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.

1 In primary unpassaged rat brain capillary endothelial cell cultures (RBECs), using reversetranscriptase PCR with primers speci®c for P2Y receptor subtypes, we detected mRNA for P2Y 2 , P2Y 4 and P2Y 6 , but not P2Y 1 receptors. 2 None of the various nucleotides tested reduced forskolin elevated cyclic AMP levels in RBECs. ATP and ATPgS, as well as adenosine, enhanced cyclic AMP accumulation in the presence of forskolin.3 Comparison of the concentration response curves to ATPgS with those for ATP and adenosine, at di erent incubation times, indicated that the response to purine nucleotides was not wholly dependent on conversion to adenosine. Adenosine deaminase abolished the response to adenosine but only reduced the response to ATP by about 50%. These results suggest the participation of a receptor responsive to nucleotides. 4 Isobutylmethylxanthine and 8-sulphophenyltheophylline prevented the cyclic AMP response, while neither 8-cyclopentyl-1,3-dipropylxanthine nor SCH58261 were e ective antagonists. 2-chloradenosine gave a robust response, but neither 2-chloro-N 6 -cyclopentyladenosine nor CGS 21680 were agonists. 5 These results show that adenosine and ATP can elevate the cyclic AMP levels of brain endothelial cells by acting on receptors which have a pharmacology apparently distinct from known P2Y and adenosine receptors.

Vascular endothelial growth factor (VEGF) is an endothelium-specific mitogen that induces angiogenesis and increases vascular permeability. These processes involve regulation of cell-cell adhesion, but molecular mechanisms have yet to be fully established. p120, also termed p120(ctn), and its variant p100 are catenins which associate with cadherins and localize to adherens junctions. VEGF was reported to stimulate tyrosine phosphorylation of catenins in endothelial cells. In contrast, we have found that VEGF potently stimulated a rapid and dose-dependent decrease in serine/threonine phosphorylation of p120 and p100. VEGF acted via VEGF receptor 2 to achieve this effect which was independent of activation of the extracellular-signal-regulated kinase pathway. Histamine and activators of protein kinase C had a very similar effect to that of VEGF on phosphorylation of p120 and p100, suggesting that these diverse stimuli may converge on a common signalling element regulating p120/p100 serine/threonine phosphorylation. These data raise the possibility that the dephosphorylation of p120 and p100 triggered by VEGF may contribute to mechanisms regulating permeability and/or motility through modulation of cadherin adhesiveness.