ATP release by mechanically loaded porcine chondrons in pellet culture

Abstract

Objective: To determine whether ATP is released from chondrocytes during mechanical stimulation and whether degradation of ATP generates inorganic pyrophosphate in chondron pellet cultures.

Methods: Chondron pellets were formed from 1.6 x 10(6) cells that had been enzymatically isolated from porcine articular cartilage. ATP was measured in media from cultures at rest and during fluid movement and cyclic compression. ATP hydrolysis was examined by high-performance liquid chromatography following the addition of gamma32P-ATP to resting cultures.

Results: Pellet cultures at rest maintained a steady-state concentration of 2-4 nM ATP in 2 ml of medium. The ATP concentration increased 5-12-fold with cyclic compression (7.5 and 15 kPa at 0.5 Hz), then decreased to preloading levels within 60 minutes despite continued loading. A subsequent increase in pressure stimulated a further increase in ATP release, suggesting that chondrocytes desensitize to load. Cell viability was similar for pellets at rest and up to 24 hours after compression. ATP released in response to mechanical stimulation was inhibited 50% by 0.5 mM octanol, suggesting a regulated mechanism for ATP release. Exogenous ATP was rapidly hydrolyzed to pyrophosphate in resting cultures.

Conclusion: The occurrence of basal levels of extracellular ATP in the presence of pyrophosphohydrolase activity indicates that ATP was continuously released by chondrocytes at rest. Considering that chondrocytes express purinoceptors that respond to ATP, we suggest a role for ATP in extracellular signaling by chondrocytes in response to mechanical load. ATP released by chondrocytes in response to mechanical load is a likely source of pyrophosphate in calcium pyrophosphate dihydrate crystal deposition diseases.