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1 The effect of suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulphonic acid (PPADS) on the stimulation of phospholipase C in 132iNI cells transfected with the human P2U-purinoceptor (h-P2U-1321N1 cells) or with the turkey P2y-purinoceptor (t-P2Y-1321N1 cells) was investigated. 2-Methylthioadenosine triphosphate (2MeSATP) was used as the agonist at t-P2Y-132INI cells and uridine triphosphate (UTP) at h-P2U-1321Nl cells.2 Suramin caused a parallel shift to the right of the concentration-response curves for 2MeSATP in the t-P2Y-132IN1 cells, yielding a Schild plot with a slope of 1.16+0.08 and a pA2 value of 5.77 +0.11. 3 Suramin also caused a shift to the right of concentration-response curves for UTP in the h-P2U-1321N1 cells, and on Schild plots gave a slope different from unity (1.57+0.19) and an apparent pA2 value of 4.32+0.13. Suramin was therefore a less potent antagonist at the P2u-purinoceptor than the P2Y-purinoceptor. 6 PPADS up to 30 giM had no effect on the concentration-response curve for UTP with the h-P2U-1321N1 cells. 7 In conclusion, suramin and PPADS show clear differences in their action at the 2 receptor types, in each case being substantially more effective as an antagonist at the P2Y-purinoceptor than at the P2U-purinoceptor. Ectonucleotidase breakdown had little influence on the nature of the responses at the two receptor types, or in their differential sensitivity to suramin.

1 The blood-brain barrier is formed by capillary endothelial cells and is regulated by cell-surface receptors, such as the G protein-coupled P2Y receptors for nucleotides. Here we investigated some of the characteristics of control of brain endothelial cells by these receptors, characterizing the phospholipase C and Ca 2+ response and investigating the possible involvement of mitogen-activated protein kinases (MAPK). 2 Using an unpassaged primary culture of rat brain capillary endothelial cells we showed that ATP, UTP and 2-methylthio ATP (2MeSATP) give similar and substantial increases in cytosolic Ca 2+, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca 2+ had little eect on the peak Ca 2+-response, but resulted in a more rapid decline to basal. There was no response to a,b-MethylATP (a,bMeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage. 3 ATP (log EC 50 75.1+0.2) also caused an increase in the total [3 H]-inositol (poly)phosphates ([ 3 H]-InsP x ) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP4ADP, with 2MeSATP and a,bMeATP giving no detectable response. 4 Stimulating the cells with ATP or UTP gave a rapid rise in the level of inositol 1,4,5-trisphosphate (Ins(1,4,5)P 3 ), with a peak at 10 s followed by a decline to a sustained plateau phase. 2MeSATP gave no detectable increase in the level of Ins(1,4,5)P 3 .5 None of the nucleotides tested aected basal cyclic AMP, while ATP and ATPgS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 mM forskolin. 6 Both UTP and ATP stimulated tyrosine phosphorylation of p42 and p44 mitogen-activated protein kinase (MAPK), while 2MeSATP gave a smaller increase in this index of MAPK activation. By use of a peptide kinase assay, UTP gave a substantial increase in MAPK activity with a concentrationdependency consistent with activation at P2Y 2 receptors. 2MeSATP gave a much smaller response with a lower potency than UTP. 7 These results are consistent with brain endothelial regulation by P2Y 2 receptors coupled to phospholipase C, Ca 2+ and MAPK; and by P2Y 1 -like (2MeSATP-sensitive) receptors which are linked to Ca 2+ mobilization by a mechanism apparently independent of agonist stimulated Ins (1,4,5)P 3 levels. A further response to ATP, acting at an unde®ned receptor, caused an increase in cyclic AMP levels in the presence of forskolin. The dierential MAPK coupling of these receptors suggests that they exert fundamentally distinct in¯uences over brain endothelial function.