Abstract
IntroductionExtracellular 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.MethodsWe 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.ResultseATP 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.ConclusionsThese 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.
Highlights
Extracellular ATP is released by articular chondrocytes under physiological and pathological conditions
Understanding the mechanisms of Extracellular ATP (eATP) efflux may result in novel therapies for calcium crystal arthritis and osteoarthritis
Considerable evidence supports its role in extracellular pyrophosphate transport [9,10]. Extracellular pyrophosphate (ePPi) is a key regulator of pathologic mineralization in cartilage and other tissues. ePPi can be generated from eATP through the action of ecto-enzymes with nucleoside triphosphate pyrophosphohydrolase (NTPPPH) activity, such as Ecto-nucleoside triphosphohydrolase 1 (ENPP1)
Summary
Extracellular ATP (eATP) is released by articular chondrocytes under physiological and pathological conditions. 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. Most cell types release ATP to the extracellular space under both physiologic and pathologic conditions [1]. Low levels of extracellular ATP (eATP) transduce mechanical signals [2]. We recently showed that stable over-expression of the progressive ankylosis gene product (ANK) dramatically increases eATP levels in articular chondrocytes [8]. Delineating mechanisms of eATP efflux in cartilage may lead to the identification of novel modulators of ePPi production
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
