Environmental particulate matter (PM) pollutants adversely affect human health, but the molecular basis is poorly understood. The ion channel transient receptor potential vanilloid-1 (TRPV1) has been implicated as a sensor for environmental PM and a mediator of adverse events in the respiratory tract. The objectives of this study were to determine whether TRPV1 can distinguish chemically and physically unique PM that represents important sources of air pollution; to elucidate the molecular basis of TRPV1 activation by PM; and to ascertain the contributions of TRPV1 to human lung cell and mouse lung tissue responses exposed to an insoluble PM agonist, coal fly ash (CFA1). The major findings of this study are that TRPV1 is activated by some, but not all of the prototype PM materials evaluated, with rank-ordered responses of CFA1 Ͼ diesel exhaust PM Ͼ crystalline silica; TRP melastatin-8 is also robustly activated by CFA1, whereas other TRP channels expressed by airway sensory neurons and lung epithelial cells that may also be activated by CFA1, including TRPs ankyrin 1 (A1), canonical 4␣ (C4␣), M2, V2, V3, and V4, were either slightly (TRPA1) or not activated by CFA1; activation of TRPV1 by CFA1 occurs via cell surface interactions between the solid components of CFA1 and specific amino acid residues of TRPV1 that are localized in the putative pore-loop region; and activation of TRPV1 by CFA1 is not exclusive in mouse lungs but represents a pathway by which CFA1 affects the expression of selected genes in lung epithelial cells and airway tissue.

Transient receptor potential vanilloid 1 (TRPV1) is a calciumselective ion channel expressed in human lung cells. We show that activation of the intracellular subpopulation of TRPV1 causes endoplasmic reticulum (ER) stress and cell death in human bronchial epithelial and alveolar cells. TRPV1 agonist (nonivamide) treatment caused calcium release from the ER and altered the transcription of growth arrest-and DNA damage-inducible transcript 3 (GADD153), GADD45␣, GRP78/BiP, ATF3, CCND1, and CCNG2) in a manner comparable with prototypical ER stress-inducing agents. The TRPV1 antagonist -328) inhibited mRNA responses and cytotoxicity. EGTA and ruthenium red inhibited cell surface TRPV1 activity, but they did not prevent ER stress gene responses or cytotoxicity. Cytotoxicity paralleled eukaryotic translation initiation factor 2, subunit 1 (EIF2␣) phosphorylation and the induction of GADD153 mRNA and protein. Transient overexpression of GADD153 caused cell death independent of agonist treatment, and cells selected for stable overexpression of a GADD153 dominant-negative mutant exhibited reduced sensitivity. Salubrinal, an inhibitor of ER stress-induced cytotoxicity via the EIF2␣K3/EIF2␣ pathway, or stable overexpression of the EIF2␣-S52A dominant-negative mutant also inhibited cell death. Treatment of the TRPV1-null human embryonic kidney 293 cell line with TRPV1 agonists did not initiate ER stress responses. Likewise, n-benzylnonanamide, an inactive analog of nonivamide, failed to cause ER calcium release, an increase in GADD153 expression, and cytotoxicity. We conclude that activation of ER-bound TRPV1 and stimulation of GADD153 expression via the EIF2␣K3/EIF2␣ pathway represents a common mechanism for cytotoxicity by cell-permeable TRPV1 agonists. These findings are significant within the context of lung inflammatory diseases where elevated concentrations of endogenous TRPV1 agonists are probably produced in sufficient quantities to cause TRPV1 activation and lung cell death.

Neurologists and emergency department physicians are frequently involved in the comprehensive evaluation of a first generalized seizure. An important aspect of this evaluation is a detailed history which can identify a provoked seizure secondary to drug toxicity and hence avoid unnecessary treatment with antiepileptic drugs. ''Spice'' is an umbrella term for a variety of synthetic cannabinoid products whose inhalation has been associated with an increasing number of toxic side effects resulting in emergency department visits. These side effects (including psychosis, tachyarrhythmia, and seizures) are not typically seen with marijuana (Cannabis sativa) use. We report 2 patients with no prior history of neurological disease that experienced their first generalized tonic-clonic seizure after smoking Spice. The mechanism behind the possible proconvulsant effect of synthetic cannabinoids is not known, but it may be due to their effects at the cannabinoid receptor CB 1 . Although the US Drug Enforcement Administration placed 5 synthetic cannabinoids into schedule 1 for a 12-month period beginning March 2011, new Spice products containing different synthetic cannabinoids continue to emerge. Because synthetic cannabinoids are not detectable on commercial drug screens it is important that neurologists and emergency department physicians consider Spice inhalation in their differential diagnosis of a first generalized seizure.

Thomas KC, Roberts JK, Deering-Rice CE, Romero EG, Dull RO, Lee J, Yost GS, Reilly CA. Contributions of TRPV1, endovanilloids, and endoplasmic reticulum stress in lung cell death in vitro and lung injury. Am J Physiol Lung Cell Mol Physiol 302: L111-L119, 2012. First published September 23, 2011 doi:10.1152/ajplung.00231.2011.-Endogenous agonists of transient receptor potential vanilloid-1 (TRPV1) (endovanilloids) are implicated as mediators of lung injury during inflammation. This study tested the hypothesis that endovanilloids produced following lipopolysaccharide (LPS) treatment activate TRPV1 and cause endoplasmic reticulum stress/GADD153 expression in lung cells, representing a mechanistic component of lung injury. The TRPV1 agonist nonivamide induced GADD153 expression and caused cytotoxicity in immortalized and primary human bronchial, bronchiolar/alveolar, and microvascular endothelial cells, proportional to TRPV1 mRNA expression. In CF-1 mice, Trpv1 mRNA was most abundant in the alveoli, and intratracheal nonivamide treatment promoted Gadd153 expression in the alveolar region. Treatment of CF-1 mice with LPS increased Gadd153 in the lung, lactate dehydrogenase (LDH) in bronchoalveolar lavage (BAL) fluid, and lung wet-to-dry weight ratio. Cotreating mice with LPS and the TRPV1 antagonist LJO-328 reduced Gadd153 induction and LDH in BAL but did not inhibit increases in lung wet-to-dry ratio. In Trpv1 Ϫ/Ϫ mice treated with LPS, Gadd153 induction and LDH in BAL were reduced relative to wild-type mice, and the wet-to-dry weight ratios of lungs from both wild-type and Trpv1 Ϫ/Ϫ mice decreased. Organic extracts of blood collected from LPS-treated mice were more cytotoxic to TRPV1-overexpressing cells compared with BEAS-2B cells and extracts from control mice, however, most pure endovanilloids did not produce cytotoxicity in a characteristic TRPV1-dependent manner. Collectively, these data indicate a role for TRPV1, and endogenous TRPV1 agonists, in ER stress and cytotoxicity in lung cells but demonstrate that ER stress and cytotoxicity are not essential for pulmonary edema.capsaicin; lipopolysaccharides; lung injury; endovanilloids; transient receptor potential vanilloid-1 LUNG INJURY CAN ARISE FROM inflammatory responses elicited by diverse stimuli, including chemical exposures, trauma, endotoxic and hemorrhagic shock, sepsis, and burn injury (24,25,44,45). Limited advances in the treatment of lung injury and the more severe clinical manifestation, acute respiratory distress syndrome (ARDS), mandate a better understanding of the underlying biochemical processes that contribute to lung injury during systemic inflammation (24,44,45).Transient receptor potential vanilloid-1 (TRPV1) is a calcium-selective ion channel that is activated by capsaicin and its synthetic analog nonivamide as well as temperatures Ͼ42°C and H ϩ (pH Ͻ6.2 at 37°C) (7). TRPV1 also is purportedly activated by various inflammatory mediators, including hydrogen sulfide (H 2 S) (1, 5, 41), anandamide (AEA), 2-arachidonoyl glycerol (2-AG), leuk...

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.