P-2-PURINOCEPTOR ACTIVATION STIMULATES PHOSPHOINOSITIDE HYDROLYSIS AND INHIBITS ACCUMULATION OF CAMP IN CULTURED VENTRICULAR MYOCYTES

Extracellular ATP modulates cardiac contraction through P2-purinoceptors on cardiac myocytes. To elucidate the molecular mechanism of this response, we examined the effects of P2-purinoceptor activation on phosphoinositide (PI) hydrolysis and the cAMP system in cultured ventricular myocytes of fetal mice. In a concentration-dependent manner, ATP stimulated accumulations of [H-3]inositol monophosphate, bisphosphate, and trisphosphate with the half-maximum effective concentration of approximately 1-mu-M in the myocytes labeled with [H-3]inositol. The order of efficacy of a series of adenyl compounds for stimulation of PI hydrolysis was adenosine 5'-O-(3-thiotriphosphate) (ATP-gamma-S), ATP > ADP, 5'-adenylylimidodiphosphate (APPNP) > alpha,beta-methyleneadenosine 5'-triphosphate (APCPP) > beta,gamma-methyleneadenosine 5'-triphosphate, AMP > adenosine. On the other hand, 100-mu-M ATP-gamma-S inhibited isoproterenol-induced accumulation of cAMP by approximately 70% without decreasing the time to maximal cAMP levels, as measured by radioimmunoassay. This response was also concentration dependent, with a half-maximum inhibitory concentration (IC50) of approximately 1-mu-M. All of the tested ATP, ADP, and ATP analogues inhibited the cAMP system, and the responses to ATP-gamma-S, APPNP, and APCPP were insensitive to an A1-purinoceptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine. Pertussis toxin attenuated the ATP-induced PI hydrolysis by no more than 25% at 100 ng/ml but completely suppressed the AtP-gamma-S-induced inhibition of the cAMP system. Protein kinase C-activating phorbol ester, 4-beta-phorbol 12-beta-myristate 13-alpha-acetate, inhibited the ATP-induced PI hydrolysis with an IC50 of approximately 1 nM and also attenuated the ATP-gamma-S-induced inhibition of the cAMP system at greater-than-or-equal-to 1 nM, although a biologically inactive phorbol ester, 4-alpha-phorbol 12,13-didecanoate, did not. From these data, P2-purinoceptor activation stimulates PI hydrolysis by activating phospholipase C primarily through pertussis toxin-insensitive G proteins and attenuates cAMP accumulation by inhibiting adenylate cyclase through pertussis toxin-sensitive G proteins. Protein kinase C is likely to negatively regulate both the signal transduction systems.