Dephosphorylation of L-pyruvate kinase during rat liver hepatocyte isolation

In isolated rat liver cells, the inhibition of L-pyruvate kinase (L-PK) by a cyclic AMP-dependent phosphorylation mechanism is involved in the hormonal control of glycolysis and gluconeogenesis. The aim of this study was to ascertain whether or not the in vivo phosphorylation state of the enzyme was maintained during the liver perfusion used to prepare isolated liver cells. When the L-PK phosphorylation state was studied indirectly in liver extracts by kinetic measurement, it was found that, during the perfusion, the S0.5 of phosphoenol pyruvate (PEP) for L-PK was decreased in a time-dependent manner from 1 +/- 0.08 to 0.64 +/- 0.1 mM (P less than 0.01) and 0.58 +/- 0.06 mM in liver cells. This shift was prevented only by the addition of glucagon to the perfusion medium. The extent of phosphorylation of L-PK was also estimated by incubation of the liver extract with [gamma-32P]ATP, protein kinase, and cyclic AMP, and measurement of 32Pi incorporated in L-PK by specific immunoprecipitation. In liver extracts removed at the beginning of the perfusion, 0.4 mol Pi/mol L-PK was incorporated and there was no stimulation by cyclic AMP. In contrast, in the liver extracts removed after 30 min of perfusion, cyclic AMP stimulated 32P incorporation two to threefold, and 1.6 mol Pi/mol L-PK was incorporated. These data suggest that L-PK was activated by a dephosphorylation mechanism during rat liver perfusion. This phenomenon could be involved in the classical inactivation of gluconeogenesis observed in the perfused rat liver model.     

Induction of choline kinase by polycyclic aromatic hydrocarbon carcinogens in rat liver

The administration to rats of polycyclic aromatic hydrocarbons such as 3-methylcholanthrene, 3,4-benzo(a) pyrene and β-naphthoflavone caused a significant elevation of hepatic choline kinase activity. On the other hand, phenobarbital-type inducers (phenobarbital, 1,1,1-trichloro 2,2-bis (ρ-chlorophenyl) ethane (DDT) and hexachlorobenzene) did not stimulate the activity at all. The administration of either cycloheximide or actinomycin D completely depressed the elevation of choline kinase activity induced by polycyclic aromatic hydrocarbons, indicating that the elevated activity by these chemicals could be due to the change in the enzyme level. These results strongly suggest that induction of choline kinase are involved in the sequence of events leading to the induction of hepatic drug metabolism by polycyclic aromatic hydrocarbons.     

Uridine kinase in embryonic rat liver. Modulation of enzyme activity by 5-azacytidine

Uridine kinase activity in rat liver decreases during embryonic and postnatal development. Administration of 5-azacytidine enhances liver uridine kinase activity in adult rats, but depresses it in embryos. The liver enzymes from the foetus and the adult are precipitated at different (NH(4))(2)SO(4) concentrations although they are eluted at about the same position on chromatography on a column of Sepharose 6B.     

Immunohistochemical demonstration of glycogen phosphorylase in rat brain slices

Paraffin-embedded sections from paraformaldehyde-fixed rat brain were stained immunocytochemically for glycogen phosphorylase brain isozyme BB, using a monoclonal mouse antibody and the biotin-strept-avidin method, with either horseradish peroxidase or beta-galactosidase as marker enzymes. Two cell types showed strong glycogen phosphorylase-immunoreactivity: Astrocytes and ependymal cells. Most intensive staining was observed in the cerebellar cortex, the neocortex and the hippocampus. Astrocytes in the cerebellar white matter stained positively. The choroid plexus cells stained poorly or not at all. Neurons throughout the brain were negative, as well as oligodendrocytes and bundles of myelinated nerve fibers. These data are consistent with the immunocytochemical localization of glycogen phosphorylase in astroglia-rich primary cultures derived from rat brain.     

Immunohistochemical demonstration of EC cells in rat gastrointestinal tract

Summary Selective immunohistochemical demonstration of enterochromaffin (EC) cells in the rat gastrointestinal tract has been carried out, using a highly specific rabbit antiserotonin (5-HT) serum and the unlabeled peroxidase-antiperoxidase complex (PAP), technique. The daily profile of the EC cell density was also studied using this method. No significant change was recorded.     

Immunohistochemical demonstration of glycogen phosphorylase in rat brain slices

Summary Paraffin-embedded sections from paraformaldehyde-fixed rat brain were stained immunocytochemically for glycogen phosphorylase brain isozyme BB, using a monoclonal mouse antibody and the biotin-streptavidin method, with either horseradish peroxidase or -galactosidase as marker enzymes. Two cell types showed strong glycogen phosphorylase-immunoreactivity: Astrocytes and ependymal cells. Most intensive staining was observed in the cerebellar cortex, the neocortex and the hippocampus. Astrocytes in the cerebellar white matter stained positively. The choroid plexus cells stained poorly or not at all. Neurons throughout the brain were negative, as well as oligodendrocytes and bundles of myelinated nerve fibers. These data are consistent with the immunocytochemical localization of glycogen phosphorylase in astroglia-rich primary cultures derived from rat brain.     

Immunohistochemical demonstration of histamine N-methyltransferase-like structures in rat kidney

Summary Histamine N-methyltransferase (S-adenosylmethionine: histamine N-methyltransferase, E.C. 2.1.1.8) was purified to homogeneity from rat kidney, and antibody was raised against it in guinea pigs. The antibody immunoprecipitated histamine N-methyltransferase. Immunofluorescent histochemical studies with anti-histamine N-methyltransferase antibody as the first antibody and goat antiguinea pig IgG conjugated with fluorescein isothiocyanate as the second, showed the presence of immunoreactive structures in the proximal tubules of rat kidney. The brain showed no immunoreaction with the antibody.     

Immunohistochemical demonstration of cytochrome b5 and hemopexin in rat liver parenchymal cells using horseradish peroxidase

The membrane-bound hemoprotein, cytochrome b₅, and the serum protein, hemopexin, were demonstrated in rat liver parenchymal cells using an immunohistochemical approach. Other liver cells contained neither protein. The immunological “sandwich” technique employs a complex of horseradish peroxidase with anti-horseradish peroxidase sequentially coupled to other antibodies including those to rat cytochrome b₅ and hemopexin.     

Induction by butylated hydroxytoluene of rat liver gamma-glutamyl transpeptidase activity in comparison to expression in carcinogen-induced altered lesions

Butylated hydroxytoluene (BHT) at concentrations of 300-6000 ppm in the diet caused a dose-dependent increase in gamma-glutamyl transpeptidase (GGT) activity in normal F344 male rat liver at 18 weeks. However, the activities of glutathione S-transferases (GSTs) of rat liver cytosol were enhanced only at concentrations of 3000 or 6000 ppm BHT. Histochemically, the enhanced GGT activity was localized to hepatocytes surrounding the portal areas. Autoradiographic measurements of DNA synthesis showed that dietary BHT did not increase the level of cell proliferation and the GGT-positive hepatocytes did not exhibit different rates of DNA synthesis from those of GGT-negative cells. Feeding of the liver carcinogen N-2-fluorenylacetamide (FAA) induced foci and nodules of GGT-positive altered cells which exhibited higher rates of DNA synthesis than those of surrounding GGT-negative hepatocytes. Following iron loading, the periportal GGT-positive hepatocytes produced by BHT accumulated cellular iron, whereas the cells in FAA-induced lesions excluded iron. These results suggest that dietary BHT induces GGT activity in periportal hepatocytes without proliferation of the cells and that induction does not represent fetal expression or a preneoplastic alteration.     

Immunohistochemical demonstration of prostaglandins in various tissues of the rat

To demonstrate the tissue localization of prostaglandin (PG) E₂, PGF₂ and 6-keto-PGF₁ (a stable metabolite of PGI₂) various tissues, including decalcified periodontal tissue of 7-week-old male Wistar strain rats, were immunohistochemically examined using a streptavidin-biotin complex method. Besides tissue macrophages and endothelial cells in various tissues, hepatocytes, renal tubular cells, and parietal and chief cells in the gastric mucosa showed a positive reaction for the various PGs examined. PGs were demonstrated in the cytoplasm or in association with the cell membrane. We generally observed no difference between the localization patterns of PGE₂-, PGF₂-, and 6-keto-PGF₁-positive cells in these tissues. However, in the periodontal ligament and alveolar bone, 6-keto-PGF₁ was localized in the cytoplasm of osteocytes, osteoblasts, cementocytes, and cementoblasts, while no reaction for PGE₂ or PGF₂ was revealed in these cells. We demonstrated the immunohistochemical localization of PGs in various rat tissues including decalcified periodontal tissue and discuss the important roles of PGs in the modulation of their normal functions in these tissues.     

Immunohistochemical demonstration of calbindin-containing nerve endings in the rat esophagus

Immunoreactivity for calbindin was found in nerve endings with irregular laminar shapes in the rat esophagus. In the myenteric ganglia, laminar endings of a range of sizes formed a complex network and appeared to lie at the surface of the ganglion. The myenteric ganglia that contained nerve endings were most abundant in the upper portion of the eosphagus, their number decreasing orally to anally. Calbindin-immunoreactive nerve cell bodies were scattered throughout the esophagus. Laminar terminals were found in the connective tissue of the lamina propria immediately beneath the epithelium and in the muscularis mucosae. Occasional nerve branches formed a network of aborizing endings that surrounded part of the submucosal arterioles. Immunoreactive nerve endings in the mucosa and submucosa were present only in the upper part of the cervical esophagus. Unilateral vagotomy caused a remarkable decrease in the number of the myenteric ganglia containing the calbindin-immunoreactive laminar endings after 15 days or survival; in some of ganglia, the laminar structures disappeared and nerve endings showing weak immunoreactivity had an indistinct appearance, so that the outline of the ganglia became obscure. In operated rats at 24 days, the number of innervated ganglia was about half that in normal rats. However, there was no change in the morphology and the occurrence of the immunoreactive laminar structures in the mucosa and submucosa after denervation. The results show that many of the laminar endings that are immunoreactive for calbindin in the myenteric ganglia are derived from the vagus nerve. Thus, the calbindin-immunoreactive nerve endings with laminar expansions that are found in the rat eosphageal wall could be sensory receptors.     

Immunohistochemical demonstration of a dermorphin-like peptide in the rat brain

Summary The new opioid heptapeptide dermorphin, first isolated from frog skin, has been localized immunohistochemically in nerve cell bodies and nerve fibers of the arcuate-periarcuate region, around the organum vasculosum of the lamina terminalis (OVLT) and in the subfornical organ of the rat brain. A role of dermorphin in modulating LHRH release is suggested.     

Immunohistochemical demonstration of GABAB receptors in the rat gastrointestinal tract

Immunohistochemical localization of GABA_B-receptors was demonstrated in the rat gastrointestinal tract using a monoclonal antibody (GB-1) raised against the purified GABA_B-receptor. Immunoreactive staining for GABA_B-receptors was found in some populations of endocrine, muscular and neuronal components in the stomach and gut wall. Positive mucosal epithelial, probably endocrine, cells were distributed throughout the stomach and intestine. Double immunostaining indicated that such positive cells for GABA_B-receptors often co-possessed serotonin in the small intestine but not in the gastric body. In the muscular layer of the digestive canal, positive staining was seen as dotty granules punctuated on the surface of muscle fibers. In the enteric nervous system, positive neuronal somata were found in both submucosal and myenteric ganglia throught the entire canal extending from the stomach to the rectum. This is the first report to visualize the cellular localization of GABA_B-receptors in the gastrointestinal system of the rat, and should provide a fundamental basis for future studies on gastrointestinal functions regulated by GABA_B-receptors. Special issue dedicated to Dr. Kinya Kuriyama.     

Increased UDP-glucuronyltransferase and gamma-glutamyltranspeptidase in enzyme-altered rat liver lesions produced by low doses of aflatoxin B1

Preneoplastic liver lesions were produced in female Wistar rats by low doses of aflatoxin B1 (Model 1: administration of 37.5 micrograms/kg 12 and 24 h after partial hepatectomy; Model 2: continuous application of 3.5 micrograms/kg in tap water daily for 28 days with partial hepatectomy after 14 days. The animals then received sodium phenobarbital, 0.1% in tap water, for 180 to 400 days). In both models numerous altered hepatic foci (AHF) and hyperplastic nodules (HN) were detected enzyme histochemically by their negative ATPase and positive gamma-glutamyltranspeptidase reactions. Immunohistochemically these lesions were also UDP-glucuronyltransferase positive. Increased UDP-glucuronyltransferase adds to permanent alterations of a number of drug metabolizing enzymes observed in a variety of different tumor models. These alterations are responsible for the toxin-resistant phenotype (Faber 1984b). Increased gamma-glutamyltranspeptidase was detected both enzyme histochemically and immunohistochemically; whereas gamma-glutamyltranspeptidase activity was present in both AHF/HN and in periportal areas by enzyme histochemistry, the immunohistochemical method selectively stained gamma-glutamyltranspeptidase in AHF and HN. Immunohistochemically detectable UDP-glucuronyltransferase and gamma-glutamyltranspeptidase are markers of putative precancerous liver lesions which may be useful in the analysis of the prestages of liver carcinogenesis.     

Immunohistochemical demonstration of androgen-binding protein in the rat prostatic gland.

Androgen-binding protein (ABP) is one of the best-characterized products of synthesis by the Sertoli cells in the rat. Although the exact physiological role of ABP remains to be determined, it has been widely used to study Sertoli cells and testicular function in this species. Since this protein is the principal carrier for testosterone in rat testis and epididymis, we decided to investigate ABP immunoreactivity (ABP-I) in androgen-dependent organs, including testicle, epididymides, prostate, and seminal vesicles. The location of ABP was investigated by immunohistochemistry using specific antisera against rat ABP. As previously described in the testis, rat ABP-I was identified in the seminiferous tubules within the cytoplasm of the Sertoli cells and the tubular luminae. The epididymis showed ABP-I only in epithelial cells of the proximal caput. We demonstrated ABP-I in the apical portions of epithelial cells of the rat prostate. Short-term castration and/or ligation of the efferent ducts did not suppress prostatic ABP-I. ABP-I was not present in seminal vesicles of control rats nor under any of the experimental conditions used throughout this study. The results also indicate the presence of ABP-I in prostatic epithelium, probably because of a mechanism similar to that described in epididymis. Our data support and enhance the concept that ABP may serve as a transmembrane carrier protein for androgens in androgen target organs in the male reproductive tract.