Many physiological and pathological processes in the cardiac tissue have been shown to be associated with a release of endothelin (ET) peptides and with induction of specific ET-receptors and G-protein-coupled ion channels. However, the exact mechanism regulating ET-receptors in the myocardium is controversial. The response to ET-1, the most important member of the ET family, is rapidly attenuated by down-regulation of ET-receptors. The internalization of ET-1 bound to two subclasses of specific receptors (ETA and ETB) that are abundant in the myocardium has been hypothesized to activate and/or inhibit a variety of intracellular signal transducing systems. The [125I]ET-1, BQ-3020 and selective ET-antagonists were used to study the subtype-selective component of regulation of ET-1 receptors in myocardial membranes. We determined the characteristics of [125I]ET-1 binding and [3H]thymidine incorporation in whole cell saturation studies and measured Ca 2+ channel induction and the total number of inactive Ca2+ channels in photoaffinity studies with [3H]azidopine. Here we demonstrate four important components of the complex ET-1 response in human, porcine and rat myocardium, leading to aberrant responses of cells. After ET-1 induction, adaptive subtype-ETB selective down-regulation predominated in human embryonic fibroblasts, in porcine membrane vesicles and in microsomal membranes of renal hypertensive rats, with preferential high affinity ET-1 binding to ETA receptors and with the resultant ETA mediated proliferative and mitogenic activation of human fibroblasts. The ET-1 induction was also accompanied by profound inactivation of Ca2+ channels in myocardial membranes., J. Dřímal, M. Mislovičová, A. Ismail, F. Monček., and Obsahuje bibliografii
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 AZl-labelled saturable population of sites on VSMC with Bmax = 1.59±0.10 pmol/mg of protein and Kd = 5.40±1.70 nmol/1; and 1.32±0.11 pmol/mg w.w. and Kd = 1.09±0.20 nmol/1 in isolated rings of the rat aorta. Preincubation with ET-1 (0.1, 1.0 and 10 nmol/1) 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 photolabclling 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 prcincubation with S-(+)-niguldipine.