Heme oxygenase 1 overexpression increases iron fluxes in caco-2 cells

Heme oxygenase-1 is a microsomal enzyme that, when induced by stress, protects the cells from oxidative injury. Heme oxygenase-1 participates in the cleavage of the heme ring producing biliverdin, CO and ferrous Fe. The released Fe becomes part of intracellular Fe pool and can be stored in ferritin or released by an iron exporter. The mechanism by which heme enters cells is not completely understood, although it had been suggested that it might be internalized by an endocytosis process. In this study, we expressed a full-length Heme oxygenase-1 cDNA in Caco-2 cells and measured intracellular iron content, heme-iron uptake and transport and immunolocalization of heme oxygenase-1 in these cells. We found that heme oxygenasc-1 expressing cells showed increased apical heme iron uptake and transepithelial transport when compared to control cells. These results suggested that heme oxygenase-1 mediates heme iron influx and efflux in intestinal cells.     

Role of membrane surface potential and other factors in the uptake of non-transferrin-bound iron by reticulocytes

Reticulocytes suspended in low ionic strength media such as isotonic sucrose solution efficiently take up non-transferrin-bound iron and utilize it for heme synthesis. The present study was undertaken to determine how such media facilitate iron utilization by the cells. The effects of changes in membrane surface potential, membrane permeability, cell size, transmembrane potential difference, oxidation state of the iron, and lipid peroxidation were investigated. Iron uptake to heme, cytosol, and stromal fractions of cells was measured using rabbit reticulo-cytes incubated with ⁵⁹Fe-labelled Fe(II) in 0.27 M sucrose, pH 6.5. Suspension of the cells in sucrose led to increased membrane permeability, loss of intracellular K⁺, decreased cell size, and increased transmembrane potential difference. However, none of these changes could account for the high efficiency of iron uptake which was observed. The large negative membrane surface potential which occurs in sucrose was modified by the addition of mono-, di-, tri-, and polyvalent cations to the solution. This inhibited iron uptake to a degree which for many cations varied with their valency. Other cations (Mn²⁺, Co²⁺, Ni²⁺, Zn²⁺) were also very potent inhibitors, probably due to direct action on the transport process. Ferricyanide inhibited iron uptake, while ferrocyanide and ascorbate increased the uptake of Fe(III) but not Fe(II). It is concluded that the high negative surface potential of reticulocytes suspended in sucrose solution facilitates iron uptake by aiding the approach of iron to the transport site on the cell membrane. The iron is probably transported into the cell in the ferrous form. © 1994 wiley-Liss, Inc.     

Iron transport mechanisms in reticulocytes and mature erythrocytes

The mechanism of iron transport into erythroid cells was investigated using rabbit reticulocytes and mature erythrocytes incubated with ⁵⁹Fe-labelled Fe(II) in isotonic sucrose or in solutions in which the sucrose was replaced with varying amounts of isotonic NaCl or KCl. Iron uptake was inhibited at all concentrations of NaCl, in a concentration-dependent manner, but with KCl inhibition occurred only at concentrations up to 10 mM. Higher KCl concentrations stimulated iron uptake to the cytosol of the cells, but inhibited its incorporation into heme. This effect became more marked as the iron concentration was raised. It was found that KCl inhibits iron incorporation into heme and stimulates iron uptake by mature erythrocytes, as well as by reticulocytes. It is concluded that erythroid cells can take up nontransferrin-bound Fe(II) by two mechanisms. One is a high-affinity mechanism that is limited to reticulocytes, saturates at a low iron concentration, and is inhibited by metabolic inhibitors. The other is a low-affinity process that is found in both reticulocytes and erythrocytes, becomes more prominent at higher iron concentrations, and is stimulated by KCl, as well as RbCl, LiCl, CsCl, and choline Cl. The KCl stimulation is inhibited by amiloride, but not by metabolic inhibitors, and its operation is not dependent on changes in cell volume or membrane potential, but it does require the presence of a permeant extracellular anion. Iron uptake by this process appears to occur by facilitated transport and is possibly assoicated with exchange of Na⁺. A further aspect of this study was a comparison of iron uptake by reticulocytes from Fe(II)-sucrose and Fe(II)-ascorbate using a variety of incubation conditions. No major differences were observed. © 1995 Wiley-Liss, Inc.     

Mechanisms of neuroprotection by hemopexin: modeling the control of heme and iron homeostasis in brain neurons in inflammatory states

Hemopexin provides neuroprotection in mouse models of stroke and intracerebral hemorrhage and protects neurons in vitro against heme or reactive oxygen species (ROS) toxicity via heme oxygenase‐1 (HO1) activity. To model human brain neurons experiencing hemorrhages and inflammation, we used human neuroblastoma cells, heme–hemopexin complexes, and physiologically relevant ROS, for example, H₂O₂ and HOCl, to provide novel insights into the underlying mechanism whereby hemopexin safely maintains heme and iron homeostasis. Human amyloid precursor protein (hAPP), needed for iron export from neurons, is induced ~twofold after heme–hemopexin endocytosis by iron from heme catabolism via the iron‐regulatory element of hAPP mRNA. Heme–hemopexin is relatively resistant to damage by ROS and retains its ability to induce the cytoprotective HO1 after exposure to tert‐butylhydroperoxide, although induction is impaired, but not eliminated, by exposure to high concentrations of H₂O₂ in vitro. Apo‐hemopexin, which predominates in non‐hemolytic states, resists damage by H₂O₂ and HOCl, except for the highest concentrations likely in vivo. Heme–albumin and albumin are preferential targets for ROS; thus, albumin protects hemopexin in biological fluids like CSF and plasma where it is abundant. These observations provide strong evidence that hemopexin will be neuroprotective after traumatic brain injury, with heme release in the CNS, and during the ensuing inflammation. Hemopexin sequesters heme, thus preventing unregulated heme uptake that leads to toxicity; it safely delivers heme to neuronal cells; and it activates the induction of proteins including HO1 and hAPP that keep heme and iron at safe levels in neurons.     

Non-transferrin-bound iron uptake in Belgrade and normal rat erythroid cells

Belgrade (b) rats have an autosomal recessive, microcytic, hypochromic anemia. Transferrin (Tf)-dependent iron uptake is defective because of a mutation in DMT1 (Nramp2), blocking endosomal iron efflux. This experiment of nature permits the present study to address whether the mutation also affects non-Tf-bound iron (NTBI) uptake and to use NTBI uptake compared to Tf-Fe utilization to increase understanding of the phenotype of the b mutation. The distribution of ⁵⁹Fe²⁺ into intact erythroid cells and cytosolic, stromal, heme, and nonheme fractions was different after NTBI uptake vs. Tf-Fe uptake, with the former exhibiting less iron into heme but more into stromal and nonheme fractions. Both reticulocytes and erythrocytes exhibit NTBI uptake. Only reticulocytes had heme incorporation after NTBI uptake. Properly normalized, incorporation into b/b heme was ∼20% of +/b, a decrease similar to that for Tf-Fe utilization. NTBI uptake into heme was inhibited by bafilomycin A1, concanamycin, NH₄Cl, or chloroquine, consistent with the endosomal location of the transporter; cellular uptake was uninhibited. NTBI uptake was unaffected after removal of Tf receptors by Pronase or depletion of endogenous Tf. Concentration dependence revealed that NTBI uptake into cells, cytosol, stroma, and the nonheme fraction had an apparent low affinity for iron; heme incorporation behaved like a high-affinity process, as did an expression assay for DMT1. DMT1 serves in both apparent high-affinity NTBI membrane transport and the exit of iron from the endosome during Tf delivery of iron in rat reticulocytes; the low-affinity membrane transporter, however, exhibits little dependence on DMT1. J. Cell. Physiol. 178:349–358, 1999. © 1999 Wiley-Liss, Inc.     

Iron utilization in rabbit reticulocytes: A study using succinylacetone as an inhibitor of heme synthesis

We have investigated the effect of succinylacetone (4,6-dioxoheptanoic acid) on hemoglobin synthesis and iron metabolism in reticulocytes. Succinylacetone, 0.1 and 1 mM, inhibited [2-¹⁴C]glycine incorporation into heme by 91.2 and 96.4%, respectively, and into globin by 85 and 90.2%, respectively. 60 μM hemin completely prevented the inhibition of globin synthesis by succinylacetone, indicating that succinylacetone inhibits specifically the synthesis of heme. Added porphobilinogen, but not δ-aminolevulinic acid, partly overcame the inhibition of ⁵⁹Fe incorporation into heme caused by succinylacetone suggesting that the drug inhibits δ-aminolevulinic acid dehydratase in reticulocytes. Succinylacetone, 10 μM, 0.1 and 1 mM, inhibited ⁵⁹Fe incorporation into heme by 50, 90 and 93%, respectively, but stimulated reticulocyte ⁵⁹Fe uptake by about 25–30%. In succinylacetone-treated cells ⁵⁹Fe accumulates in a fraction containing plasma membranes and mitochondria as well as cytosol ferritin and an unidentified low molecular weight fraction obtained by Sephacryl S-200 chromatography. Reincubation of washed succinylacetone- and ⁵⁹Fe-transferrin-pretreated reticulocytes results in the transfer of ⁵⁹Fe from the particulate fraction (plasma membrane plus mitochondria) into hemoglobin and this process is considerably stimulated by added protoporphyrin. Although the nature of the iron accumulated in the membrane-mitochondria fraction in succinylacetone-treated cells is unknown some of it is utilizable for hemoglobin synthesis, while cytosolic ferritin iron would appear to be mostly unavailable for incorporation into heme.     

The gills as an important uptake route for the essential nutrient iron in freshwater rainbow trout Oncorhynchus mykiss

Short-term (3h) acquisition of iron (16 nmol ⁵⁹FeCl₃ l⁻¹) from oxic, alkaline fresh water was assessed in rainbow trout Oncorhynchus mykiss in the presence or absence of a range of iron chelators, all of which had differing binding affinities for ferric iron [100 μmol l⁻¹ of desferrioxamine (DFO), Log₁₀K₁ 32·5; citric acid Log₁₀K₁ 11·9; nitrilotriacetic acid (NTA) Log₁₀K₁ 15·9, CP20 and CP94 (Log₁₀K₁ > 30), as well as humic acid (HA), Log₁₀K₁ 5·04, 5 mg l⁻¹]. In the absence of chelators (control conditions) O. mykiss acquired iron from the water under laboratory lights (wavelength range of the lights 440–650 nm, peak intensity 548–626 nm) via the gill. In these conditions iron uptake onto the gill had a maximum transport capacity (J_max) of 11·2 pmol Fe g⁻¹ h⁻¹ (gill organ mass) and a K_m of 21·3 nmol Fe l⁻¹ h⁻¹. Furthermore, there were two components to iron accumulation into the carcass of these fish, a slow rate of aqueous iron uptake at low concentrations (6–24 nmol Fe l⁻¹), followed by a faster rate of uptake at higher iron concentrations (48–96 nmol Fe l⁻¹), suggesting that the rate-limiting step of iron uptake at low iron concentrations is the apical entry step. O. mykiss also acquired iron in the presence of HA, although the majority of the other chelators prevented iron uptake. Ultraviolet light (354 nm) treatment of Fe-DFO increased iron bioavailability. Results suggest that rainbow trout are able to access either the predicted very low concentrations (picomolar) of ferrous iron present in fresh water or the ferric oxide complexes present in oxic environments. The iron uptake rate measured (0·75 pmol g⁻¹ h⁻¹) would be sufficient to provide a substantial proportion (c. 85%) of the daily iron requirements of growing salmonid fry.     

Role of clathrin‐mediated endocytosis in the use of heme and hemoglobin by the fungal pathogen Cryptococcus neoformans

Heme is a major source of iron for pathogens of humans, and its use is critical in determining the outcome of infection and disease. Cryptococcus neoformans is an encapsulated fungal pathogen that causes life‐threatening infections in immunocompromised individuals. C. neoformans effectively uses heme as an iron source, but the underlying mechanisms are poorly defined. Non‐iron metalloporphyrins (MPPs) are toxic analogues of heme and are thought to enter microbial cells via endogenous heme acquisition systems. We therefore carried out a mutant screen for susceptibility against manganese MPP (MnMPP) to identify new components for heme uptake in C. neoformans. We identified several genes involved in signalling, DNA repair, sugar metabolism, and trafficking that play important roles in susceptibility to MnMPP and in the use of heme as an iron source. We focused on investigating the role of clathrin‐mediated endocytosis (CME) and found that several components of CME including Chc1, Las17, Rvs161, and Rvs167 are required for growth on heme and hemoglobin and for endocytosis and intracellular trafficking of these molecules. We show that the hemoglobin uptake process in C. neoformans involves clathrin heavy chain, Chc1, which appears to colocalise with hemoglobin‐containing vesicles and to potentially assist in proper delivery of hemoglobin to the vacuole. Additionally, C. neoformans strains lacking Chc1, Las17, Rvs161, or Rvs167 were defective in the elaboration of several key virulence factors, and a las17 mutant was avirulent in a mouse model of cryptococcosis. Overall, this study unveils crucial functions of CME in the use of heme iron by C. neoformans and reveals a role for CME in fungal pathogenesis.     

Iron-uptake in the Euryarchaeon Halobacterium salinarum

Iron-uptake is well studied in a plethora of pro- and eukaryotic organisms with the exception of Archaea, which thrive mainly in extreme environments. In this study, the mechanism of iron transport in the extremely halophilic Euryarchaeon Halobacterium salinarum strain JW 5 was analyzed. Under low-iron growth conditions no siderophores were detectable in culture supernatants. However, various xenosiderophores support growth of H. salinarum. In [⁵⁵Fe]–[¹⁴C] double-label experiments, H. salinarum displays uptake of iron but not of the chelator citrate. Uptake of iron was inhibited by cyanide and at higher concentrations by Ga. Furthermore, a K_M for iron uptake in cells of 2.36 μM and a V_max of approximately 67 pmol Fe/min/mg protein was determined. [⁵⁵Fe]-uptake kinetics were measured in the absence and presence of Ga. Uptake of iron was inhibited merely at very high Ga concentrations. The results indicate an energy dependent iron uptake process in H. salinarum and suggest reduction of the metal at the membrane level.     

Heme and non-heme iron transporters in non-polarized and polarized cells

Background  

Heme and non-heme iron from diet, and recycled iron from hemoglobin are important products of the synthesis of iron-containing molecules. In excess, iron is potentially toxic because it can produce reactive oxygen species through the Fenton reaction. Humans can absorb, transport, store, and recycle iron without an excretory system to remove excess iron. Two candidate heme transporters and two iron transporters have been reported thus far. Heme incorporated into cells is degraded by heme oxygenases (HOs), and the iron product is reutilized by the body. To specify the processes of heme uptake and degradation, and the reutilization of iron, we determined the subcellular localizations of these transporters and HOs.     

Transferrin receptors,iron transport and ferritin metabolism in friend erythroleukemia cells

Four aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of erythroid differentiation by dimethyl sulfoxide. (1) The binding of ¹²⁵I-labeled transferrin was determined over a range of transferrin concentrations from 0.5 to 15 μM. Scatchard analysis of the binding curves demonstrated equivalent numbers of transferrin binding sites per cell: 7.78 ± 2.41 · 10⁵ in non-induced cells and 9.28 ± 1.57 · 10⁵ after 4 days of exposure to dimethyl sulfoxide. (2) The rate of iron transport was determined by measuring iron uptake from ⁵⁹Fe-labeled transferrin. Iron uptake in non-induced cells was approx. 17 000 molecules of iron/cell per min; 24 h after addition of dimethyl sulfoxide it increased to 38 000, and it rose to maximal levels of approx. 130 000 at 72 h. (3) Heme synthesis, assayed qualitatively by benzidine staining and measured quantitatively by incorporation of ⁵⁹Fe or [2-¹⁴C]glycine into cyclohexanone-extracted or crystallized heme, was not detected until 3 days after addition of dimethyl sulfoxide, when 12% of the cells were stained by benzidine and 6 pmol ⁵⁹Fe and 32 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. After 4 days, 60% of the cells were benzidine positive and 34 pmol ⁵⁹Fe and 90 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. (4) The rate of incorporation of ⁵⁹Fe into ferritin, measured by immunoprecipitation of ferritin by specific antimouse ferritin immunoglobulin G, rose from 4.4 ± 0.6 cells to 18.4 ± 1.3 pmol ⁵⁹Fe/h per 10⁸ cells 3 days after addition of dimethyl sulfoxide, and then fell to 11.6 ± 3.1 pmol 4 days after dimethyl sulfoxide when heme synthesis was maximal. These studies indicate that one or more steps in cellular iron transport distal to transferrin binding is induced early by dimethyl sulfoxide and that ferritin may play an active role in iron delivery for heme synthesis.     

SIDEROPHORE‐INDEPENDENT IRON UPTAKE BY IRON‐LIMITED CELLS OF THE CYANOBACTERIUM ANABAENA FLOS‐AQUAE1

Iron acquisition by iron‐limited cyanobacteria is typically considered to be mediated mainly by siderophores, iron‐chelating molecules released by iron‐limited cyanobacteria into the environment. In this set of experiments, iron uptake by iron‐limited cells of the cyanobacterium Anabaena flos‐aquae (L.) Bory was investigated in cells resuspended in siderophore‐free medium. Removal of siderophores decreased iron‐uptake rates by ～60% compared to siderophore‐replete conditions; however, substantial rates of iron uptake remained. In the absence of siderophores, Fe(III) uptake was much more rapid from a weaker synthetic chelator [N‐(2‐hydroxyethyl)ethylenediamine‐N,N′,N′‐triacetic acid (HEDTA); log K_cond = 28.64 for Fe(III)HEDTA(OH)^?] than from a very strong chelator [N,N′‐bis(2‐hydroxybenzyl)‐ethylenediamine‐N,N′‐diacetic acid (HBED); log K_cond = 31.40 for Fe(III)HBED^?], and increasing chelator:Fe(III) ratios decreased the Fe(III)‐uptake rate; these results were evident in both short‐term (4 h; absence of siderophores) and long‐term (116 h; presence of siderophores) experiments. However, free (nonchelated) Fe(III) provided the most rapid iron uptake in siderophore‐free conditions. The results of the short‐term experiments are consistent with an Fe(III)‐binding/uptake mechanism associated with the cyanobacterial outer membrane that operates independently of extracellular siderophores. Iron uptake was inhibited by temperature‐shock treatments of the cells and by metabolically compromising the cells with diphenyleneiodonium; this finding indicates that the process is dependent on active metabolism to operate and is not simply a passive Fe(III)‐binding mechanism. Overall, these results point to an important, siderophore‐independent iron‐acquisition mechanism by iron‐limited cyanobacterial cells.     

Interactions of free copper (II) ions alone or in complex with iron (III) ions with erythrocytes of marine fish Dicentrarchus labrax

As a consequence of human activity, various toxicants - especially metal ions - enter aquatic ecosystems and many fish are exposed to considerable levels. As the free ion and in some complexes, there is no doubt that copper promotes damage to cellular molecules and structures through radical formation. Therefore, we have investigated the influence of copper uptake by the red blood of the sea bass (Dicentrarchus labrax), and its oxidative action and effects on cells in the presence of complexed and uncomplexed Fe³⁺ ions.Erythrocytes were exposed to various concentrations of CuSO₄, Fe(NO₃)₃, and K₃Fe(CN)₆ for up to 5 h, and the effects of copper ions alone and in the combination with iron determined. The results show that inside the cells cupric ion interacts with hemoglobin, causing methemoglobin formation by direct electron transfer from heme Fe²⁺ to Cu²⁺. Potassium ferricyanide as a source of complexed iron decreases Met-Hb formation induced by copper ions unlike Fe(NO₃)₃. We also found that incubation of fish erythrocytes with copper increased hemolysis of cells. But complexed and uncomplexed iron protected the effect of copper. CuSO₄ increased the level of lipid peroxidation and a protective effect on complexed iron was observed. Incubation of erythrocytes with copper ions resulted in the loss of a considerable part of thiol content at 10 and 20 μM. This effect was decreased by potassium ferricyanide and Fe(NO₃)₃ only after 1 and 3 h of incubation. The level of nuclear DNA damage assayed by comet assay showed that 20 μM CuSO₄ as well as 20 μM Fe(NO₃)₃ and 10 mM K₃Fe(CN)₆ induce single- and double-strand breaks. The lower changes were observed after the exposure of cells to K₃Fe(CN)₆. The data suggest that complexed iron can act protectively against copper ions in contrast to Fe(NO₃)₃.     

High specific activity heme-Fe and its application for studying heme-Fe metabolism in Caco-2 cell monolayers

Heme-Fe is an important source of dietary iron in humans. Caco-2 cells have been used extensively to study human iron absorption with an emphasis on factors affecting nonheme iron absorption. Therefore, we examined several factors known to affect heme iron absorption. Cells grown in bicameral chambers were incubated with high specific activity [59Fe]heme alone or with 1% globin, BSA, or fatty acid-free BSA (BSA-FA) to examine the effect of protein source on absorption. Heme iron absorption was enhanced by globin and inhibited by BSA and BSA-FA. Absorption of heme iron in cells pretreated for 7 days with serum-free medium containing 1, 25, 50, or 100 microM Fe was higher in the 1-microM-Fe pretreatment group than in all other groups (P < 0.05), showing an effect of iron status. Increased heme concentrations resulted in decreased percent absorbed but increased total heme iron absorption and increased transport rate across the basolateral membrane. Finally, cells treated with 10 microM CdCl2, which induces heme oxygenase, demonstrated higher absorption of [59Fe]heme than control cells (P < 0.05). Our results from Caco-2 cells are in agreement with human studies and make this a promising model for examining intestinal heme iron absorption.     

Role of heme oxygenase in heme-mediated inhibition of rat brain Na+−K+-ATPase: Protection by tin-protoporphyrin

Hemoglobin has been shown to inhibit brain Na⁺–K⁺-ATPase through an iron-dependent mechanism. Both hemoglobin and iron cause spontaneous peroxidation of brain lipids. Release of iron from the heme molecule in animal tissues is dependent on the activity of heme oxygenase. We hypothesized that inhibition of heme catabolism by heme oxygenase prevents the iron-mediated inhibition of Na⁺–K⁺-ATPase and might subsequently reduce the tissue damage. Therefore, we studied the effect of heme and tin-protoporphyrin, an inhibitor of heme oxygenase, on the activity of partially purified Na⁺–K⁺-ATPase from rat brain in the presence and absence of purified hepatic heme oxygenase. Heme alone at a concentration of 30 M did not inhibit Na⁺–K⁺-ATPase. However, in the presence of heme oxygenase, heme inhibited Na⁺–K⁺-ATPase by 75%. Pretreatment of rats with SnCl₂, a known inducer of heme oxygenase, reduced the basal activity of the brain Na⁺–K⁺-ATPase by 50%. Inhibition of heme oxygenase by tin-protoporphyrin (30 M) prevented the inhibition of Na⁺–K⁺-ATPase which occurred in the presence of heme and heme oxygenase. It is concluded that suppression of heme oxygenase by tin-protoporphyrin might be a therapeutic approach to management of hemoglobin-associated brain injury following CNS hemorrhage.     

Lack of Plasma Protein Hemopexin Results in Increased Duodenal Iron Uptake

Purpose

The body concentration of iron is regulated by a fine equilibrium between absorption and losses of iron. Iron can be absorbed from diet as inorganic iron or as heme. Hemopexin is an acute phase protein that limits iron access to microorganisms. Moreover, it is the plasma protein with the highest binding affinity for heme and thus it mediates heme-iron recycling. Considering its involvement in iron homeostasis, it was postulated that hemopexin may play a role in the physiological absorption of inorganic iron.

Methods and Results

Hemopexin-null mice showed elevated iron deposits in enterocytes, associated with higher duodenal H-Ferritin levels and a significant increase in duodenal expression and activity of heme oxygenase. The expression of heme-iron and inorganic iron transporters was normal. The rate of iron absorption was assessed by measuring the amount of ⁵⁷Fe retained in tissues from hemopexin-null and wild-type animals after administration of an oral dose of ⁵⁷FeSO₄ or of ⁵⁷Fe-labelled heme. Higher iron retention in the duodenum of hemopexin-null mice was observed as compared with normal mice. Conversely, iron transfer from enterocytes to liver and bone marrow was unaffected in hemopexin-null mice.

Conclusions

The increased iron level in hemopexin-null duodenum can be accounted for by an increased iron uptake by enterocytes and storage in ferritins. These data indicate that the lack of hemopexin under physiological conditions leads to an enhanced duodenal iron uptake thus providing new insights to our understanding of body iron homeostasis.     

Transferrin-receptor interaction and iron uptake by reticulocytes of vertebrate animals — a comparative study

Summary Transferrin-receptor interactions and iron uptake were studied in eleven different species of vertebrate animals (3 eutherian mammals, 3 marsupials, 2 reptiles and 1 bird, amphibian and bony fish). In the initial experiments it was shown that the uptake of transferrin-bound iron by immature erythroid cells from marsupial and reptilian species occurs by receptor-mediated endocytosis as in other vertebrate animals.Reticulocytes were incubated with¹²⁵I-⁵⁹Fe-labelled transferrins from heterologous species and the results for iron and transferrin uptake compared with those obtained with the homologous protein. Cells from eutherian mammals were able to take up transferrin and iron from other eutherians and from the bob-tailed lizard but not from marsupials and other submammalian species. With marsupials and reptiles a similar specificity was observed, and the marsupial cells could also utilize chicken transferrin but not vice versa.The results were extended by performing competition experiments in which the cells were incubated with radiolabelled homologous transferrin in the presence of increasing concentrations of non-radioactive heterologous transferrins. From the ability of the heterologous proteins to inhibit uptake of the homologous protein relative association constants (K _a ¹) for the transferrin-receptor interactions could be calculated. TheseK _a ¹ values reflected the patterns observed in the first series of experiments.These studies demonstrate that, although specificity exists in transferrin-receptor interactions throughout the range of vertebrate animals, in several instances reactivity between widely divergent species is also observed. Hence, structural similarities have been maintained throughout evolution. Nevertheless, no evidence of interaction between transferrin and its receptor from the two divisions of the Mammalia, the eutherians and the marsupials, was observed.Abbreviations BSS Hanks balanced salt solution - PBS phosphate-buffered saline - RRS Rana Ringer solution     

NTAGONISM BETWEEN CADMIUM AND IRON IN THE MARINE DIATOM THALASSIOSIRA WEISSFLOGII1

Cadmium inhibits iron uptake and assimilation in the coastal diatom Thalassiosira weissflogii Grun. The effect of cadmium on short term Fe uptake fits ft competitive binding model: where (Fe³+) and (Cd²++) tire the free ferric and cadmium ion concentrations, respectively. The apparent binding constant K_cds, is calculated to be ca. lO^8.2M^-1 compared to a K^fe of lO¹⁹ M^-1. At low free ferric ion concentrations. interference of cadmium with iron transport (at pCd = 8 and pFe* < 20) results in a simultaneous decrease in growth rate and Fe accumulation to a level known 1o limit growth. Upon decreasing the free cadmium ion concentration, cells accumulate a large amount oj iron prior to resumption of normal growth. At higher free ferric ion concentrations (pFe* < 20) normal or elevated Fe quotas are absented but “luxury consumption” of iron still occurs upon reversal of toxicity. Evidence that these algae with high cellular iron quotas are effectively Fe deficient is provided by a decrease in the cytochrome f/chlorophyll a ratio and a much greater decrease in NO₃- reductase activity than in aldolase activity or H¹⁴C0₃ assimilation. Under the conditions of this study, cadmium had little effect on Si accumulation. The transport of methylamine (an analog of NH⁺₄) is unaffected by short term exposure to high free cadmium ion concentration but is greatly inhibited upon long term (97 h) exposure.     

Mechanisms of phosphate uptake into brush-border membrane vesicles from goat jejunum

This study concerns the uptake of inorganic phosphate into brush-border membrane vesicles prepared from jejunal tissues of either control or Ca-and/or P-depleted goats. The brush-border membrane vesicles showed a time-dependent accumulation of inorganic phosphate with a typical overshoot phenomenon in the presence of an inwardly directed Na⁺ gradient. The Na⁺-dependent inorganic phosphate uptake was completely inhibited by application of 5 mmol·l^-1 sodium arsenate. Half-maximal stimulation of inorganic phosphate uptake into brush-border membrane vesicles was found with Na⁺ concentrations in the order of 5 mmol·l^-1. Inorganic phosphate accumulation was not affected by a K⁺ diffusion potential (inside negative), suggesting an electroneutral transport process. Stoichiometry suggested an interaction of two or more Na ions with one inorganic phosphate ion at pH 7.4. Na⁺-dependent inorganic phosphate uptake into jejunal brush-border membrane vesicles from normal goats as a function of inorganic phosphate concentration showed typical Michaelis-Menten kinetic with V _max=0.42±0.08 nmol·mg^-1 protein per 15 s^-1 and K _m=0.03±0.01 mmol·l^-1 (n=4, x ±SEM). Long-term P depletion had no effect on these kinetic parameters. Increased plasma calcitriol concentrations in Ca-depleted goats, however, were associated with significant increases of V _max by 35–80%, irrespective of the level of P intake. In the presence of an inwardly directed Na⁺ gradient inorganic phosphate uptake was significantly stimulated by almost 60% when the external pH was decreased to 5.4 (pH_out/pH_in=5.4/7.4). The proton gradient had no effect on inorganic phosphate uptake in absence of Na⁺. In summary, in goats Na⁺ and calcitriol-dependent mechanisms are involved in inorganic phosphate transport into jejunal brush-border membrane vesicles which can be stimulated by protons.Abbreviations AP activity of alkaline phosphatase - BBMV brush-border membrane vesicles - EGTA ethyleneglycol-triacetic acid - n _app apparent Hill coefficient - P _i inorganic phosphate - PTH parathyroid hormone