Beschreibung
UCCC act as sentinels at the entrance of the urogenital tract and initiate protective measures upon perception of potentially harmful substances through of the canonical taste cascade (including ?-gustducin, PLC?2 and TRPM5). The transcription factor Pou2f3 is essential for the development of UCCC. CCC of the trachea and the gastrointestinal tract express the complete synthetic pathways for prostaglandins and leukotrienes. Relevant proteins here are FLAP for cysteinyl-leukotriene synthesis and Cox-1 for prostaglandin synthesis. Here, we asked whether UCCC also express the components of prostaglandin and leukotriene synthesis pathways. Expression analyses were carried out on murine urethra using NGS-data and RT-PCR. The presence of the proteins FLAP, ChAT and of the components of the taste transduction cascade as well as of Cox-1, ChAT and FLAP in UCCC was investigated by immunohistochemistry in C57BL/6Rj- and ChAT-eGFP mice. Immunohistochemical differentiation from neuroendocrine cells was carried out using a genetically modified mouse strain in which the transcription factor Pou2f3, a central developmental factor of UCCC, was deleted. Labelling with antibodies against serotonin as a marker for neuroendocrine cells and FLAP was performed. In supernatants of explanted urethrae from Pou2f3-/-- and C57BL/6Rj-mice, cysteinyl-leukotriene concentration was measured by ELISA after stimulation with the bitter substance denatonium to investigate a possible UCCC-mediated cysteinyl-leukotriene release.
Serotonin-immunoreactive neuroendocrine cells showed no FLAP+ labelling in Pou2f3-/-- and C57BL/6Rj-animals. In the urethra, NGS-data and RT-PCR showed mRNA expression of proteins relevant for prostaglandin and leukotriene synthesis, such as FLAP and Cox-1. Immunohistochemically, FLAP+-cells could be found in the urethral epithelium of ChAT-eGFP-mice. ChAT+/FLAP+-cells showed the typical club-shaped UCCC morphology, whereas ChAT-/FLAP+-cells did not. In addition, FLAP+-cells, which are most likely immune cells, could be detected in the lamina propria. In ChAT-eGFP-mice, ChAT and FLAP were colocalised in 63% of cells in females and 49% of cells in males with typical UCCC morphology. Only 40% of cells in females and 35% of cells in males of (Ad-)Villin+-cells were also FLAP+. 53.9% of ?-Gust+-cells, 71% of PLC?2+-cells in males and females, and 53% in females and 63% in males of TRPM5+-cells were FLAP+ in C57BL/6Rj-mice. ChAT and Cox-1 were colocalised in 59% of cells in females and 62% of cells in males, Cox-1 and FLAP were colocalised in 68% of cells in females and 92% of cells in males. FLAP+-cells in Pou2f3-/--mice did not show typical UCCC morphology and were Cox-1-.
Stimulation of explanted urethras with denatonium did not result in a significant increase in cysteinyl-leukotriene concentration from a baseline value in the supernatant above 5000 pg/ml. Compared to other organs (such as the gallbladder), this represents a markedly elevated value. A masking of UCCC-mediated leukotriene release by other sources, e.g. immune cells, seems possible. In studies on tissues from human body donors, no labelling with characteristic CCC markers and FLAP could be shown.
This study identified two UCCC phenotypes: UCCC type 1 exhibit the components of eicosanoid synthesis such as FLAP and Cox-1 in addition to classical brush cell markers such as ChAT and the components of the taste transduction cascade. UCCC type 1 were more abundant. UCCC type 2 showed only ChAT and components of taste transduction. UCCC-released eicosanoids could play a role in the urethra in UCCC-mediated neurogenic inflammation or UCCC-mediated contraction of the urethra after contact with pathogens. The specific function of prostaglandins or leukotrienes released from UCCC remains unclear and requires further investigation.
