Comparative effects of substituted phenothiazines and their free radicals on (Na +,K +)-activated adenosine triphosphatase

Abstract

The inhibitory effects of free radicals of various substituted phenothiazines on (Na +,K +)-activated ATPase [Mg 2+-dependent, (Na +,K +)-activated ATP phosphohydrolase; EC 3.6.1.3] were studied in vitro. Enzyme preparations were obtained from rat brain microsomal fractions after deoxycholic acid and NaI treatments. Free radicals were produced by either ultraviolet (253.7 nm) irradiation or enzymatic oxidation with peroxidase. Without enzymatic or photo-oxidation, phenothiazine derivatives failed to inhibit (Na +,K +)-activated ATPase activity significantly. Photo-oxidative intermediates of thioridazine, triflupromazine and trifluoperazine were potent inhibitors of (Na +,K +)-activated ATPase activity. Those from chlorpromazine, perphenazine and promazine were less potent. Peroxidase-hydrogen peroxide treatment of promazine, thioridazine, perphenazine and chlorpromazine produced free radical intermediates which significantly inhibited (Na +,K +)-activated ATPase activity. The same treatment of triflupromazine and trifluoperazine, however, failed to produce detectable amounts of free radical intermediates. Concomitantly, no inhibition of (Na +,K +)-activated ATPase activity was observed under these conditions. It was concluded that free radical intermediates of various substituted phenothiazines are differentially potent inhibitors of (Na +,K +)-activated ATPase, and that the formation of free radicals from substituted phenothiazines is dependent upon the oxidizing conditions and nature of substituent groups.