The characterisation of cloned P2Y receptors transfected into 1321N1 astrocytoma cells
thesisposted on 15.12.2014, 10:35 by Steven J. Charlton
Work in this thesis was primarily involved with the characterisation of four receptors, the turkey and bovine P2Y1, and the human P2Y2 and P2Y4 receptors, transfected into the human astrocytoma cell line, 1321N1. It also describes the preliminary characterisation of two vascular smooth muscle cell types, and the generation of P2Y1-GST fusion proteins for the production of antibodies.;1321N1 cells were found to release nucleotides in response to sheer stress, but this was overcome by adaptation of the stimulation method. 1321N1 cells expressing the four receptors were assayed for PLC activity, and the rank order of agonist potency for each receptor was generally consistent with reports in the literature. Nucleotide interconversion at the surface of 1321N1 cells was also examined indirectly using the ecto-ATPase inhibitor ARL 67156 and hexokinase. Although there was no significant effect of nucleotide breakdown on the agonist concentration-responses, it was found that upstream conversion of nucleotide diphosphates to triphosphates occurred, giving the false impression of activity of nucleoside diphosphates.;The antagonists suramin, PPADS and NF023 were examined. Suramin was a competitive antagonist at the P2Y1 receptor, had weaker effects at the P2Y2 receptor, and was inactive at the P2Y4 receptor. PPADS was selective for the P2Y1 receptor over the P2Y2 and P2Y4 receptors, and NF023 appeared to have non-competitive actions. ATP was found to antagonise the response to UTP at the P2Y4 receptor. The time course of PLC activation appeared to rapidly desensitise with each of the receptors. Pertussis toxin reduced the responses to the P2Y2 receptors, but not to the P2Y1 and P2Y4 receptors, and responses to each of the receptors was inhibited by activation of protein kinase C.;A preliminary study with human saphenous vein smooth muscle cells showed that responses to nucleotides were small and difficult to characterise. In contrast, nucleotides elicited a robust response at spontaneously hypertensive rat smooth muscle cells, consistent with action at a P2Y4 receptor.