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Real-time detection of acetylcholine release from the human endocrine pancreas

Nature Protocols volume 7, pages 1015–1023 (2012)Cite this article

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Abstract

Neurons, sensory cells and endocrine cells secrete neurotransmitters and hormones to communicate with other cells and to coordinate organ and system function. Validation that a substance is used as an extracellular signaling molecule by a given cell requires a direct demonstration of its secretion. In this protocol we describe the use of biosensor cells to detect neurotransmitter release from endocrine cells in real-time. Chinese hamster ovary cells expressing the muscarinic acetylcholine (ACh) receptor M3 were used as ACh biosensors to record ACh release from human pancreatic islets. We show how ACh biosensors loaded with the Ca2+ indicator Fura-2 and pressed against isolated human pancreatic islets allow the detection of ACh release. The biosensor approach is simple; the Ca2+ signal generated in the biosensor cell reflects the presence (release) of a neurotransmitter. The technique is versatile because biosensor cells expressing a variety of receptors can be used in many applications. The protocol takes ∼3 h.

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Figure 1: Imaging setup for real-time imaging of neurotransmitter release with biosensor cells.
Figure 2: Perfusion setup to detect neurotransmitter release from human islets with biosensor cells.
Figure 3: Screening of biosensor cells with acetylcholine (ACh).
Figure 4: Biosensor cell approach.
Figure 5: Real-time detection of ACh release.

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References

  1. Purves, D. et al. Neuroscience 88–91 (Sinauer Associates, 2008).

  2. Jacques-Silva, M. et al. ATP-gated P2X(3) receptors constitute a positive autocrine signal for insulin release in the human pancreatic beta cell. Proc. Natl. Acad. Sci. USA 107, 6465–6470 (2010).

    Google Scholar 

  3. Rodriguez-Diaz, R. et al. Alpha cells secrete acetylcholine as a non-neuronal paracrine signal priming beta cell function in humans. Nat. Med. 17, 888–892 (2011).

    Google Scholar 

  4. Hayashi, M. et al. Vesicular inhibitory amino acid transporter is present in glucagon-containing secretory granules in alphaTC6 cells, mouse clonal alpha-cells, and alpha-cells of islets of Langerhans. Diabetes 52, 2066–2074 (2003).

    Google Scholar 

  5. Kim, H. et al. Serotonin regulates pancreatic beta cell mass during pregnancy. Nat. Med. 16, 804–808 (2010).

    Google Scholar 

  6. Cabrera, O. et al. Automated, high-throughput assays for evaluation of human pancreatic islet function. Cell Transplant. 16, 1039–1048 (2008).

    Google Scholar 

  7. Perry, M., Li, Q. & Kennedy, R.T. Review of recent advances in analytical techniques for the determination of neurotransmitters. Anal. Chim. Acta 653, 1–22 (2009).

    Google Scholar 

  8. Huang, Y. et al. Mouse taste buds use serotonin as a neurotransmitter. J. Neurosci. 25, 843–847 (2005).

    Google Scholar 

  9. MacDonald, P.E., Braun, M., Galvanovskis, J. & Rorsman, P. Release of small transmitters through kiss-and-run fusion pores in rat pancreatic beta cells. Cell Metab. 4, 283–290 (2006).

    Google Scholar 

  10. Huang, Y.A., Maruyama, Y. & Roper, S.D. Norepinephrine is coreleased with serotonin in mouse taste buds. J. Neurosci. 28, 13088–13093 (2008).

    Google Scholar 

  11. Tachibana, M. & Okada, T. Release of endogenous excitatory amino acids from ON-type bipolar cells isolated from the goldfish retina. J. Neurosci. 11, 2199–2208 (1991).

    Google Scholar 

  12. Kataoka, Y. & Ohmori, H. Of known neurotransmitters, glutamate is the most likely to be released from chick cochlear hair cells. J. Neurophysiol. 76, 1870–1879 (1996).

    Google Scholar 

  13. Braun, M. et al. Regulated exocytosis of GABA-containing synaptic-like microvesicles in pancreatic beta-cells. J. Gen. Physiol. 123, 191–204 (2004).

    Google Scholar 

  14. Braun, M. et al. Corelease and differential exit via the fusion pore of GABA, serotonin, and ATP from LDCV in rat pancreatic beta cells. J. Gen. Physiol. 129, 221–231 (2007).

    Google Scholar 

  15. Obermüller, S. et al. Selective nucleotide-release from dense-core granules in insulin-secreting cells. J. Cell Sci. 118, 4271–4282 (2005).

    Google Scholar 

  16. Hollins, B. & Ikeda, S.R. Heterologous expression of a P2x-purinoceptor in rat chromaffin cells detects vesicular ATP release. J. Neurophysiol. 78, 3069–3076 (1997).

    Google Scholar 

  17. Whim, M.D. & Moss, G.W. A novel technique that measures peptide secretion on a millisecond timescale reveals rapid changes in release. Neuron 30, 37–50 (2001).

    Google Scholar 

  18. Cabrera, O. et al. Glutamate is a positive autocrine signal for glucagon release. Cell Metab. 7, 545–554 (2008).

    Google Scholar 

  19. Rubí, B. et al. Dopamine D2-like receptors are expressed in pancreatic beta cells and mediate inhibition of insulin secretion. J. Biol. Chem. 280, 36824–36832 (2005).

    Google Scholar 

  20. Huang, Y.A., Dando, R. & Roper, S.D. Autocrine and paracrine roles for ATP and serotonin in mouse taste buds. J. Neurosci. 29, 13909–13918 (2009).

    Google Scholar 

  21. Huang, Y.A., Pereira, E. & Roper, S.D. Acid stimulation (sour taste) elicits GABA and serotonin release from mouse taste cells. PLoS ONE 6, e25471 (2011).

    Google Scholar 

  22. Nguyen, Q.T. et al. An in vivo biosensor for neurotransmitter release and in situ receptor activity. Nat. Neurosci. 13, 127–132 (2010).

    Google Scholar 

  23. Speier, S. et al. Noninvasive high-resolution in vivo imaging of cell biology in the anterior chamber of the mouse eye. Nat. Protoc. 3, 1278–1286 (2008).

    Google Scholar 

  24. Speier, S. et al. Noninvasive in vivo imaging of pancreatic islet cell biology. Nat. Med. 14, 574–578 (2008).

    Google Scholar 

  25. Karlsson, S., Myrsén, U., Nieuwenhuizen, A., Sundler, F. & Ahrén, B. Presynaptic sympathetic mechanism in the insulinostatic effect of epinephrine in mouse pancreatic islets. Am. J. Physiol. 272, R1371–R1378 (1997).

    Google Scholar 

  26. Huang, Y.J. et al. The role of pannexin 1 hemichannels in ATP release and cell-cell communication in mouse taste buds. Proc. Natl. Acad. Sci. USA 104, 6436–6441 (2007).

    Google Scholar 

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Acknowledgements

This work was funded by the Diabetes Research Institute Foundation (DRIF); US National Institutes of Health grants R56DK084321 (A.C.), R01DK084321 (A.C.), R01DC000374 (S.D.R.) and R01DC007630 (S.D.R.); the Juvenile Diabetes Research Foundation; the Swedish Research Council; the Novo Nordisk Foundation; the Swedish Diabetes Association, the Stichting af Jochnick Foundation and The Erling-Persson Family Foundation; and the World Class University program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (R31-2008-000-10105-0).

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Author notes

  1. Rayner Rodriguez-Diaz and Robin Dando: These authors contributed equally to this work.

Authors and Affiliations

  1. Diabetes Research Institute, Miller School of Medicine, University of Miami, Miami, Florida, USA

    Rayner Rodriguez-Diaz, Per-Olof Berggren & Alejandro Caicedo

  2. The Rolf Luft Research Center for Diabetes and Endocrinology, Karolinska Institutet, Stockholm, Sweden

    Rayner Rodriguez-Diaz & Per-Olof Berggren

  3. Department of Physiology and Biophysics, Miller School of Medicine, University of Miami, Miami, Florida, USA

    Robin Dando, Y Anthony Huang, Stephen D Roper & Alejandro Caicedo

  4. Division of Integrative Biosciences and Biotechnology, WCU Program, University of Science and Technology, Pohang, Korea

    Per-Olof Berggren

  5. Program in Neuroscience, Miller School of Medicine, University of Miami, Miami, Florida, USA

    Stephen D Roper & Alejandro Caicedo

  6. Division of Endocrinology, Department of Medicine, Diabetes and Metabolism, Miller School of Medicine, University of Miami, Miami, Florida, USA

    Alejandro Caicedo

Authors
  1. Rayner Rodriguez-Diaz
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  2. Robin Dando
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  3. Y Anthony Huang
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  4. Per-Olof Berggren
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  5. Stephen D Roper
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  6. Alejandro Caicedo
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Contributions

R.D. and Y.A.H. performed experiments with biosensor cells to detect acetylcholine secretion; R.D., R.R.-D. and A.C. analyzed and quantified data; R.R.-D., R.D., P.-O.B., S.D.R. and A.C. designed the study and wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding authors

Correspondence to Per-Olof Berggren, Stephen D Roper or Alejandro Caicedo.

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The authors declare no competing financial interests.

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Rodriguez-Diaz, R., Dando, R., Huang, Y. et al. Real-time detection of acetylcholine release from the human endocrine pancreas. Nat Protoc 7, 1015–1023 (2012). https://doi.org/10.1038/nprot.2012.040

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