A CALCIUM-RELEASE ACTIVATED CALCIUM INFLUX IN PRIMARY CULTURES OF RATOSTEOBLAST-LIKE CELLS

Osteoblast-like (OBL) cells in primary culture were tested for their a bility to generate a calcium release activated calcium flux (CRAC). In flux of Ca2+ was optically detected by fura-2. Intracellular calcium s tores (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 cou ld be blocked by flufenamic acid (100 mu M) but not verapamil (20 mu M ). Ni2+ (1 mM), although reversibly blocking CRAC? accelerated the ini tial [Ca2+], influx rate. Induction of CRAC enhanced the influx of Mn2 + 4.3-fold, as measured by quenching of fura-2 fluorescence. In summar y, OBL cells exhibit a CRAC which allows for the permeation of ions ot her than Ca2+. This Ca2+ flux may be activated by transmembraneous gra dients of Ca2+ and Ni2+.