Isolated Brush Border Membrane Research Articles

#1448 Regulation of renal phosphate excretion independent of FGF23 and PTH

Abstract Background and Aims The phosphaturic hormones fibroblast growth factor 23 (FGF23) and parathyroid hormone (PTH) promote renal phosphate excretion through FGFR1/Klotho and PTH1R-mediated ERK1/2 activation, respectively, leading to inhibition of the sodium-phosphate cotransporters NPT2a (SLC34A1) and NPT2c (SLC34A3) in proximal tubule (PT) cells. In bone cells, high extracellular phosphate can directly activate ERK1/2 via the sodium-phosphate cotransporters PiT-1 (SLC20A1) and PiT-2 (SLC20A2). To date, it is unclear to what extent phosphate can also regulate its own excretion in the kidney independently of FGF23 and PTH. Method To further investigate the role of phosphate in the regulation of renal phosphate homeostasis, male C57BL/6 mice were placed on a 0.8% control phosphate diet (CPD) or a 2% high phosphate diet (HPD) for 6 months. After four months, one HPD group was additionally treated with a calcimimetic (etelcalcetide) to lower FGF23 and PTH. In vitro, PT cells were stimulated with phosphate, FGF23 or PTH ± the phosphate transporter inhibitor foscarnet. Results HPD resulted in increased plasma concentrations of FGF23 and PTH, which were associated with reduced tubular phosphate reabsorption and increased fractional phosphate excretion. RNAseq analyses from isolated PT cells showed a reduction of Fgfr1, Kl, Slc34a1 and Slc34a3 and an induction of Slc20a2 in the HPD group compared to CPD. qPCR and immunoblot analyses of whole kidney tissue confirmed the reduction of the FGF23 co-receptor Klotho in the HPD group, while Fgfr1 was not significantly altered. Protein expression of NPT2a was decreased in isolated brush border membrane vesicles from HPD-fed mice, and immunofluorescence staining showed internalization of NPT2a from the apical brush border membrane. Treatment with etelcalcetide resulted in a reduction of circulating FGF23 and PTH, whereas urinary phosphate excretion in HPD mice remained elevated. In cultured PT cells, high phosphate induced phosphorylation of ERK1/2 and mRNA expression of Slc20a2, while Slc34a1 was suppressed. Phosphate-mediated PiT-2/ERK1/2 activation could be blocked by simultaneous treatment with foscarnet. Conclusion Our data suggest that high phosphate leads to internalization of NPT2a via direct activation of the PiT-2/ERK1/2 signaling cascade, independent of FGF23 and PTH, resulting in increased renal phosphate excretion.

Potential of Ablation Therapy during Hepatocellular Carcinoma

Background: This study explored the potential of ablation therapy on membrane fluidity changes in diethylnitrosamine (DEN) induced hepatic cancer.Methods: Male Wistar rats were segregated into four groups viz., normal control, DEN treated, ablation therapy treated, and DEN ablation therapy treated. We assessed the viscosities as well as fluidity parameters in isolated brush border membranes using the membrane extrinsic fluorophore pyrene.Results: DEN treatment successfully induced hepatic cancer in the livers of rats and ablation therapy worked well in terms of therapy. DEN treatment resulted in a significant rise in lipid peroxidation (LPO) and significant decrease in the, reduced glutathione levels (GSH). A significant decrease was also noticed in the activities of glutathione reductase (GR), glutathione transferase (GST), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) following DEN treatment. On the other hand, ablation therapy treatment to DEN-treated rats resulted in a significant decrease in the LPO levels but caused a significant rise in the levels of GSH. Moreover, the activities of GR, GST, SOD, CAT, and GPx showed significant improvement after ablation therapy treatment. The results further demonstrated a marked decrease in membrane microviscosity following DEN treatment. On the other hand, a significant increase was noticed in both excimer/monomer ratio and fluidity parameter in DEN-treated rats. However, membrane microviscosity and the fluidity alterations were significantly restored back to near normal with ablation therapy treatment.Conclusions: The study, therefore, concluded that ablation therapy holds good therapeutic potential against DEN-induced hepatic cancer.

Oral Nigella sativa oil and thymoquinone administration ameliorates the effect of long-term cisplatin treatment on the enzymes of carbohydrate metabolism, brush border membrane, and antioxidant defense in rat intestine.

We have previously shown that oral administration of Nigella sativa oil (NSO) ameliorates the deleterious gastrointestinal effects of cisplatin (CP), administered as a single dose. Since a typical clinical CP dosing regimen involves multiple cycles of CP administration in lower doses, in the present study we investigate the protective efficacy of NSO and its major bioactive constituent, thymoquinone (TQ), against multiple-dose CP treatment-induced deleterious biochemical and histological changes in rat intestine. Rats were divided into six groups, viz., control, CP, CP+NSO, CP+TQ, NSO, and TQ. Animals in CP+NSO and CP+TQ groups were pre-administered NSO (2ml/kg bwt, orally) and TQ (1.5mg/kg bwt, orally), respectively, daily for 14days and were then treated with five repeated doses of CP (3mg/kg bwt, i.p.), every fourth day for 20days while still receiving NSO/TQ. CP treatment alone led to a significant decline in specific activities of brush border membrane (BBM) enzymes while NSO or TQ administration to CP-treated rats significantly prevented the decline in BBM enzyme activities in the isolated brush border membrane vesicles (BBMV) as well as in mucosal homogenates. Furthermore, both NSO and TQ administration markedly ameliorated CP-induced alterations on carbohydrate metabolism enzymes and the enzymatic and non-enzymatic parameters of antioxidant defense system in the intestinal mucosa. However, NSO appeared to be more efficacious than TQ in protecting against CP-induced gastrointestinal dysfunction. Histopathological findings corroborated the biochemical results. Thus, NSO and TQ may prove clinically useful in amelioration of the intestinal toxicity associated with long-term CP chemotherapy.

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.

Comprehensive cytotoxic evaluation of morin, a bioflavonoid against verapamil on rat gastrointestinal epithelium for novel pharmaceutical application involving P-glycoprotein inhibition

In this study, a comprehensive and comparative cytotoxic evaluation of morin against verapamil on rat intestinal epithelium as P-gp inhibitors through in-vitro gastrointestinal short-term toxicity assays involving permeability studies for safety evaluation was investigated. In this study, the effect of morin (1 mM or 10 mM) or verapamil (1 mM or 10 mM) or sodium deoxycholate (10 mM) was investigated on intestinal epithelium and isolated brush border membrane using biomarker assays. Cytotoxicity was determined using 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) assay. The nutrients transport was assessed using everted sacs studies. Paracellular permeability was measured using Lucifer yellow, followed by morphometric analysis of intestinal sacs. Our results indicated that morin was effective in maintaining cell viability with no significant changes (P > 0.05) in the activity of intestinal brush border markers, membrane integrity and morphometric analysis as compared with control. On the contrary, dramatic (P < 0.01) changes were noticed in the release of membrane markers, cell viability and surface characteristics of intestinal segments when treated with verapamil or sodium deoxycholate as compared with control or morin. Our findings confirm that morin is non-toxic to rat intestinal epithelium against verapamil demonstrating the potential use of bioflavonoid as safe and novel pharmaceutical adjuvant as P-gp inhibitor.

Safety evaluation of naringenin upon experimental exposure on rat gastrointestinal epithelium for novel optimal drug delivery

Objective: To assess the effect of naringenin on the intestinal biochemical composition, function and histology for gastrointestinal toxicity since it has not yet been adequately exploited for safety through standard assays.Methods: Here, we describe naringenin (1 mM, 10 mM and 100 mM, respectively) or sodium deoxycholate (10 mM) effects on isolated brush border membrane from intestinal segments with single pass intestinal perfusion using lactate dehydrogenase, alkaline phosphatase and protein assays. MTT assay was used for cytotoxicity studies. Everted gut sac studies were used for evaluating the transport of nutrients across the intestinal segments. Lucifer yellow was used for paracellular permeability, followed by histological changes and surface characteristic studies of intestinal sacs.Results: The results indicated no significant alterations with naringenin, although significant (p < 0.01) changes were noticed with sodium deoxycholate in the activity of the rat intestinal brush border associated enzymes such as LDH, followed by intact cell viability with marked decrease in the villi height of the intestinal segments.Conclusions: These observations indicate that naringenin was harmless upon exposure to rat gastrointestinal epithelium, clearly demonstrating the potential use of naturally occurring bioflavonoid as safe and novel pharmaceutical adjuvant in oral dosage forms as P-gp inhibitor.

Adaptive responses of calcium and phosphate homeostasis in goats to low nitrogen intake: renal aspects

In a previous study, in goats, we showed that apart from variations in dietary calcium (Ca) and phosphorus intake, also low dietary nitrogen (N) intake altered plasma concentrations of hormones, which regulate Ca and phosphate (Pi ) homeostasis. These hormonal responses in goats were in accordance with findings in monogastric animals and humans with low protein intake. In the aforementioned studies, alterations of electrolyte transport in the kidneys were also observed. However, whether renal electrolyte transport in goats is also involved in the adaptation of Ca and Pi homeostasis to low N intake remains unknown. Thus, the aim of the present study was to investigate whether in addition to the hormonal changes, as observed in our former study, renal Ca transport and renal Pi transport were also altered by low N intake in goats. Therefore, in kidney samples from the goats used in our former study, the protein expression of Ca and Pi transporters and of related regulatory proteins was examined. Furthermore, the uptake of Pi into isolated brush border membrane vesicles (BBMV) was detected. The results showed that the protein amount of the renal sodium-dependent Pi transporter NaPi IIa was elevated, and concomitantly, protein expression of its upstream regulators, the parathyroid hormone receptor and the extracellular signal-regulated kinases 1 and 2 was decreased. However, Pi uptake into renal BBMV was not enhanced. Furthermore, protein expression of the renal Ca channel, the transient receptor potential cation channel subfamily V member 5 (TRPV5) and of the vitamin D receptor was not influenced by dietary N reduction. We conclude that regulation of renal Pi transporter expression in goats is involved in the adaptation of electrolyte homeostasis to low N intake.

Performance, organ zinc concentration, jejunal brush border membrane enzyme activities and mRNA expression in piglets fed with different levels of dietary zinc

This study aimed at investigating the effect of dietary zinc on performance, jejunal brush border membrane enzyme activities and mRNA levels of enzymes and two zinc transporters in piglets. A total of 126 piglets were weaned at 26 ±1 days of age and randomly allocated into three groups fed with diets 50, 150 and 2500 mg zinc/kg. Performance was recorded and at weekly intervals, eight piglets per group were killed. The activities of isolated brush border membrane enzymes including lactase, maltase, sucrase, aminopeptidase-N and intestinal alkaline phosphatase (IAP), and the relative transcript abundance of aminopeptidase-N (APN), sucrase–isomaltase (SUC), IAP and the two zinc transporters SLC39A4 (ZIP4) and SLC30A1 (ZnT1) were investigated in the jejunum. Feeding pharmacological zinc levels increased weight gain (p < 0.001) during the first week, but performance was lower (p < 0.05) in the third week. Organ zinc concentrations were increased by high dietary zinc level. The activity of IAP was higher (p < 0.05) with the highest dietary zinc level, no effects were determined for other enzymes. Dietary zinc level had no effect on transcript abundance of digestive enzymes. The mRNA levels decreased (p < 0.001) for ZIP4, and increased for ZnT1 (p < 0.05) with pharmacological zinc levels. In conclusion, pharmacological zinc levels improved performance in the short-term. Intestinal mRNA level of zinc transporters changed with high zinc supply, but this did not prevent zinc accumulation in tissues, suggesting hampered homoeostatic regulation. This might cause impaired performance during longer supply.

Rescue of epithelial HCO3− secretion in murine intestine by apical membrane expression of the cystic fibrosis transmembrane conductance regulator mutant F508del

This study investigated whether expression of the common cystic fibrosis transmembrane conductance regulator (CFTR) mutant F508del in the apical membrane of enterocytes confers increased bicarbonate secretory capacity on the intestinal epithelium of F508del mutant mice compared to that of CFTR knockout (KO) mice. CFTR KO mice, F508del mutant mice (F508del) and wild-type (WT) littermates were bred on the FVB/N background. F508del isolated brush border membrane (BBM) contained approximately 5-10% fully glycosylated band C protein compared to WT BBM. Similarly, the forskolin (FSK)-induced, CFTR-dependent short-circuit current (I(sc)) of F508del mucosa was approximately 5-10% of WT, whereas the HCO(3)(-) secretory response ( ) was almost half that of WT in both duodenum and mid-colon studied in vitro and in vivo. While WT intestine retained full FSK-induced in the absence of luminal Cl(-), the markedly higher than I(sc) in F508del intestine was dependent on the presence of luminal Cl(-), and was blocked by CFTR inhibitors. The Ste20-related proline-alanine-rich kinases (SPAK/OSR1), which are downstream of the with-no-lysine (K) protein kinases (WNK), were rapidly phosphorylated by FSK in WT and F508del, but significantly more slowly in CFTR KO intestine. In conclusion, the data demonstrate that low levels of F508del membrane expression in the intestine of F508del mice significantly increased FSK-induced HCO(3)(-) secretion mediated by Cl(-)/HCO(3)(-) exchange. However, in WT mucosa FSK elicited strong SPAK/OSR1 phosphorylation and Cl(-)-independent HCO(3)(-) efflux. This suggests that therapeutic strategies which deliver F508del to the apical membrane have the potential to significantly enhance epithelial HCO(3)(-) secretion.

Intestinal glucose absorption in calves as affected by different carbohydrate sources

From numerous recent studies, it has been demonstrated that the development of the forestomach system in ruminants and thus microbial carbohydrate fermentation do not exclude the potential of the small intestines for enzymatic carbohydrate digestion and subsequent monosaccharide absorption. However, the role of regulatory nutritional factors is still under discussion. Therefore, we investigated the kinetic parameters of intestinal Na(+) -dependent glucose absorption and SGLT1 expression using isolated brush border membrane vesicles (BBMV) from the jejunum of 10-week-old calves kept on either hay, concentrate or corn silage-based diets in addition to milk replacer. While the maximal transport capacity was significantly higher for concentrate and corn silage-fed animals, SGLT1 protein expression was highest in BBMV isolated from hay-fed animals. This observation differs from the prevalent conception that induction of Na(+) -dependent glucose uptake via SGLT1 is based on an increased number of transporters at the brush border membrane.

PKB/SGK-Resistant GSK3 Enhances Phosphaturia and Calciuria

Insulin and IGF1-dependent signaling activates protein kinase B and serum and glucocorticoid inducible kinase (PKB/SGK), which together phosphorylate and inactivate glycogen synthase kinase GSK3. Because insulin and IGF1 increase renal tubular calcium and phosphorus reabsorption, we examined GSK3 regulation of phosphate transporter activity and determined whether PKB/SGK inactivates GSK3 to enhance renal phosphate and calcium transport. Overexpression of GSK3 and the phosphate transporter NaPi-IIa in Xenopus oocytes decreased electrogenic phosphate transport compared with NaPi-IIa-expressing oocytes. PKB/SGK serine phosphorylation sites in GSK3 were mutated to alanine to create gsk3(KI) mice resistant to PKB/SGK inactivation. Compared with wildtype animals, gsk3(KI) animals exhibited greater urinary phosphate and calcium clearances with higher excretion rates and lower plasma concentrations. Isolated brush border membranes from gsk3(KI) mice showed less sodium-dependent phosphate transport and Na-phosphate co-transporter expression. Parathyroid hormone, 1,25-OH vitamin D levels, and bone mineral density were decreased in gsk3(KI) mice, suggesting a global dysregulation of bone mineral metabolism. Taken together, PKB/SGK phosphorylation of GSK3 increases phosphate transporter activity and reduces renal calcium and phosphate loss.

The plasma carnitine concentration regulates renal OCTN2 expression and carnitine transport in rats

Previous findings in rats and in human vegetarians suggest that the plasma carnitine concentration and/or carnitine ingestion may influence the renal reabsorption of carnitine. We tested this hypothesis in rats with secondary carnitine deficiency following treatment with N-trimethyl-hydrazine-3-propionate (THP) for 2 weeks and rats treated with excess l-carnitine for 2 weeks. Compared to untreated control rats, treatment with THP was associated with an approximately 70% decrease in plasma carnitine and with a 74% decrease in the skeletal muscle carnitine content. In contrast, treatment with l-carnitine increased plasma carnitine levels by 80% and the skeletal muscle carnitine content by 50%. Treatment with l-carnitine affected neither the activity of carnitine transport into isolated renal brush border membrane vesicles, nor renal mRNA expression of the carnitine transporter OCTN2. In contrast, in carnitine deficient rats, carnitine transport into isolated brush border membrane vesicles was increased 1.9-fold compared to untreated control rats. Similarly, renal mRNA expression of OCTN2 increased by a factor of 1.7 in carnitine deficient rats, whereas OCTN2 mRNA expression remained unchanged in gut, liver or skeletal muscle. Our study supports the hypothesis that a decrease in the carnitine plasma and/or glomerular filtrate concentration increases renal expression and activity of OCTN2.

Evidence of an essential role of NHERF1 and NHEF2 in G‐protein coupled receptor regulation of CFTR‐mediated intestinal anion secretion in vivo

Heterologous expression studies have demonstrated that the PDZ‐proteins NHERF1 and NHERF2 modulate CFTR interaction with other proteins. To study their biological roles in vivo in an epithelium that highly expresses CFTR as well as NHERF1 and 2, we investigated the effect of NHERF1 and 2 ablation on CFTR‐dependent duodenal HCO3− secretion. Proximal duodenum of anesthetized mice was perfused in situ, and HCO3− secretion was determined by back‐titration. Co‐localization of CFTR, β2‐adrenergic receptor and LPA2 receptors, as well as PDZ proteins in the luminal membrane of crypt duodenocytes were established by Western analysis of isolated brush border membranes, immunohistochemistry and laser micro‐dissection followed by q‐PCR (quantitative‐PCR). NHERF1 ablation strongly reduced basal as well as forskolin (FSK)‐stimulated HCO3− secretory rates, and completely prevented β2‐adrenergic stimulation. NHERF2 deletion significantly augmented FSK‐stimulated HCO3− secretion, and completely prevented the inhibitory effect of LPA. We conclude that NHERF1 and 2 differentially modulate basal and agonist‐mediated duodenal HCO3− secretion in vivo in a CFTR‐dependent fashion. NHERF1 is an obligatory linker for β2‐adrenergic stimulation of CFTR, and strongly augments cAMP‐mediated stimulation. NHERF2 confers inhibitory signals, i.e. as a coupling factor between inhibitory LPA receptor signalling and CFTR.

Cyclooxygenase-2 Inhibition by Etoricoxib Modulates Plasma Membrane Fluidity in Rat Colon

The present study was designed to investigate the effects of a selective cycloogenase-2 (COX-2) inhibitor, etoricoxib, on the membrane-specific enzymes, lipid composition, and changes in fluidity parameters of rat colonic plasma membrane. Two doses of the drug were used, one within its therapeutic anti-inflammatory range as based on the ED50 value in rats (Eto-1), while the other at 10 times higher dose relating to the toxicity studies (Eto-2), which have not been reported so far. The activity of the membrane alkaline phosphatase was found to be increased after treatment with both the doses of etoricoxib as compared to the control. The total lipid and the cholesterol content showed a decrease while an increase in ganglioside sialic acid content was noted. Phospholipid content and the cholesterol-phospholipid molar ratio showed no change in either of the treatment groups. Fluorescence polarization studies with diphenylhexatriene showed that membrane fluidity was least altered in the isolated brush border membrane from colon or in the liposomes prepared from the membrane lipid extracts. Also, the translational diffusion studied with pyrene showed that the fluidity parameter was decreased as measured by the excimer formation. It is concluded that etoricoxib, a new generation COX-2 inhibitor (called the coxibs), appeared to be a safe nonsteroidal anti-inflammatory drug (NSAID) in the colonic membranes.

Structure of the Claudin-binding Domain of Clostridium perfringens Enterotoxin

Clostridium perfringens enterotoxin is a common cause of food-borne and antibiotic-associated diarrhea. The toxin's receptors on intestinal epithelial cells include claudin-3 and -4, members of a large family of tight junction proteins. Toxin-induced cytolytic pore formation requires residues in the NH(2)-terminal half, whereas residues near the COOH terminus are required for binding to claudins. The claudin-binding COOH-terminal domain is not toxic and is currently under investigation as a potential drug absorption enhancer. Because claudin-4 is overexpressed on some human cancers, the toxin is also being investigated for targeting chemotherapy. Our aim was to solve the structure of the claudin-binding domain to advance its therapeutic applications. The structure of a 14-kDa fragment containing residues 194 to the native COOH terminus at position 319 was solved by x-ray diffraction to a resolution of 1.75A. The structure is a nine-strand beta sandwich with previously unappreciated similarity to the receptor-binding domains of several other toxins of spore-forming bacteria, including the collagen-binding domain of ColG from Clostridium histolyticum and the large Cry family of toxins (including Cry4Ba) of Bacillus thuringiensis. Correlations with previous studies suggest that the claudin-4 binding site is on a large surface loop between strands beta8 and beta9 or includes these strands. The sequence that was crystallized (residues 194-319) binds to purified human claudin-4 with a 1:1 stoichiometry and affinity in the submicromolar range similar to that observed for binding of native toxin to cells. Our results provide a structural framework to advance therapeutic applications of the toxin and suggest a common ancestor for several receptor-binding domains of bacterial toxins.

Regulation of Intestinal Phosphate Transport I. Segmental expression and adaptation to low-Pidiet of the type IIb Na+-Picotransporter in mouse small intestine

The Na(+)-P(i) cotransporter NaPi-IIb (SLC34A2) has been described to be involved in mouse small intestinal absorption of P(i) and to be regulated by a number of hormones and metabolic factors. However, a possible segmental expression of NaPi-llb in small intestine has not been addressed so far. Here, we describe that the NaPi-IIb cotransporter is highly abundant in the ileum of mouse small intestine, whereas it is almost absent in the duodenum and in the jejunum. Na(+)-P(i) cotransport studies with isolated brush border membranes confirmed that NaPi-IIb cotransport is highest in the ileum. Upregulation by a low-phosphate diet of NaPi-IIb and NaPi-IIb cotransport was observed both in the jejunum and the ileum. Furthermore, evidence is provided that a low-phosphate diet provokes an increase of the NaPi-IIb mRNA abundance along the entire small intestine. These data suggest that in mouse small intestine, phosphate is absorbed transcellulary by an Na(+)-dependent pathway in the ileum, whereas in the duodenum and jejunum, this pathway is of minimal importance. Furthermore, we conclude that along the entire mouse small intestine, low-phosphate diet affects transcription and/or the stability of NaPi-IIb mRNA.

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.

NHERF-1 uniquely transduces the cAMP signals that inhibit sodium–hydrogen exchange in mouse renal apical membranes

Sodium–hydrogen exchanger regulatory factor isoform-1 (NHERF-1) and NHERF-2 are two structurally related PDZ-domain-containing protein adapters that effectively transduce cyclic AMP (cAMP) signals that inhibit NHE3, the sodium–hydrogen exchanger isoform present at the apical surface of kidney and gut epithelia. The mouse renal proximal tubule expresses both NHERF isoforms, suggesting their redundant functions as regulators of renal electrolyte metabolism. To define the role of NHERF-1 in the physiological control of NHE3, we analyzed NHE3 activity in isolated brush border membrane (BBM) preparations from renal proximal tubules of wild-type (WT) and NHERF-1 (−/−) mice. Basal Na +–H + exchange was indistinguishable in BBMs from WT and NHERF-1 (−/−) mice (0.96±0.08 and 0.95±0.10 nmol/mg protein/10 s, respectively). Activation of membrane bound cAMP-dependent protein kinase (PKA) by cAMP inhibited NHE3 activity in WT BBMs (0.55±0.07 nmol/mg protein/10 s or 40±9%, P<0.01) but had no discernible effect on Na +–H + exchange in the NHERF-1 (−/−) BBM (0.97±0.07 nmol/mg protein/10 s; P=not significant). This was associated with a significant decrease in cAMP-stimulated phosphorylation of NHE3 immunoprecipitated from solubilized NHERF-1 (−/−) BBMs. As the protein levels for NHE3, NHERF-2, PKA and ezrin were not changed in the NHERF-1 (−/−) BBMs, the data suggest a unique role for NHERF-1 in cAMP-mediated inhibition of NHE3 activity in the renal proximal tubule of the mouse.

Mechanisms of Vitamin B12 Absorption in Breast‐Fed Infants

ABSTRACTObjectivesThe mechanisms of vitamin B12 absorption in infants are unknown. We investigated whether haptocorrin (HC), a vitamin B12‐binding protein in human milk, facilitates vitamin B12 absorption during the neonatal period or if it occurs by a process similar to that in adults involving another vitamin B12‐binding protein, intrinsic factor (IF).MethodsTo determine whether HC or IF can deliver vitamin B12 to the enterocyte, binding studies using Caco‐2 intestinal cells in culture and purified human milk HC‐[57Co]vitamin B12 or [125I]IF‐vitamin B12 were performed. Determination of IF secretion by infant stomach was investigated by a competitive ELISA on fecal extracts from breast‐fed infants. Determination of receptors specific for IF‐vitamin B12 or HC‐vitamin B12 in infant intestine was achieved by ligand blot analysis using isolated brush border membrane vesicles (BBMV) from fetal and adult intestine and Caco‐2 cells. PCR was performed to identify the IF receptor gene transcript in Caco‐2 cells and fetal intestine.ResultsLimited binding of both HC and IF to Caco‐2 cells was observed; however, HC displayed affinity to low molecular weight proteins in BBMV from fetal intestine and Caco‐2 cells while IF showed affinity for a 240 kDa protein in BBMV from fetal intestine and Caco‐2 cells. IF receptor gene transcript was identified in fetal intestine and Caco‐2 cells. An increase in IF excretion from breast‐fed infants throughout early life was observed.ConclusionsAn IF‐dependent vitamin B12 absorption mechanism appears to be in place in breast‐fed infants. However, IF levels may be too low in early life to participate in vitamin B12 absorption; therefore, haptocorrin may mediate vitamin B12 absorption until the absorption function can be taken over by a more mature IF system.

Mechanisms of vitamin B(12) absorption in breast-fed infants.

The mechanisms of vitamin B(12) absorption in infants are unknown. We investigated whether haptocorrin (HC), a vitamin B(12) -binding protein in human milk, facilitates vitamin B(12) absorption during the neonatal period or if it occurs by a process similar to that in adults involving another vitamin B(12) -binding protein, intrinsic factor (IF). To determine whether HC or IF can deliver vitamin B(12) to the enterocyte, binding studies using Caco-2 intestinal cells in culture and purified human milk HC-[ (57)Co]vitamin B(12) or [(125)I]IF-vitamin B(12) were performed. Determination of IF secretion by infant stomach was investigated by a competitive ELISA on fecal extracts from breast-fed infants. Determination of receptors specific for IF-vitamin B(12) or HC-vitamin B(12) in infant intestine was achieved by ligand blot analysis using isolated brush border membrane vesicles (BBMV) from fetal and adult intestine and Caco-2 cells. PCR was performed to identify the IF receptor gene transcript in Caco-2 cells and fetal intestine. Limited binding of both HC and IF to Caco-2 cells was observed; however, HC displayed affinity to low molecular weight proteins in BBMV from fetal intestine and Caco-2 cells while IF showed affinity for a 240 kDa protein in BBMV from fetal intestine and Caco-2 cells. IF receptor gene transcript was identified in fetal intestine and Caco-2 cells. An increase in IF excretion from breast-fed infants throughout early life was observed. An IF-dependent vitamin B(12) absorption mechanism appears to be in place in breast-fed infants. However, IF levels may be too low in early life to participate in vitamin B(12) absorption; therefore, haptocorrin may mediate vitamin B(12) absorption until the absorption function can be taken over by a more mature IF system.