title: Constitutive expression of P-glycoprotein as a determinant of loading with fluorescent calcium probes.
abstract: Determination of intracellular calcium levels in Chinese hamster ovary (CHO) cells using the fluorescent calcium probe indo-1AM was hindered by the low level of accumulation of indo-1 in these cells. CHO cells are known to express basal levels of the multidrug resistance efflux pump P-glycoprotein (P-gp). Rhodamine-123, which is a known substrate of P-gp, was used to confirm the presence of P-gp in CHO cells. Verapamil and cyclosporin (CsA), both inhibitors of P-gp, enhanced accumulation of indo-1 in these cells and therefore allowed for improved intracellular calcium measurements. P-gp overexpressing colchicine-resistant CHO cells (CHRC5) also displayed enhanced indo-1AM loading with P-gp inhibitors. Nondetectable levels of P-gp activity were found in wild-type CEM-CCRF cells (human T lymphoblasts), and these cells did not show any difference in indo-1AM loading in the presence or absence of P-gp inhibitors. Loading of a second calcium fluorescent probe fluo-3AM was improved in CHO cells by P-gp inhibition, whereas the structurally related pH probe BCECF-AM was minimally affected. Because low levels of P-gp may be expressed by a range of cell lines and normal tissues, it is suggested that this be considered if difficulties are encountered in loading fluorescent calcium probes.
authors.affiliation: Ontario Cancer Institute, University of Toronto, Canada.
authors.forename: C B
authors.initials: CB
authors.lastname: Brezden
authors.forename: D W
authors.initials: DW
authors.lastname: Hedley
authors.forename: A M
authors.initials: AM
authors.lastname: Rauth
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors / Q000378:metabolism*; D000818:Animals; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism*; D006224:Cricetinae; D003412:Cricetulus; D016572:Cyclosporine / Q000494:pharmacology; D003600:Cytosol / Q000378:metabolism*; D005260:Female; D005434:Flow Cytometry; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006801:Humans; D007211:Indoles / Q000378:metabolism; D020112:Rhodamine 123; D012235:Rhodamines; D014700:Verapamil / Q000494:pharmacology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007211:Indoles; D012235:Rhodamines; D020112:Rhodamine 123; D016572:Cyclosporine; C043829:2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein; D014700:Verapamil; C048960:indo-1; D002118:Calcium