Molecular Pharmacology, Vol 4, 600-612, Copyright © 1968 by the American Society for Pharmacology and Experimental Therapeutics

Inhibition of Brain Sodium- and Potassium-Stimulated Adenosine Triphosphatase Activity by Chlorpromazine Free Radical

¹ Department of Pharmacology, Michigan State University, East Lansing, Michigan 48823

Ultraviolet light was observed to have a profound effect on the inhibition in vitro of rat brain microsomal sodium- and potassium-stimulated ATPase activity [Mg²⁺-dependent, (Na⁺ + K⁺)-stimu1ated ATP phosphohydrolase, EC. 3.6.1.3] by chlorpromazine.

The inhibition of the enzyme activity was minimal when the experiments were carefully performed, avoiding exposure to light. However, a significant inhibition of (Na⁺ + K⁺)-ATPase activity was observed when the chlorpromazine free radical was added to the enzyme mixture after it had been generated either by ultraviolet irradiation or by chemical oxidation (with sulfuric acid) of chlorpromazine solutions. The concentration required for 50% inhibition of enzyme activity (I₅₀) was 40 µM. Even greater inhibition was observed when the chlorpromazine free radical was generated by ultraviolet irradiation in a mixture containing enzyme. Under these experimental conditions, the I₅₀ was 3.5 µM. The ultraviolet irradiation of the drug-enzyme mixture failed to enhance the inhibition of (Na⁺ + K⁺)-ATPase activity when inhibitory concentrations of ouabain or p-hydroxymercuribenzoate were used. A positive correlation was observed between the amount of chlorpromazine transformed by ultraviolet irradiation and the enhancement of inhibition of (Na⁺ + K⁺)-ATPase activity by a corresponding amount of irradiation of the chlorpromazine-enzyme mixture. An enhanced inhibition (I₅₀ = 15 µM) was also observed when the chlorpromazine free radical was generated in the ATPase assay system in the simultaneous presence of peroxidase and hydrogen peroxide. The inhibition of (Na⁺ + K⁺)-ATPase activity by chlorpromazine sulfoxide was minimal even in the presence of peroxidase and hydrogen peroxide. It is concluded that a semiquinone free radical of chlorpromazine, rather than chlorpromazine itself, may be responsible for the inhibition of sodium- and potassium-stimulated ATPase activity in vitro.

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ACKNOWLEDGMENT We thank Mrs. Lillian Edney for expert technical assistance.