RESUMO
L-Arginine and L-ornithine stimulate insulin release from pancreatic islets exposed to D-glucose. This coincides with an increased outflow of 86Rb and 45Ca from prelabelled islets and an increased net uptake of 45Ca by the islets. In the presence of D-glucose, L-lysine stimulates insulin secretion to the same extent as L-arginine or L-ornithine, but the hormonal release is not further enhanced by combinations of these cationic amino acids. L-Arginine or L-ornithine failed to enhance insulin release evoked by either L-leucine or 2-ketoisocaproate. The inhibitor of ornithine decarboxylase D,L-alpha-difluoromethyl ornithine failed to affect the metabolism and insulinotropic action of D-glucose in pancreatic islets, and only caused a partial inhibition of the secretory response to either L-arginine or L-ornithine. The latter amino acids inhibited modestly but significantly D-glucose utilization and oxidation by pancreatic islets. These and complementary findings suggest that the secretory response to L-arginine and L-ornithine is not attributable to any major change in the overall oxidative catabolism of nutrients, but involves mainly a biophysical component, such as the depolarization of the plasma membrane by these cationic amino acids.
Assuntos
Arginina/farmacologia , Insulina/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ornitina/farmacologia , Animais , Arginina/metabolismo , Cálcio/metabolismo , Relação Dose-Resposta a Droga , Eflornitina/farmacologia , Glucose/farmacologia , Inositol/metabolismo , Secreção de Insulina , Ilhotas Pancreáticas/metabolismo , Lisina/farmacologia , Ornitina/metabolismo , Oxirredução , Poliaminas/biossíntese , RatosRESUMO
Exogenous L-arginine and L-ornithine rapidly accumulate in rat pancreatic islets. L-Arginine is converted to L-ornithine and urea. Endogenous or exogenous L-ornithine generates di- and polyamines, the putrescine turnover being faster than that of spermidine and spermine. However, the major pathway for L-ornithine metabolism consists of its transamination to L-glutamaldehyde and further conversion to L-glutamate. The amines and L-glutamate derived from exogenous L-ornithine are incorporated into islet proteins at the intervention of transglutaminase and cycloheximide-sensitive biosynthetic processes, respectively. These findings suggest the hypothesis that the insulinotropic action of L-arginine and L-ornithine could somehow be related to the metabolism of these cationic amino acids in islet cells.
Assuntos
Arginina/metabolismo , Insulina/metabolismo , Ilhotas Pancreáticas/metabolismo , Ornitina/metabolismo , Animais , Arginase/metabolismo , Arginina/farmacologia , Glucose/farmacologia , Glutamatos/metabolismo , Ilhotas Pancreáticas/efeitos dos fármacos , Ilhotas Pancreáticas/enzimologia , Modelos Biológicos , Ornitina/análogos & derivados , Ornitina/farmacologia , Oxirredução , Poliaminas/biossíntese , Ratos , Ureia/biossínteseRESUMO
Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of tissues and cell types. This investigation explores the response of murine embryonic palate mesenchymal (MEPM) cells to epidermal growth factor (EGF) in terms of biosynthesis of putrescine and its transport across the plasma membrane and tests the hypothesis that polyamine transport can serve as an alternative mechanism (other than biosynthesis) for elevating intracellular polyamines during stimulation of MEPM cellular proliferation. MEPM cells treated with EGF were stimulated to proliferate and showed a dose- and time-dependent stimulation of ornithine decarboxylase (ODC) which was maximal at 4-6 hours. EGF also stimulated the initial rate of putrescine transport in a dose- and time-dependent manner. This stimulation was found to be maximal 3 hours after treatment and specific for the putrescine transport system. The kinetic parameters of putrescine transport shifted from 2.52 microM (Km) and 23.6 nmol/mg protein/15 minutes (Vmax) in nonstimulated cells to 4.48 microM (Km) and 39.8 nmol/mg protein/15 minutes (Vmax) in EGF-treated cells. This kinetic shift did not require de novo protein or RNA synthesis, as cycloheximide (10 micrograms/ml) and actinomycin D (50 micrograms/ml) had little effect on the ability of EGF to stimulate the initial rate of putrescine uptake. The rate of transport, however, was found to be inversely related to cell density. The addition of exogenous putrescine concomitantly with EGF blocked the induction of ODC, while in the presence of difluoromethylornithine (DFMO) (irreversible inhibitor of ODC) the initial rate of putrescine transport remained elevated throughout the time course studied. This stimulation of putrescine uptake caused by polyamine deprivation was reversed by exogenous putrescine and Ca++ while alpha-aminoisobutyric acid (AIB) further stimulated the rate of uptake. EGF's ability to stimulate cellular DNA synthesis was inhibited by DFMO. If DFMO-treated cells were stimulated with EGF in the presence of exogenous putrescine, this stimulatory effect was preserved. These studies indicate that the rate of polyamine transportation is highly responsive to a signal which initiates biosynthesis of polyamines. Further, this transportation system provides a compensatory mechanism allowing the cell to increase intracellular levels of polyamines when environmental conditions inhibit biosynthesis or when polyamines are abundant.
Assuntos
Fator de Crescimento Epidérmico/fisiologia , Mesoderma/citologia , Poliaminas/biossíntese , Animais , Divisão Celular/efeitos dos fármacos , Cicloeximida/farmacologia , DNA/biossíntese , Dactinomicina/farmacologia , Relação Dose-Resposta a Droga , Eflornitina/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Feminino , Masculino , Mesoderma/enzimologia , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos , Ornitina Descarboxilase/metabolismo , Poliaminas/metabolismo , Poliaminas/farmacocinética , Putrescina/biossíntese , Putrescina/farmacocinética , Putrescina/farmacologia , Fatores de TempoRESUMO
1. Intestinal epithelial cell proliferation was measured in rats maintained on total parenteral nutrition (TPN), in TPN rats given 300 micrograms of recombinant human epidermal growth factor (urogastrone-epidermal growth factor, URO-EGF) day-1 kg-1, and in further groups given URO-EGF and difluoromethylornithine (DFMO), an inhibitor of the enzyme ornithine decarboxylase (ODC). 2. URO-EGF significantly increased intestinal cell proliferation throughout the gastrointestinal tract. The proliferative response of the colon was particularly pronounced. 3. DFMO reduced the proliferative effect of urogastrone in the stomach and small intestine. DFMO also reduced URO-EGF-stimulated intestinal cell proliferation in the colon, but to a lesser extent. 4. It is concluded that ODC is essential for effecting the proliferative response of the stomach and small intestine to URO-EGF, but this role may be less important in the colon.
Assuntos
Fator de Crescimento Epidérmico/fisiologia , Intestinos/fisiologia , Poliaminas/biossíntese , Animais , Divisão Celular/efeitos dos fármacos , Colo/efeitos dos fármacos , Colo/fisiologia , Eflornitina/farmacologia , Células Epiteliais , Epitélio/fisiologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/fisiologia , Masculino , Ratos , Ratos EndogâmicosRESUMO
Treatment of HeLa cells with the polyamine biosynthesis inhibitors, alpha-difluoromethylornithine (DFMO) or methylglyoxal bis(guanylhydrazone) (MGBG), results in, depending on the conditions, partial or complete depletion of the cellular polyamines: putrescine, spermidine and spermine. In this compromised state cells exhibited a distinct deficiency in repair of X-ray-induced DNA strand breaks. The half-time for return of normal DNA sedimentation following 1.6 Gy was 9.5 min for untreated control cells and 22, 32 and 50 min for cells treated with MGBG, DFMO + MGBG and DFMO, respectively. Normal repair kinetics were restored to these cells upon a short incubation in media containing all three polyamines. The rapid early phase of repair following higher X-ray doses (16 Gy) was also delayed in polyamine-depleted cells but later repair occurring 1-4 h post-irradiation, representing chromatin reconstitution, was apparently normal.
Assuntos
Reparo do DNA , DNA de Neoplasias/efeitos da radiação , Poliaminas/biossíntese , Eflornitina/farmacologia , Células HeLa , Humanos , Mitoguazona/farmacologia , Putrescina/biossíntese , Espermidina/biossíntese , Espermina/biossínteseRESUMO
The diamine, putrescine, and polyamines, spermidine and spermine, are low molecular weight organic cations with documented regulatory roles in cell growth and differentiation. Multiple lines of direct and indirect evidence suggest that these organic cations also may function in stimulus-response coupling processes regulating cellular injury and repair. For example, recent studies in monocrotaline-treated rats, hyperoxic rats, and in cultured pulmonary endothelial cells suggest that polyamines regulate pulmonary endothelial integrity and may thus participate in development and/or regression of acute edematous lung injury. To determine if the polyamines are involved in a well-characterized animal model of acute lung injury, the present experiments assessed the relation between changes in polyamine synthesis and development of edema in lungs from rats treated with alpha-naphthylthiourea (ANTU). ANTU caused dose- and time-dependent increases in the lung activity of the initial and rate-limiting enzyme in polyamine biosynthesis, ornithine decarboxylase (ODC) and in the lung contents of the polyamines putrescine, spermidine, and spermine. ANTU also caused dose- and time-dependent increases in the lung wet-to-dry weight ratio indicative of pulmonary edema formation. Changes in lung polyamine biosyntheic activity after ANTU did not relate temporally to changes in the lung wet-to-dry weight ratio: ODC activity was depressed during the 3-h period immediately following ANTU administration, a period when the wet-to-dry weight ratio was increasing, and markedly elevated at 18 h after ANTU administration when the wet-to-dry weight ratio had returned to control levels. Pretreatment of the animals with alpha-difluoromethylornithine, a highly specific inhibitor of ODC, failed to attenuate ANTU-induced increases in lung wet-to-dry weight ratio. These observations indicate polyamine synthesis is enhanced in rat lungs with ANTU-induced pulmonary edema but, unlike certain other models of lung injury and pulmonary edema, accumulation of polyamines probably is not essential for development of edematous lung injury. It is conceivable that in this animal model polyamines play a role in lung repair processes or some longer-term consequence of lung injury.
Assuntos
Pulmão/efeitos dos fármacos , Ornitina Descarboxilase/metabolismo , Poliaminas/biossíntese , Edema Pulmonar/induzido quimicamente , Tioureia/análogos & derivados , Animais , Eflornitina/farmacologia , Pulmão/enzimologia , Pulmão/patologia , Masculino , Modelos Biológicos , Tamanho do Órgão , Inibidores da Ornitina Descarboxilase , Poliaminas/fisiologia , Edema Pulmonar/enzimologia , Ratos , Ratos Endogâmicos , Tioureia/toxicidadeRESUMO
When bovine small lymphocytes stimulated by concanavalin A were treated with inhibitors (alpha-difluoromethylornithine (5 mM) and ethylglyoxal bis(guanylhydrazone) (100 microM)] of polyamine biosynthesis, swelling of the Golgi apparatus was observed. This was accompanied by decreases in the amount of the Golgi apparatus and of the specific activity of galactosyltransferase. Both spermidine and spermine, at physiological concentrations, stimulated galactosyltransferase activity 2-3-fold. When mice were treated with these inhibitors, the following changes were observed in the epithelial cells of small intestine: swelling of the Golgi apparatus; decrease in the amount of the Golgi apparatus; and decrease of galactosyltransferase activity.
Assuntos
Galactosiltransferases/antagonistas & inibidores , Complexo de Golgi/efeitos dos fármacos , Intestino Delgado/enzimologia , Linfócitos/enzimologia , Poliaminas/biossíntese , Animais , Bovinos , Células Cultivadas , Concanavalina A/farmacologia , Eflornitina/farmacologia , Epitélio/enzimologia , Complexo de Golgi/ultraestrutura , Mucosa Intestinal/enzimologia , Intestino Delgado/efeitos dos fármacos , Intestino Delgado/ultraestrutura , Ativação Linfocitária/efeitos dos fármacos , Linfócitos/efeitos dos fármacos , Linfócitos/ultraestrutura , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica , Mitoguazona/análogos & derivados , Mitoguazona/farmacologia , Espermidina/farmacologia , Espermina/farmacologiaAssuntos
Poliaminas/biossíntese , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Parede Celular/metabolismo , Centrifugação com Gradiente de Concentração , DNA Fúngico/biossíntese , Proteínas Fúngicas/biossíntese , Mutação , RNA Fúngico/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/ultraestruturaRESUMO
Inhibition of polyamine synthesis by 2-difluoromethylornithine (DFMO) treatment had no apparent effect on the initial manifestation of Epstein-Barr virus (EBV) infection in human B lymphocytes, because the expression of EBV-encoded nuclear antigens (EBNA) occurred normally. However, many subsequent steps in the transformation process were inhibited by DFMO treatment. These include cellular DNA synthesis and immunoglobulin (IgM, IgG and IgA) synthesis and secretion. Consequently, DFMO treatment blocked the progression of the transformation process of EBV-infected B lymphocytes. EBV-carrying marmoset B lymphocytes (B95-8 cells) were also blocked in their DNA synthesis when treated with DFMO. At variance with other DNA synthesis inhibitors, which induce virus production very effectively in B95-8 cells, DFMO caused no increase in the number of cells expressing the early antigens associated with the lytic cycle.
Assuntos
Antígenos Virais/genética , Linfócitos B/efeitos dos fármacos , Transformação Celular Viral , Replicação do DNA/efeitos dos fármacos , Eflornitina/farmacologia , Herpesvirus Humano 4/genética , Poliaminas/biossíntese , Linfócitos B/citologia , Linfócitos B/imunologia , Linhagem Celular , Células Cultivadas , Criança , Antígenos Nucleares do Vírus Epstein-Barr , Herpesvirus Humano 4/imunologia , Humanos , Imunoglobulinas/biossíntese , Poliaminas/antagonistas & inibidoresRESUMO
Contrary to previous findings, ornithine decarboxylase (ODC) was stabilized by treatment of cells with DL-alpha-difluoromethylornithine, an enzyme-activated irreversible inhibitor of ODC. Both this inhibitor and cyclohexylamine, a spermidine synthase inhibitor known to stabilize ODC, caused decreases in the antizyme/ODC ratio by increasing ODC content and conversely decreasing antizyme content. The relationship between cellular polyamine levels and antizyme content indicated that spermidine is the most important polyamine for antizyme induction. These results suggest that antizyme is involved in the mechanism underlying the stabilization of ODC by inhibitors of polyamine synthesis and support the hypothesis that cellular polyamines regulate ODC degradation via antizyme.
Assuntos
Ornitina Descarboxilase/metabolismo , Poliaminas/biossíntese , Biossíntese de Proteínas , Proteínas , Animais , Linhagem Celular , Cicloexilaminas/farmacologia , Eflornitina/farmacologia , Indução Enzimática/efeitos dos fármacos , Estabilidade Enzimática , Humanos , Inibidores da Ornitina Descarboxilase , Poliaminas/farmacologia , Espermidina/farmacologiaRESUMO
A bisbenzyl polyamine analogue, MDL 27695, rapidly repressed ornithine decarboxylase (ODC) and S-adenosyl-L-methionine decarboxylase (AdoMet DC) activity and depleted polyamines in rat hepatoma (HTC) cells. The suppression of ODC and AdoMet DC activity was temporally related to metabolism of MDL 27695 by intracellular polyamine oxidase to a free-amine analogue, MDL 26752, which, when added directly to HTC cells, suppressed ODC activity and polyamine biosynthesis more rapidly and to a greater extent than did the bisbenzyl analogue. The ODC suppression caused by MDL 27695 was completely blocked by the addition of a polyamine oxidase inhibitor to the HTC-cell cultures along with MDL 27695. These data suggested that MDL 27695 acted as a prodrug, with metabolism to an active analogue being necessary for ODC repression to occur. MDL 27695 and MDL 26752 completely abolished division of HTC cells when added to cultures at 1 microM. This established them as being among the most potent antiproliferative polyamine analogues yet described. MDL 27695 has also been shown to possess significant antimalarial effects both in vitro and in vivo, and it is possible that the marked suppression of polyamine biosynthesis described herein may contribute to its anti-malarial effects as well as its antiproliferative effects in mammalian cells.
Assuntos
Neoplasias Hepáticas Experimentais/metabolismo , Poliaminas/biossíntese , Poliaminas/farmacologia , Adenosilmetionina Descarboxilase/metabolismo , Animais , Dexametasona/farmacologia , Ornitina Descarboxilase/metabolismo , Ratos , Espermina/análogos & derivados , Espermina/farmacologia , Células Tumorais Cultivadas/efeitos dos fármacosRESUMO
A review of recent papers dealing with the role of protein kinase C (PK C) in regulation of normal and tumour cell proliferation contains the data on the interaction of PK C with the tumour promoters and oncoproteins as well as the data which characterize changes of the PK C activity in tumour cells and during carcinogenesis. Probable mechanisms of the PK C influence on the functioning of the growth factors receptors are discussed. It is proposed that one of the PK C functions which is substantial for induction of the mitogenic signal is the polyamine synthesis stimulation. Participation of PK C in the tumour promotion may play a key role in multistep processes of the carcinogenesis.
Assuntos
Neoplasias Experimentais/fisiopatologia , Proteína Quinase C/fisiologia , Animais , Carcinógenos/farmacologia , Divisão Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Proteínas de Neoplasias/fisiologia , Neoplasias Experimentais/enzimologia , Poliaminas/biossíntese , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/fisiologiaRESUMO
An early increase in ornithine decarboxylase (ODC) activity and polyamine levels in rat cerebral capillaries was previously implicated in the mediation of blood-brain barrier (BBB) breakdown in cold-injured brain. A time course study in rat cerebrum indicated that cold injury evokes a biphasic increase in ODC activity and polyamine levels in perilesional cortex. ODC activity rose sharply (fourfold) within 1 min, remained elevated for 5 min, and then returned to the basal level by 10 min. A transient rise in polyamine concentration followed in the rank order of putrescine greater than spermidine greater than spermine. A secondary rise in ODC activity commenced in perilesional tissue at 2-6 h and peaked (8.8-fold) at 48 h. Major increases in the content of putrescine (330%), spermidine (103%), and spermine (50%) developed at 48-72 h. alpha-Difluoromethylornithine (DFMO), a specific irreversible inhibitor of ODC, suppressed the evoked increase in ODC activity and abolished the associated increase in content of polyamines, findings indicating that the accumulation of polyamines in cryoinjured brain reflects enhanced synthesis resulting from an ODC-mediated increase in putrescine content. Cycloheximide and actinomycin D were without effect on the early increase in ODC activity but inhibited the delayed increase in ODC activity, an observation suggesting that the initial increase in activity reflects an activation of a cryptic ODC via a posttranslational process, whereas the delayed increase in activity results from ODC synthesis mainly under transcriptional control.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Aspirina/farmacologia , Barreira Hematoencefálica , Lesões Encefálicas/enzimologia , Temperatura Baixa , Dexametasona/farmacologia , Ornitina Descarboxilase/metabolismo , Poliaminas/biossíntese , Verapamil/farmacologia , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Lesões Encefálicas/metabolismo , Cicloeximida/farmacologia , Dactinomicina/farmacologia , Eflornitina/farmacologia , Ratos , Ratos EndogâmicosRESUMO
Diamino butanone (DAB), a competitive inhibitor of ornithine decarboxylase (ODC) a key enzyme in polyamine biosynthesis, inhibited the yeast to hyphae transition in Mucor rouxii, induced by transfer from anaerobiosis to aerobiosis, but not the opposite phenomenon. Addition of DAB to anaerobic yeast cells brought about a decrease in ODC and polyamine levels. In these conditions, the aerobic shift produced only a weak increase in ODC activity and no change in polyamine levels. DAB also blocked phorogenesis in M. rouxii and in Phycomyces blakesleeanus. At the effective concentrations DAB did not affect cell growth of either fungus. It is suggested that low, constant levels of ODC and polyamines are necessary for cell growth, and that high transient levels are required during the differentiative steps. DAB, at the concentrations used, affects this last process, but does not interfere with the maintenance level of polyamines.
Assuntos
Mucorales/efeitos dos fármacos , Putrescina/análogos & derivados , Diferenciação Celular/efeitos dos fármacos , Mucor/efeitos dos fármacos , Mucor/crescimento & desenvolvimento , Mucor/metabolismo , Mucorales/crescimento & desenvolvimento , Mucorales/metabolismo , Inibidores da Ornitina Descarboxilase , Phycomyces/efeitos dos fármacos , Phycomyces/crescimento & desenvolvimento , Phycomyces/metabolismo , Poliaminas/biossíntese , Putrescina/farmacologiaRESUMO
The purpose of this study was to confirm the photoprotective effect on skin of vitamins A and E, due to inhibition of polyamine synthesis and production of free radicals. These variables were measured in the lumbar epidermis of the female hairless mouse subjected to UVA + B irradiation. Polyamines were assayed in epidermal homogenate by HPLC, and production of oxygenated free radicals was determined by spectrofluorometric assay of malonyl dialdehyde. It was determined that butyl-hydroxy-toluene and vitamin E inhibited production of free radicals (56% and 60%, respectively) and caused a significant reduction in polyamine biosynthesis (P less than 0.01), whereas the inhibitory effect of malonyl dialdehyde induced by vitamin A (30%) had no associated effect on polyamine metabolism.
Assuntos
Epiderme/metabolismo , Oxigênio/metabolismo , Poliaminas/biossíntese , Protetores contra Radiação/farmacologia , Vitamina A/farmacologia , Vitamina E/farmacologia , Animais , Hidroxitolueno Butilado/administração & dosagem , Hidroxitolueno Butilado/farmacologia , Carotenoides/administração & dosagem , Carotenoides/farmacologia , Epiderme/efeitos dos fármacos , Epiderme/efeitos da radiação , Feminino , Radicais Livres , Humanos , Malondialdeído/biossíntese , Camundongos , Camundongos Pelados , Raios Ultravioleta , beta CarotenoRESUMO
Polyamines are essential for cell growth and differentiation. Trifluoperazine (TFP) is a potent, competitive inhibitor of the calcium-calmodulin complex. TFP, when given to rats after partial hepatectomy, causes a significant decrease in DNA synthesis. The purpose of this study was to examine the effects of TFP on polyamine biosynthesis and on liver regeneration after partial hepatectomy. TFP (60 mg/kg, bodyweight) or saline control was administered to 80 male Sprague-Dawley rats 2 h before, 2 h after, or at the time of hepatectomy. Polyamines (putrescine, spermidine and spermine) were measured at the time of hepatectomy, and at 6, 24, 48, and 72 h after hepatectomy. TFP, when it was administered either 2 h before or at time of hepatectomy, blocked increases in putrescine that are seen normally at 6 h after hepatectomy. When TFP was given at the time of partial hepatectomy, putrescine was increased at 24 h, and then returned to normal levels at 72 h. Spermidine was inhibited at 24 h, but not at 48 and 72 h. Spermine was not significantly altered at any time. The administration of TFP 2 h after hepatectomy did not significantly alter concentrations of polyamines. The weight of regenerating liver was decreased by TFP at 48 h (23 per cent) and 72 h (22 per cent) after hepatectomy. These findings provide evidence that the calcium-calmodulin complex is required for the synthesis of liver polyamines before liver regeneration can proceed.
Assuntos
Calmodulina/antagonistas & inibidores , Regeneração Hepática/efeitos dos fármacos , Fígado/metabolismo , Poliaminas/biossíntese , Trifluoperazina/farmacologia , Animais , Hepatectomia , Fígado/efeitos dos fármacos , Masculino , Putrescina/biossíntese , Ratos , Ratos Endogâmicos , Espermidina/biossíntese , Espermina/biossíntese , Fatores de TempoRESUMO
Growth of Trichomonas vaginalis in a semi-defined medium was inhibited by 5 mM DL-alpha-difluoromethylornithine (DFMO). Using high pressure liquid chromatography (HPLC) analysis, putrescine and cadaverine levels were found to be 90 and 100% reduced, respectively after 120 h exposure, whilst spermidine and spermine levels were unchanged. Putrescine (40 microM) and cadaverine (6 microM) were detected in the spent media from control cultures. Neither of these diamines was detected in spent media from 72 h DFMO-treated cultures. Changes in intracellular levels of amine precursors were also determined by HPLC. There was a transient increase in ornithine to 39 nmol (mg protein)-1 at 48 h in the DFMO-treated cells while it remained undetectable in control cells throughout the experiment. Arginine and citrulline levels remained high, decreasing to control levels only after 72 h. Only spermine (1 mM) rescued DFMO-treated cells, and this is discussed with respect to the presence of a putative spermine-specific oxidase designated by its sensitivity to aminoguanidine. Aerobic incubation of growing (normal) cells with [14C]spermine resulted in the production of an unknown metabolite (19% of total label), whose content was reduced to 5% under anaerobic conditions. Decarboxylated S-adenosylmethionine remained undetectable in DFMO-treated cells, and the methylation index (ratio of S-adenosylmethionine to S-adenosylhomocysteine) did not change from the control value of 9.3. Ornithine decarboxylase, S-adenosylmethionine synthetase, S-adenosylmethionine:L-homocysteine methyltransferase, and S-adenosylhomocysteine hydrolase enzyme activities were detected. However, S-adenosylmethionine decarboxylase, spermidine synthase or spermine synthase were not detected. These findings are discussed with reference to the arginine dihydrolase pathway whose end products are putrescine and ATP.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Eflornitina/farmacologia , Poliaminas/biossíntese , Trichomonas vaginalis/efeitos dos fármacos , Adenosina/metabolismo , Animais , Cadaverina/biossíntese , Cadaverina/metabolismo , Cromatografia Líquida de Alta Pressão , Meios de Cultura , Ornitina Descarboxilase/metabolismo , Polarografia , Poliaminas/metabolismo , Putrescina/biossíntese , Putrescina/metabolismo , S-Adenosilmetionina/metabolismo , Espermidina/biossíntese , Espermidina/metabolismo , Espermina/biossíntese , Espermina/metabolismo , Trichomonas vaginalis/enzimologiaRESUMO
Ornithine decarboxylase, the rate-limiting enzyme in the polyamine biosynthetic pathway has been purified 7,600 fold from Plasmodium falciparum by affinity chromatography on a pyridoxamine phosphate column. The partially purified enzyme was specifically tagged with radioactive DL-alpha-difluoromethylornithine and subjected to polyacrylamide gel electrophoresis under denaturing conditions. A major protein band of 49 kilodalton was obtained while with the purified mouse enzyme, a typical 53 kilodalton band, was observed. The catalytic activity of parasite enzyme was dependent on pyridoxal 5'-phosphate and was optimal at pH 8.0. The apparent Michaelis constant for L-ornithine was 52 microM. DL-alpha-difluoromethylornithine efficiently and irreversibly inhibited ornithine decarboxylase activity from P. falciparum grown in vitro or Plasmodium berghei grown in vivo. The Ki of the human malarial enzyme for this inhibitor was 16 microM. Ornithine decarboxylase activity in P. falciparum cultures was rapidly lost upon exposure to the direct product, putrescine. Despite the profound inhibition of protein synthesis with cycloheximide in vitro, parasite enzyme activity was only slightly reduced by 75 min of treatment, suggesting a relatively long half-life for the malarial enzyme. Ornithine decarboxylase activity from P. falciparum and P. berghei was not eliminated by antiserum prepared against purified mouse enzyme. Furthermore, RNA or DNA extracted from P. falciparum failed to hybridize to a mouse ornithine decarboxylase cDNA probe. These results suggest that ODC from P. falciparum bears some structural differences as compared to the mammalian enzyme.
Assuntos
Ornitina Descarboxilase , Plasmodium falciparum/enzimologia , Animais , Cromatografia de Afinidade , DNA , Eflornitina/metabolismo , Células Eucarióticas/enzimologia , Humanos , Soros Imunes , Mamíferos , Hibridização de Ácido Nucleico , Ornitina Descarboxilase/imunologia , Ornitina Descarboxilase/isolamento & purificação , Ornitina Descarboxilase/metabolismo , Inibidores da Ornitina Descarboxilase , Plasmodium berghei/enzimologia , Poliaminas/biossíntese , RNA MensageiroRESUMO
Ornithine decarboxylase (ODC) is a rate-limiting enzyme in polyamine synthesis, and polyamines are required for cell growth. As an approach to clarifying the mechanism of action IL-1, the effects of IL-1 on ODC activity were examined in various cell lines whose proliferation was either suppressed or enhanced by IL-1. The proliferation of all cell types used in these experiments was markedly suppressed by a specific ODC inhibitor, alpha-difluoromethyl ornithine (DFMO), substantiating the crucial role of ODC activity for cell proliferation. ODC activity also was considerably suppressed by IL-1 in those cells on which IL-1 exerts an antiproliferative effect, such as a human melanoma cell line (A375) and malignant human mammary cell lines (MCF-7 and T-47D). On the other hand, ODC activity was stimulated in cells that are stimulated to proliferate in response to IL-1, such as a mouse helper T cell line (D10.G4.1), a NK cell-like cell line (YT), and a human glioblastoma cell line (U373 MG). The effect of IL-1 on ODC activity preceded and directly correlated in a dose-dependent manner with its effect on DNA synthesis. Furthermore, putrescine, a product of the ODC reaction and a precursor of polyamines, was able to overcome most, but not all, the antiproliferative action of IL-1 in A375 melanoma cells, which were the most sensitive to suppression by IL-1. However, putrescine did not reverse the cytostatic effect of IL-1 on MCF-7 and T-47D cell lines. In contrast, putrescine, like IL-1, exhibited some co-mitogenic activity on D10.G4.1 cells. Because the biological activities of TNF and IL-1 show considerable overlap, the effect of TNF on ODC activity also was examined. TNF had an antiproliferative effect on A375 cells and stimulated the proliferation of U373 MG cells. The ODC activity in A375 cells was suppressed by TNF, and the ODC activity in U373 MG cells was stimulated by TNF. Putrescine also partially overcame the inhibitory effect of TNF. These results suggest that the regulation of ODC activity may be a key component in the antiproliferative and proliferative action of IL-1 and TNF in some tumor cell types.
Assuntos
Divisão Celular/efeitos dos fármacos , Interleucina-1/farmacologia , Ornitina Descarboxilase/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Animais , Linhagem Celular , DNA/biossíntese , Eflornitina/farmacologia , Glioma/enzimologia , Glioma/metabolismo , Glioma/patologia , Inibidores do Crescimento/farmacologia , Humanos , Interleucina-1/fisiologia , Melanoma/enzimologia , Melanoma/metabolismo , Melanoma/patologia , Camundongos , Inibidores da Ornitina Descarboxilase , Poliaminas/biossíntese , Putrescina/farmacologia , Linfócitos T Auxiliares-Indutores/efeitos dos fármacos , Linfócitos T Auxiliares-Indutores/enzimologia , Linfócitos T Auxiliares-Indutores/metabolismoRESUMO
The effect of a protein-free diet (PF) or a restricted intake of chow (RI) and subsequent host repletion with total parenteral nutrition (PF-TPN, RI-TPN) on tumor growth and polyamine metabolism of fibrosarcoma-bearing rats was examined. Host weight was significantly reduced by PF and RI. Tumor growth was reduced in malnourished rats with the PF regimen resulting in the greatest decrease. Rats receiving TPN after 14 days of the RI or PF regimens had higher host weight and plasma albumin levels than malnourished rats. Tumor growth during TPN was evaluated as the percent increase and compared with that of the respective malnourished rats. The percent increase for RI-TPN rats was significantly greater although a trend toward an increase was also evident for PF-TPN rats. Tumor ornithine decarboxylase (ODC) activity and putrescine levels were increased for PF rats and decreased for RI rats while tumor ODC activity was consistently increased by TPN. Tumor growth, ODC activity, and putrescine levels were simultaneously increased only for those rats fed the RI regimen prior to TPN. These results show a disparity in tumor ODC activity, putrescine levels, and tumor growth in malnourished rats. The results of this study suggest that the nutritional origin of cachexia influences the response of the tumor to TPN and emphasizes the importance of considering the methods to induce malnutrition in designing therapuetic regimens.