Effects of des-aspartate-angiotensin I on angiotensin II-induced incorporation of phenylalanine and thymidine in cultured rat cardiomyocytes and aortic smooth muscle cells

Des-aspartate-angiotensin I, a pharmacologically active nine-amino acid angiotensin peptide, and losartan, an AT(1) angiotensin receptor antagonist, but not angiotensin-(1-7), another active angiotensin peptide, completely attenuated the angiotensin II-induced incorporation of [3H]phenylalanine in cultured rat cardiomyocytes. The attenuation by des-aspartate-angiotensin I but not that of losartan was inhibited by indomethacin. The data support an earlier suggestion that the nonapeptide attenuates cardiac hypertrophy in rats via an indomethacin-sensitive angiotensin AT(1) receptor subtype. In rat aortic smooth muscle cells, both des-aspartate-angiotensin I and angiotensin-(1-7) had no effect on the angiotensin II-induced [3H]phenylalanine incorporation. However, the two peptides significantly attenuated the angiotensin II-induced [3H]thymidine incorporation in the smooth muscle cells. The attenuation by angiotensin-(1-7) but not by des-aspartate-angiotensin I was inhibited by (D-Ala(7))-angiotensin-(1-7), a specific angiotensin-(1-7) antagonist. Des-aspartate-angiotensin I also attenuated FCS-stimulated [3H]thymidine incorporation. This attenuation was inhibited by the peptide angiotensin receptor antagonist, (Sar(1), Ile(8))-angiotensin II, but not by losartan. These data indicate that des-aspartate-angiotensin I and angiotensin-(1-7) do not participate in the process of protein synthesis in vascular smooth muscle cells and that the nonapeptide and heptapeptide act on different non-AT(1) receptors to mediate their anti-hyperplasic action. Although the exact mechanisms of action remain to be elucidated, the findings indicate that des-aspartate-angiotensin I acts as an agonist on angiotensin AT(1) and non-AT(1) receptor subtypes and induces responses that oppose the actions of angiotensin II.     

Angiotensin-(1-12) is an alternate substrate for angiotensin peptide production in the heart

Identification of angiotensin-(1-12) as an intermediate precursor derived directly from angiotensinogen led us to explore whether the heart has the capacity to process angiotensin-(1-12) into biologically active angiotensin peptides. The generation of angiotensin I, angiotensin II, and angiotensin-(1-7) from exogenous angiotensin-(1-12) was evaluated in the effluent of isolated perfused hearts mounted on a Langendorff apparatus in three normotensive and two hypertensive strains: Sprague-Dawley, Lewis, congenic mRen2.Lewis, Wistar-Kyoto, and spontaneously hypertensive rats. Hearts were perfused with Krebs solution for 60 min before and after the addition of angiotensin-(1-12) (10 nmol/l). Angiotensin-(1-12) caused the rapid appearance of both angiotensin I and angiotensin II in the perfusate that peaked between 30 and 60 min of recirculation. Production of angiotensin-(1-7) from exogenous angiotensin-(1-12) rose steadily over the course of the 60-min experiment. These data directly demonstrate that angiotensin-(1-12) is a substrate for the formation of angiotensin peptides in cardiac tissue. This finding further suggests that this angiotensinogen-derived product is a previously unrecognized important precursor peptide to the renin-angiotensin system cascade.     

AT(1) and AT(2) receptor expression and blockade after acute ischemia-reperfusion in isolated working rat hearts

We assessed ANG II type 1 (AT(1)) and type 2 (AT(2)) receptor (R) expression and functional recovery after ischemia-reperfusion with or without AT(1)R/AT(2)R blockade in isolated working rat hearts. Groups of six hearts were subjected to global ischemia (30 min) followed by reperfusion (30 min) and exposed to no drug and no ischemia-reperfusion (control), ischemia-reperfusion and no drug, and ischemia-reperfusion with losartan (an AT(1)R antagonist; 1 micromol/l), PD-123319 (an AT(2)R antagonist; 0.3 micromol/l), N(6)-cyclohexyladenosine (CHA, a cardioprotective adenosine A(1) receptor agonist; 0.5 micromol/l as positive control), enalaprilat (an ANG-converting enzyme inhibitor; 1 micromol/l), PD-123319 + losartan, ANG II (1 nmol/l), or ANG II + losartan. Compared with controls, ischemia-reperfusion decreased AT(2)R protein (Western immunoblots) and mRNA (Northern immunoblots, RT-PCR) and impaired functional recovery. PD-123319 increased AT(2)R protein and mRNA and improved functional recovery. Losartan increased AT(1)R mRNA (but not AT(1)R/AT(2)R protein) and impaired recovery. Other groups (except CHA) did not improve recovery. The results suggest that, in isolated working hearts, AT(2)R plays a significant role in ischemia-reperfusion and AT(2)R blockade induces increased AT(2)R protein and cardioprotection.     

Binding and signaling of angiotensin-(1-7) in bovine kidney epithelial cells involves the AT(4) receptor

Angiotensin-(1-7) decreased mitogen-activated protein (MAP) kinase (Erks) activation in cultured Mardin-Darby bovine kidney (MDBK) epithelial cells. Also, saturable, high-affinity (125)I-angiotensin-(1-7) binding was detected in MDBK cell membranes. Together, the data suggested the possible presence of an angiotensin-(1-7) receptor. However, ligand structure-binding studies revealed that angiotensin-(3-7) and AT(4) receptor ligands competed with high-affinity for (125)I-angiotensin-(1-7) binding. Furthermore, angiotensin-(3-7) and AT(4) receptor ligands decreased MAP kinase activation in MDBK cells. These results demonstrate that NH(2)-terminal-deleted metabolites of angiotensin-(1-7) can bind with high affinity to the AT(4) receptor and regulate the MAP kinase/Erk signaling pathway in renal epithelial cells.     

Effects of angiotensin analogues and angiotensin receptor antagonists on paraventricular neurones.

In a previous study we observed that most neurones in the paraventricular nucleus are excited by angiotensin-(1-7). In comparison with angiotensin III this excitatory action was significantly delayed. The aim of the present microiontophoretic study of angiotensin II-sensitive rat paraventricular neurones was to compare the effect of the angiotensin-analogues angiotensin-(1-7), angiotensin-(2-7), angiotensin II and angiotensin III on the spontaneous activity of these neurones and to test angiotensin receptor subtype 1 antagonists (CGP 46027 or DuP 753) and subtype 2 selective antagonists (CGP 42112A and PD 123177) in order to acquire more evidence of the receptor subtype present. As previously observed angiotensin II, angiotensin III and angiotensin-(1-7) excited most neurones. The effect of angiotensin-(1-7) was usually weaker than that of angiotensin II, and in contrast to angiotensin III the latencies were not significantly different. Angiotensin-(1-7) seemed to be active by itself, because its effect was antagonised by angiotensin receptor antagonists. Angiotensin-(2-7) was mostly inactive, although a few cells were excited. Whereas the excitatory effects of angiotensin-(1-7), angiotensin II and angiotensin III could always be inhibited with both angiotensin receptor subtype antagonists 1 and 2, that produced by angiotensin-(2-7) was only weakly antagonised, if at all. Subtype 1 selective antagonists were effective at lower concentrations than selective subtype 2 antagonists.     

Differences in myocardial ischemic tolerance between 1- and 7-day-old rabbits.

Between 1 and 7 days of life, the newborn rabbit heart shifts from predominantly using carbohydrates to predominantly using fatty acids as an energy substrate. We therefore used isolated working hearts from 1- or 7-day-old rabbits to determine the effects of fatty acids on myocardial glucose use and the ability of hearts to recover following various periods of transient no-flow ischemia. One-day-old hearts were perfused via the inferior vena cava and ejected buffer through the cannulated aorta and pulmonary artery. Seven-day-old hearts were perfused via the left atrium and ejected buffer through the cannulated aorta. To measure glucose use, hearts were perfused with 11 mM [3H, 14C]glucose, 3% albumin, and 500 microU insulin/mL, in the presence or absence of 0.4 mM palmitate. In the absence of fatty acids, glycolytic rates were similar in 1- and 7-day-old hearts, whereas glucose oxidation rates were 5 times greater in 7-day-old hearts. Palmitate did not have any major effects on overall glucose use in 1-day-old hearts, but did markedly inhibit glycolysis and glucose oxidation in 7-day-old hearts. A series of hearts were also subjected to periods (25-60 min) of no-flow ischemia, followed by 30 min of aerobic reperfusion. In the absence of palmitate, 1-day-old hearts subjected to ischemic periods of up to 60 min recovered some degree of mechanical function during reperfusion, whereas 7-day-old rabbit hearts failed to recover if hearts were subjected to ischemic periods of 35 min or longer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Angiotensin-(1-7) prevents diabetes-induced cardiovascular dysfunction

The aim of this study was to test the hypothesis that treatment with angiotensin-(1-7) [ANG-(1-7)] or ANG-(1-7) nonpeptide analog AVE-0991 can produce protection against diabetes-induced cardiovascular dysfunction. We examined the influence of chronic treatment (4 wk) with ANG-(1-7) (576 microg.kg(-1).day(-1) ip) or AVE-0991 (576 microg.kg(-1).day(-1) ip) on proteinuria, vascular responsiveness of isolated carotid and renal artery ring segments and mesenteric bed to vasoactive agonists, and cardiac recovery from ischemia-reperfusion in streptozotocin-treated rats (diabetes). Animals were killed 4 wk after induction of diabetes and/or treatment with ANG-(1-7) or AVE-0991. There was a significant increase in urine protein (231 +/- 2 mg/24 h) in diabetic animals compared with controls (88 +/- 6 mg/24 h). Treatment of diabetic animals with ANG-(1-7) or AVE-0991 resulted in a significant reduction in urine protein compared with vehicle-treated diabetic animals (183 +/- 16 and 149 +/- 15 mg/24 h, respectively). Treatment with ANG-(1-7) or AVE-0991 also prevented the diabetes-induced abnormal vascular responsiveness to norepinephrine, endothelin-1, angiotensin II, carbachol, and histamine in the perfused mesenteric bed and isolated carotid and renal arteries. In isolated perfused hearts, recovery of left ventricular function from 40 min of global ischemia was significantly better in ANG-(1-7)- or AVE-0991-treated animals. These results suggest that activation of ANG-(1-7)-mediated signal transduction could be an important therapeutic strategy to reduce cardiovascular events in diabetic patients.     

Actions of angiotensin peptides on the rabbit pulmonary artery

The presence of the angiotensin AT1A-like receptor subtype in the pulmonary artery and AT1B-like receptor subtype in the pulmonary trunk of the rabbit has been reported in two earlier studies. The present study further investigated these receptor subtypes using five other angiotensins (namely angiotensin II, angiotensin III, angiotensin IV, angiotensin-(1-7) and angiotensin-(4-8)). The direct action of the angiotensins on the rabbit pulmonary arterial and trunk sections and the ability of each angiotensin to further contract or relax preconstricted sections of the pulmonary artery and trunk were studied using the organ bath set-up. The effects of angiotensin III on the 3H overflow from re-uptaken [3H]noradrenaline in the electrically-contracted rabbit pulmonary arterial and trunk sections were also studied. The contractile response of the arterial and trunk section had the following rank order potency: angiotensin II > angiotensin III > angiotensin IV. The contractile response to these angiotensins was greatly reduced or absent in the pulmonary trunk. Angiotensin II further contracted the preconstricted arterial and trunk sections. In contrast, angiotensin III further contracted the preconstricted arterial section but relaxed the preconstricted trunk section. Angiotensin IV similarly relaxed the preconstricted trunk section but had minimum effect on the preconstricted arterial section. Angiotensin-(1-7) and angiotensin-(4-8) had no effect on both sections. The actions of the three angiotensins were inhibited by losartan, an AT1-selective antagonist. Indomethacin, a cyclo-oxygenase inhibitor, inhibited the relaxation caused by angiotensin III and angiotensin IV in the trunk section. The effects of angiotensin III on the electrically preconstricted sections of the pulmonary trunk and artery were not accompanied by any significant changes in 3H overflow. The differential responses produced by angiotensin II and its immediate metabolites via two positionally located and functionally opposing receptor subtypes suggest that the pulmonary trunk and artery is not a passive conduit but an important regulator of blood flow from the heart to the lung.     

Effect of angiotensin-(1-7) on jejunal absorption of water in rats

The effect of angiotensin-(1-7) on jejunal water absorption in rats was investigated. The jejunal sac of anesthetized rats was filled with two ml of tyrode solution containing 3.7 MBq of tritiated water. A femoral vein was cannulated for administration of peptides and drugs. Infusion of Ang-(1-7) at the dose of 0.7 ng/kg.min produced a significant increase in jejunal water absorption compared to control (32% increase). The Ang-(1-7) antagonist A-779 abolished the effect of Ang-(1-7) on water absorption. A reduction of the Ang-(1-7) effect was also produced by treatment with the AT(1) receptor antagonist, losartan or the AT(2) receptor antagonist, PD123.177. The increase in jejunal water absorption produced by Ang-(1-7) was blocked by the nitric oxide synthase inhibitor, L-NAME and by indomethacin. These data suggest that the effect of Ang-(1-7) on the jejunal loop is mediated by activation of a multiple angiotensin receptors and/or by an atypical angiotensin receptor. Furthermore, the effect of Ang-(1-7) on jejunal water absorption is mediated by nitric oxide and by a cyclooxygenase-dependent mechanism.     

Mechanisms of angiotensin-(1-7)-induced inhibition of angiogenesis

Angiotensin-(1-7) [ANG-(1-7)], an endogenous bioactive peptide constituent of the renin-angiotensin system, acts as an inhibitory growth factor in vitro and in vivo. In this study, we evaluated whether the antiangiogenic effect of ANG-(1-7) in the mouse sponge model of angiogenesis might be receptor mediated and involved in the release of nitric oxide (NO). The hemoglobin content (microg/mg wet tissue) of 7-day-old sponge implants was used as an index of the vascularization and showed that daily injections of ANG-(1-7) (20 ng) inhibited significantly the angiogenesis in the implants relative to the saline-treated group. The specific receptor antagonist D-Ala(7)-ANG-(1-7); A-779 prevented ANG-(1-7)-induced inhibition of angiogenesis. The antiangiogenic effect was also abolished by pretreatment with NO synthase inhibitors aminoguanidine (1 mg/ml) or N(G)-nitro-L-arginine methyl ester (0.3 mg/ml). Selective AT1 and AT2 angiotensin-receptor antagonists and an angiotensin-converting enzyme inhibitor, in combination with ANG-(1-7) or alone, did not alter angiogenesis in the implants. These results establish that the regulation of the vascular tissue growth by ANG-(1-7) is associated with NO release by activation of an angiotensin receptor distinct from AT1 and AT2.     

Angiotensin-(1-7)-induced activation of ERK1/2 is cAMP/protein kinase A-dependent in glomerular mesangial cells

The renin-angiotensin system (RAS) plays an important role in renal physiology and kidney injury. Although the cellular effects of the RAS activation are generally attributed to angiotensin II (ANG II), the recent identification of angiotensin-converting enzyme 2 has shifted the focus to other peptides including Ang-(1-7). The G protein-coupled receptor for Ang-(1-7), mas, is expressed by mesangial cells (MC) but the signal transduction pathways activated by Ang-(1-7) in MC have not been fully elucidated. Accordingly, we studied the effect of Ang-(1-7) on extracellular signal-related kinase (ERK)1/2 activation in rat MC. Ang-(1-7)-induced ERK1/2 phosphorylation in MC is time- and concentration-dependent. Pretreatment of MC with the mas receptor antagonist A-779 but not the AT(1) antagonist losartan or the AT(2) antagonist PD123319 abrogated ERK1/2 activation. Neither pretreatment with the NADPH oxidase inhibitors diphenyleneiodonium and apocynin nor pretreatment with the epidermal growth factor (EGF) receptor antagonists AG1478 and PD158780 attenuated Ang-(1-7)-induced activation of ERK1/2. Even though each of these compounds abolished ANG II-induced activation of ERK1/2. Ang-(1-7) increased intracellular cAMP levels and activated protein kinase A (PKA) and inhibition of either adenylyl cyclase or PKA activity attenuated Ang-(1-7)-induced ERK1/2 activation. In conclusion, Ang-(1-7)-induced activation of ERK1/2 is cAMP/PKA-dependent in MC, but independent of NADPH oxidase and the EGF receptor.     

Effects of genetic deletion of angiotensin-(1-7) receptor Mas on cardiac function during ischemia/reperfusion in the isolated perfused mouse heart

In this study we investigated the role of Mas on cardiac function during ischemia/reperfusion in isolated perfused mouse heart. Following a stabilization period of 30 min, hearts from WT and Mas KO mice were subjected to global ischemia. After 20 min of ischemia, the flow was restarted and the hearts were reperfused for 30 min. An additional group of WT mice was perfused with solution containing the Ang-(1-7) receptor Mas antagonist A-779. Isolated heart of Mas KO and WT treated with A-779 presented an increase in the perfusion pressure in the baseline period. This difference increased with 5 min of reperfusion reaching similar values to baseline period at the end of the reperfusion. Isolated hearts of Mas KO and WT treated with A-779 also presented a decreased systolic tension, +/-dT/dt, and HR. Upon global ischemia WT hearts showed a significant decrease in systolic tension and an increase in diastolic tension. During reperfusion an increase in systolic and diastolic tension was observed in WT mice. Deletion or blockade of Mas markedly attenuated these changes in isolated hearts. These results indicate that Mas plays an important role in cardiac function during ischemia/reperfusion which is in keeping with the cardiac and coronary effects previously described for Ang-(1-7).     

Effects of captopril and angiotensin II receptor blockers (AT1, AT2) on myocardial ischemia-reperfusion induced infarct size

The renin-angiotensin system (RAS) plays a major role in regulating the cardiovascular system, and disorders of the RAS contribute largely to the cardiac pathophysiology, including myocardial ischemia-reperfusion (MI/R) injury. Two subtypes of angiotensin II (Ang II) receptors have been defined on the basis of their differential pharmacological properties. The current study was undertaken to address the question as to whether the inhibition of the angiotensin converting enzyme (ACE) by captopril and the AT1 and AT2 receptor blockers losartan and PD123319 modulate MI/R-induced infarct size in an in vivo rat model. To produce necrosis, a branch of the descending left coronary artery was occluded for 30 min followed by two hours of reperfusion. ECG changes, blood pressure, and heart rate were measured during the experiment. Captopril (3 mg/kg), losartan (2 mg/kg), and PD123319 (20 μg/kg/min) were given in an IV 10 min before ischemia and were continued during the ischemic period. The infarcted area was measured by TTC staining. The volume of infarct and the risk zone was determined by planimetry. Compared to the control group (55.62±4.00%) both captopril and losartan significantly reduced the myocardial infarct size (30.50±3.26% and 37.75±4.44%), whereas neither PD123319 nor PD123319+losartan affected the infarct size volume (46.50±3.72% and 54.62±2.43%). Our data indicates that captopril and losartan exert cardioprotective activity after an MI/R injury. Also, infarct size reduction by losartan was halted by a blockade of the AT2 receptor. Therefore, the activation of AT2 receptors may be potentially protective and appear to oppose the effects mediated by the AT1 receptors.     

Angiotensin II (AT1) receptors and NADPH oxidase regulate Cl- current elicited by beta1 integrin stretch in rabbit ventricular myocytes

Direct stretch of beta1 integrin activates an outwardly rectifying, tamoxifen-sensitive Cl(-) current (Cl(-) SAC) via focal adhesion kinase (FAK) and/or Src. The characteristics of Cl(-) SAC resemble those of the volume-sensitive Cl(-) current, I(Cl,swell). Because myocyte stretch releases angiotensin II (AngII), which binds AT1 receptors (AT1R) and stimulates FAK and Src in an autocrine-paracrine loop, we tested whether AT1R and their downstream signaling cascade participate in mechanotransduction. Paramagnetic beads coated with mAb for beta1-integrin were applied to myocytes and pulled upward with an electromagnet while recording whole-cell anion current. Losartan (5 microM), an AT1R competitive antagonist, blocked Cl(-) SAC but did not significantly alter the background Cl(-) current in the absence of integrin stretch. AT1R signaling is mediated largely by H(2)O(2) produced from superoxide generated by sarcolemmal NADPH oxidase. Diphenyleneiodonium (DPI, 60 microM), a potent NADPH oxidase inhibitor, rapidly and completely blocked both Cl(-) SAC elicited by stretch and the background Cl(-) current. A structurally unrelated NADPH oxidase inhibitor, 4-(2-aminoethyl) benzenesulfonyl fluoride (AEBSF, 0.5 and 2 mM), also rapidly and completely blocked Cl(-) SAC as well as a large fraction of the background Cl(-) current. With continuing integrin stretch, Cl(-) SAC recovered upon washout of AEBSF (2 mM). In the absence of stretch, exogenous AngII (5 nM) activated an outwardly rectifying Cl(-) current that was rapidly and completely blocked by DPI (60 microM). Moreover, exogenous H(2)O(2) (10, 100, and 500 microM), the eventual product of NADPH oxidase activity, also activated Cl(-) SAC in the absence of stretch, whereas catalase (1,000 U/ml), an H(2)O(2) scavenger, attenuated the response to stretch. Application of H(2)O(2) during NADPH oxidase inhibition by either DPI (60 microM) or AEBSF (0.5 mM) did not fully reactivate Cl(-) SAC, however. These results suggest that stretch of beta1-integrin in cardiac myocytes elicits Cl(-) SAC by activating AT1R and NADPH oxidase and, thereby, producing reactive oxygen species. In addition, NADPH oxidase may be intimately coupled to the channel responsible for Cl(-) SAC, providing a second regulatory pathway.     

Ischemia-reperfusion alters gene expression of Na+-K+ ATPase isoforms in rat heart

The present study investigated whether oxidative stress plays a role in ischemia-reperfusion-induced changes in cardiac gene expression of Na(+)-K(+) ATPase isoforms. The levels of mRNA for Na(+)-K(+) ATPase isoforms were assessed in the isolated rat heart subjected to global ischemia (30 min) followed by reperfusion (60 min) in the presence or absence of superoxide dismutase (5 x 10(4)U/L) plus catalase (7.5 x 10(4)U/L), an antioxidant mixture. The levels of mRNA for the alpha(2), alpha(3), and beta(1) isoforms of Na(+)-K(+) ATPase were significantly reduced in the ischemia-reperfusion hearts, unlike the alpha(1) isoform. Pretreatment with superoxide dismutase+catalase preserved the ischemia-reperfusion-induced changes in alpha(2), alpha(3), and beta(1) isoform mRNA levels of the Na(+)-K(+) ATPase, whereas the alpha(1) mRNA levels were unaffected. In order to test if oxidative stress produced effects similar to those seen with ischemia-reperfusion, hearts were perfused with an oxidant, H(2)O(2) (300 microM), or a free radical generator, xanthine (2mM) plus xanthine oxidase (0.03 U/ml) for 20 min. Perfusion of hearts with H(2)O(2) or xanthine/xanthine oxidase depressed the alpha(2), alpha(3), and beta(1) isoform mRNA levels of the Na(+)-K(+) ATPase, but had lesser effects on alpha(1) mRNA levels. These results indicate that Na(+)-K(+) ATPase isoform gene expression is altered differentially in the ischemia-reperfusion hearts and that antioxidant treatment appears to attenuate these changes. It is suggested that alterations in Na(+)-K(+) ATPase isoform gene expression by ischemia-reperfusion may be mediated by oxidative stress.     

Prostaglandin production in response to angiotensin-(1-7) in rabbit isolated vasa deferentia

Angiotensin-(1-7) is a predominant metabolite of angiotensin I in brain tissue. Its neuromodulatory and prostaglandin (PG) synthesizing capabilities were investigated in the rabbit isolated vas deferens. This metabolite had no significant effect as a neuromodulator, however it increased PGE synthesis in the vasa deferentia with a potency equivalent to that of angiotensin II. The angiotensin-(1-7) has a unique spectrum of activity among the angiotensin peptides to selectively increase PG synthesis. It could be useful in defining the relevance of angiotensin-induced PG synthesis in various systems, particularly in neuronal tissue. Angiotensin-(1-7) potentially could be useful in defining angiotensin receptor subtypes, as well.     

Evidence for a new angiotensin-(1-7) receptor subtype in the aorta of Sprague-Dawley rats

We have recently described, in the mouse aorta, the vasodilator effect of angiotensin-(1-7) (Ang-(1-7)) was mediated by activation of the Mas Ang-(1-7) receptor and that A-779 and D-Pro7-Ang-(1-7) act as Mas receptor antagonists. In this work we show pharmacological evidence for the existence of a different Ang-(1-7) receptor subtype mediating the vasodilator effect of Ang-(1-7) in the aorta from Sprague-Dawley (SD) rats. Ang-(1-7) induced an endothelium-dependent vasodilator effect in aortic rings from SD rats which was inhibited by removal of the endothelium and by L-NAME (100 microM) but not by indomethacin (10 microM). The Ang-(1-7) receptor antagonist D-Pro7-Ang-(1-7) (0.1 microM) abolished the vasodilator effect of the peptide. However, the other specific Ang-(1-7) receptor antagonist, A-779 in concentrations up to 10 microM, did not affect vasodilation induced by Ang-(1-7). The Ang II AT1 and AT2 receptors antagonists CV11974 (0.01 microM) and PD123319 (1 microM), respectively, the bradykinin B2 receptor antagonist HOE 140 (1 microM) and the inhibitor of ACE captopril (10 microM) did not change the effect of Ang-(1-7). Our results show that in the aorta of SD rats, the vasodilator effect of Ang-(1-7) is dependent on endothelium-derived nitric oxide. This effect is mediated by the activation of Ang-(1-7) receptors sensitive to D-Pro7-Ang-(1-7), but not to A-779, which suggests the existence of a different Ang-(1-7) receptor subtype.     

Involvement of the Gi/o/cGMP/PKG pathway in the AT2-mediated inhibition of outer cortex proximal tubule Na+-ATPase by Ang-(1-7)

The molecular mechanisms involved in the Ang-(1-7) [angiotensin-(1-7)] effect on sodium renal excretion remain to be determined. In a previous study, we showed that Ang-(1-7) has a biphasic effect on the proximal tubule Na+-ATPase activity, with the stimulatory effect mediated by the AT1 receptor. In the present study, we investigated the molecular mechanisms involved in the inhibition of the Na+-ATPase by Ang-(1-7). All experiments were carried out in the presence of 0.1 nM losartan to block the AT1 receptor-mediated stimulation. In this condition, Ang-(1-7) at 0.1 nM inhibited the Na+-ATPase activity of the proximal tubule by 54%. This effect was reversed by 10 nM PD123319, a specific antagonist of the AT2 receptor, and by 1 muM GDP[beta-S] (guanosine 5'-[beta-thio]diphosphate), an inhibitor of G protein. Ang-(1-7) at 0.1 M induced [35S]GTP[S] (guanosine 5'-[gamma-[35S]thio]triphosphate) binding and 1 mug/ml pertussis toxin, an inhibitor of G(i/o) protein, reversed the Ang-(1-7) effect. Furthermore, it was observed that the inhibitory effect of Ang-(1-7) on the Na+-ATPase activity was completely reversed by 0.1 microM LY83583, an inhibitor of guanylate cyclase, and by 2 muM KT5823, a PKG (protein kinase G) inhibitor, and was mimicked by 10 nM d-cGMP (dibutyryl cGMP). Ang-(1-7) increased the PKG activity by 152% and this effect was abolished by 10 nM PD123319 and 0.1 microM LY83583. Taken together, these data indicate that Ang-(1-7) inhibits the proximal tubule Na+-ATPase by interaction with the AT2 receptor that subsequently activates the G(i/o) protein/cGMP/PKG pathway.     

The angiotensin II-AT1 receptor stimulates reactive oxygen species within the cell nucleus

We and others have reported significant expression of the Ang II Type 1 receptor (AT1R) on renal nuclei; thus, the present study assessed the functional pathways and distribution of the intracellular AT1R on isolated nuclei. Ang II (1 nM) stimulated DCF fluorescence, an intranuclear indicator of reactive oxygen species (ROS), while the AT1R antagonist losartan or the NADPH oxidase (NOX) inhibitor DPI abolished the increase in ROS. Dual labeling of nuclei with antibodies against nucleoporin 62 (Nup62) and AT1R or the NADPH oxidase isoform NOX4 revealed complete overlap of the Nup62 and AT1R (99%) by flow cytometry, while NOX4 was present on 65% of nuclei. Treatment of nuclei with a PKC agonist increased ROS while the PKC inhibitor GF109203X or PI3 kinase inhibitor LY294002 abolished Ang II stimulation of ROS. We conclude that the Ang II-AT1R-PKC axis may directly influence nuclear function within the kidney through a redox sensitive pathway.     

Effect of the NADPH oxidase inhibitor apocynin on ischemia-reperfusion lung injury

Apocynin (4-hydroxy-3-methoxy-acetophenone) inhibits NADPH oxidase in activated polymorphonuclear (PMN) leukocytes, preventing the generation of reactive oxygen species. To determine if apocynin attenuates ischemia-reperfusion lung injury, we examined the effects of apocynin (0.03, 0.3, and 3 mM) in isolated in situ sheep lungs. In diluent-treated lungs, reperfusion with blood (180 min) after 30 min of ischemia (ventilation 28% O(2), 5% CO(2)) caused leukocyte sequestration in the lung and increased vascular permeability [reflection coefficient for albumin (sigma(alb)) 0.47 +/- 0.10, filtration coefficient (K(f)) 0.14 +/- 0.03 g. min(-1). mmHg(-1). 100 g(-1)] compared with nonreperfused lungs (sigma(alb) 0.77 +/- 0. 03, K(f) 0.03 +/- 0.01 g. min(-1). mmHg(-1). 100 g(-1); P < 0.05). Apocynin attenuated the increased protein permeability at 0.3 and 3 mM (sigma(alb) 0.69 +/- 0.05 and 0.91 +/- 0.03, respectively, P < 0. 05); K(f) was decreased by 3 mM apocynin (0.05 +/- 0.01 g. min(-1). mmHg(-1). 100 g(-1), P < 0.05). Diphenyleneiodonium (DPI, 5 microM), a structurally unrelated inhibitor of NADPH oxidase, worsened injury (K(f) 0.32 +/- 0.07 g. min(-1). mmHg(-1). 100 g(-1), P < 0.05). Neither apocynin nor DPI affected leukocyte sequestration. Apocynin and DPI inhibited whole blood chemiluminescence and isolated PMN leukocyte-induced resazurin reduction, confirming NADPH oxidase inhibition. Apocynin inhibited pulmonary artery hypertension and perfusate concentrations of cyclooxygenase metabolites, including thromboxane B(2). The cyclooxygenase inhibitor indomethacin had no effect on the increased vascular permeability, suggesting that cyclooxygenase inhibition was not the explanation for the apocynin results. Apocynin prevented ischemia-reperfusion lung injury, but the mechanism of protection remains unclear.