Stimulatory effect of calmodulin antagonists on phospholipid base-exchange reactions in rabbit platelet membranes

Abstract The properties of Ca 2+ -dependent incorporation of [ 3 H]serine, [ 3 H]ethanolamine and [ 3 H]choline into the corresponding phospholipids mediated by base-exchange enzymes in rabbit platelet membranes were studied in the presence or absence of the calmodulin antagonists chlorpromazine, trifluoperazine and N -(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), all of which markedly activate three base-exchange reactions. The base-exchange activities were dependent on Ca 2+ both in the presence and absence of the drugs. Other metal ions tested did not stimulate the base-exchange reactions, even in the presence of the drugs. Apparent K m values for serine, ethanolamine and choline were not affected significantly by the concentration of Ca 2+ , with or without the drugs. [ 3 H]Serine incorporation into phospholipid was competitively inhibited by ethanolamine and choline, [ 3 H]choline incorporation was competitively inhibited by serine and ethanolamine, whereas [ 3 H]ethanolamine incorporation was competitively inhibited by serine and noncompetitively by choline. These competitive and noncompetitive relations between each base were also not affected by the drugs. The amount of 45 Ca 2+ binding to platelet membranes was decreased by the drugs dose dependently. A weaker calmodulin antagonist, N -(6-aminohexyl)-1-naphthalenesulfonamide (W-5), only slightly stimulated the base-exchange reactions, but did clearly inhibit 45 Ca 2+ binding to the membranes, in the same manner as that of the other calmodulin antagonists used. The concentration of chlorpromazine, trifluoperazine, W-7 and W-5, required to produce half-maximal inhibition of Ca 2+ binding, was approximately 30 μM. These results suggest that the calmodulin antagonists used activate the base-exchange reactions only in the presence of Ca 2+ without changing the affinity of each free base to base-exchange enzymes. The activation of the base-exchange reactions was not due to the increase in free Ca 2+ caused by the drug-induced inhibition of Ca 2+ binding to platelet membranes.