Bernward Scholkens

Protective effects of HOE642, a selective sodium-hydrogen exchange subtype 1 inhibitor, on cardiac ischaemia and reperfusion

OBJECTIVE The aim was to characterise the new compound HOE642 as a selective and cardioprotective Na+/H+ exchange inhibitor in various models. METHODS The effect of HOE642 was tested in the osmotically activated Na+/H+ exchange of rabbit erythrocytes and in propionate induced swelling of human thrombocytes. Recovery of pH after an NH4Cl prepulse and effects on other ion transport systems by patch clamp technique were investigated in rat cardiomyocytes. NHE subtype specifity of the compound was determined by 22Na+ uptake inhibition in a fibroblast cell line separately expressing subtype isoforms 1-3. Protective effects of HOE642 in cardiac ischaemia and reperfusion by ligation of coronary artery were investigated in isolated working rat hearts and in anaesthetised rats. RESULTS HOE642 concentration dependently inhibited the amiloride sensitive sodium influx in rabbit erythrocytes, reduced the swelling of human platelets induced by intracellular acidification, and delayed pH recovery in rat cardiomyocytes. In the isolated working rat heart subjected to ischaemia and reperfusion HOE642 dose dependently reduced the incidence and the duration of reperfusion arrhythmias. It also reduced the the release of lactate dehydrogenase and creatine kinase, and preserved the tissue content of glycogen, ATP, and creatine phosphate. In anaesthetised rats undergoing coronary artery ligation intravenous and oral pretreatment with HOE642 caused a dose dependent reduction or a complete prevention of ventricular premature beats, ventricular tachycardia, and ventricular fibrillation. The compound was well tolerated and neutral to circulatory variables. Other cardiovascular agents tested in this model were not, or were only partly, effective at doses showing marked cardiodepressive effects. CONCLUSIONS HOE642 is a very selective NHE subtype 1 inhibitor showing cardioprotective and antiarrhythmic effects in ischaemic and reperfused hearts. Further development of well tolerated compounds like HOE642 could lead to a new therapeutic approach in clinical indications related to cardiac ischaemia and reperfusion.

A specific B2-bradykinin receptor antagonist HOE 140 abolishes the antihypertrophic effect of ramipril

To evaluate the role of bradykinin in the antihypertrophic effect of the angiotensin‐converting enzyme (ACE) inhibitor, ramipril, we investigated the influence of HOE 140, a specific B2‐receptor antagonist, on the effects of ramipril on left ventricular hypertrophy (LVH) in rats with aortic banding. Ramipril at a dose of 1 mg kg−1 day−1 for 6 weeks prevented the increase in blood pressure and development of LVH after aortic banding; plasma ACE activity was significantly inhibited. A lower dose of ramipril (10 μg kg−1 May−1 for 6 weeks) had no effect on the increase in blood pressure or on plasma ACE activity, but prevented LVH after aortic banding. The antihypertrophic effects of the higher and the lower dose ramipril, as well as the antihypertensive action of the higher dose of ramipril were abolished by the coadministration of HOE 140 (500 μg kg−1 day−1). The present data show for the first time that the beneficial effects of an ACE‐inhibitor on LVH in rats with hypertension caused by aortic banding can be prevented by a specific B2‐receptor antagonist.

Reduction of infarct size by local angiotensin-converting enzyme inhibition is abolished by a bradykinin antagonist.

We investigated the role of local (cardiac) bradykinin in the infarct-limiting effect of the ACE inhibitor, ramiprilat, by using the novel bradykinin antagonist, HOE 140

Hoe 694, a new Na+/H+ exchange inhibitor and its effects in cardiac ischaemia

1 The benzoylguanidine derivative Hoe 694 ((3‐methylsulphonyl‐4‐piperidino‐benzoyl) guanidine methanesulphonate) was characterized as an inhibitor of Na+/H+ exchange in rabbit erythrocytes, rat platelets and bovine endothelial cells. The potency of the compound was slightly lower or comparable to ethylisopropyl amiloride (EIPA). 2 To investigate a possible cardioprotective role of the Na+/H+ exchange inhibitor Hoe 694, rat isolated working hearts were subjected to ischaemia and reperfusion. In these experiments all untreated hearts suffered ventricular fibrillation on reperfusion. Addition of 10−7 m Hoe 694 to the perfusate almost abolished reperfusion arrhythmias in the rat isolated working hearts. 3 Hoe 694 reduced the release of lactate dehydrogenase (LDH) and creatine kinase (CK), which are indicators of cellular damage during ischaemia, into the venous effluent of the hearts by 60% and 54%, respectively. 4 The tissue content of glycogen at the end of the experiments was increased by 60% and the high energy phosphates ATP and creatine phosphate were increased by 240% and 270% respectively in the treated hearts as compared to control hearts. 5 Antiischaemic effects of the Na+/H+ exchange inhibitor, Hoe 694, were investigated in a second experiment in anaesthetized rats undergoing coronary artery ligation. In these animals, pretreatment with Hoe 694 caused a dose‐dependent reduction of ventricular premature beats and ventricular tachycardia as well as a complete suppression of ventricular fibrillation down to doses of 0.1 mg kg−1, i.v. Blood pressure and heart rate remained unchanged. 6 We conclude that the new Na+/H+ exchange inhibitor, Hoe 694, shows cardioprotective and antiarrhythmic effects in ischaemia and reperfusion in rat isolated hearts and in anaesthetized rats. In view of the role which Na+/H+ exchange seems to play in the pathophysiology of cardiac ischaemia these effects could probably be attributed to Na+/H+ exchange inhibition.

Beneficial Effects of the Converting Enzyme Inhibitor, Ramipril, in Ischemic Rat Hearts

Components of the renin-angiotensin system (RAS) have been found in heart tissue and it is likely that angiotensin II (ANG II) is generated locally in the heart as in other organs. Pharmacological interference with converting enzyme (CE) inhibitors reduced CE activity and ANG II generation in the heart. To investigate whether local inhibition of CE in the heart with the CE inhibitor ramipril might contribute to the therapeutic effects, experiments were performed in isolated perfused working rat hearts. Acute regional myocardial ischemia was induced by occlusion of the left coronary artery followed by reperfusion. In ischemic isolated rat hearts, both single oral pretreatment with ramipril (1 mg/kg) or perfusion with the active moiety, ramiprilat (10 micrograms/ml), protected against ventricular fibrillation, which invariably occurred in control hearts during reperfusion. Reperfusion arrhythmias were aggravated by perfusion with ANG I and ANG II, but prevented by bradykinin. ANG I-enhanced ventricular fibrillations were completely eliminated during local CE inhibition with ramipril. The CE inhibitor also improved cardiodynamics. Coronary flow, left ventricular pressure, dp/dtmax, and myocardial oxygen consumption were increased in comparison to controls without changes in heart rate. In the perfusate of treated hearts, lactate dehydrogenase, and creatine kinase activities and lactate production, were reduced. Myocardial tissue levels of glycogen, ATP, and creatine phosphate were increased in ramipril-pretreated hearts whereas lactate was decreased. The results of these experiments in rat hearts suggest that local inhibition of CE by ramipril exerts protective effects after ischemia and reperfusion by reducing arrhythmias and improving cardiac function and metabolism, thus probably contributing to the therapeutic effects of CE inhibitors in cardiovascular diseases.

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.

Persistent tissue converting enzyme inhibition following chronic treatment with Hoe498 and MK421 in spontaneously hypertensive rats.

Inhibition of angiotensin II generation in the plasma does not fully explain the antihypertensive effects of converting enzyme (CE) inhibitors. Thus, we investigated the role of CE inhibition in the tissue for the antihypertensive action of these drugs. After discontinuation of 4 weeks of oral treatment with either Hoe498 (3 mg/kg/day) or MK.421 (30 mg/kg/day) in spontaneously hypertensive rats (SHRSP), the reduced pressor response to intravenous angiotensin 1 was normalized within 1 day. although systolic and diastolic blood pressure remained decreased during a 1 week post-treatment follow-up period. Two weeks of oral treatment with Hoe498 (1 mg/kg day) and MK421 (30 mg/kg/day) in SHRSP lowered blood pressure markedly and inhibited CE in the plasma (43% and 22%). lung (85% and 33%). kidney (76% and 76%). aortic wall (75% and 48%). and (with Hoe498) in the heart (55%). After drug discontinuation, blood pressure remained decreased for an additional 2 weeks, whereas plasma CE was normal or elevated during the follow-up period. However, tissue CE activity measured 1 week after drug withdrawal was still inhibited in the aortic wall (67% and 30%) and in the kidney (48% and 41%). These results support the hypothesis that the prolonged antihypertensive action of CE inhibitors may be related to persistent CE inhibition in tissues such as vascular wall and kidney. Further, the data support the importance of CE inhibition at target sites other than plasma and lung vascular endothelium.

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.

Brain converting enzyme inhibition: A possible mechanism for the antihypertensive action of captopril in spontaneously hypertensive rats

The effects of various doses of the converting enzyme inhibitor captopril injected into the lateral brain ventricle (i.c.v.) and intravenously (i.v.) on blood pressure (BP) and on converting enzyme activity were tested in stroke prone conscious spontaneously hypertensive rats (SHR-sp) and in normotensive Wistar Kyoto rats (WKY). Injection of 500 micrograms captopril i.c.v. produced a marked biphasic BP effect in SHR-sp, an initial increase followed by a long-lasting decrease. Only the initial BP increase was observed in WKY. The pressor responses to i.c.v. angiotensin I (ANG I) were completely blocked after i.c.v. captopril injection and this effect lasted for 24 h. The pressor responses to i.v. ANG I were also inhibited immediately after 500 micrograms captopril i.c.v. and gradually returned to control values within 5 h. Intravenous injections of 500 micrograms captopril almost completely inhibited the pressor responses to i.v. ANG I; they caused a moderate BP decrease in SHR-sp and had no significant BP effects in WKY. In SHR-sp, 5 micrograms captopril i.c.v. caused a reduction of BP with a concomitant inhibition of the pressor effects of i.c.v. ANG I. Both effects lasted about 30 min. The pressor responses to i.v. ANG I were not inhibited. In WKY, 5 micrograms captopril i.c.v. had no effect on BP. It is concluded that captopril can reduce BP by action on the brain without peripheral inhibition of converting enzymes. Following high doses injected i.c.v., the inhibitor leaks into the periphery but this cannot explain the marked hypotensive effect in SHR-sp.