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+.
Heinroth-Hoffmann
I
I
Institute of Pharmacology and Toxicology, University of Halle-Wittenberg, Halle (Saale), Germany.
Vogelsang
M
M
Schiewe
P
P
Morawietz
H
H
Holtz
J
J
Pönicke
K
K
Brodde
O E
OE
D000318:Adrenergic beta-Agonists / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D004347:Drug Interactions; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1 / Q000378:metabolism / Q000494:pharmacology; D019333:Endothelin-2 / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D007295:Inositol Phosphates / Q000096:biosynthesis; D007457:Iodine Radioisotopes; D007545:Isoproterenol / Q000494:pharmacology; D009447:Neuroblastoma / Q000378:metabolism* / Q000648:ultrastructure; D010456:Peptides, Cyclic / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin / Q000819:agonists* / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured; D014662:Vasoconstrictor Agents / Q000494:pharmacology; D014757:Viper Venoms / Q000494:pharmacology
D000318:Adrenergic beta-Agonists; D065128:Endothelin Receptor Antagonists; D019332:Endothelin-1; D019333:Endothelin-2; D007295:Inositol Phosphates; D007457:Iodine Radioisotopes; D010456:Peptides, Cyclic; D012333:RNA, Messenger; D044022:Receptor, Endothelin A; D017466:Receptors, Endothelin; D014662:Vasoconstrictor Agents; D014757:Viper Venoms; C057354:sarafotoxins s6; D000242:Cyclic AMP; D007545:Isoproterenol; C072247:cyclo(Trp-Asp-Pro-Val-Leu); D002118:Calcium