Reactive oxygen species (ROS) generated during ischemia-reperfusion (I/R) enhance myocardial injury, but brief periods of myocardial ischemia followed by reperfusion [ischemic preconditioning (IP)] induce cardioprotection. Ischemia is reported to stimulate glucose uptake through the translocation of GLUT-4 from the intracellular vesicles to the sarcolemma. In the present study we demonstrated involvement of ROS in IP-mediated GLUT-4 translocation along with increased expression of caveolin (Cav)-3, phospho (p)-endothelial nitric oxide synthase (eNOS), p-Akt, and decreased expression of Cav-1. The rats were divided into the following groups: 1) control sham, 2) N-acetyl-L-cysteine (NAC, free radical scavenger) sham (NS), 3) I/R, 4) IP + I/R (IP), and 5) NAC + IP (IPN). IP was performed by four cycles of 4 min of ischemia and 4 min of reperfusion followed by 30 min of ischemia and 3, 24, 48 h of reperfusion, depending on the protocol. Increased mRNA expression of GLUT-4 and Cav-3 was observed after 3 h of reperfusion in the IP group compared with other groups. IP increased expression of GLUT-4, Cav-3, and p-AKT and p-eNOS compared with I/R. Coimmunoprecipitation demonstrated decreased association of Cav-1/eNOS in the IP group compared with the I/R group. Significant GLUT-4 and Cav-3 association was also observed in the IP group. This association was disrupted when NAC was used in conjunction with IP. It clearly documents a significant role of ROS signaling in Akt/eNOS/Cav-3-mediated GLUT-4 translocation and association in IP myocardium. In conclusion, we demonstrated a novel redox mechanism in IP-induced eNOS and GLUT-4 translocation and the role of caveolar paradox in making the heart euglycemic during the process of ischemia, leading to myocardial protection in a clinically relevant rat ischemic model.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18882号 / 医博第3993号 / 新制||医||1009(附属図書館) / 31833 / 京都大学大学院医学研究科医学専攻 / (主査)教授 宮本 享, 教授 小泉 昭夫, 教授 村井 俊哉 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The purpose of this study was to characterize a novel technique to study neurogenic control of cutaneous vasodilation. We monitored skin blood flow (SkBF) during in-vivo electrical stimulation (e-stim) intended to activate cutaneous nerves and used intradermal microdialysis to deliver receptor antagonists to characterize their contribution to cutaneous vasodilation. We examined the role of acetylcholine receptors (RACh), nitric oxide (NO), and vasoactive intestinal peptide receptors (RVIP) on the cutaneous vasodilation induced by e-stim in the absence of the sympathetic adrenergic nervous system. Six men and three women participated in the study. Three intradermal microdialysis probes were placed in the skin of the dorsal side of their forearm. The adrenergic nervous system was eliminated by delivery of a cocktail of phentolamine (0.01 mg/ml), propranolol (1 mM), and BIBP-3226 (10 µM). At one skin site atropine (0.1 mg/ml) was delivered to block RACh. At a second site we blocked nitric oxide synthase (NOS, 10 mM L-NAME) and RACh. Finally at the third site, we blocked RACh, NOS, and RVIP (0.47 mg/ml VIP10-28). The SkBF response to 1 minute stages of graded increases in frequency (0.2, 1, 2, 4, 8, and 32 Hz) at a current of 1.0 ± 0.1 mA was used to generate a stimulus-response curve before and after drug delivery. At skin site 1 RACh blockade decreased the area under curve (AUC) by 4% from 614 ± 279 to 591 ± 331 (p>0.05). Nitric oxide synthase and RACh blockade reduced the vasodilator response to e-stim by 23% from 1036 ± 457 to 801 ± 448 AUC (p>0.05). Combined NOS, RACh, and RVIP blockade reduced the vasodilator response by 48% from 802 ± 412 to 418 ± 268 AUC (p=0.07). RACh blockade had no effect on the vasodilator response to e-stim. However, in these preliminary studies both NOS and RVIP blockade provided some attenuation of the cutaneous vasodilator response associated with e-stim. Additional studies will focus on these two neurotransmitters as this novel method is refined. Advantages of e-stim include activating the sympathetic nervous system without activating local and humoral factors and studying neurotransmitters in an in-vivo setting during rest, thermal stress, or exercise.

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