Alterations In Membrane Lipid Composition Research Articles

Anesthetics modulate phospholipase C hydrolysis of monolayer phospholipids by surface pressure

Anesthetics are believed to produce anesthesia through the reversible inhibition of synaptic transmission but how this is accomplished is unknown. Based on earlier studies of anesthetic-enzyme-phospholipid interaction, we surmised that anesthetics may inhibit synaptic transmission by increasing synaptic membrane lateral pressure thereby inhibiting phospholipid hydrolysis, membrane transduction and synaptic transmission. As a first approximation towards investigating this concept, we hypothesized that anesthetics modulate the rate of phospholipase C hydrolysis of a lipid monolayer through its effects on surface pressure. The relationship between the hydrolysis rate of a monolayer of dipalmitoylphosphatidylcholine [ 14C-choline] (DPPC) by phospholipase C (Plase C) and monolayer surface pressure (SP) as altered by either halothane, isoflurane, or by physical compression at 37°C was studied. The decline in surface 14C-activity as the [ 14C]choline diffuses into the Krebs-Ringer bicarbonate buffer aqueous subphase is estimated as the rate of DPPC hydrolysis measured by the initial slope method. DPPC hydrolysis was about 300 cpm/min and constant between SP of 0 to 20 dynes/cm. Higher SP between 25 and 30 dyne/cm, whether induced by halothane, isoflurane or physical compression, increased the rate of hydrolysis by 5-fold to a peak rate of about 1600 cpm/min at 25–30 dynes/cm. At a SP above 32 dynes/cm, DPPC hydrolysis abruptly ceased. We conclude that anesthetics can reversibly inhibit synaptic transmission through their effects on synaptic membrane lateral pressure. We also speculate that membrane lateral pressure may be a highly sensitive means of controlling membrane function through alteration in membrane lipid composition, membrane enzyme activity, receptor affinity and ion channel permeability.

Modifications of hepatic drug metabolizing enzyme activities in rats fed baobab seed oil containing cyclopropenoid fatty acids

The effects on drug metabolizing enzymes of cyclopropenoid fatty acids present in baobab seed oil were evaluated in rats fed either a diet with baobab seed oil (1.27% cyclopropenoid fatty acids in the diet) or a diet with heated baobab seed oil (0.046% cyclopropenoid fatty acids in the diet). Comparison was made with rats fed a mixture of oils that contained no cyclopropenoid fatty acid. Rats fed baobab oil showed retarded growth. In comparison with the other groups, the relative liver weights were markedly increased whereas cytochrome P-450 content and NADPH cytochrome c reductase and NADH cytochrome c reductase activities were decreased. In rats fed the heated baobab oil the relative liver weight was decreased and the cytochrome P-450 level and reductase activities were increased relative to levels in rats fed the unheated oil. Ethoxycoumarin deethylase, ethoxyresorufin deethylase and pentoxyresorufin depentylase activities, expressed on the basis of cytochrome P-450, were greater in the group fed unheated baobab seed oil. Cytosolic glutathione transferase activity was markedly decreased in rats fed fresh baobab seed oil and heating the oil, which reduced the content of cyclopropenoid fatty acids, led to a considerable increase of this activity. UDP-glucuronyl transferase activities were not modified by the type of oil included in the diet. It is possible that the mechanisms of action of cyclopropenoid fatty acids are related to alterations of membrane lipid composition or microsomal proteins.

Polyamines alter intestinal glucose transport.

Polyamines are required for the growth of all eukaryotic cells. Enterocytes respond to luminal nutrients with large increases in polyamine synthesis, even though they are mature, nonproliferating cells. The role of polyamines in these cells is unknown. The current experiments examined whether polyamines affected intestinal transport of glucose, since absorption is the primary activity of enterocytes and since polyamines are known to affect membrane function and stability. Glucose transport was examined in rabbit brush-border membrane vesicles (BBMV). BBMV from rabbits given 5% alpha-difluoromethylornithine (DFMO) in their drinking water 24 h before they were killed transported significantly less glucose than control vesicles [38% decrease in maximal transport rate (Jmax)]. Orogastric administration of spermine, spermidine, or putrescine to DFMO-treated animals 24 h before they were killed prevented the decrease. In rabbits receiving only orogastric spermine, glucose transport was significantly increased (64% increase in Jmax), whereas in vivo spermidine and putrescine decreased Jmax. This increase in Jmax caused by in vivo administration of spermine was not dependent on protein synthesis. Addition of polyamines whether in vivo or in vitro decreased Michaelis constant in vesicles from control and DFMO-treated animals. The change in glucose transport induced by DFMO or polyamines was not related to altered membrane lipid composition or fluidity.(ABSTRACT TRUNCATED AT 250 WORDS)

Apical Dominance in Apple (Malus domestica Borkh): The Possible Role of Indole-3-Acetic Acid (IAA)

The effect of IAA on apical dominance in apple buds was examined in relation to changes in proton density (free water) and membrane lipid composition in lateral buds. Decapitation induced budbreak and enhanced lateral bud growth. IAA replaced apical control of lateral buds and maintained paradormancy. Maximal inhibition was obtained when IAA was applied immediately after the apical bud was removed; delaying application reduced the effect of IAA. An increase in proton density in lateral buds was observed 2 days after decapitation, whereas the change in membrane lipid composition occurred 4 days later. Removing the terminal bud increased membrane galacto- and phospholipids and the ratio of unsaturated to corresponding saturated fatty acids. Decapitation also decreased the ratio of free sterols to phospholipids in lateral buds. Applying thidiazuron to lateral buds of decapitated shoots enhanced these effects, whereas applying IAA to the terminal end of decapitated shoots inhibited the increase of proton density and prevented changes in membrane lipid composition in lateral buds. These results suggest that change in water movement alters membrane lipid composition and then induces lateral bud growth. IAA, presumably produced by the terminal bud, restricts the movement of water to lateral buds and inhibits their growth in apple.

Protein Kinase C Activity Is Reduced in Epidermal Cells from Energy-Restricted SENCAR Mice ,

Female SENCAR mice were pre-fed a control or 40% energy-restricted (ER) diet with energy removed from fat and carbohydrate, or a control, balanced high fat (BHF, with similar energy from fat and carbohydrate), 35% energy restricted from fat (HCR) or 35% energy restricted from carbohydrate (HFR) diet. Epidermal cells were isolated by trypsin digestion for measurement of protein kinase C (PKC) activity, lipid composition or lipid metabolism. Dietary restriction of fat or carbohydrate energy (HFR or HCR group) reduced particulate PKC activity in epidermal cells compared with cells from control mice. The ratio of soluble particulate PKC activity was higher in epidermal cells from mice fed the HCR diet compared with those fed the HFR diet. Diet did not affect soluble PKC activity. Inositol accumulation was measured in the water- or lipid-soluble fractions of prelabeled ([3H]inositol) epidermal cells following a 1-h incubation in media with LiCl. Phosphatidylinositol, inositol biphosphate and inositol triphosphate fractions were more heavily labeled in cells from mice fed the ER diet. Energy restriction did not modify epidermal total lipid or phospholipid composition, but 1,2-diacylglycerol levels were elevated in relation to cell number in epidermal cells from mice fed the ER diet. These data suggest that dietary energy restriction modified PKC activity through a pathway other than alteration in membrane lipid composition or inositol lipid metabolism.

Altered membrane lipid composition in a human meningosarcoma

In a sample of meningosarcoma, obtained at the time of surgery, the amount of total gangliosides and phospholipids was examined, together with the cholesterol content and the distribution of different ganglioside and phospholipid species. The phosphatidylinositol, phosphatidylinositol-4-phosphate, phosphatidylinositol-4, 5-bisphosphate and phosphatidylcholine fatty acid composition was also analyzed. The ganglioside pattern in the meningosarcoma was different from the previously reported pattern in meningiomas of different histological origin, showing a higher concentration of GD3, indicating that the so-called b pathway of ganglioside biosynthesis was the preferred one in this type of tumor; moreover the percentage content of polysialylated gangliosides was very low. Cholesterol and phospholipid content was lower than in meningiomas; the phosphatidylcholine increase and the sphingomyelin decrease would indicate a lower membrane microviscosity, a characteristic of tumor cells. Phosphoinositide and phosphatidylcholine fatty acid analysis revealed a considerable amount of docosahexaenoic acid. This abnormal presence of this fatty acid could lead to the production, after receptor stimulation, of a diacylglycerol containing docosahexaenoic acid, which, in turn, could be responsible for an altered activation pattern of protein kinase C, in this way promoting carcinogenesis.

Effect of ethinylestradiol and epomediol on bile flow and biliary lipid composition in rat

Epomediol (1,3,3-trimethyl-2-oxabicyclo(2.2.2.)octan-6,7-endo,endo-diol) (EPO) is a terpenoid compound shown to reverse 17α-ethinylestradiol (EE)-induced cholestasis in rat. The effect is related to the restoration of normal liver plasma membrane fluidity values. To further characterize the effect of EPO, bile flow and biliary lipid composition were measured in rats treated either with EE or EE associated with EPO. EE significantly reduced the bile flow; this reduction was prevented by concomitant treatment with EPO with an increase in the bile salt secretion rate. EPO alone showed a choleretic effect. The biliary secretion rate of cholesterol was also significantly reduced by EE while being comparable to controls in EE-EPO-treated animals. Phospholipid (PL) biliary excretion was significantly (P < 0.002) increased by EE either alone or combined with EPO. After EE treatment, the biliary PL composition showed a reduction in phosphatidylcholine (PC) concentration with a parallel increase in lyso-phosphatidylcholine (LPC) when compared to control animals (PC:LPC ratio 5.0 ± 2.5 vs 26.8 ± 9.9, mean ± SD, P < 0.005). EPO administration to EE-treated rats restored the biliary PC:LPC ratio to control values (27.6 ± 10.6). EPO alone did not show any appreciable effect as compared to both control and EE-EPO treated animals. As increased concentrations of LPC have been reported to induce an alteration in the function of membrane lipids and membrane-associated proteins, such as regulatory enzymes for bile acid, cholesterol and phospholipid metabolism, these results suggest that the protective effect of EPO in EE-induced cholestasis may be related to the reversal of the alterations in membrane lipid composition and function induced by EE.

Lipid composition and peroxide levels of mucosal cells in the rat large intestine in relation to dietary fat

To examine whether dietary fat alters membrane lipid composition and peroxidation of polyunsaturated fatty acids in "non-proliferative" and "proliferative" cells in the large intestine, Sprague-Dawley rats were fed diets providing a polyunsaturated-to-saturated fatty acid ratio of 1.2 or 0.3 at a high or low level of fat intake for a 25-day period. Cell populations were isolated and the effect of dietary fat on membrane polyunsaturated fatty acid content and peroxide levels was determined. Neither fat level nor fatty acid composition of diet influenced total cholesterol, total phospholipids, and percentage of phospholipid classes in membrane phospholipids. Feeding the high fat and/or high polyunsaturated-to-saturated fatty acid ratio diet increased polyunsaturated fatty acid content of mucosal cell phospholipids. Increase in polyunsaturated fatty acid content was paralleled by a decrease in the monounsaturated fatty acid content of mucosal cell phospholipids. Membrane content of total saturated fatty acids was not significantly affected by diet. Variation in phospholipid fatty acid composition between "non-proliferative" and "proliferative" cells was observed. Lipid peroxide levels in mucosal cell lipid fractions were altered by dietary fat treatment. Animals fed high fat diets, compared to groups fed low fat diets, exhibited higher membrane peroxide levels when results are expressed as nmol/mg protein. Higher peroxide levels were observed in mucosal cells for rats fed high polyunsaturated-to-saturated fatty acid ratio diets when results were expressed per nmol of phospholipid. It is concluded that changes in fat level and fatty acid composition of the diet alters the mucosal cell membrane lipid composition in the rat large intestine and influences susceptibility of mucosal cell lipid to peroxidation. Further research is required to delineate which dietary factors--fat level, polyunsaturated-to-saturated fatty acid ratio, or both--have a primary influence on the degree of lipid peroxidation.

Protonophore-resistance and cytochrome expression in mutant strains of the facultative alkaliphile Bacillus firmus OF4

Two protonophore-resistant mutants, designated strains CC1 and CC2, of the facultative alkaliphile Bacillus firmus OF4 811M were isolated. The ability of carbonyl cyanide m-chlorophenylhydrazone (CCCP) to collapse the protonmotive force (delta mu H+) was unimpaired in both mutants. Both resistant strains possessed elevated respiratory rates when grown at pH 7.5, in either the presence or absence of CCCP. Membrane cytochromes were also elevated: cytochrome o in particular in strain CC1, and cytochromes aa3, b, c and o in strain CC2. Strain CC2 also maintained a higher delta mu H+ than the others when grown in the absence of CCCP. When grown in the presence of low concentrations of CCCP, strains CC1 and CC2 both maintained higher values of delta mu H+ than the wild-type parent and correspondingly higher capacities for ATP synthesis. In large-scale batch culture at pH 10.5, both mutant strains grew more slowly than the parent and contained significantly reduced levels of cytochrome o. Cells of stran CC1 also displayed a markedly altered membrane lipid composition when grown at pH 10.5. Unlike previously characterized protonophore-resistant strains of B. subtilis and B. megaterium, neither B. firmus mutant possessed any ability above that of the parent strain to synthesize ATP at given suboptimal values of delta mu H+. Instead, both resistant alkaliphile strains maintained a higher delta mu H+ and a correspondingly higher delta Gp than the parent strain when growing in sublethal concentrations of CCCP, apparently as a result of mutational changes affecting respiratory chain composition. Also of note in both the mutant and the wild-type strains was a marked elevation in the level of one of the multiple terminal oxidases, an aa3-type cytochrome, during growth at pH 7.5 in the presence of CCCP or during growth at pH 10.5, i.e. two conditions that reduce the bulk delta mu H+.

Plant Lipids: Metabolism, Mutants, and Membranes

The mechanisms that regulate plant lipid metabolism determine the dietary and industrial value of storage oils found in economically important species and may control the ability of many plants to survive exposure to temperature extremes. Many of the problems researchers have in defining the pathways, enzymes, and genes involved in plant lipid metabolism appear to be amenable to analysis by genetic approaches. Mutants with alterations in membrane lipid composition have also been used to study the structural and adaptive roles of lipids. The application of genetic engineering methods affords opportunities for researchers to apply knowledge gained about plant lipid metabolism toward enhanced use of plant oils as abundant and renewable sources of reduced carbon.

Effects of lovastatin treatment on red blood cell and platelet cation transport.

Hypercholesterolemia frequently accompanies hypertension, and it has been suggested that by affecting membrane lipid composition, hypercholesterolemia may cause or accentuate abnormalities in several red blood cell transports associated with hypertension. Such an effect might obfuscate the relation of membrane markers to hypertension and decrease their usefulness in genetic studies of the heritable basis of hypertension. To determine if changing plasma lipids affects membrane transport, we studied the effects of the cholesterol-lowering agent lovastatin on red blood cell lithium-sodium countertransport and sodium-potassium-chloride cotransport, red blood cell sodium and water content, and platelet amiloride-sensitive volume responsiveness to cytoplasmic acidification, an indirect measure of sodium-proton exchange that has been proposed as a new membrane marker for hypertension. In a 24-week, placebo-controlled, double-blinded, randomized trial, lovastatin significantly lowered total and low density lipoprotein cholesterol and raised high density lipoprotein cholesterol. Red blood cell lithium-sodium countertransport and sodium-potassium-chloride cotransport were not significantly altered. Red blood cell sodium content decreased significantly in the lovastatin-treated group, probably as a result of an increase in red blood cell sodium-potassium pump activity. Platelet amiloride-sensitive responses to cytoplasmic acidification were significantly depressed by lovastatin treatment, suggesting that lowering plasma cholesterol may suppress platelet sodium-proton exchange. It has been hypothesized that the hyperlipidemias frequently observed in essential hypertensive patients may alter membrane lipid composition and affect membrane cation transport activities.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of membrane fatty acyl composition on LDL metabolism in Hep G2 hepatocytes

The mechanism by which dietary cis-unsaturated fatty acids lower plasma levels of low-density lipoprotein (LDL) cholesterol is unknown. Since plasma membrane incorporation of dietary cis-unsaturated fatty acids is known to alter the function of plasma membrane associated proteins, perhaps by increasing membrane fluidity, we examined LDL receptor function in Hep G2 hepatocytes that were unmodified, enriched with the cis-unsaturated fatty acids oleate or linoleate, or enriched with the saturated fatty acids stearate or palmitate. Hepatocytes enriched in cis-unsaturated fatty acids exhibited augmented LDL binding, uptake, and degradation in comparison to unmodified cells. In contrast, Hep G2 hepatocytes enriched in saturated fatty acids had decreased LDL binding, uptake, and degradation. Enrichment with oleate or linoleate resulted in a decrease in the calculated fatty acyl mole-weighted melting point of the plasma membrane and an increase in plasma membrane fluidity, as measured by the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene incorporated into the plasma membrane. Conversely, stearate or palmitate enrichment resulted in an increased plasma membrane fatty acyl mole-weighted melting point and decreased plasma membrane fluidity. LDL binding, uptake, and degradation varied with plasma membrane fluidity in a highly correlated manner. Thus, one mechanism by which dietary cis-unsaturated fatty acids lower LDL cholesterol may possibly involve an alteration in membrane lipid composition or membrane fluidity that promotes enhanced LDL receptor function, thereby leading to increased hepatic clearance of LDL.

Plasma membrane enrichment with cis-unsaturated fatty acids enhances LDL metabolism in U937 monocytes.

The mechanism by which dietary cis-unsaturated fatty acids lower low density lipoprotein (LDL) cholesterol is unknown. Because cis-unsaturated fatty acids incorporated into cell membranes increase membrane fluidity and, as a result, can alter membrane-dependent cell functions, we examined LDL binding, uptake, and degradation in upregulated U937 monocytes enriched in membrane oleate, a monounsaturated fatty acid, and membrane linoleate, a polyunsaturated fatty acid. The same parameters were also examined in upregulated U937 monocytes enriched in membrane stearate, a saturated fatty acid, and in upregulated, unmodified U937 monocytes. Monocytes enriched in cis-unsaturated fatty acids exhibited augmented LDL binding, internalization, and degradation compared with both stearate-enriched monocytes and unmodified monocytes. The molar potency of linoleate in augmenting LDL metabolism was 50% greater than that of oleate. Enrichment with oleate and linoleate resulted in a decrease in the fatty acyl mole-weighted melting point of the plasma membrane and an increase in plasma membrane fluidity, as indicated by a reduction in the steady-state fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene incorporated into the membrane. Stearate-enriched monocytes exhibited a slight increase in the plasma membrane fatty acyl mole-weighted melting point and essentially no change in plasma membrane fluidity. Thus, one mechanism by which cis-unsaturated fatty acids lower LDL cholesterol may involve alteration in membrane lipid composition and physical properties, thereby leading to an increase in cellular clearance of this atherogenic lipoprotein.

Enhanced Thermal Tolerance in a Mutant of Arabidopsis Deficient in Palmitic Acid Unsaturation

A mutant of Arabidopsis thaliana, deficient in the activity of a chloroplast omega9 fatty acid desaturase, accumulates high amounts of palmitic acid (16:0), and exhibits an overall reduction in the level of unsaturation of chloroplast lipids. Under standard conditions the altered membrane lipid composition had only minor effects on growth rate of the mutant, net photosynthetic CO(2) fixation, photosynthetic electron transport, or chloroplast ultrastructure. Similarly, fluorescence polarization measurements indicated that the fluidity of the membranes was not significantly different in the mutant and the wild type. However, at temperatures above 28 degrees C, the mutant grew more rapidly than the wild type suggesting that the altered fatty acid composition enhanced the thermal tolerance of the mutant. Similarly, the chloroplast membranes of the mutant were more resistant than wild type to thermal inactivation of photosynthetic electron transport. These observations lend support to previous suggestions that chloroplast membrane lipid composition may be an important component of the thermal acclimation response observed in many plant species which are photosynthetically active during periods of seasonally variable temperature extremes.

Lipids are major components of human immunodeficiency virus (HIV): Modification of HIV lipid composition, membrane organization, and protein conformation by AL‐721®

AbstractThe lipid composition of human immunodeficiency virus (HIV) was investigated. Several strains of HIV were found to contain considerable amounts of lipid. The major lipid constituents were cholesterol and phospholipid with a molar ratio close to one. This lipid composition and membrane apparent microviscosity, as determined by diphenylhexatriene fluorescent polarization, indicate that the HIV envelope is an extremely rigid membrane. A previous in vitro study and two recent clinical reports have indicated that AL‐721®, a lipid mixture designed to alter membrane lipid composition and organization by extraction of membrane cholesterol, has anti‐HIV activity. We found that AL‐721® modifies the lipid composition of HIV, reducing the cholesterol content and the cholesterol to phospholipid molar ratio. Both DPH fluorescent probe and endogenous protein fluorescent studies suggest that AL‐721® alters HIV envelope organization and/or apparent microviscosity. Membrane perturbation of HIV by AL‐721® may represent a new approach to AIDS therapy.

1,25-Dihydroxycholecalciferol stimulates renal phosphate transport by directly altering membrane phosphatidylcholine composition

Controversy exists regarding the mechanisms by which 1,25-dihydroxycholecalciferol (1,25(OH 2)D 3) alters membrane lipid composition and ion transport. Recent studies have demonstrated stimulation of the transfer of 1-acyl-2-( N-4-nitrobenz-2-oxa-1,3-diazole)aminocaproylphosphatidylcholine (NBD-PC) by 1,25(OH) 2D 3. In the present studies, brush border membrane vesicle phosphatidylcholine content was increased after incubation with liposomes composed of dioleoylphosphatidylcholine or β-inoleyl, γ-palmitoyl phosphatidylcholine and 1,25(OH) 2D 3 (10 −7 M). Vesicular phosphatidylcholine content was increased from control levels of 49.5 μg/mg protein to 56.9 and 58.5, respectively, after treatment with the liposomes containing 1,25(OH) 2D 3, P < 0.05. When the vesicles were incubated with liposomes composed of β-inoleyl, γ-palmitoyl phosphatidylcholine, phosphate transport was stimulated from 231 ± 20 to 431 ± 41 pmol/ mg protein per 15 s in the presence of 1,25(OH) 2D 3 if the vesicles were derived from vitamin D deficient rats and from 443 ± 33 to 601 ± 42 pmol/ mg protein per 15 s if the vesicles were prepared from normal rats. Despite phosphatidylcholine transfer, incubation with liposomes composed of dioleoylphosphatidylcholine and 1,25(OH) 2D 3 did not stimulate phosphate transport. Furthermore, incubation of vesicles with liposomes and 1,25(OH) 2D 3 did not alter glucose transport.

Butyrate effects on normal and adapted hepatoma cells: Morphological response and implications for vectoral cholesterol transport

The cell line 4IC6, adapted for growth in 6 m m sodium butyrate from Hepatoma Tissue Culture cells [R. Chalkley, and A. Shires (1985) J. Biol. Chem. 260, 7698–7704] , exhibits a fourfold increase in histone acetate turnover. The 4IC6 cells were about 25 times more resistant to butyrate relative to the parental cell line as measured by cloning efficiency. This line also maintains a flatter and more extended morphology when growing in the presence of 6 m m sodium butyrate relative to the parental line. Both cell lines maintain similar intracellular butyrate levels and incorporate [1- 14C]butyrate into lipids to similar extents when incubated in medium containing high levels of the fatty acid. These results show that 4IC6 cells have not attained butyrate resistance through acquiring the ability to metabolize butyrate more efficiently or in a significantly different manner when compared with the parental cell line. The membrane lipid composition was nearly identical between the two cell types. Thus the different morphologies exhibited by each cell line were not a consequence of altered membrane lipid composition. The resistant line, 4IC6, maintains about 10-fold higher cholesterol ester levels and half the level of triglycerides found in the parental line. The butyrate-resistant cells also synthesize cholesterol at about a 1.8-fold higher rate than do the parental cells. This difference in de novo synthesis is reflected by a difference of a similar factor in the amount of radioactive cholesterol the two cell lines accumulate over 12 generations. These results are discussed with respect to models for equilibration of serum lipoprotein-derived and newly synthesized Cholesterol.

Modifications of membrane lipid structure and their influence on cell growth, passive permeability, and enzymatic and transport activities in Acholeplasma laidlawii B

Acholeplasma laidlawii B is a simple procaryotic microorganism, without a cell wall, whose membrane lipid fatty acid composition and cholesterol content can be dramatically modified. It is thus possible to markedly vary the temperature and cooperativity of the gel to lipid-crystalline phase transition, thereby altering the phase state and "fluidity" of the A. laidlawii membrane lipids. By varying the chain length of the exogenous fatty acids supplied, it is also possible to modify the thickness of the membrane lipid bilayer. Acholeplasma laidlawii B cell growth and most membrane functions are strongly dependent on the phase state of the membrane lipids. When gel-state lipid predominates at physiological temperatures, cell growth declines sharply, nonelectrolyte permeability exhibits a pronounced local maximum, net glucose transport is markedly reduced, and ATPase activity declines moderately. Upon complete conversion of the lipid to the gel state, cell growth ceases, passive permeability is markedly reduced, and net glucose transport is abolished, but appreciable ATPase activity remains. Provided that the membrane lipid is predominantly or exclusively in the liquid-crystalline state, cell growth and ATPase activity are almost independent of membrane lipid fatty acid composition and cholesterol content, except that cell growth is inhibited by the presence of "hyperfluid" lipid. In contrast, passive permeability and active glucose uptake are influenced by alterations in membrane lipid composition, with increases in lipid fluidity resulting in higher nonelectrolyte translocation rates. Alterations in the length of the membrane lipid hydrocarbon chains per se also appear to affect A. laidlawii growth and membrane function. Thus lipids containing fatty acids with effective chain lengths of 13 carbons or less do not support cell growth or normal enzymatic and transport activities, and lipids with effective hydrocarbon chain lengths of 19 carbons or more do not support normal cell growth.

Ethynylestradiol impairs bile salt uptake and Na-K pump function of rat hepatocytes.

Ethynylestradiol impairs bile flow and bile salt maximum secretory rate in rats, implying a secretory defect. In addition, Na-K-ATPase activity is decreased in liver surface membranes, suggesting abnormalities at the sinusoidal as well as the canalicular membrane. We investigated whether ethynylestradiol pretreatment affects bile salt uptake and Na-K pump function in isolated rat hepatocytes. Ethynylestradiol-treated cells were functionally intact as assayed with trypan blue exclusion, lactate dehydrogenase release, and oxygen consumption. Initial taurocholate uptake velocity was reduced by 73% in ethynylestradiol-treated hepatocytes [Vmax, 1.0 +/- 0.1 vs. 3.7 +/- 0.2 mumol X min-1 X (10(6) cells)-1; P less than 0.001; Km, 34 +/- 5 vs. 33 +/- 3 microM]. Na-K-ATPase activity in cell homogenates (36 +/- 5 vs. 27 +/- 4 mumol Pi X h-1 X mg prot-1; P less than 0.05), ouabain-suppressible rubidium-86 influx [6.8 +/- 1.1 vs. 4.8 +/- 1.0 nmol K+ X min-1 X (10(6) cells)-1; P less than 0.05], and intracellular potassium concentration (126 +/- 10 vs. 110 +/- 16 mmol/l; P less than 0.05) were reduced after ethynylestradiol. Taurocholate uptake measured at different temperatures between 25 degrees and 37 degrees was linear when plotted according to Arrhenius. The energy of activation was increased by 40% in ethynylestradiol-treated hepatocytes [17 +/- 4 vs. 23 +/- 4 kcal X mol-1 X (10(6) cells)-1; P less than 0.05], consistent with decreased membrane fluidity. These data suggest the possibility that during ethynylestradiol-induced cholestasis a disorder of the sinusoidal domain, caused perhaps by ethynylestradiol-induced alterations in membrane lipid composition, is an important contributing factor.

Action of bacterial cytotoxins on normal mammalian cells and cells with altered membrane lipid composition

Cytotoxic proteins produced by a number of bacteria, as well as one from a marine invertebrate, were tested for their ability to disrupt the permeability barrier of mammalian cells. Agents were tested individually and in combinations shown to have synergistic disruptive actions on erythrocytes. Toxins included the lipid-hydrolyzing enzymes sphingomyelinases C and D and cholesterol oxidase, as well as the non-enzymatic agents, helianthus toxin, streptolysin O and saponin. Cells treated included cultured human skin fibroblasts, normal human erythrocytes and erythrocytes enhanced and depleted in membrane cholesterol. Fibroblasts were disrupted by helianthus toxin and by the combination of sphingomyelinase C and cholesterol oxidase. Thin layer chromatographic analysis of the treated cells confirmed the enzymatic alteration of membrane lipids by the lipid hydrolases. Human erythrocytes having an increased content of membrane cholesterol were more sensitive than normal cells to agents which interact with membrane sterol. Conversely, cholesterol-depleted cells were more resistant to these as well as other agents. Results are discussed in relation to biochemical mechanisms of action of the agents tested, and to their possible significance in bacterial pathogenesis.