Binding and internalization of p1,p4-diadenosine 5'-tetraphosphate by bovine aortic endothelial cells

p1,p4-Diadenosine 5'-tetraphosphate (Ap4A) has been implicated as a modulator of blood vessel tone. We have recently demonstrated that the infusion of Ap4A into swine induces vasodilation (Hilderman et al., Am. J. Hypertension 10 (1997) 94A) and that Ap4A induces the release of nitric oxide (NO) from bovine aortic endothelial cells (BAEC) (Hilderman and Christensen, FEBS Lett. 427 (1998) 320-324). However, the interaction of Ap4A with endothelial cells is incompletely understood. Therefore, we determined the characteristics of [3H]-Ap4A binding to BAEC in normal and ATP-depleted cells. These binding studies demonstrate that the interaction of Ap4A with BAEC involves two distinct steps: an ATP independent step and a second ATP dependent step leading to internalization of Ap4A. The initial interaction of Ap4A with BAEC is not affected by either EGTA or iodoacetate; however, both agents block the second step. These data suggest that calcium ions and sulfhydryl groups are required for Ap4A internalization but not for an initial binding event.