Characterisation of serum‐induced intracellular Ca 2+ oscillations in primary bone marrow stromal cells

Intracellular Ca 2+ signalling is pivotal to cell function and [Ca 2+ ] i oscillations permit precise and prolonged modulation of an array of Ca 2+ ‐sensitive processes without the need for extended, global elevations in [Ca 2+ ] i . We have studied [Ca 2+ ] i signalling in primary rat marrow stromal cells exposed to foetal calf serum (FCS) constituents at concentrations up to those required to promote growth and differentiation in culture. Spontaneous [Ca 2+ ] i signalling was not observed, but exposure to 1% FCS induced regular, sustained Ca 2+ oscillations in 41 ± 3% of cells. Incidence of FCS‐induced oscillations was dose‐dependent, saturating at 0.5%. These oscillations were arrested by disruption of Ca 2+ stores with 100 nM–1 µM thapsigargin or discharge of mitochondrial membrane potential and were sensitive to blockade of IP 3 ‐receptors by 50 µM 2‐amino‐ethoxydiphenyl borate (2‐APB) and inhibition of phospholipase C with 5 µM U73122. The oscillations decreased in frequency and amplitude following inhibition of Ca 2+ influx with EGTA or La 3+ but were poorly sensitive to nifedipine (1–10 µM) and Bay K 8644 (300 nM). The factor(s) responsible for inducing [Ca 2+ ] i oscillations are heat stable, insensitive to disulphide bond reduction with 20 mM dithioerythritol and retained by a 30 kDa molecular weight filter. Serum is routinely present in culture medium at 10%–15% [v/v] and marrow stromal cells maintained under culture conditions exhibited sustained oscillations. This is the first demonstration of agonist‐induced complex Ca 2+ signals in marrow stromal cells. We conclude that Ca 2+ oscillations occur constantly in these cells in culture and are potentially important regulators of cell proliferation and differentiation. J.Cell.Physiol. © 2005 Wiley‐Liss, Inc.