COX-2 mediates morphine-induced delayed cardioprotection via an iNOS-dependent mechanism☆
Introduction
Exposure of the heart to a sublethal ischemic stress renders the myocardium relatively resistant to a subsequent ischemic insult, which is known as ischemic preconditioning. As a potent endogenous form of cardioprotection against ischemia–reperfusion injury, ischemic preconditioning has been described to be a biphasic event: the acute phase is limited to 1–3 h after a brief ischemic stimulus, and the delayed phase emerges 24 h later and may last up to 72 h (Murry et al., 1986, Tang et al., 1996). Opioids have been shown to confer both the acute and the delayed phase of cardioprotection similar to ischemic preconditioning (Schultz et al., 1996, Patel et al., 2003). As one of the most widely used opioids for the treatment of pain, morphine has been shown to induce acute cardioprotection in cardiac myocytes (McPherson and Yao, 2001), isolated working hearts (Shi et al., 2003) and in vivo models (Schultz et al., 1996, Ludwig et al., 2003). We have previously reported that morphine also produces delayed cardioprotection in mice 24 h after administration (Jiang et al., 2004).
Cardioprotection induced by preconditioning is a complex process that involves a network of intricate regulatory mechanisms at the levels of cell signaling and gene expression. Pharmacological and genetic evidence has convincingly shown that the inducible nitric oxide synthase (iNOS) plays a necessary role in mediating the cardioprotective effects conferred by ischemic preconditioning. In our previous work, we have also demonstrated an essential role for iNOS in mediating morphine-induced delayed cardioprotection (Jiang et al., 2004). Besides iNOS, recent evidence indicates that cardioprotection induced by ischemic preconditioning and δ-opioid receptor agonists is mediated by cyclooxygenase-2 (COX-2) and that COX-2-dependent synthesis of prostanoids is necessary for the observed cardioprotective effects (Shinmura et al., 2000, Shinmura et al., 2002a, Shinmura et al., 2002b, Kodani et al., 2002). Since both iNOS and COX-2 are obligatory mediators of ischemic preconditioning, the questions arise as to whether COX-2 is also involved in mediating the cardioprotection induced by morphine and whether there is an interaction between iNOS and COX-2 or they act as independent effectors of morphine-induced cardioprotection.
Therefore the current study was designed to define the function of COX-2 in morphine-induced biphasic cardioprotection in a murine model of coronary artery occlusion in vivo and to investigate the effect of iNOS on COX-2 in mediating the cardioprotection using an iNOS-selective inhibitor and iNOS gene-knockout mice.
Section snippets
Animals
Adult male wild type (WT) mice (B6129PF2/J) and iNOS gene-knockout (−/−) mice (B6, 129) (Jackson Laboratory, Bar Harbor, ME) weighing 25 to 30 g were used in this study (Guo et al., 1999). The experimental protocol was approved by the Ethics Review Committee for Animal Experimentation of Hamamatsu University School of Medicine and in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
Drugs and chemicals
Morphine was purchased from Shionogi Co. (Osaka, Japan). NS-398, S-methylthiourea sulfate
Results
A total of 45 mice were initially included in the protocol for the infarct-size study. Five mice were excluded because of a marked hypotension (MAP < 30 mm Hg) during coronary occlusion and reperfusion. Another two mice were omitted as a result of technical difficulties with the experimental preparation. Complete data were obtained in the remaining 38 mice. Final numbers in the groups are as follows: Control n = 6; M 1 h, n = 6; M 24 h, n = 7; NS, n = 6; M 1 h + NS, n = 6; M 24 h + NS, n = 7.
Discussion
The salient findings of the current study can be summarized as follows: (1) morphine induces biphasic cardioprotection in mice. Only the delayed cardioprotection, but not the acute phase, is associated with the up-regulation of myocardial content of PGs and expression of COX-2, and is abolished by selective inhibition of COX-2. (2) Selective inhibition of iNOS or targeted abrogation of iNOS gene blocks the enhancement of myocardial PGs but does not inhibit the up-regulation of COX-2 expression.
Conclusion
In conclusion, the current study demonstrates that the delayed cardioprotection induced by morphine, but not the acute phase, is mediated by COX-2 via an iNOS-dependent pathway. To the best of our knowledge, this is the first study which provides a direct link between COX-2 pathway and iNOS pathway in mediating morphine-induced delayed cardioprotection.
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Xiaojing Jiang and Enyi Shi contributed equally to this work. Dr. Shi is supported by Japan Society for the Promotion of Science. Xiaojing Jiang and Enyi Shi are currently affiliated with China Medical University.