Probenecid blocks human P2X7 receptor-induced dye uptake via a pannexin-1 independent mechanism

Archana Bhaskaracharya; Phuong Dao-Ung; Iman Jalilian; Mari Spildrejorde; Kristen K Skarratt; Stephen J Fuller; Ronald Sluyter; Leanne Stokes

doi:10.1371/journal.pone.0093058

Probenecid blocks human P2X7 receptor-induced dye uptake via a pannexin-1 independent mechanism

PLoS One. 2014 Mar 26;9(3):e93058. doi: 10.1371/journal.pone.0093058. eCollection 2014.

Authors

Archana Bhaskaracharya¹, Phuong Dao-Ung¹, Iman Jalilian², Mari Spildrejorde², Kristen K Skarratt¹, Stephen J Fuller¹, Ronald Sluyter², Leanne Stokes³

Affiliations

¹ Sydney Medical School Nepean, University of Sydney, Nepean Hospital, Penrith, New South Wales, Australia.
² School of Biological Sciences, Illawarra Health and Medical Research Institute, University of Wollongong, Wollongong, New South Wales, Australia.
³ Sydney Medical School Nepean, University of Sydney, Nepean Hospital, Penrith, New South Wales, Australia; Health Innovations Research Institute, School of Medical Sciences, RMIT University, Bundoora, Melbourne, Victoria, Australia.

Abstract

P2X7 is a ligand-gated ion channel which is activated by ATP and displays secondary permeability characteristics. The mechanism of development of the secondary permeability pathway is currently unclear, although a role for the hemichannel protein pannexin-1 has been suggested. In this study we investigated the role of pannexin-1 in P2X7-induced dye uptake and ATP-induced IL-1β secretion from human monocytes. We found no pharmacological evidence for involvement of pannexin-1 in P2X7-mediated dye uptake in transfected HEK-293 cells with no inhibition seen for carbenoxolone and the pannexin-1 mimetic inhibitory peptide, 10Panx1. However, we found that probenecid inhibited P2X7-induced cationic and anionic dye uptake in stably transfected human P2X7 HEK-293 cells. An IC50 value of 203 μM was calculated for blockade of ATP-induced responses at human P2X7. Probenecid also reduced dye uptake and IL-1β secretion from human CD14+ monocytes whereas carbenoxolone and 10Panx1 showed no inhibitory effect. Patch clamp and calcium indicator experiments revealed that probenecid directly blocks the human P2X7 receptor.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Adenosine Triphosphate / physiology
Biological Transport, Active
Calcium Signaling
Connexins / metabolism*
Ethidium / metabolism
Fluorescent Dyes / metabolism
HEK293 Cells
Humans
Inhibitory Concentration 50
Interleukin-1beta / metabolism
Isoquinolines / metabolism
Lipopolysaccharides / physiology
Monocytes / immunology
Monocytes / metabolism
Nerve Tissue Proteins / metabolism*
Probenecid / pharmacology*
Purinergic P2X Receptor Antagonists / pharmacology*
Receptors, Purinergic P2X7 / metabolism*

Substances

Connexins
Fluorescent Dyes
Interleukin-1beta
Isoquinolines
Lipopolysaccharides
Nerve Tissue Proteins
PANX1 protein, human
Purinergic P2X Receptor Antagonists
Receptors, Purinergic P2X7
Adenosine Triphosphate
lucifer yellow
Ethidium
Probenecid

Grants and funding

This work was supported by a project grant from the National Health and Medical Research Council of Australia (LS, ID632687) and the Nepean Medical Research Foundation (LS). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.