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

Abstract

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 \-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 32010. 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 \-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 34010 (at 1 mmol/l carbachol). The number and affinity of \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%. Exposure of the cells to 1 mmol/l carbachol for 3 days increased ADP-ribosylation of elongation factor 2 by 40% concomitant with a slight (about 20%) increase in the total protein content of the cardiomyocytes. The partial protein synthesis inhibition by Pseudomonas exotoxin A had no influence on the carbachol-induced decrease in the level of pertussis toxin-sensitive G-proteins. Similarly, the carbachol-induced increase in adenylyl cyclase responsiveness also remained unaltered by Pseudomonas exotoxin A. The data presented indicate that chronic muscarinic cholinoceptor agonist treatment decreases the level of α-subunits of Gi- proteins. This decrease in Giα- subunits is apparently at least in part responsible for the observed increase in adenylyl cyclase responsiveness after chronic carbachol treatment.

DOI: 10.1007/BF00169060

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@article{Reithmann1995ChronicMC, title={Chronic muscarinic cholinoceptor stimulation increases adenylyl cyclase responsiveness in rat cardiomyocytes by a decrease in the level of inhibitory G-protein α-subunits}, author={Christopher Reithmann and Karl Werdan}, journal={Naunyn-Schmiedeberg's Archives of Pharmacology}, year={1995}, volume={351}, pages={27-34} }