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Chronic muscarinic cholinoceptor stimulation increases adenylyl cyclase responsiveness in rat cardiomyocytes by a decrease in the level of inhibitory G-protein alpha-subunits.

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Medizinische Klinik I, Klinikum Grosshadern, Universität München, Germany.


Exposure of neonatal rat cardiomyocytes for 3 days to the muscarinic cholinoceptor agonist carbachol led to a concentration-dependent increase in adenylyl cyclase stimulation by the beta-adrenoceptor agonist isoproterenol by up to 115% (at 1 mmol/l carbachol). In addition, direct adenylyl cyclase stimulation by forskolin was increased in carbachol (1 mmol/l)-treated cells by 32%. Pretreatment of the rat cardiomyocytes with pertussis toxin, which enhances adenylyl cyclase activity by a functional inactivation of the inhibitory G-protein (Gi), was performed to investigate the possible role of Gi-proteins in carbachol-induced sensitization of adenylyl cyclase stimulation. After pretreatment of the cells with pertussis toxin, the carbachol-mediated increase in forskolin-stimulated adenylyl cyclase activity was lost and the carbachol-mediated increase in beta-adrenoceptor-stimulated adenylyl cyclase activity was attenuated. Labelling of the 40 kDa pertussis toxin substrates in cardiomyocyte membranes was decreased by carbachol in a concentration-dependent manner by up to 34% (at 1 mmol/l carbachol). The number and affinity of beta 1-adrenoceptors was unaltered following the chronic carbachol treatment. The specific protein synthesis inhibitor Pseudomonas exotoxin A was used to study whether the carbachol-induced decrease in the level of pertussis toxin-sensitive G-proteins and increase in adenylyl cyclase activity depend on de-novo protein synthesis. Pseudomonas exotoxin A inhibits peptide chain elongation by ADP-ribosylating elongation factor 2. Treatment of the cells with 1 ng/ml Pseudomonas exotoxin A for 3 days led to a reduction in the subsequent ADP-ribosylation of elongation factor 2 in the cytosol of the heart muscle cells by 57%.(ABSTRACT TRUNCATED AT 250 WORDS).

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