Desensitization of a g-Aminobutyric Acid Type A Receptor in Rat Is Increased by Chronic Treatment with Chlordiazepoxide: A Molecular Mechanism of Dependence

When rats were made tolerant to the benzodiazepine tranquilizer chlordiazepoxide (CDPX) by its steady administration, a particular g-aminobutyric acid type A (GABAA) receptor in cerebral cortex was modified. Its rate of desensitization in the absence of CDPX was enhanced (3-fold with 10 mM GABA) below saturation with GABA, and the dependence of this rate on GABA concentration was changed from sigmoid to hyperbolic. This mimicked the effect of the presence of CDPX on desensitization of the naive receptor. This receptor has been characterized by its rapid desensitization (t1⁄2 5 30 msec at saturation). In contrast, a different, slower desensitizing GABAA receptor, on the same membrane, was unaffected, and the initial transmembrane halide exchange rate of the faster desensitizing receptor was unaltered. In the presence of CDPX, the initial halide exchange rate of the modified receptor was enhanced, but the already enhanced desensitization rate was not altered. During chronic presence of CDPX and the development of tolerance, the total signal due to this receptor remained constant at the value before exposure. After discontinuation, the total signal decreased but could be restored to the original value by the presence of CDPX. It was postulated that dependence and withdrawal syndromes result from a decreased ratio of initial chloride flux rate to desensitization rate, caused by an increase in desensitization. The contribution of this effect in vivo would depend on desensitization making a contribution to signal termination [or the fraction of receptors that are inactive (desensitized)]. In the quench flow experiments, the total signal due to this receptor from naive rat did not depend much on GABA concentration or the presence of CDPX because the result of increased channel opening was counterbalanced by increased desensitization. In contrast, the total signal of this receptor from tolerant rat was significantly increased by CDPX or increased GABA concentration. Differences between these experiments and measurements reported with other drugs could be explained if, in those experiments, the halide exchange rate, as well as its desensitization rate, retained an enhanced value in the absence of the drug. Benzodiazepine and its derivatives are widely used as anxiolytics (Haefely et al., 1990; Leonard, 1993; Olsen et al., 1991; Ticku, 1990). Their pharmacological action is primarily due to their effect on GABAA receptors (Barnard et al., 1992; DeLorey and Olsen, 1992; Kardos, 1993; Macdonald and Olsen, 1994; Tobin et al., 1991). These receptors respond to the neurotransmitter GABA by opening transmembrane channels for chloride and, much more slowly, by losing the ability to form open channels (desensitization). Channel opening increases the membrane permeability to chloride, increasing the contribution of chloride channels to the control of membrane potential, thereby hyperpolarizing a cell and making a neuron less excitable. But the role of desensitization is not established, although it is phylogenetically conserved in many channel-forming receptors. Benzodiazepines and related drugs enhance both channel opening and desensitization. For example, the tranquilizer CDPX enhances transmembrane conductance (Choi et al., 1977; Macdonald and Barker, 1978), permeability to chloride (Serf özö and Cash, 1992) and receptor desensitization (Cash and Serf özö, 1995; Farrant et al., 1990; Mierlak and Farb, 1988). Benzodiazepine drugs can give rise to dependency and withdrawal effects in humans and animals. During, and shortly after, continuous presence of the drug, there is a decreased effect of a given dose (tolerance), and after discontinuation, there are behavioral withdrawal symptoms (dependence) (Auta et al., 1994; Byrnes et al., 1993; Grimm and Hershkowitz, 1981; Little et al., 1987; Miller et al., 1988; Stephens and Schneider, 1985; Wilson and Gallager, 1988). Received for publication January 22, 1997. 1 This work was supported in part by a grant from the Research Council of the University of Missouri Medical School, the Missouri Agricultural Experiment Station (BCHB0307) (D.J.C.) and a United States Public Health Service Grant (AA08219) (A.M.A.). P.S. held a Missouri Institute of Psychiatry Fellowship. ABBREVIATIONS: GABA, g-aminobutyric acid; CDPX, chlordiazepoxide; HEPES, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid;. 0022-3565/97/2832-0704$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 283, No. 2 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 283:704–711, 1997 704 at A PE T Jornals on Sptem er 3, 2017 jpet.asjournals.org D ow nladed from Chronic administration of these drugs causes neurochemical as well as behavioral changes (Gallager and Primus, 1993; Gallager and Tallman, 1990; Miller, 1991; Rosenberg and Chiu, 1985). In particular, in rats and mice, continuous presence of these drugs produces changes in ligand binding as well as channel opening properties of GABA receptor (Gallager et al., 1985; Hernandez et al., 1990; Tietz et al., 1993). The magnitude of these effects increases with the pharmacological efficacy of the benzodiazepine. These observations were complex. (a) Measurements of various biochemical and functional properties of the receptor changed with different time courses. (b) The changes in receptor properties depended on the protocol of the chronic administration (e.g., continuous or intermittent, injected or inserted). (c) The changes varied in different brain regions. (d) They varied with the individual drug chronically administered. (e) They varied with the testing protocol and with the ligand used to assay the effects on the receptor. Evidently, tolerance and dependence are complex responses including series of different events at different types of receptor. Observations of changes in rates of protein subunit synthesis suggested that a change in subunit composition might occur but at a rate too slow to account for the early changes of channel function observed (Heninger et al., 1990; Kang et al., 1994; Kang and Miller, 1991; Primus and Gallager, 1992;). The rates of the initial changes caused by chronic administration and also by discontinuation suggested that modification of the receptor in the membrane occurs. A measure of GABAA receptor function has been the GABA-mediated uptake of Cl into sealed vesicles formed from membrane of disrupted cells (Allan et al., 1985; Harris and Allan, 1985; Subbarao and Cash, 1985). In these measurements, radiotracer ion transport is due to specific anion exchange through the receptor channel and is a function of two different responses, channel opening and desensitization of the receptor. Using a rapid chemical kinetics technique, quench flow, the initial halide-exchange rate through open channel of GABAA receptor and the rate of its desensitization have been resolved (Cash and Subbarao, 1987b, 1987c). Quench-flow ion flux methods are suitable for investigating changes in a receptor due to drug administration, learning or disease because the membrane suspension can be made directly from brain and can be mixed rapidly with solutions of known and controlled concentrations. We are investigating changes accompanying tolerance to CDPX in a native membrane preparation from rat cerebral cortex, in which two GABAA receptors have been distinguished by their desensitization rates (Cash and Subbarao, 1987a, 1987c). After the addition of GABA, these receptors mediate transmembrane Cl exchange, which proceeds in two phases, each terminated by a desensitization process. This is described by equations 1 to 3, in which JA and JB are the initial rate constants for ion exchange, a measure of open channel concentration, and a and b are rate constants for desensitization of the faster desensitizing and slower desensitizing receptors, respectively: [*X]t/[*X ]` is the fractional transmembrane equilibration of isotopic specific activity at time t (e.g., see figs. 1–3). The faster desensitizing receptor exhibits higher initial activity (;80% of total channel opening activity; JA/JB ; 5), such that the initial signal intensity is desensitized to reveal a slower desensitizing signal due to the second receptor (equation 4). These two phases of halide exchange are sufficiently separated in time for the four rate constants (equations 2b and 3b) to be determined. The ion-exchange rate constant Jt (equation 4) is a measure of the number of open channels at time t and initially has the value JA 1 JB. The functions A and B (equations 2b and 3b) pertaining to the two receptors, respectively, are given directly by the quench flow measurements (equation 1) and are equal to the area under the curve of Jt plotted against time, shown in fig. 6 for the single receptor type, A. The value of A represents the open channel integrated over time and is a dimensionless factor that determines the size of the signal up to time t and the total signal. Total signal is relevant where a number of signals contribute to a summed membrane potential. @*X #t @*X #` 5 1 2 e (1)