Characterization and Channel Coupling of the P2Y12Nucleotide Receptor of Brain Capillary Endothelial Cells

Abstract

B10 cells are an endothelial clonal line derived spontaneously by culture and selection of rat brain microvascular endothelial cells (1), a source of considerable pharmacological potential. The B10 cells respond to extracellular adenine (but not uracil) nucleotides with intracellular Ca 2ϩ mobilization, mediated by a G-protein-coupled P2Y 1 receptor (2). Whereas the known rat P2Y 1 receptor cDNA could be isolated from the B10 cells (2), no transcript for any other then-known P2Y receptor subtype was detectable in them. However, the B10 cells were found to exhibit another second messenger response, namely the inhibition of stimulated adenylyl cyclase, but with a nucleotide agonist specificity very similar to that of the P2Y 1 receptor (2). It was further demonstrated (3) that selective antagonists of the P2Y 1 receptor, such as adenosine 3Ј-phosphate 5Ј-phosphate, were unable to affect that response, although it showed high sensitivity to 2-propylthio-D-␤,␥-difluoromethylene-ATP (AR-C66096), 1 a specific antagonist (4, 5) of the adenylyl cyclase-inhibitory P2Y T or "P2T" receptor for ADP, which was known as an important functional component of blood platelets. That study (3) demonstrated that a second P2Y receptor activity is present in the B10 cell, with some of its functional features in common with the platelet P2Y T receptor.Recently, a cDNA encoding a nucleotide receptor with a novel seven-transmembrane sequence was identified independently by two groups, starting from a human platelet cDNA library or an orphan human DNA sequence, and shown to be distantly but significantly related to the known P2Y receptors (6, 7). This was designated as the P2Y 12 receptor. Its amino acid sequence lies on a previously unrecognized separate branch of the P2Y family (8). It was characterized in both studies to show its adenine nucleotide specificity and its inhibition of forskolin-stimulated adenylyl cyclase, in which it corresponds to the platelet P2Y T receptor (6, 7). That human P2Y 12 receptor cDNA was expressed again in cell line hosts by other groups, confirming the reported series of agonists but with higher potencies in the cAMP decrease (9) and showing their affinities by competition with the binding of radiolabeled 2-MeSADP (9, 10). In the original identification of the human P2Y 12 receptor, a similar cDNA was also derived from a rat platelet library (6), and its RNA was shown to express in Xenopus oocytes, but this receptor was not further characterized.The question, therefore, obviously arises as to whether the cyclase inhibitory receptor for adenine nucleotides of the B10 capillary endothelial cell is in fact the P2Y 12 receptor. However, this cannot necessarily be assumed, because the P2T receptor has commonly been described in the literature as specific to the platelet (11,12). Furthermore, the adenylyl cyclase-inhibitory P2Y receptor activity in the B10 cell differs in an important

Purinergic receptor P2RY12-dependent microglial closure of the injured blood–brain barrier

“…Although most relevant studies have highlighted the relative specificity of brain P2RY12 expression to microglial cells, some controversy on the point exists, in that studies have variably reported that P2RY12 might also be expressed in brain endothelial cells (35), human smooth muscle vascular cells (36), and astrocytes (37). However, these reports have without exception been based on analysis of cultured cells or ex vivo preparations.…”

Regulation of death and survival in astrocytes by ADP activating P2Y1 and P2Y12 receptors

“…It was found that adenine nucleotides activate P2Y 12 receptors to inhibit voltage-gated Ca 2+ currents in PC12 cells via a voltage-dependent and PTX-sensitive mechanism [36,37]. P2Y 12 receptors in rat sympathetic neurons were shown to mediate a voltage-dependent and PTX-sensitive inhibition of N-type calcium channels [38,39].…”

Role of G protein–gated inwardly rectifying potassium channels in P2Y12 receptor–mediated platelet functional responses

“…Because GIRK channels are activated by G i signaling upon P2Y 12 receptor activation in other systems, 29,30 we investigated whether GIRK channels are functional effectors of the P2Y 12 receptor-mediated G i signaling in platelets.…”

“…29 Subsequently, the rat P2Y 12 receptor was shown to activate GIRK channels in sympathetic neurons. 30 GIRK channels are expressed in the brain and heart, where they modulate resting membrane potential and cellular excitability. [31][32][33] GIRK channels are tetrameric complexes formed by the variable assembly of 4 subunits (GIRK1-4).…”