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.     

Ex Vivo Intestinal Sacs to Assess Mucosal Permeability in Models of Gastrointestinal Disease

The epithelial barrier is the first innate defense of the gastrointestinal tract and selectively regulates transport from the lumen to the underlying tissue compartments, restricting the transport of smaller molecules across the epithelium and almost completely prohibiting epithelial macromolecular transport. This selectivity is determined by the mucous gel layer, which limits the transport of lipophilic molecules and both the apical receptors and tight junctional protein complexes of the epithelium. In vitro cell culture models of the epithelium are convenient, but as a model, they lack the complexity of interactions between the microbiota, mucous-gel, epithelium and immune system. On the other hand, in vivo assessment of intestinal absorption or permeability may be performed, but these assays measure overall gastrointestinal absorption, with no indication of site specificity. Ex vivo permeability assays using "intestinal sacs" are a rapid and sensitive method of measuring either overall intestinal integrity or comparative transport of a specific molecule, with the added advantage of intestinal site specificity. Here we describe the preparation of intestinal sacs for permeability studies and the calculation of the apparent permeability (P_app)of a molecule across the intestinal barrier. This technique may be used as a method of assessing drug absorption, or to examine regional epithelial barrier dysfunction in animal models of gastrointestinal disease.     

Interaction exists between matriptase inhibitors and intestinal epithelial cells

The type II trypsin-like transmembrane serine protease matriptase, is mainly expressed in epithelial cells and one of the key regulators in the formation and maintenance of epithelial barrier integrity. Therefore, we have studied the inhibition of matriptase in a non-transformed porcine intestinal IPEC-J2 cell monolayer cultured on polyester membrane inserts by the non-selective 4-(2-aminoethyl)-benzosulphonylfluoride (AEBSF) and four more selective 3-amidinophenylalanine-derived matriptase inhibitors. It was found that suppression of matriptase activity by MI-432 and MI-460 led to decreased transepithelial electrical resistance (TER) of the cell monolayer and to an enhanced transport of fluorescently labelled dextran, a marker for paracellular transport between apical and basolateral compartments. To this date this is the first report in which the inhibition of matriptase activity by synthetic inhibitors has been correlated to a reduced barrier integrity of a non-cancerous IPEC-J2 epithelial cell monolayer in order to describe interaction between matriptase activity and intestinal epithelium in vitro.     

Transcellular and paracellular elements of salt chemosensation in toad skin

Dehydrated toads absorb water by pressing a specialized (seat patch) area of the skin to moist surfaces. This behavior, the water absorption response (WR), is preceded by periods of more limited skin contact (seat patch down, SPD) in which the suitability of the rehydration source is evaluated. WR and SPD behaviors were suppressed on 250 mM NaCl and 200 mM KCl solutions. Ten micromolar amiloride partially restored SPD and WR on NaCl solutions. The addition of 5 mM La(3+) also partially restored the initiation of WR and this effect was additive to the effect of amiloride, suggesting transcellular and paracellular pathways exist in parallel. Similarly, 5 mM La(3+) partially restored the initiation of WR on KCl solutions, to levels comparable to those with K(+)gluconate, suggesting a paracellular pathway for detection of K(+). Hyperosmotic (250 mM) NaCl solutions bathing the mucosal surface rapidly and reversibly increased the paracellular conductance of isolated skin and this increase was partially inhibited by 5 mM La(3+). These results suggest that the regulation of tight junctions has a chemosensory role in toad skin.     

Uncoupling of gate and fence functions of MDCK cells by the actin-depolymerizing reagent mycalolide B

The tight junction serves as a paracellular gate to seal the paracellular space of apposing cells and as a molecular fence to prevent diffusion of membrane proteins and lipids in epithelial cells. Although involvement of the actin cytoskeleton has been considered to be important in these two functions, it remains to be elucidated whether both functions are regulated in a coupled manner or differentially by actin. Treatment of highly polarized MDCK cells with mycalolide B (MB), a recently developed actin-depolymerizing reagent, induced a decrease of transepithelial resistance in a dose- and time-dependent manner with reversibility when the reagent was washed out. Changes in cytoskeletal actin, such as a reduction of cortical actin, irregularity of stress fibers, and punctated actin aggregates, were observed after MB treatment. However, the fence function, as studied by diffusion of apically labeled sphingomyelin/BSA complex, remained intact in the MB-treated MDCK cells. Localization of junctional molecules and apical marker proteins such as E-cadherin, ZO-1, and 114-kDa protein was shown to be unaffected. Furthermore, freeze-fracture study showed apparent tight junction strands. Collectively, MB treatment abolished the paracellular gate but not the fence function of MDCK cells, suggesting that cytoskeletal actin may play differential roles in the gate and fence functions of the tight junction.     

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.     

Claudin-4 is required for AMPK-modulated paraceliular permeability in submandibular gland ceils

Tight junction plays an important rote in mediating paraceUular permeability in epithelia. We previously found that activation of AMP- activated protein kinase （AMPK） increased saliva secretion by modulating paraceUular permeability in submandibular glands. However, the molecular mechanisms underlying AMPK-modulated paraceUular permeability are unknown. In this study, we found that AICAR, an AMPK agonist, increased saliva secretion in the isolated rat submandibular glands, decreased transepithelial electrical resistance （TER）, and increased 4 kDa FITC-dextran flux in cultured SMG-C6 cells. AICAR also induced redistribution of tight junction protein claudin-4, but not claudin-1, claudin-3, occtudin, or ZO-1, from the cytoplasm to the membrane. Moreover, knockdown of claudin-4 by shRNA suppressed while claudin-4 re-expression restored the TER and 4 kDa FITC-dextran flux responses to AICAR. Additionally, AICAR increased ERK1/2 phosphorylation, and inhibition of ERK1/2 by U0126, an ERK1/2 kinase inhibitor, or by siRNA decreased AICAR-induced TER responses. AICAR induced the serine S199 phosphorylation of claudin-4 and enhanced the inter- action of claudin-4 and occludin. Furthermore, pretreatment with U0126 significantly suppressed AMPK-modulated phosphorytation, redistribution, and interaction with occludin of claudin-4. Taken together, these results indicated that claudin-4 played a crucial role in AMPK-rnodutated paraceUular permeability and ERK1/2 was required in AMPK-modulated tight junction barrier function in subman- dibular gland.     

A Double‐Edged Sword: Complement Component 3 in Toxoplasma gondii Infection

Sprague Dawley rats and Kunming (KM) mice are artificially infected with type II Toxoplasma gondii strain Prugniaud (Pru) to generate toxoplasmosis, which is a fatal disease mediated by T. gondii invasion of the central nervous system (CNS) by unknown mechanisms. The aim is to explore the mechanism of differential susceptibility of mice and rats to T. gondii infection. Therefore, a strategy of isobaric tags for relative and absolute quantitation (iTRAQ) is established to identify differentially expressed proteins (DEPs) in the rats’ and the mice's brains compared to the healthy groups. In KM mice, which is susceptible to T. gondii infection, complement component 3 (C3) is upregulated and the tight junction (TJ) pathway shows a disorder. It is presumed that T. gondii‐stimulated C3 disrupts the TJ of the blood–brain barrier in the CNS. This effect allows more T. gondii passing to the brain through the intercellular space.     

Cell membrane and transepithelial voltages and resistances in isolated rat hepatocyte couplets

Summary The basic electrical properties of an isolated rat hepatocyte couplet (IRHC) system have been analyzed using classical techniques of epithelial electrophysiology, including measurement of electric potentials, resistances and intracellular ion activities. Applications of these techniques are discussed with respect to their limitations in small isolated cells. Mean intracellular and intracanalicular membrane potentials ranged from –23.7 to –46.7 and –4.3 to –5.9 mV, respectively. Membrane resistances were determined using an equivalent circuit analysis modified according to the geometry of the IRHC system. Resistances of the sinusoidal (basolateral) and canalicular (luminal) cell membranes and tight junctions averaged 0.15 and 0.78 G and 25m, respectively. The cells are electrically coupled via low resistance intercellular communications (58 M). Intracellular ion activities for Na⁺, K⁺ and Cl^– averaged 12.2, 88.1 and 17.7 mmol/liter, respectively. The basolateral membrane potential reveals a permeability sequence ofP _K>P _Cl>P _Na. The luminal potential showed minimal dependence on changes in transjunctional ion gradients, indicating a poor ion selectivity of the paracellular pathway. The electrogenic (Na⁺–K)-ATPase contributes little to the luminal and cellular negative electric potential. Therefore, the luminal potential probably results from the secretion of impermeant ions and a Donnan distribution of permeant ions, a mechanism which provides the osmotic driving force for bile formation. By providing the unique opportunity to measure luminal potentials, this isolated hepatocyte system permits study of secretory mechanisms for the first time in a mammalian gland using electrophysiologic techniques.