Angiotensin II type 2 receptor-dependent increases in nitric oxide synthase expression in the pulmonary endothelium is mediated via a Gα_i3/Ras/Raf/MAPK pathway

Abstract

SC. Angiotensin II type 2 receptor-dependent increases in nitric oxide synthase expression in the pulmonary endothelium is mediated via a G␣i3/Ras/Raf/MAPK pathway. Am J Physiol Cell Physiol 292: C2185-C2196, 2007. First published February 28, 2007 doi:10.1152/ajpcell.00204.2006.-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␣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 AT2 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␣i3, Shc, Grb2, Ras, and Raf-1 link Src to activation of MAPK and to the AT2-dependent increase in eNOS expression in PAECs.Src; mitogen-activated protein kinase ANGIOTENSIN II (ANG II), a key regulator of fluid homeostasis, vascular tone, and cell growth (7), exerts its physiological affects by binding to two major G protein-coupled receptor subtypes, type 1 (AT 1 ) and type 2 (AT 2 ). The AT 1 receptor, which mediates vasoconstriction, water and sodium intake, and induction of cell growth, is linked to the activation of many signaling molecules (7, 18) including GTP-binding proteins, PLC, PKC, phosphatidylinositol 3-kinase, ERK1/2, and tyrosine kinases. While the AT 2 receptor is linked to neuronal differentiation, apoptosis, inhibition of cell growth, and regulation of blood pressure (6), there is much controversy in the literature regarding the signaling pathways downstream of the AT 2 receptor. Investigators have demonstrated that the AT 2 receptor is linked to both the activation and inhibition of protein phosphatases (15,16,38) and as well as ERK1/2 phosphorylation (11, 37). Originally, the AT 2 ...

International Union of Basic and Clinical Pharmacology. XCIX. Angiotensin Receptors: Interpreters of Pathophysiological Angiotensinergic Stimuli

“…Evidence of ICL3 involvement was also shown in AT 2 receptor induction of apoptotic responses in the PC12W neuronal lineage cells and in AT 2 receptor-mediated inhibition of IP 3 generation in Xenopus oocytes (Kumar et al, 2002). More recent studies implicated the involvement of G i in AT 2 receptor-dependent increases in nitric oxide synthase expression (Li et al, 2007a) and the inhibition of proximal tubule Na + -ATPase by Ang(1-7) Gohlke et al, 1998;Siragy, 2000). The production of prostacyclin in differentiated adipocytes was blocked by PD123177 and not by losartan, which suggests that this is an AT 2 receptor-mediated signal (Darimont et al, 1994).…”

Preconditioning via Angiotensin Type 2 Receptor Activation Improves Therapeutic Efficacy of Bone Marrow Mononuclear Cells for Cardiac Repair

“…Furthermore, it appears that up-regulated eNOS expression and subsequently enhanced NO production are mediated via AT2R activation, involving G alpha i3/Ras/Raf/MAPK (ERK) pathway[17,18]. Therefore, we hypothesized that AT2R/p-ERK/eNOS/NO pathway activation would be account for the cardioprotective effects conferred by preconditioned BMMNCs.…”

“…Such effects can be abolished when AT2R antagonist was added, suggesting that AT2R signaling could also promote the paracrine action of BMMNCs which support the survival of NRCMs. Several studies has clearly shown that AT2R stimulation can induce pulmonary artery relaxation through enhanced eNOS expression and NO releasing[17,18]. Pretreatment with eNOS enhancer AVE9488 increases NO generation of bone marrow mononuclear cells and improves their functional activity in a murine model of unilateral hind limb ischemia[29].…”

Intracellular Angiotensin‐II Interacts With Nuclear Angiotensin Receptors in Cardiac Fibroblasts and Regulates RNA Synthesis, Cell Proliferation, and Collagen Secretion

“…AT1R signaling occurs principally via Ga q11 and involves the production of inositol trisphosphate and mobilization of intracellular Ca 2+ ; in contrast, AT2Rs commonly signal through Gai3 to activate the NO/ cGMP pathway . 30,31 In nuclei isolated from atrial fibroblasts, AT2Rs were responsible for Ang-II-dependent NO production, since the AT1R-selective antagonist valsartan had no effect on DAF-2 fluorescence, whereas the AT2R-selective antagonist PD 123,319 reduced DAF-2 fluorescence to levels similar to those observed when nuclei were pretreated with NOS inhibitor, L-NAME. Consistent with previous findings in fibroblast nuclei isolated from whole heart, endothelial NOS immunoreactivity was detected in nuclei isolated from fibroblasts.…”