CONSTITUTIVE EXPRESSION OF P-GLYCOPROTEIN AS A DETERMINANT OF LOADINGWITH FLUORESCENT CALCIUM PROBES

Determination of intracelluIar 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 p ump P-glycoprotein (P-gp), Rhodamine-123, which is a known substrate o f P-gp, was used to confirm the presence of P-gp in CHO cells, Verapam il and cyclosporin (CsA), both inhibitors of P-gp, enhanced accumulati on of indo-1 in these cells and therefore allowed for improved intrace llular calcium measurements. P-gp overexpressing colchicine-resistant CHO cells (CH(R)C5) 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 sh ow 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 encountere d in loading fluorescent calcium probes. (C) 1994 Wiley-Liss, Inc.