Characterisation Of Whole‐Cell Properties In Chondrocytes From An In Vitro Model Of Arthritis

Abstract

Chondrocytes express a rich complement of ion channels with a range of functions including regulation of membrane potential (Vm) and cell volume. Transcript studies show changes in ion channel expression with osteoarthritis (OA) and in vitro studies show that pro‐inflammatory cytokines can induce cell apoptosis. Using an in vitro model of OA (DMEM + 10ng/ml TNFα and Il1β) we examined changes in cellular properties with patch‐clamp and intracellular Ca2+ measurements. Healthy chondrocytes have depolarised Vm (~‐10mV) so, using a whole cell tail current voltage protocol, we investigated if Vm was affected in our model of OA. Cells received a 1s hyperpolarising step with 10mV increments for 10 cycles. Cells from the model had a whole cell reversal potential (Vrev) 16±2mV more positive than healthy cells (n=5, p<0.05). Healthy cells had a greater whole‐cell conductance than cytokine treated cells (1.8±0.6nS vs 0.9±0.1nS; n=4 and 5, respectively; p<0.05). Given the positive shift in Vrev, we investigated Ca2+ handling of these cells using hypotonic challenge to increase intracellular Ca2+ entry. Healthy cells responded to the challenge with a peak Ca2+ increase of 115±15nM (n=16). When applied to cytokine treated cells the same challenge caused a greater Ca2+ increase (328±45nM; n=11; p蠄0.01). This Ca2+ increase was inhibited by a TRPC channel antagonist, PYR3 (n=69). Intracellular Ca2+ increases greater than 300nM have been shown to lead to cell apoptosis; the changes we observe here could implicate important pathological changes in cytokine‐stimulated chondrocytes. Further work is necessary to identify the mechanism by which this Ca2+ increase occurs. Funded by DBOARD (EU FP7/2007‐2013).