ENDOSOMAL ACCUMULATION OF PH INDICATOR DYES DELIVERED AS ACETOXYMETHYL ESTERS

Intracellular distributions of the putative cytosolic pH indicator dye s BCECF [2',7'-bis-(2-carboxyethyl)-5(and 6)-carboxyfluorescein], C.SN ARF [5(and 6)-carboxy- seminaphthorhodafluor-1], and C.SNARF-calcein h ave been examined in Neurospora crassa and in murine fibroblasts (NIH- 3T3 cells) under conditions in which both kinds of cells produce visib le microscopic vacuoles. All three dyes were administered in electrone utral forms, with the hydroxyl and carboxyl groups esterified (designa ted as AM esters). As judged qualitatively from fluorescence levels, h ydrolytic derivatives of the two heavily esterified dyes (BCECF-AM and C.SNARF-calcein-AM) accumulated in the vacuoles after exposures of ap proximately 15 min or more, while the simpler dye (C.SNARF-AM) and its derivatives were almost excluded from visible vacuoles. Fluorescence from this dye, alone among the three, also washed out of Neurospora ra pidly upon removal of extracellular dye. There was no evidence for sta ble accumulation of any of the dyes in cytosol per se. For BCECF(-AM), comparison of the distribution of fluorescence with the size distribu tion of vacuoles in Neurospora strongly suggests that the dyes are als o accumulated by endomembranal vesicles (EMVs) which lie below the lim it of resolution in the light microscope, and the same inference can b e drawn for the fibroblasts. Uptake of -AM dyes by EMVs, including fra nk vacuoles, probably results from the action of intravesicular estera ses, following diffusional entry of lipophilic neutral molecules or pa rtially de-esterified anions. Calculations of actual cytosolic pH valu es, or even changes of pH, based on intracellular fluorescence of thes e dyes, clearly depend upon quantitative knowledge of the subcellular dye distribution. Therefore, until the problem is reliably solved of h ow to visualize submicroscopic vesicles in living cells, the safest ap proach to the use of BCECF, C-SNARF and their congeners for cytosolic pH measurement would be to devise methods for coaxing uptake of the io nic forms of these dyes and to abandon use of the esterified forms.