Apical membrane localization of ouabain-sensitive K(+)-activated ATPase activities in rat distal colon

1991 ◽  
Vol 261 (6) ◽  
pp. G1005-G1011 ◽  
Author(s):  
J. R. Del Castillo ◽  
V. M. Rajendran ◽  
H. J. Binder
Keyword(s):  
Atpase Activity ◽  
Parietal Cell ◽  
Colonic Mucosa ◽  
Apical Membrane ◽  
Distal Colon ◽  
Proximal Colon ◽  
Gastric Parietal Cell ◽  
Fold Enrichment ◽  
Rat Distal Colon ◽  
Apical Membranes

This study sought to establish the presence of K(+)-activated adenosinetriphosphatase (ATPase) activity in the colonic mucosa of the rat distal colon. K(+)-activated ATPase activity was present in apical membranes but not in basolateral membranes. K(+)-activated ATPase activity in apical membranes represented an approximate 10-fold enrichment compared with that in the homogenate. Na(+)-K(+)-activated ATPase activity was also present in homogenate but was enriched less than fourfold in apical membranes. K(+)-activated ATPase activity in apical membranes had both ouabain-sensitive and ouabain-insensitive components. In contrast, Na(+)-K(+)-activated ATPase activity was completely inhibited by ouabain. Similar half-maximal concentrations for K+ and pH activation curves were found for both ouabain-sensitive and ouabain-insensitive fractions. In addition to K+, the ouabain-sensitive fraction of K(+)-activated ATPase activity was stimulated by Rb+, NH+4, and Cs+, whereas the ouabain-insensitive fraction was activated only by Rb+. K(+)-activated ATPase activity was significantly inhibited by vanadate but not by N-ethylmaleimide or omeprazole. In the proximal colon, in contrast to the distal colon, active K+ absorption is not present, and K(+)-activated ATPase is approximately 20% of that in the distal colon. These studies demonstrate that K(+)-activated ATPase is present in apical membranes of rat distal colon and permit the speculation that this enzyme represents a unique and distinct ATPase (compared with either Na(+)-K(+)-ATPase or gastric parietal cell K(+)-ATPase) and is likely linked closely to the active K+ absorptive process present in this epithelium.

Differential localization of colonic H+-K+-ATPase isoforms in surface and crypt cells

1998 ◽  
Vol 274 (2) ◽  
pp. G424-G429 ◽  
Author(s):  
Vazhaikkurichi M. Rajendran ◽  
Satish K. Singh ◽  
John Geibel ◽  
Henry J. Binder
Keyword(s):  
Spatial Distribution ◽  
Atpase Activity ◽  
Distal Colon ◽  
Compelling Evidence ◽  
Α Subunit ◽  
Colonic Crypts ◽  
Rat Distal Colon ◽  
Acid Load ◽  
Apical Membranes ◽  
Crypt Cells

Two distinct colonic H+-K+-adenosinetriphosphatase (H+-K+-ATPase) isoforms can be identified in part on the basis of their sensitivity to ouabain. The colonic H+-K+-ATPase α-subunit (HKcα) was recently cloned, and its message and protein are present in surface (and the upper 20% of crypt) cells in the rat distal colon. These studies were performed to establish the spatial distribution of the ouabain-sensitive and ouabain-insensitive components of both H+-K+-ATPase activity in apical membranes prepared from surface and crypt cells and K+-dependent intracellular pH (pHi) recovery from an acid load both in isolated perfused colonic crypts and in surface epithelial cells. Whereas H+-K+-ATPase activity in apical membranes from surface cells was 46% ouabain sensitive, its activity in crypt apical membranes was 96% ouabain sensitive. Similarly, K+-dependent pHi recovery in isolated crypts was completely ouabain sensitive, whereas in surface cells K+-dependent pHi recovery was insensitive to ouabain. These studies provide compelling evidence that HKcα encodes the colonic ouabain-insensitive H+-K+-ATPase and that a colonic ouabain-sensitive H+-K+-ATPase isoform is present in colonic crypts and remains to be cloned and identified.

Three distinct mechanisms of HCO3−secretion in rat distal colon

2004 ◽  
Vol 287 (3) ◽  
pp. C612-C621 ◽  
Author(s):  
Sadasivan Vidyasagar ◽  
Vazhaikkurichi M. Rajendran ◽  
Henry J. Binder
Keyword(s):  
Colonic Mucosa ◽  
Short Chain Fatty Acid ◽  
Channel Blocker ◽  
Apical Membrane ◽  
Anion Channel ◽  
Distal Colon ◽  
Chain Fatty Acid ◽  
Direct Examination ◽  
Rat Distal Colon ◽  
Ph Stat

HCO3−secretion has long been recognized in the mammalian colon, but it has not been well characterized. Although most studies of colonic HCO3−secretion have revealed evidence of lumen Cl−dependence, suggesting a role for apical membrane Cl−/HCO3−exchange, direct examination of HCO3−secretion in isolated crypt from rat distal colon did not identify Cl−-dependent HCO3−secretion but did reveal cAMP-induced, Cl−-independent HCO3−secretion. Studies were therefore initiated to determine the characteristics of HCO3−secretion in isolated colonic mucosa to identify HCO3−secretion in both surface and crypt cells. HCO3−secretion was measured in rat distal colonic mucosa stripped of muscular and serosal layers by using a pH stat technique. Basal HCO3−secretion (5.6 ± 0.03 μeq·h−1·cm−2) was abolished by removal of either lumen Cl−or bath HCO3−; this Cl−-dependent HCO3−secretion was also inhibited by 100 μM DIDS (0.5 ± 0.03 μeq·h−1·cm−2) but not by 5-nitro-3-(3-phenylpropyl-amino)benzoic acid (NPPB), a Cl−channel blocker. 8-Bromo-cAMP induced Cl−-independent HCO3−secretion (and also inhibited Cl−-dependent HCO3−secretion), which was inhibited by NPPB and by glibenclamide, a CFTR blocker, but not by DIDS. Isobutyrate, a poorly metabolized short-chain fatty acid (SCFA), also induced a Cl−-independent, DIDS-insensitive, saturable HCO3−secretion that was not inhibited by NPPB. Three distinct HCO3−secretory mechanisms were identified: 1) Cl−-dependent secretion associated with apical membrane Cl−/HCO3−exchange, 2) cAMP-induced secretion that was a result of an apical membrane anion channel, and 3) SCFA-dependent secretion associated with an apical membrane SCFA/HCO3−exchange.

Ouabain-sensitive K(+)-ATPase in epithelial cells from guinea pig distal colon

1990 ◽  
Vol 258 (4) ◽  
pp. G506-G511 ◽  
Author(s):  
T. Watanabe ◽  
T. Suzuki ◽  
Y. Suzuki
Keyword(s):  
Guinea Pig ◽  
Atpase Activity ◽  
Apical Membrane ◽  
Distal Colon ◽  
Proximal Colon ◽  
Maximal Effect ◽  
Colonic Epithelial Cell ◽  
Entire Colon ◽  
Cell Homogenate ◽  
Crude Membrane Fraction

We have previously shown that an ouabain-sensitive H(+)-K+ exchange mechanism may be present in the apical membrane of guinea pig distal colon [Y. Suzuki and K. Kaneko. Am. J. Physiol. 256 (Gastrointest. Liver Physiol. 19): G979-G988, 1989]. The present study is aimed to demonstrate the presence of an ATPase responsible for this exchange. ATPase activity was determined in the crude membrane fraction of the colonic epithelial cell homogenate. ATPase activity under Na(+)-free conditions was increased by the addition of K+, with a half-maximal effect at 55 microM. This increase was completely abolished by 1 mM ouabain, suggesting the presence of an ouabain-sensitive K(+)-ATPase. The ouabain-sensitive K(+)-ATPase activity was inhibited by vanadate (100 microM) and N,N'-dicyclohexylcarbodiimide (100 microM) but was resistant to oligomycin (4.5 micrograms/ml) and NaN3 (1 mM). The ouabain-sensitive K(+)-ATPase activity was observed in the distal but not in the proximal colon, whereas Na(+)-K(+)-ATPase activity was distributed along the entire colon. Omeprazole (40 microM) reduced the colonic K(+)-ATPase activity by 31 +/- 6%, whereas it reduced the gastric K(+)-ATPase activity by 78 +/- 8%. These results suggest that the ouabain-sensitive K(+)-ATPase as demonstrated here is responsible for the colonic H(+)-K+ exchange. This ATPase could be similar to but is not identical with either Na(+)-K(+)-ATPase or gastric H(+)-K(+)-ATPase.

Colonic H-K-ATPase β-subunit: identification in apical membranes and regulation by dietary K depletion

1999 ◽  
Vol 276 (2) ◽  
pp. C350-C360 ◽  
Author(s):  
Pitchai Sangan ◽  
Sarah S. Kolla ◽  
Vazhaikkurichi M. Rajendran ◽  
Michael Kashgarian ◽  
Henry J. Binder
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Distal Colon ◽  
Proximal Colon ◽  
Mrna Abundance ◽  
Β Subunit ◽  
Physical Association ◽  
Rat Distal Colon ◽  
Apical Membranes ◽  
K Depletion

P-type ATPases require both α- and β-subunits for functional activity. Although an α-subunit for colonic apical membrane H-K-ATPase (HKcα) has been identified and studied, its β-subunit has not been identified. We cloned putative β-subunit rat colonic H-K-ATPase (HKcβ) cDNA that encodes a 279-amino-acid protein with a single transmembrane domain and sequence homology to other rat β-subunits. Northern blot analysis demonstrates that this HKcβ is expressed in several rat tissues, including distal and proximal colon, and is highly expressed in testis and lung. HKcβ mRNA abundance is upregulated threefold compared with normal in distal colon but not proximal colon, testis, or lung of K-depleted rats. In contrast, Na-K-ATPase β1 mRNA abundance is unaltered in distal colon of K-depleted rats. Na depletion, which also stimulates active K absorption in distal colon, does not increase HKcβ mRNA abundance. Western blot analyses using a polyclonal antibody raised to a glutathione S-transferase-HKcβ fusion protein established expression of a 45-kDa HKcβ protein in both apical and basolateral membranes of rat distal colon, but K depletion increased HKcβ protein expression only in apical membranes. Physical association between HKcβ and HKcα proteins was demonstrated by Western blot analysis performed with HKcβ antibody on immunoprecipitate of apical membranes of rat distal colon and HKcα antibody. Tissue-specific upregulation of this β-subunit mRNA in response to K depletion, localization of its protein, its upregulation by K depletion in apical membranes of distal colon, and its physical association with HKcα protein provide compelling evidence that HKcβ is the putative β-subunit of colonic H-K-ATPase.

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.

Corticosteroid alteration of active electrolyte transport in rat distal colon

1983 ◽  
Vol 245 (5) ◽  
pp. G668-G675 ◽  
Author(s):  
E. S. Foster ◽  
T. W. Zimmerman ◽  
J. P. Hayslett ◽  
H. J. Binder
Keyword(s):  
Colonic Mucosa ◽  
Short Circuit ◽  
Short Circuit Current ◽  
Distal Colon ◽  
Transport Processes ◽  
Electrolyte Transport ◽  
Sodium Absorption ◽  
Rat Distal Colon ◽  
Potassium Absorption ◽  
Potassium Secretion

To determine the effect of corticosteroids on active transport processes, unidirectional fluxes of 22Na, 36Cl, and 42K were measured under short-circuit conditions across isolated stripped distal colonic mucosa of the rat in control, secondary hyperaldosterone, and dexamethasone-treated animals. In controls net sodium and chloride fluxes (JNanet and JClnet) and short-circuit current (Isc) were 6.6 +/- 2.2, 7.6 +/- 1.6, and 1.3 +/- 0.2 mu eq X h-1 X cm-2, respectively. Although aldosterone increased Isc to 7.3 +/- 0.5 mu eq X h-1 X cm-2, JNanet (6.9 +/- 0.7 mu eq X h-1 X cm-2) was not altered and JClnet was reduced to 0 compared with controls. Dexamethasone also stimulated Isc but did not inhibit JClnet. In Cl-free Ringer both aldosterone and dexamethasone produced significant and equal increases in JNanet and Isc. Theophylline abolished JNanet in control animals but not in the aldosterone group. Aldosterone reversed net potassium absorption (0.58 +/- 0.11 mu eq X h-1 X cm-2) to net potassium secretion (-0.94 +/- 0.08 mu eq X h-1 X cm-2). Dexamethasone reduced net potassium movement to 0 (-0.04 +/- 0.12 mu eq X h-1 X cm-2). These studies demonstrate that 1) corticosteroids stimulate electrogenic sodium absorption and 2) aldosterone, but not dexamethasone, inhibits neutral NaCl absorption and stimulates active potassium secretion. The effects of mineralocorticoids and glucocorticoids on electrolyte transport are not identical and may be mediated by separate and distinct mechanisms.

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.

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.

Cl- channels of the gastric parietal cell that are active at low pH

1993 ◽  
Vol 264 (6) ◽  
pp. C1609-C1618 ◽  
Author(s):  
J. Cuppoletti ◽  
A. M. Baker ◽  
D. H. Malinowska
Keyword(s):  
Gastric Mucosa ◽  
Atpase Activity ◽  
Parietal Cell ◽  
Asymmetric Reduction ◽  
Ion Selectivity ◽  
Channel Activity ◽  
Open Probability ◽  
Gastric Parietal Cell ◽  
Cl Channel ◽  
Cl Channels

HCl secretion across mammalian gastric parietal cell apical membrane may involve Cl- channels. H(+)-K(+)-ATPase-containing membranes isolated from gastric mucosa of histamine-stimulated rabbits were fused to planar lipid bilayers. Channels were recorded with symmetric 800 mM CsCl solutions, pH 7.4. A linear current-voltage (I-V) relationship was obtained, and conductance was 28 +/- 1 pS at 800 mM CsCl. Conductance was 6.9 +/- 2 pS at 150 mM CsCl. Reversal potential was +22 mV with a fivefold cis-trans CsCl concentration gradient, indicating that the channel was anion selective with a discrimination ratio of 6:1 for Cl- over Cs+. Anion selectivity of the channel was I- > Cl- > or = Br- > NO3-, and gluconate was impermeant. Channels obtained at pH 7.4 persisted when pH of medium bathing the trans side of the bilayer (pHtrans) was reduced to pH 3, without a change in conductance, linearity of I-V relationship, or ion selectivity. In contrast, asymmetric reduction of pH of medium bathing the cis side of the bilayer from 7.4 to 3 always resulted in loss of channel activity. At pH 7.4, open probability (Po) of the channel was voltage dependent, i.e., predominantly open at +80 mV but mainly closed at -80 mV. In contrast, with low pHtrans, channel Po at -80 mV was increased 3.5-fold. The Cl- channel was Ca2+ indifferent. In absence of ionophores, ion selectivity for support of H(+)-K(+)-ATPase activity and H+ transport was consistent with that exhibited by the channel and could be limited by substitution with NO3-, whereas maximal H(+)-K(+)-ATPase activity was indifferent to anion present, demonstrating that anion transport can be rate limiting. Cl- channels with similar characteristics (conductance, linear I-V relationship, and ion selectivity) were also present in H(+)-K(+)-ATPase-containing vesicles isolated from resting (cimetidine-treated) gastric mucosa, exhibiting at -80 mV a pH-independent approximately 3.5-fold lower Po than stimulated vesicle channels. At -80 mV, reduction of pHtrans increased Po of both resting and stimulated Cl- channels by five- to sixfold. Changing membrane potential from 0 to -80 mV across stimulated vesicles increased Cl- channel activity an additional 10-fold.(ABSTRACT TRUNCATED AT 400 WORDS)

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