Despite an intensive research effort, very little is known about the cellular mechanism underlying osteoarthritis. As the aged population continues to increase, the number of individuals afflicted with arthritis will increase emphasizing the critical need for insights into the pathophysiology of this costly disease. The investigators believe that a better understanding of biophysical signal transduction in chondrocytes is a critical first step in understanding osteoarthritis. They propose to characterize mechano-chemical signal transduction, in response to fluid flow, in chondrocytes isolated from bovine articular cartilage. Their central hypothesis is that biophysical signal transduction in chondrocytes is at least partly defined by stretch activated (SA) channels which activate cytosolic Ca2+ mobilization. Their long term goals are to characterize fluid flow effects on cytosolic Ca2+ and proteoglycan synthesis and the role SA channels play in linking these two responses. These goals will be accomplished through the completion of four specific aims. 1) Quantify, in real time, the cytosolic Ca2+ concentration of bone articular chondrocytes (BAC) exposed to steady and oscillatory fluid flow; 2) Examine membrane stretch-induced channel activity BAC; 3) Quantify [Ca2+]i in BAC exposed to fluid flow in the presence and absence of factors which regulate specific signalling pathways; and 4) Quantify aggrecan mRNA and proteoglycan synthesis in BAC exposed fluid flow. BAC will be isolated and used after only subculture. Type II collagen expression will be monitored with indirect immunofluorescence and type I and II collagen mRNA by nuclease protection assays. Fluid flow effects on [Ca2+]i will be quantified by microspectrofluometry. SA channel function and expression will be quantified by patch clamp electrophysiology and nuclease protection assays, respectively. Aggrecan mRNA expression and proteoglycan synthesis, in response to fluid flow, will be quantified by nuclease protection assays and 35S-sulfate incorporation. These experiments will be repeated in the presence of specific inhibitors of fluid flow-induced cytosolic CA2+ mobilization. The results of this project will provide insights into the mechanism by which biophysical signals regulate chondrocyte metabolism.
