epidermal-growth-factor and 2-aminoisobutyric-acid

EGFR-T17 cells were pretreated with oleic acid and 5-10 minutes later stimulated with EGF, to study if early ionic signals are instrumental in inducing metabolic cellular response. Oleic acid blocks EGF-induced [Ca2+]i rise and Ca2+ influx without altering 2-deoxyglucose and 2-aminobutiryc acid uptake nor acute, nor chronically. Oleic acid it is shown, in the first minutes favors the entrance of both molecules to modify the physico-chemical membrane state. On the other hand, oleic acid is unable to block protein synthesis. The results suggest that EGF-induced Ins(1,4,5)P3/Ca2+ pathway does not seem to be decisive in the control of cellular metabolic activity.

Topics: 3T3 Cells; Aminoisobutyric Acids; Animals; Biological Transport; Calcium; Cell Line; Cell Membrane; Deoxyglucose; Epidermal Growth Factor; ErbB Receptors; Fibroblasts; Humans; Inositol 1,4,5-Trisphosphate; Kinetics; Mice; Oleic Acid; Oleic Acids; Recombinant Proteins; Time Factors; Transfection

We examined the effects of epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I) on the uptake of AIB by and its transfer across near-term human placental syncytiotrophoblast in two-sided culture. Pre-incubation of the trophoblast cell layer with either EGF (50ng/ml) or IGF-I (100ng/ml) on the apical (microvillous, in vivo maternal-facing) side reduced rates of unidirectional microvillous-to-basal transtrophoblast AIB transfer, increasing AIB retention within the cells. EGF on the basal (fetal-facing) side of the cell layer enhanced AIB uptake from the microvillous side but also increased overall mediated permeability in both directions. IGF-I at the basal surface, however, increased AIB uptake across the microvillous membrane, and induced a backflux from the cells into the basal medium dependent upon basal AIB concentration, suggesting that in vivo IGF-I on the fetal side enhances maternal-to-fetal placental transfer. The ideas are consistent with current concepts of maternal-placental and fetal-placental interactions regulating pregnancy and fetal development.

Topics: Aminoisobutyric Acids; Biological Transport; Cells, Cultured; Dose-Response Relationship, Drug; Epidermal Growth Factor; Female; Humans; Insulin-Like Growth Factor I; Kinetics; Microvilli; Pregnancy; Trophoblasts

[1-14C]-2-aminoisobutyric acid (AIB) uptake and signal transduction pattern after epidermal growth factor (EGF) stimulation were examined in freshly isolated hepatocytes from 20-day-old fetuses and 3-month-old rats. EGF induced a transient increase of AIB transport after 10 min only in adult animals; the observed unresponsiveness of fetal liver is not dependent on a lack of EGF receptors which are present though to a lesser extent on the plasma membrane in this period. As far as the production of the second messengers, inositol trisphosphate (IP3) and calcium, is concerned, substantial differences were found: EGF increased IP3 production in adult hepatocytes, whereas it had no effect in fetal ones. Moreover, the addition of EGF induced a calcium transient in hepatocytes from adult animals, while there was no increase in fetal cells. The lack of EGF effect on amino acid transport in fetal cells could be due to its inability to produce both IP3 and calcium transients, suggesting that this transduction pathway is not activated during fetal life.

Topics: Amino Acids; Aminoisobutyric Acids; Animals; Biological Transport; Calcium; Epidermal Growth Factor; Inositol Phosphates; Kinetics; Liver; Rats; Rats, Wistar; Second Messenger Systems; Signal Transduction

Amino acid uptake mediated by system A was studied in cultured fetal and adult hepatocytes, subjected to growth stimulation by EGF and insulin, or to growth inhibition by high cell density. The mitogenic stimulation induced a strong transport increase only in fetal cells, while the cell density-dependent growth inhibition, probably mediated by molecules present on adult hepatocyte membranes, provoked the decrease of amino acid uptake only in the adult cells. The results indicate that the different modulation of amino acid transport by cell growth is dependent on the age and the differentiation stage of hepatocytes.

Topics: Age Factors; Amino Acids; Aminoisobutyric Acids; Animals; Biological Transport; Cell Differentiation; Cell Division; Cell Membrane; Cells, Cultured; DNA; Epidermal Growth Factor; Gestational Age; Humans; Liver; Rats; Rats, Wistar

The epidermal growth factor (EGF) receptor tyrosine kinase activity is required for both the earliest EGF-stimulated post-binding events (enhancement of inositol phosphate formation and Ca2+ influx, activation of Na+/H+ exchange), and the ultimate EGF-induced mitogenic response. To assess the role of EGF receptor kinase in EGF-induced metabolic effects (2-deoxyglucose and 2-aminoisobutyric acid uptake), we used NIH3T3 cells (clone 2.2), which do not possess endogenous EGF receptors and which were transfected with cDNA constructs encoding either wild type or kinase-deficient human EGF receptor (HER). In addition, we tested the importance of three HER autophosphorylation sites (Tyr-1068, Tyr-1148, and Tyr-1173) in transduction of EGF-stimulated 2-deoxyglucose uptake. Taking our data together, we conclude the following: (i) HER tyrosine kinase activity is required to elicit EGF stimulation of both 2-deoxyglucose and 2-aminoisobutyric acid uptake; (ii) mutations on individual HER autophosphorylation sites, Tyr-1068, Tyr-1148, and Tyr-1173 do not impair EGF-stimulated 2-deoxyglucose uptake.

Topics: Aminoisobutyric Acids; Animals; Biological Transport; Deoxy Sugars; Deoxyglucose; DNA Mutational Analysis; Epidermal Growth Factor; ErbB Receptors; Kinetics; Mice; Protein-Tyrosine Kinases; Structure-Activity Relationship; Transfection; Tyrosine

Topics: Adult; Aminoisobutyric Acids; Biological Transport, Active; Carcinoma, Squamous Cell; Cell Line; Cell Transformation, Viral; Cells, Cultured; Epidermal Cells; Epidermal Growth Factor; Epidermis; Humans; Infant, Newborn; Insulin; Kinetics; Simian virus 40; Sodium; Tumor Cells, Cultured

It was previously reported that the lectins wheat germ agglutinin (WGA) and concanavalin A (Con A) inhibit the mitogenic actions of multiple peptide growth factors in human fibroblasts without having a significant effect on mitogen binding. The current studies were designed to further examine the mechanisms of this antimitogenic action of lectins. Addition of WGA at progressively later times after stimulation of fibroblasts with peptide mitogens revealed significant inhibition of DNA synthesis even when the lectin was added 16-20 h after growth factors. This suggests an inhibitory effect on a pathway occurring late in G1, or close to the G1/S boundary. WGA also inhibited stimulation of DNA synthesis by non-peptide agents such as colchicine and vanadate ion, indicating that the lectin inhibits a common distal step in the mitogenic response, rather than acting primarily on events occurring at the level of the growth factor-receptor interaction. WGA had a rapid (within 30 min) inhibitory effect on insulin-stimulated amino acid uptake, but Con A, which like WGA blocked mitogen-stimulated 3H-dT incorporation, had little effect on stimulation of amino acid uptake. Thus the inhibition of DNA synthesis and amino acid uptake by lectins appear to be mediated by distinct mechanisms. WGA binding to fibroblasts persisted even when the lectin was removed from the incubation medium, but unlike 125I-EGF, which was rapidly internalized at 24 degrees C, little 125I-WGA was internalized. Incubation of fibroblasts for 20 h with WGA or Con A was not toxic to cells, since reversal of lectin binding by the appropriate saccharide allowed normal subsequent stimulation of DNA synthesis by EGF and insulin. However, the observation that cells exposed to antimitogenic lectins undergo a marked decrease in cell spreading suggests that changes in cell shape may be relevant to the mechanism by which lectin-treated fibroblasts become unresponsive to mitogenic stimulation.

Topics: Aminoisobutyric Acids; Cell Division; Cell Survival; Cells, Cultured; Concanavalin A; Epidermal Growth Factor; Fibroblasts; Humans; Lectins; Microscopy, Electron; Thymidine; Wheat Germ Agglutinins

The effect of plant lectins on amino acid uptake and DNA synthesis in cultured human skin fibroblasts stimulated by various peptide mitogens was studied. Wheat germ agglutinin (WGA), at a concentration of 5 micrograms/ml, which by itself had little effect on 3H-aminoisobutyric acid (AIB) uptake, markedly inhibited stimulation of 3H-AIB uptake by somatomedin-C, insulin, epidermal growth factor (EGF) and platelet-derived growth factor. This inhibition could not be overcome by increasing the concentration of peptide added. Neither WGA nor concanavalin A (Con A) significantly affected basal 3H-thymidine incorporation. However both lectins, at concentrations of 5-20 micrograms/ml, decreased EGF- and insulin-stimulated DNA synthesis while succinyl Con A, a divalent lectin derivative, did not. The inhibitory effects of lectins on mitogenic stimulation were reversed by alpha-methyl mannose (Con A) or N-acetylglucosamine (WGA), and were not due to a reduction in the binding of growth factors to their receptors. It is concluded that certain lectins noncompetitively inhibit the response of human fibroblasts to multiple peptide mitogens at the post-receptor level, possibly by interfering with lateral mobility and aggregation of mitogen-receptor complexes.

Topics: Acetylglucosamine; Adult; Aminoisobutyric Acids; Binding Sites; Concanavalin A; DNA Replication; Epidermal Growth Factor; Fibroblasts; Growth Substances; Humans; Insulin; Insulin-Like Growth Factor I; Lectins; Male; Platelet-Derived Growth Factor; Receptors, Cell Surface; Skin; Time Factors; Wheat Germ Agglutinins

The polypeptide growth factors, nerve growth factor, epidermal growth factor, and platelet-derived growth factor, as well as insulin do not induce ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) unless a minimal concentration of an ornithine decarboxylase-inducing amino acid, such as asparagine, is present in the medium. The effects of the growth factors were studied in appropriately responsive cell lines: pheochromocytoma (PC12) cells for nerve and epidermal growth factors, fibroblasts (NIH 3T3) for platelet-derived growth factor, and fibroblasts and hepatoma (KRC-7) cells for insulin. The nonmetabolizable amino acid analog alpha-aminoisobutyric acid can replace asparagine, indicating that the covalent modification of the inducing amino acid is not necessary for the induction of ornithine decarboxylase by these growth factors. For the same intracellular concentration of the inducing amino acid, the presence of the growth factors induces higher levels of ornithine decarboxylase. The evidence indicates that these growth factors do not induce ornithine decarboxylase by raising the intracellular concentration of amino acids but rather act synergistically with the inducing amino acid. Evidence is provided that the induction of polyamine-dependent growth by these growth factors is mediated by amino acids. The relationship of these results to the A and N amino acid transport systems and to the Na+ influxes in relation to growth is discussed.

Topics: Amino Acids; Aminoisobutyric Acids; Animals; Asparagine; Cells, Cultured; Enzyme Induction; Epidermal Growth Factor; Fibroblasts; Growth Substances; Insulin; Liver Neoplasms, Experimental; Mice; Nerve Growth Factors; Ornithine Decarboxylase; Platelet-Derived Growth Factor

EGF inhibits the cAMP stimulation of amino acid transport in primary cultures of rat hepatocytes. This effect is not observed in freshly isolated cells incubated in suspension but only in cells that have been allowed to attach to culture dishes. Sequential addition of cAMP and EGF to monolayer cultures of hepatocytes revealed that EGF does not affect the half-life of the carrier proteins induced by cAMP but rather prevents the induction of new amino acid transport proteins. The EGF effect is inhibited by drugs interfering with protein synthesis. These results indicate that EGF promotes the synthesis of specific proteins that are able to prevent the cAMP induction of amino acid carriers.

Topics: Amino Acids; Aminoisobutyric Acids; Animals; Biological Transport, Active; Carrier Proteins; Cells, Cultured; Cyclic AMP; Epidermal Growth Factor; Kinetics; Liver; Male; Rats; Rats, Inbred Strains