ATTENUATION OF ENDOTHELIN-1-INDUCED CALCIUM RESPONSE BY TYROSINE KINASE INHIBITORS IN VASCULAR SMOOTH-MUSCLE CELLS

Although tyrosine kinases play an important role in cell growth and ha ve been implicated in regulation of smooth muscle contraction, their r ole in agonist-induced myoplasmic Ca2+ responses is unclear. We examin ed effects of the tyrosine kinase inhibitors genistein and methyl 2,5- dihydroxycinnamate (MDHC) on the endothelin-l (ET-l)-induced Ca2+ resp onse and determined underlying mechanisms for the effects. Freshly iso lated smooth muscle cells from porcine coronary arteries were loaded w ith fura 2 ester, and myoplasmic free Ca2+ (Ca-m(2+)) concentration wa s estimated with fura 2 microfluorometry. Both genistein and MDHC inhi bited the initial transient Ca-m(2+) response to ET by 54 and 81%, res pectively (P < 0.05), in the presence of extracellular Ca2+. Genistein also significantly delayed the Ca-m(2+) response, with the latent per iod from ET-1 application to the beginning of the Ca-m(2+) response be ing increased from 1.08 +/- 0.17 to 2.65 +/- 0.52 min (P < 0.05). In t he absence of extracellular Ca2+, genistein inhibited the ET-l-induced Ca-m(2+) response by 93% (P < 0.05). The Ca-m(2+) responses to caffei ne (5 mM) or inositol trisphosphate (IP3) applied intracellularly via a patch-clamp pipette were not affected by genistein. Both genistein a nd MDHC also abolished the sustained Ca-m(2+) response to ET-1. Howeve r, the Ca-m(2+) response to depolarization by 80 mM K+ was not inhibit ed by MDHC and only inhibited 22% by genistein (P < 0.05). These resul ts indicate that I) activation of tyrosine kinases is an important reg ulatory mechanism for the ET-1-induced Ca-m(2+) response in vascular s mooth muscle and 2) tyrosine kinases mediate ET-l-induced Ca2+ release with no direct effect on IP3-mediated Ca2+ release. Thus ET-1-mediate d signaling upstream of IP3 interaction with the Ca2+ stores is regula ted by tyrosine kinases.