A calcium release activated calcium influx in primary cultures of rat osteoblast-like cells

Osteoblast-like (OBL) cells in primary culture were tested for their ability to generate a calcium release activated calcium flux (CRAC). Influx of Ca2+ was optically detected by fura-2. Intracellular calcium stores (ICS) were emptied in the absence of extracellular calcium ([Ca2+](e)) by 5 mu M thapsigargin (TG) or 2 mu M A23187, Readdition of 1.8 mM [Ca2+](e) increased the free intracellular Ca2+ ([Ca2+](i)) after a delay of 30-60 seconds at a rate of 2.3 nM/s due to CRAC. This rate depended on [Ca2+](e) and was substantially lowered if readdition of 1.8 mM [Ca2+], was preceded by, e.g., 0.72 mM [Ca2+](e). CRAC-induced [Ca2+](i) peaks were correlated (r= 0.543) with [Ca2+](i) peaks during the complete depletion of ICS with A23187. Ca2+ influx due to CRAC could be blocked by flufenamic acid (100 mu M) but not verapamil (20 mu M). Ni2+ (1 mM), although reversibly blocking CRAC? accelerated the initial [Ca2+], influx rate. Induction of CRAC enhanced the influx of Mn2+ 4.3-fold, as measured by quenching of fura-2 fluorescence. In summary, OBL cells exhibit a CRAC which allows for the permeation of ions other than Ca2+. This Ca2+ flux may be activated by transmembraneous gradients of Ca2+ and Ni2+.