No abstract

We measured intracellular free calcium concentrations ([Ca++]i) in the subcellular compartments of Toxoplasma gondii infected living cells using microspectrofluorometry and Indo-1 staining. [Ca++]i mapping was defined in infected and uninfected cells and in the neoformed parasitophorous vacuole (PV) 24 and 48 hr after parasite inoculation. At 24 hr after infection, a [Ca++]i gradient (PV/cytoplasm) was observed in favor of the PV in 72% of infected cells (p<0.001). Inside of the PV (lumen and parasites), [Ca++]i values appeared to be homogeneously distributed. At 48 hr after infection, the parasites had replicated and formed typical rosettes of more than 16 parasites. At this step, a positive [Ca++]i gradient (PV/cytoplasm) was detected in all analyzed cells (p<0.001). This result suggests that the PV (lumen and parasites) represents an individual subcellular compartment within the host cell that includes an independent [Ca++]i. Moreover, after 48 hr the cytoplasmic [Ca++]i decreased significantly (39 nM) compared with that measured from uninfected cells (53 nM) (p <0.05). Furthermore, the exit of Toxoplasma mediated by the calcium ionophore 4BrA23187 was preceded by a rise of [Ca++]i to 1 mM in the PV. The [Ca++]i rise and the liberation of parasites from their host appear to be correlated. On the basis of these observations, we suggest that the increase of [Ca++]i in the vacuole may act as a signal that triggers the egress of T. gondii.

Scanning microspectrofluorometry has been developed to perform the mapping of fluorescence spectra from all locations in a living cell. This new method has been applied to study the molecular environment of rhodamine 123 (R123) in sensitive (K562, CEM) and multidrug-resistant (K562-R, CEM/VLBIoo) tumor cells.All cells exposed to R123 showed a similar distribution of fluorescence in the perinuclear region. A lower cytoplasmic fluorescence intensity corresponding to a reduced drug accumulation was observed in resistant cells, as expected in the multidrug resistance process.Fluorescence emission spectra of R123 are useful to probe the polarity of the R123 environment. Thus, fluorescence spectra of Rl23-treated cells have been analyzed as a linear combination of model spectra: R123 in water and R123 in tensio-active Triton X-100. In sensitive cells, emission spectra of R123 underwent a red shift, equivalent to those observed in isolated coupled mitochondria. This suggests the formation of a complex in hydrophobic sites. In contrast, R123 spectra were less shifted in resistant cells, showing two types of both hydrophobic and hydrophilic binding sites. This could be related to an intracellular redistribution of R123 in resistant cells.

Multidrug-resistant cells are believed to contain a plasmamembrane-efflux pump which is hypothesized to expel anticancer drugs from the cytosol to the cell exterior. Many of these drugs are classified as weak bases whose binding to intracellular targets is pH-dependent. Slight alterations in intracellular pH gradients have been shown to affect accumulation, endocytosis and secretion of drugs. In this study, we developed a new method based on confocal spectral imaging analysis to determine intracellular pH gradients in sensitive and MDR tumor cells. Fluorescein isothiocyanate (FITC) and tetramethylrhodamine conjugated to dextran (FRD) and SNAFL-calcein-AM were used to determine pH in acidic compartments. Carboxy-SNARF1-AM was used to examine cytosolic pH. We observed that sensitive (HL60, K562, CEM and MCF7) cells exhibit lower acidity of the subcellular organelles than that corresponding to drug-resistant derivatives. Moreover, results obtained with carboxy-SNARF1-AM show that resistant cells display a more alkaline cytosolic pH. This results in a considerably larger pH gradient between the vesicular compartments and the cytosol of resistant cells than of sensitive cells. The lower pH gradient observed in sensitive cells may be related to a disruption in the organization of the trans-Golgi network (TGN). In drug-resistant cells, the organization of TGN appears compact. In addition, confocal microscopic analysis of cells labelled with FRD and SNAFL-calcein showed that sensitive cells contain a lower number of acidified vesicles. This suggest a diminished capacity of these cells to remove protonated drugs from the cytoplasm to secretory compartments followed by their secretion through the activity of the secretory and recycling pathways. Int.