Klaus Deckmann

Bitter triggers acetylcholine release from polymodal urethral chemosensory cells and bladder reflexes

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. 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.

Chemosensory epithelial cells in the urethra: sentinels of the urinary tract

Abstract A peculiar cell type of the respiratory and gastrointestinal epithelia, originally termed “brush cell” or “tuft cell” by electron microscopists because of its apical tuft of microvilli, utilizes the canonical bitter taste transduction cascade known from oropharyngeal taste buds to detect potential hazardous compounds, e.g. bacterial products. Upon stimulation, this cell initiates protective reflexes and local inflammatory responses through release of acetylcholine and chemokines. Guided by the understanding of these cells as sentinels, they have been newly discovered at previously unrecognized anatomical locations, including the urethra. Solitary cholinergic urethral cells express canonical taste receptors and are polymodal chemosensors for certain bitter substances, glutamate (umami) and uropathogenic Escherichia coli. Intraurethral bitter stimulation triggers cholinergic reflex activation of bladder detrusor activity, which is interpreted as cleaning flushing of the urethra. The currently known scenario suggests the presence of at least two more urethral chemosensory cell types: non-cholinergic brush cells and neuroendocrine serotonergic cells. The potential implications are enormous and far reaching, as these cells might be involved in monitoring and preventing ascending urinary tract infection and triggering of inappropriate detrusor activity. However, although appealing, this is still highly speculative, since the actual number of distinct chemosensory cell types needs to be finally clarified, as well as their embryological origin, developmental dynamics, receptor equipment, modes of signalling to adjacent nerve fibres and other cells, repertoire of chemo- and cytokines, involvement in pathogenesis of diseases and many other aspects.

Cholinergic urethral brush cells are widespread throughout placental mammals

We previously identified a population of cholinergic epithelial cells in murine, human and rat urethrae that exhibits a structural marker of brush cells (villin) and expresses components of the canonical taste transduction signaling cascade (α-gustducin, phospholipase Cβ2 (PLCβ2), transient receptor potential cation channel melanostatin 5 (TRPM5)). These cells serve as sentinels, monitoring the chemical composition of the luminal content for potentially hazardous compounds such as bacteria, and initiate protective reflexes counteracting further ingression. In order to elucidate cross-species conservation of the urethral chemosensory pathway we investigated the occurrence and molecular make-up of urethral brush cells in placental mammals. We screened 11 additional species, at least one in each of the five mammalian taxonomic units primates, carnivora, perissodactyla, artiodactyla and rodentia, for immunohistochemical labeling of the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), villin, and taste cascade components (α-gustducin, PLCβ2, TRPM5). Corresponding to findings in previously investigated species, urethral epithelial cells with brush cell shape were immunolabeled in all 11 mammals. In 8 species, immunoreactivities against all marker proteins and ChAT were observed, and double-labeling immunofluorescence confirmed the cholinergic nature of villin-positive and chemosensory (TRPM5-positive) cells. In cat and horse, these cells were not labeled by the ChAT antiserum used in this study, and unspecific reactions of the secondary antiserum precluded conclusions about ChAT-expression in the bovine epithelium. These data indicate that urethral brush cells are widespread throughout the mammalian kingdom and evolved not later than about 64.5millionyears ago.

Brush cells, the newly identified gatekeepers of the urinary tract.

Purpose of review A specialized epithelial cell with chemosensory properties of taste cells known from the mouth has been newly identified in the urethra and linked to pathogen recognition. We here describe its properties and its link to defence mechanisms, showing parallels to similar sentinel cells in the respiratory and gastrointestinal tract. Recent findings In the urethra, slender epithelial cells with apical microvilli (‘brush cells’) express bitter and umami taste receptors and the downstream signalling cascade known from oropharyngeal gustation, utilizing it to monitor for bacterial products and bacterial growth facilitating conditions. Upon stimulation, they release acetylcholine, and their sensitivity is subjected to cholinergic feedback. They are approached by cholinoceptive sensory nerve fibres, and intraurethral bitter application evokes reflex detrusor activity. Similar cells in the respiratory and gastrointestinal mucosa additionally regulate immune function through local neurogenic inflammation and cytokine release, triggered by bacterial products and parasites. Summary This cell is interpreted to serve as chemosensory sentinel for potential hazardous compounds in the urethral lumen, triggering a protective mechanism (flushing through micturition) against further ascent. Dysfunction may be related to higher risk of infection or inadequate detrusor activity, pharmacological intervention may be considered to combat infection or detrusor overactivity.

Muscarinic receptors 2 and 5 regulate bitter response of urethral brush cells via negative feedback

We have recently identified a cholinergic chemosensory cell in the urethral epithelium, urethral brush cell (UBC), that, upon stimulation with bitter or bacterial substances, initiates a reflex detrusor activation. Here, we elucidated cholinergic mechanisms that modulate UBC responsiveness. We analyzed muscarinic acetylcholine receptor (M1–5 mAChR) expression by using RT‐PCR in UBCs, recorded [Ca2+]i responses to a bitter stimulus in isolated UBCs of wild‐type and mAChR‐deficient mice, and performed cystometry in all involved strains. The bitter response of UBCs was enhanced by global cholinergic and selective M2 inhibition, diminished by positive allosteric modulation of M5, and unaffected by M1, M3, and M4 mAChR inhibitors. This effect was not observed in M2 and M5 mAChR‐deficient mice. In cystometry, M5 mAChR‐deficient mice demonstrated signs of detrusor overactivity. In conclusion, M2 and M5 mAChRs attenuate the bitter response of UBC via a cholinergic negative autocrine feedback mechanism. Cystometry suggests that dysfunction, particularly of the M5 receptor, may lead to such symptoms as bladder overactivity.—Deckmann, K., Rafiq, A., Erdmann, C., Illig, C., Durschnabel, M., Wess, J., Weidner, W., Bschleipfer, T., Kummer, W. Muscarinic receptors 2 and 5 regulate bitter response of urethral brush cells via negative feedback. FASEB J. 32, 2903–2910 (2018). www.fasebj.org

ENaC in Cholinergic Brush Cells

Cholinergic polymodal chemosensory cells in the mammalian urethra (urethral brush cells = UBC) functionally express the canonical bitter and umami taste transduction signaling cascade. Here, we aimed to determine whether UBC are functionally equipped for the perception of salt through ENaC (epithelial sodium channel). Cholinergic UBC were isolated from ChAT-eGFP reporter mice (ChAT = choline acetyltransferase). RT-PCR showed mRNA expression of ENaC subunits Scnn1a, Scnn1b, and Scnn1g in urethral epithelium and isolated UBC. Scnn1a could also be detected by next generation sequencing in 4/6 (66%) single UBC, two of them also expressed the bitter receptor Tas2R108. Strong expression of Scnn1a was seen in some urothelial umbrella cells and in 65% of UBC (30/46 cells) in a Scnn1a reporter mouse strain. Intracellular [Ca2+] was recorded in isolated UBC stimulated with the bitter substance denatonium benzoate (25 mM), ATP (0.5 mM) and NaCl (50 mM, on top of 145 mM Na+ and 153 mM Cl− baseline in buffer); mannitol (150 mM) served as osmolarity control. NaCl, but not mannitol, evoked an increase in intracellular [Ca2+] in 70% of the tested UBC. The NaCl-induced effect was blocked by the ENaC inhibitor amiloride (IC50 = 0.47 μM). When responses to both NaCl and denatonium were tested, all three possible positive response patterns occurred in a balanced distribution: 42% NaCl only, 33% denatonium only, 25% to both stimuli. A similar reaction pattern was observed with ATP and NaCl as test stimuli. About 22% of the UBC reacted to all three stimuli. Thus, NaCl evokes calcium responses in several UBC, likely involving an amiloride-sensitive channel containing α-ENaC. This feature does not define a new subpopulation of UBC, but rather emphasizes their polymodal character. The actual function of α-ENaC in cholinergic UBC—salt perception, homeostatic ion transport, mechanoreception—remains to be determined.