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 (I BMX), a phosphodiesterase inhibitor, by reducing protein phosphorylati on without altering cAMP levels. In the present study, we tested the h ypothesis that the cAMP-independent inhibitory action of ACH is also e vident in isolated cardiomyocytes. cAMP-dependent protein kinase (PKA) activity ratio (-cAMP/+cAMP) and phosphorylation of phospholamban (PL B) were determined in unlabeled and P-32-labeled guinea pig ventricula r 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 mu M), AC H attenuated PLB phosphorylation stimulated by low concentration(10-10 0 mu M) but not by high concentrations (>200 mu M) of IBMX. EC,, value for IBMX-induced phosphorylation of PLB was 32 +/- 6 mu M and increas ed nearly 3-fold after addition of ACH while PKA activity ratio remain ed unchanged. The rank order of cyclic nucleotide derivatives to phosp horylate PLB was 8 bromo-cAMP > dibutyryl cAMP > 8 bromo-cGMP > dibuty ryl cGMP. ACH reduced phosphorylation of PLB stimulated by 8 bromo-cAM P. We conclude that in isolated cardiomyocytes (1) ACH inhibits phosph orylation of PLB stimulated by either IBMX or 8 bromo-cAMP and (2) ACH does not lower IBMX-stimulated PKA activity ratio. These effects of A CH on PLB phosphorylation cannot be explained by a reduction in IBMX-s timulated cAMP levels but may involve the activation of protein phosph atases.