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CELLULAR AND MOLECULAR

Compounds Exhibiting Selective Efficacy for Different Subunits of Human Recombinant -Aminobutyric Acid_A Receptors

Sharp and Dohme Research Laboratories, Merck, Neuroscience Research Centre, Terlings Park, Harlow, Essex, United Kingdom

Inhibitory GABA_A receptor modulators are widely used therapeutic agents for a variety of central nervous system disorders. Ltk^– cells stably expressing human recombinant GABA_A subunits (11–32s) were seeded into 96-well plates, loaded with chlorocoumarin-2-dimyristoyl phosphatidylethanolamine and bis(1,3-diethyl-2-thiobarbiturate)trimethineoxonol, and rapid fluorescence resonance energy transfer technique (FRET) measurements were made of GABA-evoked depolarizations in low-Cl^– buffer using a voltage/ion probe reader. The influence of different subunits on the ability of agents to modulate and directly activate the ion channel was examined. GABA evoked concentration-dependent decreases in FRET, increasing fluorescence emission ratio (460/580 nm) at 112, 122, and 132 receptors with similar maximal amplitude (P > 0.05, n = 17) and EC₅₀ values of 2.4 ± 0.2, 2.5 ± 0.2, and 1.3 ± 0.1 µM, respectively. Piperidine-4-sulfonic acid and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol were less potent, with EC₅₀ values of 8.7 ± 0.9, 9.2 ± 0.5, and 11.7 ± 1.2, and 43.7 ± 6.4, 24.8 ± 1.6, and 26.1 ± 2.4 µM, respectively. Potency and maximal efficacy of propofol, methyl 6,7-dimethoxy-4-ethyl--carboline-3-carboxylate, pentobarbital, and steroids, 5-pregnan-3-ol-20-one and 5-pregnan-3-ol-20-one, were unaffected by the isoform present in the receptor complex. However, several compounds displayed 2/3 subunit selectivity, notably loreclezole, R(–)-etomidate, and a group of anti-inflammatory agents including mefenamic acid, flufenamic acid, meclofenamic acid, tolfenamic acid, niflumic acid, and diflunisal. The anti-inflammatories exhibited varying levels of efficacy at 2/3 subunits, with micromolar potency, while having antagonist or weak inverse agonist profiles at 112. Diflunisal was the most efficacious compound, eliciting greater potentiation than loreclezole (90 ± 14% and 109 ± 14% at 3 and 2, respectively, compared with 62 ± 6% and 56 ± 3%), whereas niflumic acid exhibited the lowest efficacy. An additional agent, olsalazine, weakly potentiated responses at all three receptors without any selectivity. This study identifies and characterizes a variety of allosteric modulators for which subunits are an important determinant of efficacy and potency.

Address correspondence to: Alison J. Smith, Sharp and Dohme Research Laboratories, Merck, Neuroscience Research Centre, Terlings Park, Eastwick Road, Harlow, Essex CM20 2QR, UK. E-mail: alison_smith{at}merck.com.

This article has been cited by other articles:

				R. W. Olsen and W. Sieghart International Union of Pharmacology. LXX. Subtypes of {gamma}-Aminobutyric AcidA Receptors: Classification on the Basis of Subunit Composition, Pharmacology, and Function. Update Pharmacol. Rev., September 1, 2008; 60(3): 243 - 260. [Abstract] [Full Text] [PDF]