Endothelin-induced myoplasmic Ca2+ responses and tyrosine phosphorylation in coronary smooth muscle.

This study investigated the role of tyrosine phosphorylation and source of Ca 2+ in prolonged endothelin-1 (ET-1)-induced potentiation of myoplasmic free Ca 2+ ([Ca 2+ ] m ) responses to depolarization in coronary smooth muscle cells Fura-2 microfluorometry showed typical increases in [Ca 2+ ] m in response to 80 mM K+ (80K) and 0.01 μM endothelin. After washout of ET-1 80K-induced [Ca 2+ ] m increases were augmented (potentiated) 31%. Time to peak [Ca 2+ ] m response to 80K was less after ET-1 exposure than before. ET-1 potentiation of 80K-induced [Ca 2+ ] m responses by decreased sarcoplasmic reticulum (SR) buffering of [Ca 2+ ] m or Ca 2+ -induced Ca 2+ release was ruled out by lack of potentiation by 5 mM caffeine and I μM thapsigargin. Diltiazem abolished potentiation, providing evidence for Ca 2+ influx through voltage-gated Ca 2+ channels (VGCC). Genistein (30 μM) and methyl 2,5-dihydroxycinnamate ( 1 μM, MDHC) abolished potentiation of Ca 2+ 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 α 1C subunit of the VGCC. Tyrosine phosphorylation of proteins other than VGCC is necessary for prolonged potentiation by ET-I of depolarization-induced Ca 2+ influx.