Mechanism of noradrenaline-induced heterologous desensitization of adenylate cyclase stimulation in rat heart muscle cells: increase in the level of inhibitory G-protein α-subunits

Abstract

The mechanism of heterologous desensitization of adenylate cyclase stimulation was studied in cultured neonatal rat heart muscle cells. After culturing of the cells for 3 days in the presence of 1 μM noradrenaline there was in addition to a 52% decrease in isoproterenol-stimulated adenylate cyclase activity, a lessening of the stimulation of ß-adrenoceptor-independent adenylate cyclase by guanosine-5′-O-(thiotriphosphate) and forskolin by 24 and 34%, respectively. The decrease in receptor-independent adenylate cyclase stimulation by forskolin, but not the attenuation of isoproterenol-stimulated adenylate cyclase activity, was abolished by pertussis toxin (PTX) pretreatment of the cells. G i, the inhibitory G-protein of adenylate cyclase was therefore quantitated. Labelling of the M r ≈ 40 kDa PTX substrates in membranes of noradrenaline-treated cells was increased by 70% as shown by pertussis toxin-catalyzed ADP ribosylation of heart cell membranes. This increase was also seen in the presence of an excess of purified ßγ-subunits of transducin and of GTP, suggesting that the increased labelling was not due to elevation of the level of ßγ-subunits or increase in the concentration of GTP in the membranes of noradrenaline-treated cells. Analysis of the PTX substrates on high resolution urea/SDS-polyacrylamide gels revealed that at least two distinct PTX substrates (40 and 41 kDa) were present in rat heart cell membranes. The labelling of both substrates was increased in membranes of desensitized cells. Immunoblotting of heart cell membranes with anti-G iα-antibodies demonstrated a marked increase in the amount of G iα in membranes of noradrenaline-treated cells. In contrast, immunoblotting with anti-ß-antibodies showed that the level of the ß-subunit of G-proteins (36 kDa) was unchanged after noradrenaline exposure. The data indicate that prolonged treatment of rat heart muscle cells with noradrenaline leads to an increase in the level of α-subunits of G i-proteins. This suggests that this increase is responsible for the observed heterologous desensitization of adenylate cyclase stimulation.