Arterioscler Thromb Vasc Biol 1997 Apr 01;174:769-77. doi: 10.1161/01.atv.17.4.769.

Functional expression of a P2T ADP receptor in Xenopus oocytes injected with megakaryocyte (CMK 11-5) RNA.

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Since the P2T purinergic (ADP) receptor is unique to the megakaryocytic/platelet lineage, cells of this lineage were screened for the relative effects of ADP and ATP in intracellular Ca2+ levels. Like platelets, CMK 11-5 cells responded with an increase in intracellular Ca2+ mobilization in response to ADP but not to ATP or adenosine. In contrast, both nucleotides increased intracellular Ca2+ mobilization in the megakaryoblastic cell lines MO7E and Meg-01, indicating that they contain P2Y receptors or a mixed complement of purinergic receptors. Pharmacological responsiveness of CMK 11-5 cells to nucleotides paralleled those of platelets, in which ADP and ADP-alpha-S are active as agonists and ATP and ATP-alpha-S are inactive as agonists but act as antagonists. [3H]ADP and 35S-ATP-alpha-S bound to CMK 11-5 cells at a high-affinity site (Kd1 and Ki1, 262 and 125 nmol/L, respectively) and a low-affinity site (Kd2 and Ki2, 10,100 and 5400 nmol/L, respectively) with 2 x 10(6) to 6 x 10(6) sites per cell. ADP bound at both sites was competed with ADP, ATP, and ATP-alpha-S with affinities in a rank order similar to that found for platelets (ATP-alpha-S approximately ATP approximately ADP > or = ADP-beta-S approximately adenosine), suggesting the presence of a P2T receptor on CMK 11-5 cells. Photoaffinity labeling of intact CMK 11-5 cells with 35S-ATP-alpha-S resulted in the labeling of the alpha-subunit of GP IIb as found with platelets, although this was confirmed to be independent of ADP receptors. After RNA from CMK 11-5 cells was microinjected into Xenopus oocytes, only ADP and ADP-alpha-S stimulated 45Ca2+ efflux, which was not observed with ATP, 2-methylthio-ATP, alpha, beta-methylene-ATP, ATP-gamma-S, ATP-alpha-S, or adenosine. In addition, incubation of RNA-injected oocytes with ATP or ATP-alpha-S but not adenosine blocked the 45Ca2+ response to ADP. These experiments demonstrate that a nascent receptor that responded specifically to ADP but not to other P1, P2Y, P2X, and P2U agonists was expressed in functional form on Xenopus oocytes.

???displayArticle.pubmedLink??? 9108793
???displayArticle.link??? Arterioscler Thromb Vasc Biol
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