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

Abstract

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 [Ca2+](i). ATP, UTP, and 2-methylthio ATP (2-MeSATP) increased [Ca2+](i) to similar levels, while 2-MeSADP, ADP and adenosine gave 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 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 α,β-methyleneATP. Cells grown on rat tail collagen instead of ECM gave smaller and less uniform [Ca2+](i) responses, suggesting that the differentiating effect of the ECM contributed to a more uniform receptor profile. 5. The [Ca2+](i) response to the P2Y1-selective agonist 2-MeSADP was abolished in the presence of the subtype-selective antagonist adenosine 3'-phosphate 5'-phosphosulphate (PAPS). 6. The P2Y2 antagonist suramin completely blocked the response to ATP and inhibited the response to UTP by 66%. 7. The A1 subtype-selective adenosine receptor agonist N6-Cyclopentyladenosine (CPA) gave a small but characteristic [Ca2+](i) response, while A(2A) and A(2B) subtype-selective agonists failed to generate [Ca2+](i) changes. 8. The results are consistent with the presence on RBCEC of a P2Y2-like receptor coupled to phospholipase C, and a P2Y1-like receptor mobilizing intracellular Ca2+. The role of multiple nucleotide receptors in the function of the brain endothelium is discussed.