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D Belelli

Publications and source records attributed to D Belelli.

At least 19 recordsLinked to original sources
PubMed2005

Neuroactive steroids and inhibitory neurotransmission: mechanisms of action and physiological relevance.

Dysfunction of GABA(A) receptor-mediated inhibition is implicated in a number of neurological and psychiatric conditions including epilepsy and affective disorders. Some of these conditions have been associated with abnormal levels of certain endogenously occurring neurosteroids, which potently and selectively enhance the function of the brain's major inhibitory receptor, the GABA(A) receptor. Consistent with their ability to enhance neuronal inhibition, such steroids exhibit in animals and humans anxiolytic, anticonvulsant and anesthetic actions. Neurosteroids, exemplified by the potent progesterone metabolite, 5alpha-pregnan-3alpha-ol-20-one can be synthesized de novo in the CNS both in neurones and glia in levels sufficient to modulate GABA(A) receptor function. Neurosteroid levels are not static, but are subject to dynamic fluctuations, for example during stress, or the later stages of pregnancy. These observations suggest that these endogenous modulators may refine the function of the brain's major inhibitory receptor and thus, play an important physiological and pathophysiological role. However, given the ubiquitous expression of GABA(A) receptors throughout the mammalian CNS, changes in neurosteroid levels should be widely experienced, causing a generalized enhancement of neuronal inhibition. Such a non-specific action would seem incompatible with a physiological role. However, neurosteroid action is both brain region and neurone selective. This specificity results from a variety of molecular mechanisms including receptor subunit composition, local steroid metabolism and phosphorylation. This paper will evaluate the relative contribution these mechanisms play in defining the interaction of neurosteroids with synaptic and extra-synaptic GABA(A) receptors.

Animals↗
PubMed2004

The interaction of anaesthetic steroids with recombinant glycine and GABAA receptors.

BACKGROUND: Anaesthetic steroids are established positive allosteric modulators of GABAA receptors, but little is known concerning steroid modulation of strychnine-sensitive glycine receptors, the principal mediators of fast, inhibitory neurotransmission in the brain stem and spinal cord. This study compared the modulatory actions of five anaesthetic pregnane steroids and two non-anaesthetic isomers at human recombinant alpha1 glycine and alpha1beta2gamma2L GABAA receptors. METHODS: Recombinant alpha1 glycine or alpha1beta2gamma2L GABAA receptors were expressed in Xenopus laevis oocytes and agonist-evoked currents recorded under voltage-clamp. Steroid modulation of currents evoked by GABA, or glycine, was quantified by determining the potency (EC50) and maximal effect of the compounds. RESULTS: The anaesthetics minaxolone (EC50=1.3 microM), Org20599 (EC50=1.1 microM) and alphaxalone (EC50=2.2 microM) enhanced currents mediated by GABAA receptors. The anaesthetics also enhanced currents mediated by glycine receptors, although with higher EC50 values (minaxolone 13.1 microM; Org20599=22.9 microM and alphaxalone=27.8 microM). The maximal enhancement (to 780-950% of control) produced by the three steroids acting at the GABAA receptor was similar, but currents evoked by glycine were potentiated with increasing effectiveness by alphaxalone (199%) <Org20599 (525%) <minaxolone (1197%). The anaesthetic isomers, 5alpha-pregnan-3alpha-ol-20-one and 5beta-pregnan-3alpha-ol-20-one (eltanolone) enhanced GABAA receptor-mediated currents with similar potency and efficacy, but only the former enhanced glycine, the latter causing inhibition. The non-anaesthetic steroids 5alpha-pregnan-3beta-ol-20-one and 5beta-pregnan-3beta-ol-20-one modulated neither GABAA, nor glycine, receptors. CONCLUSIONS: The data demonstrate that structure-activity relationships for steroid modulation at glycine and GABAA receptors differ. Comparing the EC50 values reported here with free plasma concentrations during steroid-induced anaesthesia indicates that a selective modulation of GABAA receptor activity is likely to occur in vivo.

Anesthetics↗
PubMed2003

GABAA receptor modulation by the novel intravenous general anaesthetic E-6375.

E-6375 (4-butoxy-2-[4-(2-cyanobenzoyl)-1-piperazinyl] pyrimidine hydrochloride) is a new intravenous general anaesthetic with an anaesthetic potency, in mice, comparable to propofol, or etomidate. Here, we examined the effect of E-6375 upon the GABAA receptor, a putative target of intravenous anaesthetic action. E-6375 reversibly enhanced GABA-evoked currents mediated by recombinant GABAA (alpha1beta2gamma2L) receptors expressed in Xenopus laevis oocytes, with little effect on NMDA- and kainate-evoked currents mediated by NR1a/NR2A and GluR1o/GluR2o glutamate receptors, respectively. E-6375 prolonged the decay of GABA-evoked miniature inhibitory postsynaptic currents recorded from rat Purkinje neurones demonstrating the anaesthetic also enhanced the activity of synaptic GABAA receptors. The GABA enhancing action of E-6375 on recombinant GABAA receptors was unaffected by the subtype of the alpha isoform (i.e. alphaxbeta2gamma2L; x=1-3) within the receptor, but was increased by the omission of the gamma2L subunit. Receptors incorporating beta2, or beta3, subunits were more sensitive to modulation by E-6375 than those containing the beta1 subunit. The selectivity of E-6375 was largely governed by the identity (serine or asparagine) of a single amino acid residue within the second transmembrane domain of the beta-subunit. The various in vivo actions of general anaesthetics may be mediated by GABAA receptor isoforms that have a differential distribution within the CNS. The identification of agents, such as E-6375, that discriminate between GABAA receptor subtypes may augur the development of general anaesthetics with an improved therapeutic profile.

Amino Acid Sequence↗
PubMed2001

Alpha-amino acid phenolic ester derivatives: novel water-soluble general anesthetic agents which allosterically modulate GABA(A) receptors.

In the search for a novel water-soluble general anesthetic agent the activity of an alpha-amino acid phenolic ester lead, identified from patent literature, was markedly improved. In addition to improving in vivo activity in mice, good in vitro activity at GABA(A) receptors was also conferred. Within the series of compounds good enantioselectivity for both in vitro and in vivo activity was found, supporting a protein-mediated mechanism of action for anesthesia involving allosteric modulation of GABA(A) receptors. alpha-Amino acid phenolic ester 19, as the hydrobromide salt Org 25435, was selected for clinical evaluation since it retained the best overall anesthetic profile coupled with improved stability and water solubility. In the clinic it proved to be an effective intravenous anesthetic in man with rapid onset of and recovery from anesthesia at doses of 3 and 4 mg/kg.

Allosteric Regulation↗
PubMed2001

Neurosteroid modulation of recombinant and synaptic GABAA receptors.

Certain pregnane steroids are now established as potent, positive allosteric modulators of the gamma-aminobutyric acid type A (GABAA) receptor. These compounds are known to be synthesized in the periphery by endocrine glands, such as the ovaries and the adrenal glands, and can rapidly cross the blood-brain barrier. Therefore, such steroids could act as endogeneous modulators of the major inhibitory receptor in the mammalian central nervous system. However, the demonstration that certain neurons and glia can synthesize the pregnane steroids (i.e., neurosteroids) additionally suggests that they may serve a paracrine role by influencing GABAA-receptor function through their local release in the brain itself. Here, we demonstrate that these neurosteroids are highly selective and extremely potent modulators of the GABAA receptor. The subunit composition of the GABAA receptor may influence the actions of the neurosteroids, particularly when considering concentrations of these agents thought to occur physiologically, which may underlie their reported differential effects at certain inhibitory synapses. However, recent work suggests that the phosphorylation status of either the synaptic GABAA receptor or its associated proteins may also influence neurosteroid sensitivity; these findings are discussed. Upon administration, the neurosteroids exhibit clear behavioral effects, including sedation, anticonvulsant actions, and behaviors predictive of anxiolysis; when given at high doses, they induce general anesthesia. Numerous synthetic steroids have been synthesized in an attempt to therapeutically exploit these properties, and these data are reviewed in this chapter. However, targeting the brain enzymes that synthesize and metabolize the neurosteroids may offer a new approach to exploit this novel endocrine-paracrine neurotransmitter interaction.

Animals↗
PubMed2001

Modulation of native and recombinant GABA(A) receptors by endogenous and synthetic neuroactive steroids.

Upon administration, certain pregnane steroids produce clear behavioural effects including, anxiolysis, sedation, analgesia, anaesthesia and are anti-convulsant. This behavioural profile is characteristic of compounds that act to enhance the actions of GABA acting at the GABA(A) receptor. In agreement, numerous studies have now demonstrated these steroids to be potent, positive allosteric modulators of the GABA(A) receptor. The pregnane steroids are synthesized in the periphery by endocrine glands such as the adrenals and the ovaries, but are also made by neurons and glial cells in the central nervous system itself. Hence, these compounds could play both an endocrine and a paracrine role to influence neuronal excitability by promoting inhibition. Here we review evidence that the pregnane steroids are highly selective and extremely potent GABA(A) receptor modulators and that their effects at 'physiological' concentrations (low nanomolar) may be influenced by the subunit composition of the GABA(A) receptor. This feature may underlie recent findings demonstrating the effects of the neurosteroids on inhibitory synaptic transmission to be brain region dependent, although recent reports suggest that phosphorylation mechanisms may additionally influence neurosteroid sensitivity of the GABA(A) receptor. Numerous synthetic steroids have been synthesized in an attempt to therapeutically exploit the behavioural effects of the pregnane steroids and progress with this approach will be discussed. However, the demonstration that the steroids may be made within the central nervous system offers the alternative strategy of targeting the enzymes that synthesize/metabolise the neurosteroids to exploit this novel endocrine/paracrine interaction.

Animals↗
PubMed1999

Complementary regulation of anaesthetic activation of human (alpha6beta3gamma2L) and Drosophila (RDL) GABA receptors by a single amino acid residue.

1. The influence of a transmembrane (TM2) amino acid located at a homologous position in human beta1 (S290) and beta3 (N289) GABAA receptor subunits and the RDL GABA receptor of Drosophila (M314) upon allosteric regulation by general anaesthetics has been investigated. 2. GABA-evoked currents mediated by human wild-type (WT) alpha6beta3gamma2L or WT RDL GABA receptors expressed in Xenopus laevis oocytes were augmented by propofol or pentobarbitone. High concentrations of either anaesthetic directly activated alpha6beta3gamma2L, but not RDL, receptors. 3. GABA-evoked currents mediated by human mutant GABAA receptors expressing the RDL methionine residue (i.e. alpha6beta3N289Mgamma2L) were potentiated by propofol or pentobarbitone with approximately 2-fold reduced potency and, in the case of propofol, reduced maximal effect. Conspicuously, the mutant receptor was refractory to activation by either propofol or pentobarbitone. 4. Incorporation of the homologous GABAA beta1-subunit residue in the RDL receptor (i.e. RDLM314S) increased the potency, but not the maximal effect, of GABA potentiation by either propofol or pentobarbitone. Strikingly, either anaesthetic now activated the receptor, an effect confirmed for propofol utilizing expression of WT or mutant RDL subunits in Schnieder S2 cells. At RDL receptors expressing the homologous beta3-subunit residue (i.e. RDLM314N) the actions of propofol were similarly affected, whereas those of pentobarbitone were unaltered. 5. The results indicate that the identity of a homologous amino acid affects, in a complementary manner, the direct activation of human (alpha6beta3gamma2L) and RDL GABA receptors by structurally distinct general anaesthetics. Whether the crucial residue acts as a regulator of signal transduction or as a component of an anaesthetic binding site per se is discussed.

Amino Acids↗
PubMed1999

General anaesthetic action at transmitter-gated inhibitory amino acid receptors.

Research within the past decade has provided compelling evidence that anaesthetics can act directly as allosteric modulators of transmitter-gated ion channels. Recent comparative studies of the effects of general anaesthetics across a structurally homologous family of inhibitory amino acid receptors that includes mammalian GABAA, glycine and Drosophila RDL GABA receptors have provided new insights into the structural basis of anaesthetic action at transmitter-gated channels. In this article, the differential effects of general anaesthetics across inhibitory amino acid receptors and the potential relevance of such actions to general anaesthesia will be discussed.

Allosteric Regulation↗
PubMed1999

The interaction of general anaesthetics and neurosteroids with GABA(A) and glycine receptors.

The positive allosteric effects of four structurally distinct general anaesthetics (propofol, pentobarbitone, etomidate and 5alpha-pregnan-3alpha-ol-20-one [5alpha3alpha]) upon recombinant GABA(A) (alpha6beta3gamma2L), invertebrate GABA (RDL) and glycine (alpha1) receptors expressed in Xenopus laevis oocytes have been determined. Propofol and pentobarbitone enhanced agonist (GABA or glycine as appropriate) evoked currents at GABA(A), glycine, and RDL receptors, whereas etomidate and 5alpha3alpha were highly selective for the GABA(A) receptor. Utilizing site-directed mutagenesis, we demonstrate that the nature of the interaction of propofol, pentobarbitone and etomidate (but not 5alpha3alpha) with mammalian and invertebrate ionotropic GABA receptors depends critically upon the nature of a single amino acid located in the second transmembrane region (TM2) of these receptors. These data are discussed in relation to the specificity of action of general anaesthetics.

Anesthetics↗
PubMed1999

A single amino acid confers barbiturate sensitivity upon the GABA rho 1 receptor.

Many structurally diverse general anaesthetics enhance inhibitory neurotransmission in the central nervous system by interacting with the GABAA receptor. By contrast, GABA receptors composed of the rho 1 subunit are anaesthetic-insensitive. Here, we demonstrate that both delta-hexachlorocyclohexane (delta-HCH; 1-100 microM), a positive allosteric modulator of the GABAA receptor, and the anaesthetic pentobarbitone (10-600 microM) have no effect on GABA-evoked currents mediated by wild-type rho 1 recombinant receptors (expressed in Xenopus laevis oocytes). By contrast, these agents produce up to a 10 fold enhancement of GABA responses transduced by a rho 1 receptor in which a transmembrane located isoleucine residue is replaced by serine. However, not all general anaesthetics were similarly influenced by this mutation, because propofol and 5 beta-pregnan-3 alpha-ol-20-one (5 beta 3 alpha) remained ineffective. These data are discussed in relation to the specificity of general anaesthetic action.

Anesthetics, General↗
PubMed1999

Molecular determinants of (+)-tubocurarine binding at recombinant 5-hydroxytryptamine3A receptor subunits.

The 5-hydroxytryptamine type 3 (5-HT3) receptor is a transmitter-gated ion channel mediating neuronal excitation. The receptor native to neurons, or as a homopentameric assembly of 5-HT3A receptor subunits, displays a species-dependent pharmacology exemplified by a 1800-fold difference in the potency of (+)-tubocurarine [(+)-Tc] as an antagonist of the current response mediated by mouse and human receptor orthologs. Here, we attempt to identify amino acid residues involved in binding (+)-Tc by use of chimeric and mutant 5-HT3A subunits of mouse and human expressed in Xenopus laevis oocytes. Replacement of the entire extracellular N-terminal domain of the mouse 5-HT3A (m5-HT3A) subunit by that of the human ortholog and vice versa exchanged the differential potency of (+)-Tc, demonstrating the ligand binding site to be contained wholly within this region. Mutagenesis of multiple amino acid residues within a putative binding domain that exchanged nonconserved residues between mouse and human receptors shifted the apparent affinity of (+)-Tc in a reciprocal manner. The magnitude of the shift increased with the number of residues (3, 5, or 7) exchanged, with septuple mutations of m5-HT3A and human 5-HT3A subunits producing a 161-fold decrease and 53-fold increase in the apparent affinity of (+)-Tc, respectively. The effect of point mutations was generally modest, the exception being m5-HT3A D206E, which produced a 9-fold decrease in apparent affinity. We conclude that multiple amino acids within a binding loop of human and mouse 5-HT3A subunits influence the potency of (+)-Tc.

Amino Acid Sequence↗
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↗
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

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↗