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.     

Calcium, asparagine and cAMP are required for ornithine decarboxylase activation in intact Chinese hamster ovary cells.

Asparagine specifically activated ornithine decarboxylase activity 5–7 fold by 7–8 h in confluent cultures maintained with a salts/glucose medium. When dibutyryl cAMP was added with asparagine, a 40–50 fold stimulation of ornithine decarboxylase activity was produced. Ornithine decarboxylase activation in the salts/glucose medium was not sensitive to actinomycin D. Omission of Ca⁺⁺ and Mg⁺⁺ from the medium abolished the ability of asparagine and/or dibutyryl cAMP to stimulate enzyme activity. Calcium was essential for the asparagine and dibutyryl cAMP mediated stimulation of ornithine decarboxylase activity.     

The differential activation of ornithine decarboxylase in synchronized cultures maintained with a salts/glucose medium

Asparagine and cAMP caused differential activation of ornithine decarboxylase activity that was dependent upon the cell cycle phase of Chinese Hamster Ovary cells maintained with a salts/glucose medium. Prior to incubation in the salts/glucose medium, stationary phase cultures were stimulated to proliferate by refeeding with fresh complete growth medium. Induction of ornithine decarboxylase activity was dependent upon two conditions: (1) the salts/ glucose medium required supplementation with an appropriate amino acid and cAMP; (2) cultures had to be in late G1/early S phase when placed in the salts/glucose medium. Cultures in G0 phase had moderate capacity for ornithine decarboxylase enhancement but lost this capacity as they traversed into early G1 or into mid S phase. These results demonstrate the importance of the cell cycle in modulating the activation of ornithine decarboxylase by asparagine and cAMP.     

Calcium stimulates ornithine decarboxylase activity in cultured mammalian epithelial cells

Ornithine decarboxylase (ODC) activity usually rises to a peak a few hours after a trophic stimulus. The stimulation of ODC has been shown to depend on extracellular calcium in several in vitro eukaryotic systems. We have investigated the effect of calcium concentration on ODC activity and have found that ODC is stimulated when CaCl2 alone is added to calcium-deprived cells. Epithelial cells from calf esophagus were cultured and grown until stratified. Replacement of medium with fresh serum-free medium resulted in stimulation of ODC activity, which peaked at 4 hours and declined to basal level by 10 hours. Subsequent depletion of Ca2+ either by addition of ethylene glycol bis (beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA) or by replacement of medium with Ca2+-free medium, resulted in obliteration of ODC activity 4 hours later. Conversely, cultures in which medium was replaced with Ca2+-free medium and at 10 hours were repleted with Ca2+ (either by addition of CaCl2 or by replacement of medium with Ca2+-containing medium) exhibited a pronounced elevation of ODC activity 4 hours later. ODC activity peaked at 6 hours after the addition of CaCl2 and declined by 8 hours. The effect was elicited by a wide range of concentrations of added Ca2+ from 0.1 mM to 4.0 mM, but was maximal at 1.0 mM. ODC activity was totally abolished if either cycloheximide (10 micrograms/ml) or putrescine (10 mM) was added to cultures immediately prior to Ca2+ addition. Actinomycin D (2, 5, or 10 micrograms/ml) added 30 minutes before Ca2+ did not prevent the stimulation of ODC by added Ca2+. Stimulation by Ca2+ is dependent on (1) absence of Ca2+ during the initial 10-hour incubation and (2) duration of incubation in Ca2+-free medium prior to Ca2+ replenishment. The results indicate that Ca2+ can increase ODC in epithelial cells exposed to Ca2+-depleted medium and that the increase in ODC depends on protein synthesis but is not inhibited by actinomycin D.     

Glucose elevates ornithine decarboxylase expression in Vero cells

The addition of Earle's balanced salt solution (EBSS) of amino acids that are transported by a Na+-dependent cotransport system was not required by Vero cells for ornithine decarboxylase (ODC:EC 4.1.1.17) amplification. Vero cell ODC activity was elevated tenfold above basal levels when confluent cells were incubated for 5 hr in EBSS alone. ODC activity increased as a function of the incubation time in EBSS and was not elevated above basal enzyme levels when cells were incubated in EBSS minus glucose. ODC expression increased as a function of the glucose concentration in EBSS, with 20 mM glucose producing a 90-fold increase in ODC activity. ODC expression is more responsive to glucose in high-density quiescent cultures than in low-density growing cultures. Enhanced ODC expression by glucose depended on Na+ and K+ concentrations. The specific activity of ODC was also elevated above basal levels when mannose or fructose replaced glucose in EBSS. The addition of alanine or asparagine to EBSS enhanced ODC activity above levels obtained with EBSS containing standard (5.5 mM) glucose concentrations. In the absence of glucose, alanine was more effective than asparagine in enhancing ODC expression. These results suggest that the transport of amino acids is not an absolute requirement for Vero cell ODC expression and that ODC expression is linked to changes in cellular energetics and/or ion fluxes.     

Effect of cyclic nucleotides on the induction of ornithine decarboxylase in BHK cells by serum and insulin

Quiescent baby hamster kidney cells in 0.5% serum synthesize little DNA and have low levels of ornithine decarboxylase (ODC), the rate-limiting enzyme in polyamine synthesis. After adding serum to 5%, ODC activity is increased 30 fold, reaching a maximum at 6 hr, whereas DNA synthesis is reinitiated at 12 hr. Five μg/ml insulin also increases ODC activity 3 fold by 4 hr. In quiescent 3T3 cells and mouse embryo fibroblasts, serum and insulin may trigger many metabolic events by causing a transient drop in intracellular cyclic AMP and a rise in cyclic GMP. To test this hypothesis in BHK cells, cAMP levels were raised by adding dibutyryl cAMP and/or theophylline, or by stimulating adenylate cyclase with Prostaglandin E₁. cAMP blocks the serum stimulation of DNA synthesis, but increases ODC activity, both in quiescent cells and in cells treated with serum and insulin. These results suggest that serum and insulin control ODC activity through a mechanism independent of a drop in cAMP.     

Interaction of alpha and beta adrenergic stimulation on aortic ornithine decarboxylase activity

The interaction between beta and alpha adrenergic agonists on regulation of cockerel aortic ornithine decarboxylase (ODC) activity was examined. The beta adrenergic agonist isoproterenol both reduced basal aortic ODC activity and prevented induction of the decarboxylase by the alpha adrenergic agonist methoxamine. 3-Isobutyl-1- methylxanthine (IBMX) similarly reduced basal ODC activity and blocked induction of the enzyme by methoxamine. When given ten minutes before or after methoxamine, isoproterenol prevented aortic ODC induction, but not large sustained increases in blood pressure evoked by the alpha adrenergic agonist. In contrast, when injected three hours after methoxamine, isoproterenol had no effect on already elevated levels of enzyme activity. Addition of isoproterenol (10(-7)M), IBMX (1 mM) or dibutyryl cAMP (2.5 mM) to isolated aortic segments cultured in minimal salts-glucose media evoked decreases in basal levels of ODC activity resembling those observed in the intact animal. These results suggest that the balance between alpha and beta adrenergic stimulation may be an important feature of the regulation of polyamine biosynthesis in artery wall cells.     

Maintenance of pancreatic endocrine cells of the neonatal rat: IV. The effect of 3-amino-3-deoxyglucose

Monolayer islet cells prepared from neonatal rat pancreases were cultured in media with 5.5 mM glucose alone or further supplemented with 5 mM 3-amino-3-deoxyglucose (3A3dG) for a total of 7 days. After culture for 7 days, 3A3dG-supplementation maintained the recovery of insulin released into the medium during the last 2 days of a 7-day culture at a level 2.9 fold higher that at day 0. Similarly, the insulin content of the cells was significantly higher than the initial level at day 0 (2.8-fold) and that of the cells grown in medium with glucose alone (4.5-fold). The maximum secretory responses to glucose (2.8-16.7 mM), leucine (2.5-10 mM) and 2-ketoisocaproate (2.5-10 mM) were several times as high as the initial. Further, 3A3dG caused a selective deletion of fibroblasts mostly consisting of endocrine cells. In these monolayer cells, either the cAMP response to glucose or the cellular cAMP content were significant. In conclusion, it is suggested that the beneficial effect of 3A3dG may be associated with an increase in either the oxidative catabolism of amino acids or the activity of adenylate cyclase in the B cell.     

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.     

Bacterial lipopolysaccharide induction of ornithine decarboxylase in the macrophage-like cell line RAW264: requirement of an inducible soluble factor

Ornithine decarboxylase (ODC, EC 4.1.1.17) activity is induced in the RAW264 macrophage-like cell line by bacterial lipopolysaccharide (LPS). As little as 0.1 ng/ml LPS promoted an increase in ODC activity, while maximal ODC activity (30-fold above control) was induced with 1.0 microgram/ml LPS. An increase in ODC activity was detectable within 90 min of LPS addition. The LPS-induced increase in ODC activity was prevented by inhibitors of protein and RNA synthesis. The induction of the enzyme by LPS was not dependent on prostaglandin production. However, PGE2 (1 microgram/ml) and 8-bromo-cyclic AMP (1 mM), neither of which had an effect on ODC activity when added alone, each acted synergistically to enhance the LPS induction of ODC activity. Enzyme induction was not associated with an alteration in Km for ornithine, which remained constant at 0.04 mM. The extent of the increase in ODC in response to LPS increased with increasing cellular density. This relationship was dependent not on absolute cell density of the monolayer but on the cell number in relation to medium volume, and this dependence could be extrapolated to the origin. Addition of conditioned media from LPS-stimulated but not unstimulated cultures enhanced the ODC increase in sparsely plated cultures in response to a maximal concentration of LPS. The addition of polymyxin B, a reagent that blocks the effects of LPS, including the increase in ODC activity, did not totally inhibit the conditioned medium stimulation. This data indicates that two signals, LPS and a LPS-induced mediator, are involved in the induction of ODC activity in RAW264 cells.     

Modulatory function of protein kinase C in the activation of ornithine decarboxylase and in cAMP production in rat osteoblasts

The effect of activation of protein kinase C on stimulation of ornithine decarboxylase (ODC) activity and cAMP production was studied in fetal rat osteoblasts. Both phorbol 12-myristate, 13-acetate (PMA), an activator of protein kinase C, and 4 alpha-phorbol, ineffective in activating protein kinase C, failed to stimulate ODC activity and cAMP production. We tested the effect of protein kinase C on stimulation of ODC activity by parathyroid hormone (PTH) and forskolin. In contrast to PTH-stimulated ODC activity, which was not affected by PMA, forskolin-stimulated (1 and 10 microM) ODC activity was dose dependently reduced. PMA (400 nM) reduced both 1 and 10 microM forskolin-stimulated ODC activity to the same level, approximately 3 nmol CO2/mg protein, which suggests a controlling role of protein kinase C in forskolin-stimulated ODC activity. The study of the effect of protein kinase C on PTH- and forskolin-stimulated cAMP production also revealed differences between PTH and forskolin. When PMA was added simultaneously with PTH (4 and 20 nM) or forskolin (1 and 10 microM) the PTH-stimulated cAMP production was dose-dependently potentiated by PMA, whereas forskolin-stimulated cAMP production was not affected. However, both PTH- and forskolin-stimulated cAMP production was dose-dependently augmented when PMA was added 3 min prior to PTH or forskolin. With increasing preincubation periods (up to 24 h) with PMA instead of a potentiation an inhibition was observed. This inhibition is not due to PTH receptor desensitization, although, on basis of the present results desensitization can not completely be excluded. In all cases 4 alpha-phorbol was without effect. The present results show that protein kinase C modulates stimulation of ODC activity and cAMP production in fetal rat osteoblasts. The modulation of both ODC activity and cAMP production appears to be dependent on the nature of the stimulator. The present data suggest a role for protein kinase C in limiting the cAMP-mediated stimulation of ODC activity in these cells. Furthermore, it is suggested that protein kinase C can interfere at more than one site in the cAMP-generating system.     

Production of Solvents by Clostridium acetobutylicum Cultures Maintained at Neutral pH

The formation of acetone and n-butanol by Clostridium acetobutylicum NCIB 8052 (ATCC 824) was monitored in batch culture at 35°C in a glucose (2% [wt/vol]) minimal medium maintained throughout at either pH 5.0 or 7.0. At pH 5, good solvent production was obtained in the unsupplemented medium, although addition of acetate plus butyrate (10 mM each) caused solvent production to be initiated at a lower biomass concentration. At pH 7, although a purely acidogenic fermentation was maintained in the unsupplemented medium, low concentrations of acetone and n-butanol were produced when the glucose content of the medium was increased (to 4% [wt/vol]). Substantial solvent concentrations were, however, obtained at pH 7 in the 2% glucose medium supplemented with high concentrations of acetate plus butyrate (100 mM each, supplied as their potassium salts). Thus, C. acetobutylicum NCIB 8052, like C. beijerinckii VPI 13436, is able to produce solvents at neutral pH, although good yields are obtained only when adequately high concentrations of acetate and butyrate are supplied. Supplementation of the glucose minimal medium with propionate (20 mM) at pH 5 led to the production of some n-propanol as well as acetone and n-butanol; the final culture medium was virtually acid free. At pH 7, supplementation with propionate (150 mM) again led to the formation of n-propanol but also provoked production of some acetone and n-butanol, although in considerably smaller amounts than were obtained when the same basal medium had been fortified with acetate and butyrate at pH 7.     

Effect of prostaglandins on ornithine decarboxylase activity in the testis of immature rat

Intratesticular injection of prostaglandin E2 (PGE2) and F2 alpha (PGF2 alpha) caused stimulation of ornithine decarboxylase (ODC) activity in the testis of immature rats. PGE2 at a dose of 10 microgram per testis was maximally effective 2 hours after the injection. Dibutyryl cyclic AMP (cAMP) and 1 methyl, 3-isobutyl xanthine (MIX), a phosphodiesterase inhibitor, also stimulated ODC activity. Simultaneous injection of PGE2 and FSH or LH caused additional stimulation of ODC activity. Similarly injection of PGE2 in addition to cAMP or MIX also caused increased stimulation of ODC. Indomethacin (IM, 60 microgram/testis) inhibited LH, FSH or cAMP induced ODC activity. However, IM at the same dose inhibited the synthesis of total proteins. These results suggest that PGE2 and PGF2 alpha stimulate the activity of ODC. The action of prostaglandins may be independent of the action of gonadotropic hormones. cAMP appears to mediate the action of prostaglandins in the testis of rat.     

REGULATION OF A GLUCOSE TRANSPORT SYSTEM IN STICHOCOCCUS BACILLARIS1

Glucose and related non-metabolizable analogs were transported into cells of Stichococcus bacillaris Naeg. By a specific and active transport system. Glucose transport capacity was stimulated eight-fold by incubation in medium of low osmotic potential (0.09 osM). Stimulation occurred over 24 h in the dark and over 72 h in low osmotic medium. Inhibition of protein synthesis prevented any transport, stimulation from occurring. Kinetic studies revealed that the stimulation caused an increase in Ike maximal velocity of transport and did not affect the half-saturation constant for transport. It was concluded that incubation of cells in the dark or in low osmolar medium induces a synthesis of the transport system. The glucose analog 2-deoxy-D-glucose was only phosphorylated to a limited extent upon entry into the cells, and the free sugar accumulated linearly in dark pre-incubated tells for a period of at least six minutes to reach an intracellular/extracellular concentration ratio of almost 300. Glucose, in contrast, was rapid h converted to sucrose and other cell constituents. Cells incubated 24 h with, glucose or 6-deoxy-D-glucose did not exhibit any altered transport system activity. Cells incubated 24 h with 7 mM dibutyryl cAMP exhibited a 2.5-fold stimulation of transport activity. No stimulation was observed in cells treated only 30 min with dibutyryl cAMP.     

Effects of dexamethasone and dibutyryl cyclic AMP on polyamine synthesizing enzymes in mouse lymphoma cells

S49.1 Lymphoma cells were arrested in G₁ phase of the cell cycle when treated with either 1 μM dexamethasone (Dex) or 0.5 mM N⁶, O²-dibutyryl cyclic adenosine 3′ :5′ -monophosphate (Bt₂cAMP) plus 0.2 mM theophylline. However, the two agents had markedly different effects on aspects of polyamine and cyclic nucleotide metabolism within the arrested cells. Bt₂cAMP had an early and pronounced inhibitory effect on ornithine decarboxylase (ODC) activity causing a decrease to 40% of control within 1 h. However, there was no significant inhibition of ODC activity in the Dex-treated cells until 4 h of exposure, at which time ODC activity was reduced to approximately 60% of the control value. Sadenosyl-L-methionine decarboxylase (SAMD) activity was reduced by both agents, Bt₂cAMP having the more pronounced inhibitory effect. The activity of SAMD was reduced to 40% of control after 10 h of Dex, whereas Bt₂cAMP reduced the activity to approximately 25% of control within 4 h. Intracellular polyamine pools were decreased rapidly in Dex-treated cells but not in those exposed to Bt₂cAMP. Bt₂cAMP decreased the amount of type I (PK_I) and type II (PK_II) cyclic AMP-dependent protein kinase (cAMP-PK) activity to 30% of control or less within 2 h. In contrast, Dex had very little effect on either PK_I or PK_II until 24 h, when cell viability was affected. The specific activity of both PK_I and PK_II remained significantly decreased in cells exposed to Bt₂cAMP for 6 h and then resuspended in fresh medium. The rapid decrease in ODC activity in response to Bt₂cAMP and the slow recovery after washout may be due to the marked decreases in total PK_I and PK_II activities. Dex, which had no effect on PK_I and PK_II specific activities, only slowly inhibited ODC activity and recovery of enzyme activity was rapid upon resuspension in fresh medium. These data further stress the importance of the maintenance of the cellular protein kinase pools in the regulation of the recovery time to growth inhibition in response to naturally occurring steroids and second messengers.     

Cyclic AMP and serum arrest of the mitotic activity of human diploid fibroblasts.

Mitotic activity in confluent cultures of human diploid fibroblasts was arrested by the reduction of the serum concentration of the incubation medium to 0.5% or by the addition of 0.5 mM 6-N, 2'-O-dibutyryl-adenosine 3':5'-cyclic monophosphate (db cAMP). Under either of these conditions, cultures maintained a constant cell number for 14 days; cultures continuously exposed to medium containing 10% serum doubled their cell number during this 14-day period. The protein cotent per cell decreased by 20% when cells were maintained with 0.5% serum whereas that of cells exposed to db cAMP remained constant. Ultrastructural studies revealed that cells exposed to db cAMP exhibited a morphology typical of cells cultures with 10% serum alone, whereas cells incubated with 0.5% serum showed the ultrastructural changes in mitochondria, endoplasmic reticulum and Golgi complex previously identified with low-serum arrest. Cellular adenosine 3':5'-cyclic monophosphate (cAMP) levels remained constant during the 7-day growth period in which confluency was attained, as well as during the 14-day arrested period with 0.5% serum. These results indicated that the mitotic inhibition induced by reducing the serum concentration of the incubation medium was not mediated by increased intracellular levels of cAMP and differed from that induced by the addition of exogenous db cAMP.     

Role of cAMP in modulating intrafollicular progesterone levels and oocyte maturation in amphibians (Rana pipiens)

The role of cAMP in regulating follicular progesterone levels and oocyte maturation was investigated following in vitro culture of amphibian (Rana pipiens) ovarian follicles. Intrafollicular levels of cAMP were manipulated with the use of a stimulator of cAMP synthesis (forskolin) or by exogenous addition of cAMP alone or either of these in combination with an inhibitor of cAMP catabolism (3-isobutyl-1-methyl xanthine, IBMX). Follicular progesterone content was determined by RIA and oocyte maturation was assessed cytologically. In the presence of increasing doses of forskolin (0-3 microM), cAMP (0-3 mM), or dibutyryl cAMP (dbcAMP, 0-2.5 mM) increasing but low levels of progesterone were detected. Increasing doses of IBMX (0-0.09 mM) alone had no significant effect on follicular steroid content. Exogenous cAMP, dbcAMP, or IBMX (0.09 mM) suppressed hormone-induced oocyte maturation. Simultaneous exposure of follicles to increasing doses of both forskolin (0-3 microM) and IBMX (0-0.09 mM) markedly increased intrafollicular progesterone levels to those produced by frog pituitary homogenate (FPH). A marked increase in progesterone levels also occurred when follicles were exposed to exogenous cAMP (3 mM) and IBMX (0.09 mM). These results indicate that exogenous cAMP is incorporated by follicle cells and that forskolin effects are mediated through cAMP. Changes in follicular progesterone levels (increase and decrease) over time following FPH or cAMP manipulation (cAMP + IBMX or forskolin + IBMX) were essentially identical. In contrast to cAMP, cGMP was inactive in inhibiting hormone induced GVBD or stimulating follicular progesterone accumulation. Elevation of follicular and medium levels of progesterone resulting from FPH or cAMP stimulation required the presence of the somatic follicular cells. The decrease in follicular progesterone levels with prolonged culture was not associated with a corresponding increase in progesterone levels in the medium. The decrease in follicular progesterone levels appears to reflect steroid catabolism rather than loss of steroid to the culture medium. The results suggest that the level of intracellular cAMP in the follicle cells is modulated by the relative activity of the adenylate cyclase system and phosphodiesterase and that FPH can affect both components. Thus, intracellular levels of cAMP play a key role in regulating follicular progesterone levels and FPH action on the follicle cells. The steroidogenic capacity of follicle cells can be manipulated independently of FPH stimulation.