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.

Drug glucuronidation by human renal UDP-glucuronosyltransferases.

The UDP-glucuronosyltransferases catalyse the conjugation of glucuronic acid to a wide variety of endobiotics and xenobiotics, representing one of the major conjugation reactions in the conversion of both exogenous (e.g. drugs and pesticides) and endogenous compounds (e.g. bilirubin and steroid hormones). The liver is the major site of glucuronidation, however a number of extrahepatic tissues exhibit particular UDP-glucuronosyltransferase activities. The present study was undertaken to assess the human renal UDP-glucuronosyltransferase system. Enzymatic analysis of human kidney showed that a limited number of UDP-glucuronosyltransferase isoforms were expressed in this tissue. However the level of renal UGT activity towards the anaesthetic propofol was higher compared with human liver. The glucuronidation of propofol is catalysed by UGT1A8/9 suggesting higher levels of this isoform in the kidney. Immunoblot analysis revealed two major UDP-glucuronosyltransferase immunopositive bands to be present in human kidney as compared to four major bands in human liver. The human kidney was capable of conjugating various structurally diverse drugs and xenobiotics.

Reactivity of mefenamic acid 1-o-acyl glucuronide with proteins in vitro and ex vivo.

Mefenamic acid is a nonsteroidal anti-inflammatory drug commonly used in analgesia. The use of this drug has been implicated in several cases of nephrotoxicity including acute renal failure and tubulointerstitial nephritis. One theory of drug-induced tubulointerstitial nephritis is that the drug or a derivative of the drug becomes irreversibly bound to certain sites in renal tissue and an immune response is directed against the hapten-host conjugate. Previous studies have shown that in humans the nonsteroidal anti-inflammatory drug mefenamic acid is metabolized by both phase I enzymes and the phase II enzyme family UDP-glucuronosyltransferase. Indeed, three glucuronides were identified and isolated from human urine by semipreparative HPLC after oral administration of mefenamic acid. This study focuses on mefenamic acid glucuronide and further characterizes this acyl glucuronide in terms of stability and its ability to bind irreversibly to proteins. Stability studies of mefenamic acid glucuronide in aqueous buffer highlighted the relative stability of this acyl glucuronide at physiological pH. The half-life at 37 degrees C, pH 7.4, was 16.5 +/- 3.1 hr, which is considerably longer than those reported for many acyl glucuronides. The degradation of mefenamic acid glucuronide was accelerated under alkaline conditions, decreasing the half-life to 5 +/- 1.6 hr at pH 8.0. Mefenamic acid glucuronide, although extremely stable in buffer at physiological pH, was found to bind irreversibly to human serum albumin in vitro. Irreversible binding to cellular proteins in culture was also evident with the addition of mefenamic acid to the heterologous Chinese hamster lung fibroblast cell line V79 expressing the human UDP-glucuronosyltransferase isoenzyme UGT1*02. This binding was directly related to glucuronide formation, because irreversible binding was not evident in the untransfected cell line V79.