3.2 Activation of TRPA1 channels is involved in H2O2-induced enhancement effects
Studies have shown that H2O2 can target TRPA1 channels[28, 32, 36]. To determine the involvement of TRPA1 activation in H2O2-induced effects, HC-030031 (30μM), a potent and selective inhibitor of TRPA1[37], was applied 5–10 min before and during H2O2application. HC-030031 applied alone had no effects on contractile activity. H2O2-induced enhancement was attenuated significantly in the presence of HC-030031 (Fig. 1B), and the concentration–response curve of AUC and Δg was shifted to the right (Fig. 1C). In the presence of HC-030031, EC50 was 1.29 mM and 743.9μM for AUC and Δg, respectively, which was much larger than that of the control. The maximum response (Emax) was reduced by 37.8% and 15.3% for AUC and Δg, respectively, in the presence of HC-030031. These observations suggested that TRPA1 activation was involved in H2O2-induced effects.
Similar to H2O2, AITC (10μM, 30μM, 100μM, 300μM, 1mM), a specific agonist of TRPA1, also produced a concentration-dependent increase in the SBCs of human-bladder strips (Fig. 2A). The concentration–response curve of AITC revealed an EC50 of 195.3μM for AUC (Fig. 2B). However, a reliable EC50 for Δg could not be obtained because saturation was not reached even at 1 mM of AITC. The enhancement effect of AITC (300μM) could be blocked by HC-030031 (30μM) (Fig. 2C). The AUC and Δg were reduced by 73% and 62.4%, respectively, in the presence of HC-030031 (Fig. 2D). Because H2O2 at 100μM and AITC at 300μM produced a prominent increase in the SBCs of human-bladder strips, they were used as the test concentrations in subsequent experiments.
3.3 TRPA1 expressed on sensory nerves is involved in H2O2-inducedenhancement effects
Nicholas et al showed that H2O2increased the firing activity of bladder sensory afferents mainly by activating TRPA1 channels[28]. It is accepted that sensory afferents regulate SBCs by releasing neurotransmitter CGRP or SP, a process termed “efferent actions of sensory afferents”[23, 24, 26, 38, 39]. To examine the involvement of TRPA1 on bladder sensory nerves in H2O2-induced effects, bladder strips were treated with a high dose of capsaicin (10μM) 10–15 min before and during H2O2 to desensitize bladder sensory nerves. Capsaicin applied alone did not impact the contractile activity. Desensitizing sensory afferents with capsaicin reduced the H2O2 (100μM)-induced increase in AUC and Δg significantly by 68.1% and 71.5%, respectively (p< 0.05) (Fig. 3A and B). After desensitization of sensory nerves, the AITC (300μM)-induced increase in AUC and Δg was reduced by 71.1% and 73.7%, respectively (p < 0.05) (Fig. 3C and D).
To further confirm the involvement of activation of sensory afferents, TTX (1μM) was applied to block nerve firing. TTX applied alone did not impact the contractile activity. However, application of TTX (1μM) reduced the H2O2 (100μM)-induced increase in the AUC and Δg significantly by 61.3% and 72.9%, respectively (Fig. 3A and B). Application of TTX (1μM) reduced AITC (300μM)-induced increase in AUC and Δg significantly by 73.5% and 70.9%, respectively (Fig. 3C and D).
Immunofluorescence experiments revealed TRPA1 channels on sensory afferents located in sub-urothelial and intramuscular layers. These sensory afferents also expressed SP (Fig. 4A and B).