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

最近显示没有供体产生双相 心脏组织的收缩作用，在低NO时增强 在高NO水平下的抑郁症。我们研究了 亚细胞机制参与了NO的相反作用， 心脏收缩，并研究是否NO调节 收缩仅通过鸟苷酸环化酶（GC）激活，或者如果 一些贡献通过cGMP/PKG非依赖性发生 在indo-1负载的成年心肌细胞中，而高 NO供体S-亚硝基N-乙酰基青霉胺的浓度 (SNAP，100 μ M），显著减弱收缩幅度 (TA)24.4 +/- 4.5%（不改变Ca 2+瞬态（CaT）） 或总cAMP），低浓度SNAP（1 μ mol/L） 显著增加TA（38 +/- 10%）、CaT（26 +/- 10%）和 cAMP水平（6.2至8.5 pmol/mg蛋白质）。负 100 μ mol/L SNAP的收缩反应完全 在PKG特异性阻滞剂KT 5823存在下消除 （1 μ mol/L）; 1 μ mol/L SNAP的正性收缩反应 尽管存在GC的选择性抑制剂ODQ， (10 umol/L），但在存在 ODQ加特异性抑制性cAMP类似物Rp-8-CPT-cAMPS (100 umol/L）。在细胞悬浮液中的平行实验表明， 腺苷酸环化酶（AC）活性显著增加， SNAP浓度（0.1-1 μ mol/L）（AC，高于基础浓度18- 20 活动）。我们的结论是，NO可以调节AC和GC， 心肌细胞高水平的NO诱导大幅度增加， cGMP和负性肌力作用介导的 肌丝对Ca 2+反应性的PKG依赖性降低。 低水平的NO至少部分地通过一种新的 AC的cGMP非依赖性激活并诱导阳性 收缩反应

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.