Subcellular localization of glycogen-UDP-glucosyltransferase in the hepatocytes of intact and fasting rats

Electron microscope cytochemical techniques by Takeuchi et al. were used to study the localization of glycogen-UDP-glucosyl-transferase (GUDPG) in hepatocytes of intact and fasting rats. Glycogen was found in the cytoplasm, nuclei, mitochondria, microbodies and lysosomes of the hepatocytes of intact and fasting animals after special cytochemical procedures for determination of GUDPG.     

Topographic distribution of dividing hepatocytes in the regenerating liver lobule in the period of maximum mitotic activity

Topographic distribution of dividing hepatocytes was studied in the liver lobule after hepatectomy performed at 10-11.30 a. m. The studies were conducted 20-32 h after surgery during the first increase in mitotic activity. It has been established that dividing hepatocytes appear simultaneously in all lobule areas, i. e. that all the hepatocytes are capable of responding to regenerating stimuli. However, further behaviour of dividing hepatocytes becomes different. It is concluded that hepatocytes from outer and intermediate lobule areas possess a more pronounced proliferative potential during peak mitotic activity after partial hepatectomy.     

Variations in immunoreactivity of angiotensinogen and cathepsins B and H in rat hepatocytes over 24 hours

To examine variations in immunoreactivity of angiotensinogen and cathepsins B and H in hepatocytes over 24 hr, rat liver was examined immunohistochemically. Immunoreactivity of angiotensinogen and cathepsins B and H in periportal and perivenous hepatocytes varied significantly over 24 hr, when analyzed by an image analyzer. In periportal and perivenous hepatocytes, immunoreactivity of angiotensinogen was highest at 0800 hr and lowest at 2000 hr or 0000 hr, whereas that of cathepsins B and H was maximal at 1600 hr and minimal at 0400 hr or 0800 hr. Proteolytic activities of cathepsins B and H in liver extracts varied in parallel to the variations in immunoreactivity of these enzymes. Localization of angiotensinogen in the liver acinus was inversely correlated to that of cathepsins B and H; angiotensinogen was predominantly localized in periportal hepatocytes, but cathepsins B and H were in perivenous hepatocytes at each time point examined. These results suggest that angiotensinogen in hepatocytes is actively synthesized and secreted early in the light period, whereas proteolytic activities in lysosomes of hepatocytes are augmented late in the light period.     

Action of hepatic chalones on the proliferation of hepatocytes from intact and denervated livers

The paper is concerned with the action of chalones, tissue-specific inhibitors of cell proliferation, on DNA synthesis and mitotic activity of hepatocytes in the intact and denervated liver during regeneration. Experiments were made on Wistar rats. Liver denervation was performed by bilateral subdiaphragmal vagotomy. In control and vagotomized animals, two thirds of the liver was resected. The data obtained indicate that chalones noticeably reduce the number of DNA-synthesizing cells and mitoses in the regenerating liver of intact animals. During regeneration of the denervated liver, chalones do not produce any inhibitory action on the intensity of proliferation. Analysis of the data obtained allows a conclusion that preservation of adequate innervation of the organ is needed for realization of the action of hepatic chalones.     

Protein denaturation in intact hepatocytes and isolated cellular organelles during heat shock

There is circumstantial evidence that protein denaturation occurs in cells during heat shock at hyperthermic temperatures and that denatured or damaged protein is the primary inducer of the heat shock response. However, there is no direct evidence regarding the extent of denaturation of normal cellular proteins during heat shock. Differential scanning calorimetry (DSC) is the most direct method of monitoring protein denaturation or unfolding. Due to the fundamental parameter measured, heat flow, DSC can be used to detect and quantitate endothermic transitions in complex structures such as isolated organelles and even intact cells. DSC profiles with common features are obtained for isolated rat hepatocytes, liver homogenate, and Chinese hamster lung V79 fibroblasts. Five main transitions (A-E), several of which are resolvable into subcomponents, are observed with transition temperatures (Tm) of 45-98 degrees C. The onset temperature is approximately 40 degrees C, but some transitions may extend as low as 37-38 degrees C. In addition to acting as the primary signal for heat shock protein synthesis, the inactivation of critical proteins may lead to cell death. Critical target analysis implies that the rate limiting step of cell killing for V79 cells is the inactivation of a protein with Tm = 46 degrees C within the A transition. Isolated microsomal membranes, mitochondria, nuclei, and a cytosolic fraction from rat liver have distinct DSC profiles that contribute to different peaks in the profile for intact hepatocytes. Thus, the DSC profiles for intact cells appears to be the sum of the profiles of all subcellular organelles and components. The presence of endothermic transitions in the isolated organelles is strong evidence that they are due to protein denaturation. Each isolated organelle has an onset for denaturation near 40 degrees C and contains thermolabile proteins denaturing at the predicted Tm (46 degrees C) for the critical target. The extent of denaturation at any temperature can be approximately by the fractional calorimetric enthalpy. After scanning to 45 degrees C at 1 degree C/min and immediately cooling, a relatively mild heat shock, an estimated fraction denaturation of 4-7% is found in hepatocytes, V79 cells, and the isolated organelles other than nuclei, which undergo only 1% denaturation because of the high thermostability of chromatin. Thus, thermolabile proteins appear to be present in all cellular organelles and components, and protein denaturation is widespread and extensive after even mild heat shock.     

Dynamic glycogen synthesis in the hepatocytes of different areas of the hepatic lobes in rats

Using image analyser Magiscan, a quantitative analysis of the total glycogen and of its two fractions was made in hepatocytes of portal and central zones of the liver lobule of rats after a 48 hour starvation and 15, 30, 60, 120 minutes after refeeding. Glycogen content was the lowest after a 48 hour starvation and only a few cells of the central zone contained a noticeable glycogen quantity. Glycogen synthesis initiation began 15 minutes after refeeding. Glycogen synthesis is characterized by a higher glycogen content in the portal zone of liver lobule, and further this difference was even more increased. Different changes were observed in the content of glycogen fractions in the process of glycogen resynthesis after starvation of rats.     

Estrogen receptor levels in the liver of intact and gonadectomized rats and in cultured hepatocytes]

The concentrations of hepatic estrogen receptor were determined in intact and gonadectomized male and female rats. The hydroxylapatite assay demonstrated that the ablation of gonads induces, in the liver, an increase of estrogen receptors. The data obtained suggests that the synthesis of these receptors in the liver might be estrogen- and androgen-dependent. The same analysis performed on hepatocyte cytosol, derived from ovariectomized females, and cultured for 24, 48 and 72 hours, showed that time of culture is an important factor in determining a decrease of estrogen receptor concentrations in the cells. The results obtained allowed us to conclude that in the maintenance of the normal levels of the hepatic estrogen receptors, either sexual and non-sexual hormones are involved.     

Increased oxidation of p-nitrophenol and aniline by intact hepatocytes isolated from pyrazole-treated rats

Induction of cytochrome P-450 IIE1 by pyrazole has been shown in a variety of studies with isolated microsomes or reconstituted systems containing the purified P-450 isozyme. Experiments were conducted to document induction by pyrazole in intact hepatocytes by studying the oxidation of p-nitrophenol to 4-nitrocatechol or of aniline to p-aminophenol. Hepatocytes prepared from rats treated with pyrazole for 2 days oxidized p-nitrophenol or aniline at rates which were 3- to 4-fold higher than saline controls. To observe maximal induction in hepatocytes, it was necessary to add metabolic substrates such as pyruvate, sorbitol or xylitol, which suggests that availability of the NADPH cofactor may be rate-limiting in the hepatocytes from the pyrazole-treated rats. Carbon monoxide inhibited the oxidation of p-nitrophenol and aniline by hepatocytes from the pyrazole-treated rats and controls, demonstrating the requirement for cytochrome P-450. The oxidation of both substrates by the hepatocyte preparations was inhibited by a variety of agents that interact with and are effective substrates for oxidation by P-450 IIE1 such as ethanol, dimethylnitrosamine, pyrazole and 4-methylpyrazole. Microsomes isolated from pyrazole-treated rats oxidized aniline and p-nitrophenol at elevated rats compared to saline controls. These results indicate that induction by pyrazole of the oxidation of drugs which are effective substrates for P-450 IIE1 can be observed in intact hepatocytes. The extent of induction and many of the characteristics of aniline or p-nitrophenol oxidation observed with isolated microsomes from pyrazole-treated rats can also be found in the intact hepatocytes.     

Relationship between resting metabolism and hepatic metabolism: effect of hypothyroidism and 24 hours fasting.

In this paper, we have examined the relationship between the changes in the resting metabolic rate (RMR) and the changes in hepatic metabolism induced by hypothyroidism and 24 h fasting. The results show that hypothyroidism induces a significant decrease in RMR, while 24 h fasting reduces RMR in euthyroid but not in hypothyroid rats. We have also measured oxygen consumption in isolated hepatocytes from euthyroid and hypothyroid rats, fed or fasted for 24 h. The results show that hypothyroidism is characterized by reduced hepatic oxygen consumption. On the other hand, 24 h fasting induces an increase in oxygen consumption in both euthyroid and hypothyroid rat liver cells, although the respiratory rates of hypothyroid rats were lower than those of euthyroid rats. The above findings, as a whole, show that hypothyroidism and 24 h fasting have similar effects on RMR but opposite effects on hepatic metabolism. In addition, a normal thyroid state does not appear to be necessary for the observed changes in hepatic metabolism due to 24 h fasting.     

Determining the repair rate for radiation-induced chromatid deletions during the first 24 hours after irradiation

The number of chromatid plus isochromatid deletions present in the testes and bone marrow of the Chinese hamster was measured as a function of time following acute exposure to cobalt-60 gamma irradiation. The number of breaks remaining scorable at any time after irradiation exposure could be represented by a simple exponential equation (N = Noe-kt). The values of No and k, calculated after 100 roentgens of cobalt-60 gamma irradiation, were 1.15 breaks/cell and 0.16/hour, respectively, for the testes, and 0.53 break/cell and 0.14/hour, respectively, for the bone marrow. The average length of time that the breaks remain scorable, 1/k, was found to be 6.3 hours for the testes and 7.1 hours for the bone marrow. By suitable formulation, it was possible to predict the number of breaks present by using varied radiation exposure rates and times after exposure. This formulation could be used for both single and multiple radiation exposures.