Localization of the novel angiotensin peptide, angiotensin-(1-12), in heart and kidney of hypertensive and normotensive rats

A low expression of angiotensinogen in the heart has been construed as indicating a circulating uptake mechanism to explain the local effects of angiotensin II on tissues. The recent identification of angiotensin-(1-12) in an array of rat organs suggests this propeptide may be an alternate substrate for local angiotensin production. To test this hypothesis, tissues from 11-wk-old spontaneously hypertensive rats (SHR) and Wistar-Kyoto (WKY) rats (n = 14) were stained with purified antibodies directed to the COOH terminus of angiotensin-(1-12). Robust angiotensin-(1-12) staining was predominantly found in ventricular myocytes with less staining found in the medial layer of intracoronary arteries and vascular endothelium. In addition, angiotensin-(1-12) immunoreactivity was present in the proximal, distal, and collecting renal tubules within the deep cortical and outer medullary zones in both strains. Preadsorption of the antibody with angiotensin-(1-12) abolished staining in both tissues. Corresponding tissue measurements by radioimmunoassay showed 47% higher levels of angiotensin-(1-12) in the heart of SHR compared with WKY rats (P < 0.05). In contrast, renal angiotensin-(1-12) levels were 16.5% lower in SHR compared with the WKY rats (P < 0.05). This study shows for first time the localization of angiotensin-(1-12) in both cardiac myocytes and renal tubular components of WKY and SHR. In addition, we show that increased cardiac angiotensin-(1-12) concentrations in SHR is associated with a small, but statistically significant, reduction in renal angiotensin-(1-12) levels.     

Olmesartan medoxomil treatment potently improves cardiac myosin-induced dilated cardiomyopathy via the modulation of ACE-2 and ANG 1-7 mas receptor

Angiotensin converting enzyme-2 (ACE-2) is a monocarboxypeptidase that metabolises angiotensin (ANG)-II into angiotensin 1-7 (ANG 1-7), thereby functioning as a negative regulator of the renin-angiotensin system. We investigated whether treatment with ANG-II type 1 receptor blocker, olmesartan medoxomil is associated with the attenuation of cardiac myosin-induced dilated cardiomyopathy (DCM) through recently established new axis of ACE-2/ANG 1-7 mas receptor. DCM was elicited in Lewis rats by immunisation with cardiac myosin, and 28 days after immunisation, the surviving Lewis rats were divided into two groups and treated with either olmesartan medoxomil (10 mg/kg/day) or vehicle. Myocardial protein and mRNA levels of ACE-2, ANG 1-7 mas receptor were upregulated in the olmesartan-treated group compared with those of vehicle-treated DCM rats. In contrast, Olmesartan treatment effectively suppressed the myocardial protein and mRNA expressions of inflammatory markers in comparison to the vehicle-treated DCM rats. Olmesartan treatment significantly reduced fibrosis, hypertrophy and their marker molecules (OPN, CTGF, ANP and GATA-4, respectively), as well as matrix metalloproteinases compared with those of vehicle-treated DCM rats. Enhanced myocardial protein levels of phospho-p38 MAPK, phospho-JNK and phospho MAPKAPK-2 in the vehicle-treated DCM rats were prevented by olmesartan treatment. In addition, olmesartan treatment significantly lowered the protein expressions (Nitrotyrosine, p47phox and p67phox) and superoxide radical production compared with those of vehicle-treated DCM rats. Our present study might serve as a new therapeutic target of DCM in cardiovascular diseases and cardiac myosin-induced DCM via the modulation of ACE-2/ANG 1-7 mas receptor axis in rats with DCM after myosin-immunisation.     

Cardioprotective effects of telmisartan against heart failure in rats induced by experimental autoimmune myocarditis through the modulation of angiotensin-converting enzyme-2/angiotensin 1-7/mas receptor axis

Angiotensin-converting enzyme-2 (ACE-2) is a homolog of ACE that preferentially forms angiotensin-(ANG)-1-7 from angiotensin II (ANG II). We investigated the cardioprotective effects of telmisartan, a well-known angiotensin receptor blockers (ARBs) against experimental autoimmune myocarditis (EAM). EAM was induced in Lewis rats by immunization with porcine cardiac myosin. The rats were divided into two groups and treated with telmisartan (10 mg/kg/day) or vehicle for 21 days. Myocardial functional parameters were significantly improved by treatment with telmisartan compared with vehicle-treated rats. Telmisartan lowered myocardial protein expressions of NADPH oxidase subunits 3-nitrotyrosine, p47phox, p67 phox, Nox-4 and superoxide production significantly than vehicle-treated rats. In contrast myocardial protein levels of ACE-2, ANG 1-7 mas receptor were upregulated in the telmisartan treated group compared with those of vehicle-treated rats. The myocardial protein expression levels of tumor necrosis factor receptor (TNFR)-associated factor (TRAF)-2, C/EBP homologous protein (CHOP) and glucose-regulated protein (GRP) 78 were decreased in the telmisartan treated rats compared with those of vehicle-treated rats. In addition, telmisartan treatment significantly decreased the protein expression levels of phospho-p38 mitogen-activated protein kinase (MAPK), phospho-JNK, phospho-ERK and phospho (MAPK) activated protein kinase-2 than with those of vehicle-treated rats. Moreover, telmisartan significantly decreased the production of proinflammatory cytokines, myocardial apoptotic markers and caspase-3 positive cells compared with those of vehicle-treated rats. Therefore, we suggest that telmisartan was beneficial protection against heart failure in rats, at least in part by suppressing inflammation, oxidative stress, ER stress as well as signaling pathways through the modulation of ACE2/ANG1-7/Mas receptor axis.     

Effect of hypertension on angiotensin-(1-7) levels in rats with different angiotensin-I converting enzyme polymorphism

To determine circulating angiotensin-(1-7) [Ang-(1,7)] levels in rats with different angiotensin converting enzyme (ACE) genotypes and to evaluate the effect of hypertension on levels of this heptapeptide, plasma levels of angiotensin II (Ang II) and Ang-(1-7) were determined by HPLC and radioimmunoassay in (a) normotensive F0 and F2 homozygous Brown Norway (BN; with high ACE) or Lewis (with low ACE) rats and (b) in hypertensive F2 homozygous male rats (Goldblatt model). Genotypes were characterized by PCR and plasma ACE activity measured by fluorimetry. Plasma ACE activity was 2-fold higher (p < 0.05) in homozygous BN compared to homozygous Lewis groups. In the Goldblatt groups, a similar degree of hypertension and left ventricular hypertrophy was observed in rats with both genotypes. Plasma Ang II levels were between 300-400% higher (p < 0.05) in the BN than in the Lewis rats, without increment in the hypertensive animals. Plasma Ang-(1-7) levels were 75-87% lower in the BN rats (p < 0.05) and they were significantly higher (p < 0.05) in the hypertensive rats from both genotypes. Plasma levels of Ang II and Ang-(1-7) levels were inversely correlated in the normotensive rats (r = -0.64; p < 0.001), but not in the hypertensive animals. We conclude that there is an inverse relationship between circulating levels of Ang II and Ang-(1-7) in rats determined by the ACE gene polymorphism. This inverse relation is due to genetically determined higher ACE activity. Besides, plasma levels of Ang-(1-7) increase in renovascular hypertension.     

Primary role of angiotensin-converting enzyme-2 in cardiac production of angiotensin-(1-7) in transgenic Ren-2 hypertensive rats

Angiotensin-converting enzyme-2 (ACE2) converts angiotensin II (ANG II) to angiotensin-(1-7) [ANG-(1-7)], and this enzyme may serve as a key regulatory juncture in various tissues. Although the heart expresses ACE2, the extent that the enzyme participates in the cardiac processing of ANG II and ANG-(1-7) is equivocal. Therefore, we utilized the Langendorff preparation to characterize the ACE2 pathway in isolated hearts from male normotensive Sprague-Dawley [Tg((-))] and hypertensive [mRen2]27 [Tg((+))] rats. During a 60-min recirculation period with 10 nM ANG II, the presence of ANG-(1-7) was assessed in the cardiac effluent. ANG-(1-7) generation from ANG II was similar in both the normal and hypertensive hearts [Tg((-)): 510 +/- 55 pM, n=20 vs. Tg((+)): 497 +/- 63 pM, n=14] with peak levels occurring at 30 min after administration of the peptide. ACE2 inhibition (MLN-4760, 1 microM) significantly reduced ANG-(1-7) production by 83% (57 +/- 19 pM, P<0.01, n=7) in the Tg((+)) rats, whereas the inhibitor had no significant effect in the Tg((-)) rats (285 +/- 53 pM, P>0.05, n=10). ACE2 activity was found in the effluent of perfused Tg((-)) and Tg((+)) hearts, and it was highly associated with ACE2 protein expression (r=0.78). This study is the first demonstration for a direct role of ACE2 in the metabolism of cardiac ANG II in the hypertrophic heart of hypertensive rats. We conclude that predominant expression of cardiac ACE2 activity in the Tg((+)) may be a compensatory response to the extensive cardiac remodeling in this strain.     

Antihypertensive treatment differentially affects vascular sphingolipid biology in spontaneously hypertensive rats

Background

We have previously shown that essential hypertension in humans and spontaneously hypertensive rats (SHR), is associated with increased levels of ceramide and marked alterations in sphingolipid biology. Pharmacological elevation of ceramide in isolated carotid arteries of SHR leads to vasoconstriction via a calcium-independent phospholipase A₂, cyclooxygenase-1 and thromboxane synthase-dependent release of thromboxane A₂. This phenomenon is almost absent in vessels from normotensive Wistar Kyoto (WKY) rats. Here we investigated whether lowering of blood pressure can reverse elevated ceramide levels and reduce ceramide-mediated contractions in SHR.

Methods and Findings

For this purpose SHR were treated for 4 weeks with the angiotensin II type 1 receptor antagonist losartan or the vasodilator hydralazine. Both drugs decreased blood pressure equally (SBP untreated SHR: 191±7 mmHg, losartan: 125±5 mmHg and hydralazine: 113±14 mmHg). The blood pressure lowering was associated with a 20–25% reduction in vascular ceramide levels and improved endothelial function of isolated carotid arteries in both groups. Interestingly, losartan, but not hydralazine treatment, markedly reduced sphingomyelinase-induced contractions. While both drugs lowered cyclooxygenase-1 expression, only losartan and not hydralazine, reduced the endothelial expression of calcium-independent phospholipase A₂. The latter finding may explain the effect of losartan treatment on sphingomyelinase-induced vascular contraction.

Conclusion

In summary, this study corroborates the importance of sphingolipid biology in blood pressure control and specifically shows that blood pressure lowering reduces vascular ceramide levels in SHR and that losartan treatment, but not blood pressure lowering per se, reduces ceramide-mediated arterial contractions.     

Advances in biochemical and functional roles of angiotensin-converting enzyme 2 and angiotensin-(1-7) in regulation of cardiovascular function

Angiotensin-converting enzyme 2 (ACE2) is the first human homologue of ACE to be described. ACE2 is a type I integral membrane protein that functions as a carboxypeptidase, cleaving a single hydrophobic/basic residue from the COOH-terminus of its substrates. Because ACE2 efficiently hydrolyzes the potent vasoconstrictor angiotensin II to angiotensin (1-7), this has changed our overall perspective about the classical view of the renin angiotensin system in the regulation of hypertension and heart and renal function, because it represents the first example of a feedforward mechanism directed toward mitigation of the actions of angiotensin II. This paper reviews the new data regarding the biochemistry of angiotensin-(1-7)-forming enzymes and discusses key findings such as the elucidation of the regulatory mechanisms participating in the expression of ACE2 and angiotensin-(1-7) in the control of the circulation.     

Differential response of angiotensin peptides in the urine of hypertensive animals

Urinary excretion rates of angiotensin I (Ang I), angiotensin II (Ang II), and angiotensin-(1-7) [Ang-(1-7)] were determined in normotensive Sprague Dawley (SD), spontaneously hypertensive (SHR), and mRen-2 transgenic hypertensive animals before and following blockade of Ang II synthesis or activity for two weeks. This study was performed to determine for the first time whether inhibition of Ang II alters the excretion of angiotensin peptides in the urine. Rats were given either tap water or water medicated with lisinopril, losartan or both agents in combination. Blood pressure was monitored at regular intervals during the experiment by the tail-cuff method, and once again at the end of the study with a catheter implant into a carotid artery. Metabolic studies and 24 h urinary excretion variables and angiotensin peptides were determined before and during the procedures. While all three treatments normalized the blood pressure of hypertensive animals, therapy with either lisinopril or the combination of lisinopril and losartan had a greater antihypertensive effect in both SHR and [mRen-2]27 transgenic hypertensive rats. In the urine, the concentration of the angiotensins (normalized by 24-h creatinine excretion) was several-fold higher in the untreated hypertensive animals than in normotensive SD rats. In SD rats, lisinopril or lisinopril and losartan produced a sustained rise in urinary levels of Ang-(1-7) without changes in the excretion of Ang I and Ang II. In contrast, Ang I and Ang-(1-7) were significantly elevated in SHR medicated with lisinopril alone or in combination with losartan. Only losartan, however, augmented urinary levels of Ang II in the SHR. The antihypertensive effects of the three separate regimens had no effect on the urinary excretion of angiotensin peptides in [mRen-2]27 transgenic hypertensive rats. These data show that Ang I and Ang-(1-7) are excreted in large amounts in the urine of SD, SHR and [mRen-2]27 hypertensive rats. The unchanged Ang-(1-7) excretion in transgenic hypertensive (Tg+) rats after inhibition of the renin-angiotensin system agrees with the previous finding of a reduced plasma clearance of the peptide in this model of hypertension. The data suggest that this form of hypertension may be associated with increased activity of an endogenous converting enzyme inhibitor.     

微小RNA在自发性高血压大鼠主动脉的差异表达

微小RNAs(microRNAs,miRNAs)是一类基因组编码、非蛋白质编码的小RNA,在转录后水平负性调节靶基因表达.本研究探讨miRNAs在自发性高血压大(spontaneously hypertensive rats,SHR)主动脉的表达特征及其与高血压的关系.取4、8、16和24周龄雄性SHR大鼠及同龄正常血压对照(Wistar-Kyoto,WKY)大鼠.MiRanda、TargetScan和PicTar用于候选miRNAs及靶基因预测分析.通过实时定量RT-PCR检测大鼠主动脉miR-1、miR-133a、miR-155及miR-208的表达,并进一步通过实时定量RT-PCR检测呈差异表达的miR-155和miR-208的预测靶基因mRNA表达.结果显示,SHR大鼠主动脉miR-155表达在4、8、24周时与同龄WKY大鼠无显著差异,但在16周时明显低于同龄WKY大鼠(P<0.05),且大鼠主动脉miR-155表达量与血压呈负相关(r=-0.525,P<0.05).MiR-208表达在4周龄时最高,随年龄增长明显下降(P<0.05),其表达水平与血压和年龄呈负相关(r=-0.400,P<0.05;r=-0.684,P<0.0001),但在SHR和WKY大鼠之间无显著差异.miR-1和miR-133a在各年龄组SHR和WKY大鼠间未呈现差异表达.MiR-155和miR-208表达与相应预测靶基因mRNA表达无显著负相关性.以上结果表明,miR-155表达在成年SHR大鼠主动脉明显低于WKY,并与血压呈负相关,提示miR-155可能参与高血压的发生发展,主动脉miR-155表达异常可能是SHR大鼠血压升高的原因之一.大鼠主动脉miR-208表达在幼年时最高,随年龄增长而明显下降,提示其可能与血管发育有关.     

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.     

Effect of acute oral administration of captopril and MK-421 on vascular angiotensin converting enzyme activity in the spontaneously hypertensive rat

Vascular angiotensin converting enzyme (ACE) may be important as a potential site of action for the antihypertensive effect of ACE inhibitors. ACE activity, estimated by hydrolysis of [³H] HipGlyGly, was similar in the aorta, mesenteric and carotid arteries of SHR. ACE activity in veins was not as consistent and was significantly lower in the jugular veins and vena cava of SHR than in the arteries. Nevertheless, ACE activity in all the blood vessels examined, although less than lung ACE activity, was higher than ACE activity found in other tissues such as brain, heart and kidney. Equieffective antihypertensive doses of captopril (10 mg/kg p.o.) and MK-421 (1.0 mg/kg p.o.) dramatically inhibited ACE activity in all the arteries and veins examined. Maximal ACE inhibition occured within 15 minutes after the oral administration of captopril. In contrast, maximal ACE inhibition was slower in onset and of longer duration after MK-421 than after captopril for all the vessels. Thus, relatively high ACE activity can be measured in both arteries and veins from the spontaneously hypertensive rat (SHR) and ACE was dramatically inhibited after antihypertensive oral doses of captopril or MK-421. Furthermore, vascular ACE inhibition can be used to compare the onset and duration of activity of ACE inhibitors; MK-421 has a longer onset and duration in SHR than captopril based on inhibition of ACE in blood vessels.     

Reciprocal changes in renal ACE/ANG II and ACE2/ANG 1-7 are associated with enhanced collecting duct renin in Goldblatt hypertensive rats

Alterations in the balance between ANG II/ACE and ANG 1-7/ACE2 in ANG II-dependent hypertension could reduce the generation of ANG 1-7 and contribute further to increased intrarenal ANG II. Upregulation of collecting duct (CD) renin may lead to increased ANG II formation during ANG II-dependent hypertension, thus contributing to this imbalance. We measured ANG I, ANG II, and ANG 1-7 contents, angiotensin-converting enzyme (ACE) and ACE2 gene expression, and renin activity in the renal cortex and medulla in the clipped kidneys (CK) and nonclipped kidneys (NCK) of 2K1C rats. After 3 wk of unilateral renal clipping, systolic blood pressure and plasma renin activity increased in 2K1C rats (n = 11) compared with sham rats (n = 9). Renal medullary angiotensin peptide levels were increased in 2K1C rats [ANG I: (CK = 171 ± 4; NCK = 251 ± 8 vs. sham = 55 ± 3 pg/g protein; P < 0.05); ANG II: (CK = 558 ± 79; NCK = 328 ± 18 vs. sham = 94 ± 7 pg/g protein; P < 0.001)]; and ANG 1-7 levels decreased (CK = 18 ± 2; NCK = 19 ± 2 pg/g vs. sham = 63 ± 10 pg/g; P < 0.001). In renal medullas of both kidneys of 2K1C rats, ACE mRNA levels and activity increased but ACE2 decreased. In further studies, we compared renal ACE and ACE2 mRNA levels and their activities from chronic ANG II-infused (n = 6) and sham-operated rats (n = 5). Although the ACE mRNA levels did not differ between ANG II rats and sham rats, the ANG II rats exhibited greater ACE activity and reduced ACE2 mRNA levels and activity. Renal medullary renin activity was similar in the CK and NCK of 2K1C rats but higher compared with sham. Thus, the differential regulation of ACE and ACE2 along with the upregulation of CD renin in both the CK and NCK in 2K1C hypertensive rats indicates that they are independent of perfusion pressure and contribute to the altered content of intrarenal ANG II and ANG 1-7.     

Olmesartan, an AT1 antagonist, attenuates oxidative stress, endoplasmic reticulum stress and cardiac inflammatory mediators in rats with heart failure induced by experimental autoimmune myocarditis

Studies have demonstrated that angiotensin II has been involved in immune and inflammatory responses which might contribute to the pathogenesis of immune-mediated diseases. Recent evidence suggests that oxidative stress may play a role in myocarditis. Here, we investigated whether olmesartan, an AT(1)R antagonist protects against experimental autoimmune myocarditis (EAM) by suppression of oxidative stress, endoplasmic reticulum (ER) stress and inflammatory cytokines. EAM was induced in Lewis rats by immunization with porcine cardiac myosin, were divided into two groups and treated with either olmesartan (10 mg/kg/day) or vehicle for a period of 21 days. Myocardial functional parameters measured by hemodynamic and echocardiographic analyses were significantly improved by the treatment with olmesartan compared with those of vehicle-treated rats. Treatment with olmesartan attenuated the myocardial mRNA expressions of proinflammatory cytokines, [Interleukin (IL)-1β, monocyte chemoattractant protein-1, tumor necrosis factor-α and interferon-γ)] and the protein expression of tumor necrosis factor-α compared with that of vehicle-treated rats. Myocardial protein expressions of AT(1)R, NADPH oxidase subunits (p47phox, p67phox, gp91phox) and the expression of markers of oxidative stress (3-nitrotyrosine and 4-hydroxy-2-nonenal), and the cardiac apoptosis were also significantly decreased by the treatment with olmesartan compared with those of vehicle-treated rats. Furthermore, olmesartan treatment down-regulated the myocardial expressions of glucose regulated protein-78, growth arrest and DNA damage-inducible gene, caspase-12, phospho-p38 mitogen-activated protein kinase (MAPK) and phospho-JNK. These findings suggest that olmesartan protects against EAM in rats, at least in part via suppression of oxidative stress, ER stress and inflammatory cytokines.     

Chronic hydralazine treatment alters the acute pressure-natriuresis curve in young spontaneously hypertensive rats

The pressure-natriuresis relationship was studied in anesthetized, 7- to 9-week-old control spontaneously hypertensive rats (SHR) and in SHR that had been treated with hydralazine (20 mg.kg-1.day-1 in drinking water) starting at 4-5 weeks of age. To minimize reflex changes in kidney function during changes in renal artery pressure, neural and hormonal influences on the kidney were fixed by surgical renal denervation, adrenalectomy, and infusion of a hormone cocktail (330 microL.kg-1.mikn-1) containing high levels of aldosterone, arginine vasopressin, hydrocortisone, and norepinephrine dissolved in 0.9% NaCl containing 1% albumin. Changes in renal function were measured using standard clearance techniques, while renal artery pressure was varied between 136 +/- 1 and 186 +/- 2 mmHg (1 mmHg = 133.32 Pa) in control SHR (n = 10) and between 113 +/- 1 and 162 +/- 2 mmHg in treated SHR (n = 11). Mean arterial pressure (+/- SE) under Inactin anesthesia was 172 +/- 3 mmHg in control SHR and 146 +/- 3 mmHg in treated SHR (p less than 0.05). Where renal artery pressure overlapped between groups, there were no significant differences in glomerular filtration rate. Renal blood flow was also similar in both groups, although at 160 mmHg blood flow was slightly but significantly reduced in treated SHR. Urine flow and total and fractional sodium excretion increased similarly with increases in renal artery pressure in both groups, but the pressure-natriuresis curve in hydralazine-treated SHR was displaced to the left along the pressure axis. The data indicate that chronic administration of hydralazine in young SHR enhances fractional sodium excretion, suggesting that tubular reabsorption of sodium is decreased by hydralazine.     

Metabolism of bradykinin in aorta of hypertensive rats

Alterations in the formation and metabolism of bradykinin (Bk) are hypothesized to play a role in the pathophysiology of hypertension, atherosclerosis and vascular complications of diabetes. However, despite its prominent role in cardiovascular regulation, studies on bradykinin have been limited by various difficulties in accurate measurements of this peptide in biological samples. In this study, using the LC-ESI-MS method we estimated the conversion of exogenous Bk to its main metabolites - Bk-(1-5) and Bk-(1-7) - in endothelial cell culture and in fragments of aorta of normotensive (WKY) and hypertensive rats (SHR). The effects of angiotensin converting enzyme (ACE) and neutral endopeptidase (NEP) inhibitors were more pronounced in SHR: perindoprilat inhibited Bk-(1-5) formation by 49 % and 76 % in WKY and SHR rats, respectively, and tiorphan tended to decrease formation of Bk-(1-5) in both groups of animals. The degradation of bradykinin and generation of both metabolites were significantly higher in the aorta of SHR rats than in WKY controls. Our results show that even in relatively early hypertension (in 4-month old SHR rats) inactivation of Bk by aorta wall is enhanced.     

Reducing arginase activity via dietary manganese deficiency enhances endothelium-dependent vasorelaxation of rat aorta

L-Arginine is a common substrate for the enzymes arginase and nitric oxide synthase (NOS). Acute inhibition of arginase enzyme activity improves endothelium-dependent vasorelaxation, presumably by increasing availability of substrate for NOS. Arginase is activated by manganese (Mn), and the consumption of a Mn-deficient (Mn-) diet can result in low arginase activity. We hypothesize that endothelium-dependent vasorelaxation is greater in rats fed Mn- versus Mn sufficient (Mn+) diets. Newly weaned rats fed Mn+ diets (0.5 microg Mn/g; n = 12) versus Mn+ diets (45 microg Mn/g; n = 12) for 44 +/- 3 days had (i) lower liver and kidney Mn and arginase activity (P < or = 0.05), (ii) higher plasma L-arginine (P < or = 0.05), (iii) similar plasma and urine nitrate + nitrite, and (iv) similar staining for endothelial nitric oxide synthase in thoracic aorta. Vascular reactivity of thoracic aorta (approximately 720 microm i.d.) and small coronary arteries (approximately 110 microm i.d.) was evaluated using wire myographs. Acetylcholine (ACh; 10(-8)-10(-4) M) produced greater (P < or = 0.05) vasorelaxation in thoracic aorta from Mn- rats (e.g., maximal percent relaxation, 79 +/- 7%) versus Mn + rats (e.g., maximal percent relaxation, 54 +/- 9%) at 5 of 7 evaluated doses. Tension produced by NOS inhibition using N(G) monomethyl-L-arginine (L-NMMA; 10(-3) M) and vasorelaxation evoked by (i) arginase inhibition using difluoromethylornithine (DFMO; 10(-7) M), (ii) ACh (10(-8)-10(-4) M) in the presence of DFMO, and (iii) sodium nitroprusside (10(-9)-10(-4) M) were unaffected by diet. No differences existed between groups concerning these responses in small coronary arteries. These findings support our hypothesis that endothelium-dependent vasorelaxation is greater in aortic segments from rats that consume Mn- versus Mn+ diets; however, responses from small coronary arteries were unaffected.     

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.