Monoclonal antibody MRK16 was used to determine the location of P-glycoprotein, the product of the multidrug-resistance gene (MDRJ), in normal human tissues. The protein was found to be concentrated in a small number of specific sites. Most tissues examined revealed very little Pglycoprotein. However, certain cell types in liver, pancreas, kidney, colon, and jejunum showed specific localization of P-glycoprotein. In liver, P-glycoprotein was found exclusively on the biliary canalicular front of hepatocytes and on the apical surface of epithelial cells in small biliary ductules. In pancreas, P-glycoprotein was found on the apical surface of the epithelial cells of small ductules but not larger pancreatic ducts. In kidney, P-glycoprotein was found concentrated on the apical surface of epithelial cells of the proximal tubules. Colon and jejunum both showed high levels ofP-glycoprotein on the apical surfaces of superficial columnar epithelial cells. Adrenal gland showed high levels of P-glycoprotein diffusely distributed on the surface ofcells in both the cortex and medulla. These results suggest that the protein has a role in the normal secretion of metabolites and certain anti-cancer drugs into bile, urine, and directly into the lumen of the gastrointestinal tract.

MAb C219 showed intense localization in selected skeletal muscle fibers and all cardiac muscle fibers in rat and human tissues. ATPase cytochemistry showed

Multidrug-resistant cells contain a plasma membrane efflux pump, the multidrug transporter, which actively expels certain hydrophobic drugs from the cytosol to the cell exterior. These drugs are usually positively charged at physiological pH. Because one might predict that this efflux of positively charged molecules might deplete the cytosol of protons, raising the cytosolic pH, we examined the cytosolic pH of multidrug-resistant cells directly using a pH-sensitive dye coupled to a membrane-impermeable molecule. The dye (SNARF), covalently coupled to 10,000 MW dextran, was mechanically microinjected into the cytosol of cultured multidrug-resistant mouse NIH3T3 cells which express the human multidrug transporter. The fluorescence emission of the dye in living cells was measured using epifluorescence microscopy at different wavelengths to provide a measure of the pH of the cytosolic environment. Multidrug-resistant cells had a higher cytosolic pH than drug-sensitive normal parental cells. As the pH of the culture medium was increased, normal cells maintained their cytosolic pH below 7.0, whereas the cytosolic pH of multidrug resistant cells rose. The difference in cytosolic pH between the two cell types was more than 0.2 pH units at an external culture medium pH of 8.2. Treatment with agents that inhibit multidrug transporter-mediated efflux, such as verapamil and vinblastine, essentially eliminated the elevation of cytosolic pH, presumably because they are good substrates for the pump which overwhelm its capacity to pump other materials. These results suggest that the multidrug transporter is indirectly a proton pump, and that cells may contain an endogenous substrate or substrates for this transporter in the absence of added drugs.

We have previously shown that forskolin and 3-isobutyl-

We isolated an IgG2a murine monoclonal antibody (MAb) termed MAb57, specifically reactive with multi-drug-resistant (MDR) human cells. Its specificity toward the MDRI gene product (P-glycoprotein) has been demonstrated by the concordant segregation of the MAb57 epitope with the MDRI gene in interspecific mouse x human cell hybrids, and the reactivity of several different MDRI gene-expressing cells with MAb57, particularly insect cells acutely infected with a baculovirus encoding the MDRI gene. MAb57 can be used to detect, by flow cytometry, variations in the relative drug-resistance levels of several MDR KB and CEM cell variants. This immunological probe has also proven useful in selectively destroying MDR target cells in an antibody-dependent cell-mediated (ADCC) assay system as well as in detecting P-glycoprotein expression in normal and malignant tissues and cells.