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

1. 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.
2. Confluent rat airway smooth muscle cells in culture possessed a mixed ET receptor population (30% ET_A : 70% ET_B), with a density of approximately 3400±280 ET_A and 8000±610 ET_B receptors/cell (n=3 experiments). The density of ET_B, but not ET_A receptors increased substantially in serum-containing medium. However, a 2-day period of serum deprivation, which inhibited cellular growth, substantially reduced ET_B receptor density such that the ET receptor subtype proportions were approximately equal (55% ET_A; 45% ET_B) and similar to those previously observed in intact rat tracheal smooth muscle.
3. 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 ET_A receptor activation, although a modest and inconsistent ET_B response was observed. The ET_A-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, ET_B receptor activation was exclusively coupled to extracellular calcium influx.
4. Somewhat surprisingly, human airway smooth muscle cells in culture contained a homogeneous population of ET_A 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 ET_A 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 ET_A-mediated calcium response in these human airway smooth muscle cells in culture was entirely dependent upon the mobilization of calcium from intracellular stores.
5. In summary, rat cultured tracheal airway smooth muscle cells contained both ET_A and ET_B receptors. ET_A receptors, the numbers of which remained constant during cell growth, were linked to the release of Ca²⁺ from intracellular stores and a strong rise in [Ca²⁺]_i in the majority of airway smooth muscle cells. In stark contrast, the numbers of ET_B receptors increased significantly during cell growth, an effect that was diminished substantially by incubation in serum-free medium. Moreover, despite the greater number of ET_B receptors, their activation in a small number of airway smooth muscle cells produced only a weak rise in [Ca²⁺]_i, which appeared to be attributable to the influx of extracellular Ca²⁺. In contrast, the populations of ET receptors and their linkage to [Ca²⁺]_i were markedly different in the human cultured airway smooth muscle cells used in the current study compared to that previously observed in intact human isolated bronchial smooth muscle.