Determination of Glutamate and GABA Released by Mouse Embryonic Stem Cells Using HILIC-ESI-MS/MS

Author(s): Haoyu Lv, Yabin Tang, Fan Sun, Shimin An, Xinjie Yang, Wenbin Li, Xiaosheng Wang, Liang Zhu*

Journal Name: The Natural Products Journal

Volume 10 , Issue 2 , 2020

Become EABM
Become Reviewer

Graphical Abstract:


Abstract:

Background: In recent years, more and more researches have shown that neurotransmitters can also be synthesized and released by peripheral non-neural cells. However, specificity and high sensitivity detection means were required for confirming ESCs autocrine glutamate and γ - aminobutyric acid (GABA). Glutamate and GABA are water-soluble and polar compounds which cannot be retained on a reversed phase C18 column, and their contents are often at a trace level. On the other hand, the biological matrix such as cell culture fluid contains a large number of amino acids, vitamins, carbohydrates, inorganic ions and other substances. Therefore, the main problem is the selection of the chromatographic column to avoid matrix interference.

Objective: To establish a rapid and reliable method for the simultaneous determination of glutamate and GABA released from embryonic stem cells based on analytical chemistry.

Methods: Glutamate and GABA released from mouse embryonic stem cells were determined on the basis of hydrophilic interaction chromatography coupled with electrospray ionization tandem Mass Spectrometry (HILIC- ESI- MS/MS), using isotope internal standards and substitution matrix method.

Results: Undifferentiated embryonic stem cells autocrine glutamate and GABA and will reach releasing- reuptacking dynamic equilibriums at different time points. In contrast, neither glutamate nor GABA releasing could be detected from the MEFs, indicating the specificity release of the mESCs in the applied analytic method.

Conclusion: A novel, simple, sensitive, selective and quantitative method was developed for determination of the glutamate and GABA from mouse embryonic stem cells.

Keywords: Glutamate, γ-aminobutyric acid, HILIC, LC-MS/MS, mouse embryonic stem cells, neurotransmitter.

[1]
Tam, P.P.; Loebel, D.A. Gene function in mouse embryogenesis: Get set for gastrulation. Nat. Rev. Genet., 2007, 8, 368-381.
[2]
Nedergaard, M.; Takano, T.; Hansen, A.J. Beyond the role of glutamate as a neurotransmitter. Nat. Rev. Neurosci., 2002, 3, 748-755.
[3]
Fallarino, F.; Volpi, C.; Fazio, F.; Notartomaso, S.; Vacca, C.; Busceti, C.; Bicciato, S.; Battaglia, G.; Bruno, V.; Puccetti, P.; Fioretti, M.C.; Nicoletti, F.; Grohmann, U.; Di Marco, R. Metabotropic glutamate receptor-4 modulates adaptive immunity andrestrains neuroinflammation. Nat. Med., 2010, 16, 897-902.
[4]
Cabrera, O.; Jacques-Silva, M.C.; Speier, S.; Yang, S.N.; Kohler, M.; Fachado, A. Vieira. E.; Zierath, J.R.; Kibbey, R.; Berman, D.M.; Kenyon, N.S.; Ricordi, C.; Caicedo, A.; Berggren, P.O. Glutamate is a positive autocrine signal for glucagon release. Cell Metab., 2008, 7, 545-554.
[5]
LoTurco, J.J.; Owens, D.F.; Heath, M.J.; Davis, M.B.; Kriegstein, A.R. GABA and glutamate depolarize cortical progenitor cells and inhibit DNA synthesis. Neuron, 1995, 15, 1287-1298.
[6]
Fernando, R.N.; Eleuteri, B.; Abdelhady, S.; Nussenzweig, A.; Andang, M.; Ernfors, P. Cell cycle restriction by histone H2AX limits proliferation of adult neural stem cells. Proc. Natl. Acad. Sci. USA, 2011, 108, 5837-5842.
[7]
Hayashi, M.; Yamada, H.; Uehara, S.; Morimoto, R.; Muroyama, A.; Yatsushiro, S.; Takeda, J.; Yamamoto, A.; Moriyama, Y. Secretory granule-mediated co-secretion of L-glutamate and glucagon triggers glutamatergic signal transmission in islets of Langerhans. J. Biol. Chem., 2003, 278(3), 1966-1974.
[8]
Morimoto, R.; Uehara, S.; Yatsushiro, S.; Juge, N.; Hua, Z.; Senoh, S.; Echigo, N.; Hayashi, M.; Mizoguchi, T.; Ninomiya, T.; Udagawa, N.; Omote, H.; Yamamoto, A.; Edwards, R.H.; Moriyama, Y. Secretion of L-glutamate from osteoclasts through transcytosis. EMBO J., 2006, 25, 4175-4186.
[9]
Andang, M.; Hjerling-Leffler, J.; Moliner, A.; Lundgren, T.K.; Castelo-Branco, G.; Nanou, E.; Pozas, E.; Bryja, V.; Halliez, S.; Nishimaru, H.; Wilbertz, J.; Arenas, E.; Koltzenburg, M.; Charnay, P.; Manira, A.E.; Ibanez, C.F.; Ernfors, P. Histone H2AX-dependent GABA(A) receptor regulation of stem cell proliferation. Nature, 2008, 451, 460-464.
[10]
Schwirtlich, M.; Emri, Z.; Antal, K.; Mate, Z.; Katarova, Z.; Szabo, G. GABA(A) and GABA(B) receptors of distinct properties affect oppositely the proliferation of mouse embryonic stem cells through synergistic elevation of intracellular Ca(2+). FASEB, 2010, 24, 1218-1228.
[11]
Teng, L.; Tang, Y.B.; Sun, F.; An, S.M.; Zhang, C.; Yang, X.J.; Lv, H.Y.; Lu, Q.; Cui, Y.Y.; Hu, J.J.; Zhu, L.; Chen, H.Z. Non-Neuronal Release of Gamma-Aminobutyric Acid by Embryonic Pluripotent Stem Cells. Stem Cells Dev., 2013, 22, 2944-2953.
[12]
Teng, L.; Lei, H.M.; Sun, F.; An, S.M.; Tang, Y.B.; Meng, S.; Wang, C.H.; Shen, Y.; Chen, H.Z.; Zhu, L. Autocrine glutamatergic transmission for the regulation of embryonal carcinoma stem cells. Oncotarget, 2016, 7(31), 49552-49564.
[13]
Macinnes, N.; Duty, S. Group III metabotropic glutamate receptors act as heteroreceptors modulating evoked GABA release in the globuspallidus in vivo. Eur. J. Pharmacol., 2008, 580, 95-99.
[14]
Bianchi, L.; Della, C.L.; Tipton, K.F. Simultaneous determination of basal and evoked output levels of aspartate, glutamate, taurine and 4-aminobutyric acid during microdialysis and from superfused brain slices. J. Chromatogr. B Biomed. Sci. Appl., 1999, 723, 47-59.
[15]
Ma, D.; Zhang, J.; Sugahara, K.; Ageta, T.; Nakayama, K.; Kodama, H. Simultaneous determination of gamma-aminobutyric acid and glutamic acid in the brain of 3- mercaptopropionic acid-treated rats using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry. J. Chromatogr. B Biomed. Sci. Appl., 1999, 726, 285-290.
[16]
Eckstein, J.A.; Ammerman, G.M.; Reveles, J.M.; Ackermann, B.L. Analysis of glutamine, glutamate, pyroglutamate, and GABA in cerebrospinal fluid using ion pairing HPLC with positive electrospray LC/MS/MS. J. Neurosci. Methods, 2008, 171, 190-196.
[17]
Zhang, X.; Rauch, A.; Lee, H.; Xiao, H.; Rainer, G.; Logothetis, N.K. Capillary hydrophilic interaction chromatography/mass spectrometry for simultaneous determination of multiple neurotransmitters in primate cerebral cortex. Rapid Commun. Mass Spectrom., 2007, 21, 3621-3628.
[18]
Buck, K.; Voehringer, K.; Ferger, B. Rapid analysis of GABA and glutamate in microdialysis samples using high performance liquid chromatography and tandem mass spectrometry. J. Neurosci. Methods, 2009, 182, 78-84.
[19]
Stokvis, E.; Rosing, H.; Beijnen, J.H. Stable isotopically labeled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry: necessity or not? Rapid Commun. Mass Spectrom., 2005, 19, 401-407.
[20]
Peng, L.; Jiang, T.; Rong, Z.X.; Liu, T.; Wang, H.; Shao, B.Y.; Ma, J.; Yang, L.; Kang, L.; Shen, Y.F.; Li, H.F.; Qi, H.; Chen, H.Z. Surrogate based accurate quantification of endogenous acetylcholine in murinebrain by hydrophilic interaction liquid chromatography–tandem massspectrometry. J. Chromatogr. B, 2011, 879, 3927-3931.


Rights & PermissionsPrintExport Cite as

Article Details

VOLUME: 10
ISSUE: 2
Year: 2020
Page: [122 - 129]
Pages: 8
DOI: 10.2174/2210315509666190211123132
Price: $25

Article Metrics

PDF: 18
HTML: 2