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.     

Role of solute drag in intestinal transport

This study presents experiments related to the role of solvent drag and solute drag in the transmembrane movement of nonelectrolytes in a perfused rat intestine preparation. Conditions were chosen to simulate the effects of luminal hyperosmolarity on the permeability of tracer solutes. Data are presented on net water flux, transepithelial potentials, and lumen-to-blood and blood-to-lumen tracer solute movements during control electrolyte perfusion and after making the perfusate hyperosmotic. The results indicate that both solvent drag and solute drag can play significant roles in the transepithelial movement of solute and solute permeabilities in the rat ileum preparation. It is suggested that the potential roles of solvent drag and solute drag should be accounted for or considered during the characterization of the mechanisms of biological membrane function.     

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.     

Actions of external hypertonic urea, ADH, and theophylline on transcellular and extracellular solute permeabilities in frog skin

Increases in transepithelial solute permeability were elicited in the frog skin with external hypertonic urea, theophylline, and vasopressin (ADH). In external hypertonic urea, which is known to increase the permeability of the extracellular (paracellular) pathway, the unidirectional transepithelial fluxes of Na (passive), K, Cl, and urea increased substantially while preserving a linear relationship to each other. The same linear relationship was also observed for the passive Na and urea fluxes in regular Ringer and under stimulation with ADH or 10 mM theophylline, indicating that their permeation pathway was extracellular. A linear relationship between Cl and urea fluxes could be demonstrated if the skins were separated according to their open circuit potentials; parallel lines were obtained with increasing intercepts on the Cl axis as the open circuit potential decreased. The slopes of the Cl vs. urea lines were not different from that obtained in external hypertonic urea, indicating that this relationship described the extracellular movement of Cl. The intercept on the ordinate was interpreted as the contribution from the transcellular Cl movement. In the presence of 0.5 mM theophylline or 10 mU/ml of ADH, mainly the transcellular movement of Cl increased, whereas 10 mM theophylline caused increases in both transcellular and extracellular Cl fluxes. These and other data were interpreted in terms of a possible intracellular control of the theophylline-induced increase in extracellular fluxes. The changes in passive solute permeability were shown to be independent of active transport. The responses of the active transport system, the transcellular and paracellular pathways to theophylline and ADH could be explained in terms of the different resulting concentrations of cyclic 3'-5'-AMP produced by each of these substances in the tissue.     

Mechanisms of glucose absorption at a high carbohydrate level in the rat small intestine in vivo

At low maltose concentations, the rates of maltose hydrolysis and glucose absorption increased with increasing substrate concentration in the infusate. A significant fluid secretion occurred in the isolated intestinal loop perfused with hypertonic maltose solution, but water fluxes were close to zero in case of almost isotonic perfusion solutions. The findings suggest that the active transport remains a main mechanism of glucose absorption at super-high maltose concentration as well. The rates of maltose hydrolysis and released glucose absorption in the isolated intestinal loops perfused with super-high maltose concentration, enhance mainly due to an increase of effective digestive-absorptive surface of the villi and, in part, to substrate diffusion across the intestinal epithelium rather than paracellular solvent drag.     

Effects of solvent and solute drag on transmembrane diffusion

The present study compares and quantitates both solvent drag and solute drag forces in a system with both heteropore and homopore membranes. It is shown that tracer solute permeability can be increased if solution flow or driver solute flux is in the direction of tracer diffusion. Either force can decrease tracer permeability if the force can decrease tracer permeability if the force is opposite to the direction of tracer diffusion. The two forces can be additive or one force may reduce the effect of the other force. In the particular system quantitated, solute drag is shown to be some 300 times more effective than solvent drag on a mole-to-mole basis. The use of a number of solute pairs on other homopore and heteropore membranes confirms the finding that the two drag forces can be analyzed or manipulated in a variety of systems.     

A mathematical model of solute coupled water transport in toad intestine incorporating recirculation of the actively transported solute

A mathematical model of an absorbing leaky epithelium is developed for analysis of solute coupled water transport. The non-charged driving solute diffuses into cells and is pumped from cells into the lateral intercellular space (lis). All membranes contain water channels with the solute passing those of tight junction and interspace basement membrane by convection-diffusion. With solute permeability of paracellular pathway large relative to paracellular water flow, the paracellular flux ratio of the solute (influx/outflux) is small (2-4) in agreement with experiments. The virtual solute concentration of fluid emerging from lis is then significantly larger than the concentration in lis. Thus, in absence of external driving forces the model generates isotonic transport provided a component of the solute flux emerging downstream lis is taken up by cells through the serosal membrane and pumped back into lis, i.e., the solute would have to be recirculated. With input variables from toad intestine (Nedergaard, S., E.H. Larsen, and H.H. Ussing, J. Membr. Biol. 168:241-251), computations predict that 60-80% of the pumped flux stems from serosal bath in agreement with the experimental estimate of the recirculation flux. Robust solutions are obtained with realistic concentrations and pressures of lis, and with the following features. Rate of fluid absorption is governed by the solute permeability of mucosal membrane. Maximum fluid flow is governed by density of pumps on lis-membranes. Energetic efficiency increases with hydraulic conductance of the pathway carrying water from mucosal solution into lis. Uphill water transport is accomplished, but with high hydraulic conductance of cell membranes strength of transport is obscured by water flow through cells. Anomalous solvent drag occurs when back flux of water through cells exceeds inward water flux between cells. Molecules moving along the paracellular pathway are driven by a translateral flow of water, i.e., the model generates pseudo-solvent drag. The associated flux-ratio equation is derived.     

Effects of metabolic inhibition on ion transport by dog bronchial epithelium

Mammalian bronchial epithelium absorbs Na+ under basal conditions, but Cl- secretion can be induced. We studied the effects of several modes of metabolic inhibition on the bioelectric properties and solute permeability of dog bronchial epithelium mounted in Ussing chambers. Net Na+ absorption and short-circuit current were inhibited by approximately 75% by hypoxia or by 10(-3) M NaCN. The reduced net Na+ absorption was characterized by a decrease in absorptive flux and an increase in backflux. The latter change was proportional to an increase in permeability to [14C]mannitol, implying that solute flow through a paracellular shunt was increased. In contrast, the reduction of conductance expected from exposure to amiloride (0.94 +/- 0.15 ms/cm2 or 12%) was abolished by NaCN pretreatment. Metabolic inhibition also decreased epithelial conductance and unidirectional Cl- fluxes by approximately 25%. NaCN rapidly and reversibly inhibited the hyperpolarization of potential difference (PD) induced by low luminal bath [Cl-]. This effect was mimicked by the Cl- channel blocker, 5-nitro-2-(3-phenylpropylamino) benzoic acid. Because the transepithelial Cl- diffusion PD reflects, in part, the depolarization of the Cl- -conductive apical cell membrane, metabolic inhibition appears to affect this path. We conclude that metabolic inhibition not only decreased net ion transport by dog bronchial epithelium but also inhibited cellular Na+- and Cl- -conductive pathways and increased paracellular permeability.     

A new internal perfusion method for transport studies in squid giant axons

A new internal perfusion method has been developed which allows control of the internal solute composition in squid axons. The superiority of this technique compared to the old perfusion methods is shown by the experiments performed which have reproduced, both qualitatively and quantitatively, the Na+ and Ca2+ fluxes observed in intact and dialyzed axons. Compared with the internal dialysis, the perfusion method has the advantage that the permeability barrier give by the porous capillary has been eliminated. This allows the introduction into the axon of solutes with very high molecular weight, at the same time that a fast and reliable internal control can be achieved.     

Theoretical Analysis of Net Tracer Flux Due to Volume Circulation in a Membrane with Pores of Different Sizes : Relation to solute drag model

When osmotic pressure across an artificial membrane, produced by a permeable electrically neutral solute on one side of it, is balanced by an external pressure difference so that there is no net volume flow across the membrane, it has been found that there will be a net flux of a second electrically neutral tracer solute, present at equal concentrations on either side of the membrane, in the direction that the "osmotic" solute diffuses. This has been ascribed to solute-solute interaction or drag between the tracer and the osmotic solutes. An alternative model, presented here, considers the membrane to have pores of different sizes. Under general assumptions, this "heteroporous" model will account for both the direction of net tracer flux and the observed linear dependence of unidirectional tracer fluxes on the concentration of the osmotic solute. The expressions for the fluxes of solutes and solvent are mathematically identical under the two models. An inequality is derived which must be valid if the solute interaction model and/or the heteroporous model can account for the data. If the inequality does not hold, then the heteroporous model alone cannot explain the data. It was found that the inequality holds for most published observations except when dextran is the osmotic solute.     

A review of experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms

Experimental measurements of effective diffusive permeabilities and effective diffusion coefficients in biofilms are reviewed. Effective diffusive permeabilities, the parameter appropriate to the analysis of reaction-diffusion interactions, depend on solute type and biofilm density. Three categories of solute physical chemistry with distinct diffusive properties were distinguished by the present analysis. In order of descending mean relative effective diffusive permeability (De/Daq) these were inorganic anions or cations (0.56), nonpolar solutes with molecular weights of 44 or less (0.43), and organic solutes of molecular weight greater than 44 (0.29). Effective diffusive permeabilities decrease sharply with increasing biomass volume fraction suggesting a serial resistance model of diffusion in biofilms as proposed by Hinson and Kocher (1996). A conceptual model of biofilm structure is proposed in which each cell is surrounded by a restricted permeability envelope. Effective diffusion coefficients, which are appropriate to the analysis of transient penetration of nonreactive solutes, are generally similar to effective diffusive permeabilities in biofilms of similar composition. In three studies that examine diffusion of very large molecular weight solutes (>5000) in biofilms, the average ratio of the relative effective diffusion coefficient of the large solute to the relative effective diffusion coefficient of either sucrose or fluorescein was 0.64, 0.61, and 0.36. It is proposed that large solutes are effectively excluded from microbial cells, that small solutes partition into and diffuse within cells, and that ionic solutes are excluded from cells but exhibit increased diffusive permeability (but decreased effective diffusion coefficients) due to sorption to the biofilm matrix.     

Convection and Diffusion in Charged Hydrated Soft Tissues: A Mixture Theory Approach

The extracellular matrix of cartilage is a charged porous fibrous material. Transport phenomena in such a medium are very complex. In this study, solute diffusive flux and convective flux in porous fibrous media were investigated using a continuum mixture theory approach. The intrinsic diffusion coefficient of solute in the mixture was defined and its relation to drag coefficients was presented. The effect of mechanical loading on solute diffusion in cartilage under unconfined compression with a frictionless boundary condition was analyzed numerically using the model developed. Both strain-dependent hydraulic permeability and diffusivity were considered. Analyses and results show that (1) In porous media, the convective velocity for each solute phase is different. (2) The solute convection in tissue is governed by the relative convective velocity (i.e., relative to solid velocity). (3) Under the assumption that all the frictional interactions among solutes are negligible, the relative convective velocity for α-solute phase is equal to the relative solvent velocity multiplied by its convective coefficient (H ^α) which is also known as the hindrance factor in the literature. The relationship between the convective coefficient and the relative diffusivity of solute is presented. (4) Solute concentration profile within the cartilage sample depends on the phase of dynamic compression.     

A new internal perfusion method for transport studies in squid giant axons

A new internal perfusion method has been developed which allows control of the internal solute composition in squid axons. The superiority of this technique compared to the old perfusion methods is shown by the experiments performed which have reproduced, both qualitatively and quantitatively, the Na⁺ and Ca²⁺ fluxes observed in intact and dialyzed axons. Compared with the internal dialysis, the perfusion method has the advantage that the permeability barrier given by the porous capillary has been eliminated. This allows the introduction into the axon of solutes with very high molecular weight, at the same time that a fast and reliable internal control can be achieved.     

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.     

Effects of raised osmolarity on canine tracheal epithelial ion transport function

Evaporation of water from upper airway surfaces increases surface liquid osmolarity. We studied the effects of raised osmolarity of the solution bathing the luminal surface of excised canine tracheal epithelium. Osmolarity was increased by adding NaCl or mannitol. NaCl addition induced a concentration-dependent fall in short-circuit current and a rise in transepithelial conductance (-33% and +14% per 100 mosM, respectively). Unidirectional isotopic fluxes of 22Na, 36Cl, and [14C]mannitol were measured in short-circuited tissues in the base-line state and after addition of NaCl or mannitol to an isotonic mucosal solution. NaCl addition (75 mM) caused a 50% increase in conductance (G) and a parallel increase in [14C]mannitol permeability (Pmann), indicating an increase in paracellular permeability. Net Cl- secretion was reduced 50%, and net Na+ absorption was unchanged despite an increased chemical gradient for absorption, indicating an inhibition of active ion transport. Mannitol addition (150 mM) abolished net Na+ absorption but did not increase G or Pmann or change net Cl- secretion. These results suggest that responses to increased tracheal surface liquid osmolarity during spontaneous breathing may occur in both the cellular (inhibition of active Na+ and Cl- transport) and paracellular (increased [14C]mannitol permeability) compartments of the mucosa.     

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.     

Absorption kinetics for leucine, cycloleucine and alpha-aminoisobutyric acid by rat small intestine in vivo

The intestinal absorption kinetics of three neutral amino acids, leucine, cycloleucine and alpha-aminoisobutyric acid, has been studied in rat jejunum in vivo, with luminal perfusion during successive periods, by measuring the passive component and the active transport. The mass-transfer coefficients of the passive process, are similar for the three amino acids and increase with the perfusion rate. The transport component, obtained from the difference between total absorption and passive diffusion, shows saturation kinetics and also increases with the perfusion rate. The apparent Michaelis constants, Km, and the maximal transport rates for the three amino acids have been determined. The Km values are greater than those reported for in vitro studies, a result imputable to greater thickness of the unstirred layers in vivo and to the unequal signification of the constant in both conditions. Passive flux has proved to be an important component for in vivo absorption, even at low substrate concentrations (1-5 mM), so that its evaluation cannot be neglected for the calculation ot the kinetic constants of the mediated transport.     

Physiological regulation of transepithelial impedance in the intestinal mucosa of rats and hamsters

Isolated intestinal segments from rats or hamsters were recirculated with balanced salt solutions containing fluorocarbon emulsion to provide 6 vpc oxygen. The lumen contained an axial Ag-AgCl electrode, and the serosal surface was surrounded by a cylindrical shell of Ag-AgCl. Transmural impedances were measured at frequencies from 0.01-30 kHz before and after removal of the mucosal epithelium. The resistance of intercellular junctions, RJ, the distributed resistance of the lateral spaces, RL, and the distributed membrane capacitance, CM, were computed from the relations between frequency and impedance. Activation of Na-coupled solute transport by addition of glucose, 3-0-methyl glucose, alanine or leucine caused two- to threefold decreases of transepithelial impedance. Typical changes induced by glucose in hamster small intestine were RJ 30----13 omega, RL 23----10 omega, and CM 8----20 microF (per cm length of segment). Half maximal response occurred at a glucose concentration of 2-3 mM. The area per unit path length of the junctions (Ap/delta chi = specific resistance divided by RJ) in glucose activated epithelium was 3.7 cm in hamster midgut and 6.8 cm in rat. These values are close to the 4.3 cm estimated independently from coefficients of solvent drag and hydrodynamic conductance in glucose-activated rat intestine in vivo. The transepithelial impedance response to Na-coupled solute transport was reversibly dependent upon oxygen tension. It is proposed that activation of Na-coupled solute transport triggers contraction of circumferential actomyosin fibers in the terminal web of the microvillar cytoskeletal system, thereby pulling apart junctions and allowing paracellular absorption of nutrients by solvent drag as described in the previous accompanying paper. Anatomical evidence in support of this hypothesis is presented in the following second accompanying paper.