Cardioprotection with angiotensin converting enzyme inhibitor and angiotensin II type 1 receptor antagonist is not abolished by nitric oxide synthase inhibitor in ischemia-reperfused rabbit hearts.

Although angiotensin converting enzyme (ACE) inhibitor and/or angiotensin II type 1 (AT1) receptor antagonist can protect the myocardium against ischemia-reperfusion injury, the mechanisms of the effect have not yet been characterized at the cellular level. We here examined the effect of the combination of an ACE inhibitor, temocaprilat, an AT1 receptor antagonist, CV-11974 and/or a nitric oxide synthase inhibitor, L-NAME, on the myocardial metabolism and contraction during ischemia and reperfusion by using phosphorus 31-nuclear magnetic resonance (31P-NMR) in Langendorff rabbit hearts. After normothermic 20 min global ischemia, postischemic reperfusion of 30 min was carried out. Twenty-one hearts were divided into three experimental groups consisting of 7 hearts each: a Tem+CV group perfused with a combination of temocaprilat and CV-11974; a Tem+CV+L-NAME group perfused with a combination of temocaprilat and CV-11974 plus L-NAME, and a control group. During ischemia, both the Tem+CV group and Tem+CV+L-NAME group showed a significant inhibition of the decrease in adenosine triphosphate (ATP) compared with the control group (p<0.01); the increase in ATP was 50+/-3%, 42+/-4%, and 19+/-4% in the Tem+CV group, Tem+CV+L-NAME group, and control group, respectively. Both experimental groups also showed a significant inhibition of the increase in left ventricular end-diastolic pressure (LVEDP) compared with the control group (p<0.01). After postischemic reperfusion, the Tem+CV group and Tem+CV+L-NAME group again showed a significant improvement of ATP as compared with the control group (p<0.01); the increase in ATP was 73+/-3%, 64+/-3%, and 47+/-4% in the Tem+CV group, Tem+CV+L-NAME group, and control group, respectively, and a significant decrease of LVEDP as compared with the control group (p<0.01). There were no differences in ATP, or LVEDP during ischemia and reperfusion between the Tem+CV group and Tem+CV+ L-NAME group. In conclusion, the combination of temocaprilat and CV-11974 showed significant potential for improving myocardial energy metabolism and relaxation during both myocardial ischemia and reperfusion. This beneficial effect was not dependent on NO synthase.     

雌激素对血管内皮细胞一氧化氮合酶活性调控的受体机制研究

目的　观察 17β 雌二醇对大鼠肺血管内皮细胞一氧化氮合酶 (NOS)活性的影响及雌激素受体在其中的作用。方法　用贴壁法和无酚红 16 40培养基培养大鼠肺血管内皮细胞 ,在不同浓度的 17β 雌二醇 (伴或不伴有雌激素受体拮抗剂代莫昔芬 )作用下 ,观察一定时间内内皮细胞的NOS活性及一氧化氮 (NO)的产量。放射配体结合分析技术检测内皮中的雌激素受体。结果　 (1) 1～ 10nmol的 17β 雌二醇作用 8～ 2 4h ,内皮细胞的NO产量显著增加 (vs对照 P<0 .0 5 ) ,10nmol的 17β 雌二醇作用 8～ 2 4hNOS活性显著增强 (与对照比 ,8h ,P<0 .0 5 ;16h ,2 4h ,P<0 .0 1)。 (2 )大鼠肺血管内皮细胞中存在雌激素受体。 (3)雌激素受体拮抗剂他莫昔芬能显著抑制雌激素的上述作用 (P <0 .0 1)。结论　 17β 雌二醇能增强大鼠血管内皮细胞的NOS活性和NO产量 ,该作用由雌激素受体介导 ,可能是雌激素降低血管阻力、抑制动脉粥样硬化作用的重要机理之一。     

Effects of an HMG-CoA reductase inhibitor in combination with an ACE inhibitor or angiotensin II type 1 receptor antagonist on myocardial metabolism in ischemic rabbit hearts.

We investigated the effects of a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, pravastatin, an angiotensin converting enzyme (ACE) inhibitor, temocaprilat, and an angiotensin II type 1 (AT1) receptor antagonist, CV-11974, on myocardial metabolism during ischemia in isolated rabbit hearts using phosphorus 31-nuclear magnetic resonance (31P-NMR) imaging. Forty-five minutes of continuous normothermic global ischemia was carried out. Pravastatin, temocaprilat, CV-11974 or a nitric oxide synthase inhibitor, L-NAME was administered from 60 min prior to the global ischemia. Japanese white rabbits were divided into the following experimental groups, a control group (n=7), a group treated with pravastatin (P group; n=7), a group treated with pravastatin and temocaprilat (P+T group; n=7), a group treated with pravastatin and CV-11974 (P+CV group; n=7), and a group treated with pravastatin and L-NAME (P+L-NAME group; n=7). During ischemia, P group, as well as either P+T group or P+CV group, showed a significant inhibition of the decreases in adenosine triphosphate (ATP) and intracellular pH (pHi) (p<0.01, respectively, at the end of ischemia compared to the control group as well as P+L-NAME group), and a significant inhibition of the increase in inorganic phosphate (Pi) (p<0.01, respectively, compared with the control group as well as P+L-NAME group). These results suggest that pravastatin significantly improved myocardial energy metabolism during myocardial ischemia. This beneficial effect was dependent on NO synthase. However, this beneficial effect was not enhanced by either temocaprilat or CV-11974.     

Effects of angiotensin II on nitric oxide generation in proliferating and quiescent rat coronary microvascular endothelial cells.

Nitric oxide (NO) and the mitogenic peptide angiotensin II (Ang II) have been implicated in endothelial cell growth. However, the putative relationship between these two opposing agents with respect to endothelial cell growth remains unknown. In this study, proliferating and confluent rat coronary microvascular endothelial cells (CMEC) were treated with different doses of Ang II, Ca2+ ionophore A23187, or valsartan (an Ang II type 1 (AT1) receptor inhibitor) alone or in combination for 24 h before measuring the nitrite levels as an index of NO generation. NO production and endothelial NO synthase (eNOS) mRNA/protein expression were found to be 3-fold greater in proliferating vs. quiescent CMEC. Treatments of CMEC with Ang II or Ca2+ ionophore A23187 equally increased NO production without altering the fold-difference in the basal release of NO from proliferating vs. confluent CMEC. Valsartan abolished NO production in CMEC treated with Ang II but not Ca2+ ionophore A23187. Treatments of endothelium-intact vascular rings with Ang II (1 nmol/l to 10 micromol/l) plus valsartan or PD-123319, an Ang II type 2 (AT2) receptor inhibitor, attenuated vascular responses to acetylcholine in an Ang II dose-dependent manner. In these rings, phenylephrine produced significant increases in contractile responses only at nmol/l concentrations of Ang II. In contrast, pharmacological and mechanical inactivation of endothelium enhanced contractile responses to phenylephrine at micromol/I concentrations of Ang II. These data demonstrate that Ang II stimulates NO production in CMEC in both an AT1- and an AT2 receptor-regulated manner, and that this stimulation of NO may be beneficial in counterbalancing the direct vasoconstrictor effect of Ang II on underlying smooth muscle cells.     

CV11974阻断血管紧张素Ⅱ对培养心肌细胞活力的影响

目的　本研究应用新型的非肽类血管紧张素Ⅱ (ANGⅡ )受体拮抗剂CV1 1 974,特异性地阻断ANGⅡ受体(AT1 ) ,观察它对培养心肌细胞活力的影响 ,旨在探讨心肌细胞功能下降的细胞机理 ,为临床正确应用ANGⅡ受体拮抗剂提供实验依据。方法　本研究利用培养的乳鼠心肌细胞 ,根据活细胞线粒体在能量代谢过程中可作用于甲基噻唑基四唑 (MTT)产生蓝紫色结晶产物 (formazan)这一原理 ,应用比色法测定ANGⅡ对培养心肌细胞的活力的影响和CV1 1 974的干预作用。结果 :ANGⅡ在 1 2h之内使formazan产物的OD值逐渐上升 ,1 2h达到高峰 ,此后开始下降 ,至 2 4h已低于正常水平 ,它们的OD值之间均差异有极显著性意义 (P <0 .0 1 ) ,CV1 1 974可以显著地拮抗ANGⅡ对心肌细胞活力的影响 ,应用CV1 1 974后所有时相点MTT产物的OD值之间差异无显著性意义 ,(P >0 .0 5)。结论　该结果提示ANGⅡ在早期具有提高心肌细胞活力的效应 ,CV1 1 974可以显著地拮抗ANGⅡ对心肌细胞活力的影响。这一发现对了解ANGⅡ在心功能不全代偿和心衰发生中的作用及临床如何恰当使用ANGⅡ受体拮抗剂具有重要参考价值     

Blockade of endogenous angiotensin-(1-7) in the hypothalamic paraventricular nucleus reduces renal sympathetic tone

In this study, we tested the hypothesis that angiotensin-(1-7) [Ang-(1-7)] acting in the neurons of paraventricular hypothalamic nucleus (PVN) contributes to the maintenance of sympathetic activity and blood pressure. For this purpose, the effects of microinjection of the A-779, the receptor Mas antagonist, into the PVN on mean arterial pressure (MAP), heart rate (HR), and renal sympathetic nerve activity (RSNA) were evaluated. In rats anesthetized with urethane (1.2 to 1.4 g/kg IP), bilateral microinjections of A-779 (0.1 nmol) into the PVN resulted in a selective and significant decrease in RSNA (-26+/-6% versus -2+/-3% vehicle; saline 0.9%). The magnitude of the decrease in RSNA produced by A-779 was comparable to that observed after microinjection of muscimol (1 nmol; -26+/-4%), a powerful neuronal inhibitor. A higher dose of A-779 (1 nmol) caused a reduction in RSNA (-21+/-4%) that was comparable in magnitude to the reduction observed with the lower dose. When compared with vehicle solution, no significant changes in MAP or HR were observed with both doses of A-779 tested. A decrease in RSNA was also observed after microinjections into the PVN of the angiotensin II type 2 (AT2) receptor antagonist PD123319 (1 nmol; -18+/-4%). Microinjections of the AT1 antagonist losartan but not CV 11974 reduced MAP without changing RSNA. These results suggest that Ang-(1-7) Mas receptors and AT2 receptors in the PVN neurons play a role in mediating the tonic maintenance of RSNA.     

Divergent roles of angiotensin II AT1 and AT2 receptors in modulating coronary microvascular function

Angiotensin II (Ang II) is a potent vasoconstrictor in the peripheral circulation and has been implicated in many cardiovascular diseases associated with elevated oxidative stress. However, its direct vasomotor action and its linkage to oxidative stress-induced vascular dysfunction in the coronary microcirculation remain elusive. In this study, we directly assessed the vasomotor action of Ang II in isolated porcine coronary arterioles and also examined whether Ang II can modulate endothelium-dependent nitric oxide (NO)-mediated dilation via superoxide production. Ang II evoked vasoconstriction at a low concentration (1 nmol/L) and dilations at higher concentrations (>10 nmol/L). Ang II type 1 (AT(1)) receptor antagonist losartan abolished vasoconstriction, whereas Ang II type 2 (AT(2)) receptor antagonist PD 123319 eliminated vasodilation. Adenosine stimulated a significant arteriolar NO production and dilation. NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA) abolished stimulated NO production and attenuated vasodilation. Pretreating vessels with a subvasomotor concentration of Ang II (0.1 nmol/L, 60 minutes) mimicked inhibitory effects of L-NMMA. Ang II-mediated inhibition was not observed in the presence of L-NMMA or after endothelial removal but was prevented by losartan, superoxide scavenger TEMPOL, or NADPH oxidase inhibitor apocynin. Dihydroethidium staining showed that Ang II elicited losartan- and TEMPOL-sensitive superoxide production in arterioles. These results demonstrate that Ang II evokes AT1 receptor-mediated vasoconstriction and AT2 receptor-mediated vasodilation of coronary arterioles. Ang II at a subvasomotor level impairs endothelium-dependent NO-mediated dilation attributable to elevated superoxide production via AT1 receptor activation of NADPH oxidase. These data may partly explain the impaired coronary flow regulation in heart diseases associated with an upregulated renin-angiotensin system.     

AVE 0991, a nonpeptide mimic of the effects of angiotensin-(1-7) on the endothelium

Recently, we demonstrated that the heptapeptide angiotensin-(1-7) (Ang-[1-7]) exhibits a favorable kinetic of nitric oxide (NO) release accompanied by extremely low superoxide (O2-) production. In this report we describe AVE 0991, a novel nonpeptide compound that evoked effects similar to Ang-(1-7) on the endothelium. AVE 0991 and unlabeled Ang-(1-7) competed for high-affinity binding of [125I]-Ang-(1-7) to bovine aortic endothelial cell membranes with IC50 values of 21+/-35 and 220+/-280 nmol/L, respectively. Stimulated NO and O2- release from bovine aortic endothelial cells was directly and simultaneously measured on the cell surface by selective electrochemical nanosensors. Peak concentrations of NO and O2- release by AVE 0991 and Ang-(1-7) (both 10 micromol/L) were not significantly different (NO: 295+/-20 and 270+/-25 nmol/L; O2-: 18+/-2 and 20+/-4 nmol/L). However, the released amount of bioactive NO was approximately 5 times higher for AVE 0991 in comparison to Ang-(1-7). The selective Ang-(1-7) antagonist [D-Ala(7)]-Ang-(1-7) inhibited the AVE 0991-induced NO and O2- production by approximately 50%. A similar inhibition level was observed for the Ang II AT1 receptor antagonist EXP 3174. In contrast, the Ang II AT2 receptor antagonist PD 123,177 inhibited the AVE 0991-stimulated NO production by approximately 90% but without any inhibitory effect on O2- production. Both NO and O2- production were inhibited by NO synthase inhibition ( approximately 70%) and by bradykinin B2 receptor blockade (approximately 80%). AVE 0991 efficiently mimics the effects of Ang-(1-7) on the endothelium, most probably through stimulation of a specific, endothelial Ang-(1-7)-sensitive binding site causing kinin-mediated activation of endothelial NO synthase.     

Angiotensin II stimulates calcium and nitric oxide release from Macula densa cells through AT1 receptors

A fluorescent nitric oxide (NO) indicator, 4,5-diaminofluorescein diacetate, and the calcium indicator, indo-1, with 488 nm and 364 nm UV confocal laser scanning microscopy were used to detect NO and calcium concentration in rabbit macula densa (MD) cells challenged by angiotensin II (Ang II). Glomeruli with attached thick ascending limbs with the MD plaque were isolated and perfused. Ang II concentration from 10(-9) to 10(-5) progressively increased MD cell calcium and NO to peak values at 10(-6) and 10(-7), respectively. Ang II (10(-6) M) caused the cytosolic calcium concentration ([Ca(2+)](i)) to increase by 125.8+/-16.3 nM (n=17) from the bath and by 52.3+/-11.5 nM (n=18) from the lumen. AT(1) antagonist CV-11974 (10(-6) M) blocked the Ang II-induced calcium responses from bath and lumen, but AT(2) antagonist PD-123319 (10(-6) M) did not. AT(2) agonist CGP-42112A (10(-6) M) did not affect [Ca(2+)](i) in MD cells from either side. Ang II (10(-6) M) increased the NO production by 16%+/-3.4% (n=26) from the bath and by 18%+/-3.1% (n=24) from the lumen. CV-11974 (10(-6) M) blocked the NO responses from both sides, but PD-123319 (10(-6) M) did not on either side. CGP-42112A (10(-6) M) had no effect on NO in MD cells. In calcium-free experiments there was no difference from the result in normal calcium solutions. In conclusion, we found that Ang II increased [Ca(2+)](i) and stimulated NO production in MD cells from the basolateral and luminal sides through AT(1) receptors.     

Effects of angiotensin converting enzyme inhibitor and angiotensin II receptor antagonist combination on nitric oxide bioavailability and atherosclerotic change in Watanabe heritable hyperlipidemic rabbits.

We investigated the effects of co-administration of an angiotensin-converting enzyme inhibitor (ACEI) and angiotensin type 1 receptor blocker (ARB) on nitric oxide (NO) bioavailability in genetically hyperlipidemic rabbits with our newly developed NO sensor. Plasma NO was measured using the new NO sensor in the abdominal aorta of anesthetized Watanabe heritable hyperlipidemic (WHHL) rabbits. Acetylcholine (ACh)-stimulated (20 microg in 5 min into the aortic arch) NO production was recorded after an 8 week per os pretreatment with 1) vehicle (control), 2) the ACEI enalapril (E: 3 mg/kg/day), 3) the ARB losartan (L: 30 mg/kg/day) and 4) enalapril (1.5 mg/kg/day)+losartan (15 mg/kg/day) (E+L). Intra-aortic infusion of ACh produced an increase in plasma NO concentration, which was significantly greater with all the drug treatments than with the control. E increased ACh-induced NO significantly more than L (by 6.9 nmol/L, and 4.7 nmol/L, respectively). E+L increased ACh-induced NO by 9.5 nmol/L, significantly more than either E or L. Plasma peroxynitrite concentration was 1.2 pmol/mg protein in the control group and significantly less than in the E- and L-group. The lowest peroxynitrite concentration was observed in the E+L group (0.5 pmol/mg protein), which was significantly lower than in the E-group and the L-group. Optical coherence tomography and histology of the thoracic aorta revealed that the plaque area decreased significantly more with the combination than with the monotherapy (p<0.01). In conclusion, the combined treatment with an ACEI and an ARB may have additive protective effects on endothelial function as well as atherosclerotic change.     

Role of cardiac ATP-sensitive K+ channels induced by angiotensin II type 1 receptor antagonist on metabolism, contraction and relaxation in ischemia-reperfused rabbit heart

The role of cardiac adenosine triphosphate-sensitive K+ (K(ATP)) channels induced by angiotensin II type 1 (AT1) receptor antagonist, CV-11974, on myocardial metabolism and contraction during ischemia, and reperfusion by the phosphorus 31-nuclear magnetic resonance in Langendorff-perfused rabbit hearts was investigated. After 20 min of continuous normothermic global ischemia, 30 min of postischemic reperfusion was carried out. CV-11974 with or without the K(ATP) channel blocker, glibenclamide, or the bradykinin B2 receptor antagonist, D-Arg-[Hyp3,D-Phe7]bradykinin, was administered 40 min prior to the global ischemia. Adenosine triphosphate (ATP), creatine phosphate (PCr), inorganic phosphate (Pi), intracellular pH (pHi), left ventricular systolic developed pressure, left ventricular end-diastolic pressure (LVEDP), and coronary flow were measured. Twenty-eight hearts were divided into 4 experimental groups consisting of 7 hearts each. Group I consisted of controls, Group II perfused with CV-11974 (10(-6) mol/L), Group III perfused with CV-11974 (10(-6) mol/L) in combination with glibenclamide (10(-6) mol/L), and Group IV perfused with CV-11974 (10(-6) mol/L) in combination with D-Arg-[Hyp3,D-Phe7]bradykinin (10(-6) mol/L). Group II showed a significant inhibition of the decrease in ATP during ischemia and reperfusion compared with Group I (p<0.01), being 42+/-3% and 19+/-4% at ischemia, 69+/-3% and 47+/-4% at reperfusion in Group II and Group I, respectively. Group II also showed a significant inhibition of the increase in LVEDP during ischemia and reperfusion compared with Group I (p<0.01), being 13+/-4 mmHg and 52+/-8 mmHg at ischemia, 8+/-2 mmHg and 26+/-5 mmHg at reperfusion in Group II and Group I, respectively. However, Group II did not inhibit the decrease in ATP and the increase in LVEDP during ischemia and reperfusion. Group IV also showed no inhibition of the aforementioned parameters during the same period. These results suggest that CV-11974 has a significant beneficial effect for improving myocardial energy metabolism and relaxation during both myocardial ischemia and reperfusion, which is provided by K(ATP) channels and bradykinin B2 receptor. The cardioprotective quality of the AT1 receptor antagonist is caused by the K(ATP) channels that are mediated by the bradykinin B2 receptor.     

Coronary microvascular endothelial cell redox state in left ventricular hypertrophy : the role of angiotensin II

Left ventricular hypertrophy (LVH) is associated with elevated plasma angiotensin II (Ang II) levels and endothelial dysfunction. The relationship between Ang II and endothelial dysfunction remains unknown, however, but it may involve an alteration in endothelial cell redox state. We therefore investigated the effect of Ang II on NADH/NADPH oxidase-mediated superoxide anion (O(2)(-)) production by cultured guinea pig coronary microvascular endothelial cells (CMVEs) and CMVEs freshly isolated from a guinea pig, pressure-overload model of LVH. Lucigenin chemiluminescence was used to measure O(2)(-) production in the particulate fraction of CMVE lysates. In cultured cells, incubation with Ang II (0.1 nmol/L to 1 micromol/L for 18 hours) resulted in significant (P<0.01) increases in both NADH- and NADPH-dependent O(2)(-) production, with a peak effect at 1 nmol/L. The latter was significantly (P<0.01) inhibited by the AT(1) receptor antagonist losartan (1 micromol/L for 18 hours). In contrast, the O(2)(-) response to Ang II (0.1 nmol/L to 1 micromol/L for 18 hours) was largely unaffected by concomitant exposure to the AT(2) antagonist PD 123319 (1 micromol/L). In freshly isolated CMVEs from nonoperated animals, NADH- and NADPH-dependent O(2)(-) production was not different from that in sham-operated animals but was significantly (P<0.05) elevated in the aortic-banded animals. Plasma Ang II levels were significantly (P<0.001) elevated in the aortic-banded (1.25+/-0.12 microg/L, n=12) compared with sham-operated animals (0.63+/-0.06 microg/L, n=12). These data suggest that the endothelial dysfunction associated with LVH may be due, at least in part, to the Ang II-induced upregulation of NADH/NADPH oxidase-dependent O(2)(-) production.     

Angiotensin II modulates renal sympathetic neurotransmission through nitric oxide in AT2 receptor knockout mice

OBJECTIVE: Angiotensin (Ang) II enhances renal sympathetic neurotransmission and stimulates nitric oxide (NO) release. The present study investigates whether Ang II-mediated modulation of sympathetic neurotransmission is dependent on NO production in the kidney. AT2 -/y receptor-deficient mice are used to identify the Ang II receptor subtype involved. METHODS: Mice kidneys were isolated and perfused with Krebs-Henseleit solution. Drugs were added to the perfusion solution in a cumulative manner. Release of endogenous noradrenaline (NA) was measured by high-performance liquid chromatography (HPLC). AT1 receptor expression was analysed by real-time polymerase chain reaction (PCR). RESULTS: Ang II (0.01-30 nmol/l) dose dependently increased pressor responses in kidneys of AT2 -/y mice and wild-type (AT2 +/y) mice. Maximal pressor responses and EC50 values for Ang II was greater in AT2 -/y than in AT2 +/y mice. L-NAME (N(omega)-nitro-L-arginine methyl ester; 0.3 mmol/l) enhanced Ang II-induced pressor responses in both strains. In AT2 -/y mice, Ang II-induced facilitation of NA release was more pronounced than in AT2 +/y mice. L-NAME reduced Ang II-mediated facilitation of NA release in both strains. This reduction was more potent in AT2 -/y mice. In kidneys of AT2 -/y mice the AT1 receptor expression was significantly upregulated. CONCLUSION: These results suggest that activation of AT1 receptors by Ang II releases NO in mouse kidney to modulate sympathetic neurotransmission. Since AT1 receptors are upregulated in AT2 -/y mice kidneys, NO-dependent effects were greater in these mice. Thus, NO seems to play an important modulatory role for renal sympathetic neurotransmission.     

Angiotensin II type 2 receptor inhibits vascular endothelial growth factor-induced migration and in vitro tube formation of human endothelial cells

Endothelial cell migration and tube formation in response to vascular endothelial growth factor (VEGF) play an important role in the process of angiogenesis. Recent data indicate that angiotensin type 2 (AT2) receptor stimulation is antiangiogenic. Therefore, we studied the effect of angiotensin II (Ang II) on VEGF-induced migration and in vitro tube formation of human endothelial cells. Ang II inhibited VEGF-induced migration of EA.hy926 cells, human coronary artery (HCA) and human dermal microvascular (HDM) endothelial cells (ECs) as well as tube formation by HDMECs. The AT2 receptor antagonist PD123,319 but not the AT1 receptor antagonist losartan blocked the inhibitory effect of Ang II. The inhibitory effect of Ang II on VEGF-induced migration of endothelial cells was mimicked by the specific AT2 receptor agonist CGP-42112A. The phosphorylation of Akt and its downstream effector endothelial NO synthase (eNOS) is pivotal to VEGF-induced angiogenesis. We therefore investigated the effect of Ang II on VEGF-induced Akt and eNOS phosphorylation. Ang II diminished the VEGF-induced phosphorylation of Akt and eNOS in endothelial cells, whereas the autophosphorylation of VEGF receptors was unaffected. CGP-42112A again mimicked and PD123,319 but not losartan blocked the inhibitory effect of Ang II. Treatment of endothelial cells with pertussis toxin (PTX) totally abolished the AT2 receptor-mediated inhibition of VEGF-induced endothelial cell migration and blocked the inhibition of Akt and eNOS phosphorylation. In conclusion, this study indicates that AT2 receptor stimulation inhibits VEGF-induced endothelial cell migration and tube formation via activation of a PTX-sensitive G protein. These findings may explain the reported antiangiogenic properties of the AT2 receptor.     

Contrasting effects of angiotensin type 1 and 2 receptors on nitric oxide release under pressure.

This study was designed to test the hypothesis that increased pressure itself could cause endothelial dysfunction and lead to decreased nitric oxide (NO) release, partly through effects on the tissue renin angiotensin system in hypertension. Cultured endothelial cells (ECs) isolated from the aortas of WKY rats were continuously exposed to a pressure of 150 mmHg in a CO2 incubator for 72 h using a pressure system, and the NOx (NO2 and NO3) and angiotensin II (Ang II) concentrations in the supernatant were measured. An Ang II type 1 receptor (AT1R) antagonist (losartan) and an Ang II type 2 receptor (AT2R) antagonist (PD123319) were added to the medium. The expression of AT1R and AT2R mRNAs was also examined. Pressure loading significantly decreased the NO release from ECs. Concomitant administration of losartan restored NO release to the level before the application of pressure (p<0.001). This effect of losartan was blocked by simultaneous administration of PD123319, bradykinin type 2 receptor antagonist, and NO synthase inhibitor (p<0.05). The Ang II concentration was increased by pressure and was further increased by losartan. The gene expression of AT1R was not changed by pressure, but AT2R mRNA was increased almost 2-fold. These results indicate that high pressure itself attenuates NO release from ECs, and that losartan improves NO release by activating the bradykinin system via AT2R stimulation. In addition, the increase of AT2R gene expression in ECs during exposure to pressure may compensate for the reduction of NO.     

Cardiac angiotensin II type 2 receptor activates the kinin/NO system and inhibits fibrosis

We have previously demonstrated that stimulation of the angiotensin (Ang) II type 2 receptor in vascular smooth muscle cells caused bradykinin production by activating kininogenase in transgenic mice. The aim of this study was to determine whether overexpression of AT2 receptors in cardiomyocytes attenuates Ang II-induced cardiomyocyte hypertrophy or interstitial fibrosis through a kinin/nitric oxide (NO)-dependent mechanism in mice. Ang II (1.4 mg/kg per day) or vehicle was subcutaneously infused into transgenic mice and wild-type mice for 14 days. The amount of cardiac AT2 receptor relative to AT1 receptor in transgenic mice was 22% to 37%. Ang II caused similar elevations in systolic blood pressure (by approximately 45 mm Hg) in transgenic mice and wild-type mice. Myocyte hypertrophy assessed by an increase in myocyte cross-sectional area, left ventricular mass, and atrial natriuretic peptide mRNA levels were similar in transgenic and wild-type mice. Ang II induced prominent perivascular fibrosis of the intramuscular coronary arteries, the extent of which was significantly less in transgenic mice than in wild-type mice. Inhibition of perivascular fibrosis in transgenic mice was abolished by cotreatment with HOE140, a bradykinin B2 receptor antagonist, or L-NAME, an inhibitor of NO synthase. Cardiac kininogenase activity was markedly increased (approximately 2.6-fold, P<0.001) after Ang II infusion in transgenic mice but not in wild-type mice. Immunohistochemistry indicated that both bradykinin B2 receptors and endothelial NO synthase were expressed in the vascular endothelium, whereas only B2 receptors were present in fibroblasts. These results suggest that stimulation of AT2 receptors present in cardiomyocytes attenuates perivascular fibrosis by a kinin/NO-dependent mechanism. However, the effect on the development of cardiomyocyte hypertrophy was not detected in this experimental setting.     

体外研究胰岛素及磷脂酰肌醇3-激酶途径对一氧化氮生成的影响

目的探讨胰岛素及磷脂酰肌醇3-激酶(PI3-K)途径对NO生成的影响。方法检测胰岛素、葡萄糖以及PI3-K活性不可逆的抑制剂(Wortmannin)对培养的人脐静脉内皮细胞(HUVECs)PI3-K表达以及NO、超氧阴离子(O_2~-)产生和内皮型一氧化氮合酶(eNOS)活性的影响。实验分为对照组、10 mU/L胰岛素组、100 mU/L胰岛素组、甘露醇组、5 mmol/L葡萄糖+10 mU/L胰岛素组(5 mmol/L G1组)、25 mmol/L葡萄糖+100 mU/L胰岛素组(25 mmol/L G2组)、50 nmol/L Wortmannin组(50 nmol/L W组)、50 nmol/L Wortmannin+10 mU/L胰岛素组(50 nmol/L W1组)和50 nmol/L Wortmannin+100 mU/L胰岛素组(50 nmol/L W2组)。结果与对照组比较,不同浓度胰岛素组eNOS活性及NO水平显著升高(P<0.01);25 mmol/L G2组、50 nmol/L W组、50 nmol/LW1组和50 nmol/L W2组eNOS活性及NO水平均显著降低,O_2~-生成明显增加(P<0.01);与对照组比较,不同浓度胰岛组、50 nmol/L W组、50 nmol/L W1组和50 nmol/L W2组PI3-K蛋白表达显著升高(P<0.05,P<0.01)。结论 PI3-K信号途径对于促进NO产生、维持血管内皮细胞的正常功能具有重要作用,在高糖、高胰岛素状态下该条途径受损并由此引发内皮功能障碍。     

Plasmatic nitric oxide,but not von Willebrand Factor,is an early marker of endothelial damage,in type 1 diabetes mellitus patients without microvascular complications

Type 1 diabetes mellitus (IDDM) is associated with coronary artery disease and microvascular damage. Long-term glycemic control reduces but not fully prevents such complications. Recent evidence suggests that microvascular disease associated to IDDM begins with endothelial dysfunction. In this study, we evaluated changes in levels of nitric oxide (NO) and von Willebrand Factor (vWF) to detect early endothelial dysfunction in IDDM patients recently diagnosed. Subjects were included in one of the following groups: Group 1 (n=14): healthy subjects; Group 2 (n=14): IDDM patients recently diagnosed (<1 year), with no clinical evidence of microvascular disease; Group 3 (n=14): IDDM patients with microvascular disease (retinopathy and nephropathy). Urinary NO metabolites were similar in Group 1 (1.45+/-0.13) and Group 2 (1.6+/-0.2 micromol/mg creatinine) (P>.05), as well as vWF (99.6+/-5.7% and 84.3+/-5.1%, Groups 1 and 2, respectively, P>.05). Plasmatic NO metabolites were lower in Groups 2 and 3 (54.6+/-5.1 and 50.02+/-13.65 nmol/ml, respectively) compared with Group 1 (91.1+/-6.6 nmol/ml) (P=.0005). Also, in Group 3, urinary NO metabolites were lower (0.27+/-0.03 micromol/mg creatinine) and vWF was higher (184+/-25%) than Groups 1 and 2. There is evidence of early endothelial dysfunction even in IDDM patients recently diagnosed, with good glycemic control and without systemic hypertension, dyslipidemia or microvascular disease; this endothelial damage was detected as a decrease in plasmatic NO metabolite levels, before appearance of any clinical evidence of microvascular disease.