Structural basis for physiological regulation of paracellular pathways in intestinal epithelia

Isolated segments of hamster small intestine were perfused with oxygenated salt-fluorocarbon emulsions with or without 10-25 mM glucose, alanine or leucine. Resistances of intercellular occluding junctions and of lateral spaces and the distributed capacitance of epithelial plasma membranes were estimated from steady-state transepithelial impedances at frequencies from 0.01-10 kHz. The segments were then fixed in situ with isorheic 2.5% glutaraldehyde while continuing to measure impedance. This method of fixation increased the resistance of lateral spaces but had little effect on the resistance of occluding junctions or on membrane capacitance. The large decreases of impedance induced by glucose or amino acids were preserved in fixed tissue and could therefore be correlated with changes in structure. The observed changes of impedance were interpreted as decreased resistance of occluding junctions and lateral spaces together with increased exposed surface of lateral membranes (capacitance). Glucose, alanine or leucine induced expansion of lateral intercellular spaces as seen by light and electron microscopy. Large dilatations within absorptive cell occluding junctions were revealed by electron microscopy. Freeze-fracture analysis revealed that these dilatations consisted of expansions of compartments bounded by strands/grooves. These solute-induced structural alterations were also associated with condensation of microfilaments in the zone of the perijunctional actomyosin ring, typical of enhanced ring tension. Similar anatomical changes were found in epithelia fixed in situ at 38 degrees C during luminal perfusion with glucose in blood-circulated intestinal segments of anesthetized animals. These structural changes support the hypothesis that Na-coupled solute transport triggers contraction of perijunctional actomyosin, thereby increasing junctional permeability and enhancing absorption of nutrients by solvent drag as described in the two accompanying papers.     

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.     

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

Summary 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,R _J , the distributed resistance of the lateral spaces,R _L , and the distributed membrane capacitance,C _M , 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 wereR _J 3013 ,R _L 2310 , andC _M 820 F (per cm length of segment). Half maximal response occurred at a glucose concentration of 2–3mm. The area per unit path length of the junctions (Ap/x=specific resistance ÷R _J ) 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.     

Structural basis for physiological regulation of paracellular pathways in intestinal epithelia

Summary Isolated segments of hamster small intestine were perfused with oxygenated salt-fluorocarbon emulsions with or without 10–25mm glucose, alanine or leucine. Resistances of inter-cellular occluding junctions and of lateral spaces and the distributed capacitance of epithelial plasma membranes were estimated from steady-state transepithelial impedances at frequencies from 0.01–10 kHz. The segments were then fixedin situ with isorheic 2.5% glutaraldehyde while continuing to measure impedance. This method of fixation increased the resistance of lateral spaces but had little effect on the resistance of occluding junctions or on membrane capacitance. The large decreases of impedance induced by glucose or amino acids were preserved in fixed tissue and could therefore be correlated with changes in structure. The observed changes of impedance were interpreted as decreased resistance of occluding junctions and lateral spaces together with increased exposed surface of lateral membranes (capacitance). Glucose, alanine or leucine induced expansion of lateral intercellular spaces as seen by light and electron microscopy. Large dilatations within absorptive cell occluding junctions were revealed by electron microscopy. Freeze-fracture analysis revealed that these dilatations consisted of expansions of compartments bounded by strands/grooves. These solute-induced structural alterations were also associated with condensation of microfilaments in the zone of the perijunctional actomyosin ring, typical of enhanced ring tension. Similar anatomical changes were found in epithelia fixedin situ at 38°C during luminal perfusion with glucose in blood-circulated intestinal segments of anesthetized animals. These structural changes support the hypothesis that Na-coupled solute transport triggers contraction of perijunctional actomyosin, thereby increasing junctional permeability and enhancing absorption of nutrients by solvent drag as described in the two accompanying papers.     

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.     

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.     

Transepithelial resistance of ciliary epithelial cells in culture: functional modification by protamine and extracellular calcium.

1. Bovine pigmented and human non-pigmented ciliary epithelial cells were cultured on porous filter supports to obtain measurements of transepithelial electrical parameters. 2. The non-pigmented cells showed maximal transepithelial resistance of 15-30 omega cm2 from the third to seventh day in culture. 3. The pigmented ciliary cells reached maximal resistances of 9-20 omega cm2 after the fourth day in culture. 4. The transepithelial resistances of the cultured epithelia were functionally increased by protamine. This effect could be reversed by heparin. 5. We conclude that the range of resistances in cultured ciliary epithelial cells is the same as in whole ciliary preparations. Thus, cultured ciliary epithelial cells can be used for studies on transepithelial transport.     

Improved electrical coupling in uterine smooth muscle is associated with increased numbers of gap junctions at parturition

We have studied some passive electrical properties of uterine smooth muscle to determine whether a change in electrical parameters accompanies gap junction formation at delivery. The length constant of the longitudinal myometrium increased from 2.6 +/- 0.8 mm (X +/- SD) before term to 3.7 +/- 1 mm in tissues from delivering animals. The basis of the change was a 33% decrease in internal resistance and a 46% increase in membrane resistance. Axial current flow in an electrical syncytium such as myometrium is impeded by the cytoplasm of individual cells plus the junctions between cells. Measurement of the longitudinal impedance indicated that the specific resistance of the myoplasmic component was constant at 319 +/- 113 omega . cm before term and 340 +/- 93 omega . cm at delivery. However, a decrease in junctional resistance was apparent from 323 +/- 161 omega . cm to 134 +/- 64 omega . cm at delivery. 1.5-2 d after delivery, the junctional resistance was increased, as was the myoplasmic resistance. Thin-section electron microscopy of some of the same muscle samples showed that gap junctions were present in significantly greater numbers in the delivering tissues. Therefore, our results support the hypothesis that gap junction formation at delivery is associated with improved electrical coupling of uterine smooth muscle.     

Na+ Recirculation and Isosmotic Transport

The Na(+) recirculation theory for solute-coupled fluid absorption is an expansion of the local osmosis concept introduced by Curran and analyzed by Diamond & Bossert. Based on studies on small intestine the theory assumes that the observed recirculation of Na(+) serves regulation of the osmolarity of the absorbate. Mathematical modeling reproducing bioelectric and hydrosmotic properties of small intestine and proximal tubule, respectively, predicts a significant range of observations such as isosmotic transport, hyposmotic transport, solvent drag, anomalous solvent drag, the residual hydraulic permeability in proximal tubule of AQP1 (-/-) mice, and the inverse relationship between hydraulic permeability and the concentration difference needed to reverse transepithelial water flow. The model reproduces the volume responses of cells and lateral intercellular space (lis) following replacement of luminal NaCl by sucrose as well as the linear dependence of volume absorption on luminal NaCl concentration. Analysis of solvent drag on Na(+) in tight junctions provides explanation for the surprisingly high metabolic efficiency of Na(+) reabsorption. The model predicts and explains low metabolic efficiency in diluted external baths. Hyperosmolarity of lis is governed by the hydraulic permeability of the apical plasma membrane and tight junction with 6-7 mOsm in small intestine and < or = 1 mOsm in proximal tubule. Truly isosmotic transport demands a Na(+) recirculation of 50-70% in small intestine but might be barely measurable in proximal tubule. The model fails to reproduce a certain type of observations: The reduced volume absorption at transepithelial osmotic equilibrium in AQP1 knockout mice, and the stimulated water absorption by gallbladder in diluted external solutions. Thus, it indicates cellular regulation of apical Na(+) uptake, which is not included in the mathematical treatment.     

Tight junction structure and ZO-1 content are identical in two strains of Madin-Darby canine kidney cells which differ in transepithelial resistance

The relationship of tight junction permeability to junction structure and composition was examined using two strains of Madin-Darby canine kidney (MDCK) cells (I and II) which differ greater than 30-fold in transepithelial resistance. This parameter is largely determined by paracellular, and hence junctional, permeability under most conditions. When these two strains of cells were grown on permeable filter supports, they formed monolayers with equivalent linear amounts of junction/area of monolayer. Ultrastructural analysis of these monolayers by thin section EM revealed no differences in overall cellular morphology or in tight junction organization. Morphometric analysis of freeze-fractured preparations indicated that the tight junctions of these two cell strains were similar in both number and density of junctional fibrils. Prediction of transepithelial resistance for the two strains from this freeze-fracture data and a published structure-function formulation (Claude, P. 1978, J. Memb. Biol. 39:219- 232) yielded values (I = 26.5 omega/cm2, II = 35.7 omega/cm2) that were significantly lower than those observed (I = 2,500-5,000 omega/cm2, II = 50-70 omega/cm2). Consistent with these structural studies, a comparison of the distribution and cellular content of ZO-1, a polypeptide localized exclusively to the tight junction, revealed no significant differences in either the localization of ZO-1 or the amount of ZO-1 per micron of junction (I = 1,415 +/- 101 molecules/micron, II = 1,514 +/- 215 molecules/micron).     

Heptaminol hydrochloride as an epithelium transporter

Electrophysiological and transport effects induced by heptaminol hydrochloride were studied in frog epithelium. This tissue, which can easily be maintained in vitro, is a valuable model for studying sodium active transport with hormone-dependent characteristics that reproduce mammalian nephron behavior (notably in areas with tight gap junctions). The two following techniques were used: the Ussing short-circuit current method and the swept-frequency impedance measurement method. Our findings indicate the following. (i) Heptaminol hydrochloride significantly increases the short-circuit current and transepithelial polarization. (ii) This effect develops progressively as the molecule is introduced on the serous side (3Na+/2K+ active countertransport sites). Time to maximum development is approximately 20 min and the electrophysiological effect lasts from 60 to 90 min. (iii) The mean equivalent cationic current rise is larger in sulfate-Ringer (+23 +/- 4.6 microA, p less than 0.01) than in chloride-Ringer (+14 +/- 4.9 microA, p less than 0.05). The increase in short-circuit current is approximately 0.9 muequiv. cm-2 h-1 in sulfate-Ringer. (iv) The increase in mean polarization is greater in chloride (+21 +/- 6.2 mV, p less than 0.02) than in sulfate (+6 +/- 1.5 mV, p less than 0.01) following a diphasic effect on potential. (v) Changes in apical impedance Z are small (-454 +/- 323 omega, nonsignificant) compared with transepithelial resistance in sulfate (-1065 +/- 359 omega, p less than 0.05). (vi) Changes in membrane capacitance reflect changes in the membrane surface. However, no significant capacitance changes are produced in sulfate and chloride solution by heptaminol hydrochloride (-0.04 +/- 0.11 microF and 0.05 +/- 0.11 microF, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrical properties and active solute transport in rat small intestine

Summary The transepithelial resistance, the cell membrane resistance and the ratio of resistances of the serosal (baso-lateral) to the mucosal (brush border) cell membrane were measured in rat duodenum, jejunum and ileum by means of microelectrode techniques. These measured values were not affected in the presence of actively transported solutes in the mucosal bathing fluid.Contribution of an electrical conductance through the extracellular shunt pathway to the total transepithelial conductance was quantitatively estimated using an electrically equivalent circuit analysis. These values estimated in respective tissues of small intestine were approx. 95% of the total transepithelial conductance, remaining unaffected by an active solute transport.From these data, the changes in emf's of the mucosal and serosal membrane induced byd-glucose or glycine were separately evaluated.     

Solute transport process in intestinal epithelial cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na+ and K+ concentrations, significant gains of Na+ and K+ occurred with glucose transport. The quantitative relationships among net gains of Na+, K+ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na+ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na+-efflux and K+-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

Characterization of sodium transport in gustatory epithelia from the hamster and rat

The transduction of sodium salts occurs through a variety of mechanisms, including sodium influx through amiloride-sensitive sodium channels, anion-dependent sodium movement through intercellular junctions and unidentified amiloride-insensitive mechanisms. Characterizations of sodium transport in lingual epithelium mounted in Ussing chambers have focused almost exclusively on epithelia containing only fungiform taste buds. In the present study we have investigated sodium transport by measuring NaCl-induced short-circuit current from lingual epithelia containing fungiform, foliate, vallate and palatine taste buds in the hamster and the rat. All areas show measurable sodium transport, yet significant differences were noted between the epithelia from the rat and the hamster and among the different epithelia within a single species in terms of current density, transepithelial resistance and mucosal amiloride sensitivity. In general, epithelia from the anterior tongue were of a lower resistance and transported sodium more effectively than from the posterior tongue. Moreover, fungiform- and vallate-containing epithelia in the rat had a greater current density than did the corresponding tissues in the hamster. Amiloride sensitivity also differed between the rat and the hamster. In the hamster all gustatory areas showed some amiloride sensitivity, while in the rat the vallate-containing epithelia were devoid of amiloride- sensitive sodium transport. The results are consistent with the interpretation that all chemosensitive areas may participate in the detection of salts but the degree of salt transport and the mechanism of transport is variable among different lingual epithelia and different species.      

The Chloride Conductance of Tight Junctions of Rat Ileum Can Be Increased by cAMP But Not by Carbachol

It is well known, that in mammalian small intestine, cAMP increases Cl⁻ permeability of the apical membrane of enterocytes as part of its secretory action. Paradoxically, this is usually accompanied by an increase of the transepithelial resistance. In the present study we report that in the presence of bumetanide (to block basolateral Cl⁻ uptake) cAMP always decreased the transepithelial resistance. We examined whether this decrease in resistance was due to a cAMP-dependent increase of the paracellular electrolyte permeability in addition to the increase of the Cl⁻ permeability of the apical cell membrane. We used diffusion potentials induced by serosal replacement of NaCl, and transepithelial current passage to evoke transport number effects. The results revealed that cAMP (but not carbachol) could increase the Cl⁻ permeability of the tight junctions in rat ileum. Moreover, we observed a variation in transepithelial resistance of individual tissue preparations, inversely related to the cation selectivity of the tissue, suggesting that Na⁺ permeability of the tight junctions can vary between preparations. Received: 7 September 1996/Revised: 5 November 1996     

Symmetric infection of rotavirus on polarized human intestinal epithelial (Caco-2) cells.

When rotavirus infects the mature villus tip cells of the small intestine, it encounters a highly polarized epithelium. In order to understand this virus-cell interaction more completely, we utilized a cell culture-adapted rhesus rotavirus (RRV) to infect human intestinal (Caco-2) and Madin-Darby canine kidney (MDCK-1) polarized epithelial cells grown on a permeable support. Filter-grown Caco-2 cells and MDCK-1 cells, producing a transepithelial resistance of 300 to 500 and greater than 1,000 omega . cm2, respectively, were infected from either the apical or basolateral domain with RRV or Semliki Forest virus. Whereas Semliki Forest virus infection only occurred when input virions had access to the basolateral domain of MDCK-1 or Caco-2 cells, RRV infected MDCK-1 and Caco-2 monolayers in a symmetric manner. The effect of rotavirus infection on monolayer permeability was analyzed by measuring the transepithelial electrical resistance. Rotavirus infection on filter-grown Caco-2 cells caused a transmembrane leak at 18 h postinfection, before the development of the cytopathic effect (CPE) and extensive virus release. Electrical resistance was completely abolished between 24 and 36 h postinfection. Although no CPE could be detected on RRV-infected MDCK cells, the infection caused a transmembrane leak that totally abolished the electrical resistance at 18 to 24 h postinfection. Cell viability and the CPE analysis together with immunohistochemistry and immunofluorescence data indicated that the abolishment of resistance across the monolayer was due not to an effect on the plasma membrane of the cells but to an effect on the paracellular pathway limited by tight junctions. Attachment and penetration of rotavirus onto Caco-2 cells caused no measurable transmembrane leak during the first hour of infection.     

Solute Transport Process in Intestinal Epithelial Cells

In rat small intestine, the active transport of organic solutes results in significant depolarization of the membrane potential measured in an epithelial cell with respect to a grounded mucosal solution and in an increase in the transepithelial potential difference. According to the analysis with an equivalent circuit model for the epithelium, the changes in emf's of mucosal and serosal membranes induced by active solute transport were calculated using the measured conductive parameters. The result indicates that the mucosal cell membrane depolarizes while the serosal cell membrane remarkably hyperpolarizes on the active solute transport. Corresponding results are derived from the calculations of emf's in a variety of intestines, using the data that have hitherto been reported. The hyperpolarization of serosal membrane induced by the active solute transport might be ascribed to activation of the serosal electrogenic sodium pump. In an attempt to determine the causative factors in mucosal membrane depolarization during active solute transport, cell water contents and ion concentrations were measured. The cell water content remarkably increased and, at the same time, intracellular monovalent ion concentrations significantly decreased with glucose transport. Net gain of glucose within the cell was estimated from the restraint of osmotic balance between intracellular and extracellular fluids. In contrast to the apparent decreases in intracellular Na⁺ and K⁺ concentrations, significant gains of Na⁺ and K⁺ occurred with glucose transport. The quantitative relationships among net gains of Na⁺, K⁺ and glucose during active glucose transport suggest that the coupling ratio between glucose and Na⁺ entry by the carrier mechanism on the mucosal membrane is approximately 1:1 and the coupling ratio between Na⁺-efflux and K⁺-influx of the serosal electrogenic sodium pump is approximately 4:3 in rat small intestine. In addition to the electrogenic ternary complex inflow across the mucosal cell membrane, the decreases in intracellular monovalent ion concentrations, the temporary formation of an osmotic pressure gradient across the cell membrane and the streaming potential induced by water inflow through negatively charged pores of the cell membrane in the course of an active solute transport in intestinal epithelial cells are apparently all possible causes of mucosal membrane depolarization.     

Streptomycin toxicity in primary cultures of flounder renal proximal tubule cells

Summary The aminoglycoside antibiotic streptomycin is a known nephrotoxin in vivo and a common component of cell culture media. The effects of streptomycin (100 μg/ml) on transepithelial electrical properties, glucose transport, glycolytic metabolism, and morphology were examined in primary proximal tubule cell cultures from winter flounder (Pseudopleuronectes americanus) kidney. Streptomycin treatment on either Days 2 to 12 or Days 8 to 13 abolished the transepithelial potential difference and short-circuit current across the monolayer but had no effect on transepithelial resistance in confluent 12 to 13-dcultures, suggesting the loss of active transepithelial transport. Consistent with these findings, mucosal-to-serosal glucose fluxes were greatly reduced in streptomycin-treated cultures and insensitive to the transport inhibitor phlorizin, indicating the absence of the apical Na-dependent glucose transport system associated with net glucose reabsorption. In addition to transport processes, antibiotic treatment also interfered with cellular energy metabolism as judged by the rapid reduction in glycolytic lactate production observed in the presence of streptomycin. Scanning and transmission electron microscopy revealed that streptomycin-treated culture were composed of cuboidal-to-columnar shaped cells which maintained intact tight junctions similar to control cultures. However, apical microvilli, the presumed sites of mucosal transport systems, were severely reduced in number in streptomycin-treated cultures. We concluded that streptomycin, at a dose commonly used in cell culture, inhibited the expression of differentiated function by flounder proximal tubule cell cultures. These cell cultures may provide a suitable model system for examination of the mechanisms of aminoglycoside nephrotoxicity. This investigation was supported by the University of Connecticut Research Foundation and by grant PCM-8003452 from the National Science Foundation, Washington, DC.     

Type I cell-like morphology in tight alveolar epithelial monolayers

The pulmonary alveolar epithelium separates air spaces from a fluid-filled interstitium and might be expected to exhibit high resistance to fluid and solute movement. Previous studies of alveolar epithelial barrier properties have been limited due to the complex anatomy of adult mammalian lung. In this study, we characterized a model of isolated alveolar epithelium with respect to barrier transport properties and cell morphology. Alveolar epithelial cells were isolated from rat lungs and grown as monolayers on tissue culture-treated Nuclepore filters. On Days 2-6 in primary culture, monolayers were analyzed for transepithelial resistance (Rt) and processed for electron microscopy. Mean cell surface area and arithmetic mean thickness (AMT) were determined using morphometric techniques. By Day 5, alveolar epithelial cells in vitro exhibited morphologic characteristics of type I alveolar pneumocytes, with thin cytoplasmic extensions and protruding nuclei. Morphometric data demonstrated that alveolar pneumocytes in vitro develop increased surface area and decreased cytoplasmic AMT similar to young type I cells in vivo. Concurrent with the appearance of type I cell-like morphology, monolayers exhibited high Rt (greater than 1000 omega.cm2), consistent with the development of tight barrier properties. These monolayers of isolated alveolar epithelial cells may reflect the physiological and morphological properties of the alveolar epithelium in vivo.     

Effects of Bacillus cereus var. toyoi as probiotic feed supplement on intestinal transport and barrier function in piglets

The objective of the study was to assess the effects of feed supplementation with the probiotic Bacillus cereus var. toyoi on transport and barrier properties of pig jejunum. Sows and their respective piglets were randomly assigned to two feeding groups: a control group and a probiotic group in which the standard diet was supplemented with Bacillus cereus var. toyoi. At the age of 14, 28, 35 and 56 days, 5 piglets per subgroup were killed and tissue samples from the mid jejunum were mounted in conventional Ussing chambers. Absorptive and secretory properties of the jejunum epithelia were assessed by stimulation of Na-coupled glucose and L-glutamine transport and stimulation of ion secretion by PGE2. Kinetic parameters maximal transport velocity (Vmax) and Michaelis Menten constant (Km) were calculated for glucose and PGE2-stimulated ion secretion. Mannitol fluxes and tissue resistance were measured to evaluate barrier function. With respect to absorption, glucose transport was not changed by treatment and only a slightly higher L-glutamine transport was observed in the probiotic group compared with the control group. The PGE2-stimulated the short circuit current (DeltaIsc) in the small intestine and Vmax were higher in the probiotic group at days 28 and 35 compared with the control group. The probiotic seems to have a stabilising (decreasing) effect on the variability of the data. Changes of absorptive and secretory transport properties dependent on age were observed.