Intracellular Spermidine Research Articles

Effects of novel C-methylated spermidine analogs on cell growth via hypusination of eukaryotic translation initiation factor 5A

The polyamines, putrescine, spermidine, and spermine, are ubiquitous multifunctional cations essential for cellular proliferation. One specific function of spermidine in cell growth is its role as a butylamine donor for hypusine synthesis in the eukaryotic initiation factor 5A (eIF5A). Here, we report the ability of novel mono-methylated spermidine analogs (α-MeSpd, β-MeSpd, γ-MeSpd, and ω-MeSpd) to function in the hypusination of eIF5A and in supporting the growth of DFMO-treated DU145 cells. We also tested them as substrates and inhibitors for deoxyhypusine synthase (DHS) in vitro. Of these compounds, α-MeSpd, β-MeSpd, and γ-MeSpd (but not ω-MeSpd) were substrates for DHS in vitro, while they all inhibited the enzyme reaction. As racemic mixtures, only α-MeSpd and β-MeSpd supported long-term growth (9-18 days) of spermidine-depleted DU145 cells, whereas γ-MeSpd and ω-MeSpd did not. The S-enantiomer of α-MeSpd, which supported long-term growth, was a good substrate for DHS in vitro, whereas the R-isomer was not. The long-term growth of DFMO-treated cells correlated with the hypusine modification of eIF5A by intracellular methylated spermidine analogs. These results underscore the critical requirement for hypusine modification in mammalian cell proliferation and provide new insights into the specificity of the deoxyhypusine synthase reaction.

Overexpression of spermidine/spermine N 1-acetyltransferase or treatment with N 1-N 11-diethylnorspermine attenuates the severity of zinc-induced pancreatitis in mouse

Depletion of pancreatic intracellular polyamine pools has been observed in acute pancreatitis both in the animal models and in humans. In this study, the wild-type mice, polyamine catabolic enzyme spermidine/spermine N(1)-acetyltransferase overexpressing (SSAT mice) and SSAT-deficient mice were used to characterize the new zinc-induced acute pancreatitis mouse model and study the role of polyamines and polyamine catabolism in this model. Intraperitoneal zinc injection induced acute necrotizing pancreatitis in wild-type mice as well as in SSAT-overexpressing and SSAT-deficient mice. Serum α-amylase activity was significantly increased in all zinc-treated mice compared with the untreated controls. However, the α-amylase activities in SSAT mice were constantly lower than those in the other groups. Histopathological examination of pancreatic tissue revealed edema, acinar cell necrosis and necrotizing inflammation, typical for acute pancreatitis. Compared with the other zinc-treated mice less damage according to the histopathological analysis was observed in the pancreatic tissue of SSAT mice. Levels of intracellular spermidine, and occasionally spermine, were significantly decreased in pancreases of all zinc-treated animals and SSAT enzyme activity was enhanced both in wild-type and SSAT mice. Interestingly, a spermine analog, N(1), N(11)-diethylnorspermine (DENSpm), enhanced the proliferation of pancreatic cells and reduced the severity of zinc-induced pancreatitis in wild-type mice. The results show that in mice a single intraperitoneal zinc injection causes acute necrotizing pancreatitis accompanied by decrease of intracellular polyamine pools. The study supports the important role of polyamines for the integrity and function of the pancreas. In addition, the study suggests that whole body overexpression of SSAT obtained in SSAT mice reduces inflammatory pancreatic cell injury.

Growth-related changes in intracellular spermidine and its effect on efflux pump expression and quorum sensing in Burkholderia pseudomallei

The Burkholderia pseudomallei BpeAB-OprB resistance-nodulation-division (RND) family pump effluxes aminoglycoside and macrolide antibiotics as well as acylhomoserine lactones (AHLs) involved in quorum sensing. Expression of bpeA-lacZ was cell density-dependent and was inducible in the presence of these compounds. Intracellular levels of spermidine and N-acetylspermidine increased with cell density in wild-type B. pseudomallei KHW, but were always lower in the bpeAB pump mutant at all growth phases. The significance of changes in intracellular spermidine on efflux pump expression was demonstrated by the disruption of the binding of the BpeR repressor protein to the bpeABoprB regulatory region in vitro in the presence of increasing spermidine concentrations. This was supported by dose-dependent activation of bpeA-lacZ transcription in vivo in the presence of exogenous spermidine and N-acetylspermidine, thus implicating the involvement of the BpeAB-OprB pump in spermidine homeostasis in B. pseudomallei. Consequently, inhibition of intracellular spermidine synthesis reduced the efflux of AHLs by BpeAB-OprB. Other potential therapeutic applications of spermidine synthase inhibitors include the reduction of swimming motility and biofilm formation by B. pseudomallei.

Spermine analogue-regulated expression of spermidine/spermine N1-acetyltransferase and its effects on depletion of intracellular polyamine pools in mouse fetal fibroblasts

SSAT (Spermidine/spermine N1-acetyltransferase, also known as SAT1), the key enzyme in the catabolism of polyamines, is turned over rapidly and there is only a low amount present in the cell. In the present study, the regulation of SSAT by spermine analogues, the inducers of the enzyme, was studied in wild-type mouse fetal fibroblasts, expressing endogenous SSAT, and in the SSAT-deficient mouse fetal fibroblasts transiently expressing an SSAT-EGFP (enhanced green fluorescent protein) fusion gene. In both cell lines treatments with DENSpm (N(1),N(11)-diethylnorspermine), CPENSpm (N(1)-ethyl-N(11)-[(cyclopropyl)-methy]-4,8-diazaundecane) and CHENSpm (N(1)-ethyl-N(11)-[(cycloheptyl)methy]-4,8-diazaundecane) led to high, moderate or low induction of SSAT activity respectively. The level of activity detected correlated with the presence of SSAT and SSAT-EGFP proteins, the latter localizing both in the cytoplasm and nucleus. RT-PCR (reverse transcription-PCR) results suggested that the analogue-affected regulation of SSAT-EGFP expression occurred, mainly, after transcription. In wild-type cells, DENSpm increased the amount of SSAT mRNA, and both DENSpm and CHENSpm affected splicing of the SSAT pre-mRNA. Depleted intracellular spermidine and spermine levels inversely correlated with detected SSAT activity. Interestingly, the analogues also reduced polyamine levels in the SSAT-deficient cells expressing the EGFP control. The results from the present study show that the distinct SSAT regulation by different analogues involves regulatory actions at multiple levels, and that the spermine analogues, in addition to inducing SSAT, lower intracellular polyamine pools by SSAT-independent mechanisms.

Inactivation of KsgA, a 16S rRNA Methyltransferase, Causes Vigorous Emergence of Mutants with High-Level Kasugamycin Resistance

The methyltransferases RsmG and KsgA methylate the nucleotides G535 (RsmG) and A1518 and A1519 (KsgA) in 16S rRNA, and inactivation of the proteins by introducing mutations results in acquisition of low-level resistance to streptomycin and kasugamycin, respectively. In a Bacillus subtilis strain harboring a single rrn operon (rrnO), we found that spontaneous ksgA mutations conferring a modest level of resistance to kasugamycin occur at a high frequency of 10(-6). More importantly, we also found that once cells acquire the ksgA mutations, they produce high-level kasugamycin resistance at an extraordinarily high frequency (100-fold greater frequency than that observed in the ksgA(+) strain), a phenomenon previously reported for rsmG mutants. This was not the case for other antibiotic resistance mutations (Tsp(r) and Rif(r)), indicating that the high frequency of emergence of a mutation for high-level kasugamycin resistance in the genetic background of ksgA is not due simply to increased persistence of the ksgA strain. Comparative genome sequencing showed that a mutation in the speD gene encoding S-adenosylmethionine decarboxylase is responsible for the observed high-level kasugamycin resistance. ksgA speD double mutants showed a markedly reduced level of intracellular spermidine, underlying the mechanism of high-level resistance. A growth competition assay indicated that, unlike rsmG mutation, the ksgA mutation is disadvantageous for overall growth fitness. This study clarified the similarities and differences between ksgA mutation and rsmG mutation, both of which share a common characteristic--failure to methylate the bases of 16S rRNA. Coexistence of the ksgA mutation and the rsmG mutation allowed cell viability. We propose that the ksgA mutation, together with the rsmG mutation, may provide a novel clue to uncover a still-unknown mechanism of mutation and ribosomal function.

Degrading Polyamines for Migration

Integrins are heterodimeric transmembrane proteins that mediate cell adhesion and migration through various mechanisms. Binding of the cytoplasmic domain of the integrin α9 subunit to the spermidine/spermine- N 1 -acetyltransferase (SSAT)--which induces the breakdown of the higher-order polyamines spermidine and spermine to the lower-order polyamine putrescine--is required for migration mediated by the α9β1 integrin. deHart et al. (see commentary by Vandenberg) report that α9 recruitment of SSAT to focal adhesions affects cell migration by modulating the activity of an inward rectifier K + (Kir) channel. Both wild-type and catalytically inactive forms of in vitro-translated SSAT bound to tagged α9 in a pull-down assay. Expression of any of the catalytically inactive forms in α9-expressing Chinese hamster ovary (CHO) cells prevented migration on an α9-specific ligand in a dominant-negative fashion. This effect was specific to α9, because migration mediated by a chimeric α9α5 protein, in which the intracellular domain of α9 was replaced with the intracellular domain of α5, was not affected by expression of the catalytically inactive SSATs. Depleting intracellular spermidine concentrations, using a combination of the ornithine decarboxylase inhibitor DFMO and putrescine, inhibited migration of CHO cells that overexpressed wild-type α9 but not those expressing the α9α5 chimera. Unexpectedly, restoring spermidine concentration by addition of putrescine in DFMO-treated cells restored migration. Furthermore, in mouse embryo fibroblasts (MEFs) overexpressing α9, but not those overexpressing the chimeric α9α5, migration was reduced when the expression of polyamine oxidase (PAO), an enzyme required for polyamine metabolism downstream of SSAT, was knocked down by small interfering RNAs (siRNAs). Adding putrescine did not restore migration to the PAO-deficient cells. Thus, the results from experiments in which global intracellular polyamine concentrations were altered with pharmacological agents were complex but may indicate that local changes in cellular polyamine concentrations at the focal adhesion regulate migration. K + efflux is implicated in cell migration, and Kir channels are inhibited by spermidine and spermine; thus, SSAT may modulate polyamine concentrations to stimulate migration. Indeed, treating α9- (but not α9α4- or α9α5-) expressing MEFs with Ba 2+ to inhibit Kir channels or with a α9β1-blocking antibody decreased migration of cells on an α9-specific ligand but had no effect on adhesion of cells to nonintegrin-specific substrates. Of the eight Kir subunits detected in MEF cells, only Kir4.2 appeared to have a role in α9-mediated migration. Knockdown of Kir4.2 by a short hairpin RNA (shRNA) or inhibition of Kir channels with Ba 2+ in either α9-expressing MEFs or human microvascular endothelial cells, which natively express Kir4.2 and α9β1, impaired migration on α9 substrates. In the MEFs expressing α9, the effects of DFMO and Kir4.2 shRNA treatment were not additive, suggesting that polyamines regulate cell migration through Kir4.2. In an in vitro wound assay, α9-expressing MEF cells treated with Ba 2+ , Kir4.2 shRNA, or both migrated shorter distances than untreated cells and extended multiple leading-edge cytoplasmic projections, whereas untreated cells usually extended only one lamellopodium. Fluorescently tagged α9 and Kir4.2 colocalized to structures that looked like focal adhesions, but only at the leading edge of MEFs. These results suggest that the α9 subunit of integrins recruits SSAT to focal adhesions, where it decreases the local concentration of higher-order polyamines; this may relieve repression of Kir channels, thus initiating an outward K + flow. The authors model that the K + efflux may cause the influx of Ca 2+ , which is known to stimulate migration in other contexts. G. W. deHart, T. Jin, D. E. McCloskey, A. E. Pegg, D. Sheppard, The α9β1 integrin enhances cell migration by polyamine-mediated modulation of an inward-rectifier potassium channel. Proc. Natl. Acad. Sci. U.S.A. 105 , 7188-7193 (2008). [Abstract] [Full Text] C. A. Vandenberg, Integrins step up the pace of cell migration through polyamines and potassium channels. Proc. Natl. Acad. Sci. U.S.A. 105 , 7109-7110 (2008). [Full Text]

Hypusine modification for growth is the major function of spermidine in Saccharomyces cerevisiae polyamine auxotrophs grown in limiting spermidine

Spermidine and its derivative, hypusinated eIF5A, are essential for the growth of Saccharomyces cerevisiae. Very low concentrations of spermidine (10(-8) M) are sufficient for the growth of S. cerevisiae polyamine auxotrophs (spe1Delta, spe2Delta, and spe3Delta). Under these conditions, even though the growth rate is near normal, the internal concentration of spermidine is <0.2% of the spermidine concentration present in wild-type cells. When spe2Delta cells are grown with low concentrations of spermidine, there is a large decrease in the amount of hypusinated eukaryotic initiation factor 5A (eIF5A) (1/20 of normal), even though there is no change in the amount of total (modified plus unmodified) eIF5A. It is striking that, as intracellular spermidine becomes limiting, an increasing portion of it (up to 54%) is used for the hypusine modification of eIF5A. These data indicate that hypusine modification of eIF5A is a most important function for spermidine in supporting the growth of S. cerevisiae polyamine auxotrophs.

Validation of spermidine synthase as a drug target in African trypanosomes.

The trypanocidal activity of the ODC (ornithine decarboxylase) inhibitor DFMO (difluoromethylornithine) has validated polyamine biosynthesis as a target for chemotherapy. As DFMO is one of only two drugs used to treat patients with late-stage African trypanosomiasis, the requirement for additional drug targets is paramount. Here, we report the biochemical properties of TbSpSyn (Trypanosoma brucei spermidine synthase), the enzyme immediately downstream of ODC in this pathway. Recombinant TbSpSyn was purified and shown to catalyse the formation of spermidine from putrescine and dcSAM (decarboxylated S-adenosylmethionine). To determine the functional importance of TbSpSyn in BSF (bloodstream form) parasites, we used a tetracycline-inducible RNAi (RNA interference) system. Down-regulation of the corresponding mRNA correlated with a decrease in intracellular spermidine and cessation of growth. This phenotype could be complemented by expressing the SpSyn (spermidine synthase) gene from Leishmania major in cells undergoing RNAi, but could not be rescued by addition of spermidine to the medium due to the lack of a spermidine uptake capacity. These results therefore genetically validate TbSpSyn as a target for drug development and indicate that in the absence of a functional biosynthetic pathway, BSF T. brucei cannot scavenge sufficient spermidine from their environment to meet growth requirements.

Regulation of polyamine synthesis in human hepatocytes by hepatotrophic factor augmenter of liver regeneration

Different stages of liver regeneration are regulated by a variety of factors such as the liver growth associated protein ALR, augmenter of liver regeneration. Furthermore, small molecules like polyamines were proven to be essential for hepatic growth and regeneration. Therefore, using primary human hepatocytes in vitro we investigated the effect of ALR on the biosynthesis of polyamines. We demonstrated by HPLC analysis that recombinant ALR enhanced intracellular hepatic putrescine, spermidine, and spermine levels within 9–12 h. The activation of polyamine biosynthesis was dose dependent with putrescine showing the strongest increase. Additionally, ALR treatment induced mRNA expression of ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase, both key enzymes of polyamine biosynthesis. Further, ALR induced c-myc mRNA expression, a regulator of ODC expression, and therefore we assume that ALR exerts its liver regeneration augmenting effects through stimulation of its signalling pathway leading in part to enhanced polyamine synthesis.

Protective effect of gamma‐aminobutyric acid (GABA) against cytotoxicity of ethanol in isolated rat hepatocytes involves modulations in cellular polyamine levels

Introduction GABA is a potent amino acid neurotransmitter with various physiologic effects throughout the body. Serum concentration of GABA is known to be increased in liver failure. However, the function of GABA in liver is not clear. In this study, we investigated the involvement of modulations in cellular polyamine levels on the protective effect of GABA against cytotoxicity of ethanol in isolated rat hepatocytes. Method Hepatocytotoxicity was induced by the addition of ethanol. Viability of hepatocytes with or without GABA and polyamine was evaluated by Neutral Red assay. We used HPLC method with fluorometric detection to measure levels of intracellular putrescine (PUT), spermidine (SPD) and spermine (SPM) which are major polyamines ubiquitously present in eukaryotic cells. Result The addition of ethanol to hepatocyte induced a decrease in cell viability. However, incubation with GABA significantly prevented this decrease in a dose-dependent manner. Ethanol also resulted in a loss of PUT and SPD levels and the loss was significantly returned to control levels by the addition of GABA. Furthermore, the decrease in cell viability induced by ethanol was significantly suppressed by supplementation of PUT and SPD. Conclusion These results suggest that GABA has protective effect against cytotoxicity of ethanol in isolated rat hepatocytes. This effect may be modulated by the maintenance of intracellular polyamine levels.

Different intracellular polyamine concentrations underlie the difference in the inward rectifier K+ currents in atria and ventricles of the guinea‐pig heart

The outward component of the strong inward rectifier potassium current, I(K1), is significantly larger in ventricles than in atria of the heart, resulting in faster repolarization at the final phase of the action potential in ventricles. However, the underlying mechanism of the difference in I(K1) remains poorly understood. I(K1) channels are composed of subunits from the Kir2 subfamily, and I(K1) amplitude is determined by the voltage-dependent blockade of the channel by the intracellular polyamines spermine and spermidine, and by Mg(2+). Using a perforated patch-clamp method, which minimizes changes in the intracellular polyamine and Mg(2+) concentrations, we detected repolarization-induced outward I(K1) transients, which are caused by competition between Mg(2+) and spermine to block the channel, in ventricular but not in atrial myocytes from guinea-pig heart. The contribution of the Kir2.3 subunit to the I(K1) channel was found to be minor in the guinea-pig heart, because the activation time course of the Kir2.3 currents was approximately 10-fold slower than those of I(K1), and the marked external pH sensitivity of the Kir2.3 currents was not found in I(K1). Both the Kir2.1 and Kir2.2 currents recorded from inside-out patches exhibited outward transients similar to those of ventricular I(K1) in the presence of 5-10 microM spermine and 0.6-1.1 mM Mg(2+), and their amplitudes were diminished by increasing the spermine or spermidine concentrations. The total and free polyamine concentrations in guinea-pig cardiac tissues were higher in atria than ventricles. These results strongly suggest that different intracellular polyamine concentrations are responsible for the difference in atrial and ventricular I(K1) of the guinea-pig heart.

Two Kir2.1 channel populations with different sensitivities to Mg2+ and polyamine block: a model for the cardiac strong inward rectifier K+ channel

The strong inward rectification of the whole cell Kir2.1 current, which is very similar to the cardiac inward rectifier K(+) current (I(K1)), is caused by voltage-dependent blockade of outward currents by the intracellular polyamines spermine and spermidine. We recently showed that macroscopic Kir2.1 currents obtained from inside-out patches in the presence of various concentrations of cytoplasmic polyamines are well explained by the sum of the currents through two populations of channels that show differing susceptibilities to polyamine blockade. The outward currents obtained with 5-10 microM cytoplasmic spermine showed current-voltage relationships similar to those of I(K1) and were considered to flow mostly through a small population of channels exhibiting lower spermine sensitivity. Here we used inside-out patches to examine the blockade of macroscopic Kir2.1 currents by cytoplasmic Mg(2+) in the absence and presence of cytoplasmic spermine. Outward currents were blocked by 0.6 and 1.1 microM Mg(2+) in a concentration-dependent manner, but a small fraction ( approximately 0.1) of the macroscopic conductance was resistant to Mg(2+) at those concentrations, suggesting there are two populations of Kir2.1 channels with different sensitivities to Mg(2+). Furthermore, at those concentrations, Mg(2+) blocked inward currents by inducing a shallow blocked state that differed from the deeper state causing the inward rectification. In the presence of 1.1 microM Mg(2+) + 5 microM spermine, Mg(2+) blocked a substantial current component during depolarizing pulses and generated transient outward components, which is consistent with findings from earlier whole-cell experiments. In the steady state, Mg(2+) blocked the currents at voltages around and negative to the reversal potential and induced sustained outward components. The steady-state and time-dependent current amplitudes and the fractional blockades caused by spermine and Mg(2+) could be quantitatively explained by a model in which Mg(2+) competes with spermine to block the high-affinity channel and induces three conductance states. The present results suggest that the outward I(K1) flows through two populations of channels with different sensitivities to cytoplasmic blockers.

Metabolic and Antiproliferative Consequences of Activated Polyamine Catabolism in LNCaP Prostate Carcinoma Cells

Depletion of intracellular polyamine pools invariably inhibits cell growth. Although this is usually accomplished by inhibiting polyamine biosynthesis, we reasoned that this might be more effectively achieved by activation of polyamine catabolism at the level of spermidine/spermine N(1)-acetyltransferase (SSAT); a strategy first validated in MCF-7 breast carcinoma cells. We now examine the possibility that, due to unique aspects of polyamine homeostasis in the prostate gland, tumor cells derived from it may be particularly sensitive to activated polyamine catabolism. Thus, SSAT was conditionally overexpressed in LNCaP prostate carcinoma cells via a tetracycline-regulatable (Tet-off) system. Tetracycline removal resulted in a rapid approximately 10-fold increase in SSAT mRNA and an increase of approximately 20-fold in enzyme activity. SSAT products N(1)-acetylspermidine, N(1)-acetylspermine, and N(1),N(12)-diacetylspermine accumulated intracellularly and extracellularly. SSAT induction also led to a growth inhibition that was not accompanied by polyamine pool depletion as it was in MCF-7 cells. Rather, intracellular spermidine and spermine pools were maintained at or above control levels by a robust compensatory increase in ornithine decarboxylase and S-adenosylmethionine decarboxylase activities. This, in turn, gave rise to a high rate of metabolic flux through both the biosynthetic and catabolic arms of polyamine metabolism. Treatment with the biosynthesis inhibitor alpha-difluoromethylornithine during tetracycline removal interrupted flux and prevented growth inhibition. Thus, flux-induced growth inhibition appears to derive from overaccumulation of metabolic products and/or from depletion of metabolic precursors. Metabolic effects that were not excluded as possible contributing factors include high levels of putrescine and acetylated polyamines, a 50% reduction in S-adenosylmethionine, and a 45% decline in the SSAT cofactor acetyl-CoA. Overall, the study demonstrates that activation of polyamine catabolism in LNCaP cells elicits a compensatory increase in polyamine biosynthesis and downstream metabolic events that culminate in growth inhibition.

Polyamine depletion switches the form of 2-deoxy- d-ribose-induced cell death from apoptosis to necrosis in HL-60 cells

Our previous studies demonstrated that intracellular polyamine depletion blocked HL-60 cell apoptosis triggered by exposure to 2-deoxy- d-ribose (dRib). Here, we have characterized the intracellular events underlying the apoptotic effects of dRib and the involvement of polyamines in these effects. Treatment of HL-60 cells with dRib induces loss of mitochondrial transmembrane potential, radical oxygen species production, intracellular glutathione depletion and translocation of Bax from cytosol to membranes. These effects are followed by cell death. However, the mode of cell death caused by dRib depends on intracellular levels of polyamines. d-Rib-treated cells with normal polyamine levels, progressing through the G 1 into the S and G 2/M phases, undergo apoptosis, while in polyamine-depleted cells, being blocked at the G 1 phase, cell death mechanisms are switched to necrosis. The present study points to a relationship between the cell cycle distribution and the mode of cell death, and suggests that the level of intracellular spermidine, essential to cell cycle progression, may determine whether a cell dies by apoptosis or necrosis in response to a death stimulus.

Effects of dihydrotestosterone on cardiac inward rectifier K(+) current.

There are well-described sexually dimorphic differences both in the electrocardiogram and in the propensity to develop drug-induced arrhythmias. The QT interval and the risk of ventricular proarrhythmia are reduced in males compared with females. Inward rectifier potassium current (IK(1)) is a primary determinant of the ventricular resting membrane potential, and an important contributor to myocardial excitability. Using the whole-cell patch-clamp technique, we evaluated the effects of dihydrotestosterone (DHT) on IK(1) in ventricular myocytes from castrated rabbits that were treated with either replacement DHT or vehicle-control for 3 weeks. Compared with the DHT-treated group, myocytes from the control animals had a significant reduction in inward IK(1) conductance (p < 0.005) and rectification ratio (RR) (p < 0.04) with no significant change in peak outward current. Acute DHT superfusion of the myocytes increased inward IK(1) conductance from baseline (p < 0.05) and increased the RR (p < 0.05). Testosterone has been reported to increase intracellular ornithine decarboxylase activity in ventricular tissue, which would increase intracellular polyamines, known modulators of IK(1) rectification. We found that inclusion of the intracellular polyamines spermidine and putrescine in the pipette solution caused a decrease in inward IK(1), accompanied by an increase in peak outward current and a reduction in the RR. In summary, DHT modulates IK(1) in a chronic, as well as, an acute fashion. These effects are not because of altered intracellular polyamines. DHT may modulate myocardial excitability through effects on IK(1).

Effect of drugs inhibiting spermidine biosynthesis and metabolism on the in vitro development of Plasmodium falciparum.

Treatment of Plasmodium falciparum with the potent inhibitor dicyclohexylamine completely arrests in vitro cell proliferation of the chloroquine-susceptible P. falciparum strain NF54 and the R strain, which shows less sensivity to chloroquine. The average inhibitory concentration (IC50) values determined for both strains revealed different inhibition profiles. The IC50 value for the chloroquine-sensitive NF54 strain was 97 microM and 501 microM for the R strain. Monitoring polyamine pools after treatment with dicyclohexylamine leads to a significant decrease in the intracellular spermidine content, which was nearly reversed by supplementation with spermidine. Since spermidine is an important precursor for the biosynthesis of hypusine and homospermidine in eukaryotes, we studied the developmental effect on both P. falciparum strains of 1,7-diaminoheptane as an inhibitor of deoxyhypusine synthase (EC 1.1.1.249) in mammalian cells, and agmatine as a moderate inhibitor of homospermidine synthase (EC 2.5.1.44). Inhibition profiles with 1,7-diaminoheptane resulted in an IC50 value of 466 microM for the NF54 strain and 319 microM for the R strain. Spermidine pools changed significantly. Inhibition with agmatine caused a strong decrease in parasitemia for the chloroquine-susceptible NF54 strain, with a determined IC50 value of 431 microM and an IC50 value of 340 microM for the less chloroquine-susceptible R strain. Spermidine was not detectable after inhibition. The uncommon triamine homospermidine occurred in both P. falciparum strains. To our knowledge this is the first evidence of homospermidine in P. falciparum. The use of specific inhibitors of spermidine metabolism might be a novel strategy for the design of new antimalarials, and suggests the occurrence of both enzymes in the parasite.

Effect of spermidine on the in vivo degradation of ornithine decarboxylase in Saccharomyces cerevisiae.

As part of our studies on the regulation of polyamine biosynthesis in Saccharomyces cerevisiae, we have investigated the effect of spermidine on the degradation of ornithine decarboxylase in this organism. We have found that in S. cerevisiae, as in other eukaryotic cells, the rate of degradation of ornithine decarboxylase, measured either enzymatically or immunologically, is increased by the addition of spermidine to a yeast culture. It is noteworthy that this effect of added spermidine is found even when the experiments are conducted with strains in which the ornithine decarboxylase is overexpressed several hundred-fold more than the wild-type level. The effect of added spermidine in the overexpressed SPE1 strains is best seen in spe2 mutants in which the initial intracellular spermidine is very low or absent. Experiments with cycloheximide show that new protein synthesis is required to effect the breakdown of the ornithine decarboxylase. These results indicate that S. cerevisiae contains an antizyme-like mechanism for the control of the level of ornithine decarboxylase by spermidine, even though, as contrasted with other eukaryotic cells, no specific antizyme homologue has been detected either in in vitro experiments or in the S. cerevisiae genome.

Genetic analysis of spermidine synthase from Leishmania donovani

The polyamine biosynthetic pathway of protozoan parasites has been validated as a target in antiparasitic chemotherapy. To investigate this pathway at the biochemical and genetic level in a model parasite, the gene encoding spermidine synthase (SPDSYN), a key polyamine biosynthetic enzyme, has been cloned and sequenced from Leishmania donovani. The L. donovani SPDSYN gene encodes a polypeptide of 300 amino acids that exhibits 56% amino acid identity with the human counterpart. SPDSYN is present as a single copy gene in the leishmanial genome and encodes a 1.6 kb transcript. Employing SPDSYN flanking sequences to construct drug resistance cassettes, a Δ spdsyn knockout strain of L. donovani was created by double targeted gene replacement. This Δ spdsyn line could not convert putrescine to spermidine and was auxotrophic for polyamines. The polyamine auxotrophy could be circumvented by exogenous spermidine but not by putrescine (1,4-diaminobutane), cadaverine (1,5-diaminopentane), 1,3-diaminopropane, or spermine. Incubation of the null mutant in polyamine-deficient medium resulted in a rapid depletion in the intracellular spermidine level with a concomitant elevation of the putrescine pool. In addition, the level of trypanothione, a spermidine-containing thiol, was reduced, whereas the glutathione pool increased 3–4-fold. These data establish that SPDSYN is an essential enzyme in L. donovani promastigotes. The molecular and cellular reagents created in this investigation provide a foundation for subsequent structure-function and inhibitor design studies on this key polyamine biosynthetic enzyme.

Involvement of Polyamines in B Cell Receptor–Mediated Apoptosis: Spermine Functions as a Negative Modulator

The B cell lymphoma WEHI231 has been used as a model for studying clonal deletion of B cells on the basis of its ability to undergo growth arrest and apoptosis by B cell antigen receptor (BCR) cross-linking. To comprehensively analyze the genes involved in BCR-mediated apoptosis, we applied the technique of serial analysis of gene expression (SAGE) to WEHI231. Comparison of expression patterns revealed that BCR cross-linking caused coordinate changes in the expression of genes involved in polyamine metabolism. Polyamines are ubiquitous compounds required for cell proliferation and homeostasis. The coordinate expression of the polyamine-related genes was confirmed by semiquantitative reverse transcriptase–polymerase chain reaction analysis. During apoptosis, the genes involved in polyamine biosynthesis were downregulated, whereas those involved in polyamine catabolism were upregulated, suggesting that intracellular polyamines play a role in BCR-mediated apoptosis. Levels of intracellular putrescine, spermidine, and spermine were reduced after BCR cross-linking. These effects were prevented by concurrent CD40 stimulation, which blocked BCR-mediated apoptosis. Furthermore, addition of spermine could repress the BCR-mediated apoptosis by attenuating the mitochondrial membrane potential (Δψm) loss and activation of caspase-7 induced by BCR signaling. These findings strongly suggest that polyamine regulation is involved in apoptosis during B cell clonal deletion.

Polyamine regulation of ornithine decarboxylase and its antizyme in intestinal epithelial cells.

Ornithine decarboxylase (ODC) is feedback regulated by polyamines. ODC antizyme mediates this process by forming a complex with ODC and enhancing its degradation. It has been reported that polyamines induce ODC antizyme and inhibit ODC activity. Since exogenous polyamines can be converted to each other after they are taken up into cells, we used an inhibitor of S-adenosylmethionine decarboxylase, diethylglyoxal bis(guanylhydrazone) (DEGBG), to block the synthesis of spermidine and spermine from putrescine and investigated the specific roles of individual polyamines in the regulation of ODC in intestinal epithelial crypt (IEC-6) cells. We found that putrescine, spermidine, and spermine inhibited ODC activity stimulated by serum to 85, 46, and 0% of control, respectively, in the presence of DEGBG. ODC activity increased in DEGBG-treated cells, despite high intracellular putrescine levels. Although exogenous spermidine and spermine reduced ODC activity of DEGBG-treated cells close to control levels, spermine was more effective than spermidine. Exogenous putrescine was much less effective in inducing antizyme than spermidine or spermine. High putrescine levels in DEGBG-treated cells did not induce ODC antizyme when intracellular spermidine and spermine levels were low. The decay of ODC activity and reduction of ODC protein levels were not accompanied by induction of antizyme in the presence of DEGBG. Our results indicate that spermine is the most, and putrescine the least, effective polyamine in regulating ODC activity, and upregulation of antizyme is not required for the degradation of ODC protein.