Active absorption of hypoxanthine by lamb jejunum in vitro

The kinetics of [³H]hypoxanthine entry into lamb mid-jejunum were measured using 2-min mucosal exposures. Mucosal hypoxanthine uptake occurred by a Na⁺-and energy-dependent saturable mechanism indicating that a carrier-mediated active transport process is involved. Inhibitory measurements indicate that hypoxanthine, adenosine, uracil and thymine compete for a common entry mechanism. Adenine, uric acid, allantoine and thymidine produced no significant inhibition of hypoxanthine entry. In additional experiments hypoxanthine was transported against a high concentration gradient from the mucosal to the serosal side of everted sacs of mid-jejunum.Therefore hypoxanthine appears to be absorbed by active transport from the lamb jejunum.     

Membrane potentials of epithelial cells in rat small intestine

1. Stripped sacs of rat jejunum in which the outer muscle layers had been removed were found to maintain substantial transport and electrical activities.2. Mucosal and serosal membrane potentials of epithelial cells of normal and stripped everted sacs of rat jejunum were recorded in vitro together with the transmural potential difference.3. The cell interior was negative relative to both serosal and mucosal fluids, the transmural potential being the sum of the two membrane potentials.4. Changes in the transmural potentials in the presence of actively transferred hexoses and amino acids were entirely due to variations in the serosal potential, the mucosal potential being unchanged.5. Serosal and transmural potential increases on the addition of galactose were consistent with Michaelis-Menten kinetics, giving apparent K(m) values of 14.9 and 14.1 mM respectively.6. Phlorrhizin, ouabain, 2,4-dinitrophenol and sodium fluoroacetate inhibited serosal potential changes in the presence of galactose.7. Osmotic potentials resulting from transmural osmotic gradients originated from the serosal layers of the tissue.8. The results are consistent with the concept of a serosally located, electrogenic sodium pump which is stimulated by actively transferred hexoses and amino acids. The sodium-dependent entry mechanism at the mucosal membrane is non-electrogenic.     

Uptake of 5-methyltetrahydrofolic acid by the rat jejunum.

1. Everted sacs of rat jejunum were used to study the transport of 5-methyltetrahydrofolic acid at various concentrations and at different pHs. 2. The transport of 5-methyltetrahydrofolic acid appeared to be linear with increasing incubation time at a 5-methyltetrahydrofolic acid concentration of 10(-5) M in the incubating medium. Tissue uptake was much higher than serosal uptake. 3. At 'zero' concentration gradient of 5-methyltetrahydrofolic acid, the transport from mucosal to serosal sides was negligible but tissue uptake was appreciable. 4. Reducing the incubation temperature from 37 to 27 degrees C gave a Q10 value of 1-8. 5. The characteristics of 5-methyltetrahydrofolic acid uptake suggested a non-saturable transport mechanism. 6. Studies were also carried out with isolated mucosal epithelial cells at different incubation times and at different pH values. 7. The over-all results suggest that 5-methyltetrahydrofolic acid transport is most likely a passive diffusion of its zwitterion and also a solvent drag with water flow. 8. The 5-methyltetrahydrofolic acid may be converted to its zwitterion in an acid microclimate at the surface of the intestinal absorbing cell.     

Drug uptake into everted intestinal sacs. II. Inhibition of secretion by hypertonicity.

Mucosal hypertonicity, metabolic inhibitors, or absence of glucose and oxygen enhance mucosal-to-serosal influx of the cationic drug, pralidoxime (PAM), into sacs of everted rat jejunum in vitro. Conversely, efflux of PAM, which is twice the influx rate, is inhibited by mucosal hypertonicity or cyanide and iodoacetate. When sacs containing PAM, 0.87 mM, and glucose, 10 mM, were placed in identical drug- and sugar-containing mediums, the inside (serosal) concentration of PAM fell by over half in 120 min, whereas that of glucose more than doubled. Mucosal hypertonicity depressed PAM efflux and glucose influx regardless of serosal osmolarity. Although azide and mucosal hypertonicity each depressed glucose uptake and oxygen consumption while accelerating net PAM influx, azide more effectively depressed glucose and oxygen uptake, whereas hypertonicity caused greater acceleration of PAM uptake. Hypertonicity did not affect PAM binding to intestinal tissue. Varying mucosal pH did not change PAM or glucose uptake. Thus, mucosal hypertonicity apparently enhances net mucosal-to-serosal transfer of PAM by blocking its active secretion from serosa to mucosa.     

The use of dietary-restricted rat intestine for active transport studies

1. The effect of dietary restriction (sufficient to produce a loss of about 32% of initial body weight) on intestinal active transport has been studied in the rat by the use of sacs of everted mid-small intestine. Eight D-sugars, four L-sugars and two D-amino acids were employed.2. Dietary restriction enhanced the normally occurring active transport of D-galactose, 3-O-methyl-D-glucose and D-methionine. In addition, sacs of dietary-restricted small intestine were able to concentrate in the serosal fluid D-fucose, D-xylose and D-histidine, which sacs of normal rat intestine could not do. The final (1 hr) serosal/mucosal concentration ratios produced for these actively transported substances were independent of net water movement.3. Sugars which were not concentrated in the serosal fluid of sacs of fully fed or dietary-restricted intestine were D-arabinose, D-fructose, D-glucosamine, D-mannose, L-arabinose, L-fucose, L-sorbose and L-xylose.4. The characteristics of D-fucose and D-xylose active transport suggest that they are transported by the mechanism which actively transports D-glucose. The comparatively low content of D-glucose in dietary-restricted intestine, compared with fully fed intestine, may be part of the explanation for observable active transport of D-fucose and D-xylose by dietary-restricted sacs.5. Thinning of the intestinal wall is believed not to be the cause of the enhanced active transport found during dietary restriction.6. The results show that dietary-restricted rat small intestine may, at times, be more useful than fully fed rat small intestine in the study of intestinal active transport.     

Kinetics of glucose transport by the perfused mid-gut of the freshwater prawn Macrobrachium rosenberg ii.

1. Mucosal influx of [3H]glucose was examined in the mid-gut of a freshwater prawn, Macrobrachium rosenbergii, using an in vitro perfusion technique. 2. [3H]glucose transfer across the apical cell membrane of the epithelium exhibited Michaelis-Menten kinetics (Jmax.in = 0-15 mumole glucose equiv/g. min, Kt = 0-17 mM). Under Na-free conditions, glucose influx was significantly reduced and a linear function of substrate concentration, indicative of either slow cellular diffusion (KD = 7-6 X 10(3) mumole glucose equiv/g. min. mM) or a facilitated process with a low carrier affinity for the sugar. 3. Phlorizin was a potent competitive inhibitor of glucose influx (K1 = 3-6 X10(-3) mM), galactose and 3-O-methylglucose (3-O-MG) were weak inhibitors, and fructose had no evident effect on glucose uptake. Azide, but not iodoacetate (IAA), significantly depressed influx. 4. Absorbed [3H]glucose was rapidly metabolized by the mid-gut. The majority of accumulated activity within the tissue was in the form of phosphorylated compounds and tritiated water (THO), while only 0-3% was recovered as a free-glucose. 5. Preliminary studies examining transmural [3-H]glucose transport, however, demonstrated a significant net mucosal to serosal free-glucose flux across the prawn mid-gut which was Na-dependent and IAA- and phlorizin-sensitive. Two alternative interpretations of the data are advanced as possible mechanisms for transepithelial glucose transport: (1) group translocation, or (2) the operation of an energized, high affinity, baso-lateral sugar transport carrier.     

The kinetics of influx of calcium and strontium into rat intestine in vitro

1. The role of uptake across the brush border in the intestinal absorption of calcium has been studied by examining the kinetics of influx into slices of rat intestine in vitro. Both mucosal and serosal surfaces were exposed to the medium.2. The rate of influx was accurately defined by a two-component expression comprising a saturable (Michaelis-Menten) term and a second term linear with concentration. Influx across the mucosal surface of closed sacs was similar, and the saturable component for slice influx could be ascribed mainly to transport across the mucosal surface. The half-saturation constant for Ca was near 1 mM. This component was predominant at normal luminal concentrations of free Ca in the duodenum of young rats, but less so in jejunum and ileum and in older rats.3. The same kinetic expression applied to Sr influx, with a half-saturation constant of 2-3 mM, and possibly also to Ba with an even higher value.4. The saturable component of Ca influx was greatly reduced by 2,4: dinitrophenol (DNP); influx was also inhibited by iodoacetate, cyanide and at 0 degrees C. Inhibition commenced soon after exposure of the slices. A high concentration of DNP also caused an increase in the linear component of Ca influx.5. The kinetics of Ca influx across the mucosal surface agreed closely with the kinetics of steady-state absorption of Ca either across the whole mucosal epithelium in vivo or across the entire intestinal wall in vitro. This agreement supports the hypothesis that Ca entry across the brush border is the rate-limiting step in absorption; such a hypothesis would allow net Ca translocation while preserving a low intracellular concentration of ionic Ca in the mucosal epithelial cells.     

Concentration-dependent preferences of absorptive and excretive transport cause atypical intestinal absorption of cyclic phenylalanylserine: small intestine acts as an interface between the body and ingested compounds

Intestinal absorption of cyclic phenylalanylserine (cyclo(Phe-Ser)), a precursor of gliotoxin, was studied in isolated rat small intestine as a model cyclic dipeptide. Absorption clearance (CLabs) decreased in the presence of glycylsarcosine, cephalexin or cephradine, substrates for H+/oligopeptide cotransporter (PEPT1). CLabs of cyclo(Phe-Ser) also decreased at 4 degrees C. These indicate that cyclo(Phe-Ser) is in part transported by PEPT1. However, Eadie-Hofstee plot of absorption revealed an atypical profile at lower concentrations of cyclo(Phe-Ser) (around 0.1 mM). Moreover, comparative experiments of absorptive and excretive transport showed that excretive transport from the serosal to mucosal side of isolated intestinal tissue at a 0.1 mM cyclo(Phe-Ser) was superior to absorptive transport from the mucosal side to the serosal side, and vise versa at a 1 mM cyclo(Phe-Ser). These results as well as the results of kinetic analysis indicate that intestinal absorption consists of passive transport, carrier-mediated absorptive transport by PEPT1 and carrier-mediated excretive transport, resulting in atypical absorption. Although cyclic dipeptides have potentials for drug, their intestinal absorption may be complex. The results of this study lead us conclude that absorptive and excretive transport by the small intestine acts as an interface between the body and ingested compounds.     

p-Aminohippuric acid transport into brush border vesicles isolated from flounder kidney.

p-Aminohippuric acid (PAH) transport was investigated in brush border vesicles isolated from renal proximal tubules of the winter flounder. Three characteristics of carrier-mediated transport were demonstrated: 1) unlabeled PAH inhibited the uptake of [3H]PAH; 2)[3H]PAH efflux from the vesicles was stimulated in the presence of unlabeled PAH in the extravesicular medium; and 3) PAH influx was inhibited by 2,4-dinitrophenol (DNP) and 4-acetamido-4'-isothiocyano-2,2'-disulfonic stilbene (SITS). D-Glucose plus a sodium gradient stimulated PAH uptake, as did a K2SO4 gradient plus valinomycin, suggesting that PAH is transported as an anion. In contrast, PAH uptake into a membrane fraction containing mainly basal-lateral plasma membranes exhibited a larger inhibition by probenecid but a smaller inhibition by unlabeled PAH and SITS. Thus, carrier-mediated transfer of PAH driven by the electrochemical potential difference for PAH is demonstrated in the brush border membrane of the flounder kidney.     

Measurement of sugar transport in single living cells

 A fluorometric method that allows repeatable measurement of sugar transport rates and parameters in single living cells is presented. Intracellular sugar concentrations were estimated in real time from changes in cell volume that occur secondary to permeation of sugars across the plasma membrane. In turn, the cell volume changes were estimated from variations of intracellular calcein fluorescence measured by confocal microscopy. Using HeLa cells, the assay allowed reproducible measurement of the uptake and exit of d-galactose and 3-O-methyl-d-glucose. The rate of zero-trans uptake (i.e. at an intracellular concentration of zero) of galactose at an extracellular concentration of 200 mM was 0.34±0.05 mM/s (n=8). Apparent V _max and K _m for galactose exit were 0.32±0.05 mM/s (n=9) and 30±7.2 mM (n=9), respectively. The apparent affinity of infinite-trans (i.e. at a very high intracellular concentration) uptake of 3-O-methyl-d-glucose was 3.8±0.47 mM (n=8). Galactose uptake was 93±8% (n=8) inhibited in the presence of 50 μM phloretin, whereas galactose exit was 96±6% (n=5) trans-inhibited by 100 mM 4,6-ethylidine-d-glucose. This technique may help to characterize sugar transport in freshly isolated cells, co-cultures and heterogeneous cell explants. It may also allow available cell microinjection technology to be used to study the regulation of sugar transporters’ intrinsic activity. In principle, similar approaches might also be applied in functional studies of other transporters for which non-metabolized substrates are available. Received: 5 August 1998 / Received after revision: 2 October 1998 / Accepted: 10 November 1998     

Aspects of intestinal folate transport in the rat.

1. In vitro preparations of rat jejunum were used to study the metabolic dependence, the structural specificity, and the pH sensitivity of the folic acid absorption process. 2. The presence of 2:4 dinitrophenol, and the absence of oxygen, in the muscosal bathing medium both led to a decrease in the tissue accumulation and serosal transfer of folic acid by everted sacs. 3. 10-formylfolic acid, present in the mucosal medium at a molar ratio of 10:1 with labelled folic acid, appears to compete for tissue accumulation sites but has no significant effect on serosal transfer. 4. The efflux of folic acid from pre-loaded jejunal sacs is stimulated by the presence in the mucosal medium of the folic acid and related compounds. Pteroyl-L-glutamic acid, 10-formylfolic acid and methotrexate elicit a significant increase in the efflux rate; pteroyl-D-glutamic acid was significantly less effective and pteroic acid had no effect. 5. The uptake of folic acid by isolated jejunal cells prepared by enzymic disaggregation of the mucosa, was found to be influenced by the pH of the bathing medium, uptake being enhanced at a pH of between 5 and 6. 6. It is concluded that the effect of metabolic inhibitors and acid pH conditions on the uptake of folic acid in vitro is consistent with a passive absorption mechanism, influenced in intact preparations and in vivo by the jejunal pH microclimate. However the occurrence of competitive inhibition and stimulated efflux may indicate the existence of a structurally specific accumulation process at some site, or sites, within the mucosa.     

Developmental changes of sugar transport in the ovine small intestine

Summary Intestinal monosaccharide transport was studied in young lambs (age: up to 1 week) and in older lambs (age: 2.5–4 months) with well developed forestomach system employing everted sacs of small intestine.Both glucose and galactose were transported against a high concentration gradient from the mucosal to the serosal side of the intestinal wall in young lambs.In the older lambs glucose was transported only against a small concentration gradient, when intestinal glucose metabolism was diminished by reducing the pH of the incubation medium from 7 to 5. Galactose and -methyl-glucoside, which are not markedly metabolized by the intestine, were transported against a small but similar concentration gradient at both pH values in these animals. In young lambs, however, at pH 5 intestinal galactose transport was lower than at pH 7. These results indicate that active intestinal monosaccharide transport becomes rudimentary in the maturing sheep.Some results were reported in a preliminary form (Scharrer, 1975)     

Na-K-ATPase localization in teleost urinary bladder by [3H]ouabain autoradiography.

Previous studies on the urinary bladder of the seawater-acclimated winter flounder (pseudopleuronectes americanus) demonstrated that active Na and Cl transport were ouabain sensitive. This suggested a relationship between the Na pump and Na-K-ATP-ase. The specific binding of [H]ouabain to Na-K-ATPase provides a means of localizing the site of active Na transport. In isolated bladders, a positive linear correlation (r= 0.89) was found between the active Na transport rate and the Na-K-ATPase activity. Ouabain binding by the bladder surface appeared to be saturable and relatively specific, e.g., was reduced by a high K concentration. When only the mucosal side of the bladder was exposed to 5 muM ouabain, both inhibitory effects and binding were small and are explained by finite permeability of the bladder to ouabain. In contrast, binding and inhibitory effects from the serosal side were much greater. Autoradiographs demonstrated that [3H]ouabain was bound only to the serosal side of the epithelial cells. Ultrastructural examination revealed that the area of ouabain binding coincided with the basal and lateral plasma membranes.     

Relationship between volatile fatty acids and magnesium absorption in mono- and polygastric species.

In monogastric animals magnesium is absorbed from the small and large intestine. In ruminants the forestomach system, in particular the rumen, is the most important site of magnesium absorption. Various mechanisms are involved in intestinal magnesium absorption (solvent drag, diffusion, carrier-mediated transport). In the large intestine and rumen an active transepithelial magnesium transport from the mucosal to the serosal side of the epithelium was recently demonstrated. Since in the large intestine and in the rumen, volatile fatty acids (VFA, mainly acetate, propionate, butyrate) deriving from fermentation of carbohydrates represent the major anions, the influence of VFA on magnesium absorption from these parts of the gut was recently investigated. VFA at physiological concentrations stimulated magnesium absorption in both cases. In the rat large intestine VFA enhanced only magnesium absorption by the distal colon, sodium and water absorption remaining unaffected. Both in sheep rumen and in the distal colon of the rat butyrate was most effective in this regard, followed in descending order by propionate and acetate. Sodium absorption by the rat proximal colon and caecum, and by the sheep rumen, was similarly enhanced by VFA. It has been suggested that the latter effect is due to the function of VFA as intracellular proton donators for the Na+/H+ exchanger located in the apical membrane of the epithelial cells. In analogy a Mg2+/H+ exchanger, located in the apical membrane of the epithelium in the distal colon and rumen, is fully consistent with the stimulatory effects of VFA on magnesium absorption at these sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Epithelial organic cation transporters ensure pH-dependent drug absorption in the airway

Most inhaled beta(2)-adrenergic agonist and anticholinergic bronchodilators have low lipid solubility because of their transient or permanent positive net charge at physiologic pH. Airway absorption of these cationic drugs is incompletely understood. We examined carrier-mediated mechanisms of cationic drug uptake by human airway epithelia. Airway tissues and epithelial cells, obtained from lung donors without preexisting lung disease, were evaluated for organic cation transporter expression by quantitative RT-PCR and immunofluorescence. For in vitro functional studies on primary airway epithelial cells, uptake of the cationic fluorophore 4-[4-(dimethylamino)-styryl]-N-methylpyridinium (ASP+) was characterized. Quantitative RT-PCR analysis demonstrated high mRNA levels for two polyspecific organic cation/carnitine transporters, OCTN1 and OCTN2, in human airway epithelia. Immunofluorescence of human airway sections confirmed OCTN1/2 protein expression, with a predominant localization to the apical portion of epithelial cells. Primary airway epithelial cells showed a carrier-mediated, temperature-sensitive and saturable uptake of ASP(+). Seventy-five to eighty percent of ASP(+) uptake was inhibited by L-carnitine, an OCTN2-carried zwitterion. The uptake was pH dependent, with approximately 3-fold lower rates at acidic (pH 5.7) than at alkaline (pH 8.2) extracellular pH. Albuterol and formoterol inhibited ASP(+) uptake, suggesting that all these molecules are carried by the same transport mechanism. These findings demonstrate the existence and functional role of a pH-dependent organic cation uptake machinery, namely OCTN1 and OCTN2, in human airway epithelia. We suggest that epithelial OCTN1/2 are involved in the delivery of inhaled cationic bronchodilators to the airway tissue.     

Basolateral choline transport in isolated rabbit renal proximal tubules

 Choline can undergo both net secretion and net reabsorption by renal proximal tubules, but at physiological plasma levels net reabsorption occurs. During this process, choline enters the cells at the luminal side down an electrochemical gradient via a specific transporter with a high affinity for choline. It appeared likely that choline was then transported out of the cells against an electrochemical gradient at the basolateral membrane by countertransport for another organic cation. This possibility was examined by studying net transepithelial reabsorption and basolateral uptake and efflux of [¹⁴C]choline in isolated S2 segments of rabbit renal proximal tubules. Basolateral uptake, which was inhibited by other organic cations such as tetraethylammonium (TEA), appeared to occur by the standard organic cation transport pathway. However, the addition of TEA to the bathing medium not only failed to trans-stimulate net transepithelial reabsorption and basolateral efflux of [¹⁴C]choline but it actually inhibited transepithelial reabsorption by @60%. The results do not support the presence of a countertransport step for choline against an electrochemical gradient at the basolateral membrane. Instead, they suggest that choline crosses this membrane by some form of carrier-mediated diffusion even during the reabsorptive process. Received: 24 March 1998 / Received after Revision: 15 June 1998 / Accepted: 2 July 1998     

Selectivity of the 2-deoxyglucose transport system in human and guinea pig polymorphonuclear leukocytes.

To determine whether the deleterious action of D-galactose upon phagocyte function could be related to inhibition of glucose uptake, the properties of glucose transport were investigated by following the incorporation of [G-3H]2-deoxyglucose into human and guinea pig polymorphonuclear leukocytes (PMN). Uptake of [G-3H]2-deoxyglucose by guinea pig PMN proceeded in vitro with a Km of 1.8 mM and Vmax of 0.67 nmol/min per 10(6) cells. This system was competitively inhibited by glucose and mannose but was not significantly affected by galactose, fructose, or 3-0-methylglucose. Maximal uptake of 2-deoxyglucose occurred at 41 degrees C, and phosphorylation was necessary for its intracellular concentration. Transport of 2-deoxyglucose, although not altered by uncouplers of oxidative phosphorylation, was sensitive to inhibitors of glycolysis. Preincubation of cells with 2 mM iodoacetate for 30 min significantly reduced the uptake of 2-deoxyglucose and the intracellular levels of adenosine-5'-triphosphate without decreasing cell viability. These results indicated that uptake of 2-deoxyglucose in guinea pig PMN occurred by facilitated diffusion with subsequent phosphorylation. Similar results were obtained with PMN isolated from human peripheral blood.     

Glucose carriers at maternal and fetal sides of the trophoblast in guinea pig placenta.

Trophoblast uptake and unidirectional influx of 3H-labeled hexoses were measured relative to L-[14C]glucose (extracellular marker) using a single-circulation, paired-tracer dilution technique. Successive runs were performed in the fetal and maternal circulations of isolated dually perfused guinea pig placentas, obtained from anesthetized dams and perfused for 60--140 min. The leakiness, estimated from the percentage of the L-glucose dose that crossed the trophoblast, varied (25 +/- 3% (SE), n = 28). On the injection side the maximal sugar uptake (Umax) was measured from early venous concentration ratios, since rapid tracer backflux occurred: Umax = (1 -- 3H/14C) x 100. Umax was independent of the leakiness. In all 14 placentas studied, stereospecific saturable transport of D-glucose was demonstrated at fetal (Umax = 56 +/- 4% (SE), n = 14) and maternal (62 +/- 1% (SE), n = 14) surfaces. The mean unidirectional influxes were 3.3 and 3.5 mumol.min-1.g-1, respectively. Uptakes were inhibited by phloretin and less effectively by phlorizin. D-glucose, 3-O-methylglucose, D-mannose and D-galactose had similar Umax values, about four times that of D-fructose. Tracer backflux and transplacental flux were also equal from both sides. It is concluded that similar hexose carriers, which resemble the human erythrocyte carrier, exist at the membrane on both sides of the trophoblast. The nondestructive technique employed characterizes carriers and receptors at the blood side of cells and could be applied to the placenta or other organs in the intact animal.     

Absorption of folic acid by everted segments of rat jejunum

1. Everted rings of rat intestine were used to study the initial uptake rate of folic acid at various concentrations and incubation temperatures in vitro.2. Folic acid was accumulated linearly for periods of at least 30 min.3. The initial uptake rate was found to reach a constant value as the concentration of folic acid in the incubation medium was increased above 5 x 10(-6)M.4. Reducing the temperature of incubation from 37 to 27 degrees C gave small Q(10) values at either end of the concentration range.5. Transfer of folic acid into the serosal compartment of everted sacs was shown to undergo a rate reduction in the same concentration range.6. A mechanism for folic acid transport is suggested in which folic acid is converted to the neutral species at the mucosal surface in an acid microclimate.