Mechanism of ET A ‐receptor stimulation‐induced increases in intracellular Ca 2+ in SK‐N‐MC cells

• Published in: British journal of pharmacology Vol. 125; no. 6; pp. 1202 - 1211
• Main Authors: Heinroth‐Hoffmann, Ingrid, Vogelsang, Magdalene, Schiewe, Pia, Morawietz, Henning, Holtz, Jürgen, Pönicke, Klaus, Brodde, Otto‐Erich
• Format: Journal Article
• Language: English
• Published: 10.02.2009
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1038/sj.bjp.0702208

The mechanism underlying endothelin‐1 (ET‐1)‐induced increases in intracellular Ca 2+ 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 [ 3 H]‐IPs in [ 3 H]‐myo‐inositol prelabelled cells) and intracellular Ca 2+ (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 p K i ‐values of 8.3 and 8.6, respectively, while the ET B ‐receptor antagonist BQ‐788 did not. Pretreatment of the cells with pertussis toxin (PTX, 500 ng ml −1 overnight) reduced ET‐1‐induced Ca 2+ increases by 46±5%, but rather enhanced ET‐1‐induced IP‐formation. Chelation of extracellular Ca 2+ by 5 m m EGTA did not affect ET‐1‐induced IP‐formation. However, in the presence of 5 m m EGTA or SKF 96365, an inhibitor of receptor mediated Ca 2+ influx (1.0–3.0×10 −5   m ) ET‐1‐induced Ca 2+ increases were inhibited in normal, but not in PTX‐treated cells. [ 125 I]‐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. 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 ET A ‐receptors that couple to IP‐formation and inhibition of cyclic AMP formation. Stimulation of these ET A ‐receptors increases intracellular Ca 2+ by at least two mechanisms: a PTX‐insensitive IP‐mediated Ca 2+ mobilization from intracellular stores and a PTX‐sensitive influx of extracellular Ca 2+ . British Journal of Pharmacology (1998) 125 , 1202–1211; doi: 10.1038/sj.bjp.0702208