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.


Supplementary Figure 3. HNO-induced TRPA1 activation is reversed by DTT treatment
Effects of HC030031 and dithiothreitol (DTT) on AS-induced calcium transients in DRG neurons: ASinduced (300 µM, 45 s) increases in intracellular calcium are slow in reversal (upper lane) but can be accelerated by application of HC030031 (50 µM, 10 min, middle lane) or DTT (5 mM, 10 min, lower lane) measured as the percentage of decrease within the first minute of HC/DTT application in (a). This results in a lower area under the curve of intracellular calcium calculated for the tenth minute (b) after AS application in cells treated with HC030031 or DTT. A rebound increase of intracellular calcium was observed upon washout of HC030031, but not DTT (c), suggesting that DTT deactivated TRPA1, while HC just impeded further calcium entry (all ANOVA following HSD post hoc tests; * p < 0.001 each; n = 67 responding DRG neurons per group; all error bars represent SEM).

Supplementary Figure 4. HNO activates TRPA1 by modifying cysteine residues a)
Purification of mTRPA1 expressed in HEK cells. Representative chromatogram from IMAC purification step. The cell lysate was mixed with Ni-beads and incubated at 0 °C for 30 min. The column was then packed and the poly-His tagged mTRPA1 eluted with 50 mM His in 50 mM phosphate buffer pH 7.4. b-c) SDS-PAGE and anti-V5 immunoblot of the purified protein. d) Detection of disulfide bond formation on purified mTRPA1 channel protein (100 µM) treated with AS, using modified biotin-switch assay. Lane 1: TRPA1 treated with 1.5 mM AS, lane 2: TRPA1 treated with 0.5 mM AS, lane 3: untreated protein, lane 4: TRPA1 pretreated with IA and then treated with 1.5 mM AS. e) MALDI-TOF mass spectra of AS-treated synthetic peptide: No differences could be seen between AS-treated (red) and control (black) sample at low-mass ranges (m/z 450-1000).

Supplementary Figure 5. Deconvoluted MS spectra of CGRP 8-37 before and after the treatment with AS
While the lower spectrum shows the peaks corresponding to the peptide in cluster with different number of Na + ions, the upper spectrum, corresponding to the CGRP 8-37 treated with AS shows presence of additional peaks implying that the peptide gets chemically modified, which can account for the moderate effects of this inhibitor as observed in experiments shown in Fig. 4a.
Supplementary Ffigure 6. AS-induced axon reflex erythema a) Complete series of laser Doppler images taken before and every 2.5 minutes after double-blind intracutaneous injection of Angeli's salt (0.7 µmol), decomposed Angeli's salt or DEA NONOate (0.23 µmol) to human volunteers' volar forearms. This vasodilatation in human skin is induced by antidromic action potential conduction into collaterals of wide-branching mechano-insensitive C-nociceptors that release CGRP. DEA NONOate induced restricted, local vasodilatation which probably accounts for a direct NO/cGMP effect on the cutaneous blood vessels. Decomposed AS did neither induce a widespread axon-reflex erythema, nor a marked localized vasodilatation, but only a short-lasting weak erythema as with physiological saline injection. b-c) Photograph of the soft injection blebs (1clemastine, 2-AS) and surrounding axon flare erythema and (c) comparison of AS-induced maximal flare size without and with pre-treatment with clemastine. 150 µL of clemastine was injected and after 10 min, 0.7 µmol AS was injected into the border of clemastine injection bleb. (repeated measures ANOVA, LSD post hoc tests; * p ≤ 0.05; n = 6; all error bars represent SEM).

Supplementary Ffigure 7. Intracellular generation of HNO and co-localization of TRPA1 and CBS. a)
Kinetics of H 2 S (50 M) decay upon addition of NO solution (100 M) in the presence (red) and absence (green) of 2 mM glutathione in 300 mM potassium phosphate buffer (pH 7.4). Black line represents the spontaneous decay/removal of H 2 S in the same time scale. The obtained kinetic traces (red and green) suggest the same initial reaction rate in the presence and absence of glutathione. Addition of glutathione only results in a shift of a baseline. b) Rate of HNO formation (determined from the slope of the HNO electrode signal vs time plots) vs H 2 S (blue) and NO (red) concentration, while the other reactant concentration is maintained constant and in large excess. The points represent the mean of at least three measurments. c) Basal fluorescence of the HNO sensor CuBOT1 in sensory neurons was reduced by inhibition of NO-synthase by L-NMMA (1 mM, 2 h of pretreatment) and cystathionine beta synthase (CBS) by oxamic acid (1 mM, 2 h of pretreatment, ANOVA HSD post hoc test; p < 0.001, treated vs. control respectively; n > 150 per group; error bars represent SEM, scale bar = 25 µm). b) Confocal images of a cross-section through the rat spinal trigeminal nucleus caudalis immunohistochemically double-stained with antibodies against TRPA1 (Cy3, red) and CBS (FITC, green) combined with nuclear DAPI staining (blue). Bundles of immunostained afferent nerve fibers run through the trigeminal tract (right side of images) and into the superficial laminae of the trigeminal nucleus (lamina 1 seen on left side of images). Most of the nerve fibers show the signal for TRPA1 and CBS, producing the yellow colour in the combined image. Neuronal cell bodies are not TRPA1 or CBS immunopositive (scale bar = 50 µm). Figure 8. H 2 S alone does not activate TRPA1 in the physiological concentration range a) Na 2 S providing H 2 S does not activate TRPA1 in concentrations below 500 µM in sensory neurons. Upper panel: 100 µM H 2 S did not induce increases in intracellular calcium in DRG neurons even if applied for four minutes (n = 328), nor did H 2 S at 300µM (n = 146). Lower panel: H 2 S 500 µM for four minutes led to a mean increase in intracellular calcium in 11% of DRG neurons (yellow trace: ΔCa 2+ > 50 nM, black trace: all cells, n = 100). b) However, H 2 S at 1 mM applied to hTRPA1 transfected HEK cells for 120 s induced increases in intracellular calcium (25% out of 249 cells), but not in cells expressing hTRPA1-3CK, a mutant lacking essential cysteines (n = 566; mean ± SEM). Nevertheless, high concentrations of H 2 S have been described to induce cytotoxicity by inhibition of cytochrome c oxidases. Inhibition of aerobic metabolism depletes ATP and causes lactic acid accumulation and furthermore block of the mitochondrial electron transport chain will increase formation of reactive oxygen species. Lactic acid and ROS have been described to activate TRPA1, the latter one by modification of critical cysteines. Therefore indirect mechanisms of TRPA1 activation by high concentrations of H 2 S seem possible. Figure 9. Combination of NO and H 2 S activates TRPA1 channels a) NO + H 2 S-evoked inward currents in CHO cells expressing mTRPA1 could be strongly potentiated by switching from calcium free to external solution containing 2 mM calcium. b) HC030031 and dithiothreitol (DTT) had similar effects on NO + H 2 S-induced calcium transients as on transients evoked by AS in DRG neurons: NO + H 2 S (75 µM each, 45 s)-evoked increases in intracellular calcium are slow in reversal (upper lane) but can be accelerated by application of HC030031 (50 µM, 10 min, middle lane) or DTT (5 mM, 10 min, lower lane) measured as the percentage of decrease within the first minute of HC/DTT application in (i). This results in a lower area under the curve of intracellular calcium calculated for the tenth minute (ii) after NO and H 2 S application in cells treated with HC030031 or DTT. A rebound increase of intracellular calcium was observed upon washout of HC030031, but not DTT (iii), suggesting that DTT deactivated TRPA1, while HC just impeded further calcium entry (all ANOVA following HSD post hoc tests; * p < 0.001 each; n = 101 responding DRG neurons per group; all error bars represent SEM).