Ca2+ signalling by endothelin receptors in rat and human cultured airway smooth muscle cells

The aim of the current study was to characterize the ET receptor subtypes in cultured airway smooth muscle cells derived from rat trachea and human bronchus using radioligand binding techniques and to investigate the coupling of ET receptors to intracellular calcium signalling mechanisms using endothelin receptor‐selective agonists (sarafotoxin S6c) and antagonists (BQ‐123, BQ‐788) and digital image fluorescence microscopy. Confluent rat airway smooth muscle cells in culture possessed a mixed ET receptor population (30% ETA: 70% ETB), with a density of approximately 3400±280 ETA and 8000±610 ETB receptors/cell (n = 3 experiments). The density of ETB, but not ETA receptors increased substantially in serum‐containing medium. However, a 2‐day period of serum deprivation, which inhibited cellular growth, substantially reduced ETB receptor density such that the ET receptor subtype proportions were approximately equal (55% ETA; 45% ETB) and similar to those previously observed in intact rat tracheal smooth muscle. Challenge of rat airway smooth muscle cells in culture with endothelin‐1 elicited a concentration‐dependent biphasic increase in [Ca2+]i (EC50: 16 nM), that comprised an initial transient peak [Ca2+]i increase (typically 350 nM) followed by a modest sustained component. The endothelin‐1‐induced biphasic [Ca2+]i increase was primarily due to ETA receptor activation, although a modest and inconsistent ETB response was observed. The ETA‐mediated [Ca2+]i increase was due primarily to the mobilization of IP3‐sensitive and to a lesser extent ryanodine‐sensitive intracellular calcium stores. In contrast, ETB receptor activation was exclusively coupled to extracellular calcium influx. Somewhat surprisingly, human airway smooth muscle cells in culture contained a homogeneous population of ETA receptors at a density of 6100±800 receptors cell−1 (n = 3 experiments). Serum deprivation was without effect on either ET receptor subtype proportion or ETA receptor density. Challenge of human airway smooth muscle cells with endothelin‐1 provoked a concentration‐dependent increase in [Ca2+]i (EC50: 15 nM), with a peak [Ca2+]i increase to greater than 700 nM. Furthermore, the ETA‐mediated calcium response in these human airway smooth muscle cells in culture was entirely dependent upon the mobilization of calcium from intracellular stores. In summary, rat cultured tracheal airway smooth muscle cells contained both ETA and ETB receptors. ETA receptors, the numbers of which