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

1 The mechanism underlying endothelin-l (ET-l)-induced increases in intracellular Ca2+ concentrations in the human neuroblastoma cell-line SK-N-MC was investigated. 2 ET-receptor agonists increased inositol phosphate (IP)-formation (assessed as accumulation of total [H-3]-IPs in [H-3]-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 pK(i)-values of 8.3 and 8.6, respectively, while the ETB-receptor antagonist BQ-788 did not. 3 Pretreatment of the cells with pertussis toxin (PTX, 500 ng ml(-1) overnight) reduced ET-l-induced Ca2+ increases by 46+/-5%, but rather enhanced ET-l-induced IF-formation. 4 Chelation of extracellular Ca2+ by 5 mM EGTA did not affect ET-l-induced IF-formation. However, in the presence of 5 mM EGTA or SKF 96365, an inhibitor of receptor mediated Ca2+ influx (1.0-3.0x10(-5) M) ET-l-induced Ca2+ increases were inhibited in normal, but not in PTX-treated cells. 5 [I-125]-ET-1 binding studies as well as mRNA expression studies (by RT-FCR) detected only ET(A-)receptors whereas expression of ETB-receptor mRNA was marginal. 6 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. 7 We conclude that SK-N-MC cells contain a homogeneous population of ETA-receptors that couple to IF-formation and inhibition of cyclic AMP formation. Stimulation of these ETA-receptors increases intracellular Ca2+ by at least two mechanisms: a PTX-insensitive IF-mediated Ca2+ mobilization from intracellular stores and a PTX-sensitive influx of extracellular Ca2+.