AuPS Logo Programme
Previous Next PDF

Probenecid, a gout treatment, blocks the human P2X7 receptor

A. Bhaskaracharya,1 P. Dao-Ung,1 I. Jalilian,2 M. Spildrejorde,2 K. Skarratt,1 S. Fuller,1 R. Sluyter2 and L. Stokes,1 1Sydney Medical School Nepean, University of Sydney, Nepean Hospital, Penrith, NSW 2750, Australia and 2Illawarra Health and Medical Research Institute, School of Biological Science, University of Wollongong, NSW 2522, Australia.

Activation of the ligand-gated ion channel P2X7 induces uptake of large organic cations and anions, a feature known as secondary permeability and measured by the rapid uptake of dye molecules. The hemichannel pannexin-1 is thought to be responsible for P2X7-induced secondary permeability. Our aim in this study was to determine whether the increased dye uptake response observed in gain-of-function 348Thr-P2X7 receptors was due to increased signalling through pannexin-1 using pharmacological tools.

Methods: ATP-induced dye uptake was measured using a fluorescent plate reader and ethidium/YOPRO-1 dyes. Calcium responses were measured using Fluo-4AM calcium indicator dye and a CoolSnap HQ2 CCD camera coupled to a fluorescent microscope. ATP-induced inward currents were measured by whole cell patch clamp. CD14+ monocytes were isolated from mononuclear cells and primed with LPS for IL-1β secretion assays. Pannexin-1 inhibitors tested were carbenoxolone (CBX), a peptide blocker (10Panx1), flufenamic acid, mefloquine and probenecid.

Results: Unexpectedly we found no pharmacological evidence for pannexin-1 involvement in dye uptake in HEK-293 cells expressing human P2X7 receptors or in primary human monocytes. The pannexin-1 inhibitor carbenoxolone did show an inhibitory effect on ATP-induced dye uptake in J774 mouse macrophages. Probenecid dose-dependently blocked YOPRO dye uptake in HEK cells expressing human P2X7 and blocked ATP-induced IL-1β secretion from primary human monocytes. Probenecid also blocked P2X7-induced calcium responses and ATP-induced inward currents in HEK-hP2X7 cells suggesting that this compound actually interacts with and blocks the P2X7 receptor.