MECHANISMS OF SIGNAL TRANSDUCTION OF CARDIAC OPIOID RECEPTOR STIMULATION

虽然阿片肽(OP)与儿茶酚胺从 心脏中的神经末梢；由心脏产生和分泌 肌细胞；能够激活驻留在其上的OP受体 心脏细胞膜；OP在心脏功能中的生理作用 病理生理学，包括信号转导机制，已经 只得到了很少的调查。因此，我们最近已经证明了OP的 具有突触后心脏细胞内信号传递作用，这涉及一种 与β_1-肾上腺素能受体(β_1-AR)的强“串扰” 刺激途径。这项工作确定了串扰在 Delta-OPR和Beta1-AR通过百日咳毒素敏感(PTX)发生 完整分离的参与腺酰环化酶抑制的Gi蛋白 心脏制剂和分离的心肌细胞。在非心脏细胞中， 虽然 Delta-OPR发挥直接作用，涉及激活 磷脂酶C对肌醇磷脂代谢和IP3形成的直接作用 证据表明 Delta-OPR偶联百日咳毒素不敏感的Gq蛋白或 到目前为止，对PTX敏感的Gi蛋白还不可用。本实验室以前曾 显示剂量依赖的负性肌力作用，包括 细胞内[Ca~(2+)]和全细胞电压抑制发生在心脏 肌细胞。我们之前还展示了OP及其 前脑啡肽原信使核糖核酸随着年龄的增加而显著增加。根据…… 这些发现，是一项关于 -亮氨酸脑啡肽(LE)对6-羟基多巴胺中OPR的刺激 年轻大鼠(6个月)和老年大鼠(24个月)的预处理离体心在 进步。LE以剂量依赖的方式降低峰值收缩压 在6个月和24个月大鼠中，但在24个月大鼠中程度更大。EC50是 在6个月内约10-8M le，但在24个月内约10-9M le(p 大于0.05，n=4)。PTX敏感蛋白作用的初步评价 Gi-蛋白在LE的直接作用表明，PTX预处理有 不抑制LE诱导的负性肌力作用 LE与β1-AR信号转导通路的相互作用。 人离体心cAMP、IP3和PKC的测定 所有小组都在进行中。两个人之间的串音 -OPR和Beta1-AR信号转导通路是重要的 心脏兴奋-收缩偶联的调节机制。 目前，我们的数据表明，有两种类型的G蛋白与 这个 Delta-OPR，参与互动和直接影响 阿片肽。完全阐明这些细胞内信号 转导途径及其作用机制，特别是在 衰老或疾病的过程仍然是这个项目的重点。

Although opioid peptides (OP) are: coreleased with catecholamines from neuronal endings in the heart; are produced and secreted by cardiac myocytes; capable of activating OP receptors (OPR) which reside on cardiac cell membranes; the physiological role of OP's in cardiac function and pathophysiology,including the mechanism of signal transduction,has received meager investigation. Thus we have recently shown that OP's have a postsynaptic cardiac intracellular signalling role which involves a potent "cross-talk" with the beta1-adrenergic receptor (beta1-AR) stimulation pathway. This work identified that the cross-talk between delta-OPR and beta1-AR occurs via a pertussis toxin sensitive (PTX) Gi-protein involved with adenylyl cyclase inhibition in intact isolated heart preparations and isolated cardiac myocytes. In non-cardiac cells, although delta-OPR exert direct effects which involve the activation of phospholipase C for phosphoinositide metabolism and IP3 formation, direct evidence that demonstrates delta-OPR coupling to either pertussis toxin-insensitive Gq-protein or PTX-sensitive Gi-protein is unavailable to date. This lab has previously shown that a dose-dependant negative inotropic effect, including inhibition of intracellular [Ca2+]and whole cell voltage, occurs in cardiac myocytes. We have also previously shown that OP's and their preproenkephalin mRNA are significantly increased with age. In light of these findings, a study of the direct effects of -OPR stimulation by leucine-enkephalin (LE) in 6-hydroxydopamine pretreated isolated hearts from young adult (6mo)and old rats (24mo)is in progress. LE reduced peak systolic pressure in a dose dependent manner in 6 and 24mo rats but to a greater extent in 24mo rats. The EC50 was approximately 10-8 M LE in 6 mo but 10-9M LE in 24mo hearts (p less than 0.05,n=4). A preliminary assessment of the role of PTX-sensitive Gi-protein in the direct effects of LE indicated that PTX pretreatment did not inhibit the LE-induced negative inotropy in either age group, as is LE's interactive effect with the beta1-AR signalling transduction pathway. Assays for the measurement of cAMP, IP3 and PKC in isolated hearts from all groups are in progress. The cross-talk between -OPR and beta1-AR signal transduction pathways is an important mechanism in the regulation of cardiac excitation-contraction coupling. Presently, our data indicates that two types of G-protein are coupled to the delta-OPR that are involved with the interactive and direct effects of opioid peptides. Complete elucidation of these intracellular signal transduction pathways and their mechanism of action, particularly during the process of aging or disease remains the focus of this project.