α-CGRP Mediated Delayed Preconditioning Induced By Nitroglycerin In Rat Intestine

Calcitonin gene-related peptide(CGRP), a principal transmitter in capsaicin-sensitive sensory nerves, is distributed widely in cardiovascular tissues and has a broad variety of biological effects including the most potent vasodilatory action known to date. In the present study, it has been shown that CGRP participates in the early and delayed preconditioning of myocardium, intestine, liver, and so on. Recently, Zhou et al reported that delayed cardioprotection induced by nitroglycerin(NTG) is related to stimulation of the release and synthesis of α-CGRP. Based on the case that CGRP mediates the protective effects of nitroglycerin-induced preconditioning in rat small intestine, we want to examine in this study weather it is in rat small intestine that delayed preconditioning induced by nitroglycerin is related to stimulation of the release and synthesis of α-CGRP.In this study, we first investigated the protective effects in the anesthetized rat small intestine of nitroglycerin (120μg/㎏,i.v.)in vivo experiment. The ischemic/reperfusion(I/R)injury was made by 30min occlusion of the super mesenteric artery followed by 60min reperfusion. The NTG group was intravenous (i.v.) injected with nitroglycerin 24 hours before the experiment. From the results we could see that the release of dehydsogenase(LDH)was reduced but the CGRP content in the plasma was increased significantly in the NTG group. Pretreatment with nitroglycerin produced less pronounced small intestine mucosal damage than in the I/R group(P<0.05). Pretreatment with capsaicin which specifically depletes the transmitters in capsaicin-sensitive sensory nerves, abolished the protection of NTG. All these show NTG could induce the delayed preconditioning in rat small intestine and the protective effect is positive correlative with the content of CGRP in plasma. The results reveal CGRP involves in the delayed preconditioning induced by NTG in rat small intestine.The second step we examined the possibility that the delayed preconditioning afforded by NTG in rat small intestine is related to stimulation of the release and synthesis of CGRP. Total RNA was isolated from the rat lumbar dorsal root ganglia(DRG)treated for 0h,8h and 24h with NTG. A previously validated semi-quantitative RT-PCR method(β－actin as the endogenous control sequence)was employed to quantify the mRNA expression ratio ofα- and β-CGRP isoform against ubiquitously expression β－actin. From the results we could see that NTG caused a significant increase in the expression of α-CGRP mRNA, but not β-CGRP mRNA, concomitantly with an increase in concentration of CGRP. Pretreatment with capsaicin which specifically exhausted the transmitter content of sensory nerves, abolished the increase effect of NTG. The result show NTG stimulate the synthesis of α-CGRP. The results reveal that the delayed preconditioning afforded by NTG in rat small intestine is related to stimulation of the release and synthesis ofα-CGRP.In order to examine the effect of NO-cGMP pathway in our study, methylene blue (MB,30mg/kg i.p.), an inhibitor of guanylate cyclase, was applied. From the results we could see that NTG could markedly elevate the level of serum nitric oxide(NO)(P<0.05). Pretreated with methylene blue, the increase of plasma CGRP andα-CGRP mRNA in DRG by NTG, but not the elevation of serum nitric oxide concentration, was abolished completely. All these show that nitroglycerin, as a donor of NO, inducing delayed preconditioning in rat small intestine is mediated by the α-CGRP isoform via the NO-cGMP pathway.