Opioid-induced potentiation of the exercise pressor reflex via acid-sensing ion channels (ASIC3) in health and simulated peripheral artery disease

项目总结 肌肉(骨骼或心脏)的慢性缺血会导致乳酸和其他炎症的积聚。 代谢物，随后间质pH值下降。随之而来的组织酸中毒在触发 跛行(行走引起的疼痛)和胸痛。高亲和力和选择性的MU阿片受体(MOR) 在缺血和炎症条件下，也会释放激动剂，即内吗啡素。酸敏离子 通道(ASIC)是感知与组织酸中毒相关的pH变化的关键角色， 炎症和疼痛。ASIC在感觉(即背根神经节，DRG)和中枢均有高表达 神经元是电压不敏感的去极化阳离子通道。内吗啡素-1(E-1)和-2(E-2)是 已知的激活MOR和发挥止痛作用的四肽。然而，我们最近的发现表明，E- 1和E-2可显著增强酸诱导的L细胞和小鼠成纤维细胞ASIC3电流 急性分离的背根节神经元，不依赖于吗啡的激活。两种阿片类药物的增强作用都是 结扎股动脉(外周动脉疾病)大鼠背根神经节神经元数量显著增加 模型)。重要的是，我们的体内数据显示，E-2显著增强了乳酸诱导的 大鼠平均动脉压升高。E-2介导的增强作用被显著减弱 ASIC3阻滞剂APETx2，但对纳洛酮不敏感。我们的长期目标是了解 内吗啡和临床使用阿片类药物(羟考酮、芬太尼、瑞芬太尼)的作用机制 调节ASIC3电流，通道上的相互作用部位，以及它们如何调节运动升压 由肌肉收缩引起的反射(EPR)。我们的总体假设是慢性肌肉 缺血-伴随着酸化和炎症的环境，以及ASIC3表达增强- 增加内吗啡素的释放，导致ASIC3电流增强。总体效果是过度兴奋 产生夸大EPR的主要传入。这一假设将使用互补性在 体外和体内的方法。我们将确定内吗啡的生物物理和药理作用。 处方阿片类药物对L细胞异源表达ASIC3的影响。我们将确定ASIC3通道 功能上否定E-1和E-2作用的残基，但离开通道的基本生理 完好无损。我们还将比较阿片类激动剂对背根节ASIC通道电流的药理作用 “自由灌流”或“结扎”股动脉大鼠的神经元。我们将考察E-1、E-2的影响 处方阿片类药物对野生型和ASIC3基因敲除大鼠诱发的EPR的影响 动脉被“自由灌流”或“结扎”。总体而言，这些实验将提供关于如何 阿片肽在缺血条件下增强ASIC电流，从而增强升压反应 运动和与长期使用处方阿片类药物(即阿片类药物诱导的)相关的可能恶化的疼痛 痛觉过敏)。

PROJECT SUMMARY Chronic ischemia in muscle (skeletal or cardiac) leads to accumulation of lactic acid and other inflammatory metabolites with subsequent drop in interstitial pH. The ensuing tissue acidosis plays a major role in triggering claudication (walking-induced pain) and chest pain. The high-affinity and selective mu opioid receptor (MOR) agonists, endomorphins, are also released under ischemic and inflammatory conditions. Acid-sensing ion channels (ASIC) are key players in the perception of pH changes associated with tissue acidosis, inflammation and pain. ASIC are expressed highly in sensory (i.e. dorsal root ganglion, DRG) and central neurons and are voltage-insensitive, depolarizing cationic channels. Endomorphin-1 (E-1) and -2 (E-2) are tetrapeptides known to activate MOR and exert analgesic effects. However, our recent findings indicate that E- 1 and E-2 significantly potentiated the acid-induced ASIC3 currents in transfected fibroblast L-cells and in acutely isolated DRG neurons independent of MOR activation. The potentiation by both opioids was significantly greater in DRG neurons isolated from rats with ligated femoral arteries (peripheral artery disease model). Importantly, our in vivo data demonstrated that E-2 significantly enhanced the lactic acid-induced increase in mean arterial pressure in rats. The E-2-mediated enhancement was significantly attenuated by the ASIC3 blocker, APETx2, but was insensitive to naloxone. Our long-term goal is to understand the mechanisms by which endomorphins and clinically employed opiates (oxycodone, fentanyl, remifentanil) modulate ASIC3 currents, the interacting site on the channel, and how they regulate the exercise pressor reflex (EPR) which is evoked by muscle contraction. Our overall hypothesis is that chronic muscle ischemia—accompanied by an acidified and inflamed environment, and enhanced ASIC3 expression— elevates endomorphin release, leading to enhanced ASIC3 currents. The overall effect is hyperexcitability of primary afferents that produces an exaggerated EPR. This hypothesis will be tested using complementary in vitro and in vivo approaches. We will determine the biophysical and pharmacological effects of endomorphins and prescription opiates on heterologously expressed ASIC3 in L-cells. We will identify ASIC3 channel residues that functionally negate the actions of E-1 and E-2, but leave the basic physiology of the channel intact. We also will compare the opioid agonist pharmacological profiles on ASIC channel currents in DRG neurons from rats with “freely perfused” or “ligated” femoral arteries. We will examine the effects of E-1, E-2 and prescription opiates on the EPR evoked in both wild-type and ASIC3 knockout rats in which the femoral arteries are “freely perfused” or “ligated”. Overall, these experiments will provide novel information about how opioid peptides potentiate ASIC currents under ischemic conditions, thereby enhancing pressor responses to exercise and possibly worsening pain associated with chronic use of prescription opiates (i.e., opioid-induced hyperalgesia).