Characterization of calcium translocation across the plasma membrane of primary osteoblasts using a lipophilic calcium-sensitive fluorescent dye, calcium green C18.
The synthesis of Calcium Green C18, a lipophilic fluorescent calcium-sensitive dye, and its use as a monitor of Ca2+ 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 Kd of the indicator for Ca2+ in aqueous solution (pH 7.2, 22 degrees C, ionic strength 0.1 M) is 0.23 +/- 0.04 microM and in the presence of liposomes is 0.062 +/- 0.007 microM. 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 microM for 10 min at 37 degrees 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 approximately 30% of the cells was observed following 10(-8) M 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 microM quercetin and 10 microM vanadate, partially specific inhibitors of plasma membrane Ca(2+)-ATPase, to (b) 0.1 mM trifluoperazine, an agent which renders calmodulin ineffective, and to (c) 10 mM neomycin sulfate, which blocks release of Ca2+ from intracellular stores. Thapsigargin (5 microM), an inhibitor of Ca(2+)-ATPase of the endoplasmic reticulum, prolonged fluorescence. These observations indicate that cell surface fluorescence was due to the capture of Ca2+ by Calcium Green C18 after Ca2+ had been translocated across osteoblast plasma membranes. Involvement of the plasma membrane Ca(2+)-ATPase, known to be present in osteoblasts in substantial amounts, is implicated.
Lloyd
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Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.
Kuhn
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MA
Gay
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CV
D000818:Animals; D001692:Biological Transport; D002118:Calcium / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002645:Chickens; D004533:Egtazic Acid / Q000031:analogs & derivatives / Q000737:chemistry / Q000494:pharmacology; D005110:Extracellular Space / Q000378:metabolism; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D066298:In Vitro Techniques; D007202:Indicators and Reagents / Q000138:chemical synthesis*; D008055:Lipids / Q000737:chemistry; D009930:Organic Chemicals; D010006:Osteoblasts / Q000378:metabolism*; D010281:Parathyroid Hormone / Q000494:pharmacology; D012995:Solubility; D013050:Spectrometry, Fluorescence
D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D008055:Lipids; D009930:Organic Chemicals; D010281:Parathyroid Hormone; C095636:calcium green C18; C071336:calcium green; D004533:Egtazic Acid; C025603:1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid; D002118:Calcium