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

Endothelin-1 (ET-1) activates two types of Ca -permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca channel (SOCC) in Chinese hamster ovary cells expressing endothelinA receptors (CHO-ETAR). These channels can be distinguished by their sensitivity to Ca channel blockers 1-( -[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 Ca 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-ETAR 385) or an unpalmitoylated (Cys 383 Cys3SerSer) ETAR (CHO-SerETAR), each of which is coupled with Gq or Gs/G12, respectively. In CHOSerETAR, a dominant-negative mutant of G12 inhibited AA release. SK&F 96365 inhibited ET-1–induced AA release in CHOETAR 385, whereas LOE 908 inhibited it in CHO-SerETAR. These results indicate the following: 1) ET-1–induced AA release depends on Ca 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. The release of arachidonic acid (AA) from the membrane lipids is catalyzed by phospholipase A2 (PLA2) in mammalian cells (Dennis, 1997). Hormones and growth factors including endothelin-1 (ET-1) stringently regulate PLA2 activity (Dennis, 1997; Leslie, 1997; Trevisi et al., 2002). AA is converted into other biologically active metabolites such as leukotrienes, lipoxins, prostaglandins, and thromboxanes by different enzymes. These metabolites seem to play significant roles in several important processes, including vascular contraction and cell growth (Gong et al., 1995; Anderson et al., 1997). Previous reports indicate that the key enzyme responsible for agonist-induced AA release is cytosolic PLA2 (cPLA2) (Lin et al., 1992; Roshak et al., 1994). ET-1 also induces AA release through cPLA2 activation (Trevisi et al., 2002). cPLA2 is a cytosolic 85-kDa Ca -dependent PLA2 and is activated by both an increase in intracellular free Ca concentration ([Ca ]i) and Ser-505 phosphorylation by mitogen-activated protein kinase or protein kinase C (Leslie, 1997). Extracellular Ca influx plays critical roles in the ET-1–induced AA release (Stanimirovic et al., 1994; Wu-Wong et al., 1996). However, it remains unclear what types of Ca channels are This study was supported by a grant from the Smoking Research Foundation, Japan, and by the Uehara Memorial Foundation Fellowship, Tokyo, Japan. ABBREVIATIONS: AA, arachidonic acid; AACOCF3, arachydonyl trifluoromethyl ketone; CHO, Chinese hamster ovary; [Ca 2 ]i, intracellular free Ca concentration; CHO-ETAR, Chinese hamster ovary cells expressing endothelinA receptors; CHO-ETAR 385, Chinese hamster ovary cells that express human endothelinA receptor truncated at the carboxyl-terminal downstream of Cys385; CHO-SerETAR, Chinese hamster ovary cells that express an unpalmitoylated (CysCys3SerSer) human endothelinA receptor; cPLA2, cytosolic phospholipase A2; ET-1, endothelin-1; G12G228A, dominant-negative mutant of G12; NSCC, nonselective cation channel; PI3K, phosphoinositide 3-kinase; PLA2, phospholipase A2; SOCC, store-operated Ca 2 channel; VICC, voltage-independent Ca channel; SK&F 96365, 1-( -[3-(4-methoxyphenyl) propoxy]4-methoxyphenethyl)-1H-imidazole hydrochloride; LOE 908, (R,S)-(3,4-dihydro-6,7-dimethoxy-isochinolin-1-yl)-2-phenyl-N,N-di[2-(2,3,4-trimethoxyphenyl)ethyl]acetamid mesylate; LY294002, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one; BQ123, cyclo(D-Trp-D-Asp-Pro-D-ValLeu-)Na ; BQ788, 2-6-dimethylpiperidinecarbonyl-methyl-Leu-Nin-[methoxycarbonyl]-D-Trp-D-Nle. 0026-895X/03/6403-689–695$7.00 MOLECULAR PHARMACOLOGY Vol. 64, No. 3 Copyright © 2003 The American Society for Pharmacology and Experimental Therapeutics 2542/1084481 Mol Pharmacol 64:689–695, 2003 Printed in U.S.A. 689 at A PE T Jornals on Sptem er 0, 2017 m oharm .aspeurnals.org D ow nladed from involved in ET-1–induced AA release. These uncertainties are mainly caused by the lack of specific Ca -channel blockers. We have recently shown that a sustained increase in [Ca ]i caused by ET-1 results from Ca 2 entry through three types of voltage-independent Ca channel (VICC) into CHO cells expressing ETAR (CHO-ETAR): two types of Ca 2 permeable nonselective cation channels (designated NSCC-1 and NSCC-2) and a store-operated Ca channel (SOCC) (Kawanabe et al., 2001). In particular, these channels can be distinguished using Ca -channel blockers such as SK&F 96365 and LOE 908. NSCC-1 is sensitive to LOE 908 and resistant to SK&F 96365; NSCC-2 is sensitive to both LOE 908 and SK&F 96365, and the SOCC is resistant to LOE 908 and sensitive to SK&F 96365 (Kawanabe et al., 2001). Thus, SK&F 96365 and LOE 908 may be useful for identifying which Ca channels are involved in ET-1–induced AA release in CHO-ETAR. Moreover, phosphoinositide 3-kinase (PI3K) was reported to be involved in the angiotensin IIinduced cPLA2 activation and AA release in vascular smooth muscle cells (Silfani and Freeman, 2002). PI3K plays essential roles in the activation of NSCC-2 and SOCC by ET-1 in CHO-ETAR (Kawanabe et al., 2002a). Therefore, we examined the effects of PI3K on ET-1–induced AA release in CHO-ETAR. Biological actions of ET-1 are mediated by two distinct receptor subtypes, ETAR and ETBR, that belong to a family of G protein-coupled receptors (Arai et al., 1990; Sakurai et al., 1990). ETAR are functionally coupled with Gq, Gs, and G12 in CHO cells (Aramori and Nakanishi, 1992; Kawanabe et al., 2002c). Therefore, in the present study, we investigated which G protein subtypes were involved in ET-1–induced AA release. For this purpose, we used a dominant-negative mutant of G12 (G12G228A) and two types of mutated ETAR designated ETAR 385 and SerETAR to clarify the involvement of Gq, Gs, and G12 in ET-1–induced AA release. ETAR 385 lacks a C terminus downstream of Cys and couples only with Gq in CHO cells (Kawanabe et al., 2002c). SerETAR is unpalmitoylated because of substitution of all of the cysteine-to-serine residues (CysCys3SerSer) and couples with Gs and G12 in CHO cells (Kawanabe et al., 2002c). Moreover, ET-1 activates SOCC in CHO-ETAR 385 and NSCC-1 in CHOSerETAR (Kawanabe et al., 2002b). Materials and Methods Cell Culture. We used CHO-ETAR, CHO-ETAR 385, and CHOSerETAR, which were constructed as described previously (Kawanabe et al., 2002b,c). CHO cells were maintained in Ham’s F-12 medium supplemented with 10% fetal calf serum under a humidified 5% CO2/95% air atmosphere. [H]Arachidonic Acid Release. The level of [H]arachidonic acid release was determined as described previously (Perez et al., 1993). Briefly, cells in 100-mm dishes were incubated overnight with [H]arachidonic acid (final concentration, 1 Ci/ml). After washing, ET-1 was added for 5 min. The medium was then removed, acidified with 100 l of 1 N formic acid, and extracted with 3 ml of chloroform. The extracts were evaporated to dryness, resuspended in 50 l of chloroform, and applied to silica gel plates for thin-layer chromatography (Merck, Darmstadt, Germany). The plates were developed in heptane/diethyl ether/acetic acid/water (v/v, 75:25:4). The distance of movement was visualized with iodine vapor. The location of arachidonic acid was verified with the use of a purified arachidonic acid (PerkinElmer Life Sciences, Boston, MA). The plate was scraped, and the radioactivity was counted with use of a liquid scintillation