| Summary: | Chondrocytes are the cells of articular cartilage responsible for the production and maintenance of their extracellular matrix (ECM). They exist in a unique environment of low partial pressures of oxygen, low pH and avascularity (Urban, 1994). The chondrocyte environment is also challenged by constant osmotic fluxes during osmolarity changes induced by joint loading. This thesis investigates the ion channels expressed by chondrocytes and examines the possible functions for these channels. Whole cell and single channel electrophysiological experiments identified chloride, sodium, potassium and non-selective cation conductances in isolated canine chondrocytes. Whole-cell current clamp measurements of membrane potential (Vm) showed that chondrocytes have very depolarised Vm compared to other cell types (in the range of -8 to -14mV, measured from bovine, ovine, equine and canine chondrocytes). Permeability experiments carried out in whole-cell voltage clamp mode determined that the non-selective cation current was selective for calcium over sodium and potassium at a level similar to that of the transient receptor potential vanilloid type five and six channels (TRPVS and 6). RT-PCR detected mRNA for TRPV4 and TRPVS channels. The effect of these channels on chondrocyte membrane potential (Vm) was determined using whole-cell current-clamp electrophysiology. Experiments with the selective TPRVS inhibitor econazole suggest that TRPVS is constitutively active and important for setting the depolarised Vm. Conversely, TRPV4 appeared to be inactive at rest and when activated by the selective agonist 4 a-phorbol 12,13-didecanoate indirectly caused an increase in potassium conductance and thus membrane hyperpolarisation. A low conductance sodium channel was also identified. Sensitivity to low concentrations of amiloride and benzamil indicate that this channel is the epithelial sodium channel (ENaC). Whole-cell current-clamp showed that this ENaC had a small but significant contribution to the Vm. A mixed population of chloride channels was found in the chondrocyte using single channel and whole-cell current and voltage clamp recordings. This population consisted of a maxi-chloride channel and a calcium-activated chloride channel. A model of chondrocyte Vm was then developed combining the approach of Hodgkin and Huxley (1952b) with parameters measured in the patch clamp experiments. This model was able to simulate many of the effects of inhibition of channel conductances on the resting membrane potential. Whole-cell current clamp experiments using inhibitors of the ion conductances discovered previously, verified the models predictions. This thesis then tested the hypothesis that the depolarised Vm of chondrocytes facilitates volume regulation. Cell volume experiments discovered that at more negative Vm chondrocytes were unable to regulate their volume upon hypotonic challenge. At more positive potentia Is, cell volume recovery was significantly greater. This is likely to be due to the greater driving force for potassium efflux and suggests that the depolarised Vm is an adaptation which allows the chondrocyte to effectively regulate its volume.
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