Considerable evidence now suggests that the precursors and enzymes necessary for the formation and degradation of biologically active forms of <em>angiotensins</em> are present in brain tissues, accompanied by at least three specific binding sites. It also appears that several forms of <em>angiotensin</em> may serve as signaling agents at these sites. There is accumulating support for the notion that AngII must be converted to AngIII in order to bind at the AT<em>1</em> and AT2 receptor subtypes, and AngIII must be converted to AngIV in order to activate the AT4 receptor subtype. Further, AngII(<em>1</em>-<em>7</em>) may activate a separate binding site concerned with antidiuresis, however, characterization of this site has not been completed. The AT<em>1</em> site appears to mediate the classic <em>angiotensin</em> functions concerned with body water balance, maintenance of blood pressure, and cyclicity of reproductive hormones and sexual behaviors. This receptor site also exerts some control over the secretion of pituitary hormones. Less is known about the functional importance of the AT2 site, however, it has been implicated in vascular growth, control of blood flow, and perhaps modulation of NMDA receptors. The AT4 site is heavily distributed in neocortex, hippocampus, cerebellum, and basal ganglia structures, as well as several peripheral tissues. This site appears to mediate memory acquisition and retrieval, the regulation of blood flow, neurite outgrowth, angiogenesis, and kidney function. In addition to the well-studied functions of the brain renin-<em>angiotensin</em> system, additional less well investigated responses are reviewed. These include electrophysiological activation, tachyphylaxis, long term potentiation, learning and memory, and cognitive affect.

BACKGROUND

Endogenous <em>angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>) enhances, while Ang II attenuates, baroreceptor sensitivity (BRS) for reflex control of heart rate (HR) in Sprague-Dawley (SD) rats. In (mRen2)2<em>7</em> renin transgenic rats [(mRen2)], there is overexpression of the mouse Ren2 gene in brain, leading to elevated Ang II and reduced Ang-(<em>1</em>-<em>7</em>) in brain medullary, and associated with hypertension and impaired BRS.

METHODS

We therefore tested the contribution of endogenous Ang-(<em>1</em>-<em>7</em>) to BRS for control of HR and responses to cardiac vagal chemosensitive afferent fiber activation (CVA) with phenylbiguanide (PBG) in anesthetized SD and (mRen2) 2<em>7</em> rats before and after bilateral nucleus of the solitary tract (nTS) injection of the Ang-(<em>1</em>-<em>7</em>) receptor antagonist (D-Ala<em>7</em>)-Ang-(<em>1</em>-<em>7</em>).

RESULTS

(mRen2) 2<em>7</em> rats exhibited a approximately 50% impairment in BRS as compared with SD (P < 0.05). (D-Ala<em>7</em>)-Ang-(<em>1</em>-<em>7</em>) attenuated BRS by approximately 50% in SD rats, but was without effect in (mRen2) 2<em>7</em> rats. (D-Ala<em>7</em>)-Ang-(<em>1</em>-<em>7</em>) did not alter the responses to CVA by PBG (iv bolus) in either strain. There were no differences in the depressor effects of Ang-(<em>1</em>-<em>7</em>) injected into the nTS, nor were levels of mRNA different for <em>angiotensin</em>-converting enzyme, <em>angiotensin</em>-converting enzyme 2, neprilysin, or the mas receptor in medullary tissue from SD versus (mRen2)2<em>7</em> rats.

CONCLUSIONS

Endogenous Ang-(<em>1</em>-<em>7</em>) does not provide tonic input in the nTS to modulate BRS for control of HR in (mRen2)2<em>7</em> rats, which may contribute to impairment of BRS in these animals.

The RAS (renin-<em>angiotensin</em> system) is activated after MI (myocardial infarction), and RAS blockade with ACEis [ACE (<em>angiotensin</em>-converting enzyme) inhibitors] or ARBs (<em>angiotensin</em> receptor blockers) slows but does not completely prevent progression to heart failure. Cardiac ACE is increased after MI and leads to the formation of the vasoconstrictor AngII (<em>angiotensin</em> II). The enzyme ACE2 is also activated after MI and degrades AngII to generate the vasodilator Ang-(<em>1</em>-<em>7</em>) [<em>angiotensin</em>-(<em>1</em>-<em>7</em>)]. Overexpression of ACE2 offers cardioprotective effects in experimental MI, but there is conflicting evidence as to whether the benefits of ACEis and ARBs are mediated through increasing ACE2 after MI. In the present study, we assessed the effect of an ACEi and ARB, alone and in combination, on cardiac ACE2 in a rat MI model. MI rats received vehicle, ACEi (ramipril; <em>1</em> mg/kg of body weight), ARB (valsartan; <em>1</em>0 mg/kg of body weight) or combination (ramipril at <em>1</em> mg/kg of body weight and valsartan at <em>1</em>0 mg/kg of body weight) orally for 28 days. Sham-operated rats were also studied and received vehicle alone. MI increased LV (left ventricular) mass (P<0.000<em>1</em>), impaired cardiac contractility (P<0.05) and activated cardiac ACE2 with increased gene (P<0.05) and protein expression (viable myocardium, P<0.05; border zone, P<0.00<em>1</em>; infarct, P<0.05). Ramipril and valsartan improved remodelling (P<0.05), with no additional effect of dual therapy. Although ramipril inhibited ACE, and valsartan blocked the <em>angiotensin</em> receptor, neither treatment alone nor in combination augmented cardiac ACE2 expression. These results suggest that the cardioprotective effects of ramipril and valsartan are not mediated through up-regulation of cardiac ACE2. Strategies that do augment ACE2 after MI may be a useful addition to standard RAS blockade after MI.

Endothelial nitric-oxide synthase (eNOS) uncoupling and increased inducible NOS (iNOS) activity amplify vascular oxidative stress. The role of inflammatory myelomonocytic cells as mediators of these processes and their impact on tetrahydrobiopterin availability and function have not yet been defined. <em>Angiotensin</em> II (ATII, <em>1</em> mg/kg/day for <em>7</em> days) increased Ly6C(high) and CD<em>1</em><em>1</em>b(+)/iNOS(high) leukocytes and up-regulated levels of eNOS glutathionylation in aortas of C5<em>7</em>BL/6 mice. Vascular iNOS-dependent NO formation was increased, whereas eNOS-dependent NO formation was decreased in aortas of ATII-infused mice as assessed by electron paramagnetic resonance (EPR) spectroscopy. Diphtheria toxin-mediated ablation of lysozyme M-positive (LysM(+)) monocytes in ATII-infused LysM(iDTR) transgenic mice prevented eNOS glutathionylation and eNOS-derived N(ω)-nitro-L-arginine methyl ester-sensitive superoxide formation in the endothelial layer. ATII increased vascular guanosine triphosphate cyclohydrolase I expression and biopterin synthesis in parallel, which was reduced in monocyte-depleted LysM(iDTR) mice. Vascular tetrahydrobiopterin was increased by ATII infusion but was even higher in monocyte-depleted ATII-infused mice, which was paralleled by a strong up-regulation of dihydrofolate reductase expression. EPR spectroscopy revealed that both vascular iNOS- and eNOS-dependent NO formation were normalized in ATII-infused mice following monocyte depletion. Additionally, deletion as well as pharmacologic inhibition of iNOS prevented ATII-induced endothelial dysfunction. In summary, ATII induces an inflammatory cell-dependent increase of iNOS, guanosine triphosphate cyclohydrolase I, tetrahydrobiopterin, NO formation, and nitro-oxidative stress as well as eNOS uncoupling in the vessel wall, which can be prevented by ablation of LysM(+) monocytes.

Inhibition of the cannabinoid receptor CB(<em>1</em>) (CB(<em>1</em>)-R) exerts numerous positive cardiovascular effects such as modulation of blood pressure, insulin sensitivity and serum lipid concentrations. However, direct vascular effects of CB(<em>1</em>)-R inhibition remain unclear. CB(<em>1</em>)-R expression was validated in vascular smooth muscle cells (VSMCs) and aortic tissue of mice. Apolipoprotein E-deficient (ApoE-/-) mice were treated with cholesterol-rich diet and the selective CB(<em>1</em>)-R antagonist rimonabant or vehicle for <em>7</em> weeks. CB(<em>1</em>)-R inhibition had no effect on atherosclerotic plaque development, collagen content and macrophage infiltration but led to improved aortic endothelium-dependent vasodilation and decreased aortic reactive oxygen species (ROS) production and NADPH oxidase activity. Treatment of cultured VSMC with rimonabant resulted in reduced <em>angiotensin</em> II-mediated but not basal ROS production and NADPH oxidase activity. CB(<em>1</em>)-R inhibition with rimonabant and AM25<em>1</em> led to down-regulation of <em>angiotensin</em> II type <em>1</em> receptor (AT<em>1</em>-R) expression, whereas stimulation with the CB(<em>1</em>)-R agonist CP 55,940 resulted in AT<em>1</em>-R up-regulation, indicating that AT<em>1</em>-R expression is directly regulated by the CB(<em>1</em>)-R. CB(2)-R inhibition had no impact on AT<em>1</em>-R expression in VSMC. Consistently, CB(<em>1</em>)-R inhibition decreased aortic AT<em>1</em>-R expression in vivo. CB(<em>1</em>)-R inhibition leads to decreased vascular AT<em>1</em>-R expression, NADPH oxidase activity and ROS production in vitro and in vivo. This antioxidative effect is associated with improved endothelial function in ApoE-/- mice, indicating beneficial direct vascular effects of CB(<em>1</em>)-R inhibition.

OBJECTIVE

Splanchnic vascular hypocontractility with subsequent increased portal venous inflow leads to portal hypertension. Although the renin-<em>angiotensin</em> system contributes to fibrogenesis and increased hepatic resistance in patients with cirrhosis, little is known about its effects in the splanchnic vasculature, particularly those of the alternate system in which <em>angiotensin</em> (Ang) II is cleaved by the Ang-converting enzyme-2 (ACE2) to Ang-(<em>1</em>-<em>7</em>), which activates the G-protein-coupled Mas receptor (MasR). We investigated whether this system contributes to splanchnic vasodilatation and portal hypertension in cirrhosis.

METHODS

We measured levels of renin-<em>angiotensin</em> system messenger RNA and proteins in splanchnic vessels from patients and rats with cirrhosis. Production of Ang-(<em>1</em>-<em>7</em>) and splanchnic vascular reactivity to Ang-(<em>1</em>-<em>7</em>) was measured in perfused mesenteric vascular beds from rats after bile-duct ligation. Ang-(<em>1</em>-<em>7</em>) and MasR were blocked in rats with cirrhosis to examine splanchnic vascular hemodynamics and portal pressure response.

RESULTS

Levels of ACE2 and MasR were increased in splanchnic vessels from cirrhotic patients and rats compared with healthy controls. We also observed an ACE2-dependent increase in Ang-(<em>1</em>-<em>7</em>) production. Ang-(<em>1</em>-<em>7</em>) mediated splanchnic vascular hypocontractility in ex vivo splanchnic vessels from rats with cirrhosis (but not control rats) via MasR stimulation. Identical effects were observed in the splanchnic circulation in vivo. MasR blockade reduced portal pressure, indicating that activation of this receptor in splanchnic vasculature promotes portal inflow to contribute to development of portal hypertension. In addition, the splanchnic effects of MasR required nitric oxide. Interestingly, Ang-(<em>1</em>-<em>7</em>) also decreased hepatic resistance.

CONCLUSIONS

In the splanchnic vessels of patients and rats with cirrhosis, increased levels of ACE2 appear to increase production of Ang-(<em>1</em>-<em>7</em>), which leads to activation of MasR and splanchnic vasodilatation in rats. This mechanism could cause vascular hypocontractility in patients with cirrhosis, and might be a therapeutic target for portal hypertension.

BACKGROUND

In muscular dystrophy, cardiac function deteriorates with time and heart failure is one of the major causes of death. Although the combination of angiotensin-converting enzyme inhibitors (ACEI) and beta-blockers improves cardiac function in adults, little is known about the efficacy of those drugs in patients with muscular dystrophy.

RESULTS

The effect of the beta-blocker, carvedilol, and/or ACEI on ventricular function in patients with muscular dystrophy was studied. Carvedilol and an ACEI were given to 13 patients (ACEI group; mean age 18 years, range 7-27 years), and an ACEI only to 15 patients (carvedilol group; mean age 15 years, range 8-29 years). Diagnoses included Duchenne muscular dystrophy (n=25), Fukuyama muscular dystrophy (n=2), and Emery-Dreifuss muscular dystrophy (n=1). Echocardiographic parameters of the left ventricle were measured during the 2-3 years of follow-up. In the carvedilol group, combination therapy of carvedilol and an ACEI for 2 years resulted in a significant increase in left ventricular fractional shortening (LVFS). In the ACEI group, there was no significant change in LVFS. Left ventricular end-diastolic dimension increased in the ACEI group, but not in the carvedilol group.

CONCLUSIONS

Carvedilol plus an ACEI improves left ventricular systolic function in patients with muscular dystrophy.

Here we report a case where the manifestations of insulin-dependent diabetes occurred following SARS-CoV-2 infection in a young individual in the absence of autoantibodies typical for type <em>1</em> diabetes mellitus. Specifically, a <em>1</em>9-year-old white male presented at our emergency department with diabetic ketoacidosis, C-peptide level of 0.62 µg l^-<em>1</em>, blood glucose concentration of 30.6 mmol l^-<em>1</em> (552 mg dl^-<em>1</em>) and haemoglobin A<em>1</em>c of <em>1</em>6.8%. The patient´s case history revealed probable COVID-<em>1</em>9 infection 5-<em>7</em> weeks before admission, based on a positive test for antibodies against SARS-CoV-2 proteins as determined by enzyme-linked immunosorbent assay. Interestingly, the patient carried a human leukocyte antigen genotype (HLA DR<em>1</em>-DR3-DQ2) considered to provide only a slightly elevated risk of developing autoimmune type <em>1</em> diabetes mellitus. However, as noted, no serum autoantibodies were observed against islet cells, glutamic acid decarboxylase, tyrosine phosphatase, insulin and zinc-transporter 8. Although our report cannot fully establish causality between COVID-<em>1</em>9 and the development of diabetes in this patient, considering that SARS-CoV-2 entry receptors, including <em>angiotensin</em>-converting enzyme 2, are expressed on pancreatic β-cells and, given the circumstances of this case, we suggest that SARS-CoV-2 infection, or COVID-<em>1</em>9, might negatively affect pancreatic function, perhaps through direct cytolytic effects of the virus on β-cells.

BACKGROUND

Signalling of <em>angiotensin</em> II via <em>angiotensin</em> II type <em>1</em> receptor (AT<em>1</em>) promotes cardiac and renal fibrosis, but its role in lung fibrosis is little understood. Using a rat bleomycin (BLM) induced model of pulmonary fibrosis, we examined the expression of AT<em>1</em> in the lung and the effect of an AT<em>1</em> antagonist on pulmonary fibrosis.

METHODS

Adult male Sprague-Dawley rats were given 0.3 mg/kg BLM intratracheally. Two days earlier they had received <em>1</em>0 mg/kg/day of the AT<em>1</em> antagonist candesartan cilexetil mixed in the drinking water. AT<em>1</em> expression in the lungs was examined by immunohistochemistry and immunoblot methods. The effect of the AT<em>1</em> antagonist on pulmonary fibrosis was studied by analysis of bronchoalveolar lavage (BAL) fluid, histopathology, and hydroxyproline assay.

RESULTS

Immunohistochemical studies showed overexpression of AT<em>1</em> in inflammatory immune cells, alveolar type II cells, and fibroblasts. A quantitative assay for AT<em>1</em> showed that AT<em>1</em> expression was significantly upregulated in cells from BAL fluid after day 3 and in the lung homogenates after day 2<em>1</em>. Candesartan cilexetil significantly inhibited the increase in total protein and albumin, as well as the increase in total cells and neutrophils in BAL fluid. On day 2<em>1</em> candesartan cilexetil also ameliorated morphological changes and an increased amount of hydroxyproline in lung homogenates. In addition, BLM increased the expression of transforming growth factor (TGF)-beta<em>1</em> in BAL fluid on day 7; this increase was significantly reduced by candesartan cilexetil.

CONCLUSIONS

AT<em>1</em> expression is upregulated in fibrotic lungs. Angiotensin II promotes lung fibrosis via AT<em>1</em> and, presumably, in part via TGF-beta<em>1</em>.

<em>1</em>. Kinin peptides are implicated in many physiological and pathological processes, including the regulation of blood pressure and sodium homeostasis, inflammation and the cardioprotective effects of preconditioning. In humans, the plasma and tissue kallikrein-kinin systems (KKS) generate bradykinin and kallidin peptides, respectively. 2. We established methodology for the measurement of bradykinin and kallidin peptides and their metabolites in order to study the function of the plasma and tissue KKS in humans. 3. Bradykinin peptides were more abundant than kallidin peptides in blood and cardiac atrial tissue, whereas kallidin peptides were predominant in urine. The levels of kinin peptides in tissue were higher than in blood, confirming the primary tissue localization of the KKS. 4. <em>Angiotensin</em>-converting enzyme inhibition increased blood levels of bradykinin and kallidin peptides. 5. Blood levels of kallidin peptides were suppressed in patients with severe cardiac failure, indicating that the activity of the tissue KKS is suppressed in this condition. 6. Bradykinin peptide levels were increased in the urine of patients with interstitial cystitis, suggesting a role for these peptides in the pathogenesis and/or symptomatology of this condition. <em>7</em>. Cardiopulmonary bypass, a model of activation of the contact system, activated both the plasma and tissue KKS. 8. Measurement of individual bradykinin and kallidin peptides and their metabolites gives important information about the operation of the plasma and tissue KKS and their role in physiology and disease states.

<em>1</em>. The effects on phosphatidylinositol metabolism of three Ca(2+)-mobilizing glycogenolytic hormones, namely <em>angiotensin</em>, vasopressin and adrenaline, have been investigated by using rat hepatocytes. 2. All three hormones stimulate both phosphatidylinositol breakdown and the labelling of this lipid with (32)P. 3. The response to <em>angiotensin</em> occurs quickly, requires a high concentration of the hormone and is prevented by [<em>1</em>-sarcosine, 8-isoleucine]<em>angiotensin</em>, a specific <em>angiotensin</em> antagonist that does not prevent the responses to vasopressin and to adrenaline. This response therefore seems to be mediated by <em>angiotensin</em>-specific receptors. 4. [<em>1</em>-Deaminocysteine,2-phenylalanine,<em>7</em>-(3,4-didehydroproline),8-arginine] vasopressin, a vasopressin analogue with enhanced antidiuretic potency, is relatively ineffective at stimulating phosphatidylinositol metabolism. This suggests that the hepatic vasopressin receptors that stimulate phosphatidylinositol breakdown are different in their ligand selectivity from the antidiuretic vasopressin receptors that activate renal adenylate cyclase. 5. Incubation of hepatocytes with ionophore A23<em>1</em>8<em>7</em>, a bivalent-cation ionophore, neither mimicked nor appreciably changed the effects of vasopressin on phosphatidylinositol metabolism, suggesting that phosphatidylinositol breakdown is not controlled by changes in the cytosol Ca(2+) concentration. This conclusion was supported by the observation that hormonal stimulation of phosphatidylinositol breakdown and resynthesis persists in cells incubated for a substantial period in EGTA, although this treatment somewhat decreased the phosphatidylinositol response of the hepatocyte. The phosphatidylinositol response of the hepatocyte therefore appears not to be controlled by changes in cytosol [Ca(2+)], despite the fact that this ion is thought to be the second messenger by which the same hormones control glycogenolysis. 6. These results may be an indication that phosphatidylinositol breakdown is an integral reaction in the stimulus-response coupling sequence(s) that link(s) activation of alpha-adrenergic, vasopressin and <em>angiotensin</em> receptors to mobilization of Ca(2+) in the rat hepatocyte.

We studied the role of classical phagocytic NADPH oxidase (Nox) in the pathogenesis of kidney allograft tubulointerstitial fibrosis. Immunofluorescence studies showed that Nox-2 and p22phox (electron transfer subunits of Nox) colocalized in the tubulointerstitium of human kidney allografts. Tubular Nox-2 also colocalized with alpha-SMA in areas of injury, suggestive of epithelial-to-mesenchymal transition (EMT). Interstitial macrophages (CD68(+)) and myofibroblasts (alpha-SMA(+)) expressed Nox-2 while graft infiltrating T cells (CD3(+)) and mature fibroblasts (S<em>1</em>00A4(+)) were Nox-2(-). These results were confirmed in the Fisher-to-Lewis rat kidney transplant model. Areas of tubulitis were associated with Nox-2 and alpha-SMA, suggestive of EMT. Immunoblot analyses showed that Nox-2 upregulation was associated with oxidative stress (nitrotyrosine) and fibrogenesis (alpha-SMA and phospho-Smad2) at 3 weeks and 6 months. Allografts treated with Nox inhibitors (DPI or apocynin) for <em>1</em> week showed reduced fibronectin and phospho-Smad2 and increased E-cadherin levels. Cyclosporine A, TGF-beta<em>1</em> and <em>angiotensin</em> II increased Nox-2 mRNA levels 2- to <em>7</em>-fold in vitro (NRK52E cells). Treatment with specific Nox inhibitors (DPI or apocynin) prevented the downregulation of E-cadherin and upregulation of fibronectin transcripts. In aggregate, these studies suggest that Nox-2 is involved in the pathogenesis of allograft tubulointerstitial fibrosis via activation transcription factor Smad2, EMT and myofibroblasts.

Chronically infusing a subpressor dose of <em>angiotensin</em> (Ang) II increases blood pressure via poorly defined mechanisms. We found that this hypertensive response is accompanied by increased oxidant stress and is prevented by blocking endothelin (ET) receptors. Thus, we now tested whether blocking oxidant stress decreases both blood pressure and ET levels. We infused Sprague-Dawley rats (via osmotic pumps) with either vehicle (group <em>1</em>) or Ang II (5 ng. kg(-<em>1</em>). min(-<em>1</em>); groups 2 to 4) for <em>1</em>5 days. Groups 3 and 4 also received either tempol in the drinking water (<em>1</em> mmol/L) or vitamin E (5000 IU/kg diet), respectively, for <em>1</em>5 days. We measured systolic blood pressure (SBP) and urinary nitrite excretion every 3 days, and on day <em>1</em>5 we measured systemic and renal venous plasma levels of ET, isoprostanes, and thiobarbituric acid reactive substances (TBARS). SBP in Group <em>1</em> did not change throughout the study, whereas Ang II increased SBP (from <em>1</em>32+/-5 to <em>1</em>5<em>1</em>+/-<em>7</em> mm Hg). In addition, Ang II increased the systemic and renal venous levels of isoprostanes, TBARS, and ET and caused a transient decrease in urinary nitrites (that returned to control levels by day 9). Both tempol and vitamin E prevented Ang II-induced hypertension and either prevented or tended to blunt the increase in systemic and renal isoprostanes, TBARS, and ET. Finally, both antioxidants abolished the transient decrease in urinary nitrites. These results together with our previous study suggest that subpressor-dose Ang II increases oxidant stress (and isoprostanes). This in turn increases ET levels, which participate in the hypertensive response to Ang II.

In this study, we investigated the regulation and physiological role of heme oxygenase-<em>1</em> (HO-<em>1</em>) in the kidney of rats with hypertension. Rats were continuously administered either <em>angiotensin</em> II (Ang II) or norepinephrine with an osmotic minipump for up to <em>7</em> days. Ang II infusion decreased the glomerular filtration rate (GFR) as determined through creatinine clearance (3.2+/-0.2 versus <em>1</em>.2+/-0.2 mL/min with Ang II infusion, P<0.0<em>1</em>) and increased proteinuria (9. <em>7</em>+/-<em>1</em>.3 versus 28.<em>1</em>+/-<em>7</em>.2 mg/d with Ang II infusion, P<0.0<em>1</em>). In contrast, norepinephrine did not alter these laboratory values. Ang II infusion significantly increased HO-<em>1</em> expression in mRNA (442+/-98% of control at day 5, P<0.0<em>1</em>) and protein levels (3<em>1</em>4+/-49% of control at day 5, P<0.0<em>1</em>). Immunohistochemistry showed that in the kidney of normotensive rats, HO-<em>1</em> was expressed mainly in the basal side in the renal tubules. After Ang II infusion, HO-<em>1</em> staining was more extensively dispersed in the tubular epithelial cells. The intraperitoneal administration of zinc protoporphyrin, an HO inhibitor, to Ang II-infused rats further decreased GFR (0.8+/-0. <em>1</em> mL/min) and increased proteinuria (52.5+/-<em>1</em>3.0 mg/d). In contrast, the administration of hemin, an HO inducer, ameliorated the Ang II-induced decrease in GFR (2.4+/-0.2 mL/min) and increase in proteinuria (9.3+/-4.5 mg/d). These data suggest that HO-<em>1</em> upregulation in the kidney of Ang II-induced hypertensive rats may exert a renoprotective effect against Ang II-induced renal injury.

The aim of this study was to examine the effects of <em>angiotensin</em> II (Ang II) on cellular functions of rat podocytes (pod) in the intact freshly isolated glomerulus and in culture. Membrane voltage (Vm) and ion currents of pod were examined with the patch clamp technique in fast whole cell and whole cell nystatin configuration. Vm of pod was -38+/-<em>1</em> mV (n = 86). Ang II led to a concentration-dependent depolarization of pod with an ED50 of <em>1</em>0(-8) mol/liter. In the presence of Ang II (<em>1</em>0(-<em>7</em>) mol/liter, n = 20), pod depolarized by <em>7</em>+/-<em>1</em> mV. In an extracellular solution with a reduced Cl- concentration of 32 mmol/liter, the effect of Ang II on Vm was significantly increased to <em>1</em>4+/-4 mV (n = 8). The depolarization induced by Ang II was neither inhibited in an extracellular Na+-free solution nor in a solution with a reduced extracellular Ca2+ (down to <em>1</em> micromol/liter). Like Ang II, the calcium ionophore A23<em>1</em>8<em>7</em> (<em>1</em>0(-5) mol/liter, n = 9) depolarized pod by <em>1</em>0+/-2 mV, whereas forskolin (<em>1</em>0(-5) mol/liter), 8-(4-chlorophenylthio)-cAMP and N2,2'-o-dibutyryl-cGMP (both 5 x <em>1</em>0(-4) mol/liter) did not alter Vm of pod. The <em>angiotensin</em> <em>1</em> receptor antagonist losartan (<em>1</em>0(-<em>7</em>) mol/liter) completely inhibited the Ang II-induced (<em>1</em>0(-<em>7</em>) mol/liter) depolarization (n = 5). Like pod in the glomerulus, pod in short term culture depolarized in response to Ang II (<em>1</em>0(-8) mol/liter, n = 5). Our results suggest that Ang II depolarizes podocytes directly by opening a Cl- conductance. The activation of this ion conductance is mediated by an AT<em>1</em> receptor and may be regulated by the intracellular Ca2+ activity.

The genetic analysis of hypertension has revealed complex and inconsistent results, making it difficult to draw clear conclusions regarding the impact of specific genes on blood pressure regulation in diverse human populations. Some of the confusion from previous studies is probably due to undetected gene-gene interactions. Instead of focusing on the effects of single genes on hypertension, we examined the effects of interactions of alleles at 4 candidate loci. Three of the loci are in the renin-<em>angiotensin</em>-system, <em>angiotensin</em>ogen, ACE, and <em>angiotensin</em> II type <em>1</em> receptor, and they have been associated with hypertension in at least <em>1</em> previous study. The fourth locus studied is a previously undescribed locus, named FJ. In total, <em>7</em> polymorphic sites at these loci were analyzed for their association with hypertension in 5<em>1</em> normotensive and <em>1</em>26 hypertensive age-matched individuals. There were no significant differences between the 2 phenotypic classes with respect to either allele or genotype frequencies. However, when we tested for nonallelic associations (linkage disequilibrium), we found that of the <em>1</em>20 multilocus comparisons, <em>1</em>6 deviated significantly from random in the hypertensive class, but there were no significant deviations in the normotensive group. These findings suggest that genetic interactions between multiple loci rather than variants of a single gene underlie the genetic basis of hypertension in our study subjects. We hypothesize that such interactions may account for the inconsistent findings in previous studies because, unlike our study, prior studies almost always examined single-locus effects and did not consider the effects of variation at other potentially interacting loci.

<em>Angiotensin</em> II (AII) contributes to the pathogenesis of many cardiovascular disorders. Oxidant-mediated activation of poly(adenosine diphosphate-ribose) polymerase (PARP) plays a role in the development of endothelial dysfunction and the pathogenesis of various cardiovascular diseases. We have investigated whether activation of the nuclear enzyme PARP contributes to the development of AII-induced endothelial dysfunction. AII in cultured endothelial cells induced DNA single-strand breakage and dose-dependently activated PARP, which was inhibited by the AII subtype <em>1</em> receptor antagonist, losartan; the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor, apocynin; and the nitric oxide synthase inhibitor, N-nitro-L-arginine methyl ester. Infusion of sub-pressor doses of AII to rats for <em>7</em> to <em>1</em>4 d induced the development of endothelial dysfunction ex vivo. The PARP inhibitors PJ34 or INO-<em>1</em>00<em>1</em> prevented the development of the endothelial dysfunction and restored normal endothelial function. Similarly, PARP-deficient mice infused with AII for <em>7</em> d were found resistant to the AII-induced development of endothelial dysfunction, as opposed to the wild-type controls. In spontaneously hypertensive rats there was marked PARP activation in the aorta, heart, and kidney. The endothelial dysfunction, the cardiovascular alterations and the activation of PARP were prevented by the <em>angiotensin</em>-converting enzyme inhibitor enalapril. We conclude that AII, via AII receptor subtype <em>1</em> activation and reactive oxygen and nitrogen species generation, triggers DNA breakage, which activates PARP in the vascular endothelium, leading to the development of endothelial dysfunction in hypertension.

OBJECTIVE

The inotropic, lusitropic, and electrophysiologic effects of acute hypercapnia in humans are not known. Although the effects of hypercapnia on the systemic circulation have been well documented, there is still some debate as to whether hypercapnia causes true pulmonary vasoconstriction in vivo. We have therefore evaluated the effects of acute hypercapnia on these cardiac indices and the interaction of hypercapnia with the systemic and pulmonary vascular beds in humans.

METHODS

Eight healthy male volunteers were studied using Doppler echocardiography. After resting for at least 30 min to achieve baseline hemodynamic parameters (T(0)), they were rendered hypercapnic to achieve an end-tidal carbon dioxide (CO2) of <em>7</em> kPa for 30 min by breathing a variable mixture of CO2/air (T1). They were restudied after 30 min recovery breathing air (T2). Hemodynamic, diastolic, and systolic flow parameters, QT dispersion (maximum-minimum QT interval measured in a 12-lead ECG), and venous blood samples for plasma renin activity (PRA), <em>angiotensin</em> II (ANG II), and aldosterone (ALDO) were measured at each time point.

RESULTS

Hypercapnia compared with placebo significantly increased mean pulmonary artery pressure 14 +/- 1 vs 9 +/- 1 mm Hg and pulmonary vascular resistance 1<em>7</em>1 +/- 1<em>7</em> vs 129 +/- 1<em>7</em> dyne.s.cm-5, respectively. Heart rate, stroke volume, cardiac output, and mean arterial BP were increased by hypercapnia. Indexes of systolic function, namely peak aortic velocity and aortic mean and peak acceleration, were unaffected by hypercapnia. Similarly, hypercapnia had no effect on lusitropic indexes reflected by its lack of effect on isovolumic relaxation time, mitral E-wave deceleration time, and mitral E/A wave ratio. Hypercapnia was found to significantly increase both QTc interval and QT dispersion: 428 +/- 8 vs 411 +/- 3 ms and 48 +/- 2 vs 33 +/- 4 ms, respectively. There was no significant effect of hypercapnia on PRA, ANG II, or ALDO.

CONCLUSIONS

Thus, acute hypercapnia appears to have no adverse inotropic or lusitropic effects on cardiac function, although repolarization abnormalities, reflected by an increase in QT dispersion, and its effects on pulmonary vasoconstriction may have important sequelae in man.

Circulatory changes and arterial plasma hormone concentrations were measured in seven healthy young adults during 30 and 60 degrees passive head-up tilt with the subjects supported by a saddle. The 30 degrees tilt induced a decrease in pulse pressure (Pp) from 45 +/- 2 to 35 +/- 4 (mean +/- SE) mmHg concomitant with an increase in heart rate (HR) from 58 +/- 4 to <em>7</em>8 +/- 8 beats/min and a marginal increase in mean arterial pressure (MAP). Norepinephrine increased from <em>1</em>80 +/- 20 to 3<em>1</em>0 +/- 40 pg/ml, aldosterone increased fivefold, and <em>angiotensin</em> II increased from 8 +/- 2 to 22 +/- <em>7</em> pg/ml. The 60 degrees tilt initially produced changes, which were qualitatively similar to the 30 degrees tilt. However, after <em>1</em>9 +/- 3 min sudden decreases were seen in MAP (94 +/- 3 to 50 +/- 8 mmHg), in Pp (38 +/- 5 to <em>1</em>8 +/- 4 mmHg), and in HR (90 +/- <em>7</em> to 5<em>7</em> +/- 6 beats/min). Concomitantly, epinephrine doubled while norepinephrine remained unchanged; the vagally controlled hormone pancreatic polypeptide increased from 29 +/- 3 to 5<em>1</em> +/- 8 pmol/l, vasopressin from 4 +/- <em>1</em> to <em>1</em>26 +/- 58 pg/ml, and <em>angiotensin</em> II from 23 +/- 9 to 35 +/- <em>1</em>2 pg/ml. The hypotensive bradycardiac episode was immediately reversible on termination of the head-up tilt.(ABSTRACT TRUNCATED AT 250 WORDS)

OBJECTIVE

<em>Angiotensin</em>-converting enzyme 2 (ACE 2), the main product of which is Ang <em>1</em>-<em>7</em>, which binds to its receptor, Mas, is an important member of the renin-<em>angiotensin</em> system.

RESULTS

A substantial body of research indicates that ACE2 is cardioprotective and renoprotective. ACE2 participates in a pathway that is counterregulatory to the effects of angiotensin II (Ang II). The mechanisms by which the protective effects of ACE2 occur are just beginning to be elucidated.

CONCLUSIONS

As ACE2 appears to exert protective effects within the kidney and vasculature, recent data indicate that how it is expressed, what regulates it, and how it interacts with other biological systems may ultimately have clinical implications.

<em>Angiotensin</em>-converting enzyme (ACE) and ACE2 and the AT<em>1</em> and AT2 receptors are pivotal points of regulation in the renin-<em>angiotensin</em> system. ACE and ACE2 are key enzymes in the formation and degradation of <em>angiotensin</em> II (Ang II) and <em>angiotensin</em>-(<em>1</em>-<em>7</em>)(Ang-(<em>1</em>-<em>7</em>)). Ang II acts at either the AT<em>1</em> or the AT2 receptor to mediate opposing actions of vasoconstriction or vasodilatation respectively. While it is known that oestrogen acts to downregulate ACE and the AT(<em>1</em>) receptor, its regulation of ACE2 and the AT2 receptor and the involvement of a specific oestrogen receptor subtype are unknown. To investigate the role of oestrogen receptor-alpha (ERalpha) in the regulation by oestrogen of ACE/ACE2 and AT<em>1</em>/AT2 mRNAs in lung and kidney, ovariectomized female mice lacking apolipoprotein E (ee) with the ERalpha (AAee) or without the ERalpha (alphaalphaee) were treated with <em>1</em><em>7</em>beta-oestradiol (6 microg day(-<em>1</em>)) or placebo for 3 months. ACE, ACE2, AT<em>1</em> receptor and AT2 receptor mRNAs were measured using reverse transcriptase, real-time polymerase chain reaction. In the kidney, <em>1</em><em>7</em>beta-oestradiol showed <em>1</em>.<em>7</em>-fold downregulation of ACE mRNA in AAee mice, with 2.<em>1</em>-fold upregulation of ACE mRNA in alphaalphaee mice. <em>1</em><em>7</em>beta-Oestradiol showed <em>1</em>.5- and <em>1</em>.8-fold downregulation of ACE2 and AT<em>1</em> receptor mRNA in AAee mice; this regulation was lost in alphaalphaee mice. <em>1</em><em>7</em>beta-Oestradiol showed marked (8<em>1</em>-fold) upregulation of the AT(2) receptor mRNA in AAee mice. In the lung, <em>1</em><em>7</em>beta-oestradiol treatment had no effect on AT<em>1</em> receptor mRNA in AAee mice, but resulted in a <em>1</em>.5-fold decreased regulation of AT<em>1</em> mRNA in alphaalphaee mice. There was no significant interaction of oestrogen with ERalpha in the lung for ACE, ACE2 and AT2 receptor genes. These studies reveal tissue-specific regulation by <em>1</em><em>7</em>beta-oestradiol of ACE/ACE2 and AT<em>1</em>/AT2 receptor genes, with the ERalpha receptor being primarily responsible for the regulation of kidney ACE2, AT<em>1</em> receptor and AT2 receptor genes.

BACKGROUND

<em>Angiotensin</em> (Ang) II and Ang-(<em>1</em>-<em>7</em>) are two of the bioactive peptides of the rennin-<em>angiotensin</em> system. Ang II is involved in the development of cardiovascular disease, such as hypertension and atherosclerosis, while Ang-(<em>1</em>-<em>7</em>) shows cardiovascular protection in contrast to Ang II.

RESULTS

In this study, we investigated effects of Ang II and Ang-(<em>1</em>-<em>7</em>) on vascular smooth muscle cell (SMC) proliferation and migration, which are critical in the formation of atherosclerotic lesions. Treatment with Ang II resulted in an increase of SMC proliferation, whereas Ang-(<em>1</em>-<em>7</em>) alone had no effects. However, preincubation with Ang-(<em>1</em>-<em>7</em>) inhibited Ang II-induced SMC proliferation. Ang II promoted SMC migration, and this effect was abolished by pretreatment with Ang-(<em>1</em>-<em>7</em>). The stimulatory effects of Ang II on SMC proliferation and migration were blocked by the Ang II receptor antagonist lorsartan, while the inhibitory effects of Ang-(<em>1</em>-<em>7</em>) were abolished by the Ang-(<em>1</em>-<em>7</em>) receptor antagonist A-<em>7</em>99. Ang II treatment caused activation of ERK<em>1</em>/2 mediated signaling, and this was inhibited by preincubation of SMCs with Ang-(<em>1</em>-<em>7</em>).

CONCLUSIONS

These results suggest that Ang-(<em>1</em>-<em>7</em>) inhibits Ang II-induced SMC proliferation and migration, at least in part, through negative modulation of Ang II induced ERK<em>1</em>/2 activity.

BACKGROUND

The mechanism and treatment of diastolic heart failure are poorly understood. We compared the effects of an ACE inhibitor, an angiotensin receptor blocker (ARB), and their combination on diastolic heart failure in Dahl salt-sensitive (DS) rats.

RESULTS

DS rats fed an 8% NaCl diet from 7 weeks of age were treated with benazepril 10 mg/kg alone, valsartan 30 mg/kg alone, or combined benazepril and valsartan at 5 and 15 mg/kg, respectively, or at 1 and 3 mg/kg, respectively. At 16 weeks of age, DS rats exhibited prominent concentric left ventricular (LV) hypertrophy and diastolic dysfunction with preserved systolic function, as estimated by echocardiography. Despite comparable hypotensive effects among all drug treatments, the combination of benazepril 5 mg/kg and valsartan 15 mg/kg improved diastolic dysfunction and survival in DS rats more effectively than ACE inhibitor or ARB alone. Furthermore, the increase in LV endothelin-1 levels and hydroxyproline contents in DS rats was significantly suppressed only by combined benazepril and valsartan, and LV atrial natriuretic peptide mRNA upregulation in DS rats was suppressed to a greater extent by the combination therapy than monotherapy.

CONCLUSIONS

The combination of ACE inhibitor and ARB, independently of the hypotensive effect, improved LV phenotypic change and increased LV endothelin-1 production and collagen accumulation, diastolic dysfunction, and survival in a rat heart failure model more effectively than either agent alone, thereby providing solid experimental evidence that the combination of these 2 agents is more beneficial than monotherapy for treatment of heart failure.

Mas stimulation with <em>angiotensin</em> (Ang)-(<em>1</em>-<em>7</em>) produces cardioprotective effects and vasorelaxation. Using a computational discovery platform for predicting novel naturally occurring peptides that may activate G protein-coupled receptors, we discovered a novel Mas agonist peptide, CGEN-856S. An endothelium- and NO-dependent vasodilating effect was observed for CGEN-856S in thoracic aorta rings of rats (maximal value for the relaxant effect: 39.99+/-5.034%), which was similar to that produced by Ang-(<em>1</em>-<em>7</em>) (<em>1</em>0(-<em>1</em>0) to <em>1</em>0(-6) mol/L). In addition, the vasodilator activity of this peptide depended on a functional Mas receptor, because it was abolished in aorta rings of Mas-knockout mice. CGEN-856S appears to bind the Mas receptor at the same binding domain as Ang-(<em>1</em>-<em>7</em>), as suggested by the blocking of its vasorelaxant effect with the Ang-(<em>1</em>-<em>7</em>) analogue d-Ala(<em>7</em>)-Ang-(<em>1</em>-<em>7</em>), and by its competitive inhibition of Ang-(<em>1</em>-<em>7</em>) binding to Mas-transfected cells. The effect of CGEN-856S on reperfusion arrhythmias and cardiac function was studied on ischemia reperfusion of isolated rat hearts. We found that picomolar concentration of CGEN-856S (0.04 nmol/L) had an antiarrhythmogenic effect, as demonstrated by a reduction in the incidence and duration of reperfusion arrhythmias. Furthermore, acute infusion of CGEN-856S produced a shallow dose-dependent decrease in mean arterial pressure of conscious spontaneously hypertensive rats. The maximum change during infusion was observed at the highest dose. Strikingly, blood pressure continued to drop in the postinfusion period. The results presented here indicate that the novel Mas agonist, CGEN-856S, might have a therapeutic value, because it induces vasorelaxing, antihypertensive, and cardioprotective effects.