Polyamine metabolism in Pneumocystis carinii

Alpha-difluoromethylornithine (DFMO) is being used to treat Pneumocystis carinii pneumonia despite a lack of in vitro evidence supporting its antipneumocystis activity. DFMO is a specific inhibitor of ornithine decarboxylase, the rate-limiting enzyme of polyamine biosynthesis. To investigate polyamine metabolism in P. carinii, extracts of the organism were analyzed for polyamine content and ornithine decarboxylase activity, and [3H]ornithine and [14C]arginine incorporation into polyamines during short-term culture was determined. P. carinii extracts contained putrescine and spermidine in a ratio of 0.17:1; traces of spermine were detected. Although ornithine decarboxylase activity was not detected, P. carinii incorporated ornithine and arginine into putrescine and spermidine but not into spermine, suggesting that the spermine detected derived from contaminating host cells. Uninfected rat lung incorporated ornithine minimally. Pentamidine, DFMO, and alpha-monofluoromethyldehydroornithine methyl ester inhibited ornithine incorporation by up to 86% at clinically achievable concentrations. These data provide a rationale for using polyamine synthesis antagonists in P. carinii pneumonia and a method for screening antipneumocystis drugs in vitro.     

Involvement of polyamines in apoptosis of cardiac myoblasts in a model of simulated ischemia

Apoptotic cell death of cardiomyocytes is involved in several cardiovascular diseases including ischemia, hypertrophy, and heart failure. The polyamines putrescine, spermidine, and spermine are polycations absolutely required for cell growth and division. However, increasing evidence indicates that polyamines, cell growth, and cell death can be tightly connected. In this paper, we have studied the involvement of polyamines in apoptosis of H9c2 cardiomyoblasts in a model of simulated ischemia. H9c2 cells were exposed to a condition of simulated ischemia, consisting of hypoxia plus serum deprivation, that induces apoptosis. The activity of ornithine decarboxylase, the rate limiting enzyme of polyamine biosynthesis that synthesizes putrescine, is rapidly and transiently induced in ischemic cells, reaching a maximum after 3 h, and leading to increased polyamine levels. Pharmacological inhibition of ornithine decarboxylase by alpha-difluoromethylornithine (DFMO) depletes H9c2 cardiomyoblasts of polyamines and protects the cells against ischemia-induced apoptosis. DFMO inhibits several of the molecular events of apoptosis that follow simulated ischemia, such as the release of cytochrome c from mitochondria, caspase activation, downregulation of Bcl-xL, and DNA fragmentation. The protective effect of DFMO is lost when exogenous putrescine is provided to the cells, indicating a specific role of polyamine synthesis in the development of apoptosis in this model of simulated ischemia. In cardiomyocytes obtained from transgenic mice overexpressing ornithine decarboxylase in the heart, caspase activation is dramatically increased following induction of apoptosis, with respect to cardiomyocytes from control mice, confirming a proapoptotic effect of polyamines. It is presented for the first time evidence of the involvement of polyamines in apoptosis of ischemic cardiac cells and the beneficial effect of DFMO treatment. In conclusion, this finding may suggest novel pharmacological approaches for the protection of cardiomyocytes injury caused by ischemia.     

The role of polyamine biosynthesis in hematopoietic precursor cell proliferation in mice

Under the influence of a selective irreversible inhibitor of ornithine decarboxylase (ODC), DL-alpha-difluoromethylornithine (DFMO), early hematopoiesis was enhanced. In the bone marrow, the absolute number of cells that give rise to spleen colonies in lethally irradiated mice (CFU-S), granulocytic colonies in diffusion chambers in mice (CFU-DG), and granulocyte-monocyte colonies in agar in vitro (CFU-C) was increased 2-4 fold. This could be abrogated by administration of putrescine, confirming the association of the stimulatory effect with polyamine biosynthesis most likely via depression of ornithine decarboxylase activity and subsequent synthesis of putrescine. Analysis of cell cycle characteristics by 3H-TdR suicide technique demonstrated that the proportion of CFU-S, CFU-DG, and CFU-C in S-phase was significantly increased. Additionally, the stimulatory effect was reflected by enhanced colony formation in diffusion chambers implanted intraperitoneally in mice receiving DFMO. This could also be eliminated by treatment of the host animal with putrescine, again suggesting that polyamine biosynthesis plays an important role at the early stages of hematopoiesis in vivo. Effect of DFMO on colony formation in vitro (CFU- C) was inhibitory and not reversible with putrescine. It could be partially eliminated by aminoguanidine, which neutralizes diamine oxidase present in fetal calf serum used in the CFU-C assay. These data suggest that the effect of DFMO in vitro was nonspecific.     

Dose-dependent inhibition by cyclosporine A of the induction of pancreatic ornithine decarboxylase (ODC) in rats

The effect of cyclosporine A (CsA) and alpha-difluoromethylornithine (DFMO) on the camostate-induced increase in pancreatic ornithine decarboxylase (ODC) activity and polyamine biosynthesis has been studied in vivo. Six hours after application of the synthetic trypsin inhibitor camostate (200 mg/kg b wt orally) pancreatic ODC activity increased about 140-fold and putrescine concentration about ninefold. CsA inhibited the elevation of both parameters in a dose-dependent manner. CsA pretreatment for 3 d with doses of 7.5, 10.0, and 12.5 mg/kg b wt orally once a day and consecutive CsA blood levels 24 h after the last CsA application of 751 +/- 62, 968 +/- 70, and 1,395 +/- 79 ng/mL, respectively, resulted in a complete inhibition of the camostate-stimulated increase in pancreatic ODC activity and putrescine concentration in vivo. DFMO (2% in drinking water and additionally 300 mg/kg b wt intraperitoneally at 8 AM, 12 noon, and 4 PM) inhibited the increase in both, ODC activity, and putrescine, significantly in an equipotent degree as 2.5 mg CsA/kg b wt, whereas higher doses of CsA proved to be more effective than DFMO in the chosen subtoxic dose. In all cases, no significant changes in pancreatic spermidine and spermine concentration, DNA and protein content, or pancreatic and body weight were observed. It is concluded that CsA in doses used for immunosuppression in clinical practice is a very potent and more effective inhibitor of ODC activity and polyamine synthesis in vivo than DFMO. This ODC inhibitory effect of CsA is a further detail to elucidate the up to now incompletely understood mechanisms of action of this immunosuppressive agent.     

Polyamines and growth regulation of cultured human breast cancer cells by 17 beta-oestradiol

The growth of ZR-75-1 cells, a line of human breast cancer cells in culture, is stimulated by oestradiol and inhibited by anti-oestrogens. Changes in growth rate caused by these agents are accompanied by changes in activity of ornithine decarboxylase, a rate-limiting enzyme for polyamine synthesis. Furthermore, the growth inhibition caused by tamoxifen, an anti-oestrogen, can be reversed by the addition of spermine, spermidine or putrescine to the cells. Insulin can also stimulate ZR-75-1 cell growth and this is again accompanied by an increase in ODC activity. The reduced cell growth rate observed when the cells become confluent is associated with a marked decrease in ornithine decarboxylase activity. Experiments performed with DFMO, a specific and irreversible inhibitor of ODC, show that this compound can prevent the stimulation of growth by oestradiol and that this may be overcome by the addition of putrescine to the cells. It would appear that increased ODC activity and polyamine synthesis are necessary components of the stimulation of breast cancer cell growth by oestradiol but that other growth regulatory stimuli also may act via this enzyme.     

Enhancement of the antiproliferative activity of human interferon by polyamine depletion

Recombinant leukocyte type A human interferon and human lymphoblastoid interferon in combination with alpha-difluoromethylornithine (DFMO), an inhibitor of polyamine biosynthesis, have synergistic antiproliferative activity against colony formation in vitro by human tumor cells differing greater than 1000-fold in their intrinsic sensitivity to inhibitory effects of interferon. Interferon and DFMO in combination with doxorubicin have greater antiproliferative activity than is expected on the basis of additive effects based on the activity of doxorubicin alone and the synergistic activity of interferon plus DFMO. The addition of the polyamine putrescine to the cell cultures eliminates the synergistic interactions of interferon and DFMO and does not inhibit the activity of interferon itself. Aspirin and indomethacin at concentrations in vitro greater than those required for anti-inflammatory activity in vivo did not inhibit the antiproliferative activity of interferon alone and did not inhibit the synergistic activity of the combination of interferon and DFMO. Combination regimens including interferon and DFMO merit clinical evaluation for therapeutic activity against advanced cancers. Nonsteroidal anti-inflammatory agents should be studied for their ability to ameliorate symptomatic toxicity from interferon.     

Polyamine synthesis in bone marrow granulocytes: effect of cell maturity and early changes following an inflammatory stimulus

Enriched fractions of mature and immature neutrophil granulocytes, isolated from guinea pig bone marrow, were assayed for ornithine decarboxylase activity and polyamine content. The results show that immature granulocytes contain at least ten times more ornithine decarboxylase activity and two times more spermidine than mature granulocytes. The incorporation of 14C-ornithine into putrescine and spermidine of intact immature granulocytes was three to four times and ten times, respectively, that of mature granulocyte preparations. Six hours after an inflammatory stimulus, transient increases of 14-fold and 3-fold in the activities of ornithine decarboxylase and S-adenosyl- L-methionine decarboxylase, respectively, were observed in immature bone marrow granulocytes. At this time the incorporation of 14C- ornithine into putrescine and spermidine in bone marrow granulocytes from stimulated animals was 14 times that of cells from controls. A maximum increase in DNA synthesis in these cells during the inflammatory response occurred 6 hr after the maximum increase in the polyamine synthetic activity. Together these data suggest that polyamine synthesis in the granulocyte compartment of the bone marrow is associated chiefly with immature proliferating cells and that increased polyamine synthesis precedes increased granulocyte proliferation in the bone marrow following an inflammatory stimulus.     

The effects of 2-deoxy-D-glucose and alpha-difluoromethylornithine on the growth of pancreatic cancer in vivo

The glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), inhibits growth of some cancers. alpha-Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase, the rate-limiting enzyme in polyamine biosynthesis. We and others have previously shown that DFMO inhibits cancer growth in a number of models. The present study was designed to investigate the effects of 2-DG alone and combined with DFMO on the growth of H2T hamster pancreatic ductal adenocarcinoma. Twenty-eight male Syrian golden hamsters were inoculated with 500,000 H2T cells, and then randomized into four groups of seven each: group 1 served as control; group 2 received DFMO (3% in drinking water); group 3 received 2-DG (500 mg/kg/day) intraperitoneally; group 4 received a combination of 2-DG and DFMO. Treatment began 5 days after tumor cell inoculation and continued for 28 days. At the end of the treatment period, the area of the H2T tumor was reduced 31% by DFMO compared with a 22% reduction caused by 2-DG. Tumor weight was significantly reduced (31%) by DFMO but not by 2-DG. Tumor contents of DNA, RNA, and protein were also reduced by DFMO but not 2-DG. Tumor concentration of the polyamines, putrescine and spermidine, were reduced by DFMO, but 2-DG did not alter levels of polyamines. The combination of DFMO and 2-DG caused a significantly greater reduction in tumor weight and putrescine content compared with DFMO alone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Prevention of a plant disease by specific inhibition of fungal polyamine biosynthesis.

DL-alpha-Difluoromethylornithine (DFMO), an inhibitor of the polyamine biosynthetic enzyme ornithine decarboxylase (EC 4.1.1.17), strongly retards the growth of several species of phytopathogenic fungi in vitro. Such inhibition can be completely reversed by putrescine or spermidine, confirming the essentiality of polyamines for growth of fungal hyphae. We now show that DFMO can protect bean plants (Phaseolus vulgaris Linnaeus cv. Pinto) against infection by uredospores of the bean rust fungus, Uromyces phaseoli Linnaeus, race O. Unifoliolate leaves of 10-day-old greenhouse-grown seedlings were sprayed with 400 microliter per leaf of DFMO at various concentrations in 0.01% Tween 20 at pH 7.0 before or after inoculation with uredospores of Uromyces. After 16 hr in darkness in dew chambers to facilitate spore germination, plants were transferred to the greenhouse, arranged randomly, and examined for local lesions 7 days later. All concentrations of DFMO 0.50 mM or higher gave complete protection against the pathogen; at lower concentrations, postinoculation treatments with DFMO were generally more effective than preinoculation. The appearance of lesions on plants treated with lower concentrations of DFMO was retarded 2-6 days. DFMO also confers protection on unsprayed parts of treated plants, indicating the translocation of some protective effect from sprayed areas. DL-alpha-Difluoromethylarginine, an analogous inhibitor of arginine decarboxylase (EC 4.1.1.19), which is the rate-limiting enzyme in an alternative pathway for polyamine biosynthesis in higher plants, confers no protection even at 5 mM. This emphasizes ornithine decarboxylase as the biochemical locus of choice for the prevention of plant diseases by inhibiting polyamine metabolism.     

The role of the urea cycle and polyamines in albumin synthesis

Albumin synthesis is stimulated by those amino acids which increase urea synthesis and membrane bound polysome aggregation. Ornithine, an amino acid not incorporated into protein and produced from arginine in the urea cycle, is an albumin-stimulating amino acid and is the precursor of the polyamines, and we have shown that the polyamine spermine promotes bound polysome aggregation. To test the concept that ureogenesis with its generation of ornithine might play a key role in albumin synthesis regulation via the polyamine pathway, isolated livers from fasted donors were perfused with ornithine, alpha-difluoromethyl ornithine (DFMO), and spermine. In control experiments, albumin synthesis was 13.4 +/- 0.8 mg per 100 gm liver per hr and polysome aggregation was 47%. These were increased in the presence of ornithine (26.0 +/- 2.6 mg and 59%); if the livers were preperfused with DFMO before the addition of ornithine, then the expected increase in albumin synthesis and polysome reaggregation did not occur (16.3 +/- 1.4 mg and 47%). However, if spermine was present with DFMO during the preperfusion, then the addition of ornithine had the expected effect (albumin synthesis = 26.1 +/- 1.2 mg and polysome aggregation = 62%). This suggests that if the ornithine to putrescine pathway is blocked, ornithine does not stimulate albumin synthesis and offers support to the concepts that (a) ornithine stimulation of albumin production is via polyamine synthesis and (b) that the urea cycle plays a more important role in protein metabolism than simply the pathway for nitrogen disposal.     

Role of the arginine-nitric oxide pathway in the regulation of vascular smooth muscle cell proliferation

The objective of this study was to elucidate the mechanisms by which nitric oxide (NO) inhibits rat aortic smooth muscle cell (RASMC) proliferation. Two products of the arginine-NO pathway interfere with cell growth by distinct mechanisms. N(G)-hydroxyarginine and NO appear to interfere with cell proliferation by inhibiting arginase and ornithine decarboxylase (ODC), respectively. S-nitroso-N-acetylpenicillamine, (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate, and a nitroaspirin derivative (NCX 4016), each of which is a NO donor agent, inhibited RASMC growth at concentrations of 1-3 microM by cGMP-independent mechanisms. The cytostatic action of the NO donor agents as well as alpha-difluoromethylornithine (DFMO), a known ODC inhibitor, was prevented by addition of putrescine but not ornithine. These observations suggested that NO, like DFMO, may directly inhibit ODC. Experiments with purified, recombinant mammalian ODC revealed that NO inhibits ODC possibly by S-nitrosylation of the active site cysteine in ODC. DFMO, as well as the NO donor agents, interfered with cellular polyamine (putrescine, spermidine, spermine) production. Conversely, increasing the expression and catalytic activity of arginase I in RASMC either by transfection of cells with the arginase I gene or by induction of arginase I mRNA with IL-4 resulted in increased urea and polyamine production as well as cell proliferation. Finally, coculture of rat aortic endothelial cells, which had been pretreated with lipopolysaccharide plus a cytokine mixture to induce NO synthase and promote NO production, caused NO-dependent inhibition of target RASMC proliferation. This study confirms the inhibitory role of the arginine-NO pathway in vascular smooth muscle proliferation and indicates that one mechanism of action of NO is cGMP-independent and attributed to its capacity to inhibit ODC.     

Uptake of Extracellular, Dietary Putrescine Is an Important Regulatory Mechanism of Intracellular Polyamine Metabolism DuringCamostate-Induced Pancreatic Growth in Rats

Aim of the present study was to evaluate therole of cellular uptake of dietary [ H]putrescine forthe regulation of pancreatic, hepatic, and smallintestinal polyamine metabolism during normal andcamostate-induced pancreatic growth in rats in vivo. Initiallydose-response and time-course studies of[³H]putrescine uptake were performed. MaleWistar rats were either treated with the synthetictrypsin inhibitor camostate (200 mg/kg body wt orally twice daily),camostate plus the ornithine decarboxylase inhibitoralpha-difluoromethylornithine (DFMO) (2% in drinkingwater plus 3 × 300 mg/kg body wt intraperitoneallyduring daytime) or saline as controls. After 4, 8, 12,24, 36, 48, or 120 hr, five to seven animals per groupwere killed, respectively. Orally fed [ H]putrescine (10nmol/kg body wt. 2 hr prior to death) is rapidly taken up and further metabolized tospermidine in normal growing pancreas, liver, and smallintestine. Feeding of camostate significantly enhanceddietary [³H]putrescine uptake, whilesimultaneous inhibition of de novo synthesis ofintracellular polyamines by DFMO resulted in a highlysignificant further increase in cellular uptake oforally fed [³H]putrescine, which isimmediately metabolized to spermidine. The present in vivo data confirmthe important role of dietary putrescine uptake for themaintenance of intracellular polyamine pool in normaland stimulated pancreatic growth. Furthermore, dietary putrescine uptake is an importantregulatory mechanism to maintain the normal andgrowth-stimulated cellular polyamine pool in thepancreas after potent simultaneous inhibition ofintracellular de novo polyamine synthesis.     

Polyamines and the calcium paradox in rat hearts

Polyamine levels were measured by means of high-performance liquid chromatography in Langendorff-perfused rat hearts subjected to the calcium paradox protocol. The concentrations of putrescine, spermidine and spermine did not change significantly during calcium-free perfusion but decreased when calcium was readmitted. This decrease was due to membrane disruption and release of the polyamines into the coronary effluent. The sum of released and remaining spermidine exceeded the concentration of spermidine in control hearts, but, for spermine, this sum was lower than the control level. The addition of 0.5 mM EGTA to the calcium-free solution raised the myocardial concentrations of putrescine and spermidine and enhanced the net increase of spermidine on calcium repletion. DL-alpha-Difluoromethylornithine (DFMO) inhibited these increases and lowered the putrescine level during all perfusion stages. External polyamines had a negative inotropic effect and inhibited the loss of myoglobin on calcium repletion (order of effectiveness: spermine greater than spermine greater than putrescine). Inhibition of contractions by the combined action of verapamil and ryanodine or by potassium depolarization did not prevent myoglobin loss. External polyamines had no effect on high K/low Na contractures, which were mediated mainly by Na-Ca exchange. Calcium-free perfusion in the presence of 0.5 to 1 mM EGTA improved the membrane protection by polyamines or by diamines and analogues, like ornithine, 1,3-diaminopropane, or DFMO, which, in the absence of EGTA, gave no clear protection. It is concluded that calcium depletion and repletion influences myocardiaal polyamine concentrations by (1) membrane disruption and release of polyamines into the coronary effluent, and (2) probably by a stimulation of ornithine decarboxylase activity. The changes in polyamine concentrations do not seem to have any causal role in calcium overload and cell death. Exogenous polyamines protect against membrane damage.     

Role of polyamines in isoproterenol-induced cardiac hypertrophy: effects of alpha-difluoromethylornithine, an irreversible inhibitor of ornithine decarboxylase

DFMO is a selective irreversible inhibitor of ornithine decarboxylase (ODC), the initial enzyme in the polyamine biosynthetic pathway. DFMO was utilized to determine the role of polyamines in isoproterenol (ISO)-induced cardiac hypertrophy. Daily subcutaneous administration of 200 mg/kg of DFMO reduced cardiac putrescine levels but did not significantly alter the basal levels of spermidine or spermine, nor was normal cardiac growth affected. ISO-induced cardiac hypertrophy was accompanied by increased putrescine and spermidine levels but spermine was not significantly altered. DFMO reversed the ISO-induced increases in putrescine and inhibited or attenuated both the increases in spermidine content and the cardiac hypertrophy. Although normal ODC activity appears not to be necessary for the maintenance of basal levels of polyamines or for normal cardiac growth, sustained inhibition of ODC interferes with ISO-induced elevations of putrescine, spermidine and heart weight.     

Alterations in polyamine levels induced by phorbol diesters and other agents that promote differentiation in human promyelocytic leukemia cells

Polyamine levels were evaluated in human HL-60 promyelocytic leukemia cells after treatment with inducers of terminal differentiation. Differentiation in these cells was determined by increases in the percentage of morphologically mature cells and in lysozyme activity. Treatment of the HL-60 cells with phorbol 12-myristate-13-acetate (PMA), phorbol 12,13-didecanoate or other inducers of terminal differentiation such as dimethylsulfoxide and retinoic acid resulted in increased levels of putrescine. However, no increase in putrescine could be detected after PMA treatment of a HL-60 cell variant that exhibited a decreased susceptibility to PMA-induced terminal differentiation. Similarly, no increase in putrescine was observed with two non-tumor-promoters (phorbol 12,13-diacetate and 4-O-methyl-PMA) or with anthralin, a non-phorbol tumor promoter. In addition to enhancing putrescine levels, PMA also increased the amount of spermidine and decreased the amount of spermine. The increase in putrescine and spermidine preceded the expression of the various differentiation markers. Unlike the changes observed in the polyamine levels after PMA treatment, the activities of ornithine and S-adenosylmethionine decarboxylases, which are polyamine biosynthetic enzymes, did not significantly change. α-Methylornithine and α-difluoromethylornithine and methylglyoxal bis(guanylhydrazone), which are inhibitors of the polyamine biosynthetic enzymes, did not affect differentiation in control or PMA-treated cells. Because of these observations, we suggest that the change in polyamine levels involve biochemical pathways other than the known biosynthetic ones. By-products of these pathways may perhaps be the controlling factors involved in the induction of terminal differentiation in the HL-60 and other cell types as well.     

Growth inhibition of the androgen responsive DDT(1)MF-2 cell line by glucocorticoids: the role of ornithine decarboxylase

While testosterone (T) stimulates the growth of DDT₁MF-2 cells, glucocorticoids arrest the growth of these cells in the G₀/G₁ stage of the cycle. Ornithine decarboxylase (ODC), the first and rate-limiting enzyme in the polyamine biosynthetic pathway, is highly sensitive both to growth and inhibitory stimuli. To assess the mechanism of glucocorticoid inhibition of cell growth, the effect of triamcinolone acetonide (TA) on growth and ODC was studied. DDT₁-MF-2 cell growth was inhibited by TA and difluoromethyl ornithine (DFMO), an irreversible inhibitor of ODC. TA (10nm) inhibited the ODC activity to 10% of the control levels by 12 h and inhibition was maintained at all later intervals studied. Ten μm DFMO inhibited ODC activity to a maximum of 50% of control. The concentration of ODC mRNA was maximally decreased at 15 h after TA administration. Though TA and DFMO inhibited cell growth and ODC activity in DDT₁-MF2 cells, growth inhibition by DFMO, but not by TA, was overcome by the addition of putrescine, the product of ODC reaction. Thus, inhibition of ODC is one pathway through which glucocorticoids inhibit DDT₁MF-2 cell growth. ODC inhibition, however, is not the only pathway through which glucocorticoids act.     

Decreased ornithine decarboxylase activity in the kidneys of Dahl salt-sensitive rats.

To assess the roles of polyamines (putrescine, spermidine, and spermine) and ornithine decarboxylase (ODC), the rate-limiting enzyme of polyamine synthesis, in the development of salt-sensitive hypertension, we evaluated activity and expression of ODC, urinary polyamine excretion, and antizyme (endogenous ODC inhibitor protein) expression in Dahl salt-sensitive (SS) and salt-resistant (SR) rats after they were fed on a low (0.3%) or high (4%) salt diet for 4 weeks. We also examined the effects of spermidine and difluoromethylornithine (DFMO: a specific inhibitor of ODC) on the systolic blood pressure and ODC protein expression in SS rats fed a high salt diet. Renal ODC activity and urinary polyamine excretion in SS rats were lower than those in SR rats after 4 weeks treatment with a low or high salt diet. The renal ODC protein expression of SS rats was paradoxically increased as compared to the SR group. A high salt diet did not alter ODC activity but increased ODC protein only in SS rats. ODC mRNA and antizyme protein expressions were not significantly different among the four groups. Spermidine supplementation attenuated and DFMO exaggerated hypertension in SS rats fed a high salt diet. Spermidine down-regulated and DFMO up-regulated renal ODC protein in SS rats on a high salt diet. ODC activity was decreased but protein was paradoxically increased in kidneys of SS rats. ODC protein was suggested to increase in compensation for the inhibition of its activity. Impaired ODC activity and polyamine production in the kidney may exaggerate salt-sensitive hypertension in SS rats.     

Involvement of spermidine in proliferation and differentiation of human promyelocytic leukemia cells

The polyamines putrescine, spermidine, and spermine have been implicated in the regulation of cell proliferation and differentiation. Previous studies, however, have demonstrated that the polyamines are essential for proliferation, but not differentiation, of HL-60 human promyelocytic leukemia cells. We have extended these findings by demonstrating a highly significant relationship between intracellular spermidine levels and HL-60 proliferation. However, in contrast to previous studies, we have also demonstrated that induction of HL-60 differentiation with dimethyl sulfoxide, hexamethylene bisacetamide, butyric acid, or retinoic acid is inhibited by alpha-difluoromethyl ornithine (DFMO) depletion of intracellular putrescine and spermidine. Further, the addition of exogenous spermidine abrogates DFMO inhibition of HL-60 differentiation, thus confirming the involvement of this polyamine in the expression of a differentiated phenotype. The discrepancy between our results and those of previous studies probably stems from the nearly complete, rather than partial, depletion of intracellular spermidine achieved in the present work. The results of the present study thus demonstrate the involvement of spermidine in both proliferation and induction of HL-60 differentiation with certain agents.     

Hyperoxic lung injury and polyamine biosynthesis. Age-related differences

To determine if lung cell replication and repair might be different between younger (30-day-old) and older (60-day-old) rats, we studied polyamine and DNA biosynthesis in rats exposed to 1.0 atm oxygen for 24, 48, 56, or 72 h. By 24 h, no statistically significant changes were observed, but by 48 h, ornithine decarboxylase and putrescine increased; S-adenosylmethionine decarboxylase activity increased by 56 h in the younger rats but not in the older rats. By 72 h, spermidine, [3H]thymidine incorporation, and the labeling index of cells in the alveolar zone had increased only in the younger rats. During the first 56 h, hyperoxia inhibited DNA synthesis. We conclude that hyperoxia initially suppresses lung cell replication but subsequently, if the rat survives, there are increases in polyamine biosynthesis and cell replication that may be important for the development of oxygen tolerance.     

Changes in polyamine metabolism of rat liver after administration of D-galactosamine. Favorable effects of putrescine administration on galactosamine-induced hepatic injury.

There are many reports showing a close relation between polyamine metabolism and tissue growth or recovery of damaged tissues, such as regenerating liver. Thus, changes in polyamine metabolism in the livers from rats treated with D-galactosamine, an inducer of experimental hepatitis, were studied. The activity of ornithine decarboxylase started to increase 14 hr after administration of galactosamine and reached 30 times the normal activity at about 25 hr, the time of maximum severity of hepatitis. The content of putrescine increased to about 10 times the control value. After increases in the putrescine content and ornithine decarboxylase activity, the hepatitis started to diminish. Increases in the activity of S-adenosylmethionine decarboxylase and the content of spermidine were observed 33-37 hr after administration of galactosamine. The maximum values of these parameters, which were significantly higher than the control values, were observed after the healing process had started.