Interaction of epidermal growth factor with adult rat liver parenchymal cells in primary culture

Moriarity, Debra M.; Savage, C. Richard

doi:10.1016/0003-9861(80)90208-8

Cited by 74 publications

(28 citation statements)

References 32 publications

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“…Two hours of incubation at 4°C is sufficient to reach steady-state binding conditions for lz51-EGF in primary hepatocyte cultures (data not shown). This optimum binding time is in agreement with that found by other investigators for EGF binding in hepatocyte cultures (Moriarty and Savage, 1980). Nonspecific binding was determined as counts bound in the presence of 1,000-fold excess unlabeled EGF.…”

Section: L%-egf Binding Assaysupporting

confidence: 90%

Norepinephrine decreases EGF binding in primary rat hepatocyte cultures

Cruise

Cotecchia

Michalopoulos

1986

Journal Cellular Physiology

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Norepinephrine (NE) produced a dose-dependent inhibition of 125I-epidermal growth factor (EGF) binding to adult rat hepatocytes in primary culture. This effect was maximal after 1 hr of incubation with NE and could be blocked by the presence of an alpha 1-specific adrenergic receptor antagonist. The inhibition of binding correlates with the ability of NE to enhance hepatocyte DNA synthesis in the presence of EGF and appears to be mediated by a reduction in EGF receptor number, without a significant change in receptor affinity.

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Section: L%-egf Binding Assaysupporting

confidence: 90%

Norepinephrine decreases EGF binding in primary rat hepatocyte cultures

Cruise

Cotecchia

Michalopoulos

1986

Journal Cellular Physiology

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“…Vdref dt= -kon-(PT -X2)'C1 -Q.C1 + koffX2 [4] dX2= ko -(PT -X2)-C1 -(koff + ks)'X2, [5] dt where Vdref iS the volume of the extracellular space, C1 is the concentration of the peptide in the extracellular space, Q is the perfusion rate, and X2 is the amount of the peptide bound to the receptors. The plasma flow rate at each dose was in the range of 0.85-1.13 ml of plasma per min per g of liver.…”

Section: Determination Of Kinetic Parameters For the Interactionmentioning

confidence: 99%

“…The interaction between peptide hormones and their specific receptors has been analyzed principally by in vitro studies with isolated or cultured hepatocytes (4) and liver plasma membranes (5). These systems, however, neither maintain cell polarity nor include ligand delivery by the blood flow, thus creating difficulty when the physiological significance of the peptide-receptor interaction is being experimentally evaluated.…”

mentioning

confidence: 99%

Dynamic determination of kinetic parameters for the interaction between polypeptide hormones and cell-surface receptors in the perfused rat liver by the multiple-indicator dilution method.

Sato

Sugiyama

Sawada

et al. 1988

Proc. Natl. Acad. Sci. U.S.A.

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Hepatic elimination of epidermal growth factor (EGF) via receptor-mediated endocytosis was studied by a multiple-indicator dilution method in the isolated perfused rat liver, in which cell polarity and spatial organization are maintained. In this method EGF was given with inulin, an extracellular reference, as a bolus into the portal vein, and dilution curves of both compounds in the hepatic vein effluent were analyzed. Analysis of the dilution curve for EGF, compared with that for somatostatin, which showed no specific binding to isolated liver plasma membranes, resulted as follows: (i) both extraction ratio and distribution volume of '2MI-labeled EGF decreased as the injected amount of unlabeled EGF increased; (ii) the ratio plot [In (inulin/EGF) versus time] ofthe dilution curve for EGF exhibited an upward straight line initially for a short period of time (-10 sec), whereas the ratio plot [In (inulin/somatostatin) versus time] of somatostatin gradually decreased. The multiple-indicator dilution method was used for other peptides also. Insulin and glucagon, known to have hepatocyte receptors, behaved similarly to EGF in shape of their ratio plots. Thus, analysis of dilution curves can reveal whether or not the cell surface has receptors for certain peptides. In addition, the dilution curves for EGF at various doses (tracer 30 jag) were analyzed simultaneously based on a kinetic model incorporating the perfusion rate, the association rate constant of EGF to surface receptors (kon), the dissociation rate constant of EGF from the EGF-receptor complex (ko)9 and the sequestration rate constant ofthe complex. The kinetic parameters [the dissociation constant (Kd = kl/k,) and the number of surface receptors] calculated by this analysis were comparable with reported values obtained by in vitro direct binding measurements at equilibrium using liver homogenates. We conclude that the multiple-indicator dilution method is a good tool for analyzing the dynamics of peptide hormones-cell-surface receptor interaction under a condition in which spatial architecture of the liver is maintained.Many peptide hormones in the circulating blood are eliminated by the liver. Above all, for hormones taken up via receptor-mediated endocytosis, the liver is a major homeostatic regulator in controlling the concentration of peptide hormones in circulating blood (1-3).The interaction between peptide hormones and their specific receptors has been analyzed principally by in vitro studies with isolated or cultured hepatocytes (4) and liver plasma membranes (5). These systems, however, neither maintain cell polarity nor include ligand delivery by the blood flow, thus creating difficulty when the physiological significance of the peptide-receptor interaction is being experimentally evaluated. Use of the liver-perfusion method may effectively resolve this problem, because cell polarity and spatial architecture between hepatocytes and the capillary bed are maintained in this system. However, determination of the microscopic kinetic constan...

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“…Recently, we demonstrated the specific interaction of EGF with cell surface receptors on normal adult rat liver cells in primary culture (10). These cells are maintained as nondividing cultures.…”

mentioning

confidence: 99%

“…Exactly what those mechanisms are, however, is not clear. OrnDCase decarboxylase has a short halflife, 10 min, in regenerating liver (32), with its levels regulated mainly by its rates ofsynthesis and degradation (33). The ability of EGF to nearly double OrnDCase levels in the presence of concentrations of cycloheximide that inhibit more than 90% of protein synthesis (34) suggests that EGF may be either activating an inactive form of the enzyme or decreasing its degradation rate.…”

mentioning

confidence: 99%

Epidermal growth factor stimulation of ornithine decarboxylase activity in a human hepatoma cell line.

Moriarity¹,

DiSorbo²,

Litwack³

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

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Epidermal growth factor (EGF) bound specifically to the human hepatoma cell line PLC/PRF/5. Treatment of these cells with nanomolar concentrations of EGF for 4-6 hr resulted in a 2-to 6-fold increase in ornithine decarboxylase (L-or-nithine carboxy-lyase, EC 4.1.1.17) activity. 12-0-Tetradecanoylphorbol 13-acetate also produced an increase in enzyme activity in these cells and exhibited an additive effect with EGF Epidermal growth factor (EGF) influences a number ofcellular processes such as nutrient transport; protein, RNA, and DNA synthesis; and cell multiplication in many normal and abnormal cell types (1). One enzyme whose cellular level is increased by EGF treatment is ornithine decarboxylase (OrnDCase; L-ornithine carboxy-lyase, EC 4.1.1.17), which catalyzes the ratelimiting step in polyamine biosynthesis (2). This enzyme is of special interest because polyamines have been implicated in the regulation ofcell growth (3,4). EGF elevated OrnDCase levels in the testes, kidney, liver, stomach, and duodenum of mice injected with the growth factor (5, 6). In vitro, EGF increased OrnDCase levels in mouse and human skin fibroblasts (7), porcine granulosa cells (8), and normal rat kidney cells (9).Recently, we demonstrated the specific interaction of EGF with cell surface receptors on normal adult rat liver cells in primary culture (10). These cells are maintained as nondividing cultures. To assess the EGF effects on a dividing population of liver cells, we have used the human hepatoma cell line PLC/ PRF/5. This cell line was established in 1976 and was found to produce hepatitis B surface antigen (11,12). DiSorbo et al. (13) demonstrated the presence ofthe hepatic glucocorticoid receptor and the ability of glucocorticoids to induce tyrosine aminotransferase activity in these cells. As they do not synthesize albumin or alpha fetoprotein but do synthesize several other plasma proteins (14), it appears that only partially differentiated liver cell function is present in these cells. Elucidation of the hormonal factors that may be involved in the growth of tumorderived cells is of key importance in understanding the conditions that initiate and sustain malignant growth.We have examined the binding of EGF to the PLC/PRF/ 5 cells and the effect of EGF treatment on their OrnDCase activity level. The human hepatoma cells were found to have specific high-affinity binding sites for EGF and to exhibit increased OrnDCase activity after exposure to EGF for several hours.Thus, EGF may be one humoral factor involved in the regulation of liver cell growth. The tumor promotor, 12-0-tetradecanoylphorbol 13-acetate (TPA), had an effect similar to EGF on OrnDCase activity in this cell line. MATERIALS AND METHODSCell Cultures. The human hepatoma cell line PLC/PRF/5 was obtained from the Wistar Institute (Philadelphia, PA). The cells were grown in an air incubator using Eagle's minimal essential medium-alpha growth medium (GIBCO)/4 mM glutamine/7% fetal bovine serum (North American Biologicals, Miami, FL)/50 mM N-2-hydroxyeth...

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Interaction of epidermal growth factor with adult rat liver parenchymal cells in primary culture

Cited by 74 publications

References 32 publications

Norepinephrine decreases EGF binding in primary rat hepatocyte cultures

Norepinephrine decreases EGF binding in primary rat hepatocyte cultures

Dynamic determination of kinetic parameters for the interaction between polypeptide hormones and cell-surface receptors in the perfused rat liver by the multiple-indicator dilution method.

Epidermal growth factor stimulation of ornithine decarboxylase activity in a human hepatoma cell line.

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