Absorption and paracellular visualization of fluorescein, a hydrosoluble probe, in intact house sparrows (Passer domesticus)

We describe a method to visualize the cellular location of compounds during absorption by the small intestine in intact animals. First, we employed pharmacokinetic methodology to measure the fractional absorption of sodium fluorescein, a small (MW = 376) water-soluble molecule that is widely used as hydrophilic marker molecule for paracellular permeability studies. Based on the hypothesis that the paracellular pathway acts as a sieve, we predicted that fluorescein absorption would be considerable, but less than that of passively absorbed L-glucose which is a smaller molecule (MW = 180). When the two compounds were gavaged into house sparrows simultaneously, the birds absorbed significantly less fluorescein (42 +/- 8%) than L-glucose (82 +/- 7%), as predicted, and absorptions of the two were correlated as one would predict if they shared the same pathway. We removed intestinal tissue 10 min after gavage with sodium fluorescein and determined the cellular location of the compound's fluorescence using confocal laser microscopy. The fluorescent signal was found primarily in the paracellular space. In contrast, in the same type of experiment using instead the similar-sized fluorescent lipophilic compound rhodamine 123 (MW = 381), most fluorescence appeared inside enterocytes, as expected for a compound that diffuses across the apical membrane. Thus, results from all the experiments are consistent with the hypothesis that hydrophilic fluorescein is absorbed primarily via a paracellular pathway. These methods could be applied to visualize absorption pathways of other compounds in other intact animals.     

Gastrointestinal digestion and absorption of Pen j 1, a major allergen from Kuruma prawn, Penaeus japonicus

Tropomyosin had been identified as a major allergen in shrimp. The digestion and absorption of tropomyosin (Pen j 1) from kuruma prawn were investigated by ex vivo, in vitro, and in vivo techniques in order to elucidate the relationship between the allergenicity of the allergen and its gastrointestinal behavior. Pen j 1 transported the Caco-2 monolayer in a dose-dependent manner, and also enhanced the permeability of lucifer yellow, a marker of paracellular transportation, at high concentrations of the allergen. Studies with everted sacs revealed that Pen j 1 was rapidly degraded to small peptides (MW<3.5 kDa) and amino acids by intestinal proteases and absorbed from enterocytes. Furthermore, Pen j 1 orally administered to rats tended to remain in the stomach rather than in the small intestine, after which the allergen moved to the epithelial cells. These observations suggest that Pen j 1 may be absorbed via the gastric mucosa prior to its digestion in the intestines.     

The sweet life: diet sugar concentration influences paracellular glucose absorption

Small birds and bats face strong selection pressure to digest food rapidly in order to reduce digesta mass carried during flight. One mechanism is rapid absorption of a high proportion of glucose via the paracellular pathway (transfer between epithelial cells, not mediated by transporter proteins). Intestinal paracellular permeability to glucose was assessed for two nectarivorous passerines, the Australian New Holland honeyeater (Phylidonyris novaehollandiae) and African white-bellied sunbird (Cinnyris talatala) by measuring the bioavailability of radiolabelled, passively absorbed l -glucose. Bioavailability was high in both species and increased with diet sugar concentration (honeyeaters, 37 and 81% and sunbirds, 53 and 71% for 250 and 1000mmoll-1 sucrose diets, respectively). We conclude that the relative contribution of paracellular to total glucose absorption increases with greater digesta retention time in the intestine, and paracellular absorption may also be modulated by factors such as intestinal lumen osmolality and interaction with mediated glucose uptake. The dynamic state of paracellular absorption should be taken into account in future studies.     

Electroaffinity in paracellular absorption of hydrophilic d-dipeptides by sparrow intestine

We previously demonstrated size selectivity in the absorption of nonelectrolyte hydrosoluble probes in birds, presumably by the paracellular pathway. Our goal in this study was to determine the charge selectivity in the absorption of hydrosoluble d-dipeptides, because there have been no studies of the electroaffinity of this absorption pathway in birds. For this purpose isosmotic solutions with two hydrophilic d-dipeptides: serine-lysine (positive at pH 7.4) and serine-aspartic (negative at pH 7.4) were gavaged into the stomach in nonanesthetized house sparrows (Passer domesticus), and injected into the pectoralis with a syringe in different trials. Fractional absorption was calculated as F = [AUC by gavage)]/[AUC by injection] (AUC = area under the curve of plasma probe concentration vs. time). Fractional absorption was significantly higher for the positively charged than negatively charged dipeptide (respectively, F=0.30±0.05 vs. F=0.17±0.03). These findings give the first evidence of cation selectivity by the paracellular route in the absorption of hydrosoluble solutes in the small intestine in birds.     

Allometry of paracellular absorption in birds

Water-soluble nutrients can be absorbed across the intestinal epithelium by transcellular and paracellular processes. Recent studies suggest that small birds (<180 g) have more extensive paracellular absorption of glucose than nonflying mammals. This may be a feature that compensates for a reduced small intestine size because small birds have smaller mass-corrected intestinal length than do nonflying mammals, but the difference diminishes in larger birds. We hypothesized that if this explanation were correct, there would be a negative correlation between paracellular absorption and body mass in birds and that larger birds would have paracellular absorption comparable to that of nonflying mammals. We tested this hypothesis, using consistent methodology, by measuring the extent of absorption of a series of inert carbohydrate probes in heavier bird species (>300 g) selected from diverse taxa: American coots, mallards, pheasants, and pigeons. Absorption of carbohydrate probes was inversely related to body mass in birds, and absorption of these probes in large birds (>500 g) was comparable to absorption measurements in nonflying mammals. Higher paracellular uptake in the smaller avian species may offer a physiologically inexpensive means of nutrient absorption to compensate for a reduced small intestine size but may make those species more vulnerable to toxicant absorption.     

Gossypol affects ion transport in the isolated intestine of the seawater adapted eel, Anguilla anguilla

Cottonseed (Gossypium sp.) meals are protein rich and inexpensive, but the presence of the polyphenolic dialdehyde, gossypol, is responsible of many toxic effects in animals including fishes. Recently an effect on the transepithelial ion transport in rat colon has been demonstrated. In this study we investigated the effect of gossypol on the transepithelial electrical parameters of the isolated intestine of seawater adapted eel, Anguilla anguilla, by employing a Ussing chamber technique. We showed that the addition of gossypol to the perfusion media reduced short circuit current (I(sc)), a measure of Cl- active absorption in this tissue, and increased tissue conductance (g(t)). The observation that the effect of gossypol on both I(sc) and g(t) was modified by the pretreatment with TFP, a calmodulin inhibitor, suggests that the substance acts via a Ca2+ calmodulin pathway and excludes the possibility that the observed effects were due to a cytotoxic action. In addition, experiments performed in the presence of verapamil suggest that the polyphenolic pigment increases Ca2+ influx. It is likely that gossypol stimulates a basolateral quinine sensitive K+ conductance producing a K+ flux in absorptive direction that explains the reduction of I(sc). In addition dilution potential experiments showed that the polyphenolic aldehyde increases the anion conductance of the paracellular pathway. In conclusion our study suggests that gossypol alters ion transport in eel intestine by acting on both transcellular and paracellular pathways. Since the intestine is an important organ for maintaining the water and ion balance in seawater adapted fish, it is conceivable that gossypol could impair the ability of the animals to adapt to the environment.     

Role of the Paracellular Pathway in Isotonic Fluid Movement Across the Renal Tubule

Evidence for a highly permeable paracellular shunt in the proximal tubule is reviewed. The paracellular pathway is described as a crucial site for the regulation of net absorption and for solute-solvent interaction. Available models for the coupling of salt and water transport are assessed with respect to the problem of isotonic water movement. Two new models are proposed taking into account that the tight junctions are permeable to salt and water and that active transport sites for sodium are distributed uniformly along the lateral cell membrane. The first model (continuous model) is a modification of Diamond and Bossert''s proposal using different assumptions and boundary conditions. No appreciable standing gradients are predicted by this model. The second model (compartmental model) is an expansion of Curran''s double membrane model by including additional compartments and driving forces. Both models predict a reabsorbate which is not isosmotic. For the particular case of the proximal tubule it is shown that in the presence of a leaky epithelium these deviations from isotonicity might have escaped experimental observation.     

Structure of zonulae occludentes and the permeability of the epithelium to short-chain fatty acids in the proximal and the distal colon of guinea pig

Summary Absorption of short-chain fatty acids has been studied in the proximal and the distal colon of anaesthetized guinea pigs. Segments were perfused with a solution similar in chemical composition to that of normal colonic fluids. In the proximal colon the permeability of the mucosa was similar for acetate, propionate and butyrate. For acetate the permeability was significantly higher in the proximal than in the distal colon, and the reverse was seen for butyrate. In the distal colon the short-chain fatty acids seem to be absorbed mainly in the undissociated form due to their lipid solubility: a paracellular pathway for the dissociated molecules is of no major importance. In the proximal colon, on the other hand, a considerable portion of acetate and propionate disappears in the ionized form. Light microscopy (semithin sections) and electron microscopy (freeze-fracture replicas) showed remarkable morphological differences between the proximal and the distal colon. Leaky spots with only few strands were present in the zonulae occludentes between the epithelial cells at the surface of the proximal colon. In the distal colon the junctions between the cells were more compact, and significantly more strands separated the lumen from the intercellular space. These results suggest that short-chain fatty acids could be absorbed by a paracellular pathway in the proximal colon, and not in the distal colon. In the proximal colon the number of strands of the zonulae occludentes between surface cells and that between cryptal cells was similar. On the contrary, in the distal colon significantly more strands were present between surface cells than between cryptal cells. Morphological and physiological considerations suggest that absorption of short-chain fatty acids in the crypts is negligible.     

The effect of gastric inhibitory polypeptide on intestinal glucose absorption and intestinal motility in mice

Gastric inhibitory polypeptide (GIP) is released from the small intestine upon meal ingestion and increases insulin secretion from pancreatic β cells. Although the GIP receptor is known to be expressed in small intestine, the effects of GIP in small intestine are not fully understood. This study was designed to clarify the effect of GIP on intestinal glucose absorption and intestinal motility. Intestinal glucose absorption in vivo was measured by single-pass perfusion method. Incorporation of [¹⁴C]-glucose into everted jejunal rings in vitro was used to evaluate the effect of GIP on sodium-glucose co-transporter (SGLT). Motility of small intestine was measured by intestinal transit after oral administration of a non-absorbed marker. Intraperitoneal administration of GIP inhibited glucose absorption in wild-type mice in a concentration-dependent manner, showing maximum decrease at the dosage of 50 nmol/kg body weight. In glucagon-like-peptide-1 (GLP-1) receptor-deficient mice, GIP inhibited glucose absorption as in wild-type mice. In vitro examination of [¹⁴C]-glucose uptake revealed that 100 nM GIP did not change SGLT-dependent glucose uptake in wild-type mice. After intraperitoneal administration of GIP (50 nmol/kg body weight), small intestinal transit was inhibited to 40% in both wild-type and GLP-1 receptor-deficient mice. Furthermore, a somatostatin receptor antagonist, cyclosomatostatin, reduced the inhibitory effect of GIP on both intestinal transit and glucose absorption in wild-type mice. These results demonstrate that exogenous GIP inhibits intestinal glucose absorption by reducing intestinal motility through a somatostatin-mediated pathway rather than through a GLP-1-mediated pathway.     

Transport of chlorine in the proximal tubule. Its effects on water-electrolyte absorption

Several studies in rat kidney have established that an appreciable fraction of proximal absorption is passive in nature and occurs across the highly conductive paracellular pathway. Passive absorption is generally ascribed to the transepithelial Cl- distribution, luminal Cl- activity (alpha lCl) being higher than plasma Cl- activity (alpha pCl). The inequality alpha lCl greater than alpha pCl generates a transepithelial diffusion potential, lumen positive, which taken together with the chemical potential differences of Cl- and Na+ across the epithelium gives rise to transepithelial electrochemical potential differences for Cl- and Na+ favoring their absorption. The alpha lCl greater than alpha pCl distribution is traditionally ascribed to preferential bicarbonate absorption. We argue that HCO3- absorption alone cannot generate a non equilibrium transepithelial Cl- distribution. Other mechanisms are necessary. Our measurements in amphibian proximal tubule demonstrate that the intracellular Cl- activity, alpha cCl, is higher than the theoretical value predicted for equilibrium. This distribution is the result of two basolateral coupled transport processes (Cl-/HCO3- exchange and Cl-/Na+ cotransport). It contributes to the exit of Cl- from cell to lumen (by passive diffusion and K+/Cl- cotransport), yielding alpha lCl values higher than the theoretical value for equilibrium with regard to plasma. Thus, a small transcellular flux of Cl- (without solvent) proceeds from interstitium to lumen. It compensates the dissipative tendency of a much higher paracellular Cl- absorptive flux (in association with water) on the transepithelial Cl- gradient. The result is a steady-state luminal Cl- distribution above equilibrium, along the major part of the proximal tubule.     

Electrophysiology of flounder intestinal mucosa. I. Conductance properties of the cellular and paracellular pathways

We evaluated the conductances for ion flow across the cellular and paracellular pathways of flounder intestine using microelectrode techniques and ion-replacement studies. Apical membrane conductance properties are dominated by the presence of Ba-sensitive K channels. An elevated mucosal solution K concentration, [K]m, depolarized the apical membrane potential (psi a) and, at [K]m less than 40 mM, the K dependence of psi a was abolished by 1-2 mM mucosal Ba. The basolateral membrane displayed Cl conductance behavior, as evidenced by depolarization of the basolateral membrane potential (psi b) with reduced serosal Cl concentrations, [Cl]s. psi b was unaffected by changes in [K]s or [Na]s. From the effect of mucosal Ba on transepithelial K selectivity, we estimated that paracellular conductance (Gp) normally accounts for 96% of transepithelial conductance (Gt). The high Gp attenuates the contribution of the cellular pathway to psi t while permitting the apical K and basolateral Cl conductances to influence the electrical potential differences across both membranes. Thus, psi a and psi b (approximately 60 mV, inside negative) lie between the equilibrium potentials for K (76 mV) and Cl (40 mV), thereby establishing driving forces for K secretion across the apical membrane and Cl absorption across the basolateral membrane. Equivalent circuit analysis suggests that apical conductance (Ga approximately equal to 5 mS/cm2) is sufficient to account for the observed rate of K secretion, but that basolateral conductance (Gb approximately equal to 1.5 mS/cm2) would account for only 50% of net Cl absorption. This, together with our failure to detect a basolateral K conductance, suggests that Cl absorption across this barrier involves KCl co-transport.     

Basolateral aromatic amino acid transporter TAT1 (Slc16a10) functions as an efflux pathway

Basolateral efflux is a necessary step in transepithelial (re)absorption of amino acids from small intestine and kidney proximal tubule. The best characterized basolateral amino acid transporters are y+LAT1-4F2hc and LAT2-4F2hc that function as obligatory exchangers and thus, do not contribute to net amino acid (re)absorption. The aromatic amino acid transporter TAT1 was shown previously to localize basolaterally in rat's small intestine and to mediate the efflux of L-Trp in the absence of exchange substrate, upon expression in Xenopus oocytes. We compared here the amino acid influx and efflux via mouse TAT1 in Xenopus oocytes. The results show that mTAT1 functions as facilitated diffusion pathway for aromatic amino acids and that its properties are symmetrical in terms of selectivity and apparent affinity. We show by real-time RT-PCR that its mRNA is highly expressed in mouse small intestine mucosa, kidney, liver, and skeletal muscle as well as present in all other tested tissues. We show that mTAT1 is not N-glycosylated and that it localizes to the mouse kidney proximal tubule. This expression is characterized by an axial gradient similar to that of the luminal neutral amino acid transporter B0AT1 and shows the same basolateral localization as 4F2hc. mTAT1 also localizes to the basolateral membrane of small intestine enterocytes and to the sinusoidal side of perivenous hepatocytes. In summary, we show that TAT1 is a basolateral epithelial transporter and that it can function as a net efflux pathway for aromatic amino acids. We propose that it, thereby, may supply parallel exchangers with recycling uptake substrates that could drive the efflux of other amino acids.     

Modulations of food-derived substances on intestinal permeability in Caco-2 cell monolayers

The effects of more than 300 kinds of food extracts on intestinal permeability were investigated in Caco-2 cells with the use of model compounds: Lucifer Yellow (LY) for the paracellular pathway, Fluorescein (FC) for the monocarboxylic acid transporter-mediated pathway, and Rhodamine 123 (RH) for the p-glycoprotein-mediated efflux pathway. With several extracts of increasing or decreasing LY permeation, increasing FC or RH permeation was also observed, indicating modulation by dietary substances in several pathways for intestinal absorption.     

Increased zinc absorption but not secretion in the small intestine of metallothionein-null mice

Metallothionein (MT) has been assigned a role in intestinal Zn absorption and secretion. The influence of MT was investigated in isolated segments of the small intestine from mice lacking the expression of MT I and II genes (MT−/−). To measure Zn absorption, washed 10- to 12-cm segments of the proximal and distal small intestine of MT−/− and control MT+/+ mice were filled with ⁶⁵Zn as ZnSO₄ (10 μg/mL), and the amount of ⁶⁵Zn appearing in the external buffer was measured over 4 h. To measure Zn secretion, the same procedure was followed using everted gut segments. The ⁶⁵Zn absorption from the small intestine was significantly greater in MT−/− mice, but only in the absence of albumin. In the proximal small intestine, the inclusion of 2% albumin in the external buffer significantly increased Zn absorption from 6.8% (no albumin) to 13.2% (with albumin) for MT−/−, and from 4.9% (no albumin) to 14.2% (with albumin) for MT+/+. In the distal segment, the respective values, with and without albumin respectively were 9.5% and 15.1% for MT−/− mice and 4.3% and 16.1% for MT+/+ mice. Regarding ⁶⁵Zn secretion, there was no difference between MT+/+ and MT−/− in either segment. However, the rate of secretion was higher in the proximal small intestine for both genotypes. Although it can be demonstrated that MT limits Zn absorption under controlled conditions in vitro, the ability of albumin to overcome this effect emphasizes the importance of circulating ligands in Zn transport.     

Heligmosomoides polygyrus: inoculum size and glucose, ion, and gas fluxes in the small intestine of mice

Female CDI mice were inoculated with 10, 50, 100, 250, or 500 larvae of Heligmosomoides polygyrus. At Days 7, 9, and 12 after infection, the anterior third of the small intestine was perfused using an in vivo technique. The distribution of worms in the mouse intestine was determined after 7, 9, and 12 days. All worms that were recovered were from the proximal half of the small intestine. When compared to uninfected controls, there was a significant increase (+56%) in glucose absorption of the small intestine at Day 7 after infection with inocula of 50 and 100 larvae; at Day 9, glucose absorption was significantly increased with a 10-larvae inoculum. A decrease in glucose absorption occurred at Days 7 and 9 after infection with a 500-larvae inoculum. Net water absorption was significantly increased (+183%) with the 50- and 100-larvae inocula at Day 7, but was significantly reduced at Day 9 after infection with the 50-, 100-, 250-, and 500-larvae inocula. Both Cl- and Na+ absorption were significantly increased with the 50-, 100-, and 250-larvae inocula at Day 7 after infection; at 9 and 12 days, there was significant net secretion of both ions. In control mice, there was net secretion of K+, while with the 50-, 100-, and 250-larvae inocula on Day 7 there was significant net absorption of K+ ions.(ABSTRACT TRUNCATED AT 250 WORDS)     

Recent advances in our understanding of 1,25-dihydroxyvitamin D(3) regulation of intestinal calcium absorption

Calcium is required for many cellular processes including muscle contraction, nerve pulse transmission, stimulus secretion coupling and bone formation. The principal source of new calcium to meet these essential functions is from the diet. Intestinal absorption of calcium occurs by an active transcellular path and by a non-saturable paracellular path. The major factor influencing intestinal calcium absorption is vitamin D and more specifically the hormonally active form of vitamin D, 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). This article emphasizes studies that have provided new insight related to the mechanisms involved in the intestinal actions of 1,25(OH)(2)D(3). The following are discussed: recent studies, including those using knock out mice, that suggest that 1,25(OH)(2)D(3) mediated calcium absorption is more complex than the traditional transcellular model; evidence for 1,25(OH)(2)D(3) mediated active transport of calcium by distal as well as proximal segments of the intestine; 1,25(OH)(2)D(3) regulation of paracellular calcium transport and the role of 1,25(OH)(2)D(3) in protection against mucosal injury.     

Comparative in situ study of the intestinal absorption of aluminum, manganese, nickel, and lead in rats

This comparative study of the intestinal absorption of four toxic metals (aluminum, manganese, nickel, and lead) carried out in rats using the in situ intestinal perfusion technique was able to measure the partition of each metal between the intestine (intestinal retention), the blood circulation, and target tissues after 1 h. The perfused metal solutions were at concentrations likely to occur during oral intoxication. It was found that aluminum (48 and 64 mM), even as a citrate complex, crossed the brush border with difficulty (0.4% of the perfused amount); about 60% of this was retained in the intestine and the remainder was found in target tissues (about 36%). Conversely, lead (4.8–48 μM) penetrated the intestine more easily (about 35% of the perfused amount), was slightly retained (about 12% of the input), and was soon found in the tissues (about 58% of the input) and to a lesser degree in circulation (about 29%). Within the same concentration range, nickel and manganese showed certain similarities, such as a reduced crossing of the brush border proportional to the increase in the concentration perfused (0.17–9.5 mM). There was similar intestinal retention and absorption (about 80% and 20% of the input, respectively). Manganese crossed the brush border more easily and was diffused more rapidly into tissues. Finally, the addition of equimolar amounts of iron (4.7 mM) produced opposite effects on the absorption of the two elements, inhibiting manganese and showing a trend to increase in nickel absorption. This could be the result of competition between Fe²⁺ and Mn²⁺ for the same transcellular transporters and the slight predominance of paracellular mechanism in the event of “Fe²⁺-Ni²⁺” association.     

A Comparison of mucosal surface area and villous histology in small intestines of the Brazilian free‐tailed bat (Tadarida brasiliensis) and the mouse (Mus musculus)

Studies on birds have led to the hypothesis that increased intestinal absorption between enterocytes (paracellular) evolved as a compensation for smaller intestinal size in fliers, which was perhaps selected to minimize the mass of digesta carried. This hypothesis predicts that bats will also exhibit relatively reduced intestinal size and high paracellular absorption, compared with nonflying mammals. Published studies on three bat species indicate relatively high paracellular absorption. One mechanism for increasing paracellular absorption per cm² small intestine (SI) is increased number of tight junctions (TJs) across which paracellular absorption occurs. To our knowledge, we provide the first comparative analysis of enterocyte size and number in flying and nonflying mammals. Intestines of insectivorous bats Tadarida brasiliensis were compared with Mus musculus using hematoxylin and eosin staining method. Bats had shorter and narrower SIs than mice, and after correction for body size difference by normalizing to mass^3/4, the bats had 40% less nominal surface area than the mouse, as predicted. Villous enhancement of surface area was 90% greater in the bat than in the mouse, mainly because of longer villi and a greater density of villi in bat intestines. Bat and mouse were similar in enterocyte diameter. Bats exceeded mice by 54.4% in villous area per cm length SI and by 95% in number of enterocytes per cm² of the nominal surface area of the SI. Therefore, an increased density of TJs per cm² SI may be a mechanistic explanation that helps to understand the high paracellular absorption observed in bats compared to nonflying mammals. J. Morphol. 276:102–108, 2015. © 2014 Wiley Periodicals, Inc.     

An in vitro examination of intestinal iron absorption in a freshwater teleost, rainbow trout (Oncorhynchus mykiss)

This study investigated the physiological characteristics of intestinal iron absorption in a freshwater teleost, rainbow trout (Oncorhynchus mykiss). Using an in vitro gastro-intestinal sac technique, we evaluated the spatial pattern and concentration dependent profile of iron uptake, and also the influence of luminal chemistry (pH and chelation) on iron absorption. We demonstrated that the iron uptake rate in the anterior intestine is significantly higher than that in the mid and posterior intestine. Interestingly, absorption of iron in the anterior intestine occurs likely via simple diffusion, whereas a carrier-mediated pathway is apparent in the mid and posterior intestine. The uptake of ferric and ferrous iron appeared to be linear over the entire range of iron concentration tested (0–20 μM), however the uptake of ferrous iron was significantly higher than that of ferric iron at high iron concentrations (>15 μM). An increase in mucosal pH from 7.4 to 8.2 significantly reduced iron absorption in both mid and posterior intestine, implying the involvement of a Fe²⁺/H⁺ symporter. Iron chelators (nitrilotriacetic acid and desferrioxamine mesylate) had no effects on iron absorption, which suggests that fish are able to acquire chelated iron via intestine.