Characterization of Ca2+ channels and G proteins involved in arachidonic acid release by endothelin-1/endothelinA receptor

Endothelin-1 (ET-1) activates two types of Ca2+-permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca2+ channel (SOCC) in Chinese hamster ovary cells expressing endothelinA receptors (CHO-ETAR). These channels can be distinguished by their sensitivity to Ca2+ channel blockers 1-(beta-3-(4-methoxyphenyl) propoxy]-4-methoxyphenethyl)-1H-imidazole hydrochloride (SK&F 96365) and (R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate (LOE 908). NSCC-1 is sensitive to LOE 908 and resistant to SK&F 96365; NSCC-2 is sensitive to both blockers, and SOCC is resistant to LOE 908 and sensitive to SK&F 96365. In this study, we examined the mechanism of ET-1-induced arachidonic acid (AA) release. Both SK&F 96365 and LOE 908 inhibited ET-1-induced AA release with the IC50 values correlated to those of ET-1-induced Ca2+ influx. Moreover, combined treatment with these blockers abolished ET-1-induced AA release. Wortmannin and LY294002, inhibitors of phosphoinositide 3-kinase (PI3K), partially inhibited ET-1-induced AA release. LOE 908, but not SK&F 96365, inhibited ET-1-induced AA release in wortmannin-treated CHO-ETAR. ET-1 also induced AA release in CHO cells expressing ETAR truncated at the carboxyl terminal downstream of Cys385 (CHO-ETARDelta385) or an unpalmitoylated (Cys383 Cys385-388--> Ser383Ser385-388) ETAR (CHO-SerETAR), each of which is coupled with Gq or Gs/G12, respectively. In CHO-SerETAR, a dominant-negative mutant of G12 inhibited AA release. SK&F 96365 inhibited ET-1-induced AA release in CHO-ETARDelta385, whereas LOE 908 inhibited it in CHO-SerETAR. These results indicate the following: 1) ET-1-induced AA release depends on Ca2+ influx through NSCC-1, NSCC-2, and SOCC in CHO-ETAR; 2) Gq and G12 mediate AA release through ETAR in CHO cells; and 3) PI3K is involved in ET-1-induced AA release, which depends on NSCC-2 and SOCC.