Effects of arginine, S-adenosylmethionine and polyamines on nerve regeneration

Introduction - Axon growth and axon regeneration are complex processes requiring an adequate supply of certain metabolic precursors and nutrients. Material and methods - This article reviews the studies examining some of the processes of protein modification fundamental to both nerve regeneration and to the continuous and adequate supply of specific factors such as arginine, S-adenosylmethionine and polyamines. Results - The process of arginylation notably increases following nerve injury and during subsequent regeneration of the nerve, with the most likelyfunction of arginine-modification of nerve proteins being the degradation of proteins damaged through injury. It appears that defective methyl group metabolism may be one of the leading causes of demyelination, as suggested by the observation of reduced cerebrospinal fluid concentrations of s-adenosylmethionine (SAMe) and 5-methyltetrahydrofolate, the key metabolites in methylation processes, in patients with a reduction in myelination of corticospinal tracts. Polyamine synthesis, which depends strongly on the availability of both SAMe and arginine, markedly increases in neurons soon after an injury. This "polyamine-response" has been found to be essential for the survival ofthe parent neurons after injury to their axons. Polyamines probably exert their effects through involvement in DNA, RNA and protein synthesis, or through post-translational modifications that areindicated as the most relevant events of the "axon reaction." Conclusions - Nerve regeneration requires the presence of arginine, s-adenosylmethionine, and polyamines. Further studies are needed to explore the mechanisms involved in these processes.     

Polyamines found in gingival fluid enhance the secretory and oxidative function of human polymorphonuclear leukocytes in vitro

Many bacterial and host cells contain large amounts of polyamines that can be released at infection sites as a result of cell lysis. Consequently, the putrescine and spermidine content of gingival fluid from inflamed periodontal pockets (0.1 to 1 mM) is sharply elevated in comparison to peripheral blood. At these levels, polyamines potentiated fMet-Leu-Phe-induced Ca²⁺ signaling in polymorphonuclear leukocytes (PMNs) in vitro. Consistent with the essential role of Ca²⁺ signaling in PMN activation, secondary granule release and superoxide anion production by fMet-Leu-Phe-stimulated PMNs was enhanced in the presence of polyamines. Thus, polyamines may play a local role in modulating the antimicrobial activity of PMNs in periodontal disease.     

2-deoxy-D-glucose inhibits the antitumor effects of α-difluoromethylornithine on the growth of colon cancer in vivo

Summary The glycolytic inhibitor, 2-deoxy-D-glucose (2-DG), has been shown to inhibit the growth of certain cancers. -Difluoromethylornithine (DFMO) is an irreversible inhibitor of ornithine decarboxylase (ODC), the ratelimiting enzyme in polyamine biosynthesis. DFMO has been shown to inhibit cancer growth in a number of models. The present study was designed to investigate the effects of 2-DG alone and combined with DFMO on MC-26 mouse colon adenocarcinoma tumors growing in vivo. Twenty-eight male Balb/c mice were inoculated with 250,000 MC-26 cells, and then randomized into four groups of 7 each: group I served as control; group II received DFMO (3% in drinking water); group III received 2-DG (500 mg/kg/d IP); group IV received a combination of 2-DG and DFMO. Treatment began 5 days after tumor cell inoculation. MC-26 tumor area was reduced 73% by DFMO compared to a 24% reduction caused by 2-DG. The tumor weight was reduced 80% by DFMO and 52% by 2-DG. The tumor contents of DNA, RNA, and protein were significantly reduced by DFMO but not 2-DG. The tumor concentration of the polyamines putrescine and spermidine were reduced by DFMO alone or combined with 2-DG while spermine levels remained unchanged. 2-DG alone did not alter polyamine levels. These results indicate that both 2-DG and DFMO, when added as single agents, inhibit tumor growth. However, the addition of 2-DG to the DFMO regimen inhibited the antitumor effects of DFMO. Survival studies performed on MC-26 cells in vitro corroborated the antagonisms between DFMO and 2-DG that were shown in vivo.Dr. Upp was awarded a fellowship grant from the American Cancer Society Texas Division.     

Differential actions of exogenous and intracellular spermine on contractile activity in smooth muscle of rat portal vein

Effects of the naturally occurring polyamine spermine on electrical and contractile properties of the rat portal vein were studied. 1 mM spermine nearly abolished spike activity and spontaneous contractions and decreased the intracellular Ca²⁺ concentration ([Ca²⁺],). The phasic force responses to 0.1 and 1 μM phenylephrine were partially inhibited, but not the sustain plateau contraction caused by 5 /IM phenylephrine. The Ca²⁺-force relation in high-K⁺ (128 mM)-depolarized veins was shifted to the right, EC₅₀ for Ca²⁺ increasing from 0.50 ± 0.03 mM (control, n= 8) to 0.65 ± 0.06 and to 0.94 ± 0.03 at 1 (n – 4) and 10 (n = 3) mM spermine, respectively. However, at a Ca²⁺ concentration of 2.5 mM, giving maximal force, there was no effect of spermine (1 mM) on either force or [Ca²⁺],. Whereas extracellular spermine thus reduced contractile activity at moderate levels of stimulation, increased intracellular concentration of spermine potentiated the force response to Ca²⁺. Intracellular loading of spermine by reversible permeabilization increased its concentration by 2–3 times. The spontaneous activity and response to phenylephrine were unchanged. However, the Ca²⁺-force relation of depolarized veins was shifted to the left, EC₅₀ decreasing from 0.51 ± 0.04 mM in controls (n= 7) to 0.36 ± 0.02 mM in the loaded veins (n= 9). Spermine increased Ca²⁺-activated force in portal veins permeabilized with β-escin. The degree of potentiation was consistent with observed effects in spermine-loaded intact veins. The results suggest that spermine at physiological intracellular concentration may contribute to the determination of Ca²⁺ sensitivity in vascular smooth muscle cells.     

Effects of luminal stimuli on polyamine metabolism in the small intestine of the rat: the role of enteric nerves

The aim of this study was to investigate to what extent polyamine metabolism in the small intestine of the rat is controlled by the enteric nervous system. Polyamine metabolism was followed by measuring the activity of ornithine decarboxylase (ODC) and in some instances also the content of polyamines (putrescine, spermidine and spermine). ODC activity in the intestine was increased when intraluminal pressure was increased and 3 h after placing cholera toxin in the intestinal lumen. Cholera toxin also increased the tissue putrescine content. Atropine or hexamethonium given i.v. did not influence the evoked changes of ODC activity. The pressure induced changes were not decreased by placing lidocaine on the serosal surface. On the other hand, the ODC activity of control segments were decreased by hexamethonium or atropine. The presence of glucose in the intestinal perfusate did not augment tissue ODC activity, neither did the heat stable enterotoxin from Escherichia coli (STa). It is concluded that the effect on polyamine metabolism evoked by luminal pressure or cholera toxin seems not to be mediated via nerves, while nerves seem to influence ODC activity during control conditions. The experiments with enterotoxins suggest that cAMP is the intracellular second messenger controlling intestinal ODC activity.     

Schedule dependent potentiation of antitumor drug effects by α-difluoromethylornithine in human gastric carcinoma cells in vitro

A clone of human gastric cancer cells (AGS-6) and the parental line (AGS-P) from which it was isolated were used in cell survival studies to determine whether pretreatment for 24, 48 or 72h with -difluoromethylornithine (DFMO, 5mM) would increase the cell's sensitivity to 5-Fluorouracil (5FU), Adriamycin (Adria), 1-(2-chloroethyl)-3-(4-methyl cyclohexyl)-1-nitrosourea (MeCCNU), or Bleomycin (Bleo). Generally, the AGS parental cells were most sensitive to the anticancer agents after exposures to DFMO. However, there was no way to predict in advance from DFMO-induced changes in ornithine decarboxylase (ODC), polyamine or cell kinetics values, how long an exposure to DFMO was required before sensitization to an anticancer agent occurred. The degree of potentiation for a single drug was variable from time to time during exposure to DFMO, and broad differences in the sensitizations were demonstrated among the four anticancer drugs. The AGS-6 clone exhibited little or no increased sensitivity as a result of pretreatment with DFMO, even though the DFMO-induced reductions in ODC and polyamine values in these cells were similar to those produced in the more sensitive parental line.     

Polyamine metabolism in gliomas

Summary Biosynthesis of the polyamines putrescine, spermidine, and spermine has been found to be activated in tissues with cellular proliferation. In the present study we have investigated polyamine levels and the activity of the first rate-limiting enzyme ornithine decarboxylase (ODC) in tumour samples obtained during operation of 202 patients with gliomas. Biochemical data were closely related to the grading of malignancy and to the morphological characteristics of each sample. Mean ODC activity was significantly higher in all gliomas as compared to peritumoural non-neoplastic brain. Furthermore, it was significantly higher (p 0.001) in anaplastic gliomas who grade III and IV (9.0 ± 9.6 nmol/g/h) than in gliomas WHO grade I and II (3.3 ± 4.2 nmol/g/h). Highest enzyme activity (58.5 nmol/g/h) was found in solid and vital parts of malignant tumours, whereas predominantly necrotic areas exhibited low ODC activity (< 1 nmol/g/h). Thus, intra- and interindividual variability of ODC activity corresponded well to histomorphological heterogeneity in high-grade gliomas. Putrescine levels also increased with rising grade of malignancy, whereas spermidine and spermine levels did not correlate with the histological grading. In conclusion, high ODC activity represents a biochemical marker of malignancy in gliomas, but low values do not prove benignity. The present study reinforces the need of further and more extensive tumour sampling closely related to follow-up investigations in the heterogeneous group of gliomas.     

Polyamine involvement in the secretion and action of TGF-α in hormone sensitive human breast cancer cells in culture

These experiments were designed to test polyamine (PA)* involvement in the secretion and action of transforming growth factor (TGF-) in hormone responsive MCF-7 breast cancer cells in liquid culture. At the same time, we evaluated the influence of culture conditions (with serum vs. serum depleted) and subclonality of MCF-7 cells on PA involvement in estrogen (E₂) and TGF- stimulated cell proliferation. Despite inducing a profound suppression of cellular PA levels and inhibiting basal and E₂-stimulated growth, administration of the PA synthesis inhibitor -difluoromethylornithine (DFMO) did not influence either basal or E₂-induced TGF- secretion. In the same experiments, on the other hand, addition of DFMO completely blocked the growth stimulatory effect of exogenous TGF-. However, when the culture conditions were changed to serum-free medium, TGF- and E₂-induced cell proliferation was affected modestly or not at all by DFMO administration, despite similar suppression of cellular ornithine decarboxylase (ODC) activity and PA levels. In addition, different clones of MCF-7 cells differed in their sensitivity to the antiproliferative effect of DFMO as well as in basal levels of ODC activity and PA. We conclude that PAs are not involved in basal or E₂-stimulated TGF- secretion in MCF-7 breast cancer cells. On the other hand, PAs do seem to be important mediators of TGF- and E₂-induced breast cancer cell proliferation, though the degree of such involvement appears to be influenced by serum factors and clonal variability of MCF-7 cells.     

鹿茸多胺的抗脂质过氧化作用

目的 研究鹿茸多胺的抗氧化作用。方法 测定鹿茸多胺在体外对NADPH-维生素C和Fe^2 半胱氨酸系统诱发的微粒体脂质过氧化反应(MDA形成)的影响，对黄嘌呤-黄嘌呤氧化酶系统超氧阴离子自由基(O2^-)产生(还原型细胞色素C形成)的影响，在体内对CCI。和乙醇诱发的小鼠肝脂质过氧化反应(MDA形成)的影响。结果 鹿茸多胺在体外能明显抑制NADPH-维生素C和Fe^2 一半胱氨酸系统诱发的大鼠脑、肝、肾微粒体脂质过氧化反应(MDA形成)，及黄嘌呤一黄嘌呤氧化酶系统O2^-的产生(还原型细胞色素C形成)。在体内能抑制CCl4和乙醇诱发的小鼠肝脂质过氧化反应(MDA形成)。结论 鹿茸多胺具有抗氧化作用。