Pharmacology Biochemistry & Behavior, Vol. 22, pp. 955--960,1985. © AnkhoInternational Inc. Printedin the U.S.A. 0091-3057/85$3.00 + .00 Barbiturate Tolerance and Dependence: Effects on Synaptosomal Sodium Transport and Membrane Fluidity I MARK A. MITCHELL,T JOANNA PERIS* AND R. ADRON HARRIS .2 *Denver V.A. Medical Center, Department of Pharmacology, University of Colorado School of Medicine Denver, CO 80262 and ~rDepartment of Pharmacology, School of Medicine, University of Missouri Columbia, MO 65212 Received 10 September 1984 MITCHELL, M. A., J. PERIS AND R. A. HARRIS. Barbiturate tolerance and dependence: Effects on synaptosomal sodium transport and membrane fluidity. PHARMACOL BIOCHEM BEHAV 22(6) 955-960, 1985.--DBA mice were fed lab chow containing phenobarbital for seven or eight days. Upon withdrawal of the phenobarbital diet, dependence was evidenced by appearance of hypothermia, handling-induced convulsions and lethal seizures. Functional tolerance was determined by injecting phenobarbital into mice treated with the phenobarbital diet or a pair-fed control diet and measuring the brain concentration of phenobarbital at the time of loss of righting reflex and the time of regaining righting reflex. Both measures demonstrated that chronic consumption of phenobarbital resulted in functional tolerance. When the diet was withdrawn for two days, tolerance was no longer present, indicating a rapid reversal of the adaptive changes. The veratridine-stimulated uptake of 24Na by isolated brain synaptosomes was used as a measure of membrane function. Sodium uptake was inhibited in vitro by pentobarbital and ethanol, and the inhibitory effects of these drugs were attenuated by chronic in vivo phenobarbital treatment. The fluidity of brain synaptic plasma membranes was estimated by the fluorescence polarization of the fluorescent probe molecules 1-(4-trimethylammonium phenyl)-6-phenyl-l,3,5-hexatriene and 1,6-diphenyl-l,3,5-hexatriene. Synaptic membranes from mice treated chronically with phenobarbital did not differ from those of control mice with regard to either the baseline fluorescence polarization of the probes or the decrease in fluorescence polarization produced by in vitro exposure to phenobarbital or ethanol. Taken together, these results indicate that although chronic phenobarbital ingestion resulted in tolerance and dependence (studied in vivo), and adaptation of sodium"channels (studied in vitro), there was no evidence that these changes were due to alterations in the membrane physical properties. Membrane fluidity Barbiturate tolerance Sodium transport Ethanol Barbiturate dependence DBAJ2 mice Synaptosomes ACUTE exposure to general anesthetics is suggested to in- crease membrane fluidity thereby disrupting cellular proc- esses and producing sedation [23,25]. In support of a com- mon mechanism of anesthetic action, concentration- dependent increases in brain synaptosomal membrane (SPM) fluidity have been shown to occur following in vitro exposure to ethanol, barbiturates, and gaseous anesthetics [4, 11, 13, 14, 24]. Repeated exposure to ethanol causes a homeoviscous adapation of brain membranes that lessens the in vitro fluidizing drug effect (tolerance) [4, 15, 20, 27]. In contrast to these extensive studies of ethanol effects, investigations of the membrane effects of chronic barbiturate intoxication are scant. Johnson et al. [17] reported resistance to the fluidizing effects of ethanol in membranes from mice treated chroni- cally with pentobarbital by a pellet implantation method. Al- though these results are suggestive of cross-tolerance to ethanol, the effects of barbiturates were not assessed, and membrane tolerance to barbiturates was therefore inferred but not proven. We investigated the effects of chronic phenobarbital in- toxication on brain membrane fluidity and function to de- termine the following: First, does chronic phenobarbital ex- posure produce changes in brain membranes similar to those treated chronically with ethanol (increased initial membrane rigidity and decreased in vitro drug response)? To answer this question, mice were made tolerant to and dependent on phenobarbital by the adulterated chow method of Belknap et al. [1]. The physical properties of brain membranes from control and tolerant-dependent mice were compared in vitro using fluorescence polarization of probes specific for differ- ent membrane regions. Secondly, we determined whether chronic ingestion of phenobarbital altered a specific mem- brane function: synaptic sodium transport. The voltage- ~This work was supported by funds from the Veterans Administration and USPH grants AA-06399 and AA-05195. 2Requests for reprints should be addressed to Dr. R. Adron Harris, Department of Pharmacology-C236, University of Colorado, School of Medicine, 4200 E. 9th Avenue, Denver, CO 80262. 955