The pharmacology of nucleotide receptors on primary rat brain endothelial cells grown on a biological extracellular matrix: effects on intracellular calcium concentration

1 Brain capillary endothelial cells express a variety of nucleotide recepto rs, but differences have been reported between culture models. This study r eports examination of nucleotide receptors on primary cultured rat brain ca pillary endothelial cells (RBCEC) grown on a biological extracellular matri x (ECM) to produce a more differentiated phenotype. 2 Fura-2 fluorescence ratio imaging was used to monitor intracellular free calcium concentration [Ca2+](i). ATP, UTP, and 2-methylthioATP (2-MeSATP) i ncreased [Ca2+](i) to similar levels, while 2-MeSADP, ADP and adenosine gav e smaller responses. 3 Removal of extracellular calcium caused no significant change in the [Ca2 +]i response to 2-MeSATP, evidence that the response was mediated by a meta botropic (P2Y) receptor. 4 All cells tested responded to ATP, UTP, 2-MeSATP and ADP, while 63% respo nded to adenosine and 50% to 2-MeSADP. No cells responded to alpha,beta -me thyleneATP. Cells grown on rat tail collagen instead of ECM gave smaller an d less uniform [Ca2+](i) responses, suggesting that the differentiating eff ect of the ECM contributed to a more uniform receptor profile. 5 The [Ca2+](i) response to the P2Y(1)-selective agonist 2-MeSADP was aboli shed in the presence of the subtype-selective antagonist adenosine 3'-phosp hate 5'-phosphosulphate (PAPS). 6 The P2Y(2) antagonist suramin completely blocked the response to ATP and inhibited the response to UTP by 66%. 7 The Al subtype-selective adenosine receptor agonist N-6-Cyclopentyladenos ine (CPA) gave a small but characteristic [Ca2+]i response, while AZA and A (2B) subtype-selective agonists failed to generate [Ca2+]i changes. 8 The results are consistent with the presence on RBCEC of a P2Y(2)-like re ceptor coupled to phospholipase C, and a P2Y(1)-like receptor mobilizing in tracellular Ca2+. The role of multiple nucleotide receptors in the function of the brain endothelium is discussed.