Year

2013

Degree Name

Master of Science - Research

Department

School of Biological Sciences

Abstract

The P2X7 receptor is an adenosine 5’-triphosphate (ATP)-gated ion channel expressed on the cell-surface of many cell types, including cells of haematopoietic origin and epithelial cells. Upon prolonged activation by extracellular ATP, P2X7 has the ability to form pores permeable to large organic cations such as ethidium+ and YO-PRO-12+. P2X7 activation leads to the stimulation of the NALP3 inflammasome and the subsequent release of the proinflammatory cytokines interleukin (IL)-1β and IL-18.

The first aim of this study was to confirm the presence or absence of P2X7 and NALP3-related components in Madin-Darby canine kidney (MDCK) epithelial cells and lipopolysaccharide (LPS)-primed canine monocytes. Reverse transcriptase (RT)-PCR detected the presence of P2X7, NALP3, caspase-1, IL-1β and IL-18 mRNA in MDCK cells and monocytes. The identity of each transcript, except IL-1β due to its small size, was confirmed by sequencing. This data suggests that MDCK cells will provide a useful model cell line to study the role of P2X7 and NALP3-related components in kidney epithelial cells and renal disorders. Moreover, these results indirectly support our previous observations that P2X7 activation induces IL-1β from LPS-primed canine monocytes.

The second aim of this study was to determine if ATP or the Toll-like receptor (TLR) ligands, LPS or lipoteichoic acid (LTA), induce IL-1β release from MDCK cells and in whole canine blood. ELISA measurements failed to detect IL-1β release from MDCK cells despite examining various combinations of ATP, LPS and/or nigericin (which induces IL-1β release independently of P2X7). Further studies are required however to determine whether MDCK cells can release IL-1β. In contrast to MDCK cells, LPS and ATP but not LTA induced IL-1β release in whole blood. Moreover, ATP-induced IL-1β release in whole blood required priming with LPS. A role for P2X7 in LPS- and ATP-induced IL-1β release in blood however could not be established, as the P2X7 antagonist AZ10606120 failed to impair IL-1β release. Further studies are required to determine if either or both of these processes require P2X7 activation.

The third aim of this study was to sequence and pharmacologically characterise a recombinant canine P2X7 receptor cloned from an English Springer Spaniel. Sequencing of the cloned receptor revealed two nonsynonymous (missense) single nucleotide polymorphisms (SNPs): Leu440Phe and Pro452Ser. Immunoblotting confirmed the expression of P2X7 protein in canine P2X7-transfected HEK 293 cells. Moreover, flow cytometric measurements demonstrated that ATP induced ethidium+ (314 Da) uptake into P2X7-transfected but not mock-transfected HEK 293 cells in a time-dependent manner. The P2X7 agonists ATP, 3’-O-(4-benzoyl)benzoyl ATP (BzATP) and adenosine 5’-O-(3-thiotriphosphate) (ATPγS) induced ethidium+ uptake into P2X7-transfected HEK 293 cells in a concentration-dependent manner with EC50 values of 253 μM, 13 μM and 438 μM respectively. In contrast, adenosine 5’-diphosphate (ADP), uridine 5’-triphosphate (UTP) and α,β-methylene ATP (α,β-meATP) failed to induce ethidium+ uptake into these cells. The P2X7 antagonists A438079, AZ10606120, AZ11645373, BBG and KN-62 completely impaired ATP-induced ethidium+ uptake into P2X7-transfected HEK 293 cells with IC50 values of 190 nM, 11 nM, 7 nM, 1110 nM and 19 nM respectively. Finally, ATP induced YO-PRO-12+ (375 Da) and propidium2+ (415 Da) uptake into P2X7-transfected cells. Collectively, the recombinant canine P2X7 receptor demonstrated similar characteristics to that of native canine and recombinant human P2X7. Thus, P2X7 drugs developed to block P2X7 in humans may also be of therapeutic value in dogs.

The last aim of this thesis was to confirm if the relative monocyte P2X7 function varies between dogs and to determine if this variation is due to SNPs in the P2RX7 gene. Flow cytometric measurements of ATP-induced YO-PRO-12+ uptake into peripheral blood canine monocytes confirmed that the relative P2X7 function varied between dogs and within breeds. Flow cytometric measurements also demonstrated that the relative P2X7 function of human monocytes varies between subjects and over time. Amplification and sequencing of the canine P2RX7 gene of 19 dogs and MDCK cells identified four non-synonymous SNPs: Phe103Leu (exon 3), Arg270Cys (exon 8), Arg365Gln (exon 11), and Pro452Ser (exon 13). The dogs for which we had both functional data and genomic DNA (n = 62) were screened for the above SNPs and the resulting genotypes were compared to relative P2X7 function. Three of the SNPs (Phe103Leu, Arg365Gln and Pro452Ser) did not correspond with a change in P2X7 function. In contrast, the Arg270Cys SNP was associated with a loss-of-function in P2X7, however this finding was restricted to one dog, as well as MDCK cells which have low P2X7 function. Future studies using mutant P2X7 receptors, obtained by site-directed mutagenesis, are required to explore if any of these SNPs alter P2X7 function.

This thesis forms a part of an ongoing study investigating the role of canine P2X7 in inflammation and immunity. The confirmation of P2X7 in canine monocytes and MDCK cells, and the identification of SNPs in the canine P2RX7 gene will provide future opportunities to investigate the role of the P2X7 receptor and its gene in canine health and disease.

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