Funktionelle und molekulare Untersuchung von Mechanismen, welche zur Entfernung von Protonen aus dem Pansen beitragen

The rumen of cattle and sheep is a large fermentation chamber where symbiotic microbiota break down otherwise indigestible fibrous components of feed to short chain fatty acids (SCFA), which represent the most important energy source for ruminants. To obtain optimal conditions for ruminal fermentation, ruminal fluid needs to be buffered sufficiently to avoid acidic conditions, which involves buffering by bicarbonate (HCO3-) entering the rumen with saliva. Surprisingly, however, the buffering constants of the bicarbonate system have never been determined in ruminal fluid, compromising the ability to estimate the contribution of this system to ruminal buffering. Furthermore, ammonia can accumulate to toxic levels in the rumen due to fermentational deamination of protein and non-protein nitrogen. In part, this ammonia can be incorporated into high-grade microbial protein, but amounts exceeding microbial capacity must be absorbed from the rumen, hepatically detoxified and renally excreted, with detrimental consequences both for the protein efficiency of the ruminant and for the environment. Therefore, interest in identifying and characterizing the efflux pathway for ammonia from the rumen is considerable and enhanced by the fact that ruminal efflux of ammonia has long been known to involve absorption of the ionic form (NH4+) through a cation channel, thus contributing to elimination of protons and ruminal pH homeostasis. The present thesis aimed to answer two unsolved questions regarding to ruminal buffering: 1. What are the buffering constants for the bicarbonate buffering system in ruminal fluid? 2. What is the molecular identity of the transport protein that mediates the absorption of NH4+ from the rumen? The first part of the thesis focussed on the determination of the buffering constants of the bicarbonate system in ruminal fluid (Paper 1, Hille et al., 2016). In this open buffer system, ruminal protons are mainly buffered by HCO3- that enters the rumen with saliva or via secretion of the ruminal epithelium. To calculate the buffer capacity of ruminal fluid, the solubility of CO2 (α) and the equilibrium constant of CO2/HCO3- (pK) needed to be known, neither of which have previously been determined for ruminal fluid. Accordingly, ruminal fluid from cattle fed either hay or concentrate diets was investigated using both the classical Astrup technique and a newly developed titration technique. In both feeding scenarios, values were found to be similar to the values known for Ringer solution and human blood. The data show that SCFA- and HCO3- are the primary buffers in ruminal fluid and that at physiological levels of ruminal pH, HCO3- is almost completely converted to CO2, which leaves with eructation. This mechanism ensures both an efficient elimination of protons from the rumen, and the maintenance of the concentration gradient driving HCO3- secretion across the ruminal epithelium. Finally, the concept of base excess was introduced for ruminal fluid for the first time. The purpose of the second part of the present thesis was to identify and characterize possible transport proteins mediating the absorption of ammonium by the ruminal epithelium. Recent work shows that in vitro, ruminal transport of Na+ and NH4+ can be stimulated by certain agonists of the transient receptor potential family (TRP), and that the ruminal epithelium expresses mRNA for the bovine analogue of TRPV3 (bTRPV3) as a suitable candidate gene. The current thesis demonstrates that these modulators also stimulate the absorption of the essential nutrient Ca2+ in isolated ruminal epithelium in a dose dependent manner (Paper 2, Rosendahl et al., 2016). In a second step, the bTRPV3 channel was overexpressed in HEK-293 cells and characterized via the whole cell and single channel configuration of the patch clamp technique and via intracellular calcium imaging (Paper 3, Schrapers et al., 2018). It emerged that this channel reflects the properties of the conductance found in the native ruminal epithelium: the bTRPV3 conducts various cations, and the conductance of monovalent cations is modulated by the divalent cations and by TRPV3 agonists such as menthol and thymol. The conductance of different cations was measured using both the whole cell and single channel patch clamp technique, which revealed decreasing permeabilities in the order of NH4+ > Na+ > Mg2+ > Ca2+ > NMDG+. Furthermore, the application of various TRPV3 agonists increased Na+, NH4+ and K+ currents in the whole cell patch clamp configuration. Using intracellular calcium imaging, an increase in calcium influx after the addition of menthol could subsequently be found. The bovine TRPV3 should thus play an important role in mediating the ruminal transport of physiologically relevant cations such as Na+, K+, NH4+ and Ca2+. In summary, this thesis - determined the buffering constants for the bicarbonate buffer system in ruminal fluid for the first time, - demonstrated that TRP channel agonists can stimulate transport of Ca2+ across the native ruminal epithelium in vitro and - characterized the bovine representative of TRPV3 as an ion channel suitable for mediating the transport of Na+, K+ and NH4+ and Ca2+ across the rumen