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 μM 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 μM thapsigargin. Diltiazem abolished potentiation, providing evidence for Ca2+ influx through voltage-gated Ca2+ channels (VGCC). Genistein (30 μM) and methyl 2,5-dihydroxycinnamate (1 μM, 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 α1C subunit of the VGCC. Tyrosine phosphorylation of proteins other than VGCC is necessary for prolonged potentiation by ET-1 of depolarization-induced Ca2+ influx.
- Digital imaging microscopy
- Sarcoplasmic reticulum
- Voltage-gated Ca channels
ASJC Scopus subject areas
- Cardiology and Cardiovascular Medicine