THE P2Y PURINOCEPTOR IN RAT-BRAIN MICROVASCULAR ENDOTHELIAL-CELLS COUPLE TO INHIBITION OF ADENYLATE-CYCLASE

1 B10 cells, a clonal line of rat brain capillary cells, exhibit a sin gle P-2 purinoceptor, activation of which leads to increases in free i ntracellular calcium. In the current study the identity of this P2Y re ceptor was determined by its binding parameters for a range of purinoc eptor ligands and by its complementary DNA (cDNA) sequence. The signal transduction mechanism activated by this receptor was also investigat ed. 2 The radioligand [S-35]-dATP alpha S bound with high affinity (K- d = 9.8 nM) to the P2Y purinoreceptor expressed on B10 cells, which wa s found to be extremely abundant (B-max = 22.5 pmol mg(-1) protein). T he calculated K-i values of a range P-2 purinoreceptor agonists which competitively displaced binding of [S-35]-dATP alpha S led to the rank order of affinity: dATP alpha S (K-i 3.4 nM) > 2-chloroATP (2-CIATP) (13 nM), ATP (22 nM) > ATP gamma S (43 nM) > 2-methylthioATP (2-MeSATP ) (88 nM) > ADP (368 nM) > > UTP, L-beta,gamma-methyleneATP (both > 10 ,000 nM). The P-2 purinorecptor antagonists, Reactive blue 2 and suram in, were also to displace binding , with K-i values of 8333 and 1358 n M respectively. In contrast pyridoxal-phosphate-6-azophenyl-2',4'-disu lphonic acid 4-sodium (PPADS) was able to displace only 20% of [S-35]d ATP alpha S binding at a concentration of 100 mu M. 3 2-ClATP (EC(50) = 0.22 mu M), 2-MeSATP (0.54 mu M), ADP (7.9 mu M) and ATP (a patial a gonist), but not UTP, inhibited the cyclic AMP formation stimulated by cholera toxin, in a manner that was prevented by pertussi toxin. The purinoreceptor antagonist, PPADS, was found to be inactive at a concen tration of 100 mu M. 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 P2 Y receptor that is coupled to the inhibition of adenylate cyclase. Seq uence analysis of the entire coding region revealed that both were 100 % identical to the rat P2Y(1) purinoreceptor cDNA. No other P2Y-type r eceptor mRNA could be detected in B10 cells. Exactly the same sequence was isolated from rat brain cortical astrocytes, where 2-MeSATP has b een shown to increase phospholipase C activity. 5 Since the receptor r esponsible for the transduction shares with the aforementioned binding site significant pharmacological features, including a strong activit y of 2-MeSATP (characteristic of P2Y(1) receptors alone among all know n P2Y purinoreceptos) and an unusual insensitivity to PPADS, and since abundant mRNA is present of the P2Y(1) receptor on rat brain microvas cular endothelial cells can account for all of the observations. This single P2Y(1) receptor, therefore, appears to couple in different nati ve cell types to either adenylate cyclase inhibition or to phospholipa se C activation.