Brush Border Membrane Vesicles Uptake Research Articles

Increased phosphate absorption in mice lacking NHE3 in intestinal epithelial cells

The kidneys play a major role in maintaining phosphate (Pi) homeostasis. Patients in later stages of CKD develop hyperphosphatemia. One novel treatment option is tenapanor, an intestinal‐specific NHE3 inhibitor. To gain mechanistic insight into the role of intestinal NHE3 in Pi homeostasis, we studied tamoxifen (Tam) ‐inducible intestinal epithelial cell‐specific NHE3 knockout (NHE3IEC‐KO) mice with the hypothesis that their phenotype should reflect inhibitory effects of tenapanor on intestinal Pi uptake. Mice were challenged by high dietary Pi, and hormones as well as plasma Pi were determined. The in vivo effects of tenapanor were studied via 33P uptake from the intestine. Pi transport was measured ex vivo in everted gut sacs (EGS) and brush border membrane vesicles (BBMV). Intestinal and renal protein expression of Pi transporters were determined. No differences were found between genotypes in terms of Pi homeostasis before Tam. Two weeks after Tam, NHE3IEC‐KO mice had similar Pi homeostasis, but a ~25% reduction in FGF23 compared with control mice on standard diet. Two‐fold greater intestinal Npt2b expression in NHE3IEC‐KO mice (P<0.05) was associated with significantly enhanced Pi uptake in EGS and BBMV. One hour after acute oral Pi loading (0.5 mol/L NaH2PO4), plasma Pi levels increased to a significantly greater extent in NHE3IEC‐KO compared to control mice (2.0±0.2 vs 1.1±0.1 mmol L‐1, P<0.05). Tenapanor treatment caused ~67% lower 33P uptake (P<0.05) after 5 minutes compared with vehicle‐treated control mice. At later time points, tenapanor‐treated control mice showed an increase in intestinal Pi uptake, reaching a maximum of 33P in plasma after 60 minutes. In response to high dietary Pi, plasma Pi slightly but significantly decreased (‐0.25±0.1 mmol/L, P<0.05) in control mice, possibly the consequence of the combination of significantly elevated PTH and FGF23. In contrast to control mice, high dietary Pi significantly increased plasma Pi in NHE3IEC‐KO mice (0.5±0.1 mmol/L, P<0.05), which was associated with significantly increased plasma PTH levels (P<0.05). FGF23 levels also increased to a significantly greater extent in NHE3IEC‐KO vs control mice (552±66 vs 244±14 pg/mL, P<0.05). This was associated with ~2.25‐fold greater Npt2b expression (P<0.05). Fifty percent lower renal Npt2c expression (P<0.05) and a trend for lower Npt2a expression (~25%) were unable to correct for higher plasma Pi. In summary, intestinal NHE3 significantly contributes to Pi homeostasis. In contrast to effects described for tenapanor on Pi homeostasis, NHE3IEC‐KO mice show higher, rather than lower, intestinal Pi uptake.

Inhibition of phosphate transport by NAD+/NADH brush border membrane vesicles.

Nicotinamide is an important regulator of Pi homeostasis after conversion into NAD+/NADH. In this work, we have studied the classical inhibition of Pi transport by these compounds in the brush border membrane vesicles (BBMV) of rat kidney and rat intestine, and we examined the effects in opossum kidney (OK) cells and in phosphate transporter-expressing Xenopus laevis oocytes. In BBMV, NAD+ required preincubation at either room temperature or on ice to inhibit Pi uptake in BBMV. However, no effects were observed in the known Slc34 or Slc20 Pi transporters expressed in Xenopus oocytes, in OK cells, or in isolated rat cortical nephron segments. In BBMV from jejunum or kidney cortex, the inhibition of Pi transport was specific, dose-related, and followed a competitive inhibition pattern, as shown by linear transformation and nonlinear regression analyses. A Ki value of 538 µM NAD+ in kidney BBMV was obtained. Ribosylation inhibitors and ribosylation assays revealed no evidence that this reaction was responsible for inhibiting Pi transport. An analysis of the persistence of NAD+/NADH revealed a half-life of just 2 min during preincubation. Out of several metabolites of NAD degradation, only ADP-ribose was able to inhibit Pi uptake. Pi concentration also increased during 30 min of preincubation, up to 0.67 mM, most likely as a metabolic end product. In conclusion, the classical inhibition of Pi transport by NAD+/NADH in BBMV seems to be caused by the degradation metabolites of these compounds during the preincubation time.

Significant Species Differences in Intestinal Phosphate Absorption between Dogs, Rats, and Monkeys

A treatment for hyperphosphatemia would be expected to reduce mortality rates for CKD and dialysis patients. Although rodent studies have suggested sodium-dependent phosphate transporter type IIb (NaPi-IIb) as a potential target for hyperphosphatemia, NaPi-IIb selective inhibitors failed to achieve efficacy in human clinical trials. In this study, we analyzed phosphate metabolism in rats, dogs, and monkeys to confirm the species differences. Factors related to phosphate metabolism were measured and intestinal phosphate absorption rate was calculated from fecal excretion in each species. Phosphate uptake by intestinal brush border membrane vesicles (BBMV) and the mRNA expression of NaPi-IIb, PiT-1, and PiT-2 were analyzed. In addition, alkaline phosphatase (ALP) activity was evaluated. The intestinal phosphate absorption rate, including phosphate uptake by BBMV and NaPi-IIb expression, was the highest in dogs. Notably, urinary phosphate excretion was the lowest in monkeys, and their intestinal phosphate absorption rate was by far the lowest. Dogs and rats showed positive correlations between Vmax/Km of phosphate uptake in BBMV and NaPi-IIb expression. Although phosphate uptake was observed in the BBMV of monkeys, NaPi-IIb expression was not detected and ALP activity was low. This study revealed significant species differences in intestinal phosphate absorption. NaPi-IIb contributes to intestinal phosphate uptake in rats and dogs. However, in monkeys, phosphate is poorly absorbed due to the slight degradation of organic phosphate in the intestine.

A fluorescence method for determination of glucose transport by intestinal BBMV of common carp

Epithelial brush-border membrane vesicles (BBMVs) were isolated from the intestine of common carp and studied systematically by enzyme activity, transmission electron microscopy and immunoblotting. The uptake time course and the substrate concentration effect were assessed, and then, the ability of phlorizin and cytochalasin B to inhibit uptake was analyzed. The results show that sucrase, alkaline phosphatase and Na+-K+-ATPase activities in these vesicles were enriched 7.94-, 6.74- and 0.42-fold, respectively, indicating a relatively pure preparation of apical membrane with little basolateral contamination. The vesicular structure was in complete closure, as confirmed by electron microscopy. The presence of SGLT1 on the BBMVs was confirmed by Western blot analysis. In the time course experiment, the glucose uptake by BBMVs in Na+ medium displayed an initial accumulation (overshoot) at 5 min followed by a rapid return to equilibrium values at 60 min. Over the 2-NBDG concentration range selected, the external 2-NBDG concentration in NaSCN medium graphed as a curved line. Phlorizin and cytochalasin B had an obvious inhibitory effect on 2-NBDG transport in carp BBMVs, and the detected fluorescence intensity decreased. The inhibition rate in the 1000 μM group was the strongest at 64.18% and 63.61% of phlorizin and cytochalasin B, respectively, indicating the presence of carriers other than SGLT1. This study is the first to demonstrate that 2-NBDG can be used as a convenient and sensitive probe to detect glucose uptake in fish BBMVs. This technology will provide a convenient method to discover new effects and factors in glucose metabolism.

Uptake of benzo[a]pyrene, but not of phenanthrene, is inhibited by fatty acids in intestinal brush border membrane vesicles of rainbow trout (Oncorhynchus mykiss)

Partial replacement of fish ingredients with vegetable ingredients has elevated levels of polycyclic aromatic hydrocarbons (PAHs) in Atlantic salmon reared on these feeds. PAH uptake in the intestinal tract is postulated to occur in association with lipid absorption and could well be affected by fatty acid composition. We therefore investigated the effects of a fish oil and vegetable oil fatty acid, eicosapentaenoic acid (EPA; 20:5n−3) and oleic acid (18:1n−9) respectively, on the uptake of benzo[a]pyrene (BaP) and phenanthrene (PHE) across the intestinal brush border membrane in the salmonid species rainbow trout (Oncorhynchus mykiss).BaP and PHE were solubilized in mixed micelles composed of either EPA or oleic acid and administrated to isolated brush border membrane vesicles (BBMV) derived from the pyloric caeca, proximal intestine and distal intestine. In the absence of free fatty acids (FFA) trans-membrane uptake of BaP and PHE was 2–7 times lower than the fraction associated to or in the membrane. In the presence of FFA, trans-membrane BaP uptake had decreased by 80 and 40% at the highest EPA and oleic acid concentration, respectively, whereas PHE uptake was virtually unaffected. In the presence of BaP, but not PHE, trans-membrane EPA uptake in BBMV had decreased.This study obtained evidence for PAH-dependent interactions with FFA uptake. We conclude that intestinal BaP uptake is reduced by luminal FFA contents whereas PHE uptake is not. A large fraction of the administrated BaP and PHE remains associated with the cellular membrane of enterocytes and may interfere with uptake of nutrients.

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.

Effect of chronic alcohol feeding on vitamin B1 (thiamine) transport in rat kidneys

Renal uptake of thiamine plays an important role in regulating the vitamin body homeostasis and is mediated by thiamine transporters1 and 2 (THTR1 and THTR2). Little is known about the effect of chronic alcohol consumption on renal thiamine transport. We addressed this issue in this investigation using purified renal brush border membrane vesicles (BBMV) and basolateral membrane vesicles (BLMV) isolated from rats fed alcohol liquid diet (36% calories) for 2, 4 and 6 weeks and compared the findings to those of pair‐fed controls (alcohol free liquid diet). Results showed that chronic ethanol feeding significantly inhibits carrier mediated thiamine uptake in renal BBMV and the effect was evident as early as 2 weeks after the start of alcohol feeding. Uptake of thiamine by renal BLMV was similarly inhibited. This decrease in thiamine transport was associated with parallel decrease in mRNA level of THTR1 and 2 (detected by real‐time PCR) as well as in level of transcription of their respective genes [established from level of expression of heterogeneous nuclear RNA (hnRNA)]. These studies show for the first time that chronic alcohol consumption leads to inhibition in renal thiamine uptake and the effect appears to be mediated (at least in part) via transcriptional mechanism(s). (Supported by grants from DVA and NIH).

GABARAP deficiency modulates expression of NaPi-IIa in renal brush-border membranes.

Renal reabsorption of inorganic phosphate (P(i)) is mainly mediated by the Na(+)-dependent P(i)-cotransporter NaPi-IIa that is expressed in the brush-border membrane (BBM) of renal proximal tubules. Regulation and apical expression of NaPi-IIa are known to depend on a network of interacting proteins. Most of the interacting partners identified so far associate with the COOH-terminal PDZ-binding motif (TRL) of NaPi-IIa. In this study GABA(A) receptor-associated protein (GABARAP) was identified as a novel interacting partner of NaPi-IIa applying a membrane yeast-two-hybrid system (MYTH 2.0) to screen a mouse kidney library with the TRL-truncated cotransporter as bait. GABARAP mRNA and protein are present in renal tubules, and the interaction of NaPi-IIa and GABARAP was confirmed by using glutathione S-transferase pulldowns from BBM and coimmunoprecipitations from transfected HEK293 cells. Amino acids 36-68 of GABARAP were identified as the determinant for the described interaction. The in vivo effects of this interaction were studied in a murine model. GABARAP(-/-) mice have reduced urinary excretion of P(i), higher Na(+)-dependent (32)P(i) uptake in BBM vesicles, and increased expression of NaPi-IIa in renal BBM compared with GABARAP(+/+) mice. The expression of Na(+)/H(+) exchanger regulatory factor (NHERF)1, an important scaffold for the apical expression of NaPi-IIa, is also increased in GABARAP(-/-) mice. The absence of GABARAP does not interfere with the regulation of the cotransporter by either parathyroid hormone or acute changes of dietary P(i) content.

Glucose transport in fibroblasts is unaffected by polyamines

Objective Wound healing is characterized by a net increase in glucose utilization in wound tissues. The mediators involved in this process remain largely unknown. Because polyamines are known to stimulate d-glucose uptake in brush-border membrane vesicles, we investigated whether or not they stimulated sugar uptake in confluent cultured fibroblasts. Methods Cells (at a quiescent or growing state) were incubated for 1 h with various concentrations (0.5–4 mM) of putrescine, spermine, or spermidine or for a range of times (30 min to 3 h) with 2 mM of these same polyamines. Cultures were then incubated for 5 min at +37°C with 2-deoxy- d-[1- 3H] glucose. Results Polyamines were found to have no action on sugar uptake in any of the experimental configurations. Conclusion These data suggest that polyamines have no effect in cell types in which glucose uptake is mediated by a passive facilitated diffusion process (energy independent). This contrasts with results obtained with cells in which sugar uptake is dependent on adenosine triphosphate. Even if this model does not reflect the complexity of wound healing, these negative results are nevertheless important because they suggest that the arginine- and ornithine-mediated effects on wound healing are not related to a polyamine-mediated increase in glucose transport in fibroblasts.

GLUT2 protein at the rat proximal tubule brush border membrane correlates with protein kinase C (PKC)-βl and plasma glucose concentration

GLUT2 is the main renal glucose transporter upregulated by hyperglycaemia, when it becomes detectable at the brush border membrane (BBM). Since glucose-induced protein kinase C (PKC) activation in the kidney is linked to diabetic nephropathy, we investigated the effect of glycaemic status on the protein levels of PKC isoforms alpha, betaI, betaII, delta and epsilon in the proximal tubule, as well as the relationship between them and changes in GLUT2 production at the BBM. Plasma glucose concentrations were modulated in rats by treatment with nicotinamide 15 min prior to induction of diabetes with streptozotocin. Levels of GLUT2 protein and PKC isoforms in BBM were measured by western blotting. Additionally, the role of calcium signalling and PKC activation on facilitative glucose transport was examined by measuring glucose uptake in BBM vesicles prepared from proximal tubules that had been incubated either with thapsigargin, which increases cytosolic calcium, or with the PKC activator phorbol 12-myristate,13-acetate (PMA). Thapsigargin and PMA enhanced GLUT-mediated glucose uptake, but had no effect on sodium-dependent glucose transport. Diabetes significantly increased the protein levels of GLUT2 and PKC-betaI at the BBM. Levels of GLUT2 and PKC-betaI correlated positively with plasma glucose concentration. Diabetes had no effect on BBM levels of alpha, betaII, delta or epsilon isoforms of PKC. Enhanced GLUT2-mediated glucose transport across the proximal tubule BBM during diabetic hyperglycaemia is closely associated with increased PKC-betaI. Thus, altered levels of GLUT2 and PKC-betaI proteins in the BBM may be important factors in the pathogenic processes underlying diabetic renal injury.

Effects of Lactobacillus casei and Bifidobacterium bifidum on intestinal enzymatic activity and sugar uptake

Probiotics are live organisms, present in milk and its derived products, with beneficial effects for human health. In the present study, we have investigated the possible use of Lactobacillus casei (Lc) and Bifidobacterium bifidum (Bb) as probiotic factors, determining its effect on some aspects of the intestinal physiology. Swiss mice (n=60) were divided in three groups and fed for 28 days with a control standard diet and milk with the different bacteria strains at a concentration of 108 cfu/ml. After the animal sacrifice, a portion of jejunum was excised and analysed for the presence of Peyer's patches. Other fragment was collected and processed for the isolation of brush border membrane vesicles (BBMV). In these vesicles the activity of sucrase, maltase, D-galactose uptake and the expression of the sugar transporter (SGLT1) were measured. Addition of Bb or Lc in the diet did not alter body weight gain. In contrast, the number of jejunal Peyer's patches present in Lc- and Bb-fed-animals was significantly increased as compared to control group (p<0.01). Maltase and sucrase activities were also significantly increased (p<0.001) in both bacteria strains-fed animals. In BBMV uptake assays probiotics inhibited Na+-dependent D-galactose uptake although SGLT1 expression in the two supplemented dietary groups increased as compared with (to) control group. These data may indicate that Lc or Bb intake seems to stimulate: the immune system, the enzymatic activity and, the SGLT1 expression. It is suggested that dietary addition of these bacterial strains could help to improve intestinal sugar digestion and absorption. This work was supported by Health Department of the Government of Navarra.

Chronic, sublethal nickel acclimation alters the diffusive properties of renal brush border membrane vesicles (BBMVs) prepared from the freshwater rainbow trout

Brush border membrane vesicles (BBMVs) were prepared from the kidneys of rainbow trout exposed acutely (72 h; 13,380 μg Ni L − 1 ), chronically (11 months; 289 μg Ni L − 1 ), or chronically and acutely, to waterborne nickel (Ni). Uptake of 63Ni into renal BBMVs was temperature-dependent and fitted a two component kinetic model composed of a saturable, Michaelis–Menten component prominent at lower Ni concentrations, and a moderate linear diffusive component apparent at higher Ni concentrations. Chronic Ni exposure reduced the permeability of the BBM to Ni, evidenced by a significantly reduced slope of the linear diffusive component of BBMV uptake. Efflux of Ni from 63Ni-loaded renal BBMVs was not significantly altered by acute Ni challenge. Both Ca 2+ and Mg 2+ inhibited uptake of Ni into renal BBMVs when present at a molar ratio to Ni of 1000 : 1. Mg 2+-induced inhibition, however, was concentration-dependent and significant in BBMVs prepared from chronically Ni-acclimated fish at far lower molar ratios of 100 and 10 to 1. The data suggest that subtle, long-term modulation of membrane structure and function in the rainbow trout may be a compensatory response to chronic waterborne Ni exposure. Additionally, the data challenge the assumptions of constancy of the physiological parameters underlying physiologically based approaches to modeling metal toxicity. Such approaches are currently employed to derive water quality criteria for some metals.

Effect of ischemia reperfusion on sodium-dependent phosphate transport in renal brush border membranes

The effect of ischemia induced acute renal failure (ARF) on the transport of phosphate (Pi) after early (15–30 min) and prolonged (60 min) ischemia in the brush border membrane vesicles (BBMV) from rat renal cortex was studied. Sodium-dependent transport of Pi declined significantly and progressively due to ischemia. Western blot analysis of BBM from ischemic rats showed decreased expression of NaPi-2. A compensatory increase was observed in Pi uptake in BBMV from contralateral kidneys. There was no significant difference in NaPi-2 expression between BBMV from sham and contralateral kidneys. Early blood reperfusion for 15 min after 30 min ischemia caused further decline in Pi uptake. Prolonged reperfusion for 120 min caused partial reversal of transport activities in 30-min ischemic rats. However, no improvement in the transport of Pi was observed in 60-min ischemic rats after 120 min of blood reperfusion. Kinetic studies showed that the effect of ischemia and blood reperfusion was dependent on the V max of the Na-Pi transporter. Western blot analysis showed increased expression of NaPi-2 in the BBMs from ischemia–reperfusion animals. Further, a shift in the association of Na ions to transport one molecule of Pi was observed under different extracellular Na concentrations [Na] o. Feeding rats with low Pi diet and/or treatment with thyroid hormone (T3) prior to ischemia resulted in increased basal Pi transport. Ischemia caused similar decline in Pi transport in BBM from LPD and/or T3 animals. However, recovery in these animals was faster than the normal Pi diet fed (NPD) animals. The study suggests a change in the intrinsic properties of the Na-Pi transporter in rat kidneys due to ischemia. The study also indicates that treatment with T3 and feeding LPD prior to ischemia caused faster recovery of phosphate uptake due to ischemia–reperfusion injury.

Enhanced glucose absorption in the rat small intestine following repeated doses of 5-fluorouracil

Many studies demonstrated that 5-fluorouracil (5-FU) treatment of rodents caused the damage of small intestine, resulting in the malabsorption, while we recently found that repeated administration of 5-FU to rats increased Na +-dependent glucose absorption in the small intestine. This study investigated the cause of enhanced glucose absorption. 3- O-methyl- d-glucose (3-OMG) absorption was examined using the everted intestine technique. d-Glucose uptake, phlorizin binding, Western blot analysis and membrane fluidity were examined using small intestinal brush-border membrane vesicles (BBMV). Repeated oral administration of 5-FU to rats increased Na +-dependent 3-OMG absorption in the small intestine, while alkaline phosphatase activity in the small intestine decreased. Na +/K +-ATPase activity of 5-FU-treated rats was about three-fold higher than that of control rats. Although the amount of Na +-dependent glucose co-transporter (SGLT1) in 5-FU-treated rats decreased, the overshoot magnitude of d-glucose uptake in BBMV was not altered. Maximum binding of phlorizin in 5-FU-treated rats was 1.5-fold larger than that of control rats, but not altered the maximal rate of d-glucose absorption, Michaelis constant of d-glucose and dissociation constant of phlorizin. The membrane fluidity of 5-FU-treated rats increased. The enhanced d-glucose absorption in 5-FU-treated rats seems to occur secondarily due to the activation of Na +/K +-ATPase activity in basolateral membranes (BLM). Because the amounts of SGLT1 in 5-FU-treated rats decreased, the increase of turnover rate of SGLT1 and/or an expression of unknown Na +-dependent glucose co-transporter with high affinity for d-glucose and phlorizin sensitivity would contribute to the enhancement of d-glucose transport in 5-FU-treated rats.

Simple-sugar meals target GLUT2 at enterocyte apical membranes to improve sugar absorption: a study in GLUT2-null mice.

The physiological significance of the presence of GLUT2 at the food-facing pole of intestinal cells is addressed by a study of fructose absorption in GLUT2-null and control mice submitted to different sugar diets. Confocal microscopy localization, protein and mRNA abundance, as well as tissue and membrane vesicle uptakes of fructose were assayed. GLUT2 was located in the basolateral membrane of mice fed a meal devoid of sugar or containing complex carbohydrates. In addition, the ingestion of a simple sugar meal promoted the massive recruitment of GLUT2 to the food-facing membrane. Fructose uptake in brush-border membrane vesicles from GLUT2-null mice was half that of wild-type mice and was similar to the cytochalasin B-insensitive component, i.e. GLUT5-mediated uptake. A 5 day consumption of sugar-rich diets increased fructose uptake fivefold in wild-type tissue rings when it only doubled in GLUT2-null tissue. GLUT5 was estimated to contribute to 100 % of total uptake in wild-type mice fed low-sugar diets, falling to 60 and 40 % with glucose and fructose diets respectively; the complement was ensured by GLUT2 activity. The results indicate that basal sugar uptake is mediated by the resident food-facing SGLT1 and GLUT5 transporters, whose mRNA abundances double in long-term dietary adaptation. We also observe that a large improvement of intestinal absorption is promoted by the transient recruitment of food-facing GLUT2, induced by the ingestion of a simple-sugar meal. Thus, GLUT2 and GLUT5 could exert complementary roles in adapting the absorption capacity of the intestine to occasional or repeated loads of dietary sugars.

Fluorescein-methotrexate transport in brush border membrane vesicles from rat small intestine

The transport characteristics of fluorescein-methotrexate (F-MTX) in isolated brush border membrane vesicles (BBMVs) from rat small intestine were studied. F-MTX uptake in BBMVs was measured by a rapid filtration technique. Our results demonstrated that F-MTX uptake into vesicles was 1) significantly increased under the experimental conditions of an outwardly directed OH − gradient or an inwardly directed H +gradient, 2) sensitive to temperature, 3) increased with decreasing pH of the incubation buffer, 4) significantly inhibited by 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS) at the early stage of the uptake, and 5) significantly inhibited by methotrexate (MTX). Thus, the transport of F-MTX in BBMVs was shown to be mediated in part by the reduced folate transporter (RFC) which was known to transport MTX through the epithelium of small intestine.

Intestinal transport of monosaccharides and amino acids during postnatal development of mink.

Intestinal development is typically studied using omnivores. For comparative purposes, we examined an altricial carnivore, the mink (Mustela vison). In mink, intestinal dimensions increase up to 8 wk after birth and then remain constant (length) or decrease (mass) into maturity despite continuing gains in body mass. Rates of glucose and fructose transport decline after birth for intact tissues but increase for brush-border membrane vesicles (BBMV). Rates of absorption for five amino acids that are substrates for the acidic (aspartate), basic (lysine), neutral (leucine and methionine), and imino acid (proline) carriers increase between birth and 24 h for intact tissues before declining, but increase after 2 wk for BBMV. The proportion of BBMV amino acid uptake that is Na(+)-dependent increases during development but for aspartate is nearly 100% at all ages. Tracer uptake by BBMV can be inhibited by 100 mmol/l of unlabeled amino acid, except for lysine. BBMV uptake of the dipeptide glycyl-sarcosine does not differ between ages, is not Na(+) dependent, and is only partially inhibited by 100 mmol/l unlabeled dipeptide. Despite the ability to rapidly and efficiently digest high dietary loads of protein, rates of amino acid and peptide absorption are not markedly higher than those of other mammals.

Calcium Does Not Inhibit Iron Absorption or Alter Iron Status in Infant Piglets Adapted to a High Calcium Diet

The purpose of this study was to investigate whether a dietary calcium:iron ratio similar to that often consumed by premature human infants inhibits iron absorption in infant piglets adapted to a high calcium diet. Male Yorkshire piglets were randomized at 3 to 4 d of age to a high calcium diet (4.67 g/L = HC) or a normal calcium diet (2.0 g/L = NC) and fed for 2 to 2.5 wk. An iron dextran injection was administered in amounts to achieve a marginal state of iron repletion to simulate iron status of premature infants. In vivo iron absorption from the diet was determined using the radiotracers 55Fe and 59Fe and whole body counting. Calcium:iron interactions at absorption sites in piglets fed HC and NC were investigated by measurements of time-dependent 59Fe uptake in response to different calcium:iron ratios in vitro in brush border membrane vesicles (BBMV). In vivo iron absorption from the diet did not differ between NC and HC diet groups [57 ± 8% versus 55 ± 17% (mean ± SD), respectively]. Iron status and iron contencentrations in spleen, liver, intestine, kidney and heart did not differ between diet groups. Iron uptake in BBMV was significantly reduced by calcium in both HC and NC (P < 0.001); but there were no significant differences in iron uptake in response to different calcium:iron ratios between HC and NC. With feeding a HC diet for 2 wk there may be an adaptive response to counteract the inhibitory effects of calcium on iron absorption, thus resulting in similar in vivo iron absorption and iron status irrespective of the 1.3-fold difference in dietary calcium:iron ratio between piglet groups. However, future studies are needed to determine the specific sites of calcium:iron interactions and adaptation mechanisms. Since the calcium:iron ratios used in this study reflect the usual calcium:iron ratios in diets for premature infants, it is unlikely that interactive effects of calcium with iron will compromise iron status in this infant population when diets are supplemented with calcium.

Maturational changes in rabbit renal brush border membrane vesicle urea permeability.

Urea transport in the proximal tubule is thought to occur by passive diffusion through the lipid bilayers of the cell membranes. The lipid composition of cell membranes changes during maturation and may directly affect urea permeability of proximal tubule membranes. The present study examined the maturation of urea transport in rabbit renal brush border membrane vesicles (BBMV). BBMV from adult and neonatal (9- to 11-d-old) New Zealand white rabbits were loaded with 500 mM urea and mixed with an iso-osmotic mannitol solution using a stop-flow instrument. Vesicle shrinkage, due to efflux of urea, was followed with light scattering and urea permeability was calculated from an exponential fit of the data. Urea permeability was significantly lower in the neonatal BBMV than the adult at 25 degrees C (0.34+/-0.04 x 10(-6) versus 0.56+/-0.03 x 10(-6) cm/sec;p < 0.001, n=7) and 37 degrees C (0.45+/-0.04 x 10(-6) versus 0.66+/-0.03 x 10(-6) cm/sec; p=0.001, n=7). There was no effect of 250 microM phloretin on urea permeability in either adult or neonatal BBMV at either temperature. The activation energy for urea diffusion was higher in the neonatal than the adult BBMV. Because the maturational increase in urea permeability could potentially be due to a sodium-dependent urea transporter in the adult BBMV, the sodium dependence of urea uptake in adult BBMV was examined. There was no difference in urea permeability in the presence or absence of 20 mM NaCl. Permeability of the lipid-soluble molecule, glycerol, was also found to be the same in the neonatal and adult BBMV. Urea transport in the apical membrane of neonatal and adult proximal tubules is not phloretin sensitive, a finding consistent with diffusion of urea via the lipid bilayer. The rate of urea diffusion is lower in neonatal membranes and may be an important factor in overall urea excretion. This may also play a role in developing and maintaining a high medullary urea concentration and thus the ability to concentrate the urine during renal maturation.

Unique mechanism of inhibition of Na+-amino acid cotransport during chronic ileal inflammation

In the chronically inflamed ileum, unique mechanisms of alteration of transport processes suggest regulation by different immune-inflammatory mediator pathways. We previously demonstrated that Na+-glucose cotransport in the chronically inflamed ileum was inhibited by a decrease in cotransporter number without a change in glucose affinity. The aim of this study was to determine the alterations in Na+-amino acid cotransport in chronically inflamed ileum produced by coccidial infection in rabbits. [3H]alanine uptake was performed in cells and vesicles by rapid filtration. In villus cells from chronically inflamed ileum, Na+-K+-ATPase was reduced 50% and Na+-alanine cotransport was also reduced (5.8 +/- 1.2 in normal and 1.4 +/- 0.5 nmol/mg protein in inflamed; n = 6, P < 0.05). [3H]alanine uptake in brush-border membrane vesicles was reduced in chronically inflamed ileum (73.2 +/- 1.2 in normal and 21.5 +/- 3.2 pmol/mg protein in inflamed; n = 3, P < 0.05), suggesting a direct effect on the cotransporter itself. Na+-amino acid cotransport in chronically inflamed ileum was inhibited by a decrease in affinity without a change in the maximal rate of uptake, and unaltered steady-state mRNA levels also suggested that the number of cotransporters was unchanged. Thus the mechanisms of inhibition of Na+-amino acid cotransport and Na+-glucose cotransport in chronically inflamed ileum are different. These observations suggest that different immune-inflammatory mediators may regulate different transport pathways during chronic ileitis.