We studied the exchange of cholesterol between radioactively labeled plasma and human erythrocytes.Results from experiments in which [3H]cholesterol and ['4CJ cholesterol were exchanged sequentially into the cells and back out into unlabeled plasma, showed that transmembrane movement of cholesterol occurred with a half-time that was either less than 50 min or greater than 10 days. To obtain further information about the transmembrane movement of cholesterol, we used a technique [Jaeobson, B. S. & Branton, D. (1977) Science 195, 302-304] for exposure of the cytoplasmic surface of erythrocyte membranes. This method involved the ionic attachment of erythrocytes to derivatized glass beads followed by disruption of the cells, leaving the beads covered by membrane with the cytoplasmic surface exposed. [3HlCholesterol was exchanged into intact erythrocytes which then were attached to beads. The beads with attached membrane were incubated with phospholipid-cholesterol vesicles and the exchange of cholesterol between the membrane cytoplasmic surface and vesicles was studied. We found that [3Hlcholesterol was present at the cytoplasmic surface, indicating that transmembrane movement of cholesterol had occurred within the 2.5 hr required to complete the experiment. This result suggests that the more rapid rate of transmembrane cholesterol movement, inferred from-the experiments described above, is the one that applies.Cholesterol from erythrocyte membranes exchanges rapidly with the unesterified cholesterol of plasma lipoproteins both in vivo (1) and in vitro (2). The mechanisms involved in this exchange process are not understood. Some studies have indicated that all of erythrocyte cholesterol is exchangeable (3, 4), whereas others have shown that a significant portion does not exchange (5,6). The extent to which red cell cholesterol is available for exchange has important implications with regard both to membrane structure and to mechanisms of cholesterol exchange.It is widely believed that the lipids of the erythrocyte membrane are arranged in a bilayer that has transmembrane compositional asymmetry in its phospholipid components. The location of cholesterol in the membrane is not known and it is possible that it too is asymmetrically distributed across the bilayer. A recent study of the cholesterol content of the inner and outer halves of the erythrocyte membrane has indicated that there is cholesterol in both membrane halves, with possibly more on the outer half of the membrane (7).If cholesterol located on the cytoplasmic side of the membrane is to exchange with plasma lipoproteins, it first must move across the bilayer. Translocation across the erythrocyte membrane occurs for phosphatidylcholine. Bloj and Zilversmit (8) found a half-time for the process of 2.3 hr in rat erythrocyte ghosts; Renooij et al. (9) measured a half-time of 4.5 hr in intact rat red cells.The transbilayer movement of cholesterol in vesicles occurs with a half-time in excess of 6 days (10). In [14C]cholesterol were sequential...

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Ultraviolet radiation (UVR) is one of the risk factors for skin cancer and the main inducer of melanin pigmentation, the major protective mechanism of mammalian skin against radiation damage. The melanin pigments, eumelanin and pheomelanin, are likely to be important in protection against UVR, but their precursors are generally considered as phototoxic. The available data suggest DNA damage as the mechanism of phototoxicity. However, the effect of melanin precursors on membrane damage through lipid peroxidation, another important and probably more relevant (from the point-of-view of the melanosomal confinement of these molecules) mechanism of phototoxicity, not known. As a model system for UVR-melanin-membrane interactions, we irradiated liposomes in the presence of eumelanin, pheomelanin and two of their major precursors, 5,6-dihydroxyindole (DHI) and 5-S-cysteinyldopa (SCD). The presence of the two melanin precursors substantially reduced the formation of lipid peroxidation products resulting from UVR exposure. The antioxidant activity of the melanin precursors was diminished under strong prooxidant conditions (presence of Fe3+). These results suggest that melanin precursors may have an important role in the protection of skin against the harmful effects of UVR including photocarcinogenesis.

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The permeability of lipid bilaycrs and biological mcmbrancs to superoxidc free radicals was examined by using supcroxidc dismutasc (,SOD)-loaded lipid vesicles and SOD-loaded crythrocytc ghosts. After exposing SOD lipid vesicles and SOD ghosts to enzymatically produced superoxide radicals and using spin-trapping and electron spin resonance (ESR) techniques, WC found that SODcntrappcd withinerythrocyte ghostseffectively scavenges external O;-while SOD inside the lipid bilaycrs has no effect. These resulls confirm that O:-is able lo cross through a biological plasma membrane but not across a pure lipid bilaycr. The data provide instruction as to how and where anti-oxidant thcrdpy is to be approached rclalivc to the site of oxygen free radical production.Supcroxidc radical; Permcabilhy, Lipid vesicle; Erythrocytc ghost: Electron spin resonance IluTRODlJCTIONSince oxygen free radicals are thought to play an important role in direct cellular injury, superoxide dismutase (SOD) has been considered to be an important enzyme to strengthen cellular defence. However, because of the compartmentalized production of O:-and the localized distribution of SOD in cells [l-33, the successful use of exogenous SOD to eliminate Ot-in situ requires a thorough knowledge of the permeability of cell membranes to oxygen free radicals.To date, reports of the permeation of lipid bilayers to Ot-are not conclusive. Takahashi et al. reported that the lipid bilayers were not permeable to Ot-generated inside lipid vesicles by the illumination of flavin mononucleotide [A], while Rumyantseva et al. found that enzymatitally produced O;-could cross a lipid bilayer to cause ferricyanide-ferrocyanide transformation inside lipid vesicles [5]. Evidence for the permeability of biological membranes to OI-is limited to one available example of erythrocyte membrane [6]. By using xanthine oxidaseloaded lipid vesicles and chemically defined reaction, Lynch and Fridovich reported that erythrocyte membrane was permeable to O;-possibly via an anion channel.In order to clarify the permeability of O;-, a precise method for detecting and differentiating free radical species appears indispensable. In our laboratory, we have recently applied electron spin resonance (ESR) and spin trapping techniques, a more direct and sensi- tive method, to study the permeability of O;-. We have loaded phosphatidylcholine lipid vesicles and resealed erythrocyte ghosts with SOD. Following the incubation of SOD vesicles or SOD ghosts with an external source of O;-, the permeation of membranes to OI-was determined by measuring retained external concentration of O:-using both the ESR techniques as well as a chemically defined reaction. The results show that SOD trapped inside lipid vesicles did not scavenge external O;-suggesting that lipid bilayers are not permeable to O;-. In contrast, SOD-loaded ghosts are able to scavenge external OI-as effectively as free SOD supporting that the erythrocyte membranes are permeable to OS-. MATERIALS AND METHODS I. Prcyxmtriot~ CI/ SOD-luud~d lipid ws...

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ABSTRACT. The toxicity of bilirubin was investigated in 2 neural cell lines NBRlOA and N115 using a quantitative dye assay 3-(4,s dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium biomide (MTT) as a measure of cell viability and [%]thymidine incorporation as a measure of DNA synthesis. Short exposures (up to 2 h) to bilirubin, even up to a bilirubin-albumin molar ratio of 1.5, yielded no evidence of toxicity using these assays. At longer exposure times (24 h) a decrease in cell viability and I3H]thymidine incorporation was detected at a molar ratio of 0.8 when the bilirubin concentration was 0.1 m M or higher, whereas lower bilirubin levels a t this molar ratio showed no deleterous effect. The effect of bilirubin is more pronounced a t a molar ratio of 1.5 with longer incubation periods. The M T T assay showed the N115 cells appeared to be more resistant to bilirubin cytotoxicity than NBRlOA cells, a finding which was not obtained from ['Hlthymidine incorporation studies. This discrepancy can be explained by the fact that we are measuring two different variables; the M T T assay estimates the number of viable cells a t the end of the experiment by measuring mitochondria1 function whereas the [%)I]thymidine assay measures the rate of DNA synthesis during the last 2 h of the experiment. The concentration effect of bilirubin is evident from the ['HIthymidine studies in that at a molar ratio of 1.5 and bilirubin concentration of 0.075 m M or higher, there is both cell kill (decrease in DNA) and inhibition of [3H]-thymidine incorporation (decrease in specific activity). When the bilirubin concentration is reduced to 0.03 mM, there is little or no cell death (no change DNA) but inhibition still exists (42% decrease in specific activity).Thus, cell viability and function of these two neural lines is dependent not only on the bilirubin albumin molar ratio, but also on the absolute concentration of bilirubin and albumin as well a s the time of exposure. ciated with irreversible nuclear damage in the brain (kernicterus) (I). The toxic effect of bilirubin in tissue culture cells have been reported in a number of studies including histologic damage in dissociated rat cerebellum (2), decrease in glucose consumption in human fibroblasts (3), decrease in viability and ATP content (4), and increase in potassium leakage in modified L-929 mouse fibroblasts (5). Thaler (6) reported a decrease in DNA synthesis in hepatoma cells. Cell viability in most of these studies was assessed by the exclusion of either trypan blue or erythrocin B. The dyes will only stain those cells which are nonviable and leaky. A qualitative assessment of viable to nonviable cells is usually done by counting under the microscope.T o our knowledge, n o studies have been done on the cytotoxicity of bilirubin on neural cell lines in culture. In the present study, we evaluate the effects of bilirubin in the presence or absence of albumin on cytotoxicity in two established neural cell lines, namely N1 15, a rat neuroblastoma cell and NBRlOA a murine neuroblastoma h...

The effect of surface curvature on the spontaneous movement of cholesterol between membranes was investigated by measuring the rates of cholesterol transfer from donor vesicles of various sizes to a common acceptor vesicle. Donor vesicles of size in the range 40-240 nm were prepared by extruding multilamellar dispersions through polycarbonate filters of different pore sizes under pressure. The smallest donor vesicle and the acceptor vesicles were obtained by the normal sonication procedures. The rate of cholesterol transfer, as measured by the movement of [3H]cholesterol, decreases with increasing size of the donor vesicle in an almost linear fashion. The extrapolation of the results gave a half-time (t1/2) of 16-20 h of the desorption of cholesterol from a planar bilayer, and this can be considered as a reference value for most cellular membranes which are characterized by very low curvatures. Our earlier studies have shown that the t1/2 for cholesterol efflux is influenced by the presence of gangliosides and phosphatidylethanolamine, and the asymmetric distribution of these lipids in the plasma membrane could partially account for the large difference in the rates of cholesterol movement from the two sides of the plasma membrane. The small differences in rates arising from asymmetric distribution will be magnified by the longer t1/2 obtained here for membranes of low curvatures, so that the large difference in rates might be a coupled effect of lipid asymmetry and low curvature of the plasma membrane. This, in turn, may have a role in maintaining the large differences in cholesterol/phospholipid molar ratios observed between plasma membrane and intracellular membranes.