Mechanism of ETA‐receptor stimulation‐induced increases in intracellular Ca2+ in SK‐N‐MC cells

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Abstract
The mechanism underlying endothelin‐1 (ET‐1)‐induced increases in intracellular Ca2+ concentrations in the human neuroblastoma cell‐line SK‐N‐MC was investigated. ET‐receptor agonists increased inositol phosphate (IP)‐formation (assessed as accumulation of total [3H]‐IPs in [3H]‐myo‐inositol prelabelled cells) and intracellular Ca2+ (assessed by the FURA‐2 method) with an order of potency: ET‐1>sarafotoxin 6b (S6b)>ET‐3=S6c; the ETA‐receptor antagonist BQ‐123 inhibited both responses with apparent pKi‐values of 8.3 and 8.6, respectively, while the ETB‐receptor antagonist BQ‐788 did not. Pretreatment of the cells with pertussis toxin (PTX, 500 ng ml−1 overnight) reduced ET‐1‐induced Ca2+ increases by 46±5%, but rather enhanced ET‐1‐induced IP‐formation. Chelation of extracellular Ca2+ by 5 mm EGTA did not affect ET‐1‐induced IP‐formation. However, in the presence of 5 mm EGTA or SKF 96365, an inhibitor of receptor mediated Ca2+ influx (1.0–3.0×10−5 m) ET‐1‐induced Ca2+ increases were inhibited in normal, but not in PTX‐treated cells. [125I]‐ET‐1 binding studies as well as mRNA expression studies (by RT–PCR) detected only ETA‐receptors whereas expression of ETB‐receptor mRNA was marginal. ET‐1 (10−8 m) inhibited isoprenaline‐evoked cyclic AMP increases; this was antagonized by BQ‐123, not affected by BQ‐788 and abolished by PTX‐treatment. We conclude that SK‐N‐MC cells contain a homogeneous population of ETA‐receptors that couple to IP‐formation and inhibition of cyclic AMP formation. Stimulation of these ETA‐receptors increases intracellular Ca2+ by at least two mechanisms: a PTX‐insensitive IP‐mediated Ca2+ mobilization from intracellular stores and a PTX‐sensitive influx of extracellular Ca2+. British Journal of Pharmacology (1998) 125, 1202–1211; doi:10.1038/sj.bjp.0702208

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