The interaction of the brominated flame retardant: Tetrabromobisphenol A with phospholipid membranes

Tetrabromobisphenol A (TBBPA) is one of the most widely used members of the family of brominated flame retardants (BFRs). BFRs, including TBBPA have been shown to be widely distributed within the environment and there is growing evidence of their bio-accumulation within animals and man. Toxicological studies have shown that TBBPA can be harmful to cells by modulating a number of cell signalling processes. In this study, we employed fluorescence spectroscopy and differential scanning calorimetry to investigate the interaction of TBBPA with phospholipid membranes, as this is the most likely route for it to influence membrane-associated cellular processes. TBBPA readily and randomly partitions throughout all regions of the phospholipid bilayer with high efficacy {partition coefficient (Log K_p) = 5.7 ± 0.7}. A decrease in membrane fluidity in both liquid-crystalline and gel-phase membranes was detected at concentrations of TBBPA as low as 2.5 μM. TBBPA also decreases the phase transition temperature of dipalmitoyl phoshatidylcholine (DPPC) membranes and broadened transition peaks, in a fashion similar to that for cholesterol. TBBPA, however, also prefers to partition into membrane regions not too highly enriched with cholesterol. Our findings therefore suggests that, the toxic effects of TBBPA, may at least in part, be due to its lipid membrane binding/perturbing effects, which in turn, could influence biological processes involving cell membranes.     

Bnip3 mediates mitochondrial dysfunction and cell death through Bax and Bak

TBBPA (tetrabromobisphenol A) is currently the most widely used type of BFR (brominated flame retardant) employed to reduce the combustibility of a large variety of electronic and other manufactured products. Recent studies have indicated that BFRs, including TBBPA, are bio-accumulating within animal and humans. BFRs including TBBPA have also been shown to be cytotoxic and potentially endocrine-disrupting to a variety of cells in culture. Furthermore, TBBPA has specifically been shown to cause disruption of Ca2+ homoeostasis within cells, which may be the underlying cause of its cytotoxicity. In this study, we have demonstrated that TBBPA is a potent non-isoform-specific inhibitor of the SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) (apparent K(i) 0.46-2.3 microM), thus we propose that TBBPA inhibition of SERCA contributes in some degree to Ca2+ signalling disruption. TBBPA binds directly to the SERCA without the need to partition into the phospholipid bilayer. From activity results and Ca2+-induced conformational results, it appears that the major effect of TBBPA is to decrease the SERCA affinity for Ca2+ (increasing the K(d) from approx. 1 microM to 30 microM in the presence of 10 microM TBBPA). Low concentrations of TBBPA can quench the tryptophan fluorescence of the SERCA and this quenching can be reversed by BHQ [2,5-di-(t-butyl)-1,4-hydroquinone] and 4-n-nonylphenol, but not thapsigargin, indicating that TBBPA and BHQ may be binding to similar regions in the SERCA.     

The incorporation of cholesterol into inner mitochondrial membranes and its effect on lipid phase transition

Incubations of rat liver inner mitochondrial membranes with liposomes prepared from $\text{1,2-}\text{dipalmitoyl}\text{-sn-}\text{glycero}\text{-3-}\text{phosphocholine}$ (DPPC) and cholesterol resulted in a considerable enrichment of the cholesterol composition of these membranes. This enrichment is not accompanied by an alteration in the membrane phospholipid content or fatty acid composition. The exogenous cholesterol appears to be integrated into the membrane structure because it has effects consistent with the known properties of this sterol in other natural and artificial membrane systems.Differential scanning calorimetry on both intact membranes and extracted lipids showed that as the ratio of cholesterol to phospholipid was increased, the endotherm corresponding to the lipid phase transition was reduced. Freeze-fracture electron microscopy of the native membranes showed that intramembranous particles are randomly distributed above the phase transition temperature. Below this temperature large smooth areas, believed to correspond to lipid in the gel state from which proteins have been excluded, can be observed. In the presence of high concentrations of cholesterol the fracture faces observed below the lipid transition temperature show no regions of phase segregation, an observation consistent with previous studies using pure lipids where cholesterol was observed to prevent the lipid undergoing a cooperative phase transition.The results are discussed in terms of the observed low concentrations of cholesteorl in normal liver inner mitochondrial membranes and the distribution of cholesterol within the liver cells.     

Adaptive alteration in phospholipid composition of plasma membranes from a somatic cell mutant defective in the regulation of cholesterol biosynthesis

A somatic cell mutant (CR1) of a Chinese hamster ovary cell (CHO-K1) which has previously been shown to be defective in the regulation of cholesterol biosynthesis accumulates more cholesterol than the parental cell line in plasma membranes. Although such an increase in membrane cholesterol should lead to an increase in the order parameter of these membranes, as measured with an electron spin resonance spin probe, the order parameters of mutant and wild-type plasma membranes are identical- -apparently because of an adaptive alteration in membrane phospholipid composition. The phospholipid compositions of mutant and wild-type cell plasma membranes are compared and the mutant is shown to have a threefold higher level of oleic acid and a twofold lower level of phosphatidylethanolamine than the wild type. These results are consistent with model studies which show that these compositional changes lead to lower-order parameters for phospholipid dispersions.     

Effect of cholesterol on human erythrocyte membrane. A spin label study

The effect of cholesterol on the membrane fluidity of human erythrocytes has been studied by electron spin resonance (ESR) spectroscopy, sensing the motion of androstane and fatty acid spin labeles in the cell membrane and in vesicles made from extracted phospholipids. 1. Androstane spin label (ASL) was incorporated from ASL-containing phospholipid vesicles into the erythrocyte membrane, essentially by a partition mechanism in proportion to their phospholipid contents. 2. On increasing the cholesterol or ASl content in the cell membrane, the spin label was gradually immobilized. 3. ASL motion in the cell membrane seemed to be primarily determined by the cholesterol/phospholipid molar ratio, regardless of the membrane protein-lipid interaction, as judged from the temperature effects on the ESR spectra of both membranes. 4. However, glutaraldehyde pretreatment induced considerable changes of the cholesterol-lipid interaction in the cell membrane, i.e., strong immobilization and cluster formation of ASL were observed.     

Trans fatty acid derived phospholipids show increased membrane cholesterol and reduced receptor activation as compared to their cis analogs

The consumption of trans fatty acid (TFA) is linked to the elevation of LDL cholesterol and is considered to be a major health risk factor for coronary heart disease. Despite several decades of extensive research on this subject, the underlying mechanism of how TFA modulates serum cholesterol levels remains elusive. In this study, we examined the molecular interaction of TFA-derived phospholipid with cholesterol and the membrane receptor rhodopsin in model membranes. Rhodopsin is a prototypical member of the G-protein coupled receptor family. It has a well-characterized structure and function and serves as a model membrane receptor in this study. Phospholipid-cholesterol affinity was quantified by measuring cholesterol partition coefficients. Phospholipid-receptor interactions were probed by measuring the level of rhodopsin activation. Our study shows that phospholipid derived from TFA had a higher membrane cholesterol affinity than their cis analogues. TFA phospholipid membranes also exhibited a higher acyl chain packing order, which was indicated by the lower acyl chain packing free volume as determined by DPH fluorescence and the higher transition temperature for rhodopsin thermal denaturation. The level of rhodopsin activation was diminished in TFA phospholipids. Since membrane cholesterol level and membrane receptors are involved in the regulation of cholesterol homeostasis, the combination of higher cholesterol content and reduced receptor activation associated with the presence of TFA-phospholipid could be factors contributing to the elevation of LDL cholesterol.     

Evidence for distinct cholesterol domains in fiber cell membranes from cataractous human lenses

Previous studies in our laboratory have provided direct evidence for the existence of distinct cholesterol domains within the plasma membranes of human ocular lens fiber cells. The fiber cell plasma membrane is unique in that it contains unusually high concentrations of cholesterol, with cholesterol to phospholipid (C/P) mole ratios ranging from 1 to 4. Since membrane cholesterol content is disturbed in the development of cataracts, it was hypothesized that perturbation of cholesterol domain structure occurs in cataracts. In this study, fiber cell plasma membranes were isolated from both normal (control) and cataractous lenses and assayed for cholesterol and phospholipid. Control and cataractous whole lens membranes had C/P mole ratios of 3.1 and 1.7, respectively. Small angle x-ray diffraction approaches were used to directly examine the structural organization of the cataractous lens plasma membrane versus control. Both normal and cataractous oriented membranes yielded meridional diffraction peaks corresponding to a unit cell periodicity of 34.0 A, consistent with the presence of immiscible cholesterol domains. However, comparison of diffraction patterns indicated that cataractous lens membranes contained more pronounced and better defined cholesterol domains than controls, over a broad range of temperature (5-40 degrees C) and relative humidity (52-92%) levels. In addition, diffraction analyses of the sterol-poor regions of cataractous membranes indicated increased membrane rigidity as compared with control membranes. Modification of the membrane lipid environment, such as by oxidative insult, is believed to be one potential mechanism for the formation of highly resolved cholesterol domains despite significantly reduced cholesterol content. The results of this x-ray diffraction study provide evidence for fundamental changes in the lens fiber cell plasma membrane structure in cataracts, including the presence of more prominent and highly ordered, immiscible cholesterol domains.     

Some commonly used brominated flame retardants cause Ca2+-ATPase inhibition, beta-amyloid peptide release and apoptosis in SH-SY5Y neuronal cells

Brominated flame retardants (BFRs) are chemicals commonly used to reduce the flammability of consumer products and are considered pollutants since they have become widely dispersed throughout the environment and have also been shown to bio-accumulate within animals and man. This study investigated the cytotoxicity of some of the most commonly used groups of BFRs on SH-SY5Y human neuroblastoma cells. The results showed that of the BFRs tested, hexabromocyclododecane (HBCD), tetrabromobisphenol-A (TBBPA) and decabromodiphenyl ether (DBPE), all are cytotoxic at low micromolar concentrations (LC(50) being 2.7 ± 0.7 μM, 15 ± 4 μM and 28 ± 7 μM, respectively). They induced cell death, at least in part, by apoptosis through activation of caspases. They also increased intracellular [Ca(2+)] levels and reactive-oxygen-species within these neuronal cells. Furthermore, these BFRs also caused rapid depolarization of the mitochondria and cytochrome c release in these neuronal cells. Elevated intracellular [Ca(2+)] levels appear to occur through a mechanism involving microsomal Ca(2+)-ATPase inhibition and this maybe responsible for Ca(2+)-induced mitochondrial dysfunction. In addition, μM levels of these BFRs caused β-amyloid peptide (Aβ-42) processing and release from these cells with a few hours of exposure. These results therefore shows that these pollutants are both neurotoxic and amyloidogenic in-vitro.     

Membrane solubility of ethanol in chronic alcoholism. The effect of ethanol feeding and its withdrawal on the protection by alcohol of rat red blood cells from hypotonic hemolysis

Membranes from ethanol-fed rats are resistant to the in vitro effects of ethanol on membrane structure and function. We have proposed that the resistance arises from adaptive changes in membrane composition which lower the solubility (partition coefficient) of ethanol in these membranes. The partition of ethanol (and other alcohols and anesthetics) into red blood cells protects the cells from hypotonic hemolysis. Here, we show that the protection by alcohols and anesthetics of red blood cells from ethanol-fed rats is greatly attenuated. This finding indicates that the membrane solubility of these agents is lowered in chronic alcoholism and thus explains the resistance to the acute effects of ethanol. The protection from hemolysis decreases over 2 weeks of ethanol-feeding and returns to normal values within 1 day after ethanol withdrawal. These changes are associated with a parallel increase in total and free serum cholesterol during ethanol feeding and a return to normal values within a day after withdrawal. However, we find only a slight increase in the cholesterol/phospholipid ratio of the red blood cell membranes during the development of ethanol tolerance. In rats fed a cholesterol and saturated fat diet, the increase in serum cholesterol is also associated with an attenuation of the protection from hypotonic hemolysis.     

Characterization of the interaction of single tryptophan containing mutants of IpaC from Shigella flexneri with phospholipid membranes

Shigella flexneri causes dysentery after invading the epithelial cells of the human colon. Enterocyte invasion is induced by the bacterial effector IpaC (invasion plasmid antigen C), which triggers Shigella entry into epithelial cells by a rather poorly understood mechanism. IpaC is also involved in pathogen escape into the host cell cytoplasm following uptake, and this property may be reflected in its ability to disrupt phospholipid vesicles in vitro. Purified recombinant IpaC interacts with liposome vesicles to cause the release of small molecules trapped inside. This interaction requires that the liposomes possess an acidic phospholipid component. To better understand the events involved in the disruption of liposomes by IpaC, single tryptophan mutants were generated to permit the use of intrinsic fluorescence, circular dichroism, and ultraviolet absorption spectroscopies to examine the effect that phospholipid membrane association has on IpaC structure and stability. These mutants were also used to determine how amino acid substitutions within specific regions of IpaC influence its activity in vivo. The outcomes of this study include findings that cholesterol greatly impacts IpaC association with phospholipid membranes, tryptophan incorporation into specific regions of IpaC (especially near the C-terminus) can greatly impact its in vivo activity, and interaction with phospholipid membranes causes differing degrees of change in the fluorescence of tryptophan residues introduced at specific sites within IpaC. These data, together with fluorescence quenching analyses, provide new functional and structural information concerning IpaC and its insertion into phospholipid membranes.     

Aspirin inhibits formation of cholesterol rafts in fluid lipid membranes

Aspirin and other non-steroidal anti-inflammatory drugs have a high affinity for phospholipid membranes, altering their structure and biophysical properties. Aspirin has been shown to partition into the lipid head groups, thereby increasing membrane fluidity. Cholesterol is another well known mediator of membrane fluidity, in turn increasing membrane stiffness. As well, cholesterol is believed to distribute unevenly within lipid membranes leading to the formation of lipid rafts or plaques. In many studies, aspirin has increased positive outcomes for patients with high cholesterol. We are interested if these effects may be, at least partially, the result of a non-specific interaction between aspirin and cholesterol in lipid membranes.We have studied the effect of aspirin on the organization of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC) membranes containing cholesterol. Through Langmuir–Blodgett experiments we show that aspirin increases the area per lipid and decreases compressibility at 32.5 mol% cholesterol, leading to a significant increase of fluidity of the membranes. Differential scanning calorimetry provides evidence for the formation of meta-stable structures in the presence of aspirin. The molecular organization of lipids, cholesterol and aspirin was studied using neutron diffraction. While the formation of rafts has been reported in binary DPPC/cholesterol membranes, aspirin was found to locally disrupt membrane organization and lead to the frustration of raft formation. Our results suggest that aspirin is able to directly oppose the formation of cholesterol structures through non-specific interactions with lipid membranes.     

The effect of brominated flame retardants on neurotransmitter uptake into rat brain synaptosomes and vesicles

The environmental levels of brominated flame retardants (BFRs) are increasing, but little is known about their toxic effects. In this paper, we show that some of the most important BFRs in commercial use today, have a neurotoxicological potential. Hexabromocyclododecane (HBCD) and tetrabromobisphenol-A (TBBPA) inhibit plasma membrane uptake of the neurotransmitters dopamine, glutamate and gamma-amino-n-butyric acid (GABA) at a concentration level similar to what previously found for polychlorinated biphenyls (PCBs) and even for ecstasy. The IC(50) value for HBCD on dopamine uptake was 4 microM, and the IC(50) values for TBBPA were 9, 6 and 16 microM for dopamine, glutamate and GABA, respectively. HBCD also inhibited glutamate uptake at low concentrations, but never achieved more than 50% inhibition. The inhibition was primarily due to their effect on the membrane potential, measured by the membrane potential marker tetraphenylphosphonium bromide (TPP(+)). Other brominated flame retardants such as octaBDE and decaBDE did not have any effects on uptake. TBBPA, HBCD and even the pentabrominated diphenylether mixture (pentaBDE, DE-71, Great Lakes) also inhibited the vesicular uptake of dopamine with an IC(50) value of 3, 3 and 8 microM, respectively. The neurotoxicological consequences of these findings for environmental contaminants such as BFRs and PCBs are discussed.     

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55-1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid-cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid-cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid-cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 degrees C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.     

Functions of Cholesterol and the Cholesterol Bilayer Domain Specific to the Fiber-Cell Plasma Membrane of the Eye Lens

The most unique feature of the eye lens fiber-cell plasma membrane is its extremely high cholesterol content. Cholesterol saturates the bulk phospholipid bilayer and induces formation of immiscible cholesterol bilayer domains (CBDs) within the membrane. Our results (based on EPR spin-labeling experiments with lens-lipid membranes), along with a literature search, have allowed us to identify the significant functions of cholesterol specific to the fiber-cell plasma membrane, which are manifest through cholesterol–membrane interactions. The crucial role is played by the CBD. The presence of the CBD ensures that the surrounding phospholipid bilayer is saturated with cholesterol. The saturating cholesterol content in fiber-cell membranes keeps the bulk physical properties of lens-lipid membranes consistent and independent of changes in phospholipid composition. Thus, the CBD helps to maintain lens-membrane homeostasis when the membrane phospholipid composition changes significantly. The CBD raises the barrier for oxygen transport across the fiber-cell membrane, which should help to maintain a low oxygen concentration in the lens interior. It is hypothesized that the appearance of the CBD in the fiber-cell membrane is controlled by the phospholipid composition of the membrane. Saturation with cholesterol smoothes the phospholipid-bilayer surface, which should decrease light scattering and help to maintain lens transparency. Other functions of cholesterol include formation of hydrophobic and rigidity barriers across the bulk phospholipid-cholesterol domain and formation of hydrophobic channels in the central region of the membrane for transport of small, nonpolar molecules parallel to the membrane surface. In this review, we provide data supporting these hypotheses.     

Use of cyclodextrins to manipulate plasma membrane cholesterol content: evidence, misconceptions and control strategies

The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes. However, the mechanism of this activity of cyclodextrins is not completely established. Moreover, under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. We review the evidence for the specific and non-specific effects of cyclodextrins and what is known about the mechanisms for cyclodextrin-induced cholesterol and phospholipid extraction. Finally, we discuss useful control strategies that may help to verify that the observed effects are due specifically to cyclodextrin-induced changes in cellular cholesterol.     

Use of cyclodextrins to manipulate plasma membrane cholesterol content: Evidence, misconceptions and control strategies

The physiological importance of cholesterol in the cell plasma membrane has attracted increased attention in recent years. Consequently, the use of methods of controlled manipulation of membrane cholesterol content has also increased sharply, especially as a method of studying putative cholesterol-enriched cell membrane domains (rafts). The most common means of modifying the cholesterol content of cell membranes is the incubation of cells or model membranes with cyclodextrins, a family of compounds, which, due to the presence of relatively hydrophobic cavity, can be used to extract cholesterol from cell membranes. However, the mechanism of this activity of cyclodextrins is not completely established. Moreover, under conditions commonly used for cholesterol extraction, cyclodextrins may remove cholesterol from both raft and non-raft domains of the membrane as well as alter the distribution of cholesterol between plasma and intracellular membranes. In addition, other hydrophobic molecules such as phospholipids may also be extracted from the membranes by cyclodextrins. We review the evidence for the specific and non-specific effects of cyclodextrins and what is known about the mechanisms for cyclodextrin-induced cholesterol and phospholipid extraction. Finally, we discuss useful control strategies that may help to verify that the observed effects are due specifically to cyclodextrin-induced changes in cellular cholesterol.     

Levels and distributions of phospholipids and cholesterol in the plasma membrane of neuroblastoma cells.

Murine neuroblastoma cells (clone N-2A) grown in suspension (spinner cells) or attached on a plastic surface (monolayer cells) were used in studies of the phospholipid and cholesterol composition of whole cells, primary plasma membranes, plasma membranes internalized during phagocytosis of polystyrene latex beads, mitochondria and microsomes. Monolayer cells contained higher concentrations of total phospholipid, phosphatidylserine and phosphatidylcholine, and lower concentration of phosphatidylethanolamine than spinner cells. The cholesterol levels and the relative proportions of the various phospholipids were similar in both cell types except phosphatidylethanolamine and sphingomyelin whose proportions were lower in monolayer cells. The primary plasma membranes of the two cell types differed significantly in the relative proportions of all phospholipids, except sphingomyelin, and the phospholipid to protein and the cholesterol to protein ratios were all higher in the membranes of spinner cells. In contrast to these results, all the phospholipid to protein and the cholesterol to protein ratios of the internalized plasma membranes were higher in monolayer than in spinner cells, and the proportions of all phospholipids, except phosphatidylethanolamine, were similar in both cell types. The membrane distributions of individual phospholipids and cholesterol were inferred from comparison of the phospholipid and cholesterol compositions of primary plasma membranes and plasma membranes internalized during phagocytosis of polystyrene beads. The results are consistent with a non-random distribution of most phospholipids in both spinner and monolayer cells, but the patterns of these distributions were different in the two cell types. With regard to cholesterol the results are compatible with a random or a heterogeneous distribution. All the phospholipid to protein ratios of the mitochondrial fraction of both cell types were lower than those of the plasma membranes. However, these ratios of the microsomal fraction were higher than those of the plasma membranes of monolayer cells, whereas they were comparable, with a few exceptions, to those of spinner cell membranes. The cholesterol to phospholipid molar ratios of plasma membranes were 6.4 and 4.3 fold greater than those of the mitochondrial and microsomal fractions, respectively.     

Characterization of lipid domains in reconstituted porcine lens membranes using EPR spin-labeling approaches

The physical properties of membranes derived from the total lipid extract of porcine lenses before and after the addition of cholesterol were investigated using EPR spin-labeling methods. Conventional EPR spectra and saturation-recovery curves indicate that the spin labels detect a single homogenous environment in membranes before the addition of cholesterol. After the addition of cholesterol (when cholesterol-to-phospholipid mole to mole ratio of 1.55-1.80 was achieved), two domains were detected by the discrimination by oxygen transport method using a cholesterol analogue spin label. The domains were assigned to a bulk phospholipid-cholesterol bilayer made of the total lipid mixture and to a cholesterol crystalline domain. Because the phospholipid analogue spin labels cannot partition into the pure cholesterol crystalline domain, they monitor properties of the phospholipid-cholesterol domain outside the pure cholesterol crystalline domain. Profiles of the order parameter, hydrophobicity, and oxygen transport parameter are identical within experimental error in this domain when measured in the absence and presence of a cholesterol crystalline domain. This indicates that both domains, the phospholipid-cholesterol bilayer and the pure cholesterol crystalline domain, can be treated as independent, weakly interacting membrane regions. The upper limit of the oxygen permeability coefficient across the cholesterol crystalline domain at 35 °C had a calculated value of 42.5 cm/s, indicating that the cholesterol crystalline domain can significantly reduce oxygen transport to the lens center. This work was undertaken to better elucidate the major factors that determine membrane resistance to oxygen transport across the lens lipid membrane, with special attention paid to the cholesterol crystalline domain.     

Cholesterol affects physical properties and (Na+,K+)-ATPase in basolateral membranes of renal and intestinal epithelia from thermally acclimated rainbow trout

Previous work has shown that cholesterol levels are modulated in plasma membranes from some but not all tissues of poikilotherms over the course of temperature change. To gain a better understanding of tissue and membrane domain-specific cholesterol function during thermal adaptation we examined effects of cholesterol on membrane physical properties and (Na⁺,K⁺)-ATPase in native and cholesterol-enriched basolateral membranes from kidney and intestine of thermally acclimated trout (Oncorhynchus mykiss). Membrane order (as indicated by fluorescence depolarization studies) is increased, whereas its thermal sensitivity is decreased by elevated cholesterol levels in mem branes with relatively low endogenous amounts of cholesterol (intestinal membranes and renal membranes from cold-acclimated fish). Thermal sensitivities of membrane order in kidney are 1.5-fold higher in native compared with cholesterol-enriched basolateral membranes. For renal plasma membranes, (Na⁺,K⁺)- ATPase activity is lowest near the transition between native and surpraphysiological cholesterol levels. Endogenous cholesterol levels (relative to phospholipid contents) in intestinal basolateral membranes from cold-acclimated fish vary more than 1.5-fold; membranes with cholesterol/phospholipid molar ratios of 0.3 have activities of (Na⁺,K⁺)-ATPase that are twofold lower than native membranes having a ratio of 0.2. These results suggests that maintenance of cholesterol levels in intestinal basolateral membranes during thermal acclimation may ensure sufficient activity of (Na⁺,K⁺)-ATPase. Membrane function in kidney, with its high native cholesterol content, is less likely to be affected by temperature change. Accepted: 21 January 1997     

Cholesterol interactions with phospholipids in membranes.

Mammalian cell membranes are composed of a complex array of glycerophospholipids and sphingolipids that vary in head-group and acyl-chain composition. In a given cell type, membrane phospholipids may amount to more than a thousand molecular species. The complexity of phospholipid and sphingolipid structures is most likely a consequence of their diverse roles in membrane dynamics, protein regulation, signal transduction and secretion. This review is mainly focused on two of the major classes of membrane phospholipids in eukaryotic organisms, sphingomyelins and phosphatidylcholines. These phospholipid classes constitute more than 50% of membrane phospholipids. Cholesterol is most likely to associate with these lipids in the membranes of the cells. We discuss the synthesis and distribution in the cell of these lipids, how they are believed to interact with each other, and what cellular consequences such interactions may have. We also include a discussion about findings in the recent literature regarding cholesterol/phospholipid interactions in model membrane systems. Finally, we look at the recent trends in computer and molecular dynamics simulations regarding phospholipid and cholesterol/phospholipid behavior in bilayer membranes.