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 receptors, but differences have been reported between culture models. This study reports examination of nucleotide receptors on primary cultured rat brain capillary endothelial cells (RBCEC) grown on a biological extracellular matrix (ECM) to produce a more differentiated phenotype. 2. Fura-2 fluorescence ratio imaging was used to monitor intracellular free calcium concentration Ca(2+). ATP, UTP, and 2-methylthioATP (2-MeSATP) increased Ca(2+) to similar levels, while 2-MeSADP, ADP and adenosine gave smaller responses. 3. Removal of extracellular calcium caused no significant change in the Ca(2+) response to 2-MeSATP, evidence that the response was mediated by a metabotropic (P2Y) receptor. 4. All cells tested responded to ATP, UTP, 2-MeSATP and ADP, while 63% responded to adenosine and 50% to 2-MeSADP. No cells responded to alpha, beta-methyleneATP. Cells grown on rat tail collagen instead of ECM gave smaller and less uniform Ca(2+) responses, suggesting that the differentiating effect of the ECM contributed to a more uniform receptor profile. 5. The Ca(2+) response to the P2Y(1)-selective agonist 2-MeSADP was abolished in the presence of the subtype-selective antagonist adenosine 3'-phosphate 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 A(1) subtype-selective adenosine receptor agonist N(6)-Cyclopentyladenosine (CPA) gave a small but characteristic Ca(2+) response, while A(2A) and A(2B) subtype-selective agonists failed to generate Ca(2+) changes. 8. The results are consistent with the presence on RBCEC of a P2Y(2)-like receptor coupled to phospholipase C, and a P2Y(1)-like receptor mobilizing intracellular Ca(2+). The role of multiple nucleotide receptors in the function of the brain endothelium is discussed.