patch recording. The magnitude of rundown was comparable in alpha 1 beta 2 gamma 2 and beta 2 gamma 2 receptors. Neither stimulation nor inhibition of protein kinase A or protein kinase C had a significant effect on rundown. However, sodium metavanadate, an inhibitor of protein tyrosine phosphatase, significantly reduced rundown. In addition, inhibition of protein tyrosine kinase activity by either genistein or lavendustin A induced rundown of the GABA response. Inhibition of the Ca++/calmodulin-dependent phosphatase calcineurin with fenvalerate also prevented rundown of the response to GABA. Our results demonstrate that rundown of GABAA receptor function is concentration-dependent, due to depletion of ATP and/or unbuffered [Ca++]i, and does not depend on the presence or subtype of the alpha subunit. We propose that protein phosphorylation at a tyrosine kinase-dependent site, and a distinct unidentified site, which is dephosphorylated by calcineurin, maintains the function of GABAA receptors.     

Inhibition of GABAA receptor function by tyrosine kinase inhibitors and their inactive analogues

The effects of tyrosine kinase inhibitors which target the ATP binding site or the substrate binding site of tyrosine kinases were assessed on murine recombinant type A gamma-aminobutyric acid (GABAA) receptors expressed in Xenopus oocytes or HEK cells using two-electrode voltage clamp or patch clamp recording. Genistein inhibited in a noncompetitive manner GABA-activated currents recorded from alpha1beta1gamma2S receptor constructs by reducing the maximum normalized response from 1.83 +/- 0.04 to 0.71 +/- 0.04 and reducing the EC50 from 35.7 +/- 2.1 microM to 15.1 +/- 3.9 microM. After mutating the two "functionally active" substrate tyrosine (Y) residues in gamma2S and expressing the mutant receptor alpha1beta1gamma2S(Y365F, Y367F), genistein still noncompetitively inhibited the responses to GABA reducing the maximum current from 1. 81 +/- 0.03 to 0.26 +/- 0.01 and the EC50 from 33.1 +/- 2.3 microM to 5.8 +/- 2.2 microM. The inactive compound, daidzein, also similarly inhibited responses to GABA on these two receptor constructs. Inhibitors targeting the substrate binding site of tyrosine kinases, the tyrphostins, also inhibited both the wild-type and the tyrosine mutant GABAA receptors. Tyrphostin A25 and the inactive tyrphostin A1 reduced the maximum normalized responses for alpha1beta1gamma2S and alpha1beta1gamma2S(Y365F, Y367F) receptors by 73 and 64%, respectively. The tyrosine kinase inhibitors and their inactive controls did not display any significant voltage sensitivity to the antagonism of GABA-activated responses. Moreover, genistein or tyrphostin A25 did not affect the potentiation of responses to GABA by pentobarbitone or diazepam. Mutating the two "functionally silent" tyrosine residues, Y370 and Y372, known to be substrates for tyrosine kinases in the beta1 subunit and coexpression in the alpha1beta1(Y370F, Y372F)gamma2S(Y365F, Y367F) construct failed to affect the inhibitory action of genistein. The study concludes that tyrosine kinase inhibitors and their inactive controls can directly interact with GABAA receptors completely independent of any effects on tyrosine kinases.     

Properties and sex-specific differences of GABAA receptors in neurons expressing gamma1 subunit mRNA in the preoptic area of the rat

Gamma-aminobutyric acid type A (GABAA) receptors expressed within the medial preoptic area (mPOA) are known to play a critical role in regulating sexual and neuroendocrine functions. In the rat brain, high levels of expression of the gamma1 subunit mRNA of the GABAA receptor are restricted to a limited number of regions that mediate sexual behaviors, including the mPOA. The biophysical and pharmacological profiles of native gamma1-containing receptors in neurons are unknown. Here, we have characterized the properties of GABAA receptor-mediated spontaneous inhibitory postsynaptic currents (sIPSCs) and currents elicited by fast perfusion of GABA to isolated mPOA neurons of juvenile male and female rats. No significant sex-specific differences were evident in the mean peak amplitude, distribution of event amplitudes, kinetics of current decay, or the frequency of sIPSCs. The profile of modulation of sIPSCs by diazepam, beta-CCM and zolpidem, allosteric modulators that act at the benzodiazepine (BZ) site of the GABAA receptor, support the assertion that mPOA neurons of both sexes express functional gamma1-containing receptors. The ability of zolpidem to modulate both sIPSC amplitude and currents elicited by rapid perfusion of GABA to mPOA neurons differed significantly between the sexes. Zolpidem reversibly induced negative modulation of currents in mPOA neurons isolated from male rats, but had no effect in mPOA neurons from female rats. Concentration-response analysis of responses in neurons acutely isolated from male rats indicated an IC50 of 58 nM with maximal decreases of approximately 50% of control peak current amplitude. In situ hybridization analysis demonstrated that levels of the gamma1 subunit mRNA are significantly higher in mPOA neurons from male than female rats. No significant sex-specific differences were detected in the levels of alpha1, alpha2, or alpha5 mRNAs. These results suggest that native gamma1-containing receptors are expressed in primary neurons of the mPOA and that sex-specific differences in the expression of this subunit may contribute to sexual dimorphism in GABAA receptor modulation by compounds acting at the BZ site.     

Significance of serum ferritin and lactate dehydrogenase in benign and malignant disease of breast

We previously reported that corymine, an alkaloidal compound extracted from the leaves of Hunteria zeylanica native to Thailand, potentiated convulsions induced by either picrotoxin or strychnine. Therefore, to clarify the mechanism of action of corymine, the effects of corymine on gamma-aminobutyric acid (GABA) and glycine receptors were examined. We used Xenopus oocytes expressing these receptors and the two-electrode voltage-clamp method. The receptors expressed in oocytes injected with rat brain and spinal cord RNA showed the pharmacological properties of GABAA and glycine receptors, respectively. Corymine (1-100 microM) partially (20-30%) reduced the GABA responses in oocytes injected with rat brain RNA, while marked (up to 80%) dose-dependent reductions were observed in the glycine responses in oocytes injected with rat spinal cord RNA. These observations suggest that corymine was more effective against the glycine receptors than the GABA receptors. The ED50 of corymine on the glycine response was 10.8 microM. Corymine, at 30 microM, caused a shift to the right, with a lower maximal response, of the glycine concentration-response curve. This indicated that the action of corymine on glycine receptors is neither competitive nor purely non-competitive. These observations suggest that a binding site other than the glycine recognition site of the glycine receptors is the site of action of corymine.     

Functional heterogeneity of hippocampal GABAA receptors

gamma-Aminobutyric acid type A (GABAA) receptors were studied in cultured neurons taken from rat hippocampus at early postnatal stages. GABA-induced whole-cell currents showed a broad range of peak amplitudes and time-courses of desensitization. Dose-response curves of rapidly and slowly desensitizing cells revealed EC50 values of 8.5 and 37.3 microM GABA, respectively, with the Hill coefficient being greater than unity. The main-state conductance of GABAA receptor channels was 28-31 pS in all cells. GABA responses of low-affinity cells were more strongly affected by benzodiazepine receptor agonists (e.g. flunitrazepam, clonazepam) and inverse agonists (e.g. methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate), as compared to cells exhibiting high-affinity GABA responses. Currents were also potentiated by zolpidem, but were little affected by Ro 15-4513 and Zn2+. These data suggest the presence of physiologically and pharmacologically distinct GABAA receptor isoforms in neurons of the early postnatal hippocampus, which may subserve different inhibitory control mechanisms in this brain region.     

SB-205384: a GABA(A) receptor modulator with novel mechanism of action that shows subunit selectivity

1. SB-205384, and its (+) enantiomer (+)-SB-205384 were tested for their modulatory effects on human GABA(A) receptor subunit combinations expressed in Xenopus oocytes by electrophysiological methods. 2. The slowing of the decay rate induced by SB-205384 on native GABA-activated currents in rat neurones was also seen on GABA(A) currents in oocytes expressing human GABA(A) subunits. This temporal effect was observed for the alpha3beta2gamma2 subunit combination with little effect in subunit combinations containing either alpha1 or alpha2. 3. Potentiation of the peak amplitude of the GABA-activated currents by SB-205384 or (+)-SB-205384 was less specific for a particular subunit combination, although the greatest effect at 10 microM drug was seen on the alpha3beta2gamma2 subunit combination. 4. In contrast, zolpidem, a benzodiazepine site modulator, did not significantly slow decay rates of GABA(A) currents in oocytes expressing the alpha3beta2gamma2 subunit combination. Zolpidem, as expected, did selectively potentiate GABA-activated currents on oocytes expressing the gamma2 subunit compared to those containing the gamma1. 5. The results show that the novel kinetic modulatory profile of SB-205384 is selective for the alpha3beta2gamma2 subunit combination. This suggests that the compound is binding to a novel regulatory site on the subunit complex.     

Effects of volatile anesthetics on the kinetics of inhibitory postsynaptic currents in cultured rat hippocampal neurons

1. The effects of the volatile anesthetics enflurane, halothane, and isoflurane on gamma-aminobutyric acid (GABA) receptor-mediated inhibitory postsynaptic currents (IPSCs) were studied in cultured rat hippocampal neurons. The experimental concentrations of anesthetics were measured directly using gas chromatography. All three anesthetics increased the overall duration of IPSCs, measured as the time to half-decay (T1/2). Clinically effective concentrations of anesthetics [between 0.5 and 1.5 times MAC (minimum alveolar concentration)] produced between 100 and 400% increases in T1/2. These effects were fully reversible, and did not involve alterations in the reversal potential for the IPSC (EIPSC). 2. The decay of the IPSC was fitted as a sum of two exponential functions, yielding a fast component (tau fast = 20 ms), and a slow component (tau slow = 77 ms), such that the fast component accounted for 79% of the IPSC amplitude and 52% of the total charge transfer. All three anesthetics produced concentration-related increases in the amplitude and charge transfer of the slow component, while simultaneously decreasing the amplitude and charge transfer of the fast component. Thus T1/2 approximated tau fast under control conditions, but approximated tau slow in the presence of the anesthetics. 3. Varying the calcium chelating agents in the recording pipettes had no effect on the quality or magnitude of alterations in IPSC kinetics produced by halothane, suggesting that variations in intracellular calcium levels are not required for the effect of halothane on the time course of the IPSC. 4. The (+)-stereoisomer of isoflurane produced greater increases in the duration of the IPSC than the (-)-isomer when applied at approximately equal concentrations, suggesting that there is a structurally selective site of interaction for isoflurane that modulates the GABAA receptor. 5. These results suggest that the previously shown abilities of volatile anesthetics to potentiate responses to exogenously applied GABA and to prolong the duration of GABA-mediated synaptic inhibition may be due to an alteration in the gating kinetics of the GABAA receptor/channel complex. Prolongation of synaptic inhibition in the CNS is consistent with the physiological effects that accompany anesthesia and may contribute to the mechanism of anesthetic action.     

The effects of GABA and glycine on horizontal cells of the rabbit retina

Intracellular and patch-clamp recordings have been used to characterize GABA-activated channels in axonless horizontal cells (ALHC) of the rabbit retina. In our intracellular recordings on an everted eyecup preparation, GABA depolarized the horizontal cells (HC), diminished their light response amplitude and slowed the response rise time. Glycine showed similar effects on the HC light responses. In our whole cell patch-clamp recordings on dissociated ALHC, all HCs responded to 3 microM GABA but none to glycine, even at 100 microM. Dose-response relationship for GABA gave EC50 values around 10 microM and Hill slopes of 1.3. Whole-cell current-voltage (I-V) relationships of GABA-activated currents reversed close to the predicted Cl- equilibrium potential. Partial replacement of intracellular Cl- with isothetionate shifted the GABA reversal potential to a more negative value. Muscimol (30 microM), a GABAA agonist mimicked the effect of GABA, but baclofen (30 microM), a GABAB agonist and cis-aminocaprionic acid (30 microM), a GABAC agonist did not elicit any effect on ALHC. Responses to GABA were blocked by the GABAA receptor antagonist bicuculline (10 microM) and picrotoxin (100 microM). According to our results, we conclude that ALHC express GABA receptors coupled to ion channels, and they correspond to GABAA receptor subtypes.     

U-89843A is a novel allosteric modulator of gamma-aminobutyric acidA receptors

A group of pyrrolopyrimidine derivatives were examined for their interaction with rat recombinant gamma-aminobutyric acid (GABA)A receptors using the whole cell patch clamp and equilibrium binding techniques. In the alpha 1 beta 2 gamma 2 subtype of GABAA receptors expressed in human embryonic kidney cells, a prototype pyrrolopyrimidine, U-89843A (7H-pyrrol[2,3-d]pyrimidine,6,7-methyl-2,4-di- 1-pyrrolidinyl,hydrochloride), dose-dependently enhanced 5 microM GABA-induced Cl- currents with a maximal enhancement of 362 +/- 91%, a half-maximal concentration of 2 +/- 0.4 microM and a slope factor of 1.1 +/- 0.4. The drug also inhibited [35S]t-butylbicyclophosphorothionate binding in rat cerebrocortical membranes with a similar half-maximal inhibitory concentration. The enhancement of Cl- currents by U-89843A was insensitive to Ro 15-1788 (a benzodiazepine antagonist), was also observed in the alpha 3 beta 2 gamma 2 and alpha 6 beta 2 gamma 2 subtypes (no selectivity to different alpha-isoforms unlike many benzodiazepines), but was absent in the receptor subtypes consisting of two subunits (alpha 1 beta 2, alpha 1 gamma 2 and beta 2 gamma 2). It has been known that neurosteroids and barbiturates are uniformly active in both the two subunit receptors, substituted pyrazinones are only active in the alpha 1 beta 2 subtype and loreclezole is active in the subtypes containing beta 2. We propose that U-89843A interacts with an allosteric site on GABAA receptors distinct from the sites for benzodiazepines, barbiturates, neurosteroids, substituted pyrazinones or loreclezole.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereoselective effects of etomidate optical isomers on gamma-aminobutyric acid type A receptors and animals

BACKGROUND: The intravenous anesthetic etomidate is optically active and exists in two mirror-image enantiomeric forms. However, although the R(+) isomer is used as a clinical anesthetic, quantitative information on the relative potencies of the R(+) and S(-) isomers is lacking. These data could be used to test the relevance of putative molecular targets. METHODS: The anesthetic concentrations for a half-maximal effect (EC50) needed to induce a loss of righting reflex in tadpoles (Rana temporaria) were determined for both etomidate enantiomers. The effects of the isomers on gamma-aminobutyric acid (GABA)-induced currents in stably transfected mouse fibroblast cells was also investigated using the patch-clamp technique. In addition, the effects of the isomers on a lipid chain-melting phase transition were determined. RESULTS: The EC50 concentrations for general anesthesia for the R(+) and S(-) isomers were 3.4 +/- 0.1 microM and 57 +/- 1 microM, with slopes of n = 1.9 +/- 0.1 and n = 2.9 +/- 0.2, respectively. The R(+) isomer was also much more effective than the S(-) isomer at potentiating GABA-induced currents, although the degree of stereoselectivity varied with anesthetic concentration. R(+) etomidate potentiated the GABA-induced currents by increasing the apparent affinity of GABA for its receptor. Both isomers were equally effective at disrupting lipid bilayers. CONCLUSIONS: These data are consistent with the idea that the GABA(A) receptor plays a central role in the actions of etomidate. Etomidate exerts its effects on the receptor by binding directly to a specific site or sites on the protein and allosterically enhancing the apparent affinity of GABA for its receptor.     

Functional GABAA receptors on rat vagal afferent neurones

1. In the present study, in vitro electrophysiology and receptor autoradiography were used to determine whether rat vagal afferent neurones possess gamma-aminobutyric acid (GABA)A receptors. 2. GABA (1-100 microM) and isoguvacine (3-100 microM) caused a concentration-dependent depolarization of the rat isolated nodose ganglion preparation at room temperature. When applied to the tissue 20 min before the agonist, SR95531 (3 microM) and bicuculline (3 microM) caused a parallel shift to the right of the GABA and isoguvacine concentration-response curves, yielding shifts of 81 fold and 117 fold for SR95531 and 4 fold and 12 fold for bicuculline, respectively. 3. Baclofen (10 nM-100 microM) was unable to elicit a depolarization of the rat isolated nodose ganglion preparation at either room temperature or at 36 degrees C, whilst 5-aminovaleric acid (10 microM), a GABAB receptor antagonist, was unable to antagonize significantly the GABA-induced depolarization at either room temperature or at 36 degrees C. 4. [3H]-SR95531 (7.2 nM), a GABAA receptor-selective antagonist, bound topographically to sections of rat brainstem. Specific binding was highest in the medial nucleus tractus solitarius (NTS) and dorsal motor nucleus of the vagus nerve (DMVN). Binding was also observed in certain medullary reticular nuclei, in particular the parvocellular reticular nucleus. 5. Unilateral nodose ganglionectomy caused a reduction in GABAA binding site density in the medial NTS from 93 +/- 7 to 68 +/- 6 d.p.m./mm2. This procedure also caused a reduction in GABAA binding site density in the side of the NTS contralateral to the lesion, from 151 +/- 12 to 93 +/- 7 d.p.m./mm2. Sham surgery had no effect on the binding of [3H]-SR95531 in rat brainstem. 6. The present data provide evidence for the presence of GABAA receptors located on the soma and central terminals of rat vagal afferent neurones. Additionally, a population of GABAA receptors is evidenced postsynaptically in the rat NTS with respect to vagal afferent terminals. These data are discussed in relation to the functional pharmacology of GABA in this region of the NTS.     

Intracellular calcium stores modulate miniature GABA-mediated synaptic currents in neonatal rat hippocampal neurons

The whole-cell configuration of the patch clamp technique was used to record miniature gamma-aminobutyric acidA (GABAA) receptor-mediated currents (in tetrodotoxin, 1 microM and kynurenic acid 1 mM) from CA3 pyramidal cells in thin hippocampal slices obtained from postnatal (P) day (P6-9) old rats. Switching from a Ca2+-containing to a nominally Ca2+-free medium (in which Ca2+ was substituted with Mg2+, in the presence or in the absence of 100 microM EGTA) did not change significantly the frequency or amplitude of miniature events. Superfusion of thapsigargin induced a concentration-dependent increase in frequency but not in amplitude of tetrodotoxin-resistant currents that lasted for the entire period of drug application. Mean frequency ratio (thapsigargin 10 microM over control) was 1.8+/-0.5, (n = 9). In nominally Ca2+-free solutions thapsigargin was ineffective. When bath applied, caffeine (10 mM), reversibly reduced the amplitude of miniature postsynaptic currents whereas, if applied by brief pressure pulses, it produced an increase in frequency but not in amplitude of spontaneous GABAergic currents. Superfusion of caffeine (10 mM) reversibly reduced the amplitude of the current induced by GABA (100 microM) indicating a clear postsynaptic effect on GABAA receptor. Superfusion of ryanodine (30 microM), in the majority of the cells (n = 7) did not significantly modify the amplitude or frequency of miniature events. In two of nine cells it induced a transient increase in frequency of miniature postsynaptic currents. These results indicate that in neonatal hippocampal neurons, mobilization of calcium from caffeine-ryanodine-sensitive stores facilitates GABA release.     

The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid

The gamma-aminobutyric acid type A (GABAA) receptor is a transmitter-gated ion channel mediating the majority of fast inhibitory synaptic transmission within the brain. The receptor is a pentameric assembly of subunits drawn from multiple classes (alpha1-6, beta1-3, gamma1-3, delta1, and epsilon1). Positive allosteric modulation of GABAA receptor activity by general anesthetics represents one logical mechanism for central nervous system depression. The ability of the intravenous general anesthetic etomidate to modulate and activate GABAA receptors is uniquely dependent upon the beta subunit subtype present within the receptor. Receptors containing beta2- or beta3-, but not beta1 subunits, are highly sensitive to the agent. Here, chimeric beta1/beta2 subunits coexpressed in Xenopus laevis oocytes with human alpha6 and gamma2 subunits identified a region distal to the extracellular N-terminal domain as a determinant of the selectivity of etomidate. The mutation of an amino acid (Asn-289) present within the channel domain of the beta3 subunit to Ser (the homologous residue in beta1), strongly suppressed the GABA-modulatory and GABA-mimetic effects of etomidate. The replacement of the beta1 subunit Ser-290 by Asn produced the converse effect. When applied intracellularly to mouse L(tk-) cells stably expressing the alpha6beta3gamma2 subunit combination, etomidate was inert. Hence, the effects of a clinically utilized general anesthetic upon a physiologically relevant target protein are dramatically influenced by a single amino acid. Together with the lack of effect of intracellular etomidate, the data argue against a unitary, lipid-based theory of anesthesia.     

Selective modulation of GABAA receptors by aluminum

Aluminum has been implicated in several neurodegenerative conditions including Alzheimer's disease. Because the mammalian olfactory system has an unusual capacity for the uptake and transneuronal spread of inhaled substances such as aluminum, whole cell recording techniques were used to examine the actions of aluminum on basic membrane properties and amino acid receptors on rat olfactory bulb mitral/tufted (M/T) neurons in culture. Aluminum had little direct effects on M/T neurons. Aluminum (100 microM) did not evoke a membrane current or alter action-potential shape or duration. Aluminum also had no marked effects on the family of voltage-gated membrane currents evoked by a series of 10-mV, 50-ms depolarizing steps. However, aluminum dramatically potentiated the current evoked by 30 microM gamma-aminobutyric acid (GABA) at concentrations <100 microM. Conversely, higher concentrations of aluminum blocked the GABA-evoked current. The effects of aluminum on GABA-evoked currents were not voltage dependent. Aluminum (100 microM) equally potentiated both inward currents at -30 mV and outward currents at + 30 mV. At 300 microM, aluminum blocked both inward and outward currents to a similar extent. In some neurons, aluminum only blocked the current and potentiation was not observed. The biphasic action of aluminum on GABA-evoked currents suggests separate binding sites: a high-affinity potentiating site and a low-affinity inhibiting site. Despite its effects on GABA-evoked currents, aluminum did not alter membrane currents evoked by glutamate, N-methyl-D-aspartate, kainate, or glycine. Aluminum also did not reduce spontaneous excitatory synaptic activity, suggesting little, if any, effect on glutamate release. Although a causal role for aluminum in Alzheimer's disease and other neuropathological conditions remains controversial, it is clear that elevated aluminum concentrations in the brain are associated with a variety of cognitive impairments. The present results indicate that aluminum can alter the function of GABAA receptors and may suggest that aluminum can contribute to cognitive impairment through disruption of inhibitory circuits.