title: Estimation of intracellular calcium activity in confluent monolayers of primary cultures of quail medullary bone osteoclasts.
abstract: The present study was concerned with an investigation of the relative utilities of the fluorescent indicators fura-2 and fluo-3 for spectrofluorimetric estimation of the intracellular calcium concentration ([Ca2+]i) in confluent osteoclast monolayers that had been isolated from medullary bone of quail hens and maintained in tissue culture for 6-8 days. Additionally, we have determined the effects of raised extracellular calcium ([Ca2+]o) and chicken calcitonin (CT) on [Ca2+]i in this preparation. Relative to fura-2, fluo-3 was poorly incorporated into the osteoclasts and had a high apparent equilibrium binding constant (Kd) for Ca2+ binding (809 nM). The osteoclasts were only weakly sensitive to the calcium ionophore, ionomycin. It is concluded that fura-2 is of greater utility than furo-3 in this preparation. In contrast to its lack of effect in freshly isolated cells, elevated [Ca2+]o up to 20 mM stimulated a concentration-dependent increase in [Ca2+]i in cultured osteoclasts, but CT was without effect. These findings further support the idea that quail osteoclasts are able to acquire a Ca2+ sensor or 'receptor' and thus to respond to [Ca2+]o in a similar manner to mammalian osteoclasts when they are removed from the bone microenvironment, but retain a refractoriness to CT under these conditions.
authors.affiliation: School of Pharmacy and Biomedical Sciences, University of Portsmouth.
authors.forename: S
authors.initials: S
authors.lastname: Arkle
authors.forename: I M
authors.initials: IM
authors.lastname: Wormstone
authors.forename: Z A
authors.initials: ZA
authors.lastname: Bascal
authors.forename: C G
authors.initials: CG
authors.lastname: Dacke
mesh_terms: D000814:Aniline Compounds; D000818:Animals; D002116:Calcitonin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D002478:Cells, Cultured; D003370:Coturnix; D004305:Dose-Response Relationship, Drug; D005260:Female; D016257:Fura-2; D015759:Ionomycin / Q000494:pharmacology; D010010:Osteoclasts / Q000166:cytology / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D014966:Xanthenes
chemical_list: D000814:Aniline Compounds; D014966:Xanthenes; C059715:Fluo-3; D015759:Ionomycin; D002116:Calcitonin; D002118:Calcium; D016257:Fura-2