We have examined whether expression of angiotensin II (Ang II) type 1 (AT 1 ) and/or type 2 (AT 2 ) receptors are changed in thoracic aorta under pressure-overload by abdominal aortic banding in rats and determined whether their changes are accompanied by alteration in contractile response of thoracic aorta to Ang II. AT 2 receptor mRNA levels determined by reverse transcription-polymerase chain reaction or quantitative real-time polymerase chain reaction were increased by about 300% in aortas 4, 7, 14, and 28 days after banding without changes in AT 1 receptor mRNA levels. Contractile response of aortic rings to Ang II was decreased in thoracic aortas 7 days after banding, and AT 2 receptor antagonist PD123319 (1-[[4-(dimethulamino)-3-methylphenyl]methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo[4,5-c]pyridine-6-carboxylic acid ditrifluoroacetate) (10 Ϫ6 M) increased the Ang II responsiveness in pressure-loaded but not in sham rings. After removal of the endothelium or treatment with N G -nitro-L-arginine methyl ester (L-NAME), no differences were observed in Ang II responsiveness between sham and pressure-loaded rings. Either losartan (1 mg/kg/day i.p.) or candesartan (2 mg/kg/day p.o.) for 7 days after banding not only abolished the up-regulation of AT 2 receptor mRNA in aortas but also recovered their Ang II responsiveness. Basal cGMP levels were 2 times higher in pressureloaded than in sham rings; both levels were not affected by Ang II (10 Ϫ7 M; 5 min), but greatly decreased by L-NAME (10 Ϫ4 M, 30 min). These results suggest that pressure-overload induces the up-regulation of AT 2 receptor expression in aortas via AT 1 receptor and thereby negatively modulates the vasoconstrictor sensitivity to Ang II, probably mediated by the mechanisms independent of the nitric oxide-cGMP system.

Abstract-To evaluate the role of vascular angiotensin II (Ang II) type 2 (AT 2 ) receptor in renovascular hypertension, we investigated expressions of AT 2 receptor and endothelial nitric oxide synthase (eNOS) in thoracic aortas of mice with 2-kidney, 1-clip (2K1C) hypertension. The mRNA levels of AT 2 receptor in aortas, but not those of AT 1 and bradykinin B 2 receptors, increased 14 days but not 42 days after clipping. The contractile response to Ang II (Ͼ0.1 mol/L) was attenuated in aortic rings excised 14 days after clipping and was restored to that of rings from sham mice by antagonists of AT 2 receptor (PD123319) and B 2 receptor (icatibant). The aortic levels of total eNOS, phosphorylated eNOS at Ser 1177 (p-eNOS), total Akt, and phosphorylated Akt at Ser 473 (p-Akt) were increased in 2K1C mice on day 14, whereas only eNOS levels were increased on day 42. The aortic cGMP levels were Ϸ20-fold greater in 2K1C mice on day 14 compared with sham mice. Administration of nicardipine for 4 days before the excision of aortas 14 days after clipping not only reduced blood pressure but also decreased the aortic levels of eNOS, p-eNOS, Akt, p-Akt, and cGMP to sham levels, whereas the administration of PD123319 or icatibant to 2K1C mice decreased p-eNOS and cGMP to sham levels without affecting blood pressure and the levels of eNOS, Akt and p-Akt. These results suggest that vascular NO production is enhanced by increased eNOS phosphorylation via the activation of AT 2 receptors in the course of 2K1C hypertension. Key Words: angiotensin II Ⅲ bradykinin Ⅲ cyclic GMP Ⅲ nitric oxide synthase Ⅲ hypertension, renovascular T he 2-kidney, 1-clip (2K1C) Goldblatt model of hypertension is an experimental model that in many respects resembles human renovascular hypertension. It is welldocumented that the increased activity of the renin-angiotensin system by the renal secretion of renin plays an important role in the development and maintenance of 2K1C hypertension. 1 The critical role of the angiotensin II (Ang II) type 1 (AT 1 ) receptor in mediating 2K1C hypertension has been confirmed, 2,3 whereas the role of the Ang II type 2 (AT 2 ) receptor in the development of 2K1C hypertension remains poorly understood. Recent studies have demonstrated that the acute and chronic AT 2 receptor blockade by PD123319 does not worsen the course of hypertension in 2K1C hypertensive mice, suggesting that the AT 2 receptor does not play a major counterbalancing role against the vasoconstricting actions of Ang II in 2K1C hypertension. 2,4 In a previous study using mice with abdominal aortic banding, we demonstrated the upregulation of AT 2 receptors in thoracic aortas, and that the stimulation of AT 2 receptors by Ang II attenuated the AT 1 receptor-mediated contractile response to Ang II in the aorta via the activation of the kinin-nitric oxide (NO)-cGMP cascade. 5 In addition, we provided evidence that the elevation of plasma renin levels after the banding-induced reduction of renal blood pressure is associated with the upregulation of AT 2 re...

Abstract-Abdominal aortic banding induces upregulation of the angiotensin II (Ang II) type-2 (AT 2 ) receptor, thereby decreasing the contractile response to Ang II in the thoracic aorta of the rat. The aim of this study was to use a mouse model to clarify the mechanisms by which the banding elicits upregulation of the aortic AT 2 receptor and the subsequent attenuation of Ang II responsiveness. Concomitantly with the elevation in blood pressure and plasma renin concentration after banding, AT 2 -receptor mRNA levels in the thoracic aorta rapidly increased in mice within 4 days. Upregulation of the AT 2 receptor, as well as blood pressure elevation after banding, was abolished by losartan administration. The contractile response to Ang II was depressed in aortic rings of banding mice but not of sham mice, and was restored by either the AT 2 -receptor antagonist PD123319 or the bradykinin B 2 -receptor antagonist icatibant. cGMP content in the thoracic aorta of banding mice was 9-fold greater than that of sham mice, and the elevation was reduced to sham levels 1 hour after intravenous injection of PD123319 or icatibant. When aortic rings were incubated with Ang II, cGMP content increased in banding rings but not in sham rings; the pretreatment with PD123319 or icatibant inhibited Ang II-induced cGMP production. These results suggest that aortic banding induces upregulation of the AT 2 receptor through increased circulating Ang II via the AT 1 receptor, thereby activating a vasodilatory pathway in vessels through the AT 2 receptor via the kinin/cGMP system. Key Words: receptors, angiotensin II Ⅲ bradykinin Ⅲ nitric oxide Ⅲ cyclic GMP Ⅲ vasodilation A ngiotensin II (Ang II) is a vasoactive peptide that regulates blood pressure and fluid homeostasis, and is likely to play a key role in the pathogenesis of cardiovascular diseases in humans. 1 Ang II exerts its various actions through 2 receptor subtypes, type-1 (AT 1 ) and type-2 (AT 2 ). 1 Disruption of the AT 2 -receptor gene in the mouse increases basal blood pressure and induces pressure responsiveness to Ang II. 2,3 In contrast, overexpression of the AT 2 -receptor gene in vascular smooth muscle cells results in a depressed pressureresponse to Ang II because of the activation of the nitric oxide (NO)-cGMP system via the AT 2 -receptor-mediated vascular kallikrein-kinin system in mice. 4 In spontaneously hypertensive rats, administration of AT 2 -receptor agonists induces a depressor response during simultaneous AT 1 -receptor blockade. 5 In normotensive rats, stimulation of AT 2 receptors causes vasodilation via the local production of bradykinin in resistant arteries of the rat mesentery in a flow-dependent manner. 6 These observations suggest that the AT 2 receptor acts as a vasodilator pathway counterregulatory to AT 1 -receptor-mediated vasoconstriction under normotensive and hypertensive conditions.The AT 1 receptor is the predominant receptor subtype in the adult vasculature, whereas the AT 2 receptor is predominant during fetal development and declines af...

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The generation of kinins on the surface of vascular endothelium has been postulated in two pathways involving plasma kallikrein and tissue kallikrein; the former pathway has been well documented, but the latter is controversial. To clarify the presence of a kinin-generating system on endothelium, we examined whether human umbilical vein endothelial cells (HUVEC) synthesize and release tissue kallikrein in vitro. Kallikrein-like activity hydrolyzing a peptide Pro-Phe-Arg-4-methyl-coumaryl-7-amide was detected in the culture medium of HUVEC and was inhibited by aprotinin but not by soybean trypsin inhibitor. Western blotting of HUVEC medium using anti-human tissue kallikrein antibodies demonstrated the release of tissue kallikrein from HUVEC, and the reverse transcription-polymerase chain reaction (RT-PCR) followed by Southern blotting revealed the expression of tissue kallikrein mRNA in HUVEC. HUVEC metabolically labeled with [35S]methionine released radioactive proteins corresponding to tissue kallikrein. RT-PCR also showed the expression of low-molecular-weight kininogen (L-kininogen) mRNA in HUVEC. The cGMP levels in HUVEC were significantly elevated by the incubation with angiotensin converting enzyme inhibitor, lisinopril, and the elevation was completely inhibited by aprotinin or bradykinin B2-receptor antagonist, FR172357. These results suggest that the endothelial cells continuously release an active form of tissue kallikrein which enables generation of kinins on the vascular endothelium.

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