Transport In Brush-border Membrane Vesicles Research Articles

Characterization of Sodium‐Dependent Pi Transport Inhibition by NAD

Hyperphosphatemia during end-stage renal disease causes many adverse outcomes. To prevent them, several Pi intestinal absorption inhibitors are necessary for targeting either the paracellular (diffusion) or the transcellular (transport) absorption pathway. Nicotinamide adenine dinucleotide (NADH/NAD+) is a classical inhibitor of intestinal and renal Pi transport and is a circadian modulator of phosphatemia. The mechanism of NAD transporter inhibition remains unclear, although a competitive inhibition mechanism has been suggested. In this work, we sought to determine if this mechanism has a direct effect on Pi transporters. The five known rat Na/Pi cotransporters (NaPi2a, NaPi2b, NaPi2c, PiT1, and PiT2) were expressed in Xenopus laevis oocytes. Uptake of 32P-Pi was performed for three days post-injection of 5 ng of the corresponding cRNAs per oocyte and after 30 minutes of preincubation with 0.5 mM NAD, NADH, or NAM. None of the compounds affected the Pi uptake of any of the expressed Pi transporters in oocytes. Pi transport in the brush border membrane vesicles (BBMV) of either rat kidney cortex or jejunum epithelium was also assayed. As expected, it was significantly inhibited by NADH and NAD when BBMV were preincubated for 30 min at room or ice-cold temperature. NAM had no effect, and the effect was specific for Na-dependent Pi uptake (D-glucose uptake was not affected). Dose-response analyses on jejunum or kidney BBMV revealed IC50 values in the micromolar range for 50 µM of Pi uptake. Several Michaelis-Menten kinetics were performed in the presence of different concentrations of NAD, and a Lineweaver-Burk plot suggested changes in affinity (i.e., competitive inhibition), which contrasts with the lack of an effect on the transporters expressed in oocytes. The competitive model was confirmed by non-linear regression of a global fit with shared parameters, providing a Ki value of 538 µM NAD. When the effect of NAD was assayed in Opossum Kidney cells, a proximal tubule cell line model expressing a regulated NaPi2a, no evidence of Pi transport inhibition was observed. To check whether NAD-mediated ribosylation was involved in Pi transport inhibition, a classical ribosylation assay was performed with 32P-NAD, using jejunum and renal BBMV. No evidence of ribosylation was found for NaPi2b or NaPi2a, even after one month of exposure to an X-ray film. This was confirmed using several ribosylation inhibitors (meta-iodobenzylguanidine, novobiocin, vitamin K1, vitamin K3, or 3-methoxybenzamide), which failed to prevent NAD inhibition of Pi transport in BBMV. In conclusion, despite the kinetic findings in BBMV, NAD inhibition of Na/Pi-cotransporters is, most likely, not mediated through a direct interaction with these transporters. Instead, inhibition seems to be mediated through an indirect mechanism acting on components that are absent in Xenopus oocytes and in OK cells, which ends in the modification of Pi transport affinity (Km) and occurs at ice-cold temperature. Nevertheless, the unlikely inhibition of a novel, unknown Pi transporter cannot be completely discarded.

Intestinal phosphate absorption is mediated by multiple transport systems in rats.

Apical inorganic phosphate (Pi) transport in the small intestine seems to be mainly mediated by the sodium/Pi cotransporter NaPi2b. To verify this role, we have studied the combined effects of pH, phosphonoformate, and Pi deprivation on intestinal Pi transport. Rats were fed, ad libitum, three fodders containing 1.2, 0.6, or 0.1% Pi for 1, 5, or 10 days. Pi deprivation (0.1%) increased both sodium-activated and sodium-independent Pi transport in brush-border membrane vesicles from the duodenum and jejunum for all three times. Alkaline pH inhibited Pi transport, despite the increasing concentration of [Formula: see text] (NaPi2b substrate), whereas acidity increased transport when the concentration of the PiT1/PiT2 substrate, [Formula: see text], was at its highest. The effect of Pi deprivation was maximal at acid pH, but both basal and upregulated transport were inhibited (70%) with phosphonoformate, an inhibitor of NaPi2b. PiT2 and NaPi2b protein abundance increased after 24 h of Pi deprivation in the duodenum, jejunum, and ileum, whereas PiT1 required 5-10 days in the duodenum and jejunum. Therefore, whereas transporter expressions are partially correlated with Pi transport adaptation, the pH effect precludes NaPi2b, and phosphonoformic acid precludes PiT1 and PiT2 as the main transporters. Transport and transporter expression were also inconsistent when feeding was limited to 4 h daily, because the 1.2% Pi diet paradoxically increased Pi transport in the duodenum and jejunum, but NaPi2b and PiT1 expressions only increased with the 0.1% diet. These findings suggest the presence of a major transporter that carries [Formula: see text] and is inhibited by phosphonoformate.NEW & NOTEWORTHY The combined effects of dietary inorganic phosphate (Pi) content, pH, and phosphonoformate inhibition suggest that the resulting apical Pi transport in the small intestine cannot be fully explained by the presence of NaPi2b, PiT1, or PiT2. We provide evidence of the presence of a new sodium-coupled Pi transporter that uses [Formula: see text] as the preferred substrate and is inhibited by phosphonoformate, and its expression correlates with Pi transport in all assayed conditions.

L-leucine, l-methionine, and l-phenylalanine share a Na+/K+-dependent amino acid transporter in shrimp hepatopancreas

Hepatopancreatic brush border membrane vesicles (BBMV), made from Atlantic White shrimp (Litopenaeus setiferus), were used to characterize the transport properties of (3)H-L-leucine influx by these membrane systems and how other essential amino acids and the cations, sodium and potassium, interact with this transport system. (3)H-L-leucine uptake by BBMV was pH-sensitive and occurred against transient transmembrane concentration gradients in both Na(+)- and K(+)-containing incubation media, suggesting that either cation was capable of providing a driving force for amino acid accumulation. (3)H-L-leucine uptake in NaCl or KCl media were each three times greater in acidic pH (pH 5.5) than in alkaline pH (pH 8.5). The essential amino acid, L-methionine, at 20mM significantly (p<0.0001) inhibited the 2-min uptakes of 1mM (3)H-L-leucine in both Na(+)- and K(+)-containing incubation media. The residual (3)H-L-leucine uptake in the two media were significantly greater than zero (p<0.001), but not significantly different from each other (p>0.05) and may represent an L-methionine- and cation-independent transport system. (3)H-L-leucine influxes in both NaCl and KCl incubation media were hyperbolic functions of [L-leucine], following the carrier-mediated Michaelis-Menten equation. In NaCl, (3)H-L-leucine influx displayed a low apparent K M (high affinity) and low apparent J max, while in KCl the transport exhibited a high apparent K M (low affinity) and high apparent J max. L-methionine or L-phenylalanine (7 and 20mM) were competitive inhibitors of (3)H-L-leucine influxes in both NaCl and KCl media, producing a significant (p<0.01) increase in (3)H-L-leucine influx K M, but no significant response in (3)H-L-leucine influx J max. Potassium was a competitive inhibitor of sodium co-transport with (3)H-L-leucine, significantly (p<0.01) increasing (3)H-L-leucine influx K M in the presence of sodium, but having negligible effect on (3)H-L-leucine influx J max in the same medium. These results suggest that shrimp BBMV transport (3)H-L-leucine by a single L-methionine- and L-phenylalanine-shared carrier system that is enhanced by acidic pH and can be stimulated by either Na(+) or K(+) acting as co-transport drivers binding to shared activator sites.

Sodium-Hydrogen Exchanger Regulatory Factor 1 (NHERF-1) Transduces Signals That Mediate Dopamine Inhibition of Sodium-Phosphate Co-transport in Mouse Kidney

Dopamine inhibited phosphate transport in isolated renal brush border membrane vesicles and in cultured renal proximal tubule cells from wild-type but not from NHERF-1 null mice. Co-immunoprecipitation experiments established that NHERF-1 associated with D1-like receptors. In wild-type mice, dopamine stimulated cAMP accumulation and protein kinase C (PKC) activity in renal proximal tubule cells, an effect that was abolished by SCH-23390, a D1-like receptor antagonist. In NHERF-1 null kidney tissue; however, dopamine failed to stimulate either cAMP accumulation or PKC activity. Infection of proximal tubule cells from NHERF-1 null mice with adenovirus-green fluorescent protein-NHERF-1 restored the ability of dopamine to stimulate cAMP and PKC. Finally, in (32)P-labeled wild-type proximal tubule cells and in opossum kidney cells, dopamine increased NHERF-1 phosphorylation at serine 77 of the PDZ I domain of NHERF-1, a site previously shown to attenuate binding of cellular targets including the Npt2a (sodium-dependent phosphate transporter 2a). Together, these studies establish that NHERF-1 plays a key role in dopamine signaling and is also a downstream target of D1-like receptors in the mouse kidney. These studies suggest a novel role for the PDZ adapter protein NHERF-1 in coordinating dopamine signals that inhibit renal phosphate transport.

Identification of a novel sodium-dependent fructose transport activity in the hepatopancreas of the Atlantic lobsterHomarus americanus

[(3)H]Fructose and [(3)H]glucose transport were determined in brush-border membrane vesicles (BBMV), basolateral membrane vesicles (BLMV) and isolated cells (E, R, F, B) of H. americanus (Atlantic lobster) hepatopancreas. Glucose transport in BBMV was equilibrative in the absence of sodium and concentrative in the presence of sodium. Sodium-dependent glucose transport by BBMV was not inhibited by a tenfold molar excess of fructose. Glucose transport by BLMV was equilibrative and sodium independent. Fructose uptake by BBMV and BLMV was equilibrative in the absence of sodium and concentrative in the presence of sodium. This enhancement was not affected by a tenfold molar excess of glucose in the presence of sodium. E-, F- and B-cells showed sodium-dependent uptake of fructose, while R-cells did not. Sodium-dependent fructose uptake by E-cells was not inhibited by a tenfold molar excess of glucose or mannose. Western blot analysis of BBMV, BLMV and E-, R-, F- and B-cells using rabbit polyclonal antibodies directed against epitopes of mammalian GLUT2, GLUT5, SGLT1 and SGLT4 indicated the presence of cross-reacting lobster proteins. Sequence alignment of the mammalian proteins with translated, lobster expressed sequence tags also indicated significant identity between species. Comparison of fructose and glucose uptake in the absence and presence of sodium by BBMV, BLMV and isolated cells indicated the presence of a distinct sodium-dependent transport activity for each sugar in the Atlantic lobster.

Expression and functionality of the Na +/ myo-inositol cotransporter SMIT2 in rabbit kidney

Myo-inositol (MI) is involved in several important aspects of cell physiology including cell signaling and the control of intracellular osmolarity i.e. by serving as a “compatible osmolyte”. Currently, three MI cotransporters have been identified: two are Na +-dependent (SMIT1 and SMIT2) and one is H +-dependent (HMIT) and predominantly expressed in the brain. The goal of this study was to characterize the expression of SMIT2 in rabbit kidney and to compare it to SMIT1. First, we quantified mRNA levels for both transporters using quantitative real-time PCR and found that SMIT1 was predominantly expressed in the medulla while SMIT2 was mainly in the cortex. This distribution of SMIT2 was confirmed on Western blots where an antibody raised against a SMIT2 epitope specifically detected a 75 kDa protein in both tissues. Characterization of MI transport in brush-border membrane vesicles (BBMV), in the presence of d- chiro-inositol and l-fucose to separately identify SMIT1 and SMIT2 activities, showed that only SMIT2 is expressed at the luminal side of proximal convoluted tubules. We thus conclude that, in the rabbit kidney, SMIT2 is predominantly expressed in the cortex where it is probably responsible for the apical transport of MI into the proximal tubule.

Transport of γ-Hydroxybutyrate in Rat Kidney Membrane Vesicles: Role of Monocarboxylate Transporters

Intoxication with gamma-hydroxybutyrate (GHB) is associated with coma, seizure, and death; treatment of overdoses is symptomatic. Previous studies in our laboratory have demonstrated that L-lactate and pyruvate treatment can increase the renal clearance of GHB and increase its elimination in rats, suggesting that GHB may undergo renal reabsorption mediated by monocarboxylic acid transporters (MCTs). The goals of this study were to characterize the renal transport of GHB in rats and to determine the role of MCT in its renal transport. Brush-border membrane (BBM) and basolateral membrane (BLM) vesicles were isolated from rat kidney cortex, and the uptake of L-lactate and GHB was characterized. L-Lactate and GHB undergo both pH- and sodium-dependent transport in BBM vesicles and pH-dependent transport in BLM vesicles. A simple Michaelis-Menten equation best described the pH-dependent uptake of GHB in BBM (Km, 8.0 +/- 1.8 mM; Vmax, 838 +/- 45 pmol/mg/s) and in BLM vesicles (Km, 10.5 +/- 2.6 mM; Vmax, 806 +/- 253 pmol/mg/s). mRNA of MCT1 and MCT2 was determined in rat kidney cortex using reverse transcriptase-polymerase chain reaction; using Western blot, the protein expression of MCT1 was present mainly in BLM vesicles, with weak expression in BBM vesicles, whereas that of MCT2 was exclusively in BLM vesicles. Studies with rat MCT1 gene-transfected MDA-MB231 cells demonstrated that GHB was a substrate of MCT1. The data suggest that rat MCT1 may represent an important transporter for GHB in renal tubule cells. This investigation provides evidence for the importance of MCTs in the reabsorption of the monocarboxylic acids l-lactate and GHB in the kidney.

Nephrotoxicity of uranyl acetate: effect on rat kidney brush border membrane vesicles

Since the Gulf war exposure to depleted uranium, a known nephrotoxic agent, there is a renewed interest in the toxic effects of uranium in general and its mechanism of nephrotoxicity which is still largely unknown in particular. In order to investigate the mechanism responsible for uranium nephrotoxicity and the therapeutic effect of urine alkalization, we utilized rat renal brush border membrane vesicles (BBMV). Uranyl acetate (UA) caused a decrease in glucose transport in BBMV. The apparent K (i) of uranyl was 139+/-30 microg uranyl/mg protein of BBMV. Uranyl at 140 microg/mg protein of BBMV reduced the maximal capacity of the system to transport glucose [V (max) 2.2+/-0.2 and 0.96+/-0.16 nmol/mg protein for control and uranyl treated BBMV (P<0.001), respectively] with no effect on the apparent K (m) (1.54+/-0.33 and 1.54+/-0.51 mM for control, and uranyl treated BBMV, respectively). This reduction in V(max) is at least partially due to a decrease in the number of sodium-coupled glucose transporters as apparent from the reduction in phlorizin binding to the uranyl treated membranes, V (max) was reduced from 247+/-13 pmol/mg protein in control BBMV to 119+/-3 pmol/mg protein in treated vesicles (P<0.001). The pH of the medium has a profound effect on the toxicity of UA on sodium-coupled glucose transport in BBMV: higher toxicity at neutral pH (around pH 7.0), and practically no toxicity at alkaline pH (7.6). This is the first report showing a direct inhibitory dose and pH dependent effect of uranyl on the glucose transport system in isolated apical membrane from kidney cortex.

Formate-stimulated NaCl absorption in the proximal tubule is independent of the pendrin protein.

A significant fraction of active chloride reabsorption across the apical membrane of the proximal tubule is mediated by a chloride/formate exchange process, whereby intracellular formate drives the transport of chloride into the cell. When chloride/formate exchange operates in parallel with Na(+)/H(+) exchange and H(+)-coupled recycling of formate, the net result is electroneutral NaCl reabsorption. Pendrin is the protein product of the PDS gene (SLC26A4) and functions in several different anion exchange modes, including chloride/formate exchange. Pendrin is expressed in the kidney and may serve as the transporter responsible for formate-dependent NaCl reabsorption. In the present study, Pds-knockout mice were used to determine the role of pendrin in proximal tubule chloride reabsorption. We show that formate-dependent NaCl absorption in microperfused proximal tubules is similar between wild-type and pendrin-deficient mice. In addition, there is no difference in the rate of formate-mediated chloride transport in brush-border membrane vesicles isolated from wild-type and pendrin-deficient mice. These studies demonstrate that pendrin is not responsible for formate-dependent NaCl reabsorption in the proximal tubule.

Increased expression of ileal apical sodium-dependent bile acid transporter in postpartum rats.

The expression and activity of the apical ileal sodium-dependent bile acid transporter (asbt) was examined in the small intestine of control, pregnant, and lactating postpartum rats 2, 12, and 21 days after delivery. Western blot analysis of brush border membrane vesicles (BBMV) prepared from different regions of the small intestine demonstrated that expression of asbt was maximal in the most distal segments for all experimental groups, was not substantially affected in pregnant and 2-day postpartum rats, and was significantly increased in 12- and 21-day postpartum rats. Analysis of mRNA suggested that asbt protein was regulated at the posttranscriptional level in postpartum rats. Increased expression of asbt protein postpartum was maximal (approximately 2-fold) in the proximal region of the ileum, consistent with a 60% increase in taurocholate (TC) transport in BBMV from the proximal ileum in 14- to 21-day postpartum rats relative to control rats. Absorption of TC, determined from the intact proximal ileum using an intestinal loop model, demonstrated a 30% increase in TC uptake per unit weight of tissue in 14- to 21-day postpartum rats relative to control rats. Together with the marked increase in intestinal mass observed at peak lactation, these data indicate a significant increase in asbt-mediated reclamation of bile acids in the intestine of lactating rats.

Creatine transport in brush-border membrane vesicles isolated from rat kidney cortex.

The kidney efficiently salvages creatine from the urine; however, the mechanism(s) that mediates renal creatine reabsorption has not been investigated. This study characterizes the creatine transport mechanism in brush-border membrane vesicles isolated from the rat renal cortex. An osmolality plot revealed that creatine is transported into an osmotically active space and that it is also bound to the membranes. An inwardly directed NaCl gradient stimulated creatine uptake and the time course of uptake exhibited an overshoot phenomenon, which indicates the presence of an active process for creatine in these membranes. The uptake of creatine showed an absolute requirement for both Na(+) and Cl(-). The NaCl gradient-dependent creatine uptake was stimulated by a valinomycin-induced, inside-negative, K(+)-diffusion potential, which suggests that the uptake process is electrogenic. Stoichiometric analyses indicated a probable couple ratio of 2 Na(+):1 Cl(-):1 creatine molecule. The kinetic study showed that creatine is transported by a high-affinity system (K(m) of 15 microM). Creatine uptake was inhibited by a 100-fold excess of various compounds with the following potency order: cold creatine = guanidinopropionic acid > nipecotic acid > gamma-aminobutyric acid (GABA) = beta-alanine = betaine, whereas carnitine, glycine, taurine, and choline were without effect. This pattern of inhibition differs from that observed for GABA uptake (unlabeled GABA = GPA > beta-alanine > nipecotic acid >> creatine). The conclusion drawn was that the apical membrane of the renal cortical tubules contains an active, high-affinity, electrogenic, 2 Na(+)/1 Cl(-)/creatine cotransporter.

Integumental amino acid uptake in a carnivorous predator mollusc (Sepia officinalis, Cephalopoda)

The epithelial cells of the integument of body, arms and tentacles of Sepia officinalis present on their apical membrane a well-organised brush border and show the morphological and histochemical characteristics of a typical absorptive epithelium. The ability of the integument to absorb amino acids was investigated both in the arms incubated in vitro and in a purified preparation of brush border membrane vesicles (BBMV). Autoradiographic pictures of the integument after incubation of the arms in sea-water with or without sodium, showed that proline intake was Na+-dependent, whereas leucine intake appeared to be a largely cation- independent process. Time course experiments of labelled leucine, proline and lysine uptakes in BBMV evidenced that these amino acids are accumulated within the vesicles in the presence of an inwardly directed sodium gradient. The sodium-driven accumulation proves that cationic and neutral amino acids are taken up by the apical membrane of the epithelium of Sepia integument through a secondary active mechanism. For leucine, a 90% inhibition of the uptake was recorded in the presence of a large excess of the substrate. In agreement with the autoradiography results, an analysis of the cation specificity transport in BBMV showed that leucine uptake had a low cation specificity, whereas lysine and proline uptakes were Na+-dependent. An excess of lysine and proline, which share with alanine two different transport systems in the gill epithelium of marine bivalves, reduced eucine uptake. The possible role of the absorptive ability of the integument in a carnivorous mollusc is discussed.

Expression of monosaccharide transporters in the intestine of human diabetics

D-glucose and Dvgalactose are transported across the brush-border membrane, into enterocytes by the Na+Iglucose co-transporter (SGLTl). Fructose is transported from the intestinal lumen into the enterocytes by the fructose transporter (GLUTS). These monosaccharides exit the cell across the basolateral membrane by the monosaccharide transporter, GLUT2. It has been shown in rats with experimentally induced diabetes that the capacity of the small intestine to absorb D-glucose increases. This increase is mainly due to enhanced activity and abundance of SGLTl and GLUT2. No studies have been carried out to investigate if a similar situation occurs in the intestine of diabetic humans. We have assessed the expression of SGLTl, GLUTS, disaccharidases, and the structural proteins villin and l3-actin in the duodenum of healthy control individuals and patients with non-insulin dependent diabetes mellitus (NIDDM). Biopsy samples were removed, during endoscopy, from the second part of the duodenum of both control and diabetic subjects. Purified brush-border membrane vesicles (BBMV) were prepared from human biopsies using an established technique. A 4-fold increase in the abundance of SGLTI in the BBMV isolated from diabetic patients (85.2 ± 13.4 pmollmg protein, n=6) compared with controls (20.0 ± 5.6 pmollmg protein, n=6), was shown by quantitative western blotting. A similar increase in the initial rate of Na+-dependent Dsglucose transport in BBMV isolated from the intestine of diabetic patients compared with controls was also shown. The abundance of GLUTS, disaccharidases, villin and l3-actin were determined in the same BBMV by western blotting with appropriate antibodies. Our results indicate that levels of the fructose transporter, GLUTS, are also increased significantly in diabetics compared to controls. In contrast the abundance of lactase, sucrase, and the structural protein villin were only slightly elevated in diabetic patients. Total protein recoveries were equal in biopsies taken from both control and diabetic patients. We report here for the first time an increase in the capacity of the human intestine to absorb monosaccharides in NIDDM. This increase appears to be due to a combination of villus hypertrophy and a specific increase in the expression of SGLTl and GLUTS.

Effect of dehydration on apical Na+-H+ exchange activity and Na+-dependent sugar transport in brush-border membrane vesicles isolated from chick intestine.

The current work examines the effect of 4 days of water deprivation on Na+-H+ exchange and Na+-sugar cotransport systems in brush-border membrane vesicles isolated from either the jejunum, ileum or the colon of the chick. Apical Na+-H+ exchange activity was evaluated by measuring the pH-gradient-dependent Na+ uptake. The contribution of the Na+-H+ exchangers NHE2 and NHE3 to total Na+-H+ exchange activity was evaluated from their sensitivity to the amiloride-related drug HOE694. Dehydration increased plasma aldosterone levels from 12 to 70 pg/ml and also the activities of both Na+-H+ exchange and Na+-dependent sugar transport in the three intestinal regions tested. Na+-independent sugar transport was not modified by 4 days of water deprivation. In the ileum and colon the increase in Na+-H+ exchange activity was due to an increase in NHE2 activity, whereas in the jejunum it was due to an increase in both NHE2 and NHE3. Since we have previously reported that chronic Na+ depletion increases plasma aldosterone levels and NHE2 activity in ileum and colon, decreased small and large intestine Na+-sugar cotransport activity and had no effect on jejunal apical Na+-H+ exchange activity, it can be concluded that: (1) aldosterone does not regulate intestinal Na+-dependent sugar transport, and (2) the regulation of jejunal Na+-H+ exchange activity differs from that of either the ileum or the colon.

Taurocholate transport by brush border membrane vesicles from different regions of chicken intestine

Taurocholate transport was studied in brush border membrane vesicles (BBMV) isolated from chicken small (duodenum, jejunum, and ileum) and large (proximal cecum and rectum) intestines, using a rapid filtration technique. The purity of the BBMV was verified by the finding that the specific activity of sucrase (a brush border membrane enzyme marker) was severalfold greater in vesicles than corresponding values in mucosal homogenate. The functional integrity of isolated BBMV was evaluated by the uptake of D-glucose, which showed a transient increase in the presence of Na+. A Na+-dependence of taurocholate uptake was shown in BBMV prepared from ileum, cecum, and rectum, as taurocholate transport was transiently increased (accumulation) in the presence of a Na+ gradient between the external medium and intravesicular medium. The magnitude of the accumulation was similar among ileum, cecum, and rectum. In contrast, BBMV prepared from duodenum and jejunum did not show any Na+-dependent taurocholate transport, as the taurocholate uptake was not affected when a Na+ gradient was replaced by a K+ gradient. The use of taurochenodeoxycholate in the incubation medium inhibited Na+-dependent taurocholate transport in the ileum, cecum, and rectum. This study is the first to show the presence of a Na+-dependent bile salt transport in BBMV obtained from chicken ileum, proximal cecum, and rectum.

Extrinsic innervation modulates canine jejunal transport of glutamine, alanine, leucine, and glucose

Background: We previously showed a decrease in ileal glutamine transport in vitro and net absorption in vivo after extrinsic denervation of the canine jejunoileum. The aim was to determine whether extrinsic innervation modulates in vivo net absorption and in vitro transport of glutamine and other nutrients in canine jejunum. Methods: In vivo net jejunal uptakes of glutamine, alanine, leucine, and glucose were measured in five dogs before and 2 and 8 weeks after a model neurally isolating in situ the jejunoileum (extrinsic denervation, intestinal transection). To assess mechanisms, carrier-mediated uptakes were quantitated in jejunal brush border membrane vesicles from six dogs before and at 2 and 8 weeks after neural isolation of the jejunoileum and compared with six control dogs with fully intact extrinsic innervation. Results: In vivo net absorption of glutamine decreased at 2 weeks ( p < 0.05) and returned to normal values at 8 weeks; net absorptions of leucine, alanine, and glucose were decreased at both 2 and 8 weeks. In vitro brush border membrane vesicles transport of glutamine, leucine, and alanine followed the patterns of in vivo absorption, but glucose transport did not differ at any time point. Decreased glutamine uptake at 2 weeks resulted from a decrease in V max rather than a change in K m in sodium-dependent carrier-mediated transport. Conclusions: Extrinsic denervation down-regulated carrier-mediated transport of amino acids but not glucose. Decreased in vitro glutamine transport was mediated in part by a decrease in number rather than affinity of sodium-dependent transporters. (Surgery 1998;123:321-9.)

Extrinsic innervation modulates canine jejunal transport of glutamine, alanine, leucine, and glucose

Background: We previously showed a decrease in ileal glutamine transport in vitro and net absorption in vivo after extrinsic denervation of the canine jejunoileum. The aim was to determine whether extrinsic innervation modulates in vivo net absorption and in vitro transport of glutamine and other nutrients in canine jejunum. Methods: In vivo net jejunal uptakes of glutamine, alanine, leucine, and glucose were measured in five dogs before and 2 and 8 weeks after a model neurally isolating in situ the jejunoileum (extrinsic denervation, intestinal transection). To assess mechanisms, carrier-mediated uptakes were quantitated in jejunal brush border membrane vesicles from six dogs before and at 2 and 8 weeks after neural isolation of the jejunoileum and compared with six control dogs with fully intact extrinsic innervation. Results: In vivo net absorption of glutamine decreased at 2 weeks (p < 0.05) and returned to normal values at 8 weeks; net absorptions of leucine, alanine, and glucose were decreased at both 2 and 8 weeks. In vitro brush border membrane vesicles transport of glutamine, leucine, and alanine followed the patterns of in vivo absorption, but glucose transport did not differ at any time point. Decreased glutamine uptake at 2 weeks resulted from a decrease in Vmax rather than a change in Km in sodium-dependent carrier-mediated transport. Conclusions: Extrinsic denervation down-regulated carrier-mediated transport of amino acids but not glucose. Decreased in vitro glutamine transport was mediated in part by a decrease in number rather than affinity of sodium-dependent transporters. (Surgery 1998;123:321-9.)

Transport mechanisms of a glycoside, p-nitrophenyl- β- d-glucopyranoside, across rat small intestinal brush-border membranes

We examined the mechanism of p-nitrophenyl- β- d-glucopyranoside ( p-NP- β- d-Glc) transport in brush-border membrane vesicles from rat small intestine. The initial uptake rate showed an overshoot phenomenon in the presence of an inwardly directed sodium-ion concentration gradient. The overshoot disappeared when the sodium-ion concentration gradient was replaced with a potassium ion concentration gradient. d-Glucose and p-NP- β- d-Glc analogues inhibited the uptake, whereas uridine, leucine and disaccharide did not. Data on the concentration dependence of p-NP- β- d-Glc uptake indicated that two carrier-mediated systems are involved. The uptake via the high-affinity site required an inwardly directed sodium-ion concentration gradient, while the uptake via the low-affinity site proceeded such a gradient. d-Glucose competitively inhibited the initial uptake of p-NP- β- d-Glc via the high-affinity site with a K i value of 301 μM. The p-NP- β- d-Glc is transported in the small intestine via both the same carrier-mediated transport system that takes up d-glucose and a distinct low-affinity carrier-mediated transport system.

Peptide Mimics as Substrates for the Intestinal Peptide Transporter

4-Aminophenylacetic acid (4-APAA), a peptide mimic lacking a peptide bond, has been shown to interact with a proton-coupled oligopeptide transporter using a number of different experimental approaches. In addition to inhibiting transport of labeled peptides, these studies show that 4-APAA is itself translocated. 4-APAA transport across the rat intact intestine was stimulated 18-fold by luminal acidification (to pH 6.8) as determined by high performance liquid chromatography (HPLC); in enterocytes isolated from mouse small intestine the intracellular pH was reduced on application of 4-APAA, as shown fluorimetrically with the pH indicator carboxy-SNARF; 4-APAA trans-stimulated radiolabeled peptide transport in brush-border membrane vesicles isolated from rat renal cortex; and in Xenopus oocytes expressing PepT1, 4-APAA produced trans-stimulation of radiolabeled peptide efflux, and as determined by HPLC, was a substrate for translocation by this transporter. These results with 4-APAA show for the first time that the presence of a peptide bond is not a requirement for rapid translocation through the proton-linked oligopeptide transporter (PepT1). Further investigation will be needed to determine the minimal structural requirements for a molecule to be a substrate for this transporter.

Glucocorticoids upregulate taurocholate transport by ileal brush-border membrane.

The regulation of the enterohepatic circulation of bile acids has not been fully elucidated. Substrate availability has been shown to have a regulatory role on the ileal uptake of taurocholate (TC) by a positive feedback mechanism. Other mechanisms are likely to be involved in regulating ileal bile acid uptake. The present study was designed to test the hypothesis that the ileal bile acid transporter (iBAT) is glucocorticoid sensitive and that changes in expression are mediated by changes in iBAT synthesis. Adult Sprague-Dawley rats (300-400 g) received intraperitoneal injections with either corticosterone (5 mg/ 100 g body weight) or an equivalent vehicle (control) daily for 3 days. On day 4, ileal brush-border membrane vesicles (BBMV) and hepatic basolateral membrane vesicles (BLMV) were prepared, and TC transport was performed using the rapid filtration technique. Initial velocity was measured at selected time points, and kinetics were calculated over a range of TC concentrations. Ileal RNA was isolated, and Northern analysis of steady-state iBAT mRNA levels was determined. Western blot analysis was performed to quantitate the level of the 48-kDa iBAT protein. The initial velocity of Na(+)-dependent TC uptake at 30 s by ileal BBMV was higher in treated animals (264.3 +/- 64.6 pmol/mg protein) compared with control animals (148.3 +/- 41.1 pmol/mg protein; P = 0.07). The maximal velocity of uptake (Vmax) was significantly higher in treated vs. control animals (1,091 +/- 62.7 vs. 689.1 +/- 55.0 pmol.min-1.mg protein-1, respectively; P = 0.002), whereas there was no significant difference in the Michaelis constant (Km) between the control and treated animals (43.3 +/- 7.2 vs. 35.3 +/- 8.7 microM, respectively; P = not significant). Steady-state iBAT mRNA levels were increased twofold in the treated vs. control groups. Western blot analysis showed that the abundance of the 48-kDa iBAT protein was eightfold higher in the treated animals compared with control. Kinetic analysis of hepatic Na(+)-dependent TC uptake revealed nearly identical Vmax and Km between the study and control animals. Therefore, we conclude that TC transport by ileal BBMV is upregulated by administration of glucocorticoids. The increase in BBMV transport Vmax corresponds to an increase in both iBAT transcript and protein.