Friederike Stumpff

This study provides a comprehensive comparison between rumen tissue, primary culture, and organoid-derived cell culture inserts through detailed transcriptomic analysis. Cells grown over multiple passages in organoid culture differentiate into model epithelia that better approximate native tissue architecture and reach greater epithelial purity than primary cultures, particularly at later passages. The establishment of a sustainable organoid system with demonstrated molecular fidelity offers a valuable tool for studying rumen epithelial biology while reducing dependence on native tissue.

Ca ²⁺ and Mg ²⁺ are essential nutrients, and deficiency can cause serious health problems. Thus, lack of Ca ²⁺ and Mg ²⁺ can lead to osteoporosis, with incidence rising both in absolute and age‐specific terms, while Mg ²⁺ deficiency is associated with type II diabetes. Prevention via vitamin D or estrogen is controversial, and the bioavailability of Ca ²⁺ and Mg ²⁺ from supplements is significantly lower than that from milk products. Problems are likely to increase as populations age and the number of people on vegan diets surges. Developing new therapeutic strategies requires a better understanding of the molecular mechanisms involved in absorption by intestinal epithelia. The vitamin‐D dependent, active pathway for the uptake of Ca ²⁺ from the upper small intestine involving TRPV6 is highly efficient but only accounts for about 20% of total uptake. Instead, most Ca ²⁺ uptake is thought to occur via passive paracellular diffusion across the ileum, although sufficiently high luminal concentrations are difficult to achieve.. Interestingly, colon and caecum also have a considerable capacity for the active absorption of Ca ²⁺ and Mg ²⁺ , the molecular mechanisms of which are unclear. Intriguingly, stimulating fermentation by prebiotics enhances colonic absorption, which can rise from ~10% to ~30% of the total. Notably, fermentation releases protons, which inhibits channels highly selective for Ca ²⁺ and Mg ²⁺ (TRPV6 and TRPM6/TRPM7). Conversely, the non‐selective cation channel TRPV3 is stimulated by both intracellular acidification and by numerous herbal compounds. Spicy, fiber‐rich food, as traditionally consumed in many cultures, might enhance the uptake of Ca ²⁺ and Mg ²⁺ via this pathway.

The ruminal epithelium absorbs large quantities of NH₄⁺ and Ca²⁺. A role for TRPV3 has emerged, but data on TRPV4 are lacking. Furthermore, short-chain fatty acids (SCFA) stimulate ruminal Ca²⁺ and NH₄⁺ uptake in vivo and in vitro, but the pathway is unclear. Sequencing of the bovine homologue (bTRPV4) revealed 96.79% homology to human TRPV4. Two commercial antibodies were tested using HEK-293 cells overexpressing bTRPV4, which in ruminal protein detected a weak band at the expected ~ 100 kDa and several bands ≤ 60 kDa. Immunofluorescence imaging revealed staining of the apical membrane of the stratum granulosum for bTRPV3 and bTRPV4, with cytosolic staining in other layers of the ruminal epithelium. A similar expression pattern was observed in a multilayered ruminal cell culture which developed resistances of > 700 Ω · cm² with expression of zonula occludens-1 and claudin-4. In Ussing chambers, 2-APB and the TRPV4 agonist GSK1016790A stimulated the short-circuit current across native bovine ruminal epithelia. In whole-cell patch-clamp recordings on HEK-293 cells, bTRPV4 was shown to be permeable to NH₄⁺, K⁺, and Na⁺ and highly sensitive to GSK1016790A, while effects of butyrate⁻ were insignificant. Conversely, bTRPV3 was strongly stimulated by 2-APB and by butyrate⁻ (pH 6.4 > pH 7.4), but not by GSK1016790A. Fluorescence calcium imaging experiments suggest that butyrate⁻ stimulates both bTRPV3 and bTRPV4. While expression of bTRPV4 appears to be weaker, both channels are candidates for the ruminal transport of NH₄⁺ and Ca²⁺. Stimulation by SCFA may involve cytosolic acidification (bTRPV3) and cell swelling (bTRPV4).

Mutations of TRPV3 lead to severe dermal hyperkeratosis in Olmsted syndrome, but whether the mutants are trafficked to the cell membrane or not is controversial. Even less is known about TRPV3 function in intestinal epithelia, although research on ruminants and pigs suggests an involvement in the uptake of NH₄⁺. It was the purpose of this study to measure the permeability of the human homologue (hTRPV3) to NH₄⁺, to localize hTRPV3 in human skin equivalents, and to investigate trafficking of the Olmsted mutant G573S. Immunoblotting and immunostaining verified the successful expression of hTRPV3 in HEK-293 cells and Xenopus oocytes with trafficking to the cell membrane. Human skin equivalents showed distinct staining of the apical membrane of the top layer of keratinocytes with cytosolic staining in the middle layers. Experiments with pH-sensitive microelectrodes on Xenopus oocytes demonstrated that acidification by NH₄⁺ was significantly greater when hTRPV3 was expressed. Single-channel measurements showed larger conductances in overexpressing Xenopus oocytes than in controls. In whole-cell experiments on HEK-293 cells, both enantiomers of menthol stimulated influx of NH₄⁺ in hTRPV3 expressing cells, but not in controls. Expression of the mutant G573S greatly reduced cell viability with partial rescue via ruthenium red. Immunofluorescence confirmed cytosolic expression, with membrane staining observed in a very small number of cells. We suggest that expression of TRPV3 by epithelia may have implications not just for Ca²⁺ signalling, but also for nitrogen metabolism. Models suggesting how influx of NH₄⁺ via TRPV3 might stimulate skin cornification or intestinal NH₄⁺ transport are discussed.

Aim: Absorption of ammonia from the gut has consequences that range from encephalitis in hepatic disease to global climate change induced by nitrogenous excretions from livestock. Since patch clamp data show that certain members of the transient receptor potential (TRP) family are permeable to NH₄⁺, participation in ammonium efflux was investigated.Methods: Digesta, mucosa and muscular samples from stomach, duodenum, jejunum, ileum, caecum and colon of pigs were analysed via colourimetry, qPCR, Western blot, immunohistochemistry and Ussing chambers.Results: qPCR data show high duodenal expression of TRPV6. TRPM6 was highest in jejunum and colon, with expression of TRPM7 ubiquitous. TRPM8 and TRPV1 were below detection. TRPV2 was highest in the jejunum but almost non‐detectable in the colon. TRPV4 was ubiquitously expressed by mucosal and muscular layers. TRPV3 mRNA was only found in the mucosa of the caecum and colon, organs in which NH₄⁺ was highest (>7 mmol·L⁻¹). Immunohistochemically, an apical expression of TRPV3 and TRPV4 could be detected in all tissues, with effects of 2‐APB and GSK106790A supporting functional expression. In symmetrical NaCl Ringer, removal of mucosal Ca²⁺ and Mg²⁺ increased colonic short circuit current (I_sc) and conductance (G_t) by 0.18 ± 0.06 µeq·cm⁻²·h⁻¹ and 4.70 ± 0.85 mS·cm⁻² (P < .05, N/n = 4/17). Application of mucosal NH₄Cl led to dose‐dependent and divalent‐sensitive increases in G_t and I_sc, with effects highest in the caecum and colon.Conclusion: We propose that TRP channels contribute to the intestinal transport of ammonium, with TRPV3 and TRPV4 promising candidate proteins. Pharmacological regulation may be possible.

A therapeutic potential of the TRPA1 channel agonist cinnamaldehyde for use in inflammatory bowel disease is emerging, but the mechanisms are unclear. Semi-quantitative qPCR of various parts of the porcine gastrointestinal tract showed that mRNA for TRPA1 was highest in the colonic mucosa. In Ussing chambers, 1 mmol·L⁻¹ cinnamaldehyde induced increases in short circuit current (ΔI_sc) and conductance (ΔG_t) across the colon that were higher than those across the jejunum or after 1 mmol·L⁻¹ thymol. Lidocaine, amiloride or bumetanide did not change the response. The application of 1 mmol·L⁻¹ quinidine or the bilateral replacement of 120 Na⁺, 120 Cl⁻ or 25 HCO₃⁻ reduced ΔG_t, while the removal of Ca²⁺ enhanced ΔG_t with ΔI_sc numerically higher. ΔI_sc decreased after 0.5 NPPB, 0.01 indometacin and the bilateral replacement of 120 Na⁺ or 25 HCO₃⁻. The removal of 120 Cl⁻ had no effect. Cinnamaldehyde also activates TRPV3, but comparative measurements involving patch clamp experiments on overexpressing cells demonstrated that much higher concentrations are required. We suggest that cinnamaldehyde stimulates the secretion of HCO₃⁻ via apical CFTR and basolateral Na⁺-HCO₃⁻ cotransport, preventing acidosis and damage to the epithelium and the colonic microbiome. Signaling may involve the opening of TRPA1, depolarization of the epithelium and a rise in PGE2 following a lower uptake of prostaglandins via OATP2A1.

Large quantities of ammonia (NH₃ or NH₄⁺) are absorbed from the gut, associated with encephalitis in hepatic disease, poor protein efficiency in livestock, and emissions of nitrogenous climate gasses. Identifying the transport mechanisms appears urgent. Recent functional and mRNA data suggest that absorption of ammonia from the forestomach of cattle may involve TRPV3 channels. The purpose of the present study was to sequence the bovine homologue of TRPV3 (bTRPV3), localize the protein in ruminal tissue, and confirm transport of NH₄⁺. After sequencing, bTRPV3 was overexpressed in HEK-293 cells and Xenopus oocytes. An antibody was selected via epitope screening and used to detect the protein in immunoblots of overexpressing cells and bovine rumen, revealing a signal of the predicted ~ 90 kDa. In rumen only, an additional ~ 60 kDa band appeared, which may represent a previously described bTRPV3 splice variant of equal length. Immunohistochemistry revealed staining from the ruminal stratum basale to stratum granulosum. Measurements with pH-sensitive microelectrodes showed that NH₄⁺ acidifies Xenopus oocytes, with overexpression of bTRPV3 enhancing permeability to NH₄⁺. Single-channel measurements revealed that Xenopus oocytes endogenously expressed small cation channels in addition to fourfold-larger channels only observed after expression of bTRPV3. Both endogenous and bTRPV3 channels conducted NH₄⁺, Na⁺, and K⁺. We conclude that bTRPV3 is expressed by the ruminal epithelium on the protein level. In conjunction with data from previous studies, a role in the transport of Na⁺, Ca²⁺, and NH₄⁺ emerges. Consequences for calcium homeostasis, ruminal pH, and nitrogen efficiency in cattle are discussed.