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Characterisation Of Whole‐Cell Properties In Chondrocytes From An In Vitro Model Of Arthritis
Author(s) -
Lewis Rebecca,
Jones William,
Wegg Michaela,
BarrettJolley Richard
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.714.1
Subject(s) - intracellular , cytokine , chemistry , in vitro , patch clamp , membrane potential , reversal potential , channel blocker , microbiology and biotechnology , biophysics , medicine , endocrinology , biology , receptor , calcium , biochemistry
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 Ca 2+ 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 ( V rev ) 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 V rev , we investigated Ca 2+ handling of these cells using hypotonic challenge to increase intracellular Ca 2+ entry. Healthy cells responded to the challenge with a peak Ca 2+ increase of 115±15nM (n=16). When applied to cytokine treated cells the same challenge caused a greater Ca 2+ increase (328±45nM; n=11; p蠄0.01). This Ca 2+ increase was inhibited by a TRPC channel antagonist, PYR3 (n=69). Intracellular Ca 2+ 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 Ca 2+ increase occurs. Funded by DBOARD (EU FP7/2007‐2013).