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J Thorac Cardiovasc Surg 1994;107:505-0509
© 1994 Mosby, Inc.
Cardiopulmonary Bypass, Myocardial Management, and Support Techniques |
Rochester, Minn.
From the Section of Cardiovascular Surgery, Mayo Clinic and Mayo Foundation, Rochester, Minn.
Address for reprints: Hartzell V. Schaff, MD, Mayo Clinic, 200 First St. SW, Rochester, MN 55905.
Abstract
Reperfusion after global cardiac ischemia may injure coronary artery endothelium and lead to vasospasm and thrombosis. Oxygen-derived radicals have been implicated as mediators of this process, but the precise mechanism of injury is unknown. We hypothesized that oxygen-derived radicals impair coronary endothelial production of nitric oxide, a potent endogenous vasodilator and inhibitor of platelet adhesion. To test this theory, we developed an in vitro model of reperfusion injury in which segments of epicardial canine coronary artery were suspended in organ chambers (physiologic salt solution, 37° C, 95% oxygen and 5% carbon dioxide) and exposed to oxygen-derived radicals (generated by adding xanthine [10-4 mol/L] and xanthine oxidase [100 mU/ml] to the bathing solution for 70 minutes). After exposure to oxygen-derived radicals, epicardial coronary artery smooth muscle exhibited normal contraction to potassium ions (20 mmol/L) and prostaglandin F2 (4 x 10-6 mol/L); also, the rings relaxed normally on exposure to isoproterenol and sodium nitroprusside (10-9 to 10-4 mol/L) (n = 6). In contrast, endothelium-dependent vasodilatation to receptor-dependent agonists acetylcholine and adenosine diphosphate (10-9 to 10-4 mol/L) was impaired as compared with the reaction of control vessels not exposed to oxygen-derived radicals (n = 18, P < 0.001, and n = 10, P < 0.002, respectively). Importantly, receptor-independent, endothelium-dependent relaxation to the calcium ionophore A23187 was normal (n = 6). Further, endothelium-dependent vasodilatation to receptor-dependent agonist bradykinin (non-nitric oxide pathway) was normal after exposure to oxygen-derived radicals. This is the first study to demonstrate that oxygen-derived radicals selectively impair receptor-dependent nitric oxide production by the coronary endothelium. Diminished nitric oxide production is a likely mechanism of vasospasm and thrombosis after reperfusion of the ischemic heart. (J THORAC CARDIOVASC SURG 1994;107:505-9)
The importance of the coronary artery endothelium in maintaining vascular patency is well established.
1 Vascular patency is maintained, in large part, through release of factors that modulate vascular tone and inhibit thrombus formation. Endothelium-derived relaxing factor is the principal mediator of vasodilatation and its active component, the nitric oxide radical. 2, 3 In addition to its potent action as a vasodilator, nitric oxide also possesses antithrombogenic properties and is a promoter of platelet disaggregation. 4-6 Release of nitric oxide from the coronary endothelium is impaired after myocardial ischemia and reperfusion, 7, 8 and this impairment may contribute to the vulnerability of the coronary circulation to thrombus formation and vasospasm after extracorporeal circulation. The mechanism of this impairment is not known. Previous studies have shown that the endothelium retains the capacity to produce nitric oxide but is unable to respond to agents that act through cell surface receptors. 9
Oxygen-derived free radicals, which have been implicated in the pathogenesis of reperfusion injury, 10-14 effect tissue damage through peroxidation of lipids, oxidation of protein sulfhydryl groups, and disruption of deoxyribonucleic acid strands. 15 The role of oxygen radical scavengers has been investigated in a number of experimental models, and scavengers appear to reduce both endothelial injury and the extent of myocardial infarction. 16-18
The present study addresses two questions: (1) Does exposure of coronary artery endothelium to oxygen radicals impair release of nitric oxide? (2) What is the mechanism of impaired nitric oxide release at the cellular level?
METHODS
Heartworm-free mongrel dogs (25 to 30 kg) of either sex were anesthetized with pentobarbital sodium (30 mg/kg bolus by intravenous injection; Fort Dodge Laboratories) and exsanguinated. Beating hearts were excised and immersed in cool, oxygenated physiologic salt solution of the following milimolar composition: NaCl, 118.3; KCl, 4.7; MgSO4, 1.2; KH2PO4, 1.22; CaCl2, 2.5; NaHCO3, 25.0; and glucose, 11.1 (control solution). The procedures and handling of the animals were in compliance with the "Principles of Laboratory Animal Care" formulated by the National Society for Medical Research and the "Guide for the Care and Use of Laboratory Animals" prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication No. 86-23, revised 1985). In addition this protocol was reviewed and approved by the Insitutional Animal Care and Use Committee of the Mayo Foundation.
Coronary arteries were dissected and placed in physiologic salt solution with care taken to remove as much of the connective tissue as possible. Coronary ring segments 5 to 6 mm in length were prepared from each artery and suspended in 25 ml organ chambers filled with control solution maintained at 37° C and aerated with 95% oxygen and 5% carbon dioxide (pH 7.4). Each ring was suspended by two stainless steel clips passed through the vessel lumen. One clip was anchored to the bottom of the organ chamber, and the other was connected to a strain gauge (Statham Gould UC2; Viggo Spectramed Inc., Critical Care Division, Oxnard, Calif.) for the measurement of isometric force.
Rings were placed at the optimal point of their length-tension relationship by progressively stretching them until the contraction to potassium chloride (20 mmol/L), imposed at each level of distention, was maximal. In all experiments, the presence of functioning endothelium was confirmed by the response to acetylcholine (10-6 mol/L) of rings contracted with potassium ions (20 mmol/L). 19 After optimal tension was achieved, the rings were allowed to equilibrate for 45 minutes before the administration of drugs.
Exposure to oxygen-derived free radicals
Oxygen-derived free radicals were generated by incubating vascular ring segments with xanthine oxidase (100 mU/ml) and xanthine (10-4 mol/L final concentration) in the presence of 95% oxygen. 15 The total exposure time to xanthine oxidase and xanthine in the treatment group was 70 minutes.
A parallel group of vessels received superoxide dismutase (150 U/ml) and catalase (1200 U/ml) at the time that xanthine oxidase was added to the organ chambers to confirm that the effect of xanthine oxidase incubation was due to the generation of oxygen radicals. In all experiments, indomethacin (10-5 mol/L) was used to prevent the synthesis of endogenous prostanoids.
Vascular relaxation
After contraction with prostaglandin F2 (2 x 10-6 mol/L), rings were exposedto increasing concentrations of either acetylcholine (10-9 to 10-4 mol/L), adenosine diphosphate (ADP) (10-9 to 10-5 mol/L), bradykinin (10-9 to 10-5 mol/L), sodium fluoride (0.5 to11 mmol/L), calcium ionophore A23187 (10-9 to 10-6 mol/L), isoproterenol (10-9 to 10-4 mol/L), or sodium nitroprusside (10-9 to 10-4 mol/L). For all agonists, control and treatment groups were studied in parallel.
Drugs
The following drugs were used: acetylcholine chloride, adenosine diphosphate, calcium ionophore A23187, catalase, deferoxamine, indomethacin, isoproterenol hydrochloride, prostaglandin F2, sodium fluoride, sodium nitroprusside, superoxide dismutase (bovine), xanthine, and xanthine oxidase (all from the Sigma Chemical Company, St. Louis, Mo.). All drugs were prepared with distilled water except for indomethacin, which was dissolved in Na2CO3 (10-5 mol/L) and the calcium ionophore A23187, which was dissolved in dimethyl sulfoxide (Sigma) and diluted further in distilled water.
Data analysis
The results were expressed as means ± standard error of the mean. In all experiments, n referred to the number of animals from which rings were taken. Vascular relaxation is expressed as the negative logarithm of agonist required to reduce prostaglandin F2–induced contraction by either 50% (ED50) or 75% (ED25).*Statistical evaluation of the data was performed with the Student's t test for either paired or unpaired observations. Values were considered to be statistically significant when P was smaller than 0.05.
RESULTS
Vasoconstriction
After oxygen radical exposure, epicardial coronary artery smooth muscle exhibited normal contraction to potassium ions (20 mmol/L) and prostaglandin F2 (4 x 10-6 mol/L).
Endothelium-dependent, receptor-mediated relaxation
Endothelium-dependent vasodilatation to acetylcholine (10-9 to 10-4 mol/L) and ADP (10-9 to 10-4 mol/L) was significantly impaired after exposure to oxygen-derived radicals compared with control vessels (n = 18, P < 0.001, and n = 10, P = 0.001, respectively). Similarly, sodium fluoride (0.5 to 11.5 mmol/L) caused dose-dependent vasodilatation in control vessels but failed to cause relaxation in coronary arteries exposed to oxygen radicals (n = 12, P = 0.003). In contrast, there was no difference in endothelium-dependent vasodilatation to bradykinin (10-9 to 3 x 10-5 mol/L) between control vessels and those exposed to oxygen-derived radicals (n = 6, P = 0.66) (Table I).
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Table I).
Endothelium-independent relaxation
Both isoproterenol (10-9 to 10-6 mol/L) andsodium nitroprusside (10-9 to 10-6 mol/L) induced comparable, concentration-dependent relaxation in control coronary artery segments and coronary artery segments exposed to oxygen-derived radicals (n = 6, P = 0.71 and P = 0.29, respectively) (Table I).
Protective effect of oxygen-derived radical scavengers
Coronary artery segments exposed to xanthine oxidase (100 mU/ml) and xanthine (10-4 mol/L) in the presence of superoxide dismutase (150 U/ml) and catalase (1200 U/ml) exhibited vasodilatation to ADP (10-9 to 10-4 mol/L) that was comparable with that observed in control coronary artery segments (logED50 = 6.24 ± 0.47 and 6.61 ± 0.08, respectively, n = 6, P = 0.37).
DISCUSSION
Previous in vivo canine experiments have demonstrated that the release of nitric oxide from the coronary artery endothelium is impaired after ischemia and reperfusion. 7-9 These previous studies have also shown however, that the ability of the coronary artery endothelium to produce nitric oxide in response to the receptor-independent agonist calcium ionophore A23187 was normal. The mechanism for this pattern of impaired nitric oxide release is unknown.
In the present in vitro study, coronary artery segments exposed to oxygen-derived free radicals displayed abnormal relaxation to the endothelium-dependent, receptor-dependent agonists acetylcholine and ADP. However, exposure of coronary arteries to oxygen-derived radicals did not influence relaxation to the endothelium-dependent, receptor-independent calcium ionophore A23187. In addition, coronary arteries exposed to oxygen-derived radicals failed to relax in response to sodium fluoride but retained the capacity to relax in response to the endothelium-dependent, receptor-mediated agonist bradykinin. These results provide insight into the mechanism of impaired nitric oxide release after oxidative injury.
In canine coronary arteries, sodium fluoride is a specific tool for inducing endothelium-dependent relaxation by acting on the pertussis toxin-sensitive G-protein, 20-23 a vital component in the signal transduction pathway leading to nitric oxide synthesis (Fig. 1). The inability of sodium fluoride to induce relaxation provides evidence that oxidative injury occurs at the level of the receptor/G-protein complex. Indeed, activation of the pathway just distal to the G-protein complex, by the calcium ionophore A23187, was unimpaired after exposure to oxygen-derived radicals. Injury to the G-protein complex would render agents acting at the receptor level ineffective, explaining the impaired response of vessels to ADP and acetylcholine.
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24-27 The observation that coronary arteries exposed to oxygen-derived radicals exhibited normal relaxation to bradykinin suggests that oxidative injury to the receptor/G-protein complex may selectively disrupt receptor/G-protein function in the nitric oxide signal transduction pathway, rather than causing global receptor/G-protein injury.
This pattern of endothelium-dependent vascular relaxation after in vitro exposure to oxygen radicals is identical to that observed previously after ischemia and reperfusion in vivo. 7-9 These findings support the hypothesis that oxygen-derived radicals mediate the decrease in nitric oxide release after ischemia and reperfusion in vivo.
These results do not speak to the production of nitric oxide by the endothelium of smaller resistance vessels after exposure oxygen-derived radicals. Under normal conditions, it is the smaller vessels that control coronary vascular resistance and hence myocardial blood flow. After ischemia and reperfusion, however, the impaired production of nitric oxide by the endothelium of epicardial coronary arteries could be expected to predispose to vasospasm of these vessels, causing them to function as the primary resistance to coronary blood flow.
This is the first study to demonstrate that oxygen-derived radicals selectively impair receptor-dependent nitric oxide production by the coronary endothelium. Diminished nitric oxide production is a likely mechanism in the pathogenesis of vasospasm and thrombosis after cardiac operations.
Appendix: DISCUSSION
Dr. John H. Kennedy (Cambridge, England).
This is a very important paper and I am very interested in your results. In your presentation, you described the tissue being immersed in buffer. In the abstract you said physiologic saline. That's not so important, but was there glucose in the bathing solution? We were disappointed to find that in a study by Ono and coworkers,* cultured bovine aortic endothelial cells exposed to glucose showed depressed prostacyclin production.
Dr. Seccombe.
The glucose concentration in our solution was 11.1 mmol/L. In response to the second point, all experiments were performed in the presence of indomethacin to eliminate any effect that endogenous prostanoids might have in relaxation. We were interested only in the role of nitric oxide or other non-nitric oxide, non-prostanoid mediators.
Footnotes
Read at the Seventy-third Annual Meeting of The American Association for Thoracic Surgery, Chicago, Ill., April 25-28, 1993. 
**Ono H, Umeda F, Inoguchi T, Ibayashi H. Glucose inhibits prostacyclin production by cultured aortic endothelial cells. Thromb Haemos 1988;60:174-7.
References
This article has been cited by other articles:
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S. M. Wildhirt, S. Weismueller, C. Schulze, N. Conrad, A. Kornberg, and B. Reichart Inducible nitric oxide synthase activation after ischemia/reperfusion contributes to myocardial dysfunction and extent of infarct size in rabbits: evidence for a late phase of nitric oxide-mediated reperfusion injury Cardiovasc Res, August 15, 1999; 43(3): 698 - 711. [Abstract] [Full Text] [PDF]
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D. G Cable, T. O'Brien, I. J Kullo, R. S Schwartz, H. V Schaff, and V. J Pompili Expression and function of a recombinant endothelial nitric oxide synthase gene in porcine coronary arteries Cardiovasc Res, September 1, 1997; 35(3): 553 - 559. [Abstract] [Full Text] [PDF]
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M. Carrier, A. Khalil, A. Tourigny, B. C. Solymoss, and L. C. Pelletier Effect of L-Arginine on Metabolic Recovery of the Ischemic Myocardium Ann. Thorac. Surg., June 1, 1996; 61(6): 1651 - 1657. [Abstract] [Full Text]
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IP3), resulting in liberation of intracellular calcium. The formation of calcium-calmodulin complexes increases the activity of the enzyme nitric oxide-synthase (NOS), which catalyzes the conversion of L-arginine to nitric oxide. Sodium fluoride (NaF) activates this signal transduction pathway by selectively stimulating the G-protein. Calcium ionophore (A23187) acts at the level of the endoplasmic reticulum to effect an increase in intracellular calcium and the formation of calcium-calmodulin complexes.