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Influence of ornithine decarboxylase antizymes and antizyme inhibitors on agmatine uptake by mammalian cells

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Abstract

Agmatine (4-aminobutylguanidine), a dicationic molecule at physiological pH, exerts relevant modulatory actions at many different molecular target sites in mammalian cells, having been suggested that the administration of this compound may have therapeutic interest. Several plasma membrane transporters have been implicated in agmatine uptake by mammalian cells. Here we report that in kidney-derived COS-7cell line, at physiological agmatine levels, the general polyamine transporter participates in the plasma membrane translocation of agmatine, with an apparent Km of 44 ± 7 µM and V max of 17.3 ± 3.3 nmol h−1 mg−1 protein, but that at elevated concentrations, agmatine can be also taken up by other transport systems. In the first case, the physiological polyamines (putrescine, spermidine and spermine), several diguanidines and bis(2-aminoimidazolines) and the polyamine transport inhibitor AMXT-1501 markedly decreased agmatine uptake. In cells transfected with any of the three ornithine decarboxylase antizymes (AZ1, AZ2 and AZ3), agmatine uptake was dramatically reduced. On the contrary, transfection with antizyme inhibitors (AZIN1 and AZIN2) markedly increased the transport of agmatine. Furthermore, whereas putrescine uptake was significantly decreased in cells transfected with ornithine decarboxylase (ODC), the accumulation of agmatine was stimulated, suggesting a trans-activating effect of intracellular putrescine on agmatine uptake. All these results indicate that ODC and its regulatory proteins (antizymes and antizyme inhibitors) may influence agmatine homeostasis in mammalian tissues.

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Abbreviations

ODC:

Ornithine decarboxylase

AZ:

Antizyme

AZIN:

Antizyme inhibitor

ADC:

Arginine decarboxylase

SLC:

Solute carrier

OCT:

Organic cation transporter

MATE-1:

Multidrug and toxin extrusion transport

PTS:

Polyamine transport system

DFMO:

Alfa-difluoromethylornithine

ORF:

Open reading frame

References

  • Babál P, Ruchko M, Olson JW, Gillespie MN (2000) Interactions between agmatine and polyamine uptake pathways in rat pulmonary artery endothelial cells. Gen Pharmacol 34:255–261

    Article  PubMed  Google Scholar 

  • Burns MR, Graminski GF, Weeks RS, Chen Y, O’Brien TJ (2009) Lipophilic Lysine-Spermine conjugates are potent polyamine transport inhibitors for use in combination with a polyamine biosynthesis inhibitor. J Med Chem 52:1983–1993

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Cabella C, Gardini G, Corpillo D, Testore G, Bedino S, Solinas SP, Cravanzola C, Vargiu C, Grillo MA, Colombatto S (2001) Transport and metabolism of agmatine in rat hepatocyte cultures. Eur J Biochem 268:940–947

    Article  CAS  PubMed  Google Scholar 

  • Coffino P (2001) Regulation of cellular polyamines by antizyme. Nat Rev Mol Cell Biol 2:188–194

    Article  CAS  PubMed  Google Scholar 

  • Coleman CS, Stanley BA, Pegg AE (1993) Effect of mutations at active site residues on the activity of ornithine decarboxylase and its inhibition by active site-directed irreversible inhibitors. J Biol Chem 268:24572–24579

    CAS  PubMed  Google Scholar 

  • Coleman CS, Hu GR, Pegg AE (2004) Putrescine biosynthesis in mammalian tissues. Biochem J 379:849–855

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Dardonville C, Brun T (2004) Bisguanidine, bis(2-aminoimidazoline), and polyamine derivatives as potent and selective chemotherapeutic agents against Trypanosoma brucei rhodesiense. Synthesis and in vitro evaluation. J Med Chem 47:2296–2307

    Article  CAS  PubMed  Google Scholar 

  • Dardonville C, Goya P, Rozas I, Alsasua A, Martin MI, Borrego MJ (2000) New aromatic iminoimidazolidine derivatives as alpha1-adrenoceptor antagonists: a novel synthetic approach and pharmacological activity. Bioorg Med Chem 8:1567–1577

    Article  CAS  PubMed  Google Scholar 

  • Dardonville C, Rozas I, Callado LF, Meana JJ (2002) I(2)-imidazoline binding site affinity of a structurally different type of ligands. Bioorg Med Chem 10:1525–1533

    Article  CAS  PubMed  Google Scholar 

  • Dardonville C, Rozas I, Goya P, Giron R, Goicoechea C, Martin MI (2003) Synthesis and analgesic activity of a series of new azaalkane bis-guanidinium and bis(2-aminoimidazolinium) compounds. Bioorg Med Chem 11:1283–1291

    Article  CAS  PubMed  Google Scholar 

  • Esteban del Valle A, Paz JC, Sánchez-Jiménez F, Medina MA (2001) Agmatine uptake by cultured hamster kidney cells. Biochem Biophys Res Commun 280:307–311

    Article  Google Scholar 

  • García-Faroldi G, Rodrıguez CE, Urdiales JL, Perez-Pomares JM, Davila JC, Pejler G, Sanchez-Gimenez F, Fajardo I (2010) Polyamines are present in mast cell secretory granules and are important for granule homeostasis. PLoS ONE 5(11):e15071. doi:10.1371/journal.pone.0015071

    Article  PubMed Central  PubMed  Google Scholar 

  • Goracke-Postle CJ, Overland AC, Stone LS, Fairbanks CA (2007) Agmatine transport into spinal nerve terminals is modulated by polyamine analogs. J Neurochem 100:132–141

    Article  CAS  PubMed  Google Scholar 

  • Gründemann D, Hahne C, Berkels R, Schömig E (2003) Agmatine is efficiently transported by non-neuronal monoamine transporters extraneuronal monoamine transporter (EMT) and organic cation transporter 2 (OCT2). J Pharmacol Exp Ther 304:810–817

    Article  PubMed  Google Scholar 

  • Halaris A, Zhu H, Feng Y, Piletz JE (1999) Plasma agmatine and platelet imidazoline receptors in depression. Ann N Y Acad Sci 881:445–451

    Article  CAS  PubMed  Google Scholar 

  • Hayashi S, Murakami Y, Matsufuji S (1996) Ornithine decarboxylase antizyme: a novel type of regulatory protein. Trends Biochem Sci 21:27–30

    Article  CAS  PubMed  Google Scholar 

  • He Y, Suzuki T, Kashiwagi K, Igarashi K (1994) Antizyme delays the restoration by spermine of growth of polyamine-deficient cells through its negative regulation of polyamine transport. Biochem Biophys Res Commun 203:608–614

    Article  CAS  PubMed  Google Scholar 

  • Hiasa M, Miyaji T, Haruna Y, Takeuchi T, Harada Y, Moriyama S, Yamamoto A, Omote H, Moriyama Y (2014) Identification of a mammalian vesicular polyamine transporter. Sci Rep 4:6836. doi:10.1038/srep06836

    Article  PubMed Central  PubMed  Google Scholar 

  • Higashi K, Imamura M, Fudo S, Uemura T, Saiki R, Hoshino T, Toida T, Kashiwagi K, Igarashi K (2014) Identification of functional amino acid residues involved in polyamine and agmatine transport by human organic cation transporter 2. PLoS ONE 9(7):e102234. doi:10.1371/journal.pone.0102234

    Article  PubMed Central  PubMed  Google Scholar 

  • Kanerva K, Mäkitie LT, Pelander A, Heiskala M, Andersson LC (2008) Human ornithine decarboxylase paralogue (ODCp) is an antizyme inhibitor but not an arginine decarboxylase. Biochem J 409:187–192

    Article  CAS  PubMed  Google Scholar 

  • Kanerva K, Lappalainen J, Makitie LT, Virolainen S, Kovanen PT, Andersson LF (2009) Expression of Antizyme Inhibitor 2 in mast cells and role of polyamines as selective regulators of serotonin secretion. PLoS ONE 4(8):e6858. doi:10.1371/journal.pone.0006858

    Article  PubMed Central  PubMed  Google Scholar 

  • Kanerva K, Makitie LT, Back N, Andersson LC (2010) Ornithine decarboxylase antizyme inhibitor 2 regulates intracellular vesicle trafficking. Exp Cell Res 316:1896–1906

    Article  CAS  PubMed  Google Scholar 

  • López-Contreras AJ, López-García C, Jiménez-Cervantes C, Cremades A, Peñafiel R (2006) Mouse ornithine decarboxylase-like gene encodes an antizyme inhibitor devoid of ornithine and arginine decarboxylating activity. J Biol Chem 281:30896–30906

    Article  PubMed  Google Scholar 

  • López-Contreras AJ, Ramos-Molina B, Cremades A, Peñafiel R (2008) Antizyme inhibitor 2 (AZIN2/ODCp) stimulates polyamine uptake in mammalian cells. J Biol Chem 283:20761–20769

    Article  PubMed Central  PubMed  Google Scholar 

  • López-Contreras AJ, Sanchez-Laorden BL, Ramos-Molina B, de la Morena ME, Cremades A, Penafiel R (2009) Subcellular localization of antizyme inhibitor 2 in mammalian cells: influence of intrinsic sequences and interaction with antizymes. J Cell Biochem 107:732–740

    Article  PubMed  Google Scholar 

  • López-García C, Ramos-Molina B, Lambertos A, López-Contreras AJ, Cremades A, Penafiel R (2013) Antizyme Inhibitor 2 Hypomorphic Mice. New patterns of expression in pancreas and adrenal glands suggest a role in secretory processes. PLoS ONE 8(7):e69188. doi:10.1371/journal.pone.0069188

    Article  PubMed Central  PubMed  Google Scholar 

  • Lortie MJ, Novotny WF, Peterson OW, Vallon V, Malvey K, Mendonca M, Satriano J, Insel P, Thomson SC, Blantz RC (1996) Agmatine, a bioactive metabolite of arginine. Production, degradation, and functional effects in the kidney of the rat. J Clin Invest 97:413–420

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Lortie MJ, Ishizuka S, Schwartz D, Blantz RC (2000) Bioactive products of arginine in sepsis: tissue and plasma composition after LPS and iNOS blockade. Am J Physiol Cell Physiol 278:C1191–C1199

    CAS  PubMed  Google Scholar 

  • Makitie LT, Kanerva K, Sankila A, Andersson LC (2009) High expression of antizyme inhibitor 2, an activator of ornithine decarboxylase in steroidogenic cells of human gonads. Histochem Cell Biol 132:633–638

    Article  PubMed  Google Scholar 

  • Mitchell JLA, Judd GG, Bareyal-Leyser A, Ling SY (1994) Feedback repression of polyamine transport is mediated by antizyme in mammalian tissue-culture cells. Biochem J 299:19–22

    PubMed Central  CAS  PubMed  Google Scholar 

  • Molderings GJ, Haenisch B (2012) Agmatine (decarboxylated l-arginine): physiological role and therapeutic potential. Pharmacol Ther 133:351–365

    Article  CAS  PubMed  Google Scholar 

  • Molderings GJ, Bönisch H, Göthert M, Brüss M (2001) Agmatine and putrescine uptake in the human glioma cell line SK-MG-1. Naunyn Schmiedebergs Arch Pharmacol 363:671–679

    Article  CAS  PubMed  Google Scholar 

  • Molderings GJ, Heinen A, Menzel S, Lübbecke F, Homann J, Göthert M (2003a) Gastrointestinal uptake of agmatine: distribution in tissues and organs and pathophysiologic relevance. Ann N Y Acad Sci 1009:44–51

    Article  CAS  PubMed  Google Scholar 

  • Molderings GJ, Bruss M, Bonish H, Gother M (2003b) Identification and pharmacological characterization of a specific agmatine transport system in human tumor cell lines. Ann NY Acad Sci 1009:75–81

    Article  CAS  PubMed  Google Scholar 

  • Omote H, Moriyama Y (2013) Vesicular neurotransmitter transporters: an approach for studying transporters with purified proteins. Physiology 28:39–50

    Article  CAS  PubMed  Google Scholar 

  • Otake K, Ruggiero DA, Regunathan S, Wang H, Milner TA, Reis DJ (1998) Regional localization of agmatine in the rat brain: an immunocytochemical study. Brain Res 787:1–14

    Article  CAS  PubMed  Google Scholar 

  • Pegg AE (1987) The use of inhibitors to study the biochemistry and molecular biology of polyamine biosynthesis and uptake. In: McCann PP, Pegg AE, Sjoerdsma A (eds) Inhibition of polyamine metabolism. Biological significance and basis for new therapies. Academic Press, San Diego, pp 107–119

    Chapter  Google Scholar 

  • Piletz JE, Aricioglu F, Cheng JT, Fairbanks CA, Gilad VH, Haenisch B, Halaris A, Hong S, Lee JE, Li J, Liu P, Molderings GJ, Rodrigues AL, Satriano J, Seong GJ, Wilcox G, Wu N, Gilad GM (2013) Agmatine: clinical applications after 100 years in translation. Drug Discov Today 18:880–893

    Article  CAS  PubMed  Google Scholar 

  • Pitkanen LT, Heiskala M, Andersson LC (2001) Expression of a novel human ornithine decarboxylase-like protein in the central nervous system and testes. Biochem Biophys Res Commun 287:1051–1057

    Article  CAS  PubMed  Google Scholar 

  • Poulin R, Casero RA, Soulet D (2011) Recent advances in the molecular biology of metazoan polyamine transport. Amino Acids 42:711–723

    Article  PubMed Central  PubMed  Google Scholar 

  • Raasch W, Regunathan S, Li G, Reis DJ (1995) Agmatine, the bacterial amine, is widely distributed in mammalian tissues. Life Sci 56:2319–2330

    Article  CAS  PubMed  Google Scholar 

  • Reis DJ, Regunathan S (1998) Agmatine: an endogenous ligand at imidazoline receptors is a novel neurotransmitter. Ann N Y Acad Sci 881:65–80

    Article  Google Scholar 

  • Reis DJ, Regunathan S (2000) Is agmatine a novel neurotransmitter in brain? Trends Pharmacol Sci 21:187–193

    Article  CAS  PubMed  Google Scholar 

  • Reis DJ, Yang XC, Milner TA (1998) Agmatine containing axon terminals in rat hippocampus form synapses on pyramidal cells. Neurosci Lett 250:185–188

    Article  CAS  PubMed  Google Scholar 

  • Samal K, Zhao P, Kendzicky A, Yco LP, McClung H, Gerner E, Burns M, Bachmann A, Sholler G (2013) AMXT-1501, a novel polyamine transport inhibitor, synergizes with DFMO in inhibiting neuroblastoma cell proliferation by targeting both ornithine decarboxylase and polyamine transport. Int J Cancer 133:1323–1334

    Article  CAS  PubMed  Google Scholar 

  • Sastre M, Regunathan S, Reis DJ (1997) Uptake of agmatine into rat brain synaptosomes: possible role of cation channels. J Neurochem 69:2421–2426

    Article  CAS  PubMed  Google Scholar 

  • Satriano J, Matsufuji S, Murakami Y, Lortie MJ, Schwartz D, Kelly CJ, Hayashi S, Blantz RC (1998) Agmatine suppresses proliferation by frameshift induction of antizyme and attenuation of cellular polyamine levels. J Biol Chem 273:15313–15316

    Article  CAS  PubMed  Google Scholar 

  • Satriano J, Isome M, Casero RA Jr, Thomson SC, Blantz RC (2001) Polyamine transport system mediates agmatine transport in mammalian cells. Am J Physiol Cell Physiol 281:C329–C334

    CAS  PubMed  Google Scholar 

  • Seiler N (1983) Liquid chromatographic methods for assaying polyamines using prechromatographic derivatization. In: Tabor H, Tabor CW (eds) Methods in Enzymology, vol 94. Academic Press, New York, pp 10–25

    Google Scholar 

  • Seiler N, Dezeure F (1990) Polyamine transport in mammalian cells. Int J Biochem 22:211–218

    Article  CAS  PubMed  Google Scholar 

  • Snapir Z, Keren-Paz A, Bercovich Z, Kahana C (2008) ODCp, a brain- and testis-specific ornithine decarboxylase paralogue, functions as an antizyme inhibitor, although less eficiently than AzI1. Biochem J 410:613–619

    Article  CAS  PubMed  Google Scholar 

  • Uemura T, Yerushalmi HF, Tsaprailis G, Stringer DE, Pastorian KE, Hawel L 3rd, Byus CV, Gerner EW (2008) Identification and characterization of a diamine exporter in colon epithelial cells. J Biol Chem 283:26428–26435

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Winter TN, Elmquist WF, Fairbanks CA (2011) OCT2 and MATE1 provide bidirectional agmatine transport. Mol Pharm 8:133–142

    Article  CAS  PubMed  Google Scholar 

  • Zhang WZ, Kaye DM (2004) Simultaneous determination of arginine and seven metabolites in plasma by reversed-phase liquid chromatography with a time-controlled ortho-phthaldialdehyde precolumn derivatization. Anal Biochem 326:87–92

    Article  CAS  PubMed  Google Scholar 

  • Zhu MY, Iyo A, Piletz JE, Regunathan S (2004) Expression of human arginine decarboxylase, the biosynthetic enzyme for agmatine. Biochim Biophys Acta 1670:156–164

    Article  PubMed Central  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by grants 08681/PI/08 from Seneca Foundation (Autonomous Community of Murcia), SAF2008-03638 from the Spanish Ministry of Science and Innovation, SAF2011-29051 from Spanish Ministry of Economy and Competitiveness, and FEDER funds from The European Community. AL is recipient of a scholarship (FPU) from the Spanish Ministry of Education. We thank Dr. Mark R. Burns (Aminex Therapeutics Inc., Washington) for providing AMXT-1501 and for the critical reading of the manuscript.

Conflict of interest

The authors declare that they have no conflict of interest.

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Author notes

  1. Andrés J. López-Contreras

    Present address: Department of Cellular and Molecular Medicine, Center for Chromosome Stability, Panum Institute, University of Copenhagen, Copenhagen, Denmark

Authors and Affiliations

  1. Departamento de Bioquímica y Biología Molecular B e Inmunología, Facultad de Medicina, Universidad de Murcia, Campus de Espinardo, 30100, Murcia, Spain

    Bruno Ramos-Molina, Andrés J. López-Contreras, Ana Lambertos & Rafael Peñafiel

  2. Departamento de Farmacología, Facultad de Medicina, Universidad de Murcia, Murcia, Spain

    Asunción Cremades

  3. Instituto de Química Médica, IQM-CSIC, Juan de la Cierva 3, 28006, Madrid, Spain

    Christophe Dardonville

  4. Instituto Murciano de Investigación Biosanitaria (IMIB), Murcia, Spain

    Asunción Cremades & Rafael Peñafiel

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  1. Bruno Ramos-Molina
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  2. Andrés J. López-Contreras
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  3. Ana Lambertos
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Correspondence to Rafael Peñafiel.

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Ramos-Molina, B., López-Contreras, A.J., Lambertos, A. et al. Influence of ornithine decarboxylase antizymes and antizyme inhibitors on agmatine uptake by mammalian cells. Amino Acids 47, 1025–1034 (2015). https://doi.org/10.1007/s00726-015-1931-3

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