Factors regulating the induction of ornithine decarboxylase in fetal rat liver cells in culture

Various hormonal and non-hormonal agents were tested for their ability to induce ornithine decarboxylase (EC 4.1.1.17) in primary cultures of fetal rat liver cells that retain many of the differentiated functions of hepatocytes. The only agents to induce ornithine decarboxylase in this cell type were fetal calf serum, prostaglandin E₁ and cyclic AMP derivatives. Also, the amino acid arginine would induce ornithine decarboxylase in this cell type following arginine starvation for 24 h. These observations are in contrast to the wide range of hormones, e.g. insulin, hydrocotisone, glucagon and growth hormone, that can induce ornithine decarboxylase in vivo in the adult rat liver but which are all without effect on fetal rat liver cells.     

A human neuroblastoma cell line with a stable ornithine decarboxylase in vivo and in vitro

A human neuroblastoma cell line (Paju) grew in 10 mM difluoromethyl-ornithine, which at this concentration normally stops the growth of all mammalian cells. Ornithine decarboxylase from Paju was resistant to inhibition in vitro by difluoromethylornithine, and required 10 microM of the compound for 50% inhibition, whereas ornithine decarboxylase from SH-SY5Y cells (another human neuroblastoma) and from rat liver needed only 0.5 microM difluoromethylornithine. Paju ornithine decarboxylase also exhibited a long half-life (over eight hours) in vivo. The half-life of immunoreactive protein was significantly longer than that of the activity. The long half-life of ornithine decarboxylase in Paju cells leads to its accumulation to a specific activity of 2000 nmol/mg of protein per 30 min during rapid growth (the corresponding activity in SH-SY5Y cells was about 2.5). When partially purified ornithine decarboxylase from Paju cells was incubated with rat liver microsomes it was inactivated with a half-life of 75 min. This inactivation was accompanied by a fall in the amount of immunoreactive protein. In the same inactivating system partially purified SH-SY5Y ornithine decarboxylase had a half-life of 38 min and its half-life in vivo was 50 min. The corresponding values for rat liver ornithine decarboxylase were 45 min and 40 min, respectively. Rat liver microsomes also inactivated rat liver adenosylmethionine decarboxylase. These results suggest that Paju ornithine decarboxylase has an altered molecular conformation, rendering it resistant to (i) difluoromethylornithine and (ii) proteolytic degradation both in vivo and in vitro.     

Lack of ornithine decarboxylase activity in isolated rat liver parenchymal cells

Polyamines are associated with fundamental metabolic and functional steps in cell metabolism. The activity of ornithine decarboxylase, the key enzyme in polyamine metabolism, was followed during the preparation of rat liver parenchymal cells and in the isolated cells during incubation. In experiments in which ornithine decarboxylase was not induced in vivo, enzyme activity dropped to barely measurable values during the preparation. An even more drastic loss of enzyme activity was noted in livers in which ornithine decarboxylase activity was stimulated in vivo 20-40fold by previous injection of bovine growth hormone, or thioacetamide or elevated because of circadian rhythmical changes of the enzyme activity. Within the first 20 min of liver perfusion to disintegrate the tissue, ornithine decarboxylase activity decreased by up to 80%. The presence of bovine growth hormone during cell preparation cannot prevent the loss of enzyme activity. Incubation of the isolated cells for periods of up to 240 min did not restore the enzyme activity. Furthermore, incubation of the cells with bovine growth hormone did not induce ornithine decarboxylase, even though the medium was supplemented with amino acids in physiological concentrations. During normal liver perfusion and in contrast to the situation with isolated cells, there is no loss of enzyme activity but a small rise. Following pretreatment of the animals with bovine growth hormone or thioacetamide the highly stimulated activity of ornithine decarboxylase declined slowly during liver perfusion, but never dropped to values lower than normal for perfusion periods of up to 240 min. Moreover, in the intact perfused organ ornithine decarboxylase remains responsive to bovine growth hormone. The experiments demonstrate that enzymatic tissue dispersion by collagenase in particular or the preparation of isolated cells in general drastically alters the metabolic and functional state of rat liver parenchymal cells.     

Decarboxylation of ornithine and lysine in rat tissues.

The possibility that arginine and lysine might be decarboxylated by rat tissues was investigated. No evidence for decarboxylation of arginine could be found. Lysine decarbosylase (L-lysine carboxy-lyase, EC 4.1.1.18) activity producing CO2 and cadaverine was detected in extracts from rat ventral prostate, androgen-stimulated mouse kidney, regenerating rat liver and livers from rats pretreated with thioacetamide. These tissues all have high ornithine decarboxylase (L-ornithine carboxy-lyase, EC 4.1.1.17) activities. Lysine and ornithine decarboxylase activities were lost to similar extents on inhibition of protein synthesis by cycloheximide and on exposure to alpha-difluoromethylornithine. A highly purified ornithine decarboxylase preparation was able to decarboxylate lysine and the ratio of ornithine to lysine decarboxylase activities was constant throughout purification. Kinetic studies of the purified preparation showed that the V for ornithine was about 4-fold greater than for lysine, but the Km for lysine (9 mM) was 100-times greater than that for ornithine (0.09 mM). These experiments indicate that all of the detectable lysine decarboxylase activity in rat and mouse tissues was due to the action of ornithine decarboxylase and that significant cadaverine production in vivo would occur only when ornithine decarboxylase activity is high and lysine concentrations substantially exceed those of ornithine.     

Induction of ornithine decarboxylase in rat liver by phorbol ester is mediated by prostanoids from Kupffer cells

Administration of phorbol 12-myristate 13-acetate (PMA) to rats in vivo resulted in the induction of ornithine decarboxylase activity in the liver which could be blocked by preinjection of indomethacin, a cyclooxygenase inhibitor. In vitro administration of PMA to primary cultures of rat parenchymal cells did not lead to an induction of ornithine decarboxylase activity. It was investigated to what extent non-parenchymal liver cells could play an intermediary role in the expression of the PMA effect on ornithine decarboxylase activity in parenchymal liver cells. Addition of conditioned medium from PMA-activated Kupffer cells to cultured parenchymal cells led to the induction of ornithine decarboxylase activity in parenchymal cells. This effect was not observed with conditioned medium from untreated Kupffer cells or from Kupffer cells treated with PMA plus indomethacin. Conditioned media from PMA-treated or untreated endothelial liver cells were ineffective in the induction of ornithine decarboxylase activity in parenchymal liver cells. Prostaglandin D2, the main eicosanoid produced by Kupffer cells, was able to stimulate the synthesis of ornithine decarboxylase in parenchymal liver cells (up to 40-fold) in a dose-dependent way. Prostaglandin (PG) D2 appeared to be a more potent inducer of ornithine decarboxylase activity in parenchymal cells than PGE1 and PGE2. It is concluded that intercellular communication inside the liver mediated by prostaglandins derived from activated Kupffer cells may form a mechanism to induce synthesis of specific proteins in parenchymal cells.     

Regulation of polyamine biosynthesis by antizyme and some recent developments relating the induction of polyamine biosynthesis to cell growth

This review considers the role of antizyme, of amino acids and of protein synthesis in the regulation of polyamine biosynthesis.The ornithine decarboxylase of eukaryotic ceils and ofEscherichia coli coli can be non-competitively inhibited by proteins, termed antizymes, which are induced by di-and poly- amines. Some antizymes have been purified to homogeneity and have been shown to be structurally unique to the cell of origin. Yet, the E. c o l i antizyme and the rat liver antizyme cross react and inhibit each other's biosynthetic decarboxylases. These results indicate that aspects of the control of polyamine biosynthesis have been highly conserved throughout evolution.Evidence for the physiological role of the antizyme in mammalian cells rests upon its identification in normal uninduced cells, upon the inverse relationship that exists between antizyme and ornithine decarboxylase as well as upon the existence of the complex of ornithine decarboxylase and antizyme in vivo. Furthermore, the antizyme has been shown to be highly specific; its Keq for ornithine decarboxylase is 1.4 x 1011 M-1. In addition, mammalian ceils contain an anti-antizyme, a protein that specifically binds to the antizyme of an ornithine decarboxylase-antizyme complex and liberates free ornithine decarboxylase from the complex. In B. coli , in which polyamine biosynthesis is mediated both by ornithine decarboxylase and by arginine decarboxylase, three proteins (one acidic and two basic) have been purified, each of which inhibits both these enzymes. They do not inhibit the biodegradative ornithine and arginine decarboxylases nor lysine decarboxylase. The two basic inhibitors have been shown to correspond to the ribosomal proteins S₂₀/L₂₆ and L₃₄, respectively. The relationship of the acidic antizyme to other known B. coli proteins remains to be determined.     

Growth control in primary fetal rat liver cells in culture.

Primary fetal rat liver cells cultured in medium deficient in, but not free of, arginine in the presence of dialyzed fetal calf serum grow until the final cell density is attained and cells become quiescent in the Go phase of the cell cycle. When growing cells are transferred into arginine free medium, cells become reversibly arrested in Go. Fetal rat liver cells can be induced to synthesize DNA by addition of high levels of arginine to serum free medium. Low arginine levels in the culture medium do not induce cell growth unless serum is present. Serum stimulates arginine uptake in fetal rat liver cells suggesting that serum growth factor(s) act by increasing intracellular arginine levels high enough to initiate the growth cycle. Fractionation of fetal calf serum by gel filtration on G-200 Sephadex yields a partially purified arginine uptake stimulating activity which is eluted from the column in the same fractions that contain fetal rat liver cell growth promoting activity. Insulin induces DNA synthesis in quiescent fetal rat liver cells. Glucagon reverses the stimulatory effects of insulin. N-6,O-2-Dibutyryl adenosine 3:5-cyclic monophosphoric acid (But2c-AMP) (10-minus4 M) and theophilline (10-minus3 M) inhibit arginine uptake and the initiation of DNA synthesis by serum. The role of arginine in the control of DNA synthesis in fetal rat liver cells and the mechanism of action of serum growth factors are discussed.     

Comparison of ornithine decarboxylase from rat liver, rat hepatoma and mouse kidney.

Comparisons were made of ornithine decarboxylase isolated from Morris hepatoma 7777, thioacetamide-treated rat liver and androgen-stimulated mouse kidney. The enzymes from each source were purified in parallel and their size, isoelectric point, interaction with a monoclonal antibody or a monospecific rabbit antiserum to ornithine decarboxylase, and rates of inactivation in vitro, were studied. Mouse kidney, which is a particularly rich source of ornithine decarboxylase after androgen induction, contained two distinct forms of the enzyme which differed slightly in isoelectric point, but not in Mr. Both forms had a rapid rate of turnover, and virtually all immunoreactive ornithine decarboxylase protein was lost within 4h after protein synthesis was inhibited. Only one form of ornithine decarboxylase was found in thioacetamide-treated rat liver and Morris hepatoma 7777. No differences between the rat liver and hepatoma ornithine decarboxylase protein were found, but the rat ornithine decarboxylase could be separated from the mouse kidney ornithine decarboxylase by two-dimensional gel electrophoresis. The rat protein was slightly smaller and had a slightly more acid isoelectric point. Studies of the inactivation of ornithine decarboxylase in vitro in a microsomal system [Zuretti & Gravela (1983) Biochim. Biophys. Acta 742, 269-277] showed that the enzymes from rat liver and hepatoma 7777 and mouse kidney were inactivated at the same rate. This inactivation was not due to degradation of the enzyme protein, but was probably related to the formation of inactive forms owing to the absence of thiol-reducing agents. Treatment with 1,3-diaminopropane, which is known to cause an increase in the rate of degradation of ornithine decarboxylase in vivo [Seely & Pegg (1983) Biochem. J. 216, 701-717] did not stimulate inactivation by microsomal extracts, indicating that this system does not correspond to the rate-limiting step of enzyme breakdown in vivo.     

STUDIES ON PRIMARY CULTURES OF DIFFERENTIATED FETAL LIVER CELLS

A method for culturing non- or slowly growing, differentiated fetal rat liver cells is described. It involves the use of collagenase as a digesting agent and of a selective medium deficient in arginine which suppresses the growth of nonparenchymal liver cells. Evidence is presented that surviving cells (a) retain liver-specific urea cycle functions measured by their capacity to transform ornithine into arginine, (b) synthesize DNA in glucose-deficient medium, and (c) synthesize and secrete albumin. This primary cell culture responds to partially hepatectomized rat serum and may be an appropriate assay system for the study of mechanisms which regulate liver regeneration.     

Immortalization of normal liver functions in cell culture: rat hepatocyte-hepatoma cell hybrids expressing ornithine carbamoyltransferase activity.

Normal rat hepatocytes have been fused with highly differentiated rat hepatoma cells. Some of the hybrids express a physiologically significant level of activity of the urea cycle enzyme ornithine carbamoyltransferase (OCT), a liver-specific function not found in the hepatoma cells. These hybrids have 10% of the adult rat liver OCT specific activity, incorporate 3H-ornithine into protein arginine, and can be selectively grown in arginine-free medium supplemented with ornithine. Somatic cell hybridization of normal differentiated cells with highly differentiated neoplastic cells of the same tissue type may be useful as a general method for obtaining permanent cell lines with new tissue-specific phenotypes.     

Association of diamine oxidase and ornithine decarboxylase with maturing cells in rapidly proliferating epithelium

Diamine oxidase and ornithine decarboxylase activities are shown to have a parallel distribution across rat small intestine mucosa; levels of both enzyme activities are sharply higher in mature cells in the villus tip region than in proliferating cells in the crypt areas. Histidine decarboxylase levels were not measurable in the same cell preparations and aromatic-L-amino-acid decarboxylase activity was distributed in an opposite pattern to diamine oxidase and ornithine decarboxylase. The results suggest that intestinal diamine oxidase could be involved with polyamine metabolism. The new findings for ornithine decarboxylase suggest an in vivo role for polyamines in non-proliferative cells; rat small intestinal mucosa may be an excellent model for investigating the function of polyamines in regenerating cells.     

Association of diamine oxidase and ornithine decarboxylase with maturing cells in rapidly proliferating epithelium.

Diamine oxidase and ornithine decarboxylase activities are shown to have a parallel distribution across rat small intestine mucosa; levels of both enzyme activities are sharply higher in mature cells in the villus tip region than in proliferating cells in the crypt areas. Histidine decarboxylase levels were not measurable in the same cell preparations and aromatic-L-amino-acid decarboxylase activity was distributed in an opposite pattern to diamine oxidase and ornithine decarboxylase. The results suggest that intestinal diamine oxidase could be involved with polyamine metabolism. The new findings for ornithine decarboxylase suggest an in vivo role for polyamines in non-proliferative cells; rat small intestinal mucosa may be an excellent model for investigating the function of polyamines in regenerating cells.     

Immunochemical demonstration of increased accumulation of ornithine decarboxylase in rat liver after partial hepatectomy and growth hormone induction

Antiserum against ornithine decarboxylase (EC 4.1.1.17) was prepared in rabbits using purified ornithine decarboxylase from rat liver as the antigen. Immunoglobulins from the immune sera were covalently coupled to agarose by cyanogen bromide activation. With the aid of this immunoadsorbent against the enzyme it has been shown that following partial hepatectomy and growth hormone administration, the ornithine decarboxylase activity is elevated concomitantly with the increase in the immunoreactive enzyme protein. In addition, the rapid decay in ornithine decarboxylase activity in regenerating rat liver after cycloheximide injection is accompanied by a decrease in the immunoreactive protein. These results suggest that the activity of ornithine decarboxylase in rat liver is regulated through rapid changes in de novo synthesis and degradation of the enzyme protein.     

Rat colon ornithine and arginine metabolism: coordinated effects after proliferative stimuli

Ornithine decarboxylase (ODC) catalyzes the first step in the polyamine biosynthetic pathway, a highly regulated pathway in which activity increases during rapid growth. Other enzymes also metabolize ornithine, and in hepatomas, rate of growth correlates with decreased activity of these other enzymes, which thus channels more ornithine to polyamine biosynthesis. Ornithine is produced from arginase cleavage of arginine, which also serves as the precursor for nitric oxide production. To study whether short-term coordination of ornithine and arginine metabolism exists in rat colon, ODC, ornithine aminotransferase (OAT), arginase, ornithine, arginine, and polyamine levels were measured after two stimuli (refeeding and/or deoxycholate exposure) known to synergistically induce ODC activity. Increased ODC activity was accompanied by increased putrescine levels, whereas OAT and arginase activity were reduced by either treatment, accompanied by an increase in both arginine and ornithine levels. These results indicate a rapid reciprocal change in ODC, OAT, and arginase activity in response to refeeding or deoxycholate. The accompanying increases in ornithine and arginine concentration are likely to contribute to increased flux through the polyamine and nitric oxide biosynthetic pathways in vivo.     

L-ornithine-induced inactivation of mammalian ornithine decarboxylase in vitro

Partially purified ornithine decarboxylase, isolated from the liver of thioacetamide-treated rats, is stable in the absence of added low-molecular-mass thiols or other reducing agents. However, under these conditions, the enzyme is rapidly inactivated upon incubation with L-ornithine or L-2-methylornithine. The inactivation process follows first-order kinetics, and saturation kinetics are observed. Rapid recovery of activity is observed after subsequent addition of dithiothreitol. As distinct from L-ornithine, D-ornithine, putrescine, spermidine, or spermine do not produce inactivation of ornithine decarboxylase. Very similar results are obtained with pure ornithine decarboxylase isolated from androgen-stimulated mouse kidney, stabilized with a rat liver extract.     

Regulation of ornithine decarboxylase activity in rat ovarian cells in vitro.

Incubation of rat ovarian cell suspension with human choriogonadotropin (hCG) caused a marked enhancement of ornithine decarboxylase (EC 4.1.1.17) activity after a lag period of several hours. Even though ovarian ornithine decarboxylase could be induced in minimum essential medium by the hormone alone, supplementation of the medium with various sera greatly enhanced the stimulation of the enzyme activity. All the sera tested (human, fetal calf and horse) were able to stimulate ornithine decarboxylase activity even in the absence of hCG. Maximum stimulation of the enzyme activity by hCG and/or serum occurred in ovarian cell suspensions prepared from 30 to 33-day-old rats. There was a close correlation between the stimulation of ornithine decarboxylase activity and the accumulation fo cyclic AMP in response to the administration of the hormone (in the presence or absence of serum). However, while various sera alone markedly enhanced ovarian ornithine decarboxylase activity in vitro they, if anything, only marginally stimulated the accumulation of cyclic AMP and the secretion of progesterone in ovarian cells in the absence of gonadotropin. A similar dissociation of the stimulation of ornithine decarboxylase activity from the production of cyclic AMP and progesterone was likewise found when the ovarian cells were incubated in an enriched medium (M199) supplemented with albumin and lactalbumin hydrolysate in the absence of the hormone. Under these culture conditions ornithine decarboxylase activity was strikingly enhanced, greatly exceeding the stimulation obtained with various sera, while the accumulation of cyclic AMP and the secretion of progesterone remained virtually unchanged. Specific inhibition (up to 90%) of gonadotropin-induced ornithine decarboxylase activity by difluoromethyl ornithine or 1,3-diamino-2-propanol had little effect on the ability of the ovarian cells to respond to the hormone with increasing production of cyclic AMP and progesterone. While showing that rat ovarian ornithine decarboxylase can be induced in vitro by choriogonadotropin or various sera, our results indicate that the activation of the enzyme involves at least two different mechanisms: (i) One (in response to gonadotropin) involving a prior stimulation of cyclic AMP production, and (ii) another (in response to serum) that is not associated with increases in the accumulation of the cyclic nucleotide.     

Hydrazine stress in the diabetic: ornithine decarboxylase activity

Streptozotocin-induced diabetes of 7 weeks duration increased male Sprague-Dawley rat kidney ornithine decarboxylase activity by 4.8-fold but did not affect the liver enzyme. Hydrazine treatment of 4 hr duration stimulated equally kidney ornithine decarboxylase activities of nondiabetic and diabetic rats. Hydrazine treatment increased liver ornithine decarboxylase activity in the nondiabetic rat but did not increase it in the diabetic rat. Since hydrazine stimulates ornithine decarboxylase activity prior to polyamine and protein syntheses, we speculate that the lack of hydrazine stimulation of ornithine decarboxylase in the diabetic liver may be related in part to the unrestrained gluconeogenesis and depressed Kreb's cycle activity: the latter being required for protein synthesis.     

Polyamine synthesis in maize cell lines

Uptake of [¹⁴C]putrescine, [¹⁴C]arginine, and [¹⁴C]ornithine was measured in five separate callus cell lines of Zea mays. Each precursor was rapidly taken into the intracellular pool in each culture where, on the average, 25 to 50% of the total putrescine was found in a conjugated form, detected after acid hydrolysis. Half-maximal labeling of each culture was achieved in less than 1 minute. Within this time frame of precursor incorporation, only putrescine derived from arginine was conjugated, indicating that putrescine pools derived from arginine may initially be sequestered from ornithine-derived putrescine. The decarboxylase activities were measured in each culture after addition of exogenous polyamine to the growth medium to assess differential regulation of the decarboxylases. Arginine and ornithine decarboxylase activities were augmented by added polyamine, the effect on arginine decarboxylase being eightfold greater than on ornithine decarboxylase. Levels of extractable ornithine decarboxylase were consistently 15- to 100-fold higher than arginine decarboxylase, depending on the titer of extracellular polyamine. Taken as whole the results support the idea that there are distinct populations of polyamine that are initially sequestered after the decarboxylase reactions and that give rise to separate end products and possibly have separate functions.     

Desensitization of immature rat testicular ornithine decarboxylase to arginine vasopressin

Prior exposure of immature rat testis to arginine vasopressin caused the testis refractory at 24 h in terms of ornithine decarboxylase activity. Arginine vasopressin caused desensitization both in Leydig cells and seminiferous tubules. Arginine vasopressin induced desensitization was found to be both time and dose-dependent. Arginine vasopressin desensitized testis was refractory to luteinizing hormone, follicle stimulating hormone, norepinephrine, dibutyryl cAMP, phorbol-myristate acetate and cholera toxin at 24 h. Arginine vasopressin desensitized testis showed recovery of response to norepinephrine at 48 h after the first injection. On the contrary arginine vasopressin could stimulate ornithine decarboxylase in luteinizing hormone desensitized testis. These results indicate that in arginine vasopressin desensitized testis the block is at post cAMP step which is common to both cAMP dependent and protein kinase C-diacylglycerol system in stimulating testicular ornithine decarboxylase.     

Stimulation of testicular ornithine decarboxylase activity by arginine vasopressin

Intratesticular injection with arginine vasopressin caused stimulation of ornithine decarboxylase activity in the testes of immature rats. The increase in ornithine decarboxylase activity in response to arginine vasopressin was dose and time dependent. Maximal stimulation of ornithine decarboxylase activity occurred at 2 h after injection with 0.1 micrograms of arginine vasopressin. It was observed that stimulation of ornithine decarboxylase activity occurred in seminiferous tubules and in Leydig cells of the testis in response to arginine vasopressin.