Nicotinic acid transport mediated by pH-dependent anion antiporter and proton cotransporter in rabbit intestinal brush-border membrane

In order to determine whether the vitamin nicotinic acid is absorbed via an anion antiporter, intestinal epithelial cell membrane transport mechanisms for nicotinic acid were characterized using isolated rabbit jejunal brush-border membrane vesicles. The uptake of nicotinic acid by the membrane vesicles showed an overshoot phenomenon in the presence of an outwardly directed bicarbonate gradient or an inwardly directed proton gradient and the uptakes were two times and six times greater, respectively, than that in the absence of any ion gradient. The bicarbonate-dependent initial uptake of nicotinic acid was increased at acidic pH, showing pH-dependent transport activity. An inhibitor of anion transport, 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid, specifically reduced bicarbonate-dependent transport of nicotinic acid. The initial uptakes of nicotinic acid via the anion antiporter and the proton cotransporter were specifically inhibited by monocarboxylic acids such as acetic acid, benzoic acid, D- and L-lactic acid, pravastatin and valproic acid, but not by di- or tricarboxylic acids, bile acids or amino acids. Nicotinic acid uptake activity was, furthermore, expressed in a Xenopus laevis oocyte system after injection of messenger RNA (mRNA) derived from rabbit intestinal epithelial cells. These observations demonstrate that nicotinic acid is absorbed by two independent active transport mechanisms from small intestine, i.e. a proton cotransporter and an anion antiporter. The pH-dependence observed in the intestinal absorption of nicotinic acid might, therefore, be ascribed partly to pH-sensitive and partly to carrier-mediated transport mechanisms in the brush-border membrane.     

Genetic integrity of transforming growth factor beta (TGF-beta) receptors in cervical carcinoma cell lines: loss of growth sensitivity but conserved transcriptional response to TGF-beta

PURPOSE: Fractal kinetics was used for the analysis of the carrier-mediated transport for drugs across the intestinal epithelial cells. METHODS: The transport was examined under various agitation rates using a monolayer of Caco-2 cells and rabbit ileum sheets. RESULTS: The passive transport of antipyrine across Caco-2 cells was increased with the increasing rate of agitation and was supposed to be caused by a change in the thickness of the unstirred water layer. On the contrary, in the case of L-lactic acid transport, which follows a carrier-mediated transport mechanism, the more the agitation rate controlling the fractal dimension was increased, the more the permeability rate across the Caco-2 cells was decreased. Fractal kinetic analysis of L-lactic acid transport indicated that the permeability was caused by a single saturable process. Similar agitation effects with L-lactic acid transport were observed in the transport of phenylalanine and cephradine in Caco-2 cells. However, the permeability rates of benzoic acid and 3-O-methyl-D-glucose across Caco-2 cells and L-lactic acid transport across the rabbit ileum tissue indicated the maximum levels at a designated agitation rate. This phenomenon was likely to be caused by the agitation effects controlling not only the fractal environment but also the unstirred water layer. CONCLUSIONS: Carrier-mediated transports are well defined by fractal kinetics rather than classical kinetic analysis. Fractal kinetics are one of the important areas for understanding and confirming the properties of a carrier-mediated transport process.     

Carrier-mediated absorption of salicylic acid from hamster cheek pouch mucosa

Previously, we found that monocarboxylic acids undergo carrier-mediated transport in primary cultures of oral mucosal epithelial cells.1 In this study, we investigated whether carrier-mediated absorption of a monocarboxylic acid from the oral mucosa occurs in vivo. Salicylic acid was administered to hamster cheek pouch. At predetermined intervals, the concentration of salicylic acid in the fluid remaining in the cheek pouch lumen and the blood salicylic acid concentration were determined. The absorption of salicylic acid was saturable at high salicylic acid concentrations. Sodium azide, a metabolic inhibitor, and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP), a protonophore, significantly inhibited the absorption of salicylic acid but not the absorption of salicylamide from the oral mucosa. Various monocarboxylic acids inhibited the absorption of salicylic acid, whereas dicarboxylic acids had no such effect. Transfer of [14C]salicylic acid from the cheek pouch mucosa to the systemic circulation was observed, and the blood [14C]salicylic acid concentration in the case of coadministration with propionic acid was significantly lower than that in the case of no propionic acid coadministration. These results show that monocarboxylic acids undergo carrier-mediated absorption from the hamster cheek pouch mucosa.     

Proton-cotransport of pravastatin across intestinal brush-border membrane

PURPOSE: The purpose of the present study is to clarify the intestinal brush-border transport mechanism of a weak organic acid, pravastatin, an HMG-CoA reductase inhibitor. METHODS: The transport of pravastatin was studied by using intestinal brush-border membrane vesicles prepared from rabbit jejunum, and uptake by the membrane vesicles was measured using rapid filtration technique. RESULTS: The initial uptake of [14C]pravastatin was markedly increased with decreases in extravesicular pH and showed a clear overshoot phenomenon in the presence of a proton gradient (pHin/out = 7.5/5.5). A protonophore, carbonylcyanide p-trifluoromethoxyphenylhydrazone, significantly reduced the uptake of [14C]pravastatin. In addition, an ionophore for sodium, potassium and proton, nigericin, stimulated the uptake of [14C]pravastatin in the presence of a potassium gradient ([K+]in/[K+]out = 0/145 mM). On the other hand, neither the imposition of an inwardly directed sodium gradient nor an outwardly directed bicarbonate gradient stimulated the uptake of [14C]pravastatin. In the presence of a proton gradient (pHin/out = 7.5/5.5), the initial uptake of pravastatin was saturable with the apparent Kt of 15.2 +/- 3.2 mM and Jmax of 10.6 +/- 1.21 nmol/mg protein/10 sec. The uptake of pravastatin was significantly inhibited by monocarboxylic acid compounds such as acetic acid and nicotinic acid in a competitive manner but not by di- or tricarboxylic acids, or acidic amino acid. CONCLUSION: It was concluded that a pH-dependent transport of pravastatin across the brush-border membrane occurs by a proton-gradient dependent carrier-mediated mechanism rather than by simple diffusion of its unionized form.     

Lactate transport by cortical synaptosomes from adult rat brain: characterization of kinetics and inhibitor specificity

Since lactate released by glial cells may be a key substrate for energy in neurons, the kinetics for the uptake of L-[U-14C]lactate by cortical synaptic terminals from 7- to 8-week-old rat brain were determined. Lactate uptake was temperature-dependent, and increased by 64.9% at pH 6.2, and decreased by 43.4% at pH 8.2 relative to uptake at pH 7.3. Uptake of monocarboxylic acids was saturable with increasing substrate concentration. Eadie-Hofstee plots of the data gave evidence of two carrier-mediated uptake mechanisms with a high-affinity Km of 0.66 mM and Vmax of 3.66 mM for pyruvate, and a low-affinity system with a Km of 9.9 mM for both lactate and pyruvate and Vmax values of 16.6 and 23.1 nmol/30 s/mg protein for lactate and pyruvate, respectively. Saturable uptake was seen in the presence of 10 mM alpha-cyano-4-hydroxycinnamate. Lactate transport by synaptic terminals was much more sensitive to inhibition by sulfhydryl reagents than transport in astrocytes. Addition of 0.5 and 2 mM mersalyl decreased the uptake of 1 mM lactate by synaptic terminals by 59.3 and 66.37%, respectively. Pyruvate moderately decreased lactate transport, whereas 3-hydroxybutyrate had little effect. Quercetin, an inhibitor of lactate release, had little effect on the content of 14C lactate in synaptic terminals, supporting the concept that the majority of lactate produced within brain is from glial cells. Oxidation of L-[U-14C]lactate by synaptosomes was saturable, and yielded a Km of 1.23 mM and a Vmax of 116 nmol/h/mg protein. Overall the studies show that synaptic terminals from adult brain have a high capacity for transport and oxidation of lactate, consistent with the proposed role for this compound in metabolic trafficking in brain. Furthermore, the data provide kinetic evidence of two carrier-mediated mechanisms for monocarboxylic acid transport by synaptosomes and demonstrate that uptake of lactate by synaptic terminals is regulated differently than transport by astrocytes. Uptake of lactate by synaptic terminals also has differences from the systems described for neurons.     

The endocytosis of transferrin by rat intestinal epithelial cells

BACKGROUND/AIMS: The transferrin receptor is a prominent protein on the basal and lateral membranes of intestinal epithelial cells, yet little is known of the function of the receptor in the intestine. The aim of the present study was to determine whether intestinal transferrin receptors were capable of facilitating transferrin internalization. METHODS: Using the rat as an experimental model, the uptake of radiolabeled transferrin by cells isolated from different regions along the crypt-villus axis of the proximal small intestine was studied. RESULTS: An intestinal epithelial cell fraction highly enriched in crypt cells bound most radiolabeled transferrin. Cells in this fraction were able to internalize transferrin and recycle it back to the cell surface. A high affinity, saturable pathway of transferrin uptake by these cells predominated at transferrin concentrations below 0.3 mumol/L, whereas at higher concentrations, most uptake was via a nonsaturable process. Intravenously injected radiolabeled transferrin could be detected within intestinal crypt cells, indicating that these cells are able to internalize transferrin in vivo. CONCLUSIONS: These data suggest that intestinal crypt cells have an active transferrin/transferrin receptor system. Transferrin may play an important role in iron delivery to and/or as a growth factor for the rapidly proliferating intestinal epithelium.     

Inhibitory effects of anions and active site amino acid sequence of chicken liver L-2-hydroxyacid oxidase A, a member of the FMN-dependent alpha-hydroxyacid oxidizing enzyme family

Monocarboxylic acids with aliphatic chains were found to be mixed inhibitors of chicken liver L-2-hydroxyacid oxidase A when L-2-hydroxy-4-methylthiobutanoic acid was used as the substrate. The finding that the binding affinity of the enzyme for monocarboxylic acids was directly proportional to the number of carbon atoms in the chain strongly suggests that in addition to the electrostatic interaction due to the carboxyl moiety, hydrophobic forces may also be involved in the binding affinity of monocarboxylic acids to the enzyme's active site. Oxalate, a dicarboxylic acid, also resulted in a mixed-type inhibition of chicken liver L-2-hydroxyacid oxidase A, and, surprisingly, its binding affinity to the enzyme was found to be quite high as compared with monocarboxylic acids. This is probably due to the fact that the two carboxyl groups of oxalate give rise to electrostatic interactions with the positively charged side chains of two adjacent residues in the polypeptide chain. The inhibitory effects of other dicarboxylic acids was found to decrease as the number of carbon atoms in the chain increased. Oxamate was found however to be a novel type of potent inhibitor of the enzyme. All in all, these kinetic studies and the amino acid sequence determination in the active site region after limited proteolysis of the polypeptide chain definitely establish that chicken liver NADH/FMN containing L-2-hydroxyacid oxidase A is a member of the FMN-dependent alpha-hydroxyacid oxidizing enzyme family.     

Zinc transport across an endothelium includes vesicular cotransport with albumin

Bovine pulmonary arterial endothelial cells (BPAEC) were grown on permeable polycarbonate membrane filters suspended between two compartments representing the blood vessel lumen and the interstitium. This in vitro model of an endothelium was subjected to a battery of tests to unravel the mechanisms of zinc transport from the blood into peripheral tissues. Transport of 65Zn across BPAEC from media containing zinc concentrations up to 50 mumol/L exhibited both saturable and nonsaturable kinetics. Vmax of the saturable component was 246 +/- 43 pmol/(h x cm2) and Km was 2.3 +/- 1.3 mumol/L. Transport was pH and temperature sensitive and substantially influenced by albumin and histidine concentrations, but not influenced by analogous minerals or metabolic inhibitors. Inhibition of coated vesicle formation by depletion of intracellular potassium reduced 65Zn transport. Albumin carrying a zinc ion crossed the endothelium more rapidly than zinc-free albumin. When evaluated together, this body of evidence supports the existence of two major pathways of zinc transport across the pulmonary endothelium, but neither involves entry into the endothelial cells. One pathway involves receptor-mediated cotransport with albumin by transcytotic vesicles. The other is nonsaturable and involves cotransport with albumin and low molecular weight ligands, principally histidine, through intercellular junctions and nonselective, bulk-fluid transcytosis.     

Mechanism of valproic acid uptake by isolated rat brain microvessels

In an effort to characterize putative transport systems of valproic acid (VPA) at the blood-brain barrier, the effects of various substrates and inhibitors of known anion transporters on the equilibrium vessel-to-medium concentration (vessel/medium) ratio of VPA were investigated using isolated rat brain microvessels. The equilibrium vessel/medium ratio of VPA was decreased by the presence of high millimolar concentration of unlabeled VPA, indicating that a saturable transport system was involved in VPA transport from medium to microvessels. Short-chain monocarboxylates such as propionic acid, pyruvic acid, and L-lactic acid did not alter the vessel/medium ratio, whereas medium-chain fatty acids and unsaturated metabolites of VPA significantly inhibited the net transport of VPA. Dicarboxylates, tricarboxylate, and p-aminohippuric acid did not affect VPA accumulation in the brain microvessels. Several anionic drugs including salicylic acid, penicillin G, cefazolin, and probenecid significantly reduced the vessel/medium ratio of VPA. In addition, disulfonate inhibitors of inorganic anion exchangers, SH-group modifying reagent, and metabolic inhibitor showed remarkable inhibitory effects on the net transport of VPA between brain microvessels and medium. These results suggest that VPA may be actively transported through the antiluminal membrane via a carrier-mediated system shared by other anionic drugs.     

Carrier-mediated transport of monocarboxylate drugs in the pigmented rabbit conjunctiva

PURPOSE: To determine whether an Na+-dependent monocarboxylate transport process exists on the mucosal side of the pigmented rabbit conjunctiva and to evaluate how it may contribute to the absorption of ophthalmic monocarboxylate drugs. METHODS: L-lactate was used as a model substrate. The excised pigmented rabbit conjunctiva was mounted in a modified Ussing chamber for the measurement of short-circuit current (Isc) and 14C-L.-lactate transport. RESULTS: When added to the mucosal side at 37 degrees C and at pH 7.4, applications of as much as 40 mM L- and D-lactate increased Isc in a saturable manner. By contrast, no change in Isc was observed at 4 degrees C or under the mucosal Na+-free condition. 14C-L-lactate transport in the mucosal-to-serosal (m-s) direction at 0.01 mM revealed directionality, temperature dependency, Na+ dependency, and ouabain sensitivity, but not pH dependency. L-lactate transport in the m-s direction consisted of a saturable Na+-dependent process by the transcellular pathway and a nonsaturable process by the paracellular pathway. For the saturable process, the apparent Michaelis-Menten constant was 1.9 mM, the maximum flux was 8.9 nanomoles/cm2 per hour, and the apparent Na+ :L-lactate coupling ratio was 2:1. 14C-L-lactate transport in the m-s direction was significantly inhibited (46% to 83%) by the mucosal presence of various monocarboxylate compounds, but not by dicarboxylate compounds, zwitterionic compound, D-glucose, amino acids, and peptidomimetic antibiotics. Monocarboxylate nonsteroidal anti-inflammatory drugs and the antibacterial fluoroquinolones inhibited 14C-L-lactate transport by 40% to 85%, whereas prostaglandins and cromolyn had no effect. CONCLUSIONS: An Na+-dependent monocarboxylate transport process that may be used by non-steroidal anti-inflammatory and fluoroquinolone antibacterial drugs for transport appears to be present on the mucosal side of the pigmented rabbit conjunctiva. A possible physiologic role for the Na+-dependent monocarboxylate transport process may be to salvage tear lactate.     

Transport of L-cystine by cultivated skin fibroblasts of normal subjects and patients with cystinosis

Uptake of L-cystine at the plasma membrane of fibroblasts derived from normal and cystinotic subjects was studied. L-Cystine accumulation after a 20-min period was increased in cystinotic fibroblasts incubated in 0.08 mM L-cystine. This effect appeared to be concentration-dependent since accumulation after 20 min at 0.004 mM concentration was decreased in cystinotic cells. Kinetic data suggested that at least two nondiffusional saturable processes with widely different substrate affinities mediate initial L-cystine uptake in skin fibroblasts. In addition, the transport process with high affinity for L-cystine may itself be a two-component system, as suggested by (1) additive inhibitory effect of other neutral amino acids, and (2) preincubation studies in which preincubation with cystathionine enhanced subsequent L-cystine uptake, whereas preincubation with other neutral amino acids depressed subsequent uptake. Affinity constants and maximal velocities of initial uptake did not appear to be altered in cells derived from patients with cystinosis. After 60-sec incubation with L-[35S] cystine, cystinotic cells retained more label as cystine than did normal cells at each concentration studied. These data indicate that initial L-cystine uptakein fibroblasts of patients with cystinosis proceeds at a normal rate by means of all transport systems currently shown to be present in normal cells.     

In vitro modulation of L-arginine transport in trophoblast cells by glucose

BACKGROUND: The uptake of the semi-essential amino acid, L-arginine, into trophoblast cells was measured with the aim of determining the effect of different glucose concentrations on L-arginine influx kinetics. METHODS: This study used a novel superfused microcarrier culture system of BeWo cells (an established choriocarcinoma cell line with many characteristics of normal human trophoblast) and a rapid, paired-tracer dilution technique to measure unidirectional influx into the cells. RESULTS: At 10 mmol L-1 D-glucose, L-arginine unidirectional influx across the microvillous border of the cells was saturable with a Km of 1.14 +/- 0.14 mmol L-1 and a Vmax of 121. 36 +/- 5.89 nmol mg-1 protein min-1. When cells were preincubated for 24 h in the presence of 30 mmol L-1 D-glucose, there was a significant increase in the Vmax for L-arginine of nearly 30%. Similarly, preincubation in the presence of 1 mmol L-1 D-glucose and 12.5 mIU mL-1 human insulin reduced the Km for L-arginine influx by over 55%. CONCLUSION: These data suggest that the modulation of placental transport of L-arginine by glucose and insulin could contribute to the fetal macrosomia observed in diabetic mothers.     

Epoxygenase metabolites of arachidonic acid affect electrophysiologic properties of rat tracheal epithelial cells1

Epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids, products of the cytochrome P450 arachidonic acid epoxygenase pathway, have been shown to affect electrolyte transport in the kidney; however, the effects of these compounds on airway epithelial ion transport have not been investigated. Intact rat tracheas and primary cultures of rat tracheal epithelial cells were mounted in Ussing chambers to monitor changes in transepithelial voltage (Vt), short circuit current (Isc) and electrical resistance (Rt), with or without the addition of increasing concentrations (10(-9)-10(-6) M) of arachidonic acid, each of the four regioisomeric EETs and each of the corresponding dihydroxyeicosatrienoic acids. In intact tracheas, 11,12-EET caused dose-dependent decreases in Vt and Isc (DeltaVt = 0. 4 +/- 0.1 mV, DeltaIsc = -16.9 +/- 5.4 microA/cm2 at 10(-6) M, P < . 05 vs. vehicle), whereas changes in Rt were not significantly different than vehicle alone. 11,12-dihydroxyeicosatrienoic acid caused less impressive decreases in Vt and Isc, although arachidonic acid and the other compounds tested were without significant effects. 11,12-EET induced similar changes in cultured tracheal epithelial cell electrical parameters at concentrations as low as 10(-9) M. The effects of 11,12-EET were highly stereoselective, with activity limited to 11(R),12(S)-EET, the least abundant rat lung enantiomer. Pretreatment with amiloride or mucosal exposure to sodium free media did not significantly alter the 11,12-EET-induced changes in Vt. In contrast, pretreatment with bumetanide abolished the 11,12-EET electrophysiologic effects, suggesting that these effects may be mediated through inhibition of a chloride conductive pathway. We conclude that arachidonic acid epoxygenase metabolites cause significant changes in rat airway electrical parameters and may be involved in the control of lung fluid and electrolyte transport.     

CYP2J subfamily cytochrome P450s in the gastrointestinal tract: expression, localization, and potential functional significance

Our laboratory recently described a new human cytochrome P450 arachidonic acid epoxygenase (CYP2J2) and the corresponding rat homologue (CYP2J3), both of which were expressed in extrahepatic tissues. Northern analysis of RNA prepared from the human and rat intestine demonstrated that CYP2J2 and CYP2J3 mRNAs were expressed primarily in the small intestine and colon. In contrast, immunoblotting studies using a polyclonal antibody raised against recombinant CYP2J2 showed that CYP2J proteins were expressed throughout the gastrointestinal tract. Immunohistochemical staining of formalin-fixed, paraffin-embedded intestinal sections using anti-CYP2J2 IgG and avidin-biotin-peroxidase detection revealed that CYP2J proteins were present at high levels in nerve cells of autonomic ganglia, epithelial cells, intestinal smooth muscle cells, and vascular endothelium. The distribution of this immunoreactivity was confirmed by in situ hybridization using a CYP2J2-specific antisense RNA probe. Microsomal fractions prepared from human jejunum catalyzed the NADPH-dependent metabolism of arachidonic acid to epoxyeicosatrienoic acids as the principal reaction products. Direct evidence for the in vivo epoxidation of arachidonic acid by intestinal cytochrome P450 was provided by documenting, for the first time, the presence of epoxyeicosatrienoic acids in human jejunum by gas chromatography/mass spectrometry. We conclude that human and rat intestine contain an arachidonic acid epoxygenase belonging to the CYP2J subfamily that is localized to autonomic ganglion cells, epithelial cells, smooth muscle cells, and vascular endothelium. In addition to the known effects on intestinal vascular tone, we speculate that CYP2J products may be involved in the release of intestinal neuropeptides, control of intestinal motility, and/or modulation of intestinal fluid/electrolyte transport.     

Synthesis, structure and spectroscopic studies of europium complex with S（＋）-mandelic acid

The Eu3+ complexes with S(+) -mandelic acid were synthesized in the form of powders by mixing aqueous solutions of EuCl3,S(+) -mandelic acid and NaOH in different molar ratios.The powders were characterized by elemental analysis,X-ray powder diffraction(XRPD) method,Fourier transform infrared(FTIR) and Raman spectroscopy,UV-vis reflectance and luminescence spectra as well as lumi-nescence lifetime measurements.It was found that all studied powders of Eu3+ complexes with S(+) -mandelic acid were isostructural and crystalline and formed compounds with the formula Eu(Man) 3(H2O) 2.     

Intestinal absorption of peptides by coupling to bile acids

Poor intestinal absorption of peptides greatly limits their use as drugs for the treatment of chronic diseases. Since bile acids are efficiently absorbed by an active, Na(+)-dependent transport system in the ileum of mammals, model peptides of different chain length were attached to the 3-position of modified 3 beta-(omega-amino-alkoxy)-7 alpha, 12 alpha-dihydroxy-5 beta-cholan-24-oic acid. These peptide-bile acid conjugates inhibited Na(+)-dependent [3H]taurocholate uptake into brush-border membrane vesicles isolated from rabbit ileum in a concentration-dependent manner. Furthermore, photoaffinity labeling of the bile acid-binding proteins of M(r) 93,000 and 14,000, identified as the protein components of the ileal Na(+)-dependent bile acid transport system in rabbit ileum (Kramer, W., Girbig, F., Gutjahr, U., Kowalewski, S., Jouvenal, K., Müller, G., Tripier, D., and Wess, G. (1993) J. Biol. Chem. 268, 18035-18046) by the photoreactive taurocholate analogue, (3,3-azo-7 alpha, 12 alpha-dihydroxy-5 beta [7 beta, -12 beta-3H]cholan-24-oyl)-2-aminoethanesulfonic acid, was inhibited by the peptide-bile acid conjugates. In contrast, the parent peptides and amino acids neither had a significant effect on [3H]taurocholate uptake by ileal brush-border membrane vesicles nor on photoaffinity labeling of the ileal bile acid-binding membrane proteins. The inhibitory effect of peptide-bile acid conjugates on [3H]taurocholate transport and photoaffinity labeling of the bile acid-binding proteins in rabbit ileal vesicles decreased with increasing chain length of the attached peptide radical. By in vivo ileum perfusion in anesthetized rats an intestinal absorption of the bile acid conjugate S3744 of the fluorescent oxaprolylpeptide 4-nitrobenzo-2-oxa-1,3-diazol-beta-Ala-Phe-5-Opr-Gly (S1037) and secretion of the intact compound into bile could be demonstrated, whereas the parent peptide S1037 or its t-butylester S4404 were not absorbed. The intestinal absorption of S3744 showed a similar temperature dependence as [3H]taurocholate absorption and was inhibited by the presence of taurocholate indicating a carrier-mediated uptake of S3744 via the ileal bile acid transporter. In conclusion, these results indicate that oligopeptides can be made enterally absorable by coupling to modified bile acid molecules making use of the specific intestinal absorption pathway for bile acids. This finding may be of great importance for the design and development of orally active peptide drugs.     

Paracellular calcium transport across Caco-2 and HT29 cell monolayers

Intestinal calcium absorption has been shown to include two processes, a saturable transcellular movement and a non-saturable paracellular pathway. The potential utility of cell monolayers for studying transepithelial intestinal calcium transport has already been demonstrated; however, simultaneous evaluation of the contribution of the saturable transcellular and of the non-saturable paracellular processes to the total transepithelial transport has not yet been attempted. The aim of this study was to investigate the contribution both of transcellular and paracellular transport processes to the total transepithelial calcium transport in two cell culture monolayers. Caco-2 cells and a clone derived from HT-29 cells (HT29-Cl.19A), two cell lines derived from colon adenocarcinomas which are known to be able to exhibit typical enterocytic differentiation, were used. Cell monolayers were grown on a permeable support and used after 15 days of culture when these cells express enterocytic differentiation and high transepithelial resistance. Isotopic transport rate measurements were performed in the absence of a chemical gradient. The paracellular route was evaluated using [3H]mannitol. Calcium and [3H]mannitol transport rates across cell monolayers were not significantly different. Augmentation of calcium uptake by 200 mM sorbitol did not significantly increase calcium or mannitol transepithelial transport; however, calcium accumulation in the cells was increased by about 200%. Modulation of the monolayer permeability by addition of 10 nM vasoactive intestinal polypeptide (VIP) or 0.5 mM carbachol treatment, which respectively increased and decreased the transepithelial resistance, consequently modified calcium and mannitol transport in a parallel manner. Our results show that Caco-2 and HT29-Cl.19A cell monolayers are good models for studying the calcium paracellular transport pathway.     

The effect of Escherichia coli heat-stable (STA) enterotoxin on in vitro uptake of amino acids by small intestine of sucking rats during fluid accumulation

In vitro uptake of 14C-labelled amino acids by segments of small intestine was determined in sucking (2-4-d-old) Wistar rats. Intragastric injections of heat-stable (ST) toxin of enterotoxigenic Escherichia coli (ETEC) were given to produce fluid accumulation, defined as a gut weight: carcass weight value of > 0.085. Continued active uptake of the prototypic amino acids, leucine (by active transport system 1 for monoamino monocarboxylic (neutral) amino acids), lysine (by active transport system 2 for dibasic amino acids), and proline (by active transport system 3 for N-substituted amino acids), persisted during the active fluid accumulation response to ETEC ST toxin. The mean Kt and mean Vmax of the amino acid transport systems were similar in control (non-injected) and ST toxin-injected rats. The present study provides a scientific basis for the use of amino acids in oral rehydration solutions utilizing amino acid transport systems which are linked to absorption of Na (and water) so that reduction in diarrhoeal stools can be achieved, and emphasizes the importance of maintaining feeding during acute diarrhoea to prevent development of malnutrition.     

Production of a mixture of antimicrobial organic acids from lactose by co-culture of Bifidobacterium longum and Propionibacterium freudenreichii

The antimicrobial activities of standard solutions of three organic acids (lactic, acetic, and propionic acids) were compared using Micrococcus luteus, Pseudomonas sp. and Staphylococcus aureus as test microorganisms. At the same concentrations of the undissociated form, the antimicrobial activities of acetic and propionic acids were higher than that of lactic acid, irrespective of test microorganisms. In a single cultivation of Bifidobacterium longum, a mixture of lactic (17 g/l) and acetic (20 g/l) acids was produced from 50 g/l lactose and its antimicrobial activities against M. luteus, Pseudomonas sp., and S. aureus correspond to that of 32, 19, and 25 g/l of acetic acid, respectively. To increase the total antimicrobial activity, a co-culture of B. longum and Propionibacterium freudenreichii, in which lactic acid produced once from lactose by B. longum was converted to acetic and propionic acids by P. freudenreichii, was done using TPY medium containing commercially available peptones as a nitrogen source. By the sequential conversion of lactose using the two microorganisms, the culture supernatant containing a mixture of acetic (27 g/l) and propionic (13 g/l) acids without lactic acid was produced. The antimicrobial activities of the mixture against M. luteus, Pseudomonas sp., and S. aureus were 35, 30, and 26 g/l as a concentration of acetic acid, respectively, higher than that obtained in the cultivation of B. longum alone. When the medium containing an enzymatic hydrolyzate of whey proteins with a protease was used in the co-culture of B. longum and P. freudenreichii, the culture supernatant containing the mixture of organic acids was also obtained in the same manner as the co-culture using TPY medium and the activities were 43, 29, and 29 g/l as a concentration of acetic acid for M. luteus, Pseudomonas sp. and S. aureus, respectively.