Reestablishment of the heterogeneous distribution of hepatic glutamine synthetase during regeneration after CCl4-intoxication

Summary Intoxication of rats with CCl₄ (1 ml/kg) resulted in the almost complete loss of glutamine synthetase (GS) specific activity and immunologically detectable enzyme protein known to be expressed exclusively in some hepatocytes of the perivenous zone of the liver acinus. During regeneration the specific activity as well as the original number of GS-positive (GS⁺) hepatocytes were reestablished. However, while the GS⁺ hepatocytes in control livers were arranged in up to 3 cell layers surrounding the central veins the same number of GS⁺ hepatocytes in regenerated livers formed a single cell layer only, most likely because the central veins were enlarged in diameter. Investigation of the nuclear pattern of GS⁺ and GS^– hepatocytes of control animals in primary cultures revealed striking differences characterized by significantly more mononuclear diploid, binuclear diploid, and binuclear tetraploid cells among the GS⁺ hepatocytes and predominantly mononuclear tetraploid cells (70%) among the GS^– hepatocytes. Immediately after liver damage by CCl₄ and during regeneration small but significant changes in the nuclear pattern were noted for GS^– hepatocytes. However, the first GS⁺ cells appearing during early regeneration showed a pattern of ploidy classes close to the original one found for GS^– hepatocytes. These results indicate that new GS⁺ hepatocytes may be derived from formerly GS^– cells which are induced to express GS if they have reached the border of the central veins.     

Heterogeneous distribution of glutamine synthetase during rat liver development

Two days before birth, immunohistochemical detection of glutamine synthetase already reveals a heterogeneous distribution pattern related to the vascular architecture of the liver. Only a small number of hepatocytes in the vicinity of the efferent venules show relatively high staining intensity. Before that age, only megakaryocytes show intense staining, while liver parenchyma is only faintly stained. The developmental profile of glutamine synthetase activity shows two periods of increasing enzyme activity: one in the perinatal period and one in the second and third postnatal week. Both periods are correlated with high levels of circulating corticosteroid hormones. Although the relative number of intensely stained hepatocytes increases during the first rise in enzyme activity, the second rise is correlated with a decreasing number of glutamine synthetase-positive hepatocytes which, however, show a considerable increase in staining intensity. Carbamoylphosphate synthetase shows a homogeneous distribution pattern in the perinatal period. Conditions that lead during development to a relatively high level of glutamine synthetase expression in the pericentral compartment apparently originate before the appearance of conditions that lead to a relatively high level of carbamoylphosphate synthetase gene expression in the periportal compartment. Our results indicate that downstream localization of glutamine synthetase in liver acinus is essential from the perinatal period onwards, whereas reciprocal distribution of glutamine synthetase and carbamoylphosphate synthetase gene expression (that is found in adult rat liver) is not.     

Mathematical modelling of liver regeneration after intoxication with CCl(4)

Liver regeneration is a complex process, having evolved to protect animals from the consequences of liver loss caused by food toxins. In this study, we established a mathematical spatial-temporal model of the liver lobule regenerating after CCl(4) intoxication. The aim of modelling the regeneration process by matching experimental observations with those from a mathematical model is to gain a better understanding of the process and to recognize which parameters are relevant for specific phenomena. In order to set up a realistic minimal model, we first reconstructed a schematised liver lobule after determination of: (i) the mean number of hepatocytes between the central vein and the periphery of the lobule, (ii) the mean size of the hepatocytes and (iii) the mean number of hepatocyte columns in the inner, midzonal and peripheral ring of the lobule. In a next step, we determined the time course of cell death and BrdU incorporation after intoxication of male Sprague Dawley rats with CCl(4), thereby differentiating between inner, midzonal and peripheral hepatocytes. These parameters were used to construct a model. The basic unit of this model is the individual cell. The detailed behaviour of the cells is studied, controlled by the model parameters: (1) probability of cell division at defined positions of the lobule at a given time, (2) "coordinated cell orientation", i.e., the ability of the cells to align during the regeneration process into columns towards the central vein of a liver lobule, (3) cell cycle duration, (4) the migration activity and (5) the polarity of the hepatocytes resulting in polar cell-cell adhesion between them. In a schematised lobule, the model shows that CCl(4) initially induced cell death of a pericentral ring of hepatocytes, followed by a wave of proliferation that starts in the surviving hepatocytes next to the inner ring of dead cells and continues to the peripheral hepatocytes, finally restoring the characteristic micro-architecture of the lobule in a 7-day process. This model was used to systematically analyze the influence of parameters 1-5. Interestingly, coordinated cell orientation and cell polarity were identified to be the most critical parameters. Elimination led to destruction of the characteristic micro-architecture of the lobule and to a high degree of disorder characterized by hexagonal cell structures. Our model suggests that the ability of hepatocytes to realign after cell division by a process of coordinated cell orientation (model parameter 2) in combination with cell polarity (model parameter 5) may be at least as critical as hepatocyte proliferation (model parameter 1) itself.     

Subcellular calmodulin distribution in rat liver after CCl4 poisoning

Disturbed cellular calcium homeostasis has been observed during CCl4 poisoning, with an increase in calcium content 1 h after administration. Intracellular increase of calcium may be expected to alter membrane/cytosol distribution of calmodulin (CaM). This paper investigates changes in rat liver subcellular CaM distribution 30 min, 1 h and 2 h after CCl4 intoxication. The whole liver value remained unchanged, whereas the nuclear fraction increased and the microsomal and cytosolic fraction decreased. This may suggest that CaM is involved in the several liver cell alterations caused by CCl4 poisoning.     

Modification of glutamine synthetase in Bacillus brevis AG 4

The various forms of glutamine synthetase obtained from Bacillus brevis have been found to be antigenically identical. Alkaline phosphatase treatment of the fast moving form (GS4) reduced the electrophoretic mobility of the enzyme. Radiolabelling and autoradiographic studies have also indicated that 32P-incorporation is high in the form depicting high Rm value. Thus, it appears that these forms arise due to covalent modification of the enzyme involving a phosphate group.     

Metabolism of glycosaminoglycans in CCl4-induced liver regeneration

The incorporation of [₃₅S]-SO₄ into glycosaminoglycans of liverin vivo and in in liver slices and into the glycosaminoglycans associated with the hepatic plasma membrane of rats at different periods after a heavy dose of CC1₄ have been studied. The incorporation of [35S]-SO4 into total glycosaminoglycans decreased to as low as 40% of the control at 24 h after the administration of CC14 and later on increased reaching a maximum on the 4th day. The amount of [35S]-SO4 incorporation into heparan sulphate was also reduced to about 40% of control at 12–24 h after the onset of injury and increased thereafter reaching a maximum on the 4th day. There was only a partial reduction in the synthesis of chondroitin sulphate in the early stage of injury and then it steadily increased reaching about 3 times the control level on 4–6 days. The [³⁵S]-SO4-incorporation into dermatan sulphate, after a slight initial decrease remained at the control levels. On the 8th day after the CCl₄-induced liver injury, the rate of [³⁵S]-SO₄-incorporation was almost equal to that in normal controls. The incorporation of [³⁵S]-SO₄ into hepatic plasma membrane glycosaminoglycans showed a similar change decreasing to about 35% of control at 24h followed by an increase, reaching normal levels on the 4th day after the administration of CC1₄. About 90% of the plasma membrane glycosaminoglycans was found to be heparan sulphate. The yield of plasma membrane from normal and CCl₄-induced regenerating liver was found to be similar and therefore the results obtained were not due to difference in the yield of the membrane preparation. The data also indicate that there was no difference in the degree of sulphation. The significance of these changes in the metabolism of sulphated glycosaminoglycans particularly plasma membrane heparan sulphate in tissue regeneration has been discussed.     

Expression of carbamoylphosphate synthetase I and glutamine synthetase in hepatic organoids reconstructed by rat small hepatocytes and hepatic nonparenchymal cells

The objective of this study was to investigate the expression of carbamoylphosphate synthetase I (CPS) and glutamine synthetase (GS) in small hepatocyte colonies and whether the heterogeneous expression of the enzymes could be induced during the maturation of small hepatocytes. Small hepatocytes isolated from an adult rat liver were cultured and proliferated to form colonies. The expression of CPS and GS was examined using immunocytochemistry and immunoblotting. In this culture more than 99% of morphologically hepatic cells were positive for CPS and all small hepatocytes were negative for GS at day 5. CPS-positive cells dramatically decreased with time in culture, whereas GS-positive ones appeared and their number increased in the colonies. Two to 3 weeks after plating, colonies with rising and piled-up cells appeared and the number of such colonies reached about 25% of all colonies at day 30. In most rising and piled-up cells in colonies both proteins were strongly expressed, whereas many small hepatocytes in monolayer colonies did not express either protein. When small hepatocytes in monolayer colonies were overlayed with Matrigel, the cells gradually piled up and both CPS and GS proteins were dramatically induced. The expression of CPS and GS in small hepatocytes may interact with the extracellular matrix because the rising and piled-up cells appear to be induced by the extracellular matrix produced by hepatic nonparenchymal cells.     

Interaction of substrates with glutamine synthetase after limited proteolysis

Previous studies [Dautry-Varsat, A., Cohen, G. N., & Stadtman, E.R. (1979) J. Biol. Chem. 254, 3124-3128; Lei, M., Aebi, U., Heidner, E. G., & Eisenberg, D. (1979) J. Biol. Chem. 254, 3129-3134] have shown that Escherichia coli glutamine synthetase (GS) can be cleaved by proteases to form a limited digestion species called nicked glutamine synthetase (GS). The present study gives the amino acid sequence of the protease-sensitive region of glutamine synthetase. The present study also shows that GS is enzymatically active, but this activity is low compared to the activity of GS. The apparent Michaelis constant value for glutamate was 90 mM for GS as compared to 3 mM for GS, while the Michaelis constant values for ATP were similar for GS and GS*. The dissociation constant values for ATP, as determined by intrinsic fluorescence measurements, were similar for GS and GS*. Glutamate decreased the dissociation constant value of ATP for GS because of synergism between the two binding sites; glutamate did not decrease the dissociation constant value of ATP for GS*. The glutamate analogue methionine sulfoximine bound very tightly to GS and inactivated the enzyme in the presence of ATP. Methionine sulfoximine did not appear to bind to GS* and did not inactivate GS* in the presence of ATP. The ATP analogue 5'-[p-(fluorosulfonyl)benzoyl]adenosine bound to GS and inactivated the enzyme by forming a covalent bond with it. Glutamate accelerated this inactivation because of the synergism between the ATP and glutamate binding sites of GS.(ABSTRACT TRUNCATED AT 250 WORDS)     

o-Phosphotyrosyl glutamine synthetase: modification of the nucleotide ligation site of adenylylated glutamine synthetase

The nucleotide ligation site of adenylylated glutamine synthetase, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP, was studied by digestion with three hydrolytic enzymes. The products on micrococcal nuclease digestion were adenosine and o-phosphotyrosyl glutamine synthetase. The Km for this macromolecular substrate with the nuclease was 40 microM, at pH 8.9. The glutamine synthetase activity was not affected by deadenosylation with the nuclease, in contrast to SVPDE digestion, with which the glutamine synthetase activity was markedly increased. The Km for the native adenylylated glutamine synthetase with the SVPDE was 36 microM, i.e., similar to that for the nuclease. When the isolated o-phosphotyrosyl enzyme was incubated with alkaline phosphatase at pH 7.2, the glutamine synthetase activity rapidly increased to the same level as that of the SVPDE treated enzyme. Furthermore, kinetic properties of the o-phosphotyrosyl glutamine synthetase were compared with those of the adenylylated enzyme. The optimum pH, apparent Km for each of three substrates, glutamate, ATP, and NH3, and Vmax were in good agreement, as to either Mg2+- or Mn2+-dependent biosynthetic activity. From these results we can conclude that the regulation of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue in each subunit, without recourse to adenylylation.     

Development of the heterogeneous distribution of carbamoyl-phosphate synthetase (ammonia) in rat-liver parenchyma during postnatal development

Carbamoyl-phosphate synthetase (ammonia) is homogeneously distributed in rat-liver parenchyma at birth, as demonstrated by immunohistochemistry. A heterogeneous distribution can first be demonstrated at 6 days post partum, but can be masked by use of a too sensitive detection system. This heterogeneity is established by a decrease in enzyme content around the hepatic venules and a considerable increase in enzyme content in the remaining parenchyma. The perivenous decrease in enzyme content does not occur in all hepatocytes synchronously. The adult type of heterogeneity is characterized by a perivenous layer, only two to three cells thick, in which carbamoyl-phosphate synthetase can no longer be detected, irrespective of the sensitivity of the assay used. This situation is fully established at the age of two months.     

Distribution of glutamine synthetase and an inverse relationship between glutamine synthetase expression and intramuscular glutamine concentration in the horse

Glutamine plays important roles in the interorgan transport of nitrogen, carbon and energy but little is known about glutamine metabolism in the horse. In this study we determined the tissue distribution of glutamine synthetase expression in three Standardbred mares. Expression of glutamine synthetase was highest in kidney and mammary gland, and relatively high in liver and adipose tissue. Expression was lower in gluteus muscle, thymus, colon and lung, and much lower in small intestine, pancreas and uterus. The pattern of glutamine synthetase expression in the horse is similar to that of other herbivores and it is likely that skeletal muscle, liver, adipose tissue and lungs are the major sites of net glutamine synthesis in this species. Expression did not differ between adipose tissue depots but did vary between different muscles. Expression was highest in gluteus and semimembranous muscles and much lower in diaphragm and heart muscles. The concentration of intramuscular free glutamine was inversely correlated with expression of glutamine synthetase (r=-0.81, p=0.0017). The concentration of free glutamine was much higher in heart muscle (21.6+/-0.9 micromol/g wet wt) than in gluteus muscle (4.19+0.33 micromol/g wet wt), which may indicate novel functions and/or regulatory mechanisms for glutamine in the equine heart.     

Multiple forms of glutamine synthetase in Bacillus brevis AG 4

Glutamine synthetase in Bacillus brevis AG 4, a Gram-positive spore forming bacteria, has been found to exist in multiple molecular forms. It was purified to electrophoretic homogeneity by single-step Blue Sepharose affinity chromatography. The native enzyme has a molecular weight of 600,000 with subunits of 50,000. The enzyme samples purified from different stages of growth differed in Mg2+ sensitivity and other kinetic properties. Four different enzyme samples selected on the basis of Mg2+ sensitivity showed distinct mobilities at pH 6.3 on PAGE using discontinuous buffer system. A correlation amongst Mg2+ sensitivity, electrophoretic mobility, and kinetic properties was highly suggestive of multiple forms of glutamine synthetase in Bacillus brevis arising due to modification.     

Glutamate dehydrogenase and glutamine synthetase activities during the development of triticale grains

Glutamate dehydrogenase (GDH, EC 1.4.1.2–4) and glutamine synthetase (GS, EC 6.3.1.2) activities as well as protein content and dry matter in developing kernels of winter Triticale were determined. The relatively low level of GS activity compared to high level of NAD(P)H-dependent GDH activity during intensive filling of grains with storage compounds may indicate the participation of GDH in reductive amination of 2-oxoglutarate. The amination activity of this enzyme in all grain development phases exceeded the deaminating activity several fold. Moreover, the dynamics in the change of NAD(P)H-GDH and NAD(P)⁺-GDH activities were analysed in various tissues of the developing grains. The high amination activity of the enzyme in the seed coat, where the intensive protein synthesis occurs would also be an indication of the anabolic function of this enzyme.     

Inhibition of the adenylylation of glutamine synthetase by methionine sulfone during nitrogenase derepression

Adenylylation of glutamine synthetase was suppressed during derepression of nitrogenase synthesis in the presence of methionine sulfone and an excess of NH₄⁺. Deadenylylation of glutamine synthetase was also promoted during nitrogenase derepression under the same conditions. These results are consistent with the hypothesis that the unadenylylated form of glutamine synthetase is required for derepression of nitrogenase.     

Systemic histopathology of rats with CCl4-induced hepatic cirrhosis

Systemic histopathological examinations were carried out on rats with CCl4-induced hepatic cirrhosis. Moderate congestion in the spleen, prominent oedema in the focal acinar cell degeneration in the pancreas, marked haemorrhage and phagocytosis of haemosiderin by macrophages in the pancreaticoduodenal lymph node, appearance of monocytes bearing haemosiderin-like granules in the pulmonary arteries and cardiac right atrium, and focal segmental glomerulosclerosis were consistently observed in rats with hepatic cirrhosis. In addition, a marked increase in number of target cells and the appearance of a small number of monocytes bearing haemosiderin-like granules were also commonly found in the peripheral blood smears of these animals. These findings are considered to be important in the use of the CCl4-induced model of hepatic cirrhosis in the rat.