Characterisation of serum‐induced intracellular Ca2+ oscillations in primary bone marrow stromal cells

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Abstract
Intracellular Ca2+ signalling is pivotal to cell function and [Ca2+]i oscillations permit precise and prolonged modulation of an array of Ca2+-sensitive processes without the need for extended, global elevations in [Ca2+]i. We have studied [Ca2+]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 [Ca2+]i signalling was not observed, but exposure to 1% FCS induced regular, sustained Ca2+ 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 Ca2+ stores with 100 nM–1 µM thapsigargin or discharge of mitochondrial membrane potential and were sensitive to blockade of IP3-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 Ca2+ influx with EGTA or La3+ but were poorly sensitive to nifedipine (1–10 µM) and Bay K 8644 (300 nM). The factor(s) responsible for inducing [Ca2+]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 Ca2+ signals in marrow stromal cells. We conclude that Ca2+ oscillations occur constantly in these cells in culture and are potentially important regulators of cell proliferation and differentiation. J.Cell.Physiol.
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