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Strong earthquakes at active ocean margins can remobilize vast amounts of surficial slope sediments and dynamically strengthen the margin sequences. Current process understanding is obtained from resulting event deposits and low-resolution shear strength data, respectively. Here we directly target a site offshore Japan where both processes are expected to initiate, that is, at the uppermost part (15 cm) of a sedimentary slope sequence. Based on a novel application of short-lived radionuclide data, we identified, dated, and quantified centimeter-scale gaps related to surficial remobilization. Temporal correlation to the three largest regional earthquakes attest triggering by strong earthquakes ( M w >8). Also, extremely elevated shear strength values suggest a strong influence of seismic strengthening on shallow sediments. We show that despite enhanced slope stability by seismic strengthening, earthquake-induced sediment transport can occur through surficial remobilization, which has large implications for the assessment of turbidite paleoseismology and carbon cycling at active margins.

Raloxifene (CAS 84449-90-1, RAL), a selective estrogen receptor modulator (SERM) effective for the prevention of post-menopausal osteoporosis, also has been shown to acutely stimulate nitric oxide (NO) synthesis associated with improved endothelium-dependent relaxation. The effect of a 3-month RAL treatment (10 mg/kg/d) on basal blood pressure, measured via the carotid artery, and its challenge with increasing doses of intravenous bradykinin (1, 3 and 10 nmol/kg) was investigated. Furthermore, aortic NO bioavailability and relaxation in 9-month-old male and female ovarectomized (OVX) spontaneously hypertensive rats (SHR) was tested. Calcium ionophore stimulated NO release from aortic endothelial cells and aortic superoxide (O2-) production was directly assessed by using electrochemical nanosensors. Relaxation studies were performed with acetylcholine (10(-8) to 10(-5) mol/L) following precontraction with phenylephrine (10(-7) mmol/L). Whereas basal blood pressure (BP) was not significantly decreased in RAL treated SHR, the dose-dependent challenge with bradykinin induced an enhanced BP reduction in either gender. In contrast to female animals, aortic segments from RAL treated male animals showed significantly improved relaxaSHR) to 360 +/- 15 nmol/L. Vice versa, O2- was decreased from 110 +/- 15 to 22 +/- 1 nmol/L. In female SHR, ovarectomy led to an increase/decrease of NO/O2- from 130 +/- 5 to 180 +/- 10 nmol/L and 82 +/- 7 to 68 +/- 3 nmol/L, respectively. These effects were significantly amplified by RAL treatment (NO: 370 +/- 10 and O2-: 25 +/- 2 nmol/L). The results show that long-term treatment with RAL has beneficial affects on the cardiovascular system in old male and female OVX SHR via an increased NO bioavailability.

A markedly decreased aortic nitric oxide (NO) availability, probably due to impaired endothelial nitric oxide synthase activity with enhanced O2- and peroxynitrite production, seems to be attributable to endothelial dysfunction in spontaneously hypertensive rats (SHR) with severe congestive heart failure (CHF). In this study, we investigated the chronic effect of the angiotensin-converting enzyme inhibitor, ramipril (RA) and the loop diuretic, frusemide (FU), as well as the combination of both on endothelial NO, O2- and peroxynitrite production in aortae from SHR with failing hearts after myocardial infarction (MI). Heart failure was induced by permanent occlusion of the left coronary artery. SHR were randomised to receive either placebo, RA, (1 mg/kg/day), FU (4 mg/kg/day) or RA+FU (1 and 4 mg/kg/day, respectively). Treatments were started two weeks following MI and continued for six weeks. Reduced aortic and coronary flow indices in the working heart, which can be considered as markers for endothelial function, were significantly normalised and improved, respectively, by RA, FU or RA+FU-treatment. Similarly, all three treatment regimens significantly enhanced the reduced calcium ionophore (CaI)-induced NO-release (assessed by a NO-sensitive microsensor) from aortic endothelial cells of placebo-treated animals with CHF. Concomitantly, the increased CaI-stimulated O2- production (assessed by an electrochemical sensor) in aortic endothelial cells of placebo-treated animals with CHF was significantly reduced by RA and RA+FU-treatment. Treatment with RA and RA+FU also attenuated the dramatic increase in endothelial peroxynitrite concentration (chemiluminescence method), which was observed in placebo-treated rats with CHF. FU did not counteract improved haemo- and cardiodynamic parameters by RA. Thus, RA and FU act synergistically to enhance bioavailability of endothelium-derived NO, and this may contribute to the clinical usefulness of the combination of these drugs in treatment of heart failure.

Aim of the present study was to investigate the influence of the angiotensin II (ANG II) subtype 1 (AT(1)) receptor blockers fonsartan and losartan on blood pressure, cardiac -dynamics and -metabolism as well as functional and morphological changes in the kidney of rats after long-term inhibition of the nitric oxide (NO) synthase by N(G)-nitro-L-arginine methyl ester (L-NAME). Oral chronic treatment with L-NAME in a dose of 25 mg/kg/d over 6 weeks caused a significant increase in systolic blood pressure (198+/-13 mmHg) when compared to untreated rats (144+/-4 mmHg). Animals receiving simultaneously L-NAME and fonsartan (10 mg/kg/d) or losartan (30 mg/kg/d) were protected against blood pressure increase. L-NAME treatment caused a significant decrease in glomerular filtration rate (GFR) from 4.52+/-0.81 to 1.34+/-0.26 ml/kg(-1)/min(-1) and renal plasma flow (RPF) from 10.52+/-1.29 ml/kg(-1)/min(-1) to 5.66+/-1.06 ml/kg(-1)/min(-1). Co-treatment with fonsartan and losartan prevented L-NAME-induced reduction in GFR and RPF. There was no difference in urine, sodium and potassium excretion in groups under investigation. Plasma renin activity (PRA) was further stimulated by fonsartan and losartan treatment. L-NAME produced a significant elevation in urinary protein excretion which was antagonised by both AT(1) blockers. Isolated hearts from animals treated with L-NAME showed a significant prolongation in the duration of ventricular fibrillation and a significant decrease in coronary flow as compared to control hearts. Treatment with fonsartan and losartan significantly decreased the duration of ventricular fibrillation as compared to L-NAME group. In addition, both AT(1) blockers given alone significantly reduced the duration of ventricular fibrillation as compared to hearts from untreated controls. During ischemia the cytosolic enzymes lactate dehydrogenase and creatine kinase as well as lactate in the coronary effluent were significantly increased in the L-NAME group. Myocardial tissue values of glycogen, ATP, and creatine phosphate were decreased, whereas lactate was increased. Fonsartan and losartan treatment totally abolished these effects. Histological examination of kidneys revealed that simultaneous administration of fonsartan and losartan with L-NAME abolished L-NAME-induced arteriolar hyalinosis, segmental sclerosis of glomerular capillaries and focal tubular atrophies. In conclusion, long-term blockade of ANG II subtype AT(1) receptors by fonsartan and losartan prevented L-NAME-induced hypertension, renal insufficiency, as well as cardio-dynamic, cardio-metabolic, and morphological deteriorations.

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.

Endothelial NO synthase, being deficient in arginine and/or tetrahydrobiopterin, produces in addition to NO a significant concentration of superoxide (O2)(-)). We investigated whether such an imbalance between O2(-) and NO production is present in dysfunctional aortas of Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) with failing hearts after myocardial infarction. Heart failure was induced by permanent occlusion of the left coronary artery, resulting in a large infarction of the free left ventricular wall. Eight weeks after myocardial infarction, when WKY and SHR had compensated heart failure and congestive heart failure, respectively, calcium ionophore-induced NO release (assessed by a NO-sensitive microsensor) from aortic endothelial cells was significantly reduced from 478+/-48 to 216+/-16 nmol/L and 693+/-131 to 257+/-53 nmol/L in WKY and SHR, respectively. Concomitantly, significant increases in calcium ionophore-stimulated O2(-) production (assessed by an electrochemical sensor) could be observed in aortic endothelial cells from infarcted WKY rats (22+/-3.2 versus sham, 10.1+/-1.2 nmol/L) and SHR (102+/-8 versus sham, 67+/-5 nmol/L). A dramatic increase in endothelial peroxynitrite concentration (chemiluminescence method) from 35+/-4 to 90+/-3 nmol/L for WKY and from 60+/-5 to 170+/-10 nmol/L for SHR also was detected. Thus, the markedly decreased NO availability probably caused by impaired endothelial NO synthase activity with enhanced O2(-) and peroxynitrite production appears to be attributable to endothelial dysfunction in normotensive rats with chronic heart failure and especially in hypertensive rats with severe congestive heart failure.

-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 µmol/L, which was attenuated by NO synthase inhibition. [D-Ala(7)]Ang-(1-7) (5 µmol/L), described as a selective antagonist of Ang-(1-7) receptors, inhibited Ang-(1-7)-induced NO release only by approximately 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 µmol/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 µmol/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 (O(2)(-)) detected by an O(2)(-)-sensitive microsensor revealed that the moderately Ang-(1-7)-stimulated NO release was accompanied by a very slow concomitant O(2)(-) production with a relative low peak concentration in comparison to the O(2)(-) 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 O(2)(-).

In this study, we investigated the outcome of lifelong treatment with the angiotensin II type 1 receptor (AT(1)) blocker fonsartan (HR 720) in young stroke-prone spontaneously hypertensive rats (SHR-SP). In addition to the primary end point, lifespan, and to determine the mechanisms involved in the treatment-induced effects, parameters such as left ventricular hypertrophy, cardiac function/metabolism, endothelial function, and the expression/activity of endothelial nitric oxide synthase and of angiotensin-converting enzyme (ACE) were also investigated. Ninety 1-month-old SHR-SP were allotted to 2 groups and treated via drinking water with an antihypertensive dose of fonsartan (10 mg. kg(-1). d(-1)) or placebo. Fonsartan doubled the lifespan to 30 months in SHR-SP, which was comparable to the lifespan of normotensive Wistar-Kyoto rats. After 15 months, a time when approximately 80% of the placebo group had died, left ventricular hypertrophy was completely prevented in fonsartan-treated animals. Furthermore, cardiac function and metabolism as well as endothelial function were significantly improved. These effects were correlated with increased endothelial nitric oxide synthase expression in the heart and carotid artery and with markedly decreased tissue ACE expression/activities. Lifespan extension and cardiovascular protection by long-term AT(1) blockade with fonsartan led to similar beneficial effects, as observed with long-term ACE inhibition.

1 In this study we examined the endothelium-dependent effect of YC-1 - a benzyl indazole derivative which directly activates soluble guanylyl cyclase (sGC) - on vascular relaxation and nitric oxide (NO) and guanosine-3',5'-cyclic monophosphate (cyclic GMP) in endothelial cells. 2 In preconstricted rat aortic rings with intact endothelium, YC-1 produced a concentration-dependent relaxation. However, the concentration response curve was shifted rightward to higher concentrations of YC-1, when (i) the aortas were pre-treated with L-NG-nitroarginine methylester (L-NAME) or (ii) the endothelium was removed. 3 Incubation of bovine aortic endothelial cells (BAEC) with YC-1 produced a concentration-dependent NO synthesis and release as assessed using a porphyrinic microsensor. Pre-incubating cells with L-NAME or with 8-bromo-cyclic GMP decreased this effect indicating that the YC-1 stimulation of NO synthesis is due to an activation of nitric oxide synthase, but not to an elevation of cyclic GMP. No direct effect of YC-1 on recombinant endothelial constitutive NO synthase activity was observed. 4 The YC-1 stimulated NO release was reduced by 90%, when extracellular free calcium was diminished. 5 In human umbilical vein endothelial cells (HUVEC), YC-1 stimulated intracellular cyclic GMP production in a concentration- and time-dependent manner. Stimulation of cyclic GMP was greater with a maximum concentration of YC-1 compared to calcium ionophore A23187. Similar effects were observed in BAEC and rat microvascular coronary endothelial cells (RMCEC). 6 When HUVEC and RMCEC were pre-treated with L-NG-nitroarginine (L-NOARG), the maximum YC-1 stimulated cyclic GMP increase was reduced by >/=50%. 7 These results indicate, that beside being a direct activator of sGC, YC-1 stimulates a NO-synthesis and release in endothelial cells which is independent of elevation of cyclic GMP but strictly dependent on extracellular calcium. The underlying mechanism needs to be determined further.

The aim of the study was to investigate the influence of long-term ACE inhibition with ramipril on myocardial hypertrophy and its molecular background in spontaneously hypertensive stroke-prone rats (SHR-SP). Therefore, 1-month-old pre-hypertensive SHR-SP were randomized into three groups and exposed lifelong via drinking water to 1 mg/kg/day ramipril (anti-hypertensive dose, RHI), 10 micrograms/kg/day ramipril (non-anti-hypertensive dose, RLO) or placebo. After 15 months cardiac tissue was collected from ten rats each for immunohistochemistry and Northern blot analysis of structural proteins, proteins of the extracellular matrix and several growth factors. Results showed that RHI, but not RLO, treatment prevented development of myocyte hypertrophy (ANP). Furthermore, unlike placebo-treated rats, the ramipril-treated animals had no evidence of degeneration and loss of structural proteins (alpha -actinin), inflammatory infiltrates (CD45) and deposition of extracellular matrix proteins (collagen, fibronectin, vimentin). Only in RHI-treated animals, mRNA levels for TGF- beta(1)as well as of collagen alpha(1)(I) and fibronectin were downregulated compared to placebo-treated animals. In contrast, VEGF mRNA levels increased significantly in both groups of ramipril-treated animals v. placebo-treated SHR-SP. Thus, the reported life prolonging effect of high doses of ramipril which is associated with prevention of hypertension and hypertrophy is accompanied by prevention of the development of necrosis and fibrosis. The role of VEGF, however, seems to be independent of this effect., (Copyright 1999 Academic Press.)

Background: The effect of life-long treatment with the ACE inhibitor ramipril on hypertension-induced histological changes in the kidney was tested in stroke-prone spontaneously hypertensive rats (SHR-SP)., Methods: One-month-old pre-hypertensive SHR-SP were randomized into three groups of 45 animals each, and exposed via drinking water for their lifetime to a dose of: 1 mg.kg-1.d-1 ramipril (antihypertensive dose, HRA); 10 micrograms.kg-1.d-1 slight dose of ramipril (non-antihypertensive dose, LRA); or placebo. Histological and biochemical assessments were conducted after 15 months in ten rats each, when about 80% of the placebo group had died., Results: Kidneys from placebo treated SHR-SP showed pronounced arterial wall hypertrophy and sclerosis, arterial fibrinoid necrosis, glomerulopathy and tubular interstitial injury that were, in concert with normalized blood pressure, completely prevented by HRA treatment. LRA treatment did not affect any blood pressure increase, and also attenuated the development of arterial wall hypertrophy, sclerosis and arterial fibrinoid necrosis, though to a minor extent only, but did not change glomerular and tubulointerstitial degeneration. These effects of ramipril were associated with a dose-dependent inhibition of plasma and renal tissue ACE activities as well as lower serum concentrations of creatinine, but there were no changes in serum potassium., Conclusions: Life-long HRA-induced ACE inhibition protects against hypertension-induced renal damages in SHR-SP. This is associated with a doubling of the lifespan in these animals.

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.

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.

Young (approximately 1 month old) male normotensive Wistar-Kyoto rats (n=26) and spontaneously hypertensive rats (n=38) were randomized into three groups treated via drinking water for approximately 2 years with, respectively, placebo, low doses, or high doses of an angiotensin-converting enzyme inhibitor, ramipril (10 microg x kg[-1] x d[-1], non-blood pressure-lowering dose, or 1 mg x kg[-1] x d[-1], blood pressure-lowering dose). Relative to placebo treatment in each respective rat strain, both ramipril dosages increased endothelial constitutive nitric oxide synthase expression (Western blot) and resultant synthesis of nitric oxide (porphyrinic sensor) in freshly excised carotids and thoracic aortas, respectively. Paradoxically, this activity was associated with an increased/decreased superoxide accumulation (chemiluminescence) in freshly excised aortas from 24-/22-month-old normotensive/hypertensive rats. In normotensive rats, relative to placebo treatment, the threefold increase in superoxide accumulation with antihypertensive ramipril treatment is most likely from the >300% increase in endothelial constitutive nitric oxide synthase expression (some of which may be disarranged by local insufficiencies in L-arginine or tetrahydrobiopterin). In hypertensive rats, relative to placebo treatment, the 35% increase in nitric oxide availability by long-term antihypertensive ramipril treatment may contribute to the preservation of the endothelium and prevent its dysfunction by inhibiting superoxide production. Increased nitric oxide production with concomitant decreased superoxide accumulation (approximately one third of placebo levels) correlates positively with the previously reported +40% life span extension for rats with genetic hypertension that were treated with antihypertensive doses of ramipril.

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.

There is growing evidence for a local kallikrein-kinin system in the heart. In the ischemic heart the enhanced generation and release of kinins seem to have cardioprotective actions. In isolated rat hearts with ischemia-reperfusion injuries, perfusion with bradykinin reduces the duration and incidence of ventricular fibrillations, improves cardiodynamics, reduces release of cytosolic enzymes, and preserves energy-rich phosphates and glycogen stores. In anesthetized animals, intracoronary infusion of bradykinin is followed by comparable beneficial changes and limits infarct size. Inhibition of breakdown of bradykinin and related peptides induces similar beneficial cardiac effects. Treatment with angiotensin-converting enzyme (ACE) inhibitors such as ramipril increases cardiac kinins and reduces postischemic reperfusion injuries in isolated rat hearts as well as infarct size and remodeling in postinfarcted animals, respectively. Blockade of B2 kinin receptors increases ischemia-induced effects. In isolated hearts, ischemia-reperfusion injuries intensify with the B2 kinin receptor antagonist icatibant, which also abolishes the cardioprotective effects of ACE inhibitors and of exogenous bradykinin. Infarct size reduction by ACE inhibitors and bradykinin in anesthetized animals is reversed by icatibant. Kinins contribute to the cardioprotective effects associated with ischemic preconditioning. Preconditioning or bradykinin-induced antiarrhythmic and infarct size-limiting effects are attenuated by icatibant.

During the past decade it was recognised that kinins are potent endogenous vasoactive peptides with important cardioprotective actions which are of therapeutic relevance in the acute and chronic beneficial effects of ACE inhibitors. The effect of kinins is mediated through the release of autocoids from the endothelium, particularly nitric oxide (NO). In this review article the cardioprotective effects of kinins and their importance for the therapeutic effects of ACE inhibitors are highlighted.

In bovine aortic endothelial cells (BAECs), we previously demonstrated B1 and B2 kinin receptor-mediated increases in intracellular guanosine-3',5'-cyclic monophosphate (cGMP). In this study, the B2 kinin receptor agonist bradykinin increased cGMP in rat microvascular coronary endothelial cells (RMCECs) and human umbilical vein endothelial cells (HUVECs), which could be prevented with the specific B2 kinin receptor antagonist icatibant but not with the B1 kinin receptor antagonist des-Arg9-[Leu8]bradykinin or with the nonpeptide kinin receptor antagonist WIN 64338. B2 kinin receptor mRNA could be detected in all three cell types using reverse transcription-polymerase chain reaction and subsequent Southern blotting. The B1 kinin receptor agonist des-Arg9-bradykinin increased cGMP in RMCECs but not in HUVECs. The response in RMCECs could be prevented by des-Arg9-[Leu8]bradykinin as well as by WIN 64338 but not by icatibant. In BAECs, the B1 kinin receptor-mediated cGMP synthesis could be prevented by icatibant and desensitized by preincubation with des-Arg9-bradykinin as well as bradykinin. We detected B1 kinin receptor mRNA in RMCECs and HUVECs but not in BAECs. In HUVECs, the detection of B1 kinin receptor mRNA is in contradiction to the cGMP measurements. In BAECs, the atypical B1 kinin receptor pharmacology, the heterologous desensitization of the receptor and the failure to detect B1 kinin receptor mRNA cannot be explained by a typical B1 kinin receptor subtype. Thus, B2 kinin receptors with similar pharmacology are constitutively expressed in each of the three endothelial cell types. However, the endothelial cell types are heterogeneous in the expression of typical B1 kinin receptors and the pharmacology of the B1 kinin receptor-mediated responses.

In previous studies in stroke-prone spontaneously hypertensive rats (SHRSP), we demonstrated that early-onset, long-term angiotensin-converting enzyme inhibitor treatment improved cardiac function and metabolism and increased aortic cGMP content even at sub-antihypertensive doses. These effects could be prevented by bradykinin type 2 (B2) receptor blockade with icatibant. In the present study, we studied the effects of long-term oral treatment with the angiotensin type 1 (AT1) receptor antagonist losartan (30 mg/kg per day) on functional and biochemical parameters of the heart and on cGMP content in the aorta in SHRSP treated prenatally and subsequently up to the age of 20 weeks. Losartan prevented the development of hypertension and left ventricular hypertrophy. Cardiac function measured ex vivo in isolated perfused hearts was improved, as demonstrated by significant increases in left ventricular pressure (22.4%), differentiated left ventricular pressure (dP/dtmax) (35.1%), and coronary flow (38%). The release of the intracellular enzymes lactate dehydrogenase and creatine kinase and of lactate into the coronary effluent was reduced by 46.4%, 47.2%, and 63.6%, respectively. In myocardial tissue, the concentrations of glycogen and the energy-rich phosphates ATP and creatine phosphate were increased by 43.2%, 33.1%, and 42.4%, respectively, whereas lactate was decreased by 57.0%. The aortic tissue content of cGMP was increased fivefold. Our results demonstrate that chronic blockade of AT1 receptors with losartan improved cardiac function and metabolism and increased aortic cGMP content in SHRSP to an extent similar to that observed previously after long-term angiotensin-converting enzyme inhibitor treatment at a comparably antihypertensive dose. Prevention of hypertension and cardiac hypertrophy as well as stimulation of non-AT1 receptors are discussed to explain the cardiac and vascular actions of losartan.

Unlabelled: In male Sprague Dawley rats with left ventricular hypertrophy (LVH) and hypertension induced by aortic constriction (AC) and subsequent myocardial infarction (MI) by occlusion of the left coronary artery the effects of ACE inhibition with ramipril (RA 1 mg/kg/day via the drinking water during 6 weeks) on survival as well as cardiac function and metabolism were investigated. Respective groups (sham AC; AC; AC + sham MI; normotensive animals with sham MI; MI; MI + RA) served as comparisons. Following MI hypertensive rats with AC and LVH revealed an increased postoperative mortality (68%) when compared to normotensives without AC (28%). ACE inhibition with ramipril significantly reduced mortality in hypertensive rats by 26%. Untreated hypertensive animals with LVH clearly showed reduced MI size (6.2 +/- 2.3%) in comparison with untreated normotensive animals and MI (31.0 +/- 3.3%). In hypertensive rats with MI which died during the study a significant increase in infarct size was found compared to those which survived MI. In normotensive animals ramipril reduced infarct size by 50%. Due to the quite small infarct size observed in hypertensive rats, ACE inhibition did not further reduce MI in these animals. LVH as well as hydroxyproline/proline ratio was diminished by ACE inhibitor treatment. In the isolated hearts of ramipril treated rats contractility was improved when compared to the respective untreated groups with MI. In the coronary effluent of isolated hearts from rats with AC and MI lactate dehydrogenase and creatine kinase activities as well as lactate levels were increased. Ramipril treatment starting one week before MI normalized these parameters and in addition increased prostacyclin output. Hearts with MI from treated normotensive animals contained increased energy rich phosphates when compared to hearts from untreated rats with MI., Conclusions: Hypertensive rats with LVH undergoing MI experience increased postoperative mortality probably due to a reduced tolerance to myocardial ischemia and occurrence of arrhythmias. In these animals ACE inhibition with ramipril increased survival. Both, increased survival in hypertensive and reduction in infarct size in normotensive rats by ACE inhibition with ramipril was accompanied by an improved myocardial metabolism.

The present study examined whether alterations of endothelium-dependent vasorelaxation in spontaneously hypertensive rats (SHR) in response to the endothelium-dependent vasodilators acetylcholine and bradykinin ran parallel. We tried to find out the age at which endothelium-dependent vasorelaxation in response to each agonist became impaired and compared three different groups of SHR aged 7, 21 and 51 weeks. To be able to separate hypertension-induced alterations from age-dependent changes age-matched normotensive Wistar rats were included. Endothelium-dependent vasorelaxation was studied in the mesenteric vascular bed precontracted with noradrenaline, a typical resistance vessel, which showed relaxation to both acetylcholine and bradykinin, and the precontracted thoracic aorta, which only responded to acetylcholine. There were major differences in the agonist-dependent vasorelaxation between bradykinin and acetylcholine in SHR as a function of age. A surprising finding was that acetylcholine-induced relaxation was preserved, even slightly improved not only in young SHR (7 weeks) with developing hypertension but also in adult SHR (21 weeks) with established hypertension, which can be interpreted as a compensatory mechanism. As expected, in old SHR (51 weeks) acetylcholine-induced vasorelaxation was impaired as a consequence of the detrimental effects of long-term hypertension on endothelium. The parallel changes observed with acetylcholine in the mesenteric vascular bed and thoracic aorta provided mutual confirmation. In clear contrast to acetylcholine bradykinin-induced vasorelaxation was already imparied in young SHR with developing hypertension suggesting that bradykinin-induced vasorelaxation is either much more sensitive to detrimental effects of (even slightly) increased blood pressure or, more likely, that there is a basic deficiency in the action of bradykinin in SHR. Thus, our study allows to conclude that impairment of acetylcholine-induced endothelium-dependent vasorelaxation in the mesenteric vascular bed of SHR is a secondary phenomenon developing as a consequence of long-term hypertension while the impaired bradykinin-induced vasorelaxation seems to be a primary phenomenon that could be closely related to the development of hypertension.

From pharmacological investigations and clinical studies, it is known that angiotensin converting enzyme (ACE) inhibitors exhibit additional local actions, which are not related to hemodynamic changes and which cannot be explained only by interference with the renin angiotensin system (RAS) by means of an inhibition of angiotensin II (ANG II) formation. Since ACE is identical to kininase II, which inactivates the nonapeptide bradykinin (BK) and related kinins, potentiation of kinins might be responsible for these additional effects of ACE inhibitors. a) In rats made hypertensive by aortic banding, the effect of ramipril in left ventricular hypertrophy (LVH) was investigated. Ramipril in the antihypertensive dose of 1 mg/kg/day for 6 weeks prevented the increase in blood pressure and the development of LVH. The low dose of ramipril (10 micrograms/kg/day for 6 weeks) had no effect on the increase in blood pressure or on plasma ACE activity but also prevented LVH after aortic banding. The antihypertrophic effect of the higher and lower doses of ramipril, as well as the antihypertensive action of the higher dose of ramipril, was abolished by coadministration of the kinin receptor antagonist icatibant. In the regression study the antihypertrophic actions of ramipril were not blocked by the kinin receptor antagonist. Chronic administration of BK had similar beneficial effects in a prevention study which were abolished by icatibant and NG-nitro-L-arginine (L-NNA). In a one year study the high and low dose of ramipril prevented LVH and fibrosis. Ramipril had an early direct effect in hypertensive rats on the mRNA expression for myocardial collagen I and III, unrelated to its blood pressure lowering effect.(ABSTRACT TRUNCATED AT 250 WORDS)

WIN 64338 (phosphonium, [[4-[[2-[[bis(cyclohexylamino) methylene] amino]-3-(2-naphthalenyl) 1-oxopropyl]amino]-phenyl]- tributyl, chloride, monohydrochloride) is the first potent nonpeptide competitive bradykinin B2 receptor antagonist as shown in classical pharmacological preparations with no activity in the rabbit aorta stimulated by the bradykinin B1 receptor agonist des-Arg9-bradykinin. In primary cultured bovine aortic endothelial cells, both bradykinin and des-Arg9-bradykinin stimulate the production of intracellular cyclic GMP, an index for the production of nitric oxide. Surprisingly, WIN 64338 did not inhibit bradykinin but abolished the effect of des-Arg9-bradykinin suggesting that kinin receptor antagonists do not necessarily discriminate between kinin receptor subtypes in an identical way in different tissues and species.

To delineate the cardioprotective actions of bradykinin (BK) and the contribution of endogenous kinins to the cardiac effects of the ACE inhibitor ramipril, we used the specific B2 kinin receptor antagonist icatibant (HOE 140) during myocardial ischemia and left ventricular hypertrophy (LVH). In isolated working rat hearts, perfusion with ramiprilat (10 nM to 10 microM) reduced the incidence and duration of ventricular fibrillation, and improved cardiodynamics and myocardial metabolism. BK perfusion (0.1 nM to 10 nM) induced comparable cardioprotective effects. In addition, perfusion with ramiprilat (0.1 microM) markedly increased kinin outflow measured by RIA. The beneficial effects of ramiprilat and BK were abolished by the addition of the specific NO synthase inhibitor NG-nitro-L-arginine (L-NNA 1 microM) or icatibant (1 nM). Similar results were obtained in dogs, rabbits and rats with myocardial infarction induced by ligation of the left descending coronary artery. The influence of the icatibant on the antihypertrophic effect of ramipril and BK in the LVH was investigated in rats made hypertensive by aortic banding. Ramipril at the antihypertensive dose of 1 mg kg-1 day-1 for 6 weeks prevented the increase in blood pressure and the development of LVH. The lower non-antihypertensive dose of ramipril (10 micrograms kg-1 day-1 for 6 weeks) had no effect on the increase in blood pressure or on plasma ACE activity but also prevented LVH after aortic banding. The antihypertrophic effect of the higher and the lower dose of ramipril as well as the antihypertensive action of the higher dose of ramipril were abolished by coadministration of the icatibant.(ABSTRACT TRUNCATED AT 250 WORDS)

ACE inhibitors induce an increase in kinin levels with subsequent release of nitric oxide (NO) and prostacyclin, as shown in cultured endothelial cells and isolated rat hearts. Isolated perfused working rat hearts continuously release kinins and prostacyclin. During ischemia after ligation of the left coronary artery kinin and prostacyclin concentrations in the venous effluent of the hearts are increased. ACE inhibition with ramiprilat increases kinin concentrations during normoxia, ischemia and reperfusion, whereas deendothelialization markedly reduces kinin and prostacyclin outflow in controls as well as in ACE inhibitor-treated hearts. Rat hearts with postischemic reperfusion arrhythmias are protected by ramiprilat- and bradykinin perfusion, cardiodynamics and metabolism of treated hearts are improved. These effects are observed in concentrations too low to increase coronary flow. The cardioprotective effects of ramiprilat and bradykinin are abolished by the specific B2-kinin receptor antagonist icatibant and by an inhibitor of NO-synthase. Long-term treatment (20 weeks) with ramipril in a blood-pressure-lowering dose (1 mg/kg/day) and a subantihypertensive dose (10 micromg/kg/day) protects spontaneously hypertensive rats (stroke prone) against hypertension and left ventricular hypertrophy in the high dose. In addition, both treatment regimens induce myocardial capillary growth. Isolated hearts of these animals show increased myocardial contractility and coronary flow, reduced release of cytosolic enzymes into the coronary effluent, and improved myocardial metabolism. These changes are observed even at a dose of ramipril which does not affect blood pressure and left ventricular hypertrophy. They are abolished by chronic blockade of kinin receptors with icatibant.(ABSTRACT TRUNCATED AT 250 WORDS)

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. The presents data show that bradykinin is continuously formed in the rat isolated heart. Ischaemia increases bradykinin outflow from the heart. Presumably by inhibiting degradation of kinins, ACE inhibition significantly increased the bradykinin concentration during normoxia, ischaemia and reperfusion.

From pharmacologic investigations and clinical studies it is known that angiotensin-converting enzyme (ACE) inhibitors exhibit additional local actions, which are not related to hemodynamic changes and which cannot be explained simply by interference with the renin-angiotensin system with subsequent inhibition of angiotensin II formation. Because ACE is identical to kininase II, which inactivates the nonapeptide bradykinin (BK), potentiation of BK might be responsible for these additional effects of ACE inhibitors. To prove the specificity of BK-mediated effects by ACE inhibition, we used the specific B2 kinin receptor antagonist HOE 140 in different models: endothelial cell cultures; atherosclerosis in high-cholesterol-fed rabbits; neointima formation with smooth cell proliferation and migration after endothelial denudation in rats; myocardial ischemia in rats, rabbits, and dogs; and left ventricular hypertrophy in rats. The beneficial effects of ramipril or BK given in non-blood pressure-lowering doses in these models were abolished by HOE 140 (icatibant). Ramipril exerts cardioprotective effects in different experimental models. The formation of the endothelial autacoids nitric oxide and prostacyclin, enhanced when BK degradation is inhibited by ACE inhibition, may contribute to the observed beneficial effects.

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 micrograms kg-1 daily (RA 10 micrograms) 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 micrograms 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.