Bernward A. Schölkens

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.

Long-Term Angiotensin II Type 1 Receptor Blockade With Fonsartan Doubles Lifespan of Hypertensive Rats

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.

The K(ATP) channel blocker HMR 1883 does not abolish the benefit of ischemic preconditioning on myocardial infarct mass in anesthetized rabbits.

Abstract. Previous experimental studies showed that the benefit of ischemic preconditioning (IPC) is abolished by KATP channel blockade with glibenclamide. However, the newly discovered KATP channel blocker HMR 1883 (1-[[5-[2-(5-chloro-o-anisamido)ethyl]-methoxyphenyl]sulfonyl]-3-methylthiourea) shows marked antifibrillatory activity in the dose range of 3xa0mg/kg to 10xa0mg/kg i.v. in various experimental models without affecting blood glucose levels. In order to investigate in a head to head comparison glibenclamide and HMR 1883 with respect to their influence on IPC, experiments were performed in rabbits with ischemia-reperfusion using myocardial infarct mass as final read out.Male New Zealand White rabbits (2.6–3.0xa0kg) were subjected to 30-min occlusion of a branch of the left descending coronary artery (LAD) followed by 2-h reperfusion. For IPC experiments the LAD was additionally occluded for two periods of 5xa0min, each followed by 10-min reperfusion, before the long-term ischemia. Infarct mass was evaluated by TTC staining and expressed as a percentage of area at risk. Rabbits (n=7/group) were randomly selected to receive (i.v.) saline vehicle 5xa0min prior to the 30-min occlusion period in infarct studies without IPC or to receive glibenclamide (0.3xa0mg/kg) or HMR 1883 (3xa0mg/kg) in IPC experiments, these substances being given 5xa0min prior to the first preconditioning or 5xa0min prior to the long-term ischemia of 30xa0min. Myocardial risk mass as a percentage of left ventricular mass did not differ between groups. The same was true for the ratio of left ventricular mass to 100xa0g body weight. Myocardial infarct mass as a percentage of the area at risk in the saline vehicle group without IPC was 41±3%. Whereas glibenclamide significantly increased infarct mass (from 41±3% to 55±4%), HMR 1883 did not affect it. IPC reduced infarct mass from 41±3% to 21±4% (P<0.05 vs. control without IPC). Glibenclamide given prior to IPC or prior to the long-term ischemia totally abolished the IPC effect (42±2% and 55±4%, respectively; P<0.05 vs. control). In contrast, HMR 1883 under the same conditions did not affect infarct size when given prior to IPC or prior to the long-term ischemia (21±3% and 26±2%, respectively). The monophasic action potential duration (MAP50) was reduced from 103±3xa0ms under normoxic conditions to 82±2xa0ms, 5xa0min after ischemia in the absence of drugs. This ischemia-induced shortening of the MAP was prevented by both HMR 1883 (MAP50 103±3xa0ms) and glibenclamide (MAP50 106±3xa0ms).In conclusion, although both KATP channel blockers prevented ischemia-induced shortening of MAP, HMR 1883 did not abolish the beneficial effects of IPC on myocardial infarct mass in rabbits, whereas glibenclamide totally reversed this cardioprotective effect of IPC. This suggests that the sarcolemmal ATP-sensitive potassium channels are not involved in the mechanism of IPC.

K(ATP) channel blocker HMR 1883 reduces monophasic action potential shortening during coronary ischemia in anesthetised pigs.

Abstract. ATP-sensitive potassium channels (KATP) open during myocardial ischemia. The ensuing repolarising potassium efflux shortens the action potential. Accumulation of extracellular potassium is able to partially depolarise the membrane, reducing the upstroke velocity of the action potential and thereby impairing impulse conduction. Both mechanisms are believed to be involved in the development of reentrant arrhythmias during cardiac ischemia. The sulfonylthiourea HMR 1883 (1-[[5-[2-(5-chloro-o-anisamido)ethyl]-methoxyphenyl]sulfonyl]-3-methylthiourea) was designed as a cardioselective KATP channel blocker for the prevention of arrhythmic sudden death in patients with ischemic heart disease. The aim of this study was to show that this compound, which has already shown antifibrillatory efficacy in dogs and rats, is able to inhibit ischemic changes of the action potential induced by coronary artery occlusion in anesthetised pigs. Action potentials were taken in situ with the technique of monophasic action potential (MAP) recording.In a control group (n=7), three consecutive occlusions of a small branch of the left circumflex coronary artery resulted in reproducible reductions in MAP duration and a decrease in upstroke velocity. In a separate group (n=7), HMR 1883 (3xa0mg/kg i.v.) significantly (P<0.05) reduced the ischemia-induced shortening of the MAP: during the first and second control occlusion of the coronary artery in the HMR 1883-group, MAP50 duration shortened from 218.5±3.0xa0ms to 166.7±3.3xa0ms and from 219.7±4.5xa0ms to 164.9±1.8xa0ms, respectively. After HMR 1883, during the third occlusion, MAP duration decreased from 226.9±3.6xa0ms to 205.3±4.3xa0ms only corresponding to 59% inhibition. HMR 1883 also improved the upstroke velocity of the MAP, which was depressed by ischemia: in the two preceding control occlusions ischemia prolonged the time to peak of the MAP, an index for upstroke velocity, from 10.83±0.43xa0ms to 39.42±1.60xa0ms and from 12.97±0.40xa0ms to 37.17±2.98xa0ms, respectively. With HMR 1883, time to peak during ischemia rose from 12.42±0.51xa0ms to 25.53±2.51xa0ms only, corresponding to an average inhibitory effect of 53.4%. The irregular repolarisation contour of the ischemic MAP was also improved.In conclusion, the present results indicate that HMR 1883 effectively blocks myocardial KATP channels during coronary ischemia in anesthetised pigs, preventing an excessive shortening of the action potential and improving excitation propagation.

HMR 1883, a cardioselective KATP channel blocker, inhibits ischaemia- and reperfusion-induced ventricular fibrillation in rats

Ventricular fibrillation (VF) is a major cause of sudden cardiac death in which myocardial ischemia plays a leading role. During ischaemia activation of ATP-sensitive potassium channels (KATP) occurs, leading to potassium efflux from cardiomyocytes and shortening of the action potential favoring the genesis of ventricular fibrillation. In confirmation of this concept the sulfonylurea glibenclamide, which stimulates insulin release by inhibition of pancreatic KATP channels, has been shown to inhibit VF in different models of ischaemia by inhibition of myocardial KATP channels.HMR 1883 (1-[[5-[2-(5-chloro-o-anisamido)ethyl]-methoxyphenyl]sulfonyl]-3-methylthiourea) was designed as a cardioselective KATP channel blocker. The aim of this study was to show that with this compound it is possible to separate the antifibrillatory from the insulin-releasing effect for the treatment of patients at risk of ischaemia-induced arrhythmias and sudden death. In the present study HMR 1883 reduced VF in Sprague-Dawley rats during prolonged ischaemia and also diminished mortality and the duration of VF in a separate reperfusion experiment at 3xa0mg/kg and 10xa0mg/kg with no effect on blood glucose or insulin. Glibenclamide, which was antifibrillatory at 0.3xa0mg/kg and 1xa0mg/kg, increased plasma insulin and lowered blood glucose already at a dose as low as 0.01xa0mg/kg.In conclusion, based on its antifibrillatory action and the absence of significant pancreatic effects at therapeutic doses, HMR 1883 is of potential clinical utility for the prevention of severe arrhythmias in patients with ischaemic heart disease.

Crosstalk between ACE inhibitors, B2 kinin receptor and nitric oxide in endothelial cells

The endothelium is an important target organ of angiotensin converting enzyme (ACE) inhibitors. Within these monolayered cells establishing the interface between blood and vasculature, a complex signal transduction machinery is the basis for a major role of ACE inhibition in the regulation of vascular homoestasis. This chapter will focus on the endothelial aspects of ACE-inhibition, on its interaction with components of the kallikrein-kinin system.