TRPV3 and TRPV4 as candidate proteins for intestinal ammonium absorption

Abstract

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 (Isc) and conductance (Gt) 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 Gt and Isc, 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.

Beyond Ca2+ signalling: the role of TRPV3 in the transport of NH4+

“…In protein from a model of human keratinocytes (human skin equivalent, 12 replicates from 2 donors), the ~ 95 kDa band was weak, requiring high exposure times. The intense band at ~ 60 kDa may correspond to a previously described splice variant first reported in human keratinocytes [74] but also found in bovine rumen [39], porcine intestine, and porcine and murine skin [46] or to a degradation product.…”

“…In our previous study of the rumen [39] and in an unrelated study of cultured keratinocytes [74], the ~ 60 kDa band was also stronger than the ~ 95 kDa band, although the difference in staining intensity was not as pronounced. Furthermore, we observed that the ~ 60 kDa band was always more pronounced than the ~ 95 kDa band in various parts of the porcine intestine, the porcine skin and in murine skin [46]. While this band may reflect a breakdown product, sequences for a ~ 60 kDa splice variant of TRPV3 are available for the bovine species (AAI46079.1) and for mice (XP_006533411.1) and suggest that the protein is truncated after amino acid 527 (mouse) or 526 (bovine) (for alignment, see the supplement of [46]).…”

“…Possibly owing to the initial discovery in the visual system of Drosophila, the family was initially associated exclusively with sensory perception. However, many members of the TRP channel family are highly expressed by non-sensory organs such as epithelial cells of the skin or the intestine [46,52,56,74,75], raising the question if sensory signalling is the end of the story.…”

“…Apart from the skin, TRPV3 is also highly expressed by the apical membrane of enterocytes in intestinal epithelia such as the colon and the caecum, suggesting a role in the apical uptake of cations [46,75]. A role in inflammatory signalling has been postulated, but attempts to correlate clinical findings in ulcerative colitis with expression of TRPV3 have been inconclusive [63].…”

“…Instead, it has been suggested that transport of ammonia (NH 3 ) is facilitated by transport proteins such as the Rhesus-like glycoproteins or certain types of aquaporins [26,30,51]. Furthermore, functional studies have shown that both the colon and the caecum express an electrogenic pathway for the uptake of NH 4 + via divalent-sensitive, non-selective cation conductances with permeability to NH 4 + [46,70], correlating with the apical expression of TRPV3 by both tissues [46,75].…”