CHARACTERIZATION OF ACIDIC VESICLES IN MULTIDRUG-RESISTANT AND SENSITIVE CANCER-CELLS BY ACRIDINE-ORANGE STAINING AND CONFOCAL MICROSPECTROFLUOROMETRY

To study the pH gradient status through membranes of acidic vesicles, either in sensitive or in multidrug-resistant living cancer cells, we monitored the fluorescence-emission spectra of acridine orange. Succes sive stainings with a pH-sensitive dye and AO showed that low-pH organ elles were stained red by AO. In these compartments, high AO concentra tions are driven by the pH gradient through membrane vesicles. The res ulting rise in the dye's oligomeridmonomeric ratio induced an increase in the red/green (655-nm/530-nm) emission intensity ratio. Therefore, the accumulation of AO in acidic organelles was appraised by determin ation of the contribution of the red emission intensity (R%) in each e mission spectrum, using laser scanning confocal microspectrofluorometr y. In vesicles of multidrug-resistant K562-R cells, R% is significantl y higher (72 +/- 10%) than the value (48 +/- 8%) from K562-sensitive c ells (p<0.001). This result is interpreted as a more important accumul ation of AO in acidic cytoplasmic structures of resistant cells, which induces a shift from AO monomers (green emission) to self-associated structures (red emission). Equilibration of the pH gradient through ac idic organelles was performed by addition of weak bases and carboxylic ionophores. Ammonium chloride (0.1 mM), methylamine (0.1 mM), monensi ne (10 mu M), or nigericine (0.3 mu M) all suppressed the initial diff erence of local AO accumulation between both cell lines. These agents decreased the red emission intensity for the resistant cell line but n ot far the sensitive one. The same effects were induced by 50 mu M ver apamil, a pleiotropic drug-resistance modulator. Our data allow the hy pothesis of a higher pH gradient through membranes of acidic organelle s, which would be a potential mechanism of multidrug resistance via th e sequestration of weak bases inside these organelles.