Effects of polyamines on voltage-activated calcium channels in guinea-pig intestinal smooth muscle

Effects of polyamines on the spontaneous mechanical and electrical activity of guinea-pig intestinal smooth muscle were studied. Spermine and spermidine inhibited action potential generation and contractions, while putrescine had no effect. Single smooth muscle cells were isolated from the longitudinal muscle layer of the guinea-pig ileum. Whole-cell voltage-clamp experiments were carried out to investigate the effects of polyamines on current through voltage-activated Ca²⁺ channels. Spermine and spermidine (0.1–1 mM) reduced the inward current in a concentration-dependent manner. Spermine blocked current activated by the dihydropyridine agonist BAY K 8644 (1 M), whereas no additional inhibition by spermine was seen after blockage of dihydropyridine-sensitive channels by nifedipine (0.1 M). Inhibition by spermine or spermidine did not shift the peak of the current voltage relation of the inward current. Steady-state activation and inactivation relationships were not affected and thus the amplitude, but not the voltage dependence, of the window current responsible for Ca²⁺ inflow during sustained depolarization was affected. Putrescine (1 mM) had no significant effect on the inward current. These results suggest that spermine and spermidine inhibit contraction in spontaneously active intestinal smooth muscle by inhibiting Ca²⁺ current responsible for generation of action potentials.     

Effects of α-difluromethylornithine on the development of deeply invasive urinary bladder carcinomas in mice

Summary -Difluoromethylornithine (DFMO), was examined for its ability to suppress the development of invasive urinary bladder carcinoma in C3H/He male mice. Continuous administration of 0.2% DFMO in water following carcinogen treatment (0.05% N-bytyl-N-(4-hydroxybutyl) nitrosamine, BHBN, in drinking water for 8 weeks) was effective in suppressing urinary bladder carcinomas (P<0.05) as compared with the control group. However, when comparison was made based on tumors involving the entire urinary tract, protective effects could not be demonstrated. Coadministration of DFMO (0.2%) and BHBN (0.01%) did not alter tumor induction by the latter. These results were in sharp contrast to the protective effects in rats. Since bladder tumors in rats are of low grade and superficial whereas those in mice are of high grade and deeply invasive, our data indicate that DFMO has little to no effects against the development of aggressive forms of bladder carcinoma.Supported by NIH Grant CA 33511     

UV-Induced changes in urinary polyamine excretion in the mouse

Summary Exposure of hairless mice to the light of a germicidal lamp (254 nm) under conditions which are known to induce epidermal DNA synthesis, cell proliferation, and polyamine metabolism produced a marked increase of polyamine excretion in the urine which lasted for many days. The increase was about the same for free and acetylated polyamines. Although the ratio of N¹-acetylspermidine/N⁸-acetylspermidine increased somewhat in the urine of animals exposed to UV, the increase was not significant enough to be useful as a marker of enhanced cell proliferation. A single topical dose of -difluoromethylornithine, a selective inhibitor of ornithine decarboxylase, prevented the UV-induced increase of polyamine excretion in agreement with its effect on UV-induced epidermal polyamine turnover.     

Polyamines,myosin heavy chains,and collagen specific amino acids after a repeated bout of eccentric exercise

We investigated alternatives to commonly used biomarkers of exercise-induced tissue damage. Over 5 days following two bouts of 100 drop-to-vertical jumps (inter-bout rest period of 3 weeks), myosin heavy chain 1, hydroxylysine (HYL), hydroxyproline (HYP), spermine (SPM) and spermine synthase (SMS) were measured in the serum of 10 participants. HYL significantly increased from 5.92 ± 1.49 ng/mL to 6.48 ± 1.47 ng/mL at 24 h. A similar trend was observed for bout 2, but without reaching significance. SPM significantly increased only after bout 1 from 0.96 ± 0.19 ng/mL at pretest to a peak level of 1.12 ± 0.26 ng/mL at 24 h, while B2 increments remained non-significant. Myosin heavy chain 1, HYP and SMS values remained below the detection limit of the applied enzyme-linked immunosorbent assay (ELISA) kit. Though HYL and SM increased after the intervention, both markers showed a large standard deviation (SD) combined with small increments. Therefore, none of the investigated biomarkers provides a meaningful alternative to commonly used damage markers.     

Ethanol-Associated Alterations in the Kinetics of Putrescine Uptake and Metabolism by the Regenerating Liver

Biosynthesis of the polyamines, putrescine, spermidine, and spermine is required for DNA synthesis and liver regeneration after partial hepatectomy. We have previously reported that chronic ethanol consumption impairs polyamine synthesis and significantly retards liver regeneration after partial hepatectomy. In those studies, supplementation with putrescine restored hepatic DNA synthesis in ethanol-fed rats but exerted no effect in pair-fed controls. These differences in the response to putrescine treatment may have resulted from ethanol-associated differences in hepatic uptake, release, or metabolism of putrescine. To resolve these issues and define more completely how putrescine treatment affects DNA synthesis, we now assess the kinetics of putrescine uptake and metabolism after intraperitoneal or intravenous injection of radiolabeled putrescine (1.2 mmol/kg, specific activity 1 microCi/mmol) into rats fed 36% ethanol diets or isocaloric, nonethanol diets for 6 weeks prior to partial hepatectomy. After putrescine treatment, hepatic putrescine concentrations were greater in ethanol-fed rats than controls. Differences in post-treatment hepatic putrescine levels between ethanol and pair-fed groups could not be explained by differences in the rates of hepatic putrescine uptake or excretion into bile; residual de novo synthesis of putrescine from ornithine or metabolism of hepatic putrescine to its polyamine products, spermidine and spermine. Indeed, supplemental putrescine was not appreciably converted to spermidine or spermine in either ethanol or control rats. Hence, these latter polyamines are unlikely to be responsible for the treatment-associated improvement in DNA synthesis that has been noted in ethanol-fed rats. This suggests that putrescine itself acts to restore hepatic DNA synthesis in ethanol-fed rats.     

血管活性肠肽对人胰腺癌细胞生长的自分泌调节作用及其机制的研究

采用３Ｈ－胸腺嘧啶掺入研究了血管活性肠肽（ＶＩＰ）对我院所建一株人胰腺癌细胞生长的影响，并对该细胞株进行ＶＩＰ受体、受体后细胞内介质ｃＡＭＰ和多胺产生的研究。发现ＶＩＰ在１０－８Ｍ和１０－７Ｍ对具有ＶＩＰ受体的人胰腺癌细胞有明显促生长作用，且成浓度依赖关系，这种作用可被其受体拮抗剂（４－Ｃｌ－Ｄ－ｐｈｅ６，Ｌｅｕ１７）ＶＩＰ所阻断；ＶＩＰ能显著促进本株人胰腺癌细胞细胞内ｃＡＭＰ及多胺的产生，表明ＶＩＰ的促生长作用可能与细胞内ｃＡＭＰ和多胺的生物合成增加有关；本株细胞在培养过程中可向培养基中分泌一定量的ＶＩＰ，ＶＩＰ抗体能抑制其在无血清培养基中生长，提示ＶＩＰ对本株人胰腺癌细胞的生长有自分泌调节作用。     

Acamprosate has no effect on NMDA-induced toxicity but reduces toxicity induced by spermidine or by changing the medium in organotypic hippocampal slice cultures from rat

BACKGROUND: It has been suggested that the antirelapse drug acamprosate can inhibit or potentiate glutamate/NMDA receptor-mediated responses via a polyamine site. Additionally, subchronic exposure to acamprosate increases expression of some NMDA receptor subunits. These effects on NMDA receptors imply that the drug may have neurotoxic or neuroprotective actions under different conditions, and these studies were undertaken to evaluate this possibility in hippocampal neuronal cultures. METHODS: Organotypic hippocampal cultures from 8-day-old neonatal rats were maintained in medium for 28 days. The effects of acamprosate (100 microM) alone or on neurotoxic challenges induced by either 50 microM NMDA or 100 microM spermidine were studied. Neurotoxicity was assessed by uptake of propidium iodide 24 hr after challenge. Calcium entry was measured by uptake of 45Ca2+ into the culture during the challenge. RESULTS: Acamprosate produced no neurotoxicity in these cultures after acute or subchronic exposure. In contrast, the presence of acamprosate significantly reduced "basal" propidium iodide uptake caused by the medium change procedure; similar effects were obtained with dizocilpine (MK-801; 30 microM) and, to a lesser extent, with ifenprodil (50 microM). Acamprosate did not significantly potentiate or inhibit NMDA-induced neurotoxicity, but the presence of acamprosate significantly reduced spermidine-induced neurotoxicity. CONCLUSION: No evidence was obtained that the putative agonist or coagonist effects of acamprosate on the NMDA receptor are able to cause neurotoxicity. Similarly, no evidence for inhibitory effects of acamprosate on NMDA-induced toxicity was observed under any of these conditions. However, acamprosate significantly inhibited the toxicity associated with changing medium and the toxicity induced by spermidine in these hippocampal cultures. The mechanism is unknown but is compatible with previously reported inhibition of polyamine-mediated effects.     

Intestinal luminal putrescine is produced by collective biosynthetic pathways of the commensal microbiome

The intestinal microbiome produces various metabolites that may harm or benefit the host. However, the production pathways of these metabolites have not been well characterised.

The polyamines putrescine and spermidine required for physiological process are also produced by intestinal microbiome. The production and release of these polyamines by microbiome are poorly understood, though we have confirmed that intestinal bacteria produced putrescine from arginine. In this study, we characterised polyamine synthesis by analysing the collective metabolic functions of the intestinal microbiome. In particular, we analysed polyamines and their intermediates in faecal cultures, as well as the colonic contents of rats injected with isotope-labelled arginine through a colon catheter, using mass spectrometry.

Isotope-labelled putrescine was detected in faecal cultures and colonic contents of rats injected with isotope-labelled arginine. Putrescine is produced through multiple pathways, and its extracellular intermediates are exchanged between bacterial species. Additionally, we demonstrated that the collective metabolic pathway depends on a complex exchange of metabolites released into the colonic lumen.

This study demonstrates the existence of putrescine biosynthetic pathways based on the collective metabolic functions of the intestinal microbial community. Our findings provide knowledge to manipulate the levels of intestinal microbial products, including polyamines, that may modulate host health.     

Eflornithine Treatment in SHR: Potential Role of Vascular Polyamines and Ornithine Decarboxylase in Hypertension

This study was performed to assess the potential role of polyamines in the alterations in vascular structure and function in spontaneously hypertensive rats (SHR). The effects of chronic administration of eflornithine (α-difluoromethylornithine; DFMO), a highly specific inhibitor of ornithine decarboxylase (the rate limiting enzyme in polyamine biosynthesis), on vascular polyamine contents, vascular structure and function, and blood pressure was studied. Male SHR (16-17 weeks of age) with an average systolic blood pressure (SBP) of 161 ± 3 mmHg were used. The rats were divided into two groups and received either tap water or a 1% DFMO solution to drink for 6 weeks. SBP and body weight were recorded prior to and once-a-week during the experiment. Standard in vitro vascular reactivity studies on ring segments of aorta and tail artery were performed. Ring segment weight, arterial medial thickness, and vascular polyamine contents were also determined. Body weights were not significantly affected by the DFMO treatment. SBP in control SHR rose progressively to an average value of 185 ± 5 mmHg by the sixth experimental week. Although DFMO treatment did not cause a significant decrease in SBP compared to pretreatment values, it did prevent a further increase in SBP. Aortic and tail artery responsiveness to norepinephrine and electrical stimulation, respectively, ring segment weight, arterial medial thickness, and vascular polyamine contents were all significantly less in SHR receiving the DFMO treatment. These data are the first to demonstrate the effectiveness of DFMOto lower polyamine contents in the vasculature of hypertensive SHR. Importantly, chronic DFMO treatment prevented the further rise in SBP as well as the changes in vascular structure and function associated with the elevation in blood pressure. Thus, it appears that polyamines may play an important role in mediating the vascular alterations in SHR.