Cellular and Subcellular Localization of the Nramp2 Iron Transporter in the Intestinal Brush Border and Regulation by Dietary Iron

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4406-4417 ◽  
Author(s):  
F. Canonne-Hergaux ◽  
S. Gruenheid ◽  
P. Ponka ◽  
P. Gros
Keyword(s):  
Small Intestine ◽  
Subcellular Localization ◽  
Brush Border ◽  
Iron Acquisition ◽  
Basolateral Membrane ◽  
Microcytic Anemia ◽  
The Body ◽  
Protein Isoforms ◽  
Dietary Iron ◽  
Iron Starvation

Abstract Genetic studies in animal models of microcytic anemia and biochemical studies of transport have implicated the Nramp2gene in iron transport. Nramp2 generates two alternatively spliced mRNAs that differ at their 3′ untranslated region by the presence or absence of an iron-response element (IRE) and that encode two proteins with distinct carboxy termini. Antisera raised against Nramp2 fusion proteins containing either the carboxy or amino termini of Nramp2 and that can help distinguish between the two Nramp2 protein isoforms (IRE: isoform I; non-IRE: isoform II) were generated. These antibodies were used to identify the cellular and subcellular localization of Nramp2 in normal tissues and to study possible regulation by dietary iron deprivation. Immunoblotting experiments with membrane fractions from intact organs show that Nramp2 is expressed at low levels throughout the small intestine and to a higher extent in kidney. Dietary iron starvation results in a dramatic upregulation of the Nramp2 isoform I in the proximal portion of the duodenum only, whereas expression in the rest of the small intestine and in kidney remains largely unchanged in response to the lack of dietary iron. In proximal duodenum, immunostaining studies of tissue sections show that Nramp2 protein expression is abundant under iron deplete condition and limited to the villi and is absent in the crypts. In the villi, staining is limited to the columnar absorptive epithelium of the mucosa (enterocytes), with no expression in mucus-secreting goblet cells or in the lamina propria. Nramp2 expression is strongest in the apical two thirds of the villi and is very intense at the brush border of the apical pole of the enterocytes, whereas the basolateral membrane of these cells is negative for Nramp2. These results strongly suggest that Nramp2 is indeed responsible for transferrin-independent iron uptake in the duodenum. These findings are discussed in the context of overall mechanisms of iron acquisition by the body.

Cellular and Subcellular Localization of the Nramp2 Iron Transporter in the Intestinal Brush Border and Regulation by Dietary Iron

Blood ◽  
1999 ◽  
Vol 93 (12) ◽  
pp. 4406-4417 ◽  
Author(s):  
F. Canonne-Hergaux ◽  
S. Gruenheid ◽  
P. Ponka ◽  
P. Gros
Keyword(s):  
Small Intestine ◽  
Subcellular Localization ◽  
Brush Border ◽  
Iron Acquisition ◽  
Basolateral Membrane ◽  
Microcytic Anemia ◽  
The Body ◽  
Protein Isoforms ◽  
Dietary Iron ◽  
Iron Starvation

Genetic studies in animal models of microcytic anemia and biochemical studies of transport have implicated the Nramp2gene in iron transport. Nramp2 generates two alternatively spliced mRNAs that differ at their 3′ untranslated region by the presence or absence of an iron-response element (IRE) and that encode two proteins with distinct carboxy termini. Antisera raised against Nramp2 fusion proteins containing either the carboxy or amino termini of Nramp2 and that can help distinguish between the two Nramp2 protein isoforms (IRE: isoform I; non-IRE: isoform II) were generated. These antibodies were used to identify the cellular and subcellular localization of Nramp2 in normal tissues and to study possible regulation by dietary iron deprivation. Immunoblotting experiments with membrane fractions from intact organs show that Nramp2 is expressed at low levels throughout the small intestine and to a higher extent in kidney. Dietary iron starvation results in a dramatic upregulation of the Nramp2 isoform I in the proximal portion of the duodenum only, whereas expression in the rest of the small intestine and in kidney remains largely unchanged in response to the lack of dietary iron. In proximal duodenum, immunostaining studies of tissue sections show that Nramp2 protein expression is abundant under iron deplete condition and limited to the villi and is absent in the crypts. In the villi, staining is limited to the columnar absorptive epithelium of the mucosa (enterocytes), with no expression in mucus-secreting goblet cells or in the lamina propria. Nramp2 expression is strongest in the apical two thirds of the villi and is very intense at the brush border of the apical pole of the enterocytes, whereas the basolateral membrane of these cells is negative for Nramp2. These results strongly suggest that Nramp2 is indeed responsible for transferrin-independent iron uptake in the duodenum. These findings are discussed in the context of overall mechanisms of iron acquisition by the body.

scholarly journals (Na+ + K+)-ATPase and plasma membrane polarity of intestinal epithelial cells: presence of a brush border antigen in the distal large intestine that is immunologically related to beta subunit.

1989 ◽  
Vol 109 (3) ◽  
pp. 1057-1069 ◽  
Author(s):  
A Marxer ◽  
B Stieger ◽  
A Quaroni ◽  
M Kashgarian ◽  
H P Hauri
Keyword(s):  
Monoclonal Antibody ◽  
Small Intestine ◽  
Epithelial Cells ◽  
Brush Border ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Distal Colon ◽  
Proximal Colon ◽  
Beta Subunit ◽  
Cell Pole

The previously produced monoclonal antibody IEC 1/48 against cultured rat intestinal crypt cells (Quaroni, A., and K. J. Isselbacher. 1981. J. Natl. Cancer Inst. 67:1353-1362) was extensively characterized and found to be directed against the beta subunit of (Na+ + K+)-ATPase as assessed by immunological and enzymatic criteria. Under nondenaturing conditions the antibody precipitated the alpha-beta enzyme complex (98,000 and 48,000 Mr). This probe, together with the monoclonal antibody C 62.4 against the alpha subunit (Kashgarian, M., D. Biemesderfer, M. Caplan, and B. Forbush. 1985. Kidney Int. 28:899-913), was used to localize (Na+ + K+)-ATPase in epithelial cells along the rat intestinal tract by immunofluorescence and immunoelectron microscopy. Both antibodies exclusively labeled the basolateral membrane of small intestine and proximal colon epithelial cells. However, in the distal colon, IEC 1/48, but not C 62.4, also labeled the brush border membrane. The cross-reacting beta-subunit-like antigen on the apical cell pole was tightly associated with isolated brush borders but was apparently devoid of (Na+ + K+)-ATPase activity. Subcellular fractionation of colonocytes in conjunction with limited proteolysis and surface radioiodination of intestinal segments suggested that the cross-reacting antigen in the brush border may be very similar to the beta subunit. The results support the notion that in the small intestine and proximal colon the enzyme subunits are exclusively targeted to the basolateral membrane while in the distal colon nonassembled beta subunit or a beta-subunit-like protein is also transported to the apical cell pole.

Transport of citrate across the brush border and basolateral membrane of rat small intestine

1994 ◽  
Vol 109 (1) ◽  
pp. 39-52 ◽  
Author(s):  
Siegfried Wolffram ◽  
Rene Unternährer ◽  
Beat Grenacher ◽  
Erwin Scharrer
Keyword(s):  
Small Intestine ◽  
Brush Border ◽  
Basolateral Membrane ◽  
Rat Small Intestine

Factors that influence absorption and secretion of calcium in the small intestine and colon

1985 ◽  
Vol 248 (2) ◽  
pp. G147-G157 ◽  
Author(s):  
M. J. Favus
Keyword(s):  
Small Intestine ◽  
Brush Border ◽  
Calcium Transport ◽  
Basolateral Membrane ◽  
Distal Colon ◽  
Phosphate Transport ◽  
Dihydroxyvitamin D3 ◽  
Distal Small Intestine ◽  
Electrical Gradient ◽  
Calcium Secretion

Intestinal epithelium absorbs calcium by an energy-dependent cellular process that is stimulated by 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]. Calcium entry across the brush border is driven by existing electrochemical gradients; exit across the basolateral membrane against these same gradients is driven by a calcium-activated ATPase, sodium-calcium exchange, or both. The specific cellular sites of 1,25(OH)2D3 action remain to be identified. Calcium transport is independent of phosphate and influenced by sodium. Sodium may alter calcium transport at the brush border through alterations of the transmembrane electrical gradient and at the basolateral membrane by exchange with intracellular calcium. The segmental distribution of calcium active transport is heterogeneous, with maximal flux rates in the proximal portions of small intestine and colon and net secretion in mid- and distal small intestine and mid-and distal colon. 1,25(OH)2D3 converts regions of net secretion in ileum and colon to net absorption. 1,25(OH)2D3-stimulated active phosphate transport is largely confined to areas of low-calcium transport, with maximal phosphate absorption in jejunum, the site of maximal calcium secretion. Calcium secretion, primarily in jejunum and ileum, is nonsaturable, may follow the paracellular pathway, and is stimulated by mucosal sodium and somatostatin.

Is intestinal peptide transport energized by a proton gradient?

1985 ◽  
Vol 249 (2) ◽  
pp. G153-G160 ◽  
Author(s):  
Vadivel Ganapathy ◽  
Frederick H. Leibach
Keyword(s):  
Small Intestine ◽  
Membrane Potential ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Basolateral Membrane ◽  
Membrane Vesicles ◽  
Proton Gradient ◽  
Peptide Transport ◽  
Direct Role ◽  
Mucosal Cells

Transport of intact peptides, followed by intracellular hydrolysis in the intestinal mucosal cells, plays an important role in the absorption of protein digestion products in the mammalian small intestine. Even though earlier studies on peptide absorption in intact-tissue preparations have indicated that peptides are transported by an active Na+-dependent mechanism, recent studies with purified brush-border membrane vesicles have unequivocally demonstrated that Na+ does not play a direct role in the translocation of peptides across the membrane. Like most amino acids, peptides are also transported as zwitterions. However, peptide transport causes depolarization of the brushborder membrane in intact mucosal cells as well as in purified membrane vesicles, and the depolarization is the result of a net transfer of positive charge across the membrane during peptide transport. This electrogenic nature of peptide transport is observed even in the absence of Na+. Peptide transport is enhanced by an interior-negative membrane potential and inhibited by an interior-positive membrane potential. An inward proton gradient stimulates peptide transport, and this stimulation is reduced when the proton gradient is subjected to rapid dissipation by the presence of a proton ionophore. These observations strongly suggest that peptides are cotransported with protons in the intestine. There is substantial evidence for the existence of an inward proton gradient in the mammalian small intestine, and therefore it is very likely that this proton gradient is the in vivo energy source for the uphill transport of peptides. The Na+-H+ exchanger in the brush-border membrane, in conjunction with Na+-K+-ATPase at the basolateral membrane, is probably responsible for the generation and maintenance of the proton gradient and may thus be involved indirectly in the intestinal absorption of peptides.

Regulation of Intestinal Phosphate Transport II. Metabolic acidosis stimulates Na+-dependent phosphate absorption and expression of the Na+-Pi cotransporter NaPi-IIb in small intestine

2005 ◽  
Vol 288 (3) ◽  
pp. G501-G506 ◽  
Author(s):  
Annina Stauber ◽  
Tamara Radanovic ◽  
Gerti Stange ◽  
Heini Murer ◽  
Carsten A. Wagner ◽  
...  
Keyword(s):  
Small Intestine ◽  
Metabolic Acidosis ◽  
Brush Border ◽  
Brush Border Membrane ◽  
Membrane Vesicles ◽  
Fractional Excretion ◽  
Phosphate Transport ◽  
The Body ◽  
Mrna Levels ◽  
Fractional Excretion Of Phosphate

During metabolic acidosis, Pi serves as an important buffer to remove protons from the body. Pi is released from bone together with carbonate buffering protons in blood. In addition, in the kidney, the fractional excretion of phosphate is increased allowing for the excretion of more acid equivalents in urine. The role of intestinal Pi absorption in providing Pi to buffer protons and compensating for loss from bone during metabolic acidosis has not been clarified yet. Inducing metabolic acidosis (NH4Cl in drinking water) for 2 or 7 days in mice increased urinary fractional Pi excretion twofold, whereas serum Pi levels were not altered. Na+-dependent Pi transport in the small intestine, however, was stimulated from 1.89 ± 3.22 to 40.72 ± 11.98 pmol/mg protein (2 days of NH4Cl) in brush-border membrane vesicles prepared from total small intestine. Similarly, the protein abundance of the Na+-dependent phosphate cotransporter NaPi-IIb in the brush-border membrane was increased 5.3-fold, whereas mRNA levels remained stable. According to immunohistochemistry and real-time PCR NaPi-IIb expression was found to be mainly confined to the ileum in the small intestine, and this distribution was not altered during metabolic acidosis. These results suggest that the stimulation of intestinal Pi absorption during metabolic acidosis may contribute to the buffering of acid equivalents by providing phosphate and may also help to prevent excessive liberation of phosphate from bone.

NHE2 and NHE3 are human and rabbit intestinal brush-border proteins

1996 ◽  
Vol 270 (1) ◽  
pp. G29-G41 ◽  
Author(s):  
W. A. Hoogerwerf ◽  
S. C. Tsao ◽  
O. Devuyst ◽  
S. A. Levine ◽  
C. H. Yun ◽  
...  
Keyword(s):  
Small Intestine ◽  
Epithelial Cells ◽  
Brush Border ◽  
Basolateral Membrane ◽  
Goblet Cells ◽  
Basolateral Membranes ◽  
Western Analysis ◽  
Rabbit Jejunum ◽  
Colon And Rectum ◽  
Descending Colon

Rabbit NHE2 and NHE3 are two epithelial isoform Na+/H+ exchangers (NHE), the messages for which are found predominantly and entirely, respectively, in renal, intestinal, and gastric mucosa. The current studies used Western analysis and immunohistochemistry to identify and characterize the apical vs. basolateral membrane distribution of NHE2 and NHE3 in intestinal epithelial cells. Based on Western analysis, NHE2 and NHE3 both are present in brush-border but not basolateral membranes of small intestine. Both NHE2 and NHE3 are 85-kDa proteins. Consistent with Western analysis, NHE2 and NHE3 are immunolocalired to the brush-border but not basolateral membranes of villus epithelial cells, but not goblet cells, in human jejunum and ileum and in surface epithelial cells in the ascending and descending colon and rectum. In addition, NHE2 and NHE3 are present in small amounts in the crypt cell brush border of human jejunum, ileum, ascending and descending colon, and rectum. In rabbit jejunum, ileum, and ascending colon, NHE2 and NHE3 are present in the brush border of epithelial and not goblet cells, again much more in the villus (small intestine)/ surface cells (colon) than the crypt. NHE2 but not NHE3 is present in the brush border of rabbit descending colon surface cells and in small amounts in crypt cells. NHE2 and NHE3 are both human and rabbit small intestinal and colonic epithelial cell brush-border Na+/H+ exchanger isoforms that colocalize in all intestinal segments except rabbit descending colon, which lacks NHE3.

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