Cellular and Molecular Neurobiology

, Volume 35, Issue 5, pp 651–660 | Cite as

Ouabain and BDNF Crosstalk on Ganglion Cell Survival in Mixed Retinal Cell Cultures

  • Gustavo de Rezende Corrêa
  • Vinicius Henrique Pedrosa Soares
  • Leandro de Araújo-Martins
  • Aline Araujo dos Santos
  • Elizabeth Giestal-de-Araujo
Original Research


Brain-derived neurotrophic factor (BDNF) is a well-known and well-studied neurotrophin. Most biological effects of BDNF are mediated by the activation of TrkB receptors. This neurotrophin regulates several neuronal functions as cell proliferation, viability, and differentiation. Ouabain is a steroid that binds to the Na+/K+ ATPase, inducing the activation of several intracellular signaling pathways. Previous data from our group described that ouabain treatment increases retinal ganglion cells survival (RGC). The aim of the present study was to evaluate, if this cardiac glycoside can have a synergistic effect with BDNF, the classical trophic factor for retinal ganglion cells, as well as investigate the intracellular signaling pathways involved. Our work demonstrated that the activation of Src, PLC, and PKCδ participates in the signaling cascade mediated by 50 ng/mL BDNF, since their selective inhibitors completely blocked the trophic effect of BDNF. We also demonstrated a synergistic effect on RGC survival when we concomitantly used ouabain (0.75 nM) and BDNF (10 ng/mL). Moreover, the signaling pathways involved in this synergistic effect include Src, PLC, PKCδ, and JNK. Our results suggest that the synergism between ouabain and BDNF occurs through the activation of the Src pathway, JNK, PLC, and PKCδ.


Ouabain BDNF Retina Neuronal survival Synergistic effect 



We would like to thank Alexandre José Fernandes, Bernardino Matheus dos Santos and Arnaldo de Sá for technical assistance. We also thank Arnaldo Paes de Andrade for critical reading of our manuscript. Gustavo de Rezende Corrêa, Vinícius Henrique Pedrosa Soares and Leandro de Araujo Martins received fellowships from CAPES. This work was supported by Grants from CAPES, PRONEX-MCT, PROPPi and FAPERJ.

Conflict of interest

None of the authors declares competing interests.


  1. Araujo EG, Linden R (1993) Trophic factors produced by retinal cells increase the survival of retinal ganglion cells in vitro. Eur J Neurosci 5(9):1181–1188PubMedCrossRefGoogle Scholar
  2. Bagayogo PI, Dreyfus FC (2009) Regulated release of BDNF by cortical oligodendrocytes is mediated through metabotropic glutamate receptors and the PLC pathway. ASN Neuro 1:1–12CrossRefGoogle Scholar
  3. Barde YA, Edgar D, Thoenen H (1982) Purification of a new neurotrophic factor from mammalian brain. EMBO J 1(5):549–553PubMedCentralPubMedGoogle Scholar
  4. Blanco G, Mercer RW (1998) Isozymes of the Na-K-ATPase: heterogeneity in structure, diversity in function. Am J Physiol 275(5 Pt 2):F633–F650PubMedGoogle Scholar
  5. Bradford MM (1976) A rapid and sensitive method of the quantification of a microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  6. Chen H, Weber AJ (2001) BDNF enhances retinal ganglion cell survival in cats with optic nerve damage. Invest Ophthalmol Vis Sci 42(5):966–974PubMedGoogle Scholar
  7. Chung JY, Kim MW, Bang MS, Kim M (2013) Increased expression of neurotrophin 4 following focal cerebral ischemia in adult rat brain with treadmill exercise. PLoS ONE 8(3):e52461PubMedCentralPubMedCrossRefGoogle Scholar
  8. Corrêa GR, Santos AA, Fonte CFL, Araujo EG (2005) Ouabain induces an increase of retinal ganglion cell survival in vitro: the involvement of the protein kinase C. Brain Res 1049:89–94CrossRefGoogle Scholar
  9. Corrêa GR, Cunha KCS, Santos AA, Araujo EG (2010) The trophic effect of ouabain on retinal ganglion cell is mediated by EGF receptor and PKC delta activation. Neurochem Res 35:1343–1352CrossRefGoogle Scholar
  10. Davies C, Tournier C (2012) Exploring the function of the JNK (c-Jun N-terminal kinase) signalling pathway in physiological and pathological processes to design novel therapeutic strategies. Biochem Soc Trans 40(1):85–89PubMedCrossRefGoogle Scholar
  11. de Almeida AA, Gomes da Silva S, Fernandes J, Peixinho-Pena LF, Scorza FA, Cavalheiro EA, Arida RM (2013) Differential effects of exercise intensities in hippocampal BDNF, inflammatory cytokines and cell proliferation in rats during the postnatal brain development. Neurosci Lett 11(553C):1–6CrossRefGoogle Scholar
  12. Dvela M, Rosen H, Ben-Ami HC, Lichtstein D (2012) Endogenous ouabain regulates cell viability. Am J Physiol Cell Physiol 302(2):C442–C452PubMedCrossRefGoogle Scholar
  13. Dvela-Levitt M, Ami HC, Rosen H, Shohami E, Lichtstein D (2014) Ouabain improves functional recovery following traumatic brain injury. J Neurotrauma 31(23):1942–1947PubMedCrossRefGoogle Scholar
  14. Ebel C, Brandes G, Radtke C, Rohn K, Wewetzer K (2013) Clonal in vitro analysis of neurotrophin receptor p75-immunofluorescent cells reveals phenotypic plasticity of primary rat olfactory ensheathing cells. Neurochem Res 38(5):1078–1087PubMedCrossRefGoogle Scholar
  15. Goto A, Yamada K, Yagi N, Hiu C, Terano V, Sugimoto T (1992) Ouabain as endogenous digitalis-like factor in animal? Clin Chem 38:161–162PubMedGoogle Scholar
  16. Gottlieb SS, Rogowski AC, Weinberg M et al (1992) Elevated concentration of endogenous ouabain in patients with congestive heart failure. Circulation 86:420–425PubMedCrossRefGoogle Scholar
  17. Granado-Serrano AB, Martín MA, Goya L, Bravo L, Ramos S (2009) Time-course regulation of survival pathways by epicatechin on HepG2 cells. J Nutr Biochem 2:115–124CrossRefGoogle Scholar
  18. Haas M, Wang H, Tian J, Xie Z (2002) Src-mediated inter-receptor cross-talk between the Na +/K + -ATPase and the epidermal growth factor receptor relays the signal from ouabain to mitogen-activated protein kinases. J Biol Chem 277(24):18694–18702PubMedCrossRefGoogle Scholar
  19. Hamlyn JM, Blaustein MP, Bova S, DuCharme DW, Harris DW, Mandel F, Mathews WR, Ludens JH (1991) Identification, and characterization of a ouabain-like compound from human plasma. Proc Natl Acad Sci 88:6259–6263Google Scholar
  20. Isenmann S, Kretz A, Cellerino A (2003) Molecular determinants of retinal ganglion cell development survival, and regeneration. Prog Retin Eye Res 22:483–543PubMedCrossRefGoogle Scholar
  21. Mesulan MM (1982) Tracing neural connections with horseradish peroxidase, 2nd edn. Willey, Hoboken, p 251Google Scholar
  22. Mohammadi K, Liu L, Tian J, Kometiani P, Xie Z, Askari A (2003) Positive inotropic effect of ouabain on isolated heart is accompanied by activation of signal pathways that link Na +/K + -ATPase to ERK1/2. J Cardiovasc Pharmacol 41(4):609–614PubMedCrossRefGoogle Scholar
  23. Nibuya M, Nestler EJ, Duman RS (1996) Chronic antidepressant administration increases the expression of cAMP response element binding protein (CREB) in rat hippocampus. J Neurosci 16(7):2365–2372PubMedGoogle Scholar
  24. Pan M, Zhang C (2013) Stimulatory effect of gonadal hormones on fetal rat hippocampal neural proliferation requires neurotrophin receptor activation in vitro. Neurosci Lett 24(546):1–5CrossRefGoogle Scholar
  25. Perígolo-Vicente R, Ritt K, Pereira MR, Torres PM, Paes-de-Carvalho R, Giestal-de-Araujo E (2013) IL-6 treatment increases the survival of retinal ganglion cells in vitro: the role of adenosine A1 receptor. Biochem Biophys Res Commun 430(2):512–518PubMedCrossRefGoogle Scholar
  26. Pinzon-Guzman C, Zhang SS, Barnstable CJ (2011) Specific protein kinase C isoforms are required for rod photoreceptor differentiation. J Neurosci 31(50):18606–18617PubMedCentralPubMedCrossRefGoogle Scholar
  27. Ramos-Languren LE, Escobar ML (2013) Plasticity and metaplasticity of adult rat hippocampal mossy fibers induced by neurotrophin-3. Eur J Neurosci 37(8):1248–1259PubMedCrossRefGoogle Scholar
  28. Sabapathy K (2012) Role of the JNK pathway in human diseases. Prog Mol Biol Trans Sci 106:145–169Google Scholar
  29. Sanna MG, Correia JS, Ducrey O, Lee J, Nomoto K, Schrantz N, Deveraux QL, Ulevitch RJ (2002) IAP suppression of apoptosis involves distinct mechanisms: the TAK1/JNK1 signaling cascade and caspase inhibition. Mol Cell Biol 22:1754–1766PubMedCentralPubMedCrossRefGoogle Scholar
  30. Santos AA, Araujo EG (2000) The effect of PKC activation on survival of rat retinal ganglion cells in culture. Brain Res 853:338–343PubMedCrossRefGoogle Scholar
  31. Scheiner-Bobis G, Schoner W (2001) A fresh facet for ouabain action. Nat Med 7:1288–1289PubMedCrossRefGoogle Scholar
  32. Schoner W, Scheiner-Bobis G (2007) Endogenous and exogenous cardiac glycosides: their roles in hypertension, salt metabolism, and cell growth. Am J Physiol Cell Physiol 293:509–536CrossRefGoogle Scholar
  33. Schoner W, Bauer N, Mülle-Ehmsen J et al (2003) Ouabain as a mammalian hormone. Ann N Y Acad Sci 986:678–684PubMedCrossRefGoogle Scholar
  34. Tomimatsu N, Arakawa Y (2008) Protein kinase C-mediated protection of motoneurons from excitotoxicity. Neurosci Lett 439:143–146PubMedCrossRefGoogle Scholar
  35. Uchida H, Matsushita Y, Ueda H (2013) Epigenetic regulation of BDNF expression in the primary sensory neurons after peripheral nerve injury: implications in the development of neuropathic pain. Neuroscience 14(240):147–154CrossRefGoogle Scholar
  36. von Bartheld CS (1998) Neurotrophins in the development and regenerating visual system. Histol Histopathol 13:437–459Google Scholar
  37. Vries LDE, Finana F, Cachoux F, Vacher B, Sokoloff P, Cussac D (2010) Cellular BRET assay suggests a conformational rearrangement of preformed TrkB/Shc complexes following BDNF-dependent activation. Cell Signal 22:158–165PubMedCrossRefGoogle Scholar
  38. Waetzig V, Loose K, Haeusgen W, Herdegen T (2008) c-Jun N terminal kinases mediates Fas-induced neurite regeneration in PC12 cells. Biochem Pharmacol 76:1476–1484PubMedCrossRefGoogle Scholar
  39. Xie Z, Askari A (2002) Na+ K+ ATPase as a signal transducer. Eur J Biochem 269:2434–2439PubMedCrossRefGoogle Scholar
  40. You Y, Li W, Gong Y, Yin B, Qiang B, Yuan J, Peng X (2010) ShcD interacts with TrkB via its PTB and SH2 domains and regulates BDNF-induced MAPK activation. BMB Rep 43:485–490PubMedCrossRefGoogle Scholar
  41. Zhang Z, Fan J, Ren Y, Zhou W, Yin G (2013) The release of glutamate from cortical neurons regulated by BDNF via the TrkB/Src/PLC-γ1 pathway. J Cell Biochem 114(1):144–151PubMedCrossRefGoogle Scholar
  42. Zhao Y, Koebis M, Suo S, Ohno S, Ishiura S (2012) Regulation of the alternative splicing of sarcoplasmic reticulum Ca2+-ATPase1 (SERCA1) by phorbol 12-myristate 13-acetate (PMA) via a PKC pathway. Biochem Biophys Res Commun 423(2):212–217PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  • Gustavo de Rezende Corrêa
    • 1
  • Vinicius Henrique Pedrosa Soares
    • 1
  • Leandro de Araújo-Martins
    • 1
  • Aline Araujo dos Santos
    • 1
    • 2
  • Elizabeth Giestal-de-Araujo
    • 1
  1. 1.Departamento de Neurobiologia, Programa de Neurociências, Instituto de BiologiaUniversidade Federal FluminenseNiteróiBrazil
  2. 2.Departamento de Fisiologia e Farmacologia, Instituto BiomédicoUniversidade Federal FluminenseNiteróiBrazil

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