Vol. 62, Issue 3, 756-761, September 2002

Effects of Phosphoinositide 3-Kinase on the Endothelin-1-Induced Activation of Voltage-Independent Ca²⁺ Channels and Mitogenesis in Chinese Hamster Ovary Cells Stably Expressing Endothelin_A Receptor

Yoshifumi Kawanabe, Nobuo Hashimoto, and Tomoh Masaki

Departments of Neurosurgery (Y.K., N.H.) and Pharmacology (Y.K., T.M.), Kyoto University Faculty of Medicine, Kyoto, Japan

We recently demonstrated that endothelin-1 (ET-1) activates two types of Ca²⁺-permeable nonselective cation channel (designated NSCC-1 and NSCC-2) and a store-operated Ca²⁺ channel (SOCC) in Chinese hamster ovary cells expressing endothelin_A receptor (CHO-ET_AR). In addition, these channels can be discriminated using Ca²⁺ channel blockers (R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate (LOE 908) and 1-(-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl)-1H-imidazole (SK&F 96365). LOE 908 is a blocker of NSCC-1 and NSCC-2, whereas SK&F 96365 is a blocker of SOCC and NSCC-2. In this study, we investigated the effects of phosphoinositide 3-kinase (PI3K) on the ET-1-induced activation of these channels and mitogenesis in CHO-ET_AR using wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY 294002), inhibitors of phosphoinositide 3-kinase (PI3K). ET-1-induced Ca²⁺ influx was partially inhibited in CHO-ET_AR pretreated with wortmannin or LY 294002. In contrast, addition of wortmannin or LY 294002 after stimulation with ET-1 did not suppress Ca²⁺ influx. The Ca²⁺ channels activated by ET-1 in wortmannin or LY 294002-treated CHO-ET_AR were sensitive to LOE 908 and resistant to SK&F 96365. Wortmannin also partially inhibited ET-1-induced mitogenesis. LOE 908, but not SK&F 96365, abolished the wortmannin-resistant part of mitogenesis. The IC₅₀ values (~30 nM) of wortmannin for the ET-1-induced Ca²⁺ influx and mitogenesis were similar to those for the ET-1-induced PI3K activation. In conclusion, NSCC-2 and SOCC are stimulated by ET-1 via PI3K-dependent cascade, whereas NSCC-1 is stimulated via PI3K-independent cascade. Moreover, PI3K seems to be required for the activation of the Ca²⁺ entry, but not for its maintenance. In addition, PI3K is involved in the ET-1-induced mitogenesis that depends on the extracellular Ca²⁺ influx through SOCC and NSCC-2.