Presence of functional P2T and P2U purinoceptors on the human megakaryoblastic cell line, Meg-01 characterization by functional and binding studies.
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.
INSERM Unite 311, Biologie et Pharmacologie des Interactions du Sang avec les Vaisseaux et les Biomateriaux, Strasbourg, France.
B
B
Hechler
J P
JP
Cazenave
D
D
Hanau
C
C
Gachet
D001667:Binding, Competitive; D002118:Calcium / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D006801:Humans; D007947:Leukemia, Megakaryoblastic, Acute / Q000378:metabolism*; D015539:Platelet Activation; D011869:Radioligand Assay; D011983:Receptors, Purinergic / Q000378:metabolism / Q000502:physiology*; D018048:Receptors, Purinergic P2 / Q000378:metabolism; D014407:Tumor Cells, Cultured
D011983:Receptors, Purinergic; D018048:Receptors, Purinergic P2; D002118:Calcium