Abstract
Activation of intracellular transient receptor potential vanilloid-1 (TRPV1) in human lung cells causes endoplasmic reticulum (ER) stress, increased expression of proapoptotic GADD153 (growth arrest- and DNA damage-inducible transcript 3), and cytotoxicity. However, in cells with low TRPV1 expression, cell death is not inhibited by TRPV1 antagonists, despite preventing GADD153 induction. In this study, chemical variants of the capsaicin analog nonivamide were synthesized and used to probe the relationship between TRPV1 receptor binding, ER calcium release, GADD153 expression, and cell death in TRPV1-overexpressing BEAS-2B, normal BEAS-2B, and primary normal human bronchial epithelial lung cells. Modification of the 3-methoxy-4-hydroxybenzylamide vanilloid ring pharmacophore of nonivamide reduced the potency of the analogs and rendered several analogs mildly inhibitory. Correlation analysis of analog-induced calcium flux, GADD153 induction, and cytotoxicity revealed a direct relationship for all three endpoints in all three lung cell types for nonivamide and N-(3,4-dihydroxybenzyl)nonanamide. However, the N-(3,4-dihydroxybenzyl)nonanamide analog also produced cytotoxicity through redox cycling/reactive oxygen species formation, shown by inhibition of cell death by N-acetylcysteine. Molecular modeling of binding interactions between the analogs and TRPV1 agreed with data for reduced potency of the analogs, and only nonivamide was predicted to form a “productive” ligand-receptor complex. This study provides vital information on the molecular interactions of capsaicinoids with TRPV1 and substantiates TRPV1-mediated ER stress as a conserved mechanism of lung cell death by prototypical TRPV1 agonists.
Footnotes
This work was supported in part by the National Institutes of Health National Heart, Lung, and Blood Institute [Grant HL069813] (to C.A.R.), the National Institutes of Health National Institute of Environmental Health Sciences [Grant ES017431] (to C.A.R.), and a seed grant from the University of Utah Department of Anesthesiology (to C.A.R.). Support for the molecular modeling studies was provided by the National Institutes of Health National Institute for General Medical Sciences [Grant GM079383] (to T.E.C.). Computer time was provided by the Center for High Performance Computing at the University of Utah and supported by the National Science Foundation TeraGrid [Grant MCA01S027]. J.L. was supported by the National Research Foundation of Korea [Grant R11-2007-107-02001-0].
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.110.178491.
↵ The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material.
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ABBREVIATIONS:
- TRPV1
- transient receptor potential vanilloid 1
- GADD153
- growth arrest- and DNA damage-inducible transcript 3
- NHBE
- normal human bronchial epithelial
- SAR
- structure-activity relationship
- RT
- reverse transcription
- PCR
- polymerase chain reaction
- qPCR
- quantitative PCR
- LC50
- concentration at which 50% loss in viability (lethality) is observed
- MeO
- methoxy
- OH
- hydroxyl
- ER
- endoplasmic reticulum
- LJO-328
- N-(4-tert-butylbenzyl)-N′-(1-[3-fluoro-4-(methylsulfonylamino)-phenyl]ethyl)thiourea
- ANOVA
- analysis of variance
- TM
- transmembrane
- NAC
- N-acetylcysteine
- β2M
- β2 macroglobulin.
- Received December 20, 2010.
- Accepted February 18, 2011.
- Copyright © 2011 by The American Society for Pharmacology and Experimental Therapeutics
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