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Sensitivity of G‐protein involved in endothelin‐1‐induced Ca 2+ influx to pertussis toxin in porcine endothelial cells in situ
Author(s) -
Aoki Hiroki,
Kobayashi Sei,
Nishimura Junji,
Kanaide Hideo
Publication year - 1994
Publication title -
british journal of pharmacology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.432
H-Index - 211
eISSN - 1476-5381
pISSN - 0007-1188
DOI - 10.1111/j.1476-5381.1994.tb14841.x
Subject(s) - pertussis toxin , toxin , endothelin receptor , g protein , bordetella pertussis , chemistry , in situ , microbiology and biotechnology , biology , biochemistry , receptor , bacteria , genetics , organic chemistry
1 We designed a new method to determine quantitatively the intracellular Ca 2+ concentration ([Ca 2+ ] 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 [Ca 2+ ] i of endothelial cells in situ . 2 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, [Ca 2+ ] i signal, was exclusively endothelial cells. 3 ET‐1 (10 −7 m ) induced an elevation of [Ca 2+ ] 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 Ca 2+ ‐independent and ‐dependent components, while the second phase was exclusively extracellular Ca 2+ ‐dependent. The extracellular Ca 2+ ‐independent component of the first phase was due to the release of Ca 2+ from intracellular storage sites. The second phase and part of the first phase of [Ca 2+ ] i elevation were attributed to the influx of extracellular Ca 2+ . The Ca 2+ influx component was completely inhibited by 10 −3 m Ni 2+ but was not affected by 10 −5 m diltiazem. 4 Pertussis toxin (IAP) markedly inhibited the extracellular Ca 2+ ‐dependent elevation of [Ca 2+ ] i , but had no effect on the extracellular Ca 2+ ‐independent elevation of [Ca 2+ ] i caused by ET‐1 (10 −7 m ). 5 Bradykinin (10 −7 m ) or ATP (10 −5 m ) elevated [Ca 2+ ] i and these responses also consisted of extracellular Ca 2+ ‐independent and extracellular Ca 2+ ‐dependent components. IAP had no effect on either component of the [Ca 2+ ] i elevation induced by bradykinin or ATP. 6 From these findings we conclude that, in porcine endotheliel cells in situ , ET‐1 elevates [Ca 2+ ] i as a result of a Ca 2+ influx component from the extracellular space and release of intracelluarly stored Ca 2+ . The Ca 2+ influx is regulated by an IAP‐sensitive G‐protein, while the release of Ca 2+ from the intracellular store is not.