Evidence for a nucleotide receptor on adrenal medullary endothelial cells linked to phospholipase C and phospholipase D

1. We have investigated whether the 'atypical' P2-purinoceptor previously described on adrenal microvasculature endothelial cells is a nucleotide receptor (responds to pyrimidines and purines) and is linked to phospholipase D as well as phospholipase C. 2. Cultured bovine adrenal medullary endothelial (BAME) cells responded to the pyrimidine UTP, as well as the purines. The total [3H]-inositol phosphate responses were with a rank order of UTP > ATP- = adenosine 5'-O-(3-thio-triphosphate) (ATP gamma S) >> 2MeSATP. The selective P2x agonist beta, gamma-methylene ATP was inactive. 3. Construction of dose-response curves to ATP, ATP gamma S and UTP in the presence and absence of additional agonists showed that responses to ATP gamma S and UTP were not additive, nor were those to UTP and ATP. This suggests that purines and pyrmidines acted via a common nucleotide receptor. 4. 32P-labelled BAME cells, in the presence of butanol, produced [32P]-phosphatidylbutanol (PBut) when stimulated with ATP gamma S or the protein kinase C activator, tetradecanoyl phorbol acetate (TPA). 5. Cells labelled with [3H]-palmitate and stimulated in the presence of butanol generated [3H]-PBut with the same order of agonist potencies seen for inositol phosphate responses. 6. The protein kinase C inhibitor, Ro 31-8220, abolished TPA and agonist stimulation of [3H]-PBut production. 7. These observations, and our related studies on bovine aortic endothelial cells, provide the first demonstration of a phospholipase C linked nucleotide receptor on vascular endothelial cells. It is concluded that BAME cells express a nucleotide receptor linked to phospholipase C and phospholipase D, but that activation of phospholipase D is probably down-stream of phospholipase C.