Roles of nitric oxide and polyamines in brain tumor growth

Nitric oxide (NO) and polyamines: putrescine, spermidine and spermine, are key arginine metabolites in mammalian tissues that play critical roles i.a. in regulation of vascular tone (NO), and cell cycle regulation (polyamines). In the brain, both classes of molecules additionally have neuromodulatory and neuroprotective potential, and NO also a neurotoxic potential. Here we review evidence that brain tumors use the NO- and polyamine-synthesizing machineries to the benefit of their differentiation and growth from healthy glia and neurons. With a few exceptions, brain tumors show increased activities of one or all of the three arginine (Arg) to NO-converting nitric oxide synthase (NOS) isoforms (iNOS, eNOS, nNOS), but also elevated activities of polyamines-generating and modifying enzymes: arginase I/II, ornithine decarboxylase and spermidine/spermine N¹-acetyltransferase. The degree of stimulation of NO- and polyamine synthesis often correlates with brain tumor malignancy. Excess NO, but also spermine, spermidine and their N¹-acetylated forms, are tumor- and context-dependently involved in angiogenesis, tumor initiation and growth, and resistance to chemo- or radiotherapy. Hypothetically, increased demand for NO and/or polyamines is likely to contribute to Arg auxotrophy of malignant brain tumors, albeit the causal nexus awaits experimental verification.     

Substance P and vasoactive intestinal peptide influence polyamine metabolism in salivary glands of the rat

In parotid, sublingual and submaxillary glands stimulated by continuous intravenous infusion of the neuropeptides substance P or vasoactive intestinal peptide at various doses for 3 h, the concentrations of the polyamines putrescine, spermidine, spermine and N1-acetylspermidine as well as the activity of ornithine decarboxylase were determined. This enzyme catalyses the synthesis of putrescine and is the key enzyme in polyamine formation. Vasoactive intestinal peptide induced the most marked effects, and the most conspicuous findings were made in the sublingual glands, where the ornithine decarboxylase activity was found to have increased more than 100-fold, accompanied by an increased level of putrescine in those glands which were removed immediately after the end of the infusion. When, instead, the glands were removed 5 h after the end of the infusion there was no longer any increase in the activity of ornithine decarboxylase or in putrescine concentration, but now spermidine and spermine were found to be increased. Interestingly, the parasympathetic non-adrenergic, non-cholinergic regulation of polyamine metabolism in the major salivary glands of the rat is most predominant in the sublingual glands.     

On the role of polyamines in bone resorption induced by parathyroid hormone

In order to elucidate the possible role of polyamines in the mobilization of mineral from long-term bone cultures stimulated with parathyroid hormone we have measured the activity of ornithine decarboxylase in osteoblasts, the levels of polyamines in calvarial bone and determined the effect of added polyamines and inhibitors of polyamine biosynthesis on calcium mobilization. Parathyroid hormone (10 nmol l-1) stimulated omithine decarboxylase activity by approximately 50% in both cultured bone cells of osteoblastic phenotype, UMR 106 and in mouse calvarial osteoblast-like cells. In mouse calvaria the levels of putrescine and spermidine were increased by parathyroid hormone after 24 hours. The levels of spermine were very low and were unchanged by parathyroid hormone. The two polyamine synthesis inhibitors alpha-difluoromethylornithine (DFMO; 2 mmol l-1) and methylglyoxal-bis-guanylhydrazone (MGBG; 50 mu mol l-1) did not significantly affect the mobilization of 45Ca from parathyroid hormone-stimulated bones. All three polyamines, putrescine, spermidine and spermine, inhibited the mobilization of 45Ca induced by parathyroid hormone in a dose-dependent manner. The inhibition induced by putrescine was reversible. In summary, we have shown that parathyroid hormone increases the accumulation of polyamines in bone, but the effect is small. Furthermore, inhibition of polyamine biosynthesis does not reduce parathyroid hormone-induced mineral mobilization and the addition of polyamines leads to a reduced rather than a stimulated mineral mobilization. Thus, polyamines do not seem to be critically involved in the changes in bone resorption induced by parathyroid hormone.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyamine metabolism inSetaria cervi,the bovine filarial worm

Spermine and spermidine were found to be the principal polyamines in the bovine filarial parasiteSetaria cervi, whereas putrescine was observed in very low amounts. Studies conducted on the enzymes of polyamine biosynthesis revealed low activity for S-adenosyl-methionine decarboxylase, questionable and negligible activities for the decarboxylation of ornithine and arginine, and appreciable activity for ornithine aminotransferase. Uptake studies with radiolabeled putrescine, spermidine and spermine showed that these amines are rapidly taken up from the medium by an active uptake process. The uptake was temperature-sensitive and abolished at 0–4°C. The questionable presence of biosynthetic enzymes such as ornithine and arginine decarboxylase and, on the other hand, an effective uptake mechanism indicate that the parasite may depend on the host for its polyamine requirement, thereby indicating a possible target for chemotherapy.Communication No. 4114     

Métabolisme et fonctions des polyamines

Polyamines are molecules derived from ornithine (i.e. putrescine, spermine, spermidine) or from arginine (agmatine). Key regulatory steps of their metabolism are represented by ornithine decarboxylase (tightly regulated by antizyme and antizyme inhibitor), acetyl-CoA:spermidine/spermine N¹-acetyl-transferase, responsible for polyamine interconversion, and membrane transport systems. Polyamines being essential for gene expression, cell cycle and cell proliferation, they are involved for example in immune response, gut trophicity and wound healing. While potentially involved in cell transformation, high level of polyamine are toxic for the cell. As such their measurement has been proposed in the follow up of cancer patients and inhibitors of their synthesis have been proposed for anticancer therapy, but with limited success probably because of the complexity of their metabolism. From a nutritional point of view, polyamines could be of interest in the restoration and maintenance of gut trophicity.     

Spermidine/spermine-N1-acetyltransferase activity in isoprenaline-stimulated rat salivary glands

In parotid, sublingual and submaxillary glands stimulated by continuous intravenous infusion of isoprenaline at various doses for 3 h, the concentrations of the polyamines putrescine, spermidine, spermine and N1-acetylspermidine as well as the activities of ornithine decarboxylase and spermidine/spermine-N1-acetyltransferase were determined. Ornithine decarboxylase is the enzyme that catalyses the formation of putrescine from ornithine. The most striking effect was observed in parotid glands. Here, the beta-adrenergic agonist increased the activity of spermidine/spermine-N1-acetyltransferase markedly, but only slightly (if at all) that of ornithine decarboxylase. It also increased the concentrations of N1-acetylspermidine and putrescine markedly. The result obtained in this gland indicates interconversion of higher polyamines to putrescine.     

Transgenic animals as models in the study of the neurobiological role of polyamines

Natural polyamines, putrescine, spermidine and spermine, exhibit a number of neurophysiological and metabolic effects in brain preparations. In the in vitro studies, several specific sites of action have been identified such as ion channels, transmitter release and Ca²⁺ homeostasis. Polyamines have been linked to the development of neuronal degeneration caused by, for instance, epileptic seizures and stroke. The role of endogenous polyamines in the functioning brain is not clear, however. We review the work carried out using state-of-the-art transgenic animal models for polyamine research. A number of transgenic decarboxylase gene have been generated. Of these animals those with ornithine decarboxylase transgene show an extensive and constitutive expression of the enzyme in the brain with an exceedingly high putrescine concentration, a phenotype that is not encountered under physiological conditions. In this article we review the neurometabolic, behavioural and histological data that has been obtained from these transgenic mice.     

Polyamines and long-term disuse of rat parotid glands.

A decrease in nerve reflex activation for 7-14 days, induced by a liquid diet, caused the rat parotid gland to lose weight, involving reduction in both cell size and number. In the atrophied glands, the activity of ornithine decarboxylase, the key enzyme in polyamine formation, and the levels of the polyamines putrescine, spermidine and spermine were found to be lowered. The present results are compatible with a role for polyamines in cellular growth.     

Polyamine metabolism in filarial worms

The human and animal filarial parasites Onchocerca volvulus, Dirofilaria immitis, Brugia patei and Litomosoides carinii contained low levels of putrescine but much higher levels of spermidine and spermine as estimated by ion-pair high pressure liquid chromatography; N-acetylated polyamines were present only in minute amounts. Enzyme activities of ornithine decarboxylase (EC 4.1.1.17) and arginine decarboxylase (EC 4.1.1.19), respectively, were not detectable. Experiments carried out with O. volvulus and D. immitis demonstrated the uptake and bioconversion of labeled polyamines. There is evidence for the existence of a complete reverse pathway generating putrescine from spermidine and spermine, respectively, in both worms. N-Acetylating enzyme activities were detected in 100,000 X g preparations of homogenates from D. immitis which were capable to acetylate putrescine, spermidine and spermine. Long term incubation of the worms in the presence of labeled polyamines resulted in the excretion of putrescine and N-acetylputrescine.     

运动训练对老龄大鼠心肌多胺代谢的影响

目的:通过观察运动训练对老龄大鼠心肌多胺代谢、心肌抗炎及抗氧化能力的影响,探讨多胺代谢在运动训练延缓心肌衰老中的作用。方法:实验分为3组:老年运动组(Old+Ex),18月龄Wistar大鼠梯度跑台运动6周;老年组(Old),与Old+Ex组月龄相同的Wistar大鼠;青年组(Young),3月龄Wistar大鼠。高效液相色谱测定心肌组织中多胺(腐胺、精脒和精胺)含量;[¹⁴C]标记液闪计数方法测定心肌组织中多胺合成限速酶鸟氨酸脱羧酶(ODC)与多胺分解限速酶精脒/精胺乙酰转移酶(SSAT)活性;比色法检测心肌组织中超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量;酶联免疫吸附(ELISA)法检测心肌组织中肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)含量;超声心动图记录大鼠心脏功能;透射电镜观察心肌组织超微结构变化。结果:与Young组比,Old组大鼠心肌组织中ODC活性下降, SSAT活性升高, 精脒、精胺及总多胺池水平显著降低;心肌SOD活性下降,MDA、TNF-α和IL-1β水平升高(P<0.05);超声心动图显示,Old组大鼠左心室收缩末期直径(LVESD)与舒张末期直径(LVEDD)均明显增大,左心室射血分数(LVEF)与缩短分数(LVFS)降低(与Young组比,P<0.01)。Old+Ex组大鼠心肌组织中ODC活性增加,SSAT活性下降,精脒及总多胺池水平明显增加;心肌SOD活性升高,MDA、TNF-α和IL-1β水平均显著降低(与Old组比,P<0.05);左室功能有明显改善。超微结构观察可见Old组心肌肌丝排列不整齐,可见大量脂褐素颗粒沉积,线粒体基质疏松;Old+Ex组心肌肌节结构清晰,线粒体基质致密,嵴排列整齐。结论:运动训练通过上调多胺合成代谢、抑制其分解代谢对抗衰老引起的心肌多胺水平降低;运动训练可提高老龄心肌抗炎、抗氧化能力。维持老龄心肌多胺池在适当水平可能是运动延缓心肌衰老的部分机制之一。     

Identification of polymorphisms and balancing selection in the male infertility candidate gene, ornithine decarboxylase antizyme 3

Background  

The antizyme family is a group of small proteins that play a role in cell growth and division by regulating the biosynthesis of polyamines (putrescine, spermidine, spermine). Antizymes regulate polyamine levels primarily through binding ornithine decarboxylase (ODC), an enzyme key to polyamine production, and targeting ODC for destruction by the 26S proteosome. Ornithine decarboxylase antizyme 3 (OAZ3) is a testis-specific antizyme paralog and the only antizyme expressed in the mid to late stages of spermatogenesis.     

鸟氨酸脱羧酶/多胺系统在大鼠缺血预适应心肌保护中的作用

目的：探讨鸟氨酸脱羧酶（ODC）/多胺系统在缺血预适应（IPC）心肌保护中的作用。方法:应用离体灌流大鼠心脏复制模拟心肌缺血/再灌注模型。心脏随机分为6组：对照组 (control)、缺血/再灌注组 (IR)、弱缺血预适应组 (IPCw)、强缺血预适应组 (IPCs)、IPCw+多胺抑制剂组 (DF-EG-IPCw)和IPCs+多胺抑制剂组(DF-EG-IPCs)。免疫印迹法定量分析多胺合成限速酶鸟氨酸脱羧酶 (ODC)的表达;高效液相色谱测定心肌组织中的多胺（腐胺、精脒、精胺）含量;Powerlab多导生理记录系统记录心脏功能;氯化三苯基四氮唑 (TTC) 染色检测心肌梗死面积;TUNEL方法检测细胞凋亡率,比较其中的差异性。结果:（1）与对照组比,IR组ODC表达下调,腐胺含量增加,精胺含量减少,总多胺池减少（P<0.05）,此时心功能下降（LVDP、HR、CF均低于对照组,P<0.05）,心肌梗死面积及心肌细胞凋亡率增加（P<0.05）;（2）与IR组比,弱及强缺血预处理组（IPCw、IPCs）心肌ODC表达上调,腐胺含量减少,精胺含量增加,总多胺池增加（P<0.05或P<0.01）,此时大鼠心功能有明显改善（LVDP、HR、CF与IR组比,P<0.05）,心肌梗死面积及心肌细胞凋亡率均明显降低（P<0.01）;（3）给予多胺抑制剂后,心肌ODC表达,腐胺、精脒、精胺及总多胺池含量均明显降低（DF-EG-IPCw组 vs IPCw组;DF-EG-IPCs组vs IPCs组,P<0.05或P<0.01）,心功能明显下降（P<0.05）,心肌梗死面积及细胞凋亡率均明显增加（P<0.05）。结论:缺血预适应能明显上调大鼠心肌ODC/多胺系统,减轻缺血/再灌注心肌损伤;多胺合成代谢抑制剂取消了预适应介导的心功能改善、缩小心肌梗死面积及减少心肌细胞凋亡的作用,提示ODC/多胺系统可能参与了缺血预适应介导的心肌保护作用。     

Polyamines increase in sympathetic neurons and non-neuronal cells after axotomy and enhance neurite outgrowth in nerve growth factor-primed PC12 cells

Following axonal damage, sympathetic neurons are capable of regenerating and reinnervating their target tissues. Some years ago exogenous administration of polyamines was shown to enhance this regeneration. Recently, it was found that axonal injury leads to a dramatic up-regulation of the expression of arginase I in sympathetic neurons. This enzyme catalyzes the conversion of arginine to ornithine, which can subsequently be converted to the diamine putrescine and, ultimately, to the polyamines spermidine and spermine. In the present study, using an antiserum that reacts with both spermidine and spermine, we have found an increase in polyamine levels in both neurons and non-neuronal cells in the superior cervical ganglion 2 and 5 days following transection of the ganglion's postganglionic trunks. Using PC12 cells primed with nerve growth factor and then stripped off the culture dish and replated as a model system for axotomized sympathetic neurons, we found that spermidine treatment, with or without nerve growth factor, resulted in an increased percentage of cells with a neurite whose length was at least twice the diameter of the neuron's cell body. These increases could be seen within 48 h and were still evident after 8 days. Together, these data support the possibility that endogenous polyamines are involved in the normal regeneration which occurs following sympathetic axonal damage.     

Polyamine metabolism during cardiac hypertrophy

Treatment with thyroxine for 7 days to produce myocardial hypertrophy led to an increase in the content of putrescine, spermidine, and spermine in the rat heart. The content of decarboxylated S-adenosylmethionine, the source of the aminopropyl groups needed for polyamine synthesis, was increased by the thyroxine treatment as were the activities of ornithine and S-adenosylmethionine decarboxylases. The enhanced S-adenosylmethionine decarboxylase activity measured in vitro was due to an increase in the amount of enzyme protein as measured by immunotitration with a specific antiserum. In vivo, decarboxylation of S-adenosylmethionine was, therefore, increased both by the increased amount of enzyme protein and by the elevated concentration of putrescine (which activates the enzyme) brought about by the enhanced ornithine carboxylase activity. Spermine synthase did not change significantly during the treatment and spermidine synthase increased only slightly. Therefore, the accumulation of polyamines was mediated predominantly via the increased availability of both putrescine and decarboxylated S-adenosylmethionine. Administration of 1,3-diamino-2-propanol led to a rapid reduction in the activity of ornithine decarboxylase in the heart, and continued exposure to this substance by its inclusion in the drinking water completely prevented the increase in concentration of putrescine and polyamines in response to thyroxine. However, cardiac hypertrophy as measured by the increase in cardiac mass was not prevented by such treatment with 1,3-diaminopropanol, showing that the increased content of polyamines was not essential for the hypertrophic response.     

In vivo effects of alpha-DL-difluoromethylornithine on the metabolism and morphology of Trypanosoma brucei brucei

The EATRO 110 isolate of Trypanosoma brucei brucei was grown in rats for 60 h and the animals treated with the ornithine decarboxylase inhibitor alpha-DL-difluoromethylornithine 12 h or 36 h prior to sacrifice. Control untreated animals died 72-80 h after infection. Treated parasites were shorter and broader than the predominantly long slender forms found in untreated controls and many had two or more nuclei and kinetoplasts. Trypanosomes were purified from blood and examined for disruption of polyamine metabolism. ODC activity decreased by more than 99% after 12 h treatment and putrescine and spermidine levels also decreased dramatically. Spermine, not normally present in control cells, increased to detectable, low levels (less than 1 nmol mg-1 protein) after 36 h treatment. alpha-DL-Difluoromethylornithine-treated cells were unable to synthesize putrescine from [3H]ornithine but were able to convert [3H]putrescine + methionine to spermidine. 12-h treated parasites responded to polyamine depletion by assimilating radiolabeled polyamines in vitro at 2- to 4-times the rate of untreated cells. The metabolism of S-adenosylmethionine was also altered in treated parasites: decarboxylated S-adenosylmethionine increased more than 1000-fold over untreated cells while S-adenosylmethionine decarboxylase activity, associated with the formation of spermidine and spermine in other eukaryotes, paradoxically declined in treated cells. Synthesis of macromolecules was perturbed in treated parasites: rates of DNA and RNA synthesis declined 50-100%, while protein synthesis increased up to 4-fold in 36-h treated cells. alpha-DL-Difluoromethylornithine treatment progressively limits the parasites' ability to synthesize nucleic acids and blocks cytokinesis while inducing morphological changes resembling long slender leads to short stumpy transformation.     

Age-related changes in polyamines in memory-associated brain structures in rats

Polyamines putrescine, spermidine and spermine are positively charged aliphatic amines and have important roles in maintaining normal cellular function, regulating neurotransmitter receptors and modulating learning and memory. Recent evidence suggests a role of putrescine in hippocampal neurogenesis, that is significantly impaired during aging. The present study measured the polyamine levels in memory-related brain structures in 24- (aged), 12- (middle-aged) and 4- (young) month-old rats using liquid chromatography/mass spectrometry and high performance liquid chromatography. In the hippocampus, the putrescine levels were significantly decreased in the CA1 and dentate gyrus, and increased in the CA2/3 with age. Significant age-related increases in the spermidine levels were found in the CA1 and CA2/3. There was no difference between groups in spermine in any sub-regions examined. In the parahippocampal region, increased putrescine level with age was observed in the entorhinal cortex, and age did not alter the spermidine levels. The spermine level was significantly decreased in the perirhinal cortex and increased in the postrhinal cortex with age. In the prefrontal cortex, there was age-related decrease in putrescine, and the spermidine and spermine levels were significantly increased with age. This study, for the first time, demonstrates age-related region-specific changes in polyamines in memory-associated structures, suggesting that polyamine system dysfunction may potentially contribute to aged-related impairments in hippocampal neurogenesis and learning and memory.     

多胺在异丙肾上腺素所致大鼠心肌肥厚中的作用及意义

目的： 研究多胺、鸟氨酸脱羧酶(ODC)及精脒/精胺乙酰基转移酶（SSAT）在异丙肾上腺素所致大鼠心肌肥厚中的作用及机制。方法: 异丙肾上腺素（ISO）皮下注射复制大鼠心肌肥厚模型,应用反向高效液相色谱（RP-HPLC）、RT-PCR和Western blotting结合图像分析系统,分别检测ISO作用不同时点大鼠心肌组织多胺含量、ODC和SSAT mRNA和蛋白的表达。结果: 与对照组比较,心脏重量参数在ISO注射后7 d时显著增加,ISO注射后1 d时腐胺含量增加（P<0.05）,5 d、7 d时显著增加（P<0.01）;精脒含量在ISO注射后3 d时开始增加,ISO注射后7 d时增加显著（P<0.01）,精胺含量略有增多（P<0.05）,总多胺池显著增加。心肌组织ODC和SSAT的 mRNA表达在ISO注射后1 d时升高(P<0.05或P<0.01）,并持续在较高水平。心肌组织ODC和SSAT的蛋白表达分别在ISO注射后1 d和ISO注射后5 d时升高,ISO注射后7 d时显著升高（P<0.01）。结论: 大鼠心肌组织多胺含量增加和ODC、SSAT的表达增强可能参与ISO所致心肌肥厚的病理过程。     

Effects of neurotoxic and mechanical lesions of the mesostriatal dopamine pathway on striatal polyamine levels in the rat: modulation by chronic ganglioside GM1 treatment

In male rats, partial hemitransections but not 6-hydroxydopamine (6-OHDA)-induced lesions of the mesostriatal dopamine (DA) pathway produce after 7 days a marked and a modest increase of striatal putrescine and spermidine levels, respectively, on the lesioned side. Following chronic ganglioside GM1 treatment of partially hemitransected rats, an increase of striatal polyamine levels was observed also on the intact side. It is suggested that retrograde cell body changes produced by hemitransection may induce striatal ornithine decarboxylase activity and in this way increase striatal putrescine levels, favoring regenerative mechanisms. The increase of striatal polyamine levels by GM1 treatment on the intact side of both 6-OHDA and mechanically lesioned rats compared with intact unoperated rats may also reflect an increased synthesis of striatal polyamines.     

Regulation of polyamine content in cultured fibroblasts

The content of putrescine and of the polyamines (spermidine and spermine) and the activities of their biosynthetic enzymes were measured in 3T3 mouse fibroblasts and SV40-transformed mouse fibroblasts over the entire period from subculturing in fresh medium until confluence. The transformed cells had a substantially higher content of putrescine and spermidine than the 3T3 cells and higher activities of all of the biosynthetic enzymes. However, the ratio of spermine synthase to spermidine synthase was higher in the 3T3 cells, which correlated with their higher spermine-to-spermidine ratio. All of the biosynthetic enzymes increased in activity during cell growth. Ornithine decarboxylase increased 20-fold with a maximum at 24-36 h after culturing whereas S-adenosylmethionine decarboxylase increased 3-fold at the same time. Spermidine synthase increased 10- to 16-fold during the growth period whereas spermine synthase increased 2- to 3-fold. The relative enzyme activities and the changes in total polyamine content suggested that 1) the activity of S-adenosylmethionine decarboxylase limited the production of the polyamines and 2) the relative amounts of spermidine and spermine synthase determined the predominant polyamine that the available decarboxylated S-adenosylmethionine is used to synthesize. When 3T3 cells become quiescent at confluence, there was a substantial fall in the intracellular spermidine level because of a greatly increased excretion of spermidine into the medium. Spermine content also fell because there was an increased conversion of spermine into spermidine, which was then excreted. The specific excretion of spermidine did not occur with the transformed SV-3T3 cells.     

外源多胺对大鼠早期再生肝鸟氨酸脱羧酶蛋白水平的调节

目的 研究多胺(腐胺、精脒和精胺)对大鼠再生肝鸟氨酸脱羧酶(ODC)蛋白水平的调节,分析多胺在肝再生中的作用.方法 外源多胺(溶于0.9%NaCl)皮下注射雄性SD大鼠(180～220 g),部分肝切除(PH)诱导的大鼠再生肝中ODC蛋白水平的分析采用免疫印迹(Western blotting)方法.结果 高剂量的腐胺(20 ms/kg体重)、精脒(0.15 mg/kg体重)和精胺(6 mg/kg体重)处理后,ODC蛋白水平在PH后12 h内均低于对照组,且各组间变化趋势相近;而低剂量腐胺(0.02 mg/kg体重)、精脒(0.03 mg/kg体重)及精胺(0.06 ms/kg体重)处理组在PH后4h ODC水平迅速升高,分别比对照组高15.1%、29.5%和30.3%.结论 一定剂量的多胺对大鼠早期再生肝ODC蛋白水平有反馈调节作用,其中精脒、精胺的作用较强,腐胺较弱.