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 have been implicated in regulation of smooth muscle contraction, their role in agonist-induced myoplasmic Ca2+ responses is unclear. We examined effects of the tyrosine kinase inhibitors genistein and methyl 2,5-dihydroxycinnamate (MDHC) on the endothelin-1 (ET-1)-induced Ca2+ response and determined underlying mechanisms for the effects. Freshly isolated smooth muscle cells from porcine coronary arteries were loaded with fura 2 ester, and myoplasmic free Ca2+ (Ca2+ (m)) concentration was estimated with fura 2 microfluorometry. Both genistein and MDHC inhibited the initial transient Cam2+ response to ET by 54 and 81%, respectively (P < 0.05), in the presence of extracellular Ca2+. Genistein also significantly delayed the Cam2+ response, with the latent period from ET-1 application to the beginning of the Cam2+ response being increased from 1.08 +/- 0.17 to 2.65 +/- 0.52 min (P < 0.05). In the absence of extracellular Ca2+, genistein inhibited the ET-1-induced Cam2+ response by 93% (P < 0.05). The Cam2+ responses to caffeine (5 mM) or inositol trisphosphate (IP3) applied intracellularly via a patch-clamp pipette were not affected by genistein. Both genistein and MDHC also abolished the sustained Cam2+ response to ET-1. However, the Cam2+ response to depolarization by 80 mM K+ was not inhibited by MDHC and only inhibited 22% by genistein (P < 0.05). These results indicate that 1) activation of tyrosine kinases is an important regulatory mechanism for the ET-1-induced Cam2+ response in vascular smooth muscle and 2) tyrosine kinases mediate ET-1-induced Ca2+ release with no direct effect on IP3-mediated Ca2+ release. Thus ET-1-mediated signaling upstream of IP3 interaction with the Ca2+ stores is regulated by tyrosine kinases.
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Dalton Cardiovascular Research Center, School of Medicine, University of Missouri, Columbia 65211, USA.
Sturek
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D000818:Animals; D002110:Caffeine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002934:Cinnamates / Q000494:pharmacology; D003331:Coronary Vessels / Q000166:cytology / Q000378:metabolism; D016232:Endothelins / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005456:Fluorescent Dyes; D005470:Fluorometry; D016257:Fura-2; D019833:Genistein; D015544:Inositol 1,4,5-Trisphosphate / Q000494:pharmacology; D007529:Isoflavones / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D018408:Patch-Clamp Techniques; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors*; D012519:Sarcoplasmic Reticulum / Q000378:metabolism; D015398:Signal Transduction; D013552:Swine
D002121:Calcium Channel Blockers; D002934:Cinnamates; D016232:Endothelins; D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007529:Isoflavones; D002110:Caffeine; C067472:methyl 2,5-dihydroxycinnamate; D015544:Inositol 1,4,5-Trisphosphate; D019833:Genistein; D011505:Protein-Tyrosine Kinases; D002118:Calcium; D016257:Fura-2