Ammonia inhibits sodium and chloride absorption in rat distal colon

It was recently demonstrated that ammonia inhibits sodium absorption in the proximal colon of rats. In order to investigate the effect of luminal ammonia in the distal colon, sodium and chloride transport were measured in Ussing chambers. Under short-circuit conditions, distal colon absorbed sodium and chloride. When luminal ammonia (30 mmol l(-1)) was present, sodium and chloride absorption was diminished. Inhibition of the two Na(+)-H(+) exchanger isoforms NHE2 and NHE3, which are known to be located in the apical membrane of the distal colon epithelium, failed to influence the effect of ammonia on transepithelial sodium and chloride fluxes. The inhibitory effect of ammonia was eliminated under the following conditions: after block of carbonic anhydrases with acetazolamide, in the presence of an unspecific blocker of Na(+)-H(+) exchangers, and under chloride-free conditions. Ammonia did not alter electrogenic sodium absorption. These results demonstrate that luminal ammonia inhibits sodium and chloride absorption in rat distal colon. We suggest that ammonia inhibits NaCl absorption by interfering with a Na(+)-H(+) exchanger that is not NHE2 or NHE3     

Protective effect of HOE642, a selective blocker of Na+-H+ exchange, against the development of rigor contracture in rat ventricular myocytes

The objective of this study was to investigate the effect of Na+-H+ exchange (NHE) and HCO3--Na+ symport inhibition on the development of rigor contracture. Freshly isolated adult rat cardiomyocytes were subjected to 60 min metabolic inhibition (MI) and 5 min re-energization (Rx). The effects of perfusion of HCO3- or HCO3--free buffer with or without the NHE inhibitor HOE642 (7 microM) were investigated during MI and Rx. In HCO3--free conditions, HOE642 reduced the percentage of cells developing rigor during MI from 79 +/- 1% to 40 +/- 4% (P < 0.001) without modifying the time at which rigor appeared. This resulted in a 30% reduction of hypercontracture during Rx (P < 0.01). The presence of HCO3- abolished the protective effect of HOE642 against rigor. Cells that had developed rigor underwent hypercontracture during Rx independently of treatment allocation. Ratiofluorescence measurement demonstrated that the rise in cytosolic Ca2+ (fura-2) occurred only after the onset of rigor, and was not influenced by HOE642. NHE inhibition did not modify Na+ rise (SBFI) during MI, but exaggerated the initial fall of intracellular pH (BCEFC). In conclusion, HOE642 has a protective effect against rigor during energy deprivation, but only when HCO3--dependent transporters are inhibited. This effect is independent of changes in cytosolic Na+ or Ca2+ concentrations.     

Regulation of intracellular pH in rat renal inner medullary thin limbs of Henle's loop

Regulation of intracellular pH (pHi) was studied in isolated rat renal inner medullary thin limbs of Henle's loop in bicarbonate/phosphate-buffered medium with high pCO2, high osmolality ( congruent with670 mosmol/kg H2O; 270 mM urea; 180 mM NaCl), organic osmolytes, and a pH of 6.8 to approximate the physiological in vivo environment. The pH-sensitive fluorescent dye 2',7'-bis(2-carboxyethyl)-5,6-carboxyfluorescein (BCECF) was used to measure pHi. Resting pHi was always acid and significantly more acid in descending thin limb (DTL) cells than in ascending thin limb (ATL) cells from pure or mixed-type thin limbs. Resting pHi was slightly but significantly higher in both DTLs and ATLs in high osmolality ( approximately 670 mosmol/kg H2O) than in low osmolality ( approximately 290 mosmol/kg H2O) medium but not when sucrose replaced urea. In both DTLs and ATLs the rate of recovery of pHi following additional acidification with an NH4Cl pulse was reduced by Na+ removal from the medium and by the addition of 60 microM HOE642 (an inhibitor of the Na+/H+ exchanger, NHE1), 55 microM S1611 (inhibitor of Na+/H+ exchanger, NHE3), 1 microM bafilomycin A1 (an inhibitor of vacuolar H+ -ATPase), or 20 microM Schering 28080 (an inhibitor of H+ -K+ -ATPase) to the medium. Resting pHi was also reduced by 60 microM HOE642, 55 microM S1611, and 20 microM Schering 28080. In both DTLs and ATLs, RT-PCR revealed message for NHE1, NHE3, and vacuolar H+ -ATPase; immunocytochemistry demonstrated the expression of the protein for NHE1 (basolateral membrane), NHE3 (luminal membrane), and H+ -K+ -ATPase (luminal membrane). These data suggest that pHi in rat inner medullary thin limbs is regulated by urea and by basolateral and luminal H+ extrusion via NHE1, NHE3, vacuolar H+ -ATPase, and H+ -K+ -ATPase.     

Na+/H+ exchanger inhibition at the onset of reperfusion decreases myocardial infarct size: role of reactive oxygen species.

BACKGROUND: A burst of reactive oxygen species and activation of Na+/H+ exchanger take place at the beginning of reperfusion. The aim of this study was to assess the possible interrelation of the inhibition of Na+/H+ exchanger and reactive oxygen species about the determination of myocardial infarct size. METHODS: Isolated rat hearts were submitted to 40 min of coronary occlusion and 2 h of reperfusion. Infarct size was determined through triphenyltetrazolium chloride staining technique and was expressed as a percentage of risk area. Lipid peroxidation, as a marker of oxidative stress, was estimated by the concentration of thiobarbituric reactive substances. RESULTS: Treatment during the first 20 min of reperfusion with a selective inhibitor of Na+/H+ exchanger 1 isoform, HOE 642 (cariporide; 10 microM), significantly diminished infarct size (15.1+/-2.4% vs. 31+/-2% in untreated hearts). The administration of a "scavenger" of hydroxyl radical, N-(2-mercaptopropionyl)-glycine (2 mM), decreased infarct size in an extent similar to that of cariporide (18+/-3%). The combination cariporide+N-(2-mercaptopropionyl)-glycine did not produce additional protection (17+/-1.7%). Each intervention [HOE 642 or N-(2-mercaptopropionyl)-glycine] and its combination improved the postischemic recovery of myocardial systolic and diastolic functions in a similar extent. The content of the thiobarbituric reactive substances of untreated hearts (1012+/-144 nmol/g) decreased to 431+/-81, 390+/-82, and 433+/-41 after cariporide, N-(2-mercaptopropionyl)-glycine, and cariporide+N-(2-mercaptopropionyl)-glycine treatments, respectively. CONCLUSIONS: The present data support the conclusion that the cardioprotective effect of cariporide is associated with diminution of oxidative stress.     

Preconditioning with Na+/H+ exchange inhibitor HOE642 reduces calcium overload and exhibits marked protection on immature rabbit hearts

Inhibition of Na/H exchanger isoform-1 (NHE1) has shown significant protection in adult myocardium during ischemia/reperfusion injury; however, the effect is unclear in immature myocardium. We evaluated the effects of HOE642 (a potent, highly selective NHE1 inhibitor) preconditioning on immature rabbit hearts. Twenty immature (2-3 weeks old) New Zealand white rabbits were randomly divided into the control group (n = 10) and the HOE642 preconditioning group (n = 10). The immature isolated hearts were subjected to 45 minutes of normothermic global ischemia plus 60 minutes of reperfusion after being established on the Langendorff apparatus. During reperfusion, the recovery rates of cardiac function (LVDP, +dp/dtmax, -dp/dtmax, and coronary flow) were about 90% in the HOE642 treated group and about 50% in the control group (p < 0.05). HOE642 preconditioning can significantly decrease the release of cardiac specific enzymes CK, CK-MB and LDH (p < 0.05) and the myocardial water content (p < 0.05). Meanwhile, HOE642 markedly attenuated intracellular calcium overload (265.8 +/- 41.1 vs. 500.7 +/- 60.8 mg/kg dry wt) (p < 0.01). The blinded ultrastructural assessment under transmission electron microscopy illustrated that preconditioning with HOE642 produced evident myocyte salvage. This study demonstrates that preconditioning with HOE642 provides a significant protection during ischemia/reperfusion injury in immature myocardium, mostly by reducing myocardial calcium overload.     

Relationship between volatile fatty acids and magnesium absorption in mono- and polygastric species.

In monogastric animals magnesium is absorbed from the small and large intestine. In ruminants the forestomach system, in particular the rumen, is the most important site of magnesium absorption. Various mechanisms are involved in intestinal magnesium absorption (solvent drag, diffusion, carrier-mediated transport). In the large intestine and rumen an active transepithelial magnesium transport from the mucosal to the serosal side of the epithelium was recently demonstrated. Since in the large intestine and in the rumen, volatile fatty acids (VFA, mainly acetate, propionate, butyrate) deriving from fermentation of carbohydrates represent the major anions, the influence of VFA on magnesium absorption from these parts of the gut was recently investigated. VFA at physiological concentrations stimulated magnesium absorption in both cases. In the rat large intestine VFA enhanced only magnesium absorption by the distal colon, sodium and water absorption remaining unaffected. Both in sheep rumen and in the distal colon of the rat butyrate was most effective in this regard, followed in descending order by propionate and acetate. Sodium absorption by the rat proximal colon and caecum, and by the sheep rumen, was similarly enhanced by VFA. It has been suggested that the latter effect is due to the function of VFA as intracellular proton donators for the Na+/H+ exchanger located in the apical membrane of the epithelial cells. In analogy a Mg2+/H+ exchanger, located in the apical membrane of the epithelium in the distal colon and rumen, is fully consistent with the stimulatory effects of VFA on magnesium absorption at these sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Na+/H+ exchange inhibitor HOE642 prevents stress-induced epithelial barrier dysfunction

Recently, evidence has been obtained that the Na+/H+ exchange (NHE) inhibitor HOE642 may stabilize endothelial and epithelial barrier function in vivo. However, the underlying mechanisms are not known. Therefore, we studied the influence of HOE642 on the barrier function of the epithelial cell line CaCo2. The phorbolester phorbol 12-myristate 13-acetate (PMA) was used to induce hyperpermeability of the epithelial layer which was indirectly determined by measuring the transepithelial electrical resistance (TER). Confocal laser scan microscopy (LSM) served to analyze the intracellular localization of adherens and tight junction molecules. In five independent experiments we found that HOE642 increased TER in non-treated CaCo2 cells (control: 350 +/- 28 Omega/cm2; HOE642: 444 +/- 53 Omega/cm2) and prevented PMA-induced barrier dysfunction (PMA: 33 +/- 12 Omega/cm2; PMA plus HOE642: 496 +/- 47 Omega/cm2). LSM showed that HOE642 prevented the PMA-induced disassociation of the zonula adherens molecule beta-catenin from the cell membrane and the decreased expression of the zonula occludens molecule ZO-1. From our data we conclude that HOE642 may prevent stress-induced epithelial dysfunction by stabilization of cell membrane-associated junction molecules.     

pH regulation and buffering power in gastric smooth muscle

Intracellular pH can have profound effects on tissue function, but little is known about how pH is regulated, buffered or affects the function of gastric smooth muscle. As the pH of gastric myocytes may alter with pathophysiological disturbance of the gastric lining, or reduction in blood flow to the stomach, these parameters were investigated. Intracellular pH was measured in strips of corpus from rats and guinea-pigs and pH perturbed by the addition of Na butyrate. pH regulation was investigated using pharmacological inhibitors and ionic substitutions. Resting pH was found to be around 7.0, and buffering power relatively high, compared to other muscles in both species. In the guinea-pig amiloride, EIPA and HOE694 prevented pH regulation from an acid load, but amiloride- and EIPA-insensitive pH-regulating mechanisms were found in the rat. The pH-regulatory mechanism present in the rat was also insensitive to DIDS, SITS and removal of external Cl-, but inhibited by Na+ substitution and HOE694. Acidification reduced gastric tone in both species. We conclude that pH alteration will significantly affect gastric contractility, despite a high capacity of the tissue to buffer and regulate pH change. The sensitivity to NHE inhibitors differs between rat and guinea-pig, suggesting that Na+/H+ exchanger isoform expression differs between gastric tissue.     

Actions of the Cl- channel blocker NPPB on absorptive and secretory transport processes of Na+ and Cl- in rat descending colon

The effects of the Cl- channel blocker, NPPB (5-nitro-2-(3-phenylpropylamino)-benzoate), on the transport of Na+ and Cl- in the descending colon of the rat were studied in the Ussing chamber. In control tissue, NPPB administered at the mucosal side of the epithelium increased the short-circuit current (Isc) and inhibited the unidirectional mucosa-to-serosa fluxes of Na+ and Cl-. In HCO3- - or Cl- -free media for in the presence of SITS (4-acetamido-4'-isothiocyanato-stilbene-2,2'-disulphonic acid), this increase in Isc caused by mucosal NPPB was not observed. The serosal administration of NPPB was without effect. Mucosal NPPB (10(-4) mol l-1) decreased the forskolin-induced increase in Isc by only about 60%. However, the activation of the serosa-to-mucosa flux of Cl- caused by forskolin was inhibited completely. NPPB decreased the mucosa-to-serosa fluxes of Na+ and Cl- reduced additionally by forskolin. Serosal NPPB decreased Isc and FNasm, but had no effect on FNams or FClmas. In HCO3- -free buffer the increase in Isc induced by forskolin was inhibited completely by NPPB. The inhibition of Cl- secretion by NPPB fits well with the capacity of the drug to block Cl- channels. For the inhibition of neutral NaCl absorption two sites of action are discussed: an interaction with the Cl-/HCO3- exchanger or an interference with the extrusion of Cl- through the basolateral membrane.     

Effect of short-chain fatty acids on calcium absorption by the rat colon

An in vivo luminal perfusion technique was used to investigate whether short-chain fatty acids influence the absorption of Ca by the rat colon. Na and water absorption were also determined. In the distal colon, acetate and butyrate caused a significant increase in Ca absorption, while the absorption of Na and water were not affected. In the proximal colon, butyrate did not influence Ca absorption, but significantly enhanced Na and water absorption. These results are in part consistent with the presence of a Ca-H exchanger in the apical membrane of the distal colon mediating Ca uptake into the epithelial cell.     

Adaptation of electrolyte transport in rat large intestine after proximal resection

Electrolyte transport was studied in rat colon adapting to 50% small intestinal resection with cecectomy. Four weeks after surgery, colonic gross surface area, dry and wet weight were increased compared to sham-operated controls. Net absorption in vivo of sodium, chloride and volume was stimulated per organ and per unit tissue mass, and net potassium secretion was diminished. The electrical potential difference, mucosal Na-K-ATPase specific activity and cAMP concentration remained unaffected. In vitro, measurements of unidirectional fluxes and electrical parameters across isolated mucosa indicated enhanced electrically neutral sodium chloride absorption in the proximal and possibly diminished bicarbonate secretion in the distal colon. Thus, removal of the cecum in addition to partial jejunoilectomy induces profound adaptive changes in the colon, involving not only mucosal growth but also a functional reaction of the individual epithelial cell.     

Dietary fructose, salt absorption and hypertension in metabolic syndrome: towards a new paradigm

The worldwide increase in the incidence of metabolic syndrome correlates with marked increase in total fructose intake in the form of high-fructose corn syrup, beverage and table sugar. Increased dietary fructose intake in rodents has been shown to recapitulate many aspects of metabolic syndrome by causing hypertension, insulin resistance and hyperlipidaemia. Recent studies demonstrated that increased dietary fructose intake stimulates salt absorption in the small intestine and kidney tubules, resulting in a state of salt overload and thus causing hypertension. The absorption of salt (sodium and chloride) in the small intestine is predominantly mediated via the chloride/base exchangers DRA (Down Regulated in Adenoma) (SLC26A3) and PAT1 (Putative Anion Transporter 1) (SLC26A6), and the Na(+) /H(+) exchanger NHE3 (Sodium Hydrogen Exchanger3) (SLC9A3). PAT1 and NHE3 also co-localize on the apical membrane of kidney proximal tubule. Luminal fructose stimulated salt absorption in the jejunum and kidney tubules, responses that were significantly diminished in PAT1 null mice. These studies further demonstrated that Glut5 (SLC2A5) is the major fructose-absorbing transporter in the small intestine (and kidney proximal tubule) and plays an essential role in the systemic homeostasis of fructose. Increased dietary fructose intake for several weeks upregulated the expression of NHE3, PAT1 and Glut5 in the intestine and resulted in hypertension in wild-type mice, a response that was almost abolished in PAT1 null mice and abrogated in Glut5 null mice. This article will discuss the interaction of Glut5 with salt-absorbing transporters and review the role of dietary fructose in enhanced salt absorption in intestine and kidney as it relates to the pathogenesis of hypertension in metabolic syndrome.     

The effect of aldosterone and the renin-angiotensin system on sodium, potassium and chloride transport by proximal and distal rat colon in vivo.

1. The roles of aldosterone and angiotensin in the direct control of epithelial sodium transport in vivo have been investigated by measurement of electrical p.d. changes and of the fluxes of sodium, potassium and chloride in rat colon, an organ actively involved in electrolyte homoeostasis. Exogenous angiotensin and aldosterone were given by both short- and long-term infusions and endogenous secretion of the hormones was varied by dietary sodium variation and by nephrectomy and/or adrenalectomy. 2. In vitro angiotensin has been shown to influence colonic salt and water absorption but in the present in vivo experiments administered angiotensin had no significant action on p.d. or on the ionic fluxes of the proximal or distal colon. The increase in p.d. produced by infusing aldosterone was unaffected by giving angiotensin concurrently. The effect of sodium depletion in stimulating sodium absorption and potassium secretion was completely abolished by adrenalectomy but was unaffected by nephrectomy. 3. During prolonged infusion of angiotensin into adrenalectomized rats, a small fall in faecal fluid and sodium content was observed, but this change would have little significance in sodium homoeostasis. 4. Aldosterone and sodium depletion stimulated sodium absorption in both proximal and distal colon but significant increase in potassium secretion was demonstrable only in the distal colon. Bicarbonate secretion (by calculation) was unaffected. In the proximal colon, the increased sodium absorption appeared to be accompanied by increased chloride absorption while in the distal colon it was principally the sodium-potassium exchange that was increased. 5. Adrenalectomy reduced potassium secretion in both proximal and distal colon but sodium absorption was only significantly reduced in the proximal colon. 6. It was concluded that there is no evidence that angiotensin in the living animal has a role as an important salt retaining hormone by direct epithelial action. Aldosterone has a considerable effect which is independent of the presence of angiotensin, and which differs in proximal and distal colon in regard to the relative effects on chloride absorption and potassium secretion.     

Intracellular Na concentration and Rb uptake in proximal convoluted tubule cells and abundance of Na/K-ATPase alpha1-subunit in NHE3-/- mice

Proximal solute and fluid absorption is greatly reduced in mice in which the gene encoding the Na/H exchanger isoform 3 has been ablated (NHE3-/-). To obtain information on the intracellular functional consequences of such selective NHE3 deficiency, Na, Cl and K concentrations and cell Rb uptake were measured using electron microprobe analysis after a 30-s infusion of Rb (an index of basolateral Na/K-ATPase activity) in proximal convoluted tubule (PCT) cells of NHE3-/- and wild-type (NHE3+/+) mice. In addition, the relative abundance of the alpha1-subunit of the Na/K-ATPase in the outer cortex was determined by Western blot analysis. PCT cell Na concentration in NHE3-/- mice was slightly but significantly lower than in NHE3+/+ [13.1+/-0.6 ( n=64) vs. 14.9+/-0.6 ( n=62) mmol/kg wet wt.; means +/-SEM]. The lower intracellular Na concentration was associated with significantly reduced Rb uptake rates [9.7+/-0.6 ( n=59) vs. 14.8+/-0.8 ( n=50) mmol/kg wet wt./30 s], but the abundance of the alpha1-subunit of the Na/K-ATPase was not different between NHE3-/- and NHE3+/+ mice. Intracellular Cl concentration was higher (14.2+/-0.4 vs. 12.8+/-0.4 mmol/kg wet wt.) and K concentration unchanged (122.7+/-2.7 vs. 121.6+/-2.5 mmol/kg wet wt.) in PCT cells in NHE3-/- compared with NHE3+/+ mice. These findings suggest that the elimination of apical NHE3 in PCT cells of NHE3-/- mice reduces apical Na entry and, due to lower cell Na concentrations, Na/K-ATPase activity. The observed changes in intracellular Na concentration did not affect the expression of Na/K-ATPase in the renal cortex of NHE3-/- mice. There were no significant changes of cell Na concentration and Rb uptake in distal convoluted tubule, connecting tubule, principal and intercalated cells.     

The protein tyrosine kinase pathway is not involved in the regulation of K+ transport across the rat colon.

The protein tyrosine kinase inhibitor, genistein, is known to activate the cystic fibrosis transmembrane regulator (CFTR) Cl- channel and to inhibit K+ currents across the rat colonic epithelium. The aim of the present study is to answer the question whether these effects are involved in the regulation of transepithelial K+ transport. Therefore, the action of genistein on K+ transport in rat proximal and distal colon was studied by measuring unidirectional fluxes, uptake and efflux of Rb+ in mucosa-submucosa preparations. All effects of genistein (5 x 10(-5) mol L(-1)) were tested in the presence of a low concentration of forskolin (2 x 10(-7) mol L(-1)), because prestimulation of the cAMP pathway has been shown to be a prerequisite for a secretory action of genistein. Forskolin caused an increase in the serosa-to-mucosa flux of Rb+ (J(Rb)sm) thereby stimulating net K+ secretion in the proximal and distal colon. None of these effects was further enhanced after administration of genistein. Neither mucosal uptake of Rb+, representing mainly the activity of the H+-K+-ATPase in the distal colon, nor serosal Rb+ uptake, representing, e.g. the activity of the Na+-K+-2Cl- cotransporter, were affected by genistein. Also the efflux of Rb+ across the apical or the basolateral membrane, an indicator for the apical and basolateral K+ conductance, was unchanged in the presence of genistein. These results demonstrate that the K+ channels inhibited by genistein are not involved in transepithelial K+ transport.     

Regulatory role of cAMP in transport of Na+, Cl- and short-chain fatty acids across sheep ruminal epithelium

Sodium is absorbed in considerable amounts across the ruminal epithelium, whilst its transport is strongly interrelated with the permeation of chloride and short-chain fatty acids (SCFAs). However, regulation of ruminal Na+, Cl-, and SCFA absorption is hardly understood. The present study was therefore performed to characterize the influence of cAMP on sodium and sodium-coupled transport mechanisms in short-circuited, stripped ruminal epithelia of sheep. Elevation of intracellular cAMP concentrations by theophylline (10 mM) or theophylline in combination with forskolin (0.1 mM) significantly reduced mucosal-to-serosal sodium transport, leading to a reduction of net transport. The theophylline- or theophylline-forskolin-induced reduction of sodium transport was accompanied by a decrease in chloride net transport but revealed no effect on propionate flux. Short-chain fatty acids stimulated Na+ transport but their stimulatory effect was almost completely blocked by theophylline-forskolin. In solutions with and without SCFAs, the inhibitory effect of 1 mM amiloride on sodium transport was strongly reduced after theophylline-forskolin pretreatment of the tissues. Blocking the production of endogenous prostaglandins by addition of indomethacin (10 microM) led to a theophylline-sensitive stimulation of unidirectional and net fluxes of sodium. The findings indicate that apical, amiloride-sensitive Na+-H+ exchange and/or basolateral Na+-K+-ATPase can effectively be blocked by cAMP, leading to a decrease in sodium and chloride transport. In the ruminal epithelium, cAMP is a second messenger of prostaglandins, which are released spontaneously under in vitro conditions.     

Permeation of NH3/NH4 + and cell pH in colonic crypts of the rat

Colon cells are subjected to high concentrations of NH3 and NH4+, and a sizeable portion of this buffer is absorbed. The flux of these components into cells causes opposite effects on their pH; this effect is largely used to induce an acid load and to observe the mechanism of acid extrusion from cells. We studied cells of microdissected colon crypts loaded with BCECF and superfused with NH4Cl-containing Krebs-Ringer solution. We found a marked transient reduction in pH measured by ratiometric fluorescence microscopy, from a control value of 7.51 +/- 0.041 to 7.15 +/- 0.041 (n = 21), instead of the initial alkalinization found in most cells. This pH was reached at a rate of 0.95 +/- 0.07 pH units/min. Addition of 1 mmol/l furosemide, a blocker of Na+,K+,2Cl- cotransport, to the ammonium solution inverted this acidification toward alkalinization (pH 7.89 +/- 0.041, n = 5), and superfusion with furosemide plus 0.1 mmol/l hexamethylene amiloride, a specific blocker of Na+/H+ exchange, increased this initial alkalinization further to 8.10 +/- 0.117 (n = 7). When Krebs-Ringer with 0 Cl- containing (NH4)2SO4 instead of NH4Cl was superfused, the acid transient was also reverted to alkalinization; however, a higher degree of alkalinization was observed either when 1 mmol/l furosemide was added to the superfusing sulfate solution (when a pH of 7.78 +/- 0.010 was reached), or when ammonium gluconate was used instead of ammonium sulfate. The addition of Ba2+ to the superfusion solution did not alter the initial acidification. These data indicate that in colon crypt cells, basolateral membrane transporters, in particular the Na+,K+,2Cl- cotransporter and the Na+/H+ exchanger (but not Ba(2+)-sensitive K+ channels), mediate the predominant influx of NH4+ ions leading to the initial transient acidification.