Estimation of intracellular calcium activity in confluent monolayers of primary cultures of quail medullary bone osteoclasts

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.