Nitric oxide synthase (NOS3) and contractile responsiveness to adrenergic and cholinergic agonists in the heart. Regulation of NOS3 transcription in vitro and in vivo by cyclic adenosine monophosphate in rat cardiac myocytes.

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.

[1]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[2]  J. Balligand,et al.  Nitric Oxide-dependent Parasympathetic Signaling Is Due to Activation of Constitutive Endothelial (Type III) Nitric Oxide Synthase in Cardiac Myocytes (*) , 1995, The Journal of Biological Chemistry.

[3]  P. Lelkes,et al.  Regulation of the adenylyl cyclase signaling system in various types of cultured endothelial cells , 1995, Journal of cellular biochemistry.

[4]  C. Lowenstein,et al.  Induction of NO synthase in rat cardiac microvascular endothelial cells by IL-1 beta and IFN-gamma. , 1995, The American journal of physiology.

[5]  C. Lowenstein,et al.  Cytokine-inducible nitric oxide synthase (iNOS) expression in cardiac myocytes. Characterization and regulation of iNOS expression and detection of iNOS activity in single cardiac myocytes in vitro. , 1994, The Journal of biological chemistry.

[6]  M. Lewis,et al.  Modulation of left ventricular relaxation in isolated ejecting heart by endogenous nitric oxide. , 1994, The American journal of physiology.

[7]  E. Lalli,et al.  Signal transduction and gene regulation: the nuclear response to cAMP. , 1994, The Journal of biological chemistry.

[8]  W. Giles,et al.  An obligatory role for nitric oxide in autonomic control of mammalian heart rate. , 1994, The Journal of physiology.

[9]  S. Weremowicz,et al.  Isolation and chromosomal localization of the human endothelial nitric oxide synthase (NOS3) gene. , 1994, Genomics.

[10]  R Fischmeister,et al.  Nitric oxide regulates cardiac Ca2+ current. Involvement of cGMP-inhibited and cGMP-stimulated phosphodiesterases through guanylyl cyclase activation. , 1993, The Journal of biological chemistry.

[11]  M. Yokoyama,et al.  Cyclic AMP-elevating agents induce an inducible type of nitric oxide synthase in cultured vascular smooth muscle cells. Synergism with the induction elicited by inflammatory cytokines. , 1993, Journal of Biological Chemistry.

[12]  S. Scherer,et al.  Structure and chromosomal localization of the human constitutive endothelial nitric oxide synthase gene. , 1993, The Journal of biological chemistry.

[13]  P. Poole‐Wilson,et al.  Nitric oxide attenuates cardiac myocyte contraction. , 1993, The American journal of physiology.

[14]  M. Yoshizumi,et al.  Tumor necrosis factor downregulates an endothelial nitric oxide synthase mRNA by shortening its half-life. , 1993, Circulation research.

[15]  M. Gerritsen,et al.  Isolation and characterization of human and rat cardiac microvascular endothelial cells. , 1993, The American journal of physiology.

[16]  R M Nerem,et al.  Molecular cloning and characterization of the constitutive bovine aortic endothelial cell nitric oxide synthase. , 1992, The Journal of clinical investigation.

[17]  P. Tempst,et al.  Endothelial nitric oxide synthase: molecular cloning and characterization of a distinct constitutive enzyme isoform. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[18]  S. Snyder,et al.  Nitric oxide synthase regulatory sites. Phosphorylation by cyclic AMP-dependent protein kinase, protein kinase C, and calcium/calmodulin protein kinase; identification of flavin and calmodulin binding sites. , 1992, The Journal of biological chemistry.

[19]  M. Endoh,et al.  Effects of a Cardiotonic Quinolinone Derivative Y-20487 on the Isoproterenol-Induced Positive Inotropic Action and Cyclic AMP Accumulation in Rat Ventricular Myocardium: Comparison with Rolipram, Ro 20-1724, Milrinone, and Isobutylmethylxanthine , 1992, Journal of cardiovascular pharmacology.

[20]  Terry D. Lee,et al.  Cloning and characterization of inducible nitric oxide synthase from mouse macrophages. , 1992, Science.

[21]  S. Snyder,et al.  Cloned and expressed nitric oxide synthase structurally resembles cytochrome P-450 reductase , 1991, Nature.

[22]  J. Heyse,et al.  Beneficial effects of milrinone and enalapril on long-term survival of rats with healed myocardial infarction. , 1988, European journal of pharmacology.

[23]  P. Chomczyński,et al.  Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. , 1987, Analytical biochemistry.

[24]  C. Lowenstein,et al.  Induction of NO synthase in rat cardiac microvascular endothelial cells by IL-1β and IFN-γ , 1995 .

[25]  R. Kelly,et al.  Continual electric field stimulation preserves contractile function of adult ventricular myocytes in primary culture. , 1994, The American journal of physiology.

[26]  J. Balligand,et al.  Control of cardiac muscle cell function by an endogenous nitric oxide signaling system. , 1993, Proceedings of the National Academy of Sciences of the United States of America.

[27]  A. Cavalié,et al.  Direct and indirect regulation of cardiac L-type calcium channels by beta-adrenoreceptor agonists. , 1990, Advances in second messenger and phosphoprotein research.

[28]  L. Brunton,et al.  Resolution of soluble rat cardiac phosphodiesterases by high performance liquid chromatography. , 1988, Second messengers and phosphoproteins.