Lipid peroxidation causes an increase of lipid order and a decrease of 5'-nucleotidase activity in the liver plasma membrane.

The effect of peroxidation on 5'-nucleotidase activity as well as on membrane microviscosity has been investigated in liver plasma membranes from Wistar rats. The peroxidation was performed with 100 microM H2O2 and 200 microM FeSO4 and/or with 5 mM t-butylhydroperoxide. Treatment of the membranes with these oxidizing agents resulted in an elevation of the transition temperatures of the polarization of the lipid fluorescent probes 1,6 diphenyl-1,3,5 hexatriene (DPH), 3-p-(6-phenyl) 1,3,5 hexatriene phenylpropionic acid (PA-DPH) as well as of the fluorescent thiol reagent N-(1-pyrene) maleimide (1-PM). The peroxidation resulted in a decrease of the activity of 5'nucleotidase. Our data support that the increase of membrane microviscosity of the lipid domain regulates the activity of 5'-nucleotidase.     

Effects of aging on the relation of adenyl purine release with plasma membrane fluidity of arterial endothelial cells

Age-related changes in adenyl purine release from rat arteries and endothelial cell (EC) plasma membrane (PM) fluidity were studied. High performance liquid chromatography-fluorescence revealed that aging significantly decreased the release of adenyl purines. Pyrene-excimer spectroscopy disclosed that EC PM fluidity of aged rats decreased more significantly than that of young rats. An increase in cholesterol content and a decrease in the unsaturation index (USI) of fatty acids in cholesterol-enriched ECs reduced PM fluidity and 5'-nucleotidase (5'-ND) activity (measured by coupled assay of adenosine deaminase and glutamate dehydrogenase). Moreover, a decrease in cholesterol content and an increase in the USI of fatty acyl chains of the PM in docosahexaenoic acid-enriched ECs concurrently increased enzyme activity and extracellular adenosine. Therefore, decreases in PM fluidity, observed with age-dependent increased cholesterol and decreased USI, induce a decrease in 5'-ND activity, decrease extracellular adenosine levels, and might relate to hypertension in aged rats.     

Catalytic characteristics of 5'-nucleotidase in the structure of cell membrane rafts in smooth muscles

5'-nucleotidase (EN 3.1.3.5) is widely distributed enzyme occurring in vertebrate, bacterial and plant cells. The main physiological function of 5'-nucleotidase is hydrolysis of 5'-AMP to adenosine and Pi. It was found that the detergent-insoluble membrane domains (rafts) are enriched by proteins possessing high 5'-AMPase activity. This study is aimed to investigate some physical and chemical properties of 5'-nucleotidase, which is present in detergent insoluble membrane domains isolated from pig stomach and lung. It was shown for the first time that catalytic properties of the raft-associated 5'-nucleotidase and of the pure enzyme described in literature differ. Our results demonstrate that the greatest activity of the raft-associated enzyme takes place in the physiological conditions contrary to the pure enzyme. Our data suggest that such changes of 5'-nucleotidase catalytic activity might be due to the disruption of its interaction with membrane rafts.     

Alterations in hepatic 5'-nucleotidase in the tumor-bearing rat

The effect of the growth of the Walker 256 carcinoma on the level of 5'-nucleotidase and alkaline phosphatase in the whole liver and in an isolated hepatocyte membrane preparation of its host was investigated. Alkaline phosphatase activities of whole liver and plasma membrane were increased approximately 5-fold by tumor growth. A 50% decrease in whole liver 5'-nucleotidase activity was observed in tumor-bearing rats while the 5'-nucleotidase activity per milligram membrane protein was unaltered. Tumor growth would therefore appear to affect a pool of 5'-nucleotidase which is not associated with the plasma membrane.     

Activity of cell-detached 5'-nucleotidase in thymocytes of various densities

The rat thymocytes submitted to heating at 45 degrees C for 1 hr liberate plasma membrane fragments containing 5'-nucleotidase activity in the supernatant. The thymocytes were separated by ficoll density gradient centrifugation. High activity of 5'-nucleotidase per 10(6) cells was found in the supernatant of low density (1.069) subset of thymocytes. Thymocyte supernatant of rats treated with hydrocortisone demonstrated higher 5'-nucleotidase activity per 10(6) cells than in intact animals. This is due to an increase of the low density population of thymocytes in treated rats since the 5'-nucleotidase activity per 10(6) cells of the supernatant obtained from this density fraction is the same both in treated with hydrocortisone and intact rats. Hydrocortisone seems to induce a selection of the thymocytes with high 5'-nucleotidase activity.     

Characterization of the membrane proteins of rat liver lysosomes. Composition, enzyme activities and turnover.

Lysosomes prepared from the livers of untreated rats and from the livers of rats injected with either Triton WR-1339 or dextran yielded membranes that were similar in both polypeptide composition and activities of ATPase and acid 5'-nucleotidase. The administration of Triton WR-1339 (and dextran) resulted in an increase in ATPase activity of liver homogenates that was associated with a parallel increase in the ATPase activity of the lysosomal membrane. On the other hand, plasma membranes appear to be different from lysosomal membranes with respect to polypeptide composition and enzyme activities. The ATPase activity of lysosomal membranes is not affected by ouabain and suramin, inhibitors of the plasma-membrane ATPase. The plasma-membrane alkaline 5'-nucleotidase has little activity at acid pH. Pulse-labelling of lysosomal membranes with [3H]fucose and with [3H]- and [14C]-leucine occurred rapidly, faster than labelling of plasma membranes. The labelling kinetics indicate that lysosomal membranes may be assembled independently of plasma membranes. These data suggest that, in liver, little bulk transport of plasma membrane to lysosomes takes place, and lysosomal-membrane proteins may not be derived from those of plasma membranes.     

A histochemical study about changes in rat liver plasma membrane enzyme activities after galactosamine administration.

In rats changes in plasma membrane enzyme activities due to Gal-N intoxication were studied by enzymehistochemical methods. The bile canalicular 5'-nucleotidase and nucleoside polyphosphatase activities decreased; the sinusoidal 5'-nucleotidase remained unchanged. The bile canalicular leucyl-beta-naphthyl-amidase showed an increase in activity; the alkaline phosphatase activity remained unchanged. In contrast to the spotty necrosis, changes in plasma membrane enzyme activities were seen in all liver cells, suggesting that changes of these activities, occurring after Gal-N treatment, do not correlate with cell death. The conclusion was drawn that the deviations of the enzyme activities might be due to changes in the lipid environment of the enzyme proteins in the membrane. With the exception of alkaline phosphatase, partial hepatectomy caused the same changes in enzyme activities as did Gal-N intoxication. Nevertheless Gal-N administration to partial hepatectomized rats did not lead to hepatic necrosis. Galactose given simultaneously or within two hours after Gal-N prevented both changes in plasma membrane enzyme activities and hepatocellular damage. This suggests an important role of galactolipids and galactoproteins in the plasma membrane alterations.     

Modulation of prolactin binding sites in vitro by membrane fluidizers II. Age-dependent effects on rat ventral prostatic membranes

The objectives of this study were to determine (i) if the age-related changes in ¹²⁵I-labeled ovine prolactin specific binding of rat ventral prostate was correlated with changes in membrane lipid microviscosity and (ii) if membrane fluidizers produced age-dependent effects on prolactin binding of prostatic membranes. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Membrane preparations of ventral prostate glands obtained from immature (24–25 days old), young-adult (80–90 days old) and aged (550–610 days old) male rats were used for prolactin binding and membrane lipid microviscosity measurements. Relative to immature rats, prostatic prolactin binding decreased approximately 50% in young-adult rats and 75% in aged rats. Membrane lipid microviscosity, relative to immature rats, was increased 72% in young-adult rats and 140% in aged rats. Prostatic membranes obtained from immature animals exhibited no significant effects of in vitro alcohol treatment on prolactin binding, whereas, those obtained from aged animals exhibited maximal increase in prolactin binding. The value of the microviscosity parameter, after in vitro alcohol exposure, exhibited no significant changes in immature animals, whereas, this parameter was decreased approximately 15% in young-adults and approximately 30% in aged animals. These data suggest that in vitro fluidization of prostatic membrane exhibits an age-dependent modification of prolactin binding.     

Modulation of prolactin binding sites in vitro by membrane fluidizers. II. Age-dependent effects on rat ventral prostatic membranes

The objectives of this study were to determine (i) if the age-related changes in 125I-labeled ovine prolactin specific binding of rat ventral prostate was correlated with changes in membrane lipid microviscosity and (ii) if membrane fluidizers produced age-dependent effects on prolactin binding of prostatic membranes. The degree of fluidization was monitored by a fluorescence polarization method using 1,6-diphenylhexatriene. Membrane preparations of ventral prostate glands obtained from immature (24-25 days old), young-adult (80-90 days old) and aged (550-610 days old) male rats were used for prolactin binding and membrane lipid microviscosity measurements. Relative to immature rats, prostatic prolactin binding decreased approximately 50% in young-adult rats and 75% in aged rats. Membrane lipid microviscosity, relative to immature rats, was increased 72% in young-adult rats and 140% in aged rats. Prostatic membranes obtained from immature animals exhibited no significant effects of in vitro alcohol treatment on prolactin binding, whereas, those obtained from aged animals exhibited maximal increase in prolactin binding. The value of the microviscosity parameter, after in vitro alcohol exposure, exhibited no significant changes in immature animals, whereas, this parameter was decreased approximately 15% in young-adults and approximately 30% in aged animals. These data suggest that in vitro fluidization of prostatic membrane exhibits an age-dependent modification of prolactin binding.     

Cardiac adenosine production in rat genetic models of low and high exercise capacity

We previously demonstrated that Copenhagen (COP) and DA inbred rat strains show a wide difference in a test for aerobic treadmill running that correlated positively with isolated cardiac function. The purpose of this study was to test adenosine production as a candidate intermediate phenotype that may explain part of the difference in running and cardiac performance in these genetic models for low and high aerobic capacity. Adenosine production was measured as the activity of soluble 5'-nucleotidase and membrane-bound ecto-5'-nucleotidase in the membrane pellet and supernatant fractions of left and right ventricular muscle and gracilis muscle taken from 10 DA and 10 COP rats. Ecto-5'-nucleotidase activity in the membrane pellet of hearts from both DA and COP accounted for the vast majority of the total tissue adenosine production (>90% in the left ventricle and >80% in the right ventricle). Ecto-5'-nucleotidase activity in the pellet fraction was significantly higher in the left (22.4%) and right (46.1%) ventricles of DA rats compared with COP rats, with no differences in total protein content. There were no significant differences between the strains for 5'-nucleotidase activity in the cardiac supernatant, the gracilis pellet, or the gracilis supernatant. These data support the hypothesis that an increase in cardiac adenosine production may contribute to the greater aerobic running capacity of the DA rats.     

Chronic changes in plasma triglyceride levels do modify platelet membrane microviscosity in rats

Lipid metabolism disorders were proposed to mediate numerous cell membrane alterations in various forms of hypertension. Elevated plasma triglycerides were found to be associated with changes in membrane structure and function related to altered microviscosity in particular domains of the cell membrane. The aim of our study was to determine if an abnormal triglyceride metabolism might play a causal role in these alterations of membrane dynamics. Using genetically hypertensive rats of the Prague hereditary hypertriglyceridemic (HTG) strain we investigated whether the elevation of circulating triglycerides induced by high fructose intake and/or their lowering by chronic gemfibrozil treatment (for 10 weeks starting at the age of 6 weeks) are followed by reciprocal changes in membrane microviscosity. Two different fluorescent probes exploring either the outer membrane leaflet (TMA-DPH anisotropy) or the membrane lipid core (DPH anisotropy) were used in platelets of HTG rats. DPH (diphenylhexatriene) fluorescence anisotropy was decreased in platelets of fructose-treated HTG animals with highly elevated plasma triglyceride levels, whereas it was increased in gemfibrozil-treated HTG rats in which triglyceride levels were almost normalized. On the contrary, TMA-DPH (trimethylamino-diphenylhexatriene) anisotropy was not substantially altered in platelets from HTG rats by the above modifications of circulating triglycerides. No changes of plasma cholesterol or blood pressure were associated with the triglyceride-dependent modifications of membrane core microviscosity. Our interventional study demonstrates a major causal role of circulating triglycerides in the control of the microviscosity of membrane lipid core.     

Insulin-induced decrease in 5'-nucleotidase activity in skeletal muscle membranes

Insulin releases inositol phosphoglycans from myocytes in culture [(1986) Science 233, 967-972], which display insulinomimetic activity. Because 5'-nucleotidase is anchored to the membrane through inositol-containing phospholipid glycans, we investigated whether insulin could release the enzyme from the membrane. Membranes prepared from hindquarter muscles of rats perfused with insulin showed a 23% decrease in 5'-nucleotidase activity. Isolated membranes from muscle exposed to insulin in vitro also showed a small but reproducible decrease (9%) in 5'-nucleotidase activity relative to unexposed controls. Phospholipase C from Staphylococcus aureus released 60% of the membrane-bound 5'-nucleotidase. We propose that insulin may activate an endogenous phospholipase C that cleaves phospholipid-glycan-anchored proteins.     

Effect of nitric oxide on 5'-nucleotidase activity of the membrane rafts in the smooth muscle cells

We investigated the effect of nitric oxide on the catalytic activity of 5'-nucleotidase associated with insoluble membrane domains (rafts) of pig stomach smooth muscle. The low concentration (0.1-10.0 microM) of nitric oxide donor sodium nitroprusside led to essential increase of catalytic activity of 5'-nucleotidase. Maximal increase was observed at concentration of sodium nitroprusside of 1 microM. The enzyme's catalytic activity decreased to about control value at higher concentration of this substance. The catalytic activity of 5'-nucleotidase was also increased at presence of NaNO2, but only at high concentration (10 mM).The specific thiol-alkylating agent N-ethylmaleinimide (1-100 microM) led to essential decrease of enzyme catalytic activity. Our data shows that nitric oxide changes the AMP-ase activity of 5'-nucleotidase, that is thought to be due to direct effect of this substance on protein. We suppose, that such effect of nitric oxide could be physiologicaly important in functioning of smooth muscle.     

Subfractionation of rat liver plasma membrane. Uneven distribution of plasma membrane-bound enzymes on the liver cell surface.

Plasma membranes were isolated from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5'-nucleotidase and (Na+ + K+)-ATPase were used. The yield of plasma membrane was 0.6-0.9 mg protein per g wet weight of liver. The recovery of 5'-nucleotidase and (Na+ +K+)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the activity of glucose-6-phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5'-nucleotidase, alkaline phosphatase, (Na+ +K+)-ATPase and Mg2+-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na2+ +K+)-ATPase and Mg2+-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphate was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.     

Effects of docosahexaenoic acid on annular lipid fluidity of the rat bile canalicular plasma membrane.

The role of docosahexaenoic acid (DHA) in the fluidity of the annular lipid regions and their associated membrane-bound proteins is still not as well understood as that in the global (bulk) lipid regions. We therefore studied the effects of dietary DHA on the relationship between annular and global lipid fluidity and membrane-bound enzymes such as 5'-nucleotidase and Mg(2)+-ATPase in the rat bile canalicular membrane. Dietary DHA caused significant increases in 5'-nucleotidase and Mg(2)+-ATPase activity and in global and annular lipid fluidity, a higher increase in fluidity in the annular lipids than the global lipids, and a decrease in the cholesterol-to-phospholipid molar ratio in the canalicular membrane. Plasma total cholesterol and LDL cholesterol decreased, and fecal cholesterol increased in the DHA-fed rats. No changes were observed in oxidative markers, but glutathione peroxidase increased in the liver with DHA feeding. Annular lipid fluidity, but not global lipid fluidity, correlated remarkably well with DHA, synchronously with the activities of 5'-nucleotidase and Mg(2)+-ATPase. The data indicate that the DHA-induced increase in annular lipid fluidity is responsible for the increases observed in the enzyme activity. We therefore concluded that the increased activity of membrane-bound enzymes and transporters induced by DHA and the concomitant increase in annular lipid fluidity comprise one of the mechanisms involved in DHA-induced clearance of plasma cholesterol.     

A histochemical study about the influence of lytic enzymes on plasma membrane enzyme activities in rat liver and kidney.

1. The effect of lipolytic, glycolytic and proteolytic enzymes on the activities of plasma membrane enzyme activities in rat liver and kidney has been investigated by a pretreatment of tissue sections with the lytic enzymes. 2. The action of the proteolytic enzymes causes a very strong decrease of leucyl-beta-naphthylamidase activity, whereas the activities of ATP-ase, 5'-nucleotidase and alkaline phosphatase show a lesser decrease. This indicates a different membrane anchorage of leucyl-beta-naphthylamidase as compared to that of the phosphatases. 3. Treatment with glycolytic enzymes results in a decrease of 5'-nucleotidase and ATP-ase activity, whereas liver alkaline phosphatase and leucyl-beta-naphthylamidase show an increase in activity. 4. Treatment with phospholipase C gives about the same results. The very strong decrease of 5'-nucleotidase activity indicates a great dependence on phospholipids.     

Cytochemical localization of plasma membrane enzyme markers during interiorization of tachyzoites of Toxoplasma gondii by macrophages

The enzyme activity of Mg++-ATPase, Na+-K+-ATPase, 5'-nucleotidase and NAD(P)H-oxidase was cytochemically detected at the ultrastructural level in mouse peritoneal macrophages infected with untreated and with specific antibody-coated Toxoplasma gondii tachyzoites. The Mg++-ATPase and 5'-nucleotidase were distributed throughout the macrophages' plasma membrane but were not observed in the membrane lining endocytic vacuoles containing ingested parasites; however, Na+-K+-ATPase activity was detected in the macrophages' plasma membrane as well as in the parasitophorous vacuoles that contained untreated or specific antibody-coated parasites. Reaction product, indicative of NAD(P)H-oxidase, was detected in the parasitophorous vacuoles that contained only specific antibody-coated parasites.     

Contact-mediated changes in the fluidity of membrane lipids in normal and malignant transformed mammalian fibroblasts

The degree of microviscosity, gh, (fluidity/rigidity behavior) of membrane lipids of normal and transformed mammalian fibroblasts obtained from mice, hamsters and rats was quantitatively monitored by fluorescence polarization, P, analysis of the fluorescent probe 1,6-diphenyl 1,3,5-hexatriene (DPH) when embedded in lipid regions of cellular membranes of intact viable cells. Analysis of membrane microviscosity of six different cell populations and of individual cells in each cell population have indicated that the membrane microviscosity of all cell types, both normal and transformed fibroblasts, changes as a function of the cell density in the growing cultures. The membrane microviscosity was found to be low (high lipid fluidity) in sparse conditions but high (high lipid rigidity) in dense conditions. The induced changes in membrane microviscosity are practically reversible for all cell types and a complete reversion can be obtained within a few hours after changing the cell density conditions from sparse to dense and vice versa.Comparative studies with normal and transformed fibroblasts have shown that transformed fibroblasts have a more rigid lipid layer in their cellular membranes than normal or untransformed fibroblasts. The difference in membrane microviscosity between transformed and normal fibroblasts is higher in confluent conditions as compared with subconfluent cultures. These differences in the degree of fluidity of membrane lipids that are controlled by possible differences in the cellto-cell contact in normal and transformed fibroblasts may play a major role in determining the growth behavior of normal and malignant cells that are growing as a solid tissue and may have a direct effect on the control mechanisms that determine the presence or absence of the “density dependent inhibition” of growth.     

Changes in the membrane lipids and lymphocyte function in staphylococcal infection and the development of delayed hypersensitivity

Experimental staphylococcal infection was reproduced in rats by the intraperitoneal injection of S. aureus strain 75. The degree of the development of delayed hypersensitivity (DH) to staphylococci, microviscosity, the levels of free radical oxidation and antioxidation resistance were evaluated in the dynamics of the infectious process by the methods of chemiluminometry and fluorescent probing with pyrene. The functional activity of lymphocytes was determined by the inclusion of 3H-thymidine into DNA as the consequence of stimulation with phytohemagglutinin. The development of DH was found to depend on the microviscosity and antioxidation resistance of membrane lipids. The increase of microviscosity and the simultaneous decrease of the induction time of chemifluorescent rapid flash inhibit the development of DH, leading to the aggravation of the infectious process. The increase of fluidity and the accumulation of antioxidants facilitate the development of DH and lead to a milder course of the infectious process.     

Dependence between 5'-nucleotidase activity and phosphatidylcholine content in pig adipocyte plasma membrane. Absence of relationship with membrane physical state

1. 5'-Nucleotidase activity has been examined in pig adipocyte plasma membrane, in two different adipose sites. 2. Membrane fluidity has been investigated using 1,6-diphenylhexatriene (DPH). 3. In this work, evidence was found for a tissue specificity of 5'-nucleotidase (E.C. 3.1.3.5) activity. 4. The enzyme activity was highly correlated to the membrane phosphatidylcholine content. 5. The latter parameters remained unmodified when large alterations in membrane composition and physical state were produced by means of the dietary n-6 PUFA content manipulations.