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Journal of Molecular Neuroscience

, Volume 41, Issue 3, pp 329–339 | Cite as

Alpha7 Nicotinic Acetylcholine Receptor Expression and Activity During Neuronal Differentiation of PC12 Pheochromocytoma Cells

  • Arthur A. Nery
  • Rodrigo R. Resende
  • Antonio H. Martins
  • Cleber A. Trujillo
  • Vesna A. Eterovic
  • Henning Ulrich
Article

Abstract

Nicotinic acetylcholine receptors (nAChR) exert pivotal roles in synaptic transmission, neuroprotection and differentiation. Particularly, homomeric α7 receptors participate in neurite outgrowth, presynaptic control of neurotransmitter release and Ca2+ influx. However, the study of recombinant α7 nAChRs in transfected cell lines is difficult due to low expression of functional receptor channels. We show that PC12 pheochromocytoma cells induced to differentiation into neurons are an adequate model for studying differential nAChR gene expression and receptor activity. Whole-cell current recording indicated that receptor responses increased during the course of differentiation. Transcription of mRNAs coding for α3, α5, α7, β2 and β4 subunits was present during the course of differentiation, while mRNAs coding for α2, α4 and β3 subunits were not expressed in PC12 cells. α7 subunit expression was highest following 1 day of induction to differentiation. Activity of α7 nAChRs, however, was most elevated on day 2 as revealed by inhibition experiments in the presence of 10 nM methyllycaconitine, rapid current decay and receptor responsiveness to the α7 agonist choline. Increased α7 receptor activity was noted when PC12 were induced to differentiation in the presence of choline, confirming that chronic agonist treatment augments nAChR activity. In summary, PC12 cells are an adequate model to study the role and pharmacological properties of this receptor during neuronal differentiation.

Keywords

Nicotinic acetylcholine receptors PC12 pheochromocytoma cells Alpha7 subtypes Whole-cell recording Nicotinic receptor expression during differentiation 

Abbreviations

nAChR

nicotinic acetylcholine receptor

MLA

Methyllycaconitine citrate

CCh

Carbamoylcholine

b-FGF

basic fibroblast growth factor

dbcAMP

dibutyril cAMP

Notes

Acknowledgments

The work was supported by research grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP), project no.: 2006/61285-9, and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Brazil, awarded to H.U.; A.A. N.'s and C.A.T.'s Ph.D. theses are supported by fellowships from FAPESP, Brazil. A.H.M and V.A.E. acknowledge the NIH grant support (UPR-PRAABREP20RR016470 and G12RR03035-24), R.R.R is grateful for grants from CNPq and FAPEMIG.

References

  1. Adams CE, Broide RS, Chen Y, Winzer-Serhan UH, Henderson TA, Leslie FM et al (2002) Development of the alpha7 nicotinic cholinergic receptor in rat hippocampal formation. Brain Res Dev Brain Res 139:175–187CrossRefPubMedGoogle Scholar
  2. Alkondon M, Albuquerque EX (1994) Presence of alpha-bungarotoxin-sensitive nicotinic acetylcholine receptors in rat olfactory bulb neurons. Neurosci Lett 176:152–156CrossRefPubMedGoogle Scholar
  3. Alkondon M, Pereira EF, Cortes WS, Maelicke A, Albuquerque EX (1997) Choline is a selective agonist of alpha7 nicotinic acetylcholine receptors in the rat brain neurons. Eur J Neurosci 9:2734–2742CrossRefPubMedGoogle Scholar
  4. Alkondon M, Braga MF, Pereira EF, Maelicke A, Albuquerque EX (2000) Alpha7 nicotinic acetylcholine receptors and modulation of gabaergic synaptic transmission in the hippocampus. Eur J Pharmacol 393:59–67CrossRefPubMedGoogle Scholar
  5. Al-Robaiy S, Rupf S, Eschrich K (2001) Rapid competitive PCR using melting curve analysis for DNA quantification. Biotechniques 31(1382–1386):1388Google Scholar
  6. Angelastro JM, Ignatova TN, Kukekov VG, Steindler DA, Stengren GB, Mendelsohn C et al (2003) Regulated expression of ATF5 is required for the progression of neural progenitor cells to neurons. J Neurosci 23:4590–4600PubMedGoogle Scholar
  7. Avila AM, Dávila-García MI, Ascarrunz VS, Xiao Y, Kellar KJ (2003) Differential regulation of nicotinic acetylcholine receptors in PC12 cells by nicotine and nerve growth factor. Mol Pharmacol 64:974–986CrossRefPubMedGoogle Scholar
  8. Bray C, Son JH, Meizel S (2005) Acetylcholine causes an increase of intracellular calcium in human sperm. Mol Hum Reprod 11:881–889CrossRefPubMedGoogle Scholar
  9. Brehm P, Henderson L (1988) Regulation of acetylcholine receptor channel function during development of skeletal muscle. Dev Biol 129:1–11CrossRefPubMedGoogle Scholar
  10. Brenner HR, Witzemann V, Sakmann B (1990) Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses. Nature 344:544–547CrossRefPubMedGoogle Scholar
  11. Buisson B, Bertrand D (2002) Nicotine addiction: the possible role of functional upregulation Trends Pharmacol Sci 23:130–136Google Scholar
  12. Cho CH, Song W, Leitzell K, Teo E, Meleth AD, Quick MW et al (2005) (2005) Rapid upregulation of alpha7 nicotinic acetylcholine receptors by tyrosine dephosphorylation. J Neurosci 25:3712–3723CrossRefPubMedGoogle Scholar
  13. Corringer PJ, Sallette J, Changeux JP (2006) Nicotine enhances intracellular nicotinic receptor maturation: a novel mechanism of neural plasticity? J Physiol Paris 99:162–171CrossRefPubMedGoogle Scholar
  14. El Kouhen R, Hu M, Anderson DJ, Li J, Gopalakrishnan M (2009) Pharmacology of alpha7 nicotinic acetylcholine receptor mediated extracellular signal–regulated kinase signalling in PC12 cells. Br J Pharmacol 156:638–648CrossRefPubMedGoogle Scholar
  15. Falk L, Nordberg A, Seiger A, Kjaeldgaard A, Hellström-Lindahl E (2003) Higher expression of alpha7 nicotinic acetylcholine receptors in human fetal compared to adult brain. Brain Res Dev Brain Res 142:151–160CrossRefPubMedGoogle Scholar
  16. Gotti C, Fornasari D, Clementi F (1997) Human neuronal nicotinic receptors. Prog Neurobiol 53:199–237CrossRefPubMedGoogle Scholar
  17. Greene LA, Tischler AS (1976) Establishment of a noradrenergic clonal line of rat adrenal pheochromocytoma cells which respond to nerve growth factor. Proc Natl Acad Sci U S A 73:2424–2428CrossRefPubMedGoogle Scholar
  18. Grønlien JH, Håkerud M, Ween H, Thorin-Hagene K, Briggs CA, Gopalakrishnan M, Malysz J (2007) Distinct profiles of alpha7 nAChR positive allosteric modulation revealed by structurally diverse chemotypes. Mol Pharmacol 72:715–724CrossRefPubMedGoogle Scholar
  19. Hatton GI, Yang QZ (2002) Synaptic potentials mediated by alpha 7 nicotinic acetylcholine receptors in supraoptic nucleus. J Neurosci 22:29–37PubMedGoogle Scholar
  20. Ho PL, Raw I (1992) Cyclic AMP potentiates bFGF-induced neurite outgrowth in PC12 cells. J Cell Physiol 150:647–656CrossRefPubMedGoogle Scholar
  21. Huang CM, Tsay KE, Kao LS (1996) Role of Ca2+ in differentiation mediated by nerve growth factor and dibutyryl cyclic AMP in PC12 cells. J Neurochem 67:530–539PubMedCrossRefGoogle Scholar
  22. Kalamida D, Poulas K, Avramopoulou V, Fostieri E, Lagoumintzis G, Lazaridis K et al (2007) Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity. FEBS J 274:3799–3845CrossRefPubMedGoogle Scholar
  23. Liu Z, Zhang J, Berg DK (2007) Role of endogenous nicotinic signaling in guiding neuronal development. Biochem Pharmacol 74:1112–1119CrossRefPubMedGoogle Scholar
  24. Magdesian MH, Nery AA, Martins AH, Juliano MA, Juliano L, Ulrich H et al (2005) Peptide blockers of the inhibition of neuronal nicotinic acetylcholine receptors by amyloid beta. J Biol Chem 280:31085–31090CrossRefPubMedGoogle Scholar
  25. Matsubayashi H, Inoue A, Amano T, Seki T, Nakata Y, Sasa M et al (2004) Involvement of alpha7- and alpha4beta2-type postsynaptic nicotinic acetylcholine receptors in nicotine-induced excitation of dopaminergic neurons in the substantia nigra: a patch clamp and single-cell PCR study using acutely dissociated nigral neurons. Brain Res Mol Brain Res 129:1–7CrossRefPubMedGoogle Scholar
  26. McGehee DS, Role LW (1995) Physiological diversity of nicotinic acetylcholine receptors expressed by vertebrate neurons. Annu Rev Physiol 57:521–546CrossRefPubMedGoogle Scholar
  27. Michel PP, Vyas S, Agid Y (1995) Synergistic differentiation by chronic exposure to cyclic AMP and nerve growth factor renders rat phaeochromocytoma PC12 cells totally dependent upon trophic support for survival. Eur J Neurosci 7:251–260CrossRefPubMedGoogle Scholar
  28. Papke RL, Dwoskin LP, Crooks PA (2007) The pharmacological activity of nicotine and nornicotine on nAChRs subtypes: relevance to nicotine dependence and drug discovery. J Neurochem 101:160–167CrossRefPubMedGoogle Scholar
  29. Peng JH, Fryer JD, Hurst RS, Schroeder KM, George AA, Morrissy S et al (2005) High-affinity epibatidine binding of functional, human alpha7-nicotinic acetylcholine receptors stably and heterologously expressed de novo in human SH-EP1 cells. J Pharmacol Exp Ther 313:24–35CrossRefPubMedGoogle Scholar
  30. Resende RR, Gomes KN, Adhikari A, Britto LR, Ulrich H (2008a) Mechanism of acetylcholine-induced calcium signaling during neuronal differentiation of P19 embryonal carcinoma cells in vitro. Cell Calcium 43:107–121CrossRefPubMedGoogle Scholar
  31. Resende RR, Alves AS, Britto LR, Ulrich H (2008b) Role of acetylcholine receptors in proliferation and differentiation of P19 embryonal carcinoma cells. Exp Cell Res 314:1429–1443CrossRefPubMedGoogle Scholar
  32. Richter-Landsberg C, Jastorff B (1986) The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells. J Cell Biol 102:821–829CrossRefPubMedGoogle Scholar
  33. Role LW, Berg DK (1996) Nicotinic receptors in the development and modulation of CNS synapses. Neuron 16:1077–1085CrossRefPubMedGoogle Scholar
  34. Rydel RE, Greene LA (1987) Acidic and basic fibroblast growth factors promote stable neurite outgrowth and neuronal differentiation in cultures of PC12 cells. J Neurosci 7:3639–3653PubMedGoogle Scholar
  35. Sallette J, Bohler S, Benoit P, Soudant M, Pons S, Le Novère N et al (2004) An extracellular protein microdomain controls up-regulation of neuronal nicotinic acetylcholine receptors by nicotine. J Biol Chem 279:18767–18775CrossRefPubMedGoogle Scholar
  36. Séguéla P, Wadiche J, Dineley-Miller K, Dani JA, Patrick JW (1993) Molecular cloning, functional properties, and distribution of rat brain alpha 7: a nicotinic cation channel highly permeable to calcium. J Neurosci 13:596–604PubMedGoogle Scholar
  37. Sokolova E, Matteoni C, Nistri A (2005) Desensitization of neuronal nicotinic receptors of human neuroblastoma SH-SY5Y cells during short or long exposure to nicotine. Br J Pharmacol 146:1087–1095CrossRefPubMedGoogle Scholar
  38. Taly A, Delarue M, Grutter T, Nilges M, Le Novère N, Corringer PJ et al (2005) Normal mode analysis suggests a quaternary twist model for the nicotinic receptor gating mechanism. Biophys J 88:3954–3965CrossRefPubMedGoogle Scholar
  39. Tischler AS, Greene LA (1975) Nerve growth factor-induced process formation by cultured rat pheochromocytoma cells. Nature 258:341–342CrossRefPubMedGoogle Scholar
  40. Trujillo CA, Schwindt TT, Martins AH, Alves JM, Mello LE, Ulrich H (2009) Novel perspectives of neural stem cell differentiation: from neurotransmitters to therapeutics. Cytometry A 75:38–53PubMedGoogle Scholar
  41. Udgaonkar JB, Hess GP (1987) Acetylcholine receptor: channel-opening kinetics evaluated by rapid chemical kinetic and single-channel current measurements. Biophys J 52:873–883CrossRefPubMedGoogle Scholar
  42. Ulrich H, Akk G, Nery AA, Trujillo CA, Rodriguez AD, Eterović VA (2008) Mode of cembranoid action on embryonic muscle acetylcholine receptor. J Neurosci Res 86:93–107CrossRefPubMedGoogle Scholar
  43. Vallejo YF, Buisson B, Bertrand D, Green WN (2005) Chronic nicotine exposure upregulates nicotinic receptors by a novel mechanism. J Neurosci 25:5563–5572CrossRefPubMedGoogle Scholar
  44. Whiteaker P, Christensen S, Yoshikami D, Dowell C, Watkins M, Gulyas J et al (2007) Discovery, synthesis, and structure activity of a highly selective alpha7 nicotinic acetylcholine receptor antagonist. Biochemistry 46:6628–6636CrossRefPubMedGoogle Scholar
  45. Wonnacott S, Sidhpura N, Balfour DJ (2005) Nicotine: from molecular mechanisms to behaviour. Curr Opin Pharmacol 5:53–59CrossRefPubMedGoogle Scholar
  46. Zhao L, Kuo YP, George AA, Peng JH, Purandare MS, Schroeder KM et al (2003) Functional properties of homomeric, human alpha 7-nicotinic acetylcholine receptors heterologously expressed in the SH-EP1 human epithelial cell line. J Pharmacol Exp Ther 305:1132–1141CrossRefPubMedGoogle Scholar
  47. Zoli M, Le Novère N, Hill JA Jr, Changeux JP (1995) Developmental regulation of nicotinic ACh receptor subunit mRNAs in the rat central and peripheral nervous systems. J Neurosci 15:1912–1939PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  • Arthur A. Nery
    • 1
  • Rodrigo R. Resende
    • 2
    • 3
  • Antonio H. Martins
    • 4
  • Cleber A. Trujillo
    • 1
  • Vesna A. Eterovic
    • 4
  • Henning Ulrich
    • 1
  1. 1.Departamento de Bioquímica, Instituto de QuímicaUniversidade de São PauloSão PauloBrazil
  2. 2.Department of Physics, Institute of Exact SciencesFederal University of Minas GeraisBelo HorizonteBrazil
  3. 3.Federal University of São João Del-Rei-Campus Centro-OesteDivinópolis-MGBrazil
  4. 4.Department of BiochemistryUniversity Central del CaribeBayamonPuerto Rico

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