Ca 2+ and Mg 2+ are essential nutrients, and deficiency can cause serious health problems. Thus, lack of Ca 2+ and Mg 2+ can lead to osteoporosis, with incidence rising both in absolute and age-specific terms, while Mg 2+ deficiency is associated with type II diabetes. Prevention via vitamin D or estrogen is controversial, and the bioavailability of Ca 2+ and Mg 2+ from supplements is significantly lower than that from milk products. Problems are likely to increase as populations age and the number of people on vegan diets surges. Developing new therapeutic strategies requires a better understanding of the molecular mechanisms involved in absorption by intestinal epithelia. The vitamin-D dependent, active pathway for the uptake of Ca 2+ from the upper small intestine involving TRPV6 is highly efficient but only accounts for about 20% of total uptake. Instead, most Ca 2+ uptake is thought to occur via passive paracellular diffusion across the ileum, although sufficiently high luminal concentrations are difficult to achieve.. Interestingly, colon and caecum also have a considerable capacity for the active absorption of Ca 2+ and Mg 2+ , the molecular mechanisms of which are unclear. Intriguingly, stimulating fermentation by prebiotics enhances colonic absorption, which can rise from ~10% to ~30% of the total. Notably, fermentation releases protons, which inhibits channels highly selective for Ca 2+ and Mg 2+ (TRPV6 and TRPM6/TRPM7). Conversely, the non-selective cation channel TRPV3 is stimulated by both intracellular acidification and by numerous herbal compounds. Spicy, fiber-rich food, as traditionally consumed in many cultures, might enhance the uptake of Ca 2+ and Mg 2+ via this pathway.

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 NH4 Cl 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.

A therapeutic potential of the TRPA1 channel agonist cinnamaldehyde for use in inflammatory bowel disease is emerging, but the mechanisms are unclear. Semi-quantitative qPCR of various parts of the porcine gastrointestinal tract showed that mRNA for TRPA1 was highest in the colonic mucosa. In Ussing chambers, 1 mmol·L−1 cinnamaldehyde induced increases in short circuit current (ΔIsc) and conductance (ΔGt) across the colon that were higher than those across the jejunum or after 1 mmol·L−1 thymol. Lidocaine, amiloride or bumetanide did not change the response. The application of 1 mmol·L−1 quinidine or the bilateral replacement of 120 Na+, 120 Cl− or 25 HCO3− reduced ΔGt, while the removal of Ca2+ enhanced ΔGt with ΔIsc numerically higher. ΔIsc decreased after 0.5 NPPB, 0.01 indometacin and the bilateral replacement of 120 Na+ or 25 HCO3−. The removal of 120 Cl− had no effect. Cinnamaldehyde also activates TRPV3, but comparative measurements involving patch clamp experiments on overexpressing cells demonstrated that much higher concentrations are required. We suggest that cinnamaldehyde stimulates the secretion of HCO3− via apical CFTR and basolateral Na+-HCO3− cotransport, preventing acidosis and damage to the epithelium and the colonic microbiome. Signaling may involve the opening of TRPA1, depolarization of the epithelium and a rise in PGE2 following a lower uptake of prostaglandins via OATP2A1.

Mucosal surfaces pose a challenging environment for efficient drug delivery. Various delivery strategies such as nanoparticles have been employed so far; yet, still yielding limited success. To address the need of efficient transmucosal drug delivery, this report presents the synthesis of novel disulfide-containing dendritic polyglycerol (dPG)-based nanogels and their preclinical testing. A bifunctional disulfide-containing linker is coupled to dPG to act as a macromolecular crosslinker for poly-N-isopropylacrylamide (PNIPAM) and poly-N-isopropylmethacrylamide (PNIPMAM) in a precipitation polymerization process. A systematic analysis of the polymerization reveals the importance of a careful polymer choice to yield mucus-degradable nanogels with diameters between 100 and 200 nm, low polydispersity, and intact disulfide linkers. Absorption studies in porcine intestinal tissue and human bronchial epithelial models demonstrate that disulfide-containing nanogels are highly efficient in overcoming mucosal barriers. The nanogels efficiently degrade and deliver the anti-inflammatory biomacromolecule etanercept into epithelial tissues yielding local anti-inflammatory effects. Over the course of this work, several problems are encountered due to a limited availability of valid test systems for mucosal drug-delivery systems. Hence, this study also emphasizes how critical a combined and multifaceted approach is for the preclinical testing of mucosal drug-delivery systems, discusses potential pitfalls, and provides suggestions for solutions.

Mutations of TRPV3 lead to severe dermal hyperkeratosis in Olmsted syndrome, but whether the mutants are trafficked to the cell membrane or not is controversial. Even less is known about TRPV3 function in intestinal epithelia, although research on ruminants and pigs suggests an involvement in the uptake of NH 4 + . It was the purpose of this study to measure the permeability of the human homologue (hTRPV3) to NH 4 + , to localize hTRPV3 in human skin equivalents, and to investigate trafficking of the Olmsted mutant G573S. Immunoblotting and immunostaining verified the successful expression of hTRPV3 in HEK-293 cells and Xenopus oocytes with trafficking to the cell membrane. Human skin equivalents showed distinct staining of the apical membrane of the top layer of keratinocytes with cytosolic staining in the middle layers. Experiments with pH-sensitive microelectrodes on Xenopus oocytes demonstrated that acidification by NH 4 + was significantly greater when hTRPV3 was expressed. Single-channel measurements showed larger conductances in overexpressing Xenopus oocytes than in controls. In whole-cell experiments on HEK-293 cells, both enantiomers of menthol stimulated influx of NH 4 + in hTRPV3 expressing cells, but not in controls. Expression of the mutant G573S greatly reduced cell viability with partial rescue via ruthenium red. Immunofluorescence confirmed cytosolic expression, with membrane staining observed in a very small number of cells. We suggest that expression of TRPV3 by epithelia may have implications not just for Ca 2+ signalling, but also for nitrogen metabolism. Models suggesting how influx of NH 4 + via TRPV3 might stimulate skin cornification or intestinal NH 4 + transport are discussed.

A 15-year-old donkey gelding was referred to the Equine Clinic of the Freie Universitat Berlin because of acute onset of anorexia, bradycardia and multiple syncopes. The clinical examination revealed an irregular heartbeat (11-18 bpm) and syncopes. A continuous ECG revealed absence of P-waves and an irregular ventricular escape rhythm. An echocardiography revealed a pericardial effusion with reduction of left and right ventricular function. Laboratory abnormalities revealed increased troponin I, alpha hydroxybutyrate dehydrogenase and symmetric as well as asymmetric dimethylarginine. Increased concentrations of the cardiac glycoside digitoxin was evident in serum. Clinical findings were consistent with myocarditis with pericardial effusion and atrial standstill. Because of increasing severity of symptoms and grave prognosis the donkey was humanely euthanized. Histopathological examination of the cardiac atria including the sinoatrial node revealed severe, subacute, diffuse, suppurative myocarditis.

Magnesium (Mg) is an essential mineral without a specific hormonal regulation system. Mg-homeostasis and blood Mg-concentration depends on a continuous and sufficient absorption from the rumen. Ruminal absorption is essential for Mg-homeostasis. The apparent Mg-digestibility exhibits large variations and is influenced by the K content of the diet, the Na-status and the fermentation products short chain fatty acids (SCFA), CO2, NH4+ and pH. A meta-analysis of Mg-digestibility in dairy cows has shown that only the K-content reduces Mg-digestibility under practical feeding conditions of dairy cows. The obtained results allow an approach for an assessment of Mg-intake in dairy cows according to the milk yield and K content of the diet. The effect of Na-deficiency on Mg-absorption and the possible consequences of fermentation products are meaningful under special conditions. Mg-concentration in the blood is a "set point" for renal handling and Mg is excreted by the kidneys according to the actual requirement. Plasma concentrations < 0.75 mmol 'l(-1)- Mg cause a strong reduction of urinary Mg excretion and must be seen as critical. Concentrations between 0.75 und 0.85 mmol 'l(-1) - are borderline while a Mg-concentration > 0.85 mmol 'l(-1) - is accompanied by an almost linear increase of renal excretion as a sign of a sufficient supply of Mg with the diet.

Large quantities of ammonia (NH3 or NH4+) are absorbed from the gut, associated with encephalitis in hepatic disease, poor protein efficiency in livestock, and emissions of nitrogenous climate gasses. Identifying the transport mechanisms appears urgent. Recent functional and mRNA data suggest that absorption of ammonia from the forestomach of cattle may involve TRPV3 channels. The purpose of the present study was to sequence the bovine homologue of TRPV3 (bTRPV3), localize the protein in ruminal tissue, and confirm transport of NH4+. After sequencing, bTRPV3 was overexpressed in HEK-293 cells and Xenopus oocytes. An antibody was selected via epitope screening and used to detect the protein in immunoblots of overexpressing cells and bovine rumen, revealing a signal of the predicted ~ 90 kDa. In rumen only, an additional ~ 60 kDa band appeared, which may represent a previously described bTRPV3 splice variant of equal length. Immunohistochemistry revealed staining from the ruminal stratum basale to stratum granulosum. Measurements with pH-sensitive microelectrodes showed that NH4+ acidifies Xenopus oocytes, with overexpression of bTRPV3 enhancing permeability to NH4+. Single-channel measurements revealed that Xenopus oocytes endogenously expressed small cation channels in addition to fourfold-larger channels only observed after expression of bTRPV3. Both endogenous and bTRPV3 channels conducted NH4+, Na+, and K+. We conclude that bTRPV3 is expressed by the ruminal epithelium on the protein level. In conjunction with data from previous studies, a role in the transport of Na+, Ca2+, and NH4+ emerges. Consequences for calcium homeostasis, ruminal pH, and nitrogen efficiency in cattle are discussed.

Introduction/objectives: Asymmetric dimethylarginine (ADMA) is a cardiac biomarker in humans, symmetric dimethylarginine (SDMA) a renal biomarker in humans, cats, and dogs. The purpose of this prospective study was to investigate if measuring serum ADMA and SDMA concentrations via ELISA allows detection of cardiac disease in horses in a routine laboratory setting. In this context, reference values in horses were established. Animals, materials, and methods: Seventy-eight horses with no known medical history were compared to 23 horses with confirmed structural cardiac disease with/or without arrhythmias. Horses underwent physical examination, electrocardiography, echocardiography and venous blood sampling and were staged based on the severity of cardiac disease from 0 to II. Asymmetric dimethylarginine and SDMA were measured via ELISA and crosschecked using liquid chromatograph triple quadrupole mass spectrometry. Reference intervals with 90th percent confidence intervals were evaluated and standard software was used to test for significant differences in ADMA, SDMA, and the L-arginine/ADMA ratio between groups. Results: The reference ranges were 1.7-3.8 mmol/L and 0.3-0.8 mmol/L for ADMAand SDMA, respectively. Serum ADMA was higher in horses with heart disease compared to healthy horses (p < 0.01) and highest in horses with stage II heart disease (p = 0.02). The L-Arginine/ADMA ratio was significantly higher in healthy animals than those with cardiac disease (p = 0.001). Conclusions: Reference values for serum ADMA and SDMA using ELISA methods are presented in horses. This study confirms the association between heart disease and increased serum ADMA concentration as well as a decreased L-Arginine/ADMA ratio in horses. (c) 2020 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

To date, no specific hormonal regulation system has been identified for homoeostatic control of the essential mineral Mg. In cattle, the maintenance of physiological plasma Mg concentration depends on gastrointestinal absorption, primarily from the rumen, which serves as a pool for covering the requirement. Whereas a possible surplus (absorption greater than requirement) is rapidly excreted by the kidneys, a shortage (absorption lower than requirement) cannot be compensated for by mobilization from the large Mg pool in bones or soft tissue, so that the maintenance of the necessary physiological Mg concentration in plasma relies on continuous and sufficient absorption. Our knowledge concerning the site and mechanisms of Mg absorption has improved during the last few decades, and meta-analyses of the absorption of Mg in dairy cows have shown that the K content has a pronounced negative effect on Mg digestibility. The current recommendations of Mg intake propose a constant percentage of Mg and emphasize the depressive effect of high potassium (K) intake on Mg absorption. The current knowledge about the antagonism between K intake and Mg absorption allows a more flexible solution which includes the K content of the diet. An assessment of Mg intake is proposed that incorporates the improved knowledge of Mg absorption, metabolism and requirement. Within this framework, an equation is derived that allows a prediction of the amount of Mg required to compensate for dietary K content, the goal being to avoid both possible undernutrition or an unnecessary surplus of dietary Mg.

As nanoparticles (NPs) are readily absorbed across gastrointestinal tract, it is hypothesized that Zinc oxide nanoparticles (ZnO-NPs) will not interfere with intestinal glucose transport. The current study aims to investigate the in vitro effects of ZnO-NPs on Na linked glucose transport across jejunal mucosa in laying hens. A total of 12 White Leghorn laying hens (1.73 ± 0.176 kg), 40 wk of age, housed in individual cages were killed and mid-jejunum was divided into 4 segments to be mounted on the Ussing chambers. The mucosal side of jejunum was bathed with either no ZnO, ZnO (70 μM), ZnO-NPs (70 μM), or ZnO-NPs (35 μM) followed by addition of D-glucose (10 mM) mucosally in all the 4 experimental groups. Peak electrical response was measured 2 min after the addition of D-glucose. Results demonstrate that there was a decrease (P < 0.05) in short-circuit current (Isc) of tissues treated with ZnO compared to either control or ZnO-NPs suggesting that ZnO and not ZnO-NPs appears to interfere with electrogenic current across jejunal tissues of the laying hens. The increases in Isc following the addition of D-glucose was higher (P < 0.05) in the tissues pretreated with ZnO-NPs compared with the tissues mucosally bathed with only ZnO while the tissue conductance (Gt) remained unchanged. In conclusion, ZnO-NPs did not interfere with glucose transport and can be used as a feed supplement.

Magnesium (Mg2+) is an essential mineral without known specific regulatory mechanisms. In ruminants, plasma Mg2+ concentration depends primarily on the balance between Mg2+ absorption and Mg2+ excretion. The primary site of Mg2+ absorption is the rumen, where Mg2+ is apically absorbed by both potential-dependent and potential-independent uptake mechanisms, reflecting involvement of ion channels and electroneutral transporters, respectively. Transport is energised in a secondary active manner by a basolateral Na+/Mg2+ exchanger. Ruminal transport of Mg2+ is significantly influenced by a variety of factors such as high K+ concentration, sudden increases of ammonia, pH, and the concentration of SCFA. Impaired Mg2+ absorption in the rumen is not compensated for by increased transport in the small or large intestine. While renal excretion can be adjusted to compensate precisely for any surplus in Mg2+ uptake, a shortage in dietary Mg2+ cannot be compensated for either via skeletal mobilisation of Mg2+ or via up-regulation of ruminal absorption. In such situations, hypomagnesaemia will lead to decrease of a Mg2+ in the cerebrospinal fluid and clinical manifestations of tetany. Improved knowledge concerning the factors governing Mg2+ homeostasis will allow reliable recommendations for an adequate Mg2+ intake and for the avoidance of possible disturbances. Future research should clarify the molecular identity of the suggested Mg2+ transport proteins and the regulatory mechanisms controlling renal Mg excretion as parameters influencing Mg2+ homeostasis.

Absorption of ammonia from the rumen of cattle decreases nitrogen availability for fermentational protein synthesis, leading to increased competition of cattle with humans for protein and enhancing the release of toxic nitrogenous compounds into the environment. Given that differences in feeding and breeding might induce differences in ruminal ammonia transport, we compared electrophysiological, histological, and molecular biological characteristics of ruminal epithelia of Bos indicus crossbreds (Sahiwal-Mix, SWM) with those of Bos taurus (Holstein-Friesian, HF). As in HF, the stratified cornified epithelium of SWM expressed claudin 1 and 4. Measurements of ammonia flux (HF) and serosal pH (both breeds) suggested that at a mucosal pH of 6.4, net transport primarily occurred as NH4 + . As shown previously for HF, NH4 + induced a concentration-dependent rise in short circuit current (Isc ) in SWM that could be further stimulated by the TRP channel agonist menthol. Relative mRNA expression levels for TRPV3, TRPV4, TRPM6, and TRPM7 were significantly lower in SWM than in HF, with TRPA1 expression near the limit of detection. We conclude that uptake of ammonia from the rumen of both breeds occurs electrogenically as NH4 + with functional and molecular biological evidence pointing towards involvement of TRPV3 and TRPV4.

Results of recent in vitro experiments suggest that essential oils (EO) may not only influence ruminal fermentation but also modulate the absorption of cations like Na+, Ca2+ and NH4 + across ruminal epithelia of cattle and sheep through direct interaction with epithelial transport proteins, such as those of the transient receptor potential family. The aim of the current study was to examine this hypothesis by testing the effect of a blend of essential oils (BEO) on cation status and feed efficiency in lactating dairy cows. In the experiment, 72 dairy cows in mid-to-end lactation were divided into two groups of 36 animals each and fed the same mixed ration with or without addition of BEO in a 2×2 cross-over design. Feed intake, milk yield and composition, plasma and urine samples were monitored. Feeding BEO elevated milk yield, milk fat and protein yield as well as feed efficiency, whereas urea levels in plasma and milk decreased. In addition, plasma calcium levels increased significantly upon BEO supplementation, supporting the hypothesis that enhanced cation absorption might contribute to the beneficial effects of these EO.