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The potential of metabolomic analysis techniques for the characterisation of α1-adrenergic receptors in cultured N1E-115 mouse neuroblastoma cells

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Abstract

Several studies of neuropathic pain have linked abnormal adrenergic signalling to the development and maintenance of pain, although the mechanisms underlying this are not yet fully understood. Metabolomic analysis is a technique that can be used to give a snapshot of biochemical status, and can aid in the identification of the mechanisms behind pathological changes identified in cells, tissues and biological fluids. This study aimed to use gas chromatography-mass spectrometry-based metabolomic profiling in combination with reverse transcriptase-polymerase chain reaction and immunocytochemistry to identify functional α1-adrenergic receptors on cultured N1E-115 mouse neuroblastoma cells. The study was able to confirm the presence of mRNA for the α1D subtype, as well as protein expression of the α1-adrenergic receptor. Furthermore, metabolomic data revealed changes to the metabolite profile of cells when exposed to adrenergic pharmacological intervention. Agonist treatment with phenylephrine hydrochloride (10 µM) resulted in altered levels of several metabolites including myo-inositol, glucose, fructose, alanine, leucine, phenylalanine, valine, and n-acetylglutamic acid. Many of the changes observed in N1E-115 cells by agonist treatment were modulated by additional antagonist treatment (prazosin hydrochloride, 100 µM). A number of these changes reflected what is known about the biochemistry of α1-adrenergic receptor activation. This preliminary study therefore demonstrates the potential of metabolomic profiling to confirm the presence of functional receptors on cultured cells.

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Acknowledgments

This study was supported by the National Health and Medical Research Council and National Collaborative Research Infrastructure Strategy BioPlatforms Australia. Thanks to A/Prof. Robert Trengove for his expertise on metabolomic analysis and the use of laboratory equipment, and to Dr. Monika Tschochner for performing mycoplasma analysis. Also thanks to Dr. Eleanor Drummond for her support with the immunocytochemistry, Dr. Sarah Etherington for her input on the written material and Dr. Philip Stumbles for providing us with mouse brain tissue.

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Authors and Affiliations

  1. School of Veterinary and Life Sciences, Murdoch University, Perth, WA, Australia

    Maria I. Wenner, Garth L. Maker, Linda F. Dawson & Ian Mullaney

  2. Metabolomics Australia, Murdoch University, Perth, WA, Australia

    Garth L. Maker

  3. Separation Science and Metabolomics Laboratory, Murdoch University, Perth, WA, Australia

    Garth L. Maker

  4. School of Psychology and Exercise Science, Murdoch University, Perth, WA, Australia

    Linda F. Dawson & Peter D. Drummond

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  1. Maria I. Wenner
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Correspondence to Garth L. Maker.

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10616_2015_9915_MOESM1_ESM.tif

Supplementary material Fig. 1 Reverse transcriptase PCR image showing presence or absence of mycoplasma contamination in several cell lines. The image show absence of mycoplasma contamination in N1E-115 cells, by lack of a 300 bp PCR product in this lane (NIE). (TIFF 3863 kb)

10616_2015_9915_MOESM2_ESM.tif

Supplementary material Fig. 2 Reverse transcriptase PCR image acquired from 8 to 12 weeks old female C57BL/6 mice brains, stored in RNAlater. M, molecular lane marker (100 bp); 1, mouse brain α1A-AR (333 bp); 2, mouse brain α1B-AR (139 bp); 3, mouse brain α1D-AR (281 bp); 4, mouse brain GAPDH (167 bp). Abbrevations: α1-AR, α1-adrenergic receptor (TIFF 344 kb)

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Wenner, M.I., Maker, G.L., Dawson, L.F. et al. The potential of metabolomic analysis techniques for the characterisation of α1-adrenergic receptors in cultured N1E-115 mouse neuroblastoma cells. Cytotechnology 68, 1561–1575 (2016). https://doi.org/10.1007/s10616-015-9915-4

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