Molecular Pharmacology, Vol 18, 253-258, Copyright © 1980 by the American Society for Pharmacology and Experimental Therapeutics

Calmodulin Activation of Red Blood Cell (Ca²⁺ + Mg²⁺)-ATPase and Its Antagonism by Phenothiazines

¹ Department of Pharmacology, School of Medicine, University of Washington, Seattle, Washington 98195

Plasma membranes prepared from human red blood cells exhibit a Ca²⁺-activated, Mg²⁺-dependent adenosine triphosphatase activity which is a biochemical expression of the Ca²⁺ pump. The activity of this (Ca²⁺+Mg²⁺-ATPase was increased severalfold by the Ca²⁺-binding protein, calmodulin, whether purified or present in a crude red blood cell hemolysate. The phenothiazines chlorpromazine (CPZ) and trifluoperazine (TFP) antagonized calmodulin activation of (Ca²⁺+Mg²⁺-ATPase in an apparently competitive manner. CPZ and TFP were less effective in antagonizing (Ca²⁺+Mg²⁺)-ATPase activity in the absence of added calmodulin. TFP antagonized the calmodulin-dependent portion of (Ca²⁺+Mg²⁺)-ATPase activity relatively specifically at 10 and 30 µM. Up to 100 µM neither haloperidol nor d - or l-butaclamol significantly antagonized calmodulin-induced activation of the (Ca²⁺+Mg²⁺-ATPase. Basal ATPase activity was also not inhibited. In contrast to the lack of inhibition by nonphenothiazine neuroleptics, ruthenium red nonspecifically antagonized both basal and calmodulin-activated (Ca²⁺+Mg²⁺)-ATPase activity. It was concluded that phenothiazines antagonize activation of the (Ca²⁺+Mg²⁺)-ATPase by binding to calmodulin. Ruthenium red appears to have a different mechanism of action. Because the potent neuroleptic nonphenothiazines, haloperidol and butaclamol, fail to antagonize calmodulin activation of red blood cell (Ca²⁺+Mg²⁺-ATPase it appears that general anti-calmodulin activity does not correlate with neuroleptic activity.

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ACKNOWLEDGMENTS We would like to thank Dr. T. R. Hinds for his help in the preparation and purification of calmodulin. Dr. H. Green at Smith, Kline and French Laboratories kindly provided a generous gift of [³H]trifluoperazine.