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

摘要

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: approximately 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.