ADP and ATP activate distinct signaling pathways in human promonocytic U-937 cells differentiated with 1,25-dihydroxy-vitamin D3

We investigated the effects of purinergic P2 agonists on cell adhesion, as well as the signaling pathways involved, in U-937 human promonocytic cells differentiated to a more mature monocytic phenotype with 1,25-(OH)2-vitamin D3. In cell adhesion studies, ATP was more potent than ADP, whereas ADP showed greater efficacy. The time course of the intracellular calcium concentration ([Ca2+]i) response to ATP was biphasic (a spike followed by a plateau), whereas ADP evoked a plateau after a time lag of several seconds. The spike [Ca2+]i response evoked by ATP was due to both myo-inositol-1,4,5-trisphosphate-induced Ca2+ mobilization and influx, whereas the ADP effect was Ca2+ influx dependent. Moreover, ADP and ATP activated distinct Ca2+ influx mechanisms, as shown by Mn2+ quenching of the fura-2 signal, which was slow and long-lasting for ADP but rapid for ATP. Treatment with phorbol dibutyrate shifted the EC50 of the [Ca2+]i spike response to the right and abolished the plateau response. Accordingly, phorbol dibutyrate inhibited the ADP-evoked response but only partly inhibited the ATP-evoked Ca2+ influx. cAMP-raising agents had no effect on the [Ca2+]i spike or on Ca2+ influx but delayed or transiently inhibited the plateau phase. The plateau response thus appears to be independent of the spike response, because it can be directly evoked by ADP and undergoes distinct regulation. This suggests that ADP and ATP activate U-937 cells through distinct signaling pathways, probably involving specific receptors.