Mechanism of ET(A)-receptor stimulation-induced increases in intracellular Ca2+ in SK-N-MC cells.

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 x 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+.