Publikationsdatenbank

Functional and molecular biological studies of the absorption of ammonia across porcine intestinal epithelia (2022)

Art

Hochschulschrift

Autor

Manneck, David (WE 2)

Quelle

Berlin: Mensch und Buch Verlag, 2022 — IV, 131 Seiten

Sprache

Englisch

Verweise

URL (Volltext): https://refubium.fu-berlin.de/handle/fub188/38025

Kontakt

Institut für Veterinär-Physiologie

Oertzenweg 19 b
14163 Berlin
+49 30 838 62600
physiologie@vetmed.fu-berlin.de

Abstract / Zusammenfassung

The high ammonia emissions associated with pig fattening have serious consequences for the health of the animals, the people who care for them, and the global climate. For example, about 50 % of fattening pigs undergo severe pneumonia with identifiable scarring of the lung tissue in the course of their lives (Hillen et al. 2014; Maes et al. 2001; Grest et al. 1997). The source of this ammonia is degradation of protein within the gut, which is absorbed, detoxified by the liver and renally excreted. Most steps of this process have been investigated in detail. However, the mechanism by which ammonia is absorbed from the intestine continues to be unclear. Previous studies suggest that certain members of the TRP channel family contribute to uptake of ammonia in the form of NH4+ from the rumen. The aims of the present thesis were to investigate evidence for a channel-mediated uptake of NH4+ from the various parts of the porcine intestine, to study the expression of certain relevant members of the TRP family by the gut and to explore the effects of selected phytogenic agonists with a potential effect on these channels. A better understanding of the absorption mechanisms from the gastrointestinal tract of the pig is a prerequisite for the development of rational strategies in breeding and feeding with the aim of reducing ammonia excretion in fattening farms. Within the context of the two studies presented in this thesis, the expression levels of nine different TRP channels were studied and compared at mRNA level in the stomach (cardia and fundus), duodenum, jejunum, ileum, caecum and colon, always distinguishing between tissues of epithelial or muscular origin. While TRPV2, TRPV3, TRPV4, TRPV6, TRPM6, TRPM7, and TRPA1 could be detected in various segments of the porcine gastrointestinal tract, no evidence could be found for the expression of TRPV1 and TRPM8. Two channels with potential relevance for NH4+ transport, TRPV3 and TRPV4, were investigated further at protein level by immunoblot and immunohistochemistry, showing a predominant staining of the apical membrane of the epithelium in all segments. While TRPV4 was expressed throughout the gastrointestinal tract, mRNA data suggest that expression of the full-length TRPV3 protein is limited to those segments with the highest absorption of ammonia, namely caecum and colon. Via the Ussing chamber technique, functional evidence of TRPV3 and TRPV4 was obtained using the selective agonists 2-APB and GSK106790A. In line with a functional expression of these channels, electrogenic transport of NH4+ could be observed and was found to be further enhanced by the removal of divalent cations. In conjunction, the results suggest that the transport of NH4+ across the caecum and colon of the pigs may involve TRPV3, with possible further contributions of TRPV4. In the second study, the electrophysiological effects of the TRPA1 agonist cinnamaldehyde and the TRPV3 and TRPA1 agonist thymol were investigated in the colon of pigs. For this purpose, the Ussing chamber technique and the use of different blockers and solutions were used to investigate the mechanism of action in more detail. It was shown that the effect of cinnamaldehyde in the colon is most likely due to HCO3- mediated secretion by a prostaglandin-mediated signalling cascade following activation of TRPA1. In contrast, the electrophysiological effects after thymol addition were diverse, suggesting different mechanisms of action, which need to be investigated in more detail in further studies. The results presented provide an overview of the expression pattern of nine different TRP channels in seven different parts of the porcine gastrointestinal tract. Furthermore, evidence is presented for the electrogenic transport of ammonia in the form of NH4+. Functional data suggest a direct involvement of TRPV3 and TRPV4 in colonic transport, while TRPA1 modulates transport via a prostaglandin-dependent signalling cascade. By targeted modulation of these channels, e.g. by phytogenic agents, it seems possible to influence the transport and metabolism of cations, including the physiologically relevant NH4+ ion. Compounds with a selective inhibitory effects on the absorption of NH4+ from the hindgut are thus conceivable, but have yet to be developed. However, the use of currently available phytogenic agents, such as cinnamaldehyde, may result in other positive effects, such as the stimulation of bicarbonate secretion with buffering of protons in the lumen.