Received: 7 September 2020 | Revised: 23 October 2020 | Accepted: 5 November 2020 DOI: 10.1002/nau.24579 ORIGINAL BASIC SCIENCE ARTICLE The transient receptor potential A1 ion channel (TRPA1) modifies in vivo autonomous ureter peristalsis in rats Philipp Weinhold1 | Luca Villa2 | Frank Strittmatter1 | Christian Gratzke3 | Christian G. Stief1 | Fabio Castiglione4,5 | Francesco Montorsi2 | Petter Hedlund6,7 1Department of Urology, Ludwig‐ Maximilians‐University, Munich, Abstract Germany Aims: The current study aimed to explore the expression of transient receptor 2 Department of Urology, San Rafaele potential A1 ion channels (TRPA1) in the rat ureter and to assess if TRPA1‐active University, Milan, Italy compounds modulate ureter function. 3Department of Urology, Albert Ludwigs University, Freiburg, Germany Methods: The expression of TRPA1 in rat ureter tissue was studied by im- 4Department of Urology, Leuven munofluorescence. The TRPA1 distribution was compared to calcitonin gene‐ University, Leuven, Belgium related peptide (CGRP), α‐actin (SMA1), anoctamin‐1 (ANO1), and c‐kit. For 5 Department of Urology, University in vivo analyses, a catheter was implanted in the right ureter of 50 rats. Ureter College of London, London, UK peristalsis and pressures were continuously recorded by a data acquisition set‐ 6Department of Clinical and Experimental Pharmacology, Lund University, Lund, up during intraluminal infusion of saline (baseline), saline plus protamine Sweden sulfate (PS; to disrupt the urothelium), saline plus PS with hydrogen sulfide 7Department of Drug Research and (NaHS) or cinnamaldehyde (CA). Comparisons were made between rats Pharmacology, Linköping University, treated systemically with vehicle or a TRPA1‐antagonist (HC030031). Linköping, Sweden Results: TRPA1‐immunoreactive nerves co‐expressed CGRP and were mainly Correspondence located in the suburothelial region of the ureter. Immunoreactivity for TRPA1 Philipp Weinhold, Department of Urology, ‐ ‐ ‐ Ludwig‐Maximilians‐Universität was also encountered in c kit positive but ANO1 negative cells of the ureter München, Marchioninistraße 15, 81377 suburothelium and wall. Munich, Germany. In vivo, HC030031‐treated rats had elevated baseline peristaltic frequency Email: [email protected] muenchen.de (p < 0.05) and higher intraluminal pressures (p < 0.01). PS increased the frequency of ureter peristalsis versus baseline in vehicle‐treated rats (p <0.001)butnotin Funding information HC030031‐treated rats. CA (p <0.001) and NaHS (p < 0.001) decreased ureter Gester Foundation, Helsingborg, Sweden peristalsis. This was counteracted by HC030031 (p <0.05and p <0.01). Conclusions: In rats, TRPA1 is expressed on cellular structures considered of importance for peristaltic and mechanoafferent functions of the ureter. Functional data indicate that TRPA1‐mediated signals regulate ureter peri- stalsis. This effect was pronounced after mucosal disruption and suggests a role for TRPA1 in ureter pathologies involving urothelial damage. KEYWORDS ankyrin 1, cinnamaldehyde, H2S, interstitial cell, nerve, pacing ------------------------------------------------------------------------------------------------------------------------------------------- This is an open access article under the terms of the Creative Commons Attribution‐NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2020 The Authors. Neurourology and Urodynamics published by Wiley Periodicals LLC Neurourology and Urodynamics. 2021;40:147–157. wileyonlinelibrary.com/journal/nau | 147 148 | WEINHOLD ET AL. 1 | INTRODUCTION 2 | MATERIALS AND METHODS The ureters ensure unidirectional transport of urine 2.1 | Ethical considerations from the kidneys to the urinary bladder by peristalsis that is described to involve activities of pacemaker All experiments were approved by the Institutional Animal cells, mechanosensory signals, and electromechanical Ethics Care and Use Committees of the Lund and Linköping ‐ coupling of myogenic functions.1 3 The interface be- Universities, Sweden, and San Raffaele Scientific Institute, tween components of the ureter peristalsis system is Milan, Italy. Experiments were carried out in accordance not fully known. Electrical recordings during with the ARRIVE Guidelines.11 pyeloureteric motility have been correlated to peri- stalsis in various species and the pacing of such signals are proposed to arise from atypical smooth muscle cells 2.2 | Animals and interstitial cells of Cajal‐like cells (ICC‐LC).1,3,4 Plasticity in the expression or function of these cells are Fifty male Sprague–Dawley rats (250–300 g; Charles proposed to cause discordant regulation of ureter River) were used. Animals were maintained under stan- peristalsis in relation to vesicoureteral reflux, con- dard laboratory conditions with a 12:12 h light:dark cycle, genital ureteropelvic junction obstruction, and free access to food, water, and enrichments. Isoflurane obstructive megaureter.2,3 (alveolar concentration of 5% for induction and 2% for Besides its barrier function, the mucosa of the ur- maintenance) was used as anesthesia. After experiments, inary tract is a sensory unit that responds to mechan- rats were killed during anesthesia by carbon dioxide as- ical or chemical provocation with local release of phyxia. Ureters were harvested for immunofluorescence factors that regulate activities of other cell types that investigations from six naive separate male rats. are involved in, for example, motor functions, cell turn‐over, inflammation, or pain perception.5 As such, pressure‐dependent signals from the urothelium has 2.3 | Immunofluorescence been reported to modify the activity of sensory neurons of the perfused ureter, and capsaicin‐dependent sig- Ureter specimens were processed for immuno- nals and tachykinins are reported to modify sponta- fluorescence as previously described.12 Primary anti- neous contractile activities of upper urinary tract bodies: rabbit anti‐TRPA1 (1:500; Alomone Labs), guinea preparations.3,6 Cellular structures within the ureter pig anti‐calcitonin gene‐related peptide (CGRP; a marker mucosa, that is, the urothelium, interstitial cells, and for sensory nerves, 1:750; Euro‐Diagnostica), mouse anti‐ neurons, are express multimodal transient receptor smooth muscle α‐actin (SMA; 1:1000; Abcam), goat anti‐ potential ion channels (TRPs) that respond to chemi- anoctamin‐1 (ANO1; a marker for interstitial cells, 1:250; cal, mechanical, and thermal stimuli.7,8 Information is Santa Cruz), and a mouse anti‐c‐kit (a marker for inter- scarce on the functional role of TRPs in normal ureter stitial cells, 1:250; Santa Cruz). Secondary antibodies: peristalsis or ureter disease. The ankyrin 1 subtype Alexa Fluor, Molecular Probes Inc. (1:800). Sections were TRP (TRPA1) has been located to sensory neurons, and analyzed using a laser fluorescence microscope (Olympus interstitial cells of the proximal human ureter, and Corporation). Images were acquired using Viewfinder TRPA1‐mediated signals have been proposed involved Lite version 2.0 (Pixera Corp.). in modulation of neuromuscular functions of the isolated human and pig ureters.8,9 The TRPA1 is a cold‐ and mechanoreceptor that also acts as a sensor to 2.4 | In vivo experimental procedures irritants and bacterial lipopolysaccharides, is activated by hypoxia, and is described to be involved in neu- Animals were randomized to experimental groups roinflammatory responses of viscera.8,10 If TRPA1‐ (QuickCalcs; GraphPad Software Inc.). Briefly, during related transmission is involved in the regulation of anesthesia, an abdominal incision was performed. A ureter peristalsis has, to the best of our knowledge, not polyethylene catheter (PE‐10; Clay‐Adams) was introduced been explored. Therefore, the aim of this study was to into the ureteropelvic junction of the left ureter.13 investigate the effects by various TRPA1‐active com- The catheter was attached to a microsyringe pump (CMA pounds on ureter peristalsis in vivo in a rat model. In 100; Carnegie Medicine AB) and connected to a pressure addition, we assessed the expression of TRPA1 in the transducer (see Supplementary Information). Room tem- rat ureter in comparison to our previous findings of the perature physiological saline was infused at a speed of − human ureter.8 0.4 ml h 1 simulating normal urine production.13 Ureteral WEINHOLD ET AL. | 149 intraluminal pressure was recorded with an MP100 data also expressed immunoreactivity for CGRP (Figure 2). acquisition system (Biopac Systems Inc.). The bladder Intense TRPA1 immunoreactivity was also located in cells was incised to avoid interference on ureter pressures from interspersed in the suburothelial region (Figures 1 and 2). detrusor contractions.13 Single TRPA1‐positive cells were encountered in the In pilot experiments (data not shown), the TRPA1 muscular layer of the ureter wall (Figure 1). Co‐stainings of agonists did not effect ureter peristalsis or pressures when sectioned rat ureter tissue with antibodies for TRPA1 given intraluminally in naive rats. Therefore, after a base- and ANO1 disclosed no overlap of immunoreactivities line registration (20–30 min) with a saline infusion to (Figure 2) In contrast, TRPA1‐immunoreactive cells also achieve stable autonomous peristaltic ureter activity, an expressed c‐kit‐immunoreactivity (Figure 2). infusion with a protamine sulfate (PS)‐containing (10 mg/ ml) saline solution to disrupt the