Pre-steady state study of beta-adrenergic and purinergic receptor interaction in C6 cell membranes: undelayed balance between positive and negative coupling to adenylyl cyclase

Interactions between beta-adrenergic and ADP purinergic receptors in C6 glioma cell membrane preparations were investigated under steady state and then pre-steady state conditions of adenylyl cyclase (EC 4.6.1.1) activity, in order to determine how fast the second receptor antagonizes the transduction mechanism of the first. Cell membranes were washed to deplete them as thoroughly as possible of low molecular weight compounds, especially ATP and ADP, and to ensure better control of both substrate and agonist nucleotide concentrations. ATP concentrations were kept constant with the use of an ATP-regenerating system; the C6 cell line exhibited very active ectonucleotidases. The purinergic agonist ADP was replaced by its nonhydrolyzable congener adenosine 5'-O-(2-thio)diphosphate (ADP beta S), which was demonstrated, like ADP, to inhibit isoproterenol-stimulated adenylyl cyclase activity in intact cells (IC50 for ADP, 0.5 +/- 0.1 microM; IC50 for ADP beta S, 25 +/- 2 microM) and in membrane preparations (IC50 for ADP beta S, 79 +/- 20 microM). In the case of membrane preparations, ADP beta S did not compete with ATP, the substrate of the cyclase-catalyzed reaction, and behaved apparently as a non-competitive inhibitor of the enzyme. The pre-steady state kinetics of isoproterenol-stimulated adenylyl cyclase activity measured with a pulsed quenched-flow apparatus have previously been shown to include two steps, the first very rapid (taking place within 1-2 sec) and giving rise to a burst of cAMP synthesis and the second much slower and corresponding to the steady state reaction. ADP beta S inhibited the occurrence of both steps with comparable IC50 values (mean value, 55 +/- 20 microM). In the presence of increasing concentrations of the purinergic receptor agonist, the time constant of the exponential burst reaction was not affected, but its amplitude progressively decreased to zero. These results showed that the extinction of the beta receptor cAMP response by the purinergic ADP receptor occurred within the dead-time of the pulsed quenched-flow apparatus, which was 50 msec. Such a rapid inhibition of cAMP production excluded modulation of isoproterenol-stimulated adenylyl cyclase activity by the ADP receptor by a pathway other than its direct negative coupling to the cyclase via a Gi protein. In this respect, the P2 purinergic ADP receptor of the C6 glioma cell line appears comparable to the P2t receptor of platelets.