Oxidation and phosphorylation in rat liver nuclei and nuclear membranes in postnatal development and regeneration after partial hepatectomy]

Specific oxygen consumption by isolated nuclei of liver cells of newborn rats is higher and phosphorylation is lower as compared to adult animals. This is correlated with a higher free cytochrome oxidase activity as determined in the absence of detergents or tetramethyl-p-phenylenediamine. Correspondingly, oxygen consumption by isolated nuclear membranes of rat liver 44 hrs after partial hepatectomy is also increased and the P/O ratio is decreased 2.2-fold as compared to the controls.     

Cytochrome oxidase of rat liver nuclear membranes and its interaction with mitochondrial cytochrome oxidase]

The percent of mitochondrial protein contamination in nuclei decreased 10-fold (from 18 to 1.8%) under purification of protein-labelled mitochondria before their introduction into nuclei-free homogenate, cytochromoxidase activity being unchanged. Thus, cytochromoxidase activity of nuclei does not correlate with the amount of nuclei-adsorbed mitochondrial protein, which demonstrates the presence of nuclear cytochromoxidase independent on mitochondrial protein. Radioactivity of protein-labelled mitochondria is proportially distributed between globuline, deoxyribonucleoprotein, acid and residual nuclear proteins, as it is shown under fractionation of nuclei isolated from protein-labeled mitochondria containing homogenate. The comparison of mitochondrial protein contamination of nuclear membranes and their possible contamination with cytochromoxidase and suecinate-cytochrome-c-reducatase activities revealed that cytochromoxidase activity of nuclear membranes is twice higher and succinate-cytochrome-c-reductase activity is considerably lower than it can be referred to mitochondrial protein contamination. The ratio of cytochrome-c-oxidase and succinate-cytochrome-c-reductase activities in isolated nuclear membranes is 4-7 times as high as that in mitochondrial membranes under the same isolation procedure. The data obtained make possible to consider the cytochromoxidase activity of nuclear membranes to be really nuclear enzyme, and not a contominant of nucleipreparation with mitochondrial membranes.     

Enhancement of neutral phosphatase activity in the cytosol and nuclei of regenerating rat liver: role of endogenous regucalcin.

The role of endogenous regucalcin (RC) in the regulation of neutral phosphatase activity in regenerating rat liver was investigated. The liver weight reduced by a partial hepatectomy (about 70%) was completely restored at 72 h after surgery. Phosphotyrosine, phosphoserine, and phosphothreonine were used as the substrate for the assay of phosphatase activity. Phosphatase activity toward phosphotyrosine in the hepatic cytosol and nuclei was significantly increased at 24-72 h after hepatectomy. Such an increase was not seen in the case of phosphoserine and phosphothreonine. However, the presence of anti-RC monoclonal antibody (200 ng/ml) in the enzyme reaction mixture caused a remarkable elevation of phosphatase activity toward three phosphoaminoacids in the hepatic cytosol at 24 and 48 h after hepatectomy. In the liver nuclei after sham operation or hepatectomy, phosphatase activity toward three phosphoaminoacids was significantly raised by the addition of anti-RC antibody (150 ng/ml). The nuclear phosphatase activity toward phosphothreonine in regenerating liver was significantly enhanced in the presence of anti-RC antibody (100 and 150 ng/ml). The effect of anti-RC antibody to increase phosphatase activity toward three phosphoaminoacids in the cytosol and nuclei of regenerating liver was completely blocked by the addition of exogenous RC (1.0 microM). The present study demonstrates that protein phosphatase activity in the cytoplasm and nuclei is enhanced in regenerating rat liver. This enhancement may be suppressed by endogenous RC.     

Calcium-binding protein regucalcin inhibits deoxyribonucleic acid synthesis in the nuclei of regenerating rat liver

The effect of regucalcin, a Ca²⁺-binding protein isolated from rat liver cytosol, on deoxyribonucleic acid (DNA) synthesis in the nuclei of regenerating rat liver was investigated. At 1 day after partial hepatectomy, the liver weight was increased about 50% of that of sham-operated rats, and it reached to the same levels as sham operation at 3 days after hepatectomy. Nuclear DNA synthesis was markedly increased at 1 day after hepatectomy, and this increase was also seen at 3 days. Nuclear DNA synthesis was clearly enhanced in the presence of EGTA (0.4 mM) in the incubation mixture. The presence of Ca²⁺ ( 1.0–25 M) caused a significant decrease in the nuclear DNA synthesis of normal rat liver. Regucalcin (0.25 and 0.5 M) clearly inhibited the nuclear DNA synthesis of normal rat liver. This inhibition was also seen in the presence of Ca²⁺ (1.0 M). Moreover, in the liver nuclei obtained at 1 day after partial hepatectomy, the presence of regucalcin (0.05–0.5 M) caused a remarkable inhibition of nuclear DNA synthesis. This effect was also revealed in the presence of EGTA (0.4 mM). Thus, the inhibitory effect of regucalcin was remarkable in regenerating rat liver nuclei in comparison with that of normal rat liver. The present results demonstrate that regucalcin can suppress nuclear DNA synthesis in regenerating rat liver. We suppose that regucalcin may have a role in the regulation of nuclear DNA synthesis in liver cell proliferation.     

Dimethyl sulfoxide elicited increase in cytochrome oxidase activity in rat liver mitochondria in vivo and in vitro

A single intraperitoneal injection of dimethyl sulfoxide (275 mg/100 g body wt.) to rats stimulated cytochrome oxidase activity in liver mitochondria 2-5-fold. The enzyme activity remained at this level for as long as 5 days post-injection. There was however only 10.5% increase in the content of cytochromes a and a3 (as determined spectrophotometrically) in the same period in response to DMSO injection. The addition of either DMSO or dimethyl sulfate (a metabolite of DMSO) to isolated liver mitochondria also caused 2-3-fold increase in cytochrome oxidase activity. The results indicate that enhancement in cytochrome oxidase activity in liver mitochondria after administration of DMSO to rats is on account of activation of cytochrome oxidase caused by structural alterations in mitochondrial membranes rather than de novo synthesis of cytochrome oxidase.     

Aging-related decrease in hepatic cytochrome oxidase of the Fischer 344 rat

The effect of aging on the hepatic mitochondrial population has been determined using a rigorously defined group of Fischer 344 rats with known survivorship data. The age groups studied included mature adult controls (8.5 months; 100% survivorship), an intermediate aged group (17.5 months; 90% survivorship), and an aged group (29 months; 20% survivorship). Cytochrome oxidase activity and content were determined in homogenates and mitochondrial fractions. The mitochondrial fractions were characterized by determination of respiratory activity, and monoamine oxidase activity as well as evaluation of the polypeptide composition by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and two-dimensional electrophoresis. The yield of protein in the isolated mitochondrial fraction as well as the mitochondrial specific content decreased significantly as a function of aging. Mitochondrial specific content was determined from the specific activities of cytochrome oxidase in the homogenate (per gram liver) and in the isolated mitochondrial fraction (per mg protein). Specific activity of hepatic cytochrome oxidase decreased approximately 15% (P = 0.035) in homogenates from the 17.5-month animals with a further, highly significant (P = 0.0002) decrease (29%) in the 29-month animals. In contrast, there was no statistically significant difference among the age groups in the cytochrome oxidase specific activity in the isolated hepatic mitochondrial fractions. However, the percentage of the total homogenate cytochrome oxidase activity recovered in the isolated mitochondrial fraction decreased significantly in the 29-month animals (P = 0.0063 vs the 8.5-month controls; P = 0.022 vs the 17.5-month group). Cytochrome aa₃ content of total liver homogenates from aged animals decreased (P = 0.00064) which is in agreement with the decline in cytochrome oxidase specific activity in this age group. In the mitochondrial fraction from the aged animals, cytochrome aa₃ content was essentially unchanged which is consistent with the lack of aging-related change in mitochondrial cytochrome oxidase specific activity. In freshly isolated mitochondrial fractions, no aging-related alterations were observed in respiratory control and $\text{ADP}\text{O}$ ratios. The addition of exogenous NADH and cytochrome c did not change significantly the respiratory rate of hepatic mitochondria from control or aged animals. These results demonstrate the integrity of freshly isolated mitochondrial preparations from both control and aged Fischer 344 rats. In addition, there was no aging-related alteration in either monoamine oxidase specific activity or polypeptide composition. The similarities observed in the specific activities of cytochrome oxidase and monoamine oxidase, as well as in the cytochrome aa₃ content and polypeptide composition of the isolated mitochondrial fraction, suggest a generalized decrease in hepatic mitochondrial content as a function of aging rather than a selective loss of mitochondrial components.     

STUDIES ON ISOLATED CELL COMPONENTS : XVII. The Distribution of Cytochrome Oxidase Activity in Rat Liver Brei Fractionated in the Zonal Ultracentrifuge

The zonal ultracentrifuge was used to separate the subcellular components of rat liver brei into soluble phase, microsomal, mitochondrial, membranous fragments, and nuclear fractions during a single centrifugation. The centrifuge was run at 10,000 to 30,000 RPM for 15 to 240 minutes, and the rotor contained a 1200 ml sucrose gradient, varying linearly with radius from 17 to 55 per cent sucrose with a "cushion" of 66 per cent sucrose at the rotor edge. The distribution of the mitochondria was determined using cytochrome oxidase as the marker enzyme. An automated assay system for cytochrome oxidase was developed utilizing reduced cytochrome c as substrate, modules of the Technicon Autoanalyzer, and the Beckman DB Spectrophotometer. All of the cytochrome oxidase activity was restricted to a single peak in the gradient, and no activity could be detected in the zones occupied by the microsomes and nuclei. The mitochondrial fraction was isolated from rat liver brei in 0.25 M sucrose by differential centrifugation, and then run in the zonal ultracentrifuge.This fraction behaved in the zonal ultracentrifuge in the same way as mitochondria separated directly from intact brei. Observations of the isolated fractions in the phase contrast microscope indicated that a wide variety of granules was present in the mitochondrial zone in addition to the true mitochondria. Under the conditions employed, the mitochondria were sedimented essentially to their isopycnic position in the gradient at approximately 43.8 per cent sucrose, density 1.20 gm/cc.     

Suppressive role of endogenous regucalcin in the enhancement of deoxyribonucleic acid synthesis activity in the nucleus of regenerating rat liver

The role of endogenous regucalcin in the regulation of deoxyribonucleic acid (DNA) synthesis activity in the nuclei of regenerating rat liver after partial hepatectomy was investigated. The addition of regucalcin (0.25 and 0.5 microM) in the reaction mixture caused a significant decrease in the nuclear DNA synthesis activity of normal rat liver. This decrease was also seen in the presence of Ca2+-chelator EGTA (0.4 mM), indicating that the effect of regucalcin is not related to nuclear Ca2+. Nuclear DNA activity was significantly increased in the presence of anti-regucalcin monoclonal antibody (10-50 ng/ml) in the reaction mixture. The effect was completely abolished by the addition of regucalcin (0.5 microM). Nuclear DNA synthesis activity was significantly increased at 24, 48, and 72 h after partial heptectomy. The effect of anti-regucalcin monoclonal antibody (25 ng/ml) in increasing nuclear DNA synthesis activity was significantly enhanced at 24 and 48 h after partial hepatectomy. The presence of staurospone (10(-6) M), trifluoperazine (2 x 10(-5) M), or PD98059 (10(-5) M) in the reaction mixture caused a significant decrease in DNA synthesis activity in the nuclei obtained at 24 after partial hepateactomy. The effect of these inhibitors in the presence of anti-regucalcin monoclonal antibody (25 ng/ml) was greater than that in the absence of the antibody. The present study suggests that endogenous regucalcin plays a suppressive role in the enhancement of nuclear DNA synthesis activity in regenerating liver with cell proliferation after partial hepatectomy in rats.     

Impairment of liver regeneration during inhibition of mitochondrial protein synthesis by oxytetracycline

Under standard conditions, liver regeneration is not impaired if mitochondrial protein synthesis is completely blocked. By treating rats with oxytetracycline for various periods of time directly prior to partial hepatectomy, livers were led to a condition of relative deficiency in cytochrome c oxidase and ATP synthetase. To this end, oxytetracycline was administered by means of continuous intravenous infusion up to concentrations of 20 μg/ml serum, giving a gradual decrease in cytochrome c oxidase activity. This activity was used as a marker for functionally capable mitochondria and as a tool to monitor the efficiency of inhibition of mitochondrial protein synthesis. It is shown that liver regeneration is strongly impaired after a period of pretreatment of 22 days or more and continuation of oxytetracycline treatment during regeneration. The mitochondrial respiratory capacity is reduced to 14% of the control value under these conditions. To obtain inhibitory levels within the regenerating liver, it was necessary to raise the serum levels slightly above 20 μg/ml. This measure is most likely required because of the poor vascularization of the regenerating liver. The serum levels were kept, however, far below those known to inhibit cytoplasmic protein synthesis. The results show that in normal liver the respiratory capacity must be reduced drastically before energy-requiring processes become affected. In Zajdela hepatoma cells, similar effects are found after reduction of the cytochrome c oxidase activity to 38%. This difference in sensitivity is probably based on the different mitochondrial content of liver cells and the liver-derived Zajdela cells.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.     

Different types of mitochondria in parenchymal and non-parenchymal rat-liver cells.

1. Intact and pure parenchymal and non-parenchymal cells were isolated from rat liver. The specific activities of several mitochondrial enzymes were determined in both parenchymal and non-parenchymal cell homogenates to characterize the mitochondria in these liver cell types. 2.In general the activities of mitochondrial enzymes were lower in non-parenchymal liver cells than in parenchymal cells. The specific activity of pyruvate carboxylase in non-parenchymal cells expressed as the percentage of that in parenchymal cells was onlu 2% for glutamate dehydrogenase 4.3% and for cytochrome c oxidase 79.4%. Monoamine oxidase, as an exception, has an equal specific activity in both cell types. 3. The activity ratio of pyruvate carboxylase at 10 mM pyruvate over 0.1 mM pyruvate is 3.35 for parenchymal cells and 1.50 for non-parenchymal cells. This indicates that non-parenchymal liver cells only contain the high affinity form of pyruvate carboxylase in contrast to parenchymal cells. 4. The ratio of glycerol-3-phosphate cytochrome c reductase over succinate cytochrome c reductase activity differs from parenchymal (0.01) and non-parenchymal cells (0.10). This might indicate that the glycerol-3-phosphate shuttle, which is important for the transport of reduction equivalents for cytosol to mitochondria is relatively more active in non-parenchymal cells than in parenchymal cells. 5. The activity pattern of mitochondrial enzymes in parenchymal and non-parenchymal cell homogenates indicates that these cell types contain different types of mitochondria. The presence of these different cell types in liver will therefore contribute to the heterogeneity of isolated rat liver mitochondria in which the mitochondria from non-parenchymal cells might be considered as "non-gluconeogenic".     

Quantitative isolation of liver mitochondria by zonal centrifugation

A method was developed using zonal centrifugation to recover liver mitochondria quantiatively and free of other cellular components from a sample of whole homogenate. The fractions containing mitochondria were identified by the distribution of cytochrome oxidase and these fractions contained over 90% of the total cytochrome oxidase recovered. The mitochondrial fractions were found to be only slightly contaminated by 5′-nucleotidase (plasma membranes), acid phosphatase (lysosomes), glucose-6-phosphatase (microsomes), and catalase (peroxisomes). There was no detectable contamination by nuclear DNA (nuclei). This method was used to quantitate total liver mitochondrial protein. The development of this procedure provides a means for following total changes in mitochondrial components during mitochondrial biogenesis.     

Liver lipase and high-density lipoprotein. Lipoprotein changes after incubation of human serum with rat liver lipase

Under standard conditions, liver regeneration is impaired if mitochondrial protein synthesis is completely blocked. By treating rats with oxytetracycline for various periods of time directly prior to partial hepatectomy, livers were led to a condition of relative deficiency in cytochrome c oxidase and ATP synthetase. To this end, oxytetracycline was administered by means of continuous intravenous infusion up to concentrations of 20 micrograms/ml serum, giving a gradual decrease in cytochrome c oxidase activity. This activity was used as a marker for functionally capable mitochondria and as a tool to monitor the efficiency of inhibition of mitochondrial protein synthesis. It is shown that liver regeneration is strongly impaired after a period of pretreatment of 22 days or more and continuation of oxytetracycline treatment during regeneration. The mitochondrial respiratory capacity is reduced to 14% of the control value under these conditions. To obtain inhibitory levels within the regenerating liver, it was necessary to raise the serum levels slightly above 20 micrograms/ml. This measure is most likely required because of the poor vascularization of the regenerating liver. The serum levels were kept, however, far below those known to inhibit cytoplasmic protein synthesis. The results show that in normal liver the respiratory capacity must be reduced drastically before energy-requiring processes become affected. In Zajdela hepatoma cells, similar effects are found after reduction of the cytochrome c oxidase activity to 38%. This difference in sensitivity is probably based on the different mitochondrial content of liver cells and the liver-derived Zajdela cells.     

Receptor-mediated gonadotropin action in ovary. Differential effects of various gangliosides and cholera enterotoxin on 125I-choriogonadotropin binding, production of adenosine 3':5'-monophosphate and steroidogenesis in rat ovarian cells.

Poly(A)-containing RNA isolated from liver nuclei of untreated rats and 3 h or 12 h after partial hepatectomy or sham operation was hybridized to the complementary DNAs (cDNAs). In the homologous reactions two major components could be seen. When compared to normal liver, the complexity of the least abundant class was lower in nuclei from livers 3 h after partial hepatectomy and was higher in those isolated 12 h after operation. The heterologous reactions revealed an increase of some abundant poly(A)-containing sequences and a loss or dilution of rare sequences 3 h after operation. The latter effect was not specific to the regeneration process but occurred after laparotomy as well. 12 h after partial hepatectomy, however, about 10% new poly(A)-containing sequences were detected, corresponding to about 5000 molecules of 4500 nucleotides length, which are unique to regenerating nuclei.     

运动性内源自由基对大鼠肝线粒体的影响

采用大鼠耗竭游泳作为动物运动模型,用戊巴比妥酸(TBA)法测定脂质过氧化水平,薄层色谱—定磷法测定心磷脂含量,细胞色素C还原法测定细胞色素C氧化酶活性。结果如下:耗竭运动时,肝线粒体脂质过氧化水平升高24％;心磷脂含量下降21％;细胞色素C氧化酶活性下降25％。上述结果表明:耗竭运动时,机体内源自由基的产生是运动损伤和整体疲劳的原因之一。     

The time-course development of the effects of ethanol ingestion on choline oxidase

Male rats were fed a low-fat diet containing 36% of calories as ethanol, and the time-course development of the effects of ethanol on liver mitochondrial oxidation of choline was determined. Ethanol induced an increase in choline oxidase at days 2, 5 and 7 after being introduced into the diet. Due to an observed 32% increase in total fatty acids in the whole liver, defatted bovine serum albumin was added to the buffer used to homogenize the liver. The presence of bovine serum albumim resulted in a significant decrease in choline oxidase activity at days 2 and 5; however, ethanol still induced an increase in choline oxidase activity in these mitochondria. The total fatty acid concentration of mitochondria prepared in the absence of bovine serum albumin increased steadily until day 5; however, by day 7 the fatty acid concentration had returned to control levels. The addition of bovine serum albumin to the homogenization medium prevented the increase in the total amount of fatty acids. The fatty acid composition of the bovine serum albumin-treated mitochondria, however, was not different from the mitochondria is isolated in the absence of bovine serum albumin. Further, the addition of a free fatty acid to isolated mitochondrial preparations caused about a 100% increase in choline oxidase. These data are consistent with the idea that choline oxidase may be regulated to some extent by an influx or an increase in free fatty acids in the liver as a result of ethanol ingestion. Thus, a second mechanism has been described which contributes to the increase in choline oxidase after ethanol ingestion.     

Changes in polyamine-oxidizing capacity of peroxisomes under various physiological conditions in rats

Rat liver peroxisomal polyamine oxidase activity was determined under various physiological conditions by using the peroxidase method with phenol and 4-aminoantipyrine. N1-Acetylpolyamines such as N1-acetylspermine and N1-acetylspermidine were better substrates than the free polyamines. The polyamine oxidase activity in rat peroxisomes increased significantly when cell proliferation was high. The activity began to appear in fetal liver at the 16th approximately 18th day of pregnancy and peaked in neonatal liver on the first day (approx. 1.7-times higher than in adult liver). In regenerating rat liver, only polyamine oxidase activity among the peroxisomal enzymes tested was increased considerably 12 h after partial hepatectomy (approx. 2.8-fold over the control liver). Finally, the enzyme activity was significantly increased by administration of clofibrate, a peroxisome proliferator, which also causes hepatomegaly. In all cases, the increase in polyamine oxidase activity was not more than 3-fold. Since the level of polyamine oxidase activity in the normal liver is more than adequate in relation to the level of the substrates, the slight but significant increase under conditions of cell proliferation may have a role in modulating levels of polyamines in the proliferating liver tissue.     

Immunocytochemical determination of ploidy class-dependent bromodeoxyuridine incorporation in rat liver parenchymal cells after partial hepatectomy

Summary Immunocytochemistry of bromodeoxyuridine (BrdU) incorporated in DNA was performed on cryostat sections of rat liver and on isolated hepatocytes after partial hepatectomy using a two-step labeling technique. The method enabled the detection of S-phase nuclei in both tissue preparations. Quantification of the number of labeled nuclei in sections showed that the number of nuclei in S-phase increased from 0.3% in control liver to about 36% at 24 h after partial hepatectomy. The detection of BrdU in isolated hepatocytes showed the same labeling index of binuclear diploid, mononuclear tetraploid and binuclear tetraploid cells. A special role for mononuclear diploid cells in proliferation did not seem to occur.