8-bromocyclic-gmp and Heart-Failure

The Na-Ca exchanger (NCX) is a critical calcium efflux pathway in excitable cells, but little is known regarding its autonomic regulation.. We investigated beta-adrenergic receptor and muscarinic receptor regulation of the cardiac NCX in control and heart failure (HF) conditions in atrially paced pigs. NCX current in myocytes from control swine hearts was significantly increased by isoproterenol, and this response was reversed by concurrent muscarinic receptor stimulation with the addition of carbachol, demonstrating "accentuated antagonism." Okadaic acid eliminated the inhibitory effect of carbachol on isoproterenol-stimulated NCX current, indicating that muscarinic receptor regulation operates via protein phosphatase-induced dephosphorylation. However, in myocytes from atrially paced tachycardia-induced HF pigs, the NCX current was significantly larger at baseline but less responsive to isoproterenol compared with controls, whereas carbachol failed to inhibit isoproterenol-stimulated NCX current, and 8-Br-cGMP did not restore muscarinic responsiveness. Protein phosphatase type 1 dialysis significantly reduced NCX current in failing but not control cells, consistent with NCX hyperphosphorylation in HF. Protein phosphatase type 1 levels associated with NCX were significantly depressed in HF pigs compared with control, and total phosphatase activity associated with NCX was significantly decreased.. We conclude that the NCX is autonomically modulated, but HF reduces the level and activity of associated phosphatases; defective dephosphorylation then "locks" the exchanger in a highly active state.

Topics: Adrenergic beta-Agonists; Animals; Carbachol; Cardiac Pacing, Artificial; Cell Separation; Cells, Cultured; Cyclic GMP; Disease Models, Animal; Drug Antagonism; Female; Heart Failure; Isoproterenol; Male; Muscarinic Agonists; Myocytes, Cardiac; Niflumic Acid; Patch-Clamp Techniques; Phosphoprotein Phosphatases; Phosphorylation; Receptors, Adrenergic, beta; Receptors, Muscarinic; Sodium-Calcium Exchanger; Swine; Tachycardia

Baseline function and signal transduction are depressed in hearts with hypertrophic failure. We tested the hypothesis that the effects of cGMP and its interaction with cAMP would be reduced in cardiac myocytes from hypertrophic failing hearts. Ventricular myocytes were isolated from control dogs, dogs with aortic valve stenosis hypertrophy, and dogs with pacing hypertrophic failure. Myocyte function was measured using a video edge detector. Cell contraction data were obtained at baseline, with 8-bromo-cGMP (10(-7), 10(-6), and 10(-5) M), with erythro-9-(2-hydroxy-3-nonyl)adenine [EHNA; a cAMP phosphodiesterase (PDE(2)) inhibitor] plus 8-bromo-cGMP, or milrinone (a PDE(3) inhibitor) plus 8-bromo-cGMP. Baseline percent shortening and maximal rates of shortening (R(max)) and relaxation were slightly reduced in hypertrophic myocytes and were significantly lower in failing myocytes (R(max): control dogs, 95.3 +/- 17.3; hypertrophy dogs, 88.2 +/- 5.5; failure dogs, 53.2 +/- 6.4 mum/s). 8-Bromo-cGMP dose dependently reduced myocyte function in all groups. However, EHNA (10(-6) M) and milrinone (10(-6) M) significantly reduced the negative effects of cGMP on cell contractility in control and hypertrophy but not in failing myocytes (R(max) for control dogs: cGMP, -46%; +EHNA, -21%; +milrinone, -19%; for hypertrophy dogs: cGMP, -40%; +EHNA, -13%; +milrinone, -20%; for failure dogs: cGMP, -40%; +EHNA, -29%; +milrinone, -32%). Both combinations of EHNA-cGMP and milrinone-cGMP significantly increased intracellular cAMP in control, hypertrophic, and failing myocytes. These data indicated that the cGMP signaling pathway was preserved in hypertrophic failing cardiac myocytes. However, the interaction of cGMP with the cAMP signaling pathway was impaired in these failing myocytes.

Topics: Adenine; Animals; Body Weight; Cardiotonic Agents; Cyclic AMP; Cyclic GMP; Dogs; Drug Interactions; Enzyme Inhibitors; Heart Failure; Hypertrophy, Left Ventricular; Milrinone; Myocardial Contraction; Myocytes, Cardiac; Organ Size; Signal Transduction

Congestive heart failure (CHF) is a clinical syndrome, which is the result of systolic or diastolic ventricular dysfunction. During CHF, vascular tone is regulated by the interplay of neurohormonal mechanisms and endothelial-dependent factors and is characterized by both central and peripheral vasoconstriction as well as a resistance to nitric oxide (NO)-mediated vasodilatation. At the molecular level, vascular tone depends on the level of regulatory myosin light chain phosphorylation, which is determined by the relative activities of myosin light chain kinase and myosin light chain phosphatase (MLCP). The MLCP is a trimeric enzyme with a catalytic, a 20-kDa and a myosin targeting (MYPT1) subunit. Alternative splicing of a 3' exon produces leucine zipper positive and negative (LZ+/-) MYPT1 isoforms. Expression of a LZ+ MYPT1 has been suggested to be required for NO-mediated smooth muscle relaxation. Thus, we hypothesized that the resistance to NO-mediated vasodilatation in CHF could be attributable to a change in the relative expression of LZ+/- MYPT1 isoforms. To test this hypothesis, left coronary artery ligation was used to induce CHF in rats, and both the dose response relationship of relaxation to 8-Br-cGMP in skinned smooth muscle and the relative expression of LZ+/- MYPT1 isoforms were determined. In control animals, the expression of the LZ+ MYPT1 isoform predominated in both the aorta and iliac artery. In CHF rats, LVEF was reduced to 30+/-5% and there was a significant decrease in both the sensitivity to 8-Br-cGMP and expression of the LZ+ MYPT1 isoform. These results indicate that CHF is associated with a decrease in the relative expression of the LZ+ MYPT1 isoform and the sensitivity to 8-Br-cGMP-mediated smooth muscle relaxation. The data suggest that the resistance to NO-mediated relaxation observed during CHF lies at least in part at the level of the smooth muscle and is a consequence of the decrease in the expression of the LZ+ MYPT1 isoform.

Topics: Alternative Splicing; Animals; Blotting, Western; Calcium; Carrier Proteins; Coronary Vessels; Cyclic GMP; Dose-Response Relationship, Drug; Heart Failure; Ligation; Muscle, Smooth, Vascular; Myocardial Infarction; Nitric Oxide; Phosphoprotein Phosphatases; Phosphorylation; Protein Isoforms; Protein Phosphatase 1; Protein Processing, Post-Translational; Rats; Rats, Sprague-Dawley; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Ultrasonography; Vasodilation

We studied the effects of various phosphodiesterase (PDE) III inhibitors: amrinone, pimobendan and vesnarinone: a PDE IV inhibitor (Ro 20-1724) and a PDE V inhibitor (E-4021) on the production of cytokines which have been shown to depress myocardial function. Recently developed inotropic agents which inhibit PDE III activity have produced short-term hemodynamic benefits in patients with advanced heart failure, but long-term treatment with these agents has an adverse effect on survival. However, vesnarinone, which has been shown to improve survival dramatically, has an immunomodulating effect and inhibits the production of cytokines. Peripheral blood mononuclear cells obtained from healthy human subjects were stimulated with lipopolysaccharide and each PDE inhibitor was added. After 24 h of incubation, tumor necrosis factor alpha (TNF-alpha), interleukin 1 beta (IL-1 beta) and IL-6 in the culture supernatants were measured by an enzyme-linked immunosorbent assay. All three PDE III inhibitors, amrinone, pimobendan and vesnarinone, inhibited TNF-alpha production, but vesnarinone's inhibitory effect was the most prominent. Amrinone and pimobendan enhanced IL-1 beta production, whereas vesnarinone had no effect. Vesnarinone inhibited IL-6 production and pimobendan slightly decreased IL-6 production, whereas amrinone had no significant effect on IL-6 production. The PDE IV inhibitor, Ro 20-1724, decreased the production of IL-1 beta and TNF-alpha and also tended to inhibit IL-6 production; its modulation of cytokine production was similar to the effects of vesnarinone. Because 8Br-cAMP or 8Br-cGMP did not suppress cytokine production, the modulating effects were not considered to result from an increase in cAMP or cGMP. Differential modulation of cytokine production may play a role in the therapeutic effect in heart failure patients who are treated with drugs that have PDE-inhibitory actions. It may be important to study whether the use of dual inhibitors of PDE III and PDE IV is therapeutically more useful for the treatment of heart failure due to their immunomodulating properties.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3',5'-Cyclic-GMP Phosphodiesterases; 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; 8-Bromo Cyclic Adenosine Monophosphate; Amrinone; Cells, Cultured; Cyclic GMP; Cyclic Nucleotide Phosphodiesterases, Type 3; Cyclic Nucleotide Phosphodiesterases, Type 4; Cyclic Nucleotide Phosphodiesterases, Type 5; Heart Failure; Humans; Interleukin-1; Interleukin-6; Kinetics; Leukocytes, Mononuclear; Lipopolysaccharides; Phosphodiesterase Inhibitors; Phosphoric Diester Hydrolases; Piperidines; Pyrazines; Pyridazines; Quinazolines; Quinolines; Tumor Necrosis Factor-alpha