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

Abstract

1. The mechanism underlying endothelin-1 (ET-1)-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 [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 ET_A-receptor antagonist BQ-123 inhibited both responses with apparent pK_i-values of 8.3 and 8.6, respectively, while the ET_B-receptor antagonist BQ-788 did not.

3. 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.

4. 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.

5. [¹²⁵I]-ET-1 binding studies as well as mRNA expression studies (by RT–PCR) detected only ET_A-receptors whereas expression of ET_B-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 ET_A-receptors that couple to IP-formation and inhibition of cyclic AMP formation. Stimulation of these ET_A-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⁺.

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