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.     

Capacity for absorption of water-soluble secondary metabolites greater in birds than in rodents

Plant secondary metabolites (SMs) are pervasive in animal foods and potentially influence feeding behavior, interspecies interactions, and the distribution and abundance of animals. Some of the major classes of naturally occurring SMs in plants include many water-soluble compounds in the molecular size range that could cross the intestinal epithelium via the paracellular space by diffusion or solvent drag. There are differences among species in paracellular permeability. Using Middle Eastern rodent and avian consumers of fruits containing SMs, we tested the hypothesis that avian species would have significantly higher paracellular permeability than rodent species. Permeability in intact animals was assessed using standard pharmacological methodology to measure absorption of two radiolabeled, inert, neutral water-soluble probes that do not interact with intestinal nutrient transporters, L-arabinose (M_r = 150.1 Da) and lactulose (M_r = 342.3 Da). We also measured absorption of labeled 3-O-methyl-D-glucose (3OMD-glucose; M_r = 194.2 Da), which is a nonmetabolized analogue of D-glucose that is passively absorbed through the paracellular space but also transported across the enterocyte membranes. Most glucose was absorbed by all species, but arabinose fractional absorption (f) was nearly three times higher in birds (1.03±0.17, n = 15 in two species) compared to rodents (0.37±0.06, n = 10 in two species) (P<0.001). Surprisingly, the apparent rates of absorption in birds of arabinose exceeded those of 3OMD-glucose. Our findings are in agreement with previous work showing that the paracellular pathway is more prominent in birds relative to nonflying mammals, and suggests that birds may be challenged by greater absorption of water-soluble, dietary SMs. The increased expression of the paracellular pathway in birds hints at a tradeoff: the free energy birds gain by absorbing water-soluble nutrients passively may be offset by the metabolic demands placed on them to eliminate concomitantly absorbed SMs.     

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.     

Paracellular absorption: a bat breaks the mammal paradigm

Bats tend to have less intestinal tissue than comparably sized nonflying mammals. The corresponding reduction in intestinal volume and hence mass of digesta carried is advantageous because the costs of flight increase with load carried and because take-off and maneuverability are diminished at heavier masses. Water soluble compounds, such as glucose and amino acids, are absorbed in the small intestine mainly via two pathways, the transporter-mediated transcellular and the passive, paracellular pathways. Using the microchiropteran bat Artibeus literatus (mean mass 80.6+/-3.7 g), we tested the predictions that absorption of water-soluble compounds that are not actively transported would be extensive as a compensatory mechanism for relatively less intestinal tissue, and would decline with increasing molecular mass in accord with sieve-like paracellular absorption. Using a standard pharmacokinetic technique, we fed, or injected intraperitoneally the metabolically inert carbohydrates L-rhamnose (molecular mass = 164 Da) and cellobiose (molecular mass = 342 Da) which are absorbed only by paracellular transport, and 3-O-methyl-D-glucose (3OMD-glucose) which is absorbed via both mediated (active) and paracellular transport. As predicted, the bioavailability of paracellular probes declined with increasing molecular mass (rhamnose, 90+/-11%; cellobiose, 10+/-3%, n = 8) and was significantly higher in bats than has been reported for laboratory rats and other mammals. In addition, absorption of 3OMD-glucose was high (96+/-11%). We estimated that the bats rely on passive, paracellular absorption for more than 70% of their total glucose absorption, much more than in non-flying mammals. Although possibly compensating for less intestinal tissue, a high intestinal permeability that permits passive absorption might be less selective than a carrier-mediated system for nutrient absorption and might permit toxins to be absorbed from plant and animal material in the intestinal lumen.     

A comparison of pharmacokinetic methods for in vivo studies of nonmediated glucose absorption

Two pharmacokinetic methods are used primarily to assess systematic bioavailability of orally dosed water-soluble compounds in vivo, but there have been no direct comparisons of the estimates obtained. The "area under the curve" (AUC) method employs a single oral dose of probe compound(s) followed by multiple blood sampling to obtain plasma concentration time curves. Separate injection of probe(s) followed by multiple blood sampling is used to calculate fractional elimination rate (K(el)) and distribution pool space (S). The "steady state feeding" method relies on ad lib. feeding of a marked diet, with a single blood sample taken to measure steady state feeding concentration of probe(s); K(el) is estimated from the decline in probe concentration in excreta after injection, with a single blood sample taken to estimate S. We compared these methods directly in the Australian red wattlebird (Anthochaera carnunculata), measuring absorption of (3)H-L-glucose. The K(el) values estimated using the steady state feeding protocol were significantly higher, and estimates of S and bioavailability consequently lower, compared with the AUC protocol. The AUC method relies on fewer assumptions and allows simultaneous comparisons of absorption by mediated and nonmediated (i.e., paracellular) mechanisms but cannot be easily applied to freely feeding animals. The steady state feeding method allows work with smaller species and exploration of the effects of feeding on nutrient uptake but requires careful attention to the validity of assumptions that increase error in the calculations.     

Use of the paracellular way for the intestinal absorption of sugars

Passive absorption of D-Galactose (in the presence of 0.5 mM phlorizin), 2-deoxy-D-glucose and D-Mannitol by rat jejunum has been measured in vivo by perfusion of an intestinal segment with recirculation, along successive absorption periods of 5 or 10 min duration. In the range of 1 to 40 mM concentrations, the three solutes were absorbed at a very similar rate that varied as a lineal function of the concentrations in the perfusion solution. Absorption of 1 mM solute was not modified by the presence of 40 mM glucose or galactose. Passive absorption kinetics suggests processes of simple diffusion or solvent drag. The use of paracellular way for the passive absorption is supported by the fact that triaminopyrimidine (TAP) and protamine, which decrease the permeability through the tight junctions, also inhibit the absorption, with similar characteristics for both actions: TAP inhibition (53%) is very rapid and can be easily reversed, while that of protamine (30%) requires some time of previous exposure, lasts longer and can be reversed by heparin. The same analogy is shown by two actions that enhance the paracellular permeability: theophylline increases (30%) the passive absorption with lasting effect, while luminal hypertony enhances absorption transitorily. The passive absorption of the assayed solutes could be estimated to take place by the paracellular way in at least 50% and probably 70% or even more. The measure of net fluid fluxes reveals that solute fluxes must be prevailingly explained by simple diffusion, as the solvent drag can only play a very minor role.     

Paracellular nutrient absorption in a gum-feeding new world primate, the common marmoset Callithrix jacchus

The common marmoset is one of the few callitrichid species that is not threatened or endangered in the wild, and is widely used in biomedical research, yet relatively little is understood about its digestive physiology. Dietary specialization on plant exudates has lead to relatively reduced small intestines, yet the common marmoset has exceptional dietary breadth, allowing it to successfully utilize a variety of habitats. We predicted that passive, paracellular nutrient absorption would be used by the common marmoset to a greater extent than in other non-flying mammals. We measured the bioavailability and rates of absorption of two metabolically inert carbohydrates not transported by mediated pathways (L-rhamnose and cellobiose, molecular masses of 164 and 342, respectively) to measure paracellular uptake, and of a non-metabolized D-glucose analog (3-O-methyl-D-glucose) to measure total uptake by both mediated and paracellular pathways. We found high bioavailability of 3-O-methyl-D-glucose (83+/-5%), and much higher bioavailability of the paracellular probes than in similarly sized non-flying mammals (30+/-3% and 19+/-2% for L-rhamnose and cellobiose, respectively). Passive, paracellular nutrient absorption accounts for around 30% of total glucose absorption in common marmosets and intestinal permeability is significantly higher than in humans, the only other species of primate measured to date. This may allow the common marmoset to maintain high digestive efficiency when feeding on higher quality foods (fruit, arthropods, gums with higher proportions of simple sugars), in spite of relatively reduced small intestines correlated with adaptations for fermentative digestion of plant gums. We find no evidence to support, in primates, the hypothesis that reliance on paracellular nutrient absorption should increase with body size in mammals, but suggest instead that it may be associated with small body size and/or taxon-specific adaptations to diet.     

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.     

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.     

Digestive physiology: a view from molecules to ecosystem

Digestive physiology links physiology to applications valued by society, such as understanding ecology and ecological toxicology and managing and conserving species. Here I illustrate this applied and integrative perspective with several avian case studies. The match between digestive features and diet provides evidence of tradeoffs that preclude doing well on all possible substrates with a single digestive design, and this influences ecological niche partitioning. But some birds, such as wild house sparrow (Passer domesticus) nestlings, are digestively very flexible. Their intestinal maltase activity and mRNA for intestinal maltase glucoamylase specifically and reversibly change when they switch among foods with different starch content. Houses sparrows and many other birds absorb hydrolyzed water-soluble monomers, such as glucose, mainly passively via tight junctions between enterocytes (i.e., paracellular absorption). Such species might be good models for studying this process, which is important biomedically for absorption of drugs. High paracellular absorption may enhance absorption of low molecular weight, natural water-soluble toxins. Also, reliance of American robins (Turdus migratorius) on passive absorption makes them less sensitive to types of plant toxins that inhibit mediated glucose absorption, such as phlorizin or the flavanoid isoquercetrin. Determining absorption of environmental contaminants is another important ecological application. Common loon (Gavia immer) chicks absorbed 83% of methyl mercury in fish meals, eliminate the mercury slowly, and consequently are predicted in the wild to bioaccumulate mercury to higher concentrations than in their foods. The quantitative details can be used to set regulatory levels for mercury that will protect wildlife.     

Contribution of solvent drag through intercellular junctions to absorption of nutrients by the small intestine of the rat

The lumen of the small intestine in anesthetized rats was recirculated with 50 ml perfusion fluid containing normal salts, 25 mM glucose and low concentrations of hydrophilic solutes ranging in size from creatinine (mol wt 113) to Inulin (mol wt 5500). Ferrocyanide, a nontoxic, quadrupally charged anion was not absorbed; it could therefore be used as an osmotically active solute with reflection coefficient of 1.0 to adjust rates of fluid absorption, Jv, and to measure the coefficient of osmotic flow, Lp. The clearances from the perfusion fluid of all other test solutes were approximately proportional to Jv. From Lp and rates of clearances as a function of Jv and molecular size we estimate (a) the fraction of fluid absorption which passes paracellularly (approx. 50%), (b) coefficients of solvent drag of various solutes within intercellular junctions, (c) the equivalent pore radius of intercellular junctions (50 A) and their cross sectional area per unit path length (4.3 cm per cm length of intestine). Glucose absorption also varied as a function of Jv. From this relationship and the clearances of inert markers we calculate the rate of active transport of glucose, the amount of glucose carried paracellularly by solvent drag or back-diffusion at any given Jv and luminal glucose concentration and the concentration of glucose in the absorbate. The results indicate that solvent drag through paracellular channels is the principal route for intestinal transport of glucose or amino acids at physiological rates of fluid absorption and concentration. In the absence of luminal glucose the rate of fluid absorption and the clearances of all inert hydrophilic solutes were greatly reduced. It is proposed that Na-coupled transport of organic solutes from lumen to intercellular spaces provides the principal osmotic force for fluid absorption and triggers widening of intercellular junctions, thus promoting bulk absorption of nutrients by solvent drag. Further evidence for regulation of channel width is provided in accompanying papers on changes in electrical impedance and ultrastructure of junctions during Na-coupled solute transport.     

L-glucose absorption in house sparrows (<Emphasis Type="Italic">Passer domesticus</Emphasis>) is nonmediated

We previously demonstrated in intact house sparrows substantial absorption in vivo of L-glucose, the stereoisomer of D-glucose that is assumed not to interact with the intestines D-glucose transporter. Results of some studies challenge this assumption for other species. Therefore, we tested it in vitro and in vivo, based on the principle that if absorption of a compound (L-glucose) is mediated, then absorption of its tracer will be competitively inhibited by high concentrations of either the compound itself or other compounds (e.g., D-glucose) whose absorption is mediated by the same mechanism. An alternative hypothesis that L-glucose absorption is primarily paracellular predicts that its absorption in vivo will be increased (not decreased) in the presence of D-glucose, because the permeability of this pathway is supposedly enhanced when Na⁺-coupled glucose absorption occurs. First, using intact tissue in vitro, we found that uptake of tracer-radiolabeled L-glucose was not significantly inhibited by high concentrations (100 mM) of either L-glucose or 3-O-methyl-D-glucose, a non-metabolizable but actively transported D-glucose analogue. Second, using intact house sparrows, we found that fractional absorption of the L-glucose tracer was significantly increased, not reduced, when gavaged along with 200 mM 3-O-methyl-D-glucose. This result was confirmed in another experiment where L-glucose fractional absorption was significantly higher in the presence vs. absence of food in the gut. The greater absorption was apparently not due simply to longer retention time of digesta, because no significant difference was found among retention times. Our results are consistent with the idea that L-glucose is absorbed in a non-mediated fashion, largely via the paracellular pathway in vivo.Abbreviations AUC area under the curve - 3OMD-glucose 3-O-methyl-D-glucose Communicated by I.D. Hume     

Paracellular Absorption Is Relatively Low in the Herbivorous Egyptian Spiny-Tailed Lizard,Uromastyx aegyptia

Absorption of small water-soluble nutrients in vertebrate intestines occurs both by specific, mediated transport and by non-specific, passive, paracellular transport. Although it is apparent that paracellular absorption represents a significant route for nutrient absorption in many birds and mammals, especially small, flying species, its importance in ectothermic vertebrates has not previously been explored. Therefore, we measured fractional absorption (ƒ) and absorption rate of three paracellular probes (arabinose, l-rhamnose, cellobiose) and of 3-O-methyl d-glucose (absorbed by both mediated and paracellular pathways) by the large herbivorous lizard, Uromastyx aegyptia, to explore the relative importance of paracellular and mediated transport in an ectothermic, terrestrial vertebrate. Fractional absorption of 3-O-methyl d-glucose was high (ƒ = 0.73±0.04) and similar to other vertebrates; ƒ of the paracellular probes was relatively low (arabinose ƒ = 0.31±0.03, l-rhamnose ƒ = 0.19±0.02, and cellobiose ƒ = 0.14±0.02), and decreased with molecular mass, a pattern consistent with other vertebrates. Paracellular absorption accounted for approximately 24% of total 3-O-methyl d-glucose uptake, indicating low reliance on this pathway for these herbivorous lizards, a pattern similar to that found in other terrestrial vertebrates, and different from small flying endotherms (both birds and bats).     

Ionic conductance pathways in the mouse medullary thick ascending limb of Henle. The paracellular pathway and electrogenic Cl- absorption

Net Cl- absorption in the mouse medullary thick ascending limb of Henle (mTALH) involves a furosemide-sensitive Na+:K+:2 Cl- apical membrane symport mechanism for salt entry into cells, which occurs in parallel with a Ba++-sensitive apical K+ conductance. The present studies, using the in vitro microperfused mouse mTALH, assessed the concentration dependence of blockade of this apical membrane K+-conductive pathway by Ba++ to provide estimates of the magnitudes of the transcellular (Gc) and paracellular (Gs) electrical conductances (millisiemens per square centimeter). These studies also evaluated the effects of luminal hypertonicity produced by urea on the paracellular electrical conductance, the electrical Na+/Cl- permselectivity ratio, and the morphology of in vitro mTALH segments exposed to peritubular antidiuretic hormone (ADH). Increasing luminal Ba++ concentrations, in the absence of luminal K+, produced a progressive reduction in the transcellular conductance that was maximal at 20 mM Ba++. The Ba++-sensitive transcellular conductance in the presence of ADH was 61.8 +/- 1.7 mS/cm2, or approximately 65% of the total transepithelial conductance. In phenomenological terms, the luminal Ba++-dependent blockade of the transcellular conductance exhibited negative cooperativity. The transepithelial osmotic gradient produced by luminal urea produced blebs on apical surfaces, a striking increase in shunt conductance, and a decrease in the shunt Na+/Cl- permselectivity (PNa/PCl), which approached that of free solution. The transepithelial conductance obtained with luminal 800 mM urea, 20 mM Ba++, and 0 K+ was 950 +/- 150 mS/cm2 and provided an estimate of the maximal diffusion resistance of intercellular spaces, exclusive of junctional complexes. The calculated range for junctional dilution voltages owing to interspace salt accumulation during ADH-dependent net NaCl absorption was 0.7-1.1 mV. Since the Ve accompanying ADH-dependent net NaCl absorption is 10 mV, lumen positive, virtually all of the spontaneous transepithelial voltage in the mouse mTALH is due to transcellular transport processes. Finally, we developed a series of expressions in which the ratio of net Cl- absorption to paracellular Na+ absorption could be expressed in terms of a series of electrical variables. Specifically, an analysis of paired measurement of PNa/PCl and Gs was in agreement with an electroneutral Na+:K+:2 Cl- apical entry step. Thus, for net NaCl absorption, approximately 50% of Na+ was absorbed via a paracellular route.     

The capacity for paracellular absorption in the insectivorous bat Tadarida brasiliensis

Water-soluble nutrients are absorbed by the small intestine via transcellular and paracellular processes. The capacity for paracellular absorption seems greater in fliers than in nonfliers, although that conclusion rests mainly on a comparison of flying birds and nonflying mammals because only two frugivorous bat species have been studied. Furthermore, the bats studied so far were relatively large (>85 g, compared with most bat species which are <20 g) and were not insectivores (like about 70 % of bat species). We studied the small (11 g) insectivorous bat Tadarida brasiliensis and tested the prediction that the capacity for paracellular absorption would be as high as in the other bat and avian species studied so far, well above that in terrestrial, nonflying mammals. Using standard pharmacokinetic technique, we measured the extent of absorption (fractional absorption = f) of inert carbohydrate probes: L-arabinose (MM = 150.13) absorbed exclusively by paracellular route and 3OMD-glucose (MM = 194) absorbed both paracellularly and transcellularly. As predicted, the capacity of paracellular absorption in this insectivorous bat was high (L-arabinose f = 1.03 ± 0.14) as in other frugivorous bats and small birds. Absorption of 3OMD-glucose was also complete (f = 1.09 ± 0.17), but >80 % was accounted for by paracellular absorption. We conclude that passive paracellular absorption of molecules of the size of amino acids and glucose is extensive in this bat and, generally in bats, significantly higher than that in nonflying mammals, although the exact extent can be somewhat lower or higher depending on molecule size, polarity and charge.     

A comparative study of electrochemically and fluorometrically addressed molecular reporter groups: effects of protein microenvironment

To probe the effects of protein microenvironment on electrochemically and fluorometrically addressed molecular reporter groups, genetically engineered apo-cytochrome c peroxidase derivatives W51C, A174C, K243C, and S246C, each containing a single cysteine residue, were labeled at identical sites with two kinds of microenvironment sensitive reporters, either an electrochemically active sulfhydryl-reactive reagent, [Ru(II)(NH(3))(4)(1,10-phenanthroline-5-maleimide)](PF(6))(2) [RuPA4] or a fluorescent 6-acryloyl-2-dimethylaminonaphthalene [acrylodan] probe. Two types of sites were labeled with each probe based on their predicted solvent accessibilities from the known structure for holo-cytochrome c peroxidase. One set of sites (K243C and S246C) was selected to be completely solvent exposed, while the other two sites (W51C and A174C) were less accessible, residing in or near the heme binding site. Spectroscopic properties of the fluorescent probe were consistent with predictions for relative solvent accessibilities; however, even the less solvent accessible probes reported a quite polar environment, suggesting that this region of the apo-protein is either substantially solvent exposed or undergoes significant dynamic motion. A linear correlation was observed between the lambda(max) of the metal to ligand charge-transfer (MLCT) absorption band of the RuPA4 complex and the acrylodan emission maximum for the four labeled apo-protein variants. The same trend occurred for the formal potential of RuPA4 versus the acrylodan emission maximum, with the exception of electrochemical probe behavior at position 174, possibly due to specific probe-protein interactions.     

Paracellular absorption in laboratory mice: Molecule size-dependent but low capacity

Water-soluble nutrients are absorbed by the small intestine via transcellular and paracellular processes. The capacity for paracellular absorption seems lower in nonfliers than in fliers, although that conclusion rests largely on a comparison of relatively larger nonflying mammals (> 155 g) and relatively smaller flying birds (< 155 g). We report on paracellular absorption in laboratory mice, the smallest nonflying mammal species studied to date. Using a standard pharmacokinetic technique, we measured the extent of absorption (fractional absorption = f) of inert carbohydrate probes: l-arabinose (M_r = 150.13 Da) and cellobiose (342.3) that are absorbed exclusively by the paracellular route, and 3-O-methyl d-glucose (3OMD-glucose) (M_r = 194) absorbed both paracellularly and transcellularly. f was measured accurately in urine collection trials of 5–10 h duration. Absorption of 3OMD-glucose by mice was essentially complete (f = 0.95 ± 0.07) and much higher than that for l-arabinose (f = 0.21 ± 0.02), indicating that in mice, like other nonflying mammals, > 80% of glucose is absorbed by mediated process(es) rather than the passive, paracellular route. As in all other vertebrates, absorption of cellobiose (f = 0.13 ± 0.02) was even lower than that for l-arabinose, suggesting an equivalent molecular size cut-off for flying and nonflying animals and thus a comparable effective TJ aperture. An important ecological implication is that smaller water-soluble plant secondary metabolites that have been shown to be absorbed by the paracellular path in cell culture, such as phenolics and alkaloids, might be absorbed in substantial amounts by bats and small birds relative to nonflying mammals such as mice.     

Jejunal Creatine Absorption: What is the Role of the Basolateral Membrane?

The mechanism of the intestinal creatine absorption is not well understood. Previous studies have established the involvement of a CT1 carrier system in jejunal apical membrane. The current research was aimed at completing the picture of creatine absorption. To investigate the process supporting creatine exit from enterocyte, basolateral membrane vesicles isolated from rat jejunum were used. The presence of various symport and antiport mechanisms was searched and a NaCl-dependent electrogenic transport system for creatine was evidenced, which shares some functional and kinetic features with the apical CT1. However, Western blot and immunohistochemical experiments ruled out the presence of a CT1 transporter in the basolateral membrane. Further studies are required to identify the basolateral transport mechanism. However, in the in vivo conditions, the NaCl gradient is inwardly directed, therefore such a mechanism cannot energetically mediate the exit of creatine from the cell into the blood during the absorptive process, but rather it may drive creatine into the enterocyte. To shed more light on the creatine absorption process, a possible creatine movement through the paracellular pathway has been examined using the jejunal tract everted and incubated in vitro. A linear relationship between creatine transport and concentration was apparent both in the mucosa-to-serosa and serosa-to-mucosa directions and the difference between the two slopes suggests that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption. As a matter of fact, when transepithelial water flux is reduced by means of a mucosal hypertonic solution, the opposite creatine fluxes tend to overlap. The findings of the present study suggest that paracellular creatine movement by solvent drag may account for transintestinal creatine absorption.     

Morphological bases for intestinal paracellular absorption in bats and rodents

Flying mammals present unique intestinal adaptations, such as lower intestinal surface area than nonflying mammals, and they compensate for this with higher paracellular absorption of glucose. There is no consensus about the mechanistic bases for this physiological phenomenon. The surface area of the small intestine is a key determinant of the absorptive capacity by both the transcellular and the paracellular pathways; thus, information about intestinal surface area and micro-anatomical structure can help explain differences among species in absorptive capacity. In order to elucidate a possible mechanism for the high paracellular nutrient absorption in bats, we performed a comparative analysis of intestinal villi architecture and enterocyte size and number in microchiropterans and rodents. We collected data from intestines of six bat species and five rodent species using hematoxylin and eosin staining and histological measurements. For the analysis we added measurements from published studies employing similar methodology, making in total a comparison of nine species each of rodents and bats. Bats presented shorter intestines than rodents. After correction for body size differences, bats had ~41% less nominal surface area (NSA) than rodents. Villous enhancement of surface area (SEF) was ~64% greater in bats than in rodents, mainly because of longer villi and a greater density of villi in bat intestines. Both taxa exhibited similar enterocyte diameter. Bats exceeded rodents by ~103% in enterocyte density per cm² NSA, but they do not significantly differ in total number of enterocytes per whole animal. In addition, there is a correlation between SEF and clearance per cm² NSA of L-arabinose, a nonactively transported paracellular probe. We infer that an increased enterocyte density per cm² NSA corresponds to increased density of tight junctions per cm² NSA, which provides a partial mechanistic explanation for understanding the high paracellular absorption observed in bats compared to nonflying mammals.     

Contribution of solvent drag through intercellular junctions to absorption of nutrients by the small intestine of the rat

Summary The lumen of the small intestine in anesthetized rats was recirculated with 50 ml perfusion fluid containing normal salts, 25mm glucose and low concentrations of hydrophilic solutes ranging in size from creatinine (mol wt 113) to Inulin (mol wt 5500). Ferrocyanide, a nontoxic, quadrupally charged anion was not absorbed; it could therefore be used as an osmotically active solute with reflection coefficient of 1.0 to adjust rates of fluid absorption,J _v , and to measure the coefficient of osmotic flow,L _p . The clearances from the perfusion fluid of all other test solutes were approximately proportional toJ _v . FromL _p and rates of clearances as a function ofJ _v and molecular size we estimate (a) the fraction of fluid absorption which passes paracellularly (approx. 50%), (b) coefficients of solvent drag of various solutes within intercellular junctions, (c) the equivalent pore radius of intercellular junctions (50 Å) and their cross sectional area per unit path length (4.3 cm per cm length of intestine). Glucose absorption also varied as a function ofJ _v . From this relationship and the clearances of inert markers we calculate the rate of active transport of glucose, the amount of glucose carried paracellularly by solvent drag or back-diffusion at any givenJ _v and luminal glucose concentration and the concentration of glucose in the absorbate. The results indicate that solvent drag through paracellular channels is the principal route for intestinal transport of glucose or amino acids at physiological rates of fluid absorption and concentration. In the absence of luminal glucose the rate of fluid absorption and the clearances of all inert hydrophilic solutes were greatly reduced. It is proposed that Na-coupled transport of organic solutes from lumen to intercellular spaces provides the principal osmotic force for fluid absorption and triggers widening of intercellular junctions, thus promoting bulk absorption of nutrients by solvent drag. Further evidence for regulation of channel width is provided in accompanying papers on changes in electrical impedance and ultrastructure of junctions during Na-coupled solute transport.