Proceedings of The Physiological Society
University of Cambridge (2008) Proc Physiol Soc 11, PC92
Graded, movement-coupled hyaluronan secretion into joints and potential signal pathways in vivo.
A. K. Wann1, K. R. Ingram1, R. M. Wingate1, J. R. Levick1
1. Physiology, St Georges University of London, London, United Kingdom.
Hyaluronan (HA) governs interstitial hydraulic permeability. In synovial fluid it also contributes to joint lubrication and intra-articular (i.a.) fluid conservation via filter-cake formation. Since i.a. HA injections and exercise reportedly improve moderate osteoarthritis, we investigated the effect of graded joint movement on HA secretion and potential signal pathways in vivo. Endogenous HA was washed out from pairs of cannulated knee joint cavities in anaesthetised rabbits (pentobarbitone 30 mg kg-1, urethane 500 mg kg -1 i.v.). The joints were subjected to intermittent, passive cycling at a fixed frequency (0.5Hz) for different durations (0, 1, 3, 9min in every 15min, duration 0-60%) or 20% duration at different frequencies (0, 0.17, 0.5, 1.5Hz) to determine stimulus-response curves. Newly secreted HA was harvested by washout after 5h and analysed by HPLC. Putative signalling pathways were assessed using i.a. pharmacological agents and contralateral control vehicle in moved or static joints. Movement at 0.5Hz and 20% duration almost doubled the HA secretion rate qHA (p<0.0001, paired t test, n=35). The coupling was a graded one, with curvilinear stimulus-response curves to both frequency and duration of movement (p=0.0001, ANOVA). Since stretch stimulates HA secretion in cell culture via a Ca2+ - protein kinase C (PKC) - MEK-ERK pathway (1), we next probed the pathway’s functionality in vivo. The Ca2+ ionophore ionomycin more than doubled qHA in static joints (p=0.02, paired t test, n=5), as did PKC activation by phorbol ester (PMA) (p= 0.001). Moreover the PKC inhibitor bisindolylmaleimide I (BIM) substantially inhibited PMA-stimulated secretion in static joints (p<0.02, n=10,16, t test), as did the MEK-ERK inhibitors U0126 and PD98059 (p≤0.001, n=5 respectively, paired t test). Despite these positive results in static joints, BIM, U0126 and PD98059 each failed to inhibit movement-stimulated HA secretion significantly (p=0.96,0.23, 0.32 respectively, n=5,8,11, paired t test). By contrast, the phospholipase C (PLC) inhibitor U73122 almost totally abolished the stimulation of secretion by movement, halving qHA in moved joints (p=<0.001, paired t test, n=10). U73122 did not significant inhibit qHA in static joints (p=0.13, n=5, paired t test). The study showed for the first time that the coupling between joint usage and HA secretion reported in (2) is a graded one, and that PLC may be an obligatory step in the signal transduction pathway. Ca2+ store release, PKC activation and downstream MEK-ERK can stimulate HA secretion in vivo, as in vitro; but whereas the PKC-MEK-ERK pathway mediates the response to stretch in vitro, it did not mediate the response to movement in vivo. Further elucidation of the signalling pathways is needed, with potential therapeutic implications.
Where applicable, experiments conform with Society ethical requirements