The epithelium of the hindgut expresses several members of the transient receptor potential family (TRP), the function of which is unclear at present. While most discussions currently revolve around a sensory function for these channels, as in inflammation or pain, there is reason to believe that the non-neuronal channels may be involved in the transport of various cations, with implications for understanding the physiological function of this poorly understood segment of the gut. Thus, large quantities of ammonia are released via fermentational processes in the hindgut, contributing to the ammonia load in the portal blood. Furthermore, there is evidence to suggest that the hindgut may play a certain role in the absorption of calcium. The mechanisms are currently poorly understood, but may involve further non-selective members of the TRP channel family. For further investigations, a viable cell culture model is required. It is therefore our goal to identify a suitable model by comparing the expression of TRP channels by cell lines such as Caco-2 with those of native human and porcine tissues. In a further step, the regulation of these channels by substrates known to enhance intestinal Ca²⁺ uptake (such as calcitriol and short chain fatty acids) will be investigated. The studies will involve qPCR, Western Blot analysis and immunohistochemistry. Possible short-term effects will be studied using calcium and pH-imaging.

Studies in the rumen have shown that absorption of large organic anions may involve large conductance anion channels, with a similar role likely in the colon. Furthermore, these channels may also play a role in the release of ATP and other purinergic signalling molecules, with implications for signalling by the macula densa of the kidney or in cancer pain. Candidate genes include SLCO2A1, a putative component of the maxi-anion channel. However, verification is still required. Preliminary data from our laboratory suggest that Caco-2 cells express anion channels that may also have a certain conductance to larger anions (Fig. 3). Furthermore, SLCO2A1 has been identified in Caco-2 cells and LoVo cells by other groups. It is our goal to characterise the anion conductance of these cells using the whole-cell and single channel configuration of the patch clamp technique to investigate the relative conductance to various anions. The single channel technique will be used to search for large-conductances typical for the maxi-anion channel as a first step for identifying the candidate gene on a molecular level. In a further step, knockdown of candidate genes will be performed in collaboration with Prof. Hanley (HMU).

In the past decade, a number of novel compounds such as Elexacaftor/Tezacaftor/Ivacaftor have revolutionized the treatment of cystic fibrosis in patients suffering from certain common mutants. Before these drugs can be tried in patients suffering from less common mutants, an ex-vivo testing in patient tissues or cell cultures is required. For this reason, we are planning to set up a Ussing chamber laboratory in collaboration with Prof. E. Pouokam Kamgne (Medical School Berlin). Patient material will be supplied by Prof. C. Schwarz (Cystic Fibrosis Center Westbrandenburg). We also intend to do research on cell culture models such as Caco-2, which can be grown on filters and investigated in Ussing chambers in parallel. The latter approach will allow overexpression or knock-down of candidate genes, to be performed in collaboration with Prof. Hanley and Prof. Pfirrmann (both HMU).

For milk production, cattle need to absorb large quantities of minerals. The ruminal epithelium of cattle has the capacity to absorb Ca2+ via a menthol-sensitive pathway, most likely involving transient receptor potential channels (TRP). A likely candidate gene is TRPV3, a non-selective cation channel with the ability to transport various cations, including NH⁴⁺ and Ca²⁺. Large quantities of mRNA encoding TRPV3 were detected in bovine rumen, while any expression of the classical intestinal epithelial calcium channel TRPV6 was low or missing. However, it is often difficult to discriminate between the epithelium and the underlying structures in native epithelia. For further studies concerning the properties of this pathway and others an optimal epithelial cell culture model is thus desirable, both in order to facilitate the identification of the molecular mechanisms involved and to reduce animal testing. In collaboration with others (https://www.vetmed.fu-berlin.de/einrichtungen/zfg/we01/forschung/In-vitro-Modelle/index.html) ruminal epithelial cells are either cultivated using a classically established procedure on filters or using basement membrane extracts (BME) for several passages to establish 3D organoids. Preparations from the native rumen and from cell cultures isolated via both procedures are being compared via transepithelial electrical resistance measurements, transmission electron microscopy, immunohistochemistry, and commercial RNA sequencing.