Cholesterol modulates alkaline phosphatase activity of rat intestinal microvillus membranes

Experiments were conducted, using a nonspecific lipid transfer protein, to vary the cholesterol/phospholipid molar ratio of rat proximal small intestinal microvillus membranes in order to assess the possible role of cholesterol in modulating enzymatic activities of this plasma membrane. Cholesterol/phospholipid molar ratios from 0.71 to 1.30 were produced from a normal value of 1.05 by incubation with the transfer protein and an excess of either phosphatidylcholine or cholesterol/phosphatidylcholine liposomes for 60 min at 37 degrees C. Cholesterol loading or depletion of the membranes was accompanied by a decrease or increase, respectively, in their lipid fluidity, as assessed by steady-state fluorescence polarization techniques using the lipid-soluble fluorophore 1,6-diphenyl-1,3,5-hexatriene. Increasing the cholesterol/phospholipid molar ratio also decreased alkaline phosphatase specific activity by approximately 20-30%, whereas decreasing this ratio increased this enzymatic activity by 20-30%. Sucrase, maltase, and lactase specific activities were not affected in these same preparations. Since the changes in alkaline phosphatase activity could be secondary to alterations in fluidity, cholesterol, or both, additional experiments were performed using benzyl alcohol, a known fluidizer. Benzyl alcohol (25 mM) restored the fluidity of cholesterol-enriched preparations to control levels, did not change the cholesterol/phospholipid molar ratio, and failed to alter alkaline phosphatase activity. These findings, therefore, indicate that alterations in the cholesterol content and cholesterol/phospholipid molar ratio of microvillus membranes can modulate alkaline phosphatase but not sucrase, maltase, or lactase activities. Moreover, membrane fluidity does not appear to be an important physiological regulator of these enzymatic activities.     

Kinetics of cholesterol and phospholipid exchange between Mycoplasma gallisepticum cells and lipid vesicles. Alterations in membrane cholesterol and protein content

The kinetics of exchange of radiolabeled cholesterol and phospholipids between intact Mycoplasma gallisepticum cells and unilamellar lipid vesicles were investigated over a wide range of cholesterol/phospholipid molar ratio. The change in cholesterol/phospholipid molar ratio was achieved by adapting the sterol-requiring M. gallisepticum to grow in cholesterol-poor media, providing cells with decreased unesterified cholesterol content. At least 90% of the cholesterol molecules in unsealed M. gallisepticum membranes underwent exchange at 37 degrees C as a single kinetic pool in the presence of albumin (2%, w/v). However, we observed biphasic exchange kinetics with intact cells, indicating that cholesterol translocation from the inner to outer monolayers was rate-limiting in the exchange process. Approximately 50% of the cholesterol molecules were localized in each kinetic pool, independent of the cholesterol/phospholipid molar ratio in the cells and vesicles. A striking change in the kinetic parameters for cholesterol exchange occurred between 20 and 26 mol % cholesterol; for example, when the cholesterol/phospholipid molar ratio was decreased from 0.36 to 0.25, the half-time for equilibration of the two cholesterol pools at 37 degrees C decreased from 4.6 +/- 0.5 to 2.5 +/- 0.1 h. Phospholipid exchange rates were also enhanced on decreasing the membrane cholesterol content. The ability of cholesterol to modulate its own exchange rate, as well as that of phospholipids, is suggested to arise from the sterol's ability to regulate membrane lipid order. Extensive chemical modification of the membrane surface by cross-linking of some of the protein constituents with 1,4-phenylenedimaleimide decreased the cholesterol exchange rate. Depletion of membrane proteins by treatment of growing cultures with chloramphenicol increased the cholesterol exchange rate, possibly because of removal of some of the protein mass that may impede lipid translocation. The observations that phospholipid exchange was one order of magnitude slower than cholesterol exchange and that dimethyl sulfoxide, potassium thiocyanate, and potassium salicylate enhanced the cholesterol exchange rate are consistent with a mechanism involving lipid exchange by diffusion through the aqueous phase.     

The interaction of dietary fatty acid and cholesterol on catecholamine-stimulated adenylate cyclase activity in the rat heart

Diets supplemented with high levels of saturated or unsaturated fatty acids supplied by addition of sheep kidney fat or sunflower seed oil, respectively, were fed to rats with or without dietary cholesterol. The effects of these diets on cardiac membrane lipid composition, catecholamine-stimulated adenylate cyclase and beta-adrenergic receptor activity associated with cardiac membranes, were determined. The fatty acid-supplemented diets, either with or without cholesterol, resulted in alterations in the proportion of the (n-6) to (n-3) series of unsaturated fatty acids, with the sunflower seed oil increasing and the sheep kidney fat decreasing this ratio, but did not by themselves significantly alter the ratio of saturated to unsaturated fatty acids. However, cholesterol supplementation resulted in a decrease in the proportion of saturated and polyunsaturated fatty acids and a dramatic increase in oleic acid in cardiac membrane phospholipids irrespective of the nature of the dietary fatty acid supplement. The cholesterol/phospholipid ratio of cardiac membrane lipids was also markedly increased with dietary cholesterol supplementation. Although relatively unaffected by the nature of the dietary fatty acid supplement, catecholamine-stimulated adenylate cyclase activity was significantly increased with dietary cholesterol supplementation and was positively correlated with the value of the membrane cholesterol/phospholipid ratio. Although the dissociation constant for the beta-adrenergic receptor, determined by [125I](-)-iodocyanopindolol binding, was unaffected by the nature of the dietary lipid supplement, the number of beta-adrenergic receptors was dramatically reduced by dietary cholesterol and negatively correlated with the value of the membrane cholesterol/phospholipid ratio. These results indicate that the activity of the membrane-associated beta-adrenergic/adenylate cyclase system of the heart can be influenced by dietary lipids particularly those altering the membrane cholesterol/phospholipid ratio and presumably membrane physico-chemical properties. In the face of these dietary-induced changes, a degree of homeostasis was apparent both with regard to membrane fatty acid composition in response to an altered membrane cholesterol/phospholipid ratio, and to down regulation of the beta-adrenergic receptor in response to enhanced catecholamine-stimulated adenylate cyclase activity.     

Membrane Viscosity Correlates with α1-Adrenergic Signal Transduction of the Aged Rat Cerebral Cortex

We investigated, using adult (2-month-old) and senescent (12- and 24-month-old) rats, the effects of aging on the relationship between the alpha 1-adrenergic coupling system and the membrane viscosity of the cerebral cortex. There was no age-related difference in the KD values of [3H]prazosin binding on the membranes. The Bmax values of [3H]prazosin binding were reduced with advanced age. Norepinephrine-induced formation of 3H-labeled inositol phosphates (3H-IPs) in the slices increased with advanced age. The EC50 values for norepinephrine to stimulate the formation of 3H-IPs at advanced age were lower than that at adult age. The cholesterol content in membranes increased with advanced age. No changes in the phospholipid content in membranes were observed with advanced age. Concomitantly, an increase of the molar ratio of cholesterol to phospholipids was observed with advanced age. The membrane viscosity as measured by 1,6-diphenyl-1,3,5-hexatriene increased with advanced age. These results indicate that the altered cholesterol content and/or viscosity in cortical membranes of the aged rat may account for the loss of alpha 1-adrenergic receptor density and/or compensatory changes in the receptor-phospholipase C coupling system.     

Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decrease membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC) ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipoproteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80-85% of abetalipoproteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity (eta) between 5 and 42 degrees C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively at 10 degrees C; 4.63 vs 4.04 poise at 37 degrees C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Modulation of sarcoplasmic reticulum Ca2+-pump activity by membrane fluidity

Intramolecular excimerization of 1,3-di-1-pyrenylpropane [Py(3)Py] was used to assess the fluidity of sarcoplasmic reticulum membranes (SR); on the basis of the spectral data, the probe incorporates completely inside the membrane probably somewhere close to the polar head groups of phospholipid molecules, however not in the very hydrophobic core. The excimerization rate is very sensitive to lipid phase transitions, as revealed by thermal profiles of dimyristoyl-phosphatidylcholine (DMPC) and dipalmitoylphosphatidylcholine (DPPC) bilayers. Cholesterol abolishes pretransitions and broadens the thermal profiles of the main transitions which vanish completely at 50 mol % sterol. Excimer formation in liposomes of SR total lipid extracts does not show any sharp transitions, as in the case of DMPC and DPPC. However, the plots display discontinuities at about 20 degrees C which are broadened by cholesterol and not observed at 50 mol % sterol. Also cholesterol has been incorporated in native SR membranes by an exchange technique allowing progressive enrichment without changing the phospholipid/protein molar ratio. As in liposomes, discontinuities of excimer formation at 20 degrees C are broadened by cholesterol enrichment. The full activity of uncoupled Ca2+-ATPase is only affected by cholesterol above a molar ratio to phospholipid of 0.4. However, a significant decrease in activity (about 20%) is only noticed at a ratio of 0.6 (the highest technically achieved); at this ratio, about 28 lipid molecules per Ca2+-ATPase are expected to be relatively free from cholesterol interaction. The vesicle structure is still intact at this high ratio, as judged from the absence of basal activity (not Ca2+ stimulated).(ABSTRACT TRUNCATED AT 250 WORDS)     

Synaptic plasma membrane-bound acetylcholinesterase activity is not affected by docosahexaenoic acid-induced decrease in membrane order

We investigated the effect of administration of docosahexaenoic acid (C22:6, n-3; 300 mg/kg.day, for 12 weeks) on the degree of membrane order and membrane-bound acetylcholinesterase activity of the cerebral cortex synaptic plasma membrane in male Wistar rats. Docosahexaenoic acid levels in the synaptic plasma membrane increased significantly by 16% over levels in control rats concomitant with an increase in the molar ratio of docosahexaenoic acid to arachidonic acid. Synaptic plasma membrane order, assessed by 1,6-diphenyl-1,3,5-hexatriene, which measures order of the bulk internal hydrophobic lipid core, decreased significantly in the docosahexaenoic acid-fed rats. Lateral mobility of both global and annular lipids measured by pyrene also increased. Acetylcholinesterase activity of the synaptic plasma membrane was unaffected, and synaptic plasma membrane phospholipid contents increased in the docosahexaenoic acid-fed rats, with a concomitant decrease in the cholesterol/phospholipid molar ratio. Lipid peroxide and reactive oxygen species, indicators of tissue oxidative stress, decreased in both the cerebral cortex synaptosome and homogenate of the docosahexaenoic acid-fed rats. Arrhenius plot showed a break point in acetylcholinesterase activity at 22 degrees C and 24 degrees C in plasma membranes from docosahexaenoic acid-fed and control rats, respectively. The present experiment indicates that chronic administration of docosahexaenoic acid does not affect synaptic acetylcholinesterase activity and evoke oxidative stress, although it increases the disorder of the global and annular lipids of rat synaptic plasma membranes.     

Purification and analysis of phospholipids in the inner mitochondrial membrane fraction of bovine corpus luteum, and properties of cytochrome P-450scc incorporated into vesicles prepared from these phospholipids

Cytochrome P-450scc, which catalyses the conversion of cholesterol to pregnenolone in steroidogenic tissues, can be incorporated into artificial phospholipid vesicles and cholesterol binding to the cytochrome is affected by the composition of the vesicles. We have purified the phospholipids from the inner mitochondrial membrane fraction of the bovine corpus luteum where the cytochrome is located. The composition in mol % was 49% phosphatidylcholine, 34% phosphatidylethanolamine, 8.7% cardiolipin, 6.4% lysophosphatidylethanolamine and 1.5% phosphatidylinositol. The ratio of cholesterol to phospholipid (mol/mol) in the inner membrane fraction was 0.14 to 1. The Km for cholesterol of purified luteal cytochrome P-450scc incorporated into vesicles prepared from the total inner mitochondrial membrane phospholipids was 0.063 mol of cholesterol per mol of phospholipid. Removal of the cardiolipin component of the inner mitochondrial membrane phospholipids prior to preparation of vesicles caused a four fold increase in the Kd of cytochrome P-450 for cholesterol and a two fold increase in Km. The data suggests that in the inner mitochondrial membrane of the bovine corpus luteum the cholesterol concentration is less than saturating for cytochrome P-450scc.     

Ethinylestradiol administration selectively alters liver sinusoidal membrane lipid fluidity and protein composition

Administration of high-dose ethinylestradiol to rats decreases bile flow, Na,K-ATPase specific activity, and liver plasma membrane fluidity. By use of highly purified sinusoidal and bile canalicular membrane fractions, the effect of ethinylestradiol administration on the protein and lipid composition and fluidity of plasma membrane fractions was examined. In sinusoidal fractions, ethinylestradiol (EE) administration decreased Na,K-ATPase activity (32%) and increased activities of alkaline phosphatase (254%), Mg2+-ATPase (155%), and a 160-kDa polypeptide (10-fold). Steady-state and dynamic fluorescence polarization was used to study membrane lipid structure. Steady-state polarization of diphenylhexatriene (DPH) was significantly higher in canalicular compared to sinusoidal membrane fractions. Ethinylestradiol (5 mg/kg per day for 5 days) selectively increased sinusoidal polarization values. Similar changes were demonstrated with the probes 2- and 12-anthroyloxystearate. Time-resolved fluorescence polarization measurements indicated that EE administration for 5 days did not change DPH lifetime but increased the order component (r infinity) and decreased the rotation rate (R). However, 1 and 3 days after EE administration and with low doses (10-100 micrograms/kg per day for 5 days) the Na,K-ATPase, bile flow, and order component were altered, but the rotation rate was unchanged. Vesicles prepared from total sinusoidal membrane lipids of EE-treated rats, as well as phospholipid vesicles, demonstrated increased DPH polarization, as did intact plasma membrane fractions. Liver plasma membrane fractions showed no change in free cholesterol or cholesterol/phospholipid molar ratio, while esterified cholesterol content was increased with high-dose but not low-dose ethinylestradiol.(ABSTRACT TRUNCATED AT 250 WORDS)     

Importance of cholesterol-phospholipid interaction in determining dynamics of normal and abetalipoproteinemia red blood cell membrane

Acanthocytic red blood cells in patients with abetalipoproteinemia have a decreased membrane fluidity that is associated with increased sphingomyelin/phosphatidylcholine (SM/PC)§ ratios. Here we describe studies designed to gain better insight into (i) the interrelationship between the composition of lipoprotein and red blood cell membrane in abetalipo-proteinemia patients and normal controls; and (ii) how the differences in lipid composition of the red blood cell membrane affect its fluidity. The increased SM/PC ratio found in abetalipoproteinemia plasma high density lipoproteins (HDL) (3 times greater than controls) was paralleled by an increase in this ratio in acanthocytic red cells, but to a lesser degree (almost twice greater than control red cells). Cholesterol/phospholipid mole ratios (C/P) were increased 3-fold in abetalipoproteinemia HDL, but only slightly increased in red cells compared to controls values. As in the controls, 80–85% of abetalipo-proteinemia red cell sphingomyelin was found to be in the outer half of the erythrocyte membrane. Membrane fluidity was defined in terms of microviscosity ({ie116-1}) between 5 and 42°C by the fluorescent polarization of 1,6-diphenylhexatriene (DPH) present in erythrocyte ghost membranes. At all temperatures, membrane microviscosity was higher in abetalipoproteinemia ghosts than controls, but these differences decreased at higher temperatures (12.34 vs 9.79 poise, respectively, at 10°C; 4.63 vs 4.04 poise at 37°C). These differences were eliminated after oxidation of all membrane cholesterol to cholest-4-en-3-one by incubation with cholesterol oxidase. Following cholesterol oxidation, the membrane microviscosity decreased in patient ghosts more than in normal red blood cells so that at all temperatures no significant differences were present relative to control ghosts, in which the apparent microviscosity was also diminished but to a lesser degree. Therefore, although increased SM/PC ratios in abetalipoproteinemia may be responsible for decreased erythrocyte membrane fluidity, these effects are dependent upon normal interactions of cholesterol with red cell phospholipid.     

Sterol carrier protein-2 expression alters plasma membrane lipid distribution and cholesterol dynamics

Although sterol carrier protein-2 (SCP-2) binds, transfers, and/or enhances the metabolism of many membrane lipid species (fatty acids, cholesterol, phospholipids), it is not known if SCP-2 expression actually alters the membrane distribution of lipids in living cells or tissues. As shown herein for the first time, expression of SCP-2 in transfected L-cell fibroblasts reduced the plasma membrane levels of lipid species known to traffic through the HDL-receptor-mediated efflux pathway: cholesterol, cholesteryl esters, and phospholipids. While the ratio of cholesterol/phospholipid in plasma membranes of intact cells was not changed by SCP-2 expression, phosphatidylinositol, a molecule important to intracellular signaling and vesicular trafficking, and anionic phospholipids were selectively retained. Only modest alterations in plasma membrane phospholipid percent fatty acid composition but no overall change in the proportion of saturated, unsaturated, monounsaturated, or polyunsaturated fatty acids were observed. The reduced plasma membrane content of cholesterol was not due to SCP-2 inhibition of sterol transfer from the lysosomes to the plasma membranes. SCP-2 dramatically enhanced sterol transfer from isolated lysosomal membranes to plasma membranes by eliciting detectable sterol transfer within 30 s, decreasing the t(1/2) for sterol transfer 364-fold from >4 days to 7-15 min, and inducing formation of rapidly transferable sterol domains. In summary, data obtained with intact transfected cells and in vitro sterol transfer assays showed that SCP-2 expression (i) selectively modulated plasma membrane lipid composition and (ii) decreased the plasma membrane content cholesterol, an effect potentially due to more rapid SCP-2-mediated cholesterol transfer from versus to the plasma membrane.     

Plasma membrane cholesterol content affects nitric oxide diffusion dynamics and signaling

Nitric oxide (NO) signaling is inextricably linked to both its physical and chemical properties. Due to its preferentially hydrophobic solubility, NO molecules tend to partition from the aqueous milieu into biological membranes. We hypothesized that plasma membrane ordering provided by cholesterol further couples the physics of NO diffusion with cellular signaling. Fluorescence lifetime quenching studies with pyrene liposome preparations showed that the presence of cholesterol decreased apparent diffusion coefficients of NO approximately 20-40%, depending on the phospholipid composition. Electrochemical measurements indicated that the diffusion rate of NO across artificial bilayer membranes were inversely related to cholesterol content. Sterol transport-defective Niemann-Pick type C1 (NPC1) fibroblasts exhibited increased plasma membrane cholesterol content but decreased activation of both intracellular soluble guanylyl cyclase and vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser(239) induced by exogenous NO exposure relative to their normal human fibroblast (NHF) counterparts. Augmentation of plasma membrane cholesterol in NHF diminished production of both cGMP and VASP phosphorylation elicited by NO to NPC1-comparable levels. Conversely, decreasing membrane cholesterol in NPC1 resulted in the augmentation in both cGMP and VASP phosphorylation to a level similar to those observed in NHF. Increasing plasma membrane cholesterol contents in NHF, platelets, erythrocytes and tumor cells also resulted in an increased level of extracellular diaminofluorescein nitrosation following NO exposure. These findings suggest that the impact of cholesterol on membrane fluidity and microdomain structure contributes to the spatial heterogeneity of NO diffusion and signaling.     

Effect of lipidic factors on membrane cholesterol topology--mode of binding of theta-toxin to cholesterol in liposomes.

We have previously suggested the existence of two distinct states for cholesterol in cell membranes as revealed by high- and low-affinity binding sites for theta-toxin of Clostridium perfringens. In liposomes, phospholipid and cholesterol compositions, but not membrane protein composition, have been shown to be major determinants for the topology of membrane cholesterol. The effects of lipidic factors on cholesterol topology were investigated in detail by analyzing toxin binding to large unilamellar liposomes composed of cholesterol and phospholipids (neutral phospholipids/phosphatidylglycerol = 82:18, mol/mol). The numbers of high- and low-affinity toxin-binding sites depend strictly on the cholesterol mole percentage in liposomes. High-affinity toxin-binding sites appear only in liposomes with high cholesterol contents. Liposomes whose cholesterol/phospholipid ratio is 0.4 or less have no high-affinity sites regardless of their phospholipid compositions, while low-affinity sites appear in liposomes with lower cholesterol contents. The threshold values for the cholesterol mole percentage above which high-affinity toxin-binding sites appear were examined. The values decrease in accordance with the increase in the mole fraction of 18-carbon hydrocarbon chains among the total 14-18 carbon-hydrocarbon chains of the liposomal phospholipids. Furthermore, both the partial replacement of phosphatidylcholine with phosphatidylethanolamine and the digestion of phospholipids with phospholipase C also affect the threshold values. Thus the cholesterol mole percentage, in combination with phospholipid chain length and other factors, determines the topology of membrane cholesterol providing distinctively different affinity sites for theta-toxin.     

The influence of membrane components on regulation of alternative pathway activation by decay-accelerating factor.

Decay-accelerating factor (DAF) is a C regulatory protein which functions in membranes to inhibit autologous C activation on cell surfaces. A liposome model was used to study the mechanism of DAF action and examine the effects of membrane-bound glycophorin and LPS on the regulatory activity of DAF. Liposomes were incubated in MgEGTA-treated human serum and activation of the alternative pathway measured by C3b binding. Liposomes composed of phosphatidylcholine, phosphatidylethanolamine, and cholesterol activated the alternative pathway in proportion to their content of PE. Incorporation of 10(-7) mol/mol phospholipid of either human E or HeLa cell-derived DAF inhibited C activation by liposomes containing 40% phosphatidylethanolamine by 50%, an efficiency comparable to that observed in intact E. HeLa DAF that had been treated with phosphatidylinositol-specific phospholipase C to remove its glycolipid anchor had no effect on C activation by liposomes at concentrations as high as 10(-5) mol/mol phospholipid. Incorporation of DAF into liposomes prepared with bound C3b inhibited the deposition of additional C3b by C3bBbP. However, the incorporated DAF increased the amount of Bb generated from B in the presence of D indicating that accelerated decay of the convertase was the primary effect of DAF. Similarly, treatment of intact human E with anti-DAF decreased the amount of Bb generated by the alternative pathway convertase. To study the effects of other membrane components on DAF activity, liposomes were prepared with purified human glycophorin A or LPS. In glycophorin liposomes the presence of PE was required to activate the alternative pathway and DAF inhibited this activation. In contrast, LPS liposomes bound C3b independently of PE and the incorporation of DAF had no effect. These results demonstrate that within a membrane, DAF's inhibitory activity on the alternative pathway C3 convertase is mediated independently of other membrane proteins, that in this model the major activity of DAF is to accelerate convertase decay, and that the presence of other membrane molecules that may serve as C3 acceptors can circumvent DAF function.     

Adaptive changes in lipid composition of rat liver plasma membrane during postnatal development following maternal ethanol ingestion

The fatty acid composition of constituent phospholipids and the cholesterol content of rat liver plasma membranes were determined subsequent to maternal alcohol ingestion during pregnancy and lactation. The alcoholic group was given a liquid Metrecal diet containing 37% ethanol-derived calories. The control group was pair-fed an isocaloric sucrose/Metrecal diet. Litters were killed for lipid analyses at days 5, 15 and 25 after birth. These studies revealed that the total phospholipid phosphorus was similar and increased significantly with age in both groups. Cholesterol also increased significantly with age in both groups but was greater in the alcoholic pups, resulting in a higher cholesterol/phospholipid molar ratio. While the phosphatidylethanolamine (PE) content increased with age in both groups, that of sphingomyelin decreased. Phosphatidylserine + phosphatidylinositol (PS + PI) was significantly higher in the control group at all ages studied. A consistent increase of C22:6 in phosphatidylcholine (PC), sphingomyelin, PS + PI and in the total phospholipid fraction from alcoholic pups was observed. Although other fatty acid changes were found in PC, PS + PI and sphingomyelin, PE was not affected. These results suggest that specific adaptive changes were induced in the liver plasma membrane lipids of the progeny from alcoholic rats.     

Cholesterol cell content modulates GTPase activity of G proteins in GH4C1 cell membranes

Previous results from our laboratory showed that GH(4)C(1) cells with low-cholesterol cell content had increased adenylyl cyclase (AC) activity with a parallel increase in G protein alpha subunits associated to the plasma membrane. This effect was directly related to mevalonate availability. In the present report, we characterized the high-affinity GTPase activity present in GH(4)C(1) cell membranes and studied its regulation by cholesterol cell content. The high-affinity GTPase activity, measured as the [gamma32P]GTP hydrolysis rate, was both time-dependent and protein concentration-dependent. Cultured cells with lipoprotein-deficient serum (LPDS) showed decreased cholesterol cell content and decreased GTPase activity. The kinetic analysis, as interpreted by Lineweaver-Burk plots, indicated that low-cholesterol cell content had no effect on the apparent affinity for GTP, but resulted in a 47% decrease in the maximal velocity of the reaction. Addition of 25-hydroxycholesterol (25-HC), an inhibitor of the expression of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and synthetase to cells in LPDS, further decreased GTPase activity in a dose-dependent manner. This effect was reverted by exogenous cholesterol, but not by mevalonate. Studies with bacterial toxins revealed that neither cholera toxin (CTX) nor pertussis toxins (PTX) were able to revert the inhibition produced by low-cholesterol cell content. These results allowed us to postulate that cholesterol modulates GTPase activity in both Gs and Gi protein families. To analyse further the mechanism of modulation of GTPase activity by cholesterol cell content, [35S]GTPgammaS binding in membranes of GH(4)C(1) cells was studied. Changes in cholesterol cell content did not have any effect on GTP binding. Data demonstrated that high-affinity GTPase activity in plasma membrane of GH(4)C(1) cells is direct stimulated by cholesterol cell content and not by mevalonate availability. This example provides a mechanism by which cholesterol cell content can modulate signal transduction mediating by G proteins.     

Cholesterol superlattice modulates the activity of cholesterol oxidase in lipid membranes

Here, the interplay between membrane cholesterol lateral organization and the activity of membrane surface-acting enzymes was addressed using soil bacteria cholesterol oxidase (COD) as a model. Specifically, the effect of the membrane cholesterol mole fraction on the initial rate of cholesterol oxidation catalyzed by COD was investigated at 37 degrees C using cholesterol/1-palmitoyl-2-oleoyl-l-alpha-phosphatidylcholine (POPC) large unilamellar vesicles (LUVs, approximately 800 nm in diameter). In the three concentration ranges examined (18.8-21.2, 23.6-26.3, and 32.2-34.5 mol % cholesterol), the initial activity of COD changed with cholesterol mole fraction in a biphasic manner, exhibiting a local maximum at 19.7, 25.0, and 33.4 mol %. Within the experimental errors, these mole fractions agree with the critical cholesterol mole fractions (C(r)) (20.0, 25.0, and 33.3) theoretically predicted for maximal superlattice formation. The activity variation with cholesterol content was correlated well with the area of regular distribution (A(reg)) in the plane of the membrane as determined by nystatin fluorescence. A similar biphasic change in COD activity was detected at the critical sterol mole fraction 20 mol % in dehydroergosterol (DHE)/POPC LUVs (approximately 168 nm in diameter). These results indicate that the activity of COD is regulated by the extent of sterol superlattice for both sterols (DHE and cholesterol) and for a wide range of vesicle sizes (approximately 168-800 nm). The present work on COD and the previous study on phospholipase A(2) (sPLA(2)) [Liu and Chong (1999) Biochemistry 38, 3867-3873] suggest that the activities of some surface-acting enzymes may be regulated by the extent of sterol superlattice in the membrane in a substrate-dependent manner. When the substrate is a sterol, as it is with COD, the enzyme activity reaches a local maximum at C(r). When phospholipid is the substrate, the minimum activity is at C(r), as is the case with sPLA(2). Both phenomena are in accordance with the sterol superlattice model and manifest the functional importance of membrane cholesterol content.     

Isolation and characterization of plasma membranes from Friend erythroleukaemic cells. A study with sphingomyelinase C

Plasma membranes have been prepared from Friend erythroleukaemic cells using a Dounce homogenization technique followed by differential and sucrose gradient centrifugations. (I) A plasma membrane fraction was obtained which showed a 20- to 30-fold enrichment in 5'-nucleotidase, alkaline phosphodiesterase I, alkaline phosphatase and in 32P-labeled (poly)phosphoinositides. About 1% of the total protein, 6-7% of phospholipid, 8-9% of cholesterol and 12-15% of each of the above markers were recovered in the plasma membrane fraction with an average yield of 15-20%. The plasma membrane was characterized by a high cholesterol to phospholipid molar ratio (0.626), a 2-fold enrichment in sphingomyelin and in phosphatidylserine as compared to the whole cell and by the complete absence of diphosphatidylglycerol. (2) When compared to the phospholipid composition of the mature mouse erythrocyte membrane, the plasma membrane of the Friend cell only differs by a higher phosphatidylcholine and a lower phosphatidylethanolamine content, whereas the levels of sphingomyelin and phosphatidylinositol plus phosphatidylserine are similar. (3) Friend cells were treated with sphingomyelinase C (S. aureus) under non-lytic conditions and subsequently submitted to subcellular fractionation. The results showed that the plasma membrane accounted for 38.5% of the total phospholipid, 64.1% of the total cholesterol and about 4.4% of the total protein content of Friend cells. (4) Sphingomyelin appeared to be asymmetrically distributed in the plasma membrane of Friend cells, with about 85% of this phospholipid being present in the outer monolayer.     

Effect of free cholesterol on incorporation of triolein in phospholipid bilayers

Triacylglycerols are the major substrates for lipolytic enzymes that act at the surface of emulsion-like particles such as triglyceride-rich lipoproteins, chylomicrons, and intracellular lipid droplets. This study examines the effect of cholesterol on the solubility of a triacylglycerol, triolein, in phospholipid surfaces. Solubilities of [carbonyl-13C]triolein in phospholipid bilayer vesicles containing between 0 and 50 mol % free cholesterol, prepared by cosonication, were measured by 13C NMR. The carbonyl resonances from bilayer-incorporated triglyceride were shifted downfield in the 13C NMR spectra from those corresponding to excess, nonincorporated material. This enabled solubilities to be determined directly from carbonyl peak intensities at most cholesterol concentrations. The bilayer solubility of triolein was inversely proportional to the cholesterol/phospholipid mole ratio. In pure phospholipid vesicles the triolein solubility was 2.2 mol %. The triglyceride incorporation decreased to 1.1 mol % at a cholesterol/phospholipid mole ratio of 0.5, and at a mole ratio of 1.0 for the bilayer lipids, the triolein solubility was reduced to just 0.15 mol %. The effects of free cholesterol were more pronounced and progressive than observed previously on the bilayer solubility of cholesteryl oleate (Spooner, P. J. R., Hamilton, J. A., Gantz, D. L., & Small, D. M. (1986) Biochim. Biophys. Acta 860, 345-353]. As with cholesteryl oleate, we suggest that cholesterol also displaces solubilized triglyceride to deeper regions of the bilayer.