Ussing Chamber Experiments Research Articles

STIM1-Regulated Ca2+ Influx across the Apical and the Basolateral Membrane in Colonic Epithelium

In nonexcitable cells, store-operated Ca(2+) entry is the most important pathway for influx of extracellular Ca(2+) serving as a second messenger in the cytoplasm. The present study investigated the expression, localization and polar distribution of two key components of store-operated Ca(2+) entry identified, e.g., in lymphocytes or epithelial cell lines-STIM1 (stromal interacting molecule 1), working as a Ca(2+) sensor in the endoplasmic reticulum, and Orai1, working as the (or part of the) store-operated Ca(2+) channel in the plasma membrane-in a native intestinal epithelium, i.e., rat colon. Immunohistochemical investigations revealed expression of STIM1 and Orai1 in the rat colonic epithelium. Ca(2+) store depletion led to a translocation of STIM1 both to the basolateral as well as to the apical cell pole as observed by confocal microscopy. A Ca(2+) depletion/repletion protocol was used in Ussing chamber experiments to investigate the contribution of basolateral and apical store-operated Ca(2+) entry to the induction of anion secretion. These experiments revealed that Ca(2+)-dependent anion secretion was induced not only by basolateral Ca(2+) repletion but also, to a lesser extent, by apical Ca(2+) repletion. Both responses were suppressed by La(3+). The effect of basolateral Ca(2+) repletion was significantly inhibited by brefeldin A, a blocker of vesicular transport from the endoplasmic reticulum to the Golgi apparatus. In a final series of experiments, fura-2-loaded HT29/B6 cells were used. A carbachol-induced increase in the cytosolic Ca(2+) concentration was significantly reduced when cells were pretreated with siRNA against STIM1. In conclusion, these results demonstrate that STIM1 as a key component of intracellular Ca(2+) signaling is expressed by rat colonic epithelium and is involved in the regulation not only of basolateral but also of apical Ca(2+) influx.

Feeding barley grain-rich diets altered electrophysiological properties and permeability of the ruminal wall in a goat model

High-producing ruminants are commonly fed large amounts of concentrate to meet their high energy demands for rapid growth or high milk production. However, this feeding strategy can severely impair rumen functioning, leading to subacute ruminal acidosis. Subacute ruminal acidosis might have consequences for electrophysiological properties by changing the net ion transfer and permeability of ruminal epithelia, which may increase the uptake of toxic compounds generated in the rumen into the systemic circulation. The objective of the present study was to investigate the effects of excessive barley feeding on the electrophysiological and barrier functions of the ruminal epithelium and serum inflammation and ketogenesis markers after a long-term feeding challenge, using growing goats as a ruminant model. A feeding trial was carried out with growing goats allocated to 1 of the 3 groups (n=5–6 animals/group), with diets consisting exclusively of hay (control diet) or hay with 30 or 60% barley grain. Samples of the ventral ruminal epithelium were taken after euthanasia and instantly subjected to Ussing chamber experiments, where electrophysiological properties of the epithelium were measured in parallel with the permeability of marker molecules of different sizes [fluorescein 5(6)-isothiocyanate and horseradish peroxidase] from luminal to apical side. Additionally, ruminal fluid and blood samples were taken at the beginning of the experiment as well as shortly before euthanasia. Ruminal fluid samples were analyzed for volatile fatty acids and pH, whereas blood samples were analyzed for lipopolysaccharide, serum amyloid A, and β-hydroxybutyrate. Electrophysiological data indicated that barley feeding increased the epithelial short-circuit current compared with the control. Tissue conductance also increased with dietary barley inclusion. As shown with both marker molecules, permeability of ruminal epithelia increased with barley inclusion in the diet. Despite a lowered ruminal pH associated with increased volatile fatty acids (such as propionate and butyrate) concentrations as well as altered epithelial properties in response to high-grain feeding, no signs of inflammation became apparent, as blood serum amyloid A concentrations remained unaffected by diet. However, greater amounts of grain in the diet were associated with a quadratic increase in lipopolysaccharide concentration in the serum. Also, increasing the amounts of barley grain in the diet resulted in a tendency to quadratically augment serum concentrations of β-hydroxybutyrate and, hence, the alimentary ketogenesis. Further studies are needed to clarify the role of barley inclusion in the development of subacute ruminal acidosis in relation to ruminal epithelial damage and the translocation of toxic compounds in vivo.

Endothelin‐1 Acutely Reduces the Permeability of Visceral Sheep Peritoneum In Vitro Through Both Endothelin‐A and Endothelin‐B Receptors

Mesothelium is an important part of the peritoneal barrier for water and ion transport, essential for effective peritoneal dialysis (PD). Peritoneal fibrosis has been associated with PD treatment failure. Endothelin-1 (ET-1) is a potent vasoactive peptide, involved in pathologic fibrotic processes. Its action is mediated mainly by endothelin type A (ETA ) and type B (ETB ) receptors. The aim of this study was to investigate, by Ussing chamber experiments, the effect of ET-1 on the transmesothelial electrical resistance (RTM ) of the isolated visceral sheep peritoneum. Intact sheets of visceral peritoneum were obtained from 40 adult sheep and mounted in Ussing-type chambers. ET-1 (10(-7) M), BQ-123 (ETA receptor antagonist; 10(-6) M), BQ-788 (ETB receptor antagonist; 10(-6) M), and their combinations were added on the apical and the basolateral side of the peritoneum. RTM was measured before and serially after addition of the substances, and changes were registered as percentage (ΔRTM %). RTM increased within 1 min after addition of ET-1 apically (ΔRTM 65.03 ± 15.87%; P < 0.05) or basolaterally (ΔRTM 85.5 ± 20.86%; P < 0.05). BQ-123 and BQ-788 and their combination significantly reduced (P < 0.05) the effect of ET-1 to a similar degree in all cases. These results clearly indicate that ET-1 reduces ionic permeability of the visceral sheep peritoneum in vitro. Additionally, it is obvious that this inhibitory effect is mediated through both ETA and ETB receptors.

Intestinal Na+ Loss and Volume Depletion in JAK3-Deficient Mice

Background/Aims: The Janus kinase 3 JAK3 participates in the signaling of immune cells. Lack of JAK3 triggers inflammatory bowel disease, which in turn has been shown to affect intestinal activity of the epithelial Na⁺ channel ENaC and thus colonic sodium absorption. At least in theory, inflammatory bowel disease in JAK3-deficient mice could lead to intestinal salt loss compromizing extracellular volume maintenance and blood pressure regulation. The present study thus explored whether JAK3 deficiency impacts on colonic ENaC activity, fecal Na⁺ exretion, blood pressure and extracellular fluid volume regulation. Methods: Experiments were performed in gene-targeted mice lacking functional JAK3 (jak3^-/-) and in wild type mice (jak3^+/+). Colonic ENaC activity was estimated from amiloride-sensitive current in Ussing chamber experiments, fecal, serum and urinary Na⁺ concentration by flame photometry, blood pressure by the tail cuff method and serum aldosterone levels by immunoassay. Results: The amiloride (50 µM)-induced deflection of the transepithelial potential difference was significantly lower and fecal Na⁺ excretion significantly higher in jak3^-/- mice than in jak3^+/+ mice. Moreover, systolic arterial blood pressure was significantly lower and serum aldosterone concentration significantly higher in jak3^-/- mice than in jak3^+/+ mice. Both, absolute and fractional renal Na⁺ excretion were significantly lower in jak3^-/- mice than in jak3^+/+ mice. Conclusions: JAK3 deficiency leads to impairment of colonic ENaC activity with intestinal Na⁺ loss, decrease of blood pressure, increased aldosterone release and subsequent stimulation of renal tubular Na⁺ reabsorption.

Upregulation of Peptide Transporters PEPT1 and PEPT2 by Janus Kinase JAK2

Background/Aims: Janus-activated kinase-2 JAK2 participates in the signaling of several hormones including growth hormone, fosters tumor growth and modifies the activity of several Na⁺ coupled nutrient transporters. Peptide uptake into intestinal and tumor cells is accomplished by electrogenic peptide transporters PEPT1 and PEPT2. The present study thus explored whether JAK2 contributes to the regulation of PEPT1 and PEPT2 activity. Methods: cRNA encoding either PEPT1 or PEPT2 was injected into Xenopus oocytes with or without additional injection of cRNA encoding wild type JAK2, constitutively active ^V617FJAK2 or inactive ^K882EJAK2. The current created by the dipeptide glycine-glycine (I_gly-gly) was determined by dual electrode voltage clamp and taken as measure for electrogenic peptide transport. Results: No appreciable I_gly-gly was observed in water injected oocytes. In PEPT1 or PEPT2 expressing oocytes I_gly-gly was significantly increased by additional coexpression of JAK2. As shown in PEPT1 expressing oocytes, I_gly-gly without significantly modifying the concentration required for halfmaximal I_gly-gly (K_M). Following disruption of carrier insertion with brefeldin A (5 µM) I_gly-gly declined similarly fast in Xenopus oocytes expressing PEPT1 with JAK2 and in Xenopus oocytes expressing PEPT1 alone. In oocytes expressing both, PEPT1 and ^V617FJAK2, I_gly-gly was gradually decreased by JAK2 inhibitor AG490 (40 µM). According to Ussing chamber experiments pharmacological JAK2 inhibition similarly decreased I_gly-gly in mouse intestine. Conclusion: Regulation of the peptide transporters PEPT and PEPT2 does involve the Janus-activated kinase-2 JAK2.

The renin–angiotensin system in the esophageal mucosa of healthy subjects and patients with reflux disease

Objective. Components of the renin–angiotensin system (RAS) were recently discovered in the esophagus, which could be of interest in relation to gastroesophageal reflux disease (GERD). The present study was undertaken to confirm and further investigate the expression of RAS in healthy and refluxed exposed human esophageal mucosae. Methods.Esophageal biopsies were obtained from healthy subjects (n = 34) and individuals with erosive reflux disease (ERD, n = 28). Evaluation of general morphology and histological signs of reflux as well as investigation of gene transcript, protein expression and localization of various RAS components using RT-PCR, ELISA, western blot and immunohistochemistry were performed. Physiological effects of the AT2R were investigated in Ussing chamber experiments. Results. The study confirmed histological signs of reflux in ERD and expression of ACE, AT1R, AT2R and CatD in all examined specimens. In addition, the main effector peptide AngII, the pro-hormone AGT, the Mas receptor and the angiotensin-forming enzymes renin, CMA, CatG and NEP were present. Individuals with reflux disease had higher transcription activity of ACE and AT1R, increased protein levels of AT2R and lower levels of MasR. AT2R stimulation increased the ion currents in healthy epithelium, whereas epithelium from individuals with reflux disease exhibited no significant response. Conclusions. The study demonstrated that a local RAS is present in the human esophageal epithelium. Some RAS components were significantly altered in individuals diagnosed with ERD suggesting involvement in the pathophysiology of GERD.

Nano- and microscaled particles for drug targeting to inflamed intestinal mucosa—A first in vivo study in human patients

Most of the drugs used in the treatment of inflammatory bowel disease (IBD) become systemically bioavailable and potentially bear strong adverse effects. Targeting the inflamed areas of the intestine and keeping the drug localised at its site of action can reduce adverse effects. In animal studies, luminal uptake into inflamed mucosal areas has been shown to be size dependent. We investigated the potential of nano- and microparticle uptake into the rectal mucosa of human IBD patients. Fluorescently labelled placebo nanoparticles (NP) 250nm in size and microparticles (MP) 3.0μm in size were prepared. 2h after rectal application to patients with Crohn's disease (CD) or ulcerative colitis (UC), confocal laser endomicroscopy was performed to visualise the particles in inflamed mucosal areas. In biopsies, ex vivo mucosal transport processes were investigated in miniaturised Ussing chambers. We examined 33 patients with IBD (19 patients with CD, 14 patients with UC) and 6 healthy controls. A significantly enhanced accumulation of MP in ulcerous lesions was observed (covered area=1.28% (range 0.83%–3.45%) vs. 0% in controls; p=0.011), while NP were visible only in traces on mucosal surfaces of all patients. The Ussing chamber experiments suggest persorption of particles through cellular voids; statistical significance was only reached for NP. Drug-containing particles may have great potential to more specifically target intestinal lesions to maximise therapeutic efficacy and minimise potential side effects. Nanoparticles may not be required for local drug delivery to intestinal lesions in humans, thereby minimising the risk of unintended translocation into the blood system.

OSR1-sensitive small intestinal Na+ transport

The oxidative stress responsive kinase 1 (OSR1) contributes to WNK (with no K)-dependent regulation of renal tubular salt transport, renal salt excretion, and blood pressure. Little is known, however, about a role of OSR1 in the regulation of intestinal salt transport. The present study thus explored whether OSR1 is expressed in intestinal tissue and whether small intestinal Na(+)/H(+) exchanger (NHE), small intestinal Na(+)-glucose cotransport (SGLT1), and/or colonic epithelium Na(+) channel (ENaC) differ between knockin mice carrying one allele of WNK-resistant OSR1 (osr1(+/KI)) and wild-type mice (osr1(+/+)). OSR1 protein abundance was determined by Western blotting, cytosolic pH from BCECF fluorescence, NHE activity from Na(+)-dependent realkalinization following an ammonium pulse, SGLT1 activity from glucose-induced current, and colonic ENaC activity from amiloride-sensitive transepithelial current in Ussing chamber experiments. As a result, OSR1 protein was expressed in small intestine of both osr1(+/KI) mice and osr1(+/+) mice. Daily fecal Na(+), K(+), and H(2)O excretion and jejunal SGLT1 activity were lower, whereas small intestinal NHE activity and colonic ENaC activity were higher in osr1(+/KI) mice than in osr1(+/+) mice. NHE3 inhibitor S-3226 significantly reduced NHE activity in both genotypes but did not abrogate the difference between the genotypes. Plasma osmolarity, serum antidiuretic hormone, plasma aldosterone, and plasma corticosterone concentrations were similar in both genotypes. Small intestinal NHE3 and colonic α-ENaC protein abundance were not significantly different between genotypes, but colonic phospho-β-ENaC (ser633) was significantly higher in osr1(+/KI) mice. In conclusion, OSR1 is expressed in intestine and partial WNK insensitivity of OSR1 increases intestinal NHE activity and colonic ENaC activity.

May the Truth Be with You: Lubiprostone as EP Receptor Agonist/ClC-2 Internalizing “Inhibitor”

Chronic constipation is a common clinical problem. Lubiprostone, a bicyclic fatty acid derived from prostaglandin (PG) E1, has been clinically used for the treatment of chronic constipation in adults. Camilleri et al. [1] first reported that lubiprostone inhibited gastric emptying, but accelerated small and large intestinal transit, which is associated with postprandial fullness. Subsequently, on the basis of clinical trials, investigators have reported a beneficial effect of lubiprostone in subjects with chronic constipation and functional bowel disease [2, 3]. Lubiprostone was marketed as belonging to a new therapeutic class, working by a mechanism described as a ClC-2 chloride channel activator. Cuppoletti et al. [4] first reported that lubiprostone increased Cl secretion by activation of the intestinal epithelial cell ClC-2 channel, localized on the apical membrane of enterocytes. Since epithelial Cl secretion is mainly mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) [5], mutations of which cause the disease cystic fibrosis (CF), enhancement of CFTR-independent Cl secretion has been explored as a rescue treatment for CF. Mice with the CFTR mutation DF508, the most common mutation observed in CF patients [6], and CFTR knockout (KO) exhibit an intestinal phenotype, i.e., intestinal obstruction due to loss of Cl secretion, enhancing their utility as models of CF [7]. Nevertheless, the cellular localization and functional roles of epithelial ClC-2 channel and the mechanism of lubiprostone effects on Cl secretion have been questioned. First described in 1992 [8], ClC-2 channels are expressed in the stomach, intestine, colon, brain, heart, and muscles. ClC-2 was first proposed to provide a pathway for epithelial Cl secretion, in addition to CFTR [9]. This hypothesis, however, is not supported by the observation that combined ClC-2 KO/CFTR mutant mice survived longer than CFTR mutant mice, associated with an attenuated intestinal phenotype [10]. Ussing chamber experiments using colonic epithelia suggest that ClC-2 function is localized to the basolateral membrane, facilitating Cl reabsorption rather than Cl secretion. Ussing chamber studies using ClC-2 KO mice also demonstrate that basolateral epithelial ClC-2 has a major function promoting NaCl and fluid absorption, not secretion, in distal colon [11]. Immunohistochemical studies have localized ClC-2 to the basolateral membranes of distal colonic surface enterocytes [12–15], but not to the apical membranes. Therefore, the proposed property of lubiprostone as a ClC2 activator, on the basis of available data, seems questionable [16]. The mechanism of lubiprostone-induced Cl secretion is also controversial. Lubiprostone was originally proposed to increase the open probability of ClC-2 directly, independently of CFTR function [4]. Lubiprostone, however, activated PG E type (EP) receptors, especially EP4 Y. Akiba J. D. Kaunitz Greater Los Angles Veterans Affairs Healthcare System, Los Angeles, CA, USA

Altered response to hydrogen sulphide during experimental colitis in rats

Hydrogen sulphide (H2 S) is produced in the intestine by sulphate-reducing bacteria and during metabolism of L-cysteine within the mucosa. This gasotransmitter induces anion secretion by stimulating enteric neurons and by a direct effect on epithelial cells. As H2 S is discussed to exert both pro- and anti-inflammatory actions, we aimed to investigate the role of H2 S during experimental colitis by comparing the effects of blockade of H2 S-forming endogenous enzymes with the effect of a S-reduced diet to diminish microbial production of H2 S. Rectal application of trinitrobenzenesulfonic acid (TNBS) was used to induce chronic colitis. The level of inflammation was assessed macroscopically and histologically. In Ussing chamber experiments, colonic specimens from TNBS-treated animals exhibited a higher tissue conductance, that is, a higher epithelial permeability, and a slightly reduced basal short-circuit current (a measure of net ion transport) in relation to non-inflamed control tissue. Analgetic treatment with flupirtine, a central antinociceptive analgetic, did not interfere with the induction of the inflammatory response so that all animals were treated with flupirtine to reduce pain and distress during the development of colitis. The secretory response evoked by an exogenous H2 S donor, NaHS, was significantly decreased after induction of colitis, whereas the response to Ca(2+) - or cAMP-dependent secretagogues was unaltered. This downregulation was not observed in the colitis group fed on a S-reduced diet. The decreased NaHS response indicates a desensitization of the tissue by inflammation, which might be explained by an upregulation of colonic H2 S production as described in some models of inflammation.

Modulation of intestinal glucose transport in response to reduced nitrogen supply in young goats1

The reduction of dietary protein is a common approach in ruminants to decrease the excretion of N because ruminants are able to recycle N efficiently by the rumino-hepatic circulation. In nonruminant species an impact on other metabolic pathways such as glucose metabolism was observed when dietary protein intake was reduced. However, an impact of dietary N reduction in goats on glucose metabolism especially on intestinal glucose absorption is questionable because ruminants have very efficient endogenous recycling mechanisms. Therefore, the aim of the present study was to characterize the intestinal absorption of glucose in growing goats kept on different N supply under isoenergetic conditions. The different CP concentrations (20, 16, 10, 9, and 7% CP) of the experimental diets were adjusted by adding urea to the rations. Intestinal flux rates of glucose were determined by Ussing chamber experiments. For a more mechanistic approach, the Na(+)-dependent uptake of glucose into intestinal brush-border membrane vesicles (BBMV) and the expression patterns of the Na(+)-dependent glucose transporter SGLT1 and the glucose transporter 2 (GLUT2) were determined. Reduced N intake resulted in a decrease of plasma glucose (P < 0.001) and insulin (P = 0.004) concentrations whereas the intestinal flux rates of glucose were elevated (P < 0.001), which were inhibited by phlorizin. However, the uptake of glucose into intestinal BBMV was not changed whereas the expression of SGLT1 on mRNA (P < 0.05) and protein abundance (P = 0.03) was decreased in response to a reduced N intake. The mRNA expression of GLUT2 was not affected. From these data, it can be concluded that the intestinal absorption of glucose was modulated by changes in dietary N intake. It is suggested that intracellular metabolism or basolateral transport systems or both might be activated during this feeding regimen because the apical located SGLT1 might not be involved. Therefore, an impact of dietary N reduction on glucose metabolism in growing goats occurred as in monogastric animals.

Luminal cholinergic signalling in airway lining fluid: a novel mechanism for activating chloride secretion via Ca2+‐dependent Cl‐ and K+ channels

Recent studies detected the expression of proteins involved in cholinergic metabolism in airway epithelial cells, although the function of this non-neuronal cholinergic system is not known in detail. Thus, this study focused on the effect of luminal ACh as a regulator of transepithelial ion transport in epithelial cells. RT-PCR experiments were performed using mouse tracheal epithelial cells for ChAT and organic cation transporter (OCT) transcripts. Components of tracheal airway lining fluid were analysed with desorption electrospray ionization (DESI) MS. Effects of nicotine on mouse tracheal epithelial ion transport were examined with Ussing-chamber experiments. Transcripts encoding ChAT and OCT1-3 were detected in mouse tracheal epithelial cells. The DESI experiments identified ACh in the airway lining fluid. Luminal ACh induced an immediate, dose-dependent increase in the transepithelial ion current (EC₅₀: 23.3 µM), characterized by a transient peak and sustained plateau current. This response was not affected by the Na⁺-channel inhibitor amiloride. The Cl⁻-channel inhibitor niflumic acid or the K⁺-channel blocker Ba²⁺ attenuated the ACh effect. The calcium ionophore A23187 mimicked the ACh effect. Luminal nicotine or muscarine increased the ion current. Experiments with receptor gene-deficient animals revealed the participation of muscarinic receptor subtypes M₁ and M₃. The presence of luminal ACh and activation of transepithelial ion currents by luminal ACh receptors identifies a novel non-neuronal cholinergic pathway in the airway lining fluid. This pathway could represent a novel drug target in the airways.

Steviol, an aglycone of natural sweetener stevioside, slows MDCK cyst progression by reducing activity and expression of CFTR chloride channels

Pathogenesis of polycystic kidney disease (PKD) involved cAMP‐stimulated chloride secretion into cyst lumen. The present study was aimed to investigate an inhibitory effect and detailed mechanisms of steviol and its derivatives on cyst growth and cyst formation using Madin Darby canine kidney (MDCK) cyst models. Among 4 steviol‐related compounds, steviol was found to be the most potent compound that inhibited both MDCK cyst growth and formation. Steviol at 100 μM reversibly inhibited cyst growth and cyst formation by 38.3% and 72.4 %, respectively. Steviol at doses up to 200 μM had no cytotoxic effect on MDCK cells. Determined by Ussing chamber experiments, steviol acutely inhibited forskolin‐stimulated apical chloride current in MDCK monolayers by ~40%. Interestingly, prolonged treatment (2–24 hours) with steviol produced 50–80% inhibition of forskolin‐stimulated apical chloride current across MDCK monolayers in dose and time‐dependent manners. In addition, treatment with steviol significantly reduced cystic fibrosis transmembrane conductance regulator (CFTR) expression in MDCK cells. These findings suggest that steviol retarded MDCK cyst progression, in part, by reducing both CFTR activity and expression. Steviol and related compounds represent promising natural plant‐based drug candidates for polycystic kidney disease.This work was supported by the Thailand Research Fund and the Office of the Higher Education Commission and Mahidol University under the National Research Universities Initiative.

Multi‐parametric approach to identify coffee components that regulate mechanisms of gastric acid secretion

Chlorogenic acid (CA), caffeine (CAFF), pyrogallol (PYR), catechol (CAT), (β)N-alkanoyl-hydroxytryptamides (C5HT) and N-methylpyridinium (N-MP) were evaluated for their influence on mechanisms of gastric acid secretion as single compounds and in biomimetic mixtures. Compounds were tested in coffee representative concentrations. Human gastric cancer cells (HGT-1) were used to study the proton secretory activity by Ussing chamber experiments and FACS analysis. For activation of EGFr, Akt1, ERK1/2, ATF-2 and cAMP levels, we performed pathway screening assays. Time-dependent expression of related genes were determined by real-time PCR. Part of the data was used for neural network modeling to identify the most relevant compounds. N-MP increased the expression of the anti-secretory somatostatin receptor by 114%, whereas C5HT decreased its expression by 52%. N-MP down-regulated the pro-secretory CHRM3 receptor by 36% and the H⁺,K⁺-ATPase by 36%. CAFF stimulated the secretory activity in the functional assays, whereas N-MP and CA decreased proton secretion. After applying a pathway analysis, we were able to discriminate between CAFF, CA, CAT, C5HT, PYR and histamine-activating EGFr signaling and N-MP-associated ERK1/2 signaling. By applying a multi-parametric approach, N-MP was shown to effectively down-regulate mechanisms of gastric acid secretion in human parietal gastric cells.

Ex vivo intestinal studies on calcium and phosphate transport in growing goats fed a reduced nitrogen diet

In ruminant feeding, the reduction of dietary protein is an effective approach for decreasing the excretion of N. In non-ruminant species, the intestinal absorption of Ca was affected when dietary protein was reduced. Therefore, it was the aim of the present study to characterise the intestinal absorption of Ca and inorganic phosphate (P(i)) in goats fed different N and Ca diets. Intestinal flux rates of Ca and P(i) were determined in goats fed a reduced N and Ca diet by Ussing chamber experiments. For a more mechanistic approach, the uptake of Ca and P(i) in intestinal brush-border membrane vesicles (BBMV), the expression levels of the epithelial Ca channel transient receptor potential vanilloid channel type 6 (TRPV6), the sodium-dependent P(i) transporter (NaPi) IIb and the vitamin D receptor (VDR) were measured. In goats fed a reduced N and Ca diet, the intestinal flux rates of Ca and P(i) were elevated. However, the reduced N and Ca diet had no effect on the uptake of Ca and P(i) in intestinal BBMV, while the expression of TRPV6 and NaPi IIb protein in the corresponding intestinal segments was even decreased. The mRNA expression of NaPi IIb and VDR was not affected. Therefore, a post-transcriptional regulation of TRPV6 and NaPi IIb protein was suggested in goats fed a reduced N and Ca diet. From these data, it can be concluded that the intestinal absorption of Ca and P(i) in growing goats was affected by changes in dietary N and Ca intake like those in single-stomached animals but differently modulated.

Hypoxia Inhibits Colonic Ion Transport via Activation of AMP Kinase

Mucosal hypoxia is a common endpoint for many pathological processes including ischemic colitis, colonic obstruction and anastomotic failure. Previous studies suggest that hypoxia modulates colonic mucosal function through inhibition of chloride secretion. However, the molecular mechanisms underlying this observation are poorly understood. AMP-activated protein kinase (AMPK) is a metabolic energy regulator found in a wide variety of cells and has been linked to cystic fibrosis transmembrane conductance regulator (CFTR) mediated chloride secretion in several different tissues. We hypothesized that AMPK mediates many of the acute effects of hypoxia on human and rat colonic electrolyte transport. The fluorescent chloride indicator dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide was used to measure changes in intracellular chloride concentrations in isolated single rat colonic crypts. Ussing chamber experiments in human colonic mucosa were conducted to evaluate net epithelial ion transport. This study demonstrates that acute hypoxia inhibits electrogenic chloride secretion via AMPK mediated inhibition of CFTR. Pre-treatment of tissues with the AMPK inhibitor 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl)]-3-pyridin-4-yl-pyyrazolo [1,5-a] pyrimidine (compound C) in part reversed the effects of acute hypoxia on chloride secretion. We therefore suggest that AMPK is a key component of the adaptive cellular response to mucosal hypoxia in the colon. Furthermore, AMPK may represent a potential therapeutic target in diseased states or in prevention of ischemic intestinal injury.

Mechanisms of Chloride and Bicarbonate Secretion Across Rat Proximal Colon Evoked by a CO Donor

The aim of the present study was to investigate whether carbon monoxide induces changes in ion transport across rat proximal colon. In Ussing chamber experiments, tricarbonyldichlororuthenium(II) dimer (RuCO), a CO-donor, evoked a concentration-dependent increase in short-circuit current (Isc). Anion substitution experiments suggest that a secretion of Cl− and HCO3 − underlie the RuCO-induced current. Partial inhibition by the neurotoxin tetrodotoxin suggests the involvement of secretomotor neurons in this response. Consequently, endogeneous carbon monoxide might be a physiological modulator of colonic ion transport.

SGK1-dependent stimulation of intestinal SGLT1 activity by vitamin D

The serum- and glucocorticoid-inducible kinase SGK1 has previously been shown to mediate the glucocorticoid-dependent stimulation of several intestinal transport systems including the electrogenic glucose transporter SGLT1. In squamous carcinoma cells, SGK1 expression is stimulated by 1,25(OH)₂D₃, the biologically active metabolite of vitamin D. The present study explored whether vitamin D influences the intestinal SGLT1 activity. Jejunal SGLT1 activity was determined by Ussing chamber experiments. Under a normal diet, the electrogenic glucose transport was similar in SGK1 knockout (sgk1 ( -/- )) and wild type mice (sgk1 ( +/+ )). Following a vitamin D-rich diet (14 days 10,000 I.U. vitamin D), the SGK1 transcript levels as well as the SGLT1 protein abundance were increased in sgk1(+/+) mice. Moreover, SGLT1 activity was increased in sgk1(+/+) mice but not in sgk1(-/-) mice following a vitamin D-rich diet. Furthermore, an oral glucose load was followed by an increase in the plasma glucose concentration to significantly higher values in sgk1(+/+) mice treated with a vitamin D-rich diet than in untreated sgk1(+/+) mice. In conclusion, vitamin D treatment upregulates the expression of SGK1, which in turn enhances SGLT1 activity.

Radiation decreases murine small intestinal HCO3− secretion

Purpose: While secretagogue-induced diarrhea is rich in chloride (Cl−) and bicarbonate (HCO3−) anions, little is known about diarrhea or its anionic composition following irradiation. We performed studies to characterize the differences between cyclic adenosine monophosphate (cAMP)-stimulated anion secretions in irradiated and non-irradiated mice.Materials and methods: HCO3− secretion was examined in basal, cAMP-stimulated, and irradiated jejunal tissues from BALB/c (Bagg albino) mice. The abdomens of the mice were γ-irradiated using a caesium-137 source.Results: Ussing-chamber experiments performed in an HCO3−-containing, Cl−-free solution on the bath side showed inhibition of HCO3− in irradiated mice. Non-irradiated mice exhibited bumetanide-sensitive and insensitive current, while irradiated mice displayed bumetanide-sensitive current. pH-stat experiments showed inhibition of basal and cAMP-stimulated HCO3− secretions in irradiated mice. Immunohistochemistry and Western blot analysis displayed a sodium-bicarbonate cotransporter expression in the villus and not the crypt of non-irradiated mice, while its expression and protein levels decreased in irradiated mice.Conclusions: Anion secretions in irradiated mice, being primarily Cl− and minimally HCO3−, differ from that of secretagogue-induced anion secretions. Understanding anion loss will help us correct electrolyte imbalances, while reduced HCO3− secretion in the upper-gastrointestinal tract might also have implications for irradiation-induced nausea and vomiting.

Mapping the paracellular pore: the role of two highly conserved cysteines in the first extracellular loop of claudins

Claudins are a family of tight junction transmembrane proteins whose first extracellular loop (1st ECL) determines paracellular permeability and ion selectivity in epithelia. There are two cysteines in the 1st ECL that are highly conserved among all claudins (C54 and C64 in claudin-2) and whose function is unknown. We hypothesized that they are linked by an intramolecular disulfide bond that is necessary for correct folding, trafficking and pore function. To test this hypothesis, we mutated C54 and C64 in claudin-2 either to alanine or serine, and generated stably transfected MDCK I Tet-off cell lines expressing each mutant protein. Western blotting showed a high molecular weight band in each of the single mutants that was not seen in wild-type claudin-2 or the double mutants, consistent with the formation of claudin dimers. By immunofluorescence staining, the alanine mutants were mislocalized intracellularly while the serine mutants were correctly inserted into the tight junction. In Ussing chamber experiments, the conductance and Na+ permeability of all the serine mutants were markedly lower than wild-type claudin-2. Our findings suggest that the two highly conserved cysteines in the 1st ECL are disulfide bonded and that this disulfide bond is necessary for normal pore formation but not for protein trafficking. Supported by NIH grants R01DK062283 and R01DK062283-07S1.