Evidence against a role of inducible nitric oxide synthase in the endothelial protective effects of delayed preconditioning

Abstract

Preconditioning the heart with brief periods of ischaemia induces delayed endothelial protection against reperfusion injury, but the precise mechanisms involved in this endogenous protein are still unclear. Induction of the type II-nitric oxide synthase (iNOS) acts as a mediator of the preconditioning against myocardial infarction and stunning. The present study was designed to assess whether iNOS also contributes to the delayed endothelial protective effects of preconditioning. Rats were subjected to 20 min ischaemia followed by 60 min reperfusion 24 h after sham surgery or preconditioning (one cycle or 2 min ischaemia/5 min reperfusion and two cycles of 5 min ischaemia/5 min reperfusion). At the end of the reperfusion, coronary segments were removed distal to the site of occlusion and mounted in wire myographs. Ischaemia-reperfusion (I/R) decreased the endothelium-dependent relaxations to acetylcholine (maximal relaxations: sham, 66+/-5%; I/R, 40+/-1%; P<0.05) and this impairment was prevented by preconditioning (maximal relaxation: 61+/-6%). Administration of N-(3-aminomethyl)benzyl)acetaminide (1400W), a highly selective inhibitor for iNOS, 10 min before prolonged ischaemia did not modify the beneficial effect of preconditioning (maximal relaxation: 66+/-5%). These data show that preconditioning induces delayed protection against reperfusion-injury. However, in contrast to the myocytes, these endothelial protective effects of delayed preconditioning do not involve iNOS.

Figure 1

Experimental groups and treatment protocols. Solid boxes indicate periods of myocardial ischaemia induced by the left coronary artery occlusion. Preconditioning was performed by occlusion of the artery for 2 min followed by 5 min reperfusion, and two periods of 5 min occlusion separated by 5 min reperfusion. All animals were left 24 h before the infarct protocol was performed. This consisted of 20 min coronary occlusion followed by 60 min reperfusion. N-(3-(aminomethyl)benzyl)acetaminide (1400W) was given 10 min before the infarct coronary occlusion.

Figure 2

Contractile responses induced by increasing concentrations of serotonin. Serotonin does not induce endothelium-dependent relaxations in rat coronary arteries and thus induces only smooth muscle contraction. I/R, ischaemia-reperfusion; PC, preconditioning; 1400W, N-(3-(aminomethyl)benzyl)acetaminide. Contractions are expressed as percentage of maximal response and values are mean±s.e.mean.

Figure 3

Relaxing responses induced by increasing concentrations of the NO donor SIN-1 in coronary arteries isolated from rats of the six groups. The arteries segments were precontracted by serotonin (10⁻⁵ M). I/R, ischaemia-reperfusion; PC, preconditioning; 1400W, N-(3-(aminomethyl)benzyl)acetaminide. Relaxations are expressed as percentage of the contractile response to serotonin and values are mean±s.e.mean.

Figure 4

Relaxing responses induced by increasing concentrations of acetylcholine in coronary arteries isolated from sham rats, rats subjected to the infarct protocol and rats preconditioned (PC) 24 h before ischaemia-reperfusion (I/R). Arteries were pre-contracted by serotonin (10⁻⁵ M). Relaxations are expressed as percentage of contractile response to serotonin and values are mean±s.e.mean. ^*P<0.05 and ^**P<0.01 vs sham; #P<0.05 vs PC.

Figure 5

Effect of 1400W on relaxing responses induced by increasing concentrations of acetylcholine after pre-contraction by serotonin (10⁻⁵ M). (a) Coronary arteries isolated from sham-operated rats. (b) Coronary arteries isolated from rats subjected to ischaemia-reperfusion (I/R). (c) Coronary arteries isolated from rats preconditioned (PC) 24 h before the infarct protocol. Relaxations are expressed as percentage of contractile response to serotonin and values are mean±s.e.mean.

Figure 6

In vivo efficiency of 1400W. (a) Mean arterial blood pressure before administration of LPS. (b) Mean arterial blood pressure 3 h after administration of LPS (3 mg kg⁻¹, i.v.) and before administration of 1400W. (c) Mean arterial blood pressure 90 min after administration of 1400W (1 mg kg⁻¹) or solvent. Values are mean±s.e.mean. ^*P<0.05 vs control.