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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:769-777.)
© 1997 American Heart Association, Inc.

Articles

Functional Expression of a P_2T ADP Receptor in Xenopus Oocytes Injected With Megakaryocyte (CMK 11-5) RNA

Nicholas J. Greco

From the Platelet Biology Department, The American Red Cross, Jerome Holland Laboratory, Rockville, Md.

Correspondence to Nicholas J. Greco, PhD, American Red Cross, 15601 Crabbs Branch Way, Rockville, MD 20855. E-mail greco{at}usa.redcross.org

Abstract Since the P_2T 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 P_2Y 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--S are active as agonists and ATP and ATP--S are inactive as agonists but act as antagonists. [³H]ADP and ³⁵S-ATP--S bound to CMK 11-5 cells at a high-affinity site (K_d1 and K_i1, 262 and 125 nmol/L, respectively) and a low-affinity site (K_d2 and K_i2, 10 100 and 5400 nmol/L, respectively) with 2x10⁶ to 6x10⁶ sites per cell. ADP bound at both sites was competed with ADP, ATP, and ATP--S with affinities in a rank order similar to that found for platelets (ATP--SATPADPADP-ß-Sadenosine), suggesting the presence of a P_2T receptor on CMK 11-5 cells. Photoaffinity labeling of intact CMK 11-5 cells with ³⁵S-ATP--S resulted in the labeling of the -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--S stimulated ⁴⁵Ca²⁺ efflux, which was not observed with ATP, 2-methylthio-ATP, ,ß-methylene-ATP, ATP--S, ATP--S, or adenosine. In addition, incubation of RNA-injected oocytes with ATP or ATP--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, P_2Y, P_2X, and P_2U agonists was expressed in functional form on Xenopus oocytes.

Key Words: Xenopus oocyte • ADP • P_2T purinergic receptor

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