Transient receptor potential vanilloid-1 (TRPV1) is a mediator of lung toxicity for coal fly ash particulate material.

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 > diesel exhaust PM > 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 4α (C4α), 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 agonists cause endoplasmic reticulum stress and cell death in human lung cells.

Transient receptor potential vanilloid 1 (TRPV1) is a calcium-selective 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), GADD45alpha, GRP78/BiP, ATF3, CCND1, and CCNG2) in a manner comparable with prototypical ER stress-inducing agents. The TRPV1 antagonist N-(4-tert-butylbenzyl)-N'-(1-[3-fluoro-4-(methylsulfonylamino)-phenyl]ethyl)thiourea (LJO-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 (EIF2alpha) 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 EIF2alphaK3/EIF2alpha pathway, or stable overexpression of the EIF2alpha-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 EIF2alphaK3/EIF2alpha 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.

TRPV1 antagonists elevate cell surface populations of receptor protein and exacerbate TRPV1-mediated toxicities in human lung epithelial cells.

TRPV1 mediates cell death and pro-inflammatory cytokine production in lung epithelial cells exposed to prototypical receptor agonists. This study shows that NHBE, BEAS-2B and TRPV1 over-expressing BEAS-2B cells pre-treated with various TRPV1 antagonists become sensitized to the prototypical TRPV1 agonist, nonivamide, via a mechanism that involves translocation of existing receptor from the endoplasmic reticulum to the plasma membrane. As such, typical cellular responses to agonist treatment, as measured by calcium flux, inflammatory cytokine gene induction, and cytotoxicity were exacerbated. These data were in contrast to the results obtained when TRPV1 antagonists were co-administered with nonivamide; conditions which inhibited TRPV1-mediated effects. The antagonists LJO-328, SC0030, and capsazepine increased the cytotoxicity of nonivamide by approximately 20-fold and agonist-induced calcium flux by approximately 6-fold. Inflammatory-cytokine gene induction by nonivamide was also increased significantly by pre-treatment with the antagonists. The enhanced responses were inhibited by the co-administration of antagonists with nonivamide, confirming that increases in sensitivity were attributable to increased TRPV1-associated activity. Sensitization was attenuated by brefeldin A (a golgi transport inhibitor), but not cycloheximide (a protein synthesis inhibitor), or actinomycin D (a transcription inhibitor). Sensitized cells exhibited increased calcium flux from extracellular calcium sources, while unsensitized cells exhibited calcium flux originating primarily from intracellular stores. These results demonstrate the presence of a novel mechanism for regulating the sub-cellular distribution of TRPV1 and subsequent control of cellular sensitivity to TRPV1 agonists.

Calcium-dependent and independent mechanisms of capsaicin receptor (TRPV1)-mediated cytokine production and cell death in human bronchial epithelial cells.

Activation of the capsaicin receptor (VR1 or TRPV1) in bronchial epithelial cells by capsaicinoids and other vanilloids promotes pro-inflammatory cytokine production and cell death. The purpose of this study was to investigate the role of TRPV1-mediated calcium flux from extracellular sources as an initiator of these responses and to define additional cellular pathways that control cell death. TRPV1 antagonists and reduction of calcium concentrations in treatment solutions attenuated calcium flux, induction of interleukin-6 and 8 gene expression, and IL-6 secretion by cells treated with capsaicin or resiniferatoxin. Most TRPV1 antagonists also attenuated cell death, but the relative potency and extent of protection did not directly correlate with inhibition of total calcium flux. Treatment solutions with reduced calcium content or chelators had no effect on cytotoxicity. Inhibitors of arachidonic acid metabolism and cyclo-oxygenases also prevented cell death indicating that TRPV1 agonists disrupted basal arachidonic acid metabolism and altered cyclo-oxygenase function via a TRPV1-dependent mechanism in order to produce toxicity. These data confirm previous results demonstrating calcium flux through TRPV1 acts as a trigger for cytokine production by vanilloids, and provides new mechanistic insights on mechanisms of cell death produced by TRPV1 agonists in respiratory epithelial cells.

Oxidatively induced DNA-protein cross-linking between single-stranded binding protein and oligodeoxynucleotides containing 8-oxo-7,8-dihydro-2'-deoxyguanosine.

The formation of covalent cross-links between amino acid side chains and DNA bases in DNA-protein complexes is a significant pathway in oxidative damage to the genome, yet much remains to be learned about their chemical structures and mechanisms of formation. In the present study, DNA-protein cross-links (DPCs) were formed between synthetic oligodeoxynucleotides containing an 8-oxo-7,8-dihydro-2'deoxyguanosine (OG) or an 8-oxo-7,8-dihydro-2'-deoxyadenosine (OA) nucleotide and Escherichia coli singled-stranded binding protein (SSB) under oxidative conditions. Studies with various sequences indicated that DNA homopolymers and those lacking 8-oxopurines were less reactive toward DPC formation. DPCs were formed in the presence of HOCl, peroxynitrite, and the one-electron oxidants Na(2)IrCl(6), Na(2)IrBr(6), and Na(3)Fe(CN)(6). Protein-protein cross-linking was also observed, particularly for oxidants of high reduction potential such as Na(2)IrCl(6). The adducted oligodeoxynucleotides were sensitive to hot piperidine treatment leading to strand scission at the site of cross-linking. In addition, the covalent cross-links were somewhat heat and acid labile, which may be related to the difficulties encountered in obtaining complete characterization of trypsin digests of the DPCs. However, model reactions involving the single amino acids lysine, arginine, and tyrosine, residues known to be involved in base contacts in the DNA:SSB complex, could be studied, and the adduct formed between N(alpha)-acetyllysine methyl ester and an 18-mer containing OG was tentatively characterized by electrospray ionization mass spectrometry as analogues of spiroiminodihydantoin and guanidinohydantoin. A mechanism involving nucleophilic attack of an amino acid side chain (e.g. the epsilon-amino group of lysine) at C5 of a 2-electron oxidized form of OG is proposed.