Epidermal growth factor and cyclic AMP stimulate Na+/H+ exchange in isolated rat hepatocytes

Na+/H+ exchange in acid-loaded isolated hepatocytes was measured using the intracellular pH indicator biscarboxyethyl-carboxyfluorescein (BCECF) to follow intracellular pH (pHi). The rate of amiloride-sensitive Na(+)-dependent recovery from cytoplasmic-acid-loading was found to be increased in cells treated with epidermal growth factor (EGF), 8-(4-chlorophenylthio)adenosine 3',5'-monophosphate (ClPhScAMP) or phorbol 12-myristate 13-acetate (PMA). These three agents increased the rate of Na+/H+ exchange to similar extents and their effects were not additive. The stimulation was shown in all three cases to be due an alkaline shift of 0.1 in the set point pH of the Na+/H+ exchanger. Experiments measuring the uptake of 22Na+ into acid-loaded primary hepatocyte monolayer cultures confirmed these results. EGF, ClPhScAMP and PMA significantly increased the amiloride-inhibitable accumulation of 22Na+, thus providing further evidence that Na+/H+ exchange is stimulated by these effectors.     

Epidermal growth factor mimics insulin effects in rat hepatocytes.

Epidermal growth factor (EGF) mimicked the effect of insulin to activate glycogen synthase and stimulate glycogen synthesis in isolated rat hepatocytes. Both agents required glucose (greater than 5 mM) and had similar time courses of action. The maximum effect of EGF was approx. 70% of that of insulin, and the half-maximally effective concentrations were 9 nM and 4 nM respectively. Combinations of the two agents produced additive responses. EGF also resembled insulin in its ability to inhibit the effects of 0.1-1.0 nM-glucagon on cyclic AMP and glycogen phosphorylase in hepatocytes. The maximum effect of EGF was approx. 70% of that of insulin, and the half-maximally effective concentrations were approx. 5 nM and 0.5 nM respectively. EGF and insulin inhibited phosphorylase activation by exogenous cyclic AMP, and inhibited cyclic AMP accumulation induced by forskolin. They also inhibited phosphorylase activation provoked by phenylephrine, but not by vasopressin. EGF added alone rapidly activated phosphorylase and increased cytosolic [Ca2+], but the effects were no longer apparent at 5 min and were smaller than those of vasopressin. Insulin did not induce these changes. In hepatocytes previously incubated with myo-[3H]inositol, EGF did not significantly increase myo-inositol 1,4,5-trisphosphate. However, its ability to increase cytosolic [Ca2+] was blocked by neomycin, an inhibitor of phosphatidylinositol bisphosphate hydrolysis. It is concluded that some, but not all, of the effects of EGF in liver are strikingly similar to those exerted by insulin, suggesting that these agents may have some similar mechanisms of action in this tissue.     

Studies on lipid peroxidation in rat liver nuclei and isolated nuclear membranes

Non-enzymatic and enzymatically-driven lipid peroxidation processes were studied in rat liver nuclei and isolated nuclear membranes, by evaluating the formation of thiobarbituric acid-chromophore, free malondialdehyde, lipofuscin-like pigments, and the degradation of polyunsaturated fatty acids of the nuclear membrane lipids. The results obtained show that: (1) both non-enzymatic and enzymatically driven lipid peroxidation processes are operative in cell nuclei and isolated nuclear membranes; (2) only for isolated nuclear membranes, a good qualitative and up to a great extent quantitative correlation between malondialdehyde and lipofuscin-like pigment formation was obtained; (3) there is a qualitative but not quantitative correlation between malondialdehyde formation and polyunsaturated fatty acid degradation; (4) lipid peroxidation processes in isolated nuclear membranes and intact nuclei have an essentially identical kinetic behaviour. No statistical differences in the relative increases in the concentrations of malondialdehyde and lipofuscin-like pigments or in the degradation of polyunsaturated fatty acids were obtained, when the two systems were compared, except in the presence of NADPH-ADP-Fe3+, which induced a significantly larger degradation of polyunsaturated fatty acids in isolated nuclear membranes than in intact nuclei, and (5) no malondialdehyde-DNA fluorescent adduct formation was observed in any of the experimental groups studied, as inferred from the characteristics of the fluorescent spectra of lipofuscin-like pigments extracted from incubated nuclear preparations.     

Phosphorylation of nuclear matrix proteins in isolated regenerating rat liver nuclei

The phosphorylation of nuclear matrix proteins from normal and regenerating rat liver nuclei was examined using an $\text{in vitro}$ system of isolated nuclei and γ-³²P-ATP. Phosphorylation of the nuclear matrix proteins was 2–3 fold higher than that of the total nuclear proteins in normal nuclei. The level of phosphorylation of the matrix proteins was enhanced an additional three fold at a period in liver regeneration (12 hours) just preceding the onset of DNA synthesis.     

Epidermal growth factor accelerates the intestinal cessation of macromolecular transmission in the suckling rat

1. The effects of EGF administered subcutaneously on the intestinal cessation of macromolecular transmission and sucrase development were investigated in suckling rats and compared with those on hydrocortisone-treated pups. 2. In the EGF-treated pups, intestinal absorptive response of IgG was suppressed 50% whereas, the sucrase activity was not affected. In the hydrocortisone-treated pups, the absorptive response was inhibited completely, while sucrase activity was induced precociously. 3. The characteristics of intestinal cessation was morphologically observed at the jejunal epithelial cells in EGF and hydrocortisone-treated pups. 4. These results suggest that EGF affects the maturation of gastrointestinal function in a manner different from that of glucocorticoid hormones.     

Epidermal growth factor and 12-O-tetradecanoylphorbol 13-acetate stimulate lactate production and the pentose phosphate pathway in freshly isolated rat hepatocytes

Epidermal growth factor (EGF) and tetradecanoylphorbol acetate (TPA) rapidly stimulated the production of lactate by hepatocytes isolated from fed rats. Our results indicate that enzymes of both glycolysis and the pentose phosphate pathway are involved in these actions. EGF stimulated CO2 release from the 1-position of glucose, and caused a small but significant increase in pyruvate kinase activity. In addition, EGF caused a rise in fructose 1,6-bisphosphate and fructose 2,6-bisphosphate concentrations, indicating activation of phosphofructokinase. TPA did not alter the concentrations of these sugar phosphates, but did cause an increased lactate production and CO2 production from the 1-position of glucose similar to EGF. Furthermore, the EGF stimulation of lactate formation was independent of the presence of medium Ca2+. Phenylephrine stimulation of this process, in parallel incubations, was entirely dependent upon the presence of Ca2+ in the medium. We conclude that EGF stimulates glycolysis and the pentose phosphate pathway in isolated hepatocytes from fed rats. The duplication of these actions by TPA suggests that protein kinase C is a mediator of EGF action in hepatocytes.     

Flow cytometric analysis of isolated rat liver nuclei during growth

The development of hepatocyte polyploidy in rats aged up to 4 months was analyzed by flow cytometry using both scatter and fluorescent parameters to distinguish DNA diploid and DNA tetraploid populations and to discriminate between parenchymal and non-parenchymal compartments. The precise origin of each class of nuclei was assessed in whole liver homogenate using purified hepatocytes, obtained by liver perfusion followed by separation on Percoll gradient, and identifying the peaks corresponding to parenchymal nuclei. The results indicate that preparative procedures involving homogenization of the rat liver tissue caused loss of the DNA octaploid population. Data on the relative proportion of the different DNA ploidy elements during rat liver development, which are in good agreement with those observed by cell analysis by means of microspectrophotometry, indicate the usefulness of flow cytometry as a choice method for the analysis of ploidy distribution.     

Epidermal growth factor enhances acetylation of nuclear proteins in cultured human liver cells

Addition of epidermal growth factor (EGF) to Chang liver cells in a low serum culture causes a long-lasting, two- to three-fold increase in the acetylation of nuclear proteins. This EGF effect is manifested before an increase of cell proliferation in response to EGF. Studies by SDS-polyacrylamide gel electrophoresis and isoelectric focusing show that both histones and acid-insoluble proteins of lower molecular weights are acetylated upon administration of EGF. Tumor promoter teleocidin, which inhibits internalization and nuclear accumulation of EGF, acts antagonistically with EGF in enhancing the acetylation of nuclear proteins. Lysosomal inhibitor chloroquine enhances nuclear accumulation of EGF but has no significant effects on the stimulation of nuclear protein acetylation by EGF. These data appear to suggest that the EGF-enhancement of the acetylation of nuclear proteins is mediated by the steps such as an internalization or nuclear accumulation of EGF or EGF-receptor complex.     

Studies on nuclear acid deoxyribonucleases and proofreading exonuclease isolated from rat liver nuclei

Three kinds of deoxyribonucleases (peaks A, B, and C) were separated from purified rat liver nuclei on DEAE-cellulose column chromatography, and their characteristics were partially studied. Peaks A and B had endonuclease activities under acidic conditions with low substrate specificity and did not require divalent metal ions. Peak C had an exonuclease activity under alkaline conditions with substrate specificity for denatured DNA or single stranded homopolymer and required divalent cations. Peak C degraded 3'-terminally mismatched substrate much faster than 3'-terminally matched substrate.     

DNA degradation in isolated rat liver nuclei

Purified rat liver nuclei were incubated at 4°C. After different intervals of incubation aliquots of the nuclear suspension were taken and DNA was extracted by a common SDS-phenol-chloroform procedure. The fractionation of DNA by agar gel electrophoresis revealed large DNA fragments. It was shown that the well-known DNA degradation to monomers and its multiples is preceeded by an earlier breakdown of DNA into characteristic large fragments.     

Epidermal growth factor in the rat lung

Summary Epidermal Growth Factor (EGF) in pharmacological doses is able to induce precoccious lung maturation in rabbits and sheeps. As EGF is probably acting in a para- or autocrine way, we have searched for EGF in the lungs.We report EGF immunoreactivity to be present in the type II pneumocytes of the rat from a couple of days prior to birth and throughout life. Further, we report EGF immunoreactivity to be present in cells in the bronchi and the bronchioles from day 20–21 of gestation and throughout life. G-200 gelchromatography of lung extracts indicates that the EGF-reactive material is a high molecular weight form of EGF.Since previous studies have shown that EGF in pharmacological doses is able to promote lung maturation, our results may imply a physiological role for EGF in the lungs.     

Epidermal growth factor reactivity in rat milk

The concentration of EGF immunoreactivity in rat whey increases from 0.3 pmol/ml at lactation day 1 to 2.0 pmol/ml at lactation day 19. The concentration of EGF is not influenced when the rats undergo sialoadenectomy prior to mating. On S-200 gel chromatography, almost all EGF-reactivity in rat whey elutes as a broad peak corresponding to a Stokes radius of 4.0 nm (an approximate molecular weight of 80 kDa). Almost no 6 kDa EGF is present. Judged by gel filtration of whey pre-incubated with 125I-EGF (6 kDa), no binding protein for EGF is present in rat whey. When rat milk is incubated overnight at 37 degrees C, the 80 kDa EGF is degraded and elutes as a peak with a Stokes radius of 2.7 nm, corresponding to a molecular weight of approximately 35 kDa EGF and as a peak corresponding to 6 kDa EGF. Also, after partial purification by immuno-affinity chromatography, the EGF-reactive material in rat whey behaves as a peptide with a Stokes radius of 2.7 nm, corresponding to a molecular weight of approximately 35 kDa at gel filtration. Comparative binding studies between EGF purified from the submandibular glands and the EGF purified from rat whey confirm differences in the binding to antibodies raised against submandibular EGF, but not in binding to the EGF-receptor. Our results make it unlikely that EGF in rat whey is derived from the submandibular glands.     

Alterations in nuclear anatomy by chemical modification of proteins in isolated rat liver nuclei

Whole rat liver nuclei were treated with citraconic anhydride, a reagent specific for primary amines. Dramatic changes were observed in nuclear morphology and light scattering properties. An analysis for DNA and RNA content suggested that DNA was released from the nuclei with a short half-time, approximately 2-4s demonstrating a biphasic release profile. RNA was similarly released but with a monophasic profile. Analysis of SDS-PAGE gels of modified nuclei demonstrated a progressive enrichment of nuclear matrix (lamins) polypeptides with extent of modification. H1 histone was quantitatively lost as a function of modification reagent concentration, while approx. 50% of the nucleosomal histones cosedimented with DNA- and RNA-free nuclei. Modification in the presence of 2 mM EGTA released all the DNA and RNA [less than or equal to 1% remaining) while retaining structures characteristic of nuclear matrix, nucleoli, and ribonucleoprotein (predominantly hnRNA group A and B). These nucleic acid-deficient structures have been termed nuclear fossils to differentiate them from high salt detergent-prepared empty nuclear sacks, nuclear remnants, or nuclear scaffolds. Modification in the presence of 2% Triton X-100 results in structures similar to the nuclear fossils (EGTA treatment), but missing the double bilayer and a 51K polypeptide that is a major component of the other structures. The use of chemical modification on the nucleus provides an experimental approach for examining the role of ionic interactions in controlling nuclear structure. Citraconylation may thus serve two functions: (a) as a protein-specific perturbant of nuclei capable of simply and rapidly preparing a range of structural variants for the analysis of nuclear interactions; (b) offer a paradigm for control of nucleic acid-polypeptide interactions based on post-translational alterations in protein charge.     

Epidermal growth factor in the rat lung

Epidermal Growth Factor (EGF) in pharmacological doses is able to induce precoccious lung maturation in rabbits and sheeps. As EGF is probably acting in a para- or autocrine way, we have searched for EGF in the lungs. We report EGF immunoreactivity to be present in the type II pneumocytes of the rat from a couple of days prior to birth and throughout life. Further, we report EGF immunoreactivity to be present in cells in the bronchi and the bronchioles from day 20-21 of gestation and throughout life. G-200 gelchromatography of lung extracts indicates that the EGF-reactive material is a high molecular weight form of EGF. Since previous studies have shown that EGF in pharmacological doses is able to promote lung maturation, our results may imply a physiological role for EGF in the lungs.     

Epidermal growth factor, insulin, and estrogen stimulate development of prolactin-secreting cells in cultures of GH3 cells

Pituitary tumor GH3 cells synthesize and secrete both growth hormone (GH) and prolactin (PRL). Morphological and functional changes of GH3 cells induced by epidermal growth factor (EGF, 10 nM), insulin (300 nM), and estradiol-17beta (E2, 1 nM) were studied. Treatment of cultures of GH3 cells for 6 days with EGF, insulin, or E2 alone, and with EGF plus E2 did not affect the total number of GH3 cells, but a combination of EGF, insulin, and E2 decreased the total number of GH3 cells compared with control treatment. DNA-synthesizing cells were detected by monitoring 5-bromo-2'-deoxyuridine (BrdU) uptake. EGF, E2, or a combination of EGF, insulin, and E2 significantly decreased the proportion of BrdU-labeled cells (21.1+/-1.7%, 21.0+/-1.4%, 18.2+/-1.3%; P<0.05, P<0.05, P<0.01, respectively) compared with control treatment (28.6+/-1.5%), but insulin did not (31.4+/-2.4%). Immunocytochemical analysis of GH3 cells cultured in 5% fetal calf serum-supplemented medium (control) showed that about 70% of all GH3 cells were GH-immunoreactive cells (GH-ir cells), apparently containing only GH, and 14% were mammosomatotrophs (MS cells), containing both GH and PRL, while PRL-immunoreactive cells (PRL-ir cells), containing only PRL, were not detected. No GH or PRL immunoreactivity could be detected in the remaining cells (15%). EGF decreased the proportion of GH-ir cells. The effects of EGF were enhanced by simultaneous exposure to insulin and E2; this decreased the proportion of GH-ir cells to about 20% of the total GH3 cells and significantly increased the proportion of MS cells to 300% of controls. Treatment with EGF plus insulin, EGF plus E2, or a combination of EGF, insulin, and E2 all stimulated the appearance of PRL-ir cells. Exposure to EGF caused a significant decrease in GH mRNA (P<0.01) and a significant increase in PRL mRNA (P<0.05). These observations suggest that EGF is closely involved in differentiation of PRL-ir cells from GH-ir cells via MS cells in GH3 cell cultures. Cytosine arabinoside (10(-7) M), an inhibitor of cell division, did not affect the changes in proportion of the three cell types induced by treatment with a combination of EGF, insulin, and E2. It is therefore probable that the transdifferentiation does not require mitosis of the GH3 cells.