Regional distribution of [35S]2′-deoxy 5′-O-(1-thio) ATP binding sites and the P2Y1 messenger RNA within the chick brain
Section snippets
Materials
[35S]dATPαS (1400 Ci/mmol) and unlabelled dATPαS were supplied by New England Nuclear (Hitchin, U.K.). 2-methylthio ATP (2MeS-ATP) was from RBI (Natick, MA., U.S.A.). Reactive Blue-2 (RB-2), α,β-methylene ATP (α,β-meATP), UTP and other chemicals were purchased from Sigma (Poole, U.K.). βmax-Hyperfilm and 14C microscales for quantitative autoradiographic analysis were obtained from Amersham (Little Chalfont, U.K.). Oligonucleotides were custom-synthesized and polyacrylamide gel electrophoresis
Characterization of the [35S]dATPαS binding sites
[35S]dATPαS autoradiography was performed on horizontal sections of one-day-old chick brain in the presence of a range of P2 receptor ligands, to determine the nature of the binding sites. Representative sections, of the [35S]dATPαS binding obtained in the absence or presence of a large excess of another nucleotide or the P2 receptor antagonist[5]RB-2, are shown in Fig. 1. Substantial labelling was observed when sections were incubated with a nanomolar concentration of [35S]dATPαS (Fig. 1A). An
Distribution of the [35S]dATPαS binding sites
In the present study we have examined the distribution of [35S]dATPαS binding sites throughout the chick brain by in vitro autoradiography. Previously we have shown that this radioligand labelled a large number of binding sites (Bmax: 37 pmol [35S]dATPαS bound/mg protein) in chick brain particulate membrane fraction.[34]The binding profile at these sites was in agreement with the pharmacology established for the recombinant P2Y1 receptor, transiently expressed in COS-7 cells or in Xenopus
Conclusions
In summary, we have investigated the anatomical localization of the P2Y1 receptor protein and transcript in the chick brain. The widespread and abundant distribution of both mRNA and protein in the chick neuraxis suggests multiple and important roles for this receptor in the CNS. Indeed, the expression of this mRNA detected in neurons may indicate a role for this receptor in neurotransmission. However, further functional studies are necessary to delineate the contribution of this receptor type
Acknowledgements
We thank C. Thrasivoulou and T. Gysbers for expert technical assistance with image analysis and photography. T.E.W. and E.A.B. thank the Wellcome Trust for support.
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Studies towards the development of a PET radiotracer for imaging of the P2Y<inf>1</inf> receptors in the brain: synthesis, <sup>18</sup>F-labeling and preliminary biological evaluation
2019, European Journal of Medicinal ChemistryCitation Excerpt :In chicken and rat brain membranes, a Bmax for the nucleotidic ligand [35S]dATPαS of 37 and 39 pmol/mg membrane protein, respectively, was reported. Levels of [35S]dATPαS binding in individual structures (0.047–0.309 pmol/mg wet tissue) in chick brain are suggested to be comparable to those found for other G protein-linked receptors such as the muscarinic, dopamine and α2-adrenergic receptors and corresponded to their respective local transcripts [17,30]. The distribution of [35S]dATPαS specific binding sites was widespread throughout the rat brain, prominent in several regions including piriform cortex, ectostriatum, cerebellum and some nuclei in the telencephalon and mesencephalon [31].
Role for purinergic receptors in memory processing in young chicks
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2006, Pharmacology and TherapeuticsATP potentiates the formation of AChR aggregate in the co-culture of NG108-15 cells with C2C12 myotubes
2005, FEBS LettersCitation Excerpt :Several lines of evidence support this notion. First, P2Y1 receptor mRNA and protein are widely expressed in neurons of different regions of the brain [25], while the P2Y2 receptor mRNA has also been shown to occur in many brain regions [26]. In line with these observations, recent studies from our laboratory also suggest the expression of P2Y receptors in cultured rat cortical neurons (Tsim et al., unpublished results).
Kinetics of [<sup>35</sup>S]dATPαS interaction with P2Y<inf>1</inf> purinoceptor in rat brain membranes
2004, Neuroscience LettersP2Y receptor expression on astrocytes in the nucleus accumbens of rats
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- 1
These authors contributed equally to this study.
- 2
Permanent address: Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, U.K.