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International Journal of Impotence Research (2014) 27, 16–19 & 2014 Macmillan Publishers Limited All rights reserved 0955-9930/14 www.nature.com/ijir

ORIGINAL ARTICLE Expression and distribution of the transient receptor potential cationic channel ankyrin 1 (TRPA1) in the human vagina

SU¨ ckert1, JE Sonnenberg2, K Albrecht1, MA Kuczyk1 and P Hedlund3,4

The transient receptor potential cationic channel type A1 (TRPA1), belonging to a superfamily of cationic membrane channels, has been suggested to act as mechano- and pain sensor and, thus, to play a role in neurotransmission in the human body, including the urogenital tract. While the expression of TRPA1 has been investigated in a variety of tissues, up until today, no study has addressed the expression and distribution in the female genital tract. The present study aimed to investigate the expression and distribution of TRPA1 protein in human vaginal tissue. Reverse transcriptase PCR (RT-PCR) was applied in order to identify messenger ribonuleic acid specifically encoding for TRPA/A1. The distribution of TRPA1 in relation to the neuronal synthase (nNOS) and the signaling peptide calcitonin gene-related peptide (CGRP) was examined by means of immunohistochemical methods (double- antibody technique, laser fluorescence microscopy). RT-PCR analysis revealed the expression of mRNA encoding sequences specific for TRPA in the vaginal wall and epithelium. Immunostaining related to TRPA1 was observed in the basal epithelium and in slender varicose nerve fibers transversing the subepithelial and stromal space of the vaginal sections. In addition, these fibers presented immunoreactivity specific for nNOS or CGRP. The smooth musculature of the vaginal wall and small vessels interspersing the tissue did not present signals related to TRPA1. The findings indicate that TRPA1 might be involved in afferent neurotransmission in the vagina and work synergistically together with the nitric oxide/cyclic guanosine monophosphate pathway.

International Journal of Impotence Research (2014) 27, 16–19; doi:10.1038/ijir.2014.23; published online 24 July 2014

INTRODUCTION sulfide, enabling the channel to act as mechano- and pain sensor 5 It is assumed that the vagina plays a pivotal role in the perception at the cellular level. To date, there is growing evidence that of coital stimulation, leading to sexual arousal and, finally, orgasm. disturbances in local afferent (sensory) mechanisms might be With visual and sensory stimulation, an increase in genital blood related to the pathogenesis of dysfunctions of the lower urinary flow and the relaxation of the smooth musculature of the vagina tract. Thus, not surprisingly, current research has been devoted to occurs, resulting in an increase in vaginal luminal diameter and the TRPA channel and its potential role in sensory signaling and 9 lubrication, thus allowing penetration of the male penis during neurotransmission in the urogenital tract. The expression and sexual intercourse.1,2 Although our knowledge regarding the distribution of the TRPA1 was investigated by means of molecular physiology of the female sexual response is constantly growing, biology, western blot analysis or immunohistochemistry in the the mediators and mechanisms contributing to this process are prostate, the mucosa and smooth muscle of the human urinary still poorly understood.3,4 This slow progress is due to the bladder, as well as in the urethra. Functional studies using isolated complexity of the factors and their interactions—neuronal path- tissue of the bladder or urethra were also conducted in order to ways, endocrine and local cellular mediators—involved in the evaluate the significance of TRPA1 in the control of the outflow 10–12 biological control of the female sexuality. The transient receptor region. Up until today, no study has addressed TRPA1 in female potential cationic channel ankyrin 1 (TRPA1) is a member of the genital tissues, such as the clitoris, labia or vagina. Therefore, it TRP superfamily of membrane channels, consisting of the six sub- was the aim of the present study to evaluate in the human vagina families TRPA, TRPC, TRPM, TRPP, TRPML and TRPV. TRP channels, by means of molecular biology (reverse transcriptase PCR, RT-PCR permeable to most divalent cations, are known to play a central analysis) and conventional immunohistochemistry the expression role in smooth muscle contraction, the release of neurotransmit- and distribution of TRPA1. ters, cell proliferation and apoptosis.5 Although TRP channels are only poorly selective for Ca2 þ , they contribute mainly to changes 2 þ in cytosolic Ca by acting as entry gates into the plasma. TRPA1 MATERIALS AND METHODS is abundantly expressed in vertebrate dorsal root and trigeminal Tissue source and handling ganglia, as well as in hair cells of the inner ear.6–8 The channel protein can be activated by physical stimuli, such as noxious cold, In accordance with the regulations of the local ethical committee of the Hannover Medical School, specimens of the human vagina were obtained or chemical activators, including , , from five female cadavers (age at the time of death: 16–42 years) as well as cinnamonaldehyde, , diallyl disulfide (stinging compounds in from five women (aged 48–63 years, three menopausal, two postmeno- , , , or , respectively), pausal; mean age: 53 years) who had undergone pelvic surgery for (irritant in exhaust fumes and ) or dihydrogen gynecological malignancies. None of the female donors had a known

1Division of Surgery, Department of Urology and Urological Oncology, Hannover Medical School, Hannover, Germany; 2Sexual Function Research Unit, Institute for Biochemical Research and Analysis, Hannover, Germany; 3Faculty of Medicine, Department of Clinical Pharmacology, Linko¨ping University, Linko¨ping, Sweden and 4Department of Urology, Urological Research Institute (URI), University Vita Salute San Raffaele, Milan, Italy. Correspondence: Professor S U¨ ckert, Division of Surgery, Department of Urology and Urological Oncology, Hannover Medical School, 30625 Hannover, Germany. E-mail: [email protected] Received 10 October 2013; revised 9 January 2014; accepted 30 May 2014; published online 24 July 2014 TRPA1 in the human vagina SU¨ ckert et al 17 history of endocrinological or neurological diseases or sexual dysfunction. RT-PCRs were of the expected size of 656 base pairs (bp). Different Full wall preparations were taken from the lower mid portion of the vagina magnitudes of mRNA expression were observed in the vaginal and immediately placed in an ice-cold solution of CUSTODIOL (Dr Franz tissues: the relative degree of mRNA expression appeared to be Ko¨hler Chemie GmbH, Alsbach, Germany). more abundant in the vaginal wall than in the epithelial layer. A characteristic pattern of RT-PCR products from the experiments RT-PCR analysis using human vaginal wall and vaginal epithelium is displayed in For RT-PCR experiments, the vaginal epithelium was carefully separated Figure 1. from the smooth musculature and both tissue portions were frozen in liquid . The expression of mRNA specifically encoding for TRPA1 Immunohistochemistry was analyzed by means of the RT-PCR. Reaction mixtures were prepared in test tubes (volume: 0.2 ml) using up to 5 mg total RNA extracted from Examination of numerous sections (three to five) at different levels specimens of the vaginal wall or epithelium, 1 ml 10 mM dNTP and 1 ml revealed that the TRPA1 protein is present in the human vagina. oligo (dT)12–18 (0.5 mg ml À 1) and made-up to 10 ml with sterile water. The The findings appeared consistent in all tissue sections examined. mixtures were incubated for 5 min at 65 1C, then placed on ice for at least Immunosignals related to TRPA1 were observed in the epithelial 1 min. Nine (9) ml of a mixture comprising 2 ml10Â RT buffer, 4 ml25mM layer (basal epithelial cells and cells lining the vaginal lumen) and MgCl2,2ml 0.1 M dithiothreitol and 1 ml RaceOUT recombinant ribonuclease À 1 in slender varicose nerve fibers transversing the subepithelial space inhibitor (40 units ml ) was added followed by incubation for 2 min at and smooth muscle layer of the vaginal sections. In the epithelial 1 42 C. Fifty units of SuperScript II reverse transcriptase (Invitrogen GmbH, cells, staining was seen throughout the cytoplasm without any Karlsruhe, Germany) were added to each test tube (except for the negative RT control) followed by incubation for 50 min at 42 1C. The reaction was particular relation to subcellular structures (Figure 2a). A majority, terminated by heating for 15 min to 70 1C and then cooling the samples to but not all, TRPA1-immunoreactive (IR) subepithlial nerves pre- 0 1C. Two units of RNase H were added to each tube followed by sented immunoreactivity for CGRP (Figure 2b–d). In nerve fibers incubation for 20 min at 37 1C. PCR amplification was performed using extending through the muscle layer, TRPA1 was also co-localized 1–2 ml cDNA template (200 ng ml À 1), 1–2 ml specific primers (10 pmol ml À 1) with CGRP (Figure 2e–g). In addition, these fibers were character- and 25 ml Taq PCR Master Mix (Qiagen AG, Hilden, Germany). A negative ized by the expression of nNOS. All TRPA1-IR nerves also exhibited control (omitting the template cDNA) was included in each experiment. strong fluorescence signals specific for nNOS and vice versa PCRs were performed in a Model 9600 thermocycler (PE Biosystems GmbH, (Figure 2h–l). The smooth musculature of the vaginal wall and Weiterstadt, Germany). Cycling conditions were set as follows: initial small blood vessels interspersing the tissue did not present signals denaturation for 3 min at 94 1C, 35 cycles of denaturation (30 s at 94 1C), annealing phase (30 s at 52–60 1C), extension (1 min at 72 1C), final related to TRPA1 (results not shown). extension (10 min at 72 1C). DISCUSSION Immunohistochemistry The normal female sexual response depends on an unimpaired After immersion-fixation for 4 h in 4% in phosphate-buffered function of the peripheral autonomic nervous system and on saline (pH 7.4), tissue preparations were rinsed several times with phos- intracellular signaling pathways controlling the tone of genital phate-buffered saline containing 15% (w/w) sucrose and then embedded in Tissue-Tec (Miles Laboratories, Elkhart, IN, USA). Tissue specimens were vascular and non-vascular smooth muscle. Thus, it has been sliced with a cryostat to sections of 8–10 mm thickness and thaw-mounted assumed that, aside from an insufficient bioavailability of nitric onto glass slides. Sections were preincubated (2 h) in phosphate-buffered oxide and cyclic guanosine monophosphate, sexual dysfunction saline with 0.2% Triton X-100 and 0.1% bovine serum albumin, followed by might also be related to disturbances in local afferent (sensory) exposure for 24 h to the primary antibodies directed against TRPA1 mechanisms. In a variety of cells, the TRPA ion channel has been (working dilution 1:500), calcitonin gene-related peptide (CGRP, 1:500), and shown to act as a mechanosensor and/or nociceptor in the neuronal nitric oxide synthase (nNOS, 1:1.000). After rinsing, sections were mechanism of sensory transduction. TRPA has the potential to incubated for 90 min with Alexa Fluor-conjugated secondary antibodies modulate the electrical activity of excitable neurons in the central (1:800). Thereafter, sections were mounted in phenylendiamine and (brain) and peripheral nervous system.5 As there are lines of visualization commenced using a laser fluorescence microscope (Olympus, Osaka, Japan). Imaging was performed using the ViewFinder Lite programme (version 2.0, Pixera, Egham, UK). Negative controls omitting the primary antibodies were performed for all samples.

Antibody source Antibodies directed against TRPA1 (raised in rabbits) were purchased from Alamone Labs (Jerusalem, Israel), the mouse anti-eNOS antibodies were from Sigma Chemical Co. (St Louis, MO, USA), anti-CGRP antibodies (raised in guinea-pigs) from EuroDiagnostica (Malmoe, Sweden). Alexa Fluor secondary antibodies were obtained from Molecular Probes Europe BV (Leiden, The Netherlands). The anti-nNOS antibodies (raised in sheeps) were generously provided by Dr Piers Emson, The Babraham Institute, Molecular Neuroscience Group, Department of Neurobiology, Cambridge, UK.

RESULTS RT-PCR analysis Tissue from at least five individuals was subjected to the RT-PCR Figure 1. Reverse transcriptase (RT) PCR analysis: agarose gel analysis. The results obtained provided no basis to believe that the electrophoresis of products originating from RT-PCR. In the time interval to tissue excision may have affected the outcome of experiments, total mRNA isolated from the vaginal wall (vw) and 2 þ vaginal epithelium () and a primer specific for the TRPA1 receptor the experiments. The Mg dependency of the reactions con- was used. The size of the fragment resulting from the amplification ducted confirmed the accuracy of the procedure. RT-PCR revealed reaction is 656 bp. Lane 1 ¼ 100 bp DNA ladder (GeneRuler, that mRNA encoding for TRPA1 is expressed in the vaginal tissue. Fermentas GmbH, St Leon-Rot, Germany); lane 2 ¼ vw, 1 ml MgCl2; cDNA specifically encoding for TRPA1 was found to be present in lane 3 ¼ vw, 2 ml MgCl2; lane 4 ¼ vw, 2.5 ml MgCl2; lane 5 ¼ ve, 0.5 ml almost all specimens examined. The products resulting from the MgCl2); lane 6 ¼ ve, 1 ml MgCl2.

& 2014 Macmillan Publishers Limited International Journal of Impotence Research (2015), 16 – 19 TRPA1 in the human vagina SU¨ ckert et al 18

Figure 2. (a–l) Expression and distribution of TRPA1 in the human vaginal wall as evaluated by means of conventional fluorescence immunohistochemistry: (a) localization of TRPA1 in cells of the vaginal epithelial layer (lu ¼ vaginal lumen; magnification  10). (b) Bundle of TRPA-IR subepithelial nerve fibers in the human vaginal wall (magnification  40). Insert displays a single nerve fiber labeled for TRPA1 transversing the section (magnification  100). (c, d) Close-up of a varicose subepithelial nerve double-stained for TRPA1 (c, green) and CGRP (d, red; magnification  100). (e) TRPA1-IR varicose nerve fiber running through the smooth muscle portion of the vaginal wall (magnification  100). (f, g). Co-localization of TRPA1 (f, green) and CGRP (g, red) in a nerve fiber transversing deeper layers of the vaginal wall (magnification  100). (h, i) Varicose nerve projecting in different spatial levels of the section displaying dot-like immunosignals specific for TRPA1 (h, green) and nNOS (i, red; magnification  100). (j, k) Image of another representative section transversed by a nerve terminal presenting both TRPA1 (j, green) and nNOS (k, red) in its neurons. (l) Overlay of j and k (magnification  100, oil). CGRP, calcitonin gene- related peptide; IR, immunoreactive; nNOS, neuronal nitric oxide synthase; TRPA1, transient receptor potential cation channel type A1.

International Journal of Impotence Research (2015), 16 – 19 & 2014 Macmillan Publishers Limited TRPA1 in the human vagina SU¨ ckert et al 19 evidence suggestive of an involvement of TRP channels in the using isolated vaginal smooth muscle and compounds known to pathogenesis of various disease states, including those of the activate TRPA1) may show whether or not TRPA1 appears to play a urogenital tract, such as neurogenic bladder dysfunction, role in the maintenance of vaginal function. carcinoma of the prostate, cystitis and vulvodynia, drugs targeting the activity of TRP seem to offer great potential.13–15 For example, desensitization of the TRPV1 channel by respective CONFLICT OF INTEREST antagonists, such as , has been proposed a feasible strategy in the treatment of the overactive bladder.16 The authors declare no conflict of interest. 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& 2014 Macmillan Publishers Limited International Journal of Impotence Research (2015), 16 – 19