Articular chondrocytes synthesize and remodel the cartilage of synovial joints by maintaining the correct balance between matrix degradation and new matrix synthesis. Restoring this balance is the key to preventing or reversing the extensive cartilage damage of degenerative joint disease.From this perspective, the primary function of articular chondrocytes could be described as extracellular matrix homeostasis. The relationships between intracellular metabolic homeostasis, signalling pathways and matrix dynamics are very poorly understood. Specifically, the pivotal signalling ion, calcium, has been neglected in chondrocytes, and little is known about the regulation and possible roles of calcium in chondrocyte physiology. The applicants propose a detailed characterization of the receptor mechanisms and functional importance of calcium mobilization in mammalian articular chondrocytes, using cells in vitro, derive from human, equine and porcine cartilage. Calcium responses from cultured monolayers of mammalian chondrocytes will be analyzed at the single cell level by fluorescent ratio-imaging using Fura-2, an indicator of intracellular calcium concentration. Initially, the objectives are to build a catalogue of the precise response characteristics in each species for each stimulant. However, the focus of this proposal is the human articular chondrocyte. Using population calcium measurements as well as single cell receptor-mediated responses to bradykinin (acting through B2 receptors) and adenosine triphosphate (acting through P2 purinoceptors) will be characterized pharmacologically.Interactions between calcium-mobilizing receptors and other chondrocyte activators, such as the cytokine, interleukin 1, will be evaluated. A novel response, induction of calcium oscillations has been observed for the direct, non-receptor stimulant of calcium mobilization, thapsigargin. This reagent will be used as a receptor- independent probe of calcium dynamics and functions. Functional roles of cytosolic calcium elevation will be assessed by analyzing the roles of bradykinin, ATP and thapsigargin in growth regulation, stress protein induction, cytoprotection and prostaglandin production, both alone, in combination and together with other chondrocyte stimulants. This project will be to begin to analyze the functional responses of chondrocytes in relation to their original location within the articular cartilage. Chondrocyte subpopulations will be separated by vibratome sectioning and density gradient centrifugation. Differentiated cells maintained in alginate beads or explant culture will be analyzed using state-of-the-art single cell imaging microscopy.
