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.