Acid-base effects on intestinal Cl- absorption and vesicular trafficking

2004 ◽  
Vol 286 (5) ◽  
pp. C1062-C1070 ◽  
Author(s):  
Alan N. Charney ◽  
Richard W. Egnor ◽  
David Henner ◽  
Haroon Rashid ◽  
Nicholas Cassai ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Anion Exchanger ◽  
Apical Membrane ◽  
Ussing Chamber ◽  
Vesicular Trafficking ◽  
Acid Base ◽  
Western Blots ◽  
Rat Ileum ◽  
Apical Membranes ◽  
Type 3

In rat ileum and colon, apical membrane [Formula: see text] exchange and net Cl- absorption are stimulated by increases in Pco2 or [Formula: see text]. Because changes in Pco2 stimulate colonic Na+ absorption, in part, by modulating vesicular trafficking of the Na+/H+ exchanger type 3 isoform to and from the apical membrane, we examined whether changes in Pco2 affect net Cl- absorption by modulating vesicular trafficking of the [Formula: see text] exchanger anion exchanger (AE)1. Cl- transport across rat distal ileum and colon was measured in the Ussing chamber, and apical membrane protein biotinylation of these segments and Western blots of recovered proteins were performed. In colonic epithelial apical membranes, AE1 protein content was greater at Pco2 70 mmHg than at Pco2 21 mmHg but was not affected by pH changes in the absence of CO2. AE1 was internalized when Pco2 was reduced and exocytosed when Pco2 was increased, and both mucosal wortmannin and methazolamide inhibited exocytosis. Wortmannin also inhibited the increase in colonic Cl- absorption caused by an increase in Pco2. Increases in Pco2 stimulated ileal Cl- absorption, but wortmannin was without effect. Ileal epithelial apical membrane AE1 content was not affected by Pco2. We conclude that CO2 modulation of colonic, but not ileal, Cl- absorption involves effects on vesicular trafficking of AE1.

Acid-base effects on intestinal Na+ absorption and vesicular trafficking

2002 ◽  
Vol 283 (3) ◽  
pp. C971-C979 ◽  
Author(s):  
Alan N. Charney ◽  
Richard W. Egnor ◽  
Jesline Alexander-Chacko ◽  
Nicholas Cassai ◽  
Gurdip S. Sidhu
Keyword(s):  
Electron Microscopy ◽  
Apical Membrane ◽  
Ringer Solution ◽  
Vesicular Trafficking ◽  
Acid Base ◽  
Western Blots ◽  
Ph Values ◽  
Transmission Electron ◽  
Apical Membranes ◽  
Nhe Isoforms

We examined for vesicular trafficking of the Na+/H+ exchanger (NHE) in pH-stimulated ileal and CO2-stimulated colonic Na+absorption. Subapical vesicles in rat distal ileum were quantified by transmission electron microscopy at ×27,500 magnification. Internalization of ileal apical membranes labeled with FITC-phytohemagglutinin was assessed using confocal microscopy, and pH-stimulated ileal Na+ absorption was measured after exposure to wortmannin. Apical membrane protein biotinylation of ileal and colonic segments and Western blots of recovered proteins were performed. In ileal epithelial cells incubated in HCO[Formula: see text]/Ringer or HEPES/Ringer solution, the number of subapical vesicles, the relative quantity of apical membrane NHE isoforms 2 and 3 (NHE2 and NHE3, respectively), and apical membrane fluorescence under the confocal microscope were not affected by pH values between 7.1 and 7.6. Wortmannin did not inhibit pH-stimulated ileal Na+ absorption. In colonic epithelial apical membranes, NHE3 protein content was greater at a Pco 2 value of 70 than 21 mmHg, was internalized when Pco 2 was reduced, and was exocytosed when Pco 2 was increased. We conclude that vesicle trafficking plays no part in pH-stimulated ileal Na+absorption but is important in CO2-stimulated colonic Na+ absorption.

Characterization of an 80-kDa phosphoprotein involved in parietal cell stimulation

1989 ◽  
Vol 256 (6) ◽  
pp. G1070-G1081 ◽  
Author(s):  
T. Urushidani ◽  
D. K. Hanzel ◽  
J. G. Forte
Keyword(s):  
Membrane Protein ◽  
Parietal Cell ◽  
Apical Membrane ◽  
Peptide Mapping ◽  
Integral Membrane Protein ◽  
Gastric Glands ◽  
Low Speed ◽  
Calcium Dependent ◽  
Apical Membranes

When isolated rabbit gastric glands were stimulated with histamine plus isobutylmethylxanthine, a redistribution of H+-K+-ATPase, from microsomes to a low-speed pellet, occurred in association with the phosphorylation of an 80-kDa protein (80K) in the apical membrane-rich fraction purified from the low-speed pellet. Histamine alone or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP), but not carbachol, also stimulated both the redistribution of H+-K+-ATPase and phosphorylation of 80K. Under stimulated conditions, 80K copurified in the apical membrane fraction along with H+-K+-ATPase and actin; whereas purified microsomes from resting stomach were highly enriched in H+-K+-ATPase but contained neither 80K nor actin. Treatment of the apical membranes with detergents, salts, sonication, and so on, led us to conclude that 80K is a membrane protein, unlike actin; however, the mode of association of 80K with membrane differed from H+-K+-ATPase, an integral membrane protein. Isoelectric focusing and peptide mapping revealed that 80K consists of six isomers of slightly differing pI, with 32P occurring only in the three most acidic isomers and exclusively on serine residues. Moreover, stimulation elicited a shift in the amount of 80K isomers, from basic to acidic, as well as phosphorylation. We conclude that 80K is an apical membrane protein in the parietal cell and an important substrate for cAMP-dependent, but not calcium-dependent, pathway of acid secretion.

Quantitation and topography of membrane proteins in highly water-permeable vesicles from ADH-stimulated toad bladder

1991 ◽  
Vol 261 (1) ◽  
pp. C143-C153 ◽  
Author(s):  
H. W. Harris ◽  
M. L. Zeidel ◽  
C. Hosselet
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
Apical Membrane ◽  
Water Channel ◽  
Protein S ◽  
Water Channels ◽  
Toad Bladder ◽  
Western Blots ◽  
Vesicle Protein

Antidiuretic hormone (ADH) stimulation of toad bladder granular cells rapidly increases the osmotic water permeability (Pf) of their apical membranes by insertion of highly selective water channels. Before ADH stimulation, these water channels are stored in large cytoplasmic vesicles called aggrephores. ADH causes aggrephores to fuse with the apical membrane. Termination of ADH stimulation results in prompt endocytosis of water channel-containing membranes via retrieval of these specialized regions of apical membrane. Protein components of the ADH water channel contained within these retrieved vesicles would be expected to be integral membrane protein(s) that span the vesicle's lipid bilayer to create narrow aqueous channels. Our previous work has identified proteins of 55 (actually a 55/53-kDa doublet), 17, 15, and 7 kDa as candidate ADH water channel components. We now have investigated these candidate ADH water channel proteins in purified retrieved vesicles. These vesicles do not contain a functional proton pump as assayed by Western blots of purified vesicle protein probed with anti-H(+)-ATPase antisera. Approximately 60% of vesicle protein is accounted for by three protein bands of 55, 53, and 46 kDa. Smaller contributions to vesicle protein are made by the 17- and 15-kDa proteins. Triton X-114-partitioning analysis shows that the 55, 53, 46, and 17 kDa are integral membrane proteins. Vectorial labeling analysis with two membrane-impermeant reagents shows that the 55-, 53-, and 46-kDa protein species span the lipid bilayer of these vesicles. Thus the 55-, 53-, and 46-kDa proteins possess characteristics expected for ADH water channel components. These data show that the 55- and 53- and perhaps the 46-, 17-, and 15-kDa proteins are likely components of aqueous transmembrane pores that constitute ADH water channels contained within these vesicles.

Immunocytochemical characterization of the high-affinity thiazide diuretic receptor in rabbit renal cortex

1993 ◽  
Vol 264 (1) ◽  
pp. F141-F148 ◽  
Author(s):  
D. H. Ellison ◽  
D. Biemesderfer ◽  
J. Morrisey ◽  
J. Lauring ◽  
G. V. Desir
Keyword(s):  
Thiazide Diuretic ◽  
Apical Membrane ◽  
Distal Tubule ◽  
Rabbit Kidney ◽  
Kidney Cortex ◽  
Thick Ascending Limb ◽  
Western Blots ◽  
Intense Staining ◽  
High Affinity ◽  
Apical Membranes

Thiazide diuretics increase urinary NaCl excretion primarily by inhibiting Na and Cl transport across the apical membrane of cells in the renal distal tubule. Although these diuretics bind to a membrane protein that couples transport of Na and Cl directly, the molecular nature of this transporter and its localization in the mammalian kidney remain controversial. The present experiments were designed to develop monoclonal antibodies to the high-affinity thiazide diuretic receptor to investigate its molecular characteristics and its cellular and subcellular localization in rabbit kidney. Mice were immunized with high-affinity thiazide diuretic receptors that had been partially purified from rabbit kidney cortex. Resulting hybridomas were screened for the ability to immunoprecipitate thiazide diuretic receptors that were labeled with the thiazide-like diuretic [3H]metolazone. A single hybridoma (MAb JM5) produced antibodies capable of immunoprecipitating up to 80% of the labeled thiazide receptors from solubilized renal cortical membranes. MAb JM5 reacted with a 125-kDa protein on Western blots of solubilized renal cortical apical membranes. It stained the apical membrane of cells in the distal convoluted and connecting tubule but did not stain proximal tubules, glomeruli, or interstitial structures. Less intense staining of apical membranes of principal cells in the collecting tubule and a subpopulation of cells in the thick ascending limb were also present. These results indicate that the high-affinity thiazide diuretic receptor comprises a 125-kDa protein that localizes to the apical membrane of cells in the renal distal tubule.

scholarly journals Segmental differences in Slc26a3-dependent Cl− absorption and HCO3− secretion in the mouse large intestine in vitro in Ussing chambers

2021 ◽  
Vol 71 (1) ◽  
Author(s):  
Hisayoshi Hayashi ◽  
Hiroki Nagai ◽  
Kou-ichiro Ohba ◽  
Manoocher Soleimani ◽  
Yuichi Suzuki
Keyword(s):  
Large Intestine ◽  
Anion Exchanger ◽  
Apical Membrane ◽  
Distal Colon ◽  
Proximal Colon ◽  
Acid Base ◽  
Functional Roles ◽  
Ussing Chambers ◽  
Mouse Cecum

AbstractThe anion exchanger slc26a3 (DRA), which is mutated in congenital chloride-losing diarrhea, is expressed in the apical membrane of the cecum and middle-distal colon but not in the proximal colon of rodent large intestines. To elucidate the functional roles of DRA, we measured unidirectional 36Cl− and 22Na+ fluxes and HCO3− secretion in vitro in each of these segments using DRA-KO mice. Robust Cl− absorption, which was largely abolished after DRA deficiency, was present in the cecum and middle-distal colon but absent in the proximal colon. Na+ absorption was present in all three segments in both the control and DRA-KO mice. The luminal-Cl−-dependent HCO3− secretions in the cecum and middle-distal colon were abolished in the DRA-KO mice. In conclusion, DRA mediates Cl− absorption and HCO3− secretion in the mouse cecum and middle-distal colon, and may have roles in H2O absorption and luminal acid/base regulation in these segments.

Basolateral Cl− uptake mechanisms in Xenopus laevis lung epithelium

2010 ◽  
Vol 299 (1) ◽  
pp. R92-R100 ◽  
Author(s):  
Jens Berger ◽  
Martin Hardt ◽  
Wolfgang G. Clauss ◽  
Martin Fronius
Keyword(s):  
Xenopus Laevis ◽  
Ion Transport ◽  
Anion Exchanger ◽  
Short Circuit ◽  
Ussing Chamber ◽  
Short Circuit Current ◽  
Transport Processes ◽  
Lung Epithelium ◽  
Active Ion Transport ◽  
Uptake Mechanisms

A thin liquid layer covers the lungs of air-breathing vertebrates. Active ion transport processes via the pulmonary epithelial cells regulate the maintenance of this layer. This study focuses on basolateral Cl− uptake mechanisms in native lungs of Xenopus laevis and the involvement of the Na+/K+/2 Cl− cotransporter (NKCC) and HCO3−/Cl− anion exchanger (AE), in particular. Western blot analysis and immunofluorescence staining revealed the expression of the NKCC protein in the Xenopus lung. Ussing chamber experiments demonstrated that the NKCC inhibitors (bumetanide and furosemide) were ineffective at blocking the cotransporter under basal conditions, as well as under pharmacologically stimulated Cl−-secreting conditions (forskolin and chlorzoxazone application). However, functional evidence for the NKCC was detected by generating a transepithelial Cl− gradient. Further, we were interested in the involvement of the HCO3−/Cl− anion exchanger to transepithelial ion transport processes. Basolateral application of DIDS, an inhibitor of the AE, resulted in a significantly decreased the short-circuit current (ISC). The effect of DIDS was diminished by acetazolamide and reduced by increased external HCO3− concentrations. Cl− secretion induced by forskolin was decreased by DIDS, but this effect was abolished in the presence of HCO3−. These experiments indicate that the AE at least partially contributes to Cl− secretion. Taken together, our data show that in Xenopus lung epithelia, the AE, rather than the NKCC, is involved in basolateral Cl− uptake, which contrasts with the common model for Cl− secretion in pulmonary epithelia.

The transcytotic pathway of an apical plasma membrane protein (B10) in hepatocytes is similar to that of IgA and occurs via a tubular pericentriolar compartment

1996 ◽  
Vol 109 (6) ◽  
pp. 1215-1227 ◽  
Author(s):  
I. Hemery ◽  
A.M. Durand-Schneider ◽  
G. Feldmann ◽  
J.P. Vaerman ◽  
M. Maurice
Keyword(s):  
Electron Microscopy ◽  
Plasma Membrane ◽  
Membrane Proteins ◽  
Membrane Protein ◽  
Apical Membrane ◽  
Rat Hepatocytes ◽  
Apical Plasma Membrane ◽  
Apical Surface ◽  
Plasma Membrane Protein ◽  
Microtubule Disruption

In hepatocytes, newly synthesized apical plasma membrane proteins are first delivered to the basolateral surface and are supposed to reach the apical surface by transcytosis. The transcytotic pathway of apical membrane proteins and its relationship with other endosomal pathways has not been demonstrated morphologically. We compared the intracellular route of an apical plasma membrane protein, B10, with that of polymeric IgA (pIgA), which is transcytosed, transferrin (Tf) which is recycled, and asialoorosomucoid (ASOR) which is delivered to lysosomes. Ligands and anti-B10 monoclonal IgG were linked to fluorochromes or with peroxidase. The fate of each ligand was followed by confocal and electron microscopy in polarized primary monolayers of rat hepatocytes. When fluorescent anti-B10 IgG and fluorescent pIgA were simultaneously endocytosed for 15–30 minutes, they both uniformly labelled a juxtanuclear compartment. By 30–60 minutes, they reached the bile canaliculi. Tf and ASOR were also routed to the juxtanuclear area, but their fluorescence patterns were more punctate. Microtubule disruption prevented all ligands from reaching the juxtanuclear area. This area corresponded, at least partially, to the localization of the mannose 6-phosphate receptor, an endosomal marker. By electron microscopy, the juxtanuclear compartment was made up of anastomosing tubules connected to vacuoles, and was organized around the centrioles. B10 and pIgA were mainly found in the tubules, whereas ASOR was segregated inside the vacuolar elements and Tf within thinner, recycling tubules. In conclusion, transcytosis of the apical membrane protein B10 occurs inside tubules similar to those carrying pIgA, and involves passage via the pericentriolar area. In the pericentriolar area, the transcytotic tubules appear to maintain connections with other endosomal elements where sorting between recycled and degraded ligands occurs.

Expression of rat kidney anion exchanger 1 in type A intercalated cells in metabolic acidosis and alkalosis

1999 ◽  
Vol 277 (6) ◽  
pp. F841-F849 ◽  
Author(s):  
Saskia Huber ◽  
Esther Asan ◽  
Thomas Jöns ◽  
Christiane Kerscher ◽  
Bernd Püschel ◽  
...  
Keyword(s):  
Anion Exchanger ◽  
Direct Evidence ◽  
Rat Kidney ◽  
Enzyme Reaction ◽  
Type A ◽  
Mrna Levels ◽  
Intercalated Cells ◽  
Acid Base ◽  
Anion Exchanger 1 ◽  
Acid Base Status

By enzyme-linked in situ hybridization (ISH), direct evidence is provided that acid-secreting intercalated cells (type A IC) of both the cortical and medullary collecting ducts of the rat kidney selectively express the mRNA of the kidney splice variant of anion exchanger 1 (kAE1) and no detectable levels of the erythrocyte AE1 (eAE1) mRNA. Using single-cell quantification by microphotometry of ISH enzyme reaction, medullary type A IC were found to contain twofold higher kAE1 mRNA levels compared with cortical type A IC. These differences correspond to the higher intensity of immunostaining in medullary versus cortical type A IC. Chronic changes of acid-base status induced by addition of NH4Cl (acidosis) or NaHCO3 (alkalosis) to the drinking water resulted in up to 35% changes of kAE1 mRNA levels in both cortical and medullary type A IC. These experiments provide direct evidence at the cellular level of kAE1 expression in type A IC and show moderate capacity of type A IC to respond to changes of acid-base status by modulation of kAE1 mRNA levels.

GLP-1 is localized to the mitotic region of the C. elegans germ line

Development ◽  
1994 ◽  
Vol 120 (10) ◽  
pp. 2901-2911 ◽  
Author(s):  
S.L. Crittenden ◽  
E.R. Troemel ◽  
T.C. Evans ◽  
J. Kimble
Keyword(s):  
Membrane Protein ◽  
Germ Line ◽  
Integral Membrane Protein ◽  
Membrane Receptor ◽  
Down Regulation ◽  
Western Blots ◽  
Tip Cell ◽  
C Elegans ◽  
Cell Signal ◽  
Translational Level

In C. elegans, germline mitosis depends on induction by the somatic distal tip cell (DTC) and on activity of the glp-1 gene. Using antibodies to GLP-1 protein, we have examined GLP-1 on western blots and by immunocytochemistry. GLP-1 is tightly associated with membranes of mitotic germline cells, supporting its identification as an integral membrane protein. Furthermore, GLP-1 is localized within the germ line to the mitotic region, consistent with the model that GLP-1 acts as a membrane receptor for the distal tip cell signal. Unexpectedly, GLP-1 and the zone of mitosis extend further than the DTC processes. We present three models by which the DTC may influence GLP-1 activity and thereby determine the zone of mitosis. The spatial restriction of GLP-1 appears to be controlled at the translational level in hermaphrodites. We suggest that down-regulation of GLP-1 may be required to effect the transition from mitosis into meiosis.

Time-dependent apical membrane K+ and Na+ selectivity in cultured kidney cells

1987 ◽  
Vol 253 (1) ◽  
pp. C1-C6 ◽  
Author(s):  
S. R. Thomas ◽  
E. Mintz
Keyword(s):  
Apical Membrane ◽  
Time Dependent ◽  
Kidney Cells ◽  
K Channels ◽  
Membrane Selectivity ◽  
Conductive Pathway ◽  
Group I ◽  
A6 Epithelia ◽  
Group Ii ◽  
Apical Membranes

Intracellular microelectrodes were used to study apical membrane selectivity to Na+ and K+ of cultured toad kidney cells (A6) grown on permeable supports. Membrane selectivity was tested by responses of apical membrane potential to replacement of Na+ by K+ or tetraethylammonium and by addition of amiloride to perfusion solutions. The A6 epithelia fell into two groups: those with K+-selective apical membranes, lack of amiloride sensitivity, and near-zero transepithelial potential (group I); and those with Na+-selective apical membranes and a serosa-positive, amiloride-sensitive transepithelial potential (Vm----s; group II). The transition from group I to group II behavior appeared definitive and time dependent, occurring approximately 10 days after plating onto filters. Transepithelial measurements under sterile conditions showed that overnight incubation with aldosterone (10(-7) M), after development of tight junctions (transepithelial resistance elevated) but before development of significant Vm----s, induced the switch from group I to group II behavior. Apical addition of Ba2+, a known blocker of K+ channels, unexpectedly reduced transepithelial resistance (Rm----s) in group I and group II A6, suggesting that it not only blocked K+ channels (when they are present) but may also open a parallel conductive pathway. In summary, after approximately 10 days in culture, apical membranes of A6 epithelia undergo a switch from K+ to Na+ selectivity, overnight incubation with aldosterone can trigger this change, and finally, Ba2+ may open a paracellular conductive pathway.

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