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. © 1994 Wiley‐Liss, Inc. Copyright © 1994 Wiley‐Liss, Inc.