Abstract
The transient receptor potential vanilloid-1 ion channel (TRPV1) is a non-selective ligand-gated cation channel. It is an integrator of a wide variety of exogenous and endogenous physical and chemical stimuli, including capsaicin, noxious heat (>42 °C), and protons (pH < 5.2). TRPV1 is expressed predominantly in primary sensory neurons involved in pain sensation, but also in other neuronal cell types, in the plasma membrane of different non-neuronal cells such as immune cells, keratinocytes, smooth muscle cells, and in the urothelium. Some of these cell types are involved in inflammation. When activated, TRPV1 leads to the gating of cations, including Ca2+, thus generating changes in intracellular Ca2+ concentration. Calcium ions play fundamental roles in many cellular processes, virtually in all cells. The use of Ca2+ fluorescent indicators is a tool for monitoring intracellular Ca2+ concentration.
In this chapter, we describe a method for recording and monitoring Ca2+ signals through the single wavelength fluorescent indicator Fluo-4 acetoxymethyl (AM), and the ratiometric fluorescent indicator Fura-2 AM in HEK-293 cells transfected with TRPV1 and other TRP channels. TRPV1 pharmacological modulation may potentially represent a strategy for the control of pain and inflammatory conditions in a variety of diseases and injury states.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Paredes RM, Etzler JC, Watts LT et al (2008) Chemical calcium indicators. Methods 46:143–151
Gaudet R (2008) TRP channels entering the structural era. J Physiol 586:3565–3575
Moran MM, McAlexander MA, Bíró T et al (2011) Transient receptor potential channels as therapeutic targets. Nat Rev Drug Discov 10:601–620
Vennekens R, Owsianik G, Nilius B (2008) Vanilloid transient receptor potential cation channels: an overview. Curr Pharm Des 14:18–31
De Petrocellis L, Schiano Moriello A (2013) Modulation of the TRPV1 channel: current clinical trials and recent patents with focus on neurological conditions. Recent Pat CNS Drug Discov 8:180–204
Gees M, Colsoul B, Nilius B (2010) The role of transient receptor potential cation channels in Ca2+ signaling. Cold Spring Harb Perspect Biol 10:a003962
Bang S, Kim KY, Yoo S et al (2007) Transient receptor potential V2 expressed in sensory neurons is activated by probenecid. Neurosci Lett 425:120–125
Trevisani M, Smart D, Gunthorpe MJ et al (2002) Ethanol elicits and potentiates nociceptor responses via the vanilloid receptor-1. Nat Neurosci 5:546–551
Grynkiewicz G, Poenie M, Tsien RY (1985) A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260:3440–3450
Marini P, Schiano Moriello A, Cristino L et al (2009) Cannabinoid CB1 receptor elevation of intracellular calcium in neuroblastoma SH-SY5Y cells: interactions with muscarinic and delta-opioid receptors. Biochim Biophys Acta 1793:1289–1303
Takahashi A, Camacho P, Lechleiter JD et al (1999) Measurement of intracellular calcium. Physiol Rev 79:1089–1125
Neeper MP, Liu Y, Hutchinson TL et al (2007) Activation properties of heterologously expressed mammalian TRPV2: evidence for species dependence. J Biol Chem 282:15894–15902
Monet M, Gkika D, Lehen’kyi V et al (2009) Lysophospholipids stimulate prostate cancer cell migration via TRPV2 channel activation. Biochim Biophys Acta 1793:528–539
De Petrocellis L, Ligresti A, Schiano Moriello A et al (2011) Effects of cannabinoids and cannabinoid-enriched Cannabis extracts on TRP channels and endocannabinoid metabolic enzymes. Br J Pharmacol 163:1479–1494
Ortar G, Morera L, Schiano Moriello A et al (2012) Modulation of thermo-transient receptor potential (thermo-TRP) channels by thymol-based compounds. Bioorg Med Chem Lett 22:3535–3539
De Petrocellis L, Orlando P, Schiano Moriello A et al (2012) Cannabinoid actions at TRPV channels: effects on TRPV3 and TRPV4 and their potential relevance to gastrointestinal inflammation. Acta Physiol 204:255–266
De Petrocellis L, Ortar G, Schiano Moriello A et al (2015) Structure–activity relationships of the prototypical TRPM8 agonist icilin. Bioorg Med Chem Lett 25(11):2285–2290. doi:10.1016/j.bmcl.2015.04.032
De Petrocellis L, Starowicz K, Schiano Moriello A et al (2007) Regulation of transient receptor potential channels of melastatin type 8 (TRPM8): effect of cAMP, cannabinoid CB(1) receptors and endovanilloids. Exp Cell Res 313:1911–1920
De Petrocellis L, Vellani V, Schiano-Moriello A et al (2008) Plant-derived cannabinoids modulate the activity of transient receptor potential channels of ankyrin type-1 and melastatin type-8. J Pharmacol Exp Ther 325:1007–1015
Ortar G, De Petrocellis L, Morera L et al (2010) (-)-Menthylamine derivatives as potent and selective antagonists of transient receptor potential melastatin type-8 (TRPM8) channels. Bioorg Med Chem Lett 20:2729–2732
Del Prete D, Caprioglio D, Appendino G et al (2015) Discovery of non-electrophilic capsaicinoid-type TRPA1 ligands. Bioorg Med Chem Lett 25:1009–1011
De Petrocellis L, Davis JB, Di Marzo V (2001) Palmitoylethanolamide enhances anandamide stimulation of human vanilloid VR1 receptors. FEBS Lett 506:253–256
Ligresti A, De Petrocellis L, Hernán Pérez de la Ossa d et al (2010) Exploiting nanotechnologies and TRPV1 channels to investigate the putative anandamide membrane transporter. PLoS One 5, e10239
Di Marzo V, Fontana A, Cadas H et al (1994) Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 372:686–691
Smart D, Gunthorpe MJ, Jerman JC et al (2000) The endogenous lipid anandamide is a full agonist at the human vanilloid receptor (hVR1). Br J Pharmacol 129:227–230
Ross RA, Gibson TM, Brockie HC et al (2001) Structure-activity relationship for the endogenous cannabinoid, anandamide, and certain of its analogues at vanilloid receptors in transfected cells and vas deferens. Br J Pharmacol 132:631–640
Olah Z, Karai L, Ladarola MJ (2001) Anandamide activates vanilloid receptor 1 (VR1) at acidic pH in dorsal root ganglia neurons and cells ectopically expressing VR1. J Biol Chem 276:31163–31170
Acknowledgements
We thank Dr. Nicola De Marchi and Dr. Cristoforo Silvestri for their careful review of the manuscript and for their valuable advice.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2016 Springer Science+Business Media New York
About this protocol
Cite this protocol
Moriello, A.S., De Petrocellis, L. (2016). Assay of TRPV1 Receptor Signaling. In: Maccarrone, M. (eds) Endocannabinoid Signaling. Methods in Molecular Biology, vol 1412. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3539-0_7
Download citation
DOI: https://doi.org/10.1007/978-1-4939-3539-0_7
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-3537-6
Online ISBN: 978-1-4939-3539-0
eBook Packages: Springer Protocols