Activation of distinct cAMP- and cGMP-dependent pathways by NO in cardiomyocytes

NO 激活心肌细胞中不同的 cAMP 和 cGMP 依赖性途径

• 批准号: 6431412
• 负责人: Edward G Lakatta
• 金额: --
• 依托单位: NATIONAL INSTITUTE ON AGING
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6431412
• 关键词: aging; biological signal transduction; calcium channel; calcium flux; enzyme inhibitors; heart contraction; laboratory rat; myocardium; myofibrils; nitric oxide; receptor coupling; vascular endothelium

NO donors were recently shown to produce biphasic contractile effects in cardiac tissue, with augmentation at low NO levels and depression at high NO levels. We examined the subcellular mechanisms involved in the opposing effects of NO on cardiac contraction, and investigated whether NO modulates contraction exclusively via guanylyl cyclase (GC) activation, or if some contribution occurs via cGMP/PKG-independent mechanisms, in indo-1 loaded adult cardiac myocytes. While a high concentration of the NO donor, S-nitroso N-acetyl-penicillamine (SNAP, 100 uM), significantly attenuated contraction amplitude (TA) by 24.4 +/- 4.5% (without changing the Ca2+ transient (CaT) or total cAMP), a low concentration of SNAP (1 umol/L) significantly increased TA (38 +/- 10%), CaT (26 +/- 10%), and cAMP levels (from 6.2 to 8.5 pmol/mg protein). The negative contractile response of 100 umol/L SNAP was completely abolished in the presence of the specific blocker of PKG, KT 5823 (1 umol/L); the positive contractile response of 1 umol/L SNAP persisted despite the presence of the selective inhibitor of GC, ODQ (10 umol/L) alone, but was completely abolished in the presence of ODQ plus the specific inhibitory cAMP analog Rp-8-CPT-cAMPS (100 umol/L). Parallel experiments in cell suspensions showed significant increases in adenylyl cyclase (AC) activity at low concentrations (0.1-1 umol/L) of SNAP (AC, 18-20% above basal activity). We conclude that NO can regulate both AC and GC in cardiac myocytes. High levels of NO induce large increases in cGMP and a negative inotropic effect mediated by a PKG-dependent reduction in myofilament responsiveness to Ca2+. Low levels of NO increase cAMP at least in part by a novel cGMP-independent activation of AC and induces a positive contractile response.

最近显示，NO 供体可在心脏组织中产生双相收缩效应，在低 NO 水平时增强，在高 NO 水平时抑制。 我们检查了 NO 对心脏收缩的相反作用所涉及的亚细胞机制，并研究了在负载 indo-1 的成年心肌细胞中，NO 是否仅通过鸟苷酸环化酶 (GC) 激活来调节收缩，或者是否通过 cGMP/PKG 独立机制发生了一些贡献。 高浓度的 NO 供体 S-亚硝基 N-乙酰青霉胺 (SNAP，100 uM) 显着减弱收缩幅度 (TA) 24.4 +/- 4.5%（不改变 Ca2+ 瞬态 (CaT) 或总 cAMP），低浓度 SNAP (1 umol/L) 显着增加 TA (38 +/- 10%)、CaT (26 +/- 10%)，和 cAMP 水平 （从 6.2 到 8.5 pmol/mg 蛋白质）。 在 PKG 特异性阻断剂 KT 5823 (1 umol/L) 存在下，100 umol/L SNAP 的负收缩反应被完全消除；尽管单独存在 GC 选择性抑制剂 ODQ (10 umol/L)，1 umol/L SNAP 的正向收缩反应仍持续存在，但在 ODQ 加特异性抑制性 cAMP 类似物 Rp-8-CPT-cAMPS (100 umol/L) 存在下完全消除。 细胞悬浮液中的平行实验表明，低浓度（0.1-1 umol/L）SNAP 时腺苷酸环化酶（AC）活性显着增加（AC，比基础活性高 18-20%）。 我们得出结论，NO 可以调节心肌细胞中的 AC 和 GC。 高水平的 NO 会诱导 cGMP 大幅增加，以及由 PKG 依赖性肌丝对 Ca2+ 反应性降低介导的负性肌力作用。 低水平的 NO 至少部分通过一种新型的不依赖于 cGMP 的 AC 激活来增加 cAMP，并诱导积极的收缩反应。