ACCELERATED COMMUNICATION Molecular Targets for the Myorelaxant Action of Diazepam

Diazepam is used clinically for its myorelaxant, anxiolytic, sedative, and anticonvulsant properties. Although the anxiolytic action is mediated by a2 g-aminobutyric acid A (GABAA) receptors, the sedative action and in part the anticonvulsant action are mediated by a1 GABAA receptors. To identify the GABAA receptor subtypes mediating the action of diazepam on muscle tone, we have assessed the myorelaxant properties of diazepam in a2(H101R) and a3(H126R) knock-in mice harboring diazepam-insensitive a2 or a3 GABAA receptors, respectively. Whereas in a2(H101R) mice the myorelaxant action of diazepam was almost completely abolished at doses up to 10 mg/kg, the same dose induced myorelaxation in both wild-type and a3(H126R) mice. It was only at a very high dose (30 mg/kg diazepam) that a2(H101R) mice showed partial myorelaxation and a3(H126R) mice were partially protected from myorelaxation compared with wild-type mice. Thus, the myorelaxant activity of diazepam seems to be mediated primarily by a2 GABAA receptors and at high concentrations also by a3 GABAA receptors. Classical benzodiazepines are in wide clinical use as hypnotics, tranquilizers, muscle relaxants, and anticonvulsants. These effects are caused exclusively by their interaction with the benzodiazepine site of GABAA receptors. Based on the presence of more than a dozen subunit genes, the central nervous system contains a plethora of structurally diverse GABAA receptors (Fritschy and Mohler, 1995; McKernan and Whiting, 1995; Barnard et al., 1998). The vast majority of GABAA receptors are benzodiazepine-sensitive and can be grouped into 4 classes characterized by the type of a subunit being either a1, a2, a3 or a5. Diazepam and related classical benzodiazepines interact with equal affinity with all benzodiazepine-sensitive GABAA receptors (Benke et al., 1996; Costa and Guidotti, 1996). Recently, a promising strategy was developed to assign particular pharmacological effects of benzodiazepines to a specific GABAA receptor subtype. This approach is based on a mutation-induced molecular switch by which the respective GABAA receptor is rendered benzodiazepine-insensitive, as originally shown on recombinant receptors. When a conserved histidine residue in the benzodiazepine binding site of the respective a subunit is replaced by an arginine residue [a1(H101R); a2(H101R); a3(H126R); a5(H105R)], the respective receptor is insensitive to diazepam but remains responsive to GABA (Wieland et al., 1992; Kleingoor et al., 1993; Benson et al., 1998). This molecular switch has recently been introduced into GABAA receptors in vivo. A mutant mouse line was generated with a knock-in point mutation [a1(H101R)] in which those benzodiazepine effects mediated via a1 GABAA receptors were expected to be blunted (Rudolph et al., 1999). The behavioral analysis of this mutant mouse line demonstrated that the sedative, amnesic, and part of the anticonvulsant effects of diazepam are mediated by a1 GABAA receptors. In contrast, the anxiolytic and myorelaxant effects of diazepam were unaltered in the a1 (H101R) mice compared with wild-type mice, suggesting that these effects are mediated by other GABAA receptor subtypes (Rudolph et al., 1999). To assign the contribution of a2 and a3 GABAA receptors to the pharmacological spectrum of benzodiazepines, two further mouse lines were recently generated that contain the a2(H101R) and a3 (H126R) point mutations, respectively (Löw et al., 2000). The a2 GABAA receptor is mainly exThis work was supported by a grant from the Swiss National Science Foundation. F.C., K.L., and R.K. contributed equally to this work. 1 Present address: Department of Neurosciences, University of California, San Diego, La Jolla, California. 2 Present address: Department of Neurology, Vestibulo-Ocular Laboratory, University Hospital, Zürich, Switzerland. ABBREVIATIONS: GABA, g-aminobutyric acid. 0026-895X/01/5903-442–445$3.00 MOLECULAR PHARMACOLOGY Vol. 59, No. 3 Copyright © 2001 The American Society for Pharmacology and Experimental Therapeutics 585/889670 Mol Pharmacol 59:442–445, 2001 Printed in U.S.A. 442 at A PE T Jornals on M ay 5, 2017 m oharm .aspeurnals.org D ow nladed from pressed in the limbic system, whereas a3 GABAA receptors are prominent in neurons of the reticular activating system of the brainstem. A detailed biochemical, autoradiographical, and immunohistochemical analysis demonstrated that the distribution and cellular location of the point-mutated receptors correspond to those of wild-type mice. However, their affinity for diazepam was reduced by a factor of at least 1000. An initial pharmacological analysis showed that the anxiolytic-like effect of diazepam is specifically mediated via a2 GABAA receptors but not by a3 GABAA receptors (Löw et al., 2000). In the present investigation, an attempt is made to attribute the myorelaxant action of diazepam to a2 or a3 GABAA receptors by comparing the diazepam-induced changes in muscle tone in the a2 and a3 mutant mouse strains compared with wild-type. The muscle tone was assessed in the horizontal wire test, in which the ability of the animals to grasp and hang on to a wire is measured. As a control that is independent of the benzodiazepine site, the myorelaxant activity of the GABAB agonist baclofen was tested. The myorelaxant activity was differentiated from the diazepam-induced sedation by including measurements of the spontaneous locomotor activity of the mutant and wild-