SUMMARY: The forestomachs of cows and sheep have historically served as important models for the study of epithelial transport. Thus, the ruminal epithelium was among the first tissues in which absorption of chloride against an electrochemical gradient was observed, requiring a tight paracellular barrier to prevent back-leakage. However, little is known about ruminal barrier function, despite the considerable implications for ruminant health. The tight junction proteins of the omasum have never been investigated, and no cell culture model exists. We present a new method for the isolation of cells from forestomach epithelia. Protein expression of cells and source tissues of sheep were studied using western blot, PCR and confocal laser scanning microscopy. Cultured cells were characterized by transepithelial resistance (TER) measurements and patch clamping. Cells developed TER values of 729±134 Ω cm2 (rumen) and 1522±126 Ω cm2 (omasum). Both primary cells and source epithelia of rumen and omasum expressed cytokeratin, occludin and claudins 1, 4 and 7 (but not claudins 2, 3, 5, 8 and 10), consistent with the observed paracellular sealing properties. Staining for claudin-1 reached the stratum basale. The full mRNA coding sequence of claudins 1, 4 and 7 (sheep) was obtained. Patch-clamp analyses of isolated cells proved expression of an anion conductance with a permeability sequence of gluconate<acetate<chloride. This is in accordance with a model that ruminal and omasal transport of anions such as chloride and acetate has to occur via a transcellular route and involves channel-mediated basolateral efflux, driven by Na+/K+-ATPase.

Absorption of ammonia from the gastrointestinal tract results in problems that range from hepatic encephalopathy in humans to poor nitrogen efficiency of cattle with consequences for the global climate. Previous studies on epithelia and cells from the native ruminal epithelium suggest functional involvement of the bovine homologue of TRPV3 (bTRPV3) in ruminal NH₄⁺ transport. Since the conductance of TRP channels to NH₄⁺ has never been studied, bTRPV3 was overexpressed in HEK-293 cells and investigated using the patch-clamp technique and intracellular calcium imaging. Control cells contained the empty construct. Divalent cations blocked the conductance for monovalent cations in both cell types, with effects higher in cells expressing bTRPV3. In bTRPV3 cells, but not in controls, menthol, thymol, carvacrol, or 2-APB stimulated whole cell currents mediated by Na⁺, Cs⁺, NH₄⁺_, and K⁺, with a rise in intracellular Ca²⁺ observed in response to menthol. While only 25% of control patches showed single-channel events (with a conductance of 40.8 ± 11.9 pS for NH₄⁺ and 25.0 ± 5.8 pS for Na⁺), 90% of bTRPV3 patches showed much larger conductances of 127.8 ± 4.2 pS for Na⁺, 240.1 ± 3.6 pS for NH₄⁺, 34.0 ± 1.7 pS for Ca²⁺, and ~ 36 pS for NMDG⁺. Open probability, but not conductance, rose with time after patch excision. In conjunction with previous research, we suggest that bTRPV3 channels may play a role in the transport of Na⁺, K⁺, Ca²⁺ and NH₄⁺ across the rumen with possible repercussions for understanding the function of TRPV3 in other epithelia.