Elsevier

Neuroscience

Volume 84, Issue 3, 24 February 1998, Pages 825-837
Neuroscience

Regional distribution of [35S]2′-deoxy 5′-O-(1-thio) ATP binding sites and the P2Y1 messenger RNA within the chick brain

https://doi.org/10.1016/S0306-4522(97)00478-8Get rights and content

Abstract

The distribution of the P2Y1 receptor protein and transcript in the one-day-old chick brain were determined by quantitative in vitro ligand autoradiography and in situ hybridization histochemistry. We have previously used [35S]2′-deoxy 5′-O-(1-thio) ATP as a radioligand for the recombinant P2Y1 receptor transiently expressed in COS-7 cells and have also shown that such sites are present at high density (Bmax: ∼37 pmol radioligand bound/mg protein) in chick brain membranes. Here we report the macroscopic localization of these [35S]2′-deoxy 5′-O-(1-thio) ATP binding sites within the chick brain. They were found to be widely distributed there (within the range of 0.047±0.012 to 0.309±0.035 pmol bound/mg wet tissue). The affinities of P2 agonists and antagonists at these binding sites was comparable to that found previously for the recombinant P2Y1 receptor. In parallel experiments, the regional and cellular localization of the P2Y1 receptor messenger RNA was examined by in situ hybridization. The transcript was also found to be widely distributed throughout the brain. High levels of hybridization were detected in the cortex piriformis, ectostriatum, hippocampus, cerebellum and in a range of discrete nuclei throughout the brain, including the ovoidalis, isthmo-opticus and spiriformis lateralis nuclei. Localization at cellular level indicates that this receptor transcript is expressed in neurons and also at non-neuronal sites. Furthermore, the distribution of the P2Y1 transcript and the [35S]2′-deoxy 5′-O-(1-thio) ATP binding sites matched in a number of the regions and structures mentioned above.

The present study clarifies the anatomical distribution of the P2Y1 receptor within the chick brain. Its broad distribution coupled with its neuronal expression suggest an important role for this type of metabotropic nucleotide receptor within the 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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    1

    These authors contributed equally to this study.

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    Permanent address: Glaxo Institute of Applied Pharmacology, Department of Pharmacology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QJ, U.K.

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