Osteoblasts, which differentiate from bone marrow stromal cells, synthesise new bone. Ca²⁺ influx modulates a range of mechanisms in osteoblasts, including matrix deposition and responses to hormones (Liu et al. 2000). L-type voltage operated Ca²⁺ channels (VOCCs) are crucial to the normal response of bone cells to mechanical force (Walker et al. 2000). L-type VOCCs are ubiquitous in osteoblastic cells and are expressed in cultured primary bone marrow cells (Publicover et al. 1995).

The properties of the L-type VOCCs expressed in marrow stromal cells were characterised using whole-cell patch clamp, (V_h = -60 mV; test pulse = +20 mV. Extracellular saline contained 10 mM BaCl2,10 mM HEPES and 130 mM N-methyl glucamine (pH 7.6 with NaOH). Electrodes were backfilled with a standard saline containing 150 mM CsCl, 5 mM EGTA, 10 mM HEPES and 10mM D-glucose (pH 7.3 with CsOH). Femora were removed from male Wistar rats humanely killed by cervical dislocation, and the marrow cavity flushed with culture medium. Marrow stromal cells were cultured in α-minimal essential media with 15 % [v/v] fetal calf serum, penicillin, streptomycin, ascorbic acid and β-glycerophosphate, in a humidified atmosphere of 5 % CO₂ at 37 °C for 16 to 30 days.

Whole-cell recordings revealed an agonist-independent enhancement of VOCC current amplitude, which reached a plateau in approximately 8 min. This ‘run-up’ was followed by typical current run-down. The run-up of current amplitude was highly significant (P = <2 X 10^-5; n = 18; 2-tailed paired t test), increasing by 225.3 ± 51.6 % of initial current (mean ± S.E.M., n = 10). Dialysis occurs between the electrode saline and cytoplasm during whole-cell recording (Pusch & Neher, 1988). This may instigate removal of an inhibitor of stromal cell VOCCs. The possibility that G-proteins confer such tonic inhibition was investigated. Preliminary experiments indicate that intracellular inclusion of 0.5 mM GTP has no significant effect on run-up amplitude (P = 0.28; 2-tailed t test), or the initial rate of run-up (P = 0.757; 2-tailed t test) in stromal cells. Run-up, particularly of this magnitude, is extremely unusual and previously unreported in bone cells.

These data suggest that a large proportion of stromal cells’ Ca²⁺ channels are inactive under basal conditions as a result of tonic inhibition. Such tonic inhibition, if removed by an endogenous agonist, would afford great potential for the enhancement of voltage-operated currents. Mechanical force generates Ca²⁺ influx via L-type VOCCs in osteoblasts (discussed above). Removal of tonic inhibition could therefore provide a coincidence detection system, whereby simultaneous agonist stimulation and force-induced VOCC activation could generate larger currents than force alone.