Jordt S. TRPV4 inhibition counteracts edema and inflammation and improves pulmonary function and oxygen saturation in chemically induced acute lung injury. Am J Physiol Lung Cell Mol Physiol 307: L158 -L172, 2014. First published May 16, 2014; doi:10.1152/ajplung.00065.2014.-The treatment of acute lung injury caused by exposure to reactive chemicals remains challenging because of the lack of mechanism-based therapeutic approaches. Recent studies have shown that transient receptor potential vanilloid 4 (TRPV4), an ion channel expressed in pulmonary tissues, is a crucial mediator of pressure-induced damage associated with ventilator-induced lung injury, heart failure, and infarction. Here, we examined the effects of two novel TRPV4 inhibitors in mice exposed to hydrochloric acid, mimicking acid exposure and acid aspiration injury, and to chlorine gas, a severe chemical threat with frequent exposures in domestic and occupational environments and in transportation accidents. Postexposure treatment with a TRPV4 inhibitor suppressed acid-induced pulmonary inflammation by diminishing neutrophils, macrophages, and associated chemokines and cytokines, while improving tissue pathology. These effects were recapitulated in TRPV4-deficient mice. TRPV4 inhibitors had similar anti-inflammatory effects in chlorine-exposed mice and inhibited vascular leakage, airway hyperreactivity, and increase in elastance, while improving blood oxygen saturation. In both models of lung injury we detected increased concentrations of N-acylamides, a class of endogenous TRP channel agonists. Taken together, we demonstrate that TRPV4 inhibitors are potent and efficacious countermeasures against severe chemical exposures, acting against exaggerated inflammatory responses, and protecting tissue barriers and cardiovascular function. acute lung injury; chlorine; TRPV4 ACUTE LUNG INJURY (ALI) and its extreme manifestation, acute respiratory distress syndrome (ARDS), are associated with high levels of morbidity and mortality (28, 37). Major triggers of ALI and ARDS are pneumonia, sepsis, trauma, acid aspiration, inhalation of toxic gases or smoke, hyperoxia, high pressure ventilation, heart failure, or pancreatitis. A major hallmark of ALI and ARDS is the acute increase in permeability of the pulmonary vascular and epithelial barriers, resulting in edema and severe hypoxia (9). ALI and ARDS are often associated with exaggerated inflammatory responses due to neutrophil infiltration and increased macrophage activity in the injured lung (14,22). These inflammatory cells may aggravate injury through protease production, through generation of oxidative reactive species and proinflammatory cytokines and chemokines, and through prevention of inflammation resolution.The ion channel transient receptor potential vanilloid 4 (TRPV4) was recently identified as a major mediator of pulmonary dysfunction in animal models of ventilator-and heart failure-induced ALI, conditions associated with dramatic increases in pulmonary and vascular pressure (17,33). Among other ...

␣7 nicotinic acetylcholine receptors (nAChRs) modulate network activity in the CNS. Thus, functional regulation of ␣7 nAChRs could influence the flow of information through various brain nuclei. It is hypothesized here that these receptors are amenable to modulation by tyrosine phosphorylation. In both Xenopus oocytes and rat hippocampal interneurons, brief exposure to a broad-spectrum protein tyrosine kinase inhibitor, genistein, specifically and reversibly potentiated ␣7 nAChR-mediated responses, whereas a protein tyrosine phosphatase inhibitor, pervanadate, caused depression. Potentiation was associated with an increased expression of surface ␣7 subunits and was not accompanied by detectable changes in receptor open probability, implying that the increased function results from an increased number of ␣7 nAChRs. Soluble N-ethylmaleimide-sensitive factor attachment protein receptor-mediated exocytosis was shown to be a plausible mechanism for the rapid delivery of additional ␣7 nAChRs to the plasma membrane. Direct phosphorylation/ dephosphorylation of ␣7 subunits was unlikely because mutation of all three cytoplasmic tyrosine residues did not prevent the genisteinmediated facilitation. Overall, these data are consistent with the hypothesis that the number of functional cell surface ␣7 nAChRs is controlled indirectly via processes involving tyrosine phosphorylation.

Exposure to chlorine (Cl(2)) damages airway and alveolar epithelia, resulting in acute lung injury and reactive airway dysfunction syndrome. We evaluated the efficacy and mechanisms by which arformoterol, a long-term β(2)-agonist, administered after exposure, mitigated the extent of this injury. Exposure of C57BL/6 mice to 400 ppm Cl(2) for 30 minutes increased respiratory system resistance and airway responsiveness to aerosolized methacholine (assessed by FlexiVent) up to 6 days after exposure, and decreased Na(+)-dependent alveolar fluid clearance (AFC). Inducible Nitric Oxide Synthase (iNOS) knockout mice developed similar degrees of airway hyperreactivity as wild-type controls after Cl(2) exposure, indicating that reactive intermediates from iNOS do not contribute to Cl(2)-induced airway dysfunction in our model. Intranasal administration of arformoterol mitigated the Cl(2) effects on airway reactivity and AFC, presumably by increasing lung cyclic AMP level. Arformoterol did not modify the inflammatory responses, as evidenced by the number of inflammatory cells and concentrations of IL-6 and TNF-α in the bronchoalveolar lavage. NF-κB activity (assessed by p65 Western blots and electrophoretic mobility shift assay) remained at control levels up to 24 hours after Cl(2) exposure. Our results provide mechanistic insight into the effectiveness of long-term β(2)-agonists in reversing Cl(2)-induced reactive airway dysfunction syndrome and injury to distal lung epithelial cells.

Among the multiple organ disorders caused by the severe acute respiratory syndrome coronavirus (SARS-CoV), acute lung failure following atypical pneumonia is the most serious and often fatal event. We hypothesized that two of the hydrophilic structural coronoviral proteins (S and E) would regulate alveolar fluid clearance by decreasing the cell surface expression and activity of amiloride-sensitive epithelial sodium (Na(+)) channels (ENaC), the rate-limiting protein in transepithelial Na(+) vectorial transport across distal lung epithelial cells. Coexpression of either S or E protein with human alpha-, beta-, and gamma-ENaC in Xenopus oocytes led to significant decreases of both amiloride-sensitive Na(+) currents and gamma-ENaC protein levels at their plasma membranes. S and E proteins decreased the rate of ENaC exocytosis and either had no effect (S) or decreased (E) rates of endocytosis. No direct interactions among SARS-CoV E protein with either alpha- or gamma-ENaC were indentified. Instead, the downregulation of ENaC activity by SARS proteins was partially or completely restored by administration of inhibitors of PKCalpha/beta1 and PKCzeta. Consistent with the whole cell data, expression of S and E proteins decreased ENaC single-channel activity in oocytes, and these effects were partially abrogated by PKCalpha/beta1 inhibitors. Finally, transfection of human airway epithelial (H441) cells with SARS E protein decreased whole cell amiloride-sensitive currents. These findings indicate that lung edema in SARS infection may be due at least in part to activation of PKC by SARS proteins, leading to decreasing levels and activity of ENaC at the apical surfaces of lung epithelial cells.

An inhibitory role for strychnine-sensitive glycine-gated chloride channels (GlyRs) in mature hippocampus is beginning to be appreciated. We have reported previously that CA1 pyramidal cells and GABAergic interneurons recorded in 3- to 4-wk-old rat hippocampal slices express functional GlyRs, dispelling previous misconceptions that GlyR expression ceases in early development. However, the effect of GlyR activation on cell excitability and synaptic circuits in hippocampus has not been fully explored. Using whole cell current-clamp recordings, we show that activation of strychnine-sensitive GlyRs through exogenous glycine application causes a significant decrease in input resistance and prevents somatically generated action potentials in both CA1 pyramidal cells and interneurons. Furthermore, GlyR activation depresses the synaptic network by reducing suprathreshold excitatory postsynaptic potentials (EPSPs) to subthreshold events in both cell types. Blockade of postsynaptic GlyRs with the chloride channel blocker 4, 4'-diisothiocyanatostilbene-2-2'-disulfonic acid (DIDS) or altering the chloride ion driving force in recorded cells attenuates the synaptic depression, strongly indicating that a postsynaptic mechanism is responsible. Increasing the local glycine concentration by blocking reuptake causes a strychnine-sensitive synaptic depression in interneuron recordings, suggesting that alterations in extracellular glycine will impact excitability in hippocampal circuits. Finally, using immunohistochemical methods, we show that glycine and the glycine transporter GlyT2 are co-localized selectively in GABAergic interneurons, indicating that interneurons contain both inhibitory neurotransmitters. Thus we report a novel mechanism whereby activation of postsynaptic GlyRs can function to depress activity in the synaptic network in hippocampus. Moreover, the co-localization of glycine and GABA in hippocampal interneurons, similar to spinal cord, brain stem, and cerebellum, suggests that this property is likely to be a general characteristic of inhibitory interneurons throughout the CNS.