Faculty Collaboration Database

The progressive ankylosis gene product ANK regulates extracellular ATP levels in primary articular chondrocytes. Arthritis Res Ther 2013 Oct 17;15(5):R154

Date

11/30/2013

Pubmed ID

24286344

Pubmed Central ID

PMC3978574

DOI

10.1186/ar4337

Scopus ID

2-s2.0-84885537495 (requires institutional sign-in at Scopus site)   38 Citations

Abstract

INTRODUCTION: Extracellular ATP (eATP) is released by articular chondrocytes under physiological and pathological conditions. High eATP levels cause pathologic calcification, damage cartilage, and mediate pain. We recently showed that stable over-expression of the progressive ankylosis gene product, ANK, increased chondrocyte eATP levels, but the mechanisms of this effect remained unexplored. The purpose of this work was to further investigate mechanisms of eATP efflux in primary articular chondrocytes and to better define the role of ANK in this process.

METHODS: We measured eATP levels using a bioluminescence-based assay in adult porcine articular chondrocyte media with or without a 10 minute exposure to hypotonic stress. siRNAs for known ATP membrane transporters and pharmacologic inhibitors of ATP egress pathways were used to identify participants involved in chondrocyte eATP release.

RESULTS: eATP levels increased after exposure to hypotonic media in a calcium-dependent manner in monolayer and 3-dimensional agarose gel cultures (p < 0.001). A potent transient receptor potential vanilloid 4 (TRPV4) agonist mimicked the effects of hypotonic media. ANK siRNA suppressed basal (p < 0.01) and hypotonically-stressed (p < 0.001) ATP levels. This effect was not mediated by altered extracellular pyrophosphate (ePPi) levels, and was mimicked by the ANK inhibitor, probenecid (p < 0.001). The P2X7/4 receptor inhibitor Brilliant Blue G also suppressed eATP efflux induced by hypotonic media (p < 0.001), while ivermectin, a P2X4 receptor stimulant, increased eATP levels (p < 0.001). Pharmacologic inhibitors of hemichannels, maxianion channels and other volume-sensitive eATP efflux pathways did not suppress eATP levels.

CONCLUSIONS: These findings implicate ANK and P2X7/4 receptors in chondrocyte eATP efflux. Understanding the mechanisms of eATP efflux may result in novel therapies for calcium crystal arthritis and osteoarthritis.

Author List

Rosenthal AK, Gohr CM, Mitton-Fitzgerald E, Lutz MK, Dubyak GR, Ryan LM

Author

Ann K. Rosenthal MD Associate Dean, Professor in the Medicine department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Adenosine Triphosphate
Animals
Ankylosis
Biological Transport
Calcium
Cartilage, Articular
Cells, Cultured
Chondrocytes
Extracellular Space
Leucine
Phosphate Transport Proteins
Probenecid
Purinergic P2X Receptor Antagonists
Receptors, Purinergic P2X4
Receptors, Purinergic P2X7
Sulfonamides
Swine
TRPV Cation Channels