Katherine S. Koch

Increased sodium ion influx is necessary to initiate rat hepatocyte proliferation

Serum-free media containing 10-50 ng insulin, glucagon and epidermal growth factor (EGF) ml-1 stimulate adult rat hepatocyte proliferation in 10-15 day old primary liver cell cultures. The kinetics of this response simulate hepatocellular transitions that accompnay liver regeneration after 67% hepatectomy. Amiloride, a Na+ influx inhibitor, reversibly blocks these transitions in vitro (ID50 approximately 0.02 mM) and in vivo (ID50 approximately 25 mg kg-1). Inhibition is observed with other cation flux modulators, including ouabain (ID50 approximately 0.2 mM), 0.2 microM monensin and 0.2 microM nigericin, but not with 0.3 mM furosemide or tetrodotoxin. The prereplicative interval in culture (0-12 hr) is characterized by preferential cellular responsiveness to EGF (0-3 hr) followed by insulin plus glucagon (3-12 hr). Parallel culture and animal studies show that the amiloride-sensitive and prereplicative intervals coincide. In culture, a burst of 22Na+ influx, stimulated by peptide-supplemented media within 1 min but decreased later at 12 hr, is retarded by amiloride. This drug also blocks delayed prereplicative events involving increased amino acid A transport system function at 4-8 hr, and 3H-uridine and 3H-leucine incorporation into RNA and protein, respectively, at 8-12 hr. These findings suggest that at least two time-ordered processes are necessary to initiate hepatic growth fully: first, activation of Na+ flux systems by peptides similar or identical to EGF; and second, potentiation of these and subsequent cellular events by the combined action of insulin plus glucagon. [Amiloride: N-amidino-3,5-diamino-6-chloropyrazinecarboxamide; furosemide: 4-chloro-N-furfuryl-5-sulfamoylanthranilic acid; AIB: alpha-aminoisobutyric acid; ID50: administered dose giving 50% inhibition of a maximal response; dFBS: dialyzed fetal bovine serum; L.I.: 3H-dT nuclear labeling index.]

GROWTH CONTROL OF DIFFERENTIATED ADULT RAT HEPATOCYTES IN PRIMARY CULTURE

Much of the current knowledge about mammalian cell growth regulation comes from physiological studies of liver regeneration, maturation and carcinogenesis. To simplify the search for cellular and extracellular factors governing these phenomena, we have experimented since 197 1 with highly differentiated primary “monolayer” cultures of fetal and adult rat hepatocytes. These cells do not live in a threedimensional organ structure. They do not “see” gas tensions that normally perfuse the liver. They neither feed from nor excrete into a bathing fluid that continuously exchanges with an uninterrupted nutrient flow. And yet, with simple manipulations, such cells thrive more than a month in culture. This is seen in FIGURE 1 (bottom panel) for adult hepatocytes. Epithelial systems like these are powerful tools because they simulate proliferative and developmental events occurring in the animal.’-5 Work with the adult system suggests that transiently increased Na+ fluxes mediate early mitogenic actions of chemically-defined growth media.6 These fluxes, like those initiating DNA synthesis in fertilized eggs or electrical impulses in neurons, may comprise the first of two parts of a general membrane signalling process controlling eukaryotic cell proliferation7 (see also, Growth Regulation by Ion Fluxes, this Annals, volume 339). The second part, analogous to synaptic events that transduce nerve impulses and facilitated in hepatocytes by the pancreatic peptides glucagon and insulin (FIGURE 2), may involve a Ca++ flux linked to a burst of cyclic AMP synthesis.’ Recent findings of abnormally high Na+ levels in hepatomas implicate a defective Na+ flux system in the pathophysiology of hepatic ~ a n c e r . ~ There is evidence for transformation-related defects in the Ca++/cAMP “couple” as well.” But little is known about the mechanism of liver growth-control loss, which occurs spontaneously with aging or after detectable exposure to environmental carcinogens. In this report, the properties of “normal” adult hepatocytes cultured in this laboratory will be reviewed. New observations linking early ionic signalling events with “membrane potential” (A*) and “intracellular pH” (pHi) changes will be described. Lastly, regarding the problem of chemical carcinogenesis, the results of specific physicochemical and biological interactions of the hepatoprocarcinogen Nacetyl-2-aminofluorene (AAF) with adult cells will be summarized. If parenchymal cells are tumor precursors, these findings may provide information to transform cultured hepatocytes that truly resemble normal liver parenchyma into malignant cells.

[47] Liver cells

Publisher Summary This chapter deals with liver cells. Methods for establishing primary monolayer fetal and adult rat hepatocyte cultures are presented in the chapter. Techniques are emphasized rather than rationale or methodological validation. Fetal livers are obtained from pregnant rats 19–21 days in gestation; 14–19-day-old fetuses can be used but hepatocyte yields are reduced. Adult livers are obtained from rats, 150–300 g body weight, and fed standard Purina Chow and water ad libitum. Fetal bovine serum is purchased from standard suppliers and is pretested to ensure its suitability. Typical differentiated properties of primary monolayer rat liver cell cultures are also summarized in the chapter. The chapter discusses media and digestion buffer construction, cell isolation and plating, and cell counting. Functional properties of primary monolayer rat liver cell cultures are tabulated in the chapter.

IONIC EVENTS AT THE MEMBRANE INITIATE RAT LIVER REGENERATION

Rat liver regeneration is a prototype for studying the regulation of animal cell proliferation.’ A series of hepatocyte culture and animal experiments has vindicated the longstanding hepatotrophic theory by providing unequivocal evidence that regeneration is hormonally Physiological regulation by at least four, possibly even six blood-borne peptides has been implicatdcg Two originate from the pancreatic islets (insulin and glucagon); and two arise from the thyroparathyroid complex (calcitonin [see Whitfield er al., this volume] and parathyroid hormone). The others belong to a class of “insulin-like” substances produced by the liver (somatomedin-c) and by the gastrointestinal tract (epidermal growth factor-urogastrone-EGF). To understand how peptides might initiate liver regeneration, wc have been studying how their interactions promote the entry of “resting” hepatocytes into the DNA-synthetic (or Sphase) of the cell “cycle.”’* 2. ‘O We also are trying to identify rapidly activated cellular processes that cause the eventual growth response. Our results strongly suggest that, in response to peptides, one (perhaps the earliest) event needed to stimulate liver regeneration involves increased activation of membrane Na+ influx systems. Preldnary accounts of this hypothesis and its supporting evidence have appeared?. ”* ”

Amiloride inhibits protein synthesis in isolated rat hepatocytes

Abstract The effects of amiloride on Na + ion influx, amino acid transport, protein synthesis and RNA synthesis have been studied in isolated rat hepatocytes. The initial rate of 22 Na + uptake and the amount of 22 Na + taken up at later time points were decreased in hepatocytes incubated in the presence of amiloride. Amiloride inhibited by about 25% the influx of α-methylamino[1− 14 C]isobutyric acid, a specific substrate for the A (Alanine preferring) system of neutral amino acid transport. By contrast, the activity of system L (Leucine preferring) was not affected by amiloride. Rates of protein synthesis were determined by using high extracellular concentrations of [ 14 C]valine in order to maintain a constant amino acid precursor pool. Amiloride inhibited protein synthesis by 85% and had no effect on RNA synthesis. Half-maximal inhibition of protein synthesis occurred with amiloride at about 150 μM. In the absence of Na + in the incubation medium, the rate of protein synthesis was reduced by about 35% and no further inhibition was observed with amiloride. These results suggest that in isolated rat hepatocytes protein synthesis is partially dependent on Na + , and that amiloride is an efficient inhibitor of protein synthesis.

The effect of amiloride on hormonal regulation of amino acid transport in isolated and cultured adult rat hepatocytes

Insulin and glucagon stimulate amino acid transport in isolated rat hepatocytes. Amiloride, a specific Na+-influx inhibitor, completely inhibited the hormonal (glucagon or insulin) stimulation of alpha-aminoisobutyric acid influx by preventing the emergence of a high-affinity transport component. The drug also inhibited [14C]valine incorporation into hepatocyte protein. The half-maximal concentration of amiloride for inhibition of protein synthesis was similar to that required for inhibition of hormone-stimulated amino acid transport (approx. 0.1 mM). In primary cultured rat hepatocytes, amiloride markedly depressed the stimulation of alpha-aminoisobutyric acid transport by glucagon, or a mixture of glucagon, insulin and epidermal growth factor. These results suggest that amiloride inhibits the hormonal stimulation of hepatocyte amino acid transport by preventing the synthesis of high-affinity transport proteins. They also suggest that the hormonal stimulation of hepatocyte amino acid transport is dependent, at least partly, on Na+ influx.

Normal Liver Progenitor Cells in Culture

The stem cell nature of normal liver progenitor cells (LPCs) is addressed by studies of normal LPCs in culture. Several questions are addressed such as: What are the patterns of proliferation, lineage commitment, differentiated gene expression, plasticity, and responses to epigenetic and environmental signals? Early studies were interpreted to show that propagable LPCs were derived from dedifferentiated or retrodifferentiated mature liver cells. The recognition that oval cells seen in hepatocarcinogenesis models in rats had characteristics of LPCs suggested that these cells might actually be LPCs or descendants of LPCs. A comparison of more than 30 publications over three decades reporting explants; clonal lines; fresh isolates or strains of cells from noncarcinogen-exposed normal mouse, rat, pig, and human liver; or embryonic tissues indicates that small, immature LPCs, which have the plasticity to mature into ductal cells or hepatocytes, can be obtained from embryonic and fetal tissues, as well as adult liver. A wide variation in the methods of isolation, culture media, feeder layers, growth factors, and substrata used to study putative LPCs in vitro makes comparisons of results from different laboratories difficult. Although the liver is endodermally derived, putative coexpression of primitive hematopoietic and hepatocytic markers is consistent with LPCs in hepatic as well as in blood-forming tissues. In addition to bile duct and hepatocytic differentiation, LPCs have been reported to express markers of pancreatic and endothelial cells in vitro, and to differentiate into bile ducts, hepatocytes, pancreatic islet and acinar epithelial cells, intestinal epithelial cells, and cardiac myocytes after transplantation in vivo. Culture of LPCs on STO embryonic fibroblast feeder layers maintains primitive phenotypes, but requirements of feeder layers appear not to be absolute and are poorly understood. Emerging trends suggest HGF, Flt-3 ligand, SCF, EGF, and DMSO promote hepatocyte differentiation of LPCs, and that transforming growth factorβ, Na+-butyrate, and culture on Matrigel promote biliary differentiation; however, exceptions have been reported. Critical studies on proliferation kinetics have not convincingly shown self-renewal and asymmetric cell division expected of tissue stem cells, but long-term doublings (up to 150 generations) without spontaneous transformation suggest considerable growth potential. The source of LPCs in normal liver remains unknown and controversial. LPCs may be derived from a liver tissue progenitor cell located in the duct or periductal tissue; from retrodifferentiation of more mature hepatocytes; from bone marrow-derived cells, which circulate through the liver; or from bone marrow remnants of intrahepatic embryonic development. Given the lack of well-defined markers for LPCs, incomplete knowledge of their growth characteristics and regulation signals, their apparent heterogeneity, their apparent plasticity, the possibilities of transdifferentiation or retrodifferentiation of other cells to LPCs, or fusion of LPCs with other cells, as well as their potential for tumorigenesis, much research needs to be conducted to understand what LPCs are and how to use them.

Application of green fluorescent protein-protein A fusion protein to western blotting.

Publisher Summary Green fluorescent protein (GFP) that is isolated from the jellyfish Aequorea victoria noncatalytically produces an intense and stable greenish fluorescence. Aequorea GFP maximally absorbs blue light at 395 nm and emits green light with a peak at 509 nm. GFP is a protein of 238 amino acids with a molecular mass of 27-30 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The hexapeptide segment, beginning at residue 64, functions as a fluorescent chromophore formed on cyclization of the residues Ser· dehydro-Tyr-Gly within the hexapeptide, by posttranslational modification. GFP has a unique structure and interesting physical properties—for example, high stability to denaturing reagents or proteases. GFP fluorescence occurs without cofactors and this property allows GFP fluorescence in nonnative organisms in which GFP is expressed. Although GFP is relatively large to serve as a fusion tag, GFP-tagged proteins retain their original functions in many cases. Therefore, GFP has been used as a reporter for gene expression, as a tracer of cell lineage, and as a fusion tag to investigate protein localization and secretion systems in vivo. The reports of GFP fusions to protein A and to streptavidin, and of a simple immunoassay system using a GFP tag also indicate a wide range of in vitro applications. This chapter discusses the construction of a protein A-GFP fusion (PA-GFP) and its use as a labeled antibody-specific ligand in immunoblotting. Immunoblotting requires a labeled antibody or antibody-specific ligand (such as protein A) and a system specifically for detection of the label. Labeling reagents frequently used have been enzymes, such as peroxidase, alkaline phosphatase, and β-galactosidase; gold particles, radioisotopes, and fluorochromes have also been used.