Characterisation of P2 purinoceptors on vascular endothelial cells.

P2 purinoceptors mediate the effects of the adenine nucleotides ATP and ADP. These receptors have been classified on the basis of rank orders of agonist potency due to the lack of potent and selective antagonists. Two types of P2 purinoceptor activate phosphoinositidase C (PIC); P2y purinoceptors, 2 methylthioATP (2MeSATP) is a selective agonist; and nucleotide receptors, UTP is a selective agonist. P2x purinoceptors, where alpha,beta-methylene ATP is a selective agonist, are not linked to PIC. This thesis has attempted to classify the PIC-linked P2 purinoceptors present on endothelial cells and to study the second messenger systems associated with these receptors. Measurements of total inositol phosphate accumulation, in the presence of lithium chloride showed that bovine aortic endothelial cells (BAE cells) possesed co-existing populations of P2y purinoceptors and nucleotide receptors, whereas bovine adrenal medulla endothelial cells (BAME cells) possesed only nucleotide receptors. Suramin, an antagonist that displays no selectivity between P2x and P2y purinoceptors, was a selective antagonist for the P2y purinoceptors but not for the nucleotide receptors on BAE cells. Both receptor types on BAE cells stimulated the formation of inositol (1,4,5) trisphosphate (Ins(l,4,5)P3). A number of differences were noted; (1), UTP produced a more prolonged stimulation of Ins(l,4,5)P3 levels than did 2MeSATP; (2), activation of protein kinase C (PKC) by phorbol, 12 myristate, 13 acetate (PMA), attenuated responses to 2MeSATP but not UTP, whereas inhibition of PKC, by Ro 31-8220, augmented responses to 2MeSATP but not UTP; (3), preincubation of BAE cells with pertussis toxin (PTx), which inactivates guanine nucleotide binding proteins (G-proteins) of the Gi and GO family, reduced responses to UTP but had no effect on those to 2MeSATP. Desensitisation experiments showed that both receptors on BAE cells could undergo homologous desensitisation, however, preincubation with UTP could reduce subsequent responses to 2MeSATP, i.e. heterologous desensitisation, but 2MeSATP could not produce heterologous desensitisation of responses to UTP. Both the homologous and heterologous desensitisations produced by nucleotide receptor activation were ablated by PKC inhibition with Ro 31-8220, whereas those to 2MeSATP were not. These studies on the modulation of Ins(l,4,5)P3 formation showed that these co-existing receptors for ATP on BAE cells were mediated by different populations of G-proteins and interacted differently with PKC. Endothelial dependent relaxations of bovine aortic collateral artery rings also indicated the presence of co-existing populations of P2y purinoceptors, which were sensitive to antagonism by suramin, and nucleotide receptors. Relaxations to ADP and 2MeSATP were almost abolished in the presence of indomethacin, an agent which inhibits cyclooxygenase, whereas relaxations to UTP were unaffected. Concentration-effect curves to ATP and adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS), showed a significant rightward shift in the presence of indomethacin. Therefore, on collateral artery rings, endothelium dependent relaxations via P2y purinoceptors were due to cyclooxygenase metabolites, while those via nucleotide receptors were not. In conclusion, BAE cells and collateral artery rings posses co-existing populations of PIC-linked P2 purinoceptors, the P2y purinoceptors and the nucleotide receptors, whereas BAME cells only posses nucleotide receptors. Both of these receptors on BAE cells are activated by ATP whereas ADP only interacts with the P2y purinoceptor. This and the differential modulation of second messenger systems by these receptors may be important mechanisms in which endothelial cell function is regulated by adenine nucleotides.