Nilce Correa Meirelles

Effect of hydration upon the fluidity of intercellular membranes of stratum corneum: an EPR study.

The principal mechanisms controlling the molecular permeability through the skin are associated to the intercellular membranes of stratum corneum (SC), the outermost layer of mammalian skin. It is generally accepted that an increase in fluidity of these membranes leads to a reduction of the physical barrier exerted by SC with a consequent enhancement in permeation of different compounds. It is known that water diffusion in SC increases with the increase in the water content in SC. Using the spin labeling method we evaluate the effect of hydration on the fluidity of intercellular membranes at three depths of the alkyl chain. Increase in the water content in SC leads to a drastic increase in membrane fluidity especially in the region near the membrane/water interface; the effect decreases on going deeper inside the hydrophobic core. Analysis of electron paramagnetic resonance (EPR) parameters as a function of temperature showed that the rotational motion at depth of the 16th carbon atom of the chain experienced a phase transition at 45 and 60 degrees C. These phase transition temperatures were not altered by changes in the water content of SC. A phase transition between 28 and 48 degrees C was observed from the segmental motion in the region near the polar headgroup (up to 12th carbon in the chain) and was strongly dependent upon the hydration of SC. Our results give a better characterization of the fluidity of SC, the main parameter involved in the mechanisms that control the permeability of different compounds through skin.

Solubilization of human erythrocyte membranes by non-ionic surfactants of the polyoxyethylene alkyl ethers series.

In the present study, we investigated the interaction of the non-ionic surfactants polyoxyethylene alkyl ethers (C(n)E(m)) with erythrocyte membranes. For this purpose we have performed hemolytic assays under isosmotic conditions with five surfactants in the 8 polyoxyethylene ether series. By applying to the hemolytic curves a quantitative treatment developed for the study of surface-active compounds on biomembranes, we could calculate the surfactant/lipid molar ratios for the onset of hemolysis (R(e)(sat)) and for complete hemolysis (R(e)(sol)). This approach also allowed the calculation of the binding constants for each surfactant to the erythrocyte membrane. Results in the C(n)E(m) series were compared to those obtained for Triton X-100, a well-known non-ionic surfactant with values of cmc and HLB in the range of the alkyl ethers studied. Inside the series the lytic effect increased with the more hydrophobic homologues (C(10)E(8)<C(12)E(8)<C(14)E(8)<C(16)E(8)<C(18)E(8)), with Re values between 3:1 and 0.03:1. The effect of C(10)E(8) and C(12)E(8) was found to be in the range of that caused by Triton X-100, proving that C(n)E(m) surfactants are strongly hemolytic.

Pathways involved in trifluoperazine-, dibucaine- and praziquantel-induced hemolysis.

This work elucidates differences in the hemolytic pathway developed by the antipsychotic trifluoperazine (TFP), the local anesthetic dibucaine (DBC) and the antihelminthic praziquantel (PZQ). Their partition coefficients (P) were measured at pH 7.4 between n-octanol, microsomes, liposomes, erythrocyte ghosts and n-octanol/water. The effective drug:lipid molar ratios for the onset of membrane solubilization (ReSAT) and complete hemolysis (ReSOL) were calculated from the experimental P values and compared with a classical surface-active compound treatment Lichtenberg, D. Biochim. Biophys. Acta 821 (1985) 470-478[. The contribution of charged/uncharged forms of TFP and DBC for the hemolytic activity was also analyzed. In all cases the hemolytic phenomena could be related to the monomeric drug insertion into the membrane. Only for TFP at isosmotic condition lysis occurs at concentrations beyond the CMC of the drug, indicating that micellization facilitates TFP hemolytic effect, while DBC and PZQ reach a real membrane saturation at their monomeric form.

Effects of polyoxyethylene chain length on erythrocyte hemolysis induced by poly[oxyethylene (n) nonylphenol] non-ionic surfactants.

The effects of three different poly[oxyethylene (n) nonylphenols], n = 9.5, 20 and 100 oxyethylene (EO) units, on erythrocyte hemolysis and on the fluidity of the erythrocyte membrane were studied. The three different surfactants showed different effects. The surfactant with average n = 9.5 EO units (C9E9) shows a biphasic effect: at low concentrations it protects erythrocytes against hypotonic hemolysis, but at higher concentrations it induces hemolysis both in isotonic and hypotonic buffers. C9E20 does not affect the erythrocyte membrane resistance to hemolysis, independent of the buffer osmolarity; this detergent did not show a hemolytic effect. C9E100 is an effective protective agent against hypotonic hemolysis, in concentration > 2 x 10(-4) M. EPR spectroscopy of spin-labeled stearic acid indicated that the three different surfactants increase the fluidity of erythrocyte ghost membranes. At the higher C9E20 and C9E100 surfactant concentrations in the presence of membrane ghosts, spin-label is located in the surfactant micelles. In the case of the hemolytic concentrations of C9E9, mixed (surfactant plus phospholipid) micelles are formed. These results suggest that C9E9 has a higher affinity for membrane phospholipids, which accounts for its lytic activity. The protective effect of C9E100 is assigned to the osmotic buffering of the liquid surrounding the cell membrane, due to the large polar chains anchored to the membrane outer monolayer but other mechanisms previously considered in the literature may also be effective.

Contribution of trifluoperazine/lipid ratio and drug ionization to hemolysis

The interaction of the antipsychotic drug trifluoperazine (TFP) with membranes was investigated in terms of lipid phase perturbation. TFP partition coefficients (P) were measured by phase separation between octanol/water and model membranes/water. The profile of P values at pH 7.4 was: microsomes (7172+/-1229)>liposomes (1916+/-341)>erythrocyte ghosts (1380+/-429)>octanol (452+/-55). Hemolytic experiments showed a biphasic, protective (at lower concentrations) and hemolytic effect above the CMC (42 microM at pH 7.4) of the phenothiazine. By applying classical treatments for surface active compounds to the hemolytic curves, we could calculate P values in whole erythrocyte cells. The preferential binding of uncharged to charged TFP in the membrane was discussed, since it results in a ionization constant (pKapp) different from that observed in the aqueous phase (pK). The TFP ionization constant was decreased from 8.1 (in water) to 7.62 in the presence of membranes and almost the same ratio of charged/uncharged TFP species is present at physiologic pH. Taking into account the DeltapK, we calculated the average TFP partition coefficient between egg phosphatidylcholine liposomes and water, at pH 7.4 (Paverage=1432), which was well correlated with the measured one (Plip=1916). Paverage is highly influenced by the uncharged TFP species and the real base/acid ratio under physiologic conditions was discussed in terms of its possible role in the biological activity of TFP.

Multiple stages of detergent-erythrocyte membrane interaction—A spin label study

The various stages of the interaction between the detergent Triton X-100 (TTX-100) and membranes of whole red blood cells (RBC) were investigated in a broad range of detergent concentrations. The interaction was monitored by RBC hemolysis-assessed by release of intracellular hemoglobin (Hb) and inorganic phosphate-and by analysis of EPR spectra of a fatty acid spin probe intercalated in whole RBC suspensions, as well as pellets and supernatants obtained upon centrifugation of detergent-treated cells. Hemolysis finished at ca. 0.9mM TTX-100. Spectral analysis and calculation of order parameters (S) indicated that a complex sequence of events takes place, and allowed the characterization of various structures formed in the different stages of detergent-membrane interaction. Upon reaching the end of cell lysis, essentially no pellet was detected, the remaining EPR signal being found almost entirely in the supernatants. Calculated order parameters revealed that whole RBC suspensions, pellets, and supernatants possessed a similar degree of molecular packing, which decreased to a small extent up to 2.5mM detergent. Between 3.2 and 10mM TTX-100, a steep decrease in S was observed for both whole RBC suspensions and supernatants. Above 10mM detergent, S decreased in a less pronounced manner and the EPR spectra approached that of pure TTX-100 micelles. The data were interpreted in terms of the following events: at the lower detergent concentrations, an increase in membrane permeability occurs; the end of hemolysis coincides with the lack of pellet upon centrifugation. Up to 2.5mM TTX-100 the supernatants consist of a (very likely) heterogeneous population of membrane fragments with molecular packing similar to that of whole cells. As the detergent concentration increases, mixed micelles are formed containing lipid and/or protein, approaching the packing found in pure TTX-100 micelles. This analysis is in agreement with the models proposed by Lasch (Biochim. Biophys Acta 1241 (1995) 269-292) and by Le Maire and coworkers (Biochim. Biophys. Acta 1508 (2000) 86-111).

Membrane effects of trifluoperazine, dibucaine and praziquantel on human erythrocytes

Trifluoperazine (TFP) is a potent antipsychotic agent, dibucaine (DBC) is a local anaesthetic and praziquantel (PZQ) is a highly effective agent against schistosomiasis. The present work was conducted to (i) investigate the cytotoxic effects of TFP, DBC and PZQ on human erythrocyte membranes; and (ii) compare the alterations induced by the cationic drugs (TFP and DBC) with those induced by the uncharged compound (PZQ), in an attempt to have a better insight on the pathways of each drug-membrane interaction. The erythrocyte morphological alterations induced by sublytic concentrations of TFP, DBC and PZQ were evaluated by scanning electron microscopy and expressed quantitatively by the morphological index. Haemolysis and release of membrane lipids (phospholipids and cholesterol) produced by selected concentrations of TFP, DBC and PZQ, were compared with those resulting from the corresponding triple concentrations of each drug. Our results showed that the uncharged molecule of PZQ induces the same morphological alterations (stomatocytosis) as the cationic drugs TFP and DBC. Haemolysis was shown to vary with the drug used and to be concentration-dependent, with values approximately 10-fold more elevated for TFP and DBC than for PZQ, which revealed a maximum of 6% haemolysis for the highest concentration tested. Different concentration-response curves were obtained for lipid elution, although the profiles of cholesterol and phospholipids released were similar for all drugs. Nevertheless, at a fixed rate of 50% haemolysis, TFP induced a approximately 2-fold increment in the elution of cholesterol when compared with that produced by DBC (P<0. 05). The different effects induced by TFP, DBC and PZQ on erythrocyte morphology, haemolysis and lipid exfoliation are related to the physical and chemical characteristics of each compound. These results suggest that distinct cell membrane interaction pathways lead to drug-specific mechanisms of cytotoxicity.

Quantitative assessment of human erythrocyte membrane solubilization by Triton X-100.

Abstract We report here a quantitative analysis of the interaction of the non-ionic surfactant Triton X-100 with human erythrocyte membranes. By applying a classical treatment for the interpretation of the action of surface active compounds to the hemolytic curves, we could calculate parameters such as R e —the effective surfactant/lipid molar ratio for erythrocyte membrane saturation ( R e sat ) and total lysis ( R e sol )—and K b , the binding constant of Triton X-100 to human erythrocyte membranes. The K b (5900 M −1 ) and R e (1.58 and 2.14) values presented here are in good agreement with literature data for Triton X-100 solubilization of model phospholipid membranes.

Lipid chain dynamics in stratum corneum studied by spin label electron paramagnetic resonance.

The lipid chain motions in stratum corneum (SC) membranes have been studied through electron paramagnetic resonance (EPR) spectroscopy of stearic acid spin-labeled at the 5th, 12th and 16th carbon atom positions of the acyl chain. Lipids have been extracted from SC with a series of chloroform/methanol mixtures, in order to compare the molecular dynamics and the thermotropic behavior in intact SC, lipid-depleted SC (containing covalently bound lipids of the corneocyte envelope) and dispersion of extracted SC lipids. The segmental motion of 5- and 12-doxylstearic acid (5- and 12-DSA) and the rotational correlation time of 16-doxylstearic acid (16-DSA) showed that the envelope lipids are more rigid and the extracted lipids are more fluid than the lipids of the intact SC over the range of temperature measured. The lower fluidity observed for the corneocyte envelope, that may be caused mainly due to lipid-protein interactions, suggests a major contribution of this lipid domain to the barrier function of SC. Changes in the activation energy for reorientational diffusion of the 16-DSA spin label showed apparent phase transitions around 54 degrees C, for the three SC samples. Some lipid reorganization may occur in SC above 54 degrees C, in agreement with results reported from studies with several other techniques. This reorganization is sensitive to the presence of the extractable intercellular lipids, being different in the lipid-depleted sample as compared to native SC and lipid dispersion. The results contribute to the understanding of alkyl chain packing and mobility in the SC membranes, which are involved in the mechanisms that control the permeability of different compounds through skin, suggesting an important involvement of the envelope in the skin barrier.

Erythrocruorin of Glossoscolex paulistus (Righi) (Oligochaeta, Glossoscolecidae): effects of divalent ions, acid—Alkaline transition and alkali and urea denaturation

The oxygen affinity and cooperativity of erythrocruorin of Glossoscolex paulistus were studied as a function of cation concentration (Ca2+, Mg2+ and Mn2+). Acid-alkaline transition showed the presence of population multiplicity. Differences in t12 values were observed for native and dissociated forms when submitted to alkaline medium. Urea 6.0–8.0 M caused modifications of the protein structure.