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

University of British Columbia

The role of guanosine 3':5'-cyclic monophosphate (cGMP) and cGMPdependent protein kinase (PKG) in the regulation of cardiac contractility is controversial. There is evidence in the literature which suggests that cGMP 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 cGMP and mediates a negative inotropic effect in (3-adrenoceptor-stimulated ventricular tissue in a time- and concentration-dependent manner and b.) some of the contractile effects of muscarinic receptor agonists can be mimicked by cGMP analogues and inhibited cGMP-lowering agents. The proposal that cGMP can mediate negative inotropic effects is difficult to reconcile with reports the nitrovasodilator sodium nitroprusside (SNP) markedly increased tissue cGMP 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 cGMP in a functional pool which can specifically activate PKG and, thereby, mediate a negative inotropic effect. Cyclic GMP-elevating agents, such as SNP, which fail to elevate cGMP in the requisite pool would be unable to activate PKG 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, SNP and atrial natriuretic peptide (ANP) on contractility, cGMP content and PKG 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 cGMP content but, in isolated cardiac myocytes, with significant, although small, increases in cellular cGMP levels. SNP and ANP had no effect on isoproterenol-stimulated contractility in ventricular strips and intact hearts. Furthermore, SNP did not change the positive inotropic effect of isoproterenol in isolated cardiomyocytes. The absence of negative inotropic effects by ANP and SNP was observed in the presence of marked increases in intact ventricular cGMP levels. SNP also increased cGMP levels by up to 8-fold in isolated cardiomyocytes. The presence of PKG 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 PKG assay were very selective for measuring PKG activity. Attempts were made to confirm the reliability of the PKG assay, after which agonist-mediated PKG activation was assessed. Carbachol had no significant effect on PKG activity at a concentration which exerted a marked negative inotropic effect in isoproterenol-stimulated ventricular tissue and cardiomyocytes. Conversely, PKG was activated in the presence of SNP or ANP in ventricular tissue and was activated by SNP in myocytes and this activation occurred without changes in the positive inotropic effects of isoproterenol. PKG activation may have occurred in the presence of carbachol but may have been underestimated by the conditions of the assay. The results of this study demonstrate that the negative inotropic effects of muscarinic receptor agonists occur in the presence of a small increase in cGMP levels and in the absence of significant activation of PKG. Larger increases in ventricular cGMP content and PKG activity were not sufficient to mediate a negative inotropic effect in the presence of SNP or ANP. Under the conditions of this study, PKG activation cannot be ruled out as playing a role in the negative inotropic effects of muscarinic receptor agonists but elevation of cGMP content and activation of PKG are not sufficient to inhibit contractility in the rat ventricle.

Thesis/Dissertation

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2009-02-19

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http://hdl.handle.net/2429/4790

Pharmaceutical Sciences

University of British Columbia

UBCV

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