Muscarinic-cholinoceptor mediated attenuation of phospholamban phosphorylation induced by inhibition of phosphodiesterase in ventricular cardiomyocytes: evidence against a cAMP-dependent effect.
In intact guinea pig ventricles, acetylcholine (ACH) has been shown to attenuate the positive inotropic effects of isobutylmethylxanthine (IBMX), a phosphodiesterase inhibitor, by reducing protein phosphorylation without altering cAMP levels. In the present study, we tested the hypothesis that the cAMP-independent inhibitory action of ACH is also evident in isolated cardiomyocytes. cAMP-dependent protein kinase (PKA) activity ratio (-cAMP/+cAMP) and phosphorylation of phospholamban (PLB) were determined in unlabeled and 32P-labeled guinea pig ventricular cardiomyocytes, respectively. IBMX increased PKA activity ratio and phosphorylation of PLB in a dose-dependent manner. When cardiomyocytes were incubated simultaneously with IBMX (0-1 mM) and ACH (2 microM), ACH attenuated PLB phosphorylation stimulated by low concentration (1O-100 microM) but not by high concentrations (> 200 microM) of IBMX. EC50 value for IBMX-induced phosphorylation of PLB was 32 +/- 6 microM and increased nearly 3-fold after addition of ACH while PKA activity ratio remained unchanged. The rank order of cyclic nucleotide derivatives to phosphorylate PLB was 8 bromo-cAMP > dibutyryl cAMP > 8 bromo-cGMP > dibutyryl cGMP. ACH reduced phosphorylation of PLB stimulated by 8 bromo-cAMP. We conclude that in isolated cardiomyocytes (1) ACH inhibits phosphorylation of PLB stimulated by either IBMX or 8 bromo-cAMP and (2) ACH does not lower IBMX-stimulated PKA activity ratio. These effects of ACH on PLB phosphorylation cannot be explained by a reduction in IBMX-stimulated cAMP levels but may involve the activation of protein phosphatases.
Gupta
R C
RC
Department of Medicine, Henry Ford Heart and Vascular Institute, Detroit, Michigan 48202, USA.
Neumann
J
J
Watanabe
A M
AM
Sabbah
H N
HN
D015056:1-Methyl-3-isobutylxanthine / Q000494:pharmacology; D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000378:metabolism; D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D003994:Bucladesine / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D006168:Guinea Pigs; D006352:Heart Ventricles; D009206:Myocardium / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D009712:Nucleotides, Cyclic / Q000378:metabolism; D010726:Phosphodiesterase Inhibitors / Q000494:pharmacology*; D010766:Phosphorylation; D011976:Receptors, Muscarinic / Q000502:physiology*; D000099211:Phospholamban
D015056:1-Methyl-3-isobutylxanthine; D015124:8-Bromo Cyclic Adenosine Monophosphate; D000109:Acetylcholine; D003994:Bucladesine; D002135:Calcium-Binding Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D009712:Nucleotides, Cyclic; D010726:Phosphodiesterase Inhibitors; D011976:Receptors, Muscarinic; D000099211:Phospholamban