Mechanisms of TRPV1-mediated lung cell death

Capsaicin is the pungent compound in chili peppers. Capsaicin causes dosedependent respiratory and cardiovascular failure by all routes. The capsaicin receptor, TRPV1, is a ligand-gated calcium channel. TRPV1 is expressed in sensory neurons and various non-neuronal cells in the lung, including epithelial cells of the conducting airways and alveoli. In human lung bronchial epithelial and alveolar cells, plasma membrane and endoplasmic reticulum (ER) populations of TRPV1 differentially influence cytokine gene expression and cell death via changes in cytosolic calcium concentrations, but a precise mechanism for TRPV1-mediated cytotoxicity was previously undefined. This project investigated how prototypical and endogenous TRPV1 agonists damage lung cells. Structure activity relationships between cell death and capsaicinoids with varied potency, and the role of TRPV1 in lung injury due to unfettered systemic inflammation were studied. In vitro assays for cell viability and calcium flux, quantitative analysis of gene expression patterns, and mutagenesis of regulatory gene products demonstrated that TRPV1 activation caused extensive ER calcium efflux, ER stress, and cell death. A series of capsaicinoid analogues were developed to determine the specificity of TRPV1 activation and ER stress as a common mechanism of toxicity in various human lung cell types. Structural modifications to the vanilloid ring drastically reduced the ability of capsaicinoids to activate TRPV1 and, accordingly, both ER stress and cytotoxicity were attenuated. Molecular modeling of iv analogue-TRPV1 binding corroborated these results and highlighted key structural features of capsaicin required for TRPV1 activation and cytotoxicity. Endogenous TRPV1 agonists have recently been implicated as pneumotoxicants during systemic inflammation. Treatment of mice i.p. with lipopolysaccharides (LPS) promoted systemic inflammation and lung injury. TRPV1 knockout mice and mice co-treated with the TRPV1 antagonist LJO-328 were protected from lung injury and evidence of an ER stress response was diminished. The endovanilloid anandamide induced ER stress and lung cell death in vitro, but these effects were not blocked by TRPV1 antagonist cotreatment, suggesting that other endovanilloids may cause ER stress and lung injury in mice. The results of this project provide a concerted mechanism of TRPV1-mediated lung epithelial cell death and illustrate a potential role of TRPV1 and endovanilloids in lung injury.