Electrodiffusive magnesium transport across the intestinal brush border membrane of tilapia (Oreochromis mossambicus).

Mg2+ transport across the brush border of proximal intestinal epithelium of the teleost fish Oreochromis mossambicus was investigated, using 27Mg2+ to trace movement of Mg2+. Mg2+ uptake in brush border membrane vesicles was stimulated by a K+ diffusion potential (inside negative). Electrodiffusive Mg2+ transport obeyed simple Michaelis-Menten kinetics and was strongly temperature dependent, indicative of a carrier mechanism. The metal ion specificity of this electrodiffusive pathway (inhibition potency order: Co > Mn = Ni > La > Ca > Gd > Ba), predicts a specific role in Mg2+ transport. Competitive inhibition by Co(III) hexammine [Co(NH3)(6)(3+)] suggests that this transport system interacts with the solvated Mg ion. We propose that this novel transport system allows the uptake of Mg2+ across the apical brush border membrane, and is involved in transcellular Mg2+ transport. Consequently, the prevailing potential difference across the apical membrane represents a major driving force for intestinal Mg2+ absorption.     

Intestinal cholesterol absorption: identification of different binding proteins for cholesterol and cholesterol absorption inhibitors in the enterocyte brush border membrane

Absorption of cholesterol from the intestine is a central part of body cholesterol homeostasis. The molecular mechanisms of intestinal cholesterol absorption and the proteins mediating membrane transport are not known. We therefore aimed to identify the proteins involved in intestinal cholesterol absorption across the luminal brush border membrane of small intestinal enterocytes. By photoaffinity labeling using photoreactive derivatives of cholesterol and 2-azetidinone cholesterol absorption inhibitors, an 80-kDa and a 145-kDa integral membrane protein were identified as specific binding proteins for cholesterol and cholesterol absorption inhibitors, respectively, in the brush border membrane of small intestinal enterocytes. The 80-kDa cholesterol-binding protein did not interact with cholesterol absorption inhibitors and vice versa; cholesterol or plant sterols did not interfere with the 145-kDa molecular target for cholesterol absorption inhibitors. Both proteins showed an identical tissue distribution and were exclusively found at the anatomical sites of cholesterol absorption-duodenum, jejunum and ileum. Neither stomach, cecum, colon, rectum, kidney, liver nor fat tissue expressed the 80- or 145-kDa binding proteins for cholesterol and cholesterol absorption inhibitors. Both proteins are different from the hitherto described candidate proteins for the intestinal cholesterol transporter,-SR-BI, ABC G5/ABC G8 or ABC A1. Our data strongly suggest that intestinal cholesterol absorption is not facilitated by a single transporter protein but occurs by a complex machinery. Two specific binding proteins for cholesterol (80 kDa) and cholesterol absorption inhibitors (145 kDa) of the enterocyte brush border membrane are probable protein constituents of the mechanism responsible for the intestinal absorption of cholesterol.     

翘嘴鳜miR-222的表达特征

为了解翘嘴鳜miR-222的时空表达规律, 研究利用实时荧光定量PCR的方法检测miR-222在翘嘴鳜不同组织、胚胎发育及胚后发育中的相对表达丰度。研究结果显示, miR-222在肌肉相关的组织中表达较高, 特别是在成年翘嘴鳜的白肌中表达最高; 胚胎发育阶段结果显示, miR-222在胚胎发育的2细胞期就有表达, 而表达量在心动期达到最高。不同组织及不同发育阶段的差异性表达结果表明, miR-222很可能参与调控鳜鱼肌肉的生长发育。为研究合成代谢过程中miR-222在肌肉生长调控中的表达规律, 通过对翘嘴鳜幼鱼在饥饿一周后饱食一餐的实验处理下, 利用实时荧光定量的方法测定miR-222在骨骼肌中的相对表达变化。结果显示, miR-222的表达量在恢复喂食后的1h显著上升(P0.05), 表明miR-222很可能是调节鱼类骨骼肌生长过程中, 参与快速应答信号系统的一类miRNA。研究为miR-222在鱼类发育中的调控作用提供一些理论依据。       

Evidence suggesting that the minimal functional unit of a renal cystine transporter is a heterodimer and its implications in cystinuria

Summary Cystinuria, one of the most common genetic disorders, is characterized by excessive excretion of cystine and basic amino acids in urine. The low solubility of cystine results in formation of kidney stones which can eventually lead to renal failure. Three types of cystinurias have been described. All involve defects in a high-affinity transport system for cystine in the brush border membranes of kidney and intestinal epithelial cells. The molecular properties of proteins involved in epithelial cystine transport are incompletely understood. A protein (NBAT, neutral and basic amino acid transporter), initially cloned by us from rat kidney and shown to be localized in the renal and intestinal brush border membranes, has been implicated in this transport, and mutations in human NBAT gene have been found in several cystinurics, making it a prime candidate for a cystinuria gene. However, mutations in NBAT were found only in Type I cystinurics and not in Types II and III suggesting that defects in other, as yet uncharacterized, genes may also be involved. NBAT has an unusual (for an amino acid transporter) membrane topology. We proposed that the protein contains four membrane-spanning domains, a model disputed by other investigators. We subsequently obtained experimental data consistent with a four membrane-spanning domain model. Furthermore, recently we showed that kidney and intestinal NBAT (85kDa) is associated with another brush border membrane protein (about 50kDa) and have proposed that the heterodimer represents the minimal functional unit of the high-affinity cystine transporter in these membranes. These findings raise the tantalizing possibilities that defects in the NBAT-associated protein might account for cystinurias in individuals with normal NBAT gene (such as the Types II and III cystinurics).     

Significance of substrate hydrophobicity for recognition by an oligopeptide transporter (PEPT1).

Our previous paper [(1999) Bioconjugate Chem. 10, 24-31] pointed out that hydrophobicity of substrates/inhibitors plays an important role in the recognition by an oligopeptide transporter (PEPT1) expressed in the human intestinal epithelial cell line Caco-2. To determine the significance of that hydrophobicity, we have now synthesized dipeptide analogues conjugating the epsilon-amino group of Lys in Val-Lys with aliphatic carboxylic acids: acetic acid (C2), propanoic acid (C3), pentanoic acid (C5), hexanoic acid (C6), and decanoic acid (C10). The affinities of these conjugates were estimated by their inhibition of the accumulation rate of Gly-Sar, a well-established substrate for PEPT1. With the increase in length of the hydrocarbon chain of the conjugates, i.e., in the hydrophobicity of the conjugates, the inhibition strengthened. Dixon-Webb plot analysis of the inhibition by the C10-conjugated dipeptide showed competitive inhibition. The trans-stimulation effect of Val-Lys conjugated to C10 or C5 on the uptake of Ceftibuten was observed using rat brush border membrane vesicles. This findings showed that these conjugates are transportable substrates. These results confirmed that the hydrophobicity of substrates/inhibitor is one of the factors in the recognition by PEPT1.     

Identification of binding proteins for cholesterol absorption inhibitors as components of the intestinal cholesterol transporter

To identify protein components of the intestinal cholesterol transporter, rabbit small intestinal brush border membrane vesicles were submitted to photoaffinity labeling using photoreactive derivatives of 2-azetidinone cholesterol absorption inhibitors. An integral membrane protein of M(r) 145.3+/-7.5 kDa was specifically labeled in brush border membrane vesicles from rabbit jejunum and ileum. Its labeling was concentration-dependently inhibited by the presence of cholesterol absorption inhibitors whereas bile acids, D-glucose, fatty acids or cephalexin had no effect. The inhibitory potency of 2-azetidinones to inhibit photolabeling of the 145 kDa protein correlated with their in vivo activity to inhibit intestinal cholesterol absorption. These results suggest that an integral membrane protein of M(r) 145 kDa is (a component of) the cholesterol absorption system in the brush border membrane of small intestinal enterocytes.     

The effect of peptide absorption on PepT1 gene expression and digestive system hormones in rainbow trout (Oncorhynchus mykiss)

The present study evaluates the effect of protein source (dipeptides, free amino acids, and intact protein) on development and growth of Salmonid fish alevin. Specifically, we follow the expression of oligopeptide transporter protein PepT1 in the intestine of rainbow trout (Oncorhynchus mykiss). Fish were fed exogenously one of four diets: three formulated (lysyl–glycine dipeptide supplemented diet — PP, free lysine and glycine supplemented diet — AA, control diet with no lysine — CON) or commercial starter (Aller Futura — AF). Fish increased mean body weight 8 fold with PP- and AA-supplemented diets resulting in significantly higher weight gain than fish fed CON. Statistical analysis revealed a significant increase in relative PepT1 expression of fish fed experimental diets. Immunohistochemical staining with PepT1 antibody showed the presence of the transporter protein in the brush border membrane of the proximal intestinal enterocytes of fish from all experimental groups. Leptin immunoreactivity occurred not only in the gastric glands but also in proximal intestine and pyloric caeca of fish fed PP, AA and AF diets. Leptin immunoreactivity was also observed in hepatocyte cytoplasm and pancreatic acinar cells. Gastrin/CCK immunoreactive cells were present in the proximal intestine and pyloric caeca.     

Isolation of brush border membranes in vesicular form from the intestinal spiral valve of the small dogfish (Scyliorhinus canicula).

A simple, rapid method for the preparation of purified brush border membranes in vesicular form from rabbit kidney proximal tubules has been applied with closely similar results to the intestinal spiral valve of the small dogfish (Scyliorhinus conicula). Since the dogfish belongs to one of the most ancient species of fish, it may be suggested that the method is generally applicable to all species later evolved which possess a brush border membrane at the mucosal surface of the cells of the intestine or kidney.     

Substrate-induced changes in the density of peptide transporter PEPT1 expressed in Xenopus oocytes

The adaptation of the capacity of the intestinal peptide transporter PEPT1 to varying substrate concentrations may be important with respect to its role in providing bulk quantities of amino acids for growth, development, and other nutritional needs. In the present study, we describe a novel phenomenon of the regulation of PEPT1 in the Xenopus oocyte system. Using electrophysiological and immunofluorescence methods, we demonstrate that a prolonged substrate exposure of rabbit PEPT1 (rPEPT1) caused a retrieval of transporters from the membrane. Capacitance as a measure of membrane surface area was increased in parallel with the increase in rPEPT1-mediated transport currents with a slope of approximately 5% of basal surface per 100 nA. Exposure of oocytes to the model peptide Gly-l-Gln for 2 h resulted in a decrease in maximal transport currents with no change of membrane capacitance. However, exposure to substrate for 5 h decreased transport currents but also, in parallel, surface area by endocytotic removal of transporter proteins from the surface. The reduction of the surface expression of rPEPT1 was confirmed by presteady-state current measurements and immunofluorescent labeling of rPEPT1. A similar simultaneous decrease of current and surface area was also observed when endocytosis was stimulated by the activation of PKC. Cytochalasin D inhibited all changes evoked by either dipeptide or PKC stimulation, whereas the PKC-selective inhibitor bisindolylmaleimide only affected PKC-stimulated endocytotic processes but not substrate-dependent retrieval of rPEPT1. Coexpression experiments with human Na(+)-glucose transporter 1 (hSGLT1) revealed that substrate exposure selectively affected PEPT1 but not the activity of hSGLT1.     

Isolation of brush border membranes in vesicular form from the intestinal spiral valve of the small dogfish (Scyliorhinus canicula)

A simple, rapid method for the preparation of purified brush border membranes in vesicular form from rabbit kidney proximal tubules has been applied with closely similar results to the intestinal spiral valve of the small dogfish (Scyliorhinus canicula). Since the dogfish belongs to one of the most ancient species of fish, it may be suggested that the method is generally applicable to all species later evolved which possess a brush border membrane at the mucosal surface of the cells of the intestine or kidney.     

The effect of epidermal growth factor on the distribution of SGLT-1 in rabbit jejunum

The effect of epidermal growth factor (EGF) on the cellular and villous distribution of the sugar transporter SGLT-1 was examined. New Zealand White rabbits (1 kg) were anesthetized, and two jejunal blind loops were isolated and exposed to either 0.9% saline or EGF (60 ng/mL saline), for 1 h. In separate experiments, tissue was harvested for brush border membrane vesicles (BBMV), microsomal membranes, or fixed for immunohistochemistry. SGLT-1 was measured in membrane fractions by Western immunoblot or localized along the villus-crypt axis by immunofluorescent microscopy. EGF increased BBMV SGLT-1 content compared with paired controls. EGF stimulation also induced a corresponding decrease in microsomal SGLT-1 levels and induced the expression of additional SGLT-1 immunoreactivity further down the villus axis. The findings suggest that EGF upregulates intestinal glucose transport by stimulating the translocation of SGLT-1 from an internal microsomal pool into the brush border, thereby recruiting more villus enterocytes into the glucose transporting population.     

The aging gut

A spectrum of changes occurs in the function of the gastrointestinal tract throughout the life of the animal. With aging, there is a decline towards newborn levels of the villus surface area and brush border membrane markers, but the absorption of some nutrients continues to increase (such as glucose and vitamin), whereas the absorption of some nutrients falls (such as cholesterol) and fatty acids). Clearly there is a distinction between age-related alterations in intestinal form and function, and some of the enzyme- or carrier-mediated changes are substrate specific. With aging, the structure and brush border membrane composition of the intestine tends to regress towards newborn levels. It remains to be clarified whether these changes are a benefit to the animal, whether they represent a degeneration of normal function, and whether these changes are the cause or the effect of various age-related alterations in metabolic and physiological function.     

Thiamine intestinal transport and related issues: recent aspects

In the intestinal lumen thiamine is in free form and very low concentrations. Absorption takes place primarily in the proximal part of the small intestine by means of a dual mechanism, which is saturable at low (physiological) concentrations and diffusive at higher. Thiamine undergoes intracellular phosphorylation mainly to thiamine pyrophosphate, while at the serosal side only free thiamine is present. Thiamine uptake is enhanced by thiamine deficiency, and reduced by thyroid hormone and diabetes. The entry of thiamine into the enterocyte, as evaluated in brush border membrane vesicles of rat small intestine in the absence of H+ gradient, is Na+- and biotransformation-independent, completely inhibited by thiamine analogs and reduced by ethanol administration and aging. The transport involves a saturable mechanism at low concentrations of vitamin and simple diffusion at higher. Outwardly oriented H+ gradients enhance thiamine transport, whose saturable component is a Na+-independent electroneutral uphill process utilizing energy supplied by the H+ gradient, and involving a thiamine/ H+ 1:1 stoichiometric exchange. The exit of thiamine from the enterocyte, as evaluated in basolateral membrane vesicles, is Na+-dependent, directly coupled to ATP hydrolysis by Na+-K+-ATPase, and inhibited by thiamine analogs. Transport of thiamine by renal brush border membrane vesicles is similar to the intestinal as far as both H+ gradient influence and specificity are concerned. In the erythrocyte thiamine transport is a Na+-independent, electroneutral process yet with two components: saturable, prevailing at low thiamine concentrations, and diffusive at higher. The saturable (specific) component is missing in patients of the rare disease known as thiamine-responsive megaloblastic anaemia (TRMA), producing a general disturbance of thiamine transport up to thiamine deficiency. The TRMA gene is located in chromosome 1q23.3. Recently, the thiamine transporter has been cloned: it is a protein of 497 amino acid residues with high homology with the reduced-folate transporter.     

Increased protein level of PEPT1 intestinal H+-peptide cotransporter upregulates absorption of glycylsarcosine and ceftibuten in 5/6 nephrectomized rats

In chronic renal failure (CRF), dietary protein is one of the factors that deteriorates residual renal functions. Numerous studies have indicated that the products of protein digestion are mainly absorbed as small peptides. However, how small peptides are absorbed in CRF remains poorly understood. H(+)-coupled peptide transporter (PEPT1/SLC15A1) plays an important role in the absorption of small peptides and peptide-like drugs in the small intestine. Because dietary protein intake is one of the risk factors for renal failure, the alteration of intestinal PEPT1 might have implications in the progression of renal disease as well as the pharmacokinetics of peptide-like drugs. In this study, we examined the alteration of intestinal PEPT1 in 5/6 nephrectomized (5/6 NR) rats, extensively used as a model of chronic renal failure. Absorption of [(14)C]glycylsarcosine and ceftibuten was significantly increased in 5/6 NR rats compared with sham-operated rats, without a change in intestinal protease activity. Western blot analysis indicated that the amount of intestinal PEPT1 protein in 5/6 NR rats was increased mainly at the upper region. On the other hand, the amount of intestinal PEPT1 mRNA was not significantly different from that of sham-operated rats. These findings indicate that the increase in absorption of small peptides and peptide-like drugs, caused by the upregulation of intestinal PEPT1 protein, might contribute to the progression of renal failure as well as the alteration of drug pharmacokinetics.     

Topological studies on the hydrolases bound to the intestinal brush border membrane. I. Solubilization by papain and triton X-100

Papain digestion of closed, right side out vesicles from pig, rat and rabbit jejunum brush border induces the release of the hydrolases bound to the membrane without grossly affecting the lipid bilayer limiting the vesicles. This observation definitely proves that intestinal hydrolases are surface components attached to the external side of the membrane. All proteins released by papain could be identified by electrophoresis and immunoelectrophoresis to already known intestinal hydrolases, with the exception of an unidentified substance strongly stained by the Schiff's reagent.The early observation that the aminopeptidase form released from pig brush border by Triton X-100 is different from that released by papain was extended to other hydrolases from pig, rat and rabbit. In some cases, the Triton-released form could be converted by further proteolytic digestion into a new form similar to that liberated by papain. These facts may be related to the existence of hydrophobic anchors retaining the intestinal hydrolases to the membrane surface.     

Multiple plasma membrane receptors but not NPC1L1 mediate high-affinity, ezetimibe-sensitive cholesterol uptake into the intestinal brush border membrane

We compared cholesterol uptake into brush border membrane vesicles (BBMV) made from the small intestines of either wild-type or Niemann-Pick C1-like 1 (NPC1L1) knockout mice to elucidate the contribution of NPC1L1 to facilitated uptake; this uptake involves cholesterol transport from lipid donor particles into the BBM of enterocytes. The lack of NPC1L1 in the BBM of the knockout mice had no effect on the rate of cholesterol uptake. It follows that NPC1L1 cannot be the putative high-affinity, ezetimibe-sensitive cholesterol transporter in the brush border membrane (BBM) as has been proposed by others. The following findings substantiate this conclusion: (I) NPC1L1 is not a brush border membrane protein but very likely localized to intracellular membranes; (II) the cholesterol absorption inhibitor ezetimibe and its analogues reduce cholesterol uptake to the same extent in wild-type and NPC1L1 knockout mouse BBMV. These findings indicate that the prevailing belief that NPC1L1 facilitates intestinal cholesterol uptake into the BBM and its interaction with ezetimibe is responsible for the inhibition of this process can no longer be sustained.     

Subcellular localization of enterokinase in human small intestine

Enterokinase (enteropeptidase, EC 3.4.4.8) was found to be purified to the same extent as sucrase and alkaline phophatase when human intestinal brush border membrane was isolated. It is concluded that, in man as in other mammals, enterokinase activity occurs in close association with the brush border membrane.However, a second localization was also found. A fraction of the mucosal homogenate containing only small amounts of brush border but large amounts of endoplasmic reticulum, basolateral membranes and mitochondria (Fraction P₁) contained a disproportionately high amount of enterokinase. The enzyme in this particulate fraction occurred in a not fully active form.