Angiotensin II (Ang II) is a potent vasopressor peptide that interacts with 2 major receptor isoforms -AT1 and AT2. Although blood pressure is increased in AT2 knockout mice, the underlying mechanisms remain undefined because of the low levels of expression of AT2 in the vasculature. Here we overexpressed AT2 in vascular smooth muscle (VSM) cells in transgenic (TG) mice. Aortic AT1 was not affected by overexpression of AT2. Chronic infusion of Ang II into AT2-TG mice completely abolished the AT1-mediated pressor effect, which was blocked by inhibitors of bradykinin type 2 receptor (icatibant) and nitric oxide (NO) synthase (L-NAME). Aortic explants from TG mice showed greatly increased cGMP production and diminished Ang II-induced vascular constriction. Removal of endothelium or treatment with icatibant and L-NAME abolished these AT2-mediated effects. AT2 blocked the amiloride-sensitive Na + /H + exchanger, promoting intracellular acidosis in VSM cells and activating kininogenases. The resulting enhancement of aortic kinin formation in TG mice was not affected by removal of endothelium. Our results suggest that AT2 in aortic VSM cells stimulates the production of bradykinin, which stimulates the NO/cGMP system in a paracrine manner to promote vasodilation. Selective stimulation of AT2 in the presence of AT1 antagonists is predicted to have a beneficial clinical effect in controlling blood pressure.

Abstract-The expression pattern of angiotensin (Ang) II type 2 receptor (AT 2 -R) in the remodeling process of human left ventricles (LVs) remains poorly defined. We analyzed its expression at protein, mRNA, and cellular levels using autopsy, biopsy, or operation LV samples from patients with failing hearts caused by acute (AMI) or old (OMI) myocardial infarction and idiopathic dilated cardiomyopathy (DCM) and also examined functional biochemical responses of failing hearts to Ang II. In autopsy samples from the nonfailing heart group, the ratio of AT 1 -R and AT 2 -R was 59% and 41%, respectively. The expression of AT 2 -R was markedly increased in DCM hearts at protein (3.5-fold) and mRNA (3.1-fold) levels compared with AMI or OMI. AT 1 -R protein and mRNA levels in AMI hearts showed 1.5-and 2.1-fold increases, respectively, whereas in OMI and DCM hearts, AT 1 -R expression was significantly downregulated. AT 1 -R-mediated response in inositol phosphate production was significantly attenuated in LV homogenate from failing hearts compared with nonfailing hearts. AT 2 -R sites were highly localized in the interstitial region in either nonfailing or failing heart, whereas AT 1 -R was evenly distributed over myocardium at lower densities. Mitogen-activated protein kinase (MAPK) activation by Ang II was significantly decreased in fibroblast compartment from the failing hearts, and pretreatment with AT 2 -R antagonist caused an additional significant increase in Ang II-induced MAPK activity (36%). Cardiac hypertrophy suggested by atrial and brain natriuretic peptide levels was comparably increased in OMI and DCM, whereas accumulation of matrix proteins such as collagen type 1 and fibronectin was much more prominent in DCM than in OMI. These findings demonstrate that (1) AT 2 -R expression is upregulated in failing hearts, and fibroblasts present in the interstitial regions are the major cell type responsible for its expression, (2) AT 2 -R present in the fibroblasts exerts an inhibitory effect on Ang II-induced mitogen signals, and (3) AT 1 -R in atrial and LV tissues was downregulated during chronic heart failure, and AT 1 -R-mediated functional biochemical responsiveness was decreased in the failing hearts. Thus, the expression level of AT 2 -R is likely determined by the extent of interstitial fibrosis associated with heart failure, and the expression and function of AT 1 -R and AT 2 -R are differentially regulated in failing human hearts. (Circ Res. 1998;83:1035-1046.)Key Words: angiotensin II type 2 receptor Ⅲ AT 2 receptor Ⅲ angiotensin II type 1 receptor Ⅲ AT 1 receptor, angiotensin II T he presence of 2 isoforms of angiotensin (Ang) II receptor was originally proposed on the basis of differences in sensitivity of receptor-ligand binding to dithiothreitol. Ang type 2 receptor (AT 2 -R), which is insensitive to dithiothreitol and has a high affinity for PD123319 and CGP42112A, was isolated, and this receptor was shown to have the same seventransmembrane domain of AT 1 -R but only minimal homology (see Review i...

Abstract-The signaling cascade elicited by angiotensin II (Ang II) resembles that characteristic of a growth factor, and recent evidence indicates transactivation of epidermal growth factor receptor (EGF-R) by G protein-coupled receptors. Here, we report the involvement of EGF-R in Ang II-induced synthesis of fibronectin and transforming growth factor-␤ (TGF-␤) in cardiac fibroblasts. Ang II stimulated fibronectin mRNA levels dose dependently, with a maximal increase (Ϸ5-fold) observed after 12 hours of incubation. Fibronectin synthesis induced by Ang II or calcium ionophore was completely abolished by tyrosine kinase inhibitors and intracellular Ca 2ϩ chelating agents. Ang II-induced fibronectin mRNA was not affected by protein kinase C inhibitors or protein kinase C depletion, whereas specific inhibition of EGF-R function by a dominant negative EGF-R mutant and tyrphostin AG1478 abolished induction of fibronectin mRNA. We isolated the rat fibronectin gene, including the 5Ј-flanking region, and found that the activator protein-1 (AP-1) binding site present in the promoter region was responsible for the Ang II responsiveness of this gene. A gel retardation assay revealed the binding of nuclear protein to the AP-1 site, which was supershifted with anti-c-fos and anti-c-jun but not anti-activating transcription factor (ATF)-2 antibodies. Conditioned medium from Ang II-treated cells contained TGF-␤ bioactivity, and addition of neutralizing TGF-␤ antibody modestly (46%) inhibited induction of fibronectin. Ang II-induced synthesis of TGF-␤ was also abolished by inhibition of EGF-R function. The effect of TGF-␤ was exerted by stabilizing fibronectin mRNA without affecting the promoter activity and required de novo protein synthesis. We concluded that Ang II-induced expression of fibronectin and TGF-␤ is mediated by downstream signaling of EGF-R transactivated by Ca 2ϩ -dependent tyrosine kinase and that Ang II-induced fibronectin mRNA expression is regulated by 2 different mechanisms, which are transcriptional control by binding of the c-fos/c-jun complex to the AP-1 site and posttranscriptional control by mRNA stabilization due to autocrine or paracrine effects of TGF-␤. Thus, this study suggests that the action of Ang II on extracellular matrix formation should be interpreted in association with the EGF-R signaling cascade. (Circ Res. 1999;84:1073-1084.) Key Words: angiotensin II receptor Ⅲ angiotensin II type 1 receptor Ⅲ angiotensin II type 2 receptor Ⅲ angiotensin II Ⅲ epidermal growth factor receptor C ardiac fibroblasts isolated from neonatal rat hearts have abundant high-affinity angiotensin II (Ang II) receptors, which are classified pharmacologically as belonging to the Ang II type 1 receptor (AT 1 ) subtype. 1,2 These cells have been used to examine AT 1 -mediated signaling 2-4 and extracellular matrix remodeling in heart failure. 5 AT 1 stimulation was found to stimulate DNA synthesis and cell proliferation 1 and also to increase the synthesis of extracellular matrix proteins, 4,6 which suggests that cardi...

Abstract-In cardiac fibroblasts, angiotensin II (Ang II) induced a rapid increase in extracellular signal-regulated kinase (ERK) activity in a pertussis toxin-insensitive manner. This ERK activation was abolished by the G q -associated phospholipase C inhibitor U73122 but was insensitive to protein kinase C (PKC) inhibitors or PKC downregulation by phorbol ester. Intracellular Ca 2ϩ chelation by BAPTA-AM or TMB-8 abolished Ang II-induced ERK activation, whereas treatment with EGTA or nifedipine did not affect it. Ca 2ϩ ionophore A23187 also induced a rapid increase in ERK activity to an extent similar to that of Ang II stimulation. Calmodulin inhibitors (W7 and calmidazolium) and tyrosine kinase inhibitors (genistein and ST638) completely blocked ERK activation by Ang II and A23187. Both Ang II and A23187 caused a rapid increase in the binding of GTP to p21Ras , which was nearly abolished by genistein and calmidazolium. Transfection with the dominant negative mutant of Ras and the Ras inhibitor manumycin completely inhibited Ang II-induced ERK activation. It was also found for the first time that cardiac fibroblasts abundantly expressed Ca 2ϩ -sensitive tyrosine kinase Pyk2/CAK␤/RAFTK and that Ang II markedly induced its activation in a Ca 2ϩ /calmodulin-sensitive manner. Overexpression of the dominant negative mutant of Pyk2 significantly attenuated Ang II-or A23187-induced ERK activities (36% and 38% inhibition compared with that in mock-transfected cells, respectively) and ERK tyrosine phosphorylation levels, as well as an increase in the binding of GTP to p21Ras . These findings demonstrate that in cardiac fibroblasts, Ang II-induced Ras/ERK activation is dominantly regulated by G q -coupled Ca 2ϩ /calmodulin signaling and that Pyk2 plays an important role in the signal transmission for efficient activation of the Ang II-induced Ras/ERK pathway. (Hypertension. 1998;32:668-675.)