Characterisation of a recombinant P_2Y purinoceptor

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• 作者：Joseph Simon ; Tania E. Webb ; Brian F. King ; Geoffrey Burnstock ; Eric A. Barnard
• 刊名：European Journal of Pharmacology: Molecular Pharmacology
• 出版年：1995
• 出版时间：30 November 1995
• 年：1995
• 卷：291
• 期：3
• 页码：281-289
• 全文大小：918 K

文摘

We have previously cloned a cDNA encoding a G-protein-coupled P₂ purinoceptor from chick brain and designated this as a P_2Y1 purinoceptor (Webb, T.E., J. Simon, B.J. Krishek, A.N. Bateson, T.G. Smart, B.J. King, G. Burnstock and E.A. Barnard, 1993, FEBS Lett. 324, 219). Here, we describe the further characterisation of this recombinant receptor expressed in both simian kidney endothelial (COS-7) cells and Xenopus oocytes. In transfected COS-7 cell membranes, the recombinant receptor showed a high level of expression (B_max = 7.9 ± 2.2 pmol [³⁵S]dATPαS bound/mg protein) and affinity (K_d = 6.6 ± 0.3 nM). In these COS-7 cells, the activation of the implanted purinoceptor induced a suramin-sensitive formation of inositol 1,4,5-triphosphate (1,4,5InsP₃). Upon expression in Xenopus oocytes, ATP was the only natural nucleoside triphosphate to elicit a Ca²⁺-activated chloride current. The P₂ purinoceptor antagonists suramin and Reactive Blue-2 were both able to inhibit this evoked current. Utilizing both expression systems, the binding affinity profile and the functional pharmacological profile of the agonists, the common series found was: 2-methylthioATP (2-MeSATP) ATP > ADPβS > ADP. These two agonist series and the lack of activity of adenosine, α,β-methyleneATP (α,β-meATP), 3′-O-(4- benzoyl)benzoyl-ATP (Bz-ATP) and UTP, together confirmed that this receptor is a specific subtype of the P_2Y purinoceptors.