Effect Of Spermine Research Articles

Effect of prolactin and spermine on the zinc content of human spermatozoa.

The effect of physiological doses O-prolactin (0-10 ng) and spermine (0-2 mg) on the intracellular content of zinc of human spermatozoa was studied. The results revealed the negative correlation of the zinc content with prolactin or spermine concentration.

Effect of Spermine on Carbohydrate Metabolism in Human Semen

Zusammenfassung Der Einflus von Spermin auf den Kohlenhydratstoffwechsel im menschlichen Sperma In einer experimentellen Arbeit wird uber den Einflus eines Spermin-Zusatzes zum menschlichen Spermaplasma (1,5–3,0 mM) auf den Kohlenhydratstoffwechsel berich-tet. Zur Verwendung gelangten Spermaproben von fertilen Personen. Fur die Enzym-aktiyitatsbestimmungen (Maltase, Amylase) wurde jede Spermaprobe von Spermatozoen durch Zentrifugation (800 g/30 Min.) befreit. Das Spermaplasma wurde 24 Std. bei 4° C gegen destilliertes Wasser dialysiert. Auserdem wurde die Fructoseutilisation und der Proteingehalt bestimmt. Die Maltaseaktivitat stieg unter Sperminzusatz an, wahrend die Amylaseaktivitat unter den gleichen Versuchsbedingungen unverandert blieb. Die Fructoseutilisation (Fructolyse) sank bei Sperminzusatz. Als mogliche Erklarung fur diesen Effekt wird diskutiert, das Spermin den Transport der Fructose durch die Mem-branen von Sauger-Spermatozoen verhindert. Hierzu sollen entsprechende Untersuchungen vorgenommen werden. Resumen Efecto de la espermina sobre el metabolismo hidrocar-bonado en semen humano Se conoce la presencia de gran cantidad de espermina en el plasma seminal humano. No obstante la funcion de las poliaminas en el semen se desconoce. Se considera de interes por tanto, estudiar los efectos de la espermina sobre las enzimas del semen humano amilasa y maltasa envueltas en la degradacion del glucogeno. Tambien se investiga si la utilizacion de la fructosa por los espermatozoides es afectada por la espermina. Las muestras de semen procedian de hombres fertiles e infertiles. Cuando se empleo plasma seminal dializado en fresco, la adicion de bajas concentra-ciones de espermina estimulo la actividad de la maltasa. Esta actividad estimuladora de la espermina sobre la maltasa se perdia cuando el plasma seminal se conservaba a 10° bajo cero. La espermina a concentracion alta inhibe la actividad de la maltasa. Asimismo se demostro que la adicion de espermina inhibe la utilizacion de la fructosa por los espermatozoides. Los resultados aportados demuestran que las poliaminas pueden afectar el metabolismo hidrocarbonado. Esto concuerda con los hallazgos de HARRISON y WHITE, de que la espermina tiene un efecto toxico sobre la motilidad de los espermatozoides del hombre y del conejo.

Effect of LH, Prolactin and Spermine on ATPase Activity of Human Spermatozoa

The effect of Prolactin and LH on the activity of spermatozoal ATPase was studied. Both the hormones activated the enzyme activity suggesting that these hormones, as they are present in the seminal fluid may influence the energy metabolism of spermatozoa. The spermine, a polyamine present in large concentration in the semen, had significantly enhanced the ATPase activity of the spermatozoa in a dose related manner.

Waterlogging tolerance of Welsh onion (Allium fistulosum L.) enhanced by exogenous spermidine and spermine

Soil flooding is a seasonal factor that negatively affects plant performance and crop yields. In order to investigate the effects of spermidine (Spd) and spermine (Spm) on the waterlogging stress, it was checked that the content of relative water content (RWC), proline, chlorophyll and malondialdehyde (MDA), net photosynthesis, the rate of hydrogen peroxide (H(2)O(2)) and superoxide radicals (O(2)(-)) generation and the antioxidant enzyme activities of superoxide dismutase (SOD) (EC 1.15.1.1), catalase (CAT) (EC 1.11.1.6), ascorbate peroxidase (APX) (EC 1.11.1.11) and glutathione reductase (GR) (EC 1.6.4.2) in Welsh onion (Allium fistulosum L) plants. Pretreatment with 2 mM of Spd and Spm effectively maintained the balance of water content in plant leaves and roots under flooding stress. In addition, the data indicate that the protective role of proline should be considered minimal, as its accumulation was found to be inversely correlated with tolerance to stress; it also significantly retarded the loss of chlorophyll, enhanced photosynthesis, decreased the rate of O(2)(-) generation and H(2)O(2) content, and prevented flooding-induced lipid peroxidation. Spd and Spm helped to maintain antioxidant enzyme activities under flooding; however, APX activity was found to increase slightly in response to Spm. The antioxidant system, an important component of the water-stress-protective mechanism, can be changed by PAs, which are able to moderate the radical scavenging system and to lessen in this way the oxidative stress. The results suggest that pretreatment with Spd and Spm prevents oxidative damage, and the protective effect of Spd was found to be greater than that of Spm.

Polyamines protect rat embryo in vitro from high glucose‐induced developmental delay and dysmorphogenesis

Pregnancy in mammals with diabetes mellitus results in low birth weight, malformations, and intrauterine death. Parenteral application of natural polyamines or their precursor, L-arginine, to diabetic pregnant rats partially prevents the alterations of development caused by diabetes mellitus. This experiment has been designed to understand if this preventive action also occurs in rat whole embryo in culture. Rat embryos of gestational day 10 were cultured for 24 h in normal medium, high glucose medium, or high glucose medium supplemented with polyamines or L-arginine, and furthermore embryo growth and development were evaluated. L-arginine and putrescine partially prevents the dysmorphogenic effects of high glucose, whereas spermidine and spermine prevent these effects almost completely. Polyamines directly protect the embryo from the toxic effect of high glucose concentration on growth and development, although the mechanism remains to be elucidated.

Binding of spermine and ifenprodil to a purified, soluble regulatory domain of the N‐methyl‐d‐aspartate receptor

The binding of spermine and ifenprodil to the amino terminal regulatory (R) domain of the N-methyl-D-aspartate receptor was studied using purified regulatory domains of the NR1, NR2A and NR2B subunits, termed NR1-R, NR2A-R and NR2B-R. The R domains were over-expressed in Escherichia coli and purified to near homogeneity. The K(d) values for binding of [(14)C]spermine to NR1-R, NR2A-R and NR2B-R were 19, 140, and 33 microM, respectively. [(3)H]Ifenprodil bound to NR1-R (K(d), 0.18 microM) and NR2B-R (K(d), 0.21 microM), but not to NR2A-R at the concentrations tested (0.1-0.8 microM). These K(d) values were confirmed by circular dichroism measurements. The K(d) values reflected their effective concentrations at intact NR1/NR2A and NR1/NR2B receptors. The results suggest that effects of spermine and ifenprodil on NMDA receptors occur through binding to the regulatory domains of the NR1, NR2A and NR2B subunits. The binding capacity of spermine or ifenprodil to a mixture of NR1-R and NR2A-R or NR1-R and NR2B-R was additive with that of each individual R domain. Binding of spermine to NR1-R and NR2B-R was not inhibited by ifenprodil and vice versa, indicating that the binding sites for spermine and ifenprodil on NR1-R and NR2B-R are distinct.

RNA as a new target for toxic and protective agents

In contrast to damage of genomic DNA and despite its potential to affect cell physiology, RNA damage is a poorly examined field in biomedical research. Potential triggers of RNA damage as well as its pathophysiological implications remain largely unknown. While less lethal than mutations in genome, such non-acutely lethal insults to cells have been recently associated with underlying mechanisms of several human chronic diseases. We investigated whether RNA damage could be related to the exposure of particular xenobiotics by testing the RNA-damaging activity of a series of chemicals with different mechanisms of action. Cultured human T-lymphoblastoid cells were treated with ethyl methanesulfonate (EMS), H2O2, doxorubicin, spermine, or S-nitroso-N-acetylpenicillamine (SNAP). Furthermore, we studied the potential protective activity of a pomegranate extract against RNA damage induced by different chemicals. Special attention has been paid to the protective mechanisms of the extract. The protective effect of pomegranate can be mediated by alterations of the rates of toxic agent absorption and uptake, by trapping of electrophiles as well as free radicals, and protection of nucleophilic sites in RNA. We used two different treatment protocols (pre- and co-treatment) for understanding the mechanism of the inhibitory activity of pomegranate. We demonstrated that total RNA is susceptible to chemical attack. A degradation of total RNA could be accomplished with doxorubicin, H2O2, spermine and SNAP. However, EMS, a well-known DNA-damaging agent, was devoid of RNA-damaging properties, while spermine and SNAP, although lacking of DNA-damaging properties, were able to damage RNA. Pomegranate reduced the RNA-damaging effect of doxorubicin, H2O2, and spermine. Its inhibitory activity could be related with its ability to forms complexes with doxorubicin and H2O2, or interacts with the intracellular formation of reactive species mediating their toxicity. For spermine, an alteration of the rates of spermine absorption and uptake can also be involved.

Oligonucleotide−Oligospermine Conjugates (Zip Nucleic Acids): A Convenient Means of Finely Tuning Hybridization Temperatures

Synthesis of oligonucleotide probes and control of their hybridization temperature are key aspects of polymerase chain reaction (PCR)-based detection of genetic sequences. A straightforward means to approach the last goal is to decrease the repulsion between the polyanionic probe and target strands. To this end, we have developed a versatile automated synthesis of oligonucleotide-oligospermine derivatives that gave fast access to a large variety of compounds. Plots of their hybridization temperatures T(m) vs overall charge provided a measure of the impact of interstrand phosphate repulsion (and of spermine-mediated attraction) on the main driving force of duplex formation, i.e., base pairing. It showed that stabilization brought about by excess cationic charges can be of larger absolute magnitude than interstrand repulsion, even in high salt media. Base sequence and conjugation site (3' or 5') hardly influenced the effect of spermine on T(m). In typical PCR probe conditions, the T(m) increased linearly with the number of grafted spermines (e.g., 6.2 degrees C per spermine for a decanucleotide probe). The large data set of T(m) vs number of spermines and oligonucleotide length allowed us to empirically derive a simple mathematical relation that is accurately predicting the T(m) of any oligonucleotide-oligospermine derivative. Zip nucleic acids (ZNA) are thus providing an interesting alternative to locked nucleic acids (LNA) or minor groove binders (MGB) for raising the stability of 8-12-mer oligonucleotides up to ca. 70 degrees C, the level required for quantitative PCR experiments.

Identification of Stimulators and Inhibitors of Cdc7 Kinase in Vitro

Cdc7 is a serine-threonine kinase that regulates initiation and progression of DNA replication. The activity of purified Cdc7 kinase is significantly stimulated by polyamines such as spermine or spermidine. Positively charged polymers of lysine or arginine also stimulate its kinase activity, whereas the negatively charged substances such as polyglutamate or nucleic acids significantly inhibit the kinase activity. Spermine affects both the K(m) and V(max) of Cdc7 kinase for a minichromosome maintenance (MCM) substrate. We also found that histones, lysine- and arginine-rich basic proteins, can stimulate Cdc7 kinase activity, and a MCM complex in association with histone is a more efficient substrate of Cdc7 than the free MCM complex. These results identify potential cellular inhibitors and stimulators of Cdc7 kinase and suggest that Cdc7 may be another target of cellular polyamines and that histones may stimulate Cdc7-mediated phosphorylation of chromatin-bound substrates. Ectopic expression of an antizyme, known to reduce the cellular polyamine levels, resulted in reduction of Cdc7-mediated phosphorylation of MCM4 protein, suggesting physiological roles of polyamines in regulation of Cdc7 kinase activity in the cells.

Impact of Spermine on Down-Regulation of Th2 Cytokines in Peripheral Blood Mononuclear Cells from Cedar Pollinosis Subjectsin Vitro

The effects of spermine (SPM) on the production of Th1 and Th2 cytokines using peripheral blood mononuclear cells (PBMCs) derived from cedar pollinosis subjects were investigated. Th2 cytokines produced by PBMCs stimulated by 10 microM SPM was significantly lower than that in the controls. These findings indicate that blood SPM is an important factor in the Th1/Th2 balance.

Ca2+-independent effects of spermine on pyruvate dehydrogenase complex activity in energized rat liver mitochondria incubated in the absence of exogenous Ca2+ and Mg2 +

In the absence of exogenous Ca(2+) and Mg(2+) and in the presence of EGTA, which favours the release of endogenous Ca(2+), the polyamine spermine is able to stimulate the activity of pyruvate dehydrogenase complex (PDC) of energized rat liver mitochondria (RLM). This stimulation exhibits a gradual concentration-dependent trend, which is maximum, about 140%, at 0.5 mM concentration, after 30 min of incubation. At concentrations higher than 0.5 mM, spermine still stimulates PDC, when compared with the control, but shows a slight dose-dependent decrease. Changes in PDC stimulation are very close to the phosphorylation level of the E(1alpha) subunit of PDC, which regulates the activity of the complex, but it is also the target of spermine. In other words, progressive dephosphorylation gradually enhances the stimulation of RLM and progressive phosphorylation slightly decreases it. These results provide the first evidence that, when transported in RLM, spermine can interact in various ways with PDC, showing dose-dependent behaviour. The interaction most probably takes place directly on a specific site for spermine on one of the regulatory enzymes of PDC, i.e. pyruvate dehydrogenase phosphatase (PDP). The interaction of spermine with PDC may also involve activation of another regulatory enzyme, pyruvate dehydrogenase kinase (PDK), resulting in an increase in E(1alpha) phosphorylation and consequently reduced stimulation of PDC at high polyamine concentrations. The different effects of spermine in RLM are discussed, considering the different activities of PDP and PDK isoenzymes. It is suggested that the polyamine at low concentrations stimulates the isoenzyme PDP(2) and at high concentrations it stimulates PDK(2).

Glucose-induced inhibition of seed germination in Lotus japonicus is alleviated by nitric oxide and spermine

Seed germination is sensitive to glucose (Glc), nitric oxide (NO) and polyamine (PA). To elucidate whether cross-talk among Glc, NO and PAs occurs in mediation of seed germination, effects of Glc, NO and spermine on seed germination of Lotus japonicus were studied. Glc retarded seed germination in a concentration-dependent manner. NO donor sodium nitroprusside (SNP) alleviated Glc-induced inhibition of seed germination, whereas the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (cPTIO) diminished the SNP-dependent alleviation of seed germination. These observations indicate that Glc may inhibit seed germination by interacting with NO signaling pathways. Exogenous spermine enhanced and the inhibitor of the spermine synthase, methylglyoxal-bis-guanyl hydrazone (MGBG), inhibited seed germination, respectively. Like SNP, spermine alleviated the Glc-induced inhibition of seed germination, whereas MGBG exaggerated the Glc-induced inhibition of seed germination. These results suggest that Glc may inhibit the spermine synthesis, leading to reductions in seed germination. NO scavenger and spermine synthase inhibitor diminished the SNP-induced alleviation of Glc-induced inhibition of seed germination. These findings reveal that both NO and spermine participate in the Glc-induced inhibition of seed germination in L. japonicus.

Exogenous polyamines alleviate the lipid peroxidation induced by cadmium chloride stress in Malus hupehensis Rehd.

In order to investigate the effects of spermidine (Spd) and spermine (Spm) on the stress of cadmium chloride (CdCl 2), it was checked that the content of polyamines and malondialdehyde (MDA), the rate of superoxide anion (O 2 −) generation and the activities of superoxidase dismutase (SOD) and guaiacol peroxidase (GPX) in 1-year-old Malus hupehensis Rehd. var. pinyiensis Jiang (tea crabapple) seedlings grown in nutrient solution. The result showed that the content of free putrescine (Put) was increased, the contents of free Spd and Spm and the (Spd + Spm)/Put ratio in leaves were decreased significantly under CdCl 2 treatment. Foliar spraying with Spd or Spm under CdCl 2 treatment, the accumulation of free Put reduced, the contents of free Spd and Spm and the ratio of (Spd + Spm)/Put in leaves increased significantly. The activities of SOD and GPX in leaves were enhanced and O 2 − generation rate and MDA content were decreased significantly by foliar spraying with Spd or Spm under CdCl 2 treatment. Results suggest that both Spd and Spm can alleviate the lipid peroxidation caused by CdCl 2 in M. hupehensis var . pinyiensis.

Spermine and Endothelial Damage During Endotoxemia

Diazenolate-NO-donors like spermine-NONOate use spermine as a nitric oxide releasing carrier. As one of the naturally occurring polyamines, spermine belongs to the most potent platelet aggregation inhibitors among the polyamines. Recent publications describe a dominating role for platelets and nitric oxide in the setting of endothelial dysfunction during endotoxemia. It is unknown whether spermine as single agent, beside his function as nitric oxide releasing carrier, exerts effects on macromolecular leakage during early endotoxemia. In male Wistar rats, macromolecular efflux was determined in mesenteric postcapillary venules using intravital microscopy at baseline, 60 and 120 min after the start of the experiment. In the first part of the experiments we investigated the effects of spermine and spermine-NONOate during leukocyte-independent states of endotoxemia, using the L- and P-selectin inhibitor fucoidin to suppress leukocyte-endothelial-interaction. In the second part we investigated the effects of spermine on endothelial damage during endotoxemia without co-administration of fucoidin. Combined treatment of animals with spermine and fucoidin was as effective as treatment with spermine-NONOate and fucoidin alone. Spermine and fucoidin as single agents had no effects on macromolecular efflux during endotoxin challenge. Combined treatment of animals with spermine and fucoidin reduce macromolecular efflux to the same degree like treatment with spermine-NONOate and fucoidin. A currently unknown interaction between fucoidin and spermine leads to a significant reduction in macromolecular efflux.

Effects of polyamines and polyamine biosynthetic inhibitors on mitotic activity ofAllium ceparoot tips

The genotoxic and cytotoxic effects of exogenous polyamines (PAs), putrescine (Put), spermidine (Spd), spermine (Spm) and PA biosynthetic inhibitors, alpha-difluoromethylornithine (DFMO), cyclohexilamine (CHA), methylglioxal bis-(guanylhydrazone) (MGBG) were investigated in the root meristems of Allium cepa L. The reduction of mitotic index and the induction of chromosomal aberrations such as bridges, stickiness, c-mitotic anaphases, micronuclei, endoredupliction by PAs and PA biosynthetic inhibitors were observed and these were used as evidence of genotoxicity and cytotoxicity.

Spermine attenuates behavioral and biochemical alterations induced by quinolinic acid in the striatum of rats

Polyamines are aliphatic amines containing nucleophilic centers that are found in all eukaryotic cells, including brain cells. These compounds determine neuroprotection in experimental models of cerebral ischemia and neurotoxicity. In the current study we investigated the protective effects of spermine, an agonist of the polyamine binding site at the N-methyl- d-aspartate receptor, against the behavioral and neurochemical alterations induced by quinolinic acid. The unilateral intrastriatal injection of quinolinic acid (180 nmol/site into the dorsal striatum) induced stereotypical motor asymmetries, assessed by the open field and elevated body swing tests. Spermine modulated quinolinic acid-induced rotational behavior biphasically. While the previous intrastriatal administration of spermine at the dose of 0.1 nmol/site increased, the administration of spermine at the dose of 10 nmol/site reduced quinolinic acid-induced rotational behavior. Spermine (10 nmol/site) also decreased the contralateral swing behavior induced by quinolinic acid. Furthermore, the effect of 10 nmol of spermine was counteracted by the co-administration of arcaine (10 nmol), a selective antagonist of the polyamine binding site at the N-methyl- d-aspartate receptor. In addition, spermine (10 nmol) protected against quinolinic acid-induced protein carbonylation in the rat striatum, further suggesting an antioxidant role for this polyamine. These results provide evidence that the behavioral and biochemical alterations induced by quinolinic acid are attenuated or prevented by spermine through its interaction with N-methyl- d-aspartate receptor and/or its antioxidant function.

Effects of putrescine, cadaverine, spermine, spermidine and β-phenylethylamine on cultured bovine mammary epithelial cells

A bovine mammary epithelial cell line (BME-UV1) and three-dimensional collagen primary bovine organoids were used to evaluate the effects of cadaverine, putrescine, spermine, spermidine and β-phenylethyla-mine on mammary epithelial cells. Each biogenic amine was diluted in several concentrations (0-50 mM in BME-UV1 and 0-4 mM in primary bovine organoids) in the appropriate saline solution for the cell culture considered. In order to determine the activity of each compound tritiated thymidine incorporation was used. At low concentrations, all amines induced cell proliferation in both cultures. In BME-UV1, spermine significantly inhibited cell proliferation (P<0.001), while the other amines inhibited at higher concentrations (50mM). In primary bovine organoids, β−phenylethylamine significantly (P<0.001) inhibited cell proliferation at 4 mM. Organoids cultured in the presence of all amines, except β-phenylethylamine, had stellate projections indicating intense cell proliferation.Proliferation of mammary epithelial cells was stimulated at low concentrations, while at high concentrations it was inhibited. Our results suggested that the effects of each compound on mammary epithelial cells could be related to the compound itself and not to mediating by the bovine amino oxidase, responsible of the formation of toxic metabolites.

Improvement of intestinal absorption of water-soluble macromolecules by various polyamines: Intestinal mucosal toxicity and absorption-enhancing mechanism of spermine

The absorption-enhancing effects of three different polyamines, spermine (SPM), spermidine (SPD) and putrescine (PUT) on the intestinal absorption of water-soluble macromolecules were examined in rats. Fluorescein isothiocyanate-labeled dextrans (FDs) with different average molecular weights were chosen as models of water-soluble macromolecules and intestinal absorption of FDs with or without these polyamines was examined by an in situ closed loop method. The intestinal absorption of fluorescein isothiocyanate-labeled dextran with an average molecular weight of 4400 (FD4) was relatively low in the absence of these polyamines. However, its absorption was improved in the presence of 5–10 mM SPM and 10 mM SPD in the jejunum and 10 mM SPM in the colon, while 10 mM PUT had almost no absorption-enhancing effect on the intestinal absorption of FD4. Overall, the enhancing effects of these polyamines were greater in the jejunal membranes than in the colonic membranes. The absorption-enhancing effect of SPM decreased as the molecular weights of FDs increased. The intestinal membrane toxicity of 10 mM SPM was evaluated by measuring the amount of protein and activity of lactate dehydrogenase (LDH) released from the intestinal epithelial cells. We also observed the morphological changes of intestinal mucosa in the presence or absence of SPM. The results indicated that the amount of protein and LDH was not changed in the presence of 10 mM SPM, although we observed a significant increase in these biological markers in the presence of 3% Triton X-100, as a positive control. Furthermore, we found no significant change in the intestinal membrane with 10 mM SPM by the morphological observation. These findings suggested that 10 mM SPM did not cause any significant membrane damage to the intestinal epithelium. To investigate the absorption-enhancing mechanism of SPM, the transepithelial electrical resistance (TEER) of the rat jejunal membranes was studied by using a diffusion chamber method. SPM decreased the TEER values in a concentration dependent manner and 10 mM SPM had almost the same effect to decrease the TEER value compared with 10 mM EDTA as a positive control. These findings suggest that SPM may loosen the tight junction of the epithelium, thereby increasing the intestinal absorption of drugs via a paracellular route. In summary, polyamines, especially SPM would be one of the suitable absorption enhancers with high effectiveness and low intestinal membrane toxicity.

Effect of spermine on lipid profile and HDL functionality in the streptozotocin-induced diabetic rat model

This study aimed to investigate the effect of spermine (Spm) as a chemical chaperone and glycation inhibitor on the lipid profile and HDL functionality in the short- and long-term treatment of the STZ-induced diabetic rats. Male Wistar rats were divided into 4 groups (control, n = 7; diabetic, n = 9). Two groups (named 2 and 3) were injected intraperitoneally with streptozotocin. Control rats (named 1 and 4) were injected with vehicle alone. The treatment of diabetic and control animals (groups 3 and 4) with 60 μmol/l of Spm in drinking water was begun. The study continued up to the end of the fifth month. The serum glucose and insulin level, AGE formation, lipid profile, paraoxonase 1 (PON1), and lecithin: cholesterol acyl transferase (LCAT) activities were measured. Significantly lower plasma PON1, and LCAT activities and higher serum AGE, TG, TC and LDL-c, and lower HDL-c were seen in diabetic rats as compared to control groups ( P < 0.01). The increased AGE, TG, TC and LDL-c levels in diabetic groups decreased gradually after receiving Spm. In addition, due to Spm administration, an increase in the HDL-c level was observed after the first month of the experiment ( P < 0.01). The increase in the PON1 and LCAT activities in the diabetic group that received Spm was significant after the second and the forth month of the experiment, P < 0.02 and P < 0.05, respectively. In conclusion, spermine administration attenuated the changed parameters to near normal values in diabetic rats. Spermine, despite a lack of significant changes on glucose metabolism and insulin secretion, was found to improve diabetes complications.

Modulation of Arginase Pathway by Chloroquine and Its Congener May Underlie the Enhanced Cell Commitment towards Erythroid Differentiation.

Arginase is a nitric oxide synthase-alternative pathway enzyme for L-arginine breakdown leading to biosynthesis of urea and L-ornithine. Evidence suggests that reduction of arginine bioavailability for NO production secondary to increased plasma arginase levels in sickle cell disease, may contribute to severe pathophysiological derangements and increase mortality. Recently, we showed that Chloroquine (CQ), an anti-malarial and anti-rheumatoid drug, displays a linear competitive mode of inhibition on sickle erythrocyte arginase (Iyamu et al., Brit J. Haematol, 2007: in press). Utilizing the K562 cell line as a model system in our present study, we obtained evidence of the anti-proliferative and differentiation effects of CQ and its analog, Hydroxychloroquine (OHCQ) at pharmacologically attainable concentrations (5–20μM). Specifically, CQ (IC50= 8.0 ±1.2 μM) and OHCQ (IC50 =17.0 ± 2.4 μM) inhibited K562 cell proliferation in a dose-dependent manner. This inhibitory effect was accompanied by enhanced commitment towards erythroid maturation as assessed by hemoglobinization (Iyamu et al., Exp Hematol, 2003); this was linked to a dose-dependent inhibition of arginase activity. To assess the role of the arginine-polyamine pathway in the induction of differentiation of K562 cells by CQ or OHCQ, cells were treated with 10 μM CQ or 20 μM OHCQ in the presence or absence of spermine (5μM) or spermidine (5 μM). Our results indicate that spermine and spermidine (intermediate products of the arginine polyamine pathway) partially reverted the effects of CQ and OHCQ on arginase activity. But this reversal did not affect the commitment of K562 cells towards erythroid maturation. We also show that CQ and OHCQ enhanced fetal hemoglobin (Hb F) synthesis by 3.4 and 3.2-fold (vs control), and maximally stimulated intracellular cGMP levels (as determined by immunosorbent assay) by 6.6- and 3.0-fold at 6hrs and 3hrs respectively. The induction of these macromolecules by CQ or OHCQ correlated with arginase inhibition. Again, the partial reversal of arginase activity by spermine or spermidine could not reduce the Hb F induction by CQ or OHCQ. This observation suggests that the commitment of cells towards erythroid maturation precedes the effects of spermine or spermidine on arginase activity. We further investigated the effects of 8-Bromo-cAMP (cAMP analog) on arginase activity in lipopolysaccharide (LPS)-stimulated K562 cells. The inhibitory effect of CQ on arginase was reversed in LPS-stimulated cells but this reversal of arginase activity was not observed in Bromo-cAMP-LPS- treated cells. Moreover, the moderate increase of arginase activity in LPS-treated cells in the presence of CQ failed to reduce the CQ-dependent enhancement of total hemoglobin and Hb F production. Indeed, the combination of 8-Bromo-cAMP with CQ in LPS-treated cells resulted in a significant inhibition (>35%) of arginase activity. This inhibitory effect was associated with a 3.9-fold increase of Hb F synthesis (vs CQ alone) as well as enhancement of total Hb production. This surprising observation demonstrates the possible involvement of the arginase pathway as well as additional, but yet unidentified mechanism(s). In conclusion, these observations that CQ and OHCQ may inhibit the initial step of the arginine-polyamine pathway, resulting in the enhancement of erythroid differentiation may provide a paradigm for targeted therapy of hemoglobinopathies.