Adenosine and acetylcholine reduce isoproterenol-induced protein phosphorylation of rat myocytes

Adenosinergic and muscarinic agents have been shown to attenuate the catecholamine-induced augmentation of both protein phosphorylation and contractile state in perfused hearts. The attenuation by phenylisopropyl-adenosine (PIA) and carbamylcholine chloride (CARB) of the isoproterenol (ISO)-induced incorporation of 32P into protein substrates was examined in isolated rat ventricular myocytes. 32P-labelled myocytes exposed to ISO (0.1 microM, 30 s) demonstrated up to an eight-fold increase of 32P incorporation into three protein substrates (155, 31, 6 kD). When myocytes were pre-incubated with either PIA or CARB for 60 s, the ISO-induced 32P incorporation in the 31 kD and the 155 kD substrates was attenuated 37% and 25%, respectively by 1 microM PIA and only 23% and 11%, by 10 microM PIA. A concentration of 1 microM CARB produced a 24% and 17% reduction in these same substrates while 10 microM CARB produced a 44% and 50% reduction. The effects of ISO were antagonized by 10 microM propanolol. The inhibitory effects of PIA were antagonized by the theophylline, sulfophenyltheophylline and dipropylcyclopentylxanthine, whereas atropine antagonized the inhibitory effects of CARB. The 32P incorporation elicited by 1 microM forskolin was reduced more by CARB than PIA. Additionally, while PIA and CARB reduced the ISO-induced increase in cAMP-dependent protein kinase (PKA) activity by 48% and 41% respectively, only CARB attenuated the ISO-elicited increase in cAMP levels, attenuating this response by 58%. The results indicate that PIA was less effective in attenuating ISO-induced 32P incorporation at higher concentrations than at lower concentrations. Moreover, this compound was less potent than CARB at attenuating the effects of ISO. It is conceivable that this difference could be related to activation of stimulatory adenosine receptors (A2) and/or a greater density of muscarinic receptors including multiple inhibitory muscarinic pathways.