Sustained hypersensitivity to angiotensin II and its mechanism in mice lacking the subtype-2 (AT2) angiotensin receptor

The vast majority of the known biological effects of the renin-angiotensin system are mediated by the type-1 (AT1) receptor, and the functions of the type-2 (AT2) receptor are largely unknown. We investigated the role of the AT2 receptor in the vascular and renal responses to physiological increases in angiotensin II (ANG II) in mice with targeted deletion of the AT2 receptor gene. Mice lacking the AT2 receptor (AT2-null mice) had slightly elevated systolic blood pressure (SBP) compared with that of wild-type (WT) control mice (P < 0.0001). In AT2-null mice, infusion of ANG II (4 pmol/kg/min) for 7 days produced a marked and sustained increase in SBP [from 116 +/- 0.5 to 208 +/- 1 mmHg (P < 0.0001) (1 mmHg = 133 Pa)] and reduction in urinary sodium excretion (UNaV) [from 0.6 +/- 0.01 to 0.05 +/- 0.002 mM/day (P < 0.0001)] whereas neither SBP nor UNaV changed in WT mice. AT2-null mice had low basal levels of renal interstitial fluid bradykinin (BK), and cyclic guanosine 3', 5'-monophosphate, an index of nitric oxide production, compared with WT mice. In WT mice, dietary sodium restriction or ANG II infusion increased renal interstitial fluid BK, and cyclic guanosine 3', 5'-monophosphate by approximately 4-fold (P < 0.0001) whereas no changes were observed in AT2-null mice. These results demonstrate that the AT2 receptor is necessary for normal physiological responses of BK and nitric oxide to ANG II. Absence of the AT2 receptor leads to vascular and renal hypersensitivity to ANG II, including sustained antinatriuresis and hypertension. These results strongly suggest that the AT2 receptor plays a counterregulatory protective role mediated via BK and nitric oxide against the antinatriuretic and pressor actions of ANG II.     

Essential role of AT1A receptor in the development of 2K1C hypertension

The aims of this study were to delineate the relative contribution of angiotensin II (ANG II) subtype 1A (AT1A) and 1B (AT1B) receptors to the development of two-kidney, one-clip (2K1C) Goldblatt hypertension in mice, to examine if increased nitric oxide synthase (NOS) activity counteracts the vasoconstrictor influences of ANG II in 2K1C hypertensive mice, and to determine the role of ANG II type 2 (AT2) receptors in 2K1C hypertension in mice. AT(1A) ANG II receptor knockout (AT1A-/-) and wild-type (AT1A+/+) mice underwent clipping of the right renal artery. Systolic blood pressure (SBP) was significantly lower in AT1A-/- compared with AT1A+/+ mice, and neither clip placement nor AT2 receptor blockade with PD 123319 (PD) altered SBP in AT1A-/- mice. A significant and sustained rise in SBP from 119+/-5 to 163+/-6 mm Hg was observed in the 2K1C AT1A+/+ mice from day 10 to day 26. Chronic PD infusion did not alter the course of hypertension in 2K1C/AT1A+/+. Acute PD infusion did not alter mean arterial pressure (MAP) in AT1A+/+, PD/AT1A+/+, 2K1C/AT1A+/+, PD/2K1C/AT1A+/+, AT1A-/-, PD/AT1A-/-, and PD/2K1C/AT1A-/- mice compared with basal levels. In contrast, acute PD infusion caused significant increases in MAP in 2K1C/AT1A-/- mice. The subsequent acute NOS inhibition caused greater increases in MAP in 2K1C/AT1A+/+ and PD/2K1C/AT1A+/+ mice than in AT1A+/+ and PD/AT1A+/+ mice. These results support the essential role of AT1A receptors in mediating 2K1C hypertension and support the hypothesis that augmented NO production serves as a counteracting system in this model of hypertension.     

Localization of central angiotensin II receptors with [125I]-sar1, ile8-angiotensin II: periventricular sites of the anterior third ventricle

The radiolabeled angiotensin II (ANG II) antagonist, [N 125I]-sar1,ile8-ANG II, was used to study brain ANG II receptors by both homogenate binding and in vitro autoradiography. In homogenate preparations of the hypothalamus, thalamus, septum and midbrain (HTSM), [125I]-sar1,ile8-ANG II bound to a single class (Hill slope 0.84 +/- 0.05) of high affinity binding sites (KD 0.42 +/- 0.03 nM, Bmax 5.98 +/- fmol/mg protein). Competition for the [125I]-sar1,ile8-ANG II binding site in HTSM membranes demonstrated a rank order potency characteristic of binding to the ANG II receptor, with the unlabeled antagonist being slightly more potent than ANG II (Ki 0.22 +/- 0.03 vs 0.95 +/- 0.06 nM, respectively). Brain slices from the region of the rostral third ventricle were incubated with 0.5 nM[125I]-sar1,ile8-ANG II in the presence or absence of 1 microM ANG II and exposed to LKB Ultrofilm. Autoradiographic images of [125I]-sar1,ile8-ANG II binding revealed that structures situated within the anterior wall of the third ventricle, i.e. the lamina terminalis, were heavily labeled; including the subfornical organ, median preoptic nucleus and organum vasculosum laminae terminalis. These results show the utility of [125I]-sar1,ile8-ANG II as a probe to study brain ANG II receptors and provides pharmacological evidence for the rostral third ventricle as a possible site for central ANG II actions.     

Increased expression of regulator of G protein signaling-2 (RGS-2) in Bartter's/Gitelman's syndrome. A role in the control of vascular tone and implication for hypertension

Regulator of G protein signaling-2 (RGS-2) plays a key role in the G protein-coupled receptor (GPCR) angiotensin II (Ang II) signaling. NO and cGMP exert a vasodilating action also through activation and binding to RGS-2 of cGMP dependent protein kinase 1-alpha, which phosphorylates RGS-2 and dephosphorylates myosin light chain. In Bartter's/Gitelman's patients (BS/GS) Ang II related signaling and vasomotor tone are blunted. Experiments were planned to explore whether RGS-2 may play a role in BS/GS vascular hyporeactivity. NO metabolites and cGMP urinary excretion were also measured. Mononuclear cells (PBM) from six BS/GS patients and six healthy controls were used. PBM RGS-2 mRNA and RGS-2 protein were increased in BS/GS: 0.47 +/- 0.06 d.u. vs 0.32 +/- 0.04, (p < 0.006) (RGS-2 mRNA), and 0.692 +/- 0.02 vs 0.363 +/- 0.06 (p < 0.0001) (RGS2 protein). Incubation of PBM with Ang II increased RGS-2 protein in controls (from 0.363 +/- 0.06 d.u. to 0.602 +/- 0.05; p < 0.0001) but not in BS/GS (from 0.692 +/- 0.02 to 0.711 +/- 0.02). NO(2)(-)/NO(3)(-) and cGMP urinary excretion were increased in BS/GS (0.46 +/- 0.13 vs 0.26 +/- 0.05 micromol/micromol of urinary creatinine, p < 0.005, and 0.060 +/- 0.030 vs 0.020 +/- 0.01 p < 0.009, respectively). These results demonstrate that RGS-2 is increased and maximally stimulated in BS/GS and human RGS-2 system reacts as predicted by knockout mice experiments. This is the first report of RGS-2 level in a human clinical condition characterized by altered vascular tone, underlines the importance of RGS-2 as a key regulator element for Ang II signaling and provides insight into the links between BS/GS genetic abnormalities and abnormal vascular tone regulation.     

Angiotensin II receptor blockade does not improve left ventricular function and remodeling in subacute mitral regurgitation in the dog

OBJECTIVES: We hypothesized that angiotensin II type-1 (AT(1)) receptor blocker (AT(1)RB) would prevent adverse left ventricular (LV) remodeling and LV dysfunction when started at the outset of mitral regurgitation (MR). BACKGROUND: Little is known regarding the efficacy of AT(1)RB treatment of MR. METHODS: Mitral regurgitation was induced by chordal disruption in adult mongrel dogs. Six normal dogs (NLs) were compared to six untreated MR dogs (MR) and seven dogs treated with the receptor blocker irbesartan (MR+AT(1)RB) started 24 h after induction of MR (60 mg/kg p.o. b.i.d.) and continued for three months. RESULTS: Treatment with AT(1)RB decreased systemic vascular resistance but did not significantly improve cardiac output, LV end-diastolic dimension (LVEDD) or LVEDD/wall thickness compared to untreated MR dogs. Resting isolated cardiomyocyte length increased in MR versus NLs and was further increased in AT(1)RB dogs. Left ventricular end-systolic dimension increased to a greater extent from baseline in AT(1)RB dogs versus untreated MR dogs (29 +/- 9% vs. 12 +/- 6%, p < 0.05), despite a significantly lower LV peak systolic pressure in AT(1)RB dogs. Plasma-angiotensin (ANG) II was elevated greater than threefold in both MR and MR+AT(1)RB versus NLs. In contrast, intracardiac ANG II was increased greater than twofold in MR dogs versus NLs, but was normalized by AT(1)RB. CONCLUSIONS: The use of AT(1)RB decreased systemic vascular resistance and attenuated local expression of the renin-angiotensin system but did not prevent adverse LV chamber and cardiomyocyte remodeling. These results suggest that blockade of the AT(1) receptor does not improve LV remodeling and function in the early myocardial adaptive phase of MR.     

Vascular angiotensin II receptor and calcium signaling in toadfish.

The renin-angiotensin system evolved during the early evolution of vertebrates and regulates blood pressure/blood volume homeostasis in nonmammalian and mammalian vertebrates. Properties of vascular angiotensin (ANG) receptors and signal pathways in primitive animals are, however, not well understood. We aimed to determine whether vascular ANG II receptors in the toadfish, Opsanus tau, an aglomerular teleost, pharmacologically resemble either the ANG subtype 1 receptor (AT1) or the subtype 2 receptor (AT2) by examining (i) the effects of selective ANG receptor antagonists on ANG II-induced vasopressor action and binding and (ii) ANG II's effect on cytosolic Ca2+ signaling. [Asn1, Val5]ANG II (native teleost ANG II) dose-dependently increased the mean arterial pressure of conscious toadfish. ANG II-induced pressor responses (100-500 ng/kg) were inhibited substantially (79-83%) by [Sar1, Ile8]ANG II (5 microg x kg-1 + 5 microg x kg-1 x min-1) and moderately (34-53%) by losartan (AT1 antagonist, 10 mg/kg + 20 mg x kg-1 x h-1) and by PD 123319 (AT2 antagonist, 10 mg/kg + 20 mg x kg-1 x h-1) (36-60%). Likewise, the [Asp1, Val5, His9]ANG I-induced pressor effect was completely eliminated by an ANG I-converting enzyme inhibitor, SQ 14,225. Specific 125I-ANG II binding to vascular smooth muscle (VSM) membrane fractions was displaced completely by [Asn1, Val5]ANG II and [Sar1, Ile8]ANG II. Losartan, but not PD 123319, partly displaced ANG II binding at 10(-10)-10(-6) M. Furthermore, ANG II (10(-7) or 10(-8) M) caused a rapid, transient increase in the cytosolic Ca2+ signal (fluorescence ratio (FR) of 340/380 nm) of isolated VSM tissues measured by fura-2 and a dual wavelength fluorospectrometer, whereas extracellular K+ induced sustained, dose-dependent (P < 0.01) increases in FR. The results indicate that toadfish VSM tissues possess a rather nonselective ANG receptor; partial inhibition of ANG II binding by losartan and stimulation of cytosolic Ca2+ signaling by ANG II suggest that the receptor has some resemblance to AT1 homologous receptors.     

Genetic inactivation of the B2 receptor in mice worsens two-kidney,one-clip hypertension: role of NO and the AT2 receptor

OBJECTIVE: Previous studies have suggested that activation of angiotensin II (ANG II) type 2 (AT2) receptors results in nitric oxide (NO) release via activation of endothelial bradykinin B2 (B2R) receptors. The present study was performed to examine the interplay of AT2 and B2R in the development and maintenance of two-kidney, one-clip (2K1C) Goldblatt hypertension. METHODS: B2R knockout (B2R-/-) mice and their wild-type controls (B2R+/+) underwent clipping of the right renal artery and were infused with either saline (SAL) or PD 123319, an AT2 receptor antagonist (PD), via an osmotic pump implanted intraperitoneally. Systolic blood pressure (SBP) was measured in conscious mice. On day 27, mean arterial pressure (MAP) responses were measured in response to consecutive blockade of AT(2) receptors and NO synthase (NOS). RESULTS: A significant and sustained rise in SBP was observed in both 2K1C B2R+/+ and B2R-/- versus sham-operated groups from day 10 to day 24 after clipping. After this time, SBP rose to significantly higher levels in 2K1C/B2R-/- than in 2K1C/B2R+/+ mice. MAP on day 27 was also higher in 2K1C/B2R-/- than 2K1C/B2R+/+ mice. Chronic PD infusion did not alter the course of hypertension in 2K1C/B2R+/+ or 2K1C/B2R-/- mice as compared with saline-infused mice. Likewise, acute PD infusion did not affect MAP in any of the groups. However, acute NOS inhibition caused significantly greater increases in MAP in 2K1C/B2R+/+ and PD/2K1C/B2R+/+ than 2K1C/B2R-/- and PD/2K1C/B2R-/- mice. CONCLUSIONS: These results indicate that B2R inactivation selectively worsens the maintenance phase of 2K1C Goldblatt hypertension and support the notion that B2R-deficient mice exhibit an impaired ability to release NO in response to elevations of ANG II levels. Chronic administration of an AT2 receptor blocker did not modify the course of 2K1C Goldblatt hypertension in either B2R-/- or B2R+/+ mice. Therefore, the role of AT2 receptors in B2R-mediated protection against ANG II-dependent hypertension remains uncertain.     

Effect of the angiotensin II type 2-receptor gene (+1675 G/A) on left ventricular structure in humans

OBJECTIVES: Our study goal was to analyze whether gene variants of angiotensin II type 2-receptor (AT2-R) modulate the effects of angiotensin II on the left ventricle (LV). BACKGROUND: Experimental data suggest that angiotensin II modifies ventricular growth responses via angiotensin II type 1-receptors (AT1-R) and AT2-R. METHODS: In 120 white, young male subjects with normal or mildly elevated blood pressure, we assessed plasma angiotensin II and aldosterone concentrations (RIA), 24-h urinary sodium excretion, 24-h ambulatory blood pressure and LV structure (two-dimensional guided M-mode echocardiography). The intronic +1675 G/A polymorphism of the X-chromosomal located AT2-R gene was investigated by single-strand conformational polymorphism analysis and DNA-sequencing. RESULTS: Hypertensive subjects with the A-allele had a greater LV posterior (11.0 +/- 1.3 vs. 9.9 +/- 1.3 mm, p < 0.001), septal (11.8 +/- 1.4 vs. 10.1 +/- 1.2 mm, p < 0.001) and relative wall thickness (0.44 +/- 0.06 vs. 0.39 +/- 0.06, p < 0.01) as well as LV mass index (138 +/- 23 vs. 120 +/- 13 g/m2, p < 0.001) than those with the G-allele. Confounding factors (i.e., body mass index and surface area, plasma angiotensin II, sodium excretion, systolic and diastolic ambulatory blood pressure) were similar between the two genotypes. In normotensive subjects, relative wall thickness (0.36 +/- 0.05 vs. 0.35 +/- 0.05) and LV mass index (115 +/- 21 vs. 112 +/- 17 g/m2) were nearly identical across the two genotypes, with similar confounding variables. CONCLUSIONS: Our data indicate that the X-chromosomal located +1675 G/A-polymorphism of the AT2-R gene is associated with LV structure in young male humans with early structural changes of the heart due to arterial hypertension.     

Increased expression of cardiac angiotensin II type 1 (AT(1)) receptors decreases myocardial microvessel density after experimental myocardial infarction

OBJECTIVE: To study the effects of increased levels of myocardial angiotensin II type 1 (AT(1)) receptor on microvascular growth following myocardial infarction (MI). METHODS: MI was created in transgenic rats (TGR) with a cardioselective overexpression of the AT(1) receptor. We used Sprague-Dawley (SD) rats as controls. Some of the rats were treated with the selective AT(1) receptor blocker losartan (Los). Rats were sacrificed after 3 weeks. RESULTS: MI caused left ventricular (LV) hypertrophy and LV dysfunction in both SD and TGR, which was prevented by AT(1) receptor blockade. Furthermore, MI decreased microvessel density in the non-infarcted myocardium (SD MI: 1653+/-37/mm(2), P<0.01 vs. sham-operated controls), however, microvessel density decreased significantly more in TGR with MI (1298+/-33/mm(2), P<0.01 vs. SD MI). AT(1) receptor blockade restored microvessel density (SD MI Los: 2046+/-195/mm(2); TGR MI Los: 1742+/-47/mm(2); P<0.01 vs. untreated). The differences in microvessel density were still present after correction for LV hypertrophy. The increase in microvessel density after AT(1) receptor blockade was not accompanied by increased myocardial vascular endothelial growth factor (VEGF) levels. Microvessel density correlated with parameters of myocardial stretch, such as LV end-diastolic pressure (-0.681, P<0.001) and N-ANP (-0.424, P=0.01). CONCLUSIONS: Microvessel density after MI is decreased when the AT(1) receptor is overexpressed, and this is amenable to AT(1) receptor blockade. This suggests that efficacy of AT(1) receptor blockers post-MI may not only be due to attenuation of LV remodeling, but also to a stimulatory effect on angiogenesis.     

Role of AT2 receptors in the cardioprotective effect of AT1 antagonists in mice

Angiotensin II (Ang II) acts mainly on two receptor subtypes: AT1 and AT2. Most of the known biological actions of Ang II are mediated by AT1 receptors; however, the role of AT2 receptors remains unclear. We tested the hypothesis that the cardioprotective effects of AT1 receptor antagonists (AT1-ant) after myocardial infarction (MI) are partially mediated by activation of AT2 receptors; thus in AT2 receptor gene knockout mice (AT2-/Y), the effect of AT1-ant will be diminished or absent. MI was induced by ligating the left anterior descending coronary artery. Four weeks later, AT2-/Y and their wild-type littermates (AT2+/Y) were started on vehicle, AT1-ant (valsartan, 50 mg/kg per day), or ACE inhibitor (enalapril, 20 mg/kg per day) for 20 weeks. Basal blood pressure and cardiac function as well as remodeling after MI did not differ between AT2+/Y and AT2-/Y. AT1-ant increased ejection fraction and cardiac output and decreased left ventricular diastolic dimension, myocyte cross-sectional area, and interstitial collagen deposition in AT2+/Y, and these effects were significantly diminished in AT2-/Y. ACE inhibitors improved cardiac function and remodeling similarly in both strains. We concluded that (1) activation of AT2 during AT1 blockade plays an important role in the therapeutic effect of AT1-ant and (2) the AT2 receptor may not play an important role in regulation of cardiac function, either under basal conditions after MI remodeling or in the therapeutic effect of ACE inhibitors.     

Interaction of angiotensin II type 1 and D5 dopamine receptors in renal proximal tubule cells

Angiotensin II type 1 (AT1) receptor and D1 and D3 dopamine receptors directly interact in renal proximal tubule (RPT) cells from normotensive Wistar-Kyoto rats (WKY). There is indirect evidence for a D5 and AT1 receptor interaction in WKY and spontaneously hypertensive rats (SHR). Therefore, we sought direct evidence of an interaction between AT1 and D5 receptors in RPT cells. D5 and AT1 receptors colocalized in WKY cells. Angiotensin II decreased D5 receptors in WKY cells in a time- and concentration-dependent manner (EC50=2.7x10(-9) M; t(1/2)=4.9 hours), effects that were blocked by an AT1 receptor antagonist (losartan). In SHR, angiotensin II (10(-8) M/24 hours) also decreased D5 receptors (0.96+/-0.08 versus 0.72+/-0.08; n=12) and to the same degree as in WKY cells (1.44+/-0.07 versus 0.92+/-0.08). However, basal D5 receptors were decreased in SHR RPT cells (SHR 0.96+/-0.08; WKY 1.44+/-0.07; n=12 per strain; P<0.05) and renal brush border membranes of SHR compared with WKY (SHR 0.54+/-0.16 versus WKY 1.46+/-0.10; n=5 per strain; P<0.05). Angiotensin II decreased AT1 receptor expression in WKY (1.00+/-0.04 versus 0.72+/-0.08; n=8; P<0.05) but increased it in SHR (0.96+/-0.04 versus 1.32+/-0.08; n=8; P<0.05). AT(1) and D5 receptors also interacted in vivo; renal D5 receptor protein was higher in mice lacking the AT1A receptor (AT1A-/-; 1.61+/-0.31; n=6) than in wild-type littermates used as controls (AT1A+/+; 0.81+/-0.08; n=6; P<0.05), and renal cortical AT1 receptor protein was higher in D5 receptor null mice than in wild-type littermates (1.18+/-0.08 versus 0.84+/-0.07; n=4; P<0.05). We conclude that D5 and AT1 receptors interact with each other. Altered interactions between AT1 and dopamine receptors may play a role in the pathogenesis of hypertension.     

Bradykinin B2 receptor knockout mice are protected from thrombosis by increased nitric oxide and prostacyclin

Bradykinin (BK) liberates nitric oxide, prostacyclin, and tissue plasminogen activator from endothelial cells. We hypothesized that BK B2 receptor knockout (KO) mice (BKB2R(-/-)) have increased thrombosis risk. Paradoxically, the BKB2R(-/-) mice have long bleeding times and delayed carotid artery thrombosis, 78 +/- 6.7 minutes, versus 31 +/- 2.7 minutes in controls. The mechanism(s) for thrombosis protection was sought. In BKB2R(-/-) plasma coagulation, fibrinolysis and anticoagulant proteins are normal except for an increased prekallikrein and decreased factor XI. BKB2R(-/-) mice have elevated BK 1-5 (160 +/- 75 fmol/mL, vs 44 +/- 29 fmol/mL in controls) and angiotensin II (182 +/- 41 pg/mL, vs 49 +/- 7 pg/mL in controls). Ramipril treatment shortens vessel occlusion time. BKB2R(-/-) mice have elevated plasma 6-keto-PGF1alpha (666 +/- 232 ng/mL, vs 23 +/- 5.3 ng/mL in controls) and serum nitrate (61 +/- 5.3 microM, vs 24 +/- 1.8 microM in controls). Treatment with L-NAME (NG-mono-methyl-L-arginine ester) or nimesulide shortens the thrombosis time. BKB2R(-/-) mice have increased angiotensin receptor 2 (AT2R) mRNA and protein expression. Treatment with an AT2R antagonist, PD123 319, normalizes the thrombosis time and nitrate and 6-keto-PGF1alpha. The long bleeding times in BKB2R(-/-) mice also correct with L-NAME and nimesulide therapy. In BKB2R(-/-) mice, angiotensin II binding to an overexpressed AT2R promotes thromboprotection by elevating nitric oxide and prostacyclin. These investigations indicate a pathway for thrombosis risk reduction via the plasma kallikrein/kinin and renin angiotensin systems.     

Negative regulation of RhoA/Rho kinase by angiotensin II type 2 receptor in vascular smooth muscle cells: role in angiotensin II-induced vasodilation in stroke-prone spontaneously hypertensive rats

OBJECTIVE: To test whether angiotensin II (Ang II) through the Ang II type 2 receptor (AT2R), downregulates RhoA/Rho kinase, which plays a role in AT1 receptor (AT1R)-mediated function. METHODS: In vitro studies were performed in A10 vascular smooth muscle cells (VSMC) and in vivo studies in mesenteric arteries from Wistar-Kyoto (WKY) and stroke-prone spontaneously hypertensive (SHRSP) rats. VSMC were stimulated with Ang II (10 mol/l), CGP42112A (10 mol/l, a selective AT2R agonist) +/- valsartan (10 mol/l, an AT1R antagonist), or the Rho kinase inhibitor fasudil (10 mol/l). AT1R and AT2R expression and myosin light chain (MLC) phosphorylation were determined by immunoblotting. RhoA activity was assessed by measuring membrane translocation. Functional significance between AT2R, RhoA/Rho kinase and vasodilation was assessed in arteries from valsartan-treated (30 mg/kg per day, 14 days) WKY and SHRSP rats. Vasodilatory responses to Ang II (10-10 mol/l) were performed in norepinephrine pre-contracted vessels +/- valsartan(10 mol/l), PD123319 (10 mol/l, an AT2R antagonist) or fasudil (10 mol/l). RESULTS: A10 VSMC expressed AT1R and AT2R. In valsartan-treated cells, Ang II-induced RhoA translocation was reduced versus controls (42 +/- 6%, P < 0.05). Similar responses were obtained with CGP42112A (45 +/- 6%, P < 0.05). This was associated with decreased MLC activation. Fasudil abrogated Ang II- and CGP42112A-mediated effects. Ang II evoked a significant vasodilatory response only in valsartan-treated SHRSP (max dilation 40 +/- 7%). PD123319 blocked these effects. Fasudil increased AngII-induced relaxation in SHRSP vessels. AT2R expression was increased by valsartan (two- to three-fold) in SHRSP arteries. RhoA translocation was increased two-fold in untreated SHRSP (P < 0.05) and was reduced by valsartan (P < 0.05). These changes were associated with decreased MLC phosphorylation. CONCLUSIONS: Ang II/AT2R negatively regulates vascular RhoA/Rho kinase/MLC phosphorylation. These processes may play a role in Ang II-mediated vasodilation in conditions associated with vascular AT2R upregulation, such as in SHRSP chronically treated with AT1R blockers, which may contribute to blood pressure lowering by these antihypertensive agents.     

Intrarenal angiotensin II augmentation in angiotensin II dependent hypertension.

In several models of angiotensin II (ANG II) dependent hypertension, intrarenal ANG II levels increase to a much greater extent than the circulating levels even though the renal renin levels are decreased. The 2-kidney-1-clip (2K1C) Goldblatt rat model is particularly intriguing because hypertension develops in the presence of an intact kidney which would be expected to maintain sodium balance and protect against hypertension. Although the non-clipped kidney becomes renin depleted, it exhibits enhanced microvascular reactivity and increased tubular fractional sodium reabsorption. The non-clipped kidney ANG II content is either elevated or unchanged and proximal tubular fluid ANG II concentrations are not suppressed compared to the nanomolar concentrations found in normal rats. These results suggest that intrarenal ANG II content can be regulated independently of renal renin content. A similar hypertensive process occurs in rats infused chronically with low doses of ANG II. Renal ANG II content increases over 14 days to a greater extent than the circulating concentrations. Functionally, ANG II infused rats demonstrate reduced sodium excretion and marked suppression of pressure natriuresis. These ANG II dependent influences on kidney function contribute to the maintenance of hypertension. Renal augmentation of ANG II, hypertension, and suppressed sodium excretion are blocked by AT1 receptor blockers. To study the mechanisms responsible for intrarenal ANG II augmentation, we infused a different form of ANG II (Val5 ANG II), that can be separated from endogenous ANG II by HPLC. These results indicated that the increased renal ANG II content was due to accumulation of circulating ANG II in addition to continued production of endogenous ANG II. The renal accumulation of Val5-ANG II was markedly reduced by concomitant treatment with the AT1 receptor blocker, losartan. In addition, we found an unchanged overall ANG II-AT1 receptor protein which probably contributes to the maintained ANG II dependent influences. Collectively, the data support the concept that there is internalization of ANG II through an AT1 receptor mediated process and that some of the internalized ANG II is protected from degradation. The augmented intrarenal ANG II coupled with sustained levels of AT1 receptors contribute to the continued ANG II dependent suppression of renal function and sodium excretion thereby maintaining the hypertension.     

Angiotensin II binding sites in the heart of Scyliorhinus canicula: an autoradiographic study

Dogfish (125)I [Asn(1), Pro(3), Ile(5)] angiotensin II ((125)I dfANG II) was used to establish the specific binding patterns of the different cardiac regions of the elasmobranch Scyliorhinus canicula by in vitro autoradiography. In the ventricular myocardium Scatchard analysis of saturation and displacement binding data revealed two classes of high- and low-affinity dfANG II binding sites (K(d) = 53 +/- 10 and 1300 +/- 900 pM). Two classes of dfANG II binding sites were also detected in the atrium (K(d) = 47 +/- 13 and 4690 +/- 930 pM) and in the outer layer of the conus arteriosus (K(d) = 16 +/- 9 and 398 +/- 83 pM). Conversely, the ventricular endocardium and the inner conal layer were characterized by a single class of dfANG II binding sites with affinity values of 48 +/- 11 and 106 +/- 3.3 pM, respectively. Competition experiments with either cold dfANG II or CV11974 or CGP42112 (specific ligands for mammalian AT(1) and AT(2) receptors, respectively) demonstrated a prevalence of CGP42112-selective dfANG II binding sites in both the inner and the outer conal layers. In the atrium, the ventricular myocardium, and the outer conal layer, dfANG II high-affinity binding sites poorly discriminated among the cold ligands. These results suggest that the dogfish heart may be a target organ of ANG II with distinct ANG II receptor subtype distributions.     

Effects of changes in sodium balance on plasma and kidney angiotensin II levels in anesthetized and conscious Ren-2 transgenic rats

OBJECTIVE: Since there is as yet no general agreement regarding the role of plasma and kidney angiotensin II (ANG II) in the development of hypertension in Ren-2 transgenic rats (TGR), in the present study we evaluated plasma and kidney ANG II levels in anesthetized and conscious TGR and in normotensive Hannover-Sprague-Dawley rats (HanSD) fed a normal salt diet (NS). Given the importance of ANG II in the development of salt-sensitive hypertension, and the fact that hypertensinogenic actions of ANG II are mediated via ANG II type 1 (AT1) receptors, the effects of high salt (HS) intake and of sodium depletion on blood pressure (BP), ANG II levels and kidney AT1 receptor protein expression in TGR and HanSD were also examined. METHODS: Rats were maintained on a NS diet (0.6% NaCl) or fed a HS diet (2% NaCl) for 4 days or were sodium depleted (40 mg/l furosemide for 1 day followed by 3 days of 0.01% NaCl diet). They were sacrificed either by an overdose of anesthetic (thiopental sodium) or by decapitation (without anesthetic) and plasma and kidney ANG II levels were determined by radioimmunoassay during the prehypertensive (32 days old), the early (52 days) and the maintenance (90 days) phases of hypertension. Total kidney AT1 receptor protein levels were assessed by Western blot analysis. RESULTS: In anesthetized animals fed the NS diet, plasma ANG II levels were lower in 32-day-old TGR than in HanSD, but at 52 and 90 days of age no significant differences were noted. ANG II concentrations in kidney tissue were similar in 32- and 90-day-old TGR and HanSD, but were higher in 52-day-old TGR than in HanSD. In contrast, in conscious animals immediately after decapitation, plasma and kidney ANG II levels were higher in TGR than in HanSD at all ages. HS diet did not change BP but suppressed ANG II levels in HanSD at all ages. In contrast, HS diet increased BP but did not decrease plasma and kidney ANG II levels in TGR at all ages. Sodium restriction did not alter BP and resulted in a marked increase in ANG II levels in HanSD, but caused a significant decrease in BP in TGR without altering plasma or tissue ANG II concentrations. There were no significant differences in renal AT1 receptor protein expression between HanSD and TGR at any age of any of the experimental groups. CONCLUSIONS: On the basis of our present results we conclude that TGR exhibit a disrupted interaction between sodium homeostasis and the regulation of the renin-angiotensin system (RAS) activity which results in the loss of BP regulation in this model.     

Beta-arrestin2-mediated inotropic effects of the angiotensin II type 1A receptor in isolated cardiac myocytes

The G protein-coupled receptor kinases (GRKs) and beta-arrestins, families of molecules essential to the desensitization of G protein-dependent signaling via seven-transmembrane receptors (7TMRs), have been recently shown to also transduce G protein-independent signals from receptors. However, the physiologic consequences of this G protein-independent, GRK/beta-arrestin-dependent signaling are largely unknown. Here, we establish that GRK/beta-arrestin-mediated signal transduction via the angiotensin II (ANG) type 1A receptor (AT(1A)R) results in positive inotropic and lusitropic effects in isolated adult mouse cardiomyocytes. We used the "biased" AT(1A)R agonist [Sar(1), Ile(4), Ile(8)]-angiotensin II (SII), which is unable to stimulate G(alpha)q-mediated signaling, but which has previously been shown to promote beta-arrestin interaction with the AT(1A)R. Cardiomyocytes from WT, but not AT(1A)R-deficient knockout (KO) mice, exhibited positive inotropic and lusitropic responses to both ANG and SII. Responses of WT cardiomyocytes to ANG were dramatically reduced by protein kinase C (PKC) inhibition, whereas those to SII were unaffected. In contrast, cardiomyocytes from beta-arrestin2 KO and GRK6 KO mice failed to respond to SII, but displayed preserved responses to ANG. Cardiomyocytes from GRK2 heterozygous knockout mice (GRK2(+/-)) exhibited augmented responses to SII in comparison to ANG, whereas those from GRK5 KO mice did not differ from those from WT mice. These findings indicate the existence of independent G(alpha)q/PKC- and GRK6/beta-arrestin2-dependent mechanisms by which stimulation of the AT(1A)R can modulate cardiomyocyte function, and which can be differentially activated by selective receptor ligands. Such ligands may have potential as a novel class of therapeutic agents.     

Simvastatin attenuates cardiovascular effects and oxidative stress induced by angiotensin II]

The effects of an HMG-CoA reductase inhibitor, simvastatin (statin, 60 mg/Kg/24 h by forced feeding), were studied on the development of hypertension, cardiac hypertrophy and oxidating stress induced by chronic perfusion of angiotensin II (ANG II, 200 ng/Kg/min s.c., for 10 days) in the rat. The statin was giver 24 hours before, and during the 10 days of ANG II. At the end of the study, mean blood pressure was measured and blood sampling performed under anaesthesia (sodium pentobarbital). The cardiac mass index was measured (cardiac mass/body weight, mg/Kg). TBARS (thiobarbituric acid reactive substances), representing the index of lipid peroxidation, was assessed by fluorimetry. The statin attenuated the development of hypertension (131 +/- 9 vs 164 +/- 4 mmHg) and the increase in cardiac mass (3.13 +/- 0.09 vs 3.46 +/- 0.09 mg/g) associated with ANG II. The overproduction of TBARS induced by ANG II was partially prevented by simvastatin (598 +/- 40 vs 794 +/- 79 pmol/mL). These results indicate that simvastatin attenuates the cardiovascular effects and lipid peroxidation induced by chronic administration of angiotensin II.