A steroid recognition site is functionally coupled to an expressed GABA(A)-benzodiazepine receptor

Pregnane steroids, particularly 3 alpha-hydroxylated metabolites of progesterone, are known to have rapid and profound effects on brain excitability. Recent evidence suggests that the gamma-aminobutyric acid (GABA(A))-benzodiazepine receptor-Cl- ionophore complex may mediate these actions. The data further suggest that these steroids modulate the complex through a novel site independent of other known sites on the complex. The hypothesis that this site is on the GABA(A)-benzodiazepine receptor-Cl- ionophore complex is tested in the present study by determining its presence on transiently expressed GABAA-benzodiazepine receptors.     

Neurosteroids block Ca2+ channel current in freshly isolated hippocampal CA1 neurons.

Certain synthetic and endogenous steroids are known to modulate neuronal responses to gamma-aminobutyric acid (GABA) and also change the firing frequency of certain neurons. However, there is nothing known of the effect(s) of steroids on voltage-gated calcium currents in mammalian neurons. We show here that the steroids (0.1-100 microM) allotetrahydrocorticosterone (THCC), dehydroepiandrosterone sulfate (DHEAS) and pregnanolone can rapidly and reversibly depress voltage-gated calcium currents in freshly isolated adult hippocampal CA1 pyramidal neurons. This blocking action occurs in the presence of picrotoxin (10 microM). Tail current analysis shows that THCC appears to be a ligand that selectively and reversibly depresses the omega-conotoxin (fraction GVIA) sensitive portion of the calcium current. These results demonstrate that certain steroid metabolites have a direct membrane site of action which may influence brain excitability.     

Effects of a benz[e]indene on gamma-aminobutyric acid-gated chloride currents in cultured postnatal rat hippocampal neurons.

Benz[e]indenes (BIs) are tricyclic molecules that can be envisioned as steroids without an A-ring. Because certain steroids are known to alter gamma-aminobutyric acid (GABA) responses in central neurons, we examined the effects of a substituted BI resembling 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha-OH-DHP) on GABA-gated chloride currents in cultured postnatal rat hippocampal neurons. The compound, BI-1, reversibly potentiated GABA currents at concentrations of > 10 nM, with an EC50 value of 0.2 microM. BI-1 increased the apparent affinity of GABA for its receptor, decreasing the GABA EC50 from 9 microM to 3 microM. BI-1 had no effect on the shape of the GABA current-voltage relationship and did not alter the GABA reversal potential. The effects of BI-1 were not altered by benzodiazepine or picrotoxin site antagonists. At concentrations up to 10 microM, where maximal effects on GABA currents were seen, BI-1 did not directly activate a membrane current. This contrasts with the effects of 3 alpha-OH-DHP, which activated chloride currents at concentrations that were subsaturating for GABA potentiation. These results suggest that the BIs may be useful for determining the mechanisms by which steroids potentiate GABA responses and directly gate chloride channels.     

Studies on the mechanism of interactions between anesthetic steroids and gamma-aminobutyric acidA receptors

Functional interactions between steroidal anesthetics and gamma-aminobutyric acidA (GABAA) receptors have been examined with 36Cl- uptake measurements in rat cerebrocortical synaptoneurosomes. The primary effect of the steroids was to enhance the affinity of GABA for its receptors without much effect on the maximal uptake rate; the ED50 for GABA decreased from 66.4 +/- 5.7 to 8.9 +/- 1.2 microM in the presence of 20 microM 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one. Stimulation of 36Cl- uptake by high concentrations of the anesthetic steroid in the absence of exogenous GABA was not due to direct stimulation of GABAA receptors, as currently proposed, but is due to enhanced action of endogenous GABA, inasmuch as the steroid markedly increases GABA affinity for the receptors. Typically, endogenous GABA was maintained at near 1 microM by a Na(+)-dependent GABA transport system in the synaptoneurosomes. Elevation of its level with nipecotic acid, a specific inhibitor of the GABA transport system, or reduction with GABase, a GABA-scavenging system, increased or decreased, respectively, the steroid-induced bicuculline-sensitive 36Cl- uptake. At low concentrations of GABA (less than 2 microM), the stimulatory effect of 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one was markedly potentiated by pentobarbital but antagonized by 3 alpha,21-dihydroxy-5 alpha-pregnan-20-one, a partial agonist of higher affinity. These observations, along with the structure-activity relationships of steroid analogs, strongly suggest the existence of a specific binding site for the steroids in GABAA receptors and led us to propose a minimal model in which two key common functional groups of anesthetic steroids, 3 alpha-OH- and 17 beta-polar substituents, interact with GABAA receptors (probably through hydrogen bondings) while their hydrophobic backbone remains in contact with the fatty acyl chains of membrane phospholipids.     

A steroid recognition site is functionally coupled to an expressed GABAA-benzodiazepine receptor

Pregnane steroids, particularly 3α-hydroxylated metabolites of progesterone, are known to have rapid and profound effects on brain excitability. Recent evidence suggests that the γ-aminobutyric acid (GABA_A)-benzodiazepine receptor-Cl⁻ ionophore complex may mediate these actions. The data further suggest that these steroids modulate the complex through a novel site independent of other known sites on the complex. The hypothesis that this site is on the GABA_A-benzodiazepine receptor-Cl⁻ ionophore complex is tested in the present study by determining its presence on transiently expressed GABA_A-benzodiazepine receptors.     

Mode of action of ICS 205,930, a novel type of potentiator of responses to glycine in rat spinal neurones

The effect of a novel potentiator of glycine responses, ICS 205,930, was studied by whole-cell recordings from spinal neurones, and compared with that of other known potentiators, in an attempt to differentiate their sites of action. The ability of ICS 205,930 (0.2 microM) to potentiate glycine responses persisted in the presence of concentrations of Zn2+ (5-10 microM) that were saturating for the potentiating effect of this ion. Preincubation with 10 microM Zn2+ before application of glycine plus Zn2+ had an inhibitory effect, which did not result from Zn2+ entry into the neurone, since it persisted with either 10 mM internal EGTA or 10 microM internal Zn2+. To test whether the potentiating effects of ICS 205,930 and Zn2+ interact, both compounds were applied without preincubation. The potentiating effect of ICS 205,930 was similar for responses to glycine and for responses to glycine plus Zn2+, provided the concentrations of agonist were adjusted so as to induce control responses of identical amplitudes. ICS 205,930 remained able to potentiate glycine responses in the presence of ethanol (200 mM). ICS 205,930 also retained its potentiating effect in the presence of the anaesthetic propofol (30 90 microM), which strongly potentiated glycine responses but, in contrast with ICS 205,930, also markedly increased the resting conductance. The anticonvulsant chlormethiazole (50-100 microM) neither potentiated glycine responses nor prevented the effect of ICS 205,930, even though it increased the resting conductance and potentiated GABA(A) responses. The mechanism of action of ICS 205,930 appears to be different from those by which Zn2+, propofol or ethanol potentiate glycine responses.     

Differential modulation of the gamma-aminobutyric acid type C receptor by neuroactive steroids.

Gamma-aminobutyric acid type C receptor channels (GABA(C)Rs) composed of rho subunits are pharmacologically distinct from GABA(A) receptor channels (GABA(A)Rs). This difference is illustrated by the insensitivity of homo-oligomeric rho(1) receptor channels to many known modulators of GABA(A)Rs, such as barbiturates and benzodiazepines. A number of endogenous metabolites of corticosterone and progesterone, known as neuroactive steroids, compose yet another class of compounds that can modulate GABA(A)Rs. Here, several neuroactive steroids are shown to also modulate the rho(1) receptor channel. 5alpha-Pregnane-3alpha,21-diol-20-one (allotetrahydrodeoxycorticosterone), 5alpha-pregnane-3alpha-ol-11, 20-dione (alphaxalone), and 5alpha-pregnane-3alpha-ol-20-one (allopregnanolone) potentiated the GABA-evoked currents from rho(1) receptor channels and concomitantly altered the deactivation kinetics by prolonging the decay time. In contrast, 5beta-pregnane-3alpha-ol-20-one (pregnanolone), 5beta-pregnane-3, 20-dione (5beta-dihydroprogesterone), and 5beta-pregnane-3alpha, 21-diol-20-one (tetrahydrodeoxycorticosterone), all potentiators of GABA(A)Rs, inhibited the GABA-elicited currents of the rho(1) receptor channel. In comparison to GABA(A)Rs, the modulation of rho(1) receptor channels by these neuroactive compounds occurred with relatively high concentrations of the neuroactive steroids and was more prominent in the presence of low concentrations of GABA, equivalent to fractions of the EC(50) value of the rho(1) receptor channel. Structural comparison of these six neuroactive steroids reveals that the key parameter in determining the mode of modulation for the rho(1) receptor channel is the position of the hydrogen atom bound to the fifth carbon, imposing a trans- or cis-configuration in the backbone structure. This is the first demonstration of isomeric compounds that can differentially modulate the activity of the rho(1) receptor channel.     

Excessive and precocious glutamate release in a mouse model of amyotrophic lateral sclerosis

The release of [3H]D-aspartate ([3H]D-ASP) or [3H]GABA evoked by glycine and that of [3H]D-ASP or [3H]glycine evoked by GABA from spinal cord synaptosomes were studied in SOD1-G93A(+) mice, a transgenic model of amyotrophic lateral sclerosis, SOD1(+) mice and SOD1(-)/G93A(-) animals. Mutant mice were killed at advanced phase of pathology or during the presymptomatic period. In SOD1(-)/G93A(-) or SOD1(+) mice glycine evoked [(3)H]d-ASP and [(3)H]GABA release, while GABA caused [3H]D-ASP, but not [3H]glycine, release. The glycine-evoked release of [3H]D-ASP, but not that of [3H]GABA, and the GABA-evoked [3H]D-ASP release, but not that of [3H]glycine, were more pronounced in SOD1-G93A(+) than in SOD1(+) mice. Furthermore, these potentiations were already present in asymptomatic 30- to 40-day-old mice. Basal [3H]D-ASP release was also higher in SOD1-G93A(+) than SOD1(+) or SOD1(-)/G93A(-) mice. The release of endogenous glutamate and GABA was also enhanced in asymptomatic animals; the glycine-evoked release of endogenous glutamate, but not of endogenous GABA, was higher in SOD1-G93A(+) than in SOD1(+) animals. The effects of glycine and GABA were insensitive to receptor blockers, but sensitive to transporter inhibitors, indicating coexistence of glutamate and glycine transporters and of glutamate and GABA transporters on glutamate-releasing terminals. The glutamate release machinery seems excessively functional in SOD1-G93A(+) animals.     

Modulation of human GABAA and glycine receptor currents by menthol and related monoterpenoids

Effects of common monoterpenoid alcohols and ketones were investigated on recombinant human gamma-aminobutyric acid A (GABAA; alpha1beta2gamma2s) and glycine (alpha1 homomers) receptors expressed in Xenopus oocytes. GABA currents were enhanced by coapplications of 10-300 microM: (+)-menthol>(-)-menthol>(-)-borneol>(-)-menthone=camphor enantiomers>carvone enantiomers, with menthol acting stereoselectively. By contrast, thujone diastereomers inhibited GABAA receptor currents while glycine currents were only markedly potentiated by menthol. Positive modulation by (+)-menthol was explored given its pronounced effects (e.g., at 100 microM, GABA and glycine EC20 responses increased by 496+/-113% and 135+/-56%, respectively). (+)-Menthol, 100 microM, reduced EC50 values for GABA and glycine from 82.8+/-9.9 to 25.0+/-1.8 microM, and from 98.7+/-8.6 to 75.7+/-9.4 microM respectively, with negligible effects on maximal currents. This study reveals a novel neuroactive role for menthol as a stereoselective modulator of inhibitory ligand-gated channels.     

GABA-dependent modulation of the Cl- ionophore by steroids in rat brain

Steroids inhibit the binding of [35S]t-butylbicyclophosphorothionate ([ 35S]TBPS) to the GABAA-benzodiazepine receptor (GBR) linked Cl- ionophore in a GABA dependent manner but not through the GABAA receptor. The most potent steroid evaluated is a naturally occurring metabolite of progesterone, 3 alpha-hydroxy,5 alpha-dihydroprogesterone with an IC50 of approximately 17 nM. Structural requirements necessary for inhibitory activity coincide with those reported for anticonvulsant and anesthetic actions. Coupled with earlier evidence that these steroids do not act directly at the benzodiazepine receptor nor the [35S]TBPS labeled site to modulate the Cl- ionophore, the possibility is proposed that a distinct membrane-bound 'steroid site' coupled to the GBR-Cl- ionophore complex exists.     

The effects of benzodiazepine agonists, inverse agonists and Ro 15-1788 on the responses of the superior cervical ganglion to GABA in vitro

The effects of benzodiazepines and their antagonists on the responses to gamma-aminobutyric acid (GABA) of the superior cervical ganglion of the rat were examined using extracellular recording. Chlordiazepoxide (1 microM to 28.9 microM) and flurazepam (145-725 nM) increased the responses of the ganglion to GABA and the increases were antagonized by Ro 15-1788, at 3.34 microM. The concentration of GABA used was 9.7 microM which gave half-maximal responses. Chlordiazepoxide similarly increased the responses of the ganglion to GABA 38.8 microM in the presence of bicuculline 27.2 microM. This concentration of GABA gave, with bicuculline, responses of a similar magnitude as those to 9.7 microM in the absence of bicuculline. Bicuculline did not affect the actions of chlordiazepoxide or the antagonism by Ro 15-1788. Ro 15-1788 did not affect the increases in GABA response caused by pentobarbitone or by phenobarbitone in the presence of bicuculline. Ethyl beta-carboline-3-carboxylate (beta CCE) (207 nM to 1 microM) significantly decreased the responses to GABA in the presence and in the absence of bicuculline. The decreases were antagonized by Ro 15-1788 (3.34 microM). beta CCE at 2.1 microM and above did not significantly change the responses to GABA. Methyl beta-carboline-3-carboxylate (beta CCM) at 88 to 440 nM significantly decreased the responses to GABA. The decreases were antagonized by Ro 15-1788 (3.34 microM) and were also seen in the presence of bicuculline. High concentrations of Ro 15-1788 decreased the responses to GABA, 9.7 microM, but increased the responses to GABA 38.8 microM in the presence of 27.2 microM bicuculline. The pattern of effects of the benzodiazepines, beta-carbolines and low doses of Ro 15-1788 on the responses to GABA was similar to the effects of these compounds on seizure threshold and anxiety-related behaviour in vivo.     

Interaction between copper and zinc at GABA(A) receptors in acutely isolated cerebellar Purkinje cells of the rat

Nanomolar concentrations of Cu(2+) induce a slowly reversible block of GABA(A) receptor-mediated currents which can be removed by chelating substances. The possible interaction of Cu(2+) with the Zn(2+) binding site on the GABA(A) receptor complex was studied in acutely isolated Purkinje cells using whole-cell recording and a fast drug application system. When Zn(2+) was applied together with 2 microM GABA, the Zn(2+)-induced block of GABA-mediated currents was not additive to the Cu(2+)-induced block. In the presence of 0.1 microM Cu(2+) in the bath solution the degree of inhibition of GABA-mediated responses by Zn(2+) was strongly attenuated. Preapplication of 100 microM Zn(2+) during 10 s, terminated 1 s before exposure to 2 microM GABA did not affect the GABA current in Cu(2+)-free solution, but relieved its block by 0.1 microM Cu(2+). This effect of Zn(2+) was concentration-dependent with an EC(50) of 72 microM. When the Cu(2+)-induced block was removed by histidine, preapplication of Zn(2+) did not increase the GABA current, indicating that the relief of Cu(2+) block by Zn(2+) is the result of its ability to actively remove Cu(2+) from the GABA receptor complex. It is proposed that the inhibitory effects of Zn(2+) and Cu(2+) on GABA-induced currents result from an action of these metal ions at distinct, but conformationally linked sites on the GABA(A) receptor protein. Under physiological conditions Zn(2+) would liberate Cu(2+) from the GABA(A) receptor, thus facilitating Cu(2+) turnover and its binding by other endogenous chelating molecules.     

Modulation of GABAA receptor-mediated currents by phenazepam and its metabolites

The effects of 7-bromo-5-(2-chlorophenyl)-1,3-dihydro-2H-1,4-benzodiazepin-2-one (phenazepam, PNZ), a 1,4-benzodiazepine derivative, and its physiological metabolites on GABA-activated whole-cell currents were studied in enzymatically isolated rat Purkinje neurones. PNZ, its hydroxylated metabolite (HPNZ) and a reference benzodiazepine, diazepam, potently enhanced (up to 200% of control) peak amplitude of currents activated by 10 microM GABA with EC50s of 6.1 +/- 0.8, 10.3 +/- 1.4 and 13.5 +/- 1.9 nM respectively. Both PNZ and HPNZ caused a parallel leftwards shift of the concentration/effect relationship for GABA. Another metabolite, 6-bromo-(2-chlorophenyl) quinazoline-2-one (QNZ), augmented responses to 10 microM GABA with a maximal efficacy similar to that of the 1,4benzodiazepines tested, although its EC50 was 2.4 +/- 0.2 microM. A further metabolite, 5-bromo-(2-chlorophenyl)-2-aminobenzophenone (ABPH), had only minimal effects on the responses elicited by 10 microM GABA. Incubation with QNZ and ABPH had biphasic effects on the concentration/effect relationship for GABA. These compounds enhanced peak amplitudes of currents activated by low concentrations of GABA, but inhibited responses to saturating concentrations of the agonist. This effect could, in part, be explained by the acceleration of the desensitisation process by those substances. It is concluded that both PNZ and HPNZ can be referred to as full positive modulators of GABAA receptors and that they are primarily responsible for GABAergic effects of therapeutic doses of PNZ.     

Dual potentiating and inhibitory actions of a benz[e]indene neurosteroid analog on recombinant alpha1beta2gamma2 GABAA receptors

Benz[e]indenes are tricyclic analogs of neuroactive steroids and can be modulators of GABA(A) receptor activity. We have examined the mechanisms of action of the benz[e]indene compound [3S-(3alpha,3aalpha,5abeta,7beta,9aalpha,9bbeta)]-dodecahydro-7-(2-hydroxyethyl)-3a-methyl-1H-benz[e]indene-3-carbonitrile (BI-2) using single-channel patch-clamp and whole-cell recordings from human embryonic kidney cells transfected with rat GABA(A) receptor alpha1, beta2, and gamma2L subunits. The data demonstrate that BI-2 is a positive modulator of GABA(A) receptor activity with a peak effect at 2 microM. The mechanism of modulation is similar but not identical to that of neuroactive steroids. Similar to steroids, BI-2 acts by prolonging the mean open time duration through an effect on the duration and prevalence of the longest open time component. However, in contrast to many steroids, BI-2 does not selectively reduce the channel closing rate. The potentiating action of BI-2 seems to be mediated through interactions with the classic neuroactive steroid binding site. Mutation to the membrane-spanning region in the alpha1 subunit Q242W and the double mutation alpha1N408A/Y411F, previously shown to abolish potentiation by neurosteroids, also diminish potentiation by BI-2. At higher concentrations (>5 microM), BI-2 inhibits receptor function by enhancing the apparent rate of desensitization. From single-channel recordings, we estimate that the entry rate into the inhibited or blocked state, k(+B), is 0.50 microM(-1) s(-1). Based on the kinetic mechanism of action, and the finding that this effect is blocked by the alpha1V256S mutation, we propose that BI-2 acts through an inhibitory site first postulated for the inhibitory neurosteroid pregnenolone sulfate.     

Anticonvulsant profile of the progesterone metabolite 5 alpha-pregnan-3 alpha-ol-20-one

5 alpha-Pregnan-3 alpha-ol-20-one (3 alpha-OH-DHP) is a naturally occurring metabolite of progesterone that can modulate brain excitability through a specific steroid recognition site on the GABA/benzodiazepine receptor-chloride ionophore complex. The anticonvulsant properties of 3 alpha-OH-DHP were determined using standardized anticonvulsant screening tests in mice. This steroid was found to be effective against metrazol-, (+)-bicuculline- and picrotoxin-induced seizures. The steroid has maximum potency against (+)-bicuculline-induced convulsions and no activity against maximal electroshock and strychnine-induced seizures. These findings support the hypothesis that therapeutically useful anticonvulsant steroids active at the putative steroid recognition site associated with the GABA/benzodiazepine receptor-chloride ionophore complex can be identified.     

Endogenous digitalis-like factors: in vitro comparison of biological and immunological activities of peptide and steroid candidates

Endogenous substances that modulate the activity of (Na+ + K+)-ATPase through interaction at the cardiac glycoside site have been postulated. Reports of digitalis-like biological and immunological activity exhibited by certain ACTH/MSH peptides and 14-OH steroids make these compounds potential candidates as endogenous digitalis-like factors. We tested several ACTH/MSH peptides and 14 alpha-OH steroids in four in vitro assays and detected no significant cardiac glycoside-like activity. On the other hand, chlormadinone acetate, a progesterone derivative shown to bind with high affinity to the digitalis receptor, was nearly equipotent to digoxigenin in a [3H]ouabain radioreceptor assay. In a [3H]digoxin radioimmunoassay, however, digoxigenin and digoxin were equipotent but chlormadinone acetate was inactive. A clear dissociation between radioreceptor assay and radioimmunoassay activity was also observed using 15 beta-OH-progesterone. Our findings indicate that (a) ACTH/MSH peptides and 14 alpha-OH steroids are not viable candidates as endogenous digitalis-like factors, (b) digoxin antibodies are not necessarily directed at molecular determinants critical for biological activity, and (c) among the compounds reported to exhibit digitalis-like activity and postulated to share structural features with an endogenous steroidal digitalis-like factor, only chlormadinone acetate and its congeners appear to constitute tenable models.     

Effects of steroids on gamma-aminobutyric acid receptors expressed in Xenopus oocytes by poly(A)+ RNA from mammalian brain and retina.

Electrical recordings were made in Xenopus oocytes to study the modulatory effects of steroids on gamma-aminobutyric acid (GABA) receptors expressed by RNA from mammalian brain and retina. GABA responses expressed by rat cerebral cortex poly(A)+ RNA were bicuculline-sensitive Cl- currents mediated by GABAA receptors. GABA responses expressed by bovine retina poly(A)+ RNA also were Cl- currents but were composed of two pharmacologically distinct components, one mediated by GABAA receptors and the other by GABA receptors with novel properties, which were resistant to bicuculline but were not activated by R(+)-baclofen, a selective agonist of GABAB receptors. As reported in neurons and in other expression systems, GABAA responses expressed in oocytes by cerebral cortex RNA were strongly and stereospecifically potentiated by 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha-OH-DHP) and 5 alpha-pregnan-3 alpha,21-diol-20-one (THDOC). Threshold levels of potentiation were detectable using 1-2 nM steroid, and at concentrations of 50 and 500 nM 3 alpha-OH-DHP shifted the EC50 of cortex GABAA responses from a control value of 92 +/- 20 microM GABA to 40 +/- 4.3 microM and 13 +/- 1.8 microM, respectively. However, even at concentrations as high as 50 microM, 3 alpha-OH-DHP did not itself elicit appreciable membrane current responses through direct activation of the cortex GABAA receptors. In addition to potentiation, 3 alpha-OH-DHP and THDOC caused pronounced increases in the rate of desensitization of GABAA responses expressed by cortex RNA. Decay time courses of currents elicited by 1 mM GABA (90-95% of the maximum response) were fitted by the sum of two exponentials. Under control conditions, the time constant of the fast component was 4.4 +/- 0.6 sec and the slow component, 22.5 +/- 4.8 sec. 3 alpha-OH-DHP at 500 nM and 5 microM reduced the time constant of the fast component by 52 +/- 7% and 84 +/- 5%, respectively, but showed little effect on the slow component. Unlike the potentiation effect, actions of pregnenolones on desensitization did not show stringent stereoselectivity, and 5 microM 5 beta-pregnan-3 beta-ol-20-one (3 beta-OH-DHP) reduced the time constant of the fast component by 59 +/- 11%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Allosteric modulation of an expressed homo-oligomeric GABA-gated chloride channel of Drosophila melanogaster.

1. Functional GABA-gated chloride channels are formed when cRNA encoding the Drosophila melanogaster GABA receptor subunit RDL is injected into the cytoplasm of Xenopus oocytes. Two-electrode voltage-clamp was used to investigate allosteric modulation of GABA-induced currents recorded from the expressed, bicuculline-insensitive, RDL homo-oligomers. 2. Flunitrazepam (0.1 microM to 100 microM) had no effect on the amplitude of responses to 10 microM GABA (approximately EC10), whereas 4'chlorodiazepam (100 microM) enhanced the amplitude of submaximal responses to GABA. 3-Hydroxymethyl-beta-carboline (1 microM) and ethyl-beta-carboline-3-carboxylate (both 1 and 100 microM) had no effect on currents induced by 30 microM (approximately EC50) GABA. However 100 microM 3-hydroxymethyl-beta-carboline reduced potentiation by 4'chlorodiazepam. 3. The sodium salts of pentobarbitone (10 microM to 1 mM) and phenobarbitone (50 microM to 1 mM) dose-dependently enhanced submaximal GABA responses. Neither barbiturate activated currents in the absence of GABA. 4. At 10 microM, the steroids 5 alpha-pregnan-3 alpha-ol-20-one and alphaxalone (5 alpha-pregnan-3 alpha-ol-11,20-dione), potentiated submaximal GABA responses. The stereoselectivity of steroid action seen on vertebrate GABAA receptors was observed on RDL homo-oligomers as 5 alpha-pregnan-3 beta-ol-20-one (10 microM) was without effect. None of the three steroids tested activated currents in the absence of GABA. 5. The novel anticonvulsant, loreclezole (100 microM), potentiated the response to 10 microM GABA, but not that of saturating concentrations of GABA. delta-Hexachlorocyclohexane (0.1 microM to 30 microM) was a potent enhancer of submaximal responses to GABA of RDL. 6. The potencies of barbiturates and steroids on RDL homo-oligomers resemble those observed for several in situ insect GABA receptors, whereas those of benzodiazepine binding-site ligands are considerably reduced. The differences in the benzodiazepine pharmacology of RDL homo-oligomers and native GABA receptors, may reflect roles of other subunits in native insect receptors.     

Effects of depressant amino acids and antagonists on an in vitro spinal cord preparation from the adult rat

A mature sacrococcygeal in vitro spinal preparation from the rat has been used to demonstrate effects of neutral amino acids and their antagonists. gamma-Aminobutanoate (GABA), glycine and taurine (0.5-5 mM) produced dose-dependent depression of spontaneous paroxysmal activity generated in Mg2+ -free medium. The depressant effect of GABA was antagonised selectively by picrotoxin (25-50 microM) and the depressant effects of glycine and taurine were antagonised selectively by strychnine (0.2 microM). Glycine (0.5-5 mM) had a dose-dependent depolarizing action which was present at the central ends of isolated ventral roots. gamma-Aminobutanoate and taurine, had only weak depolarizing actions on ventral root fibres. Depolarizing responses to glycine showed a marked fading. Reduction in the fading appeared to be responsible for a paradoxical potentiation of glycine-induced depolarizations, which occurred in the presence of strychnine (0.2-2 microM). Strychnine (2-10 microM), picrotoxin (10-50 microM) or bicuculline (10 microM) had little or no effect on the amplitude, duration or latency of the monosynaptic component of ventral root reflexes evoked by supramaximal stimulation of dorsal roots (DR-VRP). However all three antagonists introduced slow, NMDA receptor mediated, components to these ventral root potentials. Picrotoxin and bicuculline, but not strychnine, reversibly depressed the dorsal root potential evoked from an adjacent dorsal root (DR-DRP). The depressant actions of 2-amino-5-phosphonopentanoate (AP5), kynurenate and 3-((+/-)-2-carboxypiperazin-4-yl)propyl-1-phosphonic acid (CPP) revealed both NMDA and non-NMDA receptor mediated components in the dorsal root potential.