Heinroth-Hoffmann I, Vogelsang M, Schiewe P, Morawietz H, Holtz J, Pönicke K, Brodde O E
Institute of Pharmacology and Toxicology, University of Halle-Wittenberg, Halle (Saale), Germany.
Br J Pharmacol. 1998 Nov;125(6):1202-11. doi: 10.1038/sj.bjp.0702208.
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+.
研究了内皮素-1（ET-1）诱导人神经母细胞瘤细胞系SK-N-MC细胞内钙离子浓度升高的机制。ET受体激动剂可增加肌醇磷酸（IP）的生成（通过[3H]-肌醇预标记细胞中总[3H]-IP的积累来评估）和细胞内钙离子浓度（通过FURA-2法评估），其效力顺序为：ET-1＞萨拉托辛6b（S6b）＞ET-3 = S6c；ETA受体拮抗剂BQ-123可抑制这两种反应，其表观pKi值分别为8.3和8.6，而ETB受体拮抗剂BQ-788则无此作用。用百日咳毒素（PTX，500 ng ml(-1) 过夜）预处理细胞可使ET-1诱导的钙离子浓度升高降低46±5%，但却增强了ET-1诱导的IP生成。5 mM EGTA螯合细胞外钙离子对ET-1诱导的IP生成无影响。然而，在存在5 mM EGTA或受体介导的钙离子内流抑制剂SKF 96365（1.0 - 3.0 x 10(-5) M）的情况下，ET-1诱导的钙离子浓度升高在正常细胞中受到抑制，但在PTX处理的细胞中则不受影响。[125I]-ET-1结合研究以及mRNA表达研究（通过RT-PCR）仅检测到ETA受体，而ETB受体mRNA的表达微乎其微。ET-1（10(-8) M）可抑制异丙肾上腺素诱发的环磷酸腺苷（cAMP）升高；这一作用可被BQ-123拮抗，不受BQ-788影响，且经PTX处理后消失。我们得出结论，SK-N-MC细胞含有同质性的ETA受体群体，这些受体与IP生成及环磷酸腺苷生成的抑制相关联。刺激这些ETA受体可通过至少两种机制增加细胞内钙离子浓度：一种是对PTX不敏感的由IP介导的细胞内钙库钙离子动员，另一种是对PTX敏感的细胞外钙离子内流。
