Differential regulation of cholera toxin-inhibited Na-H exchange isoforms by butyrate in rat ileum

2007 ◽  
Vol 293 (4) ◽  
pp. G857-G863 ◽  
Author(s):  
Sandeep B. Subramanya ◽  
Vazhaikkurichi M. Rajendran ◽  
Pugazhendhi Srinivasan ◽  
Navalpur S. Nanda Kumar ◽  
Balakrishnan S. Ramakrishna ◽  
...  
Keyword(s):  
Protein Expression ◽  
Cholera Toxin ◽  
Apical Membrane ◽  
Membrane Vesicles ◽  
Ileal Loop ◽  
Camp Content ◽  
Nhe Isoforms ◽  
Apical Membrane Vesicles

Electroneutral Na absorption occurs in the intestine via sodium-hydrogen exchanger (NHE) isoforms NHE2 and NHE3. Bicarbonate and butyrate both stimulate electroneutral Na absorption through NHE. Bicarbonate- but not butyrate-dependent Na absorption is inhibited by cholera toxin (CT). Long-term exposure to butyrate also influences expression of apical membrane proteins in epithelial cells. These studies investigated the effects of short- and long-term in vivo exposure to butyrate on apical membrane NHE and mRNA, protein expression, and activity in rat ileal epithelium that had been exposed to CT. Ileal loops were exposed to CT in vivo for 5 h and apical membrane vesicles were isolated. 22Na uptake was measured by using the inhibitor HOE694 to identify NHE2 and NHE3 activity, and Western blot analyses were performed. CT reduced total NHE activity by 70% in apical membrane vesicles with inhibition of both NHE2 and NHE3. Reduced NHE3 activity and protein expression remained low following removal of CT but increased to control values following incubation of the ileal loop with butyrate for 2 h. In parallel there was a 40% decrease in CT-induced increase in cAMP content. In contrast, NHE2 activity partially increased following removal of CT and was further increased to control levels by butyrate. NHE2 protein expression did not parallel its activity. Neither NHE2 nor NHE3 mRNA content were affected by CT or butyrate. These results indicate that CT has varying effects on the two apical NHE isoforms, inhibiting NHE2 activity without altering its protein expression and reducing both NHE3 activity and protein expression. Butyrate restores both CT-inhibited NHE2 and NHE3 activities to normal levels but via different mechanisms.

Rat proximal NHE3 adapts to chronic acid-base disorders but not to chronic changes in dietary NaCl intake

2002 ◽  
Vol 282 (5) ◽  
pp. F835-F843 ◽  
Author(s):  
Dominique Eladari ◽  
Françoise Leviel ◽  
Françoise Pezy ◽  
Michel Paillard ◽  
Régine Chambrey
Keyword(s):  
Initial Rate ◽  
Apical Membrane ◽  
Membrane Vesicles ◽  
High Dose ◽  
Protein Abundance ◽  
Sprague Dawley ◽  
Acid Base ◽  
Sprague Dawley Rats ◽  
Apical Membrane Vesicles

In the proximal tubule, the apical Na+/H+ exchanger identified as NHE3 mediates most NaCl and NaHCO3 absorption. The purpose of this study was to analyze the long-term regulation of NHE3 during alkalosis induced by dietary NaHCO3 loading and changes in NaCl intake. Sprague-Dawley rats exposed to a low-NaCl, high-NaCl, or NaHCO3 diet for 6 days were studied. Renal cortical apical membrane vesicles (AMV) were prepared from treated and normal rats. Na+/H+ exchange was assayed as the initial rate of 22Na+ uptake in the presence of an outward H+ gradient. 22Na+uptake measured in the presence of high-dose 5-( N-ethyl- N-isopropyl) amiloride was not different among models. Changes in NaCl intake did not affect NHE3 activity, whereas NaHCO3 loading inhibited22Na+ uptake by 30%. AMV NHE3 protein abundance assessed by Western blot analysis was unaffected during changes in NaCl intake. During NaHCO3 loading, NHE3 protein abundance was decreased by 65%. We conclude that proximal NHE3 adapts to chronic metabolic acid-base disorders but not to changes in dietary NaCl intake.

Chloroquine stimulates absorption and inhibits secretion of ileal water and electrolytes

1982 ◽  
Vol 243 (2) ◽  
pp. G117-G126
Author(s):  
R. Fogel ◽  
G. W. Sharp ◽  
M. Donowitz
Keyword(s):  
Cholera Toxin ◽  
Calcium Content ◽  
Intracellular Camp ◽  
Rabbit Ileum ◽  
Camp Content ◽  
Serosal Surface ◽  
Ileal Absorption ◽  
Camp Levels

The effects of chloroquine diphosphate, a drug with "'membrane-stabilizing" properties, were studied on basal ileal absorption and on ileal secretion induced by increased intracellular cAMP levels and calcium (serotonin). The studies were performed on rat (in vivo) and rabbit ileum (in vitro). Intraluminal chloroquine (10(-4) M) reversed cholera toxin- and theophylline-induced secretion in rat ileum but did not alter the cholera toxin- and theophylline-induced increases in cAMP content. Addition of chloroquine (10(-4) M) to the mucosal surface of rabbit ileum did not alter basal active electrolyte transport or the serotonin-induced decreased Na and Cl absorption but inhibited the theophylline-induced C1 secretion. Addition of chloroquine (10(-4)) M) to the serosal surface stimulated net Na and Cl absorption. This effect may involve intracellular calcium. Chloroquine increased the rabbit ileal calcium content and decreased 45Ca2+ influx from the serosal surface. Both the mucosal and serosal effects of chloroquine described led to a net increase in absorptive function of the intestine and should prove useful in developing treatment of diarrheal diseases.

Distribution and regulation of apical Cl/anion exchanges in surface and crypt cells of rat distal colon

1999 ◽  
Vol 276 (1) ◽  
pp. G132-G137 ◽  
Author(s):  
Vazhaikkurichi M. Rajendran ◽  
Henry J. Binder
Keyword(s):  
Spatial Distribution ◽  
Concentration Gradient ◽  
Apical Membrane ◽  
Membrane Vesicles ◽  
Distal Colon ◽  
Ph Homeostasis ◽  
Rat Distal Colon ◽  
Na Depletion ◽  
Crypt Cells ◽  
Apical Membrane Vesicles

Na depletion inhibits electroneutral Na-Cl absorption in intact tissues and Na/H exchange in apical membrane vesicles (AMV) of rat distal colon. Two anion (Cl/HCO3 and Cl/OH) exchanges have been identified in AMV from surface cells of rat distal colon. To determine whether Cl/HCO3 and/or Cl/OH exchange is responsible for vectorial Cl movement, this study examined the spatial distribution and the effect of Na depletion on anion-dependent 36Cl uptake by AMV in rat distal colon. These studies demonstrate that HCO3 concentration gradient-driven36Cl uptake (i.e., Cl/HCO3 exchange) is 1) primarily present in AMV from surface cells and 2) markedly reduced by Na depletion. In contrast, OH concentration gradient-driven36Cl uptake (i.e., Cl/OH exchange) present in both surface and crypt cells is not affected by Na depletion. In Na-depleted animals HCO3 also stimulates36Cl via Cl/OH exchange with low affinity. These results suggest that Cl/HCO3 exchange is responsible for vectorial Cl absorption, whereas Cl/OH exchange is involved in cell volume and/or cell pH homeostasis.

Cl-HCO3 and Cl-OH exchanges mediate Cl uptake in apical membrane vesicles of rat distal colon

1993 ◽  
Vol 264 (5) ◽  
pp. G874-G879 ◽  
Author(s):  
V. M. Rajendran ◽  
H. J. Binder
Keyword(s):  
Apical Membrane ◽  
Kinetic Constant ◽  
Membrane Vesicles ◽  
Distal Colon ◽  
Ph Gradient ◽  
Disulfonic Acid ◽  
Ph Homeostasis ◽  
Rat Distal Colon ◽  
Hyperbolic Curve ◽  
Apical Membrane Vesicles

This study describes Cl-HCO3 and Cl-OH exchanges as the mechanism for Cl uptake by apical membrane vesicles (AMV) of rat distal colon. Although HCO3 gradient-stimulated 36Cl uptake was additionally stimulated by the additional presence of a pH gradient, pH gradient-stimulated 36Cl uptake was not further enhanced by a HCO3 gradient. HCO3 gradient-stimulated and OH gradient-stimulated 36Cl uptake was not inhibited by voltage clamping, with K and its ionophore valinomycin, but was inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, an anion exchange inhibitor, with an apparent inhibitory constant of 7.8 and 106.0 microM, respectively. Increasing intravesicular OH concentration in the absence of HCO3 (with fixed extravesicular Cl concentration) yielded a sigmoidal curve for 36Cl uptake. In contrast, increasing intravesicular OH concentration in the presence of equimolar intra- and extravesicular HCO3 (25 mM) yielded a saturable hyperbolic curve. Increasing extravesicular Cl concentration saturated both HCO3 gradient-stimulated and OH gradient-stimulated 36Cl uptake, with a kinetic constant for Cl of approximately 11.9 and 22.6 mM, respectively. We conclude that Cl uptake in AMV of rat distal colon occurs via two separate anion (Cl-HCO3 and Cl-OH) exchange processes. We speculate that one of these two anion exchanges may be responsible for transcellular Cl movement, while the other may be important in the regulation of intracellular pH homeostasis.

scholarly journals SELECTIVE RELEASE OF CONTENT FROM MICROSOMAL VESICLES WITHOUT MEMBRANE DISASSEMBLY

1974 ◽  
Vol 61 (3) ◽  
pp. 789-807 ◽  
Author(s):  
Gert Kreibich ◽  
David D. Sabatini
Keyword(s):  
Serum Proteins ◽  
Specific Activity ◽  
Membrane Vesicles ◽  
Rat Serum ◽  
Coomassie Blue ◽  
Specific Subset ◽  
Low Levels ◽  
Almost All

Rough and smooth microsomes were shown to have similar sets of polypeptide chains except for the proteins of ribosomes bound to the rough endoplasmic reticulum (ER). More than 50 species of polypeptides were detected by acrylamide gel electrophoresis, ranging in molecular weight from 10,000 to approximately 200,000 daltons. The content of rough and smooth microsomes was separated from the membrane vesicles using sublytic concentrations of detergents and differential centrifugation. A specific subset of proteins which consisted of approximately 25 polypeptides was characteristic of the microsomal content. Some of these proteins showed high rates of in vivo incorporation of radioactive leucine or glucosamine, but several others incorporated only low levels of radioactivity within short labeling intervals and appeared to be long-term residents of the lumen of the ER. Seven polypeptides in the content subfractions, including serum albumin, contained almost 50% of the leucine radioactivity incorporated during 5 min and cross-reacted with antiserum against rat serum. Almost all microsomal glycoproteins were at least partly released with the microsomal content. Smooth microsomes contained higher levels of albumin than rough microsomes, but after short times of labeling with [3H]leucine the specific activity of albumin in the latter was higher, supporting the notion that newly synthesized serum proteins are transferred from rough to smooth portions of the ER. On the other hand, after labeling for 30 min with [3H]glucosamine, smooth microsomes contained higher levels of radioactivity than rough microsomes. This would be expected if glycosidation of newly synthesized polypeptides proceeds during their transit through ER cisternae. The labeling pattern of membrane proteins in microsomes obtained from animals which received three daily injections of [3H]leucine, the last administered 1 day before sacrifice, followed the intensity of bands stained with Coomassie blue, with a main radioactive peak corresponding to cytochrome P 450. After the long-term labeling procedure most content proteins had low levels of radioactivity; this was especially true of serum proteins which were highly labeled after 30 min.

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