Sensitivity of G‐protein involved in endothelin‐1‐induced Ca2+ influx to pertussis toxin in porcine endothelial cells in situ

Abstract

We designed a new method to determine quantitatively the intracellular Ca2+ concentration ([Ca2+]i) in endothelial cells in situ, using front‐surface fluorometry and fura‐2‐loaded porcine aortic valvular strips. Using this method, we investigated the characteristics of the G‐protein involved in endothelin‐1 (ET‐1)‐induced changes in [Ca2+]i of endothelial cells in situ. Endothelial cells were identified by specific uptake of acetylated‐low density lipoprotein labelled with 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethyl‐indocarbocyanine perchlorate (DiI‐Ac‐LDL). Double staining with DiI‐Ac‐LDL and fura‐2 showed that the valvular strip was covered with a monolayer of endothelial cells and that the cellular component which contributed to the fura‐2 fluorescence, [Ca2+]i signal, was exclusively endothelial cells. ET‐1 (10−7 m) induced an elevation of [Ca2+]i consisting of two components: the first was a rapid and transient elevation to reach a peak, followed by a second, sustained elevation (the second phase). The first phase was composed of extracellular Ca2+‐independent and ‐dependent components, while the second phase was exclusively extracellular Ca2+‐dependent. The extracellular Ca2+‐independent component of the first phase was due to the release of Ca2+ from intracellular storage sites. The second phase and part of the first phase of [Ca2+]i elevation were attributed to the influx of extracellular Ca2+. The Ca2+ influx component was completely inhibited by 10−3 m Ni2+ but was not affected by 10−5 m diltiazem. Pertussis toxin (IAP) markedly inhibited the extracellular Ca2+‐dependent elevation of [Ca2+]i, but had no effect on the extracellular Ca2+‐independent elevation of [Ca2+]i caused by ET‐1 (10−7 m). Bradykinin (10−7 m) or ATP (10−5 m) elevated [Ca2+]i and these responses also consisted of extracellular Ca2+‐independent and extracellular Ca2+‐dependent components. IAP had no effect on either component of the [Ca2+]i elevation induced by bradykinin or ATP. From these findings we conclude that, in porcine endotheliel cells in situ, ET‐1 elevates [Ca2+]i as a result of a Ca2+ influx component from the extracellular space and release of intracelluarly stored Ca2+. The Ca2+ influx is regulated by an IAP‐sensitive G‐protein, while the release of Ca2+ from the intracellular store is not. 1994 British Pharmacological Society