Endothelin-induced myoplasmic Ca2+ responses and tyrosine phosphorylation in coronary smooth muscle.
This study investigated the role of tyrosine phosphorylation and source of Ca2+ in prolonged endothelin-1 (ET-1)-induced potentiation of myoplasmic free Ca2+ ([Ca2+]m) responses to depolarization in coronary smooth muscle cells. Fura-2 microfluorometry showed typical increases in [Ca2+]m in response to 80 mM K+ (80K) and 0.01 microM endothelin. After washout of ET-1 80K-induced [Ca2+]m increases were augmented (potentiated) 31%. Time to peak [Ca2+]m response to 80K was less after ET-1 exposure than before. ET-1 potentiation of 80K-induced [Ca2+]m responses by decreased sarcoplasmic reticulum (SR) buffering of [Ca2+]m or Ca2+-induced Ca2+ release was ruled out by lack of potentiation by 5 mM caffeine and 1 microM thapsigargin. Diltiazem abolished potentiation, providing evidence for Ca2+ influx through voltage-gated Ca2+ channels (VGCC). Genistein (30 microM) and methyl 2,5-dihydroxycinnamate (1 microM, MDHC) abolished potentiation of Ca2+ influx. Single cell phosphotyrosine measured directly by immunofluorescence was increased 95% in cells treated with ET-1 compared to control, genistein, and MDHC treated cells. ET-1 increased tyrosine phosphorylation of an 80-85 kDa protein, but not the 240 kDa alpha1C subunit of the VGCC. Tyrosine phosphorylation of proteins other than VGCC is necessary for prolonged potentiation by ET-1 of depolarization-induced Ca2+ influx.
Lee
Dexter L
DL
Department of Physiology, School of Medicine, Dalton Cardiovascular Research Center, and Diabetes and Cardiovascular Biology Program, University of Missouri, Columbia, Missouri 65212, USA.
Sturek
Michael
M
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism; D003331:Coronary Vessels / Q000187:drug effects* / Q000378:metabolism; D019332:Endothelin-1 / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D009210:Myofibrils / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D019000:Phosphotyrosine / Q000378:metabolism*; D012519:Sarcoplasmic Reticulum / Q000187:drug effects / Q000378:metabolism; D013552:Swine
D015220:Calcium Channels; D019332:Endothelin-1; D004791:Enzyme Inhibitors; D019000:Phosphotyrosine; D002118:Calcium