Accumulation Of Putrescine Research Articles

Alterations in intestinal uptake of putrescine and tissue polyamine concentrations in tumor-bearing rats

Intestinal absorption of putrescine and tissue metabolism of polyamines were investigated in rats grafted with the rapidly growing Mat-Lylu prostatic tumor. These animals exhibited a dramatic 21% decrease in weight and protein, but not DNA, content of their intestinal mucosa, relative to healthy rats reared under similarly controlled nutritional conditions. No significant variation in the specific activities of intestinal brush-border membrane enzymes was observed, however, suggesting a comparable differentiation state of intestinal cell exists in both groups. Putrescine uptake by brush-border membrane vesicles prepared from cancerous or healthy rat intestine was a time dependent process at 25°C. Equilibrium uptake was much greater than could be explained by equilibration of the vesicle space with putrescine, indicating that the diamine was bound to membrane sites. Kinetics of putrescine uptake at 2 min revealed that the process involves two components, a saturable Michealis-Menten carrier and passive diffusion. With respect to the kinetic parameters of putrescine transport, no significant changes were observed between the tumor-bearing and the control rats. After correction for nonspecific binding to the membranes, putrescine accumulation at equilibrium (75 min) was concentration-dependent and fit a single-site saturable model. Maximum accumulation of the diamine at equilibrium ( B max) was increased by more than 46% in the cancerous rats relative to the controls, but the dissociation constant ( K d was unchanged. Efflux of putrescine from the vesicles was slightly slower in the tumor-bearing group, but the differences were generally not significant. No change was observed with respect to the specific activity of ornithine decarboxylase and the concentration of polyamines in the intestinal mucosa. In Mat-Lylu grafted rats fed a standard diet supplemented with [ 14C]putrescine, about 19% of body radioactivity was recovered in the tumor within 24 h. This was concomitant with a decrease in t he percentatge of radioactivity retained in the intestinal, renal and hepatic tissues, relative to that retained in the same tissues of healthy rats. Our findings indicate that the presence of the tumor evolves an adaptive response in the small intestine of the rat, involving an increased capacity of the brush-border membrane to accumulate putrescine.

Putrescine and paraquat uptake in human lung slices and isolated type II pneumocytes

Paraquat is accumulated into the lungs of various species by an active uptake system which also appears to mediate the uptake of endogenous polyamines, such as putrescine. The accumulation of putrescine in the human lung has been previously shown to be mainly located in the type II cells. In the present study, we have studied the mutually competitive inhibition of putrescine and paraquat in human lung slices and the inhibition of putrescine by paraquat or cystamine in isolated human type II pneumocytes. Peripheral lung tissue taken from patients undergoing pneumectomy or lobectomy was used. The initial steps of the cell isolation procedure differed from the literature in that the tissue was first sliced in 0.7 mm thick slices, which were washed in phosphate buffered saline without calcium and magnesium (PBS −), followed by incubations with trypsin. The type II cells were purified and isolated by differential adherence on plastic followed by Percoll gradient centrifugation. Uptake was determined 48 hr after cell isolation. The accumulation of radiolabelled putrescine showed saturation kinetics, with the following apparent kinetic parameters: k n 6.7 and 6.2–7.6μM and V max2.7 and 3.0–3.4μmol/g prot/hr for slices and isolated cells, respectively. In the presence of paraquat, putrescine uptake was reduced, in both systems, in a manner compatible with competitive inhibition, with calculated inhibition constants ( k i) of 549–614 and 659–895 μM paraquat for slices and isolated cells, respectively. The accumulation of putrescine in isolated human pneumocytes was strongly reduced in the presence of cystamine, with calculated k i of 3.7 μM cystamine. These data indicate that putrescine, paraquat and cystamine accumulate in the human lung by the same uptake system, but that the affinities for the three substrates differ. The presence of an uptake system for putrescine in cultured human pulmonary type II is probably useful as a functional viability test.

Activation of the Proto-oncogene c-myc and c-fos by c-ras: Involvement of Polyamines

Rat kidney cells infected with a temperature-sensitive mutant of Kirsten sarcoma virus (Ki-MSV ts 371) expressed Ki-Ras at 37°C but not at 42°C. This expression of the oncogene was accompanied by an increase in the activity of ornithine decarboxylase (ODC) and the accumulation of putrescine. Elevation of cellular polyamine content triggered the transcription of c-myc and c-fos. α-Difluoromethylornithine, a specific inhibitor of ODC, prevented the transcription of c-myc in cells grown at 37°C. Putrescine, at physiological concentrations, triggered the transcription of c-myc and c-fos in cells grown at 42°C, when Ki-ras was not expressed. It has been suggested that polyamines participate in a cascade of events leading to the communication between membrane-bound and nuclear oncogene products. These findings may attribute a new function to the naturally occurring polyamines.

Changing root system architecture through inhibition of putrescine and feruloyl putrescine accumulation

Plant roots provide anchorage and absorb the water and minerals necessary for photosynthesis in the aerial parts of the plant. Since plants are sessile organisms, their root systems must forage for resources in heterogeneous soils through differential branching and elongation [(1988) Funct. Ecol. 2, 345-351; (1991) Plant Roots: The Hidden Half, pp. 3-25, Marcel Dekker, NY]. Adaptation to drought, for instance, can be facilitated by increased root growth and penetration. Root systems thus develop as a function of environmental variables and the needs of the plant [(1988) Funct. Ecol. 2, 345-351; (1986) Bot. Gaz. 147, 137-147; (1991) Plant Roots: The Hidden Half, pp. 309-330, Marcel Dekker, NY], We show, in a model system consisting of excised tobacco roots, that both α- dl-difluoromethylornithine (an inhibitor of putrescine biosynthesis) and the rolA gene (from the root-inducing transferred DNA of Agrobacterium rhizogenes) stimulate overall root growth and cause a conversion in the pattern of root system formation, producing a dominant or ‘tap’ root. These morphological changes are correlated with a depression in the accumulation of polyamines and their conjugates.

Regulation of putrescine export in lipopolysaccharide or IFN-gamma-activated murine monocytic-leukemic RAW 264 cells.

The regulation of putrescine/polyamine export out of the cell was investigated during activation of monocytic-leukemic RAW 264 cells with LPS and IFN-gamma. The RAW 264 cells exported putrescine constitutively at a significant rate into the culture medium. This export process appeared to be selective for putrescine in that only a small amount of other polyamines (spermidine and N1-acetylspermidine) was found in the culture medium. LPS and IFN-gamma alone and in combination markedly stimulated putrescine export and nitrite production throughout a 24-h period. The efflux of putrescine but not nitrite was further increased by the addition of ornithine (the amino acid precursor of putrescine) to the culture medium. LPS and ornithine also stimulated the intracellular accumulation of putrescine in primary inflammatory macrophages and the export of putrescine into the peritoneal exudate of the mouse. A detailed comparison of the steady state rates of accumulation of intracellular putrescine/polyamines and the rate of putrescine efflux from the cells constitutively and after LPS, IFN-gamma, and ornithine indicated that a surprisingly large fraction of total polyamine biosynthesis is comprised of exported putrescine. The observed dose-dependent inhibition of putrescine export with the drug verapamil implicated the involvement of a specific membrane transport system sensitive to calcium influx in this process. The data are discussed in regard to the potential involvement of putrescine export in the regulation of intracellular polyamine levels, cell differentiation, and macrophage-mediated cytotoxicity.

Consequences of aberrant ornithine decarboxylase regulation in rat hepatoma cells.

DH23A cells, an alpha-difluoromethylornithine (DFMO)-resistant variant of rat hepatoma tissue culture cells (HTC), contain high levels of very stable ornithine decarboxylase (ODC). In the absence of DFMO, the high ODC activity results in a large accumulation of endogenous putrescine. Concomitant with the putrescine increase is a period of cytostasis and a subsequent loss of viable cells. In contrast, HTC cells with a moderate polyamine content can be maintained in exponential growth. This suggests that a moderate polyamine concentration is necessary for both optimal cell growth and survival. The cytotoxicity observed in the DH23A cells is apparently not due to byproducts of polyamine oxidation or alterations in steady state intracellular pH or free [Ca2+]. It is possible to mimic the effects of high levels of stable ODC by treatment of cells with exogenous putrescine in the presence of DFMO. This suggests that overaccumulation of putrescine is the causative agent in the observed cytotoxicity, although the mechanism is unclear. These data support the hypothesis that downregulation of ODC may be necessary to prevent accumulation of cytotoxic concentrations of the polyamines.

Lithium exerts a time-dependent and tissue-selective attenuation of the dexamethasone-induced polyamine response in rat brain and liver

It has previously been shown that chronic, but not acute, lithium treatment indirectly prevents the dexamethasone-induced increase in brain polyamine-metabolizing enzymes. In the present study we determined the effects of lithium treatment on changes in cellular polyamines, 6 h after dexamethasone challenge (3 mg/kg intraperitoneally). The findings demonstrate that chronic lithium (daily intraperitoneal 2.5 mmol/kg injections for 2 weeks) treatment completely prevents the accumulation of putrescine, in parallel to its prevention of the dexamethasone-induced increase in ornithine decar☐ylase activity. A partial attenuation of this polyamine response was also observed in the liver. Only minor and inconsistent changes were observed in the concentrations of the polyamines, spermidine and spermine. Acute lithium treatment (a single injection at times ranging from 1 to 24 h prior to dexamethasone challenge) did not attenuate the dexamethasone-induced increases in brain putrescine concentration nor in ornithine decar☐ylase activity. It is suggested that prevention of the stress-induced polyamine response in the brain may be an important mechanism through which prophylactic lithium may exert its beneficial effect in manic-depressive illness.

Kanamycin depletes cochlear polyamines in the developing rat

Developing mammals are more sensitive to aminoglycoside antibiotics and other ototoxic agents than adults, with maximum sensitivity occurring during the period of anatomic and functional maturation of the cochlea. For the aminoglycoside antibiotics, the hypersensitive period in rats occurs during the second and third postnatal weeks. Toxicity is initially expressed as outer hair cell (OHC) damage in the high-frequency, basal region of the cochlea. Distortion-product otoacoustic emissions (DPOAEs), physiologic measures of OHC function, are particularly sensitive to aminoglycoside exposure during the period of rapid cochlear physiologic development. Toxicity is characterized by increased DPOAE thresholds and decreased amplitudes. The mechanism of developmental sensitivity to aminoglycosides is unknown. A potential biochemical target of aminoglycosides is the ornithine decarboxylase (ODC)-polyamine pathway. ODC activity is elevated in the developing rat cochlea, aminoglycosides inhibit cochlear ODC in developing rats, and alpha-difluoromethylornithine (a specific ODC inhibitor) impairs development of cochlear function. In the present study we demonstrate an incomplete polyamine response to aminoglycoside damage, characterized by inhibition of the polyamines spermidine and spermine and accumulation of putrescine in the organ of Corti. Aminoglycoside inhibition of polyamine synthesis may mediate developmental ototoxic hypersensitivity by interfering with developmental and repair processes.

Effects of acid stress on polyamine levels, ion efflux, protective enzymes and macromolecular synthesis in cereal leaves

Wheat or oat leaf segments were floated on acid solutions and the changes in several parameters measured. The putrescine content of wheat leaves was 12.8-fold greater at pH 3.5 than at pH 6 after 72 h. The increase in putrescine was accompanied by an increase in the amount of K+ efflux from the leaf tissue to the external solution. The activities of superoxide dismutase and catalase of wheat leaves at pH 3.5 decreased to 51% and 40%, respectively, of their levels at pH 6 by 24 h. [3H]Uridine and [3H]leucine incorporation into macromolecules in oat leaves (with the lower epidermis removed) decreased to 58% and 28% of the control in response to acid stress in 8 h, at the same time as a 5-fold increase in putrescine. When DL-α-difluoro-methyarginine was added to the pH 3.5 buffered solution, the effect of acid was slightly less, with the incorporation into macromolecules being 64% and 35% of the pH 6 control. The results indicated that the putrescine accumulation under acid stress was concomitant with ion efflux, and a decrease in both macromolecular synthesis and the activities of superoxide dismutase and catalase in cereal leaves.

Putrescine Accumulation is Associated with Growth Inhibition in Suspension-Cultured Rice Cells under Potassium Deficiency

Putrescine Accumulation is Associated with Growth Inhibition in Suspension-Cultured Rice Cells under Potassium Deficiency

Methyl Jasmonate Induces the Accumulation of Putrescine but not Proline in Detached Rice Leaves

The effects of methyl jasmonate (MJ) and abscisic acid (ABA) on the levels of proline and putrescine in detached rice leaves were investigated. ABA induced the accumulation of proline but not putrescine in detached rice leaves incubation in darkness. In contrast to the effect of ABA, MJ induced the accumulation of putrescine but not proline in darkness. MJ also increased the levels of putrescine in detached rice leaves in the light. Both D-arginine and α-methylornithine, inhibitors of the biosynthesis of putrescine, reduced the level of putrescine induced by MJ. It is concluded that MJ can act like certain other stresses (K + and Mg 2+ deficiency, excess NH 4 + , low pH, salinity and osmotic stress) to induce substantial increases in putrescine in detached rice leaves.

Increased drought resistance of black locust seedlings via pretreatment of seeds with paclobutrazol

The drought resistance of black locust (Robiniapseudoacacia L.) seedlings was increased by pretreatment of seeds with 250 ppm paclobutrazol. Treated seeds produced seedlings with thick roots and higher root/shoot ratios. During osmotic stress, treated plants displayed greater relative water content, lower electrolyte leakage, and fewer symptoms of wilt when compared with untreated black locust seedlings. Paclobutrazol-treated seedlings accumulated more proline than untreated seedlings in response to drought. However, osmotically induced accumulation of putrescine, a possible marker of cellular stress, was reduced in the treated plants.

Interactions of polyamines and nitrogen nutrition in plants

Biogenic amines occupy an important position among the many nitrogenous plant compounds. Polyamines are part of the overall metabolism of nitrogenous compounds, yet they do not seem to function in the ‘normal’ nitrogen nutrition. Rather, these widespread polycations (e. g. putrescine, spermidine and spermine) are involved in the regulation of growth and stress, probably by binding to negatively charged macromolecules. In addition, some diamines and polyamines are metabolized to yield 'secondary ’metabolites such as nicotine and other alkaloids. Previous studies have indicated that the ratio of nitrate to ammonium nutrition affects polyamine biosynthesis and content in intact plants. Thus, an increase in putrescine accumulation was found under conditions of excess ammonium ions, relative to nitrate. Modifications of nitrogen sources in the culture medium of tobacco cell suspensions (depletion of ammonium nitrate, or potassium nitrate, or both) resulted in marked changes in the content of cellular free polyamines. Considerable changes in the content of specific polyamines were also found with exposure to specific inhibitors of polyamine biosynthesis (difluoromethyl ornithine, difluoromethyl arginine, cyclohexylamine, methylglyoxal‐bis‐guanylhydrazone). However, a combination of nitrogen depletion of the medium and some inhibitors resulted in a very marked over‐production of spermidine and spermine. The significance of these findings is discussed in relation to the assumption that polyamines act as a metabolic buffer, and maintain cellular pH under conditions where ammonium assimilation produces an excess of protons.

Putrescine (1,4-Diaminobutane) as an Indicator of Pollution-Induced Stress in Higher Plants: Barley and Rape Stressed with Cr(III) or Cr(VI)

The performance of the free polyamines as plant stress indicators is studied in barley and rape plants grown in nutrient culture, by exposure to Cr(VI) or Cr(lIl) in concentrations ranging from 0 to 100 ppm. Putrescine levels are elevated up to 10 times in the leaves of stressed plants compared to those of control plants, but neither spermidine nor spermine show any consistent reactions on the stress. Cr(VI) is more toxic than Cr(lIl) and induces putrescine accumulation quicker than Cr(llI). Chromium concentrations in leaves reach 3000-5000 ppm (dry wt) after exposure to 100 ppm Cr(VI) and 300-400 ppm (dry wt) following exposure to 100 ppm Cr(III). Simultaneously with, or following shortly after the putrescine induction, reductions in root growth, chlorosis, induction of leaf chitinase activity, and, later, reduced shoot growth and lowered water content in leaves are observed. The pattern of the effects indicates that the basal toxicity mechanism of the two chromium species is connected to disturbance of the normal function of the root. Putrescine induction is an integrated part of the response mechanism of the stressed plants, appearing as an early sign of stress. However, the chromium concentration of the leaves as a warning of chromium-induced stress is judged to be an even more sensitive indicator.

Osmotic stress and water stress have opposite effects on putrescine and proline production in excised rice leaves

The effects of water stress and osmotic stress (sorbitol treatment) on the production of putrescine and proline in excised rice leaves were compared. Osmotic stress and water stress were found to affect differentially the levels of putrescine and proline in excised rice leaves. Putrescine accumulation is induced by osmotic stress, whereas proline accumulation is induced by water stress. The effects of ABA on the levels of proline and putrescine are similar to those of water stress, whereas the effects of jasmonic acid methyl ester (JA-Me) are similar to those of osmotic stress. Water stress results in an increase of endogenous ABA is excised rice leaves. However, neither osmotic stress nor JA-Me has effect on endogenous ABA levels in excised rice leaves. Of particular interest is the finding that proline levels increase when putrescine levels induced by osmotic stress or JA-Me are reduced by D-arginine and α-methylornithine. L-arginine and L-ornithine applied exogenously also cause an increase in proline levels. It seems that L-arginine and L-ornithine are preferentially utilized as precursors for putrescine accumulation in excised rice leaves treated with osmotic stress and JA-Me, and for proline accumulation in excised rice leaves exposed to water stress and ABA.

Changes in putrescine accumulation induced by agents affecting cytosolic pH in detached rice leaves

Effects of compounds that influenced cytosolic pH on the level of putrescine in detached rice leaves were examined. Permeant weak acids, isobutyric acid and propionic acid, increased the level of putrescine in detached rice leaves. Procaine and trisodium citrate, known to be permeant weak bases, on the other hand, decreased the level of putrescine. It seems possible that the level of putrescine in detached rice leaves is regulated by the cytosolic pH.

Needle polyamine concentrations and potassium nutrition in Scots pine.

The response of free polyamines (putrescine, spermidine and spermine) in needles of Scots pine (Pinus sylvestris L.) to varying needle potassium concentrations was investigated in two potassium fertilization experiments on drained peatlands. A significant negative correlation was observed between putrescine and potassium concentrations in needles. Putrescine responded more sensitively to decreasing needle K concentrations during the growing season than during the winter. Putrescine accumulation started when needle potassium concentrations were above 5.5 mg g(dw) (-1) in summer and above 5.0 mg g(dw) (-1) in winter. A decrease in needle potassium concentrations below 4.3 mg g(dw) (-1) in summer and below 3.5 mg g(dw) (-1) in winter resulted in an exponential increase in putrescine concentrations. Putrescine accumulation was initiated well above the needle potassium concentrations generally considered indicative of deficiency. The exponential increase in putrescine concentrations in winter occurred within the range of needle potassium concentrations that has been identified as indicating severe potassium deficiency.

Regulation of Rhizogenesis by Polyamines in Tobacco Thin Layers

Summary Tobacco thin layer explants were cultured on a rhizogenic medium for 21 days in the presence or absence of methylglyoxal-bis(guanylhydrazone) (MGBG) or cyclohexylamine (CHA), competitive inhibitors of spermidine synthesis through S-adenosylmethionine decarboxylase and spermidine synthase, respectively. On day 21, explants were transferred to hormone-free medium of the same composition with or without MGBG or CHA, alone or in combination with labelled and unlabelled spermidine. The effects of these treatments on free and bound polyamine levels, on the putrescine biosynthetic activity (ornithine decarboxylase, ODC, and arginine decarboxylase, ADC), on labelled spermidine incorporation and on the rhizogenic response were studied. In MGBG-treated explants rhizogenesis was strongly inhibited while with CHA it was less affected. In the former, the addition of spermidine caused a significant reversion of the rooting inhibition (considering both rooting percentage and the mean number of roots per explant), while not in the latter. MGBG induced strong putrescine and spermidine depletion both in the free and bound forms; CHA induced strong spermidine depletion (especially on day 21), but putrescine accumulation. In both treatments, in the presence of labelled spermidine, labelled free and bound putrescine were also detected. A general enhancement in ADC activity was observed in inhibitor-treated explants; the addition of spermidine to the inhibitors caused a further strong increase in ADC activity. These results are discussed in relation to the different metabolic pathways affected by the inhibitors.

Spermidine biosynthesis as affected by osmotic stress in oat leaves

A new assay for the evaluation of spermidine (Spd) synthase activity was developed. It involves a coupled reaction and avoids the use of decarboxylated S-adenosylmethionine, which is unstable and not easily available. This assay was applied to assess changes in enzyme activity in oat leaves subjected to osmotic stress in the dark. The results indicate that osmotically-induced putrescine (Put) accumulation in cereals results not only from the activation of the arginine decarboxylase pathway, but also from the inhibition of the activity of Spd synthase, the enzyme which catalyzes the transformation of Put to Spd. Other possibilities which could contribute to the decline of Spd and spermine levels under osmotic stress are also discussed.

Polyamine Concentration and Ethylene Evolution in Tomato Plants under Nutritional Stress

Polyamine accumulation in foliage was assessed in relation to ammonium accumulation and ethylene evolution in tomato (Lycopersicon esculentum Mill.) under nutritional stress. Nutritional stresses were induced in greenhouse-grown plants in quartz sand with an NH4-based solution or with NO3-based solutions without P, K, Ca, or Mg. Plants receiving NH4-based nutrition had higher putrescine and lower spermidine concentrations than plants receiving NO3-based nutrition. Adding AOA (10-5m) to the nutrient solution of plants receiving NH4-based nutrition suppressed putrescine accumulation but had no effect on spermidine; silver thiosulfate (10-5 m) had no effect on polyamine accumulation. Deficiencies had no consistent effect on polyamine accumulation relative to its accumulation under full-nutrition conditions, but adding AOA restricted putrescine and spermidine accumulation in all nutrient-deficient regimes. Foliar spermine accumulation was not affected by nutritional regime. Ammonium-based nutrition resulted in enhanced putrescine and ammonium accumulation and accelerated ethylene evolution rates relative to plants receiving NO3-based nutrition. All nutrient-deficient plants had higher ammonium accumulation, and all but P-deficient plants had higher ethylene evolution than those receiving full NO3-based nutrition. Although some variability occurred among treatments, an association among putrescine accumulation, ammonium accumulation, ethylene evolution. and stress-induced symptoms was apparent. Chemical name used: (aminooxy) acetic acid (AOA).