Calcium release activated membrane channels of osteoblast-like cells: targets and entrance gates for metal cations?

Enossal metal implants release ions which alter e.g. the Ca2+-based signali ng of bone cells. One crucial element of this Ca2+ signaling is the calcium release activated calcium flux (CRAC) which is involved in the refilling o f the cell's intracellular Ca2+ stores (ICS). Properties of this CRAC were studied in cultured osteoblast-like (OBL) cells using the Ca2+ sensitive fl uorescent dye fura-2. CRAC channels were opened by depleting ICS in the abs ence of extracellular calcium ([Ca2+](e)) by either thapsigargin (5 mu M) o r 4-bromo-A23187 (2 mu M). Elevation of [Ca2+](e) to 1.8 mM then increased the free intracellular Ca2+ ([Ca2+](i)). This Ca2+ influx was a typical CRA C and could be blocked by flufenamic acid (100 mu M) which is a characteris tic inhibitor of cation channels with low selectivity. Induction of CRAC en hanced the influx of extracellular Mn2+ (2 mM) 4.3fold, as measured by quen ching of flura-2 fluorescence. Ni2+, on the one hand, potentiated the immed iate CRAC while, on the other hand, it reversibly inhibited Ca2+ influx at a later stage. Similarly, Pb2+ (> 5 mu M) dose dependently inhibited the im mediate CRAC. Using the high affinity of fura-2 to Pb2+ and the resulting C a2+ binding-like change of the optical signal, the permeation of Pb2+ into OBL cells could be detected. This binding could be reversed by the cell per meant heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediam ine mine (TPEN). At the single cell level, the permeation of Pb2+ correlate d with increasing amounts of CRAC. Even low concentrations of Pb2+ (1 mu M) , which have been found in the blood of human individuals, clearly increase d the fura-2 signal. These results highlight CRAC channels of OBL cells as a possible entrance gate and/or a target for metal ions which may be releas ed from metal implant materials.