Abstracts

Rationale: It is well-established that patients with epilepsy have an increase in fracture risk. However, the mechanism for this association is less well defined. We hypothesised that osteoblasts express ion channels which could be inhibited by anti-epileptic medication, potentially altering signalling during bone remodelling and repair. We aimed to investigate whether: (1) mouse primary calvarial osteoblasts express voltage-activated sodium currents (NaV); and (2) the anti-epileptic medication carbamazepine (CBZ) inhibits these currents. Methods: Primary osteoblasts isolated from neonatal C57BL6 mice calvariae were used to examine the impact of CBZ on whole-cell current recordings produced using Patchliner, an automated planar patch-clamp system (Nanion, Germany). Immunocytochemistry was performed to confirm presence of NaV channels. Currents were elicited using a voltage protocol stepping from -100 mV to +60 mV in 10 mV increments, for 20 ms, from a holding potential of -80 mV or -60 mV. CBZ (50 μM) was applied to the cells in the continued presence of potassium channel blockers external tetraethylammonium (10 mM) and internal Cs⁺. Following washout of CBZ, 10 μM tetrodotoxin (TTX) a known voltage-gated sodium channel blocker was applied. Results: Immunocytochemistry confirmed presence of NaV in the primary calvarial osteoblast cultures. Robust voltage-activated inward currents were elicited and external application of CBZ (50 μM) resulted in a significant inhibition of current amplitude 31.6 ± 5.9 % (n = 7; p<0.001), which was partially reversed upon washout. Subsequent application of TTX (10 μM) produced almost complete inhibition of current amplitude 89.96 ± 2.14 % (n = 6; p<0.0001). Conclusions: Here we demonstrate that mouse osteoblasts express native voltage-activated sodium channels, which are sensitive to CBZ. To our knowledge this is the first study to utilise a Patchliner to examine native primary osteoblast sodium currents, and to demonstrate an inhibitory effect of CBZ on this current. Further study is required to determine whether the effects on ion channel activity observed here translate to clinically-relevant changes in bone signalling and bone quality. Funding acknowledgements: Melbourne Brain Centre Postdoctoral Fellowship 2012-2014 Victor Hurley Grants in Aid, The Royal Melbourne Hospital 2013 RACP Servier Barry Young Fellowship in Neuroscience 2012 DW Keir Fellowship in Medical Research, The Royal Melbourne Hospital 2010-2011