The synthesis of Calcium Green C, a lipophilic fluorescent calcium-sensitive dye, and its use as a monitor of Ca efflux from cells is described. This indicator consists of a Calcium Green-1 molecule conjugated to a lipophilic 18-carbon alkyl chain which will intercalate into cell membranes. The K of the indicator for Ca in aqueous solution (pH 7.2, 22 °C, ionic strength 0.1 M) is 0.23 ± 0.04 µM and in the presence of liposomes is 0.062 ± 0.007 µM. Due to its high negativity, the calcium chelating fluorophore faces the cell exterior, when loaded under a defined set of conditions. The dye was found largely on the surface of the cells when loaded at a concentration of 5 µM for 10 min at 37 °C. Five minutes after introduction of EGTA, 83-95% fluorescence disappeared, indicating that most of the fluorophore was on the cell surface. Photobleaching was minimal (3-13%). A confocal laser scanning microscope was used to detect and quantify fluorescence. Internalized dye was apparent in cells loaded for longer times (30-60 min) and in membrane-impaired cells, as shown by uptake of propidium iodide. Under defined confocal laser scanning microscope settings, a transient fluorescence at the periphery of 30% of the cells was observed following 10M parathyroid hormone treatment, indicating the presence of outwardly directed calcium transport across the plasma membrane. Calcium efflux usually lasted 7-10 min, peaking at around 2-3 min. Changes in cell shape were also observed. Calcium efflux was shown to be sensitive to (a) 10 µM quercetin and 10 µM vanadate, partially specific inhibitors of plasma membrane Ca-ATPase, to (b) 0.1 mM trifluoperazine, an agent which renders calmodulin ineffective, and to (c) 10 mM neomycin sulfate, which blocks release of Ca from intracellular stores. Thapsigargin (5 µM), an inhibitor of Ca-ATPase of the endoplasmic reticulum, prolonged fluorescence. These observations indicate that cell surface fluorescence was due to the capture of Ca by Calcium Green C after Ca had been translocated across osteoblast plasma membranes. Involvement of the plasma membrane Ca-ATPase, known to be present in osteoblasts in substantial amounts, is implicated.

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