Abstract
Intercellular communication is essential for the coordination and synchronization of cellular processes. Gap junction channels play an important role to communicate between cells and organs, including the brain, lung, liver, lens, retina, and heart. Gap junctions enable a direct route for ions like calcium and potassium, and low molecular weight compounds, such as inositol 1,4,5-trisphosphate, cyclic adenosine monophosphate, and various kinds of metabolites to pass between cells. Intercellular calcium wave propagation evoked by a local mechanical stimulus is one of the gap junction assays to study intercellular communication. In experimental settings, an intercellular calcium wave can be elicited by applying a mechanical stimulus to a single cell. Here, we describe the use of monolayers of primary bovine corneal endothelial cells as a model to study intercellular communication. Calcium wave propagation was assayed by imaging fluorescent calcium in bovine corneal endothelial cells loaded with a fluorescent calcium dye using a confocal microscope. Spatial changes in intercellular calcium concentration following mechanical stimulation were measured in the mechanical stimulated cell and in the neighboring cells. The active area (i.e., total surface area of responsive cells) of a calcium wave can be measured and used for studying the function and regulation of gap junction channels as well as hemichannels in a variety of cell systems.
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References
Kar R, Batra N, Riquelme MA et al (2012) Biological role of connexin intercellular channels and hemichannels. Arch Biochem Biophys 524:2–15
Scemes E, Spray DC, Meda P (2009) Connexins, pannexins, innexins: novel roles of “hemi-channels”. Pflugers Arch 457:1207–1226
Sohl G, Willecke K (2004) Gap junctions and the connexin protein family. Cardiovasc Res 62:228–232
Leybaert L, Sanderson MJ (2012) Intercellular Ca2+ waves: mechanisms and function. Physiol Rev 92:1359–1392
Meda P (2000) Probing the function of connexin channels in primary tissues. Methods 20:232–244
El-Fouly MH, Trosko JE, Chang CC (1987) Scrape-loading and dye transfer. A rapid and simple technique to study gap junctional intercellular communication. Exp Cell Res 168:422–430
Abbaci M, Barberi-Heyob M, Stines JR et al (2007) Gap junctional intercellular communication capacity by gap-FRAP technique: a comparative study. Biotechnol J 2:50–61
Goldberg GS, Bechberger JF, Naus CC (1995) A pre-loading method of evaluating gap junctional communication by fluorescent dye transfer. Biotechniques 18:490–497
Dakin K, Zhao Y, Li WH (2005) LAMP, a new imaging assay of gap junctional communication unveils that Ca2+ influx inhibits cell coupling. Nat Methods 2:55–62
Geletu M, Guy S, Firth K et al (2014) A functional assay for gap junctional examination; electroporation of adherent cells on indium-tin oxide. J Vis Exp 92: e51710
Wilders R, Jongsma HJ (1992) Limitations of the dual voltage clamp method in assaying conductance and kinetics of gap junction channels. Biophys J 63:942–953
D’hondt C, Himpens B, Bultynck G (2013) Mechanical stimulation-induced calcium wave propagation in cell monolayers: the example of bovine corneal endothelial cells. J Vis Exp 77:e50443
Abbaci M, Barberi-Heyob M, Blondel W et al (2008) Advantages and limitations of commonly used methods to assay the molecular permeability of gap junctional intercellular communication. Biotechniques 45(33–52):56–62
Gomes P, Srinivas SP, Vereecke J et al (2005) ATP-dependent paracrine intercellular communication in cultured bovine corneal endothelial cells. Invest Ophthalmol Vis Sci 46:104–113
Gomes P, Srinivas SP, Van Driessche W et al (2005) ATP release through connexin hemichannels in corneal endothelial cells. Invest Ophthalmol Vis Sci 46:1208–1218
Ponsaerts R, De Vuyst E, Retamal M et al (2010) Intramolecular loop/tail interactions are essential for connexin 43-hemichannel activity. FASEB J 24:4378–4395
Decrock E, De Bock M, Wang N et al (2015) Flash photolysis of caged IP3 to trigger intercellular Ca2+ waves. Cold Spring Harb Protoc 3:289–292
Acknowledgements
The work has been supported by Concerted Actions of the K.U. Leuven (GOA/09/012), Research Foundation - Flanders (F.W.O.; grant G.0298.11 to GB), Interuniversity Attraction Poles Program (Belgian Science Policy; P7/13 to G.B), and a “Krediet aan Navorser” grant of the FWO (15117.14 N to CDH).
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Iyyathurai, J., Himpens, B., Bultynck, G., D’hondt, C. (2016). Calcium Wave Propagation Triggered by Local Mechanical Stimulation as a Method for Studying Gap Junctions and Hemichannels. In: Vinken, M., Johnstone, S. (eds) Gap Junction Protocols. Methods in Molecular Biology, vol 1437. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3664-9_15
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DOI: https://doi.org/10.1007/978-1-4939-3664-9_15
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Online ISBN: 978-1-4939-3664-9
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