Regenerating Rat Liver: Correlations Between Estrogen Receptor Localization and Deoxyribonucleic Acid Synthesis

Estrogen receptor activity was quantitated in the cytosol and nucleus of normal rat liver and in regenerating rat liver at several time intervals after 75% hepatectomy. Cytosolic estradiol binding in regenerating liver decreases at 12, 24, and 48 h after hepatectomy and at 48 h is 30% of that in normal rat liver. Nuclear estrogen binding 48 h after surgery is elevated fivefold over normal values. No alterations in affinity of the receptor for estrogen have been observed. Specificity studies indicate that the estrogen receptors from both normal and regenerating liver were similar and are highly specific for estrogens. These changes in cellular distribution of receptors parallel increases in nuclear deoxyribonucleic acid synthesis and mitotic indices in the liver. © 1984, American Gastroenterological Association. All rights reserved

Changes of fatty acid metabolism and oxidative phosphorylation of rat liver mitochondria during 3'-Me-DAB feeding

Respiration, activity of oleate oxidation and composition of the total fatty acids of rat liver were investigated in 3'-Me-DAB feeding. 1. Oxidative phosphorylation of rat liver mitochondria decreased temporarily at relatively earlier stages (about 2 to 3 weeks) in 3'-Me-DAB feeding. 2. The activity of oleate oxidation of rat liver mitochondria decreased rapidly to about one third of that in control groups after the start of 3'-Me-DAB feeding. 3. In the composition of the total fatty acids of rat liver, the proportion of oleic acid increased in 3'-Me-DAB groups. 4. Unknown octadecamonoenoic acid was observed in liver mitochondria of rat fed on 3'-Me-DAB. 5. Proportions of oleic and palmitoleic acids in liver tumors and mitochondria of liver tumors induced by 3'-Me-DAB feeding increased remarkably in contrast with decrease in those of palmitic and eicosapolyenoic acids. 6. A possibility was discussed about how higher level of oleate in the liver cells in azo dye feeding may be concerned with the tumor induction.</p

Nucleic acids and protein synthesis in cancer cell mitochondria. I. Nucleic acids in rat hepatoma mitochondria

The contents of nucleic acids in rat liver and hepatoma mitochondria and the physico-chemical properties on DNA's isolated from these mitochondria were comparatively investigated. The results are briefly summarized as follows. 1. The contents of DNA and RNA per mg protein of the hepatoma cell mitochondria were about 10 and 2 to 4 times higher than those of rat liver mitochondria, respectively. 2. The &#955; max. and &#955;min. values of DNA isolated from the hepatoma mitochondria were 257 m&#956; and 231 m&#956;, respectively and those of DNA isolated from the nuclei were 259 m&#956; and 233 m&#956;, respectively, in saline-citrate, pH 7.0. 3. Three fractions of mitochondrial DNA were obtained by the sucrose density gradient and these DNA fractions corresponded, probably, to about 30 S, and 20 S and 14 S DNA's. 4. There was little difference in base compositions between nuclear and mitochondrial DNA's of the hepatoma cells. 5. The degree of hybridization between the nuclear and mitochondrial DNA's of the hepatoma cells was almost the same as that between the nuclear and nuclear DNA's of the hepatoma cells, and somewhat higher than that between the nuclear DNA of rat liver and the nuclear DNA of hepatoma cells. 6. &#34;Highly twisted&#34; circular, &#34;open&#34; circular and linear forms were observed in the DNA preparations of the hepatoma mitochondria. The average values of contour lengths of rat liver and the hepatoma DNA's observed at high frequency were 5.3 &#956; and 4.5 &#956;. 7. A discussion was made on the relation between the genetic informations of mitochondrial DNA and the formation of a mitochondrion in rat liver and the hepatoma cells.</p

Microsomal superoxide anion production and NADPH-oxidation in a series of 22 aziridinylbenzoquinones

Several 2,5-bis(1-aziridinyl)-1,4-benzoquinones (BABQs) can be activated to alkylating species by reduction of the quinone moiety. On the other hand, cytotoxicity of these compounds can be induced by redox cycling. A series of BABQs and their methylated analogues (BMABQs) with different substituents at the 3- and 6-position was synthesized in order to investigate the influence of the substituents on the reduction of the quinone moiety and on the generation of superoxide anion radicals with rat liver microsomes. Superoxide anion production (SAP) ranged from 3.7±0.1 to 742±74 nmoles/min/mg protein with quinone concentrations of 10 nmoles/ml. NADPH-oxidation was measured under the same conditions and it correlated well (r = 0.88, P < 0.001) with SAP. It ranged from 1.4±0.2 to 494±60 nmoles/min/mg protein. SAP for 22 B(M)ABQs showed a good correlation with the summated electronic substituent constant θpara,total (r = 0.86, P < 0.001). It can be concluded that superoxide anion production by 22 B(M)ABQs in rat liver microsomes can be predicted from structural features of the compounds

Estrogen Binding Protein Activity in Morris Hepatoma 7777 Compared With Normal Rat Liver

Estrogen binding protein activities were determined in the cytosol from adult male Buffalo rat liver and Morris hepatoma 7777. Estrogen receptors were prepared using the protamine sulfate precipitation technique of Chamness. The ability of various unlabeled steroids competing with [3H]estradiol was examined to establish the binding specificity. Estradiol binding in Morris hepatoma 7777 cytosol was greatly decreased compared with that present in hepatic cytosol prepared from normal rat liver. The receptor concentration expressed as femtomoles per milligram of cytoplasmic protein was 31.1 ± 2.9 SD for normal rat liver and 0.41 ± 0.88 SD for the hepatoma. Gel filtration chromatography revealed the presence of an estrogen binder in hepatoma cytosol which was not present in either normal liver or in the protamine sulfate precipitates of hepatoma cytosol. The molecular weight, binding specificity, and precipitation of this protein by specific antiserum suggests that it is α-fetoprotein. © 1984, American Gastroenterological Association. All rights reserved

Mitochondrial Swelling and Uncoupling Activity of Long-Chain Fatty Acids

The effect of various fatty acids on the swelling-contraction and oxidative phosphorylation of mitochondria from rat liver and Ehrlich ascites tumor cell have been studied and the results are as follows: 1. The swelling of rat liver mitochondria is induced by fatty acid. The extent of this uncoupling action is in the descending order of myristate, laurate, parlmitate, stearate and behenate in saturated fatty acid and linoleate, linoleneate, richinoleate and oleate in the unsaturated fatty acid. This swelling action is stronger with unsaturated fatty acids than that of saturated ones and cis form is stronger than trans form. 2. The uncoupling oxidative phosphorylation of rat liver mitochondria is also observed with these fatty acids and the activities are proportional to the degree of the swelling action. 3. The degree of swelling of rat liver mitochondria is proportional to the concentration of oleate and is inhibited by anaerobiosis and respiratory inhibitor except amytal. 4. The mitochondria swollen by fatty acid can be recontracted reversibly by ATP, Mg++ and bovine serum albumin. 5. The swelling action of sodium oleate is the strongest on mitochondria from rat liver, followed by those from the liver of Ehrlich ascites tumor bearing mouse, Ehrlich ascites tumor cells and solid Ehrlich tumor cells. 6. Sodium oleate inhibits the incorporation of 32p into ATP, ADP, GTP and UDPG in mitochondria.</p

Amiloride reduces portal hypertension in rat liver cirrhosis

Objective This study aimed to investigate the effect of amiloride on portal hypertension. Amiloride is known to inhibit Na(+)/H(+) exchangers on activated hepatic stellate cells. Methods Liver cirrhosis in rats was induced by bile duct ligation (BDL) or thioacetamide (TAA) administration. The effects of zymosan for Kupffer cell (KC) activation or a thromboxane (TX) analogue (U46619) were tested in isolated perfused livers of cirrhotic rats and in vivo. Downstream mechanisms were investigated using Rho kinase inhibitor (Y-27632) or amiloride. Acute and chronic effects of amiloride and canrenoate on portal pressure were compared in perfused livers and in vivo. TXB(2) efflux was measured by ELISA. The phosphorylation state of moesin (p-moesin) as an indicator of Rho kinase activity and expression of the thromboxane synthase were assessed by western blot analyses. The activity of hepatic stellate cells was analysed by western blot and staining for alpha-smooth muscle actin (alpha-SMA). Results In BDL rats, KC activation via zymosan increased portal pressure. This was attenuated by the Rho kinase inhibitor Y-27632. Increased thromboxane efflux following zymosan infusion remained unaltered by Y-27632. The infusion of amiloride attenuated zymosan- and U46619-induced increases in portal perfusion pressure. In vivo, direct administration of amiloride, but not of canrenoate, lowered portal pressure. In TAA and BDL rats, treatment with amiloride for 3 days reduced basal portal pressure and KC-induced increases in portal pressure whereas canrenoate had no effect. In livers of amiloride-treated animals, the phosphorylation state of moesin and the number of alpha-SMA positive cells were reduced. Conclusions Amiloride lowers portal pressure in rat liver cirrhosis by inhibition of intrahepatic vasocontraction. Therefore, patients with cirrhosis and portal hypertension may benefit from amiloride therapy

Phosphorylation of purine and pyrimidine nucleosides by isolated rat liver mitochondria

Formation of 5'-AMP, 5'-GMP, 5'-CMP and 5'UMP was confirmed in isolated rat liver mitochondria incubated with alpha-ketoglutarate, inorganic phosphate, purine nucleoside and pyrimidine nucleoside. Increased incorporation of 32Pi into ATP, GTP and UTP was observed by adding purine- and pyrimidine nucleosides. The phosphorylation of nucleosides was inhibited severely by arsenite and affected slightly by the addition of nuclear or post-mitochondrial fraction.</p

An electron cytochemical demonstration and biochemical analysis of adenosine triphosphatase activity in cancer cell plasma membrane

1. The studies of structure and function of the plasma membranes of cancer cells is extremely important for the elucidation of specificity of phenotypes of cancer cells. In order to bring this subject to light, plasma membranes, mitochondria, microsomes and nuclei have been isolated from the AH 130 ascites carcinoma cells and rat liver cells. The electron cytochemical observations and biochemical assays of M g&#178;+-Na+-K+-ATPase, ADPase, AMPase, and &#946;-glycerophosphatase activities have been carried out before and after the fixation with glutaraldehyde. 2. M g&#178;+-ATPase and Mg&#178;+-N a +-K +-ATPase are present in the isolated plasma membranes, mitochondria and microsomes in both AH 130 cells and rat liver cells. ADPase and AMPase of the mitochondria and microsomes show far lower activities than those of the corresponding enzymes found in rat liver plasma membrane. ADPase and AMPase of AH 130 cell fraction exhibit activity much lower or zero. Generally, enzymatic activity of the AH 130 cell fraction is much lower than that of rat liver cell fraction. 3. Mg&#178;+-Na+-K+-ATPase is completely abolished by 5% glutaraldehyde fixation while it shows less effect on Mg&#178;+-ATPase in the plasma membrane. ADPase and AMPase activities of the mitochondria and microsomes are completely inhibited by glutaraldehyde fixation. AMPase of the plasma membrane of rat liver is completely abolished while ADPase activity is not affected in any way. 4. Only Mg&#178;+-ATPase can be demonstrated electron cytochemically. Cytochemical reaction products of Mg&#178;+-ATPase are located at the outer layer of the plasma membrane of the AH 130 cells and rat liver tissue. In the isolated membrane fractions it is located at the inner layer. 5. &#961;-Chloromercuribenzoate has only a slight effect on Mg&#178;+-ATPase and Mg&#178;+-Na+-K+-ATPase activities of the rat liver membrane, while it inhibits these enzyme activities in the AH 130 cell membrane. NaF (1 mM) and NaN3 (1 mM) inactivate ADPase of the rat liver plasma memo brane. 6. In these experimental conditions, nonenzymatic hydrolysis of ATP by lead ions is not recognized. 7. It seems most reasonable to conclude that cytochemical electron microscopic demonstration of Mg&#178;+-ATPase after fixation with glutaraldehyde may serve as the absolute marker for the plasma membrane of ascites hepatoma and liver cells.</p