The effect of transport system A and N amino acids and of nerve and epidermal growth factors on the induction of ornithine decarboxylase activity

The induction of ornithine decarboxylase (EC 4.1.1.17) (ODC) by amino acids and by the peptide hormones nerve growth factor (NGF) and epidermal growth factor (EGF) in salts-glucose media has been studied. Only those neutral amino acids taken into the cell via one of the Na+ dependent transport systems stimulate ODC activity. Asparagine and the nonmetabolizable alpha-amino-isobutyric acid (AIB) were used as representatives of this class of inducing amino acids, and their intracellular concentrations were related to the levels of ODC induced. A threshold intracellular concentration of asparagine or AIB has to be attained before ODC can be induced. Further slight increases in intracellular concentrations of asparagine or AIB produce disproportionately large increases of ODC, resulting in a sigmoidal curve of ODC induction. These results, and the fact that the decrease in ODC levels caused by valine is associated with a concurrent decrease in the intracellular level of the inducing amino acid, suggest that the intracellular amino acid level is causally related to the induction of ornithine decarboxylase. Glutamic acid, EGF, and NGF do not induce ODC except in the presence of an inducing amino acid. They act synergistically with the inducing amino acid and produce higher ODC levels at the same intracellular concentration of the inducing amino acid.     

Control of DNA synthesis and ornithine decarboxylase activity by hormones and amino acids in primary cultures of adult rat hepatocytes

The conditions for stimulation of ornithine decarboxylase (ODC) and DNA synthesis in primary monolayer cultures of non-growing, highly differentiated hepatocytes from adult rats were compared. The syntheses of ODC and DNA were not stimulated by hormones on the 1st day of culture, but they were induced markedly by insulin (10⁻⁸ M) and epidermal growth factor (EGF, 0.1 μg/ml) in cells cultured for 40 h. The effects of insulin and EGF were synergistic, and the ODC activity as well as the DNA synthesis in the presence of these hormones was comparable to that of cultured hepatocytes from partially hepatectomized liver. Other factors had different effects on the two processes. Dexamethasone induced ODC slightly, but it inhibited DNA synthesis strongly. Putrescine inhibited ODC activity, but it had no effect on DNA synthesis. Asparagine and glutamine induced ODC activity, but they inhibited DNA synthesis; their inhibitory effects on DNA synthesis were specific to primary cultured liver cells and were not seen in an established rat liver cell line or in mouse L cells. These results show that although there is some correlation between ODC induction and DNA synthesis, the former is not essential for cell growth. There was no indication of cell division under conditions where maximal ODC induction and DNA synthesis were observed. Cytofluorometry of cells treated with insulin and EGF showed that the DNA content increased from 2 N to 4 N, and to 8 N in some cells. Therefore, under the present culture conditions, mature liver cells could enter G2 phase through S phase, but could not enter M phase.     

Proline is required for the stimulation of DNA synthesis in hepatocyte cultures by EGF

Summary Epidermal growth factor (EGF) has been shown to stimulate DNA synthesis in rat parenchymal hepatocytes both in vivo and in vitro (4,9). We report here that this response in vitro is dependent on the amino acids present in the media. Of all the amino acids, proline has the strongest effect. The response to EGF is absent without proline and none of the other amino acids can substitute for it. Added proline (1 mM) to the media caused the labeling index to increase from 11% to 55% in the presence of 50 ng/ml EGF and insulin. In the presence of proline, small additional increases of the EGF effect on DNA synthesis were stimulated by phenylalanine and tyrosine. This work was supported by NIH grants CA302241 and CA35373 and EPA CRA R811687010 EDITOR'S STATEMENT This paper describes an interesting, specific interaction between EGF and proline in stimulation of DNA synthesis in hepatocytes. This finding may shed light on the well-documented effect of amino acid load on hepatocyte proliferation in vivo and interactions of growth factors and nutrients in general. Wallace L. McKeehan     

Amino acids regulate expression of antizyme-1 to modulate ornithine decarboxylase activity

In a glucose-salt solution (Earle's balanced salt solution), asparagine (Asn) stimulates ornithine decarboxylase (ODC) activity in a dose-dependent manner, and the addition of epidermal growth factor (EGF) potentiates the effect of Asn. However, EGF alone fails to activate ODC. Thus, the mechanism by which Asn activates ODC is important for understanding the regulation of ODC activity. Asn reduced antizyme-1 (AZ1) mRNA and protein. Among the amino acids tested, Asn and glutamine (Gln) effectively inhibited AZ1 expression, suggesting a differential role for amino acids in the regulation of ODC activity. Asn decreased the putrescine-induced AZ1 translation. The absence of amino acids increased the binding of eukaryotic initiation factor 4E-binding protein (4EBP1) to 5'-mRNA cap and thereby inhibited global protein synthesis. Asn failed to prevent the binding of 4EBP1 to mRNA, and the bound 4EBP1 was unphosphorylated, suggesting the involvement of the mammalian target of rapamycin (mTOR) in the regulation of AZ1 synthesis. Rapamycin treatment (4 h) failed to alter the expression of AZ1. However, extending the treatment (24 h) allowed expression in the presence of amino acids, indicating that AZ1 is expressed when TORC1 signaling is decreased. This suggests the involvement of cap-independent translation. However, transient inhibition of mTORC2 by PP242 completely abolished the phosphorylation of 4EBP1 and decreased basal as well as putrescine-induced AZ1 expression. Asn decreased the phosphorylation of mTOR-Ser(2448) and AKT-Ser(473), suggesting the inhibition of mTORC2. In the absence of amino acids, mTORC1 is inhibited, whereas mTORC2 is activated, leading to the inhibition of global protein synthesis and increased AZ1 synthesis via a cap-independent mechanism.     

Relationship of ornithine decarboxylase activity and cAMP metabolism to proliferation of normal human bronchial epithelial cells

The mechanisms of action of extracellular mitogens for normal human bronchial epithelial cells (NHBE) were investigated by observing their effects on selected biochemical pathways when the cells were incubated in serum-free media. We find that (a) epidermal growth factor (EGF) stimulates ornithine decarboxylase (ODC) activity and the rate of cell division without stimulating cAMP; (b) alone, pituitary extract (PEX) does not stimulate ODC activity, cAMP levels, or cell division; (c) when PEX is added to medium containing EGF there is a further increase in both ODC activity and the rate of cell division, again with no increase in cAMP levels; (d) in contrast, alone, L-epinephrine (EPI) stimulates an increase in both ODC and cAMP but does not stimulate cell division; (e) when EPI is added to medium containing both EGF and PEX a further increase in the rate of cell division is noted; (f) the specific inhibitor of ODC, alpha-(difluoromethyl)-ornithine (DMFO), also inhibits NHBE cell proliferation; and (g) the beta-adrenergic receptor antagonist propranolol inhibits the mitogenic action and ODC induction by EPI observed under condition e. We conclude that an increase in ODC activity is necessary but not sufficient for an increase in proliferation of NHBE cells. In contrast, cAMP stimulation is not necessary for an increase in NHBE cell division. However, in the presence of undefined factors in PEX, increases in cAMP levels result in a synergistic increase in the rate of EGF-stimulated clonal growth. By correlating the biochemical pathways invoked by EGF, PEX, EPI, and combinations thereof with their mitogenic actions, we have better defined the role each of these different mitogens plays in stimulating epithelial cell division.     

Effect of calf and rat serum on the induction of DNA synthesis and mitosis in primary cultures of adult rat hepatocytes by cyproterone acetate and epidermal growth factor

Summary Induction of hepatocyte DNA synthesis in culture by cyproterone acetate (CPA), a potent hepatomitogen in vivo, was studied. Adult rat hepatocytes were grown on collagen gels in primary culture for 3 to 10 d. Epidermal growth factor (EGF) was used as a model inducer to establish appropriate culture conditions. (a) In serum-free medium EGF stimulated a wave of DNA synthesis in 10 to 30% of the hepatocytes. CPA had only a weak effect. (b) Increasing concentrations of newborn bovine serum (NBS) at 5 to 95% progressively inhibited the stimulatory effect of EGF. A similar inhibition was obtained by adding bovine serum albumin; 20% NBS, however, had a slightly stimulatory effect on the induction of DNA synthesis by CPA. (c) Portal rat serum (RS) at concentration of 5 to 95% markedly stimulated DNA synthesis, a plateau being reached between 20 and 95%. EGF had a distinct enhancing effect on DNA synthesis in the presence of 5 and 20% RS but not at 50 and 95%. CPA stimulated DNA synthesis in the presence of 20, 50, and 95% RS in a synergistic way. (d) Mitoses were found after treatment with EGF or with CPA. These results show that CPA can induce DNA synthesis in cultured hepatocytes and that RS contains factors facilitating the response to CPA. This study was supported by Gesellschaft für Strahlen-und Umweltforschung mbH, München, Germany.     

Epidermal growth factor potentiates the induction of ornithine decarboxylase activity by prostaglandins in embryonic palate mesenchymal cells: effects on cell proliferation and glycosaminoglycan synthesis

Epidermal growth factor (EGF) and prostaglandins (PGs) have been implicated in the regulation of a number of developmental processes in the mammalian embryonic palate. Normal palatal ontogenesis is dependent on the presence and quite possibly on the interaction of various hormones and growth factors. The interaction between EGF and PGs in regulation of murine embryonic palate mesenchymal (MEPM) cell growth and differentiation was therefore investigated by monitoring the activity of ornithine decarboxylase (ODC), the principle and rate limiting enzyme of polyamine biosynthesis. ODC activity is tightly coupled to the proliferative and differentiative state of eukaryotic cells and therefore serves as a reliable indicator of such cellular functions. Treatment of confluent cultures of MEPM cells with EGF (1-50 ng/ml) resulted in a dose-related increase in ODC activity, while similar treatment with either PGE2 or PGF2 alpha (at concentrations up to 1 microM) did not elicit a dose-dependent increase in enzyme activity. Concurrent treatment of MEPM cells with EGF (20 ng/ml) and either PGE2 or PGF2 alpha (0.1-10000 nM) resulted in a marked prostaglandin dose-dependent induction of ODC activity, suggesting a strong cooperative interaction between these factors. ODC activity was maximal by 4 to 8 hr and could be completely inhibited by preincubation of the cells with actinomycin D or cycloheximide, indicating that de novo synthesis of RNA and protein is necessary for enzyme induction. Stimulation of ODC activity by EGF and PGE2 in these cells was not positively correlated with the level of cellular DNA synthesis but did result in a ninefold increase in the synthesis of extracellular glycosaminoglycans (GAGs), a key macromolecular family implicated in palatal morphogenesis. Stimulation of GAG synthesis was significantly inhibited by the administration of 5 mM DFMO (an irreversible inhibitor of ODC), indicating that the marked increase in GAG production was dependent, in part, on the induction of ODC activity by EGF and PGE2. Qualitative analysis of the palatal GAGs indicated that synthesis of several major classes of GAGs was stimulated. Collectively these data demonstrate a cooperative interaction between EGF and PGs in the induction of ODC activity. Such activity may serve to regulate the synthesis of GAGs, which are instrumental in mammalian palatal ontogenesis.     

Differential effects of lysosomotropic amines and polyamines on processing and biological activity of EGF

The effects of lysosomotropic amines and polyamines on rat fibroblasts were studied after the administration of epidermal growth factor (EGF) in order to determine whether the intracellular processing of EGF was important for transmission of its biological signal. Following the addition of EGF, cell cultures exhibited a dose-dependent increase in ornithine decarboxylase (ODC) activity. This increase in ODC activity was drastically reduced by both methylamine, a representative lysosomotropic amine, and putrescine, a polyamine precursor. However, inasmuch as methylamine inhibited EGF-induced DNA synthesis by greater than 50%, putrescine had no inhibitory effect. Lysosomotropic amines, but not polyamines, prevented EGF processing as evidenced by their ability to block the release of intracellular 125EGF and by their ability to inhibit the formation of the final intracellular processed product of EGF, as determined by isoelectric focusing. These data suggest that the processing of EGF is consistent with the induction of DNA synthesis and ODC activity. The cellular mechanisms involved in inhibition of ODC induction by polyamines appear to be distinct from those involved in lysosomotropic amines.     

Glutamic acid potentiates hepatocyte response to mitogens in primary culture

Adult rat hepatocytes in primary culture responded to epidermal growth factor (EGF) by increased DNA synthesis. When hepatocytes were cultured in Leibovitz L-15 medium, their response to EGF was low compared with that in Williams' medium E or Koga's medium L. Furthermore, female rat hepatocytes showed almost no response to the mitogenic action of EGF compared with male rat hepatocytes in L-15 medium. Addition of glutamic acid (1–20 μM) to EGF-containing L-15 medium not only enhanced DNA synthesis > tenfold in both male and female hepatocytes, but eliminated the sex differences in DNA synthesis. Aspartic acid, glutamine, or ornithine at 20 mM did not replace the glutamic acid effect on DNA synthesis. Proline also enhanced EGF-induced DNA synthesis, although it was less effective than glutamic acid. Therefore, this effect may be specific to a high concentrations of glutamic acid. Glutamic acid by itself did not stimulate DNA synthesis at any concentrations tested. In the presence of glutamic acid, EGF showed a dose-dependent (0.5–20 ng/ml) stimulation of DNA synthesis with a maximal effect at 10 ng/ml. Almost the same effect was obtained with transforming growth factor alpha (0.5–20 ng/ml). Glutamic acid also induced an expansion of the mitogenic action of angiotensin II. Since glutamic acid did not affect [¹²⁵I]EGF binding to hepatocytes or its processing, the effect may occur internal to the receptor. These results suggest that glutamic acid modulates the sensitivity of the hepatocyte response to mitogens © 1994 Wiley-Liss, Inc.     

Amino acid-rich medium (Leibovitz L-15) enhances and prolongs proliferation of primary cultured rat hepatocytes in the absence of serum.

Multiple rounds of cell division were induced in primary cultured rat hepatocytes in serum-free, modified L-15 medium supplemented with 20 mM NaHCO3 and 10 ng/ml EGF in a 5% CO2/95% air incubator. A 150% increase in cell number and DNA content was observed between day 1 and day 5. The time course of DNA synthesis of hepatocytes cultured in L-15 medium differed from that in DMEM/F12 medium in that there were four peaks of 3H-thymidine incorporation in the L-15 medium, at 60 h, 82 h, 96 h, and 120 h, but only one peak at 48 h in modified DMEM/F12 medium. Labeling studies of the hepatocytes indicated that more than 60% of the cells were stained with antibromodeoxyuridine (BrdU) antibody in the periods of 48-72 h and 72-96 h after plating at densities between 1.5 x 10(5) and 6.0 x 10(5) cells per 35-mm dish. Even at a density of 9.0 x 10(5) cells/dish, about 40% of the cell nuclei were stained with BrdU in the periods of 48-72 h and 72-96 h. In addition, about 20% of the hepatocytes in culture initiated a second round of the cell cycle between 48 and 96 h in culture. Proliferating cells, which were mononucleate with a little cytoplasm, appeared in small clusters or colonies in the culture from day 4. These proliferating cells produced albumin. The addition of essential amino acids to the DMEM/F12 medium enhanced the DNA synthesis of hepatocytes, thus indicating that the higher level of amino acids in L-15 medium may be an important factor in its enhanced ability to support the proliferation of primary cultured rat hepatocytes.     

Induction of intestinal ornithine decarboxylase by single amino acid feeding

Intestinal and hepatic ornithine decarboxylase (ODC) activities increased to a peak 4 h after administration of a diet containing casein or an amino acid mixture simulating that of casein to rats starved for 12 h. All amino acids except cysteine with a two or three carbon skeleton, including those with a D-configuration, and alpha-amino-isobutyric acid (AIB) strongly induced intestinal ODC when given in the diet or administered intragastrically. Amino acids with a four carbon skeleton were far less effective as inducers and other amino acids did not induce intestinal ODC at all. The amino acids that induced hepatic ODC showed no particular structural characteristics: glycine and cysteine were very effective, threonine, tryptophan, methionine, and phenylalanine were less effective, and serine, valine, isoleucine, and histidine were only slightly effective. Elevation of ODC activity after amino acid administration was not due to stabilization of the enzyme protein with the amino acids. Intestinal ODC was induced by intragastric but not intraperitoneal injection of glycine, although these treatments resulted in similar increases in the tissue concentration of glycine. On the contrary, hepatic ODC was induced by glycine regardless of the administration route. Intestinal ODC was also induced only in the segment of the intestine perfused with a solution of an amino acid with which the activity increased in the feeding experiment. These results suggest that the accumulation of an amino acid per se is not a trigger for induction of intestinal ODC and that an amino acid must act on the mucosal surface to induce the enzyme.     

Effects of putrescine on synthesis and degradation of ornithine decarboxylase in primary cultured hepatocytes

Changes in both synthesis rate and degradation rate of ornithine decarboxylase (ODC) were pursued in primary cultures of adult rat hepatocytes during the process of ODC induction caused by asparagine and glucagon and also during the process of rapid ODC decay caused by putrescine. The synthesis rate of ODC was determined by [35S]methionine incorporation into the enzyme, which was separated afterwards by immunoprecipitation and sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The degradation rate of ODC was determined by following the decay of prelabeled ODC. The enzyme induction caused by asparagine (10 mM) and glucagon (1 microM) was due both to an increase in the synthesis rate and to a decrease in the degradation rate. Addition of 10 mM putrescine caused a rapid decay of ODC activity, which was faster than ODC decay in the presence of cycloheximide. This rapid decay in ODC activity was accompanied by slightly slower decay in ODC protein, which was due both to partial suppression of ODC synthesis and to several fold acceleration of ODC degradation.     

Cellular effects of ornithine decarboxylase induction in cells maintained with a salts/glucose medium

Cultures preincubated in a growth restricted salts/glucose medium in the presence and absence of ornithine decarboxylase (ODC) activating factors were then incubated under ideal growth conditions to study the influence of these factors on cell growth. Incubation of confluent cultures in a salts/glucose medium alone did not induce ODC or change the other biochemical parameters investigated. However, if cultures were incubated in the salts/glucose medium supplemented with asparagine (ASN) and agents that increase cellular cAMP levels then ODC was induced after 6–8 h. This primary induction in the salts/glucose medium resulted in altered and delayed ODC induction during growth stimulation and also caused a delay in (³H) thymidine incorporation without affecting (³H) uridine and (³H) leucine incorporation. These results demonstrate that incubation of cultures in a salts/glucose media with ASN and dibutyryl cAMP (dBcAMP) causes refractory ODC induction and altered (³H) thymidine incorporation upon growth challenge with complete medium. These effects were not observed when cells were preincubated in a salts/glucose medium alone.     

Regulation of intestinal mucosal growth by amino acids

Amino acids, especially glutamine (GLN) have been known for many years to stimulate the growth of small intestinal mucosa. Polyamines are also required for optimal mucosal growth, and the inhibition of ornithine decarboxylase (ODC), the first rate-limiting enzyme in polyamine synthesis, blocks growth. Certain amino acids, primarily asparagine (ASN) and GLN stimulate ODC activity in a solution of physiological salts. More importantly, their presence is also required before growth factors and hormones such as epidermal growth factor and insulin are able to increase ODC activity. ODC activity is inhibited by antizyme-1 (AZ) whose synthesis is stimulated by polyamines, thus, providing a negative feedback regulation of the enzyme. In the absence of amino acids mammalian target of rapamycin complex 1 (mTORC1) is inhibited, whereas, mTORC2 is stimulated leading to the inhibition of global protein synthesis but increasing the synthesis of AZ via a cap-independent mechanism. These data, therefore, explain why ASN or GLN is essential for the activation of ODC. Interestingly, in a number of papers, AZ has been shown to inhibit cell proliferation, stimulate apoptosis, or increase autophagy. Each of these activities results in decreased cellular growth. AZ binds to and accelerates the degradation of ODC and other proteins shown to regulate proliferation and cell death, such as Aurora-A, Cyclin D1, and Smad1. The correlation between the stimulation of ODC activity and the absence of AZ as influenced by amino acids is high. Not only do amino acids such as ASN and GLN stimulate ODC while inhibiting AZ synthesis, but also amino acids such as lysine, valine, and ornithine, which inhibit ODC activity, increase the synthesis of AZ. The question remaining to be answered is whether AZ inhibits growth directly or whether it acts by decreasing the availability of polyamines to the dividing cells. In either case, evidence strongly suggests that the regulation of AZ synthesis is the mechanism through which amino acids influence the growth of intestinal mucosa. This brief article reviews the experiments leading to the information presented above. We also present evidence from the literature that AZ acts directly to inhibit cell proliferation and increase the rate of apoptosis. Finally, we discuss future experiments that will determine the role of AZ in the regulation of intestinal mucosal growth by amino acids.     

Induction of mouse epidermal ornithine decarboxylase by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate: dependence on calcium availability

The role of calcium in epidermal ornithine decarboxylase (ODC) induction by 12-O-tetradecanoylphorbol-13-acetate (TPA) was determined in adult mouse skin pieces incubated in serum-free minimal essential medium (MEM). Addition of TPA to skin pieces incubated in serum-free MEM, which contains 1.82 mM Ca2+ and 0.83 mM Mg2+, resulted in about a 200-fold increase in epidermal ODC activity at about 8 h after TPA treatment. TPA failed to induce epidermal ODC in skin pieces incubated in calcium-free medium. Similarly, chelation of extracellular calcium by ethyleneglycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) prevented ODC induction by TPA, which could be resumed upon calcium restoration in the medium. Furthermore, calcium ionophore A23187, which facilitates efflux of Ca2+ across cellular membranes, induced ODC activity in incubated skin pieces. Epidermal ODC activity increased by TPA appears to be the result of an increase in both the amount of ODC protein and the level of hybridizable ODC messenger. Inhibition of the induction of ODC activity by EGTA was the result of the inhibition of the amount of active ODC protein and the level of ODC mRNA.     

Synergistic stimulatory effect of glucocorticoid, EGF and insulin on the synthesis of ribosomal RNA and phosphorylation of nucleolin in primary cultured rat hepatocytes.

Effects of dexamethasone, EGF and insulin on the synthesis of rRNA and phosphorylation of nucleolin in primary cultures of adult rat hepatocytes were studied. Hepatocytes were incubated for 8 h with EGF (20 ng/ml) plus insulin (0.1 microM) and/or for 20 h with dexamethasone (1 microM) before the end of incubation. The incorporation of [3H]uridine into acid-insoluble materials and the nuclear activity of RNA polymerase I were stimulated approx. 2-fold with EGF plus insulin and these were further enhanced 2-3-times by dexamethasone, although dexamethasone alone exerted no stimulation. When hepatocytes were incubated with [32P]orthophosphate, similar enhancement by these hormones was also observed in the phosphorylation of a nucleolar protein, nucleolin, which was detected by immunoprecipitation with anti-nucleolin antibodies. The amount of nucleolin was slightly increased by EGF plus insulin in the presence of dexamethasone, but scarcely changed by treatment with EGF plus insulin or dexamethasone alone. Cycloheximide inhibited RNA synthesis to a greater or lesser degree in the case of all hepatocytes which were cultured with or without these hormonal treatments. These results indicate that the in vivo effect of glucocorticoid on rRNA synthesis and nucleolin phosphorylation in liver is primarily a direct action on parenchymal cells and requires other growth factors such as EGF and insulin.     

Control by amino acids of the activity of system A-mediated amino acid transport in isolated rat hepatocytes.

The effect of amino acids, in concentrations corresponding to those found in the portal vein of rats given a high-protein diet, was investigated on the activity of system A amino acid transport in hepatocytes from fed rats. Amino acids counteracted the induction of system A by insulin or glucagon. This effect was observed at all concentrations of hormones tested, up to 1 microM. Amino acids did not affect the basal cyclic AMP concentration in hepatocytes, or the large rise in cyclic AMP elicited by glucagon. The reversal of system-A induction was observed at relatively low concentration of amino acids, corresponding to plasma values reported in rats given a basal diet. Amino acids were separately tested: substrates of system A were particularly efficient, but so were glutamine and histidine. Non-metabolizable substrates of system A, such as 2-aminoisobutyrate, were also inhibitory, suggesting that a part of the effect of amino acids is independent of their cellular metabolism. Provision of additional energy substrates such as lactate and oleate did not affect induction of system A or the inhibitory effects of amino acids. Thus amino acids do not act by serving as an energy source and by maintaining the integrity of hepatocytes. Inhibition of mRNA synthesis by actinomycin practically abolished the effect of amino acids on the induction of system A by glucagon. The results suggest that amino acids may promote the synthesis of protein(s) affecting the activity of system A either directly at the carrier unit or at an intermediate stage of its emergence.     

Induction of tryptophan oxygenase by dexamethasone in isolated hepatocytes. Dependence on composition of medium and pH.

Hepatocytes were isolated from perfused rat livers. 4 x 10-6 cells/ml were incubated at at 37 degrees C in different media in the absence and presence of a steroid hormone, dexamethasone phosphate (2 x 10-5 M). 1. Hormonal enzyme induction occurred in cells suspended in a simple salt medium, devoid of amino acids and macromolecules. This induction was completely blocked by addition of either actinomycin D (2 mu-g/ml) or cycloheximide (50 mu-g/ml). 2. Incubation of cells in media containing defatted albumin did not enhance hormonal enzyme induction, although disintegration of cells during incubation was reduced. Addition of a crude albumin fraction reduced tryptophan oxygenase induction and dextran completely blocked enzyme induction by dexamethasone. 3. An increase of dexamethasone concentration in the presence of albumin to 9 x 10-5 M was unable to raise enzyme induction further, and a still higher concentration of hormone, 3 x 10-4 M, resulted in reduced enzyme induction. 4. The hormonal induction of tryptophan oxygenase was most pronounced when the pH of the medium was between 7.0 and 7.6, with an optium at 7.3. No induction was found when the pH of the medium was either 6.6 or 7.8. The basal tryptophan oxygenase activity was much less influenced by similar pH variations. It is concluded that hepatocytes in suspension are able to carry out hormone-stimulated enzyme synthesis and that factors influencing this process may be studied under controlled conditions in such systems.     

Multivalent Induction of Biodegradative Threonine Deaminase

To determine the inducer(s) of the biodegradative threonine deaminase in Escherichia coli, the effects of various amino acids on the synthesis of this enzyme were investigated. The complex medium used hitherto for the enzyme induction can be completely replaced by a synthetic medium composed of 18 natural amino acids. In this synthetic medium, the omission of each of the seven amino acids threonine, serine, aspartic acid, methionine, valine, leucine, and arginine resulted in the greatest loss of enzyme formation. These seven amino acids did not significantly influence the uptake of other amino acids into the cells. Furthermore, they did not stimulate the conversion of inactive enzyme into an active form, since they did not affect the enzyme level in cells in which protein synthesis was inhibited by chloramphenicol. Threonine, serine, aspartic acid, and methionine failed to stimulate enzyme production in cells in which messenger ribonucleic acid synthesis was arrested by rifampin, whereas valine, leucine, and arginine stimulated enzyme synthesis under the same conditions. Therefore, the first four amino acids appear to act as inducers of the biodegradative threonine deaminase in E. coli and the last three amino acids appear to be amplifiers of enzyme production. The term "multivalent induction" has been proposed for this type of induction, i.e., enzyme induction only by the simultaneous presence of several amino acids.