How to manage N waste in the intestine?

Field: Medicine (Health Sciences)

In this issue of Acta Physiologica, Manneck et al describe the expression of different types of TRP (transient receptor potential) cation channels in the intestinal wall of pigs, with special emphasis on TRPV3 and TRPV4 (transient receptor potential vanilloid type 3 and 4).1 Both of them are preferentially expressed in the gastrointestinal mucosa based on qPCR data comparing their expression in the mucosal and the muscle layer of different intestinal segments. Especially the mRNA for TRPV3 showed a pronounced segment specificity with predominant expression in the caecum and in the colon. TRPV3 and TRPV4 were localized on the protein level in the apical membrane of the epithelial cells in the gastrointestinal mucosa as shown by confocal microscopy after immunohistochemical staining. The known poor selectivity of these cation channels including their permeability for NH4+ (shown already for the bovine orthologue (bTRPV3)2), led the authors to hypothesize that these channels might be involved in the intestinal absorption of NH4+. Especially the expression pattern of TRPV3 in the caecum and the colon, that is, the two main fermentation compartments of non-ruminant mammals, argued in this direction. Consequently, Manneck et al performed voltage-clamp experiments in Ussing chambers to investigate the putative role of TRPV channels in NH4+ transport.1 Indeed, luminal administration of NH4Cl induced a positive short-circuit current (Isc), compatible with the electrogenic transport of cations from the mucosal to the serosal side of the mucosa. In parallel to the TRPV3/4 expression levels, the NH4+-induced current increased from oral to anal along the longitudinal axis of the intestinal tract. A manoeuvre to activate these nonselective channels is well known from previous observations in forestomach, that is, the huge fermentation chamber of ruminants, and is based on the inhibition of cation currents across the apical membrane by divalent cations. In line with these data, luminal chelation of Ca2+ and Mg2+ with EDTA (ethylenediamino-tetraacetic acid) significantly enhanced the Isc induced by NH4+ in porcine large intestine further supporting the role of TRPV channels in intestinal NH4+ absorption. The gaseous NH3 (ammonia) and the cationic NH4+ (ammonium), which form together a chemical equilibrium with a pKa of NH4+ amounting to 8.89 at 37°C (9.25 at 25°C), are produced in large amounts during the fermentation of N-containing compounds by the intestinal microbiota. These microbiota exist in symbiosis with the hosting mammals, which in contrast to some bacteria are unable to cleave β-glycosidic bonds between glucose residues in polysaccharides such as cellulose. The symbiosis allows the mammal to use the microbially digested plant fibres in form of short-chain fatty acids produced under anaerobic conditions by the intestinal microbiota. A prerequisite for this symbiosis is that the microbes are supplemented with N sources for the synthesis of microbial protein. In general, it is thought that in the caecum and the colon, that is, the compartments where the density of microbes reaches the highest values, most of the proteins arriving in these intestinal segments originate from shaded epithelial cells as well as from poorly digestible proteins not digested and absorbed in form of free amino acids or oligopeptides in the small intestine. Ammonia is generated as a by-product from the reductive deamination and/or the oxidative decarboxylation of amino acids or from the degradation, for example, of N-containing bases in nucleic acids. A part of the ammonia—or more correctly ammonium, because at the slightly acidic pH in the lumen of the large intestine NH4+ is the predominating chemical form—is excreted via the faeces. Another fraction is absorbed by the epithelium and reaches the blood stream. Quantitatively, total concentrations of 14-20 mmol·L−1 NH3/NH4+ have been measured in human colonic effluent samples; absorption rates are assumed to be in the range of 250 mmol absorbed daily in human gastrointestinal tract.3 The higher pH in the blood (7.4 in the arterial blood vs around pH 6 in the lumen of the large intestine) shifts the chemical equilibrium between both forms, so that the contribution of NH4+, the overwhelming form in the colonic lumen, is reduced by around one order of magnitude. Ammonia reaching the liver via the portal vein must be metabolized into urea before it is released into the systemic circulation. This is necessary to protect neuronal tissues from the toxic effects of NH3. This detoxification is the function of the urea cycle, during which NH3 is bound to HCO3- finally resulting in ornithine and urea (Figure 1). Indeed, hepatic encephalopathy is a deleterious consequence of hepatic insufficiency when the detoxification potential of the liver does no more cope with the amounts of NH3/NH4+ being absorbed. The question arises, whether the absorption of NH4+ from the lumen of the large intestine by the caecal and the colonic epithelium is simply a ‘bystander’ effect representing the passage of an ‘undesired’ ion, which finally has to be detoxified by the liver, via ion channels/transporters involved in the transepithelial transport of other, physiological relevant ions such as K+ or Na+. On the other hand, a limited and possibly regulated absorption of NH4+ might fulfil a physiological function. Not only microbes, but also mammals can fix NH3—being in chemical equilibrium with NH4+—into α-ketoglutaric acid via the mitochondrial enzyme glutamate dehydrogenase. This reaction can be followed by incorporation of a further NH3 moiety into glutamic acid by the cytosolic enzyme glutamine synthetase to obtain glutamine. Thus, it might be speculated that a (modest) supply of the liver with NH3 by absorption of NH4+ across the large intestinal epithelium could have been helpful evolutionary to contribute to the support of the mammal with nitrogen for NH3 fixation. This would provide an additional source of amino acids in combination with the dominant source of amino acids absorbed from digested food proteins. To date, the absorption of ammonia/ammonium from the intestine has been considered to take place mainly in form of NH3 passing the lipid part of biomembranes by simple diffusion. However, recent results from studies in expression systems and theoretical considerations about the polar molecular structure of NH3, which exhibits a relevant dipole character because of its tetraedic molecular form, suggest a dominant mediation of the transmembrane passage of NH3 and NH4+ via transport proteins.2 Especially experiments, in which cytosolic pH changes were registered, gave the first hints which partner in the chemical equilibrium between NH3 and NH4+ is in fact taken up by a cell.3 The observation that exposure of many cells such as, for example, Xenopus oocytes overexpressing bTRPV3 to NH4Cl acidifies the cytosolic pH2 because of diffusion of NH4+ into the cell and subsequent dissociation into NH3 and H+ strongly argues for a prominent uptake of the protonated form. If NH3 would be the dominant form passing the plasma membrane, a cytosolic alkalization would have to be expected, as NH3 in the cells would bind H+ to form NH4+. To date, mostly K+ channels, K+-dependent cotransporters or primarily active K+ transporting proteins such as the H+-K+-pump have been assumed to be involved in the protein-dependent transmembrane passage of NH4+. The ‘hijacking’ of K+-transporting proteins by NH4+ is assumed to be because of the similar hydrated radius (1.45 Å) of both ions.4 The present study is in general line with a change of paradigm in the view on transport of NH4+/NH3 and other substrates thought to date to be absorbed mainly by lipid diffusion. Historically, solely the non-ionic diffusion of the gas NH3 was thought to responsible for its passage across biological membranes. This was followed by data supporting the (well- documented) evidence for the hypothesis that ammonium can use K+ transporting mechanisms because of the similarity in size between NH4+ and K+ as already mentioned above. Later on, it was discovered that renal rhesus-associated glycoproteins, belonging to the SLC42 solute transporter family, are able to transport both NH4+ and NH3, too.5 The most recent player in this field are TRP channels being involved in epithelial NH4+ transport across the forestomach demonstrated experimentally for bovine TRPV3.2 In this huge compartment of the gastrointestinal tract of ruminant animals, the absorption of NH4+ plays an important role to provide the resource for hepatic urea synthesis. In contrast to monogastric mammals, most of the urea is not excreted renally in ruminants. Instead, urea recirculates mainly via the salivary glands into the forestomach, where it serves as N supply for the ruminal microbiota. The most recent data from the same research group now published in this issue of Acta Physiologica provide compelling evidence that transient receptor potential vanilloid type channels in the apical membrane are expressed also in other parts of the gastrointestinal tract of monogastric species. Especially the gradient in expression with a peak expression of TRPV3 in caecum and colon, that is, the sites with high microbial NH3/NH4+ production, would fit well to a physiological role of these channels in NH4+ absorption. Functional evidence for the expression of apical cation conductances distinct from K+ channels or the prototypical epithelial Na+ channel (ENaC) have been measured in other segments of the large intestine, for example, in rat caecum.6 It is tempting to speculate that TRPV3 or TRPV4 channels (or other members of the TRP superfamily found in the qPCR screening experiments of Manneck et al in pig intestine1) underlie these conductances. Several points remain open in the study presented in this volume of Acta Physiologica by Manneck et al, which might be subject of future experiments. One of these questions refers to the pharmacological properties of the NH4+ conductance in the apical membrane. For example, in dependence on the type of TRP channel and the concentrations applied, 2-APB (2-aminoethoxy-diphenyl borate) acts either as blocker or as activator (of some) TRP channels.7 This chemical increased transcellular conductance in porcine colon, which would be consistent with the known ability of 2-APB to activate TRPV3, but had complex effects on Isc. Consequently, it would be helpful to test whether activators of TRPV3 or TRPV4 enhance NH4+-carried currents across large intestinal epithelia. Further open and intriguing questions refer to the putative regulation of these ammonium-permeable cation channels. Are they regulated, for example, by phosphorylation and which role plays the intracellular pH, which will acidify when significant amounts of NH4+ are passing into the cytosol and dissociate intracellularly into NH3 and protons? Quite interesting is also the inhibition of this nonselective cation channels by extracellular Ca2+ and Mg2+, which implies that any changes in the free concentration of these divalent cations, for example, after binding to macromolecules in the large intestinal lumen, might lead to drastic changes in ammonia absorption. Beside the distribution along the longitudinal axis of the gastrointestinal tract with in general increasing expression in the aboral direction for TRPV3 and TRPV4 as shown on the mRNA level, a further intriguing point is the obvious differential expression of both ion channels along the surface—crypt axis, as the immunopositive signals for both proteins were strongly concentrated near the surface region and not found within the depth of the crypts. This distribution is most probably the reason, why in previous experiments with isolated perfused crypts from rabbit colon an influx of NH4+ (and subsequent acidification of the cytosol) was only observed after basolateral administration of NH4Cl, but not when NH4Cl was administered to the solution perfusing the interior of the isolated crypts.3 Indirectly, these former experiments also support the hypothesis that a predominant part of the apical uptake of ammonia/ammonium is indeed in the form of NH4+ (mediated by TRPV3 and/or TRPV4 channels missing in the depth of the crypts) and not in form of the gaseous molecule NH3, as absorption of ammonia (via lipid diffusion or NH3-transporting proteins) should have led to an alkalization of the cytosol, which was, however, not observed.3 The different members of TRP superfamily of cation channels, containing seven known subfamilies of channels such as TRPC (canonical), TRPV (vanilloid), TRPA (ankyrin), TPRN (nomp), TRPM (melastin), TRPML (mucolipin) or TRPP (polycystin) fulfil a broad spectrum of functions including thermo-, chemo-, osmo- and mechanosensing, signal transduction or in the case of TRPV5 and TRPV6 absorption of Ca2+ in renal or intestinal epithelia.8 The experiments performed by Manneck et al now add a further component to this spectrum, that is, a putative role in the absorption of NH4+ in the gastrointestinal tract.1 Intestinal ammonia/ammonium is important for several fields. Well known in medicine is the problem that a misbalance between NH3 supply from the large intestine and the ability of the liver to convert this NH3 into urea leads to encephalopathy, for example, as a consequence of hepatic failure. Based on the observations presented here suggesting a role of TRPV3 and TRPV4 channels for the passage of NH4+ into the blood stream finally drained by the portal vein, it is tempting to speculate that blockade of these cation channels might be the basis for a new strategy for the treatment or prophylaxis of NH3-induced encephalopathy. From a physiological point of view, the recent results suggest also a change in the paradigm from overwhelming passive diffusion of NH3 (which is, however, only scarcely present in the equilibrium between NH4+ and NH3 at the slightly acidic pH within the lumen of the large intestine) to channel mediated transport of NH4+. Also, the provocative question why nonruminant animals should at all equip the apical membranes of their epithelial cells in the gastrointestinal mucosa with NH4+-permeable cation channels remains to be resolved. Thus, it would be interesting to resolve the question whether a part of the NH4+/NH3 absorbed may fulfil any physiological function (such as eg fixation of NH3 to support amino acid supply of the mammalian host) (Figure 1). A further aspect—well-discussed in the study of Manneck et al—consists in the fact that also the excreted NH3/NH4+, for example, that excreted by farm animals, has dramatic effects, not for the animal itself as it is the case for the absorbed ammonium, but for the environment. Here, this N source favours the eutrophication of water and contributes to the global climate change. Thus, further studies and in-depth knowledge of intestinal NH3/NH4+ handling in man and animals are urgently needed. There is no conflict of interest.