Two microsomal-associated iron-binding proteins observed in rat small intestinal cells during iron absorption

Acrylamide gel electrophoresis of microsomal protein obtained from rat small intestinal mucosal cells, after an injection of [³H]leucine, demonstrated increased quantities of two soluble iron-binding proteins during iron absorption, one with a high molecular weight (about 400 000) and the other of intermediate molecular weight (80 000). Both proteins were present in a ribosomal-enriched sub-fraction obtained during purification of the microsomal membrame but were not identified among the purified membrane proteins.     

Olfactory ferric and ferrous iron absorption in iron-deficient rats

The absorption of metals from the nasal cavity to the blood and the brain initiates an important route of occupational exposures leading to health risks. Divalent metal transporter-1 (DMT1) plays a significant role in the absorption of intranasally instilled manganese, but whether iron uptake would be mediated by the same pathway is unknown. In iron-deficient rats, blood (59)Fe levels after intranasal administration of the radioisotope in the ferrous form were significantly higher than those observed for iron-sufficient control rats. Similar results were obtained when ferric iron was instilled intranasally, and blood levels of (59)Fe were even greater in the iron-deficient rats compared with the amount of ferrous iron absorbed. Experiments with Belgrade (b/b) rats showed that DMT1 deficiency limited ferric iron uptake from the nasal cavity to the blood compared with +/b controls matched for iron deficiency. These results indicate that olfactory uptake of ferric iron by iron-deficient rats involves DMT1. Western blot experiments confirmed that DMT1 levels are significantly higher in iron-deficient rats compared with iron-sufficient controls in olfactory tissue. Thus the molecular mechanism of olfactory iron absorption is regulated by body iron status and involves DMT1.     

Effect of orally-administered epidermal growth factor on intestinal iron absorption and mucosal permeability

A progressive increase in intestinal 59Fe3+ absorption was observed on oral feeding of mice with physiological doses of EGF/UGO. Maximal changes were apparent after 3d and appeared to be dose-dependent. In addition to a small increase in intestinal cell proliferation, as reflected by increased ornithine decarboxylase activity, EGF/UGO-feeding increased mucosal permeability (evaluated with [51Cr]-EDTA): the latter could account for the increase in iron absorption. Sialoadenectomy, to remove the major source of endogenous EGF/UGO, had no appreciable effect on the intestinal absorption of iron.     

Effect of zinc depletion/repletion on intestinal iron absorption and iron status in rats

Iron and zinc deficiencies likely coexist in general population. We have previously demonstrated that zinc treatment induces while zinc deficiency inhibits iron absorption in intestinal cell culture models, but this needs to be tested in vivo. In the present study we assessed intestinal iron absorption, iron status (haemoglobin), red blood cell number, plasma ferritin, transferrin receptor, hepcidin) and tissue iron levels in zinc depleted, replete and pair fed control rats. Zinc depletion led to reduction in body weight, tissue zinc levels, intestinal iron absorption, protein and mRNA expression of iron transporters, the divalent metal ion transporter-1, hephaestin and ferroportin, but elevated the intestinal and liver tissue iron levels compared with the pair fed control rats. Zinc repletion led to a significant weight gain compared to zinc deficient rats and normalized the iron absorption, iron transporter expression, tissue iron levels to that of pair fed control rats. Surprisingly, haemoglobin levels and red blood cell number reduced significantly in zinc repleted rats, which could be due to rapid weight gain. Together, these results indicate that whole body zinc status has profound influence on growth, intestinal absorption and systemic utilization of iron, mediated via modulation of iron transporter expression.     

Activation of rat intestinal mucosal mast cells by fat absorption

Previous studies have linked certain types of gut mucosal immune cells with fat intake. We determined whether fat absorption activates intestinal mucosal mast cells (MMC), a key component of the gut mucosal immune system. Conscious intestinal lymph fistula rats were used. The mesenteric lymph ducts were cannulated, and the intraduodenal (i.d.) tubes were installed for the infusion of Liposyn II 20% (an intralipid emulsion). Lymphatic concentrations of histamine, rat MMC protease II (RMCPII), a specific marker of rat intestinal MMC degranulation, and prostaglandin D(2) (PGD(2)) were measured by ELISA. Intestinal MMC degranulation was visualized by immunofluorescent microscopy of jejunum sections taken at 1 h after Liposyn II gavage. Intraduodenal bolus infusion of Liposyn II 20% (4.4 kcal/3 ml) induced approximately a onefold increase in lymphatic histamine and PGD(2), ～20-fold increase in lymphatic RMCPII, but only onefold increase in peripheral serum RMCPII concentrations. Release of RMCPII into lymph increased dose dependently with the amount of lipid fed. In addition, i.d. infusion of long-chain triacylglycerol trilinolein (C18:2 n-6, the major composite in Liposyn II) significantly increased the lymphatic RMCPII concentration, whereas medium-chain triacylglycerol tricaprylin (C8:0) did not alter lymph RMCPII secretion. Immunohistochemistry image revealed the degranulation of MMC into lamina propria after lipid feeding. These novel findings indicate that intestinal MMC are activated and degranulate to release MMC mediators to the circulation during fat absorption. This action of fatty acid is dose and chain length dependent.     

Intestinal iron absorption and mucosal transferrin in rats subjected to hypoxia

Three days hypoxia (0.5 atm) increased the haemoglobin and haematocrit values in rats paralleled by enhanced intestinal iron absorption. The destination of recently-absorbed iron was primarily the erythropoietic system, viz. bone marrow, spleen and red cells. Total plasma transferrin, was increased by 30%, but no significant changes in mucosal transferrin were found. No increase in labelling of mucosal transferrin by absorbed iron was observed. These results suggest that mucosal transferrin does not play a major role in the regulation of intestinal iron absorption in hypoxia.     

Mg2+-ATP-dependent sodium transport in inside-out basolateral plasma membrane vesicles from guinea-pig small intestinal epithelial cells

The transport of sodium into inside-out basolateral plasma membrane vesicles from small intestinal epithelial cells has been examined. It was found, under equilibrium conditions, that binding of 22Na represents approx. 55% of the total uptake during an equilibration period of 30 min; 45% of the total uptake correspond to passive sodium entry in the vesicle space. In addition to binding and to passive Na+ entry, two distinct mechanisms capable of accumulating sodium in the intravesicular space can be demonstrated when ATP is added to the incubation medium. One transports sodium actively in the absence of potassium, whereas the other requires the presence of potassium in the interior of the vesicles. The two mechanisms can also be differentiated by their affinities for sodium, their optimal pH and by their behaviour towards different inhibitors. Thus, the mechanism that transports sodium in the absence of potassium is refractory to ouabain, but is inhibited by ethacrynic acid and furosemide, whilst the mechanism that accumulates sodium inside the vesicles in the presence of internal potassium is strongly inhibited by ouabain, is weakly inhibited by ethacrynic acid and is insensitive of furosemide. ATP is a specific stimulator of both processes, and the requirement for magnesium is absolute in both cases.     

Distribution and metabolism of iron in muscles of iron-deficient rats

Iron-deficiency anemia leads directly to both reduced hemoglobin levels and work performance in humans and experimental animals. In an attempt to observe a direct link between work performance and insufficient iron at the cellular level, we produced severe iron deficiency in female weanling Sprague-Dawley rats following five weeks on a low-iron diet. Deficient rats were compared with normal animals to observe major changes in hematological parameters, body weight, and growth of certain organs and tissues. The overall growth of iron-deficient animals was approximately 50% of normal. The ratio of organ weight: body weight increased in heart, liver, spleen, kidney, brain, and soleus muscle in response to iron deficiency. Further, mitochondria from heart and red muscle retained their iron more effectively under the stress of iron deficiency than mitochondria from liver and spleen. Metabolism of iron in normal and depleted tissue was measured using tracer amounts of⁵⁹Fe administered orally. As expected, there was greater uptake of tracer iron by iron-deficient animals. The major organ of iron accumulation was the spleen, but significant amounts of isotope were also localized in heart and brain. In all muscle tissue examined the⁵⁹Fe preferentially entered the mitochondria. Enhanced mitochondrial uptake of iron prior to any detectable change in the hemoglobin level in experimental animals may be indicative of nonhemoglobin related biochemical changes and/or decrements in work capacity.     

Effect of massive proximal small bowel resection on intestinal brush border membrane proteins in the dog.

Dog enterocyte brush border proteins have been studied after a 75% proximal resection of the small bowel. This study was carried on microvillar membrane preparations purified from ileal mucosa sampled before and after regeneration on neighbouring intestinal segments, each animal acting as its own control. After six weeks of regeneration a statistically significant decrease of the following enzyme specific activities was observed: lactase, cellobiase, maltase, sucrase, palatinase, dextranase, trehalase, alkaline phosphatase, aminopeptidase and gamma-glutamyl transferase. Analysis of brush border proteins by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate have shown after regeneration a decreased rate for the proteins with a molecular weight higher than 100,000 daltons. Modifications of electrophoretic patterns seem to be related to the specific activity decreases observed for brush border enzymes after regeneration, since the molecular weight of these enzymes were found between 116,000 and 285,000 daltons, after gel filtration.     

Identification of signals and mechanisms of sorting of plasma membrane proteins in intestinal epithelial cells]

In epithelial cells the plasma membrane is divided into domains that are biochemically and functionally different. In intestinal cells for example the apical domain is facing the intestinal lumen and is involved in the uptake of nutriments while the basolateral domain is mediating cell-cell adhesion and signalisation. We are interested in deciphering the mechanisms underlying the creation and maintenance of such specialized domains. As an epithelial model we have used the intestinal cell line Caco-2 and we have studied the transport and sorting of the human neurotrophin receptor (p75 NTR) in these cells. Newly synthesized p75 NTR is first transported to the basolateral membrane and then is accumulated on the apical membrane after transcytosis. This final apical localization is controlled by the presence of a membrane anchor and a cluster of O-glycosylation sites located in the part of the ectodomain close to the membrane. Among the mechanisms likely to be involved in the sorting of apical components we have looked for a role of lipid-protein microdomain formation in the Golgi apparatus. These membrane microdomains are highly enriched in glycosylphosphatidyl inositol (GPI) anchored proteins, glycosphingolipids and apical proteins such as sucrase isomaltase (SI). Such a composition is also found for endocytic structures called caveolae which are made of caveolin 1. We have expressed caveolin 1 in Caco-2 cells which do not express it and also caveolin 2, a related protein of unknown function. Expression of caveolin 1 led to formation of caveolae indicating that this protein is necessary for caveolae formation while caveolin 2 is restricted to the Golgi apparatus and has no effect on caveolae formation. However Caveolin 2 increased the amount of SI incorporated in microdomains suggesting a role in recruitment into the apical pathway. The choice for a site of fusion for transport vesicles is the last step of control during exocytosis. To identify proteins involved in that step we have cloned and characterized two members of the t-SNARE family, namely syntaxin 3 and SNAP23. Syntaxin 3 is present on the apical membrane and forms a complex with SNAP23 which is also localized on the basolateral membrane where it forms a complex with syntaxin 4. Overexpression of syntaxin 3 in Caco-2 led to a decrease of SI exocytosis towards the apical membrane confirming that syntaxin 3 is involved in targeting the fusion of apical transport vesicles to the apical pole of the cells.     

Lipid peroxidation of rabbit small intestinal microvillus membrane vesicles by iron complexes

Fe(II)- and Fe(III)-induced lipid peroxidation of rabbit small intestinal microvillus membrane vesicles was studied. Ferrous ammonium sulphate, ferrous ascorbate at a molar ratio of 10:1, and ferric citrate, at molar ratios of 1:1 and 1:20, did not stimulate lipid peroxidation. Ferrous ascorbate, 1:1, induced low stimulation, while ferrous ascorbate, 1:20 gave higher stimulation of lipid peroxidation. These results show that in our experimental system, ascorbate is a promotor rather than an inhibitor of lipid peroxidation. Ferric nitrilotriacetate (at molar ratios of 1:2 and 1:10), at an iron concentration of 200 microM, was by far the most effective in inducing lipid peroxidation. Superoxide dismutase, mannitol and glutathione had no effect, while catalase, thiourea and vitamin E markedly decreased ferrous ascorbate 1:20-induced lipid peroxidation. Ferric nitrilotriacetate-induced lipid peroxidation was slightly reduced by catalase and mannitol, significantly reduced by superoxide dismutase, and completely inhibited by thiourea. Glutathione caused a 100% increase in the ferric nitrilotriacetate-induced lipid peroxidation. These results suggest that Fe(II) in the presence of trace amounts of Fe(III), or an oxidizing agent and Fe(III) in the presence of Fe(II) or a reducing agent, are potent stimulators of lipid peroxidation of microvillus membrane vesicles. Addition of deferoxamine completely inhibited both ferrous ascorbate, 1:20 and ferric nitrilotriacetate-induced lipid peroxidation, demonstrating the requirement for iron for its stimulation. Iron-induced peroxidation of microvillus membrane may have physiological significance because it could already be demonstrated at 2 microM iron concentration.     

Molecular mechanism of intestinal iron absorption

Iron is an essential trace metal in the human diet because of its role in a number of metabolic processes including oxygen transport. In the diet, iron is present in two fundamental forms, heme and non-heme iron. This article presents a brief overview of the molecular mechanisms of intestinal iron absorption and its regulation. While many proteins that orchestrate iron transport pathway have been identified, a number of key factors that control the regulation of iron absorption still remain to be elucidated. This review also summarizes new and emerging information about iron metabolic regulators that coordinate regulation of intestinal iron absorption.     

Systemic regulation of intestinal iron absorption

The intestinal absorption of the essential trace element iron and its mobilization from storage sites in the body are controlled by systemic signals that reflect tissue iron requirements. Recent advances have indicated that the liver-derived peptide hepcidin plays a central role in this process by repressing iron release from intestinal enterocytes, macrophages and other body cells. When iron requirements are increased, hepcidin levels decline and more iron enters the plasma. It has been proposed that the level of circulating diferric transferrin, which reflects tissue iron levels, acts as a signal to alter hepcidin expression. In the liver, the proteins HFE, transferrin receptor 2 and hemojuvelin may be involved in mediating this signal as disruption of each of these molecules decreases hepcidin expression. Patients carrying mutations in these molecules or in hepcidin itself develop systemic iron loading (or hemochromatosis) due to their inability to down regulate iron absorption. Hepcidin is also responsible for the decreased plasma iron or hypoferremia that accompanies inflammation and various chronic diseases as its expression is stimulated by pro-inflammatory cytokines such as interleukin 6. The mechanisms underlying the regulation of hepcidin expression and how it acts on cells to control iron release are key areas of ongoing research.     

Sorting of endogenous plasma membrane proteins occurs from two sites in cultured human intestinal epithelial cells (Caco-2)

We studied the postsynthetic sorting of endogenous plasma membrane proteins in a polarized epithelial cell line, Caco-2. Pulse-chase radiolabeling was combined with domain-specific cell surface assays to monitor the arrival of three apical and one basolateral protein at the apical and basolateral cell surface. Apical proteins were inserted simultaneously into both membrane domains. The fraction targeted to the basolateral domain was different for the three apical proteins and was subsequently sorted to the apical domain by transcytosis at different rates. In contrast, a basolateral protein was found in the basolateral membrane only. Thus, sorting of plasma membrane proteins occurred from two sites: the Golgi apparatus and the basolateral membrane. These data explain apparently conflicting results of earlier studies.     

Ca++-transport across basal-lateral plasma membranes from rat small intestinal epithelial cells

Summary Basal-lateral plasma membrane vesicles were isolated from rat duodenum and jejunum by a Percoll gradient centrifugation technique. Ca-uptake into and Ca-release from the vesicles was studied by a rapid filtration method. In the absence of Na (K-medium) at a Ca concentration of 0.05 mmol/liter and pH 7.4, addition of 5mm MgATP stimulated Ca-uptake up to 10-fold as compared to a control without ATP. Since the Ca-ionophore A23187 (2 g/ml) prevented the accumulation of Ca above the equilibrium uptake and rapidly released Ca accumulated by the vesicles in the presence of ATP, it is concluded that the ATP-dependent uptake of Ca involves accumulation of Ca inside the vesicles. The ATP-driven Ca-transport comigrates with the (Na+K)-ATPase and dissociates from the marker enzymes for mitochondrial inner membrane, endoplasmic reticulum and brush border membrane. It is not inhibited by 1 g/ml oligomicin or 0.1 mmol/liter ruthenium red. Replacing K by Na inhibits ATP-dependent Ca-uptake by 60%. Efflux of Ca from passively preloaded vesicles is strongly temperature sensitive and enhanced by A23187. An inwardly directed Na-gradient stimulates Ca-efflux as compared to a K-gradient. Addition of gramicidin reduces the Na-stimulation of Ca-efflux, indicating direct coupling of Na and Ca fluxes across basal-lateral membranes. The results suggest that basal-lateral membranes possess two distinct mechanisms for Ca-transport:a) ATP-driven Ca-transport andb) Na/Ca-exchange.