Role of cGMP-dependent protein kinase in the negative inotropic effects of cGMP-elevating agents in the mammalian ventricle

The role of guanosine 3':5'-cyclic monophosphate (cGMP) and c G M P dependent protein kinase (PKG) in the regulation of cardiac contractility is controversial. There is evidence in the literature which suggests that c G M P is involved in the muscarinic receptor agonist-mediated antagonism of the positive inotropic effects of cAMP-elevating agents in the mammalian ventricle. Support for this relationship includes observations that a. ) acetylcholine elevates c G M P and mediates a negative inotropic effect in (3-adrenoceptor-stimulated ventricular tissue in a timeand concentration-dependent manner and b.) some of the contractile effects of muscarinic receptor agonists can be mimicked by c G M P analogues and inhibited cGMP-lowering agents. The proposal that c G M P can mediate negative inotropic effects is difficult to reconcile with reports the nitrovasodilator sodium nitroprusside (SNP) markedly increased tissue c G M P levels in several ventricular preparations but had no effect on contractile activity (Rodger and Shahid, 1984; Lincoln and Keely, 1980, 1981). A possible explanation for these conflicting results was proposed by Lincoln and Keely (1980, 1981). They hypothesized that some cGMP-elevating agents, such as muscarinic receptor agonists, increase c G M P in a functional pool which can specifically activate P K G and, thereby, mediate a negative inotropic effect. Cyclic GMP-elevating agents, such as SNP, which fail to elevate c G M P in the requisite pool would be unable to activate P K G and, as a result, would not reduce ventricular contractility. The aim of the present study was to test this hypothesis. The experimental approach was to compare the effects of carbachol, S N P and atrial natriuretic peptide (ANP) on contractility, c G M P content and P K G activity in rat intact ventricular tissue and freshly isolated rat ventricular cardiomyocytes. Carbachol induced a marked negative inotropic effect in intact, perfused hearts, ventricular strips and isolated cardiomyocytes in the presence of isoproterenol. The negative inotropic effect of carbachol in the intact ventricle was associated with insignificant changes in tissue c G M P content but, in isolated cardiac myocytes, with significant, although small, increases in cellular c G M P levels. S N P and A N P had no effect on isoproterenol-stimulated contractility in ventricular strips and intact hearts. Furthermore, S N P did not change the positive inotropic effect of isoproterenol in isolated cardiomyocytes. The absence of negative inotropic effects by A N P and S N P was observed in the presence of marked increases in intact ventricular c G M P levels. S N P also increased c G M P levels by up to 8-fold in isolated cardiomyocytes. The presence of P K G in both the intact ventricle and in isolated ventricular cardiomyocytes confirmed by MonoQ anion exchange chromatography and Western blotting. The elution profile indicated that the conditions of the P K G assay were very selective for measuring P K G activity. Attempts were made to confirm the reliability of the P K G assay, after which agonist-mediated P K G activation was assessed. Carbachol had no significant effect on P K G activity at a concentration which exerted a marked negative inotropic effect in isoproterenol-stimulated ventricular tissue and cardiomyocytes. Conversely, P K G was activated in the presence of S N P or A N P in ventricular tissue and was activated by S N P in myocytes and this activation occurred without changes in the positive inotropic