ENDOTHELIN-1 SIGNIFICANTLY INCREASED NUMBER OF SPECIFIC HIGH-AFFINITY1,4-DIHYDROPYRIDINE (DHP) BINDING-SITES PHOTOLABELED ON VASCULAR SMOOTH-MUSCLE CELLS WITH (-)-[H-3]-AZIDOPINE

The effects of endothelin-1 (ET-1) on surface membrane Ca2+ channels w ere studied on cultured human embryonal vascular smooth muscle cells ( VSMC) and on isolated rat aorta using photoaffinity labelling with DHP Ca2+ channel antagonist (-)-[H-3]-azidopine (AZI). The AZI-labelled s aturable population of sites on VSMC with B-max = 1.59+/-0.10 pmol/mg of protein and K-D = 5.40+/-1.70 nmol/l; and 132+/-0.11 pmol/mg w.w. a nd K-D = 1.09+/-0.20 nmol/l in isolated rings of the rat aorta. Preinc ubation with ET-1 (0.1, 1.0 and 10 nmol/l) increased (in a concentrati on-dependent manner) the total number of sites specifically photolabel led on VSMC. The number of sites labelled with AZI on ET-1 preincubate d VSMC increased markedly when divalent cations (Ca2+ or Mg2+ in other experiments) were present in the incubation medium. Specific photolab elling also significantly increased in VSMC pretreated with intrinsica lly photoreactive nifedipine. A protein kinase C inhibitor staurospori ne, added to the incubation medium, significantly reduced the enhanced specific photolabelling after ET-1. The increase in specific photolab elling after ET-1 preincubation (+197+/-46%; P<0.05) was also observed in rings of the rat aorta and it was significantly reduced after prei ncubation with. S-(+)-niguldipine.