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Endothelin-1 Activates Endothelial Cell Nitric-oxide Synthase via Heterotrimeric G-protein βγ Subunit Signaling to Protein Kinase B/Akt

2003; Elsevier BV; Volume: 278; Issue: 50 Linguagem: Inglês

10.1074/jbc.m306930200

1083-351X

Songling Liu, Richard T. Premont, Christopher D. Kontos, Jianhua Huang, Don C. Rockey,

Neutrophil, Myeloperoxidase and Oxidative Mechanisms

Endothelin-1 has dual vasoactive effects, mediating vasoconstriction via ETA receptor activation of vascular smooth muscle cells and vasorelaxation via ETB receptor activation of endothelial cells. Although it is commonly accepted that endothelin-1 binding to endothelial cell ETB receptors stimulates nitric oxide (NO) synthesis and subsequent smooth muscle relaxation, the signaling pathways downstream of ETB receptor activation are unknown. Here, using a model in which we have utilized isolated primary endothelial cells, we demonstrate that ET-1 binding to sinusoidal endothelial cell ETB receptors led to increased protein kinase B/Akt phosphorylation, endothelial cell nitric-oxide synthase (eNOS) phosphorylation, and NO synthesis. Furthermore, eNOS activation was not dependent on tyrosine phosphorylation, and pretreatment of endothelial cells with pertussis toxin as well as overexpression of a dominant negative G-protein-coupled receptor kinase construct that sequesters βγ subunits inhibited Akt phosphorylation and NO synthesis. Taken together, the data elucidate a G-protein-coupled receptor signaling pathway for ETB receptor-mediated NO production and call attention to the absolute requirement for heterotrimeric G-protein βγ subunits in this cascade. Endothelin-1 has dual vasoactive effects, mediating vasoconstriction via ETA receptor activation of vascular smooth muscle cells and vasorelaxation via ETB receptor activation of endothelial cells. Although it is commonly accepted that endothelin-1 binding to endothelial cell ETB receptors stimulates nitric oxide (NO) synthesis and subsequent smooth muscle relaxation, the signaling pathways downstream of ETB receptor activation are unknown. Here, using a model in which we have utilized isolated primary endothelial cells, we demonstrate that ET-1 binding to sinusoidal endothelial cell ETB receptors led to increased protein kinase B/Akt phosphorylation, endothelial cell nitric-oxide synthase (eNOS) phosphorylation, and NO synthesis. Furthermore, eNOS activation was not dependent on tyrosine phosphorylation, and pretreatment of endothelial cells with pertussis toxin as well as overexpression of a dominant negative G-protein-coupled receptor kinase construct that sequesters βγ subunits inhibited Akt phosphorylation and NO synthesis. Taken together, the data elucidate a G-protein-coupled receptor signaling pathway for ETB receptor-mediated NO production and call attention to the absolute requirement for heterotrimeric G-protein βγ subunits in this cascade. Nitric oxide (NO) 1The abbreviations used are: NOnitric oxideNOSnitric-oxide synthaseET-1endothelin-1GPCRG-protein-coupled receptorPI 3-kinasephosphoinositide 3-kinaseMAPKmitogen-activated protein kinasePDGFplatelet-derived growth factorPTXpertussis toxinAdadenovirusPTENphosphatase and tensin homolog of chromosome 10GRK2CTG-protein-coupled receptor kinase 2 carboxyl terminus. is produced from l-arginine by one of three nitric-oxide synthase (NOS) isoforms, encoded by at least three different genes (1.Moncada S. Higgs A. N. Engl. J. Med. 1993; 329: 2002-2012Crossref PubMed Scopus (5758) Google Scholar, 2.Michel T. Feron O. J. Clin. Invest. 1997; 100: 2146-2152Crossref PubMed Scopus (853) Google Scholar). The isoform most important in vascular homeostasis, endothelial cell NO synthase (eNOS) (3.Huang P.L. Huang Z. Mashimo H. Bloch K.D. Moskowitz M.A. Bevan J.A. Fishman M.C. 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Lipid products of PI 3-kinase have been implicated in diverse cellular processes such as membrane ruffling and cell growth (32.Krugmann S. Welch H. Curr. Biol. 1998; 8: R828Abstract Full Text Full Text PDF PubMed Google Scholar). Downstream of PI 3-kinase is the serine-threonine kinase, protein kinase B/Akt, which like PI 3-kinase has been implicated in a wide array of cellular processes (33.Coffer P.J. Jin J. Woodgett J.R. Biochem. J. 1998; 335: 1-13Crossref PubMed Scopus (969) Google Scholar, 34.Brazil D.P. Hemmings B.A. Trends Biochem. Sci. 2001; 26: 657-664Abstract Full Text Full Text PDF PubMed Scopus (1043) Google Scholar, 35.Brazil D.P. Park J. Hemmings B.A. Cell. 2002; 111: 293-303Abstract Full Text Full Text PDF PubMed Scopus (488) Google Scholar). Moreover, recent data suggest that the Akt pathway may be involved in endothelin signaling pathways that control cell survival (36.Su X. Wang P. Ibitayo A. Bitar K.N. Am. J. Physiol. 1999; 276: G853-G861PubMed Google Scholar, 37.Del Bufalo D. Di Castro V. Biroccio A. Varmi M. Salani D. Rosano L. Trisciuoglio D. Spinella F. Bagnato A. Mol. Pharmacol. 2002; 61: 524-532Crossref PubMed Scopus (139) Google Scholar), although the mechanism of this connection has not been established. The relationship between NO and endothelins in the control of vascular tone, although physiologically important (38.Luscher T.F. Yang Z. Tschudi M. von Segesser L. Stulz P. Boulanger C. Siebenmann R. Turina M. Buhler F.R. Circ. Res. 1990; 66: 1088-1094Crossref PubMed Scopus (260) Google Scholar), is incompletely understood. In particular, the link between ETB receptor activation and NO synthesis and the signaling cascade that leads to NO genesis remains poorly characterized. Given data suggesting that GPCRs can signal to PI 3-kinase, and that ET-1 is capable of signaling to Akt, we have hypothesized that one potential link between ET-1, ETB receptor, and NO synthesis is the Akt signaling pathway. We have examined primary isolates of sinusoidal endothelial cells, which, as for vascular endothelial cells, are known to possess only ETB receptors and to express eNOS (39.Rockey D.C. Chung J.J. Gastroenterology. 1998; 114: 344-351Abstract Full Text Full Text PDF PubMed Scopus (333) Google Scholar, 40.Housset C. Rockey D.C. Bissell D.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 9266-9270Crossref PubMed Scopus (291) Google Scholar, 41.Sakurai T. Yanagisawa M. Takuwa Y. Miyazaki H. Kimura S. Goto K. Masaki T. Nature. 1990; 348: 732-735Crossref PubMed Scopus (2366) Google Scholar, 42.Lamas S. Marsden P.A. Li G.K. Tempst P. Michel T. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 6348-6352Crossref PubMed Scopus (921) Google Scholar) and show that ET-1 binding to the ETB receptor causes eNOS activation via Akt phosphorylation, with resultant downstream eNOS phosphorylation and NO synthesis. Furthermore, we find a crucial role for G-protein βγ subunits in the endothelin/NO signaling cascade. Materials—ET-1 and sarafotoxin S6C (STX) were from American Peptide Co. Inc. (Sunnyvale, CA). Recombinant human PDGF-BB was from R&D Systems (Minneapolis, MN). Pertussis toxin (PTX) was from Calbiochem (La Jolla, CA), and LY294002 was purchased from Cell Signaling Technology (St. Louis, MO). Polyclonal anti-total-Akt, antiphospho-Akt (Ser-473) antibodies were from Cell Signaling Technologies (Beverly, MA). Monoclonal anti-GRK2/3 antibody was from Upstate Cell Signaling Solutions (Waltham, MA). Anti-phospho-eNOS (Ser-1179) antibody was from BD Transduction Laboratories (Lexington, KY). Anti-rabbit IgG/horseradish peroxidase conjugate or anti-mouse IgG/horseradish peroxidase conjugate were from Promega (Madison, WI). Cell Isolation and Culture—Preparations of sinusoidal endothelial cells were from male Sprague-Dawley rat (450–500 g) (Harlan Sprague-Dawley, Indianapolis, IN) as described previously (43.Pitas R.E. Boyles J. Mahley R.W. Bissell D.M. J. Cell Biol. 1985; 100: 103-117Crossref PubMed Scopus (156) Google Scholar). In brief, after in situ perfusion of the liver with 20 mg% Pronase (Roche Molecular Biochemicals, Indianapolis, IN), followed by collagenase (Crescent Chemical, Hauppauge, NY), dispersed cell suspensions were layered on a discontinuous density gradient of 8.2% and 15.6% Accudenz (Accurate Chemical and Scientific, Westbury, NY). Endothelial cells, present in the lower layer, were further purified by centrifugal elutriation (18 ml/min flow) and were grown in medium containing 20% serum (10% horse/calf). To verify the purity of endothelial cells, we routinely document their uptake of fluorescently labeled di-I-acetoacetylated low density lipoprotein as described (43.Pitas R.E. Boyles J. Mahley R.W. Bissell D.M. J. Cell Biol. 1985; 100: 103-117Crossref PubMed Scopus (156) Google Scholar). Additionally, endothelial isolates were probed with anti-CD31 (BD Biosciences, San Diego, CA). Contamination with stellate cells and/or Kupffer cells was detected by immunolabeling with anti-desmin (Dako, Carpenteria, CA) (44.Yokoi Y. Namihisa T. Kuroda H. Komatsu I. Miyazaki A. Watanabe S. Usui K. Hepatology. 1984; 4: 709-714Crossref PubMed Scopus (345) Google Scholar) and a specific antibody (45.Bodenheimer H.C.J. Faris R.A. Charland C. Hixson D.C. Hepatology. 1988; 8: 1667-1672Crossref PubMed Scopus (22) Google Scholar) as described (46.Rockey D.C. Chung J.J. J. Investig. Med. 1994; 42: 660-670PubMed Google Scholar). These methods demonstrated that the purity of primary endothelial cell isolates was greater than 95%; all experiments were performed with primary cells. Adenoviral Gene Transfer—Recombinant adenovirus particles were purified from infected 293 cells by lysis in virus storage buffer followed by two sequential rounds of ultracentrifugation in CsCl gradients. Viral titers were measured by standard plaque assay using 293 cells. Sinusoidal endothelial cells were infected with a replication defective adenovirus that expresses myristoylated constitutively active Akt (Ad-myr.Akt), a dominant negative Akt construct (Ad-dn.Akt) containing mutations at amino acid 308 (T → A) and amino acid 473 (S → A) (provided by Dr. Ken Walsh, Boston University Medical Center) (47.Fujio Y. Guo K. Mano T. Mitsuuchi Y. Testa J.R. Walsh K. Mol. Cell. Biol. 1999; 19: 5073-5082Crossref PubMed Scopus (188) Google Scholar) or an identical adenovirus without vector DNA (Ad-EV) at a multiplicity of infection (m.o.i.) of 250. Recombinant adenovirus containing a construct encoding a cDNA corresponding to the carboxyl terminus fragment of bovine GRK2 (48.Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar) (Ad-GRK2CT) was used to infect cells (also m.o.i. of 250). Recombinant adenovirus containing a construct encoding a cDNA corresponding to PTEN, and an identical adenovirus without cDNA was as described (49.Huang J. Kontos C.D. J. Biol. Chem. 2002; 277: 10760-10766Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar) (each at an m.o.i. of 100). For cellular transduction of adenovirus, sinusoidal endothelial cell were plated at a density of 1 × 106 cells/ml in 35- or 60-mm collagen-coated culture dishes and exposed to adenovirus in 2% serum for 16 h at 37 °C. Subsequently, adenovirus-containing medium was exchanged and cells were harvested at the indicated time points. NO Measurement—Medium was collected from sinusoidal endothelial cells after indicated treatment. To assess NO production, we analyzed the release of nitrite, the stable breakdown product of NO, using a Sievers Chemiluminescence NO Analyzer (Sievers Instruments, Inc., Boulder, CO). Measurements of known concentrations of nitrite were used to generate a standard curve between 25 and 500 pmol of nitrite. Immunoblotting—Sinusoidal endothelial cells were washed twice with ice-cold phosphate-buffered saline, and total cell lysates were prepared by scraping cells in lysis buffer (137 mm NaCl, 2 mm EDTA, 10% glycerol, 1% Triton X-100, 20 mm Tris-HCl, pH 8.0) containing protease inhibitors (1 mm sodium orthovanadate, 1 mm phenylmethylsulfonyl fluoride, 1 mm NaF, 1 μg/ml leupeptin, 1 μg/ml pepstatin). Lysates were subjected to SDS-PAGE, proteins were transferred to nitrocellulose, and specific epitopes were detected with specific primary antibody. Bound primary antibody was detected using a chemiluminescent detection kit (Tropix, Inc., Bedford, MA) over a linear range. Specific bands were scanned and quantitated by densitometry (Quantity One, Bio-Rad). Statistics—All results were expressed as the mean ± S.E. All experiments were performed in replicates utilizing cell isolates from different rats. Statistical analysis was performed using the two-tailed Student's t test, and p < 0.05 was considered statistically significant. Endothelin Receptor Activation Leads to Akt Phosphorylation—We initially investigated whether endothelin receptor activation led to Akt phosphorylation in sinusoidal endothelial cells. Exposure of sinusoidal endothelial cells to ET-1 (which has equal affinity for either the ETA or ETB receptor) or sarafotoxin S6C (which binds only to the ETB receptor) (50.Sakurai T. Yanagisawa M. Masaki T. Trends. Pharmacol. Sci. 1992; 13: 103-108Abstract Full Text PDF PubMed Scopus (621) Google Scholar), led to significant increases in Akt phosphorylation at serine 473 (Fig. 1, A and B). PDGF leads to phosphorylation of tyrosine residues (Tyr-740 and Tyr-751) on the PDGF B-receptor and activates the lipid kinase activity of PI 3-kinases, formation of PI (3,4,5)-P3 and activation of Akt. Importantly, PDGF also stimulated Akt phosphorylation, consistent with previous data (9.Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3056) Google Scholar, 10.Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2239) Google Scholar). As expected, blockade of the ETB receptor with the specific ETB receptor antagonist BQ-788 inhibited ET-1-mediated Akt phosphorylation (Fig. 1B), emphasizing that in sinusoidal endothelial cells, ET-1-mediated Akt activation proceeds via ETB activation rather than ETA receptor signaling. A unique property of the endothelin and endothelin receptor interaction is tight binding and a slow rate of dissociation (51.Hirata Y. Yoshimi H. Takaichi S. Yanagisawa M. Masaki T. FEBS Lett. 1988; 239: 13-17Crossref PubMed Scopus (216) Google Scholar). This property results in prolonged physiologic activity of ET-1, despite a relatively short half-life (52.Anggard E. Galton S. Rae G. Thomas R. McLoughlin L. de Nucci G. Vane J.R. J. Cardiovasc. Pharmacol. 1989; 13 (–S49; discussion S74): S46Crossref PubMed Scopus (160) Google Scholar). Furthermore, dissociation rates and signaling activity vary among cells from different tissues (53.Galron R. Bdolah A. Kochva E. Wollberg Z. Kloog Y. Sokolovsky M. FEBS Lett. 1991; 283: 11-14Crossref PubMed Scopus (24) Google Scholar). In sinusoidal endothelial cells, Akt phosphorylation occurred within minutes of endothelin receptor binding, peaked at 30 min, and subsequently declined (Fig. 1C), consistent with rapid signaling as well as rapid induction of inhibitory pathways. ETB Activation Induces eNOS Phosphorylation and NO Production—A number of studies have indicated that endothelial nitric-oxide synthase (eNOS) is an important Akt substrate and that eNOS is activated by phosphorylated Akt (9.Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3056) Google Scholar, 10.Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2239) Google Scholar). Therefore, we examined whether ET-1-induced Akt activation led to phosphorylation of eNOS as well as NO release in sinusoidal endothelial cells. We found that ETB receptor activation led to Akt phosphorylation and caused eNOS to be phosphorylated at serine 1179 (Fig. 2A). ET-1 induced not only Akt phosphorylation, but it also led to production of nitrite, indicative of NO synthesis (Fig. 2B). Likewise, stimulation of ETB receptors with sarafotoxin S6C led to eNOS phosphorylation (not shown) and NO synthesis (Fig. 2B). Interestingly, PDGF did not induce NO production in sinusoidal endothelial cells (Fig. 2B). Furthermore, exposure to endothelins and PDGF did not alter the level of total Akt protein expression or total eNOS protein expression (Fig. 1A). These results further substantiate the finding that ET-1 causes NO production in sinusoidal endothelial cells as a result of Akt activation and eNOS phosphorylation. ET-1 Induces NO Synthesis via a PI 3-Kinase/Akt Pathway—To further characterize the role of the PI 3-kinase/Akt pathway in endothelin-mediated NO synthesis, we examined ETB-induced Akt and eNOS phosphorylation and NO synthesis after PI 3-kinase inhibition. LY29004, a specific inhibitor of PI 3-kinase, prevented ET-1-mediated Akt phosphorylation (Fig. 3A), including at all time points after exposure to ET-1 (Fig. 3B). Additionally, LY29004 efficiently inhibited NO production (Fig. 3C); of note, NO appeared to be produced rapidly after ETB receptor activation (nitrite levels were measured in conditioned medium so that total nitrite levels increased incrementally over time). As expected, LY29004 inhibited PDGF-induced Akt phosphorylation (Fig. 3A) but had no effect on total Akt expression. Modulation of PI 3-Kinase Activation by PTEN—The inositol 3′-phosphatase, PTEN, which hydrolyzes PI 3-kinase lipid products phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate, functions in opposition to PI 3-kinase (54.Myers M.P. Stolarov J.P. Eng C. Li J. Wang S.I. Wigler M.H. Parsons R. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9052-9057Crossref PubMed Scopus (738) Google Scholar, 55.Cantley L.C. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4240-4245Crossref PubMed Scopus (1754) Google Scholar). Therefore, to further assess the role of ET-1 in activation of PI 3-kinase, we examined the effect of overexpression of a wild-type PTEN construct in sinusoidal endothelial cells stimulated with ET-1. ET-1-mediated phosphorylation of Akt in sinusoidal endothelial cells was inhibited by overexpression of wild-type PTEN (Fig. 4), further confirming that ET-1-mediated activation of Akt is PI 3-kinase-dependent. A Direct Effect of Akt on NO Production—To assess the specific role of Akt activation in ETB receptor-mediated signaling to NO, we examined the effect of dominant negative and dominant active Akt constructs (47.Fujio Y. Guo K. Mano T. Mitsuuchi Y. Testa J.R. Walsh K. Mol. Cell. Biol. 1999; 19: 5073-5082Crossref PubMed Scopus (188) Google Scholar, 56.Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 639-650Crossref PubMed Scopus (2125) Google Scholar). Expression of a dominant negative Akt construct inhibited ET-1-mediated Akt phosphorylation (Fig. 5A) and NO production (Fig. 5B). As expected, overexpression of dominant active Akt led to enhanced Akt phosphorylation; this subsequently led to an increase in NO synthesis (Fig. 5, A and B). A negative control (an identical adenovirus vector without insert DNA) failed to induce Akt phosphorylation or NO synthesis. Interestingly, the additional effect of ET-1 on both Akt phosphorylation and NO synthesis was minimal when active Akt was expressed (Fig. 5, A and B). ET-1 Stimulated Akt Phosphorylation Is Pertussis Toxin-sensitive—Sequence homology and hydropathic analysis of endothelin receptor primary structure suggests that the known endothelin receptors belong to the rhodopsin class of GPCRs (41.Sakurai T. Yanagisawa M. Takuwa Y. Miyazaki H. Kimura S. Goto K. Masaki T. Nature. 1990; 348: 732-735Crossref PubMed Scopus (2366) Google Scholar, 56.Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. 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This prevents these specific G-proteins from interacting with their activating receptors and thus prevents propagation of downstream receptor signals. We found that PTX pretreatment inhibited ET-1-induced Akt phosphorylation (Fig. 6), suggesting that Akt phosphorylation after ETB receptor stimulation is linked to Gi/o. Notably, PTX did not influence basal Akt or phospho-Akt levels (Fig. 6). G-proteins Are Critically Linked to NO Signaling—We demonstrated that ET-1 stimulates Akt and eNOS through a Gi/o-coupled mechanism. Because activation of Gi/o-coupled receptors leads to dissociation of activated Gαi/o and G-protein βγ subunits (56.Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 639-650Crossref PubMed Scopus (2125) Google Scholar, 59.Ribas C. Sato M. Hildebrandt J.D. Lanier S.M. Methods Enzymol. 2002; 344: 140-152Crossref PubMed Scopus (3) Google Scholar, 60.Clapham D.E. Neer E.J. Annu. Rev. Pharmacol. Toxicol. 1997; 37: 167-203Crossref PubMed Scopus (704) Google Scholar), we hypothesized that Akt phosphorylation was dependent on G-protein βγ subunit signaling in this system. To test this postulate, we utilized a construct coding for the carboxyl terminus of G-protein-coupled receptor kinase 2 (GRK2CT), which binds to G-protein βγ subunits and prevents their downstream signaling (48.Koch W.J. Inglese J. Stone W.C. Lefkowitz R.J. J. Biol. Chem. 1993; 268: 8256-8260Abstract Full Text PDF PubMed Google Scholar). Transduction of sinusoidal endothelial cells with an adenovirus expressing this construct abrogated ET-1-induced Akt phosphorylation and NO production (Fig. 7, A and B). These findings suggest that G-protein βγ subunits rather than Gαi/o subunits contribute to the ET-1-dependent activation of Akt. ET-1 Stimulation of eNOS Is Protein-tyrosine Kinase-independent—Non-receptor tyrosine protein kinases have been shown to exhibit cross-talk with G-protein coupled receptor-signaling cascades (31.Luttrell L.M. Ferguson S.S. Daaka Y. Miller W.E. Maudsley S. Della Rocca G.J. Lin F. Kawakatsu H. Owada K. Luttrell D.K. Caron M.G. Lefkowitz R.J. Science. 1999; 283: 655-661Crossref PubMed Scopus (1264) Google Scholar). Therefore, we postulated that non-receptor tyrosine protein kinases might have a role in ET-1-mediated signaling. However, we found that, although the tyrosine kinase inhibitor, genistein, inhibited PDGF (a well known inducer of Akt)-mediated Akt phosphorylation, it had no effect on ET-1-mediated activation of Akt (Fig. 8A). Additionally, inhibition of protein-tyrosine kinases with genistein had no effect on NO production by sinusoidal endothelial cells (Fig. 8B). These data support the concept that ET-1-mediated Akt phosphorylation and NO production is G-protein-dependent and does not involve a tyrosine kinase pathway. The endothelins comprise a family of 21-amino acid peptides that activate specific G-protein-coupled receptors in a variety of cell types to regulate blood flow and multiple other physiologic effects (61.Rubanyi G.M. Polokoff M.A. Pharmacol. Rev. 1994; 46: 325-415PubMed Google Scholar). The physiologic effects of ET-1 are mediated by at least two types of endothelin receptors, designated ETA and ETB. It is commonly accepted that stimulation of ETB receptors leads to NO production in endothelial cells. However, the signaling pathways linking the ETB receptor to NO have been poorly characterized. In this study, we have elucidated a potential mechanism linking endothelin-mediated activation of ETB receptors and eNOS (and thus, NO). We show that Akt plays a central role in this process, consistent with previous data demonstrating that Akt phosphorylation leads to eNOS phosphorylation and subsequent activation of eNOS enzymatic activity (10.Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2239) Google Scholar, 62.Luo Z. Fujio Y. Kureishi Y. Rudic R.D. Daumerie G. Fulton D. Sessa W.C. Walsh K. J. Clin. Invest. 2000; 106: 493-499Crossref PubMed Scopus (179) Google Scholar). Moreover, we reveal that heterotrimeric G-protein βγ subunits released from receptors-activated Gi/o-coupled receptors represent the critical connection to Akt, and thus eNOS. Previous data have shown that ET-1 activates several intracellular signal pathways, including adenylyl cyclase, phospholipase C, protein kinase Cs, and the mitogen-activated protein kinase (MAPK) cascades (20.Simonson M.S. Herman W.H. J. Biol. Chem. 1993; 268: 9347-9357Abstract Full Text PDF PubMed Google Scholar, 21.Herman W.H. Simonson M.S. J. Biol. Chem. 1995; 270: 11654-11661Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar, 63.Mallat A. Preaux A.M. Serradeil-Le Gal C. Raufaste D. Gallois C. Brenner D.A. Bradham C. Maclouf J. Iourgenko V. Fouassier L. Dhumeaux D. Mavier P. Lotersztajn S. J. Clin. Invest. 1996; 98: 2771-2778Crossref PubMed Scopus (102) Google Scholar). Here, we have extended the ET-1 signaling paradigm to include Akt, eNOS, and NO. We utilized several different approaches to elucidate the critical role of Akt in endothelin-mediated activation of eNOS. First, we demonstrated that Akt phosphorylation paralleled closely eNOS phosphorylation after stimulation with ET-1. Second, we showed that a dominant negative Akt construct abrogated ET-1 phosphorylation of Akt and eNOS. A central role for Akt in endothelin-mediated NO production was further supported by pharmacologic inhibition of PI 3-kinase with LY294002, because Akt activation is known to be PI 3-kinase-dependent. Finally, we demonstrated that ET-1-mediated activation of Akt was inhibited by the potent lipid phosphatase, PTEN (54.Myers M.P. Stolarov J.P. Eng C. Li J. Wang S.I. Wigler M.H. Parsons R. Tonks N.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9052-9057Crossref PubMed Scopus (738) Google Scholar, 64.Tamura M. Gu J. Matsumoto K. Aota S. Parsons R. Yamada K.M. Science. 1998; 280: 1614-1617Crossref PubMed Scopus (1082) Google Scholar), which dephosphorylates the 3′-phosphoinositide products of PI 3-kinase, phosphatidylinositol 3,4-biphosphate, and phosphatidylinositol 3,4,5-triphosphate (55.Cantley L.C. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4240-4245Crossref PubMed Scopus (1754) Google Scholar) and has been shown to regulate vascular epidermal growth factor-mediated PI 3-kinase signaling in human umbilical vein endothelial cells (49.Huang J. Kontos C.D. J. Biol. Chem. 2002; 277: 10760-10766Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar). Inhibition of endothelin-mediated eNOS activation by PTEN indicates that this process is dependent on 3-phosphoinositide production by PI 3-kinase. Extensive data from previous studies (56.Pierce K.L. Premont R.T. Lefkowitz R.J. Nat. Rev. Mol. Cell. Biol. 2002; 3: 639-650Crossref PubMed Scopus (2125) Google Scholar, 61.Rubanyi G.M. Polokoff M.A. Pharmacol. Rev. 1994; 46: 325-415PubMed Google Scholar) have linked ET-1 to canonical GPCR signaling pathways. Here, we have extended this work to emphasize the central role not only of βγ subunits, but also GRK2. Additionally, we demonstrated that PTX, which catalyzes the ADP-ribosylation of the Gi family alpha subunits and thereby interrupts Gi βγ subunit function, effectively abrogates ET-1-mediated Akt phosphorylation and NO production. Taken together, these data point to a critical role for βγ subunits in the signaling cascade. Although the data using the dominant negative GRK2CT construct, which selectively binds to and inhibits the function of βγ subunits, provides convincing evidence for the involvement of βγ subunits in the signaling cascade, it should be emphasized that our studies do not unequivocally exclude a role for Gα subunits. Selective inhibition of Gαi/o would be an attractive approach; however, it is not possible to inhibit Gαi/o without also inhibiting βγ. Nonetheless, βγ subunits appear to be necessary for Akt phosphorylation and eNOS activation after endothelin binding. Our data as well as previous work emphasize a mechanism by which GPCR activation leads to Akt phosphorylation; for example, PI 3-kinase activation appears to proceed via heterotrimeric G-protein βγ signaling (Fig. 9). Supporting the presence of this signaling pathway are previous data that demonstrate a direct interaction of βγ subunits with the catalytic domain of the PI 3-kinase γ isoform (65.Lopez-Ilasaca M. Gutkind J.S. Wetzker R. J. Biol. Chem. 1998; 273: 2505-2508Abstract Full Text Full Text PDF PubMed Scopus (87) Google Scholar). In addition, cardiac hypertrophy led to βγ subunit-dependent activation of PI 3-kinase (66.Naga Prasad S.V. Esposito G. Mao L. Koch W.J. Rockman H.A. J. Biol. Chem. 2000; 275: 4693-4698Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar). Such data are highly consistent with our findings, which emphasize a specific and novel endothelin-mediated pathway involving βγ subunit recruitment and activation of PI 3-kinase with downstream phosphorylation of Akt and eNOS. Although GPCR activation has been shown to lead to Akt activation via PI 3-kinase γ (67.Naga Prasad S.V. Barak L.S. Rapacciuolo A. Caron M.G. Rockman H.A. J. Biol. Chem. 2001; 276: 18953-18959Abstract Full Text Full Text PDF PubMed Scopus (160) Google Scholar), PI 3-kinase/Akt is well known to be linked to tyrosine kinase phosphorylation (55.Cantley L.C. Neel B.G. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4240-4245Crossref PubMed Scopus (1754) Google Scholar). For example, PDGF, a classic tyrosine kinase inducer, stimulates recruitment and activation of PI 3-kinase α via its p85 regulatory subunit and subsequent Akt phosphorylation (68.van Weering D.H. de Rooij J. Marte B. Downward J. Bos J.L. Burgering B.M. Mol. Cell. Biol. 1998; 18: 1802-1811Crossref PubMed Scopus (93) Google Scholar, 69.Kohn A.D. Takeuchi F. Roth R.A. J. Biol. Chem. 1996; 271: 21920-21926Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar). We found that endothelin-mediated Akt activation and NO production were not affected by the tyrosine kinase inhibitor genistein, supporting the observation that ETB receptors signal through PI 3-kinase γ. Furthermore, in our system, PDGF activated Akt but had no effect on eNOS. To our knowledge, this is the first report documenting a mechanism linking ETB receptor activation directly to Akt and eNOS activation in an endothelial cell system. The work extends that of others in which it has been shown that ET-1 can stimulate NO synthesis in isolated endothelial cells (14.Tsukahara H. Ende H. Magazine H.I. Bahou W.F. Goligorsky M.S. J. Biol. Chem. 1994; 269: 21778-21785Abstract Full Text PDF PubMed Google Scholar). It is notable that we have failed to identify NO production in a variety of transformed endothelial cell lines. 2S. Liu, R. T. Premont, C. D. Kontos, J. Huang, and D. C. Rockey, unpublished observation. Thus, a major advantage of the current study is that it utilized only primary cell isolates. In the context of the data presented, we further postulate that primary cells and cell lines possess dissimilar endothelin signaling pathways. Finally, the current data not only emphasize an important endothelin signaling pathway but also highlight a unique system with which to examine endothelin/NO-coupled signaling. We thank Walter Koch (Duke University Medical Center) for the kind gift of adenovirus containing GRK2CT. We thank Zhiqiang Chen for assistance with NO measurement and Xi-Lin Niu and Chunming Dong (both Duke University Medical Center) for helpful discussion.