A possible mechanism underlying corymine inhibition of glycine-induced Cl- current in Xenopus oocytes

We previously reported that corymine, an alkaloid extracted from the leaves of Hunteria zeylanica native to Thailand, inhibited glycine-induced chloride current using a receptor expression model of Xenopus oocytes. In this study, we investigated the mechanism underlying the inhibitory action of this alkaloid on glycine current using the same model. Corymine inhibited glycine current in a noncompetitive fashion. Co-application with strychnine, a competitive glycine receptor antagonist, or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), a Cl- channel blocker, corymine decreased the ED50 value of strychnine, but did not change that of DIDS. Moreover, the inhibitory effects of corymine and either strychnine or DIDS were additive. The desensitization phase of glycine current showed two exponentials and corymine preferentially inhibited the fast component, whereas strychnine affected both of them to the same extent and DIDS preferentially inhibited the slow component. When these drugs were applied repeatedly, the inhibitory effects of corymine and strychnine were not use-dependent and reversible, while the effect of DIDS was use-dependent and irreversible. The inhibitory effect of corymine on gamma-aminobutyric acid (GABA) current was less potent than the effect on glycine current, while this alkaloid failed to affect acetylcholine and serotonin currents. These results demonstrate that corymine inhibits glycine-gated CI- channels by interacting with the site different from that of DIDS.     

Gp120 can revert antagonism at the glycine site of NMDA receptors mediating GABA release from cultured hippocampal neurons

The effects of the human immunodeficiency virus type 1 envelope protein gp120 on the release of GABA elicited by N-methyl-D-aspartate (NMDA) from rat hippocampal neurons in primary culture has been investigated. NMDA (1-300 microM) increased in a concentration-dependent manner (EC50 =37.9+/-12 microM) the release of [3H]-GABA. The effect of 100 microM NMDA was prevented by 30 microM of the GABA transport inhibitor N-(4,4-diphenyl-3-butenyl)guvacine (SKF 100330A). Glycine (10 microM) or gp120 (0.01 microM) affected neither the basal nor the NMDA-evoked [3H]-GABA release. The NMDA (100 microM)-evoked release was prevented by 5,7-dichloro-kynurenic acid (5,7-DCKA), a selective antagonist at the glycine site of the NMDA receptor, in a concentration-dependent manner (IC50 approximately 0.3 microM). Glycine (3-10 microM) or gp120 (0.003-0.01 microM) produced reversal of the 5,7-DCKA antagonism in a way that suggested competition at a same site; gp120 was at least 3 orders of magnitude more potent than glycine. It is suggested that gp120 may mimic glycine at NMDA receptors.     

Pharmacology of 5-chloro-7-trifluoromethyl-1,4-dihydro-2,3-quinoxalinedione: a novel systemically active ionotropic glutamate receptor antagonist

5-Chloro-7-trifluoromethyl-1,4-dihydro-2,3-quinoxalinedione (ACEA-1011) has analgesic properties in animal models of tonic pain. To investigate the mechanisms underlying this effect we used electrical recording techniques to characterize the in vitro pharmacology of ACEA-1011 at mammalian glutamate receptors. Two preparations were used: Xenopus oocytes expressing rat brain receptors and cultured rat cortical neurons. Results showed that ACEA-1011 is a competitive antagonist at NMDA receptor glycine sites. Apparent antagonist affinities (Kb values) were 0.4 to 0.8 microM in oocytes and approximately 0.6 microM in neurons. IC50 values for ACEA-1011 against four binary subunit combinations of cloned rat NMDA receptors (NR1A/NR2A, 2B, 2C or 2D) ranged from 0.4 to 8 microM (1 microM glycine). The 20-fold variation in sensitivity was due to a combination of subunit-dependent differences in glycine and antagonist affinities; EC50 values for glycine ranged between 0.08 to 0.8 microM and Kb values for ACEA-1011 between 0.2 to 0.8 microM. In addition, ACEA-1011 inhibited AMPA-preferring non-NMDA receptors by competitive antagonism at glutamate binding sites. Kb values were 4 to 9 microM in oocytes and 9 to 10 microM in neurons. The ED50 for ACEA-1011 in a mouse maximum electroshock-induced seizure model was approximately 12 mg/kg i.v.. Our results indicate that ACEA-1011 is a systemically active broad selectivity ionotropic glutamate receptor antagonist.     

Currents evoked by GABA and glycine in acutely dissociated neurons from the rat medial preoptic nucleus

The responses of acutely dissociated medial preoptic neurons to application of GABA, and glycine were studied using the perforated-patch whole-cell recording technique under voltage-clamp conditions. GABA, at a concentration of 1 mM, evoked outward currents in all cells (n = 33) when studied at potentials positive to -80 mV. The I-V relation was roughly linear. The currents evoked by GABA were partially blocked by 25-75 microM picrotoxin and were also partially or completely blocked by 100-200 microM bicuculline. Glycine, at a concentration of 1 mM, did also evoke outward currents in all cells (n = 12) when studied at potentials positive to -75 mV. The I-V relation was roughly linear. The currents evoked by glycine were largely blocked by 1 microM strychnine. In conclusion, the present work demonstrates that neurons from the medial preoptic nucleus of rat directly respond to the inhibitory transmitters GABA and glycine with currents that can be attributed to GABAA receptors and glycine receptors respectively.     

Synthesis and structure-activity relationships of 1,2,3,4-tetrahydroquinoline-2,3,4-trione 3-oximes: novel and highly potent antagonists for NMDA receptor glycine site

A series of 1,2,3,4-tetrahydroquinoline-2,3,4-trione 3-oximes (QTOs) was synthesized and evaluated for antagonism of NMDA receptor glycine site. Glycine site affinity was determined using a [3H]DCKA binding assay in rat brain membranes and electrophysiologically in Xenopus oocytes expressing 1a/2C subunits of cloned rat NMDA receptors. Selected compounds were also assayed for antagonism of AMPA receptors in Xenopus oocytes expressing rat brain poly-(A)+RNA. QTOs were prepared by nitrosation of 2,4-quinolinediols. Structure-activity studies indicated that substitutions in the 5-, 6-, and 7-positions increase potency, whereas substitution in the 8-position causes a decrease in potency. Among the derivatives evaluated, 5,6,7-trichloro-QTO was the most potent antagonist with an IC50 of 7 nM in the [3H]DCKA binding assay and a Kb of 1-2 nM for NMDA receptors expressed in Xenopus oocytes. 5,6,7-Trichloro-QTO also had a Kb of 180 nM for AMPA receptors in electrophysiological assays. The SAR of QTOs was compared with the SAR of 1,4-dihydroquinoxaline-2,3-diones (QXs). For compounds with the same benzene ring substitution pattern, QTOs were generally 5-10 times more potent than the corresponding QXs. QTOs represent a new class of inhibitors of the NMDA receptor which, when appropriately substituted, are among the most potent glycine site antagonists known.     

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.     

beta-Alanine and taurine microinjected into the rat caudal ventrolateral medulla increase blood pressure

Unilateral microinjections of GABA, glycine, beta-alanine and taurine into the caudal ventrolateral medulla (CVLM) of the rat, led to an increase in blood pressure and heart rate. The responses to glycine, beta-alanine and taurine but not to GABA could be blocked by strychnine. The responses to taurine and beta-alanine but not to GABA and glycine could be blocked by 6-aminomethyl-3-methyl-4H-1,2,4-benzothiadiazine-1,1-dioxide (TAG), an antagonist of taurine. The taurine antagonist alone injected bilaterally into the CVLM produced a decrease in blood pressure. From CVLM areas microperfused with Krebs solution, spontaneous release of GABA, glycine, beta-alanine and taurine was detected and high K+ stimulation caused a calcium-dependent release of GABA, beta-alanine and taurine. These results suggest that beta-alanine and taurine as well as GABA may be involved in modulation of the cardiovascular control within the CVLM.     

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.     

Use of subunit-specific antisense oligodeoxynucleotides to define developmental changes in the properties of N-methyl-D-aspartate receptors

Antisense oligodeoxynucleotides were used to determine whether alterations in the expression of N-methyl-D-aspartate (NMDA) receptor subunit mRNA are responsible for developmental changes in the sensitivity of receptors to agonists and antagonists. Xenopus laevis oocytes were injected with mRNA prepared from neonatal and adult rat cerebral cortex, and the effects of agonists and antagonists were determined under voltage-clamp conditions. Glycine-site antagonists like 7-chlorokynurenate and glutamate-site antagonists like CGP-39653 were more potent at NMDA receptors expressed from mRNA from adult rat cerebral cortex than those expressed from mRNA from 1-day-old rat. NMDA receptors from 1-day-old rat cerebral cortex were more sensitive to activation by glycine than were receptors from adult rat cerebral cortex. 7-Chlorokynurenate and CGP-39653 were more potent inhibitors of responses seen with heteromeric NR1/NR2A receptors than with NR1/ NR2B receptors. Conversely, heteromeric NR1/NR2B receptors were more sensitive to activation by glycine than were NR1/NR2A receptors. We previously described a delay in the expression of the NR2A subunit in developing rat brain. Anti-sense oligodeoxynucleotides were used to determine whether the delayed expression of the NR2A subunit underlies changes in pharmacological properties observed during development. The properties of receptors seen when adult brain mRNA was coinjected with antisense oligodeoxynucleotides against the NR2A subunit were similar to those found in receptors from 1-day-old rat brain. These data suggest that changes in the sensitivity of NMDA receptors to antagonists and to glycine seen during development are a result of alterations in the expression of different species of NR2 subunit mRNA.     

Effects of [3H]-BIDN, a novel bicyclic dinitrile radioligand for GABA-gated chloride channels of insects and vertebrates

1. The radiolabelled bicyclic dinitrile, [3H]-3,3-bis-trifluoromethyl-bicyclo[2.2.1]heptane-2,2-dicarbonitrile ([3H]-BIDN), exhibited, specific binding of high affinity to membranes of the southern corn rootworm (Diabrotica undecimpunctata howardi) and other insects. A variety of gamma-aminobutyric acid (GABA) receptor convulsants, including the insecticides heptachlor (IC50, 35 +/- 3 nM) and dieldrin (IC50, 93 +/- 7 nM), displaced [3H]-BIDN from rootworm membranes. When tested at 100 microM, 1-(4-ethynylphenyl)-4-n-propyl-2,6,7-trioxabicyclo[2.2.2]oct ane(EBOB), 4-t-butyl-2,6,7-trioxa-1-phosphabicy-clo[2.2.2]octane-1-thio ne (TBPS), 1-phenyl-4-t-butyl-2,6,7-trioxabicyclo[2.2.2]octane (TBOB) and picrotoxin failed to displace 50% of [3H]-BIDN binding to rootworm membranes indicating that the bicyclic dinitrile radioligand probes a site distinct from those identified by other convulsant radioligands. 2. Dissociation studies showed that dieldrin, ketoendrin, toxaphene, heptachlor epoxide and alpha and beta endosulphan displace bound [3H]-BIDN from rootworm membranes by a competitive mechanism. 3. Rat brain membranes were also shown to possess a population of saturable, specific [3H]-BIDN binding sites, though of lower affinity than in rootworm and with a different pharmacological profile. Of the insecticidal GABAergic convulsants that displaced [3H]-BIDN from rootworm, cockroach (Periplaneta americana) and rat brain membranes, many were more effective in rootworm. 4. Functional GABA-gated chloride channels of rootworm nervous system and of cockroach nerve and muscle were blocked by BIDN, whereas cockroach neuronal GABA(B) receptors were unaffected. 5. Expression in Xenopus oocytes of either rat brain mRNA, or cDNA-derived RNA encoding a GABA receptor subunit (Rdl) that is expressed widely in the nervous system of Drosophila melanogaster resulted in functional, homo-oligomeric GABA receptors that were blocked by BIDN. Thus, BIDN probes a novel site on GABA-gated Cl- channels to which a number of insecticidally-active molecules bind.     

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.     

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.     

Functionality of protective colloids affecting the formation, size uniformity and morphology of drug-free polylactic acid microspheres

Colchicine is a microtubule depolymerizing agent used extensively in the study of cytoskeleton-dependent cell functions. In studying the possible functional interaction between the GABA(A) receptor and the cytoskeleton, we found that colchicine inhibits GABA(A) receptor function by mechanisms independent of microtubule depolymerization. Human GABA(A) receptor alpha1beta2gamma2L subunits were co-expressed in Xenopus oocytes and the effects of colchicine on GABA(A) receptor function was assessed using the two-electrode voltage-clamp technique. Co-application of GABA (10 microM) with colchicine (100 microM) resulted in a 59.9% inhibition of GABA-gated chloride currents. This effect was instantaneous in onset with no pre-incubation required and reversed within seconds. Other depolymerizing agents, such as nocodazole (20 microM) and vinblastine (200 microM), did not affect GABA(A) receptor function using the same co-application protocol used with colchicine. The polymerizing agent taxol (10-50 microM) did not affect colchicine inhibition of the GABA responses and did not itself alter GABA-gated chloride currents. The inhibitory effect of colchicine was present under conditions in which the oocyte microtubules had been depolymerized by cold temperature. These results indicate that colchicine inhibits the GABA(A) receptor via mechanisms unrelated to microtubule depolymerization. To further examine the inhibitory effect of colchicine on the GABA response, GABA (10-3000 microM) concentration-response curves were performed in the absence or presence of various concentrations of colchicine (30-300 microM). In the presence of colchicine, the GABA concentration-response curve was shifted to the right in a parallel fashion. A Schild plot of this data yielded a linear slope indicating that colchicine acts as a competitive antagonist at the GABA binding site. We conclude that colchicine is a competitive antagonist at the GABA(A) receptor and that studies using colchicine to examine the functional interaction between GABA(A) receptors and microtubules should be interpreted with caution.     

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.     

Properties of GABA(A) receptors in cultured rat oligodendrocyte progenitor cells

We have studied the properties of GABA responses in oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells derived from primary cultures of the neonatal rat brain. In whole cell voltage clamp recordings, rapid application of 1-10 mM GABA elicited current responses in > 85% of the cells examined. The dose-response relationship pooled from nine progenitor cells was best fit by a logistic function of EC50=113 microM and Hill coefficient=0.9. In contrast to the rate of current deactivation, the rate of current activation exhibited marked concentration-dependence. Pharmacologically, GABA, muscimol and ZAPA ((Z)-3[(aminiiminomethyl)thio]prop-2-enoic acid sulphate) produced responses with ligand-specific kinetics, whereas glycine and the GABA(C) receptor agonist CACA were without effect; bicuculline methochloride acted as a competitive antagonist. Neither the amplitude nor the kinetics of currents produced by 100 microM GABA were affected by the benzodiazepine flunitrazepam (1 microM). Similarly the benzodiazepine receptor inverse agonist DMCM (1 microM) was also without effect. GABA-activated currents reversed polarity within 2 mV of the calculated Cl- equilibrium potential. With brief agonist pulses deactivation was monoexponential, however, unlike neurones the rate of deactivation was voltage-independent. Desensitisation of responses to 10 mM GABA was bi-exponential and accelerated at depolarised membrane potentials. Increasing the amount of GABA(A) receptor desensitisation (by increasing the duration of the agonist exposure) consistently produced a slowing of deactivation.     

Clozapine and several other antipsychotic/antidepressant drugs preferentially block the same 'core' fraction of GABA(A) receptors

Clozapine and several other antipsychotic/antidepressant drugs that fully or partially block GABA(A) receptors were tested at concentrations that reversed the inhibitory effect of 1 microM GABA on 35S-t-butylbicyclophosphorothionate ([35S]TBPS) binding to rat forebrain membranes only about 20-30%, here designated "core" fractions. Clozapine at 10 microM reverses 1 microM GABA 25+/-4.0% (n = 23) (its "core" fraction). Fourty three compounds were tested alone, and pairwise together with 10 microM Clozapine. The "core" fractions of some of the compounds yielded significant additive reversals together with 10 microM Clozapine, while others did not. A group of 14 compounds of which 7 are clinically effective antipsychotic drugs, including Chlorprothixene, Clomacran, Clopipazan, Fluotracen, Sulforidazine, Thioproperazine, and cis-Thiothixene, were statistically non-additive with 10 microM Clozapine, suggesting that all of these drugs selectively block the same core population of GABA(A) receptors as Clozapine. These non-additivities also suggest that Clozapine at 10 microM fully saturates a subset of GABA(A) receptors blocked by 1 microM GABA. Therefore, Clozapine probably blocks 2 or more types of GABA(A)receptors, but only half of the receptors that are sensitive to 1 microM GABA. A second group of 12 compounds of which 6 are clinically active antidepressant/antipsychotic drugs including Amoxapine, Clothiapine, Dibenzepine, Inkasan (Metralindole), Metiapine and Zimelidine were slightly, but significantly, additive with Clozapine suggesting that these compounds block most of Clozapine's core fraction, plus a small additional fraction. A third group consisted of ten compounds that yielded larger (R > 80) and statistically highly significant additivities with Clozapine. Complete additivity was obtained with Bathophenanthroline disulfonate, and Isocarboxazid, suggesting that they block GABA(A) receptors other than those blocked by 10 microM Clozapine. Seven "classical" GABA(A) receptor blockers, also tested at concentrations yielding 21 to 33% reversal alone, were all significantly additive with 10 microM Clozapine, but in no case was the additivity complete. The largest additivity was obtained with Pitrazepine (21%) and the smallest with Tubocurarine (9%). These results provide further support for the notion that selective blockade of the same subset of GABA(A) receptors may contribute to the clinical antipsychotic/antidepressant effects of Clozapine. The deltaB(opt) values for Clozapine are 50+/-1.7% and 26+/-2.6% (n = 3) in whole rat forebrain and cerebellum, respectively, confirming that clozapine-sensitive GABA(A) receptors are unevenly distributed in the brain. The sedative and anxiolytic properties of Clozapine and other antipsychotic drugs may be due to selective blockade of GABergic disinhibition at certain interneurons.     

Enhancement of gamma-aminobutyric acidA receptor activity by alpha-chloralose

alpha-Chloralose is widely used as an anesthetic in the laboratory due to its minimal effects on autonomic and cardiovascular systems, yet little is known about its mechanism of action. We examined the effects of alpha-chloralose on gamma-aminobutyric acid type A (GABAA) receptor activity because recent studies have shown that several classes of general anesthetics modulate the function of this receptor. GABAA receptor activity was assayed by measuring the GABA-induced current in Xenopus oocytes expressed with human GABAA receptor alpha-1, beta-1 and gamma-2L subunits. alpha-Chloralose produced a concentration-dependent potentiation of the GABA-induced current with an EC50 value of 49 microM and a maximal effect of 239% of control. Membrane current was not affected by alpha-chloralose in the absence of GABA. alpha-Chloralose (100 microM) increased the affinity for GABA 5-fold and produced a small (17%) increase in the efficacy of GABA. Measurement of the reversal potentials for the alpha-chloralose response suggested that the effect is mediated through increased Cl- conductance. Studies of alpha-chloralose interactions with other allosteric modulators determined that alpha-chloralose binds to a site on the GABAA receptor complex distinct from the benzodiazepine, neurosteroid and barbiturate sites. Chloral hydrate, trichloroethanol and urethane also augmented GABA-induced currents. alpha-Chloralose had no effect on the hydroxytryptamine-induced currents in oocytes expressed with the 5-hydroxytryptamine3 receptor. These data extend the number of classes of anesthetics that allosterically modulate GABAA receptor activity and indicate that GABAA receptors may be a common site of action for diverse classes of general anesthetics.     

Evaluation and management of men whose radical prostatectomies failed: results of an international survey

1. The responses of the electrically-driven right ventricle strip of the guinea-pig heart to diazepam were recorded in the absence and in the presence of different selective cyclic nucleotide phosphodiesterase (PDE) inhibitors. 2. Diazepam, at concentrations ranging from 1 microM to 100 microM, was devoid of effect on the contractile force in this preparation. 3. Conversely, diazepam (5 microM-100 microM) produced a consistent positive inotropic response in the presence of a concentration (1 microM), that was without effect in the absence of diazepam, of either of the selective PDE 3 inhibitors milrinone or SK&F 94120, but not in the presence of the selective PDE 4 inhibitor rolipram. 4. This effect of diazepam was not gamma-aminobutyric acid (GABA)-dependent, since it was neither mimicked nor potentiated by GABA, and was not affected by either a high concentration (5 microM) of the antagonists of the benzodiazepine/GABA/channel chloride receptor complex, picrotoxin, flumazenil and beta-carboline-3-carboxylic acid methyl ester (betaCCMe), or by the inverse agonists, beta-carboline-3-carboxylic acid N-methylamide (betaCCMa) and methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate (DMCM, 0.1 microM). Furthermore, a specific antagonist of the peripheral benzodiazepine receptors, PK 11195 (5 microM), did not influence the effect of diazepam. 5. Biochemical studies with isolated PDEs, confirmed that diazepam selectively inhibits type 4 PDE from guinea-pig right ventricle rather than the other PDEs present in that tissue. The compound inhibited this enzyme in a non-competitive manner. Diazepam was also able to inhibit PDE 5, the cyclic GMP specific PDE absent from cardiac muscle, with a potency close to that shown for PDE 4. 6. Diazepam displaced the selective type 4 PDE inhibitor, rolipram from its high affinity binding site in rat brain cortex membranes, and also potentiated the rise in cyclic AMP levels induced by isoprenaline in guinea-pig eosinophils, where only type 4 PDE is present. 7. The PDE inhibitory properties of diazepam were shared, although with lower potency, by other structurally-related benzodiazepines, that also displaced [3H]-rolipram from its high affinity binding site. The order of potency found for these compounds in these assays was not related to their potencies as modulators of the GABA receptor through its benzodiazepine binding site. 8. The pharmacological and biochemical data presented in this study indicate that diazepam behaves as a selective type 4 PDE inhibitor in cardiac tissue and this effect seems neither to be mediated by the benzodiazepine/GABA/channel chloride receptor complex nor by peripheral type benzodiazepine receptors.     

Characterization of benzodiazepine receptors in primary cultures of fetal mouse brain and spinal cord neurons

Primary cultures of fetal mouse brain and spinal cord were examined for the presence of binding sites for [3H]diazepam. Both brain and spinal cord cultures contain high affinity binding sites which resemble benzodiazepine receptors found in mammalian CNS with respect to both pharmacologic profile and response to exogenously applied GABA. These observations, coupled with the electrophysiologic properties of these cells suggest that primary cultures of fetal mouse brain and spinal cord may be valid models for studying the role and regulation of the benzodiazepine receptor.     

Novel systemically active antagonists of the glycine site of the N-methyl-D-aspartate receptor: electrophysiological, biochemical and behavioral characterization

A series of novel tricyclic pyrido-phthalazine-dione derivatives was tested for antagonistic effects at the strychnine-insensitive modulatory site of the N-methyl-D-aspartate (NMDA) receptor (glycineB). All compounds displaced [3H]MDL-105,519 binding to rat cortical membranes with IC50 values of between 90 nM and 3.6 microM. In patch-clamp experiments, steady-state inward current responses of cultured hippocampal neurons to NMDA (200 microM, glycine 1 microM) were antagonized by these same compounds with IC50 values of 0.14 to 13.8 microM. The antagonism observed was typical for glycineB antagonists, i.e., they induced desensitization and their effects were not use or voltage dependent. Moreover, increasing concentrations of glycine were able to decrease their apparent potency. Much higher concentrations (>100 microM) were required to antagonize alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid-induced currents. They were potent, systemically active NMDA receptor antagonists in vivo against responses of single neurons in the rat spinal cord to microelectrophoretic application of NMDA with ID50 values in the low milligram per kilogram i.v. range. They also inhibited pentylenetetrazol-, NMDA- and maximal electroshock-induced convulsions in mice with ED50 values ranging from 8 to 100 mg/kg i.p. The duration of anticonvulsive action was rather short but was prolonged by the organic acid transport inhibitor probenecid (200 mg/kg). The agents tested represent a novel class of systemically active glycineB antagonists with greatly improved bioavailability.