Proceedings of The Physiological Society

University of Oxford (2011) Proc Physiol Soc 23, PC141

Poster Communications

The Involvement of Adenosine and Opiods in Remote Ischaemic Pre- and Postconditioning protection of isolated rat ventricular myocytes

R. H. Ghelani1, S. A. Edroos1, H. E. Turrell1, G. Rodrigo1

1. Cardiovascular Sciences, Leicester University, Leicester, United Kingdom.


The powerful protection afforded by ischaemic pre- and post-conditioning can also be triggered by remote conditioning, where the conditioning ischaemia is applied to a remote organ. Both remote Ischaemic Preconditioning (rIPC) and Postconditioning (rIPostC) are thought to involve the release of signalling molecules by the ischaemic tissue. Adenosine and Opioids are the most documented autocoids responsible for triggering the protection in rIPC; however, their role in rIPostC is less well known. To remove the modulating influence of endothelium and smooth muscle in the heart’s response to remote conditioning, we have used an isolated ventricular myocyte model. Single ventricular myocytes were isolated from Adult Male Wistar rat hearts. Ischaemia was simulated using a cell pelleting technique with a layer of mineral oil to prevent gaseous diffusion, and reperfusion by dispersing the myocyte pellet into oxygenated Tyrode (10 mM Glucose + 5 mM pyruvate) for 10 minutes followed by exposure to hypo-osmotic solution (33%). Cell viability was determined by the ability to exclude Trypan blue (1). To simulate remote conditioning, a separate Langendorff perfused rat heart was subject to 5 minute cycles of ischaemic preconditioning and the perfusate collected during the reperfusion phase. Myocytes were treated with this perfusate for 10 minutes prior to simulated ischaemia to induce rIPC and at the time of reperfusion of the pelleted cells to induce rIPostC. Data are mean ± S.E.M (number of experiments, hearts; one-way ANOVA and Tukey’s multiple comparison post-hoc test. We found that the % viability following ischaemia/reperfusion of control myocytes was 61.1 ± 2.7% (n=35,20), which was significantly increased in rIPC to 71.6 ± 2.7% (n=28,20; p<0.05) and rIPostC 78.4 ± 2.7 % (n=31,20; p<0.001). We used this model to look at the effect of non-specific Opioid (Naloxone) and Adenosine (8-p-Sulphophenyl Theophylline, 8p-SPT) receptor inhibitors, with the inhibitor present throughout the experimental protocol. Our data show that 100µM Naloxone reversed the protective effects of rIPC, reducing viability to 60.1 ± 5.95% (n=11, 7; p>0.05) and rIPostC to 63.7 ± 3.6% (n=12, 7; p<0.05). We also found that 100µM 8p-SPT reversed the protective effects of rIPC reducing viability to 55.8 ± 6.3% (n = 9, 8; p<0.05) and rIPostC to 67.1 ± 5.3% (n=11, 8; p>0.05). Our data shows that rIPC and rIPostC protect against reperfusion injury at the cellular level. The data also shows that Opioids and Adenosine are likely triggering molecules of rIPC and rIPostC.

Where applicable, experiments conform with Society ethical requirements