ag-183 and Hemolysis

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.. 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. 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 microM) 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.. 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.

Topics: Adenine Nucleotides; Analysis of Variance; Animals; Aorta; Basilar Artery; Blotting, Western; Calcium; Calcium Signaling; Cell Extracts; Cells, Cultured; Cytophotometry; Dogs; Enzyme Inhibitors; Erythrocytes; Female; Fluorescent Dyes; Fura-2; Genistein; Hemoglobins; Hemolysis; Molecular Weight; Muscle, Smooth, Vascular; Phosphorylation; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; Signal Transduction; Tyrphostins