The role of transient receptor potential ankyrin 1 (TRPA1) receptor activation in hydrogen-sulphide-induced CGRP-release and vasodilation

E Pinter, Zs Hajna, G Pozsgai, T Bagoly, L Boros, A Kemeny, Zs Helyes, J Szolcsanyi. University of P é cs, Pharmacology and Pharmacotherapy, Hungary

Background: Capsaicin-sensitive sensory neurons express several ion channels of the TRP family, such as transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors. TRPA1 can be activated by several chemical stimuli, such as allylisothiocyanate (AITC), resulting in the release of inflammatory neuropeptides, e.g. calcitonin gene-related peptide (CGRP). Recently, the gaseous mediator hydrogen sulphide (H2S) was suggested to have effect on capsaicin-sensitive sensory neurons. H2S-evoked CGRP release from sensory neurons of isolated rat tracheae and H2S-induced changes in the microcirculation of the mouse ear have been investigated in this study. We focused on the involvement of TRPA1 receptor activation in the effects of H2S.

Methods: Male Wistar rats (180–250 g, n=10/group) were deeply anaesthetised with sodium thiopentone (100 mg·kg-1,i.p.) and exsanguinated. Isolated tracheae were stimulated by allyl-isothiocyanate (AITC) or H2S donor sodium hydrogen sulphide (NaHS). Two tracheae were placed in the same organ bath (1.8 mL) and perfused with oxygenated (95% oxygen, 5% CO2) Krebs solution (37°C, 1 mL·min-1). CGRP release was measured by radioimmunoassay. Blood flow experiments were carried out using male and female Balb/c, C57BL/6, TRPA1 (TRPA1-/-) and TRPV1 receptor gene knockout (TRPV1-/-) mice (2-4 months, 20-30 g, n = 8/group).In the first part of the experiment Balb/c mice were treated with the selective TRPA1 antagonist HC-030031 (i.p., 30 or 100 mg/kg) 30 minutes before application of AITC or H2S-donor sodium hydrogen-sulphide (NaHS). In the second phase of the investigation TRPA1 and TRPV1 gene-deleted mice and their wild-type littermates were used. In both phases, right ears of the animals were treated with 2% AITC or 5% NaHS, while left ears received respective vehicles. Skin blood flow of the ears was detected by laser Doppler imaging (Perimed), for 30 minutes in case of AITC-, and for 50 minutes in case of NaHS-application.

Results: AITC-induced CGRP release from isolated rat tracheae (EC50: 61.42 μmol·L-1). NaHS evoked a concentration-dependent increase in CGRP release (EC50: 0.96 mmol·L-1) which was inhibited by HC-030031 but was not affected by TRPV1 receptor blocker BCTC. AITC treatment of mouse ears led to 29.8±2.8% increase in blood flow. NaHS elevated cutaneous blood flow by 61±4.5%. Effect of NaHS on microcirculation was ameliorated by HC-030031. Blood flow of TRPA1 KO mice showed significantly smaller increase in response to NaHS compared to the wild-type counterparts. Vasodilatation in TRPV1-/- animals did not differ from control. Data are presented as mean ± standard error of mean. Experimental groups were compared by one way ANOVA followed by Dunnett’s or Bonferroni’s post hoc test. Values of p<0.05 were regarded as statistically significant. Conclusions: We conclude that H2S activates TRPA1 receptors causing CGRP release from sensory nerves of rat tracheae, as well as inducing cutaneous vasodilatation in the mouse ear. TRPV1 receptors were not involved in these processes. Our results highlight that TRPA1 receptor activation should be minded as a potential mechanism of vasoactive effects of H2S.

This work supported by research grants OTKA K-81984 and Baross Gábor Program.