James B. Blair

Isolated trout liver cells: Establishing short-term primary cultures exhibiting cell-to-cell interactions

SummaryComposition and interactions of cell types in rainbow trout (Oncorhynchus mykiss) liver digested with collagenase and cultured in serum-free media were investigated. Suspensions obtained after digesting trout liver with collagenase contained all the cell types present in the liver, including liver parenchymal cells (hepatocytes), biliary epithelial cells, sinusoidal endothelium, fat-storing cells of Ito, and macrophages. A major cell pellet, mainly hepatocytes but containing significant numbers of biliary epithelial cells, was obtained by centrifuging the cell suspension at 120×g for 1 min. Cells present in this pellet quantitatively attached to culture plates coated with a trout skin extract and remain attached for 4 to 6 d with good retention of intracellular enzymes and DNA. When in culture, significant changes in and among the cells were observed. Initial preparations were rounded, single cells. Within several hours, however, cellular interactions leading to aggregation became evident and aggregates increased in size for 2 to 3 d. Scanning electron microscopy (EM) showed frequent shaftlike projections from margins of the aggregates. Transmission EM indicated that these projections represent biliary ductules forming in vitro. Adjacent hepatocytes also showed plasma membrane specializations forming junctional complexes and canaliculi characteristics of normal trout liver. After 5 to 6 d in culture, significant numbers of the cell aggregates dislodged from the plate. Analysis showed the dislodged cells were viable but vacuolated. The reestablishment in vitro of morphologic relationships resembling in situ tissue components suggest these culture preparations may have significant utility in cooperative metabolic studies of cell interactions in trout liver.

Acute hormonal control of pyruvate kinase and lactate formation in the isolated rat hepatocyte

Abstract The activity of pyruvate kinase from the isolated rat hepatocyte was studied under conditions which allow investigation into the hormonal regulation of the enzyme. Incubating hepatocytes from fed or fasted rats with 1 μ m glucagon gives approximately 60% inhibition of the enzyme activity determined at 1.6 m m P-enolpyruvate. A good correlation between the regulation of pyruvate kinase and lactate formation from 10 m m dihydroxyacetone is observed in hepatocytes from fasted rats. When hepatocytes are incubated in a Krebs-Ringer phosphate buffer, the inhibition of the pyruvate kinase activity by 1 μ m glucagon is not accompanied by a marked inhibition of lactate production from fructose. Half-maximal regulation is observed at 0.26 ± 0.02 n m glucagon and 0.37 ± 0.05 n m glucagon for the enzyme and lactate formation from dihydroxyacetone respectively. Incubating hepatocytes with 10 m m l-alanine enhances inhibition of pyruvate kinase by physiological concentrations of glucagon, lowering the half-maximally effective concentration of glucagon from 0.3 n m to approximately 0.1 n m . A small but consistent inhibition of pyruvate kinase by 10 μ m epinephrine is also observed and this inhibition is enhanced by 0.5 m m theophylline and by 10 m m l-alanine. The inhibition of pyruvate kinase by epinephrine both in the absence and presence of theophylline is blocked by the α-adrenergic antagonist phenoxybenzamine. The β-adrenergic blocker propranolol has no influence on the inhibition of the enzyme by epinephrine. Adenosine 3′:5′-monophosphate, N 6 O 2 -dibutyryl adenosine 3′:5′-monophosphate, and guanosine 3′:5′-monophosphate also inhibit glycolysis from dihydroxyacetone and modulate pyruvate kinase activity in hepatocytes from fasted rats. Oleate, ethanol, and 3-hydroxybutyrate inhibit dihydroxyacetone glycolysis, but they do not influence the activity of pyruvate kinase. The divalent metal ionophore A23187 slightly stimulates lactate synthesis from dihydroxyacetone, but it has no influence on pyruvate kinase activity.

CULTURED TROUT LIVER CELLS: UTILIZATION OF SUBSTRATES AND RESPONSE TO HORMONES

SummaryThe characterization of a recently established system for the short-term culture of rainbow trout (Oncorhynchus mykiss) liver cells in chemically defined medium has been extended to studies on the metabolic competence of the cells and the characterization of their response to hormones. Three areas of metabolism have been addressed: a) the utilization of the exogenously added substrates fructose, lactate, glucose, dihydroxyacetone, and glycerol for glucose and lactate formation; b) the effects of the pancreatic hormones insulin and glucagon on cellular glucose formation, lactate formation, and fatty acid synthesis; and c) the effects of insulin and dexamethasone on the estradiol-dependent production of vitellogenin. Incubation of trout liver cells with fructose, lactate, glucose, dihydroxyacetone, or glycerol resulted in enhanced rates of cellular glucose and lactate production. Substrate-induced effects usually were more clearly expressed after extended (20 h) than after acute (5 h) culture periods. Addition of the hormones insulin or glucagon caused dose-dependent alterations in the flux of substrates to glucose and lactate. Rates of de novo synthesis of fatty acids from [14C]acetate were stimulated by insulin and inhibited by glucagon during acute and extended incubation periods. Treatment of liver cells isolated from male trout for 72 h with estradiol induced vitellogenin production and secretion into the medium. However, the addition of insulin or dexamethasone drastically reduced this estrogen-induced vitellogenesis. These results indicate that trout liver cells cultured in defined medium maintain central metabolic pathways, including glycolysis, gluconeogenesis, lipogenesis, and vitellogenesis as well as their responsiveness to various hormones, for at least 72 h. This cell culture system should provide an excellent model to further characterize metabolic processes in fish liver.

LONG-TERM PRIMARY CULTURE OF EPITHELIAL CELLS FROM RAINBOW TROUT (ONCORHYNCHUS MYKISS) LIVER

SummaryLong-term primary cultures of epithelial cells from rainbow trout (Oncorhynchus mykiss) liver have been established. Nearly homogenous (>97%) populations of hepatocytes were placed into primary culture and remained viable and proliferative for at least 70 d. In addition to hepatocytes, proliferative biliary cells persisted in the cultures for at least 30 d. Finally, a third type of epithelial cell, which we have termed a “spindle cell,” consistently appeared and proliferated to confluence in these cultures. The confluent cultures of spindle cells were successfully subcultured and passaged.The initial behavior, growth, and optimization of serum and media requirements for these cells is described. All three cell types proliferated as measured by thymidine incorporation, autoradiography, proliferating cellular nuclear antigen analysis, and propidium iodine staining. Further efforts to characterize the cells included western blotting and immunohistochemical staining with antibodies to cytokeratins previously reported in fish liver. From these data, it appears that all three cell populations are epithelial in nature. Furthermore, significant changes in actin organization, often indicative of transformation or pluripotent cells, were observed with increased time in primary culture.

Isolation and characterization of biliary epithelial cells from rainbow trout liver

SummaryLectin binding and density gradient centrifugation were explored for isolating epithelial cells from trout liver. Hepatocytes exhibited preferential attachment to coverslips coated withPhaseolus vulgaris erythroagglutinin. Biliary epithelial cells attached with glycine max agglutinin; however, significant attachment of cellular debris limited the use of glycine max agglutinin. Percoll-density gradient centrifugation separated liver cells into two distinct populations with biliary cells and hepatocytes banding at densities of 1.04 and 1.09, respectively. A discontinuous gradient composed of 13% Ficoll (wt/wt) separated biliary cells from hepatocytes. The recovery of highly enriched biliary epithelial cells from trout liver using Ficoll gradients yielded approximately 8 million cells (0.1 ml packed cells) from 10 g liver. Western blot analysis demonstrated that the cytokeratin profile for extracts from biliary epithelial cell-enriched populations differ significantly from those seen with whole liver extracts or with extracts from hepatocyte-enriched populations. Ficoll-gradient purified biliary cells and hepatocytes attached to culture plates coated with trout skin extract and carried out linear incorporation of leucine into protein and thymidine into DNA for 24 h. A mixture of growth hormones (insulin, epidermal growth factor, and dexamethasone) stimulated thymidine incorporation into DNA; however, long-term culture of dividing biliary epithelial cells was not achieved. Chemical analysis of neutral and acidic glycolipids indicated that hepatocytes and biliary cells have similar glycolipid profiles with an exception in the region of GM3 mobility, which is attributable to differences in the ceramide moiety. These studies provide a starting point for further characterization of unique cell types of the trout liver that may be important in their response to toxic and carcinogenic agents.

Gluconeogenesis in rabbit liver: IV. The effects of glucagon, epinephrine, α-and β-adrenergic agents on gluconeogenesis and pyruvate kinase in hepatocytes given dihydroxyacetone or fructose

1. Epinephrine, isoproterenol and phenylephrine each increases significantly gluconeogenesis (from dihydroxy-acetone or D-fructose) and glycogenolysis when added to hepatocytes from 48-h fasted rabbits. Such stimulation of both processes by epinephrine, isoproterenol or phenylephrine is negated by the beta-adrenergic antagonist propranolol but remains significant in the presence of the alpha-adrenergic antagonist phentolamine. Conversely, previous data suggest that catecholamine-induced stimulation of glucose formation from L-lactate is both alpha- and beta-adrenergic-sensitive. 2. Glucagon, epinephrine, isoproterenol, phenylephrine and dibutyryl cyclic AMP each inhibits significantly pyruvate kinase activity in rabbit hepatocytes. Inhibition of pyruvate kinase activity by epinephrine, isoproterenol or phenylephrine is negated by propranolol but insensitive to phentolamine. 3. These observations suggest that enhancement by epinephrine of glucose formation from either dihydroxyacetone or D-fructose is solely beta-adrenergic-regulated, just as is its inhibition of pyruvate kinase activity. Stimulation of gluconeogenesis by glucagon, epinephrine, isoproterenol, phenylephrine or dibutyryl cyclic AMP may be at least in part directly related to their ability to inhibit pyruvate kinase.

Cloning of the Retinoblastoma cDNA from the Japanese medaka (Oryzias latipes) and preliminary evidence of mutational alterations in chemically-induced retinoblastomas

We have cloned a medaka homolog of the human retinoblastoma (Rb) susceptibility gene. The medaka Rb cDNA encodes a predicted protein of 909 amino acids. DNA sequence analysis with other vertebrate Rb sequences demonstrates that the medaka Rb cDNA is highly conserved in regions of functional importance. An antibody raised against an epitope of the human pRb recognizes the protein product of the medaka Rb gene, detecting a 105 kDa protein in all tissues examined and at differential levels for the stages of embryonic development studied. The sequence reported herein, combined with the high degree of conservation observed in critical domains, has also facilitated a preliminary investigation of the molecular etiology of chemically-induced retinoblastoma. The mutational alterations characterized suggest that medaka may provide a novel model and, thus, provide additional insight into the human retinoblastoma condition.

Rat liver pyruvate kinase: Influence of ligands on activity and fructose 1,6-bisphosphate binding

The ability for various ligands to modulate the binding of fructose 1,6-bisphosphate (Fru-1,6-P2) with purified rat liver pyruvate kinase was examined. Binding of Fru-1,6-P2 with pyruvate kinase exhibits positive cooperativity, with maximum binding of 4 mol Fru-1,6-P2 per enzyme tetramer. The Hill coefficient (nH), and the concentration of Fru-1,6-P2 giving half-maximal binding [FBP]1/2, are influenced by several factors. In 150 mM Tris-HCl, 70 mM KCl, 11 mM MgSO4 at pH 7.4, [FBP]1/2 is 2.6 microM and nH is 2.7. Phosphoenolpyruvate and pyruvate enhance the binding of Fru-1,6-P2 by decreasing [FBP]1/2. ADP and ATP alone had little influence on Fru-1,6-P2 binding. However, the nucleotides antagonize the response elicited by pyruvate or phosphoenolpyruvate, suggesting that the competent enzyme substrate complex does not favor Fru-1,6-P2 binding. Phosphorylation of pyruvate kinase or the inclusion of alanine in the medium, two actions which inhibit the enzyme activity, result in diminished binding of low concentrations of Fru-1,6-P2 with the enzyme. These effectors do not alter the maximum binding capacity of the enzyme but rather they raise the concentrations of Fru-1,6-P2 needed for maximum binding. Phosphorylation also decreased the nH for Fru-1,6-P2 binding from 2.7 to 1.7. Pyruvate kinase activity is dependent on a divalent metal ion. Substituting Mn2+ for Mg2+ results in a 60% decrease in the maximum catalytic activity for the enzyme and decreases the concentration of phosphoenolpyruvate needed for half-maximal activity from 1 to 0.1 mM. As a consequence, Mn2+ stimulates activity at subsaturating concentrations of phosphoenolpyruvate, but inhibits at saturating concentrations of the substrate or in the presence of Fru-1,6-P2. Both Mg2+ and Mn2+ diminish binding of low concentrations of Fru-1,6-P2; however, the concentrations of the metal ions needed to influence Fru-1,6-P2 binding exceed those needed to support catalytic activity.

Response of rainbow trout liver to partial hepatectomy

Abstract Existing procedures for partial hepatectomy in rainbow trout (Oncorhynchus mykiss) liver were modified and a greater than 95% success rate achieved. The initial response of the rainbow trout liver to partial hepatectomy was evaluated. DNA synthesis, determined by tritiated thymidine incorporation into DNA in liver slices, increased significantly in the first 24–48 h following partial hepatectomy and remained elevated for at least 10–14 days. Protein content based on wet weight of the liver, however, appeared to remain constant in the two weeks following partial hepatectomy. The procedure was equally effective on both large (600 g) and small (6–80 g) fish and should be useful for either promotion in complete carcinogenesis regimens or other toxicological studies requiring liver cell proliferation.

Characterization of BNF-inducible cytochrome P-450IA1 in cultures of rainbow trout liver cells

Abstract We are investigating isolated rainbow trout hepatocytes as a model system for characterizing the carcinogenic and toxic effects of xenobiotics. For these studies, it will be important to characterize cytochrome P-450IA1, the β-naphthoflavone (BNF)-inducible isozyme, which is fundamental for the phase I metabolism of many xenobiotics. In this study, ethoxyresorufin O-deethylase (EROD) activity and immunoreactivity of P-450IA1 were measured in isolated trout liver cells maintained in culture for at least 3 days. Liver cells prepared from trout pretreated with BNF exhibited readily detectable EROD activity, which rapidly declined when cells were placed in culture, with a half-life of about 15 h. Addition of BNF, dexamethasone, glucagon, insulin or δ-aminolevulinic acid alone or in combinations neither prevented this decline nor induced EROD activity in isolated cells prepared from control animals. Acetaminophen in the culture media helped maintain EROD activity, immunoreactive P-450 and total cytochrome P-450 in hepatocytes prepared from BNF-treated trout. Light and electron microscopy revealed that concentrations of acetaminophen which were most effective in maintaining EROD produced distinctive toxic effects to the cells. The mechanism(s) by which acetaminophen influences EROD activity is not yet known; however, these findings establish one way to manipulate P-450IA1 in cultured trout hepatocytes.