Effect of wortmannin and phorbol ester on Paramecium fluid-phase uptake in the presence of transferrin

Abstract

SUMMARYThe kinetics of the uptake of the fluid phase marker Lucifer Yellow (LY), and its alteration by wortmannin, an inhibitor of phosphatidylinositol-3 kinase (PI-3K), and the PKC modulators: GF 109203 X, an inhibitor, and phorbol ester, an activator was studied in eukaryotic model Paramecium aurelia. Spectrophotometric quantification of LY accumulation was performed in the presence or absence of transferrin, a marker of receptor-mediated endocytosis. Internalization of LY showed a curvilinear kinetics: the high initial rate of LY uptake (575 ng LY/ mg protein /hr) decreased almost 5-fold within 15 min, reaching plateau at 126 ng/ mg protein /hr. Transferrin induced a small increase (7.5%) in the fluid phase uptake rate (after 5 min) followed by a small decrease at longer incubation times. Lucifer Yellow and transferrin (visualized by streptavidin-FITC) were localized in Paramecium by 3-D reconstruction by confocal microscopy. LY showed a scattered, diffuse fluorescence typical of fluid phase uptake whereas transferrin accumulated in membrane-surrounded endosomes. Wortmannin did not affect LY accumulation but decreased it when transferrin was present in the incubation medium. This suggests an effect on the transferrin uptake pathway, presumably on the stage of internalization in "mixing" endosomes to which transferrin and LY were targeted. 383Phorbol ester diminished LY accumulation by 22% and this effect persisted up to 25 min of incubation. PKC inhibitor did not affect LY uptake. However, in the presence of transferrin, the LY uptake increased within the first 15 minutes followed by a rapid 20% decrease in comparison to the control. Such an effect of PKC modulators suggests that PMA action on fluid phase uptake is not directly mediated by PKC.

Pharmacological Attenuation of Paramecium Fluid-Phase Endocytosis

“…LY accumulation was characterized by a very high initial uptake reaching 580.8 ng LY/mg protein/hr, which decreased by almost 80% to 124.4 ng LY/mg protein/hr during the next 20 min of incubation of untreated cells (Table 1), similar to previous observations (WIEJAK et al 2001). Such a pattern of fluid phase uptake was also observed in the treated cells in spite of the lower amount of LY accumulated.…”

“…The accumulation of Lucifer Yellow Carbohydrazide (25 Fg/ml, Sigma, Steinheim, Germany), a fluid phase marker, was quantified at 20±1 o C by estimation of fluorescence in the Perkin-Elmer Luminescence Spectrometer LS-5B at excitation 430 nm and emission at 540 nm as described elsewhere (WIEJAK et al 2001). After various times of incubation aliquots of the cells were withdrawn from the medium and immediately placed in a 150 excess of ice-cold phosphate-saline buffer (WY-ROBA 1987) followed by centrifugation (600 H g) for 30 sec.…”

“…The supernatant and the cell pellet from the final washing were collected for assay of LY. Spectrofluorimetric estimation of LY uptake and protein determination was performed as described in WIEJAK et al (2001) from 3-4 independent series of experiments. The standard curve of LY fluorescence was estimated at each series of experiments.…”

“…Fluid phase uptake and its regulation in different cell systems have focused attention (MANIAK 2001;KJEKEN et al 2001) in relation to cell permeability , motility (AYSCOUGH 2000;MICHALIK et al 2003), secretion (HAZELTON et al 2002) and other endocytic routes such as receptor-mediated endocytosis (MCKINLEY & WILEY 1988;PAGE et al 1994;RAMOINO et al 2001;WIEJAK et al 2001) and phagocytosis (WYROBA 1991). It has been documented that the blocking of phagocytic activity leads to a decrease of fluid phase endocytosis in Paramecium cells (WYROBA 1991;RAMOINO et al 2001), but it is not known how stimulation of phagocytosis affects the fluid phase uptake in this unicellular eukaryote.…”

“…Lucifer Yellow was used as the fluid-phase marker. This reagent was found to be a useful tool for monitoring this type of endocytosis both in the higher eukaryotes (WOLKERS et al 2003) and Paramecium (WIEJAK et al 2001…”