Regulation of intracellular polyamine biosynthesis and transport by NO and cytokines TNF-alpha and IFN-gamma

Joseph Satriano1,2, Shunji Ishizuka1, D. Clay Archer1, Roland C. Blantz1,2, and Carolyn J. Kelly1

1 Division of Nephrology-Hypertension and 2 Program in Molecular Pathology, Department of Medicine, University of California, San Diego, and Veterans Affairs Medical Center, La Jolla, California 92161


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Nitric oxide (NO) has been described to exert cytostatic effects on cellular proliferation; however the mechanisms responsible for these effects have yet to be fully resolved. Polyamines, conversely, are required components of cellular proliferation. In experimental models of inflammation, a relationship between these two pathways has been suggested by the temporal regulation of a common precursor, arginine. This study was undertaken to determine the effects NO and the NO synthase (NOS)-inducing cytokines, tumor necrosis factor-alpha (TNF-alpha ) and interferon-gamma (IFN-gamma ), exert on polyamine regulation. The transformed kidney proximal tubule cell line, MCT, maintains high constitutive levels of the first polyamine biosynthetic enzyme, ornithine decarboxylase (ODC). NO donors markedly suppressed ODC activity in MCT and all other cell lines examined. TNF-alpha and IFN-gamma induction of NO generation resulted in suppressed ODC activity, an effect prevented by the inducible NOS inhibitor L-N6-(1-iminoethyl)lysine (L-NIL). Dithiothreitol reversal of NO-mediated ODC suppression supports nitrosylation as the mechanism of inactivation. We also evaluated polyamine uptake, inasmuch as inhibition of ODC can result in a compensatory induction of polyamine transporters. Administration of NO donors, or TNF-alpha and IFN-gamma , suppressed [3H]putrescine uptake, thereby preventing transport-mediated reestablishment of intracellular polyamine levels. This study demonstrates the capacity of NO and inflammatory cytokines to regulate both polyamine biosynthesis and transport.

antizyme; inflammation; nitrosylation; ornithine decarboxylase; proliferation; tumor necrosis factor-alpha ; interferon-gamma


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ARGININE IS A PRECURSOR substrate for two well-described metabolic pathways, the production of nitric oxide (NO) by NO synthase (NOS) and the production of urea and ornithine by arginase. NO has been well documented as a cytostatic agent (1, 12, 26, 37, 45, 46, 48). Ornithine is the precursor of polyamines through the first rate-limiting enzyme of polyamine biosynthesis, ornithine decarboxylase (ODC). Polyamines (putrescine, spermidine, and spermine) are required components for entry into and progression of the cell cycle (3) and as such play an important role in proliferation. In inflammatory models of experimental glomerulonephritis and wound healing these two arginine-based pathways are temporally regulated (2, 11, 25). The production of NO from arginine by inducible NOS (iNOS) is an early phase response, whereas arginine is diverted to proliferative and extracellular matrix production pathways in the later repair phase response. Administering a NOS inhibitor, NG-monomethyl-L-arginine, in experimental glomerulonephritis increases both the magnitude and rapid onset of the repair phase response (11). Thus models of inflammation suggest an interrelationship of arginine metabolic pathways to maintain the correct temporal relationships between such pathways. As NO is not an effective arginase inhibitor (for review, see Ref. 24), we speculated that NO may modulate the proliferative response in the early phase of inflammation by suppressing ODC. ODC requires a cysteine in its active center for full enzymatic activity. NO has been shown to modulate the activity of several enzymes through nitrosylation of cysteines (14-16, 36, 43, 44).

Along with polyamine biosynthesis by ODC, cells can also acquire polyamines from their external milieu. Induction of polyamine transport by arginine deprivation, but not ornithine deprivation (8), strengthens the position of arginine at the crux of both the polyamine and NO pathways. Polyamine transporters are stimulated by many of the same factors that induce ODC activity. In addition, polyamine transport can substitute for de novo polyamine biosynthesis under conditions such as ODC inhibition by difluoromethylornithine (DFMO) (8). Polyamine autoregulation occurs through the induction of antizyme, a protein that inhibits both ODC and polyamine transport, exemplifying the importance of polyamine transport in vivo. Taken together, these results illustrate the need to examine polyamine transport as well as biosynthesis when examining cellular access to polyamines.

In this study we have demonstrated that NO and the iNOS-inducing cytokines tumor necrosis factor-alpha (TNF-alpha ) and interferon-gamma (IFN-gamma ) can negatively regulate ODC activity and cellular polyamine uptake.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals and supplies. Sodium nitroprusside (SNP), S-nitroso-N-acetyl-D,L-penicillamine (SNAP), S-nitroso-L-glutathione (GSNO), (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate), (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate (MAHMA NONOate), 3-morpholinosydnonimine (SIN-1), and L-N6-(1-iminoethyl)lysine (L-NIL) were purchased from Alexis Biochemicals (San Diego, CA). TNF-alpha and IFN-gamma were purchased from Boehringer Mannheim (Indianapolis, IN). DFMO was kindly supplied by Dr. E. Bohme, Hoechst Marion Roussel (Cincinnati, OH). All other chemicals were purchased from Sigma (St. Louis, MO), unless otherwise noted.

Cell preparations. A transformed proximal tubule cell line, MCT (20), was used for all experiments, except where noted. Other cell lines examined include J774 (monocyte/macrophage), mMC (mouse glomerular mesangial) (50), ENDO (rat glomerular endothelial) (27), NIH/3T3 (fibroblast like), Ras/3T3 (Ras-transformed NIH/3T3) (49), and HT-1080 (fibrosarcoma). All cells were plated and allowed to grow to near confluence in DMEM (GIBCO/BRL, Grand Island, NY) supplemented with 5% calf serum (Gemini Bio-Products, Calabasas, CA). Cell lines are from American Type Culture Collection (ATCC; Manassas, VA), unless otherwise referenced.

ODC activity. Cells were grown on 10-cm culture plates, harvested by first washing with 10 ml ice-cold PBS, placed in ice-cold ODC reaction buffer [10 mM Tris, pH 7.4, 2.5 mM dithiothreitol (DTT), 0.3 mM pyridoxyl-5-phosphate, and 0.1 mM EDTA], and scraped, collected, and homogenized. Cell preparations were then centrifuged at 30,000 g for 40 min at 4°C. The supernatant was collected and assayed for ODC activity as previously described (35). Briefly, samples were aliquoted at 250 µl in triplicate to large-bore glass tubes; reactions then were started by the addition of 10 µM, 0.1 µCi [14C]carboxyl-labeled L-ornithine (NEN, Boston, MA) to the cytosolic extracts. Tubes were capped with rubber stoppers fitted with metabolic wells (KONTES, Vineland, NJ) containing 250 µl of trapping agent (Solvable; Packard, Meriden, NJ). Incubations were for 1 h at 37°C. Reactions were stopped by injection of 200 µl 50% TCA and allowed to equilibrate for an additional 1 h before the collection of metabolic wells and counting trapped [14C]CO2 in a beta -scintillation counter.

Nitrite determination. Measurement of the NO end product nitrite production was used to assess relative values of NO released into the cell culture media. Nitrites were determined by the standard Greiss assay according to Green et. al. (17). Assays were carried out in 96-well plates and read in a Molecular Devices E max microplate reader at 550 nm.

Nitrate and nitrite determination. In assays where noted, nitrate was converted to nitrite by Escherichia coli nitrate reductase according to Bartholomew (5). The Greiss assay was then used to determine nitrite concentration, as above.

Transport studies. Cells were grown in six-well plates until nearly confluent. Wells were aspirated before addition of 1 ml DMEM containing 10 µM [3H]putrescine (NEN) at ~100,000 cpm/well. Uptake of [3H]putrescine by MCT cells was linear for >30 min (not shown). After the incubation (uptake) reaction wells were washed three times with 3 ml PBS and cells were lysed overnight in 1 ml 2 N NaOH and counted in a beta -scintillation counter. Nonspecific binding (blank) was determined as above, except the labeled [3H]putrescine addition was immediately terminated by aspiration and PBS washes.

Statistical evaluations. Variations between samples within groups were analyzed by ANOVA, with significance determined by Fisher's protected least-significant difference post hoc test. StatView software was used for these analyses.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

SNP suppresses ODC activity. We tested the possibility that the NO donor SNP could suppress ODC activity in the transformed kidney proximal tubule cell line MCT. The proximal tubule is the primary source of arginine synthesis in the kidney (28) and is known to have locally elevated arginase activity (29). These transformed cells also display constitutively elevated ODC activity. MCT cells were prepared for the ODC activity assay as described in MATERIALS AND METHODS. SNP was added to the homogenized cell preparation in the concentrations indicated in Fig. 1A at the start of the ODC assay reaction. SNP suppressed ODC activity in a dose-dependent fashion (Fig. 1A).


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Fig. 1.   Suppression of ornithine decarboxylase (ODC) activity by sodium nitroprusside (SNP). A: varying concentrations of SNP were added directly to the high-speed spin supernatant fraction of lysed MCT cell preparations in labeled ODC activity reaction. B: J774 (monocyte/macrophage), mMC (mouse glomerular mesangial), ENDO (endothelial), NIH/3T3 (fibroblast like), Ras/3T3 (Ras-transformed NIH/3T3), and HT-1080 (fibrosarcoma) cell lines were collected, homogenized, and incubated for 1 h in the absence (control) or presence of 0.5 mM SNP before a high-speed spin and the labeled ODC reaction. Values are means ± SD of 3 observations. For A and B, all SNP-treated samples were significantly different from control groups (P < 0.001).

We measured ODC activity in a number of established and transformed cell lines in the absence or presence of SNP (Fig. 1B). Primary cultures were not used because of their low inherent ODC activity. SNP markedly suppressed ODC activity in all cell lines examined.

NO donors suppress ODC activity. SNP suppression of ODC activity was previously described by Blachier et. al. (7) in a colon carcinoma cell line. To confirm that the suppressive effects of SNP are due to NO rather than other by-products of SNP, we examined the effects of several other NO donors on ODC activity in MCT cells. Three different redox groups of NO donors were represented: NONOates as NO · donors, S-nitrosothiols (SNAP is a less toxic alternative to SNP, GSNO a physiological S-nitrosothiol), which release NO · and transfer nitrosonium (NO+) to sulfhydryl centers (44), and SIN-1 as a peroxynitrite (OONO-) generator. Cell lysates were incubated for 1 h in the absence or presence of NO donors, as indicated in Fig. 2A, before the ODC assay. All NO donors displayed the capacity to suppress ODC activity. Comparative NO end product generation by the various NO donors is shown in Fig. 2B.


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Fig. 2.   Suppression of ODC activity by nitric oxide (NO) donors. A: MCT cells were collected, homogenized, and incubated for 1 h with NO donors (1 mM each) before a high-speed spin and the labeled ODC reaction. Cell preparations not incubated with NO donors served as controls. B: NO end product determination [nitrite + nitrate (NO2 + NO3)] was performed on media from NO donor incubation period. Values are means ± SD of 2 observations. For A and B, all experimental samples were significantly different from control (P < 0.005).

Cytokine generation of NO suppresses ODC activity. To determine if NO suppression of ODC activity was of biological significance, we induced iNOS generation of NO by TNF-alpha (2.5 ng/ml) and IFN-gamma (50 U/ml) in MCT cells. Cytokine stimulation significantly suppressed ODC activity (Fig. 3A). Incubating cells with L-NIL (50 mM), an iNOS selective inhibitor, markedly reduced NO generation in response to cytokines (Fig. 3B) and consequently reduced the suppressive effects of NO induction on ODC activity (Fig. 3A). Similar results were observed in the J774 cell line (not shown) or if lipopolysaccharide (20 ng/ml) and IFN-gamma were used to induce NO generation in MCT cells (not shown).


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Fig. 3.   Effect of cytokine induction of inducible NO synthase (iNOS) on ODC activity. A: MCT cells were grown in 10-cm culture dishes to ~80% confluent. Cells were then incubated for 1 h in the presence or absence of L-N6-(1-iminoethyl)lysine (L-NIL; 50 µM), an iNOS selective inhibitor, preceding an 18-h incubation in absence (Control) or presence of tumor necrosis factor-alpha (TNF-alpha ; 2.5 ng/ml) and interferon-gamma (IFN-gamma ; 50 U/ml) (TNF/IFN). TNF/IFN and TNF/IFN + L-NIL were significantly different from control (P < 0.0001 and P < 0.001, respectively). TNF/IFN + L-NIL was significantly different from TNF/IFN (P < 0.0001). B: NO2 determination was performed on media from overnight cytokine incubation period. Values are means ± SD of 3 observations.

Reversibility of ODC activity after NO treatment. To determine if NO inhibition of ODC activity involves nitrosylation of a sulfhydryl group, the effects of the thiol reductant DTT were evaluated. Cells were harvested and centrifuged as per MATERIALS AND METHODS, except that DTT was omitted from the ODC reaction buffer. Each sample was split and incubated in either the absence (control) or presence of 0.5 mM MAHMA NONOate for 10 min at room temperature. Varying concentrations of DTT, as shown in Fig. 4A, were then added to the control and NO-treated samples for 10 min before determination of ODC activity. MAHMA NONOate has a 1 min half-life, thereby alleviating the need to separate the NO donor from the sample by column purification before DTT addition. Increasing concentrations of DTT resulted in increased enzymatic activity of ODC in samples exposed to NO, relative to control samples in the presence of DTT alone (Fig. 4A).


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Fig. 4.   Assessment of nitrosylation of ODC by dithiothreitol (DTT) reversal of NO effects on ODC activity. A: MCT cells were prepared for ODC activity determination, except for exclusion of DTT in ODC reaction buffer. Cytosolic extracts were split and incubated in the absence (control) or presence of 0.5 mM (Z)-1-{N-methyl-N-[6-(N-methylammoniohexyl)amino]}diazen-1-ium-1,2-diolate (MAHMA NONOate) for 10 min. This was followed by a 10-min DTT incubation of control and NONOate-treated samples at the concentrations indicated. ODC activity was then evaluated. B: DTT reversal of TNF-alpha - and IFN-gamma -mediated ODC suppression. MCT cells were incubated with TNF-alpha and IFN-gamma for indicated times and then harvested in DTT-free ODC reaction buffer. After preparation, samples were split and incubated for 15 min in either 0.005 or 10 mM DTT before ODC activity assay. Nos. represent percent increase in ODC activity from 0.005 to 10 mM DTT, relative to non-cytokine-treated control group (set to 0%). C: NO2 determination was performed on media from B. Values are means ± SE of 3 observations. For B and C, * P < 0.01, ** P < 0.001 compared with control group.

The ability of DTT to reverse TNF-alpha - and IFN-gamma -mediated ODC suppression was also examined. Cytokines were administered to the cells for varying lengths of time, as shown in Fig. 4B. Cells were then washed in PBS and harvested in ODC reaction buffer without DTT and frozen. Thawed samples were centrifuged, and the supernatants were aliquoted into reaction tubes containing either 0.005 or 10 mM DTT for 15 min at room temperature before assessment of ODC activity. The percent increase of ODC activity from 0.005 to 10 mM DTT, relative to non-cytokine-treated samples (0-h cytokine stimulation), is shown in Fig. 4B for each time point. DTT reversal of ODC activity in cells stimulated for 24 h with TNF-alpha and IFN-gamma was ineffective (not shown). However, there was a significant effect of increased DTT concentration in cytokine-treated cells at 8, 10, and 12 h (Fig. 4B). A corresponding increase in NO end product generation was observed at these time points (Fig. 4C).

Polyamine transport is suppressed in the presence of NO. Administration of DFMO, a potent specific inhibitor of ODC, results in a compensatory increase in polyamine uptake (8). We evaluated the effects of NO suppression of ODC activity on polyamine transport to determine if non-antizyme-mediated inhibition of ODC consequently results in compensatory induction of transport. Such an effect could reestablish intracellular polyamine levels, negating polyamine limitation as a mechanism by which NO could inhibit proliferation. Although exceptions are known, most cells use a single transporter for putrescine, spermidine, and spermine (41). Competition studies in MCT cells suggest a single polyamine transporter (not shown). [3H]putrescine uptake by MCT cells was therefore used as an indicator of polyamine transport in these studies. Transformed cells commonly display increased polyamine uptake, and Fig. 5 demonstrates rapid transport of [3H]putrescine into MCT cells. The effects of DFMO require time to evolve. The presence of DFMO did not change putrescine uptake relative to control values at 6 h, but increases were observed at 24 (105% increase over control) and 48 h (270% increase over control; not shown). In the presence of NO donors a dose- and time-dependent suppression of putrescine uptake was observed (Fig. 5). ENDO cells treated with SIN-1 gave similar results (not shown).


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Fig. 5.   Effect of difluoromethylornithine (DFMO) and NO donors on polyamine transport. MCT cells were grown in 6-well plates to ~80-90% confluent. Cells were then incubated in absence (Control) or presence of DFMO (5 mM), a synthetic suicide inhibitor of ODC, or NO donors S-nitroso-L-glutathione (GSNO; 0.1 and 0.5 mM), (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA NONOate; 0.1 and 0.5 mM), or 3-morpholinosydnonimine (SIN-1; 0.5 mM) for 6 or 24 h before a 5-min uptake period of [3H]putrescine. Values are means ± SD of 3 observations. * P < 0.05, ** P < 0.001, *** P < 0.0001 compared with control group.

Cytokine administration suppresses polyamine transport. To determine the biological significance of NO suppression of polyamine transport we used TNF-alpha and IFN-gamma to induce iNOS generation of NO in MCT cells, as in Fig. 3. Cytokine stimulation significantly suppressed [3H]putrescine uptake in MCT cells (Fig. 6A). Incubating cells with L-NIL (Fig. 6), an iNOS selective inhibitor, or 2,4-diamino-6-hydroxy-pyrimidine (DAHP) (not shown), an inhibitor of the synthesis of the NOS cofactor tetrahydrobiopterin (BH4), markedly reduced NO generation in response to cytokines (Fig. 6B), but had little effect in arresting cytokine-mediated suppression of [3H]putrescine uptake (Fig. 6A). TNF-alpha and IFN-gamma appear to act additively in suppressing polyamine transport (Fig. 6A).


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Fig. 6.   Effect of TNF-alpha and IFN-gamma on polyamine transport. A: MCT cells were grown in 6-well plates to ~80% confluent. Cells were then incubated for 1 h in presence or absence of L-NIL (50 µM), an iNOS selective inhibitor, preceding a 24-h incubation in absence (Control) or presence of TNF-alpha (2.5 ng/ml) and IFN-gamma (50 U/ml) in combination (TNF/IFN) or separately (TNF, IFN). Polyamine transport was determined by a 5-min uptake period of [3H]putrescine. TNF/IFN, TNF/IFN + L-NIL, TNF, and IFN were all significantly different from control (P < 0.001). TNF/IFN + L-NIL was significantly different from TNF/IFN (P < 0.05). B: NO2 determination was performed on media from overnight cytokine incubation period. Values are means ± SD of 3 observations.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Limiting intracellular polyamine stores required for entry and progression through the cell cycle may prove a viable way of inhibiting proliferation. We have recently observed suppression of polyamine biosynthesis and transport in a transformed cell line through polyamine-independent induction of antizyme (39). Rapid depletion of putrescine and spermidine was observed with consequent inhibition of proliferation. Here we demonstrate that NO is capable of suppressing both polyamine biosynthesis and transport.

NO donors suppress the activity of the polyamine biosynthetic enzyme ODC in MCT cell extracts and in extracts of all cell lines examined (Fig. 1B), suggesting this effect is not cell type specific. NO donors differed in their ability to suppress ODC activity (Fig. 2A). Varying cellular responses to different redox forms of NO were previously noted (13). NO generated through cytokine induction of iNOS is also capable of suppressing ODC activity. This suppression is diminished in the presence of L-NIL, a selective inhibitor of iNOS (Fig. 3A). These data suggest that the effect on the enzyme is NO mediated and that cytokine-inducible levels of NO are capable of suppressing ODC activity.

Intracellular polyamine levels are autoregulated through translational frameshift induction of a unique regulatory protein, antizyme (31). Inhibition of polyamine biosynthesis occurs by antizyme binding to ODC and suppressing ODC activity as well as rendering the enzyme susceptible to proteolysis by the 26S proteosome in a ubiquitin-independent manner (21, 34). A 30- to 60-min lag phase is prerequisite to the onset of antizyme inhibition, with maximum inhibition observed by 4 h in MCT cells (39). Suppression of ODC activity by NO is rapid, as demonstrated in Fig. 1A, where the NO donor SNP is added directly to the enzymatic reaction. These results are not temporally compatible with those of antizyme. However, a rapid transition of enzymatic activity could result from cysteine nitrosylation, which has been shown to modulate the activity of several enzymes (14-16, 36, 43, 44). Active ODC is a homodimer containing a cysteine within the active site at the dimer interface (10). A C360A mutation of this cysteine in ODC has revealed this residue to be an absolute requirement for full activity (9, 10). Because of the rapid interchange of enzymatic subunits under physiological conditions (10), this cysteine may be subject to nitrosylation. DTT reversal of NO's inhibitory effects on ODC activity (Fig. 4) is consistent with nitrosylation as the mechanism of inhibition (19, 32). In our hands, TNF-alpha and IFN-gamma stimulation of MCT cells results in an observable increase in iNOS mRNA, as determined by Northern blotting (unpublished data), and an increase in NO end product generation by 6 h (Fig. 4C). DTT reversal of cytokine-mediated ODC suppression at 8, 10, and 12 h (Fig. 4B) is in temporal accord with these observations. The ability of DTT to reverse ODC inhibition by TNF-alpha and IFN-gamma shortly after induction of NO generation suggests nitrosylation as an early event in this response. The ineffective reversal of ODC activity by DTT in cells stimulated for 24 h (not shown) implies that other cytokine-mediated mechanisms suppress ODC activity by this later time point. We show here that cytokine-mediated suppression of ODC activity in cells treated for 24 h can be largely averted in the presence of the iNOS inhibitor L-NIL (Fig. 3), suggesting that NO may be required for induction of some of these other mechanisms. Cytokines may additionally suppress ODC activity through NO-independent mechanisms. The time frame and level(s) of cytokine regulation of ODC activity require further investigation.

Suppression of polyamine transporters is a second function in the regulation of intracellular polyamines ascribed to antizyme (33, 47). It is this two-pronged mechanism of antizyme that distinguishes it from synthetic ODC inhibitors, such as DFMO. DFMO inhibition of ODC causes a compensatory induction of polyamine transporters, allowing polyamine uptake to substitute for de novo biosynthesis (8) (Fig. 5). In vivo, tumor cells can access polyamines released into the circulation by normal cells, wasting cells, gut flora, and dietary sources. A compensatory increase in polyamine transport can explain why drugs targeted exclusively at inhibition of ODC often did not result in the expected levels of polyamine depletion (6, 23, 30), thus complicating experimental interpretation and yielding less than anticipated results in clinical trials (22). However, coadministration of DFMO with a polyamine-free diet (38, 42) or a polyamine transport inhibitor (4) was beneficial in experimental cancer models in vivo and in vitro, respectively. We therefore addressed the effects of NO donors on polyamine transport as well as polyamine biosynthesis. Administration of NO donors does not result in the compensatory increase observed with DFMO, but rather a suppression of polyamine uptake (Fig. 5). At 6 h, only the high concentrations of NO donors demonstrate an effect on polyamine uptake. By 24 h the effects of the NO donors, as well as DFMO, are more apparent (Fig. 5). Thus NO modulation of polyamine transport is a gradual process. The time course for the inhibition of polyamine transporters by antizyme is similar to that for ODC, that is, it occurs rapidly after a short lag phase of <1 h (39, 47). Therefore, the slow onset of polyamine transport suppression by NO occurs in a manner that appears temporally distinct from a directly mediated event, such as nitrosylation, or that described for antizyme. Whether NO indirectly affects antizyme induction has yet to be investigated.

The iNOS-inducing cytokines TNF-alpha and IFN-gamma also suppressed polyamine uptake in MCT cells. However, neither L-NIL (Fig. 6A) nor DAHP, a NOS cofactor inhibitor (not shown), was able to substantially attenuate these effects. It should be noted that neither L-NIL (Fig. 6B) nor DAHP (not shown) was able to completely inhibit cytokine-stimulated NO generation. Therefore we cannot unequivocally state that the effects of IFN-gamma on polyamine transport are NO independent. However, as shown in Fig. 6B, inhibition of polyamine transport by TNF-alpha cannot be attributed to NO generation. Although our data demonstrate that NO donors suppress polyamine transport (Fig. 5), the presence of cytokines establishes a complex biological setting where polyamine transport may be affected by a variety of mediators (for review, see Refs. 18, 40). Furthermore, the inability of DTT to effectively reverse cytokine inhibition of ODC activity at 24 h (not shown) supports a complex and redundant regulation of intracellular polyamines during inflammation.

These data present NO as the first described endogenous molecule with the capacity to inhibit both polyamine biosynthesis and transport in a manner that appears independent of antizyme. Direct inhibition of ODC by NO, without a compensatory increase in polyamine uptake, could allow depletion of intracellular polyamine levels required for replication. NO inhibition of ODC activity could also provide a direct mechanism for the temporal interregulation of these two arginine pathways during inflammation, as has been observed in wound healing and experimental glomerulonephritis (see Fig. 7) (2, 11, 25). The capacity of the cytokines TNF-alpha and IFN-gamma to suppress polyamine uptake could effectively contribute to the inhibitory effects of NO on polyamine biosynthesis in regulating intracellular polyamines.


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Fig. 7.   Arginine metabolic pathways and their temporal relationship in inflammation. Proposed role of NO and inflammatory cytokines in modulating intracellular polyamines through suppression of polyamine biosynthesis (1) and transport (2 and 3) in inflammation, and as cytostatic agents. Pathway 1 may be initially mediated by direct nitrosylation by NO, whereas 2 and 3 appear to be modulated by as yet undetermined intermediates. Oval in cell membrane represents polyamine transporters; bars represent negative regulation. OAT, ornithine aminotransferase.


    ACKNOWLEDGEMENTS

We thank E. G. Neilson (mMC), H. Holthofer (ENDO), and F. C. White and M. Kamps (Ras/3T3) for kindly donating cell lines.


    FOOTNOTES

This work was supported by National Institutes of Health Grants DK-42155, DK-28602, HL-48108, and T32HL-07261, and the Medical Research Service Veterans Affairs Central Office. C. J. Kelly is a Clinical Investigator of Medical Research Service, Veterans Affairs Central Office; M. J. Lortie is a National Kidney Foundation Fellow.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: J. Satriano, UCSD/VAMC, Div. Nephrology-Hypertension, mail code 9111 H, 3350 La Jolla Village Dr., San Diego, CA 92161 (E-mail: jsatriano{at}ucsd.edu).

Received 24 August 1998; accepted in final form 15 January 1999.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Albina, J. E., and W. L. Henry, Jr. Suppression of lymphocyte proliferation through the nitric oxide synthesizing pathway. J. Surg. Res. 50: 403-409, 1991[Medline].

2.   Albina, J. E., C. D. Mills, W. L. Henry, Jr., and M. D. Caldwell. Temporal expression of different pathways of L-arginine metabolism in healing wounds. J. Immunol. 144: 3877-3880, 1990[Abstract/Free Full Text].

3.   Auvinen, M., A. Paasinen, L. C. Andersson, and E. Holtta. Ornithine decarboxylase activity is critical for cell transformation. Nature 360: 355-358, 1992[Medline].

4.   Aziz, S. M., M. N. Gillespie, P. A. Crooks, S. F. Tofiq, C. P. Tsuboi, J. W. Olson, and M. P. Gosland. The potential of a novel polyamine transport inhibitor in cancer chemotherapy. J. Pharmacol. Exp. Ther. 278: 185-192, 1996[Abstract].

5.   Bartholomew, B. A rapid method for the assay of nitrate in urine using the nitrate reductase enzyme of Escherichia coli. Food Chem. Toxicol. 22: 541-543, 1984[Medline].

6.   Beyer, H. S., M. Ellefson, M. Stanley, and L. Zieve. Inhibition of increases in ornithine decarboxylase and putrescine has no effect on rat liver regeneration. Am. J. Physiol. 262 (Gastrointest. Liver Physiol. 25): G677-G684, 1992[Abstract/Free Full Text].

7.   Blachier, F., V. Robert, M. Selamnia, C. Mayeur, and P. H. Duee. Sodium nitroprusside inhibits proliferation and putrescine synthesis in human colon carcinoma cells. FEBS Lett. 396: 315-318, 1996[Medline].

8.   Bogle, R. G., G. E. Mann, J. D. Pearson, and D. M. Morgan. Endothelial polyamine uptake: selective stimulation by L-arginine deprivation or polyamine depletion. Am. J. Physiol. 266 (Cell Physiol. 35): C776-C783, 1994[Abstract/Free Full Text].

9.   Coleman, C. S., B. A. Stanley, and A. E. Pegg. Effect of mutations at active site residues on the activity of ornithine decarboxylase and its inhibition by active site-directed irreversible inhibitors. J. Biol. Chem. 268: 24572-24579, 1993[Abstract/Free Full Text].

10.   Coleman, C. S., B. A. Stanley, R. Viswanath, and A. E. Pegg. Rapid exchange of subunits of mammalian ornithine decarboxylase. J. Biol. Chem. 269: 3155-3158, 1994[Abstract/Free Full Text].

11.   Cook, H. T., A. Jansen, S. Lewis, P. Largen, M. O'Donnell, D. Reaveley, and V. Cattell. Arginine metabolism in experimental glomerulonephritis: interaction between nitric oxide synthase and arginase. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F646-F653, 1994[Abstract/Free Full Text].

12.  De Groote, M. A., and F. C. Fang. NO inhibitions: antimicrobial properties of nitric oxide. Clin. Infect. Dis. 1, Suppl. 2: S162-S165, 1995.

13.   De Groote, M. A., D. Granger, Y. Xu, G. Campbell, R. Prince, and F. C. Fang. Genetic and redox determinants of nitric oxide cytotoxicity in a Salmonella typhimurium model. Proc. Natl. Acad. Sci. USA 92: 6399-6403, 1995[Abstract].

14.   Dimmeler, S., J. Haendeler, M. Nehls, and A. M. Zeiher. Suppression of apoptosis by nitric oxide via inhibition of interleukin-1beta -converting enzyme (ICE)-like and cysteine protease protein (CPP)-32-like proteases. J. Exp. Med. 185: 601-607, 1997[Abstract/Free Full Text].

15.   Gergel, D., and A. I. Cederbaum. Inhibition of the catalytic activity of alcohol dehydrogenase by nitric oxide is associated with S nitrosylation and the release of zinc. Biochemistry 35: 16186-16194, 1996[Medline].

16.   Gopalakrishna, R., Z. H. Chen, and U. Gundimeda. Nitric oxide and nitric oxide-generating agents induce a reversible inactivation of protein kinase C activity and phorbol ester binding. J. Biol. Chem. 268: 27180-27185, 1993[Abstract/Free Full Text].

17.   Green, L. C., D. A. Wagner, J. Glogowski, P. L. Skipper, J. S. Wishnok, and S. R. Tannenbaum. Analysis of nitrate, nitrite, and [15N]nitrate in biological fluids. Anal. Biochem. 126: 131-138, 1982[Medline].

18.   Grillo, M. A., and S. Colombatto. Polyamine transport in cells. Biochem. Soc. Trans. 22: 894-898, 1994[Medline].

19.   Gross, W. L., M. I. Bak, J. S. Ingwall, M. A. Arstall, T. W. Smith, J. L. Balligand, and R. A. Kelly. Nitric oxide inhibits creatine kinase and regulates rat heart contractile reserve. Proc. Natl. Acad. Sci. USA 93: 5604-5609, 1996[Abstract/Free Full Text].

20.   Haverty, T. P., C. J. Kelly, W. H. Hines, P. S. Amenta, M. Watanabe, R. A. Harper, N. A. Kefalides, and E. G. Neilson. Characterization of a renal tubular epithelial cell line which secretes the autologous target antigen of autoimmune experimental interstitial nephritis. J. Cell Biol. 107: 1359-1368, 1988[Abstract].

21.   Hayashi, S., Y. Murakami, and S. Matsufuji. Ornithine decarboxylase antizyme: a novel type of regulatory protein. Trends Biochem. Sci. 21: 27-30, 1996[Medline].

22.   Horn, Y., P. J. Schechter, and L. J. Marton. Phase I-II clinical trial with alpha-difluoromethylornithine---an inhibitor of polyamine biosynthesis. Eur. J. Cancer Clin. Oncol. 23: 1103-1107, 1987[Medline].

23.   Humphreys, M. H., S. B. Etheredge, S. Y. Lin, J. Ribstein, and L. J. Marton. Renal ornithine decarboxylase activity, polyamines, and compensatory renal hypertrophy in the rat. Am. J. Physiol. 255 (Renal Fluid Electrolyte Physiol. 24): F270-F277, 1988[Abstract/Free Full Text].

24.   Jenkinson, C. P., W. W. Grody, and S. D. Cederbaum. Comparative properties of arginases. Comp. Biochem. Physiol. B Biochem. Mol. Biol. 114: 107-132, 1996[Medline].

25.   Ketteler, M., W. A. Border, and N. A. Noble. Cytokines and L-arginine in renal injury and repair. Am. J. Physiol. 267 (Renal Fluid Electrolyte Physiol. 36): F197-F207, 1994[Abstract/Free Full Text].

26.   Kurata, S., M. Matsumoto, and U. Yamashita. Concomitant transcriptional activation of nitric oxide synthase and heme oxygenase genes during nitric oxide-mediated macrophage cytostasis. J. Biochem. (Tokyo) 120: 49-52, 1996[Abstract].

27.   Laulajainen, T., I. Julkunen, A. Haltia, S. Knuutila, A. Miettinen, and H. Holthofer. Establishment and characterization of a rat glomerular endothelial cell line. Lab. Invest. 69: 183-192, 1993[Medline].

28.   Levillain, O., A. Hus-Citharel, F. Morel, and L. Bankir. Localization of arginine synthesis along rat nephron. Am. J. Physiol. 259 (Renal Fluid Electrolyte Physiol. 28): F916-F923, 1990[Abstract/Free Full Text].

29.   Levillain, O., A. Hus-Citharel, F. Morel, and L. Bankir. Localization of urea and ornithine production along mouse and rabbit nephrons: functional significance. Am. J. Physiol. 263 (Renal Fluid Electrolyte Physiol. 32): F878-F885, 1992[Abstract/Free Full Text].

30.   Luck, M. S., and P. Bass. Inhibition of ornithine decarboxylase does not prevent intestinal smooth muscle hyperplasia in the rat. Am. J. Physiol. 267 (Gastrointest. Liver Physiol. 30): G1021-G1027, 1994[Abstract/Free Full Text].

31.   Matsufuji, S., T. Matsufuji, Y. Miyazaki, Y. Murakami, J. F. Atkins, R. F. Gesteland, and S. Hayashi. Autoregulatory frameshifting in decoding mammalian ornithine decarboxylase antizyme. Cell 80: 51-60, 1995[Medline].

32.   McDonald, L. J., and J. Moss. Stimulation by nitric oxide of an NAD linkage to glyceraldehyde-3-phosphate dehydrogenase. Proc. Natl. Acad. Sci. USA 90: 6238-6241, 1993[Abstract].

33.   Mitchell, J. L., G. G. Judd, A. Bareyal-Leyser, and S. Y. Ling. Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells. Biochem. J. 299: 19-22, 1994[Medline].

34.   Murakami, Y., S. Matsufuji, T. Kameji, S. Hayashi, K. Igarashi, T. Tamura, K. Tanaka, and A. Ichihara. Ornithine decarboxylase is degraded by the 26S proteasome without ubiquitination. Nature 360: 597-599, 1992[Medline].

35.   Packham, G., and J. L. Cleveland. Ornithine decarboxylase is a mediator of c-Myc-induced apoptosis. Mol. Cell. Biol. 14: 5741-5747, 1994[Abstract].

36.   Padgett, C. M., and A. R. Whorton. S-nitrosoglutathione reversibly inhibits GAPDH by S-nitrosylation. Am. J. Physiol. 269 (Cell Physiol. 38): C739-C749, 1995[Abstract].

37.   Peunova, N., and G. Enikolopov. Nitric oxide triggers a switch to growth arrest during differentiation of neuronal cells. Nature 375: 68-73, 1995[Medline].

38.   Quemener, V., Y. Blanchard, L. Chamaillard, R. Havouis, B. Cipolla, and J. P. Moulinoux. Polyamine deprivation: a new tool in cancer treatment. Anticancer Res. 14: 443-448, 1994[Medline].

39.   Satriano, J., S. Matsufuji, Y. Murakami, M. J. Lortie, D. Schwartz, C. J. Kelly, S. Hayashi, and R. C. Blantz. Agmatine suppresses proliferation by frameshift induction of antizyme and attenuation of cellular polyamine levels. J. Biol. Chem. 273: 15313-15316, 1998[Abstract/Free Full Text].

40.   Seiler, N., J. G. Delcros, and J. P. Moulinoux. Polyamine transport in mammalian cells. An update. Int. J. Biochem. Cell Biol. 28: 843-861, 1996[Medline].

41.   Seiler, N., and F. Dezeure. Polyamine transport in mammalian cells. Int. J. Biochem. 22: 211-218, 1990[Medline].

42.   Seiler, N., S. Sarhan, C. Grauffel, R. Jones, B. Knodgen, and J. P. Moulinoux. Endogenous and exogenous polyamines in support of tumor growth. Cancer Res. 50: 5077-5083, 1990[Abstract].

43.   Simon, D. I., M. E. Mullins, L. Jia, B. Gaston, D. J. Singel, and J. S. Stamler. Polynitrosylated proteins: characterization, bioactivity, and functional consequences. Proc. Natl. Acad. Sci. USA 93: 4736-4741, 1996[Abstract/Free Full Text].

44.   Stamler, J. S. Redox signaling: nitrosylation and related target interactions of nitric oxide. Cell 78: 931-936, 1994[Medline].

45.   Stein, C. S., Z. Fabry, S. Murphy, and M. N. Hart. Involvement of nitric oxide in IFN-gamma-mediated reduction of microvessel smooth muscle cell proliferation. Mol. Immunol. 32: 965-973, 1995[Medline].

46.   Stuehr, D. J., and C. F. Nathan. Nitric oxide. A macrophage product responsible for cytostasis and respiratory inhibition in tumor target cells. J. Exp. Med. 169: 1543-1555, 1989[Abstract].

47.   Suzuki, T., Y. He, K. Kashiwagi, Y. Murakami, S. Hayashi, and K. Igarashi. Antizyme protects against abnormal accumulation and toxicity of polyamines in ornithine decarboxylase-overproducing cells. Proc. Natl. Acad. Sci. USA 91: 8930-8934, 1994[Abstract].

48.   Vincendeau, P., and S. Daulouede. Macrophage cytostatic effect on Trypanosoma musculi involves an L-arginine-dependent mechanism. J. Immunol. 146: 4338-4343, 1991[Abstract/Free Full Text].

49.   White, F. C., A. Benehacene, J. S. Scheele, and M. Kamps. VEGF mRNA is stabilized by ras and tyrosine kinase oncogenes, as well as by UV radiation---evidence for divergent stabilization pathways. Growth Factors 14: 199-212, 1997[Medline].

50.   Wolf, G., U. Haberstroh, and E. G. Neilson. Angiotensin II stimulates the proliferation and biosynthesis of type I collagen in cultured murine mesangial cells. Am. J. Pathol. 140: 95-107, 1992[Abstract].


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