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J J Lambert

Publications and source records attributed to J J Lambert.

At least 37 records · Page 2Linked to original sources
PubMed1999

Mutagenesis of the rat alpha1 subunit of the gamma-aminobutyric acid(A) receptor reveals the importance of residue 101 in determining the allosteric effects of benzodiazepine site ligands.

The gamma-aminobutyric acid(A) (GABA(A)) receptor contains a binding site (or sites) for benzodiazepines and related ligands. Previous studies have shown that the residue occupying position 101 (rat numbering) of the alpha subunit is particularly important in determining how some of these compounds interact with the receptor. We have made multiple substitutions (F, Y, K, Q, and E) of the histidine at this position of the rat alpha1 subunit and coexpressed the mutant subunits with beta2 and gamma2 subunits in Xenopus oocytes. The effects of flunitrazepam, Ro15-1788, and Ro15-4513 on GABA-gated currents were then examined using electrophysiological techniques. Three substitutions (F, Y, and Q) had little effect on the ability of flunitrazepam to potentiate GABA-induced currents and had relatively modest effects on the EC(50) value of the flunitrazepam response. Other mutations (K and E) resulted in drastic reduction of flunitrazepam recognition. All substitutions also affected the EC(50) values for Ro15-1788 and Ro15-4513, and some led to dramatic changes in their efficacy. For example, H101Y, H101K, and H101Q produced receptors at which Ro15-1788 acted as a partial agonist (maximum potentiation of 164, 159, and 130%, respectively), whereas Ro15-4513 acted as a partial agonist at H101F, H101K, and H101E (potentiation of 122, 138, and 110%, respectively) and an antagonist at H101Y and H101Q. These results indicate that the characteristics of the residue at position 101 of the alpha1 subunit play a crucial role in determining the efficacy of benzodiazepine-site ligands.

Allosteric Regulation↗
PubMed1998

Ion permeation and conduction in a human recombinant 5-HT3 receptor subunit (h5-HT3A).

1. A human recombinant homo-oligomeric 5-HT3 receptor (h5-HT3A) expressed in a human embryonic kidney cell line (HEK 293) was characterized using the whole-cell recording configuration of the patch clamp technique. 2. 5-HT evoked transient inward currents (EC50 = 3.4 microM; Hill coefficient = 1.8) that were blocked by the 5-HT3 receptor antagonist ondansetron (IC50 = 103 pM) and by the non-selective agents metoclopramide (IC50 = 69 nM), cocaine (IC50 = 459 nM) and (+)-tubocurarine (IC50 = 2.8 microM). 3. 5-HT-induced currents rectified inwardly and reversed in sign (E5-HT) at a potential of -2.2 mV. N-Methyl-D-glucamine was finitely permeant. Permeability ratios PNa/PCs and PNMDG/PCs were 0.90 and 0.083, respectively. 4. Permeability towards divalent cations was assessed from measurements of E5-HT in media where Ca2+ and Mg2+ replaced Na+. PCa/PCs and PMg/PCs were calculated to be 1.00 and 0.61, respectively. 5. Single channel chord conductance (gamma) estimated from fluctuation analysis of macroscopic currents increased with membrane hyperpolarization from 243 fS at -40 mV to 742 fS at -100 mV. 6. Reducing [Ca2+]o from 2 to 0.1 mM caused an increase in the whole-cell current evoked by 5-HT. A concomitant reduction in [Mg2+]o produced further potentiation. Fluctuation analysis indicates that a voltage-independent augmentation of gamma contributes to this phenomenon. 7. The data indicate that homo-oligomeric receptors composed of h5-HT3A subunits form inwardly rectifying cation-selective ion channels of low conductance that are permeable to Ca2+ and Mg2+.

Calcium↗
PubMed1998

The effect of a transmembrane amino acid on etomidate sensitivity of an invertebrate GABA receptor.

1. The gamma-aminobutyric acid (GABA)-modulatory and GABA-mimetic actions of etomidate at mammalian GABA(A) receptors are favoured by beta2- or beta3- versus beta1-subunit containing receptors, a selectivity which resides with a single transmembrane amino acid (beta2 N290, beta3 N289, beta1 S290). Here, we have utilized the Xenopus laevis oocyte expression system in conjunction with the two-point voltage clamp technique to determine the influence of the equivalent amino acid (M314) on the actions of this anaesthetic at an etomidate-insensitive invertebrate GABA receptor (Rdl) of Drosophila melanogaster. 2. Complementary RNA-injected oocytes expressing the wild type Rdl GABA receptor and voltage-clamped at -60 mV responded to bath applied GABA with a concentration-dependent inward current response and a calculated EC50 for GABA of 20+/-0.4 microM. Receptors in which the transmembrane methionine residue (M314) had been exchanged for an asparagine (RdlM314N) or a serine (RdlM314S) also exhibited a concentration-dependent inward current response to GABA, but in both cases with a reduced EC50 of 4.8+/-0.2 microM. 3. Utilizing the appropriate GABA EC10, etomidate (300 microM) had little effect on the agonist-evoked current of the wild type Rdl receptor. By contrast, at RdlM314N receptors, etomidate produced a clear concentration-dependent enhancement of GABA-evoked currents with a calculated EC50 of 64+/-3 microM and an Emax of 68+/-2% (of the maximum response to GABA). 4. The actions of etomidate at RdlM314N receptors exhibited an enantioselectivity common to that found for mammalian receptors, with 100 microM R-(+)-etomidate and S-(-)-etomidate enhancing the current induced by GABA (EC10) to 52+/-6% and 12+/-1% of the GABA maximum respectively. 5. The effects of this mutation were selective for etomidate as the GABA-modulatory actions of 1 mM pentobarbitone at wild type Rdl (49+/-4% of the GABA maximum) and RdlM314N receptors (53+/-2% of the GABA maximum) were similar. Additionally, the modest potentiation of GABA produced by the anaesthetic neurosteroid 5alpha-pregnan-3alpha-ol-20-one (Rdl = 25+/-4% of the GABA maximum) was not altered by this mutation (RdlM314N = 18+/-3% of the GABA maximum). 6. Etomidate acting at beta1 (S290)-containing mammalian GABA(A) receptors is known to produce only a modest GABA-modulatory effect. Similarly, etomidate acting at RdlM314S receptors produced an enhancement of GABA but the magnitude of the effect was reduced compared to RdlM314N receptors. 7. Etomidate acting at human alpha6beta3gamma2L receptors is known to produce a large enhancement of GABA-evoked currents and at higher concentrations this anaesthetic directly activates the GABA(A) receptor complex. Mutation of the human beta3 subunit asparagine to methionine (beta3 N289M found in the equivalent position in Rdl completely inhibited both the GABA-modulatory and GABA-mimetic action of etomidate (10-300 microM) acting at alpha6beta3 N289Mgamma2L receptors. 8. It was concluded that, although invertebrate and mammalian proteins exhibit limited sequence homology, allosteric modification of their function by etomidate can be influenced in a complementary manner by a single amino acid substitution. The results are discussed in relation to whether this amino acid contributes to the anaesthetic binding site, or is essential for transduction. Furthermore, this study provides a clear example of the specificity of anaesthetic action.

Allosteric Regulation↗
PubMed1998

Pharmacological characterization of a rat 5-hydroxytryptamine type3 receptor subunit (r5-HT3A(b)) expressed in Xenopus laevis oocytes.

The present study has utilized the two electrode voltage-clamp technique to examine the pharmacological profile of a splice variant of the rat orthologue of the 5-hydroxytryptamine type 3A subunit (5-HT3A(b)) heterologously expressed in Xenopus laevis oocytes. At negative holding potentials, bath applied 5-HT (300 nM - 10 microM) evoked a transient, concentration-dependent (EC50 = 1.1+/-0.1 microM), inward current. The response reversed in sign at a holding potential of -2.1+/-1.6 mV. The response to 5-HT was mimicked by the 5-HT3 receptor selective agonists 2-methyl-5-HT (EC50= 4.1+/-0.2 microM), 1-phenylbiguanide (EC50=3.0+/-0.1 microM), 3-chlorophenylbiguanide (EC50 = 140+/-10 nM), 3,5-dichlorophenylbiguanide (EC50 = 14.5+/-0.4 nM) and 2,5-dichlorophenylbiguanide (EC50 = 10.2+/-0.6 nM). With the exception of 2-methyl-5-HT, all of the agonists tested elicited maximal current responses comparable to those produced by a saturating concentration (10 microM) of 5-HT. Responses evoked by 5-HT at EC50 were blocked by the 5-HT3 receptor selective antagonist ondansetron (IC50=231+/-22 pM) and by the less selective agents (+)-tubocurarine (IC50=31.9+/-0.01 nM) and cocaine (IC50 = 2.1+/-0.2 microM). The data are discussed in the context of results previously obtained with the human and mouse orthologues of the 5-HT3A subunit. Overall, the study reinforces the conclusion that species differences detected for native 5-HT3 receptors extend to, and appear largely explained by, differences in the properties of homo-oligomeric receptors formed from 5-HT3A subunit orthologues.

Algorithms↗
PubMed1997

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.

Amino Acids↗
PubMed1997

Anesthetic activity of novel water-soluble 2 beta-morpholinyl steroids and their modulatory effects at GABAA receptors.

(3 alpha,5 alpha)-3-Hydroxypregnan-20-ones and (3 alpha,5 alpha)-3-hydroxypregnane-11,20-diones bearing a 2 beta-morpholinyl substituent were synthesized, and the utility of these steroids as anesthetic agents was evaluated through determination of their potency and duration of hypnotic activity in mice after intravenous administration. Alkylation of the morpholinyl substituent or chlorination at C-21 afforded the novel amino steroids (2 beta,3 alpha,5 alpha)-3-hydroxy-2-(2,2-dimethyl-4-morpholinyl)-pregnane-11,20-dione (19) and (2 beta,3 alpha,5 alpha)-21-chloro-3-hydroxy-2-(4-morpholinyl)pregnan-20-one (37) that were more potent and advantageously produced shorter sleep times than related compounds which were previously reported. Furthermore, salts of these and other amino steroids generally retained good aqueous solubility. In a radioligand binding assay the compounds inhibited the specific binding of [35S]-tert-butyl bicyclophosphorothionate to rat whole brain membranes, and in an electrophysiological assay they potentiated GABAA receptor-mediated currents recorded from voltage-clamped bovine chromaffin cells. These in vitro results are consistent with the anesthetic activity of the amino steroids being related to their modulatory effects at GABAA receptors.

Anesthesia↗
PubMed1997

Subunit-dependent interaction of the general anaesthetic etomidate with the gamma-aminobutyric acid type A receptor.

1. The GABA modulating and GABA-mimetic actions of the general anaesthetic etomidate were examined in voltage-clamp recordings performed on Xenopus laevis oocytes induced, by cRNA injection, to express human recombinant gamma-aminobutyric acidA (GABAA) receptor subunits. 2. Currents mediated by recombinant receptors with the ternary subunit composition alpha x beta y gamma 2L (where x = 1,2,3 or 6 and y = 1 or 2), in response to GABA applied at the appropriate EC10, were enhanced by etomidate in a manner that was dependent upon the identity of both the alpha and beta subunit isoforms. 3. For the beta 2-subunit containing receptors tested, the EC50 for the potentiation of GABA-evoked currents by etomidate (range 0.6 to 1.2 microM) was little affected by the nature of the alpha subunit present within the hetero-oligomeric complex. However, replacement of the beta 2 by the beta 1 subunit produced a 9-12 fold increase in the etomidate EC50 (6 to 11 microM) for all alpha-isoforms tested. 4. For alpha 1, alpha 2 and alpha 6, but not alpha 3-subunit containing receptors, the maximal potentiation of GABA-evoked currents by etomidate was greater for beta 2- than for beta 1-subunit containing receptors. This was most clearly exemplified by receptors composed of alpha 6 beta 1 gamma 2L compared to alpha 6 beta 2 gamma 2L subunits, where a maximally effective concentration of etomidate potentiated currents evoked by GABA at EC10 to 28 +/- 2% and 169 +/- 4% of the maximal GABA response, respectively. 5. For alpha 1 subunit-containing receptors, the potency and maximal potentiating effect of either pentobarbitone or propofol was essentially unaffected by the beta subunit isoform contained within the receptor complex. The potency of the anaesthetic neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one was marginally higher for beta 1 rather than the beta 2 subunit-containing receptor, although its maximal effect was similar at the two receptor isoforms. 6. The GABA-mimetic action of etomidate was supported by beta 2- but not beta 1-subunit containing receptors, whereas that of pentobarbitone or propofol was evident with either beta isoform. For beta 2-subunit containing receptors, both the agonist EC50 and the maximal current produced by etomidate were additionally influenced by the alpha isoform. 7. It is concluded that the subtype of beta-subunit influences the potency with which etomidate potentiates GABA-evoked currents and that the beta isoform is a crucial determinant of the GABA-mimetic activity of this compound. The nature of the alpha-subunit also impacts upon the maximal potentiation and activation that the compound may elicit. Such pronounced influences may aid the identification of the site that recognises etomidate. More generally, these results provide a clear example of structural specificity in anaesthetic action.

Anesthetics, Intravenous↗
PubMed1997

The interaction of general anaesthetics with recombinant GABAA and glycine receptors expressed in Xenopus laevis oocytes: a comparative study.

1. The effects of five structurally dissimilar general anaesthetics were examined in voltage-clamp recordings of agonist-evoked currents mediated by recombinant gamma-aminobutyric acid (GABA)A receptors composed of human alpha 1 beta 1 and gamma 2L subunits expressed in Xenopus laevis oocytes. A quantitative comparison of the effects of these agents was made upon recombinant glycine receptors expressed as a homo-oligomer of human alpha 1 subunits, or as a hetero-oligomer of human alpha 1 and rat beta subunits. 2. Complementary RNA-injected oocytes expressing GABAA receptors responded to bath applied GABA with an EC50 of 158 +/- 34 microM. Oocytes expressing alpha 1 and alpha 1 beta glycine receptors subsequent to cDNA injection displayed EC50 values of 76 +/- 2 microM and 66 +/- 2 microM, respectively, in response to bath applied glycine. 3. Picrotoxin antagonized responses mediated by homo-oligomeric alpha 1 glycine receptors with an IC50 of 4.2 +/- 0.8 microM. Hetero-oligomeric alpha 1 beta glycine receptors were at least 100-fold less sensitive to blockade by picrotoxin. 4. With the appropriate agonist EC10, propofol enhanced GABA and glycine-evoked currents to approximately the maximal response produced by a saturating concentration of either agonist (i.e. Imax). The calculated EC50 values were 2.3 +/- 0.2 microM, 16 +/- 3 microM and 27 +/- 2 microM, for GABAA alpha 1 beta 1 gamma 2L, glycine alpha 1 and alpha 1 beta receptors, respectively. At relatively high concentrations, propofol was observed to activate directly both GABAA and glycine receptors. 5. Pentobarbitone potentiated GABA-evoked currents to 117 +/- 8.5% of Imax with an EC50 of 65 +/- 3 microM. The barbiturate also produced a substantial enhancement of the glycine-evoked currents, Imax and EC50 values being 71 +/- 2% and 845 +/- 66 microM and 51 +/- 10% and 757 +/- 30 microM for homomeric alpha 1 and heteromeric alpha 1 beta glycine receptors respectively. At high concentrations, pentobarbitone directly activated GABAA, but not glycine, receptors. 6. The potentiation by propofol or pentobarbitone of currents mediated by alpha 1 homo-oligomeric glycine receptors was in both cases associated with a parallel sinistral shift of the glycine concentration-effect curve. The effects of binary combinations of pentobarbitone and propofol at maximally effective concentrations were mutually occlusive suggesting a common site, or mechanism, of action. 7. GABA-evoked currents were maximally potentiated by etomidate to 79 +/- 2% of Imax (EC50 of 8.1 +/- 0.9 microM). By contrast, glycine-induced currents mediated by alpha 1 and alpha 1 beta glycine receptor isoforms were enhanced only to 29 +/- 4% and 28 +/- 3% of Imax. Limited solubility precluded the calculation of EC50 values for the effect of etomidate at glycine receptors. None of the receptor isoforms examined were directly activated by etomidate. 8. The neurosteroid 5 alpha-pregnan-3 alpha-ol-20-one potentiated GABA-evoked currents to 69 +/- 4% of Imax, with an EC50 value of 89 +/- 6 nM. In contrast, both alpha 1 homo-oligomeric and alpha 1 beta hetero-oligomeric glycine receptors were insensitive to the action of this steroid. A direct agonist action of the steroid was discernible at GABAA, but not glycine, receptors. 9. Trichloroethanol, the active metabolite of the general anaesthetic chloral hydrate, enhanced glycine-evoked currents to 77 +/- 10% and 94 +/- 4% of Imax on alpha 1 and alpha 1 beta glycine receptors, with EC50 values of 3.5 +/- 0.1 mM and 5.9 +/- 0.3 mM respectively. On GABAA receptors, trichloroethanol had a lower maximum enhancement (52 +/- 5% of Imax), but a slightly higher potency (EC50 1.0 +/- 0.1 mM). Trichloroethanol activated neither GABAA, nor glycine, receptors. 10. The data demonstrate a variety of intravenous general anaesthetic agents, at clinically relevant concentrations, to augment preferentially GABA-evoked currents mediated by the alpha1beta1upsilon2L receptor subunit combination as compared to their effects on both alpha1 and alpha1beta glycine receptors. However, the presence on glycine receptors of lower affinity modulatory binding sites for pentobarbitone, propofol and trichloroethanol may aid in the identification of the molecular determinants of the CNS actions of these anaesthetics.

Anesthetics, Intravenous↗
PubMed1997

Characterization of the anticonvulsant properties of ganaxolone (CCD 1042; 3alpha-hydroxy-3beta-methyl-5alpha-pregnan-20-one), a selective, high-affinity, steroid modulator of the gamma-aminobutyric acid(A) receptor.

Ganaxolone (CCD 1042) is a 3beta-methyl-substituted analog of the endogenous neuroactive steroid 3alpha-hydroxy-5alpha-pregnan-20-one. Ganaxolone inhibited binding of the gamma-aminobutyric acid (GABA)A receptor-chloride channel ligand t-[35S]butylbicyclophosphorothionate (IC50 of 80 nM) and enhanced binding of the benzodiazepine site ligand [3H]flunitrazepam (EC50 of 125 nM) and the GABA site ligand [3H]muscimol (EC50 of 86 nM), consistent with activity as a positive allosteric modulator of the GABA(A) receptor. Electrophysiological recordings showed that, whereas nanomolar concentrations of ganaxolone potentiated GABA-evoked chloride currents in Xenopus oocytes expressing the human GABA(A) receptor subunits alpha1beta1gamma2L, alpha2beta1gamma2L or alpha3beta1gamma2L, direct activation of chloride flux occurred to a limited extent only at micromolar concentrations. Ganaxolone was effective in nontoxic doses against clonic convulsions induced by s.c. pentylenetetrazol administration in mice and rats (ED50 values of 4.3 and 7.8 mg/kg i.p., respectively). Ganaxolone also exhibited potent anticonvulsant activity against seizures induced by s.c. bicuculline (ED50 of 4.6 mg/kg i.p.), i.p. TBPS (ED50 of 11.7 mg/kg i.p.) and i.p. aminophylline (ED50 of 11.5 mg/kg i.p.) in mice. Although ganaxolone effectively blocked tonic seizures induced by maximal electroshock in mice (ED50 of 29.7 mg/kg i.p.), it did so only at doses that produced ataxia on the Rotorod (TD50 of 33.4 mg/kg i.p.). Conversely, ganaxolone was a potent anticonvulsant against fully kindled stage 5 seizures induced by corneal kindling in rats (ED50 of 4.5 mg/kg i.p.), producing these effects at doses well below those that resulted in ataxia (TD50 of 14.2 mg/kg i.p.). The seizure threshold, as determined by an increase in the dose of i.v. infused pentylenetetrazol required to induce clonus, was also significantly elevated by nontoxic doses of ganaxolone in mice. In summary, these data indicate that ganaxolone is a high-affinity, stereoselective, positive allosteric modulator of the GABA(A) receptor complex that exhibits potent anticonvulsant activity across a range of animal procedures. The profile of anticonvulsant activity obtained for ganaxolone supports clinical evaluation of this drug as an antiepileptic therapy with potential utility in the treatment of generalized absence seizures as well as simple and complex partial seizures.

Animals↗
PubMed1996

Neurosteroid modulation of native and recombinant GABAA receptors.

1. The pioneering work of Hans Selye over 50 years ago demonstrated that certain steroid metabolites can produce a rapid depression of central nervous system activity. 2. Research during the last 10 years has established that such effects are mediated by a nongenomic and specific interaction of these steroids with the brain's major inhibitory receptor, the GABAA receptor. 3. Here we describe the molecular mechanism of action of such steroids and review attempts to define the steroid binding site on the receptor protein. The therapeutic potential of such neurosteroids is discussed.

Animals↗
PubMed1996

Modulation of human recombinant GABAA receptors by pregnanediols.

Utilising two point voltage-clamp techniques on Xenopus laevis oocytes expressing human (alpha 1 beta 1 gamma 2L) recombinant GABAA receptors, the GABA modulatory actions of six naturally occurring neurosteroids have been determined and compared with those of known positive allosteric modulators. The anaesthetic steroids 5 alpha- and 5 beta-pregnan-3 alpha-ol-20-one produced a concentration-dependent enhancement of the GABA-evoked current. The maximal enhancement of the agonist-induced response produced by these steroids was intermediate between that of pentobarbitone and diazepam, but much greater than that caused by bretazenil. For both the 5 alpha and 5 beta steroid a reduction of the 20 ketone group to form either the corresponding 20 alpha or 20 beta hydroxy steroid produced, in all cases, a reduction in potency and a decrease in the maximal effect. The relationship of steroid structure to these two parameters is considered. The influence of the alpha subtype (alpha x beta 1 gamma 2L, where x = 1, 2 or 3) for the behaviourally active 5 alpha-pregnan-3 alpha,20 alpha-diol is also determined. Although the maximal effect of the steroid is not influenced by the alpha subtype, the alpha 2-containing receptor exhibits a modest decrease (approximately 6-fold) in potency compared to alpha 1- and alpha 3-containing receptors. The results described here are discussed in relation to the distinct behavioural actions of the neurosteroids.

Anesthetics, General↗
PubMed1996

The anaesthetic action and modulation of GABAA receptor activity by the novel water-soluble aminosteroid Org 20599.

The anaesthetic profile of a novel water-soluble aminosteroid, Org 20599 [(2 beta, 3 alpha, 5 alpha)-21-chloro-3-hydroxy-2-(4-morpholinyl)pregnan-20-one methanesulphonate], and the ability of the compound to allosterically regulate the activity of the GABAA receptor, have been studied in comparison to the properties of established intravenous general-anaesthetic agents. Intravenously administered Org 20599 produced a rapid onset, short duration loss of the righting reflex in mice. The anaesthetic potency of Org 20599 was comparable to that of the steroids 5 alpha-pregnan-3 alpha-ol-20-one or alphaxalone, and exceeded that of propofol, thiopentone or pentobarbitone. Org 20599 and the reference anaesthetic agents allosterically displaced the binding of [35S]-t-butylbicyclophosphorothionate (TBPS) from GABAA receptors of rat-brain membranes with the order of potency: 5 alpha-pregnan-3 alpha-ol-20-one > Org 20599 > alphaxalone > propofol > thiopentone > pentobarbitone. At human recombinant alpha 1, beta 2, gamma 2L subunit-containing GABAA receptors expressed in Xenopus laevis oocytes, the anaesthetic agents produced a concentration-dependent and reversible potentiation of the peak amplitude of GABA-evoked currents. A similar positive allosteric action of Org 20599 was observed for the GABAA receptors expressed by bovine adrenal chromaffin cells maintained in culture. The rank order of potency in the aforementioned assays was identical to that determined from the displacement of TBPS binding. At concentrations greater than those required for potentiation of GABA, the anaesthetics exhibited GABA-mimetic effects with a rank order of potency that paralleled their modulatory activity. Such direct agonism varied greatly in maximal effect between compounds. The modulatory and direct agonist actions of Org 20599 were additionally confirmed utilizing rat hippocampal neurones in culture. The results indicate Org 20599 to be a potent and short-acting intravenous anaesthetic agent in mice and suggest positive allosteric regulation of GABAA receptor function to be a plausible molecular mechanism of action for the drug.

Anesthetics↗
PubMed1996

Interaction of positive allosteric modulators with human and Drosophila recombinant GABA receptors expressed in Xenopus laevis oocytes.

1. A comparative study of the actions of structurally diverse allosteric modulators on mammalian (human alpha 3 beta 2 gamma 2L) or invertebrate (Drosophila melanogaster Rdl or a splice variant of Rdl) recombinant GABA receptors has been made using the Xenopus laevis oocyte expression system and the two electrode voltage-clamp technique. 2. Oocytes preinjected with the appropriate cRNAs responded to bath applied GABA with a concentration-dependent inward current. EC50 values of 102 +/- 18 microM; 152 +/- 10 microM and 9.8 +/- 1.7 microM were determined for human alpha 3, beta 1 gamma 2L, Rdl splice variant and the Rdl receptors respectively. 3. Pentobarbitone enhanced GABA-evoked currents mediated by either the mammalian or invertebrate receptors. Utilizing the appropriate GABA EC10, the EC50 for potentiation was estimated to be 45 +/- 1 microM, 312 +/- 8 microM and 837 +/- 25 microM for human alpha 3, beta 1 gamma 2L, Rdl splice variant and Rdl receptors respectively. Maximal enhancement (expressed relative to the current induced by the EC10 concentration of GABA where this latter response = 1) at the mammalian receptor (10.2 +/- 1 fold) was greater that at either the Rdl splice variant (5.5 +/- 1.3 fold) or Rdl (7.9 +/- 0.8 fold) receptors. 4. Pentobarbitone directly activated the human alpha 3 beta 1 gamma 2L receptor with an EC50 of 1.2 +/- 0.03 mM and had a maximal effect amounting to 3.3 +/- 0.4 fold of the response evoked by the EC10 concentration of GABA. Currents evoked by pentobarbitone were blocked by 10-30 microM picrotoxin and potentiated by 0.3 microM flunitrazepam. Pentobarbitone did not directly activate the invertebrate GABA receptors. 5. 5 alpha-Pregnan-3 alpha-ol-20-one potentiated GABA-evoked currents mediated by the human alpha 3 beta 1 gamma 2L receptor with an EC50 of 87 +/- 3 nM and a maximal enhancement of 6.7 +/- 0.8 fold of that produced by the GABA EC10 concentration. By contrast, relatively high concentrations (3-10 microM) of this steroid had only a modest effect on the Rdl receptor and its splice variant. 6. A small direct effect of 5 alpha-pregnan-3 alpha-ol-20-one (0.3-10 microM) was detected for the human alpha 3 beta 1 gamma 2L receptor (maximal effect only 0.08 +/- 0.01 times that of the GABA EC10). This response was antagonized by 30 microM picrotoxin and enhanced by flunitrazepam (0.3 microM). 5 alpha-Pregnan-3 alpha-ol-20-one did not directly activate the invertebrate GABA receptors. 7. Propofol enhanced GABA-evoked currents mediated by human alpha 3 beta 1 gamma 2L and Rdl splice variant receptors with EC50 values of 3.5 +/- 0.1 microM and 8 +/- 0.3 microM respectively. The maximal enhancement was similar at the two receptor types (human 11 +/- 1.8 fold; invertebrate 8.8 +/- 1.4 fold that of the GABA EC10). 8. Propofol directly activated the human alpha 3 beta 1 gamma 2L receptor with an EC50 of 129 +/- 10 microM, and at a maximally effective concentration, evoked a current amounting to 3.5 +/- 0.5 times that elicited by a concentration of GABA producing 10% of the maximal response. The response to propofol was blocked by 10-30 microM picrotoxin and enhanced by flunitrazepam (0.3 microM). Propofol did not directly activate the invertebrate Rdl splice variant receptor. 9. GABA-evoked currents mediated by the human alpha 3 beta 1 gamma 2L receptor were potentiated by etomidate (EC50 = 7.7 +/- 0.2 microM) and maximally enhanced to 8 +/- 0.8 fold of the response to an EC10 concentration of GABA. By contrast, the Rdl, or Rdl splice variant forms of the invertebrate GABA receptor were insensitive to the positive allosteric modulating actions of etomidate. Neither the mammalian nor the invertebrate receptors, were directly activated by etomidate. 10. delta-Hexachlorocyclohexane enhanced GABA-evoked currents with EC50 values of 3.4 +/- 0.1 microM and 3.0 +/- 0.1 microM for the human alpha 3 beta 1 gamma 2L receptor and the Rdl splice variant receptor respectively. The maximal enhancement was 4.5

Allosteric Regulation↗
PubMed1996

Characterization of a human 5-hydroxytryptamine3 receptor type A (h5-HT3R-AS) subunit stably expressed in HEK 293 cells.

1. A cloned cDNA encoding a human 5-hydroxytryptamine3 receptor type A subunit (h5-HT3R-As) was transfected into human embryonic kidney (HEK 293) cells maintained in cell culture and a stable cell line expressing a high density of the recombinant receptor was selected. 2. Membrane homogenates prepared from transfected, but not untransfected, cells exhibited a homogeneous and saturable population (Bmax = 4.49 +/- 0.46 pmol mg-1 protein) of sites that bound the radiolabelled 5-HT3 receptor antagonist, [3H]-granisetron with high affinity (pKD = 8.87 +/- 0.08). Kinetic studies (at 37 degrees C) revealed rapid association (kappa +1 4.76 +/- 0.3 x 10(8) M-1 min-1) and dissociation (kappa -1 = 0.21 +/- 0.003 min-1) of the radioligand. 3. Selective and non-selective 5-HT3 receptor ligands competed for [3H]-granisetron binding with a rank order of potency (granisetron > ondansetron > meta-chlorophenylbiguanide > 5-HT > 2-methyl-5-HT > metoclopramide > > phenylbiguanide > cocaine > (+)-tubocurarine) identical to that established for 5-HT3 receptors endogenous to the human CNS. 4. In electrophysiological recordings performed on transfected cells, voltage-clamped at a holding potential of -60 mV, locally applied 5-HT (10 microM) evoked transient inward current responses that reversed in sign at a potential of -1.0 +/- 1.1 mV. Such responses were antagonized in a reversible manner by granisetron (1 nM). 5. The construction of a stable cell line expressing a high density of recombinant human 5-HT3 receptors which display appropriate pharmacology and function will assist in the further characterization of this receptor subtype and the exploration of species differences in 5-HT3 receptor pharmacology.

Binding, Competitive↗
PubMed1996

Ligand-gated ion channels opened by 5-HT in molluscan neurones.

1. 5-Hydroxytryptamine (5-HT) activated a fast (70 ms to half maximum) and desensitizing inward current through non-selective channels conducting predominantly monovalent cations in neurons of Helix aspersa. 2. alpha-Methyl-5-HT was equipotent with 5-HT in activating this current, but the known selective agonists at vertebrate 5-HT3 receptors, 2-methyl-5-HT and arylbiguanides were ineffective (< 100 microM). 5-Methoxytryptamine which is inactive on vertebrate 5-HT3 receptors was a very weak agonist. 3. The responses were antagonized by the specific vertebrate 5-HT3 receptor blocker MDL-72222 (IC50 = 1 microM), but were only weakly affected by ondansetron (10 microM). The 5-HT2-type antagonist, ketanserin (< 5 microM) had no effect. The responses were also antagonized by the non-specific antagonists (+)-tubocurarine and strychnine. 4. Unitary currents through channels non-selective for monovalent cations, and with a conductance of 2pS, could be activated repeatedly by 5-HT or alpha-methyl-5-HT in outside-out patches from neurones exhibiting the fast 5-HT-activated current (I[5-HT]fast), even in the presence of 500 microM GDP-[beta S] in the recording pipette. This strongly supports direct-gating of these channels by 5-HT. The properties of these unitary currents resembled those of I[5-HT]fast. 5. The pharmacological properties of this molluscan 5-HT-operated, ligand-gated channel differed sufficiently from known vertebrate 5-HT3-type receptors to suggest that it represents a new class of 5-HT receptor.

Animals↗
PubMed1995

A patch clamp study of the effects of ciprofloxacin and biphenyl acetic acid on rat hippocampal neurone GABAA and ionotropic glutamate receptors.

The neurotoxic effects of 4-quinolones alone and in combination with certain non-steroidal anti-inflammatory drugs (NSAIDs) may be related to an interaction at GABAA and/or ionotropic glutamate receptors. In the present study, the effects of the fluoroquinolone, ciprofloxacin, alone and in combination with the NSAID, biphenyl acetic acid (BPAA), were examined on GABAA-, NMDA-, AMPA-, and kainate-evoked current responses recorded from cultured rat hippocampal neurones, using the whole cell patch clamp technique. GABA-evoked currents were reversibly inhibited by bicuculline (3 microM) and ciprofloxacin (100 microM) to 11 +/- 5 and 38 +/- 7% of control, respectively. BPAA (100 microM) had little affect on the GABA current (the response was 82 +/- 4% of control) but enhanced the inhibitory potency of ciprofloxacin by approx. 3000-fold. The antagonist effects of ciprofloxacin (30 microM) and ciprofloxacin (0.03 microM) together with BPAA (100 microM) on the GABA-evoked current were not voltage-dependent. Whole cell currents evoked by NMDA, AMPA or kainate were little influenced by ciprofloxacin (100 microM), BPAA (100 microM), or ciprofloxacin plus BPAA (both at 100 microM); the responses being > or = 90% of control in all cases. These data suggest that the proconvulsant effects of quinolones when combined with BPAA may be related to antagonism of central GABAA receptors but not to an interaction at ionotropic glutamate receptors.

Animals↗
PubMed1995

Neurosteroids and GABAA receptor function.

In 1984, a potent and selective interaction of the steroidal anaesthetic alphaxalone with the GABAA receptor was demonstrated. Subsequent studies established that certain naturally occurring steroids were potent positive allosteric modulators of the GABAA receptor. Although peripheral endocrine glands are an important endogenous source, the brain can synthesize 'neurosteroids', and these have the potential to influence the activity of the GABAA receptor in the CNS. Systemic administration of steroids have clear behavioural effects. In this article, Jeremy Lambert and colleagues review recent advances in this field and discuss the therapeutic potential of this novel, non-genomic effect of steroids and investigate whether they may influence behaviour under physiological, or pathophysiological, conditions.

Anesthetics↗