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+)](i). ATP, UTP, and 2-methylthioATP (2-MeSATP) increased [Ca(2+)](i) 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+)](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 alpha, beta-methyleneATP. Cells grown on rat tail collagen instead of ECM gave smaller and less uniform [Ca(2+)](i) responses, suggesting that the differentiating effect of the ECM contributed to a more uniform receptor profile. 5. The [Ca(2+)](i) 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+)](i) response, while A(2A) and A(2B) subtype-selective agonists failed to generate [Ca(2+)](i) 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.
Sipos
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Department of Medical Biochemistry, Semmelweis University, Budapest, H-1444, P.O. Box 262, Hungary.
Dömötör
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Abbott
N J
NJ
Adam-Vizi
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D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology*; D000818:Animals; D001812:Blood-Brain Barrier / Q000187:drug effects / Q000502:physiology*; D001921:Brain / Q000187:drug effects / Q000502:physiology; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000187:drug effects* / Q000502:physiology; D005109:Extracellular Matrix / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D011983:Receptors, Purinergic / Q000187:drug effects* / Q000502:physiology; D013873:Thionucleotides / Q000494:pharmacology*; D014544:Uridine Triphosphate / Q000494:pharmacology*
D011983:Receptors, Purinergic; D013873:Thionucleotides; D000255:Adenosine Triphosphate; D002118:Calcium; D014544:Uridine Triphosphate; C008501:2-methylthio-ATP