Distribution and Function of the Hydrogen Sulfide–Sensitive TRPA1 Ion Channel in Rat Urinary Bladder

doi:10.1016/j.eururo.2007.10.024

European Urology

Volume 53, Issue 2, February 2008, Pages 391-400

https://doi.org/10.1016/j.eururo.2007.10.024 Get rights and content

Abstract

Objectives

To investigate the distribution of the transient receptor potential (TRP) A1 ion channel in the rat urinary bladder, and to study the effects of hydrogen sulfide (H₂S) and known TRPA1 activators on micturition in conscious rats and on heterologously expressed ion channels.

Methods

The expression of TRPA1 in urinary bladder was studied with fluorescence immunohistochemistry and real-time PCR in female Sprague-Dawley rats. Cystometric investigations were performed in conscious animals subjected to intravesical administration of sodium hydrogen sulfide (NaHS, donor of H₂S), allyl isothiocyanate (AI), and cinnamaldehyde (CA). Fluorometric calcium imaging was used to study the effect of NaHS on human and mouse TRPA1 expressed in CHO cells.

Results

TRPA1 immunoreactivity was found on unmyelinated nerve fibres within the urothelium, suburothelial space, and muscle layer as well as around blood vessels throughout the bladder. All TRPA1 immunoreactive nerves fibres also expressed TRPV1 immunoreactivity and vice versa. TRPA1 was also detected in urothelial cells at both transcriptional and protein levels. AI increased micturition frequency and reduced voiding volume. CA and NaHS produced similar changes in urodynamic parameters after disruption of the urothelial barrier with protamine sulfate. NaHS also induced calcium responses in TRPA1-expressing CHO cells, but not in untransfected cells.

Conclusions

The expression of TRPA1 on C-fibre bladder afferents and urothelial cells together with the finding that intravesical TRPA1 activators initiate detrusor overactivity indicate that TRPA1 may have a role in sensory transduction in this organ. The study also highlights H₂S as a TRPA1 activator potentially involved in inflammatory bladder disease.

Introduction

The mammalian transient receptor potential (TRP) superfamily consists of 28 different proteins that may be subdivided into six main subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), and TRPA (ankyrin) [1]. Most members of this protein superfamily are nonselective cation channels, several of which are present on primary sensory neurons and involved in somatosensory processes, including the transduction of chemical, thermal, and mechanical stimuli [1]. TRPA1 is present on capsaicin-sensitive primary sensory neurons [2], [3], [4], which upon activation elicit pain, protective reflexes, and local release of neurotransmitters in the periphery [5]. TRPA1 was initially characterised as a noxious cold receptor [2], but this role has been challenged by others [3], [4], [6], [7], [8]. Furthermore, several recent studies have suggested a role for TRPA1 and its invertebrate homologues in mechanosensation [9], [10], [11].

TRPA1 is activated by a variety of plant-derived and environmental irritants, such as allyl isothiocyanate (AI), cinnamaldehyde (CA), allicin, and acrolein [3], [4], [8], [12], all of which interact with cysteine residues in the ion channel protein [13], [14]. Interestingly, acrolein and similar aldehydes are formed endogenously during inflammation [15]. Hydrogen sulfide (H₂S) is another endogenous compound and potential inflammatory mediator that may activate TRP ion channels in the bladder [16], [17]. This gaseous molecule is also an industrial pollutant and a product of L-cysteine metabolism in bacteria [18].

As shown by immunohistochemistry and in situ hybridisation, TRPA1 is present in a subset of sensory neurons in rodent dorsal root and trigeminal ganglia, where it is coexpressed with TRPV1 and calcitonin gene–related peptide (CGRP) [2], [4]. There is also a possibility that TRPA1 is expressed in a small population of capsaicin-insensitive sensory neurons [19]. TRPA1 is also found on nerve fibres in target organs [4], [7], but the distribution of TRPA1 and the type of nerves expressing this ion channel in the urinary bladder have not been determined. The TRPA1 activators AI and CA contract rat urinary bladder in vitro through stimulation of capsaicin-sensitive nerves, providing indirect evidence that TRPA1 and TRPV1 are coexpressed in bladder afferents [20], [21]. It is well established that activation of TRPV1 in the bladder wall produces detrusor overactivity [22], but whether activation of TRPA1 has the same effect has not been investigated.

In the present study, we examined the distribution of TRPA1 and its coexpression with markers of different subsets of sensory neurons, using immunohistochemistry and real-time polymerase chain reaction (PCR). To elucidate the functional role of TRPA1, we studied the urodynamic effects of different TRPA1 activators given intravesically to conscious rats undergoing continuous cystometry. In addition, we examined the possibility that H₂S causes activation of TRPA1.

Section snippets

Animals

Female Sprague-Dawley rats (Scanbur BK AB, Sweden), weighing 200–250 g, were used. The experimental protocols were approved by the local animal ethics committee. The rats were maintained under standard laboratory conditions at 12:12 light/dark cycle with free access to food pellets and tap water.

Immunohistochemistry

Rat urinary bladders were fixed with 4% formaldehyde for 1 h and cryoprotected in 15% sucrose for 2 d [4]. The fixation was prolonged to 3 h when substance P immunoreactivity was studied. Tissues were

Immunohistochemistry and messenger RNA detection

TRPA1-immunoreactive (IR) nerve fibres were found in the muscular layer throughout the bladder with the largest density of nerve fibres in the outflow region (Fig. 1). The suburothelial region contained TRPA1-IR nerve fibres, of which some extended into the urothelium (Fig. 2). TRPA1-IR nerve fibres were also seen around blood vessels in the suburothelial region and muscular layer of the bladder (not shown). A similar pattern of distribution was observed for TRPV1-IR nerve fibres. Confocal

Discussion

The current study shows that, in the rat bladder, TRPA1 is present on unmyelinated sensory nerve fibres that express TRPV1, and the sensory neuropeptides CGRP and substance P. Previous investigations have found that TRPA1 is present on a subpopulation of TRPV1-expressing neuronal cell bodies in rodent dorsal root and trigeminal ganglia [2], [3], [4]. The current study shows a complete colocalisation of TRPA1 and TRPV1 in rat bladder afferents. Interestingly, TRPA1 is also present in the

Conclusion

The current study showed that TRPA1 ion channels were present on capsaicin-sensitive afferents and urothelial cells, and that stimulation of such ion channels induced detrusor overactivity. The coexistence of TRPA1 and TRPV1 on C-fibre bladder afferents and the expression of TRPA1 on urothelial cells may indicate a role of TRPA1 in bladder chemosensation and mechanotransduction. Finally, the potential inflammatory mediator and bacterial metabolite H₂S can activate TRPA1, implying a

Conflicts of interest

The authors declare no conflict of interest.

Acknowledgements

This work was supported by the Swedish Medical Research Foundation (grants no: 6837 and 4838), the Medical Faculty of Lund (ALF), the School of Pharmaceutical Sciences (FLÄK), Gester's Foundation, Holger K Christiansen Foundation, and the UK Medical Research Council.

References (36)

G.M. Story et al.
ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures
Cell
(2003)
D.M. Bautista et al.
TRPA1 mediates the inflammatory actions of environmental irritants and proalgesic agents
Cell
(2006)
K.Y. Kwan et al.
TRPA1 contributes to cold, mechanical, and chemical nociception but is not essential for hair-cell transduction
Neuron
(2006)
W.D. Tracey et al.
Painless, a Drosophila gene essential for nociception
Cell
(2003)
M. Bandell et al.
Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin
Neuron
(2004)
R. Patacchini et al.
Pharmacological investigation of hydrogen sulfide (H2S) contractile activity in rat detrusor muscle
Eur J Pharmacol
(2005)
L. Li et al.
Hydrogen sulphide—a novel mediator of inflammation?
Curr Opin Pharmacol
(2006)
E.L. Andrade et al.
Contractile mechanisms coupled to TRPA1 receptor activation in rat urinary bladder
Biochem Pharmacol
(2006)
O. Ishizuka et al.
Capsaicin-induced bladder hyperactivity in normal conscious rats
J Urol
(1994)
P. Hedlund et al.
Effects of tolterodine on afferent neurotransmission in normal and resiniferatoxin treated conscious rats
J Urol
(2007)

P.J. Cox

Cyclophosphamide cystitis—identification of acrolein as the causative agent

Biochem Pharmacol

(1979)

S.B. McMahon et al.

A model for the study of visceral pain states: chronic inflammation of the chronic decerebrate rat urinary bladder by irritant chemicals

Pain

(1987)

M. Koltzenburg et al.

Dynamic and static components of mechanical hyperalgesia in human hairy skin

Pain

(1992)

A. Apostolidis et al.

Proposed mechanism for the efficacy of injected botulinum toxin in the treatment of human detrusor overactivity

Eur Urol

(2006)

A. Apostolidis et al.

Capsaicin receptor TRPV1 in urothelium of neurogenic human bladders and effect of intravesical resiniferatoxin

Urology

(2005)

B. Nilius et al.

Transient receptor potential cation channels in disease

Physiol Rev

(2007)

S.E. Jordt et al.

Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1

Nature

(2004)

D.M. Bautista et al.

Pungent products from garlic activate the sensory ion channel TRPA1

Proc Natl Acad Sci U S A

(2005)

Cited by (259)

H<inf>2</inf>O<inf>2</inf> enhances the spontaneous phasic contractions of isolated human-bladder strips via activation of TRPA1 channels on sensory nerves and the release of substance P and PGE2
2023, Free Radical Biology and Medicine
Several studies have indicated that reactive oxygen species (ROS) can lead to detrusor overactivity (DO), but the underlying mechanisms are not known. Hydrogen dioxide (H₂O₂) is used commonly to investigate the effects of ROS. In present study, we investigated the effects of H₂O₂ on phasic spontaneous bladder contractions (SBCs) of isolated human-bladder strips (iHBSs) and the underlying mechanisms. Samples of bladder tissue were obtained from 26 patients undergoing cystectomy owing to bladder cancer. SBCs of iHBSs were recorded in organ-bath experiments. H₂O₂ (1μM–10mM) concentration-dependently increased the SBCs of iHBSs. These enhancing effects could be mimicked by an agonist of transient receptor potential (TRP)A1 channels (allyl isothiocyanate) and blocked with an antagonist of TRPA1 channels (HC030031; 10 μM). H₂O₂ induced enhancing effects also could be attenuated by desensitizing sensory afferents with capsaicin (10 μM), blocking nerve firing with TTX (1 μM), blocking neurokinin effects with NK2 receptor antagonist (SR48968, 10 μM), and blocking PGE2 synthesis with indomethacin (10 μM), respectively. Our study: (i) suggests activation of TRPA1 channels on bladder sensory afferents, and then release of substance P or PGE2 from sensory nerve terminals, contribute to the H₂O₂-induced enhancing effects on SBCs of iHBSs; (ii) provides insights for the mechanisms underlying ROS leading to DO; (iii) indicates that targeting TRPA1 channels might be the promising strategy against overactive bladder in conditions associated with excessive production of ROS.
Mechanotransduction in the urothelium: ATP signalling and mechanoreceptors
2023, Heliyon
The urothelium, which covers the inner surface of the bladder, is continuously exposed to a complex physical environment where it is stimulated by, and responds to, a wide range of mechanical cues. Mechanically activated ion channels endow the urothelium with functioning in the conversion of mechanical stimuli into biochemical events that influence the surface of the urothelium itself as well as suburothelial tissues, including afferent nerve fibres, interstitial cells of Cajal and detrusor smooth muscle cells, to ensure normal urinary function during the cycle of filling and voiding. However, under prolonged and abnormal loading conditions, the urothelial sensory system can become maladaptive, leading to the development of bladder dysfunction. In this review, we summarize developments in the understanding of urothelial mechanotransduction from two perspectives: first, with regard to the functions of urothelial mechanotransduction, particularly stretch-mediated ATP signalling and the regulation of urothelial surface area; and secondly, with regard to the mechanoreceptors present in the urothelium, primarily transient receptor potential channels and mechanosensitive Piezo channels, and the potential pathophysiological role of these channels in the bladder. A more thorough understanding of urothelial mechanotransduction function may inspire the development of new therapeutic strategies for lower urinary tract diseases.
The urothelium: a multi-faceted barrier against a harsh environment
2022, Mucosal Immunology
All mucosal surfaces must deal with the challenge of exposure to the outside world. The urothelium is a highly specialized layer of stratified epithelial cells lining the inner surface of the urinary bladder, a gruelling environment involving significant stretch forces, osmotic and hydrostatic pressures, toxic substances, and microbial invasion. The urinary bladder plays an important barrier role and allows the accommodation and expulsion of large volumes of urine without permitting urine components to diffuse across. The urothelium is made up of three cell types, basal, intermediate, and umbrella cells, whose specialized functions aid in the bladder's mission. In this review, we summarize the recent insights into urothelial structure, function, development, regeneration, and in particular the role of umbrella cells in barrier formation and maintenance. We briefly review diseases which involve the bladder and discuss current human urothelial in vitro models as a complement to traditional animal studies.
Essential role of Ca<inf>v</inf>3.2 T-type calcium channels in butyrate-induced colonic pain and nociceptor hypersensitivity in mice
2020, European Journal of Pharmacology
Given the role of Ca_v3.2 isoform among T-type Ca²⁺ channels (T-channels) in somatic and visceral nociceptive processing, we analyzed the contribution of Ca_v3.2 to butyrate-induced colonic pain and nociceptor hypersensitivity in mice, to evaluate whether Ca_v3.2 could serve as a target for treatment of visceral pain in irritable bowel syndrome (IBS) patients. Mice of ddY strain, and wild-type and Ca_v3.2-knockout mice of a C57BL/6J background received intracolonic administration of butyrate twice a day for 3 days. Referred hyperalgesia in the lower abdomen was assessed by von Frey test, and colonic hypersensitivity to distension by a volume load or chemicals was evaluated by counting nociceptive behaviors. Spinal phosphorylated ERK was detected by immunohistochemistry. Ca_v3.2 knockdown was accomplished by intrathecal injection of antisense oligodeoxynucleotides. Butyrate treatment caused referred hyperalgesia and colonic hypersensitivity to distension in ddY mice, which was abolished by T-channel blockers and/or Ca_v3.2 knockdown. Butyrate also increased the number of spinal phosphorylated ERK-positive neurons following colonic distension in the anesthetized ddY mice. The butyrate-treated ddY mice also exhibited T-channel-dependent colonic hypersensitivity to intracolonic Na₂S, known to enhance Ca_v3.2 activity, and TRPV1, TRPA1 or proteinase-activated receptor 2 (PAR2) agonists. Wild-type, but not Ca_v3.2-knockout, mice of a C57BL/6J background, after treated with butyrate, mimicked the T-channel-dependent referred hyperalgesia and colonic hypersensitivity in butyrate-treated ddY mice. Our study provides definitive evidence for an essential role of Ca_v3.2 in the butyrate-induced colonic pain and nociceptor hypersensitivity, which might serve as a target for treatment of visceral pain in IBS patients.
Hydrogen Sulfide (H<inf>2</inf>S)/Polysulfides (H<inf>2</inf>S<inf>n</inf>) Signalling and TRPA1 Channels Modification on Sulfur Metabolism
2024, Biomolecules
Transient receptor potential ankyrin 1 channels in the bladder mediate low temperature elicited bladder overactivity in rats
2024, Neurourology and Urodynamics

View all citing articles on Scopus

¹: These authors contributed equally to this work.

View full text

Neuro-urologyDistribution and Function of the Hydrogen Sulfide–Sensitive TRPA1 Ion Channel in Rat Urinary Bladder

Abstract

Objectives

Methods

Results

Conclusions

Introduction

Section snippets

Animals

Immunohistochemistry

Immunohistochemistry and messenger RNA detection

Discussion

Conclusion

Conflicts of interest

Acknowledgements

Cell

Cell

Neuron

Cell

Neuron

Eur J Pharmacol

Curr Opin Pharmacol

Biochem Pharmacol

J Urol

J Urol

Biochem Pharmacol

Pain

Pain

Eur Urol

Urology

Transient receptor potential cation channels in disease

Physiol Rev

Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1

Nature

Pungent products from garlic activate the sensory ion channel TRPA1

Proc Natl Acad Sci U S A

Neuro-urology
Distribution and Function of the Hydrogen Sulfide–Sensitive TRPA1 Ion Channel in Rat Urinary Bladder