title: Attenuation of endothelin-1-induced calcium response by tyrosine kinase inhibitors in vascular smooth muscle cells.
abstract: 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.
authors.affiliation: Dalton Cardiovascular Research Center, School of Medicine, University of Missouri, Columbia 65211, USA.
authors.forename: C Y
authors.initials: CY
authors.lastname: Liu
authors.forename: M
authors.initials: M
authors.lastname: Sturek
mesh_terms: 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
chemical_list: 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