4-Chloropropofol enhances chloride currents in human hyperekplexic and artificial mutated glycine receptors

Abstract Background The mammalian neurological disorder hereditary hyperekplexia can be attributed to various mutations of strychnine sensitive glycine receptors. The clinical symptoms of “startle disease” predominantly occur in the newborn leading to convulsive hypertonia and an exaggerated startle response to unexpected mild stimuli. Amongst others, point mutations R271Q and R271L in the α1-subunit of strychnine sensitive glycine receptors show reduced glycine sensitivity and cause the clinical symptoms of hyperekplexia. Halogenation has been shown to be a crucial structural determinant for the potency of a phenolic compound to positively modulate glycine receptor function. The aim of this in vitro study was to characterize the effects of 4-chloropropofol (4-chloro-2,6-dimethylphenol) at four glycine receptor mutations. Methods Glycine receptor subunits were expressed in HEK 293 cells and experiments were performed using the whole-cell patch-clamp technique. Results 4-chloropropofol exerted a positive allosteric modulatory effect in a low sub-nanomolar concentration range at the wild type receptor (EC50 value of 0.08 ± 0.02 nM) and in a micromolar concentration range at the mutations (1.3 ± 0.6 μM, 0.1 ± 0.2 μM, 6.0 ± 2.3 μM and 55 ± 28 μM for R271Q, L, K and S267I, respectively). Conclusions 4-chloropropofol might be an effective compound for the activation of mutated glycine receptors in experimental models of startle disease.</p

TRPA1 and TRPV1 are required for lidocaine-evoked calcium influx and neuropeptide release but not cytotoxicity in mouse sensory neurons

Background Local anaesthetics (LA) reduce neuronal excitability by inhibiting voltage-gated Na+ channels. When applied at high concentrations in the direct vicinity of nerves, LAs can also induce relevant irritation and neurotoxicity via mechanisms involving an increase of intracellular Ca2+. In the present study we explored the role of the Ca2+-permeable ion channels TRPA1 and TRPV1 for lidocaine-induced Ca2+-influx, neuropeptide release and neurotoxicity in mouse sensory neurons. Methods Cultured dorsal root ganglion (DRG) neurons from wildtype and mutant mice lacking TRPV1, TRPA1 or both channels were explored by means of calcium imaging, whole-cell patch clamp recordings and trypan blue staining for cell death. Release of calcitonin gene-related peptide (CGRP) from isolated mouse peripheral nerves was determined with ELISA. Results Lidocaine up to 10 mM induced a concentration-dependent reversible increase in intracellular Ca2+ in DRG neurons from wildtype and mutant mice lacking one of the two receptors, but not in neurons lacking both TRPA1 and TRPV1. 30 mM lidocaine also released Ca2+ from intracellular stores, presumably from the endoplasmic reticulum. While 10 mM lidocaine evoked an axonal CGRP release requiring expression of either TRPA1 or TRPV1, CGRP release induced by 30 mM lidocaine again mobilized internal Ca2+ stores. Lidocaine-evoked cell death required neither TRPV1 nor TRPA1. Summary Depending on the concentration, lidocaine employs TRPV1, TRPA1 and intracellular Ca2+ stores to induce a Ca2+-dependent release of the neuropeptide CGRP. Lidocaine-evoked cell death does not seem to require Ca2+ influx through TRPV1 or TRPV1

H2S and NO cooperatively regulate vascular tone by activating a neuroendocrine HNO-TRPA1-CGRP signalling pathway

Nitroxyl (HNO) is a redox sibling of nitric oxide (NO) that targets distinct signalling pathways with pharmacological endpoints of high significance in the treatment of heart failure. Beneficial HNO effects depend, in part, on its ability to release calcitonin gene-related peptide (CGRP) through an unidentified mechanism. Here we propose that HNO is generated as a result of the reaction of the two gasotransmitters NO and H2S. We show that H2S and NO production colocalizes with transient receptor potential channel A1 (TRPA1), and that HNO activates the sensory chemoreceptor channel TRPA1 via formation of amino-terminal disulphide bonds, which results in sustained calcium influx. As a consequence, CGRP is released, which induces local and systemic vasodilation. H2S-evoked vasodilatatory effects largely depend on NO production and activation of HNO-TRPA1-CGRP pathway. We propose that this neuroendocrine HNO-TRPA1-CGRP signalling pathway constitutes an essential element for the control of vascular tone throughout the cardiovascular system

Lidocaine-activated inward currents are mediated by TRPA1 and TRPV1.

<p>Representative current traces of whole-cell voltage-clamp recordings from DRG neurons using a holding voltage of– 60 mV. Application of lidocaine 30 mM for 30 s produced inward currents in cells from <b>A</b>. C57Bl/6, <b>B</b>. TRPA1^-/-, <b>C</b>. TRPV1^-/-, and <b>D</b>. TRPV1/TRPA1 ^{= / =} mice. Subsequent application at intervals of 2–3 min of capsaicin (1 μM, 10 s) and/or acrolein (100 μM, 30 s) verified expression of TRPV1 and/or TRPA1 in lidocaine positive neurons. Gamma-aminobutyric acid (GABA, 50 μM, 10 s) was applied comfirming viability of TRPV1/TRPA1 ^{= / =} ganglion neurons tested. <b>E.</b> Comprehensive recordings of mean peak currents evoked by lidocaine (30 mM) in C57Bl/6 (black bar), TRPA1^-/- (dark grey bar), TRPV1^-/- (light grey bar), and TRPV1/TRPA1 ^{= / =} (white bar) DRG neurons. The largest lidocaine-activated currents were observed in C57Bl/6 cells (n = 8), while current densities were significantly reduced in all three knockout mice strains to about the same level (TRPA1^-/-: p = 0.001, n = 8, TRPV1^-/-: p = 0.002, n = 7, TRPV1/TRPA1 ^{= / =}: p = 0.0002, n = 13). Results are mean and SEM of mean peak currents by lidocaine. For statistical analysis Mann-Whitney <i>U</i> test was calculated.</p

TRPA1 and TRPV1 differentially contribute to lidocaine-stimulated CGRP release from peripheral axons.

<p>Sciatic nerves from mice were isolated, stimulated with lidocaine and/or chemical substances (where stated) for 5 min, and induced CGRP-release over baseline was measured quantitatively. <b>A.</b> Lidocaine 10 mM and <b>B.</b> 30 mM stimulated CGRP release from C57BL/6 nerves (10mM: p = 0.0004, n = 16; 30 mM: p = 0.012, n = 8). In nerves from TRPA1^-/- and TRPV1^-/- mice less CGRP was released by lidocaine 10 mM (both p = 0.012, n = 8), while stimulation was without any effect on CGRP release from TRPV1/TRPA1 ^{= / =} nerves (p = 0.674, n = 8). <b>B.</b> Stimulation with 30 mM lidocaine induced release of CGRP of comparatively the same extent in nerves from TRPA1^-/-, TRPV1^-/- (both p = 0.012) and TRPV1/TRPA1 ^{= / =} mice (p = 0.017, all n = 8). <b>C, D.</b> Effects of additional application of either the TRPV1 inhibitor BCTC (10 μM) in TRPA1^-/- (lidocaine 10 mM: p = 0.003, 30 mM: p = 0.002, both n = 8) or the TRPA1 blocker HC030031 (50 μM) in TRPV1^-/- (n.s., n = 8 each) <b>E.</b> In C57BL/6 nerves CGRP release stimulated by lidocaine (30 mM) was nearly the same in calcium free as in calcium containing solution (n = 4). <b>F.</b> Initial application of thapsigargin (20 μM, 5 min) reduced subsequent CGRP release evoked by lidocaine (30 mM) to the half in Ca²⁺-free extracellular solution (p = 0.028, n = 8). Data are presented as mean ± SEM. For intra-individual comparison the Wilcoxon matched pairs test and for group comparison the Mann-Whitney <i>U</i> test were calculated.</p

Lidocaine-induced activation of TRPA1 and TRPV1 does not result in apoptosis.

<p><b>A</b> and <b>B.</b> Representative phase contrast photographs (20x) of cultured DRG neurons stained with the Cell-APOPercentage apoptosis assay subsequent to control (A) or 10 mM lidocaine treatment (B) for 1 h. <b>C.</b> Bar columns depicting the mean numbers of apoptotic DRG neurons (counts/visual field) following application of 10 mM lidocaine. Data are presented as mean ± S.E.M. n.s. was considered not significant by unpaired t-test.</p

Areas under the curve (AUC) of increases in [Ca²⁺]_i evoked by lidocaine.

<p><b>A-B.</b> Areas under the curve of increases in intracellular calcium evoked by lidocaine 10 mM (A) and 30 mM (B) in DRG neurons of C57BL/6, TRPV1^-/-, TRPA1^-/- and TRPV1/A1^-/- mice. Calcium responses to lidocaine stimuli significantly differ among the different genotypes (p ≤ 0.001), while at 30 mM lidocaine-evoked increase in [Ca²⁺]_i is similar in neurons of C57BL/6 and TRPV1^-/- mice (p = 1.0). <b>C-D.</b> Calcium responses to 10 and 30 mM lidocaine compared between AITC-positive and AITC-negative DRG neurons from TRPV1^-/- mice (C) or between capsaicin-positive and capsaicin-negative DRG neurons from TRPA1^-/- mice (D). * indicates p≤ 0.016 each. <b>E-F.</b> Calcium responses to three subsequent lidocaine stimuli show prominent desensitization, which involves TRPA1 receptors at 30 mM lidocaine (E; p = 0.00002) and TRPV1 receptors at 10 and 30 mM (F; p = 0.0002 and p = 0.00002). <b>G.</b> Increases in intracellular calcium evoked by capsaicin (0.3μM, black) or AITC (100μM, white bars) desensitize to a larger extent following 30 mM than after 10 mM lidocaine (p≤ 0.003; each). <b>H</b>. Lidocaine evokes calcium responses even in DRG neurons of TRPV1/A1^-/- mice, which were reduced in calcium free extracellular solution (p = 0.004), but nearly abolished only by depletion of intracellular calcium stores by pretreatment with thapsigargin (2 μM, p = 0.00002). Data are normalized to a final 60 mM potassium stimulus applied to the neuros to evoke maximal calcium influx. Error bars represent S.E.M. (all ANOVA followed by HSD post hoc tests calculated for each panel separately apart from data presented in 2A+B).</p