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+.
Institut für Physiologie, Universität-GH Essen, Germany.
M
M
Wiemann
D
D
Büsselberg
K
K
Schirrmacher
D
D
Bingmann
D000818:Animals; D000831:Animals, Newborn; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000502:physiology; D002478:Cells, Cultured; D005439:Flufenamic Acid / Q000494:pharmacology; D016257:Fura-2; D008345:Manganese / Q000378:metabolism; D009532:Nickel / Q000378:metabolism / Q000494:pharmacology; D010006:Osteoblasts / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D013050:Spectrometry, Fluorescence; D019284:Thapsigargin / Q000494:pharmacology; D014700:Verapamil / Q000494:pharmacology
D015220:Calcium Channels; D000001:Calcimycin; D008345:Manganese; D005439:Flufenamic Acid; D019284:Thapsigargin; D009532:Nickel; D014700:Verapamil; D002118:Calcium; D016257:Fura-2