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.

K+ transport in isolated guinea pig colonocytes: evidence for Na(+)-independent ouabain-sensitive K+ pump

1994 ◽  
Vol 266 (6) ◽  
pp. G1083-G1089 ◽  
Author(s):  
J. R. Del Castillo ◽  
M. C. Sulbaran-Carrasco ◽  
L. Burguillos
Keyword(s):  
Small Intestine ◽  
Epithelial Cells ◽  
Guinea Pig ◽  
Distal Colon ◽  
Proximal Colon ◽  
Metabolic Inhibition ◽  
Transport Mechanisms ◽  
Transport Pathway ◽  
K Transport

K+ transport mechanisms in epithelial cells isolated from guinea pig distal colon have been studied using 86Rb as a tracer. A transport pathway has been identified that is proposed to be identical to the mechanism mediating transepithelial K+ absorption. Guinea pig colonocytes take up K+ through at least three separate mechanisms: 1) a Na(+)-dependent, ouabain-sensitive influx that is consistent with the Na(+)-K+ pump, 2) a Na(+)-dependent bumetanide-sensitive influx consistent with the Na(+)-K(+)-2Cl- cotransporter, and 3) a Na(+)-independent ouabain-sensitive influx, consistent with an apical colonic K+ pump. These transport mechanisms are sensitive to metabolic inhibition by rotenone and to vanadate, a blocker of type P adenosinetriphosphatase (ATPases). SCH-28080, an inhibitor of gastric K(+)-H(+)-ATPase, was without effect. Measurements of net K+ fluxes revealed that isolated colonocytes concentrated K+ by two processes: 1) a Na(+)-dependent ouabain-sensitive mechanism, which is compatible with the Na(+)-K+ pump and 2) a Na(+)-independent ouabain-sensitive mechanism consistent with the proposed absorptive K+ pump. These concentrative mechanisms were also inhibited by rotenone and vanadate, but not by SCH-28080. The Na(+)-independent ouabain-sensitive K+ pump was present in the distal colon, but absent in the proximal colon and the small intestine of guinea pig. It is proposed that this Na(+)-independent ouabain-sensitive K+ pump mediates K+ absorption and is related to the luminal K(+)-ATPase.

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.

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.

Localization of a novel 210 kDa protein in Xenopus tight junctions

1997 ◽  
Vol 110 (8) ◽  
pp. 1005-1012 ◽  
Author(s):  
C.S. Merzdorf ◽  
D.A. Goodenough
Keyword(s):  
Monoclonal Antibody ◽  
Epithelial Cells ◽  
Tight Junction ◽  
Tight Junctions ◽  
Basolateral Membrane ◽  
Immunofluorescent Staining ◽  
Junctional Complex ◽  
Permeability Barrier ◽  
Membrane Domains ◽  
Kidney Liver

The tight junction is the most apical member of the intercellular junctional complex. It functions as a permeability barrier between epithelial cells and maintains the integrity of the apical and basolateral membrane domains. In order to study tight junctions in Xenopus laevis, a polyclonal antibody was raised which recognized Xenopus ZO-1. Monoclonal antibody 19B1 (mAb 19B1) was generated in rats using a crude membrane preparation from Xenopus lung as antigen. mAb 19B1 gave immunofluorescent staining patterns identical to those seen with anti-ZO-1 on monolayers of Xenopus A6 kidney epithelial cells and on frozen sections of Xenopus kidney, liver, and embryos. Electron microscopy showed that the 19B1 antigen colocalized with ZO-1 at the tight junction. Western blotting and immunoprecipitation demonstrated that ZO-1 is an approximately 220 kDa protein in Xenopus, while mAb 19B1 identified an approximately 210 kDa antigen on immunoblots. Immunoprecipitates of ZO-1 were not recognized by mAb 19B1 by western analysis. The solubility properties of the 19B1 antigen suggested that it is a peripheral membrane protein. Thus, the antigen recognized by the new monoclonal antibody 19B1 is not ZO-1 and represents a different Xenopus tight junction associated protein.

Epithelial sorting of a glycosylphosphatidylinositol-anchored bacterial protein expressed in polarized renal MDCK and intestinal Caco-2 cells

1995 ◽  
Vol 108 (1) ◽  
pp. 369-377 ◽  
Author(s):  
K.L. Soole ◽  
M.A. Jepson ◽  
G.P. Hazlewood ◽  
H.J. Gilbert ◽  
B.H. Hirst
Keyword(s):  
Epithelial Cells ◽  
Cell Surface ◽  
Intestinal Epithelial Cells ◽  
Mdck Cells ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Apical Surface ◽  
Intestinal Epithelial ◽  
Gpi Anchor ◽  
Sorting Signal

To evaluate whether a glycosylphosphatidylinositol (GPI) anchor can function as a protein sorting signal in polarized intestinal epithelial cells, the GPI-attachment sequence from Thy-1 was fused to bacterial endoglucanase E' (EGE') from Clostridium thermocellum and polarity of secretion of the chimeric EGE'-GPI protein was evaluated. The chimeric EGE'-GPI protein was shown to be associated with a GPI anchor by TX-114 phase-partitioning and susceptibility to phosphoinositol-specific phospholipase C. In polarized MDCK cells, EGE' was localized almost exclusively to the apical cell surface, while in polarized intestinal Caco-2 cells, although 80% of the extracellular form of the enzyme was routed through the apical membrane over a 24 hour period, EGE' was also detected at the basolateral membrane. Rates of delivery of EGE'-GPI to the two membrane domains in Caco-2 cells, as determined with a biotinylation protocol, revealed apical delivery was approximately 2.5 times that of basolateral. EGE' delivered to the basolateral cell surface was transcytosed to the apical surface. These data indicate that a GPI anchor does represent a dominant apical sorting signal in intestinal epithelial cells. However, the mis-sorting of a proportion of EGE'GPI to the basolateral surface of Caco-2 cells provides an explanation for additional sorting signals in the ectodomain of some endogenous GPI-anchored proteins.

ClC-2 in guinea pig colon: mRNA, immunolabeling, and functional evidence for surface epithelium localization

2002 ◽  
Vol 283 (4) ◽  
pp. G1004-G1013 ◽  
Author(s):  
Marcelo Catalán ◽  
Isabel Cornejo ◽  
Carlos D. Figueroa ◽  
María Isabel Niemeyer ◽  
Francisco V. Sepúlveda ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Guinea Pig ◽  
Apical Membrane ◽  
Basolateral Membrane ◽  
Distal Colon ◽  
Surface Epithelium ◽  
Patch Clamp Technique ◽  
Chloride Currents ◽  
Kinetics Of

The principal function of the colon in fluid homeostasis is the absorption of NaCl and water. Apical membrane Na+ channels, Na+/H+ and Cl−/HCO[Formula: see text] exchangers, have all been postulated to mediate NaCl entry into colonocytes. The identity of the basolateral exit pathway for Cl− is unknown. We have previously demonstrated the presence of the ClC-2 transcript in the guinea pig intestine. Now we explore in more detail, the tissue and cellular distribution of chloride channel ClC-2 in the distal colon by in situ hybridization and immunohistochemistry. The patch-clamp technique was used to characterize Cl− currents in isolated surface epithelial cells from guinea pig distal colon and these were compared with those mediated by recombinant guinea pig (gp)ClC-2. ClC-2 mRNA and protein were found in the surface epithelium of the distal colon. Immunolocalization revealed that, in addition to some intracellular labeling, ClC-2 was present in the basolateral membranes but absent from the apical pole of colonocytes. Isolated surface epithelial cells exhibited hyperpolarization-activated chloride currents showing a Cl− > I− permeability and Cd2+ sensitivity. These characteristics, as well as some details of the kinetics of activation and deactivation, were very similar to those of recombinant gpClC-2 measured in parallel experiments. The presence of active ClC-2 type currents in surface colonic epithelium, coupled to a basolateral location for ClC-2 in the distal colon, suggests a role for ClC-2 channel in mediating basolateral membrane exit of Cl− as an essential step in a NaCl absorption process.

Identification of Na+/H+ exchange on the apical side of surface colonocytes using BCECF

1994 ◽  
Vol 267 (1) ◽  
pp. G119-G128 ◽  
Author(s):  
G. G. King ◽  
W. E. Lohrmann ◽  
J. W. Ickes ◽  
G. M. Feldman
Keyword(s):  
Apical Membrane ◽  
Basolateral Membrane ◽  
Apical Cell ◽  
Distal Colon ◽  
Transport Processes ◽  
Acetoxymethyl Ester ◽  
Apical Cell Membrane ◽  
Apical Side ◽  
Free Media ◽  
Phi Regulation

Colonocytes must regulate intracellular pH (pHi) while they transport H+ and HCO3-. To investigate the membrane transport processes involved in pHi regulation, colonocyte pHi was measured with 2,'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) in intact segments of rat distal colon mounted on a holder that fits into a standard fluorometer cuvette and allows independent superfusion of mucosal and serosal surfaces. When NCECF-acetoxymethyl ester was in the mucosal solution only, BCECF loaded surface colonocytes with a high degree of selectivity. In HEPES-buffered solutions, basal pHi was 7.31 +/- 0.01 (n = 68), and pHi was dependent on extracellular Na+. Cells acidified in Na(+)-free solution, and pHi rapidly corrected when Na+ was returned. pHi recovered at 0.22 +/- 0.01 pH/min (n = 6) when Na+ was introduced into the mucosal solution and at 0.02 +/- 0.01 pH/min (n = 7) when Na+ was absent from the mucosal solution. The presence or absence of Na+ in the serosal solution did not affect pHi. This indicated that the Na(+)-dependent pHi recovery process is located in the apical cell membrane, but not in the basolateral membrane. Because amiloride (1 mM) inhibited Na(+)-dependent pHi recovery by 75%, Na+/H+ exchange appears to be present in the apical membrane. Because Na(+)-independent pHi recovery was not affected by K(+)-free media, 50 microM SCH-28080, 100 nM bafilomycin A1, or Cl(-)-free media, this transport mechanism does not involve a gastriclike H(+)-K(+)-ATPase, a vacuolar H(+)-ATPase, or a Cl-/base exchanger. In summary, pHi was selectively measured in surface colonocytes by this technique. In these cells, the Na+/H+ exchange activity involved in pHi regulation was detected in the apical membrane, but not in the basolateral membrane.

scholarly journals Generation of an inducible colon-specific Cre enzyme mouse line for colon cancer research

2016 ◽  
Vol 113 (42) ◽  
pp. 11859-11864 ◽  
Author(s):  
Paul W. Tetteh ◽  
Kai Kretzschmar ◽  
Harry Begthel ◽  
Maaike van den Born ◽  
Jeroen Korving ◽  
...  
Keyword(s):  
Colon Cancer ◽  
Small Intestine ◽  
Epithelial Cells ◽  
Mouse Model ◽  
Human Colon ◽  
Suppressor Gene ◽  
Proximal Colon ◽  
Tumor Formation ◽  
Cell Of Origin ◽  
Human Colon Cancer

Current mouse models for colorectal cancer often differ significantly from human colon cancer, being largely restricted to the small intestine. Here, we aim to develop a colon-specific inducible mouse model that can faithfully recapitulate human colon cancer initiation and progression. Carbonic anhydrase I (Car1) is a gene expressed uniquely in colonic epithelial cells. We generated a colon-specific inducible Car1CreER knock-in (KI) mouse with broad Cre activity in epithelial cells of the proximal colon and cecum. Deletion of the tumor suppressor gene Apc using the Car1CreER KI caused tumor formation in the cecum but did not yield adenomas in the proximal colon. Mutation of both Apc and Kras yielded microadenomas in both the cecum and the proximal colon, which progressed to macroadenomas with significant morbidity. Aggressive carcinomas with some invasion into lymph nodes developed upon combined induction of oncogenic mutations of Apc, Kras, p53, and Smad4. Importantly, no adenomas were observed in the small intestine. Additionally, we observed tumors from differentiated Car1-expressing cells with Apc/Kras mutations, suggesting that a top-down model of intestinal tumorigenesis can occur with multiple mutations. Our results establish the Car1CreER KI as a valuable mouse model to study colon-specific tumorigenesis and metastasis as well as cancer-cell-of-origin questions.

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

scholarly journals Macromolecular transport across the rabbit proximal and distal colon

Gut ◽  
1999 ◽  
Vol 44 (2) ◽  
pp. 218-225 ◽  
Author(s):  
J A Hardin ◽  
M H Kimm ◽  
M Wirasinghe ◽  
D G Gall
Keyword(s):  
Nervous System ◽  
Small Intestine ◽  
Distal Colon ◽  
Distal Segment ◽  
Proximal Colon ◽  
Colonic Epithelium ◽  
Colonic Tissue ◽  
Ussing Chambers ◽  
Macromolecular Transport ◽  
Energy Dependent

BackgroundAlthough many studies have investigated macromolecular uptake in the stomach and small intestine, little is known about macromolecular uptake in the colon.AimsTo investigate the mechanisms involved in the transport of large antigenically intact macromolecules across the proximal and distal colonic epithelium in the rabbit.MethodsThe mucosal to serosal movement of bovine serum albumin (BSA) was examined in modified Ussing chambers under short circuited conditions. The mucosal surface was exposed to varying concentrations of BSA, and after a 50 minute equilibration period, the mucosal to serosal flux of immunologically intact BSA was determined by ELISA. Total BSA flux was determined by the transport of radiolabelled 125I-BSA.ResultsIntact BSA transport in proximal and distal colonic tissue showed saturable kinetics. Intact BSA transport in the proximal and distal segment was 7% and 2% of the total 125I-BSA flux respectively. Immunologically intact BSA transport in the distal segment was significantly less than that in the proximal segment. Intact BSA transport in the proximal colon was significantly reduced following treatment with sodium fluoride, colchicine, and tetrodotoxin. Cholinergic blockade had no effect on the uptake of intact BSA.ConclusionThe findings indicate that the transport of intact macromolecules across the proximal and distal large intestine is a saturable process. Further, intact BSA transport in the proximal colon is an energy dependent process that utilises microtubules and is regulated by the enteric nervous system.

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