Beneficial Effects of Angiotensin I Converting Enzyme Inhibitor on Post-ischemic Contractile Function of Perfused Rat Heart

J Mol Cell Cardiol 28, 1659–1670 (1996)

Beneficial Effects of Angiotensin I Converting Enzyme Inhibitor on Post-ischemic Contractile Function of Perfused Rat Heart Kouichi Tanonaka, Toru Kamiyama, Aya Takezono, Kimiko Sakai and Satoshi Takeo Department of Pharmacology, Tokyo University of Pharmacy and Life Science, Hachioji, Japan (Received 13 December 1995, accepted in revised form 22 March 1996) K. T, T. K, A. T, K. S  S. T. Beneficial Effects of Angiotensin I Converting Enzyme Inhibitor on Post-ischemic Contractile Function of Perfused Rat Heart. Journal of Molecular and Cellular Cardiology (1996) 28, 1659–1670. The present study was undertaken to determine whether trandolaprilat, an active form of angiotensin I converting enzyme (ACE) inhibitor, may improve ischemia/reperfusion-induced contractile dysfunction and metabolic derangement of isolated rat hearts. Ischemia (25 min) and subsequent 60-min reperfusion resulted in a small recovery of post-ischemic left ventricular developed pressure (LVDP), a sustained increase in left ventricular end-diastolic pressure, an increase in the release of creatine kinase and ATP metabolites from the perfused heart, and changes in myocardial sodium, potassium, calcium and magnesium contents. Treatment with 10–100 l of trandolaprilat for the last 10 min of pre-ischemia recovered approximately 50–90% of pre-ischemic LVDP during reperfusion, whereas that with 30–100 l of enalaprilat restored approximately 55–65% of the pre-ischemic LVDP. Treatment with either trandolaprilat or enalaprilat at these concentrations attenuated the release of creatine kinase and ATP metabolites into the perfusate during reperfusion. Treatment with 30 l trandolaprilat suppressed ischemia/reperfusion-induced changes in myocardial ion content. Treatment with bradykinin during the last 10 min of pre-ischemia also resulted in a post-ischemic contractile recovery with a degree similar to that of the trandolaprilat-treated hearts. E4177, an AT1-antagonist, showed no effect on ischemia/reperfusion-induced changes in cardiac parameters. The enhancement of post-ischemic contractile recovery by the ACE inhibitor was abolished by treatment with either Hoechst 140, a bradykinin (BK2) antagonist, or diclofenac, a cyclooxygenase inhibitor. These results suggest that trandolaprilat is capable of attenuating ischemia/reperfusion injury of isolated perfused hearts and altered BK metabolism is, at least in part, involved in this effect.  1996 Academic Press Limited K W: Angiotensin converting enzyme inhibitor; Bradykinin; Enalaprilat; Ischemia/reperfusion; Trandolaprilat.

Introduction

has been extensively studied. Recently, pre-clinical and clinical studies have shown that ACE inhibitors are the most potent agents that have been shown to prolong life expectancy in patients and experimental animals with congestive heart failure (Peffer et al., 1985, 1987; SOLVD Investigators, 1991, 1992; Fornes et al., 1992; SAVE Investigators, 1992; TRACE Study Group, 1995), and in patients with

Angiotensin converting enzyme (ACE) inhibitors are known to be useful for treatment of hypertension in patients with renovascular or essential hypertension, and its pharmacological profile including the prevention of angiotensin II formation by inhibition of ACE (kininase II; E.C. 3.4.15.1) activity

Please address all correspondence to: Satoshi Takeo, Department of Pharmacology, Tokyo University of Pharmacy and Life Science, 1432-1 Horinouchi, Hachioji, Tokyo 192-03, Japan.

0022–2828/96/081659+12 $18.00/0

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acute myocardial infarction (AIRE Study Investigators, 1993; TRACE Study Group, 1995). Trandolapril is a new ACE inhibitor that has been extensively investigated in in vitro and in vivo animals and hypertensive patients (Mancia et al., 1992; Nguyen et al., 1992; Omboni et al., 1992). Trandolaprilat, an active form of trandolapril, has a high affinity for ACE that results in a slow dissociation and a long-lasting ACE inhibitory effect (Okunishi et al., 1992). Several investigators have shown that treatment with ACE inhibitors such as captopril and ramiprilat is beneficial for protection of ischemic/reperfused hearts and/or enhancement of post-ischemic contractile recovery (Linz et al., 1986; Li and Chen, 1987). The mechanism underlying the cardioprotective action of ACE inhibitors, however, is not fully understood (Zughaib et al., 1993). In addition, it is unknown whether cardioprotective effects of captopril and ramiprilat in ischemic/reperfused hearts are generalized for all ACE inhibitors or not. The present study was undertaken to determine whether trandolaprilat improves post-ischemic contractile function and metabolism of ischemic/reperfused hearts, and to determine possible mechanisms for cardioprotective effects of trandolaprilat.

Materials and Methods Ninety male Wistar rats, weighing 240–270 g, were used in the present study. The animals were conditioned at 23±1°C with a constant humidity of 55±5%, a cycle of 12 h light and 12 h dark, and freely accessed to food and tap water, according to the Guidelines of Experimental Animal Care issued from the Prime Minister’s Office of Japan.

Perfusion of the hearts The perfusion of isolated rat hearts was carried out according to the method described previously (Liu et al., 1993). The rats were anesthetized with ether. After thoracotomy, the hearts were rapidly isolated and transferred to a Langendorff apparatus. The hearts were perfused at 37°C with a constant flow of 9 ml/min by using a microtube pump (MP-3B, Tokyo Rikakiki, Tokyo) with the Krebs–Henseleit buffer of the following composition (m): NaCl 120, KCl 4.8, KH2PO4 1.2, MgSO4 1.2, CaCl2 1.25, NaHCO3 25 and glucose 11. The perfusion buffer was equilibrated with a gas mixture 95% O2/5% CO2. A latex balloon with an uninflated diameter

of 3.7 mm, connected to a pressure transducer (TP200T, Nihonkohden, Tokyo), was inserted into left ventricular cavity through the mitral opening, and secured with a ligature that included the left atrial remnants. A 5-mmHg of the initial left ventricular end-diastolic pressure (LVEDP) was loaded to the perfused heart. After equilibration for 15 min, the heart was paced at 300 beats/min and further equilibrated for 15 min. Left ventricular developed pressure (LVDP), a convenient maker of cardiac contractile function, was monitored by a pressure transducer connected to a carrier amplifier (AP621G, Nihonkohden) and recorded on a thermal pen recorder (WT-645G, Nihonkohden) throughout the experiment. After 30 min equilibration, the perfusion was stopped and the heart was submerged in an organ bath filled with Krebs–Henseleit buffer in which 11 m glucose was replaced with 11 m Tris/HCl. The buffer was previously equilibrated with a gas mixture of 95%/N2/5%/CO2, pH 7.4, and maintained at 37°C to avoid hypothermia-induced cardioprotection. After 25 min of ischemia, the buffer in the organ bath was drained and the hearts were reperfused for 60 min at 37°C with the Krebs– Henseleit buffer equilibrated with a gas mixture of 95% O2/5% CO2. The perfused hearts were paced throughout the experiment except for the first 15 min of reperfusion, to prevent contractile irregularities that might frequently occur during this period. For the purpose of comparison, hearts were perfused for 25 min under normoxic conditions, followed by 60 min of normoxic perfusion (normoxic group). Treatment of the perfused hearts with agents was carried out by infusing the appropriate concentrations of the agents into Krebs–Henseleit buffer for the last 10 or 12 min of pre-ischemia and/or the first 10 min of reperfusion. The agents dissolved in perfusion buffer were infused through an injection port just proximal to the aortic cannula at a flow rate of 200 ll/min by means of infusionpump (STC-523, Terumo Co., Tokyo). In the present study, the following perfusion protocol was performed (Fig. 1); group 1, hearts perfused under ischemic/reperfused conditions described above without any agent (negative control group); group 2, hearts perfused with 10 to 100 l trandolaprilat during the last 10 min of pre-ischemia (Tra); group 3, hearts perfused with 10 to 100 l enalaprilat during the last 10 min of preischemia (Ena); group 4, hearts perfused with either 10 or 30 l E4177, an AT1-antagonist, for the last 10 min of pre-ischemia (E4177); group 5, hearts perfused with 1–10 l bradykinin during the last

ACE Inhibitor and Ischemia/reperfusion Injury

Figure 1 The experimental protocol in the ischemic/ reperfused heart of group 1 (treatment without any agent; negative control), group 2 (trandolaprilat), group 3 (enalaprilat), group 4 (E4177, an AT1-antagonist), group 5 (bradykinin), group 6 (trandolaprilat plus Hoe 140, a BK2-antagonist), group 7 (trandolaprilat plus diclofenac, a cyclooxygenase inhibitor). Open columns with the agents indicate periods of treatment with them. Abbreviations: Tra=trandolaprilat, Ena=enalaprilat, BK=bradykinin, Hoe 140=Hoechst 140, Dic=diclofenac sodium.

10 min of pre-ischemia (BK); group 6, hearts perfused with trandolaprilat for the last 10 min of pre-ischemia and 3 l of Hoechst 140, a BK2antagonist, for the last 12 min of pre-ischemia and the first 10 min of reperfusion (Tra plus Hoe 140); group 7, hearts perfused with 30 l of trandolaprilat for the last 10 min of pre-ischemia and 30 l of diclofenac for the last 12 min of preischemia (Tra plus Dic). The numbers of experiments in each group are shown in the legends for figures. Measurement of myocardial ion content After determination of hemodynamic parameters, the perfusion of isolated hearts treated with and without trandolaprilat was stopped at the end of either pre-ischemia or 60-min reperfusion. The vascular space of the heart was washed with 8 ml of a cold buffer containing 320 m sucrose and 20 m Tris/HCl, pH 7.4 (washing buffer), via the aortic cannula. The myocardial sodium, potassium, calcium and magnesium contents were measured accoridng to the method described previously (Takeo et al., 1991). Six pieces of the washed myocardium were dried at 120°C for 48 h. After weighing the dried myocardium, it was digested to evaporation

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at 180°C with 60% HNO3. The residue was reconstituted with 2.5 ml of 0.75 N HNO3 and used for determination of myocardial ion content by atomic absorption spectrometer (AA-680, Shimazu, Kyoto, Japan). In a preliminary study, we examined how much extracellular space can be washed out by the present experimental procedure. The hearts were perfused for 45 min under normoxic conditions and then perfused for 20 min with the Krebs–Henseleit bicarbonate buffer containing 1 m Cobalt EDTA (Co-EDTA) solution. Co-EDTA can diffuse into extracellular space, but not into cells (Bridge et al., 1982; Hoerter et al., 1986; Kawada et al., 1992). After equilibration with CoEDTA solution, the hearts were subjected to washing with 8 ml of the cold washing buffer, followed by determination of myocardial ion content as described above. The myocardial cobalt content was determined by atomic absorption method. We found that myocardial cobalt contents at the ends of perfusion with 1 m Co-EDTA and with 8 ml of the washing buffer were 2.41±0.01 (n=4) and 0.03±0.01 lmol/g dry tissue (n=4), respectively. This indicates that almost 99% of readily exchangeable extracellular space was washed by this method.

Examination of perfusate The perfusate eluted from the heart was collected to determine creatine kinase (CK) activity. The creatine kinase activity of the perfusate was determined by the method of Bergmeyer et al. (1970). The release of the enzyme was estimated as the total creatine kinase activity of the effluent from the perfused heart. The perfusate was also used for determination of nucleosides and purine (ATP metabolites) by means of HPLC method described previously (Takeo et al., 1989). ATP metabolites released from the heart were separated through a column of C18-cellurose acetate (Cosmosil 5C18, Nakarai Tesque, Kyoto) with a 4.6 mm diameter and 15 cm length, by elution with 250 m NH4H2PO4 containing 3.5% acetonitrile (pH 6.0), at a flow rate of 1 ml/min (L-6000 pump, Hitachi, Tokyo). ATP metabolites separated was monitored at 254 nm by using u.v.detector (L-4000 detector, Hitachi, Tokyo), and recorded on data-chromatoprocessor (D-2000, Hitachi). The ATP metabolites released during reperfusion were mainly inosine and hypoxanthine. The release of ATP metabolites was estimated as total ATP metabolites in the effluent from the reperfused hearts.

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Figure 2 Typical tracings of ischemic/reperfused hearts treated without (a) and with 30 l trandolaprilat (b).

Agents Trandolaprilat and enalaprilat were kindly provided from Nippon Roussel (Tokyo, Japan). Bradykinin and diclofenac were purchased from Sigma Chemical Co. (St Louis, MO, USA). Hoe 140 was generously provided from Hoechst AG (Frankfurt/M, FRG). E4177 was kindly provided from Eisai Pharmaceutical Co. (Tokyo, Japan).

Statistics The results are expressed as the means±... Statistical significance of differences in LVDP of the hearts treated with either trandolaprilat or enalaprilat at the end of reperfusion, and the release of CK and ATP metabolites from the reperfused heart was evaluated using analysis of variance (ANOVA), following by Dunnett’s multiple comparison. Differences in LVDP recovery of the hearts with combination of treatment at the end of reperfusion, and CK release during reperfusion and myocardial ion content among hearts treated with different agents were estimated using ANOVA, followed by Bonferroni’s multiple comparison. Differences with a probability of 5% or less were considered to be statistically significant (P<0.05).

Results Effects of trandolaprilat Cardiac function of perfused hearts Typical tracings of ischemic/reperfused hearts without and with 30 l trandolaprilat-treatment are shown in Figure 2. Changes in LVDP of ischemic/

reperfused hearts untreated or treated with 10–100 l trandolaprilat are shown in Figure 3(a). Ischemia induced a rapid decline in LVDP and LVDP was ceased completely within 2 min after the onset of ischemia. Thereafter, LVDP was not generated during ischemia. LVDP of the heart recovered approximately 30% of the pre-ischemic value upon reperfusion. When the hearts were treated with either 10 or 30 l trandolaprilat for 10 min of the last preischemia, LVDP significantly recovered approximately 50 or 90% of the pre-ischemic value at the end of reperfusion. Treatment with 100 l trandolaprilat elicited the same recovering rate as that with 30 l trandolaprilat. LVEDP of the untreated heart rose under the ischemic conditions, and reached the peak level of approximately 70 mmHg at 25 min of ischemia [Fig. 3(b)]. LVEDP of the heart was further increased upon reperfusion; the maximum value was approximately 105 mmHg at 5 min after the onset of reperfusion. Thereafter, this high level of LVEDP was sustained throughout reperfusion although the LVEDP was gradually declined during reperfusion. In contrast, treatment with 10, 30 and 100 l trandolaprilat attenuated the rise in LVEDP during reperfusion, but not ischemia. At the end of preperfusion (at 0 min), perfusion pressure of trandolaprilat-treated hearts decreased [Fig. 3(c)], whereas no changes in the perfusion pressure during pre-ischemia were observed in the untreated heart. Perfusion pressure of the untreated heart increased above the baseline of pre-ischemia upon reperfusion, and this increased level was sustained throughout reperfusion. The increase in perfusion pressure during reperfusion was also observed in the hearts treated with trandolaprilat, although the increase in perfusion pressure during

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Figure 4 Creatine kinase activity (a) and released amount of ATP metabolites (b) in perfusate eluted from untreated hearts (Ε), and hearts treated with 10 to 100 l trandolaprilat (Φ). Each value represents the mean±... of six experiments. ∗ Significantly different from untreated group (P<0.05).

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Figure 3 The time course of changes in left ventricular developed pressure [LVDP, (a)], left ventricular enddiastolic pressure [LVEDP, (b)] and coronary perfusion pressure [PP, (c)] of the ischemic/reperfused heart without (Χ) and with different concentrations (10, Η; 30, Β; and 100 l, Α) of trandolaprilat. Each value represents the mean±... of six experiments. Treatment with trandolaprilat was conducted during period “Tra”. The standard errors of the symbols without any bar were within 1%. In the time course study, statistical differences between untreated and treated groups of reperfused heart were evaluated only at the end of reperfusion (at 85 min), using ANOVA and Dunnett’s multiple comparison. ∗ Significantly different from untreated group (P<0.05).

reperfusion tended to be suppressed by treatment with trandolaprilat.

Determination of creatine kinase activity in perfusate To determine the release of CK from perfused hearts, the perfusate eluted from the hearts was collected and its CK activity was measured [Fig. 4(a)]. During 30-min pre-ischemic perfusion, CK activity in the perfusate was negligible regardless of the presence or absence of trandolaprilat (less than 1 nmol NADPH/min/g wet tissue, n=5). There was a negligible CK released from the heart perfused under normoxic conditions for 85 min (less than 2 nmol NADPH/min/g wet tissue, n=5). Creatine kinase activity in perfusate markedly increased during reperfusion. Treatment with either 10, 30 or 100 l trandolaprilat significantly attenuated the release of CK from perfused hearts in a concentration-dependent manner (approximately 83,

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70 or 63% of the value of the untreated hearts, respectively, P<0.05). Released amount of ATP metabolites from perfused hearts The amount of these metabolic compounds released during reperfusion was determined and used as a measure of the release of ATP metabolites [Fig. 4(b)]. A minimal release of ATP metabolites was detected during pre-ischemia (less than 0.1 lmol/ g wet tissue, n=6). No appreciable release of ATP metabolites was observed from hearts perfused under 85-min normoxic conditions (less than 0.2 lmol/g wet tissue, n=6). The release of ATP metabolites was markedly increased during reperfusion (approximately 2.3 lmol/g wet tissue). Treatment with either 10, 30 or 100 l trandolaprilat resulted in a significant suppression of the release of ATP metabolites during reperfusion (77, 66 or 69% of the value of the untreated hearts, respectively, P<0.05). Myocardial ion content Myocardial sodium, potassium, magnesium and calcium contents of ischemic/reperfused hearts without and with 30 l trandolaprilat were determined, and the results are shown in Figure 5. The preischemic values of myocardial sodium, potassium, magnesium and calcium were 54.4±4.1, 349.1±16.4, 36.7±1.5 and 1.91±0.15 lmol/g dry tissue, respectively (n=5). Myocardial sodium and calcium contents at the end of reperfusion significantly increased by approximately three- and six-fold higher than the pre-ischemic values, respectively. Myocardial potassium and magnesium contents at the end of reperfusion significantly decreased by 60 and 65% of the pre-ischemic values, respectively. When the hearts were pretreated with 30 l trandolaprilat, myocardial sodium, potassium, magnesium and calcium contents at the end of pre-ischemia were similar to the pre-ischemic values of the untreated hearts. Treatment with trandolaprilat significantly suppressed the increases in sodium and calcium contents, and significantly attenuated the decreases in potassium and magnesium contents at the end of reperfusion.

Effects of enalaprilat When the heart was treated with 10–100 l enalaprilat for the last 10 min of pre-ischemia, the

LVDP recovered in a concentration-dependent manner during reperfusion [Fig. 6(a)]. Treatment with enalaprilat also suppressed ischemia/reperfusioninduced release of CK and ATP metabolites in a concentration-dependent manner [Fig. 6(b) and (c), respectively].

Effects of AT1-antagonist and bradykinin To determine the mechanisms underlying the enhancement of post-ischemic contractile recovery by trandolaprilat, the heart was pretreated with several agents. Post-ischemic LVDP of hearts treated with 10 to 30 l E4177, an AT1-receptor antagonist, during the last 10 min of the pre-ischemia was approximately 30% of the pre-ischemic value [Fig. 7(a)]. Creatine kinase activity in the perfusate of the ischemic/reperfused heart pretreated with E4177 at these concentrations was markedly increased and reached the same level of untreated hearts [Fig. 7(b)]. These concentrations have been shown to inhibit AT1 receptor in various tissues in vitro (Okunishi et al., 1990). Treatment with 1–10 l bradykinin during preischemia significantly enhanced the recovery of cardiac function during reperfusion in a concentration-dependent manner [Fig. 8(a)]. Treatment with 3–10 l bradykinin during the last 10 min of pre-ischemia significantly suppressed the release of CK from reperfused hearts [Fig. 8(b)].

Effects of BK2-antagonist and cyclooxygenase inhibitor LVDP of the heart treated with either 30 l trandolaprilat plus 3 l Hoe 140 or 30 l trandolaprilat plus 30 l diclofenac was approximately 30% of the pre-ischemic value [Fig. 9(a)], which was similar to that of the untreated heart. Combinated treatment with either trandolaprilat plus Hoe 140 or trandolaprilat plus diclofenac failed to suppress the ischemia/reperfusion-induced release of creatine kinase [Fig. 9(b)]. Treatment with either Hoe140 or diclofenac alone did not alter postischemic recovery of LVDP and the release of CK from reperfused hearts (data not shown). In a preliminary study, the effects of varying concentrations of Hoe 140 and diclofenac ranging from 0.1 to 3 l and 1 to 30 l, respectively on the ischemia/reperfusion injury were examined. The authors found that 3 l Hoe 140 and 30 l diclofenac completely abolished trandolaprilat-induced post-ischemic LVDP recovery of the perfused heart.

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Figure 5 Myocardial sodium, potassium, calcium and magnesium contents of the ischemic/reperfused hearts untreated (Un; Ε) or treated with 30 l trandolaprilat (Tra; Φ) at the end of reperfusion, and those of pre-ischemia (Nor; ∆). Each value represents the mean±... of six experiments. ∗ Significantly different from untreated group and # significantly different from pre-ischemic group (P<0.05).

Discussion In the present study, it was observed that treatment with trandolaprilat during pre-ischemia significantly enhanced post-ischemic contractile recovery of ischemic/reperfused hearts. In a preliminary study, the hearts were treated with trandolaprilat only during reperfusion and no appreciable LVDP recovery in the hearts was found with this treatment. Therefore, pre-treatment with trandolaprilat before ischemia is necessary to elicit the improvement of LVDP recovery of reperfused heart under the present experimental conditions. Treatment with trandolaprilat also suppressed partially the ischemia/reperfusion-induced release of CK and ATP metabolites from the reperfused hearts, and attenuated changes in myocardial ion content upon reperfusion to an appreciable degree. The release of CK is a marker of myocardial cell necrosis, since CK release implicates a loss of cytosolic macromolecular components, that is, ischemia/reperfusion-induced cell membrane disruption. The myocardial cell membrane disruption is considered to cause non-specific changes in cardiac cell membrane permeability across sarcolemma and thereby

changes in myocardial ion content. In the present study, increases in myocardial calcium and sodium contents and decreases in myocardial magnesium and potassium contents were observed. The increase in myocardial calcium content is considered to be an indicator of ischemia/reperfusion-induced calcium overload that causes cardiac dysfunction. Takeo et al. (1995) have shown that the postischemic LVDP recovery of reperfused hearts was inversely related to the myocardial sodium and calcium accumulation after reperfusion. The findings of the present study suggest that trandolaprilat is capable of attenuating ischemia/reperfusion-induced cardiac contractile failure and myocardial cell necrosis associated with suppression of an increase in cell membrane permeability and changes in myocardial ions. Cardioprotective effect of enalaprilat, a typical and representative ACE inhibitor, was also examined. Treatment with enalaprilat enhanced the recovery of cardiac contractile function in a concentration-dependent manner. The improvement of post-ischemic recovery of LVDP with enalaprilat was somewhat less than that with trandolaprilat, when LVDP recovery was compared at the same

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Figure 7 Left ventricular developed pressure at the end of reperfusion (a) and CK activity (b) in perfusate eluted during reperfusion from hearts without (untreated) and with either 10 or 30 l E4177. Each value represents the mean±... of three (10 l E4177) and five experiments (30 l E4177), respectively. ∗ Significantly different from untreated group (P<0.05).

Figure 6 Left ventricular developed pressure at the end of reperfusion (a), CK activity (b) and ATP metabolites (c) in perfusate eluted during reperfusion from hearts without (untreated) and with different concentrations of enalaprilat (10–100 l). Each value represents the mean±... of six experiments. ∗ Significantly different from untreated group (P<0.05).

concentration. Trandolapril, a prodrug of trandolaprilat, has been shown to be longer acting and more potent ACE inhibitory action than enalapril, a prodrug of enalaprilat (Brown et al., 1988; Okunishi et al., 1992). Okunishi et al. (1992) have also shown that trandolaprilat has a higher affinity for the active site of ACE molecule and/or a slower dissociation rate from the enzyme-trandolaprilat complex than enalaprilat does. The results of this study suggest that cardioprotection of trandolaprilat

and enalaprilat against ischemia/reperfusion injury is parallel to the potency of ACE inhibition. Treatment with trandolaprilat at the concentrations of 30–100 l during pre-ischemia decreased LVDP to approximately 80% of the baseline value. Several cardioprotective agents such as badrenoceptor blocking agent and calcium antagonist suppress myocardial contractile function and decrease myocardial energy consumption during hypoxia or ischemia. This cardioprotective effect is known as “energy sparing effect”. Therefore, one of the mechanisms underlying cardioprotection of the ACE inhibitor may be a suppression of LVDP during pre-ischemia and thus preservation of high energy phosphates in the ischemic myocardium. Sulfhydryl-containing ACE inhibitor such as captopril and zofenopril-sulfhydryl has been shown to attenuate oxidative injury of the myocardium and to improve myocardial function during reperfusion (Westlin and Mullane, 1988; Grover et al., 1991; Chopra et al., 1992). These observations suggest

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Figure 8 Left ventricular developed pressure at the end of reperfusion (a), CK activity (b) in perfusate eluted during reperfusion from hearts without (untreated) and with different concentrations of bradykinin (1–10 l; Φ). Each value represents the mean±... of three (1 and 10 l bradykinin), and five experiments (3 l bradykinin), respectively. ∗ Significantly different from untreated group (P<0.05).

that scavenging action of reperfusion-induced free radicals plays an important role in cardioprotective action against ischemia/reperfusion injury. In contrat to the observations, it has been shown that treatment with either captopril, a sulfhydryl-containing ACE inhibitor, or ramiprilat, a non-sulfhydryl ACE inhibitor, protects the isolated rat working hearts subjected to electrolytically generated free radicals (Pi and Chen, 1991). Furthermore, Pi and Chen (1991) suggested that ACE inhibitors protect the myocardium against ischemia/reperfusion injury through another mechanism rather than radical scavenging action by sulfhydryl moieties. Thus, the mechanism underlying the effects of trandolaprilat and enalaprilat in the present study appears not to be attributed to free-radical scavenging action. To elucidate the cardioprotective mechanism of trandolaprilat in ischemic/reperfused hearts, the hearts were treated with E4177, an AT1-antagonist

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Figure 9 Left ventricular developed pressure at the end of reperfusion (a) and creatine kinase activity (b) in perfusate eluted during reperfusion from hearts without (untreated) and with either 30 l trandolaprilat (Tra), 30 l trandolaprilat plus 3 l Hoe 140 (Tta+Hoe140) or 30 l trandolaprilat plus 30 l diclofenac (Tra+Dic). Each value represents the mean±... of four (Tra+Hoe 140 and Tra+Dic) and six experiments (Tra), respectively. ∗ Significantly different from untreated group and ∗∗ significantly different from trandolaprilat-treated group (P<0.05).

(Okunishi et al., 1993), it is generally recognized that ACE inhibitors suppress production of angiotensin II (Griengling et al., 1993; Juggi et al., 1993). In contrast, no significant recovery of LVDP was observed in the hearts pretreated with E4177. Treatment with E4177 did not suppress the ischemia/reperfusion-induced release of CK during reperfusion. The present authors’ findings suggest that the cardioprotective effects of trandolaprilat are not simply attributed to an inhibition of the converting reaction from angiotensin I to angiotensin II in the myocardium. Because ACE is an enzyme responsible not only for the conversion of angiotensin I to angiotensin II but also for the breakdown of bradykinin (kininase II), one possible mechanism is a prevention of bradykinin degradation, that is, an inhibition of kininase II (Yang et al., 1970; Bhoola et al., 1992).

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Therefore, the effect of 1–10 l bradykinin on ischemic/reperfused hearts was examined. Treatment with various concentrations of bradykinin during pre-ischemia enhanced the recovery of LVDP in a concentration-dependent manner and partially suppressed the release of CK during reperfusion. These results suggest that bradykinin, like trandolaprilat, exerts the cardioprotective effect. In addition, a decrease in coronary perfusion pressure during pre-ischemia in the heart treated with either trandolaprilat or enalaprilat was observed, whereas no changes in coronary perfusion pressure of the untreated heart were observed. Hashimoto et al. (1977) showed that the concentration of bradykinin in coronary sinus blood increased after coronary artery occlusion in in vivo dogs. Baumgarten et al. (1993) showed that treatment with ramiprilat increased bradykinin levels in the outflow of the isolated, perfused rat heart after coronary artery occlusion. These observations including these here suggest that bradykinin and bradykinin-like activity in the heart are involved in the benefit of kininase II inhibition for post-ischemic contractile recovery of the heart. To determine whether kininase II inhibition by trandolaprilat is involved in the cardioprotection against ischemia/reperfusion injury, the heart was administered with both trandolaprilat and Hoe 140. The latter is known to be a potent BK2-antagonist (Feletou et al., 1994; Koller et al., 1995; Minishall et al., 1995). The beneficial effect of trandolaprilat on post-ischemic recovery of cardiac contractile function was abolished by co-administration of Hoe 140. Several investigators have shown that the cardioprotective effects of ramipril and ramiprilat were abolished by treatment with Hoe 140 or an antagonist by which the effect of bradykinin is modified (Scholckens et al., 1988; Mortorana et al., 1990; Hartman et al., 1993). In the present study, although it was not determined whether trandolaprilat can directly bind to BK2 receptor, the results suggest that the cardioprotective effect of trandolaprilat is, at least in part, related to stimulation of bradykinin system in the myocardial tissue. Bradykinin stimulates production of nitric oxide and prostaglandins in vascular endothelium (Radomski et al., 1987). Stimulation of BK2 receptor in cultured bovine and human endothelial cells has been shown to enhance the production of prostacyclin and nitric oxide, as determined by 6-keto-prostaglandin F1a and cGMP levels (Wiemer et al., 1991; Linz et al., 1992). In the experimental model, the formation of 6-keto-prostaglandin F1a and cGMP was increased by treatment with ramiplirat. The effects were prevented by treatment

with Hoe 140. Both prostacyclin and nitric oxide are postulated to be possible cardioprotective factors against ischemia/reperfusion injury (DeDecker et al., 1977; Karmazyn and Dhalla, 1983; Darius et al., 1987; Ferrari et al., 1988, 1989; Linz et al., 1992). However, cardioprotective effects of nitric oxide are still controversial in in vivo and in vitro experiments. Ehring et al. (1994) recently showed that ramiprilat attenuated the myocardial stunning through a BK2 receptor in the reperfused canine heart in vivo. They also suggested that prostaglandins were involved in the BK2 receptor-mediated signal transduction in the myocardium, because NG-nitro--arginin methyl ester (-NAME), a nitric oxide synthase inhibitor, failed to abolish the improvement in the recovery of the stunned myocardium by treatment with ramiprilat. Depre et al. (1995) showed that treatment with -Nmonomethyl arginine (-NMMA), a nitric oxide synthase inhibitor, improved post-ischemic LVDP recovery and suppressed a rise in LVEDP of isolated rabbit hearts. They also showed that cardioprotective effect of -NMMA reversed co-administration of either -arginine, a substrate of nitric oxide synthase, or sodium nitro purusside (SNP), a potent nitric oxide donor. Their results suggest that nitric oxide is involved in an induction of ischemia/ reperfusion injury rather than cardioprotection. In addition, the authors have previously demonstrated that treatment of hypoxic hearts with prostacyclin and beraprost, a stable prostacyclin derivative, is beneficial for post-hypoxic contractile recovery that is associated with suppression of hypoxia/reoxygenation-induced release of CK and ATP metabolites from the heart, restoration of high energy phosphates and suppression of increases in myocardial sodium and calcium contents (Tanonaka et al., 1991). These pathogenesis and restorations are comparable with those in the present study, suggesting that the mechanism for cardioprotective effect of trandolaprilat underlies production of prostacyclin. Therefore, the effect of co-administration with trandolaprilat and diclofenac, a potent cyclooxygenase inhibitor, on trandolaprilat-treated heart was examined. It was found that an inhibition of cyclooxygenase activity appeared to abolish the cardioprotective effect of trandolaprilat. The findings also suggest that the cardioprotective effect of trandolaprilat is attributed to the production of prostaglandins including prostacyclin. In summary, it has been demonstrated that trandolaprilat improved the post-ischemic contractile recovery and metabolic alterations of ischemic/ reperfused heart. The cardioprotective effect was abolished by co-administration of a BK2 antagonist

ACE Inhibitor and Ischemia/reperfusion Injury

and a cyclooxygenase inhibitor. An altered bradykinin metabolism is, at least in part, involved in this effect.

Acknowledgement The authors thank Dr S. Imai, Professor of Pharmacology, and Dr T. Kawada, Department of Pharmacology, School of Medicine, Niigata University, for helpful guidance for the method of measurement of Cobalt-EDTA space in the myocardium.

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