Characterization of Ca2+ Channels and G Proteins Involved in Arachidonic Acid Release by Endothelin-1/EndothelinA Receptor

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Abstract
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-(β-[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-di[2-(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-ETARΔ385) 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-ETARΔ385, 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.
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