Peter Ove

Synthesis and degradation of liver proteins in young and old rats

Abstract The report describes the study of the in vivo synthesis and degradation of ferritin as an example of an intracellular liver protein, albumin, as an example of a protein secreted by the liver, and total liver protein in young and old rats. In addition, possible age changes in the amounts of these proteins were investigated. Several biochemical changes occur with age. 1. 1. There is twice as much ferritin and about 4 times as much iron in old rat liver ferritin as in livers from young rats. 2. 2. The accumulation of ferritin in old rats appears to be due to a decreased rate of degradation. 3. 3. Albumin synthesis is increased in old rats. 4. 4. Albumin synthesis in young rats can be increased by removing blood from the animals. Old rats do not respond to the removal of blood by increased albumin synthesis. Finally, there is a labeling pattern of ferritin with time which is different from that of total liver protein suggesting two distinct steps, synthesis of subunits and assembly into ferritin molecules. The results suggest that in order to study age-related changes one should consider changes in specific proteins that might be masked if only total proteins are investigated. In addition, certain changes might become apparent only if the system is manipulated, in our case, induction of albumin synthesis by removal of blood from the animal

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.

Nonidentity of Fetuin and Protein Growth (Flattening) Factor

Fetuin, a fetal calf serum glycoprotein, appeared to possess activity with cultured mammalian cells similar to that of a protein growth factor partially purified from adult bovine and human sera. Column chromatography, however, yielded highly purified but inactive fetuin. These results leave open questions regarding the role of this interesting and readily purified protein.

In vitro synthesis of microsomal protein and albumin in young and old rats.

Abstract 1. 1. Protein and serum albumin synthesis was investigated with isolated liver microsomes from young and old rats. Albumin synthesis of old rats, based on mg protein, is 50 % higher than that of young rats, but there is no difference in total protein synthesis. 2. 2. The increased albumin synthetic activity resides in the microsomal particles and not in the cell sap. 3. 3. The albumin synthesis of young rats can be stimulated by bleeding. Increased albumin synthesis reaches its peak at 3 h after bleeding and is 40 % higher than in non-bled young rats, whereas in old rats, bleeding does not further increase albumin synthesis. 4. 4. The cell sap fraction from bled rats, either young or old, has a stimulating effect on albumin synthesis by the microsome fraction from young rats.

Pharmacologic modulation of experimental postischemic hepatic function

The present study evaluated and compared the effects of SRI 63-441, a potent platelet activating factor antagonist, superoxide dismutase (SOD), an oxygen free radical scavenger, and ibuprofen, a cyclooxygenase inhibitor on hepatic function after 90 minutes of warm ischemia. After warm ischemia, livers were harvested and underwent 90 minutes of warm, oxygenated, sanguinous perfusion on an isolated liver perfusion apparatus. Pretreatment of donor animals with 20 mg/kg intravenous (I.V.) SRI 63-441 5 minutes before induction of total hepatic ischemia resulted in significantly increased bile production, a significant decrease in transaminase release, and a higher tissue adenosine triphosphate (ATP) content when compared with ischemic nontreated controls. SOD resulted in improved bile production and decreased transaminase liberation only when present in the perfusate at the time of in vitro reperfusion. Ibuprofen did not improve postischemic hepatic function in this model. Electron microscopy revealed patchy hepatocellular vacuolization with an intact sinusoidal endothelium in all ischemic livers. However, the degree of damage was less severe in the livers from those rats pretreated with 20 mg/kg SRI 63-441. This study demonstrates that SRI 63-441 pretreatment significantly reduces hepatic warm ischemic injury, and in the present model, appears superior to two other agents that have been advanced in the treatment of ischemic injury. The use of such agents singly or in combinations have important implications as regards gaining a better understanding of the basic mechanisms in organ ischemia, and moreover, for therapeutic applications in organ ischemia and preservation.

Improved Hepatic Function in the 24-Hour Preserved Rat Liver With UW-Lactobionate Solution and SRI 63-441

The present study compares rat liver preservation for 9, 12, and 24 h in the standard Eurocollins solution with preservation for the same time periods in the new UW-lactobionate solution. Pharmacologic manipulation with a potent platelet-activating factor antagonist, SRI 63-441, was also evaluated. After cold storage in each of the test solutions, the livers underwent 90 min of warm, oxygenated, sanguinous perfusion. A significant increase in liver weight was noted in Eurocollins-stored versus UW-lactobionate-stored livers. After 90 min of perfusion, livers preserved in UW-lactobionate produced significantly more bile and liberated significantly less glucose and transaminases when compared with Eurocollins-stored livers. Significant augmentation of bile production was observed when donor animals were pretreated with SRI 63-441 and the livers were then stored in UW-lactobionate for 24 h. Eurocollins-stored livers demonstrated increased hepatocyte vacuolization and endothelial disruption when compared with UW-lactobionate-stored livers after 12 and 24 h of preservation. This study demonstrates the superiority of UW-lactobionate solution in liver preservation and suggests that SRI 63-441 may be beneficial in the further reduction of cold ischemic injury.

Different response to epidermal growth factor of hepatocytes in cultures isolated from male or female rat liver

Deoxyribonucleic acid (DNA) synthesis in hepatocytes isolated from the livers of male and female rats has been compared in monolayer culture. Plating efficiency, DNA and protein content, viability, and morphologic appearance were the same in cultures prepared with hepatocytes isolated from male or female rats. Epidermal growth factor (EGF)-induced DNA synthesis was significantly higher in hepatocytes from male rats than in hepatocytes from female rats. This was the case whether hepatocytes were isolated from normal or partially hepatectomized male or female rats. Hepatocytes isolated from regenerating liver synthesize more DNA than those isolated from normal liver in response to EGF. This increased response to EGF in hepatocytes derived from regenerating liver was relatively the same for male- and female-derived hepatocytes, but the magnitude of the response was considerably higher in male-derived hepatocytes. In contrast, in vivo DNA synthesis in the liver remnant after partial hepatectomy was similar in male and female rats if measured 24 h after the operation. A comparison of EGF binding to male- and female-derived hepatocytes maintained in primary culture indicated a lower number of high-affinity receptors for EGF in the female hepatocytes. The addition of estrogen to primary cultures of hepatocytes isolated from male rats inhibited EGF binding as well as EGF-induced DNA synthesis. Our studies show significant differences in DNA synthesis in response to EGF when male and female hepatocytes are compared in primary culture. The regenerative response after partial hepatectomy, on the other hand, was the same in male and female rats. Thus, our studies indicate that the sex of the donor, rat is important when hepatocytes in culture are used for a variety of studies, such as hepatocyte metabolism, induction and control of DNA synthesis, and hepatocarcinogenesis. In addition, our results indicate that caution is advised when inferences are made from in vitro findings for in vivo conditions.

Functional regeneration in liver of old rats after partial hepatectomy

Abstract This study suggests that changes in liver protein metabolism occurring with age are not due to changes in the genome. Regenerating rat liver in old animals has regained functional properties of young rat liver. Specifically albumin synthesis, which is elevated in old animals, returns to young rat levels in old rats for several weeks after partial hepatectomy. Both in vivo and in vitro studies support this evidence. Old rat liver ferritin also undergoes changes in regenerating liver. The amount of ferritin iron/g liver falls to one half, the amount of iron/mg ferritin protein drops, but the amount of ferritin protein/g liver remains constant in regenerated old rat liver. The half-life of ferritin in regenerated old rat liver (2·3 days) is much shorter than in old rat liver controls (3·9 days) and approaches that of young rat liver ferritin (2·1 days). Determinations were done at a time when liver weight had been fully restored following removal of 67 per cent of the liver. Functional properties of old rat liver are restored by 12 weeks after partial hepatectomy.

A comparison of DNA repair synthesis in primary hepatocytes from young and old rats

DNA repair synthesis has been compared in primary hepatocyte cultures obtained from 3-month-old and 16-20-month-old rats. Several morphological and metabolic characteristics were determined to assure cultures of comparable quality. DNA damage was induced by the addition of bleomycin or the exposure of the culture to UV irradiation. DNA repair (unscheduled DNA synthesis) was determined by measuring [3H]thymidine incorporation. After UV irradiation, there was almost twice as much [3H]thymidine incorporation in cells obtained from young rats as in those obtained from old rats. Equal amounts of bleomycin resulted in substantially greater damage to DNA in cells from old rats than from young rats. For equal amounts of DNA damage there was again diminished [3H]thymidine incorporation in cells obtained from old rats. Finally equal amounts of bleomycin resulted in equal damage to DNA when the bleomycin was added to isolated rat liver nuclei from young or old rats. Bleomycin treated nuclei from young rats incorporated substantially more [3H]thymidine triphosphate (TTP) than bleomycin treated nuclei from old rats. The results indicate that hepatocytes from old rats are much more susceptible to bleomycin than hepatocytes from young rats and that the capacity for DNA repair synthesis is impaired in hepatocytes from old rats.

Etiology of increased albumin synthesis in old rats

Abstract We have investigated albumin synthesis and excretion of albumin into the urine in young and old rats. Evidence is provided to suggest that the higher rate of excretion of albumin into the urine in old rats is not responsible for the increased synthesis. Albumin synthesis in young and old animals was determined after partial hepatectomy, injection of serum protein into the tail vein, and after aminonucleoside puromycin induced nephrosis. For several weeks after partial hepatectomy, an operation that should not effect nephrosis in old rats, albumin synthesis in old rats is decreased. Injection of serum does not decrease albumin synthesis but causes an increase in the amount of albumin excreted into the urine. Aminonucleoside puromycin induced nephrosis does not stimulate albumin synthesis to a significant extent. It is concluded that the rate of excretion of albumin into the urine is a function of the albumin levels in the serum and that synthesis of albumin is not regulated to a great extent by the rate of excretion.