ACTIVATION OF DISTINCT CAMP- AND CGMP-DEPENDENT PATHWAYS BY NO IN CARDIOMYOCYTES

最近发现，NO供体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。我们研究了NO对抗心肌收缩作用的亚细胞机制，并研究了在INDO-1负荷的成人心肌细胞中，NO是否仅通过激活鸟苷环化酶(GC)来调节收缩，或者是否通过cGMP/PKG非依赖机制来调节收缩。高浓度的NO供体S-亚硝基-N-乙酰青霉胺(SNAP，100um)使心肌细胞收缩幅度(TA)显著降低24.4+/-4.5%(对钙瞬变(CAT)和总cAMP无明显影响)，低浓度SNAP(1 umol/L)显著增加TA(38+/-10%)、CAT(26+/-10%)和cAMP水平(6.2~8.5pmol/mg蛋白)。100umol/L SNAP的负收缩反应在PKG特异性阻断剂KT5823(1 umol/L)的存在下被完全消除，而1 umol/L SNAP的正收缩反应在GC选择性抑制剂ODQ(10umol/L)单独存在时仍然存在，但在ODQ和特异性抑制cAMP类似物RP-8-CPT-cAMP(100umol/L)的存在下完全取消。细胞悬液中的平行实验表明，在低浓度(0.1-1umol/L)的SNAP(AC，比基础活性高18-20%)时，AC活性显著增加。结论：NO对心肌细胞AC和GC均有调节作用。高水平的NO导致cGMP的大量增加和负性变力作用，这种负性变力作用是由依赖PKG的肌丝对钙的反应性降低所介导的。低水平的NO至少部分通过cGMP非依赖性激活AC来增加cAMP，并诱导正性收缩反应。-心肌细胞，收缩机制，一氧化氮，cAMP，cGMP

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. - cardiac myocytes, contractile mechanisms, nitric oxide, cAMP, cGMP