Characterization of a P2Y Purinoceptor in the Brain

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Abstract
Little has been known of the abundance in the brain of any of the G protein coupled P2 purinoceptors nor their pharmacology. Here we show that [35S]dATP alpha S is a suitable radioligand for investigating these receptors and hence that they are exceptionally abundant both in one-day-old chick (Bmax: 37 pmol agonist sites/mg protein) and adult rat brain membranes (Bmax: 39 pmol/mg protein). [35S]dATP alpha S (which is selective for P2Y over the P2X types of purinoceptor) binds with high affinity to these sites in the chick (Kd: 13.3 nM) and in the rat brain membranes (Kd: 9.1 nM). The rank order of potency of purinoceptor-active agonists and antagonists displacing [35S]dATP alpha S binding is: dATP alpha S > (3'-deoxyATP, 2-methylthioATP, ATP alpha S, ATP) > 2'-deoxyATP > 2-methylthioADP > ADP >> suramin, Reactive Blue-2 >> UTP, L-beta,gamma-methyleneATP, adenosine; this defines these binding sites as P2Y subtypes of the P2 purinoceptors. This pharmacological profile of purinergic ligands is in excellent agreement with the potency order established for the recombinant P2Y1 purinoceptor from chick brain, identifying the great majority of the brain P2 purinoceptors as identical or very similar to the native P2Y1 receptor.