Vascular response to angiotensin II is exaggerated through an upregulation of AT1 receptor in AT2 knockout mice

Blood pressure is elevated and pressor response to angiotensin II (Ang II) is exaggerated in AT2 null mice. The purpose of the present study was to elucidate the mechanism for the increased responsiveness to Ang II in the mice. The contraction of aortic strips generated by Ang II was significantly greater in the AT2 gene-deleted mice than the control, which was completely abolished by AT1 antagonist losartan. The aortic content of AT1 receptor was significantly increased (P < 0.05, n = 5) in the AT2 null mice (212 +/- 58.2 fmol/mg protein) compared with the control (98.2 +/- 55.9 fmol/mg protein). While both AT1 and AT2 mRNAs were expressed in the aorta of the control mice, only AT1 mRNA was expressed in the AT2 knockout mice. The expression of AT1 mRNA in the AT2 knockout mice was significantly higher (1.5-fold, P < 0.05, n = 5) than that in the control. The present study clearly demonstrated that the increased vascular reactivity to Ang II in AT2 knockout mice is at least partly due to an increased vascular AT1 receptor expression and suggested that AT2 counteracts AT1-mediated vascular action of Ang II through downregulation of AT1 receptor by a crosstalk between these receptors by some as yet unknown mechanisms.     

Role of angiotensin II receptor subtypes in porcine basilar artery: functional, radioligand binding, and cell culture studies

We aimed to clarify responsiveness to angiotensin (Ang) II in the porcine basilar artery and the role of Ang II receptor subtypes by functional, radioligand binding, and cell culture studies. Ang II induced more potent contractions in the proximal part than in the distal part of isolated porcine basilar arteries. The contraction induced by Ang II was inhibited by the Ang II type 1 (AT1) receptor antagonist losartan, but the Ang II type 2 (AT2) receptor antagonist PD123319 enhanced it. After removal of the endothelium, the effect of losartan remained but the effect of PD123319 was abolished. The specific binding site of [3H]Ang II on the smooth muscle membrane was inhibited by losartan, but not by PD123319. Stimulation of angiotensin II increased nitric oxide (NO) production in cultured basilar arterial endothelial cells. This production was inhibited by PD123319 and the NO synthase inhibitor L-NG-nitroarginine. These results suggest that the contraction induced by Ang II might be mediated via the activation of AT1 receptors on the basilar arterial smooth muscle cells and be modulated via the activation of AT2 receptors on the endothelial cells, followed by NO production.     

Angiotensin II and III suppress food intake via angiotensin AT(2) receptor and prostaglandin EP(4) receptor in mice

Intracerebroventricularly administered angiotensin (Ang) II and III dose-dependently suppressed food intake in mice and their anorexigenic activities were inhibited by AT(2) receptor-selective antagonist. Ang II did not suppress food intake in AT(2) receptor-knockout mice, while it did significantly in wild-type and AT(1) receptor-knockout mice. The suppression of food intake in AT(1) receptor-knockout mice was smaller than that in wild-type. The anorexigenic activities of Ang II and III were also blocked by a selective antagonist for prostaglandin EP(4) receptor. Taken together, centrally administered Ang II and III may decrease food intake through AT(2) receptor with partial involvement of AT(1) receptor, followed by EP(4) receptor activation, which is a novel pathway regulating food intake.     

Negative inotropic effects of angiotensin II, endothelin-1 and phenylephrine in indo-1 loaded adult mouse ventricular myocytes

Angiotensin II (Ang II). endothelin-1 (ET-1) and phenylephrine are receptor agonists that share the signal transduction acting through acceleration of phosphoinositide hydrolysis in the heart. Because the regulation of myocardial contractility induced by these receptor agonists shows a wide range of species-dependent variation among experimental animals, we carried out experiments to elucidate the mechanism of contractile regulation induced by these agents in mice which are employed currently more as transgenic models. Effects of Ang II, ET-1 and phenylephrine on cell shortening and Ca2+ transients were investigated in single ventricular myocytes loaded with indo-1/AM. Ang II (10(-8), 10(-7) M), ET-1 (10(-10), 10(-9) M) and phenylephrine (10(-6), 10(-5) M in the presence of the beta-adrenoceptor antagonist timolol) decreased the cell shortening [Ang II: 58.4+/-9.03 (n = 8), 50.3+/-11.90% (n = 6); ET-1: 48.4+/-8.27, 31.2+/-6.45% (n = 5); phenylephrine: 45.7+/-11.60, 28.7+/-5.89% (n = 5)]. By contrast, the amplitude of Ca2+ transients was not significantly influenced by these agonists. The selective protein kinase C inhibitor chelerythrine at 10(-6) M significantly inhibited the decrease in cell shortening induced by these receptor agonists. These results indicate that Ang II, ET-1 and phenylephrine elicit a negative inotropic effect with insignificant alteration of Ca2+ transients, which may be mainly mediated by activation of protein kinase C in mouse ventricular cardiomyocytes.     

Upregulation of ERK1/2-eNOS via AT2 Receptors Decreases the Contractile Response to Angiotensin II in Resistance Mesenteric Arteries from Obese Rats

It has been clearly established that mitogen-activated protein kinases (MAPKS) are important mediators of angiotensin II (Ang II) signaling via AT1 receptors in the vasculature. However, evidence for a role of these kinases in changes of Ang II-induced vasoconstriction in obesity is still lacking. Here we sought to determine whether vascular MAPKs are differentially activated by Ang II in obese animals. The role of AT2 receptors was also evaluated. Male monosodium glutamate-induced obese (obese) and non-obese Wistar rats (control) were used. The circulating concentrations of Ang I and Ang II, determined by HPLC, were increased in obese rats. Ang II-induced isometric contraction was decreased in endothelium-intact resistance mesenteric arteries from obese compared with control rats and exhibited a retarded AT1 receptor antagonist response. Blocking of AT2 receptors and inhibition of either endothelial nitric oxide synthase (eNOS) or extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) restored Ang II-induced contraction in obese rats. Western blot analysis revealed increased protein expression of AT2 receptors in arteries from obese rats. Basal and Ang II-induced ERK1/2 phosphorylation was also increased in obese rats. Blockade of either AT1 or AT2 receptors corrected the increased ERK1/2 phosphorylation in arteries from obese rats to levels observed in control preparations. Phosphorylation of eNOS was increased in obese rats. Incubation with the ERK1/2 inhibitor before Ang II stimulation did not affect eNOS phosphorylation in control rats; however, it corrected the increased phosphorylation of eNOS in obese rats. These results clearly demonstrate that enhanced AT2 receptor and ERK1/2-induced, NO-mediated vasodilation reduces Ang II-induced contraction in an endothelium-dependent manner in obese rats.     

Role of AT2 receptor in the brain in regulation of blood pressure and water intake

The effects of intracerebroventricular (ICV) injection of angiotensin II (ANG II) on blood pressure and water intake were examined with the use of ANG II receptor-deficient mice. ICV injection of ANG II increased systolic blood pressure in a dose-dependent manner in wild-type (WT) mice and ANG type 2 AT(2) receptor null (knockout) (AT(2)KO) mice; however, this increase was significantly greater in AT(2)KO mice than in WT mice. The pressor response to a central injection of ANG II in WT mice was inhibited by ICV preinjection of the selective AT(1) receptor blocker valsartan but exaggerated by the AT(2) receptor blocker PD-123319. ICV injection of ANG II also increased water intake. It was partly but significantly suppressed both in AT(2)KO and AT(1)aKO mice. Water intake in AT(2)/AT(1)aKO mice did not respond to ICV injection of ANG II. Both valsartan and PD-123319 partly inhibited water intake in WT mice. These results indicate an antagonistic action between central AT(1)a and AT(2) receptors in the regulation of blood pressure, but they act synergistically in the regulation of water intake induced by ANG II.     

Genetic deletion of angiotensin AT2 receptor leads to increased cell numbers in different brain structures of mice

Angiotensin II (Ang II) is a potent vasoactive peptide and displays growth factor-like properties. Different high-affinity Ang II receptor subtypes (AT1A, AT1B and AT2) have been cloned. They are expressed in various brain structures. Additionally, it has been assumed that Mas could interact directly or indirectly with the renin-angiotensin system. The AT1 receptor mediates pressor and mitogenic effects of Ang II, whereas physiological function and signaling mechanisms of the AT2 receptor remain poorly understood. Recent reports have shown that Ang II could mediate apoptosis through AT2 receptors. Since the AT1A, AT2 and Mas knockout mice provide new tools for uncovering potential actions of Ang II, the cell number in different brain structures of male adult wild-type mice and mice deficient for AT1A, AT2 or Mas was evaluated to get more insight into the role of Ang II in central nervous system development. In nearly all investigated brain structures (cortex, hippocampus, amygdala, thalamus), the cell number was significantly higher in AT2-deficient mice in comparison to wild-type mice. To the contrary, in AT1A-deficient mice the cell number was significantly less than in controls in the lateral geniculate and the medial amygdaloid nucleus. However, cell numbers were not changed in Mas-knockout mice compared to their wild-types. These results show the contrary effects of both angiotensin receptors on cell growth and represent the first demonstration of their action on neuronal cell development evidenced in the adult mouse brain.     

ETA-receptor blockade impairs vasoconstriction after hemorrhage in xenon-anesthetized dogs treated with an AT1-receptor antagonist

The effects of endothelin receptor subtype A (ETA) blockade on hemodynamics and hormonal adaptation during hemorrhage were studied in xenon/remifentanil-anesthetized dogs (n=6) pretreated with an angiotensin II type 1 (AT1)-receptor blocker. Controls: after a baseline awake period, anesthesia was induced in the dogs with propofol and maintained with xenon/remifentanil (baseline anesthesia). Sixty minutes later, 20 mL x kg(-1) of blood was withdrawn within 5 min and the dogs observed for another hour (hemorrhage). AT1 group followed the same protocol as controls except the AT1-receptor blocker losartan (i.v. 100 microg x kg(-1) x min(-1)) was started at the beginning of the experiment. AT1+ETA group was the same as AT1 group but with the addition of the ETA-receptor blocker atrasentan (i.v. 1 mg x kg(-1), then 0.01 mg x kg(-1) x min(-1)). In controls, mean arterial pressure (MAP) remained unchanged during baseline anesthesia, whereas systemic vascular resistance (SVR) increased from 3282+/-281 to 7321+/-803 dyn.s.cm-5, heart rate (HR) decreased from 86+/-4 to 40+/-3 beats x min(-1), and cardiac output (CO) decreased from 2.3+/-0.2 to 0.9+/-0.1 L x min(-1) (p<0.05), with no further changes after hemorrhage. In AT1-inhibited dogs, MAP (71+/-6 mm Hg) and SVR (5939+/-611 dyn x s x cm(-5)) were lower during baseline anesthesia and after hemorrhage, but greater than those in AT1+ETA (66+/-7 mm Hg, 5034+/-658 dyn x s x cm(-5)) (p<0.05). HR and CO were not different between groups. Plasma concentration of vasopressin was highest with AT1+ETA inhibition after hemorrhage. Combined AT1+ETA-receptor blockade impaired vasoconstriction more than did AT1-receptor blockade alone, both during baseline xenon anesthesia and after hemorrhage. Even a large increase in vasoconstrictor hormones could not prevent the decrease in blood pressure and the smaller increase in SVR. Thus, endothelin is an important vasoconstrictor during hemorrhage, and both endothelin and angiotensin II are essential hormones for cardiovascular stabilization after hemorrhage.     

Differential vasoconstrictions induced by angiotensin II: role of AT1 and AT2 receptors in isolated C57BL/6J mouse blood vessels

Genetically altered mice are increasingly used as experimental models. However, ANG II responses in mouse blood vessels have not been well defined. Therefore, the aim of this study was to determine the role of ANG II in regulating major blood vessels in C57/BL6J mice with isometric force measurements. Our results showed that in mouse abdominal aorta ANG II induced a concentration-dependent contraction (EC50 4.6 nM) with a maximum contraction of 75.1 +/- 4.9% at 100 nM compared with that of 60 mM K+. Similarly, femoral artery also exhibited a contractile response of 76.0 +/- 3.4% to the maximum concentration of ANG II (100 nM). In contrast, ANG II (100 nM)-induced contraction was significantly less in carotid artery (24.5 +/- 6.6%) and only minimal (3.5 +/- 0.31%) in thoracic aorta. The nitric oxide synthase inhibitor N omega-nitro-L-arginine methyl ester and the AT2 antagonist PD-123319 failed to enhance ANG II-induced contractions. However, an AT1 antagonist, losartan (10 microM), completely inhibited ANG II (100 nM) response in abdominal aorta and carotid artery. An AT1 agonist, [Sar1]-ANG II (100 nM), behaved similarly to ANG II (100 nM) in abdominal aorta and carotid artery. RT-PCR analyses showed that mouse thoracic aorta has a significantly lower AT1 mRNA level than abdominal aorta. These results demonstrate that major mouse vessels exhibit differential contractions to ANG II, possibly because of varied AT1 receptor levels.     

Biphasic actions of angiotensin IV on renal blood flow in the rat

Our aim was to investigate the changes in renal blood flow during brief exposure of the renal vasculature to angiotensin IV (Ang IV). Total renal blood flow was measured by electromagnetic flowmetry in pentobarbital-anesthetized male Sprague Dawley rats. Intrarenal injection of Ang I, Ang II and Ang III produced a dose-dependent vasoconstriction. In contrast, Ang IV and Ang-(3-10) produced a dose-dependent rapid vasoconstriction (lasting seconds) followed by a transient vasodilatation (lasting minutes). The biphasic response to Ang IV was unchanged in rats pre-treated with captopril, whereas the Ang-(3-10) response was abolished implying that its vasoactive activity was due to the generation of Ang IV. The vasodilatory component of Ang IV was unaffected by indomethacin. The biphasic vasoactive response of Ang IV was unaffected by divalinal-Ang IV (AT(4) receptor antagonist) or PD 123319 (AT(2) receptor antagonist), but greatly reduced by losartan or L-158,809 (AT(1) receptor antagonists). These results suggest that Ang IV is distinct from other angiotensins in that it possesses non-prostaglandin mediated renal vasodilatory activity that is apparently linked to the renal vascular AT(1) receptor.     

Angiotensin II activation of focal adhesion kinase and pp60c-Src in relation to mitogen-activated protein kinases in hepatocytes

We have investigated signaling pathways leading to angiotensin II (Ang II) activation of mitogen-activated protein kinase (MAPK) in hepatocytes. MAPK activation by Ang II was abolished by the Ang II type 1 (AT1) receptor antagonist losartan, but not by the Ang II type 2 (AT2) receptor antagonist PD123319. Ang II (100 nM) induced a rapid phosphorylation of Src (peak approximately 2 min) and focal adhesion kinase (FAK, peak approximately 5 min) followed by a decrease to basal levels in 30 min. An increased association between FAK and Src in response to Ang II was detected after 1 min, which declined to basal levels after 30 min. Treatment with the Src kinase inhibitor PP-1 inhibited FAK phosphorylation. Downregulation of PKC, intracellular Ca2+ chelator BAPTA or inhibitors of PKC, Src kinase, MAPK kinase (MEK), Ca2+/calmodulin dependent protein kinase, phosphatidylinositol 3-kinase all blocked Ang II-induced MAPK phosphorylation. In contrast to other cells, there was no evidence for the role of EGF receptor transactivation in the activation of MAPK by Ang II. However, PDGF receptor phosphorylation is involved in the Ang II stimulated MAPK activation. Furthermore, Src/FAK and Ca/CaM kinase activation serve as potential links between the Ang II receptor and MAPK activation. These studies offer insight into the signaling network upstream of MAPK activation by AT1 receptor in hepatocytes.     

Selective down-regulation of AT2 receptors in uterine arteries from pregnant ewes given 24-h intravenous infusions of angiotensin II

Previously, we showed that uterine arteries from late gestation pregnant ewes infused intravenously with angiotensin II (Ang II) for 24 h, displayed heightened responsiveness to Ang II in vitro. Furthermore, we found that a small population of ewes with a "preeclampsia-like" disorder also displayed this. Therefore, we have investigated the density and affinity of Ang II receptor subtypes in the uterine arteries from these groups. Ang II receptor binding was measured using 125I [Sar1Ile8] Ang II. Proportions of AT1 and AT2 receptors were determined by inhibiting 125I [Sar1Ile8] Ang II with losartan (AT1 antagonist) or PD 123319 (AT2 antagonist). Uterine arteries from 24-h Ang II-infused ewes had a lower proportion of AT2 receptors (56.2+/-2.3%) than control (saline-infused) ewes (84.1+/-1.0%; P<0.05). The density of AT2 receptors was reduced (P<0.05) while the density of AT1 receptors was not different. Thus, 24-h infusions of Ang II selectively down-regulated AT2 receptors in the uterine artery, resulting in heightened Ang II reactivity. By contrast, the binding properties of Ang II receptor subtypes in uterine arteries from ewes with the "preeclampsia-like" disorder were not different from control ewes.     

Endogenous angiotensin II suppresses stretch-induced ANP secretion via AT1 receptor pathway

Angiotensin II (Ang II) is released by stretch of cardiac myocytes and has paracrine and autocrine effects on cardiac myocytes and fibroblasts. However, the direct effect of Ang II on the secretion of atrial natriuretic peptide (ANP) is unclear. The aim of the present study is to test whether Ang II affects stretch-induced ANP secretion. The isolated perfused beating atria were used from control and two-kidney one-clip hypertensive (2K1C) rats. The volume load was achieved by elevating the height of outflow catheter connected with isolated atria from 5 cmH₂O to 7.5 cmH₂O. Atrial stretch by volume load caused increases in atrial contractility by 60% and in ANP secretion by 100%. Ang II suppressed stretch-induced ANP secretion and tended to increase atrial contractility whereas losartan stimulated stretch-induced ANP secretion. Neither PD123319 nor A779 had direct effect on stretch-induced ANP secretion. The suppressive effect of Ang II on stretch-induced ANP secretion was blocked by the pretreatment of losartan but not by the pretreatment of PD123319 or A779. In hypertrophied atria from 2K1C rats, stretch-induced ANP concentration attenuated and atrial contractility augmented. The response of stretch-induced ANP secretion to Ang II and losartan augmented. The expression of AT1 receptor protein and mRNA increased but AT2 and Mas receptor mRNA did not change in 2K1C rat atria. Therefore, we suggest that Ang II generated endogenously by atrial stretch suppresses stretch-induced ANP secretion through the AT1 receptor and alteration of Ang II effect in 2K1C rat may be due to upregulation of AT1 receptor.     

Angiotensin II-dependent phosphorylation at Ser11/Ser18 and Ser938 shifts the E2 conformations of rat kidney Na+/K+-ATPase

Kidney plasma membranes, which contain a single α-1 isoform of Na+/K+-ATPase, simultaneously contain two sub-conformations of E2P, differing in their rate of digoxin release in response to Na+ and ATP. Treating cells with Ang II (angiotensin II) somehow changes the conformation of both, because it differentially inhibits the rate of digoxin release. In the present study we tested whether Ang II regulates release by increasing phosphorylation at Ser11/Ser18 and Ser938. Opossum kidney cells co-expressing the AT1a receptor and either α-1.wild-type, α-1.S11A/S18A or α-1.S938A were treated with or without 10?nM Ang II for 5?min, increasing phosphorylation at the three sites. Na+/K+-ATPase was bound to digoxin-affinity columns in the presence of Na+, ATP and Mg2+. A solution containing 30?mM NaCl and 3?mM ATP eluted ~20% of bound untreated Na+/K+-ATPase (Population #1). Pre-treating cells with Ang II slowed the elution of Population #1 in α-1.wild-type and α-1.S938A, but not α-1.S11A/S18A cells. Another 50% of bound Na+/K+-ATPase (Population #2) was subsequently eluted in two phases by a solution containing 150?mM NaCl and 3?mM ATP. Ang II increased the initial rate and slowed the second phase in α-1.wild-type, but not α-1.S938A, cells. Thus Ang II changes the conformation of two forms of EP2 via differential phosphorylation.     

Angiogenic factors stimulate tubular branching morphogenesis of sonic hedgehog-deficient lungs

Sonic Hedgehog (Shh)-deficient mice have a severe lung branching defect. Recent studies have shown that hedgehog signaling is involved in vascular development and it is possible that the diminished airway branching in Shh-deficient mice is due to abnormal pulmonary vasculature formation. Therefore, we investigated the role of Shh in pulmonary vascular development using Shh/Tie2lacZ compound mice, which exhibit endothelial cell-specific LacZ expression, and Pecam-1 immunohistochemistry. In E11.5-13.5 Shh-deficient mice, the pulmonary vascular bed is decreased, but appropriate to the decrease in airway branching. However, when E12.5 Shh-deficient lungs were cultured for 4-6 days, the vascular network deteriorated compared to wild-type lungs. The expression of vascular endothelial growth factor (Vegf) or its receptor Vegfr2 (KDR/Flk-1) was not different between E12.5-13.5 Shh-deficient and wild-type lungs. In contrast, angiopoietin-1 (Ang1), but not Ang2 or the angiopoietin receptor Tie2, mRNA expression was downregulated in E12.5-E13.5 lungs of Shh null mutants. Recombinant Ang1 alone was unable to restore in vitro branching morphogenesis in Shh-deficient lungs. Conversely, the angiogenic factor fibroblast growth factor (Fgf)-2 alone or in combination with Ang1, increased vascularization and tubular growth and branching of Shh-deficient lungs in vitro. The angiogenic factors did not overcome the reduced smooth muscle cell differentiation in the Shh null lungs. These data indicate that early vascular development, mediated by Vegf/Vegfr2 signaling proceeds normally in Shh-deficient mice, while later vascular development and stabilization of the primitive network mediated by the Ang/Tie2 signaling pathway are defective, resulting in an abnormal vascular network. Stimulation of vascularization with angiogenic factors such as Fgf2 and Ang1 partially restored tubular growth and branching in Shh-deficient lungs, suggesting that vascularization is required for branching morphogenesis.     

Chronic ventricular myocyte-specific overexpression of angiotensin II type 2 receptor results in intrinsic myocyte contractile dysfunction

ANG II type 2 receptor (AT(2)) is upregulated in failing hearts, but its effect on myocyte contractile function is not known. We measured fractional cell shortening and intracellular Ca(2+) concentration transients in left ventricular myocytes derived from transgenic mice in which ventricle-specific expression of AT(2) was driven by the myosin light chain 2v promoter. Confocal microscopy studies confirmed upregulation of AT(2) in the ventricular myocytes and partial colocalization of AT(2) with AT(1). Three components of contractile performance were studied. First, baseline measurements (0.5 Hz, 1.5 mmol/l extracellular Ca(2+) concentration, 25 degrees C) and study of contractile reserve at faster pacing rates (1-5 Hz) revealed Ca(2+)-dependent contractile dysfunction in myocytes from AT(2) transgenic mice. Comparison of two transgenic lines suggested a dose-dependent relationship between magnitude of contractile dysfunction and level of AT(2) expression. Second, activity of the Na(+)/H(+) exchanger, a dominant transporter that regulates beat-to-beat intracellular pH, was impaired in the transgenic myocytes. Third, the inotropic response to beta-adrenergic versus ANG II stimulation differed. Both lines showed impaired contractile response to beta-adrenergic stimulation. ANG II elicited an increase in contractility and intracellular Ca(2+) in wild-type myocytes but caused a negative inotropic effect in myocytes from AT(2) transgenic mice. In contrast with beta-adrenergic response, the depressed response to ANG II was related to level of AT(2) overexpression. The depressed response to ANG II was also present in myocytes from young transgenic mice before development of heart failure. Thus chronic overexpression of AT(2) has the potential to cause Ca(2+)- and pH-dependent contractile dysfunction in ventricular myocytes, as well as loss of the inotropic response to ANG II.     

ACE2 deficiency enhances angiotensin II-mediated aortic profilin-1 expression, inflammation and peroxynitrite production

Inflammation and oxidative stress play a crucial role in angiotensin (Ang) II-mediated vascular injury. Angiotensin-converting enzyme 2 (ACE2) has recently been identified as a specific Ang II-degrading enzyme but its role in vascular biology remains elusive. We hypothesized that loss of ACE2 would facilitate Ang II-mediated vascular inflammation and peroxynitrite production. 10-week wildtype (WT, Ace2(+/y)) and ACE2 knockout (ACE2KO, Ace2(-/y)) mice received with mini-osmotic pumps with Ang II (1.5 mg.kg?1.d?1) or saline for 2 weeks. Aortic ACE2 protein was obviously reduced in WT mice in response to Ang II related to increases in profilin-1 protein and plasma levels of Ang II and Ang-(1-7). Loss of ACE2 resulted in greater increases in Ang II-induced mRNA expressions of inflammatory cytokines monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-1β, and IL-6 without affecting tumor necrosis factor-α in aortas of ACE2KO mice. Furthermore, ACE2 deficiency led to greater increases in Ang II-mediated profilin-1 expression, NADPH oxidase activity, and superoxide and peroxynitrite production in the aortas of ACE2KO mice associated with enhanced phosphorylated levels of Akt, p70S6 kinase, extracellular signal-regulated kinases (ERK1/2) and endothelial nitric oxide synthase (eNOS). Interestingly, daily treatment with AT1 receptor blocker irbesartan (50 mg/kg) significantly prevented Ang II-mediated aortic profilin-1 expression, inflammation, and peroxynitrite production in WT mice with enhanced ACE2 levels and the suppression of the Akt-ERK-eNOS signaling pathways. Our findings reveal that ACE2 deficiency worsens Ang II-mediated aortic inflammation and peroxynitrite production associated with the augmentation of profilin-1 expression and the activation of the Akt-ERK-eNOS signaling, suggesting potential therapeutic approaches by enhancing ACE2 action for patients with vascular diseases.     

In situ demonstration of angiotensin-dependent and independent pathways for hyperaldosteronism during chronic extracellular fluid volume depletion.

In wild-type mice, 2-wk administration of losartan, an angiotensin (Ang) II type 1 (AT1) receptor antagonist, along with dietary sodium restriction, resulted in an elevation of plasma aldosterone greater than that seen with sodium restriction alone (2.75 +/- 0.35 vs. 1.38 +/- 0.16 ng/ml, P < 0.01). Plasma potassium increased in sodium-restricted, losartan-treated mice (6.0 +/- 0.2 mEq/liter), while potassium remained unchanged in mice with sodium restriction alone. To study the effect of Ang II on glomerulosa cells that may operate independently of plasma potassium in situ, we used chimeric mice made of cells with or without the intact AT1A gene (Agtr1a). When animals were fed a normal diet or chronically infused with Ang II, the aldosterone synthase mRNA was detectable only in Agtr1a+/+ but not Agtr1a-/- zona glomerulosa cells. After 2 wk of sodium restriction, plasma aldosterone increased (1.51 +/- 0.27 ng/ml) and potassium remained on average at 4.5 +/- 0.2 mEq/liter, with aldosterone synthase mRNA expressed intensively in Agtr1a+/+, but not detectable in Agtr1a-/- cells. Simultaneous sodium restriction and losartan treatment caused increases in plasma potassium (5.5 +/- 0.1 mEq/liter) and aldosterone (1.84 +/- 0.38 ng/ml), with both Agtr1a-/- and Agtr1a+/+ cells intensively expressing aldosterone synthase mRNA. Thus, aldosterone production is regulated by Ang II in the adrenal gland during chronic alterations in extracellular fluid volume when plasma potassium is maintained within the normal range. In the light of a previous observation that dietary potassium restriction superimposed on sodium restriction abolished secondary hyperaldosteronism in angiotensinogen null-mutant mice, the present findings demonstrate that when the renin-Ang system is compromised, plasma potassium acts as an effective alternative mechanism for the volume homeostasis through its capacity to induce hyperaldosteronism.