Use of Fluorescent Probes to Investigate Hepatic Microsomal ‘Drug’-Binding Sites

Abstract

0.01 %, 0.02%, 0.03 %, 0.04% and 0.05 % in weeks 1,2,3,4 and 5 respectively. The rats continued to drink the highest concentration (0.05 %) for another 55 days. The daily consumed dose of D-amphetamine was found to be increasedfrom 16mg/kg on the first day up to 90mg/kg on day 32 and remained at that value until the experiment was terminated on day 90 without any case of death. The rats, having tolerated extremely high doses of amphetamine, developed tolerance to the toxic and anorexigenic effects of the drug. During the first 5 weeks of treatment no tolerance to the increased motor activity was observed. On day 90 we injected rats intraperitoneally with 200pCi of ~-[~H]amphetamine’kg together with 4mg/kg of unlabelled drug 1 h before decapitation. The concentration of unchanged ~-[~H]amphetamine was extracted from liver and brain according to the method described by Maickel et al . (1969) and found to be significantly increased in chronically treated rats by 76% and 29 % respectively. These results suggested a possible alteration in the concentration of amphetamine-metabolizing enzymes in the liver. To examine this possibility we determined the incorporation of [14C]leucine into microsomal and cytoplasmic proteins of rat liver. Under our experimental conditions, it was evident that chronic treatment with D-amphetamine significantly decreased the incorporation of [14C]leucine into microsomal and cytoplasmic proteins by 21 % and 25 %, respectively. The concentration of microsomal protein/gram of liver was also significantly decreased. The observed inhibition of amino acid incorporation is obviously not due to decreased transport of the radioactive precursor into the liver since the concentration of acid-soluble radioactivity in amphetamine-treated rats was significantly higher than of controls. We conclude that development of tolerance to amphetamine is not due to increased metabolism of the drug. It is most likely that chronic treatment with amphetamine inhibits synthesis de nouo of its metabolizing enzymes, thus elevating the concentration of circulating amphetamine and therefore increasing its concentration in the brain.