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METHODS IN CELL PHYSIOLOGY

A novel method for assessing effects of hydrostatic fluid pressure on intracellular calcium: a study with bovine articular chondrocytes

Department of Orthopedic Surgery, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts

Submitted 8 March 2004 ; accepted in final form 8 October 2004

Chondrocytes in articular cartilage are exposed to hydrostatic pressure and distortional stress during weight bearing and joint loading. Because these stresses occur simultaneously in articular cartilage, the mechanism of mechanosignal transduction due to hydrostatic pressure alone in chondrocytes is not clear. In this study, we attempted to characterize the change in intracellular calcium concentration ([Ca²⁺]_i) in response to the application of hydrostatic fluid pressure (HFP) to cultured bovine articular chondrocytes isolated from defined surface (SZ) and middle zones (MZ) by using a fluorescent indicator (X-rhod-1 AM), a novel custom-made pressure-proof optical chamber, and laser confocal microscopy. Critical methodology implemented in this experiment involved application of high levels of HFP to the cells and the use of a novel imaging apparatus to measure the peak [Ca²⁺]_i in individual cells. The peak [Ca²⁺]_i in MZ cells cultured for 5 days showed a significant twofold increase after the application of HFP at constant 0.5 MPa for 5 min. The peak [Ca²⁺]_i in SZ cells was lower (43%) than that of MZ cells. The peak was suppressed with an inhibitor of dantrolene, gadolinium, or a calcium ion-free buffer, but not with verapamil. This study indicated that the increase in [Ca²⁺]_i in chondrocytes to HFP is dependent on the zonal origin. HFP stimulates calcium mobilization and stretch-activated channels.

real-time optical measurement; intracellular calcium; mechanosignal transduction

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