Different fixation media have been compared in order to find one that preserves the histological structure of rat liver and allows unambiguous immunohistochemical detection of carbamoyl-phosphate synthetase (ammonia). Fixation of rat liver in a mixture of methanol, acetone, and water yields the most intense immunohistochemical staining. Using a specific antiserum raised against rat liver carbamoyl-phosphate synthetase, less than 1% of the enzyme protein is extractable after this fixation procedure, and the histological structure is similar to that after fixation in Bouin's fixative. Specific immunohistochemical staining is localized exclusively in the cytoplasm of the parenchymal cells; its granular distribution is in accordance with the mitochondrial localization of carbamoyl-phosphate synthetase. Immunohistochemical staining shows a heterogeneous distribution within the liver acinus. Staining is most intense around the portal venules, decreases slowly toward the hepatic venules and is, after an abrupt decrease, virtually absent in a limited area surrounding these venules. The possible significance of the heterogeneous distribution of carbamoyl-phosphate synthetase for ammonia metabolism is discussed.
The distribution pattern of a periportal enzyme (carbamoylphosphate synthetase) and a pericentral enzyme (glutamine synthetase) in human and rat liver has provided an objective parameter to delineate the zonal boundaries of the liver acinus. On sections, the pericental zone (zone 3) is circular and discrete rather than star-like and reticular, as predicted by the acinar concept, whereas the periportal zone (zone 1) is reticular, i.e. contiguous between adjacent acini rather than discrete. Three-dimensionally, the composite of pericentral zones (the pericentral compartment) follows the branching pattern of the terminal hepatic (central) vein, whereas the composite of periportal zones (the periportal compartment) envelops the pericentral compartment as a three-dimensional network (reticulum). This modified concept that is based upon the three-dimensional distribution of hepatocyte-specific enzymes is supported by data from the literature regarding the three-dimensional angioarchitecture of the liver, the perfusion pattern of the liver and the three-dimensional pattern of tissue oxygenation. Hence, a unified concept of the liver architecture that is based upon the observed distribution pattern of blood flow, of gene expression and of metabolism can be established.
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.
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 parturn, 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.
Proteins characteristic for the adult cellular phenotype, i.e., carbamoylphosphate synthetase (CPS) for liver and small intestine, arginase for liver, glutamate dehydrogenase (GLDH) for pancreas, liver, and small intestine, and amylase for pancreas were studied immunohistochemically in rat embryos and fetuses. At distinct developmental stages, subsets of enzymes appear synchronously in the foregut derivatives, suggesting that gene expression in the different organs is regulated by common factors. In contrast to the long-held opinion that fetal hepatocytes are a homogeneous cell population, it is shown that arginase and CPS are heterogeneously distributed between ED 16 and ED 20. This heterogeneity is related to the vascular architecture of the liver and disappears perinatally as the result of strong stimulation of enzyme synthesis. In addition, an intercellular heterogeneity in CPS content that is not related to the vasculature is observed between ED 14 and ED 20. This "random" heterogeneity reflects temporal differences in the onset of CPS accumulation in individual cells.
In adult rat liver, glutamate dehydrogenase is present in high concentrations around the terminal portal (zone 1) and hepatic (zone 3) veins, whereas its concentration is low in the intermediate zone. Although the size and staining intensity of the periportal glutamate dehydrogenase-positive compartment are less than those of the pericentral compartment, it can expand under appropriate endocrine conditions, leading to a homogeneous distribution. At birth, glutamate dehydrogenase is also homogeneously distributed. Glutamate dehydrogenase disappears from the periportal compartment during the first postnatal week and reappears in that compartment after weaning. These observations indicate an independent regulation of glutamate dehydrogenase levels in the periportal and pericentral zone. The size of the periportal glutamate dehydrogenase-containing zone is appreciably smaller than that of carbamoylphosphate synthetase, whereas the pericentral glutamate dehydrogenase-containing zone is appreciably larger than that of glutamine synthetase. The heterogeneous distribution of glutamate dehydrogenase suggests the possibility that, under normal conditions, deamination of glutamate prevails in the periportal compartment and amination of glutamate in the pericentral compartment.
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