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 microM thapsigargin (TG) or 2 microM 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+]e 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 microM) but not verapamil (20 microM). Ni2+ (1 mM), although reversibly blocking CRAC, accelerated the initial [Ca2+]i 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+.