Role of protein tyrosine phosphorylation in erythrocyte lysate-induced intracellular free calcium concentration elevation in cerebral smooth-muscle cells

Object. Tyrosine kinases play an important role in the regulation of system ic vascular smooth-muscle tone. The authors studied the involvement of prot ein tyrosine kinase activity in erythrocyte lysate-mediated signal transduc tion in cerebral smooth-muscle cells. Methods. Tyrosine kinase phosphorylation and intracellular free Ca++ ([Ca+](i)) were measured in rat aortic and basilar artery smooth-muscle cells by using Western blot and fura 2-acetoxymethyl ester microfluorimetry. Erythr ocyte lysate enhanced tyrosine phosphorylation in cultured rat aortic and b asilar smooth-muscle cells and induced a rapid transient and a prolonged pl ateau phase of [Ca++], response in rat basilar smooth-muscle cells. The tyr osine kinase inhibitors genistein and tyrphostin A51 (administered at conce ntrations of 30 or 100 mu M) attenuated both phases of erythrocyte lysate-i nduced [Ca++](i) elevation. Erythrocyte lysate was separated into low- (< 1 0 kD, which contains adenine nucleotides) and high- (>10 kD, which contains hemoglobin) molecular-weight fractions; these fractions were tested separa tely in these cells. The low-molecular-weight fraction produced a similar [ Ca++](i) response to that of erythrocyte lysate and the high-molecular-weig ht fraction produced a small response. The [Ca++](i) responses from both fr actions were inhibited by tyrosine kinase inhibitors. Conclusions. To the authors' knowledge, this is the first report to show th at tyrosine phosphorylation may be involved in erythrocyte lysate-induced s ignal transduction and [Ca++](i) responses in cerebral smooth-muscle cells.