Plasma membrane changes associated with rat liver regeneration

The lipid composition and fluidity of plasma membranes have been studied at different stages of liver regeneration (4, 15 and 24 h after surgery). The phospholipid and fatty acid composition is not modified, whereas the cholesterol/phospholipid ratio is lower with respect to control membranes. The modification of the physical properties of the membranes has been studied directly by EPR analysis and indirectly by temperature dependence and cooperativity of some membrane-bound enzymes (Mg2+-ATPase, (Na+ + K+)-ATPase and 5'nucleotidase). Surgical operation or anaesthesia alone causes an early increase in fluidity; such an effect appears to be markedly reduced at a later stage. There seems to be a marked effect of regeneration on plasma membrane fluidity 15 h after partial hepatectomy when several parameters--surface fluidity, cholesterol/phospholipid ratio, and 5'-nucleotidase activity in the presence of concanavalin A -- are modified and indicate an increase in membrane fluidity. It is suggested that this modification of membrane properties could be related to the proliferative process.     

Effect of cholesterol on the physical state and function of lymphocyte membranes

Effect of gradual increase of cholesterol content in T-lymphocyte membranes on the structure and physical state of plasmic membrane lipids and activities of the membrane-bound enzymes was investigated. The increase in cholesterol content was shown to result in a two-phase change of luminescence parameters of the fluorescent probes dimethylaminochalcone and pyrene, which indicates heterogeneity of cholesterol in the membranes. With the growth of steroid content in the cell membranes, at first, we observed a sharp decrease in the lipid bilayer fluidity and inhibition of Na+, K+-ATPase activity, which at the molar ratio cholesterol/phospholipids 0.6 in thymocyte membranes, remains at the same level. With higher cholesterol concentrations ATPase activity did not change. The effect of cholesterol on ATPase activity was in a good agreement with the effect of membrane lipids on fluidity. It is suggested that two pools of cholesterol molecules exist in the membranes, differing in their effects of bilayer fluidity and functional activity of the membranes.     

Alterations in the activities of hepatic plasma-membrane and microsomal enzymes during liver regeneration.

A marked increase in the activities of rat liver plasma-membrane (Na+ + K+)-stimulated ATPase and microsomal Ca2+-stimulated ATPase was observed 18h after partial hepatectomy. Lipid analyses for both membrane preparations reveal that in partially hepatectomized rats the cholesterol and sphingomyelin content are decreased with a subsequent decrease in the cholesterol/phospholipid molar ratio compared with those of sham-operated animals. Changes in the allosteric properties of plasma-membrane (Na+ + K+)-stimulated ATPase by F- (as reflected by changes in the Hill coefficient) indicated a fluidization of the lipid bilayer of both membrane preparations in 18 h-regenerating liver. The amphipathic dodecyl glucoside incorporated into the hepatic plasma membranes evoked a marked increase in the (Na+ + K+)-stimulated ATPase and 5'-nucleotidase activities. The lack of effect of the glucoside on the Lubrol-PX-solubilized 5'-nucleotidase indicates that changes in the activities of the membrane-bound enzymes caused by the glucoside are due to modulation of the membrane fluidity. Dodecyl glucoside appears to increase the membrane fluidity, evaluated through changes in the Hill coefficient for plasma-membrane (Na+ + K+)-stimulated ATPase. The biological significance of these data is discussed in terms of the differences and changes in the interaction of membrane-bound enzymes with membrane lipids during liver regeneration.     

Lipid peroxidation-induced changes in physical properties of annular lipids in rat brain synaptosomal membranes

The effects of lipid peroxidation (LPO) on the physical state (fluidity) of the rat brain synaptosomal lipid bilayer matrix and the annular lipid domains were investigated using the fluorescent probe pyrene. The parameters of pyrene fluorescence intensity alpha = IE/IM were measured at excitation wavelengths 280 nm and 340 nm (alpha 280 and alpha 340), reflecting fluidity of lipid bilayer matrix and annular lipids, respectively. LPO induction was shown to result in changes of fluidity of both the bilayer and annular lipids. Upon reducing formation of LPO products by carnosine, fluidity changes of both the lipid bilayer matrix and annular lipids were diminished. Conformational changes of the annular lipid domain by LPO may therefore be considered as a possible cause of the functional changes in the receptor mediated responses and of the inactivation of membrane-bound enzymes by oxidative stress.     

The effect of changes of the content of membrane cholesterol and the effect of benzyl alcohol on the activity of the intrinsic Mg2+-ATPase from the erythrocyte plasma membrane of the flounder (Platichthys flesus L.)

Extraction of membrane cholesterol and incorporation of cholesteryl hemisuccinate into the membrane affect the activity of the membrane-bound Mg2+-ATPase. Increasing the ratio of cholesterol to phospholipid from 0.30 mg/mg in the control membranes to 0.45-0.90 in the enriched membranes results in a slight increase of the activity of about 20%. Diminishing the ratio of cholesterol to phospholipid to about one tenth of the ratio of the control membrane results in a decrease of the activity to about 30% of the untreated control. Benzyl alcohol inactivates the membrane-bound enzyme. Digitonin-solubilized Mg2+-ATPase is also inactivated by benzyl alcohol. For concentrations below 20 mM the dependence of the solubilized and the membrane-bound enzymes are virtually identical, and linearly dependent on alcohol concentration. This linear relationship continues up to 70 mM for the solubilized enzyme, while inhibition of the membrane-bound form shows a slightly steeper dependence on inhibitor concentration. It is suggested that the activity of the native Mg2+-ATPase depends on the organization of the lipid phase of the membrane and that addition of benzyl alcohol or depletion of cholesterol results in a disorganization of the lipid phase which in turn results in diminished activity.     

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)     

Alterations in activity and ultrastructural localization of several phosphatases on the surface of adult rat hepatocytes in primary monolayer culture

Several enzymes associated with the hepatocyte cell surface, alkaline phosphatase (AP), 5'-nucleotidase (5'N), Mg++- and total Na+K+Mg++-ATpase, were assayed and localized cytochemically in order to gain insight into alterations of the plasma membrane components during reassociation of hepatocytes in primary monolayer culture. During a period of 4 days the activities of 5'nucleotidase and alkaline phosphatase increased spontaneously up to three- and four-fold, respectively. Dexamethasone reinforce the rise of alkaline phosphatase activity but retarded the increase of that of 5'nucleotidase. However, after the third day the level of 5'nucleotidase activity converged with the untreated controls. The activities of Mg++- and Na+K+Mg++-ATPase, which closely paralleled each other, remained essentially unchanged throughout cultivation and were not affected by dexamethasone. Cytochemical demonstration of alkaline phosphatase, 5'nucleotidase and Mg++-ATPase, using the lead salt method, revealed the potential presence of reaction product on the whole cell surface. However, the cells did not react uniformly, particularly on bile canalicular membranes. This heterogeneity seems to be due to different stages of canalicular development and to different functional states of the cultured hepatocytes.     

Effect of colchicine on rat liver plasma membrane

Colchicine effect has been tested on rat liver plasma membrane-bound enzymes after in vitro or in vivo treatment. It appears that the in vitro treatment does not affect 5'-nucleotidase, Mg2+-ATPase and (Na+ + K+)-ATPase, whereas adenylate cyclase is sensitive to both in vitro and in vivo treatment, the latter condition being also effective for 5'-nucleotidase.     

The influence of changes in the phospholipid pattern of intact fibroblasts on the activities of four membrane-bound enzymes.

Human skin fibroblasts, grown to confluency in the presence of 32P for random labelling of the phospholipids, showed upon 24 h incubation in the presence of either 8 mM L-serine or 4 mM ethanolamine an increased content of phosphatidylserine (150% of control cells) or phosphatidylethanolamine (116% of control cells), respectively. Concomitantly the phosphatidylcholine correspondingly decreased. Upon cell harvesting and gentle enzyme preparation the base-treated cells demonstrated a significantly higher unstimulated, fluoride- and thyrotropin-stimulated activity of adenylate cyclase. The activities of total ATPase, ouabain-sensitive ATPase, 5'-nucleotidase and gamma-glutamyltransferase remained unaltered. When subjecting enzyme preparations from fibroblasts to ultrasonication the activity of adenylate cyclase decreased progressively with energy applied, whereas the activities of the other enzymes were unaltered ((K+ + Na+)-ATPase, 5'-nucleotidase) or even increased (Mg2+-ATPase, gamma-glutamyltransferase). The results have a bearing upon the regulatory function of the phospholipid microenvironment of membrane-bound enzymes.     

Functional changes in bladder tissue from type III collagen-deficient mice

The present work investigates the protective effects of N-acetylcysteine (NAC) on carbofuran-induced alterations in lipid composition and activity of membrane bound enzymes (Na⁺-K⁺-ATPase and Ca²⁺-ATPase) in the rat brain. Animals were exposed to carbofuran at a dose of 1 mg/kg body weight, orally, for a period of 28 days. A significant increase in lipid peroxidation in terms of TBARS was observed in brain after carbofuran exposure. NAC administration (200 mg/kg body weight) on the other hand lowered the carbofuran-induced lipid peroxidation to near normal. The increased lipid peroxidation following carbofuran exposure was accompanied by a significant decrease in the levels of total lipids, which is attributed to the reduction in phospholipid levels. Furthermore, NAC administration had a beneficial effect on carbofuran-induced alterations in lipid composition. The ratio of cholesterol to phospholipid, a major determinant of membrane fluidity, was increased in response to carbofuran exposure. This was associated with decreased activity of Na⁺-K⁺-ATPase and Ca²⁺-ATPase. NAC was observed to offer protection by restoring the cholesterol to phospholipid ratio along with the activity of Na⁺-K⁺-ATPase and Ca²⁺-ATPase. The results clearly suggest that carbofuran exerts its neurotoxic effects by increasing lipid peroxidation, altering lipid composition and activity of membrane bound enzymes. NAC administration ameliorated the effects of carbofuran suggesting its potential therapeutic effects in carbofuran neurotoxicity.     

Influence of dietary lipids on microsomal membranes from chick breast muscle

1. The effect of different dietary fat intake on the lipid composition and fluidity of microsomal membranes as well as in the enzymatic activity of the Ca2+-ATPase from chick breast muscle was investigated. 2. When a standard diet was supplemented with 10% sunflower seed oil, an increase in the relative amounts of unsaturated fatty acids and membrane fluidity and a decrease in the cholesterol content was observed. 3. The presence of 6% cholesterol in the diet does not modify the fatty acid composition and the fluidity of the membrane but increased, in a low extension, the cholesterol content. 4. The provision of the sunflower seed oil-rich diet supplemented with cholesterol just 48 hr before death promoted an increase in the relative amounts of unsaturated fatty acids and cholesterol content whereas the membrane fluidity decreased in a significant extent. 5. Despite that dietary lipids gave rise in some cases to changes in lipid composition and in the physical state of the microsomal membrane, neither the Ca2+ uptake capacity nor the ATPase activity were significantly affected.     

Role of phosphatidylethanol in membranes. Effects on membrane fluidity, tolerance to ethanol, and activity of membrane-bound enzymes.

We investigated the effect of phosphatidylethanol (PEt) on fluidity and membrane tolerance to the fluidization induced by ethanol as well as on the activity of two membrane-bound enzymes, Na+/K+ ATPase and 5'-nucleotidase. PEt was synthesized from 1,2-dimyristoylphosphatidylcholine and phosphatidylcholine from bovine brain and studies were performed to determine the optimal experimental conditions for the insertion of PEt in natural bilayers. The effects of PEt, evaluated by differential scanning calorimetry or fluorescence polarization techniques, were studied in model membranes made of synthetic phospholipids or made of total lipids extracted from rat brain crude mitochondrial fraction (P2 fraction) and from natural membranes (P2 fraction). The presence of PEt increased the fluidity of artificial as well of natural membranes, but tolerance to the addition of ethanol, displayed by dimyristoylphosphatidylcholine vesicles and by natural membranes containing PEt, was lacking in vesicles made of dimyristoylphosphatidylethanolamine and in artificial bilayers reconstituted from total P2 lipid extracts, suggesting an involvement of PC on PEt-induced ethanol resistance. Na+/K+ ATPase activity was enhanced by the addition of small amounts of ethanol (up to 50 mM) and progressively inhibited at higher concentrations, while 5'-nucleotidase was not affected up to 400 mM ethanol. The presence of PEt in the bilayer exerted the opposite effects on the two enzymes, reducing the Na+/K+ ATPase activation induced by ethanol and enhancing 5'-nucleotidase activity. The mechanisms of the PEt-induced modifications are discussed.     

Comparative Investigation on Plasmolemma Properties of Different Reed Ecotypes in Hexi Corridor

Lipid phospholipid and fatty acid compositions, fluidity, phase transition temperatures and membrane-bound Ca2+ ,Mn2+ ,Mg2+-ATPase activities of plasmolemmas extracted from four reed Phragmites communis Trin. ecotypes in Hexi Corridor of Gansu province were investigated. The results showed that all plasmolemmas of the four reed ecotypes consisted of the same six phospholipid and seven fatty acid components, but their proportions in the plasmolemma lipids of various reed ecotypes were markedly different. Index of unsaturated fatty acid (IUFA)and fluidity of plasmolemmas increased in the sequence of swamp-,dune-, meadow-dune transitional zone, salt meadow reeds. The plasmolemmas of all reed ecotypes showed phase transition in the two ranges of low-, high temperatures (4–6℃, 20–28 ℃ ), their phase transition temperatures in the latter were markedly different, Plasmolemma-bound Ca2+ ,Mn2+ ,Mg2+-ATPase activity positively related to IUFA and fluidity. Combining the membrane properties with environments,in comparison with swamp reeds, the increases of phosphatidylcholine, cardiolipin and the significant rising of IUFA in plasmolemma lipids are closely responsible for reed saR-tolerance, whereas the increase of phosphatidylglycerol and the suitable rising of IUFA make a contribution to reed drought-tolerance.     

大鼠心肌缺血／再灌及高脂血症的心肌膜损伤

通过大鼠心肌缺血／再灌及高脂血症的模型证实,两者均有明显的生物膜损伤,主要表现为膜磷脂的降低、胆固醇及胆固醇／磷脂比增高、膜脂流动性及膜酶(Ca²⁺, Mg²⁺-ATPase)活性降低,这些异常变化与氧自由基引发的脂质过氧化增强或脂质交换有关.     

Fluidity-dependent Mg2(+)-ATPase activity in membranes from Leishmania donovani promastigotes.

The state of the lipid phase of the membrane plays a key role in the exposure of various receptors, antigens and enzymes on the membrane surface. The fluidity of membranes of Leishmania donovani promastigotes was monitored by two independent methods, i.e. influx of sterol from liposomes and removal of phospholipids by treatment with phospholipase C. The altered sterol/phospholipid ratio, in both cases, provided evidence that the activity of the functionally important membrane-bound enzyme Mg2(+)-ATPase is modulated by the state of the lipid phase of the membrane.     

Abnormal properties of Mg2(+)-ATPase in transverse tubule membranes from dystrophic chicken

Purified transverse tubule membranes from normal and dystrophic chicken skeletal muscle were isolated by a calcium-loading procedure. Normal and dystrophic T-tubules were similar in cholesterol content and (Na+,K+)-ATPase and 5'-nucleotidase activities but a significant decrease of Mg2(+)-ATPase activity was observed in dystrophic membranes. A comparative analysis of the enzyme properties revealed that the kinetic parameters were altered in dystrophic T-tubules and the ATP-hydrolyzing activity was differently affected by the ionic strength. However, the influence of temperature and the regulatory effect of concanavalin A were the same as in normal T-tubules. Membrane fluidity was similar in both preparations as estimated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene and trimethylammonium diphenylhexatriene. These results point to an impairment in the function of Mg2(+)-ATPase due to structural alterations of the enzyme.     

Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver

A method has been developed for routine high yield separation of canalicular (cLPM) from basolateral (blLPM) liver plasma membrane vesicles of rat liver. Using a combination of rate zonal floatation (TZ- 28 zonal rotor, Sorvall) and high speed centrifugation through discontinuous sucrose gradients, 9-16 mg of cLPM and 15-28 mg of blLPM protein can be isolated in 1 d. cLPM are free of the basolateral markers Na+/K+-ATPase and glucagon-stimulatable adenylate cyclase activities, but are highly enriched with respect to homogenate in the "canalicular marker" enzyme activities leucylnaphthylamidase (48-fold), gamma-glutamyl-transpeptidase (60-fold), 5'-nucleotidase (64-fold), alkaline phosphatase (71-fold), Mg++-ATPase (83-fold), and alkaline phosphodiesterase I (116-fold). In contrast, blLPM are 34-fold enriched in Na+/K+-ATPase activity, exhibit considerable glucagon-stimulatable adenylate cyclase activity, and demonstrate a 4- to 15-fold increase over homogenate in the various "canalicular markers." cLPM have a twofold higher content of sialic acids, cholesterol; and sphingomyelin compared with blLPM. At least three canalicular-(130,000, 100,000, and 58,000 mol wt) and several basolateral-specific protein bands have been detected after SDS PAGE of the two LPM subfractions. Specifically, the immunoglobin A-binding secretory component is restricted to blLPM as demonstrated by immunochemical techniques. These data indicate virtually complete separation of basolateral from canalicular LPM and demonstrate multiple functional and compositional polarity between the two surface domains of hepatocytes.     

Increased oxidative stress and decreased activities of Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase in the red blood cells of the hibernating black bear

During hibernation, animals undergo metabolic changes that result in reduced utilization of glucose and oxygen. Fat is known to be the preferential source of energy for hibernating animals. Malonyldialdehyde (MDA) is an end product of fatty acid oxidation, and is generally used as an index of lipid peroxidation. We report here that peroxidation of lipids is increased in the plasma and in the membranes of red blood cells in black bears during hibernation. The plasma MDA content was about four fold higher during hibernation as compared to that during the active, non-hibernating state (P < 0.0001). Similarly, MDA content of erythrocyte membranes was significantly increased during hibernation (P < 0.025). The activity of Ca(2+)/Mg(2+)-ATPase in the erythrocyte membrane was significantly decreased in the hibernating state as compared to the active state. Na(+)/K(+)-ATPase activity was also decreased, though not significant, during hibernation. These results suggest that during hibernation, the bears are under increased oxidative stress, and have reduced activities of membrane-bound enzymes such as Ca(2+)/Mg(2+)-ATPase and Na(+)/K(+)-ATPase. These changes can be considered part of the adaptive for survival process of metabolic depression.     

Homoeostatic control of membrane cholesterol and fatty acid metabolism in the rat liver.

Experiments were designed to assess the effect of cholesterol feeding, with or without high levels of either saturated (coconut oil) or unsaturated (sunflower-seed oil) fat on the fatty acid composition of hepatic microsomal membrane lipids, as well as on the activities of several membrane-bound enzymes of cholesterol synthesis and metabolism. Administration of 2% (w/w) cholesterol in the rat diet inhibited hydroxymethylglutaryl-CoA reductase activity, and this inhibition was much more pronounced when cholesterol was fed in combination with unsaturated rather than with saturated fat. Cholesterol 7 alpha-hydroxylase activity was increased by all the high-cholesterol diets and inhibited by both the high-fat diets. Cholesterol esterification, as assessed by acyl-CoA:cholesterol acyltransferase (ACAT) activity, was enhanced after unsaturated-fat feeding. Cholesterol supplement, without any added fat, failed to elicit any significant increase in ACAT activity, whereas consumption of cholesterol in combination with unsaturated fat led to the greatest increase in ACAT activity. After cholesterol feeding, C18:1 and C18:2 fatty acids in the microsomal phospholipids were increased, with concomitant decreases in C18:0, C20:4 and C22:6 fatty acids, leading to an overall decrease in membrane unsaturation, irrespective of the particular fat supplement. It can be concluded that the inhibition of cholesterol biosynthesis and the enhancement of cholesterol utilization, either by increased bile formation or by increased cholesterol esterification, after cholesterol feeding, may not be enough to prevent cholesterol accumulation in the microsomal membranes. Then, to compensate for the altered fluidity resulting from cholesterol enrichment, the unsaturation of membrane phospholipids is decreased, which would in turn have an effect on membrane lipid fluidity opposite to that of increased cholesterol.     

Erythrocyte membrane in rats fed high erucic acid-containing mustard oil: osmotic fragility, lipid composition, and (Na+, K+)- and (Ca2+, Mg2+)-ATPases

Erythrocyte hemolytic properties, cholesterol/phospholipid ratios, fatty acid composition, and activities of the membrane-bound enzymes (Na+, K+)- and (Ca2+, Mg2+)-ATPase were studied in male and female rats fed low erucic acid rapeseed (LEAR) and high erucic acid mustard oils (HEAM) for a period of 16 months. Rats receiving groundnut oil (GNO) served as controls. Erythrocytes from HEAM-receiving male and female rats showed increased resistance to hypotonic hemolysis. In male rats this was associated with an 85% increase (P less than 0.07) in the cholesterol/phospholipid molar ratio. The fatty acid double-bond index showed an increase in male rats receiving HEAM as well as LEAR oils. In the erythrocytes from female rats, the cholesterol/phospholipid molar ratio and double bond index remained unaffected. Specific activity of ouabain-sensitive (Na+, K+)-ATPase showed a small (+20%) but significant (P less than 0.05) increase in male but not female rats of HEAM group. Total (Na+, K+)-ATPase, ouabain-insensitive component, and (Ca2+, Mg2+)-ATPase were not altered in rats receiving LEAR or HEAM.