Exploring the role and inter-relationship among nitric oxide, opioids, and KATP channels in the signaling pathway underlying remote ischemic preconditioning induced cardioprotection in rats

Document Type : Original Article


Department of Pharmaceutical Sciences and Drug Research, Punjabi University Patiala, 147002 India


Objective(s): This study explored the inter-relationship among nitric oxide, opioids, and KATP channels in the signaling pathway underlying remote ischemic preconditioning (RIPC) conferred cardioprotection.
Materials and Methods: Blood pressure cuff was placed around the hind limb of the animal and RIPC was performed by 4 cycles of inflation (5 min) followed by deflation (5 min). An ex vivo Langendorff’s isolated rat heart model was used to induce ischemia (of 30 min duration)-reperfusion (of 120 min duration) injury.
Results: RIPC significantly decreased ischemia-reperfusion associated injury assessed by decrease in myocardial infarct, LDH and CK release, improvement in postischemic left ventricular function, LVDP, dp/dtmax, and dp/dtmin. Pretreatment with L-NAME and naloxone abolished RIPC-induced cardioprotection. Moreover, preconditioning with sodium nitroprusside (SNP) and morphine produced a cardioprotective effect in a similar manner to RIPC. L-NAME, but not naloxone, attenuated RIPC and SNP preconditioning-induced increase in serum nitrite levels. Morphine preconditioning did not increase the NO levels, probably suggesting that opioids may be the downstream mediators of NO. Furthermore, glibenclamide and naloxone blocked cardioprotection conferred by morphine and SNP, respectively.
Conclusion: It may be proposed that the actions of NO, opioids, and KATP channels are interlinked. It is possible to suggest that RIPC may induce the release of NO from endothelium, which may trigger the synthesis of endogenous opioids, which in turn may activate heart localized KATP channels to induce cardioprotection.


Main Subjects

1. Hausenloy DJ, Yellon DM. Remote ischaemic preconditioning: underlying mechanisms and clinical application. Cardiovasc Res 2008; 79:377–386.
2. Murry CE, Jennings RB, Reimer KA. Preconditioning with ischemia: a delay of lethal cell injury in ischemic myocardium. Circulation 1986; 74:1124–1136.
3. Taliyan R, Singh M, Sharma PL, Yadav HN, Sidhu KS. Possible involvement of α1-adrenergic receptor and K (ATP) channels in cardioprotective effect of remote aortic preconditioning in isolated rat heart. J Cardiovasc Dis Res 2010; 1:145–151.
4. Wolfrum S, Schneider K, Heidbreder M, Nienstedt J, Dominiak P, Dendorfer A. Remote preconditioning protects the heart by activating myocardial PKCepsilon-isoform. Cardiovasc Res 2002; 55:583–589.
5. Kant R, Diwan V, Jaggi AS, Singh N, Singh D. Remote renal preconditioning-induced cardioprotection: a key role of hypoxia inducible factor-prolyl 4-hydroxylases. Mol Cell Biochem 2008; 312:25–31.
6. Tokuno S, Hinokiyama K, Tokuno K, Löwbeer C, Hansson L, Valen G. Spontaneous ischemic events in the brain and heart adapt the hearts of severely atherosclerotic mice to ischemia. Arterioscler Thromb Vasc Biol 2002; 22:995–1001.
7. Liem DA, Verdouw PD, Ploeg H, Kazim S, Duncker DJ. Sites of action of adenosine in interorgan preconditioning of the heart. Am J Physiol Heart Circ Physiol 2002; 283:H29–H37.
8. Przyklenk K, Bauer B, Ovize M, Kloner RA, Whittaker P. Regional ischemic ‘preconditioning’ protects remote virgin myocardium from subsequent sustained coronary occlusion. Circulation 1993; 87:893–899.
9. Kharbanda RK, Mortensen UM, White PA, Kristiansen SB, Schmidt MR, Hoschtitzky JA, et al. Transient limb ischemia induces remote ischemic preconditioning in vivo. Circulation 2002; 106:2881–2883.
10. Botker HE, Kharbanda R, Schmidt MR, Bøttcher M, Kaltoft AK, Terkelsen CJ, et al. Remote ischemic conditioning before hospital admission, as a complement to angioplasty, and effect on myocardial salvage in patients with acute myocardial infarction: a randomized trial. Lancet 2010; 375:727–734.
11. Ali N, Rizwi F, Iqbal A, Rashid A. Induced remote ischemic preconditioning on ischemia-reperfusion injury in patients undergoing coronary artery bypass. J Coll Physicians Surg Pak 2010; 20:427–431.
12. Cheung MM, Kharbanda RK, Konstantinov IE, Shimizu M, Meng HF, Li J, et al. Randomized controlled trial of the effects of RIPC on children undergoing cardiac surgery: first clinical application in humans. J Am Coll Cardiol 2006; 47:2277–2282.
13. Aggarwal S, Randhawa PK, Singh N, Jaggi AS. Preconditioning at a distance: involvement of endothelial vasoactive substances in cardioprotection against ischemia-reperfusion injury. Life Sci 2016; 151:250-258.
14. Kitakaze M, Node K, Minamino T, Inoue M, Hori M, Kamada T. Evidence for nitric oxide generation in the cardiomyocytes: its augmentation by hypoxia. J Am Coll Cardiol 1995; 27:2149-2154.
15. Node K, Kitakaze M, Kosaka H, Komamura K, Minamino T, Tada M, et al. Plasma nitric oxide end products are increased in the ischemic canine heart. Biochem Biophys Res Commun 1995; 211:370-374.
16. Ignarro LJ, Napoli C, Loscalzo J. Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide: an overview. Circ Res 2002; 90:21–28.
17. Bolli R. Cardioprotective function of inducible nitric oxide synthase and role of nitric oxide in myocardial ischemia and preconditioning: an overview of a decade of research. J Mol Cell Cardiol 2001; 33:1897–1918.
18. Shahid M, Tauseef M, Sharma KK, Fahim M. Brief femoral artery ischaemia provides protection against myocardial ischaemia-reperfusion injury in rats: the possible mechanisms. Exp Physiol 2008; 93:954–968.
19. Rassaf T, Totzeck M, Cotta UB, Shiva S, Heusch G, Kelm M. Circulating nitrite contributes to cardioprotection by Remote Ischemic Preconditioning. Circ Res 2014; 114:1601-1610.
20. Headrick JP, Pepe S, Peart JN. Non-analgesic effects of opioids: cardiovascular effects of opioids and their receptor systems. Curr Pharm Des 2012; 18:6090-6100.
21. Romano MA, Seymour EM, Berry JA, McNish RA, Bolling SF. Relative contribution of endogenous opioids to myocardial ischemic tolerance. J Surg Res 2004; 118:32-37.
22. Tanaka K, Kersten JR, Riess ML. Opioid-induced cardioprotection. Curr Pharm Des 2014; 20:5696–5705.
23. Schultz JJ, Hsu AK, Gross GJ. Ischemic preconditioning in the intact rat heart is mediated by d1 but not j or μ opioid receptors. Circulation 1998; 97:1282–1289.
24. Gustein HB, Rubie EA, Mansour A, Akil H, Woodgett JR. Opioid effects on mitogen-activated protein kinase signaling cascade. Anesthesiology 1997; 87:1118–1126.
25. Schultz JJ, Hsu AK, Gross GJ. Ischemic preconditioning and morphine-induced cardioprotection involve the delta-opioid receptor in the intact rat heart. J Mol Cell Cardiol 1997; 29:2187–2195.
26. Patel HH, Moore J, Hsu AK, Gross GJ. Cardioprotection at a distance: mesenteric artery occlusion protects the myocardium via an opioid sensitive mechanism. J Mol Cell Cardiol 2002; 34:1317–1323.
27. Dickson EW, Tubbs RJ, Porcaro WA, Lee WJ, Blehar DJ, Carraway RE, et al. Myocardial preconditioning factors evoke mesenteric ischemic tolerance via opioid receptors and K(ATP) channels. Am J Physiol Heart Circ Physiol 2002; 283:H22–H28.
28. Noma A. ATP-regulated K channels in cardiac muscle. Nature 1983; 305:147–148.
29. Paucek P, Mironova G, Mahdi F, Beavis AD, Woldegiorgis G, Garlid KD. Reconstitution and partial purification of the glibenclamide-sensitive ATP-dependent K channel from rat liver and beef heart mitochondria. J Biol Chem 1992; 267:26062–26069.
30. Diwan V, Jaggi AS, Singh M, Singh N, Singh D. Possible involvement of erythropoietin in remote renal preconditioning-induced cardioprotection in rats. J Cardiovasc Pharmacol  2008; 51:126–130.
31. Yadav H, Singh M, Sharma P, Mittal D, Behl T, Kaur AP. Possible role of cyclooxygenase-2 in remote aortic preconditioning induced cardioprotection in rat heart. Pharmacologia 2012; 3:1-8.
32. Sharma R, Randhawa PK, Singh N, Jaggi AS. Possible role of thromboxane A2 in remote hind limb preconditioning-induced cardioprotection. Naunyn Schmiedebergs Arch Pharmacol 2016; 389:1-9.
33. Randhawa PK, Jaggi AS. Gadolinium and ruthenium red attenuate remote hind limb preconditioning-induced cardioprotection: possible role of TRP and especially TRPV channels. Naunyn Schmiedebergs Arch Pharmacol 2016; 389:887-896.
34. Singh B, Randhawa PK, Singh N, Jaggi AS. Investigations on the role of leukotrienes in remote hind limb preconditioning-induced cardioprotection in rats. Life Sci 2016; 152:238-243.
35. Fishbein MC, Meerbaum S, Rit J, Lando U, Kanmatsuse K, Mercier JC. Early phase acute myocardial infarct size quantification: validation of the triphenyltetrazolium chloride tissue enzyme staining technique. Am Heart J 1981; 101:593–600.
36. King JA. A routine method for estimation of lactate dehydrogenase activity. J Med Lab Tech 1959; 16:291–332.
37. Sastry KV, Moudgal RP, Mohan J, Tyagi JS, Rao GS. Spectrophotometric determination of serum nitrite and nitrate by copper-cadmium alloy. Anal Biochem 2002; 306:79-82.
38. Chen XG, Wu BY, Wang JK, Xiuying Wang, Peng Lin, Ming Yang. Mechanism of the protective effects of noninvasive limbs preconditioning on myocardial ischemia-reperfusion injury. Chin Med J (Engl) 2005; 118:1723-1727.
39. Weinbrenner C, Schulze F, Sárváry L, Strasser RH. Remote preconditioning by infrarenal aortic occlusion is operative via delta1-opioid receptors and free radicals in vivo in the rat heart. Cardiovasc Res 2004; 61:591-599.
40. Eippert F, Bingel U, Schoell E, Yacubian J, Büchel C. Blockade of endogenous opioid neurotransmission enhances acquisition of conditioned fear in humans. J Neurosci  2008; 28:5465-5472.
41. Armstead WM. Nitric oxide contributes to opioid release from glia during hypoxia. Brain Res 1998; 813:398-401.
42. Moses MA, Addison PD, Neligan PC, Ashrafpour H, Huang N, Zair M, et al. Mitochondrial KATP channels in hindlimb remote ischemic preconditioning of skeletal muscle against infarction. Am J Physiol Heart Circ Physiol 2005; 288:H559-H567.
43. Wu YN, Yu H, Zhu XH, Yuan HJ, Kang Y, Jiao JJ, et al. Noninvasive delayed limb ischemic preconditioning attenuates myocardial ischemia-reperfusion injury in rats by a mitochondrial K(ATP) channel dependent mechanism. Physiol Res 2011; 60:271–279.
44. Liang BT, Gross GJ. Direct preconditioning of cardiac myocytes via opioid receptors and KATP channels. Circ Res 1999; 84:1396-1400.