Conversion of renal angiotensin II to angiotensin III is critical for AT2 receptor-mediated natriuresis in rats

In the kidney, angiotensin II (Ang II) is metabolized to angiotensin III (Ang III) by aminopeptidase A (APA). In turn, Ang III is metabolized to angiotensin IV by aminopeptidase N (APN). Renal interstitial (RI) infusion of Ang III, but not Ang II, results in angiotensin type-2 receptor (AT(2)R)-mediated natriuresis. This response is augmented by coinfusion of PC-18, a specific inhibitor of APN. The present study addresses the hypotheses that Ang II conversion to Ang III is critical for the natriuretic response. Sprague-Dawley rats received systemic angiotensin type-1 receptor (AT(1)R) blockade with candesartan (CAND; 0.01 mg/kg/min) for 24 hours before and during the experiment. After a control period, rats received either RI infusion of Ang II or Ang II+PC-18. The contralateral kidney received a RI infusion of vehicle in all rats. Mean arterial pressure (MAP) was monitored, and urinary sodium excretion rate (U(Na)V) was calculated separately from experimental and control kidneys for each period. In contrast to Ang II-infused kidneys, U(Na)V from Ang II+PC-18-infused kidneys increased from a baseline of 0.03+/-0.01 to 0.09+/-0.02 micromol/min (P<0.05). MAP was unchanged by either infusion. RI addition of PD-123319, an AT(2)R antagonist, inhibited the natriuretic response. Furthermore, RI addition of EC-33, a selective APA inhibitor, abolished the natriuretic response to Ang II+PC-18. These data demonstrate that RI addition of PC-18 to Ang II enables natriuresis mediated by the AT(2)R, and that conversion of Ang II to Ang III is critical for this response.     

Intrarenal angiotensin III is the predominant agonist for proximal tubule angiotensin type 2 receptors

In angiotensin type 1 receptor-blocked rats, renal interstitial (RI) administration of des-aspartyl(1)-angiotensin II (Ang III) but not angiotensin II induces natriuresis via activation of angiotensin type 2 receptors. In the present study, renal function was documented during systemic angiotensin type 1 receptor blockade with candesartan in Sprague-Dawley rats receiving unilateral RI infusion of Ang III. Ang III increased urine sodium excretion, fractional sodium, and lithium excretion. RI coinfusion of specific angiotensin type 2 receptor antagonist PD-123319 abolished Ang III-induced natriuresis. The natriuretic response observed with RI Ang III was not reproducible with RI angiotensin (1-7) alone or together with angiotensin-converting enzyme inhibition. Similarly, neither RI angiotensin II alone or in the presence of aminopeptidase A inhibitor increased urine sodium excretion. In the absence of systemic angiotensin type 1 receptor blockade, Ang III alone did not increase urine sodium excretion, but natriuresis was enabled by the coinfusion of aminopeptidase N inhibitor and subsequently blocked by PD-123319. In angiotensin type 1 receptor-blocked rats, RI administration of aminopeptidase N inhibitor alone also induced natriuresis that was abolished by PD-123319. Ang III-induced natriuresis was accompanied by increased RI cGMP levels and was abolished by inhibition of soluble guanylyl cyclase. RI and renal tissue Ang III levels increased in response to Ang III infusion and were augmented by aminopeptidase N inhibition. These data demonstrate that endogenous intrarenal Ang III but not angiotensin II or angiotensin (1-7) induces natriuresis via activation of angiotensin type 2 receptors in the proximal tubule via a cGMP-dependent mechanism and suggest aminopeptidase N inhibition as a potential therapeutic target in hypertension.     

Induction of angiotensin II subtype 2 receptor-mediated blood pressure regulation in synthetic diet-fed rats

OBJECTIVE: Chronic feeding of a purified synthetic diet induces renin-angiotensin system-dependent moderate high blood pressure in normal Sprague-Dawley rats. The present study was designed to characterize the angiotensin II (Ang II) receptor type 2 (AT2)-specific mechanism of blood pressure regulation in these rats. METHODS: The effect of the AT2 receptor antagonist PD123319 (PD) on blood pressure was examined in vivo in synthetic diet-fed rats. Ang II-dependent contraction of aortic rings prepared from the synthetic diet-fed rats was also investigated. RESULTS: After 8 weeks of feeding the synthetic diet, the mean arterial pressure (MAP) was significantly elevated above levels measured in control rats (117 +/- 2 versus 102 +/- 3 mmHg, P < 0.05). Intravenous administration of PD to conscious hypertensive rats elicited an immediate dose-dependent increase in MAP that was sustained for approximately 7.4 min with 3 mg/kg PD. The angiotensin converting enzyme inhibitor captopril, but not the Ang II type 1 receptor blocker losartan, significantly attenuated the effect of PD on blood pressure. PD did not increase the plasma level of catecholamines. The PD-dependent blood pressure increase was not observed in normotensive control rats. Aortic ring assays revealed that functional activation of the AT2 receptor occurs only in the hypertensive rats, and this AT2 response is abolished by indomethacin (5 micromol/l) but not by Nomega-nitro-L-arginine methyl ester (100 Fmol/l). CONCLUSION: These results clearly demonstrate that AT2 receptor-mediated blood pressure regulation is functional in this experimental model of hypertension. Furthermore, cyclooxygenase metabolites might be the key mediators for the AT2 receptor-mediated blood pressure-lowering action.     

Angiotensin III stimulates aldosterone secretion from adrenal gland partially via angiotensin II type 2 receptor but not angiotensin II type 1 receptor

Angiotensin II (Ang II) and Ang III stimulate aldosterone secretion by adrenal glomerulosa, but the angiotensin receptor subtypes involved and the effects of Ang IV and Ang (1-7) are not clear. In vitro, different angiotensins were added to rat adrenal glomerulosa, and aldosterone concentration in the medium was measured. Ang II-induced aldosterone release was blocked (30.3 ± 7.1%) by an Ang II type 2 receptor (AT2R) antagonist, PD123319. Candesartan, an Ang II type 1 receptor (AT1R) antagonist, also blocked Ang II-induced aldosterone release (42.9 ± 4.8%). Coadministration of candesartan and PD123319 almost abolished the Ang II-induced aldosterone release. A selective AT2R agonist, CGP42112, was used to confirm the effects of AT2R. CGP42112 increased aldosterone secretion, which was almost completely inhibited by PD123319. In addition to Ang II, Ang III also induced aldosterone release, which was not blocked by candesartan. However, PD123319 blocked 22.4 ± 10.5% of the Ang III-induced aldosterone secretion. Ang IV and Ang (1-7) did not induce adrenal aldosterone secretion. In vivo, both Ang II and Ang III infusion increased plasma aldosterone concentration, but only Ang II elevated blood pressure. Ang IV and Ang (1-7) infusion did not affect blood pressure or aldosterone concentration. In conclusion, this report showed for the first time that AT2R partially mediates Ang III-induced aldosterone release, but not AT1R. Also, over 60% of Ang III-induced aldosterone release may be independent of both AT1R and AT2R. Ang III and AT2R signaling may have a role in the pathophysiology of aldosterone breakthrough.     

Intrarenal aminopeptidase N inhibition augments natriuretic responses to angiotensin III in angiotensin type 1 receptor-blocked rats

The renal angiotensin angiotensin type 2 receptor has been shown to mediate natriuresis, and angiotensin III, not angiotensin II, may be the preferential angiotensin type 2 receptor activator of this response. Angiotensin III is metabolized to angiotensin IV by aminopeptidase N. The present study hypothesizes that inhibition of aminopeptidase N will augment natriuretic responses to intrarenal angiotensin III in angiotension type 1 receptor-blocked rats. Rats received systemic candesartan for 24 hours before the experiment. After a 1-hour control, cumulative renal interstitial infusion of angiotensin III at 3.5, 7, 14, and 28 nmol/kg per minute (each dose for 30 minutes) or angiotensin III combined with aminopeptidase N inhibitor PC-18 was administered into 1 kidney. The contralateral control kidney received renal interstitial infusion of vehicle. In kidneys infused with angiotensin III alone, renal sodium excretion rate increased from 0.05+/-0.01 micromol/min in stepwise fashion to 0.11+/-0.01 micromol/min at 28 nmol/kg per minute of angiotensin III (overall ANOVA F=3.68; P<0.01). In angiotensin III combined with PC-18, the renal sodium excretion rate increased from 0.05+/-0.01 to 0.32+/-0.08 mumol/min at 28 nmol/kg per minute of angiotensin III (overall ANOVA F=6.2; P<0.001). The addition of intrarenal PD-123319, an angiotensin type 2 receptor antagonist, to renal interstitial angiotensin III plus PC-18 inhibited the natriuretic response. Mean arterial blood pressure and renal sodium excretion rate from control kidneys were unchanged by angiotensin III +/- PC-18 + PD-123319. Angiotensin III plus PC-18 induced a greater natriuretic response than Ang III alone (overall ANOVA F=16.9; P=0.0001). Aminopeptidase N inhibition augmented the natriuretic response to angiotensin III, suggesting that angiotensin III is a major agonist of angiotensin type 2 receptor-induced natriuresis.     

Role of angiotensin receptor subtypes in mesenteric vascular proliferation and hypertrophy.

The aim of this study was to explore the regulation of angiotensin receptors after chronic infusion with angiotensin II (Ang II) and to clarify the relative roles of the angiotensin type 1 (AT(1)) and type 2 (AT(2)) receptors in the mediation of Ang II-induced mesenteric vascular hypertrophy. In male Sprague-Dawley rats, Ang II infusion at a dose of 58.3 ng/min by subcutaneous osmotic minipumps for 14 days led to increased mesenteric weight and wall:lumen ratio of the vessels and proliferation of smooth muscle cells. These vascular changes were attenuated by either valsartan, an AT(1) receptor antagonist, at a dose of 30 mg. kg(-1). d(-1) by gavage, or PD123319, an AT(2) receptor antagonist, at a dose of 830 ng/min by intraperitoneally implanted osmotic minipumps. Ang II infusion was associated with hypertension, which was prevented by valsartan, but not PD123319. (125)I-Sar(1), Ile(8) Ang II binding to mesenteric vasculature was increased after Ang II infusion. Valsartan treatment was associated with reduced Ang II binding to both receptor subtypes, whereas PD123319 was associated with reduced Ang II binding to only the AT(2) receptor subtype. These findings suggest that the trophic and proliferative effects of Ang II on the mesenteric vasculature are mediated by both AT(1) and AT(2) receptors.     

Tempol augments angiotensin II-induced AT2 receptor-mediated relaxation in diabetic rat thoracic aorta

OBJECTIVE: To assess angiotensin II type 2 receptor-mediated responses in thoracic aorta of streptozotocin-induced diabetic rats. METHODS: The concentration-dependent relaxation response (in the presence of an AT1 receptor blocker) to angiotensin II (Ang II) was studied in phenylephrine (PE) or potassium chloride (KCl) precontracted rat thoracic aortic rings isolated from male Sprague-Dawley rats pretreated with streptozotocin (65 mg/kg i.p.) or vehicle 8 weeks prior to the study. RESULTS: Ang II-induced relaxation response (% relaxation), evident only in the presence of an AT1 receptor blocker, was significantly enhanced in aortic rings isolated from diabetic (55%) compared to control (25%) rats. Tempol (100 micromol/l) augmented the relaxation response in aortic rings isolated from diabetic (80%) but not control (28%) rats. N-nitro-l-arginine methyl ester (L-NAME) (100-300 micromol/l) [a nitric oxide (NO) synthase inhibitor] partially inhibited the relaxation response in diabetic (25%) and control (15%) rats. However, l-NAME (100 micromol/l) and glipizide or butanedione monoxime (1 micromol/l) (ATP-sensitive K channel blockers) together completely blocked the relaxation response. [H]Ang II saturation binding at the AT2 receptor was enhanced in aortic membranes from diabetic [maximum binding capacity, (Bmax)=1.14 +/- 0.06 fmol/mg protein] compared to control rats (Bmax=0.75 +/- 0.03 fmol/mg protein), with no change in the dissociation equilibrium constant (Kd) value (2.55 +/- 0.12 versus 2.22 +/- 0.15 nmol/l). CONCLUSIONS: The results suggest enhanced AT2-receptor density and function [mediated by a nitric oxide and ATP-sensitive K channel-dependent relaxation response (in presence of an AT1 receptor blocker)] in thoracic aorta isolated from diabetic rats. This could be a compensatory mechanism, which may be therapeutically exploited.     

Effects of intracerebroventricular atrial natriuretic factor on angiotensin II- or sodium-induced blood pressure elevation and natriuresis

We examined the effects of intracerebroventricular (i.c.v.) administration of atrial natriuretic factor (ANF) on pressor and natriuretic responses induced by i.c.v. angiotensin II (Ang II) or hypertonic NaCl. Conscious male Wistar rats were given one of the following solutions into the lateral ventricle: artificial cerebrospinal fluid (CSF); rat ANF (99-126) 1.0 microgram/kg per min; Ang II 100 ng/kg per min; 0.6 mol/l NaCl; Ang II plus ANF, and 0.6 mol/l NaCl plus ANF. The i.c.v. infusion of artificial CSF or ANF alone did not cause significant changes in mean blood pressure, urinary volume or sodium excretion (UNaV). The i.c.v. infusion of Ang II or 0.6 mol/l NaCl raised mean blood pressure, decreased urinary volume and increased UNaV. When ANF was administered with Ang II, the Ang II-induced responses were diminished significantly (delta mean blood pressure, +10 +/- 3 versus +20 +/- 4 mmHg; delta urinary volume, -38 +/- 9 versus -78 +/- 5 microliters/min; delta UNaV, +0.49 +/- 0.51 versus +2.28 +/- 0.58 mumol/min). The centrally administered ANF opposed the effects of 0.6 mol/l NaCl, though the effect was significant only in respect of blood pressure. Our results indicate that the brain ANF may have an antinatriuretic role in some conditions.     

Estrogen regulates adrenal angiotensin type 1 receptors by modulating adrenal angiotensin levels

Estrogen inhibits adrenal angiotensin type 1 receptor (AT(1)R) binding sites and attenuates the adrenal responsivity to angiotensin II (Ang II). Ang II modulates AT(1)R expression. Here, we determined if estrogen-induced down-regulation of adrenal AT(1)Rs involves modulation of adrenal Ang II. Female rats were ovariectomized (OVX) and injected with 17beta-estradiol benzoate (E(2); 40 micro g/kg) or vehicle for 7 d. Adrenal Ang II was separated from other angiotensin peptides by HPLC and measured by RIA. Scatchard analysis of radioligand binding curves showed that E(2) or captopril (Cap; 0.5 g/liter water) significantly reduced adrenal AT(1)R binding (maximum binding capacity) by 22% and 19%, respectively, compared with OVX (276 +/- 2.09 fmol/mg protein). E(2) and Cap lowered adrenal Ang II levels by 39% and 21%, respectively, compared with OVX (4.10 +/- 0.44 pmol/g). E(2) caused no further reductions in adrenal AT(1)R binding or in Ang II levels in Cap-treated OVX rats. High-dose Ang II infusion (1000 ng/kg.min) increased adrenal Ang II levels by 71% and lowered AT(1)R binding by 18%. Under these infusion conditions, E(2) did not reduce adrenal Ang II or AT(1)R binding. No differences in AT(1)R affinity (dissociation constant) were observed among groups. These data suggest that E(2) regulates the number of adrenal AT(1)R binding sites indirectly by modulating adrenal Ang II.     

Importance of the angiotensin type 1 receptor in angiotensin II-induced bronchoconstriction and bronchial hyperresponsiveness in the guinea pig

Although angiotensin II (Ang II) causes bronchoconstriction and bronchial hyperresponsiveness to methacholine in mildly asthmatic patients, the responsible mechanisms for these reactions are unclear. The authors examined the effect of intravenous infusion of Ang II on airway constriction in guinea pigs. Furthermore, the effects of subthreshold concentrations of Ang II on bronchial responsiveness to methacholine were investigated. Airway opening pressure (Pao), an index of bronchoconstriction, increased dose dependently after intravenous infusion of 3 and 10 nmol/kg Ang II (72.2 and 236.5 increase above the baseline value, respectively). In another set of experiments, animals received a methacholine inhalation challenge under a constant intravenous infusion of a subthreshold dose of Ang II (2 nmol/kg/min). The Ang II infusion elicited bronchial hyperresponsiveness to methacholine. The provocative concentration of methacholine, which produced a 200% increase above the baseline Pao (PC200), decreased from 306.9 to 156.1 micrograms/mL upon Ang II infusion. Pretreatment with TCV-116, a type 1 Ang II (AT1) receptor antagonist, but not PD123319, a type 2 Ang II (AT2) receptor antagonist, dose dependently prevented both the Ang II-induced bronchoconstriction and bronchial hyperresponsiveness to methacholine. The authors conclude that Ang II caused bronchoconstriction and induced bronchial hyperresponsiveness to methacholine via the AT1 receptors and that this effect did not involve the release of other bronchoactive mediators.     

Olmesartan improves endothelin-induced hypertension and oxidative stress in rats.

Recent studies have indicated that both endothelin (ET) and angiotensin (Ang) II stimulate oxidative stress, which contributes to the development of hypertension. Here, we examined the effects of Ang II type 1 (AT1) receptor blockade on reactive oxygen species (ROS) formation in ET-dependent hypertension. Chronic ET-1 infusion (2.5 pmol/kg/min, i.v., n=7) into rats for 14 days increased systolic blood pressure from 113+/-1 to 141+/-2 mmHg. ET-1-infused rats showed greater plasma renin activity (8.1+/-0.8 Ang I/ml/h), and greater Ang I (122+/-28 fmol/ml) and Ang II levels (94+/-13 fmol/ml) than vehicle (0.9% NaCl)-infused rats (3.1+/-0.6 Ang I/ml/h, 45+/-8 and 47+/-7 fmol/ml, respectively, n=6). Angiotensin converting enzyme and AT1 receptor expression in aortic tissues were similar between the vehicle- and ET-1-infused rats. Vascular superoxide anion (O2-) production and plasma thiobarbituric acid-reactive substance (TBARS) levels were greater in ET-1-infused rats (27+/-1 counts per minutes [CPM]/mg dry tissue weight and 8.9+/-0.8 micromol/l, respectively) than vehicle-infused rats (16+/-1 CPM/mg and 5.1+/-0.1 micromol/l, respectively). The ET-1-induced hypertension was prevented by simultaneous treatment with a new AT1 receptor antagonist, olmesartan (0.01% in chow, 117+/-5 mmHg, n =7), or hydralazine (15 mg/kg/day in drinking water, 118+/-4 mmHg, n=6). Olmesartan prevented ET-1-induced increases in vascular O2- production (15+/-1 CPM/mg) and plasma TBARS (5.0+/-0.1 micromol/l). Vascular O2- production and plasma TBARS were also decreased by hydralazine (21+/-1 CPM/mg and 7.0+/-0.3 micromol/l, respectively), but these levels were significantly higher than in vehicle-infused rats. These data suggest that ET-dependent hypertension is associated with augmentation of Ang II levels and ROS formation. The combined effects of the elevations in circulating ET-1 and Ang II, as well as the associated ROS production, may contribute to the development of hypertension induced by chronic ET-1 infusion.     

Angiotensin II, type 2 receptor is not involved in the angiotensin II-mediated pro-atherogenic process in ApoE-/- mice

OBJECTIVE: Angiotensin II (Ang II) accelerates atherogenesis in ApoE mice via the angiotensin II, type 1 receptor (AT1) while the type 2 receptor (AT2) is suggested to counteract atherogenesis. To confirm and further explore this possibility, we studied the effect of AT2 receptor antagonism on Ang II-accelerated atherosclerosis. METHODS: ApoE mice were fed a standard or high cholesterol diet (1.25%) for 4 weeks. Mice on each diet were treated with either Ang II (0.5 microg/kg per min) or Ang II in combination with PD123319 (3 mg/kg per day). Plaque distribution was assessed by en face quantification of the thoracic aorta and in cross-sections of the aortic root. Mean arterial pressure (MAP) was measured. AT1 and AT2 receptor expression were analysed using real-time polymerase chain reaction (PCR) and the localization of the AT2 receptor protein confirmed with immunohistochemistry. RESULTS: Ang II infusion increased MAP only in mice on a standard diet (P < 0.001). Regardless of diet, Ang II-infused mice had 22-30 times increased plaque area in the thoracic aorta (P < 0.001 for both). Ang II had no effect on plaque in the aortic root. Plaque area was not affected by PD123319. AT2 receptor was heavily expressed in the plaques and increased six- to ninefold by a high cholesterol diet and Ang II infusion (P < 0.01). CONCLUSION: Ang II increases the extent of atherosclerosis in ApoE mice. Despite up-regulation of the AT2 receptor, we found no support for an effect of the AT2 receptor on atherogenesis in this model.     

Angiotensin II relaxations of bovine adrenal cortical arteries: role of angiotensin II metabolites and endothelial nitric oxide

Angiotensin (Ang) II regulates adrenal steroidogenesis and adrenal cortical arterial tone. Vascular metabolism could decrease Ang II concentrations and produce metabolites with vascular activity. Our goals were to study adrenal artery Ang II metabolism and to characterize metabolite vascular activity. Bovine adrenal cortical arteries were incubated with Ang II (100 nmol/L) for 10 and 30 minutes. Metabolites were analyzed by mass spectrometry. Ang (1-7), Ang III, and Ang IV concentrations were 146+/-21, 173+/-42 and 58+/-11 pg/mg at 10 minutes and 845+/-163, 70+/-14, and 31+/-3 pg/mg at 30 minutes, respectively. Concentration-related relaxations of U46619-preconstricted cortical arteries to Ang II (maximum relaxation=29+/-3%; EC(50)=3.4 pmol/L) were eliminated by endothelium removal and inhibited by the NO synthase inhibitor, nitro-L-arginine (30 micromol/L; maximum relaxation=14+/-7%). Ang II relaxations were enhanced by the angiotensin type-1 receptor antagonist losartan (1 micromol/L; maximum relaxation=41+/-3%; EC(50)=11 pmol/L). Losartan-enhanced Ang II relaxations were inhibited by nitro-L-arginine (maximum relaxation=18+/-5%) and the angiotensin type-2 receptor antagonist PD123319 (10 micromol/L; maximum relaxation=27+/-5%). Ang (1-7) and Ang III caused concentration-related relaxations with less potency (EC(50)=43 and 24 nmol/L, respectively) but similar efficacy (maximum relaxations=39+/-3% and 48+/-5%, respectively) as losartan-enhanced Ang II relaxations. Ang (1-7) relaxations were inhibited by nitro-L-arginine (maximum relaxation=16+/-4%) and the Ang (1-7) receptor antagonist 7(D)-Ala-Ang (1-7) (1 micromol/L; maximum relaxation=10+/-3%) and eliminated by endothelium removal. Thus, Ang II metabolism by adrenal cortical arteries to metabolites with decreased vascular activity represents an inactivation pathway possibly decreasing Ang II presentation to adrenal steroidogenic cells and limits Ang II vascular effects.     

AT1 receptor mediated augmentation of intrarenal angiotensinogen in angiotensin II-dependent hypertension

Angiotensin (Ang) II-infused hypertensive rats exhibit increases in renal angiotensinogen mRNA and protein, as well as urinary angiotensinogen excretion in association with increased intrarenal Ang II content. The present study was performed to determine if the augmentation of intrarenal angiotensinogen requires activation of Ang II type 1 (AT1) receptors. Male Sprague-Dawley rats (200 to 220 g) were divided into 3 groups: sham surgery (n=10), subcutaneous infusion of Ang II (80 ng/min, n=11), and Ang II infusion plus AT1 blocker (ARB), olmesartan (5 mg/d, n=12). Ang II infusion progressively increased systolic blood pressure (SBP) compared with sham (178+/-8 mm Hg versus 119+/-4 at day 11). ARB treatment prevented hypertension (113+/-6 at day 11). Twenty-four-hour urine collections were taken at day 12, and plasma and tissue samples were harvested at day 13. The Ang II+ARB group had a significant increase in plasma Ang II compared with Ang II and sham groups (365+/-46 fmol/mL versus 76+/-9 and 45+/-14, respectively). Nevertheless, ARB treatment markedly limited the enhancement of kidney Ang II by Ang II infusion (65+/-17 fmol/g in sham, 606+/-147 in Ang II group, and 288+/-28 in Ang II+ARB group). Ang II infusion significantly increased kidney angiotensinogen compared with sham (1.69+/-0.21 densitometric units versus 1.00+/-0.17). This change was reflected by increased angiotensinogen immunostaining in proximal tubules. ARB treatment prevented this increase (1.14+/-0.12). Urinary angiotensinogen excretion rates were enhanced 4.7x in Ang II group (4.67+/-0.41 densitometric units versus 1.00+/-0.21) but ARB treatment prevented the augmentation of urinary angiotensinogen (0.96+/-0.23). These data demonstrate that augmentation of intrarenal angiotensinogen in Ang II-infused rats is AT1-dependent and provide further evidence that urinary angiotensinogen is closely linked to intrarenal Ang II in Ang II-dependent hypertension.     

Effect of angiotensin II on pulse wave velocity in humans is mediated through angiotensin II type 1 (AT(1)) receptors

The objective of this study was to examine the effect of angiotensin II (Ang II) and angiotensin II type 1 (AT(1)) receptor blockade on pulse wave velocity (PWV) in healthy humans. We studied nine young male volunteers in a double-blind randomised crossover design. Carotid-femoral PWV (an index of arterial stiffness) was measured by using a Complior machine. Subjects were previously treated for 3 days with once-daily dose of either a placebo or valsartan 80 mg. On the third day, they were infused with either placebo or 5 ng/kg/min of Ang II over 30 min. Subjects thus received placebo capsule + placebo infusion (P), valsartan + placebo infusion (V), placebo + Ang II infusion (A), and valsartan + Ang II infusion (VA) combinations. Heart rate (HR), blood pressure and PWV were recorded at baseline and then every 10 min during infusion and once after the end of infusion. There were significant increases in systolic blood pressure (SBP), diastolic blood pressure (DBP) and mean arterial pressure (MAP) with A compared with P (P = 0.002, P = 0.002, P = 0.001 respectively). These rises in blood pressure were completely blocked by valsartan. A significant rise in PWV by A was seen compared with P (8.38 +/- 0.24 vs 7.48 +/- 0.24 m/sec, P = 0.013) and was completely blocked by valsartan; VA compared with P (7.27 +/- 0.24 vs 7.48 +/- 0.24 m/sec, P = NS). Multiple linear regression analysis showed that blockade of Ang II induced increase in blood pressure by valsartan contributed to only 30% of the total reduction in Ang II induced rise in PWV (R(2) = 0.306). The conclusions were that valsartan completely blocks the effect of Ang II on PWV. The effect of Ang II on PWV is mediated through AT(1)receptors. Reduction in PWV by Ang II antagonist is not fully explained by its pressure lowering effect of Ang II and may be partially independent of its effect on blood pressure.     

Angiotensin-(1-7) downregulates the angiotensin II type 1 receptor in vascular smooth muscle cells

Angiotensin (Ang)-(1-7) is a biologically active peptide of the renin-angiotensin system that has both vasodilatory and antiproliferative activities that are opposite the constrictive and proliferative effects of angiotensin II (Ang II). We studied the actions of Ang-(1-7) on the Ang II type 1 (AT(1)) receptor in cultured rat aortic vascular smooth muscle cells to determine whether the effects of Ang-(1-7) are due to its regulation of the AT(1) receptor. Ang-(1-7) competed poorly for [(125)I]Ang II binding to the AT(1) receptor on vascular smooth muscle cells, with an IC(50) of 2.0 micromol/L compared with 1.9 nmol/L for Ang II. The pretreatment of vascular smooth muscle cells with Ang-(1-7) followed by treatment with acidic glycine to remove surface-bound peptide resulted in a significant decrease in [(125)I]Ang II binding; however, reduced Ang II binding was observed only at micromolar concentrations of Ang-(1-7). Scatchard analysis of vascular smooth muscle cells pretreated with 1 micromol/L Ang-(1-7) showed that the reduction in Ang II binding resulted from a loss of the total number of binding sites [B(max) 437.7+/-261.5 fmol/mg protein in Ang-(1-7)-pretreated cells compared with 607.5+/-301.2 fmol/mg protein in untreated cells, n=5, P<0.05] with no significant effect on the affinity of Ang II for the AT(1) receptor. Pretreatment with the AT(1) receptor antagonist L-158,809 blocked the reduction in [(125)I]Ang II binding by Ang-(1-7) or Ang II. Pretreatment of vascular smooth muscle cells with increasing concentrations of Ang-(1-7) reduced Ang II-stimulated phospholipase C activity; however, the decrease was significant (81.2+/-6.4%, P<0.01, n=5) only at 1 micromol/L Ang-(1-7). These results demonstrate that pharmacological concentrations of Ang-(1-7) in the micromolar range cause a modest downregulation of the AT(1) receptor on vascular cells and a reduction in Ang II-stimulated phospholipase C activity. Because the antiproliferative and vasodilatory effects of Ang-(1-7) are observed at nanomolar concentrations of the heptapeptide, these responses to Ang-(1-7) cannot be explained by competition of Ang-(1-7) at the AT(1) receptor or Ang-(1-7)-mediated downregulation of the vascular AT(1) receptor.     

Effects of angiotensin-(1-7) and other bioactive components of the renin-angiotensin system on vascular resistance and noradrenaline release in rat kidney

OBJECTIVE: Angiotensin (Ang) is broken down enzymatically to several different metabolites which, in addition to Ang II, may have important biological effects in the kidney. This study investigates the role of Ang metabolites on vascular resistance and noradrenaline release in the rat kidney. METHODS AND RESULTS: In rat isolated kidney Ang I, Ang II, Ang III, Ang IV and des-Asp-Ang I induced pressor responses and enhanced noradrenaline release to renal nerve stimulation (RNS) in an concentration-dependent manner, with the following rank order of potency (EC(50)): Ang II >or= Ang III > Ang I = des-Asp-Ang I > Ang IV. All effects were blocked by the AT(1)-receptor antagonist EXP 3174 (0.1 micromol/l) but not by the AT(2)-receptor antagonist PD 123319 (1 micromol/l). Angiotensin-converting enzyme (ACE) inhibition by captopril (10 micromol/l) abolished the effect of Ang I and des-Asp-Ang I but had no influence on the effect of the other metabolites. Ang-(1-7) blocked the effects of Ang I and Ang II, being 10 times more potent against Ang I than Ang II. The selective Ang-(1-7) receptor blocker d-Ala7-Ang-(1-7) (10 micromol/l) did not influence the inhibitory effects of Ang-(1-7). Ang-(1-7) (10 micromol/l) by itself had no influence on vascular resistance and RNS-induced noradrenaline release. CONCLUSION: Ang I, Ang II, Ang III, Ang IV and des-Asp-Ang I regulate renal vascular resistance and noradrenaline release by activation of AT(1) receptors. In the case of Ang I and des-Asp-Ang I this depends on conversion by ACE. Ang-(1-7) may act as a potent endogenous inhibitor/antagonist of ACE and the AT(1)-receptors, respectively.     

The positive inotropic effect of angiotensin II: role of endothelin-1 and reactive oxygen species

Many effects believed to be because of angiotensin II (Ang II) are attributable to the action of endothelin (ET)-1, which is released/produced by Ang II. We investigated whether Ang II elicits its positive inotropic effect (PIE) by the action of endogenous ET-1, in addition to the role played by reactive oxygen species (ROS) in this mechanism. Cat cardiomyocytes were used for: (1) sarcomere shortening measurements; (2) ROS measurements by epifluorescence; (3) immunohistochemical staining for preproET-1, BigET-1, and ET-1; and (4) measurement of preproET-1 mRNA by RT-PCR. Cells were exposed to 1 nmol/L Ang II for 15 minutes. This low concentration of Ang II increases sarcomere shortening by 29.2+/-3.7% (P<0.05). This PIE was abrogated by Na+/H+ exchanger or Na+/Ca2+ exchanger reverse mode inhibition. The production of ROS increased in response to Ang II treatment (DeltaROS respect to control: 68+/-15 fluorescence units; P<0.05). The Ang II-induced PIE and ROS production were blocked by the Ang II type 1 receptor blocker losartan, the nonselective ET-1 receptor blocker TAK044, the selective ETA receptor blocker BQ-123, or the ROS scavenger N-(2-mercapto-propionyl)glycine. Exogenous ET-1 (0.4 nmol/L) induced a similar PIE and increase in ROS production to those caused by Ang II. Immunostaining for preproET-1, BigET-1, and ET-1 was positive in cardiomyocytes. The preproET-1 mRNA abundance increased from 100+/-4.6% in control to 241.9+/-39.9% in Ang II-treated cells (P<0.05). We conclude that the PIE after exposure to 1 nmol/L Ang II is due to endogenous ET-1 acting through the ETA receptor and triggering ROS production, Na+/H+ exchanger stimulation, and Na+/Ca2+ exchanger reverse mode activation.     

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.     

Renal interstitial cGMP mediates natriuresis by direct tubule mechanism

The objective of this study was to test the hypothesis that renal interstitial (RI) cGMP is natriuretic in vivo. In conscious rats (n=8), urinary sodium excretion (U(Na)V) was significantly greater on days 3 and 4 of RI infusion of cGMP (1.17+/-0.14 and 1.61+/-0.11 mmol/24 h, respectively) than during vehicle infusion (0.56+/-0.15 and 0.70+/-0.17 mmol/24 h, respectively) (P<0.01). Similarly, U(Na)V was greater on days 3 and 4 of RI infusion of 8-bromo-cGMP (2.15+/-0.42 and 2.16+/-0.1 mmol/24 h, respectively). Protein kinase G inhibitor Rp-8-pCPT-cGMPS reduced cGMP-induced and 8-bromo-cGMP-induced U(Na)V to control levels. Acute RI infusion of L-arginine (L-Arg, 40 mg. kg(-1). min(-1)), but not D-arginine, caused an increase in U(Na)V from 1.65+/-0.11 to 4.07+/-0.1 micromol/30 min (P<0.01). This increase was blocked by RI infusion of N(G)-nitro-L-arginine methyl ester (100 ng. kg(-1). min(-1)) by the phosphodiesterase (PDE II) activator 5,6DMcBIMP (0.01 micromol/microL), by PDE II (0.03 U. kg(-1). min(-1)) itself, or by the soluble guanylyl cyclase inhibitor 1-H-[1,2,4]oxadiazolo-[4,2-alpha]quinoxalin-1-one (ODQ, 0.12 mg. kg(-1). min(-1)). The PDE II activator also blocked L-Arg-stimulated cGMP levels. The NO donor S-nitroso-N-acetylpenicillamine (SNAP, 0.12 micromol. L(-1). kg(-1). min(-1)) increased U(Na)V from 1.65+/-0.11 to 2.93+/-0.08 micromol/30 min (P<0.01), and this response was blocked completely by ODQ. Renal arterial but not RI administration of the heat-stable enterotoxin of Escherichia coli induced natriuresis. RA infusion of cGMP (3 microg/min) increased U(Na)V, renal blood flow (RBF), and glomerular filtration rate (GFR). Renal cortical interstitial cGMP infusion increased U(Na)V with no effect on total RBF, renal cortical blood flow, or GFR. Similarly, the natriuretic actions of renal interstitial L-Arg or SNAP were not accompanied by any change in RBF or GFR. Medullary cGMP infusion had no effect on U(Na)V, total RBF, or medullary blood flow. Texas red-labeled cGMP infused via the RI space was distributed exclusively to cortical renal tubular cells. The results demonstrate that RI cGMP inhibits renal tubular sodium absorption via protein kinase G independently of hemodynamic changes. These observations indicate that the cortical interstitial compartment provides a potentially important domain for cell-to-cell signaling within the kidney.