Exposure to hookworms in patients with Crohn's disease: a case-control study.

BACKGROUND: Helminths have been used to inhibit intestinal inflammation in patients with Crohn's disease. AIM: This study was undertaken to determine if there is a protective association of prior hookworm infection with Crohn's disease, in a region where there is epidemiological transition from parasitic and infectious diseases to increased auto-inflammatory diseases. METHODS: Hookworm exposure was assessed by peripheral blood mononuclear cell (PBMC) activation by hookworm antigens in 78 patients with Crohn's disease and 75 healthy control participants. The change in proportion of T cells exhibiting CD69 after exposure to crude hookworm antigens was measured. Interferon-γ ELISPOT response to a panel of six recombinant hookworm antigens was analysed. RESULTS: Patients with Crohn's disease were more often from an urban background (P=0.005) compared to controls, while their socioeconomic status was not significantly different. T cell activation (increase in CD3(+) CD69(+) population) by hookworm antigen was significantly higher in controls compared to Crohn's disease patients (P=0.017), while activation by the nonspecific mitogen phytohemagglutinin was similar in both groups. Circulating T memory cells (CD3(+) CD45RO(+)) after exposure to hookworm antigens were not significantly different between the two groups. Mirroring these changes, interferon-γ ELISPOT responses to hookworm antigens were seen in 36 of 75 controls compared to 20 of 78 Crohn's disease patients (Fisher's exact P=0.005). Multivariate analysis indicated that CD3CD69 shifts (P=0.019), ELISPOT reactivity (P=0.039) and place of residence (P=0.024) were all independently associated with Crohn's disease. CONCLUSION: The inverse association between Crohn's disease and hookworm antigen reactivity is consistent with the hygiene hypothesis, but requires further exploration.

Morphology of isolated colonic crypts.

The traditional paradigm of colonic fluid and electrolyte transport includes a spatial separation of absorptive and secretory processes to surface and crypt cells, respectively. Recent studies of isolated microperfused colonic crypts revealed constitutive Na-dependent fluid absorption while secretion is regulated by one or more neurohumoral agonists. One obvious reason for the difference found in microdissected crypts is their separation from the lamina propria milieu. While it has been shown that isolated crypts are devoid of obvious lamina propria elements, including pericryptal fibroblasts, detailed morphologic information of the content of isolated crypts has been lacking. To characterize the morphology of the isolated crypt, we performed transmission electron microscopy (TEM) and immunofluorescence on microdissected and Ca2+ chelated crypts. Crypt cell type analysis was carried out separately on intact rat colon using light microscopy. TEM revealed a complete lack of either lamina propria cells or extracellular material in crypts isolated by either technique. TEM also revealed a subtle difference between the two isolation methods, with intact basal membranes in microdissected crypts but focal disruption of basal membranes in Ca2+- chelated crypts. Immunofluorescent stains for two basement membrane components (laminin and collagen type IV) revealed the presence of adherent basement membrane only on microdissected crypts; evidence that the plane of separation differs in these two preparations. Crypt cell type analysis on intact rat colon revealed an equal proportion of goblet cells in the right and left colon (approximately 50%) when measuring the middle 70% of the crypts - the area studied during crypt microperfusion. This morphologic analysis will increase our understanding of the observed physiology of isolated colonic crypts.

Na(+)-dependent fluid absorption in intact perfused rat colonic crypts.

BACKGROUND & AIMS: The traditional paradigm of fluid movement in the mammalian colon is that fluid absorption and secretion are present in surface and crypt cells, respectively. We have recently demonstrated Na(+)-dependent fluid absorption in isolated crypts that are devoid of neurohumoral stimulation. We now explore the mechanism of Na(+)-dependent fluid absorption in isolated rat colonic crypts. METHODS: Net fluid absorption was determined using microperfusion techniques and methoxy[(3)H]inulin with ion substitutions and transport inhibitors. RESULTS: Net fluid absorption was reduced but not abolished by substitution of either N-methyl-D-glucamine- Cl(-) or tetramethylammonium for Na(+) and by lumen addition of 5-ethylisopropyl amiloride, an amiloride analogue that selectively inhibits Na(+)-H(+) exchange. Net fluid absorption was also dependent on lumen Cl(-) because removal of lumen Cl(-) significantly (P < 0.001) reduced net fluid absorption. DIDS at 100 micromol/L, a concentration at which DIDS is an anion exchange inhibitor, minimally reduced net fluid absorption (P < 0.05). In contrast, either 500 micromol/L DIDS, a concentration at which DIDS is known to act as a Cl(-) channel blocker, or 10 micromol/L NPPB, a Cl(-) channel blocker, both substantially inhibited net fluid absorption (P < 0.001). Finally, both the removal of bath Cl(-) and addition of bath bumetanide, an inhibitor of Na-K-2Cl cotransport and Cl(-) secretion, resulted in a significant increase in net fluid absorption. CONCLUSIONS: (1) Net Na(+)-dependent net fluid absorption in the isolated colonic crypt represents both a larger Na(+)-dependent absorptive process and a smaller secretory process; and (2) the absorptive process consists of a Na(+)-dependent, HCO(3)(-)-independent process and a Na(+)-independent, Cl(-)-dependent, HCO(3)(-)-dependent process. Fluid movement in situ represents these transport processes plus fluid secretion induced by neurohumoral stimulation.

Characterization of apical membrane Cl-dependent Na/H exchange in crypt cells of rat distal colon.

A novel Cl-dependent Na/H exchange (Cl-NHE) has been identified in apical membranes of crypt cells of rat distal colon. The presence of Cl is required for both outward proton gradient-driven Na uptake in apical membrane vesicles (AMV) and Na-dependent intracellular pH recovery from an acid load in the crypt gland. The present study establishes that Cl-dependent outward proton gradient-driven (22)Na uptake 1) is saturated with increasing extravesicular Na concentration with a Michaelis constant (K(m)) for Na of approximately 24.2 mM; 2) is saturated with increasing outward H concentration gradient with a hyperbolic curve and a K(m) for H of approximately 1.5 microM; 3) is inhibited by the Na/H exchange (NHE) inhibitors amiloride, ethylisopropylamiloride, and HOE-694 with an inhibitory constant (K(i)) of approximately 480.2, 1.1, and 9.5 microM, respectively; 4) is inhibited by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, an anion exchange inhibitor at low concentration and a Cl channel blocker at high dose, and by 5-nitro-2(3-phenylpropylamino)benzoic acid, a Cl channel blocker, with a K(i) of approximately 280.6 and 18.3 microM, respectively; and 5) substantially stimulated Cl-NHE activity by dietary Na depletion, which increases plasma aldosterone and inhibits NHE in surface cell AMV. These properties of Cl-NHE are distinct from those of NHE1, NHE2, and NHE3 isoforms that are present in colonic epithelial cells; thus these results suggest that the colonic crypt cell Cl-NHE is a novel NHE isoform.

Regulation of DRA and AE1 in rat colon by dietary Na depletion.

Two distinct Cl/anion exchange activities (Cl/HCO(3) and Cl/OH) identified in apical membranes of rat distal colon are distributed in cell type-specific patterns. Cl/HCO(3) exchange is expressed only in surface cells, whereas Cl/OH exchange is localized in surface and crypt cells. Dietary Na depletion substantially inhibits Cl/HCO(3) but not Cl/OH exchange. We determined whether anion exchange isoforms (AE) and/or downregulated in adenoma (DRA) are expressed in and related to apical membrane anion exchanges by examining localization of AE isoform-specific and DRA mRNA expression in normal and Na-depleted rats. Amplification of AE cDNA fragments by RT-PCR with colonic mRNA as template indicates that AE1 and AE2 but not AE3 mRNAs are expressed. In situ hybridization study revealed that AE1 mRNA is expressed predominantly in surface but not crypt cells. In contrast, AE2 polypeptide is expressed in basolateral membranes and DRA protein is expressed in apical membranes of both surface and crypt cells. AE1 mRNA is only minimally present in proximal colon, and DRA mRNA abundance is similar in distal and proximal colon. Dietary Na depletion reduces AE1 mRNA abundance but did not alter DRA mRNA abundance. This indicates that AE1 encodes surface cell-specific aldosterone-regulated Cl/HCO(3) exchange, whereas DRA encodes aldosterone-insensitive Cl/OH exchange.

Basolateral K-Cl cotransporter regulates colonic potassium absorption in potassium depletion.

Active potassium absorption in the rat distal colon is electroneutral, Na(+)-independent, partially chloride-dependent, and energized by an apical membrane H,K-ATPase. Both dietary sodium and dietary potassium depletion substantially increase active potassium absorption. We have recently reported that sodium depletion up-regulates H,K-ATPase alpha-subunit mRNA and protein expression, whereas potassium depletion up-regulates H,K-ATPase beta-subunit mRNA and protein expression. Because overall potassium absorption is non-conductive, K-Cl cotransport (KCC) at the basolateral membrane may also be involved in potassium absorption. Although KCC1 has not been cloned from the colon, we established, in Northern blot analysis with mRNA from the rat distal colon using rabbit kidney KCC1 cDNA as a probe, the presence of an expected size mRNA in the rat colon. This KCC1 mRNA is substantially increased by potassium depletion but only minimally by sodium depletion. KCC1-specific antibody identified a 155-kDa protein in rat colonic basolateral membrane. Potassium depletion but not sodium depletion resulted in an increase in KCC1 protein expression in basolateral membrane. The increase of colonic KCC1 mRNA abundance and KCC1 protein expression in potassium depletion of the rat colonic basolateral membrane suggests that K-Cl cotransporter: 1) is involved in transepithelial potassium absorption and 2) regulates the increase in potassium absorption induced by dietary potassium depletion. We conclude that active potassium absorption in the rat distal colon involves the coordinated regulation of both apical membrane H,K-ATPase and basolateral membrane KCC1 protein.

Ouabain-sensitive H,K-ATPase functions as Na,K-ATPase in apical membranes of rat distal colon.

Na,K-ATPase activity has been identified in the apical membrane of rat distal colon, whereas ouabain-sensitive and ouabain-insensitive H,K-ATPase activities are localized solely to apical membranes. This study was designed to determine whether apical membrane Na,K-ATPase represented contamination of basolateral membranes or an alternate mode of H,K-ATPase expression. An antibody directed against the H, K-ATPase alpha subunit (HKcalpha) inhibited apical Na,K-ATPase activity by 92% but did not alter basolateral membrane Na,K-ATPase activity. Two distinct H,K-ATPase isoforms exist; one of which, the ouabain-insensitive HKcalpha, has been cloned. Because dietary sodium depletion markedly increases ouabain-insensitive active potassium absorption and HKcalpha mRNA and protein expression, Na, K-ATPase and H,K-ATPase activities and protein expression were determined in apical membranes from control and sodium-depleted rats. Sodium depletion substantially increased ouabain-insensitive H, K-ATPase activity and HKcalpha protein expression by 109-250% but increased ouabain-sensitive Na,K-ATPase and H,K-ATPase activities by only 30% and 42%, respectively. These studies suggest that apical membrane Na,K-ATPase activity is an alternate mode of ouabain-sensitive H,K-ATPase and does not solely represent basolateral membrane contamination.

Amylase-resistant starch plus oral rehydration solution for cholera.

BACKGROUND: Although standard glucose-based oral rehydration therapy corrects the dehydration caused by cholera, it does not reduce the diarrhea. Short-chain fatty acids, which are produced in the colon from nonabsorbed carbohydrates, enhance sodium absorption. We conducted a study to determine the effects of an orally administered, nonabsorbed starch (i.e., one resistant to digestion by amylase) on fecal fluid loss and the duration of diarrhea in patients with cholera. METHODS: We randomly assigned 48 adolescents and adults with cholera to treatment with standard oral rehydration therapy (16 patients), standard therapy and 50 g of rice flour per liter of oral rehydration solution (16 patients), or standard therapy and 50 g of high-amylose maize starch, an amylase-resistant starch, per liter of oral rehydration solution (16 patients). The primary end points were fecal weight (for every 12-hour period during the first 48 hours after enrollment) and the length of time to the first formed stool. RESULTS: The mean (+/-SD) fecal weights in the periods 12 to 24 hours, 24 to 36 hours, and 36 to 48 hours after enrollment were significantly lower in the resistant-starch group (2206+/-1158 g, 1810+/-1018 g, and 985+/-668 g) than in the standard-therapy group (3251+/-766 g, 2621+/-1149 g, and 2498+/-1080 g; P=0.01, P= 0.04, and P=0.001, respectively). From 36 to 48 hours after enrollment, fecal weight was also significantly lower with the resistant-starch therapy than with the rice-flour therapy (985+/-668 g vs. 1790+/-866 g, P=0.01). The mean duration of diarrhea was significantly shorter with the resistant-starch therapy (56.7+/-18.6 hours) than with standard therapy alone (90.9+/-29.8 hours, P=0.001) or the rice-flour therapy (70.8+/-20.2 hours, P=0.05). Fecal excretion of starch was higher with the resistant-starch therapy (32.6+/-30.4) than with the standard therapy (11.7+/-4.1 g, P=0.002) or the rice-flour therapy (15.1+/-8.4 g, P=0.01). CONCLUSIONS: The addition of a resistant starch to oral rehydration solution reduces fecal fluid loss and shortens the duration of diarrhea in adolescents and adults with cholera.

Colonic H-K-ATPase alpha- and beta-subunits express ouabain-insensitive H-K-ATPase.

Active K absorption in the rat distal colon is energized by an apical H-K-ATPase, a member of the gene family of P-type ATPases. The H-K-ATPase alpha-subunit (HKcalpha) has been cloned and characterized (together with the beta-subunit of either Na-K-ATPase or gastric H-K-ATPase) in Xenopus oocytes as ouabain-sensitive (86)Rb uptake. In contrast, HKcalpha, when expressed in Sf9 cells without a beta-subunit, yielded evidence of ouabain-insensitive H-K-ATPase. Because a beta-subunit (HKcbeta) has recently been cloned from rat colon, this present study was initiated to determine whether H-K-ATPase and its sensitivity to ouabain are expressed when these two subunits (HKcalpha and HKcbeta) are transfected into a mammalian cell expression system. Transfection of HEK-293 cells with HKcalpha and HKcbeta cDNAs resulted in the expression of HKcalpha and HKcbeta proteins and their delivery to plasma membranes. H-K-ATPase activity was identified in crude plasma membranes prepared from transfected cells and was 1) saturable as a function of increasing K concentration with a K(m) for K of 0.63 mM; 2) inhibited by orthovanadate; and 3) insensitive to both ouabain and Sch-28080. In parallel transfection studies with HKcalpha and Na-K-ATPase beta1 cDNAs and with HKcalpha cDNA alone, there was expression of ouabain-insensitive H-K-ATPase activity that was 60% and 21% of that in HKcalpha/HKcbeta cDNA transfected cells, respectively. Ouabain-insensitive (86)Rb uptake was also identified in cells transfected with HKcalpha and HKcbeta cDNAs. These studies establish that HKcalpha cDNA with HKcbeta cDNA express ouabain-insensitive H-K-ATPase similar to that identified in rat distal colon.

Characterization and molecular localization of anion transporters in colonic epithelial cells.

This study describes the identification and characterization of anion transporters in apical membrane (APM) and basolateral membrane (BLM) of rat distal colon. Cl-HCO3, Cl-OH, Cl-butyrate, and butyrate-HCO3 exchanges and Na-HCO3 cotransporter are present in rat distal epithelial cells. Cl-HCO3 exchange (1) is present only in APM from surface, but not from crypt cells; (2) is also present in BLM; and (3) of surface cell is encoded by anion exchange (AE)-1 isoform, whereas BLM Cl-HCO3 is encoded by AE2 isoform. Cl-OH exchange is present only in APM, but not in BLM from surface and crypt cells, and is responsible for regulation of cell functions (i.e., cell pH and cell volume regulation). Butyrate-HCO3 exchange (1) is also present in apical membrane vesicles (AMV) from surface, but not from crypt cells; (2) is present in BLM; and (3) is responsible for SCFA-dependent HCO3 secretion. By contrast, Cl-butyrate exchange: (1) is present in APM from both surface and crypt cells; (2) is not present in BLM; and (3) recycles butyrate by absorbing Cl. Na-HCO3 cotransport: (1) is present only in BLM; (2) is expressed predominantly in midcrypt regions; and (3) may be linked to HCO3 secretion. A mechanism for HCO3 movement across the crypt apical membrane has not as yet been identified.

Cl-dependent Na-H exchange. A novel colonic crypt transport mechanism.

This communication summaries a series of observations of the transport function of the crypt of the rat distal colon. Development of methods to study both 22Na uptake by apical membrane vesicles prepared from crypt cells and intracellular pHi (pHi), fluid movement (Jv), and bicarbonate secretion during microperfusion of the crypt has led to the identification of (1) a novel Cl-dependent Na-H exchange (Cl-NHE) that most likely represents the coupling of a Cl channel to a Na-H exchange isoform that has not as yet been identified and (2) bicarbonate secretion that appears to be most consistent with HCO3 uptake across the basolateral membrane by a mechanism that is closely linked to Cl transport and its movement across the apical membrane via an anion channel. Na-dependent fluid absorption is the constitutive transport process in the crypt, while fluid secretion is regulated by one or more neurohumoral agonists. Cl-NHE is responsible for both the recovery/regulation of pHi in crypt cells to an acid load and fluid absorption.

HCO(3)(-) secretion in the rat colonic crypt is closely linked to Cl(-) secretion.

BACKGROUND & AIMS: The mechanism of colonic HCO(3)(-) secretion has not been established largely because of a lack of experimental methods for its detailed study. The present studies were designed to establish whether the isolated, perfused crypt of the rat distal colon is an excellent model to study HCO(3)(-) movement and the mechanism of colonic HCO(3)(-) secretion. METHODS: HCO(3)(-) secretion was determined in isolated, microperfused crypts by measuring [HCO(3)(-)] by microcalorimetry on nanoliter samples. RESULTS: Net HCO(3)(-) absorption was observed during lumen and bath perfusion with an HCO(3)(-)-Ringer solution. Vasoactive intestinal polypeptide (60 nmol/L), acetylcholine (100 nmol/L), or dibutyryl adenosine 3',5'-cyclic monophosphate (DBcAMP, 0.5 mmol/L) induced active HCO(3)(-) secretion that required bath but not lumen HCO(3)(-)/CO(2). DBcAMP-stimulated HCO(3)(-) secretion was not affected by acetazolamide, an inhibitor of carbonic anhydrase. Removal of lumen Cl(-) did not alter DBcAMP-stimulated HCO(3)(-) secretion but reduced fluid secretion. DBcAMP-stimulated HCO(3)(-) secretion was closely linked to active Cl(-) secretion because HCO(3)(-) secretion was substantially reduced by removal of bath Cl(-), by addition of bath bumetanide, an inhibitor of Na-K-2Cl cotransport and Cl(-) secretion, and by addition of lumen NPPB, a Cl(-) channel inhibitor. CONCLUSIONS: These studies establish that colonic crypt HCO(3)(-) secretion (1) is not a result of an apical membrane Cl(-)-HCO(3)(-) exchange, (2) is tightly associated with Cl(-) secretion, and (3) primarily occurs via an apical membrane Cl(-) channel.

Stimulation of sodium chloride absorption from secreting rat colon by short-chain fatty acids.

Inhibition of electroneutral NaCl absorption by cyclic adenosine monophosphate (cAMP) results in fluid malabsorption in cholera. Short-chain fatty acids (SCFA) stimulate electroneutral NaCl absorption from the colon. The present study investigated effects of elevated cAMP on SCFA-stimulated NaCl absorption in rat distal colon. The effect of SCFA on fluxes of 22Na and 36Cl was studied under voltage-clamp conditions, in the presence and absence of secretagogues inducing mucosal cAMP elevation [ie, theophylline, cholera toxin (CT) and forskolin]. The effect of butyrate concentration on Na absorption in CT- and theophylline-treated mucosa was compared with control normal mucosa. cAMP was measured in isolated colonocytes in the presence of secretagogues with and without SCFA using a radioassay method. All secretagogues were noted to inhibit net Na absorption and to induce net Cl secretion. In the presence of SCFA, net Na absorption was normal, and net Cl secretion was partly reversed. The flux data indicated that NaCl absorption from secreting colon was stimulated by SCFA and that Cl secretion was partially inhibited. The effects of SCFA on NaCl absorption were similar regardless of the secretagogue used. The kinetics of butyrate-stimulated Na absorption were altered by theophylline and CT, which decreased Km (6.87 and 7.17, respectively, compared to 10.75 mM for control) and increased Vmax (4.55 and 8.33 compared to 3.45 mM/eq/cm2/hr for control). cAMP production by colonocytes in response to secretory stimuli was significantly reduced (34.4%) by butyrate but not by acetate or propionate. In conclusion, SCFA-stimulated Na absorption is up-regulated by cAMP and may be an absorptive pathway that can be utilized in the therapy of cholera. Effects of butyrate on cAMP generation are also likely to be useful in secretory diarrhea.

Physiological and molecular studies of colonic H+,K+-ATPase.

This article summarizes physiological and molecular studies of the colonic H+,K+-ATPase and active potassium (K) absorption in the rat distal colon. Active K absorption is energized by an apical membrane H+,K+-ATPase that is a member of the gene family of P-type ATPases. Physiological experiments of active K absorption and enzymatic analysis of H+,K+-ATPase provide compelling evidence for 2 distinct H+,K+-ATPases with a spatial distribution to surface and crypt epithelial cells: an ouabain-sensitive isoform is present in crypt cells and an ouabain-insensitive one is present in surface cells. An alpha subunit (HKcalpha1) has been cloned from the rat colon and its message and protein are selectively located in surface epithelial cells and apical membrane of surface epithelial cells, respectively. A beta subunit (HKcbeta) has also been cloned from the rat colon and may represent the beta subunit for the colonic H+,K+-ATPase. Expression studies have yielded conflicting data: studies with HKcalpha1 and HKcbeta cDNAs that have assessed H+,K+-ATPase activity have concluded that these cDNAs encode the ouabain-insensitive isoform. In contrast, studies with HKcalpha1 and other beta subunits that have expressed 86Rb uptake have yielded evidence for a ouabain-sensitive isoform. Although both aldosterone and dietary K depletion stimulate active K absorption, only the former involves the regulation of HKcalpha1 at a posttranscriptual level.

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

P-type ATPases require both alpha- and beta-subunits for functional activity. Although an alpha-subunit for colonic apical membrane H-K-ATPase (HKcalpha) has been identified and studied, its beta-subunit has not been identified. We cloned putative beta-subunit rat colonic H-K-ATPase (HKcbeta) cDNA that encodes a 279-amino-acid protein with a single transmembrane domain and sequence homology to other rat beta-subunits. Northern blot analysis demonstrates that this HKcbeta is expressed in several rat tissues, including distal and proximal colon, and is highly expressed in testis and lung. HKcbeta 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 beta1 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 HKcbeta mRNA abundance. Western blot analyses using a polyclonal antibody raised to a glutathione S-transferase-HKcbeta fusion protein established expression of a 45-kDa HKcbeta protein in both apical and basolateral membranes of rat distal colon, but K depletion increased HKcbeta protein expression only in apical membranes. Physical association between HKcbeta and HKcalpha proteins was demonstrated by Western blot analysis performed with HKcbeta antibody on immunoprecipitate of apical membranes of rat distal colon and HKcalpha antibody. Tissue-specific upregulation of this beta-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 HKcalpha protein provide compelling evidence that HKcbeta is the putative beta-subunit of colonic H-K-ATPase.

Differential regulation of NHE isoforms by sodium depletion in proximal and distal segments of rat colon.

Dietary sodium depletion has multiple diverse effects on ion transport in the rat colon, including both the induction and inhibition of electroneutral NaCl absorption in proximal and distal colon of rat, respectively. To establish the mechanism of the differential regulation of Na+ absorption by sodium depletion, this study utilized 1) HOE-694, a dose-dependent inhibitor of Na+/H+ exchanger (NHE) isoforms, in studies of proton gradient-driven 22Na uptake (i.e., Na+/H+ exchange) by apical membrane vesicles (AMV); 2) Northern blot analyses of NHE isoform-specific mRNA abundance; and 3) Western blot analyses of NHE isoform-specific protein expression. HOE-694 inhibition studies establish that 25 microM HOE-694-sensitive (NHE2) and 25 microM HOE-694-insensitive (NHE3) Na+/H+ exchange activities are present in AMV of both proximal and distal colon of normal rats. In proximal colon, dietary sodium depletion enhanced both NHE2 and NHE3 isoform-specific Na+/H+ exchange activities, protein expression, and mRNA abundance. In contrast, in distal colon both NHE2 and NHE3 isoform-specific Na+/H+ exchange activities, protein expression, and mRNA abundance were inhibited by sodium depletion. NHE1 isoform-specific mRNA abundance in proximal or distal colon was not altered by sodium depletion. Differential effects by sodium depletion on Na+/H+ exchange in rat colon are tissue specific and isoform specific; sodium depletion both induces and inhibits apical Na+/H+ exchange at a pretranslational level.

Role of Cl channels in Cl-dependent Na/H exchange.

A novel Na/H exchange activity that requires Cl was recently identified in the apical membrane of crypt cells of the rat distal colon. This study explores the nature of the coupling of Cl and Na/H exchange. A concentration of 100 microM 5-nitro-2-(3-phenylpropylamino)benzoic acid, a Cl channel blocker, inhibited the Cl dependence of both proton gradient-driven 22Na uptake from crypt cell apical membrane vesicles and Na-dependent intracellular pH recovery from an acid load during microperfusion of the crypt lumen. Cl-dependent proton gradient-driven 22Na uptake was inhibited by 94% by 500 microM DIDS but only by 1% by 10 microM DIDS, an anion exchange inhibitor at low concentrations but a Cl channel blocker at high concentrations. In addition, a polyclonal antibody to the cystic fibrosis transmembrane conductance regulator (CFTR) inhibited Cl-dependent proton gradient-driven 22Na uptake by 38%. These results indicate that the Cl dependence of Na/H exchange in the colonic crypt apical membrane involves a Cl channel and not a Cl/anion exchange and permit the speculation that this Cl channel activity represents both CFTR and the outward rectifying Cl conductance.

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

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-driven 36Cl 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-driven 36Cl 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 stimulates 36Cl 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.

Role of apical H-K exchange and basolateral K channel in the regulation of intracellular pH in rat distal colon crypt cells.

An apical membrane ouabain-sensitive H-K exchange and a barium-sensitive basolateral membrane potassium channel are present in colonic crypt cells and may play a role in both K absorption and intracellular pH (pHi) regulation. To examine the possible interrelationship between apical membrane H-K exchange and basolateral membrane K movement in rat distal colon in the regulation of pHi, experiments were designed to assess whether changes in extracellular potassium can alter pHi. pHi in isolated rat crypts was determined using microspectrofluorimetric measurements of the pH-sensitive dye BCECF-AM (2', 7'-bis(carboxyethyl-5(6)-carboxy-fluorescein acetoxy methylester). After loading with the dye, crypts were superfused with a Na-free solution which resulted in a rapid and reversible fall in pHi (7.36 +/- 0.02 to 6.98 +/- 0.03). Following an increase in extracellular [K] to 20 mm, in the continued absence of Na, there was a further decrease in pHi (0.20 +/- 0.02, P < 0.01). K-induced acidification was blocked both by 2 mm bath barium, a K channel blocker, and by 0. 5 mm lumen ouabain. K-induced acidification was also observed when intracellular acidification was induced by a NH4Cl prepulse. These observations suggest that increased basolateral K movement increases intracellular [K] resulting in a decrease in pHi that is mediated by a ouabain-sensitive apical membrane H,K-ATPase. Our results demonstrate an interrelationship between basolateral K movement and apical H-K exchange in the regulation of pHi and apical K entry in rat distal colon.