Presence of functional P2T and P2U purinoceptors on the human megakaryoblastic cell line, Meg-01 characterization by functional and binding studies.

Abstract

The platelet receptor for ADP has been classified as a P2 purinoceptor of the P2T type where ADP is the natural agonist and ATP a competitive antagonist. Since the P2T receptor seems to be specific to platelets, we investigated the possibility that the human megakaryoblastic cell line Meg-01 might express the platelet ADP receptor, using functional studies and the ADP analogue [33P]2MeSADP as a specific P2T radioligand. ADP and 2MeSADP were able to induce shape change and pseudopod formation in Meg-01 cells. [33P]2MeSADP binding to membrane preparations was saturable and specific, binding sites being of high affinity (Kd = 13 +/- 3 nM) and present at a concentration of 13 +/- 7 fmoles/mu g protein. In contrast to UTP and GTP, ADP and ATP were able to displace the specific [33P]2MeSADP binding. ADP, 2MeSADP, UTP and ATP induced a dose dependent increase in intracellular calcium concentration. Desensitization experiments demonstrated that UTP, and ADP share a common P2U purinoceptor and that Meg-01 cells also express a P2T purinoceptor for which ADP and 2MeSADP are agonists and ATP a competitive antagonist. It was concluded that the human megakaryoblastic cell line Meg-01 expresses two distinct types of functional P2 receptor; a P2U and a P2T purinoceptor. This cell line could therefore provide the necessary material to clone the as yet unidentified platelet P2T purinoceptor.