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G Bellomo

Publications and source records attributed to G Bellomo.

At least 145 records · Page 8Linked to original sources
PubMed1988

Menadione-induced bleb formation in hepatocytes is associated with the oxidation of thiol groups in actin.

Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) or the thiol oxidant, diamide (azodicarboxylic acid bis(dimethylamide)), resulted in the appearance of numerous plasma membrane protrusions (blebs) preceding cell death. Analysis of the Triton X-100-insoluble fraction (cytoskeleton) extracted from treated cells revealed a dose- and time-dependent increase in the amount of cytoskeletal protein and a concomitant loss of protein thiols. These changes were associated with the disappearance of actin and formation of large-molecular-weight aggregates, when the cytoskeletal proteins were analyzed by polyacrylamide gel electrophoresis under nonreducing conditions. However, if the cytoskeletal proteins were treated with the thiol reductants, dithiothreitol or beta-mercaptoethanol, no changes in the relative abundance of actin or formation of large-molecular-weight aggregates were detected in the cytoskeletal preparations from treated cells. Moreover, addition of dithiothreitol to menadione- or diamide-treated hepatocytes protected the cells from both the appearance of surface blebs and the occurrence of alterations in cytoskeletal protein composition. Our findings show that oxidative stress induced by the metabolism of menadione in isolated hepatocytes causes cytoskeletal abnormalities, of which protein thiol oxidation seems to be intimately related to the appearance of surface blebs.

Animals↗
PubMed1988

Meal-induced arterial blood pressure variations in the elderly.

The effect of a standard meal on arterial blood pressure (ABP) was investigated in a group of 14 healthy elderly subjects (age greater than or equal to 65 years) and 11 controls (age less than or equal to 45 years) by means of automated noninvasive ABP monitoring. The magnitude of postprandial ABP reduction was significantly greater in the elderly subjects (systolic ABP: -22.3 +/- 4.9 vs. -7.5 +/- 2.2 mm Hg; p less than 0.05; diastolic ABP: -13.7 +/- 3.1 vs. -6.2 +/- 1.4 mm Hg; p less than 0.05) and ABP decrease was not compensated by heart rate acceleration.

Adult↗
PubMed1987

Formation and reduction of glutathione-protein mixed disulfides during oxidative stress. A study with isolated hepatocytes and menadione (2-methyl-1,4-naphthoquinone).

Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) resulted in a dose-dependent depletion of intracellular reduced glutathione (GSH), most of which was oxidized to glutathione disulfide (GSSG). Menadione metabolism was also associated with a dose- and time-dependent inhibition of glutathione reductase, impairing the regeneration of GSH from GSSG produced during menadione-induced oxidative stress. Inhibition of glutathione reductase by pretreatment of hepatocytes with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) greatly potentiated both GSH depletion and GSSG formation during the metabolism of low concentrations of menadione. Concomitant with GSH oxidation, mixed disulfides between glutathione and protein thiols were formed. The amount of mixed disulfides produced and the kinetics of their formation were dependent on both the intracellular GSH/GSSG ratio and the activity of glutathione reductase. The mixed disulfides were mainly recovered in the cytosolic fraction and, to a lesser extent, in the microsomal and mitochondrial fractions. The removal of glutathione from protein mixed disulfides formed in hepatocytes exposed to oxidative stress was dependent on GSH and/or cysteine and appeared to occur predominantly via a thiol-disulfide exchange mechanism. However, incubation of the microsomal fraction from menadione-treated hepatocytes with purified glutathione reductase in the presence of NADPH also resulted in the reduction of a significant portion of the glutathione-protein mixed disulfides present in this fraction. Our results suggest that the formation of glutathione-protein mixed disulfides occurs as a result of increased GSSG formation and inhibition of glutathione reductase activity during menadione metabolism in hepatocytes.

Animals↗
PubMed1987

Alterations in inositol phosphate production during oxidative stress in isolated hepatocytes.

The level of inositol phosphates was measured in rat hepatocytes treated with 2-methyl-1,4-naphthoquinone (menadione) or tert-butyl hydroperoxide, which cause Ca2+ mobilization from intracellular stores and an increase in cytosolic free Ca2+ concentration. Although neither agent produced any apparent changes in the resting level of inositol phosphates, pretreatment of hepatocytes with either menadione or tert-butyl hydroperoxide, as well as with several sulfhydryl reagents, markedly inhibited the increase in inositol phosphates induced by both hormonal and nonhormonal stimuli. Addition of dithiothreitol to menadione- or tert-butyl hydroperoxide-treated hepatocytes reversed this inhibition and reestablished responsiveness to extracellular stimuli. Our findings suggest that the inhibition of the inositol phosphate response by menadione and tert-butyl hydroperoxide occurs through the modification of critical sulfhydryl group(s) and that the alterations in intracellular Ca2+ homeostasis occurring during the metabolism of menadione and tert-butyl hydroperoxide in hepatocytes are not mediated by inositol phosphates.

Adenosine Triphosphate↗
PubMed1987

Prolonged fructose feeding and aldose reductase inhibition: effect on the polyol pathway in kidneys of normal rats.

The effects of diets with differing carbohydrate composition on the kidney polyol pathway were investigated. The diets employed were F = fructose rich, G = glucose rich, S = cornstarch rich, and were fed for 30 days to six groups of 12 normal male Sprague-Dawley rats with and without addition of the aldose reductase inhibitor tolrestat (T). Fructose feeding resulted in higher kidney sorbitol levels (F = 0.847 +/- 0.152, G = 0.354 +/- 0.087, S = 0.207 +/- 0.041 microM/g wet wt, P less than 0.05). This was not observed in the tolrestat-treated animals (F + T = 0.182 +/- 0.024, G + T = 0.149 +/- 0.021, S + T = 0.152 +/- 0.020 microM/g wet wt). Aldose reductase activity was reduced with tolrestat administration (F = 0.0208 +/- 0.0023, F + T = 0.0048 +/- 0.0005; G = 0.0210 +/- 0.0002, G + T = 0.0059 +/- 0.0008; S = 0.0227 +/- 0.0022, S + T = 0.0062 +/- 0.0007 microU). Myoinositol levels did not differ among groups (F = 1.973 +/- 0.182, G = 2.291 +/- 0.307, S = 2.066 +/- 0.155 microM/g wet wt), but tended to increase with aldose reductase inhibition (F + T = 2.253 +/- 0.186, G + T = 2.713 +/- 0.166, S + T = 2.618 +/- 0.221 microM/g wet wt). Plasma glucose was higher in the fructose-fed rats (F = 10.78 +/- 0.55, G = 9.09 +/- 0.058, S = 9.03 +/- 0.52, F + T = 9.75 +/- 0.61, G + T = 8.42 +/- 0.64, S + T = 8.81 +/- 0.49 mM/liter). It is concluded that prolonged fructose feeding results in the accumulation of sorbitol in the kidney, caused by increased flux of glucose through the polyol pathway. This can be prevented by aldose reductase inhibition.

Aldehyde Reductase↗
PubMed1986

Cystamine induces toxicity in hepatocytes through the elevation of cytosolic Ca2+ and the stimulation of a nonlysosomal proteolytic system.

Infusion of cystamine into the isolated, perfused rat liver resulted in tissue damage preceded by the formation of cystamine-protein mixed disulfides which were mainly detected in the plasma membrane fraction. Hepatotoxicity was prevented when dithiothreitol was infused after cystamine or when the calcium antagonist, verapamil, was co-infused with the disulfide. In isolated hepatocytes, the formation of cystamine-protein mixed disulfides was associated with an inhibition of plasma membrane Ca2+-ATPase activity and a decreased rate of Ca2+ efflux from the cells. This resulted in intracellular Ca2+ accumulation which was followed by a stimulation of both phospholipid hydrolysis and proteolysis, as indicated by enhanced rates of release of radioactivity from hepatocytes prelabeled with [14C]arachidonate and [14C]valine, respectively. Preincubation of hepatocytes with the calmodulin inhibitor, calmidazolium, or with the phospholipase inhibitors, chlorpromazine and dibucaine, inhibited the stimulation of [14C]arachidonate release by cystamine. However, none of these agents prevented the onset of cystamine toxicity in hepatocytes. In contrast, pretreatment of the cells with antipain or leupeptin, two inhibitors of Ca2+-activated proteases, abolished the stimulation of proteolysis by cystamine and also protected the cells from cystamine toxicity. Our results suggest that the perturbation of intracellular Ca2+ homeostasis by cystamine is caused by the inhibition of Ca2+ efflux associated with the formation of cystamine-protein mixed disulfides in the plasma membrane and that subsequent cytotoxicity results from Ca2+-activation of a nonlysosomal proteolytic system.

Animals↗
PubMed1986

Ca2+ homeostasis and cytotoxicity in isolated hepatocytes: studies with extracellular adenosine 5'-triphosphate.

The incubation of isolated rat hepatocytes with extracellular adenosine 5'-triphosphate (ATP) resulted in an inhibition of Ca2+ efflux. The ATP-induced Ca2+ accumulation as determined by the increase in phosphorylase a activity and the Ca2+-sensitive fluorescent indicator (2-[(2-bis-[carboxymethyl]-amino-5-methylphenoxy)-methyl]-6-methoxy-8- bis-[carboxymethyl]aminoquinoline-tetrakis-[acetoxymethyl]ester) (Quin 2-AM) was associated with both the hydrolysis of ATP and the phosphorylation of a 110 kDa protein. No significant alteration in the intracellular ATP level was observed. The appearance of surface blebs and cytotoxicity followed the rise in cytosolic Ca2+, suggesting that the increased free Ca2+ may be responsible for the loss of viability. When a calmodulin inhibitor, 1-[bis(4-chlorophenyl)methyl]-3-[ 2-(2,4-dichlorophenyl)-2-[(2,4-dichlorophenyl)methoxy] ethyl]-1H- imidazolium chloride (calmidazolium), was included in the medium prior to ATP addition, bleb formation was reduced and the loss of viability was completely prevented, indicating that a Ca2+-calmodulin process may be involved in the initiation of cytotoxicity.

Adenosine Triphosphate↗
PubMed1986

tert-Butylhydroperoxide-induced toxicity in isolated hepatocytes: contribution of thiol oxidation and lipid peroxidation.

Incubation of isolated rat hepatocytes with tert-butylhydroperoxide resulted in marked cytotoxicity preceded by intracellular glutathione depletion and extensive lipid peroxidation. Addition of antioxidants delayed, but did not prevent, this toxicity. A significant decrease in protein-free sulfhydryl groups also occurred in the presence of tert-butylhydroperoxide; direct oxidation of protein thiols and mixed disulfide formation with glutathione were responsible for this decrease. The involvement of protein thiol depletion in tert-butylhydroperoxide-induced cytotoxicity is suggested by our observation that administration of dithiothreitol, which caused re-reduction of the oxidized sulfhydryl groups and mixed disulfides, efficiently protected the cells from toxicity. Moreover, depletion of intracellular glutathione by pretreatment of the hepatocytes with diethyl maleate accelerated and enhanced the depletion of protein thiols induced by tert-butylhydroperoxide and potentiated cell toxicity even in the absence of lipid peroxidation.

Animals↗
PubMed1986

Cytotoxicity of phenazine methosulfate in isolated rat hepatocytes is associated with superoxide anion production, thiol oxidation and alterations in intracellular calcium ion homeostasis.

The metabolism of phenazine methosulfate (PMS) by isolated rat hepatocytes is associated with superoxide anion production, and with a substantial decrease in intracellular levels of reduced glutathione, most of which is oxidized to GSSG. A marked loss of protein-free sulfhydryl groups also occurs when intracellular glutathione is depleted, and cytotoxicity follows. These effects are associated with the inhibition of the plasma membrane Ca2+-ATPase and with intracellular accumulation of calcium ion which is preferentially sequestered in mitochondria. Maintenance of protein sulfhydryl groups in the reduced state by dithiothreitol (DTT) prevents the alterations in intracellular calcium homeostasis and protects against toxicity.

Animals↗
PubMed1985

Mechanisms responsible for carbon tetrachloride-induced perturbation of mitochondrial calcium homeostasis.

Incubation of isolated hepatocytes with CCl4 results in early reduction of the intracellular calcium content, mostly due to loss from the mitochondrial compartment. CCl4 treatment directly affects mitochondrial functions as indicated by the inhibition of Ca2+ uptake in cells permeabilized to the ion by digitonin exposure and by the reduction of intracellular ATP content in hepatocytes incubated in a glucose-free medium. Such mitochondrial damage is not caused by CCl4-induced stimulation of lipid peroxidation since it is not prevented by alpha-tocopherol, used at a concentration able to inhibit completely peroxidative reactions without interfering with CCl4 activation. All data together are in favour of a direct action of CCl4-reactive metabolites on liver cell calcium homeostasis.

Adenosine Diphosphate↗
PubMed1985

On the role of thiol groups in the inhibition of liver microsomal Ca2+ sequestration by toxic agents.

ATP-dependent Ca2+ sequestration by rat liver microsomes was assayed using three different methods, and characterized with regard to the effect of various inhibitors. When glucose and hexokinase were added in combination to deplete ATP in the incubation, Ca2+ uptake was followed by rapid release of Ca2+ from the microsomes. Ca2+ sequestration was inhibited by reagents that cause alkylation (e.g. p-chloromercuribenzoate) or oxidation (e.g. diamide) of protein sulfhydryl groups. Moreover, pretreatment of the microsomes with cystamine, which causes formation of mixed disulfides with protein thiols, also resulted in the inhibition of Ca2+ sequestration. It is concluded that microsomal Ca2+ sequestration is critically dependent on protein sulfhydryl groups, and that modification of protein thiols may be an important mechanism for the inhibition of microsomal Ca2+ sequestration by a variety of toxic agents.

Adenosine Triphosphate↗
PubMed1985

Glutathione S-conjugates stimulate ATP hydrolysis in the plasma membrane fraction of rat hepatocytes.

Incubation of a rat hepatocyte plasma membrane fraction with micromolar concentrations of either glutathione disulfide or various glutathione S-conjugates resulted in a several-fold increase in the rate of ATP hydrolysis. This stimulation was further enhanced when the plasma membrane fraction had been pretreated with agents that arylate or oxidize sulfhydryl groups, suggesting that this ATPase activity is modulated by the protein thiol status of the plasma membrane. It is proposed that this newly discovered ATPase may function in the cellular extrusion of both glutathione disulfide and glutathione S-conjugates.

Adenosine Triphosphatases↗
PubMed1985

Demonstration and partial characterization of glutathione disulfide-stimulated ATPase activity in the plasma membrane fraction from rat hepatocytes.

A highly purified plasma membrane fraction isolated from rat hepatocytes was found to catalyze the hydrolysis of ATP in response to micromolar concentrations of glutathione disulfide (GSSG). This process exhibited distinct kinetic parameters suggesting the existence of both a high and low affinity component. The apparent Km values (GSSG) for ATP hydrolysis were 140 microM and 1 mM for the high affinity and low affinity components, respectively. Disulfides other than GSSG were also found to stimulate ATP hydrolysis. The similarity between the kinetic properties of the GSSG-stimulated ATPase and those reported for GSSG transport in erythrocytes (Kondo, T., Dale, G. L., and Beutler, E. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 6359-6362) suggests that the ATPase may function in the active extrusion of intracellular GSSG.

Adenosine Triphosphatases↗