Significance We report the presence of a previously unidentified cholinergic, polymodal chemosensory cell in the mammalian urethra, the potential portal of entry for bacteria and harmful substances into the urogenital system. These cells exhibit structural markers of respiratory chemosensory cells (“brush cells”). They use the classical taste transduction cascade to detect potential hazardous compounds (bitter, umami, uropathogenic bacteria) and release acetylcholine in response. They lie next to sensory nerve fibers that carry acetylcholine receptors, and placing a bitter compound in the urethra enhances activity of the bladder detrusor muscle. Thus, monitoring of urethral content is linked to bladder control via a previously unrecognized cell type.

Abstract Chemosensory cells in the mucosal surface of the respiratory tract (“brush cells”) use the canonical taste transduction cascade to detect potentially hazardous content and trigger local protective and aversive respiratory reflexes on stimulation. So far, the urogenital tract has been considered to lack this cell type. Here we report the presence of a previously unidentified cholinergic, polymodal chemosensory cell in the mammalian urethra, the potential portal of entry for bacteria and harmful substances into the urogenital system, but not in further centrally located parts of the urinary tract, such as the bladder, ureter, and renal pelvis. Urethral brush cells express bitter and umami taste receptors and downstream components of the taste transduction cascade; respond to stimulation with bitter (denatonium), umami (monosodium glutamate), and uropathogenic Escherichia coli; and release acetylcholine to communicate with other cells. They are approached by sensory nerve fibers expressing nicotinic acetylcholine receptors, and intraurethral application of denatonium reflexively increases activity of the bladder detrusor muscle in anesthetized rats. We propose a concept of urinary bladder control involving a previously unidentified cholinergic chemosensory cell monitoring the chemical composition of the urethral luminal microenvironment for potential hazardous content.

Mucosal surfaces of the mammalian respiratory and gastrointestinal tract contain solitary epithelial cells with characteristic microvilli at their tip, from which the name “brush cells” derives (1⇓–3). In the respiratory tract, these brush cells serve as sentinels, using the canonical taste transduction cascade to monitor the mucosal lining fluid for potential harmful substances, such as “bitter” bacterial products, and evoking reflexes aimed at combating further ingression of such compounds, such as closure of ducts leading into adjacent compartments (vomeronasal organ) or respiratory reflexes (4⇓⇓–7).

In line with such a sentinel function, brush cell abundance decreases with increasing distance to the opening to the outside world, being numerous in the nose and nearly absent in the intrapulmonary airways. Physiologically, the urinary tract allows passage in only one direction to release urine, but ascending infection by uropathogenic bacteria is not uncommon and is a major risk factor in fatal kidney disease and male infertility (8, 9). We hypothesized that the urogenital tract also may be equipped with chemosensory sentinel cells that monitor the lumen for potential hazardous content.

Discussion Up to now, solitary chemosensory or brush cells have been identified in the respiratory and gastrointestinal tracts, but not in any other mammalian organ system. Likely owing due to their anatomic restriction to the portal of entry into the urogenital tract (i.e., the urethra and glandular ducts opening into it), these chemosensory cells have escaped detection in previous searches for urogenital brush cells that focused on more centrally located organs, including the kidney, uterus, and prostate gland, before the sentinel function of solitary chemosensory cells had been proposed (14). Coexpression patterns of various components of the taste transduction cascade and ChAT-eGFP suggest the presence of more than one chemosensory cell type in the urethra, including a cholinergic type using PLCβ2 and TRPM5, proteins essential for oropharyngeal bitter and umami perception (27), for downstream signaling. Consistent with our [Ca2+] i measurements, this signaling pathway involves Gβγ stimulation of PLCβ2 with subsequent release of Ca2+ from intracellular stores (28). The role of the taste-specific Gα protein α-gustducin (29), which is expected to activate a phosphodiesterase, is less clear (28). In taste buds, α-gustducin is inconsistently coexpressed with T1R1–3, particularly in the posterior tongue (30), and its genetic ablation diminishes, but does not abrogate, bitter and umami gustation (31, 32). Similarly, the vast majority (≥90%) of cholinergic urethral brush cells respond to bitter (denatonium) and umami (L-glutamate), but only approximately one-third coexpress α-gustducin. A striking feature of cholinergic/TRPM5+ urethral chemosensory cells is the coexpression of taste receptors of the Tas1R and Tas2R families and the resulting polymodal response to both bitter and umami stimuli. Similarly, solitary nasal chemosensory cells coexpress Tas1R3 with Tas2R5 and Tas2R8 (33); however, although the responses of these cells to bitter stimuli are well documented (34), whether they also respond to umami remains to be established. This situation is in contrast to oropharyngeal gustation, where Tas1R and Tas2R receptors are not coexpressed in taste buds (33, 35), and 83% of receptor cells in murine vallate papillae respond to only a single taste quality, which provides a basis for taste coding in taste buds (36). In oropharyngeal gustation, bitter represents an aversive stimulus and umami represents a rewarding stimulus, raising the question as to the possible functional meaning of sensing of both qualities by a single cell. In contrast, on other mucosal surfaces, such as the urethral lining, these qualities represent potentially harmful (aversive) content. Bacteria produce and secrete bitter receptor-activating substances (5, 37, 38). In biofilms, such substances from the Gram-negative bacterium Pseudomonas aeruginosa, one of the predominant causative microorganisms in catheter-associated urinary tract infection (39), can reach concentrations as high as 600 µM (40). On the other hand, glutamate metabolism is positively linked to the pathogenic potential of Proteus mirabilis in the urinary tract (41), and free amino acids (i.e., umami) facilitate bacterial growth in urine (42). Thus, the bitter/umami polymodality of chemosensory cells may serve to broaden the spectrum for recognition of potential hazardous material in the urethral lumen. Most importantly, these chemosensory cells responded to heat-inactivated uropathogenic E. coli, the primary cause of urinary tract infection (43). Acetylcholine, a secretory product of these cells, may alter sensitivity of this process in an autocrine manner, as demonstrated by the enhanced tastant response after application of a muscarinic/nicotinic receptor blocker mixture. In the more complex lingual taste buds, autocrine cholinergic signaling enhances taste signaling via muscarinic receptors (44). Our cell culture experiments revealed that the amount of acetylcholine released on bitter stimulation is also sufficient to induce paracrine effects. In situ, these cholinergic cells, like those in the trachea (6), are directly approached by sensory nerve fibers expressing the nicotinic acetylcholine receptor α3 subunit, and luminal denatonium evoked reflex activation of the detrusor muscle at a concentration that stimulated urethral chemosensory cells. This reflex activation was sensitive to local application of a nicotinic receptor blocker, demonstrating the involvement of cholinergic, nicotinic transmission from chemosensory cells to sensory nerve fibers. Nonetheless, the denatonium-induced increase in detrusor activity was not entirely abrogated, possibly owing to (i) additional involvement of excitatory muscarinic acetylcholine receptors that are also expressed by urinary tract afferent neurons (45); (ii) additional involvement of a cotransmitter, such as ATP, which transmits information from taste cells to afferent fibers in taste buds (46); or (iii) insufficient access of intraluminally applied mecamylamine to the basolaterally located communication site between chemosensory cells and nerve fibers, as lower urinary tract epithelia form an extraordinary tight barrier (47). In conclusion, we propose a concept of urinary bladder control involving a previously unidentified cholinergic chemosensory cell monitoring the chemical composition of the urethral luminal microenvironment for potential hazardous content.

Materials and Methods Animals. Two independently generated ChAT(BAC)-eGFP mice were provided by M. Kotlikoff (Cornell University), and H. Monyer (University of Heidelberg) (10, 11). Tg(Chrna3-EGFP)BZ135Gsat mice with eGFP driven by the nAChR α3β4α5 cluster were provided by I. Ibanez-Tallon (MDC Molecular Medicine) (24). C57BL/6 mice were obtained from The Jackson Laboratory. Male Clr:WI Wistar rats were obtained from Charles River Deutschland. All animals were housed under standard laboratory conditions (12 h dark, 12 h light). Mice were killed by inhalation of an overdose of isoflurane (Abbott) and exsanguination. The experiments were approved by the local authorities (Rp Giessen, Germany; reference nos. A9/2011, A11/2011, A60/2012, A61/2012, and 12/2013). Cell Isolation. Urethrae were dissected, cut into small pieces, and enzymatically digested in dispase (2 mg/mL; Sigma-Aldrich) for 30 min in HBSS (Invitrogen) and 5 min in trypsin/PBS (1:1, Invitrogen) at 37 °C. After centrifugation (60 × g, 5 min) and mechanical dissociation, cells were resuspended in PBS and separated through a cell strainer (70 µM; BD Bioscience). Chemosensory cells were identified by either eGFP or a rabbit polyclonal TRPM5-antibody (ab72151, 1:125; Abcam) directed against an extracellular domain. Cells were incubated for 1 h at 37 °C with this primary antibody, followed by a 1-h incubation with FITC-conjugated donkey anti-rabbit IgG (1:125; Millipore) at 37 °C. The same procedure, but using Cy5-conjugated donkey anti-rabbit IgG (1:400; Dianova), was also applied to cells isolated from ChAT-eGFP mice. As observed in the immunohistochemistry of tissue sections (Table S3), TRPM5 labeling and eGFP expression matched nearly 1:1. For subsequent RT-PCR analysis (primer sequences given in Table S4), chemosensory cells were isolated by incubating dissociated urethra first with the TRPM5 antibody as described above and then with magnetic beads (Invitrogen) coated with goat anti-rabbit IgG (H+L) (PI65-6100; Invitrogen), followed by harvesting by magnetic cell separation. Measurement of Intracellular Calcium Concentration. Isolated cells were loaded with fluorescent calcium indicator Calcium Orange AM (3 µL) in Tyrode III solution (297 µL; 8 mM CaCl 2 , 130 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 10 mM Hepes, 10 mM glucose, 10 mM pyruvic acid, and 5 mM NaHCO 3 ) according to the manufacturer´s protocol (Invitrogen) and then plated on coverslips for 30–60 min at 37 °C. Intracellular calcium concentration was analyzed with a confocal laser scanning microscope (Zeiss LSM 710; 561-nm wavelength generated by a DPSS 561-10 laser) during continuous superfusion (3 mL/min) with Tyrode solution. Fluorescence intensities at the start of the recording period were set arbitrarily at 100%. Test stimuli and concentrations were adenosine 5′-triphosphate bis(Tris) salt dihydrate (ATP, 0.5 mM; Sigma-Aldrich), cycloheximide (0.1 mM; Sigma-Aldrich), denatonium benzoate (25 mM; Molekula), l-glutamic acid monosodium salt monohydrate (l-glutamate, 25 mM; Sigma-Aldrich), saccharin (5 mM; Fluka), acetylcholine chloride (25 µM; Sigma-Aldrich), and heat-inactivated uropathogenic E. coli [UPEC strain CFT073 (NCBI: AE014075, NC_004431), ∼2–5 × 107 cfu, provided by T. Chakraborty, JLU Giessen]. Inhibitors were TPPO (0.25 mM, Sigma-Aldrich), eserine hemisulfate (10 µM; Sigma-Aldrich), mecamylamine hydrochloride (0.02 mM; Sigma-Aldrich), and atropine sulfate (0.002 mM; RBI). Data are presented as mean ± SEM and were analyzed by the two-tailed paired t test. Patch-Clamp Rrecordings. Cells were isolated from ChAT-eGFP mice as described for [Ca2+] i recordings, and eGFP-expressing cells were identified with a fluorescence microscope (Zeiss Axiovert 10). The bath solution consisted of 140 mM NaCl, 4.5 mM KCl, 2.5 mM CaCl 2 , 1 mM MgCl, 10 mM Hepes, and 5 mM d-glucose (pH 7.4). The pipette solution consisted of 10 mM NaCl, 18 mM KCl, 92 mM K-gluconate, 0.5 mM MgCl 2 , 1 mM EDTA, and 10 mM Hepes (pH 7.2). The liquid junction potential was absorbed with the clamped voltage, producing an effective membrane potential of –60 mV. The bath solution included appropriate amounts of mannitol to compensate for osmotic changes or different concentrations of denatonium (1–25 mM), applied by a pressure-driven perfusion system. Transmembrane currents were amplified (EPC 9; Heka Electronics) and recorded continuously (sampled with 10 kHz, filtered with 3 kHz) with Pulse 8.77 software (Heka Electronics). Urodynamic Measurement. Rats were anesthetized by an s.c. injection of urethane (1.2 g/kg) at 1 h before surgery and maintained under anesthesia during the surgical preparation while on a heating pad (37 °C). A catheter (PE 50; Intramedic) was inserted into the bladder dome and connected to a pressure transducer and an infusion pump. Saline solution at room temperature was infused into the bladder at a rate of 0.04 mL/min. After a stabilization phase of 15–30 min, the intravesical bladder pressure was recorded continuously, and 50 µL of test stimuli were delivered into the urethral external orifice via a 0.9 × 25 mm cannula (Braun Vasofix G22) mounted on a 100-µL pipette. For final data analysis, areas under the curve (AUC) of equal time periods before and after stimulation were compared; data are presented as AUC/min. The urodynamic recording sessions took 3–4 h for each animal. Fluorescence microscopy and immunohistochemistry, pre-embedding immunohistochemistry, and EM, RT-PCR, and acetylcholine measurements were performed as described previously (6, 48), with minor modifications. More details are provided in SI Materials and Methods.

Acknowledgments We thank M. Bodenbenner and K. Michael for technical assistance. This work was supported by the LOEWE Program of the State of Hesse (Non-neuronal Cholinergic Systems, project A5, to W.K. and T.B.) and the Deutsche Forschungsgemeinschaft (T.G. and V.C.). This study was awarded the Eugen-Rehfisch Prize of the Forum Urodynamicum.