CHARACTERIZATION OF DIADENOSINE TETRAPHOSPHATE (AP4A) BINDING-SITES IN CULTURED CHROMAFFIN CELLS - EVIDENCE FOR A P2Y SITE

1 Diadenosine tetraphosphate (Ap4A) a dinucleotide, which is stored in secretory granules, presents two types of high affinity binding sites in chromaffin cells. A K(d) value of 8 +/- 0.65 x 10(-11) M and B(max) value of 5420 +/- 450 sites per cell were obtained for the high affinity binding site. A K(d) value of 5.6 +/- 0.53 x 10(-9) M and a B(max) value close to 70,000 sites per cell were obtained for the second binding site with high affinity. 2 The diadenosine polyphosphates, Ap3A, Ap4A, Ap5A and Ap6A, displaced [H-3]-Ap4A from the two binding sites, the K(i) values being 1.0 nM, 0.013 nM, 0.013 nM and 0.013 nM for the very high affinity binding site and 0.5-mu-M, 0.13-mu-M, 0.062-mu-M and 0.75-mu-M for the second binding site. 3 The ATP analogues displaced [H-3]-Ap4A with the potency order of the P2y receptors, adenosine 5'-O-(2 thiodiphosphate) (ADP-beta-S) > 5'-adenylyl imidodiphosphate (AMP-PNP) > alpha,beta-methylene ATP (alpha,beta-MeATP), in both binding sites. The K(i) values were respectively 0.075 nM, 0.2 nM and 0.75 nM for the very high affinity binding site and 0.125-mu-M, 0.5-mu-M and 0.9-mu-M for the second binding site.