TRPA1 and CGRP antagonists counteract vesicant-induced skin injury and inflammation.

The skin is highly sensitive to the chemical warfare agent in mustard gas, sulfur mustard (SM) that initiates a delayed injury response characterized by erythema, inflammation and severe vesication (blistering). Although SM poses a continuing threat, used as recently as in the Syrian conflict, no mechanism-based antidotes against SM are available. Recent studies demonstrated that Transient Receptor Potential Ankyrin 1 (TRPA1), a chemosensory cation channel in sensory nerves innervating the skin, is activated by SM and 2-chloroethyl ethyl sulfide (CEES), an SM analog, in vitro, suggesting it may promote vesicant injury. Here, we investigated the effects of TRPA1 inhibitors, and an inhibitor of Calcitonin Gene Related Peptide (CGRP), a neurogenic inflammatory peptide released upon TRPA1 activation, in a CEES-induced mouse ear vesicant model (CEES-MEVM). TRPA1 inhibitors (HC-030031 and A-967079) and a CGRP inhibitor (MK-8825) reduced skin edema, pro-inflammatory cytokines (IL-1ß, CXCL1/KC), MMP-9, a protease implicated in skin damage, and improved histopathological outcomes. These findings suggest that TRPA1 and neurogenic inflammation contribute to the deleterious effects of vesicants in vivo, activated either directly by alkylation, or indirectly, by reactive intermediates or pro-inflammatory mediators. TRPA1 and CGRP inhibitors represent new leads that could be considered for validation and further development in other vesicant injury models.

A sensory neuronal ion channel essential for airway inflammation and hyperreactivity in asthma.

Asthma is an inflammatory disorder caused by airway exposures to allergens and chemical irritants. Studies focusing on immune, smooth muscle, and airway epithelial function revealed many aspects of the disease mechanism of asthma. However, the limited efficacies of immune-directed therapies suggest the involvement of additional mechanisms in asthmatic airway inflammation. TRPA1 is an irritant-sensing ion channel expressed in airway chemosensory nerves. TRPA1-activating stimuli such as cigarette smoke, chlorine, aldehydes, and scents are among the most prevalent triggers of asthma. Endogenous TRPA1 agonists, including reactive oxygen species and lipid peroxidation products, are potent drivers of allergen-induced airway inflammation in asthma. Here, we examined the role of TRPA1 in allergic asthma in the murine ovalbumin model. Strikingly, genetic ablation of TRPA1 inhibited allergen-induced leukocyte infiltration in the airways, reduced cytokine and mucus production, and almost completely abolished airway hyperreactivity to contractile stimuli. This phenotype is recapitulated by treatment of wild-type mice with HC-030031, a TRPA1 antagonist. HC-030031, when administered during airway allergen challenge, inhibited eosinophil infiltration and prevented the development of airway hyperreactivity. Trpa1(-/-) mice displayed deficiencies in chemically and allergen-induced neuropeptide release in the airways, providing a potential explanation for the impaired inflammatory response. Our data suggest that TRPA1 is a key integrator of interactions between the immune and nervous systems in the airways, driving asthmatic airway inflammation following inhaled allergen challenge. TRPA1 may represent a promising pharmacological target for the treatment of asthma and other allergic inflammatory conditions.

Neuronal Ca2+ sensor protein VILIP-1 affects cGMP signalling of guanylyl cyclase B by regulating clathrin-dependent receptor recycling in hippocampal neurons.

The family of neuronal Ca2+ sensor (NCS) proteins is known to influence a variety of physiological and pathological processes by affecting signalling of different receptors and ion channels. Recently, it has been shown that the NCS protein VILIP-1 influences the activity of the receptor guanylyl cyclase GC-B. In transfected cell lines, VILIP-1 performs a Ca2+-dependent membrane association, the reversible Ca2+-myristoyl switch of VILIP-1, which leads to an increase in natriuretic peptide-stimulated cGMP levels. In this study, we have investigated the effect of VILIP-1 on cGMP signalling in C6 cells and in primary hippocampal neurons, where VILIP-1 and GC-B are co-expressed in many but not all neurons and partially co-localize in the soma and in dendrites. Our data indicate that VILIP-1 modulates GC-B activity by influencing clathrin-dependent receptor recycling. These data support a general physiological role for VILIP-1 in membrane trafficking in the intact hippocampus, where the NCS protein may affect processes, such as neuronal differentiation and synaptic plasticity e.g. by influencing cGMP-signalling.

Cellular and subcellular localization of the inhibitory glycine receptor in hippocampal neurons.

Inhibitory glycine receptors are most abundant in spinal cord and brainstem, and glycinergic synapses have a well-established role in the regulation of locomotor behavior. Little is known about the function of glycine receptors in cortex and hippocampus, where GABA plays a dominant role in synaptic inhibition. Therefore, we have investigated tissue and cellular expression of glycine receptor alpha-subunits. Western blot and immunohistochemical analyses reveal the presence of glycine receptors in hippocampal tissue. Immunocytochemical experiments in hippocampal cultures show prominent cellular expression of glycine receptors in pyramidal neurons and GAD-positive interneurons similar to the calcium-binding protein VILIP-1 with widespread hippocampal distribution. On the subcellular level we found co-staining of GlyR and the presynaptic marker synapsin I. Furthermore, co-staining with GAD at synaptic terminals indicated partial co-localization of GABA- and glycine receptors.

MGluRs regulate the expression of neuronal calcium sensor proteins NCS-1 and VILIP-1 and the immediate early gene arg3.1/arc in the hippocampus in vivo.

The metabotropic glutamate receptor (mGluR) agonist (R,S)-3,5-dihydroxyphenylglycine (DHPG) is involved in several forms of hippocampal synaptic plasticity. DHPG application can induce slow-onset potentiation, a form of long-term potentiation (LTP), in the dentate gyrus and in the CA1 region in vivo. The induction of LTP correlates with increased expression levels of neuronal calcium sensor (NCS), considered as key elements for plasticity. In this study we investigated mGluR- and time-dependent changes in the expression of two different NCS proteins. Following DHPG application in vivo NCS-1 and VILIP-1 expression increased, with significant levels reached after 8 and 24h. The effect was attenuated by treatment with the group I mGluR specific antagonist S-4-carboxyphenylglycine. The immediate early gene (IEG) arg3.1/arc showed highest expression levels 2h after DHPG-treatment. Therefore, mGluRs at concentrations which induce synaptic plasticity regulate the expression of IEGs and NCS proteins in different time frames and thus contribute to late phases of synaptic plasticity.

Expression analysis of members of the neuronal calcium sensor protein family: combining bioinformatics and Western blot analysis.

We have used in silico mining of public databases (NCBI UniGene and NCI SAGE Anatomic Viewer) as a tool to obtain the tissue distribution pattern of three members of the neuronal calcium sensor protein family, namely VILIP-1, hippocalcin, and NCS-1 in humans. The theoretical human mRNA expression profile of the calcium sensor proteins derived from expressed sequence tag (EST) and serial analysis of gene expression (SAGE) data was compared with expression data from human tissues obtained by Western blot analysis. Since the EST databank searches do not yet give comparable results for rat which is often used as model animal, we have also analyzed the protein expression in rat tissues. Similar to the human expression profile in rat tissues calcium sensor proteins are mainly detected in the nervous system, but the data consistently implicated the additional expression in peripheral tissues with remarkable differences between the calcium sensor proteins.