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Characterization of Amino Acid Profile and Enzymatic Activity in Adult Rat Astrocyte Cultures

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Abstract

Astrocytes are multitasking players in brain complexity, possessing several receptors and mechanisms to detect, participate and modulate neuronal communication. The functionality of astrocytes has been mainly unraveled through the study of primary astrocyte cultures, and recently our research group characterized a model of astrocyte cultures derived from adult Wistar rats. We, herein, aim to characterize other basal functions of these cells to explore the potential of this model for studying the adult brain. To characterize the astrocytic phenotype, we determined the presence of GFAP, GLAST and GLT 1 proteins in cells by immunofluorescence. Next, we determined the concentrations of thirteen amino acids, ATP, ADP, adenosine and calcium in astrocyte cultures, as well as the activities of Na+/K+-ATPase and acetylcholine esterase. Furthermore, we assessed the presence of the GABA transporter 1 (GAT 1) and cannabinoid receptor 1 (CB 1) in the astrocytes. Cells demonstrated the presence of glutamine, consistent with their role in the glutamate–glutamine cycle, as well as glutamate and d-serine, amino acids classically known to act as gliotransmitters. ATP was produced and released by the cells and ADP was consumed. Calcium levels were in agreement with those reported in the literature, as were the enzymatic activities measured. The presence of GAT 1 was detected, but the presence of CB 1 was not, suggesting a decreased neuroprotective capacity in adult astrocytes under in vitro conditions. Taken together, our results show cellular functionality regarding the astrocytic role in gliotransmission and neurotransmitter management since they are able to produce and release gliotransmitters and to modulate the cholinergic and GABAergic systems.

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References

  1. De Pitta M, Brunel N, Volterra A (2015) Astrocytes: orchestrating synaptic plasticity? Neuroscience. doi:10.1016/j.neuroscience.2015.04.001

    PubMed  Google Scholar 

  2. Perea G, Sur M, Araque A (2014) Neuron-glia networks: integral gear of brain function. Front Cell Neurosci 8:378. doi:10.3389/fncel.2014.00378

    Article  PubMed  PubMed Central  Google Scholar 

  3. Allen NJ, Barres BA (2009) Neuroscience: glia—more than just brain glue. Nature 457(7230):675–677. doi:10.1038/457675a

    Article  CAS  PubMed  Google Scholar 

  4. Parpura V, Heneka MT, Montana V, Oliet SH, Schousboe A, Haydon PG, Stout RF Jr, Spray DC, Reichenbach A, Pannicke T, Pekny M, Pekna M, Zorec R, Verkhratsky A (2012) Glial cells in (patho)physiology. J Neurochem 121(1):4–27. doi:10.1111/j.1471-4159.2012.07664.x

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Agulhon C, Petravicz J, McMullen AB, Sweger EJ, Minton SK, Taves SR, Casper KB, Fiacco TA, McCarthy KD (2008) What is the role of astrocyte calcium in neurophysiology? Neuron 59(6):932–946. doi:10.1016/j.neuron.2008.09.004

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Vedam-Mai V, van Battum EY, Kamphuis W, Feenstra MG, Denys D, Reynolds BA, Okun MS, Hol EM (2012) Deep brain stimulation and the role of astrocytes. Mol Psychiatry 17(2):124–131, 115. doi:10.1038/mp.2011.61

  7. Khakh BS, Sofroniew MV (2015) Diversity of astrocyte functions and phenotypes in neural circuits. Nat Neurosci 18(7):942–952. doi:10.1038/nn.4043

    Article  CAS  PubMed  Google Scholar 

  8. Booher J, Sensenbrenner M (1972) Growth and cultivation of dissociated neurons and glial cells from embryonic chick, rat and human brain in flask cultures. Neurobiology 2(3):97–105

    CAS  PubMed  Google Scholar 

  9. Skytt DM, Madsen KK, Pajecka K, Schousboe A, Waagepetersen HS (2010) Characterization of primary and secondary cultures of astrocytes prepared from mouse cerebral cortex. Neurochem Res 35(12):2043–2052. doi:10.1007/s11064-010-0329-6

    Article  CAS  PubMed  Google Scholar 

  10. Lindsay RM, Barber PC, Sherwood MR, Zimmer J, Raisman G (1982) Astrocyte cultures from adult rat brain. Derivation, characterization and neurotrophic properties of pure astroglial cells from corpus callosum. Brain Res 243(2):329–343. doi:10.1016/0006-8993(82)90257-8

    Article  CAS  PubMed  Google Scholar 

  11. Lange SC, Bak LK, Waagepetersen HS, Schousboe A, Norenberg MD (2012) Primary cultures of astrocytes: their value in understanding astrocytes in health and disease. Neurochem Res 37(11):2569–2588. doi:10.1007/s11064-012-0868-0

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Souza DG, Bellaver B, Souza DO, Quincozes-Santos A (2013) Characterization of adult rat astrocyte cultures. PLoS ONE 8(3):e60282. doi:10.1371/journal.pone.0060282

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Bellaver B, Souza DG, Souza DO, Quincozes-Santos A (2014) Resveratrol increases antioxidant defenses and decreases proinflammatory cytokines in hippocampal astrocyte cultures from newborn, adult and aged Wistar rats. Toxicol In Vitro 28(4):479–484. doi:10.1016/j.tiv.2014.01.006

    Article  CAS  PubMed  Google Scholar 

  14. de Lores Arnaiz GR, Ordieres MG (2014) Brain Na(+), K(+)-ATPase activity in aging and disease. Int J Biomed Sci 10(2):85–102

    PubMed  PubMed Central  Google Scholar 

  15. Benarroch EE (2010) Glutamate transporters: diversity, function, and involvement in neurologic disease. Neurology 74(3):259–264. doi:10.1212/WNL.0b013e3181cc89e3

    Article  PubMed  Google Scholar 

  16. Anderson CM, Swanson RA (2000) Astrocyte glutamate transport: review of properties, regulation, and physiological functions. Glia 32(1):1–14. doi:10.1002/1098-1136(200010)32:1<1:AID-GLIA10>3.0.CO;2-W

    Article  CAS  PubMed  Google Scholar 

  17. Thullbery MD, Cox HD, Schule T, Thompson CM, George KM (2005) Differential localization of acetylcholinesterase in neuronal and non-neuronal cells. J Cell Biochem 96(3):599–610. doi:10.1002/jcb.20530

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Schmidt AP, Tort AB, Silveira PP, Bohmer AE, Hansel G, Knorr L, Schallenberger C, Dalmaz C, Elisabetsky E, Crestana RH, Lara DR, Souza DO (2009) The NMDA antagonist MK-801 induces hyperalgesia and increases CSF excitatory amino acids in rats: reversal by guanosine. Pharmacol Biochem Behav 91(4):549–553. doi:10.1016/j.pbb.2008.09.009

    Article  CAS  PubMed  Google Scholar 

  19. Domanski L, Sulikowski T, Safranow K, Pawlik A, Olszewska M, Chlubek D, Urasinska E, Ciechanowski K (2006) Effect of trimetazidine on the nucleotide profile in rat kidney with ischemia-reperfusion injury. Eur J Pharm Sci 27(4):320–327. doi:10.1016/j.ejps.2005.10.012

    Article  CAS  PubMed  Google Scholar 

  20. Ellman GL, Courtney KD, Andres V Jr, Feather-Stone RM (1961) A new and rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharmacol 7:88–95

    Article  CAS  PubMed  Google Scholar 

  21. Scherer EB, da Cunha MJ, Matte C, Schmitz F, Netto CA, Wyse AT (2010) Methylphenidate affects memory, brain-derived neurotrophic factor immunocontent and brain acetylcholinesterase activity in the rat. Neurobiol Learn Mem 94(2):247–253. doi:10.1016/j.nlm.2010.06.002

    Article  CAS  PubMed  Google Scholar 

  22. Wyse AT, Streck EL, Barros SV, Brusque AM, Zugno AI, Wajner M (2000) Methylmalonate administration decreases Na + , K + -ATPase activity in cerebral cortex of rats. NeuroReport 11(10):2331–2334

    Article  CAS  PubMed  Google Scholar 

  23. Chan KM, Delfert D, Junger KD (1986) A direct colorimetric assay for Ca2+-stimulated ATPase activity. Anal Biochem 157(2):375–380

    Article  CAS  PubMed  Google Scholar 

  24. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150(1):76–85

    Article  CAS  PubMed  Google Scholar 

  25. Zorec R, Verkhratsky A, Rodriguez JJ, Parpura V (2015) Astrocytic vesicles and gliotransmitters: slowness of vesicular release and synaptobrevin2-laden vesicle nanoarchitecture. Neuroscience. doi:10.1016/j.neuroscience.2015.02.033

    Google Scholar 

  26. Schousboe A, Bak LK, Waagepetersen HS (2013) Astrocytic control of biosynthesis and turnover of the neurotransmitters glutamate and GABA. Front Endocrinol (Lausanne) 4:102. doi:10.3389/fendo.2013.00102

    Google Scholar 

  27. Rasooli-Nejad S, Palygin O, Lalo U, Pankratov Y (2014) Cannabinoid receptors contribute to astroglial Ca(2)(+)-signalling and control of synaptic plasticity in the neocortex. Philos Trans R Soc Lond B Biol Sci 369(1654):20140077. doi:10.1098/rstb.2014.0077

    Article  PubMed  PubMed Central  Google Scholar 

  28. Souza DG, Bellaver B, Raupp GS, Souza DO, Quincozes-Santos A (2015) Astrocytes from adult Wistar rats aged in vitro show changes in glial functions. Neurochem Int. doi:10.1016/j.neuint.2015.07.016

    Google Scholar 

  29. Verkhratsky A, Rodriguez JJ, Parpura V (2012) Calcium signalling in astroglia. Mol Cell Endocrinol 353(1–2):45–56. doi:10.1016/j.mce.2011.08.039

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Fundação de Amparo à Pesquisa do Estado do Rio Grande do Sul (FAPERGS), Financiadora de Estudos e Projetos (FINEP)—IBN Net (Instituto Brasileiro de Neurociências) 01.06.0842-00, Universidade Federal do Rio Grande do Sul (UFRGS), and Instituto Nacional de Ciência e Tecnologia para Excitotoxicidade e Neuroproteção (INCTEN/CNPq).

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Authors and Affiliations

  1. Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Ramiro Barcelos, 2600, Anexo, PO Box: 90035-003, Porto Alegre, RS, Brazil

    Débora Guerini Souza, Bruna Bellaver, Gisele Hansel, Bernardo Assein Arús, Gabriela Bellaver, Aline Longoni, Janaina Kolling, Angela T. S. Wyse, Diogo Onofre Souza & André Quincozes-Santos

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  1. Débora Guerini Souza
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  2. Bruna Bellaver
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  3. Gisele Hansel
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  4. Bernardo Assein Arús
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  5. Gabriela Bellaver
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  6. Aline Longoni
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  7. Janaina Kolling
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  8. Angela T. S. Wyse
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  9. Diogo Onofre Souza
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  10. André Quincozes-Santos
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Correspondence to Débora Guerini Souza.

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Souza, D.G., Bellaver, B., Hansel, G. et al. Characterization of Amino Acid Profile and Enzymatic Activity in Adult Rat Astrocyte Cultures. Neurochem Res 41, 1578–1586 (2016). https://doi.org/10.1007/s11064-016-1871-7

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