| 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 ß-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 (G i), was performed to investigate the possible role of G i-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 ß-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 ß 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 a-subunits of G i-proteins. This decrease in G ia-subunits is apparently at least in part responsible for the observed increase in adenylyl cyclase responsiveness after chronic carbachol treatment. |
