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.

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.

scholarly journals A ZONAL ROTOR METHOD FOR THE PREPARATION OF MICROPEROXISOMES FROM EPITHELIAL CELLS OF GUINEA PIG SMALL INTESTINE

1974 ◽  
Vol 61 (1) ◽  
pp. 123-133 ◽  
Author(s):  
M. J. Connock ◽  
P. R. Kirk ◽  
A. P. Sturdee
Keyword(s):  
Small Intestine ◽  
Electron Microscope ◽  
Epithelial Cells ◽  
Guinea Pig ◽  
Analytical Data ◽  
Microscope Examination ◽  
Electron Microscope Examination ◽  
Large Particles ◽  
Zonal Separation ◽  
Zonal Rotor

A method is described for the preparation of catalase particles from homogenates made from suspensions of epithelial cells of the small intestine of the guinea pig. Electron microscope examination of the preparations revealed the presence of small diaminobenzidine-positive particles measuring 0.1–0.3 nm in diameter and resembling the microperoxisomes observed by Novikoff and Novikoff (1972. J. Cell Biol. 53:532.). Analytical data upon which the method is based are presented. The technique consisted of a rate zonal separation of microperoxisomes from large particles followed by an isopycnic separation from less dense organelles. Application of the method yielded microperoxisomes purified between 20- and 30-fold.

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.

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.

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.

scholarly journals STUDIES ON EPITHELIAL CELLS ISOLATED FROM GUINEA PIG SMALL INTESTINE

1971 ◽  
Vol 51 (2) ◽  
pp. 452-464 ◽  
Author(s):  
E. M. Evans ◽  
J. M. Wrigglesworth ◽  
K. Burdett ◽  
W. F. R. Pover
Keyword(s):  
Small Intestine ◽  
Epithelial Cells ◽  
Guinea Pig ◽  
Oxygen Tension ◽  
Plasma Membranes ◽  
Structural Integrity ◽  
Cell Metabolism ◽  
Extracellular Fluid ◽  
Lactic Dehydrogenase ◽  
Low Leakage

Sheets of mucosal epithelial cells were released from guinea pig small intestine after incubation with ethylenediaminetetraacetate. Cells in sheets retained their columnar shape for 24 hr at room temperature, and exclusion of nigrosine suggested they had intact plasma membranes. When sheets were disaggregated individual cells had normal morphology for at least 4 hr. During isolation 16% of the total protein and 24% of the total lactic dehydrogenase were lost from the cells, but subsequent enzyme leakage was low. Leakage increased with shaking, incubation at 37°C, or increasing the oxygen tension of the suspending medium, but was minimal when the Na+:K+ ratio in the medium was 8:1 and the osmolarity was high. Losses of particulate enzyme activities were negligible. Respiration was constant for up to 4 hr and was insensitive to calcium, bicarbonate, oxygen tension, and pH. It was inhibited by cyanide and iodoacetate and varied with the Na+:K+ ratio of the extracellular fluid and the structural integrity of the cells. All preparations concentrated potassium and excluded sodium, but lost this ability if ouabain was added or cells were broken. Potassium-42 uptake was also sensitive to temperature, ouabain, and structural integrity. The preparations are being used to study cell metabolism in the intestinal epithelium.

scholarly journals Studies of mucus in mouse stomach, small intestine, and colon. I. Gastrointestinal mucus layers have different properties depending on location as well as over the Peyer's patches

2013 ◽  
Vol 305 (5) ◽  
pp. G341-G347 ◽  
Author(s):  
Anna Ermund ◽  
André Schütte ◽  
Malin E. V. Johansson ◽  
Jenny K. Gustafsson ◽  
Gunnar C. Hansson
Keyword(s):  
Small Intestine ◽  
Functional Organization ◽  
Distal Colon ◽  
Proximal Colon ◽  
Peyer's Patches ◽  
Peyer’S Patches ◽  
Mucus Layer ◽  
Adhesive Properties ◽  
Intestinal Mucus ◽  
Small Intestinal

Colon has been shown to have a two-layered mucus system where the inner layer is devoid of bacteria. However, a complete overview of the mouse gastrointestinal mucus system is lacking. We now characterize mucus release, thickness, growth over time, adhesive properties, and penetrability to fluorescent beads from stomach to distal colon. Colon displayed spontaneous mucus release and all regions released mucus in response to carbachol and PGE2, except the distal colon and domes of Peyer's patches. Stomach and colon had an inner mucus layer that was adherent to the epithelium. In contrast, the small intestine and Peyer's patches had a single mucus layer that was easily aspirated. The inner mucus layer of the distal colon was not penetrable to beads the size of bacteria and the inner layer of the proximal colon was only partly penetrable. In contrast, the inner mucus layer of stomach was fully penetrable, as was the small intestinal mucus. This suggests a functional organization of the intestinal mucus system, where the small intestine has loose and penetrable mucus that may allow easy penetration of nutrients, in contrast to the stomach, where the mucus provides physical protection, and the colon, where the mucus separates bacteria from the epithelium. This knowledge of the mucus system and its organization improves our understanding of the gastrointestinal tract physiology.

scholarly journals CD24 can be used to isolate Lgr5+ putative colonic epithelial stem cells in mice

2012 ◽  
Vol 303 (4) ◽  
pp. G443-G452 ◽  
Author(s):  
Jeffrey B. King ◽  
Richard J. von Furstenberg ◽  
Brian J. Smith ◽  
Kirk K. McNaughton ◽  
Joseph A. Galanko ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Stem Cells ◽  
Epithelial Cells ◽  
Distal Colon ◽  
Proximal Colon ◽  
Egfp Expression ◽  
Colonic Epithelium ◽  
Epithelial Stem Cells ◽  
Normal Colonic Epithelium ◽  
Crypt Base

A growing body of evidence has implicated CD24, a cell-surface protein, as a marker of colorectal cancer stem cells and target for antitumor therapy, although its presence in normal colonic epithelium has not been fully characterized. Previously, our group showed that CD24-based cell sorting can be used to isolate a fraction of murine small intestinal epithelial cells enriched in actively cycling stem cells. Similarly, we hypothesized that CD24-based isolation of colonic epithelial cells would generate a fraction enriched in actively cycling colonic epithelial stem cells (CESCs). Immunohistochemistry performed on mouse colonic tissue showed CD24 expression in the bottom half of proximal colon crypts and the crypt base in the distal colon. This pattern of distribution was similar to enhanced green fluorescent protein (EGFP) expression in Lgr5-EGFP mice. Areas expressing CD24 contained actively proliferating cells as determined by ethynyl deoxyuridine (EdU) incorporation, with a distinct difference between the proximal colon, where EdU-labeled cells were most frequent in the midcrypt, and the distal colon, where they were primarily at the crypt base. Flow cytometric analyses of single epithelial cells, identified by epithelial cell adhesion molecule (EpCAM) positivity, from mouse colon revealed an actively cycling CD24+ fraction that contained the majority of Lgr5-EGFP+ putative CESCs. Transcript analysis by quantitative RT-PCR confirmed enrichment of active CESC markers [leucine-rich-repeat-containing G protein-coupled receptor 5 (Lgr5), ephrin type B receptor 2 (EphB2), and CD166] in the CD24+EpCAM+ fraction but also showed enrichment of quiescent CESC markers [leucine-rich repeats and immunoglobin domains (Lrig), doublecortin and calmodulin kinase-like 1 (DCAMKL-1), and murine telomerase reverse transcriptase (mTert)]. We conclude that CD24-based sorting in wild-type mice isolates a colonic epithelial fraction highly enriched in actively cycling and quiescent putative CESCs. Furthermore, the presence of CD24 expression in normal colonic epithelium may have important implications for the use of anti-CD24-based colorectal cancer therapies.

Mode of action of ANG II on ion transport in guinea pig distal colon

2000 ◽  
Vol 278 (4) ◽  
pp. G625-G634 ◽  
Author(s):  
Yutaka Hosoda ◽  
Adi Winarto ◽  
Toshihiko Iwanaga ◽  
Atsukazu Kuwahara
Keyword(s):  
Epithelial Cells ◽  
Guinea Pig ◽  
Ion Transport ◽  
Receptor Antagonist ◽  
Distal Colon ◽  
Evoked Response ◽  
Maximal Response ◽  
Prostaglandin E ◽  
Ang Ii

The effect of ANG II on mucosal ion transport and localization of ANG type 1 receptor (AT1R) in the guinea pig distal colon was investigated. Submucosal/mucosal segments were mounted in Ussing flux chambers, and short-circuit current ( I sc) was measured as an index of ion transport. Serosal addition of ANG II produced a concentration-dependent (10− 9–10− 5M) increase in I sc. The maximal response was observed at 10− 6 M; the increase in I sc was 164.4 ± 11.8 μA/cm2. The ANG II (10− 6 M)-evoked response was mainly due to Cl− secretion. Tetrodotoxin, atropine, the neurokinin type 1 receptor antagonist FK-888, and piroxicam significantly reduced the ANG II (10− 6M)-evoked response to 28, 45, 58, and 16% of control, respectively. Pretreatment with prostaglandin E2(10− 5 M) resulted in a threefold increase in the ANG II-evoked response. The AT1R antagonist FR-130739 completely blocked ANG II (10− 6M)-evoked responses, whereas the ANG type 2 receptor antagonist PD-123319 had no effect. Localization of AT1R was determined by immunohistochemistry. In the immunohistochemical study, AT1R-immunopositive cells were distributed clearly in enteric nerves and moderately in surface epithelial cells. These results suggest that ANG II-evoked electrogenic Cl−secretion may involve submucosal cholinergic and tachykinergic neurons and prostanoid synthesis pathways through AT1R on the submucosal plexus and surface epithelial cells in guinea pig distal colon.

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