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Characterization of a P2Y purinoceptor in the brain
J Simon1, T E Webb, E A Barnard
1Molecular Neurobiology Unit, Royal Free Hospital School of Medicine, London, UK.
Insights
This study reveals that G protein-coupled P2 purinoceptors are abundant in the brain. Using [35S]dATP alpha S as a radioligand, researchers identified these receptors as primarily the P2Y1 subtype in both chick and rat brains.
Area of Science:
- Neuroscience
- Pharmacology
- Biochemistry
Background:
- The abundance and specific subtypes of G protein-coupled P2 purinoceptors in the brain remain largely uncharacterized.
- Understanding these receptors is crucial for elucidating their roles in neurological functions and potential therapeutic interventions.
Purpose of the Study:
- To determine the abundance and pharmacological characteristics of P2 purinoceptors in brain membranes.
- To identify the specific subtypes of P2 purinoceptors present in the brain.
Main Methods:
- Utilized [35S]dATP alpha S as a selective radioligand for P2Y purinoceptors.
- Performed radioligand binding assays on one-day-old chick and adult rat brain membranes.
- Analyzed the displacement of [35S]dATP alpha S binding by various purinergic ligands to establish rank order of potency.
Main Results:
- [35S]dATP alpha S demonstrated high affinity and selectivity for P2Y purinoceptors in both chick and rat brain membranes.
- Exceptionally high densities of these receptors were found (Bmax: 37 pmol/mg protein in chick, 39 pmol/mg protein in rat).
- The pharmacological profile strongly indicated that the predominant P2 purinoceptor subtype in the brain is P2Y1.
Conclusions:
- G protein-coupled P2 purinoceptors, specifically the P2Y1 subtype, are highly abundant in the brain.
- [35S]dATP alpha S is an effective radioligand for quantifying these receptors.
- The findings provide a foundation for further research into the physiological roles and therapeutic targeting of brain P2Y1 receptors.
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.