The P2Y purinoceptor in rat brain microvascular endothelial cells couple to inhibition of adenylate cyclase

• Published in: British journal of pharmacology 1996-12, Vol.119 (7), p.1385-1392
• Main Authors: Webb, Tania E., Feolde, Erick, Vigne, Paul, Neary, Joseph T., Runberg, Anna, Frelin, Christian, Barnard, Eric A.
• Format: Article
• Language: English
• Subjects: 2‐MethylthioATP; Adenosinic and purinergic receptors; adenylate cyclase inhibition; Adenylyl Cyclase Inhibitors; Adenylyl Cyclases - metabolism; Animals; ATP; Binding, Competitive - drug effects; Biological and medical sciences; Brain - cytology; Brain - enzymology; Brain Chemistry - drug effects; C6‐2B glioma; Capillaries - enzymology; Cell Line; Cell receptors; Cell structures and functions; Cerebrovascular Circulation - physiology; Chick Embryo; Cyclic AMP - biosynthesis; DNA - biosynthesis; DNA - isolation & purification; Endothelium, Vascular - cytology; Endothelium, Vascular - enzymology; Endothelium, Vascular - metabolism; Fundamental and applied biological sciences. Psychology; Molecular and cellular biology; P2Y purinoceptors; P2Y1 binding parameters; Polymerase Chain Reaction; PPADS; rat brain capillary endothelium; Rats; Signal Transduction - physiology; Thionucleotides - metabolism
• ISSN: 0007-1188; 1476-5381
• DOI: 10.1111/j.1476-5381.1996.tb16050.x

1 BIO cells, a clonal line of rat brain capillary endothelial cells, exhibit a single P2 purinoceptor, activation of which leads to increases in free intracellular calcium. In the current study the identity of this P2Y receptor was determined by its binding parameters for a range of purinoceptor ligands and by its complementary DNA (cDNA) sequence. The signal transduction mechanism activated by this receptor was also investigated. 2 The radioligand [35S]‐dATPαS bound with high affinity (Kd = 9.8nM) to the P2Y purinoceptor expressed on B10 cells, which was found to be extremely abundant (Bmax = 22.5 pmol mg−1 protein). The calculated Ki values of a range of P2 purinoceptor agonists which competitively displaced binding of [35S]‐dATPαS led to the rank order of affinity: dATPαS (Ki 3.4 nM) > 2‐chloroATP (2‐ClATP) (13 nM), ATP (22 nM) > ATPγS (43 nM) > 2‐methylthioATP (2‐MeSATP) (88 nM) > ADP (368 nM) > > UTP, L‐β,γ‐methyleneATP (both > 10,000 nM). The P2 purinoceptor antagonists, Reactive blue 2 and suramin, were also able to displace binding, with Ki values of 833 and 1358 nM respectively. In contrast pyridoxal‐phosphate‐6‐azophenyl‐2′,4′‐disulphonic acid 4‐sodium (PPADS) was able to displace only 20% of [35S]‐dATPαS binding at a concentration of 100 μm 3 2‐ClATP (EC50 = 0.22 μm), 2‐MeSATP (0.54 μm), ADP (7.9 μm) and ATP (a partial agonist), but not UTP, inhibited the cyclic AMP formation stimulated by cholera toxin, in a manner that was prevented by pertussis toxin. The purinoceptor antagonist, PPADS, was found to be inactive at a concentration of 100 μm 4 A P2Y receptor cDNA was derived from mRNA from B10 cells and from C6‐2B, a rat glioma cell line known to possess a P2Y receptor that is coupled to the inhibition of adenylate cyclase. Sequence analysis of the entire coding region revealed that both were 100% identical to the rat P2Y1 purinoceptor cDNA. No other P2Y‐type receptor mRNA could be detected in B10 cells. Exactly the same sequence was isolated from rat brain cortical astrocytes, where 2‐MeSATP has been shown to increase phospholipase C activity. 5 Since the receptor responsible for the transduction shares with the aforementioned binding site significant pharmacological features, including a strong activity of 2‐MeSATP (characteristic of P2Y1 receptors alone among all known P2Y purinoceptors) and an unusual insensitivity to PPADS, and since abundant mRNA is present of the P2Y2 receptor but not of any other type resembling the known P2Y receptors,