Effects of different detachment procedures on viability, nitroxide reduction kinetics and plasma membrane heterogeneity of V-79 cells.

Cell detachment procedures can cause severe damage to cells. Many studies require cells to be detached before measurements; therefore, research on cells that have been grown attached to the bottom of the culture dish and later detached represents a special problem with respect to the experimental results when the properties of cell membranes undergo small changes such as in spectroscopic studies of membrane permeability. We characterized the influence of three different detachment procedures: cell scraping by rubber policeman, trypsinization and a citrate buffer treatment on V-79 cells in the plateau phase of growth (arrested in G1). We have measured cell viability by a dye-exclusion test; nitroxide reduction kinetics and membrane fluidity by EPR (electron paramagnetic resonance) method using the lipophilic spin-probe MeFASL(10,3) (5-doxylpalmitoyl-methylester), which partitions mainly in cell membranes and the hydrophilic spin-probe TEMPONE (4-oxo-2,2,6,6-tetramethylpiperidine-1-oxyl). The resulting cell damage due to the detachment process was observed with SEM (scanning electron microscopy). We found out that cell viability was 91% for trypsin treatment, 85% for citrate treatment and 70% for cell scraping. Though the plasma membrane was mechanically damaged by scraping, the membrane domain structure was not significantly altered compared with other detachment methods. On the other hand, the spin-probe reduction rate, which depends both on the transport across plasma membrane as well as on metabolic properties of cells, was the highest for trypsin method, suggesting that metabolic rate was the least influenced. Only the reduction rate of trypsin-treated cells stayed unchanged after 4 h of stirring in suspension. These results suggest that, compared with scraping cells or using citrate buffer, the most suitable detachment method for V-79 cells is detachment by trypsin and keeping cells in the stirred cell suspension until measurement. This method provides the highest cell viability, less visible damage on SEM micrographs and leaves the metabolic rate of cells unchanged.

Cholesterol prevents interaction of the cell-penetrating peptide transportan with model lipid membranes.

Interaction of the cell-penetrating peptide (CPP) cysteine-transportan (Cys-TP) with model lipid membranes was examined by spin-label electron paramagnetic resonance (EPR). Membranes were labeled with lipophilic spin probes and the influence of Cys-TP on membrane structure was studied. The influence of Cys-TP on membrane permeability was monitored by the reduction of a liposome-trapped water-soluble spin probe. Cys-TP caused lipid ordering in membranes prepared from pure dimyristoylphosphatidylcholine (DMPC) and in DMPC membranes with moderate cholesterol concentration. In addition, Cys-TP caused a large increase in permeation of DMPC membranes. In contrast, with high cholesterol content, at which model lipid membranes are in the so-called liquid-ordered phase, no effect of Cys-TP was observed, either on the membrane structure or on the membrane permeability. The interaction between Cys-TP and the lipid membrane therefore depends on the lipid phase. This could be of great importance for understanding of the CPP-lipid interaction in laterally heterogeneous membranes, while it implies that the CPP-lipid interaction can be different at different points along the membrane.

Inhibition of erythrocyte acetylcholinesterase by n-butanol at high concentrations.

Erythrocyte acetylcholinesterase (AChE) is bound to the membrane by a complex glycosylphosphatidylinositol anchor, so the effect of alcohol on AChE activity may reflect direct and/or membrane-mediated effects. The indication of a direct interaction between n-butanol and AChE molecules is the activation/inhibition of AChE by occupation of the enzyme's active and/or regulatory sites by alcohol. The activation of AChE can occur only at low concentrations of alcohols, while at high concentrations AChE is inhibited. In this work the mechanism of inhibition of erythrocyte AChE by n-butanol at high concentrations was studied. The values of activity, calculated assuming parabolic competitive inhibition, which implies that one or two molecules of inhibitor bind to the enzyme, fit well to the experimental values. From the values of the inhibition constants it was concluded that at high n-butanol concentrations two alcohol molecules usually interact with AChE.

The membrane lateral domain approach in the studies of lipid-protein interaction of GPI-anchored bovine erythrocyte acetylcholinesterase.

A novel membrane lateral domain approach was used to test whether the activity of the membrane-bound enzyme acetylcholinesterase (AChE) depends on the local properties (e.g. local lipid ordering) of bovine erythrocyte-ghost membrane. This issue has an additional aspect of interest due to an alternative mode of insertion of AChE molecules into the membrane by the glycosylphosphatidylinositol (GPI) anchor. In our experiments the lateral domain membrane structure was influenced by temperature and by the addition of n-butanol, and was quantitatively characterized using the method of EPR spectrum decomposition. The activity of AChE was determined by a colorimetric assay in the same samples. The results show that the membrane stabilizes the conformation of the membrane-bound AChE compared to the isolated AChE. In addition, a correlation was observed between the temperature dependence of order parameter of the most-ordered domain type and the activity of AChE. Therefore, our findings support the idea that the function of GPI proteins can be modulated by the lipid bilayer. Based on the assumption that the overall activity of AChE depends on the order parameters of particular domain types as well as their proportions, two models for AChE activity were introduced. In the first, a random distribution of enzyme molecules was proposed, and in the second, localization of enzyme molecules in a single (cholesterol-rich) domain type was assumed. Better agreement between measured and calculated activity values speaks in favor of the second model.

Quantifying the lateral lipid domain properties in erythrocyte ghost membranes using EPR-spectra decomposition.

Using EPR spectroscopy a typical lateral domain structure was detected in the membranes of spin-labeled bovine erythrocyte ghosts. The spectral parameters were determined by decomposing the EPR spectrum into three spectral components and tuned by a hybrid-evolutionary-optimization method. In our experiments the lateral domain structure and its properties were influenced by the variation in the temperature and by the addition of n-butanol. The specific responses of the particular domain types were detected. For the most-ordered domain type a break was seen in the temperature dependence of its order parameter, while the order parameters of the two less-ordered domain types exhibited a continuous decrease. Below the break-point temperature the alcohol-induced membrane fluidity variation is mainly a consequence of the change in the proportions of the least- and the most-ordered domain type and not the change of the domain-type ordering or dynamics (with n-butanol concentration). On the other hand, the fluidity variation above the break-point temperature arises from both types of changes. Interestingly, the proportion of the domain type that has its order parameter between that of the least- and the most-ordered domain type remains almost constant with concentration as well as with temperature, which implies its stability. Such characterization of the lateral membrane domain structure could be beneficial when considering the lipid-protein interactions, because it can be assumed that the activity of the membrane-bound enzyme depends on the properties of the particular domain type.

Cell membrane fluidity and prognosis of lung cancer.

BACKGROUND: Membranes of tumor cells have been found to posses higher fluidity than membranes of non-tumor cells. Plasma membrane fluidity is significantly correlated with malignant potential of these cells. METHODS: Seventy-five patients operated on for lung cancer were studied prospectively. During the operation, lung tumor samples were taken from the resected lung for evaluation by electron paramagnetic resonance. The fluidity variable H13, which is proportional to the plasma membrane fluidity, was determined from the electron paramagnetic resonance spectra. The association between H13 and survival was determined by survival analysis using Kaplan-Meier curves and Cox regression. RESULTS: Pathologic TNM stage and the fluidity variable H13 were the only prognostic variables significantly associated with survival time in multivariate proportional hazards regression model. Thus, H13 was shown to be an independent prognostic variable for survival, which was also confirmed by a separate analysis relating the TNM stage and H13. Dividing the patients into two groups, one with an H13 value higher than the median and another with H13 below the median, resulted in significantly different survival curves (p = 0.01). CONCLUSIONS: Patients with high plasma membrane fluidity, indicated by high H13 of the resected lung tumor tissue, seem to have poorer prognosis than those with less fluid membranes. We suggest that the fluidity variable could be used as an independent additional prognostic factor and a tool to identify patients who may be helped by adjuvant postoperative therapy.