Transepithelial Flux Research Articles

Taurine secretion in primary monolayer cultures of flounder renal epithelium: stimulation by low osmolality.

Transepithelial taurine fluxes determined in short-circuited monolayer cultures of flounder renal proximal cells in Ussing chambers revealed net taurine secretion. Both unidirectional secretory and reabsorptive taurine fluxes exhibited saturation kinetics contributed by two distinct saturable transepithelial taurine transport systems operating at different taurine concentration ranges. The taurine secretory system operating below 0. 5 mM had lower affinity but higher capacity than the reabsorptive system, whereas the one operating at high concentrations (0.5-3.0 mM) had higher affinity but the same capacity as the corresponding reabsorptive system. Exposure (2 h) of the cultures to hyposmotic medium in the presence of taurine increased taurine secretory flux twofold with no effect on the reabsorptive flux. The hyposmolality-induced increase in taurine secretion was associated with a decreased peritubular taurine efflux and a concurrent increased luminal taurine efflux; the latter occurred via a pathway that was not affected by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid but inhibited by probenecid. The culture response in hyposmotic medium mimics the in vivo response of the intact marine fish kidney to dilution.

Sodium-Sulfate Symport by Aplysia californica Gut

Sulfate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Sulfate transport can be coupled to proton, sodium symport or antiport processes involving chloride or bicarbonate. It had previously been observed in Aplysia gut that sulfate was actively absorbed. To understand the mechanism for this transport, short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and sulfate were measured to see whether there was interaction between the fluxes. The net mucosal-to-serosal flux of Na(+) was enhanced by the presence of sulfate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of sulfate was dependent upon the presence of Na(+) and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetanide, added to either side, had no effect on transepithelial potential difference or short-circuit current in the Aplysia gut bathed in a Na2SO4 seawater medium. However, mucosal thiosulfate inhibited the net mucosal-to-serosal fluxes of both sulfate and Na(+) and the thiosulfate-sensitive Na(+) flux to that of sulfate was 2:1. These results suggest the presence of a Na-SO4 symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell.

Polyunsaturated fatty acids reduce non‐receptor‐mediated transcellular permeation of protein across a model of intestinal epithelium in vitro

Dietary polyunsaturated fatty acids influence the natural history of intestinal inflammatory diseases. Varying the types of long-chain fatty acids that are exposed to cells alters the physicochemical properties of cell membranes. This study aimed to determine whether such variations alter transcellular and paracellular permeability in intestinal epithelium. Monolayers of Caco-2 cells, allowed to differentiate by culturing for 7 days following confluence, were used as the model for intestinal epithelium. Paracellular permeability was assessed by measurement of transepithelial resistance, while transcellular permeability was assessed by the transepithelial flux of horseradish peroxidase. Exposure of the cells to 100 micromol/L of palmitic acid, oleic acid, eicosapentaenoic acid, or linoleic acid, was not toxic to cells (measured by leakage of lactate dehydrogenase), and altered cell membrane fatty acid composition (as measured by gas chromatography). Flux of horseradish peroxidase was significantly affected by 24 h fatty acid exposure (P= 0.038, ANOVA), being decreased by 23 +/- 6% (mean +/- SEM) by eicosapentaenoic acid and 25 +/- 3% by linoleic acid. Oleic acid, palmitic acid and butyrate, had no effect. Transepithelial resistance also varied significantly across the treatment groups (P< 0.001) due to a 28 +/- 5% increase induced by butyrate. The long-chain fatty acids had no effect. Both omega-3 and omega-6 polyunsaturated fatty acids reduce transcellular, non-receptor-mediated permeation of proteins across differentiated Caco2 cell monolayers, without altering paracellular permeability. Alteration of intestinal barrier function should be considered as a possible mechanism of action of dietary polyunsaturated fatty acids.

Effect of cholera toxin on glutamine metabolism and transport in rabbit ileum.

The aim of the present study was to evaluate the effect of cholera toxin on energy balance from intestinal glutamine metabolism and oxidation, glutamine-dependent sodium absorption, and cholera toxin-dependent ion flux. Cholera toxin-stimulated sodium and L-glutamine ileal transport and metabolism were studied in Ussing chambers. Glutamine (10 mM) transport and metabolism were simultaneously studied using (14)C flux and HPLC. In the same tissues, the flux of each amino acid was studied by HPLC, and glutamine metabolism and oxidation were studied by the determination of amino acid specific activity and (14)CO(2) production. In control tissues, glutamine stimulated sodium absorption and was mainly oxidized. The transepithelial flux of intact glutamine represented 45% of glutamine flux across the luminal membrane. The other metabolites were glutamate and, to a lesser degree, citrulline, ornithine, and proline. Cholera toxin did not alter glutamine-stimulated sodium absorption, glutamine oxidation, transport, and metabolism. In conclusion, the present results indicate that cholera toxin does not alter glutamine intestinal function and metabolism. In addition, approximately 95% of the energy provided by glutamine oxidation remains available to the enterocyte.

Characterization of the transport of the organic cation [3H]MPP+ in human intestinal epithelial (Caco-2) cells.

The aim of this study was to characterize the transport of organic cations at the intestinal level, by studying the characteristics of the transport of 1-methyl-4-phenylpyridinium (MPP+) in Caco-2 cells. Transepithelial flux as well as cellular accumulation of [3H]MPP+ were quantitatively similar when substrate was applied from the basolateral or apical cell membrane. Verapamil (100 microM) and rhodamine123 (10 microM) significantly reduced [3H]MPP+ transepithelial flux in the apical-to-basolateral direction. When cells were grown on plastic supports, [3H]MPP+ was rapidly accumulated in the cells, both by saturable and nonsaturable mechanisms. The kinetic parameters of the saturable component were: Km: 449 microM and Vmax: 2,249 pmol per mg protein and 5 min. Uptake of [3H]MPP+ was metabolic energy-dependent and Na+-, pH- and potential-independent. It was inhibited by several organic cations (verapamil, rhodamine123, daunomycin, vinblastine, tetrabutylammonium and vecuronium) but not by others (tetraethylammonium and N-methylnicotinamide). Decynium22 and corticosterone inhibited [3H]MPP+ uptake into the cells. The P-glycoprotein antibody UIC2 (20 microg/ml) had no effect. In conclusion, [3H]MPP+ is efficiently transported by Caco-2 cells in both basolateral-to-apical (secretion) and apical-to-basolateral (absorption) directions. Absorption of [3H]MPP+ at the apical membrane seems to occur through a carrier-mediated mechanism belonging to the Amphiphilic Solute Facilitator (ASF) family of transporters, but distinct from the known members of this family.

Calcium homeostasis in larval and adult Drosophila melanogaster.

Calcium homeostasis in Drosophila melanogaster was examined in response to the challenges imposed by growth, reproduction and variations in dietary calcium content. Turnover time for calcium, calculated as the time for (45)Ca(2+)to accumulate to half the steady state value of 3.46 nmol/fly, was 3.3 days. Although larvae weighed 2x as much as adults, they contained 3-4x as much calcium. Anterior Malpighian tubules (Mts) contain much more calcium than posterior Mts, accounting for 25-30% of the calcium content of the whole fly. In response to a 6.2-fold increase in dietary calcium level, calcium content of whole flies increased only 10%. Hemolymph calcium concentration ( approximately 0.5 mM) was similar in males and females and in animals raised on diets differing in calcium content. Fluid secretion rate, secreted fluid calcium concentration, and transepithelial calcium flux in tubules isolated from flies raised on high and low calcium diets did not differ significantly. Malpighian tubules secrete calcium at rates sufficient to eliminate whole body calcium content in 0.5 and 3 days for tubules secreting fluid at basal and maximal rates, respectively. It is suggested that flies absorb high quantities of calcium from the diet and maintain homeostasis through the combined effects of elimination of calcium in fluid secreted by the Malpighian tubules and the sequestration of calcium in granules, especially within the distal segment of the anterior pair of Malpighian tubules.

Cellular adaptation of the mouse cortical thick ascending limb of Henle's loop (CTAL) to dietary magnesium restriction: enhanced transepithelial Mg2+ and Ca2+ transport.

Mice aged 4 or 8 weeks were fed with a low-Mg2+ diet for 1, 2, 3 or 4 days. After 1 day of diet, the urinary excretion of Mg2+ and Ca2+ was strongly reduced in both animal groups (4 and 8 weeks), accompanied by a significant fall in plasma Mg2+ concentration and an increase in urinary volume. This profile persisted after 2, 3 or 4 days of dietary Mg2+ restriction. After 1 day of diet, transepithelial ion net fluxes of Na+, Cl-, Ca2+ and Mg2+ (JNa' JCI, JCl, JMg) measured in vitro from isolated perfused cortical thick ascending limbs (CTALs) of these animals remained unchanged. After 2 days of diet, measurements of J(Ca) and J(Mg) in isolated perfused CTALs showed that transepithelial Mg2+ and Ca2+ reabsorption were enhanced in CTALs from Mg(2+)-depleted, 8-week-old animals, whereas transepithelial Mg2+ and Ca2+ transport were not altered in 4-week-old mice. JNa and JCl and the transepithelial potential (PDte) were not modified in CTALs from either animal group. Our results suggest that a low-Mg2+ diet leads to urinary retention of Mg2+ and Ca2+ which is most likely due to increased Mg2+ and Ca2+ transport in the CTAL. Furthermore, in response to dietary Mg2+ restriction, the reabsorption of divalent cations in the CTAL of adult, but not of young, mice undergoes cellular adaptation.

ENOS mediates L-arginine-induced inhibition of thick ascending limb chloride flux.

We recently reported that the rat thick ascending limb (THAL) possesses an active isoform of nitric oxide synthase (NOS) that is substrate-limited in vitro. NO produced by THAL NOS inhibits chloride flux. Protein and transcript for each of the primary NOS isoforms-endothelial (eNOS), inducible (iNOS), and neuronal (nNOS)-have been demonstrated in THALs. However, the NOS isoform that mediates NO-induced inhibition of chloride flux is unknown. We hypothesized that NO produced from eNOS in the THAL inhibits NaCl transport. THALs from male eNOS, iNOS, and nNOS knockout mice and C57BL/6J wild-type controls were perfused in vitro and the response of transepithelial chloride flux (J(Cl)) to L-arginine (L-Arg), the substrate for NOS, and spermine NONOate (SPM), an NO donor was measured. We first tested whether isolated mouse THALs could synthesize NO and whether this NO inhibits transport. Addition of 0. 5 mmol/L L-Arg to the bath decreased J(Cl) from 105.8+/-17.5 to 79. 2+/-15.8 pmol/mm per minute (P<0.01) in C57BL/6J wild-type mice, whereas addition of D-Arginine had no effects on J(Cl.) In contrast, addition of 0.5 mmol/L L-Arg to the bath did not alter J(Cl) of THALs from eNOS knockout mice. When 10 micromol/L SPM was added to the bath of eNOS knockout THALs, J(Cl) decreased from 89.1+/-8.6 to 74.8+/-7.5 pmol/mm/min (P<0.05). Thus the lack of responsiveness of eNOS knockout THALs to L-Arg was not due to an inability to respond to NO. We next evaluated the role of iNOS and nNOS in the response to L-Arg. Addition of 0.5 mmol/L L-Arg to the bath decreased J(Cl) in THALs from iNOS and nNOS knockout mice by 37.7+/-6.4% (P<0.05) and 31.8+/-8.3% (P<0.01), respectively. We conclude that eNOS is the active isoform of NOS in the THAL under basal conditions. Mouse THAL eNOS responds to exogenous L-Arg by increasing NO production, which, in turn, inhibits J(Cl).

Sodium-Sulfate Symport by Aplysia californica Gut

Sulfate transport across plasma membranes has been described in a wide variety of organisms and cell types including gastrointestinal epithelia. Sulfate transport can be coupled to proton, sodium symport or antiport processes involving chloride or bicarbonate. It had previously been observed in Aplysia gut that sulfate was actively absorbed. To understand the mechanism for this transport, short-circuited Aplysia californica gut was used. Bidirectional transepithelial fluxes of both sodium and sulfate were measured to see whether there was interaction between the fluxes. The net mucosal-to-serosal flux of Na was enhanced by the presence of sulfate and it was abolished by the presence of serosal ouabain. Similarly, the net mucosal-to-serosal flux of sulfate was dependent upon the presence of Na and was abolished by the presence of serosal ouabain. Theophylline, DIDS and bumetanide, added to either side, had no effect on transepithelial potential difference or short-circuit current in the Aplysia gut bathed in a Na2SO4 seawater medium. However, mucosal thiosulfate inhibited the net mucosal-to-serosal fluxes of both sulfate and Na and the thiosulfate-sensitive Na flux to that of sulfate was 2:1. These results suggest the presence of a Na-SO4 symporter in the mucosal membrane of the Aplysia californica foregut absorptive cell.

Comparison of cattle and sheep colonic permeabilities to horseradish peroxidase and hamster scrapie prion protein in vitro

BACKGROUNDParacellular permeability to solutes across the descending colon is much higher in cattle than sheep. This is a possible route for transmission of infective materials, such as scrapie prion.AIMSTo compare...

Transport of human growth hormone across Caco-2 cells with novel delivery agents: evidence for P-glycoprotein involvement

Emisphere Technologies, Inc. has synthesized a series of small molecules which have been shown to improve protein absorption through mucosal tissue. This enhancement is specific between protein and a particular delivery agent. Despite the specificity of interaction, the mechanism of enhanced tissue penetration is still unclear. The purpose of this work is to understand the enhancement mechanism(s) of these delivery agents by using Caco-2 cells as a model membrane. It was found that the bidirectional transepithelial fluxes of human growth hormone (hGH) in the presence of these delivery agents across human intestinal epithelial Caco-2 cell line showed marked asymmetry. Average permeability coefficient values obtained in the apical (AP) to basolateral (BL) direction were lower than those of the reverse (BL to AP) direction. On the other hand, the fluxes for human growth hormone alone were symmetric. When P-glycoprotein inhibitors were included in the transport medium, the permeability coefficient values of BL to AP direction were significantly decreased while the transport was increased in the reverse direction in the presence of delivery agents. P-glycoprotein inhibitors had no effect on the transport of human growth hormone alone. This study shows that human growth hormone alone can be transported across Caco-2 cells in very limited quantities by passive diffusion, but in the presence of delivery agents, human growth hormone can be effluxed in a P-glycoprotein-mediated fashion. This also indirectly shows that the human growth hormone has become more lipophilic in the presence of delivery agents.

Paracellular drug transport across intestinal epithelia: influence of charge and induced water flux

The influence of drug charge and transepithelial water flux on passive paracellular drug transport was investigated in Caco-2 cell monolayers and rat ileal mucosa in vitro. Three small hydrophilic compounds with different net charges (creatinine, erythritol and foscarnet) were used as model drugs. A hypotonic glucose-rich solution was applied apically to induce epithelial absorption of water. In the Caco-2 monolayers, permeability to creatinine (positively charged) was 25-fold greater than to foscarnet (negatively charged), indicating a pronounced cation selective paracellular permeability. During apical exposure to the hypotonic glucose-rich solution, transport of all model drugs increased in both the absorptive and secretory directions. This enhanced transport coincided with a decrease in transepithelial resistance. Further, fluorescence and transmission electron microscopy indicated dilatations of the paracellular spaces but no damage to the cell membranes. These findings suggested that the enhancement in drug transport was attributable to increased paracellular tight junction permeability rather than to “solvent drag”. In the ileal segments, mucosal exposure to the hypotonic glucose-rich solution had no effect on transepithelial resistance and only a marginal increase in drug transport was observed. Taken together, the modest absorption enhancement demonstrated in the in vitro models agrees with results obtained in vivo, supporting the conclusion that a more pronounced disruption of the tight junction barrier than that obtained through stimulation of epithelial absorption of water is required for efficient enhancement of paracellular intestinal drug absorption.

Modulation of bronchial epithelial cell barrier function by in vitro jet propulsion fuel 8 exposure.

The loss of epithelial barrier integrity in bronchial and bronchiolar airways may be an initiating factor in the observed onset of toxicant-induced lung injuries. Acute 1-h inhalation exposures to aerosolized jet propulsion fuel 8 (JP-8) have been shown to induce cellular and morphological indications of pulmonary toxicity that was associated with increased respiratory permeability to 99mTc-DTPA. To address the hypothesis that JP-8 jet fuel-induced lung injury is initiated through a disruption in the airway epithelial barrier function, paracellular mannitol flux of BEAS-2B human bronchial epithelial cells was measured. Incubation of confluent cell cultures with non-cytotoxic concentrations of JP-8 or n-tetradecane (C14), a primary constituent of JP-8, for a 1-h exposure period resulted in dose-dependent increases of paracellular flux. Following exposures of 0.17, 0.33, 0.50, or 0.67 mg/ml, mannitol flux increased above vehicle controls by 10, 14, 29, and 52%, respectively, during a 2-h incubation period immediately after each JP-8 exposure. C14 caused greater mannitol flux increases of 37, 42, 63, and 78%, respectively, following identical exposure conditions. The effect on transepithelial mannitol flux reached a maximum at 12 h and spontaneously reversed to control values over a 48-h recovery period, for both JP-8 and C14 exposure. These data indicate that non-cytotoxic exposures to JP-8 or C14 exert a noxious effect on bronchial epithelial barrier function that may preclude pathological lung injury.

Amino acid fluxes in rat thin limb segments of Henle's loop during in vitro microperfusion.

Amino acids are apparently recycled between loops of Henle and vasa recta in the rat papilla in vivo. To examine more closely papillary amino acid transport, we measured transepithelial fluxes of L-[(14)C]alanine and [(14)C]taurine in thin limbs of Henle's loops isolated from rat papilla and perfused in vitro. In descending thin limbs (DTL) in vitro, unidirectional bath-to-lumen fluxes tended to exceed unidirectional lumen-to-bath fluxes for both radiolabeled amino acids, although the difference was statistically significant only for taurine. In ascending thin limbs (ATL) in vitro, unidirectional lumen-to-bath fluxes tended to exceed unidirectional bath-to-lumen fluxes, although the difference was again statistically significant only for taurine. These results are compatible with apparent directional movements of amino acids in vivo. However, none of the unidirectional fluxes was saturable or inhibitable, an observation compatible with apparent reabsorption from the ATL in vivo but not compatible with apparent movement from vasa recta to DTL in vivo. There was no evidence of net active transepithelial transport when concentrations of radiolabeled amino acids were matched on both sides of perfused tubule segments. These data suggest that regulation of amino acid movement in vivo may involve the vasa recta, not the DTL of Henle's loops. The data also suggest that transepithelial movement of amino acids in thin limbs of Henle's loop may occur via a paracellular route.

Calcium homeostasis in crustacea: The evolving role of branchial, renal, digestive and hypodermal epithelia

Crustaceans serve as an ideal model for the study of calcium homeostasis due to their natural molting cycle. Demineralization and remineralization of the calcified cuticle is accompanied by bidirectional Ca transfer across the primary Ca transporting epithelia: gills, antennal gland (kidney), digestive system, and cuticular hypodermis. The review will demonstrate how a continuum of crustaceans can be used as a paradigm for the evolution of Ca transport mechanisms. Generally speaking, aquatic crustaceans rely primarily on branchial Ca uptake and accordingly are affected by water Ca content; terrestrial crustaceans rely on intake of dietary Ca across the digestive epithelium. Synchrony of mineralization at the cuticle vs. storage sites will be presented. Physiological and behavioral adaptations have evolved to optimize Ca balance during the molting cycle in different Ca environments. Intracellular Ca regulation reveals common mechanisms of apical and basolateral membrane transport as well as intracellular sequestration. Regulation of cell Ca concentration will be discussed in intermolt and during periods of the molting cycle when transepithelial Ca flux is significantly elevated. Molecular characterization of the sarco-/endoplasmic reticular Ca pump in aquatic species reveals the presence of two isoforms that originate from a single gene. This gene is differentially expressed during the molting cycle. Gene expression may be regulated by a suite of hormones including ecdysone, calcitonin, and vitamin D. Perspectives for future research are presented. J. Exp. Zool. 283:620–640, 1999. © 1999 Wiley-Liss, Inc.

Calcium homeostasis in crustacea: the evolving role of branchial, renal, digestive and hypodermal epithelia.

Crustaceans serve as an ideal model for the study of calcium homeostasis due to their natural molting cycle. Demineralization and remineralization of the calcified cuticle is accompanied by bidirectional Ca transfer across the primary Ca transporting epithelia: gills, antennal gland (kidney), digestive system, and cuticular hypodermis. The review will demonstrate how a continuum of crustaceans can be used as a paradigm for the evolution of Ca transport mechanisms. Generally speaking, aquatic crustaceans rely primarily on branchial Ca uptake and accordingly are affected by water Ca content; terrestrial crustaceans rely on intake of dietary Ca across the digestive epithelium. Synchrony of mineralization at the cuticle vs. storage sites will be presented Physiological and behavioral adaptations have evolved to optimize Ca balance during the molting cycle in different Ca environments. Intracellular Ca regulation reveals common mechanisms of apical and basolateral membrane transport as well as intracellular sequestration. Regulation of cell Ca concentration will be discussed in intermolt and during periods of the molting cycle when transepithelial Ca flux is significantly elevated. Molecular characterization of the sarco-/endoplasmic reticular Ca pump in aquatic species reveals the presence of two isoforms that originate from a single gene. This gene is differentially expressed during the molting cycle. Gene expression may be regulated by a suite of hormones including ecdysone, calcitonin, and vitamin D. Perspectives for future research are presented.

Bidirectional Transepithelial Water Transport: Measurement and Governing Mechanisms

In the search for the mechanisms whereby water is transported across biological membranes, we hypothesized that in the airways, the hydration of the periciliary fluid layer is regulated by luminal-to-basolateral water transport coupled to active transepithelial sodium transport. The luminal-to-basolateral ( J W L→B) and the basolateral-to-luminal ( J W B→L) transepithelial water fluxes across ovine tracheal epithelia were measured simultaneously. The J W L→B (6.1 μl/min/cm 2) was larger than J W B→L (4.5 μl/min/cm 2, p < 0.05, n = 30). The corresponding water diffusional permeabilities were P d L→B = 1.0 × 10 −4 cm/s and P d B→L = 7.5 × 10 −5 cm/s. The activation energy ( E a) of J W L→B (11.6 kcal/mol) was larger than the E a of J W B→L (6.5 kcal/mol, p < 0.05, n = 5). Acetylstrophanthidin (100 μM basolateral) reduced J W L→B from 6.1 to 4.4 μl/min/cm 2 ( p < 0.05, n = 5) and abolished the PD. Amiloride (10 μM luminal) reduced J W L→B from 5.7 to 3.7 μl/min/cm 2 ( p < 0.05, n = 5) and reduced PD by 44%. Neither of these agents significantly changed J W B→L. These data indicate that in tracheal epithelia under homeostatic conditions, J W B→L was dominated by diffusion ( E a = 4.6 kcal/mol), whereas ∼30% of J W L→B was coupled to the active Na +,K +-ATPase pump ( E a = 27 kcal/mol).

Intestinal transport of beta-lactam antibiotics: analysis of the affinity at the H+/peptide symporter (PEPT1), the uptake into Caco-2 cell monolayers and the transepithelial flux.

This study on the intestinal transport of beta-lactam antibiotics was undertaken to investigate the correlation between cellular transport parameters and the bioavailability. Transport of 23 beta-lactam antibiotics was characterized by measuring their ability to inhibit the uptake of glycylsarcosine into Caco-2 cells, their uptake into the cells and their total flux across the cell monolayers. Ceftibuten and cyclacillin were recognized by PEPT1 with affinity constants comparable to those of natural dipeptides (K(i) = 0.3 and 0.5 mM, respectively). Cefadroxil, cefamandole, cephradine, cefaclor, cefuroxime-axetil, cefixime, cephalotin, cephalexin and ampicillin also interacted with PEPTI (K(i) = 7-14 mM). In contrast, cefapirin, cefodizime, cefuroxime, cefmetazole, ceftazidime, benzyl-penicillin, ceftriaxone, cefpirome, cefotaxime, cefepime, cephaloridine and cefsulodin displayed no affinity to the transport system (K(i) > 20 mM). The uptake into the cells and the transepithelial flux was highest for those beta-lactam antibiotics, which showed the strongest inhibition of [14C]Gly-Sar transport (p < 0.0001). Exceptions were cefuroximaxetil and cephalotin. The probability of oral bioavailability for beta-lactam antibiotics is mainly determined by their affinity to PEPTI. A threshold K(i) value of 14 mM with respect to Gly-Sar uptake is required.

Ochratoxin A secretion in primary cultures of rabbit renal proximal tubule cells.

Primary cultures of rabbit renal proximal tubule cells grown under improved culture conditions were used to study the transepithelial transport of the nephrotoxic mycotoxin ochratoxin A. The basal-to-apical transepithelial flux, i.e., secretion, of this fluorescence organic acid was measured in primary cultures of rabbit renal proximal tubule cells. The basal-to-apical flux of ochratoxin A increased with time and reached a steady state after 12 h. On the other hand, the apical-to-basal flux, i.e., reabsorption, of ochratoxin A was minimal over time. The secretory flux of ochratoxin A was as much as eightfold greater than the reabsorptive flux, indicating that net secretion is the primary mechanism for ochratoxin A clearance by the proximal tubule. The kinetic analysis of ochratoxin A flux revealed secretion to be a saturable and very high-affinity process with an apparent K50 of 0.33 +/- 0.21 mM. A saturating concentration of the prototypical organic anion substrate para-aminohippurate (PAH) reduced ochratoxin A secretion by approximately 75%. The kinetic analysis of PAH inhibition of ochratoxin A secretion revealed an IC50 of 195 mM, which is similar to the IC50 for PAH inhibition of peritubular ochratoxin A uptake in tubule suspensions and the Km, values for peritubular PAH uptake. The organic anions probenecid, octanoate, and alpha-ketoglutarate reduced ochratoxin A excretion to the same degree as PAH, whereas the amino acid phenylalanine had a minimal effect on ochratoxin A secretion. Thus, collectively, these observations indicate that the secretion of ochratoxin A in primary cultures of rabbit renal proximal tubules is limited to the organic anion secretory pathway. The high affinity measured for the basal-to-apical flux of ochratoxin A suggests that at concentrations typical of naturally occurring exposures, transepithelial secretion by the organic anion transport pathway represents a significant avenue for excretion of this mycotoxin by the renal proximal tubule.

Regulation of paracellular absorption of cimetidine and 5-aminosalicylate in rat intestine.

Isolating the relative contributions of parallel transcellular and paracellular transport to the intestinal absorption of small hydrophilic molecules has proven experimentally challenging. In this report, lumenal appearance of drug metabolite is utilized as a tool to assess the contribution of paracellular transport to the absorption of cimetidine and 5-aminosalicylate (5ASA) in rat small intestine. Steady-state intestinal absorption and elimination of cimetidine and 5ASA were studied in single-pass intestinal perfusions in rats. Both drugs were metabolized in intestinal epithelia with subsequent metabolite secretion into the intestinal lumen. Jejunal cimetidine absorption decreased with increasing perfusion concentration while the ratio of lumenal metabolite to lumenal drug loss increased. Cimetidine uptake at perfusion concentrations above 0.4 mM resulted in over 80% drug elimination into the jejunal lumen. Inhibition of intracellular metabolism of cimetidine by methimazole did not alter epithelial uptake but totally abolished transepithelial cimetidine flux indicating an elevation of intracellular cimetidine. Similarly, co-perfusion of 5ASA with cimetidine and methimazole totally abolished 5ASA absorption but increased lumenal levels of N-acetyl 5ASA indicating an increase in intracellular uptake of 5ASA. Cimetidine and 5ASA absorption across rat jejunal epithelia are exclusively paracellular. Elevation of intracellular cimetidine, inferred from mass balance considerations, restricts paracellular transport of both drugs.