Gabriele Wiemer

Ramiprilat enhances endothelial autacoid formation by inhibiting breakdown of endothelium-derived bradykinin.

We studied whether inhibition of angiotensin converting enzyme stimulates the formation of nitric oxide and prostacyclin in cultured human and bovine endothelial cells by an enhanced accumulation of endothelium-derived bradykinin. Nitric oxide formation was assessed in terms of intracellular cyclic GMP accumulation, prostacyclin release by a specific radioimmunoassay. Inhibition of angiotensin converting enzyme by ramiprilat dose- and time-dependently increased the formation of nitric oxide and prostacyclin. These increases, peaking within 10 minutes, were maintained for at least 60 minutes. The ramiprilat-induced cyclic GMP increase was completely abolished by the stereospecific inhibitor of nitric oxide synthase, ArG-nitro-Larginine. The B2-kinin receptor antagonist, Hoe 140 (0.1 μM), markedly attenuated the cyclic GMP accumulation and abolished the increase in prostacyclin release. The supernatant of endothelial cells, incubated with ramiprilat (0.3 μM) for 15 minutes, elicited a significant nitric oxide release (as assessed by a guanyh/1 cyclase assay) in untreated endothelial cells used as detector tissue. Preincubation of the detector cells with Hoe 140 completely abolished this nitric oxide release. These data indicate that cultured endothelial cells from different species are capable of producing and releasing bradykinin into the extracellular space in amounts that lead to a sustained stimulation of nitric oxide and prostacyclin formation, provided that bradykinin degradation is prevented by angiotensin converting enzyme inhibition. Thus, the protective effect of angiotensin converting enzyme inhibitors observed on endothelial vasomotor function in hypertension may be explained by the local accumulation of endothelium-derived bradykinin that acts in an autocrine and paracrine manner as potent stimulus for endothelial autacoid formation.

Endothelial Dysfunction Coincides With an Enhanced Nitric Oxide Synthase Expression and Superoxide Anion Production

We investigated the effects of aortic banding-induced hypertension on the endothelium-dependent vasodilator responses in the aorta and coronary circulation of Sprague-Dawley rats. We studied the influence of hypertension on the endothelial nitric oxide synthase (NOS III) expression, assessed by Western blot and reverse transcription-polymerase chain reactions experiments, and on the superoxide anion (O2-) production. Two weeks after aortic banding, the endothelium-dependent relaxations were not altered. At this time, the expression of NOS III in the aorta and in confluent coronary microvascular endothelial cells (RCMECs) exhibited no marked changes, whereas O2- production was enhanced 1.9-fold in aortas from aortic-banded rats. Six weeks after aortic banding, the endothelium-dependent dilations were markedly impaired in the heart (50% decrease) and aorta (35% decrease). Analysis of NOS III protein and mRNA levels revealed marked increases in both aortas and confluent RCMECs (2.6- to 4-fold) from aortic-banded compared with sham-operated rats. There was no further increase in O2production in both the aorta and confluent RCMECs from aortic-banded rats. An enhanced nitrotyrosine protein level was also detected in the aorta from 6-week aortic-banded rats. These findings indicate that in hypertension induced by aortic banding, an enhanced O2- production alone is not sufficient to produce endothelial dysfunction. Endothelial vasodilator hyporesponsiveness was observed only when NOS III expression and O2- production were increased and was associated with the appearance of enhanced nitrotyrosine residues. This would suggest that the development of endothelial dysfunction is linked to an overproduction of not one, but two, endothelium-derived radicals that might lead to the formation of peroxynitrite.

Angiotensin-(1-7)–Stimulated Nitric Oxide and Superoxide Release From Endothelial Cells

The stimulation of endothelium-dependent NO release by angiotensin-(1-7) [Ang-(1-7)] has been indirectly shown in terms of vasodilation, which was diminished by NO synthase inhibition or removal of the endothelium. However, direct measurement of endothelium-derived NO has not been analyzed. With a selective porphyrinic microsensor, NO release was directly assessed from single primary cultured bovine aortic endothelial cells. Ang-(1-7) caused a concentration-dependent release of NO of 1 to 10 &mgr;mol/L, which was attenuated by NO synthase inhibition. [d-Ala7]Ang-(1-7) (5 &mgr;mol/L), described as a selective antagonist of Ang-(1-7) receptors, inhibited Ang-(1-7)–induced NO release only by ≈50%, whereas preincubation of bovine aortic endothelial cells with the angiotensin II subtype 1 and 2 receptor antagonists EXP 3174 and PD 123,177 (both at 0.1 &mgr;mol/L) led to an inhibition of 60% and 90%, respectively. A complete blockade of the Ang-(1-7)–induced NO release was observed on preincubation of the cells with 1 &mgr;mol/L concentration of the bradykinin subtype 2 receptor antagonist icatibant (HOE 140), suggesting an important role of local kinins in the action of Ang-(1-7). Simultaneous direct measurement of superoxide (O2−) detected by an O2−-sensitive microsensor revealed that the moderately Ang-(1-7)–stimulated NO release was accompanied by a very slow concomitant O2− production with a relative low peak concentration in comparison to the O2− production of the strong NO releasers bradykinin and, especially, calcium ionophore. Thus, Ang-(1-7) might preserve the vascular system, among others, due to its low formation of cytotoxic peroxynitrite by the reaction between NO and O2−.

Vasodilator dysfunction in aged spontaneously hypertensive rats: changes in NO synthase III and soluble guanylyl cyclase expression, and in superoxide anion production

OBJECTIVE/METHODS Genetic hypertension is associated with an apparent endothelial dysfunction and impaired endothelium-dependent vasodilatation in response to increased flow and receptor-dependent agonists. However, the link between impaired vasodilatation and nitric oxide (NO) synthase expression is still unclear. In the present study, dilator responses were determined in the aorta and coronary circulation of 16 month old spontaneously hypertensive (SHR) and Wistar Kyoto rats (WKY). Changes in vascular reactivity were compared with alterations in superoxide anion production as well as endothelial NO synthase (NOS III) and soluble guanylyl cyclase expression. RESULTS In the isolated perfused heart both the bradykinin- and sodium nitroprusside-induced vasodilator responses were attenuated in SHR compared to WKY. Western blot analysis revealed a parallel reduction in NOS III expression in coronary microvascular endothelial cells from SHR. Superoxide anion production in aortae from SHR was markedly elevated over that of aortae from WKY, and was almost completely abolished by pretreatment with superoxide dismutase. Superoxide dismutase induced similar relaxations in phenylephrine-preconstricted aortic rings from both SHR and WKY, but failed to restore the attenuated acetylcholine- and sodium nitroprusside-induced relaxations in SHR. No difference in NOS III expression was detected in the aortae from either strain whereas soluble guanylyl cyclase expression was markedly decreased in SHR. CONCLUSIONS These results demonstrate that NOS III expression in different tissues is differentially affected by hypertension. Moreover, although an elevated superoxide anion production is apparent in the aorta, a reduced soluble guanylyl cyclase expression appears to account for the observed vasodilator dysfunction in SHR.

Interactions among ACE, kinins and NO

Time for primary review 22 days. Angiotensin converting enzyme (ACE) is a transmembrane zinc metallopeptidase that cleaves carboxy-terminal dipeptides from several peptides and is expressed in great amounts in vascular endothelial cells [1,2]. A soluble form of the enzyme is found in plasma which is presumably derived from the membrane-bound form by proteolytic cleavage [3]. ACE plays a major role in the regulation of the vascular tone by converting the biological inactive decapeptide angiotensin I (ANG I) into the vasoconstrictor and proliferative octapeptide angiotensin II (ANG II). In a similar manner, ACE inactivates the vasodilatory nonapeptide bradykinin (BK), which derives from a number of different sources [4]. Endothelium-derived or exogenously added BK exerts its vasodilatory action through stimulation of endothelial B2 kinin receptors thereby causing the synthesis and release of vasodilator substances such as endothelium-derived hyperpolarizing factor (EDHF) [5], prostacyclin and nitric oxide (NO) [6]. Many of the effects of NO on platelets [7], smooth muscle cells [8], and cardiac myocytes [9,10] are mediated by activation of soluble guanylyl cyclase to synthesize cyclic GMP. The biological function of soluble guanylyl cyclase and NO/cyclic GMP in endothelial cells is not yet completely understood. One function of endothelial cyclic GMP may be a negative feed-back mechanism to turn off further NO synthesis [11,12]. Changes in the synthesis of ACE, BK and NO are associated with a number of cardiovascular conditions including hypertension, atherosclerosis or coronary heart disease. ACE inhibitors are able to treat these diseases by both, accumulation of endothelium-derived kinins and the inhibition of ANG II [13,14]. The separate effects of ACE, kinins as well as NO on the cardiovascular system have been thoroughly investigated and described. Since only a small amount of information is available concerning the physiological/pathophysiological significance of … * Corresponding author. Tel.: +49-69-305-6868, fax: +49-69-305-81252 wolfgang.linz{at}hmrag.com

Ramiprilat increases bradykinin outflow from isolated hearts of rat

To establish that bradykinin is formed in the heart we measured bradykinin in the venous effluent from rat isolated hearts perfused with Krebs‐Henseleit buffer. In addition, we examined the effect on bradykinin outflow of the angiotensin converting enzyme (ACE) inhibitor, ramiprilat. From rat isolated normoxic hearts a bradykinin outflow of 0.85 ± 0.1 ng ml−1 perfusate g−1 wet weight was measured. Perfusion with ramiprilat increased the bradykinin concentration to 2.8 ± 0.3 ng ml−1 perfusate g−1 wet weight. During ischaemia bradykinin outflow maximally increased 8.2 fold to 7.0 ± 0.5 ng ml−1 perfusate g−1, and in ramiprilat‐perfused hearts 5.8 fold to 16.0 ± 1.8 ng ml−1 perfusate g−1. In the reperfusion period bradykinin outflow normalized to values measured in the respective pre‐ischaemic period.

ACE-inhibition induces NO-formation in cultured bovine endothelial cells and protects isolated ischemic rat hearts.

The role of NO-formation induced by accumulated endogenous bradykinin (BK) via local ACE-inhibition with ramiprilat (RT) or by adding BK exogenously was evaluated in cultured bovine aortic endothelial cells (BAEC) and in isolated rat hearts with post-ischaemic reperfusion injuries. Furthermore we used the n-octyl-ester of ramipril (RA-octil) which was shown to have no ACE-inhibitory action. In BAEC, ACE-inhibition by RT (1 x 10(-8)-1 x 10(-6) mol/l) or addition of BK (1 x 10(-8)-1 x 10(-6) mol/l) stimulated the formation of NO and prostacyclin (PGI2) as assessed by endothelial cyclic GMP- and 6-keto-PGF1a formation. Cyclic GMP and PGI2 synthesis was completely suppressed by the NO synthase inhibitor NG-nitro-L-arginine (L-NNA, 1 x 10(-5) mol/l) and by the B2 kinin receptor antagonist HOE 140 (1 x 10(-7) mol/l). RA-octil (1 x 10(-8)-1 x 10(-4) mol/l) did not affect endothelial cyclic GMP production in BAEC. In isolated working rat hearts subjected to local ischemia with reperfusion both RT (1 x 10(-8) mol/l) and BK (1 x 10(-9) mol/l) reduced the incidence and duration of ventricular fibrillation. In parallel myocardial function (left ventricular pressure, coronary flow) and metabolism (high energy rich phosphates) were improved showing a comparable fingerprint for RT and BK. Addition of L-NNA (1 x 10(-6) mol/l) or HOE 140 (1 x 10(-9) mol/l) abolished these protective effects of RT and BK. As in the BAEC studies RA-octil was without beneficial effects on the isolated ischaemic rat heart. The findings on BAEC show that inhibition of ACE localized on the luminal side of the vascular endothelium results in increased synthesis of NO and prostacyclin by local accumulation of endothelium-derived BK. Similar mechanisms may occur in the ischaemic rat heart leading to cardioprotection.

Ramipril prevents left ventricular hypertrophy with myocardial fibrosis without blood pressure reduction: a one year study in rats.

1 Angiotensin converting enzyme (ACE)‐inhibitors have been demonstrated to be effective in the treatment of cardiac hypertrophy when used in antihypertensive doses. The aim of our one year study with an ACE‐inhibitor in rats was to separate local cardiac effects produced by a non‐antihypertensive dose from those on systemic blood pressure when an antihypertensive dose was used. 2 Rats made hypertensive by aortic banding were subjected to chronic oral treatment for one year with an antihypertensive dose of the ACE inhibitor, ramipril 1 mg kg−1 daily, (RA 1 mg) or received a low dose of 10 μg kg−1 daily (RA 10 μg) which did not affect high blood pressure. 3 Chronic treatment with the ACE‐inhibitor prevented left ventricular hypertrophy in the antihypertensive rats as did the low dose which had no effects on blood pressure. Similar effects were observed on myocardial fibrosis. Plasma ACE activity was inhibited in the RA 1 mg but not in the RA 10 μg group although conversion of angiotensin (Ang) I to Ang II in isolated aortic strips was suppressed in both treated groups. Plasma catecholamines were increased in the untreated control group, but treatment with either dose of ramipril normalized the values. The myocardial phosphocreatine to ATP ratio (an indicator of the energy state in the heart) was reduced in the vehicle control group whereas the hearts from treated animals showed a normal ratio comparable to hearts from sham‐operated animals. 4 After one year, five animals were separated from each group, treatment withdrawn, and housed for additional six months. In the RA 1 mg group, blood pressure did not reach the value of the control vehicle group and surprisingly, left ventricular hypertrophy and myocardial fibrosis did not recur in animals during withdrawal of treatment. 5 These data show that long term ACE inhibitor treatment with ramipril in antihypertensive and non‐antihypertensive doses prevented cardiac hypertrophy and myocardial fibrosis. This protective effect was still present after 6 months treatment withdrawal.

Long-term ACE Inhibition Doubles Lifespan of Hypertensive Rats

BACKGROUND We compared the outcome of lifelong treatment with the ACE inhibitor ramipril in young prehypertensive stroke-prone spontaneously hypertensive rats (SHR-SP) and age-matched normotensive Wistar-Kyoto (WKY) rats. Ramipril was given in an antihypertensive and subantihypertensive dose. In addition to the primary end point, lifespan, surrogate parameters such as cardiac left ventricular hypertrophy, cardiac function and metabolism, and endothelial function were studied. METHODS AND RESULTS One-month-old SHR-SP and WKY rats, 135 of each, were randomized into 3 groups. Each group was treated via drinking water with an antihypertensive high dose of ramipril (HRA, 1 mg x kg(-1) x d(-1)), a nonantihypertensive low dose of ramipril (LRA, 10 microg x kg(-1) x d(-1)), or placebo. Body weight and blood pressure were determined every 3 months. Molecular, biochemical, and functional data were assessed in SHR-SP and WKY rats after 15 and 30 months, respectively. These were the times when approximately 80% of the corresponding placebo group had died. Early-onset long-term ACE inhibition with HRA doubled lifespan to 30 months in SHR-SP, which was identical to the lifespan of placebo-treated normotensive WKY rats. LRA treatment prolonged lifespan from 15 to 18 months. In SHR-SP, left ventricular hypertrophy was completely prevented by HRA but not by LRA treatment. Cardiac function and metabolism as well as endothelial function were significantly improved by both doses of ramipril. Carotid expression of endothelial NO synthase was moderately enhanced, whereas cardiac ACE expression and activity were decreased to values of placebo-treated WKY rats. CONCLUSIONS Lifelong ACE inhibition doubles lifespan in SHR-SP, matching that of normotensive WKY rats. This effect correlated with preservation of endothelial function, cardiac function/size, and metabolism. Thus, these data predict a beneficial outcome on survival in high-risk patients with hypertension and associated cardiovascular diseases by ACE inhibition.

Preservation of endothelial function by ramipril in rabbits on a long-term atherogenic diet

Hypertension and hypercholeslcrolemia predispose to atherosclerosis. Ramipril, known to lower blood pressure, was used to study the effect of converting-enzyme inhibition on impairment of endothelium-derived relaxation and changes in basal cGMP content in rabbits fed an atherogenic diet (0.25% cholesterol). The generation of cGMP in the presence of bradykinin and ramiprilat was studied in vitro in aortic segments from normal untreated rabbits as well as in bovine endothelial cells. The ability to relax in response io acetylcholine was almost abolished in aortic segments from the vehicle-treated rabbits fed the atherogenic diet for 4 months. The basal cGMP content was substantially reduced. Aortic segments from rabbits concomitantly treated with ramipril (0.3 and 3.0 mg/kg/day) for 3 months showed well-preserved relaxation and matching basal cGMP content compared to normal controls. The relaxation was not significantly greater in aortic segments from ramipril-treated rabbits fed the standard diet, but the cGMP content was more than doubled. In vitro studies in aortic segments and in endothelial cells showed that both the ramiprilat and bradykinin concentrations dependency stimulated cGMP formation, which serves as a biochemical marker of nitric oxide or EDRF release. Thus, the observed endothelial protection against hypercholesterolemia by ramipril may be the result of continuously increased cGMP formation due to preserved EDRF release. This is presumably produced by enhanced bradykinin activity through inhibition of degradation by converting-enzyme inhibition with ramipril.