Regulation of brain capillary endothelial cells by P2Y receptors coupled to Ca2+, phospholipase C and mitogen‐activated protein kinase

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 Ca2+ 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 Ca2+, with a rapid rise to peak followed by a slower decline towards basal or to a sustained plateau. Removal of extracellular Ca2+ had little effect on the peak Ca2+‐response, but resulted in a more rapid decline to basal. There was no response to α,β‐MethylATP (α,βMeATP) in these unpassaged cells, but a response to this P2X agonist was seen after a single passage. 3 ATP (log EC50 −5.1±0.2) also caused an increase in the total [3H]‐inositol (poly)phosphates ([3H]‐InsPx) in the presence of lithium with a rank order of agonist potency of ATP=UTP=UDP>ADP, with 2MeSATP and α,βMeATP 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)P3), 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)P3. 5 None of the nucleotides tested affected basal cyclic AMP, while ATP and ATPγS, but not 2MeSATP, stimulated cyclic AMP levels in the presence of 5 μM 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 concentration‐dependency consistent with activation at P2Y2 receptors. 2MeSATP gave a much smaller response with a lower potency than UTP. 7 These results are consistent with brain endothelial regulation by P2Y2 receptors coupled to phospholipase C, Ca2+ and MAPK; and by P2Y1‐like (2MeSATP‐sensitive) receptors which are linked to Ca2+ mobilization by a mechanism apparently independent of agonist stimulated Ins (1,4,5)P3 levels. A further response to ATP, acting at an undefined receptor, caused an increase in cyclic AMP levels in the presence of forskolin. The differential MAPK c