Year

2011

Degree Name

Master of Science - Research

Department

School of Biological Sciences

Abstract

The P2X7 receptor is a trimeric ligand-gated ion channel, present in many mammalian species including the canine. P2X7 activation by its natural ligand, extracellular adenosine 5´-triphosphate (ATP), leads to the opening of a large non-selective pore, allowing the entry of large fluorescent dyes (such as ethidium+), and the activation of the NALP3 inflammasome and the subsequent release of pro-inflammatory cytokines interleukin (IL)-1β and IL-18. P2X7 is present on haematopoietic, bone and epithelial cells. Pannexin-1 may be involved in P2X7- induced uptake and IL-1β release, but its role remains unclear. This study aims to:

1. To determine if Madin-Darby canine epithelial kidney (MDCK) cells express functional P2X7;

2. To determine if pannexin-1 is involved in P2X7-induced ethidium+ uptake;

3. To clone P2X7 from an English Springer Spaniel;

4. To determine if P2X7 function differs between dogs or breeds.

Reverse transcriptase (RT)-PCR and immunoblotting demonstrated the existence of P2X7 mRNA and protein in MDCK cells, respectively. Incubation with ATP and 2(3´)-O-(4-benzoylbenzoyl) adenosine 5´triphosphate (BzATP) induced ethidium+ uptake into MDCK cells. Two specific P2X7 antagonists, KN-62 and A-438079, inhibited ATP-induced ethidium+ uptake in MDCK cells by 60% and 92%, respectively. Collectively, this demonstrated that MDCK cells express functional P2X7. RT-PCR also demonstrated that MDCK cells express the inflammasome components, NALP3 and caspase-1, and Toll-like receptor 4 (TLR4), but not IL-1β and IL-18. Lipopolysaccharide (LPS) up-regulated the expression of caspase-1, but not P2X7 or NALP3, in a concentration-dependent manner. LPS failed to induce the expression of either IL-1β or IL-18. The up-regulation of caspase-1 suggests that MDCK cells express functional cell-surface TLR4. The presence of TLR4 on MDCK cells was confirmed using an anti-TLR4 monoclonal antibody and flow cytometry. Therefore, as MDCK cells express functional P2X7, NALP3, caspase-1 and TLR4, this cell line should provide future opportunities to study the role of P2X7 in canine epithelial cells.

RT-PCR confirmed the expression of P2X7 and pannexin-1 in murine J774 macrophages. The expression of P2X7 in J774 cells was confirmed by immunoblotting, and by flow cytometric and ELISA measurements of ATP-induced ethidium+ uptake and IL-1β release respectively in the absence or presence of KN-62 or A-438079. Pannexin-1 antagonists, carbenoxolone (CBX), melfoquine (MFQ), flufenamic acid (FFA) and probenecid (PRO), failed to inhibit ATP-induced ethidium+ uptake into J774 cells with the exception of CBX at 50 μM. In contrast, CBX, FFA and PRO impaired P2X7-induced IL-1β release from J774 cells. Thus, although the role of pannexin-1 in P2X7-induced responses remains questionable, pannexin-1 antagonists may be of value in future studies with monocytes from canines and other species.

P2X7 was cloned from the cDNA of an English Springer Spaniel, and tagged with green fluorescence protein (GFP). This resulted in the generation of two vectors with canine P2X7, without (pP2X7-Ac-N1) or with GFP (pP2X7-AcGFP-N1). Sequence comparisons of the cloned P2X7 (English Springer Spaniel) to previously sequenced or cloned canine P2RX7 genes identified two non-synonymous single nucleotide polymorphisms (SNPs). These SNPs produced amino acid changes at positions Pro462Ser and Cys507Gly. The functional significance of these SNPs remains unknown. Flow cytometric analysis demonstrated that pcP2X7-AcGFP-N1 was expressed at low amounts in transfected human 1321N1 astrocytoma cells, but not in human embryonic kidney 293 (HEK293). It remains to be determined if this cloned P2X7 can form functional channel/pores in mammalian cells. Thus, despite successful cloning of canine P2X7, the expression and function of the cloned receptor needs further study.

Finally, a fixed-time flow cytometric assay to measure the function of P2X7 in canine peripheral blood monocytes was developed. This assay required the isolation of buffy coats from whole blood, followed by the isolation of peripheral blood mononuclear leukocytes cells by density centrifugation before assessment of ATP-induced YO-PRO-12+ uptake into CD14+ monocytes by flow cytometry. ATP-induced YO-PRO-12+ uptake into canine monocytes was blocked by KN- 62 and A-438079 indicating that this process was mediated by P2X7. ATP-induced YO-PRO-12+ uptake was higher in day 1 compared to day 0 monocytes, therefore P2X7 function was only compared in fresh (day 0) blood from different dogs. After standardization of the assay, P2X7 function in monocytes from different dogs was measured to determine the relative variation in P2X7 function between these animals. There was a 2.6-fold difference in P2X7 function between dogs with the lowest and highest ATP-induced YO-PRO-12+ uptake. ATP-induced YO-PRO-12+ uptake into monocytes, B cells and T cells was also compared among five different dog breeds. P2X7 function in T cells was significantly greater than that of monocytes and B-cells. Finally, dog genomic DNA was extracted from each dog sample and stored to establish a canine DNA bank for future sequencing of the P2RX7 gene.

This study forms part of a larger group project to understand the physiological and pathophysiological role of the canine P2X7 including the identification of SNPs which may alter P2X7 function or be associated with canine disorders. Results from this thesis will help contribute to the future progress and direction of the larger study.

FoR codes (2008)

060110 Receptors and Membrane Biology, 070705 Veterinary Immunology, 110704 Cellular Immunology, 110707 Innate Immunity

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Unless otherwise indicated, the views expressed in this thesis are those of the author and do not necessarily represent the views of the University of Wollongong.