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R Fulceri

Publications and source records attributed to R Fulceri.

70 records · Page 4Linked to original sources
PubMed1985

Calcium sequestration activity in rat liver microsomes. Evidence for a cooperation of calcium transport with glucose-6-phosphatase.

Mechanisms regulating the energy-dependent calcium sequestering activity of liver microsomes were studied. The possibility for a physiologic mechanism capable of entrapping the transported Ca2+ was investigated. It was found that the addition of glucose 6-phosphate to the incubation system for MgATP-dependent microsomal calcium transport results in a marked stimulation of Ca2+ uptake. The uptake at 30 min is about 50% of that obtained with oxalate when the incubation is carried out at pH 6.8, which is the pH optimum for oxalate-stimulated calcium uptake. However, at physiological pH values (7.2-7.4), the glucose 6-phosphate-stimulated calcium uptake is maximal and equals that obtained with oxalate at pH 6.8. The Vmax of the glucose 6-phosphate-stimulated transport is 22.3 nmol of calcium/mg protein per min. The apparent Km for calcium calculated from total calcium concentrations is 31.9 microM. After the incubation of the system for MgATP-dependent microsomal calcium transport in the presence of glucose 6-phosphate, inorganic phosphorus and calcium are found in equal concentrations, on a molar base, in the recovered microsomal fraction. In the system for the glucose 6-phosphate-stimulated calcium uptake, glucose 6-phosphate is actively hydrolyzed by the glucose-6-phosphatase activity of liver microsomes. The latter activity is not influenced by concomitant calcium uptake. Calcium uptake is maximal when the concentration of glucose 6-phosphate in the system is 1-3 mM, which is much lower than that necessary to saturate glucose-6-phosphatase. These results are interpreted in the light of a possible cooperative activity between the energy-dependent calcium pump of liver microsomes and the glucose-6-phosphatase multicomponent system. The physiological implications of such a cooperation are discussed.

Adenosine Triphosphate↗
PubMed1984

[Functional coupling of the calcium pump and glucose 6-phosphatase activity in liver microsomes: preliminary results].

The addition of G-6-Pi to the incubation system for MgATP-dependent calcium transport in liver microsomes results in a marked stimulation of Ca2+ uptake. At physiological pH values (7.2-7.4), the G-6-Pi stimulated calcium uptake is maximal and equals that obtained with oxalate at pH 6.8. In the system for the G-6-Pi-stimulated calcium uptake, G-6-Pi is actively hydrolyzed by the glucose 6-phosphatase activity of liver microsomes. Such an activity is not influenced by the concomitant calcium uptake. After the incubation of the system for the MgATP-dependent microsomal calcium transport in the presence of G-6-Pi, Pi and calcium are found in equal concentrations, on a molar base, in the recovered microsomal fraction. These results are interpreted in the light of a possible cooperative activity between the energy-dependent calcium pump of liver microsomes and the glucose 6-phosphatase multicomponent system.

Adenosine Triphosphate↗
PubMed1984

[Inhibition of the calcium-sequestering activity of liver microsomes in monobromotrichloromethane poisoning: preliminary studies on possible mechanisms].

The mechanisms by which the in vivo intoxication with BrCCl3 inhibits the calcium sequestration activity of liver microsomes were studied. The initial rate of Ca2+ transport is inhibited by nearly 50% in the intoxicated rats as compared to the controls; this indicates that the active transport of Ca2+ is markedly affected by the intoxication. The microsomal ATPases activities both in the presence and in the absence of Ca2+ were not decreased at all in the intoxicated animals. However, the Ca2+-dependent extra ATP hydrolysis shows a different kinetics in the BrCCl3-poisoned rats with respect to the controls. The release of Ca2+ from Ca2+ loaded liver microsomes is higher in the intoxicated animals. It seems therefore that the increased permeability of the membrane to Ca2+ contributes to some extent to the haloalkane-induced inhibition of the calcium sequestration activity of liver microsomes.

Adenosine Triphosphatases↗
PubMed1984

Inhibition of calcium sequestration activity of liver microsomes by 4-hydroxyalkenals originating from the peroxidation of liver microsomal lipids.

Aldehydes released during peroxidation of liver microsomal lipids and identified as 4-hydroxyalkenals (4-hydroxynonenal being quantitatively the most significant) strongly inhibited the calcium sequestration activity of liver microsomes. The ID50 for 4-hydroxynonenal was 42 microM. The inhibition appeared to be correlated with the amount of the aldehyde bound to the microsomal protein. In rats intoxicated with BrCCl3 significant amounts of protein-bound aldehydes were formed at only 5 min after poisoning, a time at which the calcium sequestring capacity is markedly inhibited.

Aldehydes↗
PubMed1984

Cytotoxic aldehydes originating from the peroxidation of liver microsomal lipids. Identification of 4,5-dihydroxydecenal.

During the NADPH-Fe-induced peroxidation of liver microsomal lipids products are formed which are provided with cytopathological activities. In a previous study one of the major products was identified as an aldehyde of the 4-hydroxyalkenal class, namely 4-hydroxynonenal. In the present study another cytotoxic product has been isolated and identified as 4,5-dihydroxy-2,3-decenal. The isolation was performed by means of thin-layer chromatography and high-pressure liquid chromatography and the structure was ascertained mainly by means of mass spectroscopy of the free aldehyde and of its derivatives. In the absence of NADPH-Fe liver microsomes produced no 4,5-dihydroxydecenal. The inhibitory activity of 4,5-dihydroxydecenal on microsomal glucose-6-phosphatase is somewhat lower than that exhibited by 4-hydroxynonenal. This lower inhibitory activity correlates with the lower capacity to bind to the microsomal protein of 4,5-dihydroxydecenal as compared to 4-hydroxynonenal. The reactivities of the two aldehydes with cysteine were comparable. The production of toxic aldehydes may represent a mechanism by which lipid peroxidation causes deleterious effects on cellular functions.

Aldehydes↗
PubMed1984

Loss of lipid peroxidation as a histochemical marker for preneoplastic hepatocellular foci of rats.

Since it is known that tumor cell membranes have lost the capacity to undergo lipid peroxidation, it seemed of interest to investigate whether the loss of susceptibility to lipid peroxidation represents a tumoral marker appearing in preneoplastic cells together with the other known tumoral markers. A histochemical technique was developed to detect lipid peroxidation in individual cells of liver sections exposed to effective prooxidants. The technique was based on the detection of protein-bound aldehydes (alkenals) with the use of the Schiff's reagent. The latter reagent can also detect carbonyl function present in acyl residues of peroxidized phospholipids of cellular membranes. Liver preneoplastic foci were obtained in rats by the i.p. administration of diethylnitrosamine and of 2-acetylaminofluorene in the diet. Frozen sections of the liver, incubated in the presence of reduced nicotinamide adenine dinucleotide phosphate:iron revealed the presence of areas in which lipid peroxidation had not been induced (Schiff-negative areas). These areas corresponded strictly, in serial sections, to areas that were strongly positive to gamma-glutamyltranspeptidase.

2-Acetylaminofluorene↗
PubMed1984

On the mechanisms of the inhibition of calcium sequestering activity of liver microsomes in bromotrichloromethane intoxication.

The mechanisms by which the in vivo intoxication with BrCCl3 inhibits the calcium sequestration activity of liver microsomes were studied. The initial rate of Ca2+ transport is inhibited by nearly 50% in the intoxicated rats as compared to the controls; this indicates that the active transport of Ca2+ is markedly affected by the intoxication. The microsomal ATPase activities both in the presence and in the absence of Ca2+ were not decreased at all in the intoxicated animals. However, the Ca2+-dependent extra ATP-hydrolysis shows a different kinetics in the BrCCl3-poisoned rats with respect to the controls. The release of Ca2+ from Ca2+-loaded liver microsomes is higher in the intoxicated animals. It seems therefore that the increased permeability of the membrane to Ca2+ contributes to some extent to the haloalkane-induced inhibition of the calcium sequestration activity of liver microsomes.

Adenosine Triphosphate↗
PubMed1982

Detection of carbonyl functions in phospholipids of liver microsomes in CCl4- and BrCCl3-poisoned rats.

Since the peroxidative cleavage of unsaturated fatty acids can result in either the release of carbonyl compounds or the formation of carbonyl functions in the acyl residues, evidence for the presence of carbonyl groups in liver microsomal phospholipids was searched for in in vivo conditions (CCl4 and BrCCl3 intoxications) in which peroxidation of lipids of hepatic endoplasmic reticulum had been previously demonstrated. The spectrophotometric examination of 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes from the intoxicated animals showed absorption spectra similar to those observed for the dinitrophenylhydrazones of various carbonyls. Similar spectra, although magnified from a quantitative point of view, were also observed with 2,4-dinitrophenylhydrazine-treated phospholipids of liver microsomes peroxidized in the NADPH-Fe-dependent system. A time-course study of microsomal lipid peroxidation showed that the amount of 2,4-dinitrophenylhydrazine-reacting groups (carbonyl functions) in phospholipids of liver microsomes increases with the incubation time and is correlated to the amount of malonic dialdehyde formed in the incubation mixture. The kinetics of the production of 4-hydroxynonenal was somewhat similar to that of malonic dialdehyde formation. In both the in vivo conditions (CCl4 and BrCCl3 intoxications) the amount of carbonyl functions in microsomal phospholipids, which was higher in the BrCCl3-intoxicated animals as compared to the CCl4-poisoned ones, was close to that found in the vitro condition in which lipid peroxidation is induced by 6 microM Fe2+. The possible pathological significance of formation of carbonyl functions in membrane phospholipids is discussed.

Animals↗
PubMed1982

Evidence for aldehydes bound to liver microsomal protein following CCl4 or BrCCl3 poisoning.

Since it has been demonstrated in previous studies that peroxidation of liver microsomal lipids leads to the production of aldehydes provided with cytopathological activities--namely 4-hydroxyalkenals--evidence was searched for aldehydes bound to microsomal protein in in vivo conditions (CCl4 and BrCCl3 intoxications) in which peroxidation of lipids of hepatic endoplasmic reticulum had been demonstrated previously. The spectrophotometric analysis of 2,4-dinitrophenylhydrazine-treated non-lipoidal residues of liver microsomes from the intoxicated rats shows absorption spectra similar to those observed for the dinitrophenylhydrazones formed in the reaction of alkenals with -SH groups of proteins or low molecular weight thiols. Similar spectra, although magnified from a quantitative point of view, were obtained either with liver microsomes allowed to react with synthetic 4-hydroxynonenal or with liver microsomes peroxidized in the NADPH-Fe-dependent system. A time-course study of microsomal lipid peroxidation shows that the amount of 2,4-dinitrophenylhydrazine-reacting groups in the non-lipoidal residue of liver microsomes increases with the incubation time and is correlated to the amount of thiobarbituric acid-reacting products formed in the incubation mixture. In both the in vivo conditions (CCl4 and BrCCl3 intoxications) the amount of 2,4-dinitrophenylhydrazine-reacting groups in the non-lipoidal residue of liver microsomes increases from 15 min up to 2 h after poisoning and is higher, in every instance, in the BrCCl3-intoxicated animals compared to the CCl4-poisoned ones. Experiments carried out to ascertain the reliability of the spectrophotometric detection of protein-bound alkenals showed that in the in vitro system in which liver microsomes are allowed to react with 4-hydroxynonenal there is a good agreement between the binding value that can be calculated from the absorption spectrum and the binding value obtained by using labelled 4-hydroxynonenal.

Aldehydes↗
PubMed1981

[Mechanism of action of carbonyl compounds (4-hydroxyalkenals) derived from the peroxidation of hepatic microsomal lipids].

When carbonyl compounds released during the NADPH-Fe dependent peroxidation of liver microsomal lipids and identified as 4-hydroxyalkenals (almost entirely 4-hydroxynonenal) are incubated with liver microsomes, a substantial amount of these products can be recovered bound to the microsomal protein, by allowing the microsomal protein to react with 2,4-dinitrophenyl-hydrazine. Concomitantly with the binding a decrease in the "exposed" and total -SH groups is stechiometrically accounted for by the amount of carbonyl compounds bound to the microsomal protein. Still in concomitance with the binding, the G-6-Pase activity of the microsomes markedly decreased. Therefore a mechanism similar to that described (12) for the binding of 4-hydroxyalkenals to -SH groups of protein and enzymes can be postulated.

Aldehydes↗
PubMed1981

[Cytotoxic effects of carbonyl compounds (4-hydroxyalkenals) derived from peroxidation of hepatic microsomal lipids on isolated hepatocytes].

Carbonyl compounds released during the NADPH-Fe dependent lipid peroxidation and identified as 4-hydroxyalkenals (almost entirely as 4-hydroxynonenal), when incubated with isolated hepatocytes, produce loss of viability in 95% of the cells, as measured by the trypan blue exclusion test. They also produce an almost complete permeabilization of the plasma-membrane, as measured by the test of the permeability to NADH. concomitantly with the permeabilization of the plasma-membrane, a marked release of enzymes (lactate dehydrogenase and glutamate-pyruvate transaminase) from the hepatocytes occurs. These and other activities of the above mentioned carbonyl compounds suggest the possibility that these products represent some of the effective mediators of the liver injury produced by those toxins, such as CCL4, which promote the peroxidation of membrane lipids.

Alanine Transaminase↗
PubMed1981

Inhibition of protein synthesis by carbonyl compounds (4-hydroxyalkenals) originating from the peroxidation of liver microsomal lipids.

Carbonyl compounds released during the NADPH-Fe dependent peroxidation of liver microsomal lipids and identified as 4-hydroxyalkenals (almost entirely as 4-hydroxynonenal) inhibit protein synthesis in a rabbit reticulocyte lysate. The ID50 was 0.48 mM. The inhibitory effect was reproduced by synthetic 4-hydroxynonenal. The inhibition was already evident at 1-2 min of incubation. The addition of -SH groups to the incubation medium afforded a marked protection against the inhibition of protein synthesis. The inhibitory effect seems to be due to an interaction of the carbonyl compound with -SH groups essential for the cellular protein synthetic machinery.

Aldehydes↗
PubMed1980

[Variations in the fatty acid composition of rat erythrocyte membrane lipids at different ages].

The fatty acid composition of the erythrocyte lipids was studied in newborn, growing and adult rats. The percentage of oleic acid progressively decreased from the birth up to the adult age, while that of linoleic acid markedly increased. A smaller increase was also seen in the arachidonic acid content. These changes do not seem to be accounted for by differences in the percentages of the major phospholipid fractions or to differences in the age of the erythrocyte. Rather, variations in the fatty acid pattern of the various fractions of plasma lipids seem to be responsible for the lipid changes in the erythrocyte membrane.

Age Factors↗
PubMed1980

Effects of carbonyl compounds (4-hydroxyalkenals) originating from the peroxidation of liver microsomal lipids on various microsomal enzyme activities of the liver.

Carbonyl compounds released during the NADPH-Fe dependent peroxidation of liver microsomal lipids and identified as 4-hydroxyalkenals (almost entirely as 4-hydroxynonenal) while inhibiting microsomal enzymes (such as glucose 6-phosphatase and aminopyrine demethylase) which are affected by lipid peroxidation, have no effect on microsomal NADPH-cytochrome c reductase. The latter enzyme activity is unaffected (or even increased) when liver microsomes are allowed to peroxidize in the NADPH-Fe dependent system. NADPH-cytochrome c reductase, contrary to the other enzymes, is similarly unaffected after CCl4 poisoning, that is in a situation in which peroxidation of membrane lipids of liver endoplasmic reticulum has been unequivocally demonstrated. It appears therefore that the effects exherted by lipid peroxidation or by 4-hydroxyalkenals originating from lipid peroxidation parallel the effects of CCl4 intoxication in vivo.

Aldehydes↗
PubMedDate not supplied

Low levels of glucose-6-phosphate hydrolysis in the sarcoplasmic reticulum of skeletal muscle: involvement of glucose-6-phosphatase.

Glucose-6-phosphate hydrolysis was measured in a fraction obtained from rabbit fast-twitch skeletal muscle and corresponding to total sarcoplasmic reticulum, as well as in three subfractions containing longitudinal tubules, terminal cisternae or both structures. In all cases the levels of hydrolysis measured both in native and disrupted membranes were approximately 60-100 times lower than the microsomal glucose-6-phosphatase activity of the corresponding livers. In contrast to liver microsomes, most (up to 80%) of the glucose-6-phosphate hydrolysing activity in muscle sarcoplasmic reticulum membranes was not inactivated by pH 5.0 pre-incubation indicating that it was not catalysed by the specific glucose-6-phosphatase enzyme. Osmotically induced changes in light-scattering intensity of sarcoplasmic reticulum vesicles revealed that, in contrast to liver microsomes, sarcoplasmic reticulum vesicles were not selectively permeable to glucose-6-phosphate as mannose-6-phosphate was also permeable and in addition they were poorly permeable to glucose. Immunoblot experiments using antibodies raised against the glucose-6-phosphatase enzyme, and liver endoplasmic reticulum glucose and Pi translocases, failed to detect the presence of these protein components in sarcoplasmic reticulum membranes. Southern blot analysis of reverse transcriptase-polymerase chain reaction products from rat muscle revealed that glucose-6-phosphatase mRNA is present in muscle. Quantification of Northern blot analysis of liver and muscle mRNA indicated that muscle contains less than 2% of the amount of glucose-6-phosphate mRNA found in corresponding livers. We conclude that very low levels of specific glucose-6-phosphatase (e.g. as in liver; E.C. 3.1.3.9) are present in muscle sarcoplasmic reticulum and that the muscle and liver glucose-6-phosphatase systems have several different properties.

Animals↗