Angiotensin II type 2 receptor-dependent increases in nitric oxide synthase expression in the pulmonary endothelium is mediated via a G alpha i3/Ras/Raf/MAPK pathway.
We have previously reported that angiotensin II (ANG II) stimulated Src tyrosine kinase via a pertussis toxin-sensitive type 2 receptor, which, in turn, activates MAPK, resulting in an increase in nitric oxide synthase (NOS) expression in pulmonary artery endothelial cells (PAECs). The present study was designed to investigate the pathway by which ANG II activates Src leading to an increase in ERK1/ERK2 phosphorylation and an increase in NOS protein in PAECs. Transfection of PAECs with G alpha(i3) dominant negative (DN) cDNA blocked the ANG II-dependent activation of Src, ERK1/ERK2 phosphorylation, and increase in NOS expression. ANG II stimulated an increase in tyrosine phosphorylation of sequence homology of collagen (Shc; 15 min) that was prevented when PAECs were pretreated with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo-[3,4-d]pyrimidine (PP2), a Src inhibitor. ANG II induced a Src-dependent association between Shc and growth factor receptor-bound protein 2 (Grb2) and between Grb2 and son of sevenless (Sos), both of which were maximal at 15 min. The ANG II-dependent increase in Ras GTP binding was prevented when PAECs were pretreated with the AT(2) antagonist PD-123319 or with PP2 or were transfected with Src DN cDNA. ANG II-dependent activation of MAPK and the increase in endothelial NOS (eNOS) were prevented when PAECs were transfected with Ras DN cDNA or treated with FTI-277, a farnesyl transferase inhibitor. ANG II induction of Raf-1 phosphorylation was prevented when PAECs were pretreated with PD-123319 and PP2. Raf kinase inhibitor 1 prevented the ANG II-dependent increase in eNOS expression. Collectively, these data suggest that G alpha(i3), Shc, Grb2, Ras, and Raf-1 link Src to activation of MAPK and to the AT(2)-dependent increase in eNOS expression in PAECs.
Li
Jianyu
J
Dept. of Biochemistry, New York Medical College, Valhalla, NY 10595, USA.
Zhao
Xiangmin
X
Li
Xinmei
X
Lerea
Kenneth M
KM
Olson
Susan C
SC
D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D000804:Angiotensin II / Q000494:pharmacology; D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects / Q000378:metabolism; D004730:Endothelium, Vascular / Q000166:cytology / Q000378:metabolism*; D019206:GTP-Binding Protein alpha Subunits, Gi-Go / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic; D008168:Lung / Q000098:blood supply / Q000378:metabolism; D020935:MAP Kinase Signaling System; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D020033:Protein Isoforms; D016392:Proto-Oncogene Proteins pp60(c-src) / Q000378:metabolism; D011651:Pulmonary Artery / Q000166:cytology; D044139:Receptor, Angiotensin, Type 2 / Q000378:metabolism*; D055765:Shc Signaling Adaptor Proteins; D048490:raf Kinases / Q000378:metabolism*; D018631:ras Proteins / Q000378:metabolism*
D048868:Adaptor Proteins, Signal Transducing; D020033:Protein Isoforms; D044139:Receptor, Angiotensin, Type 2; D055765:Shc Signaling Adaptor Proteins; D000804:Angiotensin II; D009569:Nitric Oxide; D052250:Nitric Oxide Synthase Type III; D016392:Proto-Oncogene Proteins pp60(c-src); D048490:raf Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D019206:GTP-Binding Protein alpha Subunits, Gi-Go; D018631:ras Proteins