Transepithelial Flux Research Articles

Mechanisms underlying the effects of inulin-type fructans on calcium absorption in the large intestine of rats

Inulin-type fructans (inulin, oligofructose, fructooligosaccharides) in the diet do increase intestinal calcium absorption in animals and humans, but the underlying mechanism has not been identified. We therefore assessed the effects of fermentation of inulin-type fructans on transepithelial calcium transport in rat large intestine. Transepithelial calcium fluxes in vitro (Ussing chamber), effects on gene expression, mucosal morphology, and composition of luminal contents were determined in rats fed a standard diet and/or a diet containing 10% (w/w) 1/1 inulin–oligofructose mixture (INOF). Net transepithelial calcium transport in large intestine of rats fed a standard diet was increased by high mucosal calcium concentrations, the presence of 100 mmol/L mucosal short-chain fatty acids (SCFAs), the presence of 10 g/L INOF at the mucosal side, but not by reducing mucosal pH. Tissues from rats fed INOF did not show altered calcium transport when compared to controls. However, when flux data were based on the total caecal surface area, INOF-fed rats nearly doubled absorption rate in caecum. INOF feeding altered transcript levels of several mucosal genes that can be linked to transcellular and paracellular calcium transport processes. In addition, a decreased luminal pH in caecum with markedly increased caecal pools of total, soluble, and ionized calcium resulted from INOF ingestion. Thus, inulin-type fructans increase the large intestinal calcium absorption by different mechanisms including enhanced pools of soluble and ionized calcium, an increase in the absorptive surface predominantly in caecum, the increased concentrations of SCFAs, and by direct interaction with the intestinal tissue.

Expression and function of LAT1, a neutral amino acid exchanger, in renal porcine epithelial cell line LLC‐PK1

The present study examined the expression of LAT1 and the functional characteristics of the inward and outward [14C] l-leucine transporter in the renal porcine epithelial cell line LLC-PK1. LLC-PK1 cells were cultured in polycarbonate filters and accumulation and transepithelial flux of the substrate monitored with [14C] l-leucine. LAT1 transcripts were examined by RT-PCR. LAT1 protein was detected by immunoblotting. The accumulation of [14C] l-leucine in the cell and the [14C] l-leucine transepithelial flux were four- and twofold, respectively, when the substrate was added from the basal cell side, suggesting that the basolateral membrane is endowed with a high density of transport units, when compared with the apical membrane. Increases in the transepithelial flux of [14C] l-leucine by unlabelled l-leucine were also more pronounced when unlabelled l-leucine was added from the basolateral membrane. In the absence of Na+, unlabelled l-leucine increased the basal and apical fractional outflow of [14C] l-leucine, this being similar at pH 7.4 and pH 6.2. RT-PCR and immunoblotting detected LAT1 transcript and protein, respectively. LLC-PK1 cells are endowed with the LAT1 transcript and protein and transport l-leucine through the Na+-independent and pH-insensitive LAT1 transporter. The density of transporter units in LLC-PK1 cells may be higher at the basolateral membranes, although be also present in the apical membranes.

The effect of type-1 fimbrial immunization on gut pathophysiological response in rats infected with Salmonella enterica subsp. enterica serovar Typhimurium

The pathophysiological mechanism of Salmonella enterica subsp. enterica serovar Typhimurium (Salmonella typhimurium) induced gastroenteritis is controlled by interplay of various cell signaling events. Adherence of this organism through type-1 fimbriae is known to be a vital prerequisite for the establishment of infection. In the present investigation male albino Wistar rats were immunized with purified type-1 fimbriae and challenged intragastrically with S. typhimurium. Electrolyte transport and level of different second messengers were studied in four different groups of animals. Transepithelial fluxes of Na+ and Cl- revealed absorption in immunized-challenged group as observed in case of control and immunized group while secretion was observed in infected group. Ca2+ and 3-0-methyl-D-glucose fluxes did not show any change. Significant increase in the level of intracellular Ca2+, cAMP, membrane form of protein kinase C, prostaglandins, NADPH oxidase, glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, total oxygen free radicals, reactive nitrogen intermediates, citrulline and lipid peroxidation was found in the infected group. However, in the immunized-challenged group, the values of all the parameters were found to be same as that of control as well as immunized groups. Na+, K(+)-ATPase and calmodulin levels were found to be unaltered in all the groups of animals. Thus, the immunization with type-1 fimbriae has been found to be quite effective leading to the prevention of multiple physiologic derangements in isolated ileal cells suggesting the protective role of the fimbriae.

The transport of cationic amino acids in human airway cells: expression of system y+L activity and transepithelial delivery of NOS inhibitors

The transport of arginine has been characterized in human airway Calu-3 cells. As assessed with RT-PCR, Calu-3 cells express the genes for several transporters, such as the system y+-related SLC7A1, SLC7A2, and SLC7A4; the system y+L-related SLC7A6, SLC7A7, and SLC3A2; and the system B0,+-related SLC6A14. In polarized Calu-3 cell monolayers, apical arginine influx has a leucine-sensitive, sodium-dependent component and a leucine- and lysine-resistant sodium-independent fraction. At the basolateral membrane, arginine transport was fully sodium-independent and partially inhibited by leucine provided that sodium was present in the extracellular medium. Moreover, extracellular leucine trans-stimulated arginine efflux from the basolateral membrane in the presence, but not in the absence, of sodium. The transepithelial, apical to basolateral, arginine transport strictly depended on the presence of sodium and was markedly inhibited by apical leucine, but significantly trans-stimulated by the neutral amino acid added at the basolateral side. When added at the apical side, the NOS-inhibitors NMMA and NIL, CAA analogs with a free carboxyl group, markedly inhibited the apical arginine influx and the transepithelial flux of the cationic amino acid. The same compounds trans-stimulated basolateral arginine efflux. None of these effects were observed in the presence of the methyl ester analog NAME. The basolateral medium of Calu-3 cell monolayers, obtained after incubation in the presence of the three inhibitors at the apical side, inhibited the production of NO by activated murine macrophages. The inhibitory effect of the Calu-3 cell conditioned medium was time-dependent and markedly higher with NMMA and NIL than with NAME. Moreover, the NOS-inhibitory effect of the medium was significantly enhanced if NMMA and NIL, at the apical side, and basolateral leucine were simultaneously present during the conditioning procedure. These results indicate that 1) human airway epithelial cells express a functional system y+L at the basolateral membrane; 2) in this model, transepithelial arginine transport involves apical influx through system B0,+ and basolateral efflux through system y+L, and 3) the same transporters also perform an efficient transepithelial transport of amino acid-like NOS inhibitors.

Transepithelial Taurine Transport in Caco-2 Cell Monolayers

Here we characterized transepithelial taurine transport in monolayers of cultured human intestinal Caco-2 cells by analyzing kinetic apical and basolateral uptake and efflux parameters. Basolateral uptake was Na(+)- and Cl(-)- dependent and was inhibited by beta-amino acids. Uptake by this membrane showed properties similar to those of the apical TauT system. In both membranes, taurine uptake fitted a model consisting of a non-saturable plus a saturable component, with a higher half-saturation constant and transport capacity at the apical membrane (K(m), 17.1 micromol/L; V(max), 28.4 pmol.cm(-2).5 min(-1)) than in the basolateral domain (K(m), 9.46 micromol/L; V(max), 5.59 pmol.cm(-2).5 min(-1)). The non-saturable influx component, estimated in the absence of Na(+) and Cl(-), showed no significant differences between apical and basolateral membranes (K(D), 89.2 and 114.7 nL.cm(-2) . 5 min(-1), respectively). Taurine efflux from the cells is a diffusive process, as shown in experiments using preloaded cells and in trans-stimulation studies (apical K(D),72.7 and basolateral K(D), 50.1 nL.cm(-2).5 min(-1)). Basolateral efflux rates were significantly lower than passive influx rates. We conclude that basolateral taurine uptake in Caco-2 cells is mediated by a transport mechanism that shares some properties with the apical system TauT. Moreover, calculation of unidirectional and transepithelial taurine fluxes reveals that apical influx of this amino acid is higher than basolateral efflux rates, thereby enabling epithelial cells to accumulate taurine against a concentration gradient.

Diuretic factors and second messengers stimulate secretion of the organic cation TEA by the Malpighian tubules of Drosophila melanogaster

This study showed that four factors which stimulate transepithelial fluid secretion and inorganic ion transport across the main segment of the Malpighian tubules of Drosophila melanogaster also stimulate transepithelial secretion of the prototypical organic cation tetraethylammonium (TEA). TEA fluxes across the Malpighian tubules and gut were measured using a TEA-selective self-referencing (TEA-SeR) microelectrode. TEA flux across isolated Malpighian tubules was also measured using a TEA-selective microelectrode positioned in droplets of fluid secreted by tubules set up in a modified Ramsay assay. TEA flux was stimulated by the intracellular second messengers cAMP and cGMP, which increase the lumen-positive transepithelial potential (TEP), and also by tyramine and leucokinin-I (LK-I), which decrease TEP. The largest increase was measured in response to 1 μmol l −1 LK-I which increased transepithelial TEA flux by 72%. TEA flux in the lower tubule was stimulated slightly (13%) by 1 μmol l −1 tyramine but not by any of the other factors. TEA flux across the midgut was unaffected by cAMP, cGMP or tyramine. This is the first study to demonstrate the effects of insect diuretic factors and second messengers on excretion of organic cations.

Transepithelial Transport of Microbial Metabolites of Quercetin in Intestinal Caco-2 Cell Monolayers

m-Hydroxyphenylacetic acid (mHPA), 3,4-dihydroxyphenylacetic acid (DHPA), and 4-hydroxy-3-methoxyphenylacetic acid (HMPA) are major microbial metabolites of quercetin. After administration of quercetin to human subjects, these metabolites are readily detected in blood and urine. mHPA, DHPA, and HMPA are thought to exert protective biological activity within the body due to their antioxidant properties. However, very little work has been published concerning their absorption. I have examined the absorption characteristics of the quercetin metabolites in Caco-2 cells by a coulometric detection method using HPLC-ECD. All of them exhibited nonsaturable transport in Caco-2 cells up to 30 mM, whereas HMPA and mHPA also showed proton-coupled polarized absorption. The proton-coupled directional transport of HMPA and mHPA was inhibited by the substrate of the monocarboxylic acid transporter (MCT). A considerable amount of apically loaded HMPA and mHPA was taken up and transported through to the basolateral side, while almost all of the apically loaded DHPA was retained on the apical side. Furthermore, the transepithelial flux of DHPA was inversely correlated with the paracellular permeability of Caco-2 cells, although those of HMPA and mHPA were almost constant. These results indicate that transport of DHPA was mainly via paracellular diffusion, although HMPA and mHPA were absorbed to some extent by the MCT.

Ion-selective microelectrode analysis of salicylate transport by the Malpighian tubules and gut ofDrosophila melanogaster

Transport of the organic anion salicylate by the Malpighian tubules and gut of larval and adult fruit flies was studied using two salicylate-selective microelectrode methods. The first method combined the high selectivity of tridodecylmethylammonium-based electrodes for salicylate with the self-referencing ion-selective microelectrode technique for non-invasive spatial and temporal analysis of salicylate flux. Measurements with this technique revealed secretion of salicylate across the main and distal segments of the Malpighian tubule as well as the midgut, ileum and rectum. The second method used a salicylate-selective microelectrode to measure the concentration of salicylate in fluid droplets secreted by isolated Drosophila Malpighian tubules set up in a Ramsay secretion assay. Transepithelial salicylate flux was calculated as the product of fluid secretion rate and secreted fluid salicylate concentration. Measurements with this method revealed that salicylate transport was active and saturable; the kinetic parameters J(max) and K(t) were 2.72 pmol min(-1) tubule(-1) and 0.046 mmol l(-1), respectively. Measurements of transepithelial salicylate flux determined by both microelectrode methods were in good agreement. Transepithelial flux measurements measured by microelectrodes were also validated by comparing them with measurements of radiolabelled salicylate levels in secreted droplets. Salicylate concentrations in haemolymph samples were measured with salicylate-selective microelectrodes after injection of salicylate into the haemocoel or after insects were fed salicylate-rich diets. The rate of salicylate secretion by Malpighian tubules in vitro was sufficient to account for the measured rate of decline of salicylate concentration in the haemolymph in vivo.

Innate Host Defense of the Lung: Effects of Lung-lining Fluid pH

Lung-lining fluid (LLF) is a primary constituent of the pulmonary host defense system. It is distributed continuously throughout the respiratory tract but is heterogeneous regarding its chemistry and physiology between the conducting airways and alveoli. The conducting airways are lined with airway surface liquid (ASL), a mucus gel-aqueous sol complex that interacts functionally with epithelial cilia as the mucociliary escalator. The alveoli are lined with alveolar subphase fluid (AVSF) and pulmonary surfactant. AVSF sterility is maintained in part by the phagocytic activity of resident alveolar macrophages. Normal ASL and AVSF are both more acidic than blood plasma. However, the details of acid-base regulation differ between the two media. Appreciable transepithelial acid-base flux is possible across the airway epithelium, whereas the alveolar epithelium is relatively impermeable to transepithelial acid-base flux. Moreover, one must consider the influence of resident macrophages on AVSF pH. Resident macrophages occupy a sizable fraction of AVSF by volume and are a substantial source of metabolic H+. The buffering capacities of ASL and AVSF probably are largely due to secreted peptides (e.g., ASL mucins and AVSF surfactant proteins). Acid-base exchange between the extracellular hydrophase and intracellular buffering systems of resident macrophages represents an additional buffer pool for AVSF. The pH of ASL and AVSF can be depressed by disease or inflammation. Low pH is predicted to suppress microbe clearance from the airways and alveoli, increase pathogen survival in both regions, and alter mediator release by resident macrophages and recruited leukocytes thereby increasing the propensity for bystander cell injury. Overall, ASL/AVSF pH is expected to be a major determinant of lung host defense responses.

Reorganization of ZO‐1 by sodium‐dependent glucose transporter activation after heat stress in LLC‐PK1 cells

Heat stress (HS) induces activation of high-affinity sodium-dependent glucose transporter (SGLT1) in porcine renal LLC-PK(1) cells. In this study, we investigated the roles of SGLT1 activation in reorganization of zonula occludens-1 (ZO-1), a cytosolic tight junction (TJ) protein, after HS. HS (42 degrees C, 3 h) caused decrease in transepithelial electrical resistance (TER). Subsequent incubation at 37 degrees C for 12 h increased TER above pre-HS level. The treatment of phloridzin, a potent SGLT1 inhibitor, or the replacement of glucose with a nonmetabolizable glucose analog blocked the recovery of TER and increased the transepithelial flux of FITC-dextran (4,000 Da). Immunofluorescent staining of ZO-1 showed that HS diffused ZO-1 from cell contact to cytosolic sites. Furthermore, the fraction of ZO-1 was distributed from the Triton X-100 insoluble to the Triton X-100 soluble pool. After incubation at 37 degrees C for 12 h, cell contact and ZO-1 extractability with Triton X-100 returned to pre-HS conditions, but the recovery was completely prevented by phloridzin. Tyrosine kinases activity was increased by HS that was inhibited by phloridzin. Genistein and CGP77675, tyrosine kinases inhibitors, blocked the recovery of TER and increased the transepithelial flux of FITC-dextran. Furthermore, these inhibitors prevented the recovery of cell contact and ZO-1 extractability with Triton X-100 as same as phloridzin. These findings suggested that the activation of SGLT1 reorganized ZO-1 mediated by elevation of tyrosine kinases activity after heat injury.

Gastrointestinal-tract models and techniques for use in safety pharmacology

The gastrointestinal tract (GI tract), which extracts nutrients, electrolytes, minerals, and water, is prone to injury as a result of oral drug administration. Clinical assessment of the GI tract is often limited to measurements of transit time and observations of vomiting or diarrhoea, despite the existence of methods and techniques capable of assessing specific changes in GI function at the membrane, cell, and whole animal levels. Membrane studies, record the uptake of solutes, and electrolyte transport, assessing the affects of compounds on transepithelial GI transport and flux. Such methods lend themselves to permeability, immunohistochemistry, morphology, and molecular biology techniques. Isolated cells from the GI tract or cultured cell lines provide knowledge of regulation and function at a cellular level, whilst motility patterns, taken in vivo or from biopsies, provide information at a more integrated level. In anesthetised animals, ligated segments of the intestine can be infused with test compounds, providing information about absorptive and secretory processes important for the treatment of diarrhoea. Computer simulations and modelling are used to predict the disposition of a chemical and its metabolite and can, to some extent, replace animal testing, thereby reducing development costs. Indeed, software programs can be used to simulate the dissolution, absorption, distribution, metabolism, and excretion (ADME) properties of potential drugs in the human GI tract. Finally, advances in the field of imaging, combined with endoscopy, have resulted in a wireless capsule, allowing the inspection of the GI tract anatomy and pathology without surgical intervention. It is concluded that the field of safety pharmacology could rapidly, cheaply, and routinely incorporate membrane, isolated tissue, and endoscopy techniques for GI tract testing of drugs.

Transepithelial transport of chlorogenic acid, caffeic acid, and their colonic metabolites in intestinal caco-2 cell monolayers.

Both chlorogenic and caffeic acids exhibited nonsaturable transport in Caco-2 cells, whereas caffeic acid also showed proton-coupled polarized absorption. Thus, the absorption efficiency of caffeic acid was greater than that of chlorogenic acid. Polarized transport of caffeic acid was inhibited by substrates of MCT such as benzoic and acetic acids. Almost all of the apically loaded chlorogenic and caffeic acid was retained on the apical side, and the transepithelial flux was inversely correlated with the paracellular permeability of Caco-2 cells. These results indicate that transport was mainly via paracellular diffusion, although caffeic acid was absorbed to a lesser extent by the monocarboxylic acid transporter (MCT). Furthermore, m-coumaric acid and 3-(m-hydroxyphenyl)propionic acid, the main metabolites of chlorogenic and caffeic acid by colonic microflora, competitively inhibited the transport of fluorescein, a known substrate of MCT. This suggests that their absorption could also be mediated by MCT. These findings have exemplified the physiological importance of MCT-mediated absorption in both phenolic acids per se and their colonic metabolites.

Carnosine uptake in rat choroid plexus primary cell cultures and choroid plexus whole tissue from PEPT2 null mice.

PEPT2 is functionally active and localized to the apical membrane of rat choroid plexus epithelial cells. However, little is known about the transport mechanisms of endogenous neuropeptides in choroid plexus, and the role of PEPT2 in this process. In the present study, we examined the uptake kinetics of carnosine in rat choroid plexus primary cell cultures and choroid plexus whole tissue from wild-type (PEPT2(+/+)) and null (PEPT2(-/-)) mice. Our results indicate that carnosine is preferentially taken up from the apical as opposed to basolateral membrane of cell monolayers, and that basolateral efflux in limited. Transepithelial flux of carnosine was not distinguishable from that of paracellular diffusion. The apical uptake of carnosine was characterized by a high affinity (K(m) = 34 microM), low capacity (V(max) = 73 pmol/mg protein/min) process, consistent with that of PEPT2. The non-saturable component was small (K(d) = 0.063 microL/mg protein/min) and, under linear conditions, was only 3% of the total uptake. Studies in transgenic mice clearly demonstrated that PEPT2 was responsible for over 90% of carnosine's uptake in choroid plexus whole tissue. These findings elucidate the unique role of PEPT2 in regulating neuropeptide homeostasis at the blood-cerebrospinal fluid interface.

Identification and characterization of a Kidd antigen/UT-B urea transporter expressed in human colon.

We have identified a urea transporter from the mucosa of the human colon that has characteristics consistent with a Kidd antigen/UT-B urea transporter. This intestinal urea transporter encodes a 389-amino acid peptide with a sequence identical to that previously reported for the UT-B urea transporter in erythrocytes. Expression of a UT-B 2-kb mRNA transcript and of approximately 50- and approximately 98-kDa UT-B proteins is detected in human colonic mucosa by Northern and Western blot analysis. The UT-B protein is localized in the cell membrane and cytoplasm of the superficial intestinal epithelium and in the epithelial cells in the crypts. A 2-kb UT-B mRNA transcript and the UT-B protein were also identified in the intestinal cell line Caco-2. The transepithelial flux of (14)C urea was examined in Caco-2 cells growing on porous membrane support and was significantly inhibited by phloretin, 1,3-dimethylurea, and thiourea, suggesting that the transfer of urea across the Caco-2 monolayer could be mediated, at least in part, by the UT-B urea transporter. We conclude that the Kidd antigen/UT-B urea transporter is physiologically expressed in the human colon epithelium, where it could participate in the transport of urea across the colon mucosa.

Preclinical pharmacology and pharmacokinetics of GW433908, a water-soluble prodrug of the human immunodeficiency virus protease inhibitor amprenavir.

GW433908 is the water-soluble, phosphate ester prodrug of the human immunodeficiency virus type 1 protease inhibitor amprenavir (APV). A high-yield synthesis of GW433908 is achieved by phosphorylation of the penultimate precursor of APV with phosphorous oxychloride (POCl(3)) in pyridine. A single-dose pharmacokinetic study of GW433908 sodium salt in dogs showed that APV exposure was similar to that achieved with an equivalent molar dose of the APV clinical formulation (Agenerase) and that systemic exposure to the prodrug was minimal (0.3% of the APV exposure). However, the sodium salt of GW433908 was a hygroscopic, amorphous solid and thus not suitable for pharmaceutical development. The calcium salt was a developable crystalline solid, but oral dosing afforded only 24% of the APV exposure in dogs compared with Agenerase. Acidification of the dog stomach by coadministration of HCl increased the bioavailability of the calcium salt to levels near those of the sodium salt. Single-dose administration of GW433908 calcium salt in dogs and rats produced portal vein GW433908 concentrations that were maximally 1.72 and 0.79% of those of APV concentrations, respectively. Furthermore, GW433908 had poor transepithelial flux and APV showed significant flux across human-derived Caco-2 cell monolayers (a model of intestinal permeability). Taken together, these results suggest that GW433908 is primarily metabolized to APV at or in the epithelial cells of the intestine and that the prodrug is not substantially absorbed. Based in part on these findings, GW433908 was advanced to clinical development.

Transport of pharmacologically active proline derivatives by the human proton-coupled amino acid transporter hPAT1.

Several proline derivatives such as L-azetidine-2-carboxylic acid, cis-4-hydroxy-L-proline, and 3,4-dehydro-DL-proline prevent procollagen from folding into a stable triple-helical conformation, thereby reducing excessive deposition of collagen in fibrotic processes and the growth of tumors. This study was performed to investigate whether the recently discovered human proton-coupled amino acid transporter 1 (hPAT1) is capable of transporting such pharmacologically relevant proline derivatives and also GABA analogs. Uptake of L-[3H]proline and [3H]glycine in Caco-2 cells was Na+-independent but strongly H+-dependent. The L-proline uptake was saturable and mediated by a single transport system (hPAT1) with an affinity constant of 2.0 +/- 0.2 mM. The uptake of L-[3H]proline was inhibited by D-proline, trans-4-hydroxy-L-proline, cis-4-hydroxy-L-proline, cis-4-hydroxy-D-proline, 3,4-dehydro-DL-proline, L-azetidine-2-carboxylic acid, 3-amino-1-propanesulfonic acid, D- and L-pipecolic acid, l-thiaproline, and many others. Apical uptake and transepithelial flux of L-[3H]proline across Caco-2 cell monolayers were strongly inhibited by proline derivatives in proportions corresponding to their respective affinity constants at hPAT1. The basolateral to apical flux of L-[3H]proline was only 8% of that in the opposite direction. Apical uptake of unlabeled L-proline, cis-4-hydroxy-L-proline, and L-azetidine-2-carboxylic acid was stimulated by an inside directed H+ gradient 2- to 3-fold. Total apical to basolateral flux of proline derivatives was moderately correlated with their inhibitory potency for L-[3H]proline uptake and flux inhibition. We conclude that 1) the substrate specificity of hPAT1 is very much broader than so far reported and 2) the system accepts therapeutically relevant proline and GABA derivatives. hPAT1 is a promising candidate for new ways of oral drug delivery.

Water and electrolyte salvage in an animal model of dehydration and malnutrition.

Recently, a new oral rehydration solution (ORS) called Resomal has been designed specifically for children with severe malnutrition. The aim of this study was to assess the effect of malnutrition on renal and intestinal responses to dehydration, and to compare intestinal water and electrolyte absorption from Resomal and from the standard World Health Organization-Oral Rehydration Solution (WHO-ORS). Malnutrition was achieved in a rabbit model by feeding the animals daily for 30 days with half the amount of food that a well-nourished group of control animals had consumed on the previous day. Dehydration was achieved by water deprivation for 46 hours in both control and malnourished rabbits. At 46 hours, dehydration was assessed by changes in body weight, urinary volume and osmolarity, and blood urea nitrogen concentration. At that time active colonic and jejunal mucosal electrolyte transport in Ussing chambers was also measured. Small intestinal absorption of water, sodium, and potassium was measured in vivo during intestinal perfusion of the two ORSs and in vitro by measurement of mucosal electrogenic glucose-stimulated sodium absorption across intestinal patches. Compared to controls (C), well-nourished but dehydrated (C+D) animals lost 12% of their body weight, with an 87% reduction in urine volume, a 110% increase in urine osmolality, and a 94% increase in blood urea nitrogen. In the colon of C+D animals, short-circuit current (Isc) and net sodium transepithelial flux (JNa+ net) were increased. Almost identical results were obtained in malnourished and dehydrated (M+D) animals. In the jejunum, net in vivo absorption of water (JWater), sodium (JNa+), and potassium (JK+) were increased during standard ORS infusion in both dehydrated groups. During Resomal infusion, water absorption was the same as seen with WHO-ORS, but sodium absorption was reduced, and potassium absorption was increased in both well-nourished and malnourished dehydrated animals. In vitro, compared to controls, the glucose-stimulated short-circuit current (DeltaIsc), JNa+ net and G were increased in both dehydrated groups. During experimental dehydration, the kidney and large intestine salvage water and electrolytes, thus reducing the consequences of dehydration. These findings indicate that jejunal water absorption from Resomal and WHO-ORS is increased during dehydration, but Resomal allows for less sodium and more potassium to be absorbed, both in well-nourished and malnourished dehydrated rabbits.

Water and Electrolyte Salvage in an Animal Model of Dehydration and Malnutrition

ABSTRACTBackground and aimRecently, a new oral rehydration solution (ORS) called Resomal has been designed specifically for children with severe malnutrition. The aim of this study was to assess the effect of malnutrition on renal and intestinal responses to dehydration, and to compare intestinal water and electrolyte absorption from Resomal and from the standard World Health Organization‐Oral Rehydration Solution (WHO‐ORS).MethodsMalnutrition was achieved in a rabbit model by feeding the animals daily for 30 days with half the amount of food that a well‐nourished group of control animals had consumed on the previous day. Dehydration was achieved by water deprivation for 46 hours in both control and malnourished rabbits. At 46 hours, dehydration was assessed by changes in body weight, urinary volume and osmolarity, and blood urea nitrogen concentration. At that time active colonic and jejunal mucosal electrolyte transport in Ussing chambers was also measured. Small intestinal absorption of water, sodium, and potassium was measured in vivo during intestinal perfusion of the two ORSs and in vitro by measurement of mucosal electrogenic glucose‐stimulated sodium absorption across intestinal patches.ResultsCompared to controls (C), well‐nourished but dehydrated (C+D) animals lost 12% of their body weight, with an 87% reduction in urine volume, a 110% increase in urine osmolality, and a 94% increase in blood urea nitrogen. In the colon of C+D animals, short‐circuit current (Isc) and net sodium transepithelial flux (JNa+ net) were increased. Almost identical results were obtained in malnourished and dehydrated (M+D) animals. In the jejunum, net in vivo absorption of water (JWater), sodium (JNa+), and potassium (JK+) were increased during standard ORS infusion in both dehydrated groups. During Resomal infusion, water absorption was the same as seen with WHO‐ORS, but sodium absorption was reduced, and potassium absorption was increased in both well‐nourished and malnourished dehydrated animals. In vitro, compared to controls, the glucose‐stimulated short‐circuit current (ΔIsc), JNa+ net and G were increased in both dehydrated groups.ConclusionDuring experimental dehydration, the kidney and large intestine salvage water and electrolytes, thus reducing the consequences of dehydration. These findings indicate that jejunal water absorption from Resomal and WHO‐ORS is increased during dehydration, but Resomal allows for less sodium and more potassium to be absorbed, both in well‐nourished and malnourished dehydrated rabbits.

Anion exchanger 2 is essential for spermiogenesis in mice

Na+-independent anion exchangers (AE) mediate electroneutral exchange of Cl- for HCO3- ions across cell membranes, being involved in intracellular pH and cell volume regulation and in transepithelial hydroionic fluxes. Bicarbonate activation of adenylyl cyclase is known to be necessary for sperm motility and sperm capacitation, and a few studies have suggested a possible role of AE carriers in reproduction. Among the four AE genes identified in mammals thus far, only Ae2 (Slc4a2) has been determined to be expressed in the male reproductive system, especially in developing spermatozoa and in epididymal epithelium. Most AE genes drive alternative transcription, which in mouse Ae2 results in several Ae2 isoforms. Here, we generated mice carrying a targeted disruption of Ae2 that prevents the expression of the three AE2 isoforms (Ae2a, Ae2b1, and Ae2b2) normally found in mouse testes. Male Ae2-/- mice (but not female Ae2-/- mice) are infertile. Histopathological analysis of Ae2-/- testes shows an interruption of spermiogenesis, with only a few late spermatids and a complete absence of spermatozoa in the seminiferous tubules. The number of apoptotic bodies is increased in the seminiferous tubules and in the epididymis, which also shows squamous metaplasia of the epididymal epithelium. Our findings reveal an essential role of Ae2 in mouse spermiogenesis and stress the recently postulated involvement of bicarbonate in germ-cell differentiation through the bicarbonate-sensitive soluble-adenylyl-cyclase pathway.

The effect of tumor necrosis factor-alpha on D-fructose intestinal transport in rabbits.

Tumor necrosis factor-alpha (TNF-alpha) is an important immunoregulatory cytokine involved in septic responses during bacterial infection. The aim of this study was to examine the effect of TNF-alpha on the transport of D-fructose across rabbit jejunum. A sepsis condition was evoked by intravenous administration of this cytokine and hematological and plasma parameters were analyzed and body temperature was recorded. D-Fructose transport was assayed in rabbit jejunum. Sugar absorption in TNF-alpha treated rabbits was lower than in control animals. TNF-alpha decreased both the mucosal-to-serosal transepithelial flux and the transport across brush border membrane vesicles of D-fructose. The number of D-fructose transporters (GLUT5) was analyzed by Western blot in an attempt to explain this inhibition. TNF-alpha treated animals had lower levels of GLUT5, indicating a reduction in the expression of GLUT5 protein and therefore in transport capacity. The inhibition could also be related with the secretagogue effect of TNF-alpha on the gut since the intracellular tissue water was affected and the absence of chloride ion in the incubation medium partly removed the cytokine inhibition on sugar intestinal transport in treated rabbits. Finally, in terms of possible mediators involved in the TNF-alpha effect, nitric oxide and prostaglandins appeared to play a role in the inhibition of D-fructose intestinal uptake.