Biochemical and ultrastructural changes in rat liver plasma membranes after temporary ischemia

In this study we have attempted to correlate reversible and irreversible cell damage induced by in vivo or in vitro ischemia with characteristics of the plasma membranes of liver parenchymal cells, as detected biochemically and ultrastructurally. The effects of in vivo or in vitro ischemia appeared to be similar. It was virtually impossible to isolate a substantial membrane fraction from ischemic livers, probably because of changes in the physical properties of the membranes by ischemia. The isolated membranes of ischemic liver cells show ultrastructural changes including the occurrence of many vesicular profiles and alterations in junctional complexes expressed by extended and smudged electron densities along the lateral surfaces. The microvilli of the bile canaliculi disappeared after only 15 min ischemia and cytoplasmic densities associated with junctional complexes also appeared extended and smudged. These changes correspond with the alterations observed in ischemic isolated membranes. After 30 min in vivo ischemia the activity of 5'-mononucleotidase used as a marker enzyme for plasma membranes, decreased by 75%, whereas the activity of thymidine 5'-phosphodiesterase was reduced only slightly. The changes in these enzyme activities were more prominent after in vitro ischemia than after in vivo. The morphological and biochemical changes observed in rat hepatocyte plasma membrane during the early stage of injury have no value in predicting the occurrence of necrosis in a later phase of the process since profound changes occur in plasma membrane properties after even short periods of ischemia (i.e. during the reversible stage).     

Diurnal changes in succinate and D-3-hydroxybutyrate dehydrogenase activities of rat liver mitochondria after chronic alcohol consumption and withdrawal

1. The succinate dehydrogenase (SDH) and D-3-hydroxybutyrate dehydrogenase (HBDH) activities were measured over a 24-hr period in rat liver mitochondria after chronic alcohol ingestion and withdrawal. 2. The diurnal patterns of both the enzyme activities were shown to change after alcohol consumption, with 64-66% decrease in the daily mean levels. 3. The diurnal rhythms of the SDH and HBDH activities are partially restored 24-72 hr after alcohol withdrawal. 4. There was no correlation between changes in both the enzyme activities and the NAD+/NADH ratio of liver mitochondria from control, ethanol-fed and withdrawn rats over the day.     

Diurnal changes in the glucose and glycogen levels of the blood and liver of rats in chronic consumption of alcohol and after its withdrawal

Chronic ethanol ingestion by rats exerts almost no effect on the diurnal rhythms of the blood and hepatic glucose concentrations. The rhythm of liver glycogen alters substantially in ethanol-fed animals, the phase of the rhythm being shifted and the daily mean level of glycogen being reduced by a factor of 2. Much more drastic disturbances in carbohydrate metabolism occur after ethanol withdrawal than with ethanol consumption. The diurnal rhythm of liver glycogen becomes inverted in phasing, and the rhythmic amplitude reduced greatly as compared with controls. Both the blood and hepatic glucose concentrations are maintained at nearly constant levels for 18-21 h after ethanol withdrawal, but then the level of blood glucose sharply falls, while that of hepatic glucose somewhat increases. The liver cytosolic water/blood plasma water gradient of glucose 24 h after ethanol withdrawal achieves a value of 4 and remains low 24 h later. The liver glycogen level remains relatively high over the 24 h period after ethanol withdrawal despite the severe hypoglycemia, that can be a result of a limitation of the liver cell membrane permeability for glucose.     

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

Summary 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--naphthyl-amindase 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.Dedicated to Prof. Dr. E. Havinga on the occasion of his 70th birthday     

Increased acidic phospholipids in rat brain membranes after chronic ethanol administration

Two types of plasma membranes isolated from rat brain cortex were used to study the membrane-perturbing properties of ethanol. Rats administered ethanol in the form of a liquid diet showed an increase in levels of phosphatidylserines, phosphatidylinositols and phosphatidic acids as compared to controls. The results present evidence that chronic ethanol treatment results in an increase in the acidic phospholipids in brain membranes. This type of membrane modification may have important implications for the function of membrane transport enzymes such as (Na+, K+)-ATPase, which also increases in activity upon chronic ethanol administration.     

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.     

Neutral lipids of nuclear membranes and chromatin in rat liver after hydrocortisone administration

It is shown that over 80% of nuclear neutral lipids are found in the nuclear membrane preparations, whereas only 7%--in chromatin. The injection of hydrocortisone to animals significantly decreases the neutral lipids content in both fractions. These results and the data obtained previously by the authors indicate the possible participation of nuclear membrane and chromatin lipids in the process of hormonal regulation of the genetic activity.     

Different changes of plasma membrane beta-adrenoceptors in rat heart after chronic administration of propranolol, atenolol and bevantolol

Recently, tissue segment binding method with a hydrophilic radioligand [(3)H]-CGP12177 was developed to detect plasma membrane beta-adrenoceptors in rat heart (Horinouchi et al., 2006). In the present study, propranolol (40 mg kg(-1) day(-1)), atenolol (40 mg kg(-1) day(-1)) and bevantolol (200 mg kg(-1) day(-1)) were administered to rats for 6 weeks, and the changes of plasma membrane beta-adrenoceptors and their mRNA expression in rat ventricle were examined. Chronic administration of propranolol increased the beta(1)-adrenoceptors but decreased the beta(2)-adrenoceptors without changing total amount of plasma membrane beta-adrenoceptors. Atenolol increased both plasma membrane beta(1)- and beta(2)-adrenoceptors, whereas bevantolol had no effect on the beta-adrenoceptor density and their subtype proportions. In contrast, the density of beta-adrenoceptors detected in conventional homogenate binding study was extremely low (approximately 60% of plasma membrane beta-adrenoceptors detected with the tissue segment binding method) and the effects of chronic administration of beta-adrenoceptor antagonists were not necessarily in accord with those at the plasma membrane beta-adrenoceptors. The mRNA levels of beta(1)- and beta(2)-adrenoceptors were not altered by propranolol treatment, while beta(1)-adrenoceptor mRNA significantly decreased after administration of atenolol or bevantolol without changing the level of beta(2)-adrenoceptor mRNA. The present binding study with intact tissue segments clearly shows that the plasma membrane beta(1)- and beta(2)-adrenoceptors of rat heart, in contrast to the homogenate binding sites and the mRNA levels, are differently affected by chronic treatment with three beta-adrenoceptor antagonists; up- and down-regulations of beta(1)- and beta(2)-adrenoceptors, respectively, by propranolol, and up-regulation of both the subtypes by atenolol, but no significant change in both the subtypes by bevantolol.     

Developmental changes in rat liver alcohol dehydrogenase

Hepatic alcohol dehydrogenase activity and mass content change coordinately during development in male rats. Enzyme activity and mass content increase continuously after birth to 100 and 80% of maximal values within 6 weeks (2.6 ± 0.4 μmole/min/g liver and 92 ± 20 μg/g liver), respectively. When expressed per milligram of soluble proteins, both parameters peak at 3 weeks (0.052 ± 0.002 μmole/min/mg protein and 2.0 ± 0.4 μg/mg protein) and then decrease gradually to plateau levels. These decreases probably arise from a “surge” in soluble liver protein levels that occurs after weaning. Similar developmental patterns also occur in female rats. These findings are the first quantitative measurements of this enzyme in developing animals.     

D-galactosamine induced changes in rat liver plasma membranes lipid composition and some enzyme activities

The influence of D-galactosamine administration on rat liver plasma membranes lipid composition, fluidity and some enzyme activities was investigated. D-Galactosamine was found to induce an increase of the total phospholipids, the cholesterol level and membrane rigidity. In liver plasma membranes of D-galactosamine-treated rats the exogenous phospholipase A2 activity was enhanced about 2 fold, whereas the endogenous activity was slightly decreased. No alteration of the neutral sphingomyelinase activity was observed.     

Factors that modify the metabolism of ethanol in rat liver and adaptive changes produced by its chronic administration

1. 2,4-Dinitrophenol (0.1mm) increases by 100-160% the rate of ethanol metabolism by rat liver slices incubated in a medium saturated with a gas mixture containing O(2)+CO(2)+N(2) (18:5:77). Similar effects are produced by relatively low concentrations of arsenate (10mm). At higher concentrations (37.5 and 50mm) arsenate inhibits the rate of ethanol metabolism. 2. When liver slices are incubated under an atmosphere containing O(2)+CO(2) (95:5) the metabolism of ethanol increases by about 100% over that obtained with O(2)+CO(2)+N(2) (18:5:77). However, under these conditions the activating effect of dinitrophenol is no longer observed. 3. Chronic administration of ethanol to rats for 3-4 weeks, in doses from 3 to 8g/kg per day, increases by 70-90% the ability of the liver to metabolize ethanol. In the liver slices of these rats, although an O(2)+CO(2)+N(2) (18:5:77) mixture was used, dinitrophenol does not further increase the metabolism of ethanol. If the chronic administration of ethanol is discontinued for two weeks, the rate of ethanol metabolism is lowered to control values and the activating effect of dinitrophenol is recovered. 4. No change in alcohol dehydrogenase activity was found in the liver of the rats in which the metabolism of ethanol had been increased as a result of the chronic ethanol treatment; a 40% increase in the activity of succinate dehydrogenase was observed.     

Zonal changes in the rat liver after chronic administration of phenobarbitone in ultrastructural, morphometric and biochemical correlation.

Alterations in the liver of rats subjected to 24 days of continuous administration of phenobarbitone have been supplied bu subcellular fractionation, conventional electron microscopy and morphometric analysis. The increase in wet weight of the liver was found to result from a combination of cellular hypertrophy, hyperplasia and an enlarged hepatic blood space. In the centrilobular zone all the hepatocytes underwent a substantial proliferation of total ER, became enlarged and had an increased blood supply. However, in the periportal zone phenobarbitone caused changes in only 45% of the hepatocytes, the remainder being apparently resistent or tardy. An overall dramatic increase in hepatic RER was both measured and observed but the response involved hepatocytes in which the RER had proliferated as well as those which were depleted of RER or had stacks and cisternae that were severely shortened and dispersed. These alterations are discussed in relation to changes in RER after administration of agents causing hepatonecrosis. Possible reasons for the inability of other workers to detect a phenobarbitone-induced increase in RER are also put forward. After subcellular fractionation and corection for centrifugation losses into the 9500 g pellet, using the microsomal marker cytochrome P-450, phenobarbitone-induced increase in total ER was substantially less than that found by morphometric analysis. This indicates that during the preparation of microsomes a substantial proportion of intracellular membranes, having different metabolic and synthetic properties to those finally isolated, are discarded and emphasizes the need to exercise care when using microsomal preparations.     

Diurnal changes in the 3-hydroxybutyrate dehydrogenase activity of rat liver mitochondria during chronic alcohol consumption and after cessation

The activity of 3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) in the isolated rat liver mitochondria changes but slightly during 24 h. In animals which were fed 10% ethanol solution for 3.5 months the enzyme activity varies within the daily cycle: maximum--at 10 p. m., minimum--at 1-4 p. m. and at 4-7 a. m.; the average daily activity gets three times lower. The cessation of the alcohol consumption makes average daily activity of the enzyme only two times higher, but the character of daily changes in the activity is different: the maximum is observed at 4-7 p. m., the minimum at 4-7 a. m.     

The role of liver tryptophan pyrrolase in the opposite effects of chronic administration and subsequent withdrawal of drugs of dependence on rat brain tryptophan metabolism.

1. Chronic administration of morphine, nicotine or phenobarbitone has previously been shown to inhibit rat liver tryptophan pyrrolase activity by increasing hepatic [NADPH], whereas subsequent withdrawal enhances pyrrolase activity by a hormonal-type mechanism. 2. It is now shown that this enhancement is associated with an increase in the concentration of serum corticosterone. 3. Chronic administration of the above drugs enhances, whereas subsequent withdrawal inhibits, brain 5-hydroxytryptamine synthesis. Under both conditions, tryptophan availability to the brain is altered in the appropriate direction. 4. The chronic drug-induced enhancement of brain tryptophan metabolism is reversed by phenazine methosulphate, whereas the withdrawal-induced inhibition is prevented by nicotinamide. 5. The chronic morphine-induced changes in liver [NADPH], pyrrolase activity, tryptophan availability to the brain and brain 5-hydroxytryptamine synthesis are all reversed by the opiate antagonist naloxone. 6. It is suggested that the opposite effects on brain tryptophan metabolism of chronic administration and subsequent withdrawal of the above drugs of dependence are mediated by the changes in liver tryptophan pyrrolase activity. 6. Similar conclusions based on similar findings have previously been made in relation to chronic administration and subsequent withdrawal of ethanol. These findings with all four drugs are briefly discussed in relation to previous work and the mechanism(s) of drug dependence.