Linked Life Data

7673232

Source:http://linkedlifedata.com/resource/pubmed/id/7673232

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
38
pubmed:dateCreated
1995-10-17
pubmed:abstractText
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.
pubmed:grant
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0021-9258
pubmed:author
pubmed:issnType
Print
pubmed:day
22
pubmed:volume
270
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
22445-51
pubmed:dateRevised
2007-11-14
pubmed:meshHeading
pubmed:year
1995
pubmed:articleTitle
Characterization of calcium translocation across the plasma membrane of primary osteoblasts using a lipophilic calcium-sensitive fluorescent dye, calcium green C18.
pubmed:affiliation
Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park 16802, USA.
pubmed:publicationType
Journal Article, In Vitro, Research Support, U.S. Gov't, P.H.S.