Endothelin-1 significantly increased number of specific high-affinity 1,4-dihydropyridine (DHP) binding sites photolabelled on vascular smooth muscle cells with (-)-[3H]-azidopine.
The effects of endothelin-1 (ET-1) on surface membrane Ca2+ channels were studied on cultured human embryonal vascular smooth muscle cells (VSMC) and on isolated rat aorta using photoaffinity labelling with DHP Ca2+ channel antagonist (-)-[3H]-azidopine (AZI). The AZI-labelled saturable population of sites on VSMC with Bmax = 1.59 +/- 0.10 pmol/mg of protein and KD = 5.40 +/- 1.70 nmol/l; and 1.32 +/- 0.11 pmol/mg w.w. and KD = 1.09 +/- 0.20 nmol/l in isolated rings of the rat aorta. Preincubation with ET-1 (0.1, 1.0 and 10 nmol/l) increased (in a concentration-dependent manner) the total number of sites specifically photolabelled on VSMC. The number of sites labelled with AZI on ET-1 preincubated VSMC increased markedly when divalent cations (Ca2+ or Mg2+ in other experiments) were present in the incubation medium. Specific photolabelling also significantly increased in VSMC pretreated with intrinsically photoreactive nifedipine. A protein kinase C inhibitor staurosporine, added to the incubation medium, significantly reduced the enhanced specific photolabelling after ET-1. The increase in specific photolabelling after ET-1 preincubation (+197 +/- 46%; P < 0.05) was also observed in rings of the rat aorta and it was significantly reduced after preincubation with S-(+)-niguldipine.
Drímal
J
J
Institute of Experimental Pharmacology, Slovak Academy of Sciences, Bratislava, Slovak Republic.
Koprda
V
V
D000345:Affinity Labels; D000818:Animals; D001013:Aorta, Thoracic / Q000187:drug effects / Q000378:metabolism; D001386:Azides; D001665:Binding Sites / Q000187:drug effects; D002121:Calcium Channel Blockers / Q000378:metabolism; D015220:Calcium Channels / Q000187:drug effects*; D020746:Calcium Channels, L-Type; D002478:Cells, Cultured; D004095:Dihydropyridines; D019332:Endothelin-1 / Q000494:pharmacology*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009131:Muscle, Smooth, Vascular / Q000187:drug effects*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors; D051381:Rats; D019311:Staurosporine / Q000494:pharmacology
D000345:Affinity Labels; D001386:Azides; D002121:Calcium Channel Blockers; D015220:Calcium Channels; D020746:Calcium Channels, L-Type; D004095:Dihydropyridines; D019332:Endothelin-1; D004791:Enzyme Inhibitors; C041130:azidopine; D011493:Protein Kinase C; D019311:Staurosporine