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Muscarinic-cholinoceptor mediated attenuation of phospholamban phosphorylation induced by inhibition of phosphodiesterase in ventricular cardiomyocytes: Evidence against a cAMP- dependent effect

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Abstract

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 μM), ACH attenuated PLB phosphorylation stimulated by low concentration (10-100 μM) but not by high concentrations (> 200 μM) of IBMX. EC50 value for IBMX-induced phosphorylation of PLB was 32 ± 6 μM 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.

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Authors and Affiliations

  1. Department of Medicine, Division of Cardiovascular Medicine, Henry Ford Heart and Vascular Institute, Detroit, Michigan, USA

    Ramesh C. Gupta & Hani N. Sabbah1

  2. Institut Für Pharmacologie und Toxicologie, Munster, Domagksr, 12, Federal Republic of Germany

    Joachim Neumann

  3. Lilly Research Laboratories, Eli Lilly and Company, Lilly Corporate Center, Indianapolis, USA

    August M. Watanabe

Authors
  1. Ramesh C. Gupta
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  2. Joachim Neumann
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  3. August M. Watanabe
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  4. Hani N. Sabbah1
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Gupta, R.C., Neumann, J., Watanabe, A.M. et al. Muscarinic-cholinoceptor mediated attenuation of phospholamban phosphorylation induced by inhibition of phosphodiesterase in ventricular cardiomyocytes: Evidence against a cAMP- dependent effect. Mol Cell Biochem 187, 155–161 (1998). https://doi.org/10.1023/A:1006899931151

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