Belhassen L, Kelly R A, Smith T W, Balligand J L
Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, Massachusetts 02115, USA.
J Clin Invest. 1996 Apr 15;97(8):1908-15. doi: 10.1172/JCI118622.
Cardiac myocytes express the nitric oxide synthase isoform originally identified in constitutive nitric oxide synthase cells (NOS3), which mediates the attenuation by muscarinic cholinergic agonists of beta-adrenergic stimulation of L-type calcium current and contractility in these cells. However, calcium current and contractility in these cells. However, the reciprocal regulation of NOS3 activity in myocytes by agents that elevate cAMP has not been reported. In this study, we show that NOS3 and mRNA and protein levels in cardiac myocytes are reduced both in vitro after treatment with cAMP elevating drugs, and in vivo after 3 d of treatment with milrinone, a type III cAMP phosphodiesterase inhibitor. This effect on NOS3 activity by cAMP is cell type specific because treatment of cardiac microvascular endothelial cells in vitro or in vivo did not decrease NOS3 mRNA or protein in these cells. NOS3 downregulation in myocytes appeared to be at the level of transcription since there was no modification of NOS3 mRNA half-life by agents that increase intracellular cAMP. To determine the functional effects of NOS3 downregulation, we examined the contractile responsiveness of isolated electrically paced ventricular myocytes, isolated from animals that had been treated in vivo with milrinone, to the beta-adrenergic agonist isoproterenol and the muscarinic cholinergic agonist carbamylcholine. There was no difference in baseline contractile function in cells that had been pretreated with cAMP elevating agents compared to controls, but cells exposed to milrinone in vivo exhibited an accentuation in their contractile responsiveness to isoproterenol compared to controls and a loss of responsiveness to carbamylcholine. Downregulation of myocyte NOS3 by sustained elevation of cAMP may have important implications for the regulation of myocardial contractile state by the autonomic nervous system.
心肌细胞表达最初在组成型一氧化氮合酶细胞中鉴定出的一氧化氮合酶同工型（NOS3），它介导毒蕈碱型胆碱能激动剂对这些细胞中L型钙电流和收缩性的β-肾上腺素能刺激的减弱。然而，这些细胞中的钙电流和收缩性。然而，尚未报道通过升高cAMP的药物对心肌细胞中NOS3活性的相互调节。在本研究中，我们表明，在用升高cAMP的药物处理后体外培养的心肌细胞中，以及在用III型cAMP磷酸二酯酶抑制剂米力农处理3天后体内的心肌细胞中，NOS3以及mRNA和蛋白质水平均降低。cAMP对NOS3活性的这种影响具有细胞类型特异性，因为体外或体内处理心脏微血管内皮细胞均未降低这些细胞中的NOS3 mRNA或蛋白质。心肌细胞中NOS3的下调似乎发生在转录水平，因为增加细胞内cAMP的药物未改变NOS3 mRNA的半衰期。为了确定NOS3下调的功能影响，我们检查了从体内用米力农处理的动物分离的离体电刺激心室肌细胞对β-肾上腺素能激动剂异丙肾上腺素和毒蕈碱型胆碱能激动剂氨甲酰胆碱的收缩反应性。与对照相比，用升高cAMP的药物预处理的细胞的基线收缩功能没有差异，但与对照相比，体内暴露于米力农的细胞对异丙肾上腺素的收缩反应性增强，而对氨甲酰胆碱的反应性丧失。cAMP持续升高导致的心肌细胞NOS3下调可能对自主神经系统对心肌收缩状态的调节具有重要意义。
