900 MHz modulated electromagnetic fields accelerate the clathrin-mediated endocytosis pathway.

We report new data regarding the molecular mechanisms of GSM-induced increase of cell endocytosis rate. Even though endocytosis represents an important physical and biological event for cell physiology, studies on modulated electromagnetic fields (EMF) effects on this process are scarce. In a previous article, we showed that fluid phase endocytosis rate increases when cultured cells are exposed to 900 MHz EMF similar to mobile phones' modulated GSM signals (217 Hz repetition frequency, 576 micros pulse width) and to electric pulses similar to the GSM electrical component. Trying to distinguish the mechanisms sustaining this endocytosis stimulation, we exposed murine melanoma cells to Lucifer Yellow (LY) or to GSM-EMF/electric pulses in the presence of drugs inhibiting the clathrin- or the caveolin-dependent endocytosis. Experiments were performed at a specific absorption rate (SAR) of 3.2 W/kg in a wire patch cell under homogeneously distributed EMF field and controlled temperature (in the range of 28.5-29.5 degrees C). Thus, the observed increase in LY uptake was not a thermal effect. Chlorpromazine and ethanol, but not Filipin, inhibited this increase. Therefore, the clathrin-dependent endocytosis is stimulated by the GSM-EMF, suggesting that the cellular mechanism affected by the modulated EMF involves vesicles that detach from the cell membrane, mainly clathrin-coated vesicles.

Brefeldin A and filipin distinguish two globotriaosyl ceramide/verotoxin-1 intracellular trafficking pathways involved in Vero cell cytotoxicity.

In verotoxin 1 (VT1)-sensitive cells, globotriaosyl ceramide (Gb3) bound VT1 is endocytosed and transported retrogradely to the Golgi/endoplasmic reticulum (ER). The importance of the Golgi-dependent retrograde transport of VT1 is now shown to vary as a function of both VT1 exposure time and concentration. Following 3 h exposure to < 50 ng/ml VT1, Vero cell cytotoxicity and protein synthesis inhibition is absolutely dependent on intact Golgi structure. However, after 24 h incubation with concentrations of VT1 above 50 ng/ml, a filipin-sensitive (caveolae-dependent) route for cytotoxicity becomes significant. Brefeldin A (BFA), which prevents Golgi-dependent retrograde traffic, protects cells from low VT1 concentrations but not following prolonged toxin exposure at higher VT1 concentrations. Under these conditions, only a combination of BFA and filipin is sufficient to fully protect cells. Intracellular VT1 trafficking monitored using the nontoxic B subunit showed accumulation within BFA-collapsed TGN/endosomes. Considerable VT1 B was retained at the surface of filipin-treated cells, but Golgi targeting was still apparent. Filipin-sensitive VT1 cytotoxicity does not require Golgi access and may involve direct transmembrane signaling. Although cell surface VT1 does not colocalize with caveolin 1, a small fraction of endocytosed VT1 is found within caveolin 1-containing vesicles. These studies indicate both a caveolae-dependent and independent pathway for VT1 access to the TGN/Golgi from the cell surface and two noninterconverting pools of membrane Gb3.

Fibroblasts from patients with myotonic muscular dystrophy: cholesterol requirement for proliferation and sensitivity to polyene antibiotics.

The genetic defect in myotonic muscular dystrophy (MMD) remains obscure. From the evidence that drugs blocking cholesterol biosynthesis induce myotonia and increased serum concentrations of deoxycholic acids are common among patients with MMD, evidence of the abnormal sterol metabolism in MMD fibroblasts was sought by comparing them with fibroblasts from control individuals and patients with Duchenne muscular dystrophy (DMD). Although early-onset type MMD and DMD fibroblasts have lower maximal cell densities than fibroblasts from age-matched control individuals do in medium containing 10% fetal bovine serum, we could not reveal any abnormalities in exogeneous cholesterol requirements for proliferation of MMD fibroblasts. This suggests that the sterol biosynthetic pathway in MMD fibroblasts is grossly intact. Furthermore, no difference were observed in sensitivities to polyene antibiotics, which bind to membrane sterols and presumably damage the cell membrane.

Polyene macrolide antibiotic cytotoxicity and membrane permeability alterations. I. Comparative effects of four classes of polyene macrolides on mammalian cells.

The relationship between polyene macrolide-induced early membrane damage and cytotoxicity in B1 (hamster), B82 (mouse), and RAG (mouse) cells has been investigated. Filipin (FIL) induced the greatest immediate damage, as monitored by 51Cr release, followed by mediocidin (MED), amphotericin B-deoxycholate (Fungizone) (FZ) and pimaricin (PIM). For long term effect, PIM was the least toxic followed by MED, FZ, and FIL as indicated by 24-hour survival, 72-hour viability, and growth rate of cells. In evaluating polyene macrolide-induced permeability alterations and cytotoxicity two types of interactions with mammalian cells were found: (1) cell toxicity at polyene macrolide levels not eliciting immediate membrane permeability changes; and (2) immediate membrane damage without long range toxicity.

Molecular basis for the selective toxicity of amphotericin B for yeast and filipin for animal cells.

Among the polyene antibiotics, many, like filipin, cannot be used clinically because they are toxic; amphotericin B, however, is useful in therapy of human fungal infections because it is less toxic. Both the toxicity of filipin and the therapeutic value of amphotericin B can be rationalized at the cellular and molecular level by the following observations: (i) these polyene antibiotics showed differential effects on cells; filipin was more potent in lysing human red blood cells, whereas amphotericin B was more potent in inhibiting yeast cell growth; and (ii) the effects of filipin were more efficiently inhibited by added cholesterol, the major membrane sterol in human cells, whereas the effects of amphotericin B were more efficiently inhibited by ergosterol, the major membrane sterol in yeast. The simplest inference is that the toxicity and effectiveness of polyenes are determined by their relative avidities for the predominant sterol in cell membranes.