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Ann Thorac Surg 1995;60:789-792
© 1995 The Society of Thoracic Surgeons

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I: Pathophysiology of Ischemic-Reperfusion Injury

The Role of Bradykinin and Nitric Oxide in the Cardioprotective Action of ACE Inhibitors

Cardiovascular Pharmacology, Upjohn Laboratories, The Upjohn Company, Kalamazoo, Michigan

Abstract

Background. The angiotensin-converting enzyme inhibitor ramiprilat has been previously demonstrated to protect myocardium from ischemia/reperfusion injury. The objective of these investigations was to examine the roles of bradykinin, angiotensin II, and nitric oxide in the cardioprotective effects of ramiprilat.

Methods. Anesthetized, open-chest rabbits were instrumented for production of myocardial ischemia (30 minutes) and subsequent reperfusion (120 minutes), after which myocardial infarct size was measured. Animals were treated intravenously with either saline solution, ramiprilat (50 µg/kg), the bradykinin₂ receptor antagonist HOE 140 (1 µg/kg), ramiprilat + HOE 140, angiotensin II (2.5 ng • kg^-1 • min^-1), the angiotensin II receptor antagonist losartan (20 mg/kg), ramiprilat + angiotensin II, the nitric oxide synthase inhibitor N^G-nitro-L-arginine methyl ester (100 µg • kg^-1 • min^-1), or ramiprilat + N^G-nitro-L-arginine methyl ester.

Results. Among all treatment groups myocardial infarct size was reduced significantly below saline control only by ramiprilat (-54%) and ramiprilat + angiotensin II (-37%). Pretreatment with HOE 140 or N^G-nitro-L-arginine methyl ester abolished the cardioprotective effect of ramiprilat. Neither stimulation nor antagonism of angiotensin II receptors altered infarct size from the saline control level. Also, when isolated neonatal rat cardiomyocytes were exposed to hypoxia/reoxygenation, ramiprilat (100 µmol/L) and bradykinin (10 nmol/L) improved cell viability (60%), and the protective effect of both agents was reversed by administration of HOE 140 (10 µmol/L).

Conclusions. These results indicate that the in vivo cardioprotective effect of ramiprilat can be abolished by antagonizing bradykinin receptors or inhibiting nitric oxide synthase, and that the effect is not related to angiotensin II receptor activity. The potential bradykinin-sparing property of ramiprilat may promote increased bradykinin-stimulated nitric oxide production leading to cardioprotection. Part of the cardioprotective effects of ramiprilat/bradykinin/nitric oxide may occur locally as demonstrated by the in vitro results using isolated cardiomyocytes.

Angiotensin-converting enzyme (ACE) inhibitors have been demonstrated to reduce myocardial injury in cell culture [1], in isolated hearts [2], and in intact animal models [3], all of which were subjected to ischemic conditions. These effects may not only be due to ACE inhibitor action to decrease the synthesis of angiotensin II from angiotensin I, but also to the ability of these drugs to attenuate the degradation of endogenous bradykinin by inhibition of kininase II (Fig 1), an enzyme indistinguishable from ACE [4]. Although circulating concentrations of angiotensin II and bradykinin may be altered by ACE inhibition, it has been suggested that a local action to interrupt regional cellular cardiac ACE activity may participate in the cardioprotective effects of ACE inhibitors [5]. In this way, localized angiotensin II or bradykinin concentrations in ischemic/reperfused cardiac tissue could be altered to produce autocrineor paracrine-mediated beneficial activity.

View larger version (26K): [in this window] [in a new window]   Fig 1. . The dual action of angiotensin converting enzyme (ACE) to both convert angiotensin I to II and degrade bradykinin. The actions of elevated bradykinin (+) to increase () prostaglandin and nitric oxide synthesis are also represented.

The multiple objectives of the following series of investigations were (1) to determine if the action of the ACE inhibitor ramiprilat to suppress bradykinin degradation plays a role in cardioprotection by this agent, (2) to examine if angiotensin II is involved in that effect of ramiprilat, (3) to inhibit nitric oxide synthase concomitant with ramiprilat administration and thereby ascertain if protection is associated with a bradykinin-mediated stimulatory action on that enzyme, and (4) to determine if the cardioprotective effects of ramiprilat and bradykinin could be locally mediated by simulating ischemia/reperfusion in isolated cardiomyocytes via hypoxia/reoxygenation.

Material and Methods

All procedures in this investigation are in compliance with the Animal Welfare Act Regulations (9 CFR parts 1, 2 and 3), the ``Guide for the Care and Use of Laboratory Animals'' (National Institutes of Health publication 85-23, revised 1985), and the Declaration of Helsinki. Anesthetized, open-chest rabbits were first instrumented to measure lead II electrocardiogram, systemic hemodynamics, and maximum rate of increase of left ventricular pressure, after which 30 minutes of left main coronary artery occlusion was performed (marginal branch), followed by 2 hours of reperfusion, after which myocardial infarct size was measured using the tetrazolium staining technique [14]. All pharmacologic agents were administered intravenously at maximal bolus or infusion doses that minimally affected systemic hemodynamics. To examine whether the cardioprotective action of ramiprilat was associated with bradykinin-sparing or angiotensin II synthesis inhibition effects (objectives 1 and 2), rabbits were administered intravenously either saline solution, ramiprilat (50 µg/kg), the bradykinin₂ receptor antagonist HOE 140 (1 µg/kg), ramiprilat + HOE 140, angiotensin II (2.5 ng • kg^-1 • min^-1), the angiotensin II receptor antagonist losartan (20 mg/kg), or ramiprilat + angiotensin II. To examine the potential contribution of nitric oxide with respect to the action of ramiprilat (objective 3), rabbits were given intravenously either the nitric oxide synthase inhibitor N^G-nitro-L-arginine methyl ester (100 µg • kg^-1 • min^-1) or ramiprilat (50 µg/kg) + N^G-nitro-L-arginine methyl ester. Angiotensin II and N^G-nitro-L-arginine methyl ester infusions were started 15 and 30 minutes, respectively, before coronary occlusion and continued throughout the experiment.

To examine a local cellular effect by ramiprilat or bradykinin to preserve myocyte viability (objective 4), neonatal rats were sacrificed and the hearts enzymatically digested [15]. The myocytes were plated and cultured for 5 days, after which the cells were exposed to hypoxic Tyrode's solution (5 hours), and then reoxygenated viability was assessed by propidium iodide exclusion. Before hypoxia, myocytes were treated with ramiprilat (10, 30, or 100 µmol/L), bradykinin (0.001, 0.01, 0.1, or 1 µmol/L), ramiprilat or bradykinin at the above concentrations plus HOE 140 (10 µmol/L), and untreated controls. Treatment with the above agents under normoxic conditions did not alter viability.

Results

Objective 1
Myocardial infarct size in rabbits treated at the end of coronary occlusion with ramiprilat was significantly reduced to 49% of saline control, but pretreatment with the bradykinin₂ receptor antagonist HOE 140 before ramiprilat abolished the protective effect (Fig 2).

View larger version (21K): [in this window] [in a new window]   Fig 2. . Histogram depicting saline or drug effects on myocardial infarct size expressed as a percent of area at risk. Data are shown as means +/- standard error of the mean (n = 6). (Ang II = angiotensin II; Ram = ramiprilat; *significantly [p < 0.05] different from saline.)

Objective 3
When nitric oxide synthase was inhibited with N^G-nitro-L-arginine methyl ester during ischemia/reperfusion, the cardioprotective effect of ramiprilat was also abolished (Fig 3).

View larger version (22K): [in this window] [in a new window]   Fig 3. . Histogram depicting effects of saline, ramiprilat and NGnitro-L-arginine methyl ester (L-NAME) on myocardial infarct size, expressed as a percent of area at risk. Data are shown as means +/- standard error of the mean (n = 8). (Ram = ramiprilat; *significantly [p < 0.05] different from saline.)

View larger version (22K): [in this window] [in a new window]   Fig 4. . Effects of ramiprilat, HOE 140, and bradykinin on myocyte viability (control hypoxic cells stained with propidium iodide (PI) set at 100%). Data are shown as means +/- standard error of the mean. (Brady = bradykinin; Ram = ramiprilat; *significant [p < 0.05] decrease in staining [increased viability] below control hypoxia.)

The above results collectively indicate that bradykinin plays an important role in the myocardial protective action of the ACE inhibitor ramiprilat and that the cardioprotective effect is independent of the ability to inhibit angiotensin II synthesis by ramiprilat. Furthermore, the infarct-reducing effect of ramiprilat was demonstrated to be both bradykininand nitric oxide-dependent, which suggests that increased preservation of bradykinin by inhibition of kininase II due to ramiprilat may be related to enhanced generation of nitric oxide. Also, ramiprilat and bradykinin improved viability of isolated, cultured cardiomyocytes exposed to hypoxia/reoxygenation via a bradykinin-dependent mechanism, which suggests that part of the protective effects of these agents occurs at a local tissue level without the added factors of intact circulation and nervous control. This is not surprising in that alterations in local tissue rather than systemic ACE activity have been suggested to be responsible for the beneficial action of ACE inhibitors in experimentally induced disease states such as vascular neointimal proliferation [16], hypercholesterolemia-induced vascular endothelial dysfunction [17], and ventricular hypertrophy [18].

If ramiprilat elevates endogenous bradykinin levels to reduce injury within ischemic/reperfused myocardium, how does bradykinin improve myocardial viability? Enhancement of nitric oxide production is one among many cellular activities modulated by bradykinin receptor stimulation. If ACE/kininase II inhibition increases local endogenous levels of bradykinin thereby increasing nitric oxide production, then reducing nitric oxide level should attenuate the cardioprotective effect of ramiprilat as was demonstrated in the present investigation. The effects of bradykinin or nitric oxide to produce vasodilation, reduce platelet aggregation [11], prevent neutrophil activation [10], and enhance cardiac glucose uptake [12] may also contribute to the antiischemic properties of these two endogenous mediators.

The peptide bradykinin is also degraded through pathways different than the kininase II route; therefore, ACE inhibitors may also block these alternative kinin metabolizing enzymes due to binding site similarities between the various kinin degrading proteins [19]. The relative potency with which different ACE inhibitors prevent kinin metabolism at these alternative sites may eventually segregate those agents as having a broad spectrum of ``kinin sparing'' activity, which would recommend use of only certain ACE inhibitors in pathologies where bradykinin elevation would be important therapeutically. Already, specific inhibitors of the kinin-degrading enzymes endopeptidases 24.11 and 24.15 [20] have been found to reduce myocardial infarct size in a rabbit model similar to that used in the present investigation (Schriefer JA, personal communication). Future drug development of ACE inhibitors or novel agents may take the course of improving bradykinin preservation or focusing on enhancement of nitric oxide production, dependent on the balance present between these two endogenous mediators in disease states such as myocardial ischemia/reperfusion injury.

Acknowledgments

I thank Theron Wall, Thomas Hullinger, Rita Sheehy, Gary Kurc, Daniel Linseman, and Dr Ronald Shebuski for their technical and creative contributions. Thanks also to Drs Wolfgang Linz and Bernward Schölkens (Hoechst AG) for providing HOE 140.

Footnotes

Presented at the International Symposium on Myocardial Protection From Surgical Ischemic-Reperfusion Injury, Asheville, NC, Sep 25--28, 1994.

Address reprint requests to Dr Hartman, The Upjohn Company, 7243-209-3, 301 Henrietta St, Kalamazoo, MI 49001.

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