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 systemic vascular smooth-muscle tone. The authors studied the involvement of protein tyrosine kinase activity in erythrocyte lysate-mediated signal transduction in cerebral smooth-muscle cells. Methods. Tyrosine kinase phosphorylation and intracellular free Ca⁺⁺ ([Ca²⁺](i)) were measured in rat aortic and basilar artery smooth-muscle cells by using Western blot and fura 2-acetoxymethyl ester microfluorimetry. Erythrocyte lysate enhanced tyrosine phosphorylation in cultured rat aortic and basilar smooth-muscle cells and induced a rapid transient and a prolonged plateau phase of [Ca⁺⁺](i) response in rat basilar smooth-muscle cells. The tyrosine kinase inhibitors genistein and tyrphostin A51 (administered at concentrations of 30 or 100 μM) attenuated both phases of erythrocyte lysate-induced [Ca⁺⁺](i) elevation. Erythrocyte lysate was separated into low- (< 10 kD, which contains adenine nucleotides) and high- (> 10 kD, which contains hemoglobin) molecular-weight fractions; these fractions were tested separately in these cells. The low-molecular-weight fraction produced a similar [Ca⁺⁺](i) response to that of erythrocyte lysate and the high-molecular-weight fraction produced a small response. The [Ca⁺⁺](i) responses from both fractions were inhibited by tyrosine kinase inhibitors. Conclusions. To the authors' knowledge, this is the first report to show that tyrosine phosphorylation may be involved in erythrocyte lysate-induced signal transduction and [Ca⁺⁺](i) responses in cerebral smooth-muscle cells.