scholarly journals GROWTH CONTROL OF DIFFERENTIATED FETAL RAT HEPATOCYTES IN PRIMARY MONOLAYER CULTURE

1974 ◽  
Vol 62 (3) ◽  
pp. 792-801 ◽  
Author(s):  
H. L. Leffert
Keyword(s):  
Dna Synthesis ◽  
Environmental Control ◽  
Primary Cultures ◽  
Growth Control ◽  
Rat Hepatocytes ◽  
Defined Medium ◽  
Crystalline Insulin ◽  
Fetal Rat ◽  
Primary Monolayer

The initiation of DNA synthesis has been studied in quiescent primary cultures of fetal rat hepatocytes using defined hormones and chemically defined medium. Preparations of crystalline insulin (0.01–10 µg/ml) or 20,000-fold purified somatomedin (0.01–1 µg/ml) are stimulatory. Growth hormone (0.025 µg/ml) and hydroxycortisone (0.025 µg/ml), 3':5'-GMP! (10-5 M) fail by themselves to initiate DNA synthesis but added together with insulin, enhance the stimulatory response by 50–100%. Thyroid hormones (L-T3, L-T4, 7.5–30 ng/ml) are by themselves without effect, but when they are added to thyroid hormone-depleted serum, the reconstituted mixtures show an enhanced capacity to initiate DNA synthesis. In contrast, glucagon (0.01 µg/ml) inhibits the insulin-stimulated response by about 50% without reducing basal DNA synthesis rates. The results described here and in the accompanying two reports indicate that environmental control over the initiation of DNA synthesis is complex, and can involve the participation of many factors. The in vitro findings are consistent with the concept that liver regeneration is hormonally controlled and raise the possibility that alterations of the intrahepatic ratio of insulin to glucagon are growth regulatory.

scholarly journals GROWTH CONTROL OF DIFFERENTIATED FETAL RAT HEPATOCYTES IN PRIMARY MONOLAYER CULTURE

1974 ◽  
Vol 62 (3) ◽  
pp. 780-791 ◽  
Author(s):  
K. Koch ◽  
H. L. Leffert
Keyword(s):  
Dna Synthesis ◽  
Primary Cultures ◽  
Growth Control ◽  
Rat Hepatocytes ◽  
Solvent System ◽  
Serum Factors ◽  
Conditioning Factors ◽  
Cellular Processes ◽  
Fetal Rat ◽  
Fraction I

Serum-deficient ≤0.00003% vol/vol) conditioned medium (CM) obtained from primary cultures of fetal rat hepatocytes initiates DNA synthesis and mitosis in homologous quiescent cultures. CM similarly prepared from 3T3 fibroblast cultures is inactive. At least two conditioning factors are involved in initiating DNA synthesis. The first of these, arginine, is obligatory, synthesized by the cells, and released into the culture medium. The second, a lipid or lipid-containing material, is stable to pH extremes (pH 2, pH 10) and chromatographs with an apparent R1 ∼0.5 on silica gel thin-layer plates using hexane-ether (4: 1) as the solvent system. It is suggested that these cultured hepatocytes enter or leave the G0 or early G1 phase of the cell cycle as determined in part by their capacity to use available conditioning factor and nutrient components of the medium, in particular, arginine. Serum factors including serum fraction I (4), insulin, and possibly, lipid-like conditioning material appear to initiate DNA synthesis by controlling cellular processes involved with the enhanced utilization and synthesis of growth-limiting nutrients.

scholarly journals Growth control of differentiated fetal rat hepatocytes in primary monolayer culture. IX. Specific inhibition of DNA synthesis initiation by very low density lipoprotein and possible significance to the problem of liver regeneration.

1976 ◽  
Vol 70 (1) ◽  
pp. 20-32 ◽  
Author(s):  
H L Leffert ◽  
D B Weinstein
Keyword(s):  
Dna Synthesis ◽  
Low Density Lipoprotein ◽  
Growth Control ◽  
Density Lipoprotein ◽  
Rat Hepatocytes ◽  
Hepatocyte Proliferation ◽  
Very Low Density Lipoprotein ◽  
Low Density ◽  
Rat Serum ◽  
Fetal Rat

Rat serum very low density lipoprotein (VLDL) inhibits initiation of DNA synthesis in fetal rat hepatocyte cultures; cells engaged in synthesizing DNA resist inhibition. VLDL action is specific and apparently blocks prereplicative protein synthesis. These and other results, from studies of altered blood VLDL levels and [3H] thymidine incorporation into isolated liver nuclei in 70% hepatectomized normal and mutant hyperlipoproteinemic rats, as well as from infusion studies with a "mitogenic" hormone solution, suggest that hepatic VLDL metabolism is linked to the suppression of hepatocyte proliferation.

The emergence of ErbB2 expression in cultured rat hepatocytes correlates with enhanced and diversified EGF-mediated signaling

2006 ◽  
Vol 291 (1) ◽  
pp. G16-G25 ◽  
Author(s):  
Lawrence A. Scheving ◽  
Linda Zhang ◽  
Mary C. Stevenson ◽  
Eun Soo Kwak ◽  
William E. Russell
Keyword(s):  
Dna Synthesis ◽  
Tyrosine Phosphorylation ◽  
Egf Receptor ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Immunological Methods ◽  
Erbb Receptors ◽  
Erbb2 Expression ◽  
Egfr Expression

The proliferative effects of EGF in liver have been extensively investigated in cultured hepatocytes. We studied the effects of EGF, insulin, and other growth regulators on the expression, interaction, and signaling of ErbB receptors in primary cultures of adult rat hepatocytes. Using immunological methods and ErbB tyrosine kinase inhibitors, we analyzed the expression and signaling patterns of the ErbB kinases over 120 h of culture. Basal and EGF-stimulated protein tyrosine phosphorylation increased as cells adapted in vitro. EGF receptor (EGFr) expression declined in the first 24 h, whereas ErbB3 expression rose. Although ErbB2 was not present in freshly isolated hepatocytes, EGF and insulin independently induced ErbB2 while suppressing ErbB3 expression. Low concentrations of EGF and insulin synergistically stimulated ErbB2 expression and DNA synthesis. The greatest increase in ErbB2, which is normally expressed by fetal and neonatal hepatocytes, occurred shortly before the onset of DNA synthesis (>40 h). EGF promoted EGFr and ErbB2 coassociation, stimulating tyrosine phosphorylation of both proteins. In contrast, heregulin β1 (HRG-β1) did not promote ErbB2 and ErbB3 coassociation. A selective tyrphostin inhibitor of ErbB2 suppressed EGF-stimulated DNA synthesis, but maximum suppression required the blockade of the EGFr kinase as well. Maximal EGF stimulation of DNA synthesis in vitro depends on the induction of ErbB2 and involves an EGFr-ErbB2 heterodimer. The ability of insulin to induce ErbB2 suggests both a mechanism for the synergy between insulin and EGF and a possible metabolic control of ErbB2 in vivo.

Retroviral vector infection and transplantation in rats of primary fetal rat hepatocytes

1991 ◽  
Vol 99 (1) ◽  
pp. 121-130
Author(s):  
K.S. Koch ◽  
G.G. Brownlee ◽  
S.J. Goss ◽  
A. Martinez-Conde ◽  
H.L. Leffert
Keyword(s):  
Reporter Gene ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Developmental State ◽  
Retroviral Vectors ◽  
Time Interval ◽  
Gene Products ◽  
Fetal Rat ◽  
Human Factor Ix

Retroviral vectors were used to transduce recombinant DNA encoding firefly luciferase, Escherichia coli beta-galactosidase or human factor IX into fetal rat hepatocytes in primary culture. Hepatocytes were transduced optimally during a restricted time interval, 2–4 days post-plating. Although efficient and stable expression of reporter gene products was observed in vitro, it was affected differentially by culture conditions (plating density, media constituents) and chemical modulators of hepatocyte growth and differentiation (gelatin, hydrocortisone, isobutylmethylxanthine). Cultured cells, mock-infected or infected with a luciferase-expressing vector, were harvested non-enzymatically and injected subcutaneously into the dorsal neck fascia of neonatal syngeneic rats. Tissue isolated from injection sites one week later contained hepatocyte foci. In animals transplanted with infected cells, the preliminary results suggest that luciferase activity was present at these sites in proportion to the numbers of injected cells. These findings and previous observations made with hepatocytes from neonatal and adult primary cultures, indicate that from day 19 in utero through maturity the transient temporal ‘period of susceptibility’ to infection in vitro is independent of the developmental state of starting tissue. Transplantability of cultured fetal hepatocytes infected with retroviral vectors and stably expressing reporter gene products suggests that such cells might provide promising models for liver gene therapy.

Comparison of peptide release from fetal rat hypothalamic neurones cultured in defined media and serum-containing media

1988 ◽  
Vol 116 (3) ◽  
pp. 349-356 ◽  
Author(s):  
M. J. O. Clarke ◽  
G. E. Gillies
Keyword(s):  
Primary Cultures ◽  
Defined Medium ◽  
Maximal Response ◽  
Dependent Manner ◽  
Corticotrophin Releasing Factor ◽  
Fetal Rat ◽  
Concentration Step ◽  
And Control

ABSTRACT Primary cultures of rat hypothalamic neurones were maintained either in a serum-supplemented medium or in a serum-free chemically defined medium for up to 6 weeks. The release of the 41 amino acid-containing peptide, corticotrophin-releasing factor (CRF-41), vasopressin (AVP) and somatostatin (SRIF) were followed using immunoassays. In response to K+ (56 mmol/l) depolarization both the quantities of peptides released and the magnitude of responses were significantly greater from cultures maintained in the fully supplemented defined medium. As a consequence, release of CRF-41 and AVP could be measured directly, without requiring the concentration step necessary for cultures grown in serum. The response to K+ depolarization increased with the age of the culture, suggesting neuronal maturation. Responses to K+ depolarization were Ca2+-dependent, and the addition of corticosterone (100 nmol/l) to the defined medium caused a significant reduction in the response of neurones secreting CRF-41 and AVP, but not those secreting SRIF, to depolarization. This suggests the retention in vitro of the responsiveness of stress-associated neuropeptides to the negative feedback effects of corticosterone. Neurones producing CRF-41 and AVP responded significantly in a dose-dependent manner to acetylcholine stimulation, whereas those producing SRIF did not. As cultures matured, the CRF-41- and AVP-producing neurones became more sensitive to acetylcholine with the maximal response at 1 nmol acetylcholine/1. In conclusion, the culture of rat hypothalamic neurones is improved in terms of peptide output when the cultures are maintained in a defined medium. Differential responses of the peptidergic neurones may be seen in the presence of corticosterone and neurotransmitters, illustrating the retention in vitro of specific receptor-mediated responses which have been observed in vivo. This model should prove useful in the further study of the physiological, pharmacological and biochemical maturation and control of peptidergic neurones. J. Endocr. (1988) 116, 349–356

scholarly journals GROWTH CONTROL OF DIFFERENTIATED FETAL RAT HEPATOCYTES IN PRIMARY MONOLAYER CULTURE

1974 ◽  
Vol 62 (3) ◽  
pp. 767-779 ◽  
Author(s):  
H. L. Leffert
Keyword(s):  
Dna Synthesis ◽  
Cell Attachment ◽  
Gel Filtration ◽  
Rat Hepatocytes ◽  
Hepatocyte Proliferation ◽  
Cell Multiplication ◽  
Adult Rats ◽  
Fetal Rat ◽  
Fraction I

Dialyzed fetal bovine serum contains two distinct growth-controlling macromolecular fractions: one stimulates and the other inhibits proliferation of primary cultured differentiated fetal rat hepatocytes. Both fractions are precipitated by ammonium sulfate (50% saturation, pH 7.4, 4°C). Serum fraction I (SFI, mol wt ≥ 120,000 daltons estimated by gel filtration with Bio-gel P200) appears to contain at least two factors which function, respectively, to initiate DNA synthesis (activity pH 4–10 stable) and to increase the rate at which initiated cells traverse the cell cycle (activity pH 4 and pH 10 labile). Intraperitoneal injections of SFI into adult rats have produced detectable stimulation of hepatic but not renal DNA synthesis. Serum fraction II (SFII, mol wt 40,000–80,000 daltons) suppresses in vitro incorporation of CH3-[3H]thymidine into DNA under conditions which diminish neither cell viability nor cell attachment. Mixing experiments indicate that SFI and SFII mutually antagonize each other with respect to DNA synthesis and cell multiplication. Thus, both the relative and absolute serum levels of multiple factors control in vitro fetal hepatocyte proliferation.

Expression of Liver Functions in Imrnortalised Rot Hepotocyte Cell Lines

1994 ◽  
Vol 13 (6) ◽  
pp. 439-444 ◽  
Author(s):  
Caroline MacDonald ◽  
Maureen Vass ◽  
Brian Willett ◽  
Alexander Scott ◽  
Helen Grant
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Primary Cultures ◽  
Rat Hepatocytes ◽  
Hepatic Function ◽  
Glutathione S Transferase ◽  
Early Passage ◽  
Toxicological Studies ◽  
Well Differentiated

The differentiated hepatic function of two rat liver cell lines, P9 and SV40RH1, immortalised by transfection with SV40 DNA has been investigated in terms of the glutathione synthesis, and the activities of γ-glutamyltransferase, glutathione-S-transferase and UDP-glucuronosyltransferase. SV40RH1 is a highly differentiated cell line at early passage, but the expression of some aspects of its differentiated phenotype is unstable and some functions are lost by passage 12-13. P9 is a less-well differentiated cell line, with relatively stable expression of functions between passages 4 and 13. In terms of differentiated function both cell lines represent a marked improvement over primary cultures of rat hepatocytes which de-differentiate rapidly within 24-48 h in culture. This retention of liver function in proliferating cell lines offers the opportunity to use such cells in in vitro toxicological studies.

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