SPHINGOSINE, W-7, AND TRIFLUOPERAZINE INHIBIT THE ELEVATION IN CYTOSOLIC CALCIUM-INDUCED BY HIGH K+ DEPOLARIZATION IN SYNAPTOSOMES

A possible role for protein kinases in the regulation of free cytosoli c Ca2+ levels in nerve endings was investigated by testing the effect of several kinase inhibitors on the increase in cytosolic Ca2+ (monito red with the Ca2+-sensitive dye fura-2) induced by depolarization with 15 or 30 mM K+. The ability of various drugs to inhibit the cytosolic Ca2+ response appeared to correlate with their reported mechanism of action in inhibiting protein kinases. W-7 and trifluoperazine, drugs r eported to inhibit calmodulin-dependent events, were effective inhibit ors of the increase in cytosolic Ca2+ induced by high K+ depolarizatio n, as was sphingosine, a drug that inhibits protein kinase C by bindin g to the regulatory site, but which also inhibits calcium/calmodulin k inase. On the other hand, drugs that inhibit protein kinases by bindin g to the catalytic site, such as H-7 (1 mM), staurosporine (1 muM), an d K252a (1 muM), were ineffective. Activation of protein kinase C, whi ch is blocked by each of these drugs, does not appear to be essential to the maintenance of elevated cytosolic Ca2+ in depolarized synaptoso mes. All of the drugs, including sphingosine, that functionally inhibi t the depolarization-induced elevation in cytosolic Ca2+ have in commo n the ability to bind to calmodulin. Because the drugs that inhibit pr otein kinases by competing with ATP binding at the active catalytic si te did not block the response in this system, we suggest that a calmod ulin or a calmodulin-like binding site participates in the regulation of Ca2+ increases after depolarization.