Simultaneous Intracellular Calcium and Sodium Flux Imaging in Human Vanilloid Receptor 1 (VR1)-Transfected Human Embryonic Kidney Cells: A Method to Resolve Ionic Dependence of VR1-Mediated Cell Death ELFRIDA R. GRANT, 1 ADRIENNE E. DUBIN, SUI-PO ZHANG, ROBERT A. ZIVIN, and ZHONG ZHONG 2 Drug Discovery, R. W. Johnson Pharmaceutical Research Institute, Raritan, New Jersey, La Jolla, California, and Spring House, Pennsylvania Received June 15, 2001; accepted September 25, 2001 This paper is available online at http://jpet.aspetjournals.org ABSTRACT The vanilloid receptor 1 (VR1) is a ligand-gated, nonselective cation channel important for the sensory processing of painful stimuli. Activation of VR1 leads to increases in intracellular concentrations of calcium and sodium. Prolonged activation of VR1 in mammalian expression systems leads to cell death. The mechanism of VR1-mediated toxicity may have relevance to pathophysiological processes that can occur in neurons. Therefore, we have evaluated the relative contributions of in- tracellular calcium and sodium changes to VR1-mediated tox- icity in human embryonic kidney 293 cells stably transfected with the human VR1 channel. The data demonstrate that VR1 receptor agonists capsaicin and resiniferatoxin lead to a sus- tained increase in intracellular calcium and sodium in a con- centration-dependent manner, followed by cell death. Pretreat- ment with VR1 receptor antagonists capsazepine or ruthenium red block both the calcium and sodium responses to agonists, and block agonist-induced cell death in a concentration-de- pendent manner. However, addition of antagonists several min- utes after agonists selectively reverses the agonist-induced increase in intracellular calcium, but does not reverse the ele- vated intracellular sodium concentration. Nonetheless, antag- onists retain protective efficacy against capsaicin toxicity when added several minutes after capsaicin, conditions in which the cells still manifest elevated intracellular sodium, but not ele- vated intracellular calcium. In addition, a transient VR1-medi- ated increase in intracellular calcium that returns to baseline within minutes, induced by a rapid drop in pH, from pH 7.5 to pH 6.3, also does not lead to cell death. Collectively, these data demonstrate that the most important intracellular ionic change for mediating VR1-dependent toxicity is a sustained increase of calcium. The VR1 receptor is a ligand-gated, nonspecific cation chan- nel activated by vanilloid compounds, low pH, and noxious heat (Tominaga et al., 1998). The best-known natural vanilloids are capsaicin, which is the pungent ingredient in hot chili peppers, and resiniferatoxin, synthesized by a cactus-like plant called Euphorbia resinifera (Szallasi and Blumberg, 1999). High tem- peratures, 42°C, protons, and high concentrations of anand- amide gate the VR1 receptor directly (Tominaga et al., 1998). Because the VR1 receptor is permeable to numerous cations, including Ca 2+ , Na + , and K + , its activation leads to an influx of Na + and Ca 2+ , and an efflux of K + (Szallasi et al., 1999). A number of ionic mechanisms have been implicated in cell death, including Ca 2+ (Choi, 1987; Munir et al., 1995; Grant et al., 1997), Na + (Raymond et al., 1996; Itoh et al., 1998), and K + (Yu et al., 1999). In the dorsal root ganglion, capsa- icin treatment results in selective degeneration of approxi- mately 50% of the sensory neurons of neonatal rats, and approximately 18% of these neurons in adult rats (Holzer, 1991; Jancso, 1992). When capsaicin is administered to adult rats peripherally, it results in marked peripheral nerve fiber degeneration in organs such as skin, ureter, and duodenum (Hoyes and Barber, 1981; Chung et al., 1985, 1990). Nocicep- tor terminals are destroyed reliably, but complete death of the cell bodies does not always occur. In both adult and newborn rats, the capsaicin-induced neurodegeneration is restricted to the subpopulation of small C-type primary sen- sory neurons (Jancso et al., 1977, 1985), which are the pre- dominant VR1-expressing neurons (Caterina et al., 1997). Evidence suggests that capsaicin-induced damage of C-fiber neurons may lead to transganglionic degeneration (Mannion 1 Current address: Purdue Pharma, Princeton, NJ 08540. 2 Current address: Cell & Molecular Technologies, Phillipsburg, NJ 08865. ABBREVIATIONS: VR1, vanilloid receptor 1; hVR1, human vanilloid receptor 1; HEK293, human embryonic kidney 293 cells; PI, propidium iodide; SBFI, sodium binding furan isophthalate; CM-H 2 -DCFDA, carboxy-dichlorodihydrofluorescein diacetate; FLIPR, fluorometric imaging plate reader; HBSS, Hanks’ balanced salt solution; RTX, resiniferatoxin; CCD, charge-coupled device; ROI, regions of interest; ROS, reactive oxygen species; ANOVA, analysis of variance. 0022-3565/02/3001-9 –17$3.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 300, No. 1 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 4239/954199 JPET 300:9–17, 2002 Printed in U.S.A. 9 at ASPET Journals on November 8, 2016 jpet.aspetjournals.org Downloaded from