H(+)/solute-induced intracellular acidification leads to selective activation of apical Na(+)/H(+) exchange in human intestinal epithelial cells.

The intestinal absorption of many nutrients and drug molecules is mediated by ion-driven transport mechanisms in the intestinal enterocyte plasma membrane. Clearly, the establishment and maintenance of the driving forces - transepithelial ion gradients - are vital for maximum nutrient absorption. The purpose of this study was to determine the nature of intracellular pH (pH(i)) regulation in response to H(+)-coupled transport at the apical membrane of human intestinal epithelial Caco-2 cells. Using isoform-specific primers, mRNA transcripts of the Na(+)/H(+) exchangers NHE1, NHE2, and NHE3 were detected by RT-PCR, and identities were confirmed by sequencing. The functional profile of Na(+)/H(+) exchange was determined by a combination of pH(i), (22)Na(+) influx, and EIPA inhibition experiments. Functional NHE1 and NHE3 activities were identified at the basolateral and apical membranes, respectively. H(+)/solute-induced acidification (using glycylsarcosine or beta-alanine) led to Na(+)-dependent, EIPA-inhibitable pH(i) recovery or EIPA-inhibitable (22)Na(+) influx at the apical membrane only. Selective activation of apical (but not basolateral) Na(+)/H(+) exchange by H(+)/solute cotransport demonstrates that coordinated activity of H(+)/solute symport with apical Na(+)/H(+) exchange optimizes the efficient absorption of nutrients and Na(+), while maintaining pH(i) and the ion gradients involved in driving transport.

H(+)-coupled dipeptide (glycylsarcosine) transport across apical and basal borders of human intestinal Caco-2 cell monolayers display distinctive characteristics.

Transepithelial transport and intracellular accumulation of the dipeptide glycylsarcosine (Gly-Sar) were studied using intact monolayers of the human intestinal epithelial cell line, Caco-2. Gly-Sar transport was demonstrated in both absorptive (apical-to-basal) and secretory (basal-to-apical) directions. In both directions, transport and accumulation were enhanced in the presence of a pH gradient (pHo < pHi). Under conditions similar to those found at the intestinal membrane in vivo (apical pH 6.0, basolateral pH 7.4), net absorption (145.2 pmol/cm2 per h) was observed, although experimental conditions could also be manipulated (apical pH 7.4, basolateral pH 6.0) so that net secretion was observed. Transport and accumulation (in both directions) were inhibited in the presence of either 20 mM (unlabelled) Gly-Sar or 20 mM cephalexin (an aminocephalosporin antibiotic). When added to either the apical or basolateral surface of BCECF (2',7',-bis(2-carboxyethyl)-5(6)-carboxyfluorescein)-loaded Caco-2 cell monolayers Gly-Sar (20 mM), at pH 6.0, caused a marked intracellular acidification, demonstrating that dipeptide absorption is accompanied by H(+)-flow into the cells. Cephalexin (20 mM) had similar effects (as Gly-Sar) when presented at the apical surface but also caused a marked intracellular acidification when perfused into the basolateral chamber at pH 7.4. In contrast, addition of Gly-Sar (20 mM) to the basolateral chamber (at pH 7.4) had no effect. Transepithelial absorption of dipeptides (Gly-Sar) and beta-lactam antibiotics (cephalexin) at low concentrations is predominately via a transcellular route mediated by carrier mechanisms located at both apical and basolateral membranes. Interestingly, Gly-Sar and cephalexin transport across the basolateral membrane (and, therefore, exit from the cell) display both common and distinct characteristics suggesting that more than one mechanism may be responsible for exit into the basolateral space.

The isolation and chemical characterization of a novel vasoactive intestinal peptide-related peptide from a teleost fish, the cod, Gadus morhua.

An octacosapeptide that shows sequence homology to porcine vasoactive intestinal peptide (VIP) has been isolated from a teleost fish, the cod, Gadus morhua. The full primary sequence is His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Ser-Arg-Phe-Arg-Lys-Gln-Met-Ala-Ala- Lys-Lys-Tyr-Leu-Asn-Ser-Val-Leu-Ala. This peptide contains four, or five substitutions, compared with dogfish and porcine VIP, respectively. The residues in positions 13, 26 and 28 are different in all three species. These substitutions seem to have little effect on bioactivity, since cod VIP was virtually equipotent with porcine VIP in stimulating amylase release from guinea-pig pancreatic acini. During the isolation procedure an N-terminally modified form of VIP (Des-His, or 2-28 cod VIP) was also isolated. The available data suggest the sequence of VIP is well conserved in vertebrates which is consistent with an important biological role.

H(+)-coupled alpha-methylaminoisobutyric acid transport in human intestinal Caco-2 cells.

Transepithelial apical-to-basal transport and cellular uptake of the non-metabolisable amino acid alpha-methylaminoisobutyric acid (MeAIB) across confluent monolayers of the human intestinal epithelial cell line Caco-2 are enhanced by a transepithelial pH gradient (apical pH 6.0, basolateral pH 7.4). In Na(+)-free conditions (apical pH 7.4, basolateral pH 7.4), net absorption (120 +/- 58 pmol/cm2 per h, n = 13) and uptake across the apical membrane (cell/medium ratio 0.56 +/- 0.06, n = 13) are low. However, in Na(+)-free conditions with apical pH 6.0, net absorption (685 +/- 95 pmol/cm2 per h, n = 15) and intracellular accumulation (cell/medium ratio 3.63 +/- 0.29, n = 14) were marked. Continuous monitoring of intracellular pH (pHi) in BCECF (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein)-loaded Caco-2 cell monolayers indicated that apical addition of MeAIB (20 mM) was associated with H(+)-flow across the apical membrane in both Na+ and Na(+)-free conditions. This transport process is rheogenic in Na(+)-free media, stimulating an inward short-circuit current in voltage-clamped Caco-2 cell monolayers. On the basis of competition for MeAIB accumulation and pHi experiments, L-proline, glycine, L-alanine and beta-alanine are also substrates for H(+)-linked transport at the apical membrane of Caco-2 cells but L-valine, L-leucine and L-phenylalanine are not. These data are consistent with the expression, in the apical brush-border membrane of Caco-2 cells, of a H(+)-coupled, Na(+)-independent MeAIB carrier.

A novel vasoactive intestinal peptide (VIP) from elasmobranch intestine has full affinity for mammalian pancreatic VIP receptors.

A peptide that cross reacted with N-terminal, but not C-terminal, antisera to vasoactive intestinal peptide (VIP) was isolated from extracts of intestine from the dogfish Scyliorhinus canicula. Microsequence analysis gave the structure His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Ser-Arg-Ile-Arg-Lys-Gln-Met-Ala-Val-Lys - Lys-Tyr-Ile-Asn-Ser-Leu-Leu-Ala-NH2. C-terminal amidation was determined by HPLC analysis of phenylthiocarbamyl amino acid derivatives after carboxypeptidase Y digestion. The peptide differs at five positions from the porcine octacosapeptide. Dogfish VIP was equipotent with its porcine counterpart in inhibiting binding of 125I-labelled VIP to guinea pig dispersed pancreatic acini, and in stimulating amylase secretion by the same preparation. The data indicate a strong conservation of VIP during evolution and permit identification of residues crucial for bioactivity.

H(+)-coupled (Na(+)-independent) proline transport in human intestinal (Caco-2) epithelial cell monolayers.

Previously, absorption of L-proline across the apical membrane of the intestinal enterocyte has been attributed to transport via the Na(+)-dependent Imino system. However, net (absorptive) transport of proline across intact Caco-2 cell monolayers was enhanced by acidification of the apical environment, under both Na(+)-containing and Na(+)-free conditions. This Na(+)-independent pH-dependent proline flux was associated with H+ flow across the apical membrane as determined by continuous measurement of intracellular pH. H+/proline symport was associated with an inward Isc in voltage-clamped Caco-2 epithelial layers demonstrating the electrogenic nature of this transport process. In conclusion Caco-2 cells possess an apically-localised, Na(+)-independent, electrogenic H+/imino acid transporter which may play an important role in intestinal proline absorption.

Stereoselective uptake of beta-lactam antibiotics by the intestinal peptide transporter.

1. The stereoselective transport of beta-lactam antibiotics has been investigated in the human intestinal epithelial cell line, Caco-2, by use of D- and L-enantiomers of cephalexin and loracarbef as substrates. 2. The L-isomers of cephalexin, loracarbef and dipeptides displayed a higher affinity for the oligopeptide/H(+)-symporter in Caco-2 cells than the D-isomers. This was demonstrated by inhibition of the influx of the beta-lactam, [3H]-cefadroxil. 3. By measurement of the substrate-induced intracellular acidification in Caco-2 cells loaded with the pH-sensitive fluorescent dye BCECF (2',7'-bis(2-carboxyethyl)-5-(6)-carboxy-fluorescein), it was demonstrated for the first time that L-isomers of beta-lactams not only bind to the peptide transporter with high affinity but are indeed transported. 4. Efficient proton-coupled transport of L-beta-lactam antibiotics was also shown to occur in Xenopus laevis oocytes expressing the cloned peptide transporter PepT1 from rabbit small intestine. 5. Both cell systems therefore express a stereoselective transport pathway for beta-lactam antibiotics with very similar characteristics and may prove useful for screening rapidly the oral availability of peptide-derived drugs.

Substrate specificity of the di/tripeptide transporter in human intestinal epithelia (Caco-2): identification of substrates that undergo H(+)-coupled absorption.

1. pH-dependent transepithelial transport and intracellular accumulation of the hydrolysis-resistant dipeptide glycylsarcosine (Gly-Sar) have been demonstrated in the model human intestinal epithelial cell line, Caco-2. 2. Experiments with BCECF (2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein)-loaded Caco-2 cells demonstrated that dipeptide (Gly-Sar) transport across the apical membrane is coupled to proton flow into the cell. 3. A range of postulated substrates for the intestinal di/tripeptide carrier were tested for their abilities to: (a) inhibit pH-dependent [14C]Gly-Sar apical-to-basal transport and intracellular accumulation and (b) stimulate H+ flow across the apical surface of BCECF-loaded Caco-2 cell monolayers. 4. A range of compounds (including Gly-Gly, Leu-Leu, Gly-Gly-Gly, cefadroxil and cephalexin) caused marked acidification of intracellular pH when perfused at the apical surface of Caco-2 cell monolayers. In contrast leucine and D-Leu-D-Leu failed to induce proton flow. The ability to induce proton-flow across the apical surface by these compounds, in this intestinal epithelium, was directly correlated to the relative inhibitory effects on [14C]-Gly-Sar transport and accumulation. 5. The determination of substrate-induced intracellular pH change in the Caco-2 cell system may provide a useful rapid screen for candidate substrates for absorption via H(+)-coupled transport mechanisms such as the intestinal di/tripeptide carrier in an appropriate physiological context.

D-cycloserine transport in human intestinal epithelial (Caco-2) cells: mediation by a H(+)-coupled amino acid transporter.

1. The ability of D-cycloserine to act as a substrate for H+/amino acid symport has been tested in epithelial layers of Caco-2 human intestinal cells. 2. In Na(+)-free media with the apical bathing media held at pH 6.0, D-cycloserine (20 mM) is an effective inhibitor of net transepithelial transport (Jnet) of L-alanine (100 microM) and its accumulation (across the apical membrane) in a similar manner to amino acid substrates (L-alanine, beta-alanine, L-proline and glycine). In contrast L-valine was ineffective as an inhibitor for H+/amino acid symport. Both inhibition of L-alanine Jnet and its accumulation by D-cycloserine were dose-dependent, maximal inhibition being achieved by 5-10 mM. 3. Both D-cycloserine and known substrates for H+/amino acid symport stimulated an inward short circuit current (Isc) when voltage-clamped monolayers of Caco-2 epithelia, mounted in Ussing chambers, were exposed to apical substrate in Na(+)-free media, with apical pH held at 6.0. The D-cycloserine dependent increase in Isc was dose-dependent with an apparent Km = 15.8 +/- 2.0 (mean +/- s.e. mean) mM, and Vmax = 373 +/- 21 nmol cm-2h-1. 4. D-Cycloserine (20 mM) induced a prompt acidification of Caco-2 cell cytosol when superfused at the apical surface in both Na+ and Na(+)-free conditions. Cytosolic acidification in response to D-cycloserine was dependent upon superfusate pH, being attenuated at pH 8 and enhanced in acidic media. 5. The increment in Isc with 20 mM D-cycloserine was non-additive with other amino acid substrates for H+/amino acid symport.

Angiotensin-converting enzyme (ACE) inhibitor transport in human intestinal epithelial (Caco-2) cells.

1. The role of proton-linked solute transport in the absorption of the angiotensin-converting enzyme (ACE) inhibitors captopril, enalapril maleate and lisinopril has been investigated in human intestinal epithelial (Caco-2) cell monolayers. 2. In Caco-2 cell monolayers the transepithelial apical-to-basal transport and intracellular accumulation (across the apical membrane) of the hydrolysis-resistant dipeptide, glycylsarcosine (Gly-Sar), were stimulated by acidification (pH 6.0) of the apical environment. In contrast, transport and intracellular accumulation of the angiotensin-converting enzyme (ACE) inhibitor, lisinopril, were low (lower than the paracellular marker mannitol) and were not stimulated by apical acidification. Furthermore, [14C]-lisinopril transport showed little reduction when excess unlabelled lisinopril (20 mM) was added. 3. pH-dependent [14C]-Gly-Sar transport was inhibited by the orally-active ACE inhibitors, enalapril maleate and captopril (both at 20 mM). Lisinopril (20 mM) had a relatively small inhibitory effect on [14C]-Gly-Sar transport. pH-dependent [3H]-proline transport was not inhibited by captopril, enalapril maleate or lisinopril. 4. Experiments with BCECF[2',7',-bis(2-carboxyethyl)-5(6)-carboxyfluorescein]-loaded Caco-2 cells demonstrate that dipeptide transport across the apical membrane is associated with proton flow into the cell. The dipeptide, carnosine (beta-alanyl-L-histidine) and the ACE inhibitors enalapril maleate and captopril, all lowered intracellular pH when perfused at the apical surface of Caco-2 cell monolayers. However, lisinopril was without effect. 5. The effects of enalapril maleate and captopril on [14C]-Gly-Sar transport and pHi suggest that these two ACE inhibitors share the H(+)-coupled mechanism involved in dipeptide transport. The absence of pH-dependent [14C]-lisinopril transport, the relatively small inhibitory effect on [14C]-Gly-Sar transport,and the absence of lisinopril-induced pHi changes, all suggest that lisinopril is a poor substrate for thedi/tripeptide carrier in Caco-2 cells. These observations are consistent with the greater oral availability and time-dependent absorption profile of enalapril maleate and captopril, compared to lisinopril.

Gamma-Aminobutyric acid (GABA) transport across human intestinal epithelial (Caco-2) cell monolayers.

1. Transintestinal absorption of gamma-aminobutyric acid (GABA) via a pH-dependent mechanism is demonstrated in the model human intestinal epithelial cell line Caco-2. 2. Experiments with BCECF [2',7',-bis(2-carboxyethyl)-5(6)- carboxyfluorescein]-loaded Caco-2 cells demonstrate that GABA transport across the apical membrane is coupled to proton flow into the cell. 3. Short-circuit current (ISC) measurements using Caco-2 cell monolayers under voltage-clamped conditions demonstrate that pH-dependent GABA transport is a rheogenic process even in the absence of extracellular Na+, consistent with H+/GABA symport. 4. A range of GABA analogues were tested for their abilities to: (a) inhibit pH-dependent [3H]GABA uptake across the apical membrane; (b) stimulate H+ flow across the apical surface of BCECF-loaded Caco-2 cell monolayers; (c) increase inward ISC across voltage-clamped Caco-2 cell monolayers. 5. Nipecotic acid, isonipecotic acid, D,L-beta-aminobutyric acid, and 3-amino-1-propanesulphonic acid each caused a marked acidification of intracellular pH and an increase in ISC when superfused at the apical surface of Caco-2 cell monolayers. In contrast L-alpha-amino-n-butyric acid failed to induce proton flow or ISC. The ability of these compounds to induce proton or current flow across the apical surface of this intestinal epithelium was closely related to the relative inhibitory effects on [3H]GABA uptake. 6. These observations demonstrate H+/GABA symport and suggest that this transport mechanism may be accessible as a route for oral absorption of therapeutically-useful GABA analogues.

Dual modes of 5-(N-ethyl-N-isopropyl)amiloride modulation of apical dipeptide uptake in the human small intestinal epithelial cell line Caco-2.

Selective pharmacological Na+/H+ exchange (NHE) inhibitors were used to identify functional NHE isoforms in human small intestinal enterocytes (Caco-2) and to distinguish between direct and indirect effects on transport via the intestinal di/tripeptide transporter hPepT1. The relative potencies of these inhibitors to inhibit 22Na+ influx identifies NHE3 and NHE1 as the apical and basolateral NHE isoforms. The Na+-dependent (NHE3-sensitive) component of apical dipeptide ([14C] Gly-Sar) uptake was inhibited by the selective NHE inhibitors with the same order of potency observed for inhibition of apical 22Na+ uptake. However, 5-(N-ethyl-N-isopropyl) amiloride (EIPA) also reduced [14C]Gly-Sar uptake in the absence of Na+ and this inhibition was concentration and pH (maximal at pH 5.5) dependent. NHE3 inhibition by S1611 and S3226 modulates dipeptide uptake indirectly by reducing the transapical driving force (H+ electrochemical gradient). EIPA (at 100 microM) has similar effects, but at higher concentrations (> 200 microM) also has direct inhibitory effects on hPepT1.

H+-zwitterionic amino acid symport at the brush-border membrane of human intestinal epithelial (CACO-2) cells.

Transport of a number of dipolar amino acids (and the orally active antibiotic D-cycloserine) across the apical membrane of human intestinal epithelial (Caco-2) cell monolayers is mediated by a Na+-independent, pH-dependent transport mechanism. Relatively little is known about the mode of action of this transport system so to differentiate between pH dependence and proton coupling three experimental protocols were designed and tested. The results demonstrate, firstly, that it is the transapical pH gradient and its maintenance (rather than apical acidity alone) that is important in amino acid uptake. Secondly, Na+-independent uptake of seven dipolar amino acids (with pKa (-log of acid dissociation constant) values between 1 50 and 4 23) showed a similar dependence on apical pH (half-maximal uptake being observed at pH 5 99-6 20). Thirdly, the pattern of pH-dependent amino acid ([beta]-alanine) uptake is similar irrespective of whether the cationic substrate concentration is varied or constant, demonstrating no relationship between uptake and concentration of the cationic form of the amino acid. These observations demonstrate that the transport mechanism is a H+-zwitterionic amino acid symporter and suggest that the presence of a H+ gradient at the apical surface of the human small intestine (in the form of the acid microclimate) may be important in driving nutrient absorption.

Direct assessment of dipeptide/H+ symport in intact human intestinal (Caco-2) epithelium: a novel method utilising continuous intracellular pH measurement.

Direct demonstration of intact peptide transport by the intestinal H+/dipeptide carrier is limited both by luminal/cytosolic hydrolysis and the availability of suitable radiolabelled substrates. Perfusion of Val-Val (20mM) at the apical surface of human intestinal epithelial (Caco-2) cell monolayers resulted in a marked intracellular acidification, due to dipeptide-induced H(+)-flow across the apical membrane. Val-Val (20mM) also inhibited both the pH-dependent apical-to-basal transport and intracellular accumulation of [14C]Gly-Sar. Valine (20mM) had no effect on [14C]Gly-Sar transport (and intracellular accumulation) or pHi. We conclude that this novel method for studying H(+)-coupled transport clearly differentiates between the mechanisms responsible for absorption of an intact substrate and products of hydrolysis.

Passive transepithelial absorption of thyrotropin-releasing hormone (TRH) via a paracellular route in cultured intestinal and renal epithelial cell lines.

Transport studies using intestinal brush-border membrane vesicles isolated from rats and rabbits have failed to demonstrate proton- or Na(+)-dependent carrier-mediated transport of thyrotropin-releasing hormone (TRH), despite a pharmacologically relevant oral bioavailability. To examine the hypothesis that reported levels of oral bioavailability reflect predominantly a paracellular rather than transcellular route for transepithelial transport of TRH, we have studied TRH transport in cultured epithelial cell types of intestinal (Caco-2 and T84) and renal (MDCK I, MDCK II, and LLC-PK1) origin, whose paracellular pathways span the range of permeability values observed in natural epithelia. Transport of TRH across monolayers of intestinal Caco-2 cells was similar to the flux of mannitol (approximately 1-4% per 4 hr), and unlike other putative substrates for the di-/tripeptide carrier, apical-to-basolateral transport was not increased by the presence of an acidic pH in the apical chamber. TRH transport did not show saturation, being uneffected in the presence of 20 mM cold TRH. In each cell type studied TRH and mannitol transport were similar and positively correlated with the conductance of the cell layers, consistent with a passive mechanism of absorption. This evidence suggests that, providing that a peptide is resistant to luminal hydrolysis, small but pharmacologically significant amounts of peptide absorption may be achieved by passive absorption across a paracellular route.

Thyrotropin-releasing hormone (TRH) uptake in intestinal brush-border membrane vesicles: comparison with proton-coupled dipeptide and Na(+)-coupled glucose transport.

The mechanism of thyrotropin-releasing hormone (pGlu-His-Pro-NH2; TRH) uptake across the luminal membrane of intestinal enterocytes was investigated using brush-border membrane vesicles (BBMV) from rabbit duodenum and jejunum and rat upper small intestine. [14C]Glucose accumulated within the intestinal vesicles (at 10 sec), in the presence of an inwardly directed Na+ gradient, 7- to 14-fold higher than equilibrium values (65 min). The vesicles also accumulated the dipeptide [14C]Gly-Sar. Dipeptide uptake was greatest in the presence of both an inwardly directed proton gradient and an inside negative membrane potential. The H(+)-dependent Gly-Sar transport was not affected by the presence of an excess (46-fold) of cold TRH. In contrast to the observations with glucose and Gly-Sar, the uptake of [3H]TRH after 10 or 60 sec (in each of the vesicle preparations) was not enhanced by either Na+ or H+ gradient conditions. The absence of vesicular accumulation of TRH was not due to peptide hydrolysis. For example, after a 60-sec incubation with rabbit jejunal BBMV no degradation of the tripeptide was evident. After 65 min, 6% of [3H]TRH had undergone degradation, by deamidation, to form TRH-OH. These studies provide no evidence for the oral absorption of TRH by a Na(+)- or H(+)-dependent carrier system in the brush-border membrane. Previous observations of TRH absorption in vivo may be accounted for by passive absorption of the peptide combined with its relative resistance to luminal hydrolysis.

The role of the proton electrochemical gradient in the transepithelial absorption of amino acids by human intestinal Caco-2 cell monolayers.

We determined the extent of Na(+)-independent, proton-driven amino acid transport in human intestinal epithelia (Caco-2). In Na(+)-free conditions, acidification of the apical medium (apical pH 6.0, basolateral pH 7.4) is associated with a saturable net absorption of glycine. With Na(+)-free media and apical pH set at 6.0, (basolateral pH 7.4), competition studies with glycine indicate that proline, hydroxyproline, sarcosine, betaine, taurine, beta-alanine, alpha-aminoisobutyric acid (AIB), alpha-methylaminoisobutyric acid (MeAIB), tau-amino-n-butyric acid and L-alanine are likely substrates for pH-dependent transport in the brush border of Caco-2 cells. Both D-serine and D-alanine were also substrates. In contrast leucine, isoleucine, valine, phenylalanine, methionine, threonine, cysteine, asparagine, glutamine, histidine, arginine, lysine, glutamate and D-aspartate were not effective substrates. Perfusion of those amino acids capable of inhibition of acid-stimulated net glycine transport at the brush-border surface of Caco-2 cell monolayers loaded with the pH-sensitive dye 2',7'-bis(2-carboxyethyl-5(6)-carboxyfluorescein) (BCECF) caused cytosolic acidification consistent with proton/amino acid symport. In addition, these amino acids stimulate an inward short-circuit current (Isc) in voltage-clamped Caco-2 cell monolayers in Na(+)-free media (pH 6.0). Other amino acids such as leucine, isoleucine, phenylalanine, tryptophan, methionine, valine, serine, glutamine, asparagine, D-aspartic acid, glutamic acid, cysteine, lysine, arginine and histidine were without effect on both pHi and inward Isc. In conclusion, Caco-2 cells express a Na(+)-independent, H(+)-coupled, rheogenic amino acid transporter at the apical brush-border membrane which plays an important role in the transepithelial transport of a range of amino acids across this human intestinal epithelium.

Transepithelial dipeptide (glycylsarcosine) transport across epithelial monolayers of human Caco-2 cells is rheogenic.

Net transepithelial transport (and cellular accumulation) of the dipeptide glycylsarcosine (Gly-Sar), across the apical membrane of human intestinal Caco-2 epithelia, is driven by a proton gradient (Na(+)-free conditions) and displays saturation kinetics (Km 17.4 +/- 5.1 mM, Vmax of 92.8 +/- 15.6 nmol.cm-2.h-1). Net Gly-Sar transport is associated with the stimulation of an inward short-circuit current (Isc). This dipeptide-stimulated Isc is observed in both Na(+)-containing and Na(+)-free conditions, is stimulated by apical acidity, and displays saturation kinetics (in Na(+)-free media at apical pH 6.0, Km of 13.6 +/- 4.5 mM and a Vmax of 284.1 +/- 39.3 nmol.cm-2.h-1). The maximal capacities of Gly-Sar transport and Isc suggest a dipeptide/proton stoichiometry greater than unity (1:3).

Optimal absorptive transport of the dipeptide glycylsarcosine is dependent on functional Na+/H+ exchange activity.

Optimal nutrient absorption across the intestinal epithelium is dependent on the co-ordinated activity of a number of membrane transporters. Di/tripeptide transport across the luminal membrane of the intestinal enterocyte is mediated by the H(+)-coupled di/tripeptide transporter hPepT1. hPepT1 function is dependent on the existence of a pH gradient (maintained, in part, by the action of the Na(+)/H(+) exchanger NHE3) across the apical membrane of the small intestinal epithelium. The physiological problem addressed here was to determine how two transporters (hPepT1 and NHE3), involved in nutrient absorption and pH(i) homeostasis, function co-operatively to maximise dipeptide absorption when both operate sub-optimally at typical mucosal surface pH values (pH 6.1-6.8). Functional hPepT1 activity in human intestinal epithelial (Caco-2) cell monolayers was determined by measurement of apical uptake and apical-to-basolateral transport of the dipeptide glycylsarcosine. The dependence of hPepT1 on NHE3 activity was measured (either after Na(+) removal or addition of the NHE3-selective inhibitor S1611) using both Caco-2 cell monolayers and hPepT1-expressing Xenopus laevis oocytes. Apical glycylsarcosine uptake in Caco-2 cell monolayers was modulated by apical pH, extracellular Na(+), incubation time and S1611. Uptake in hPepT1-expressing oocytes was independent of Na(+) or S1611. We conclude that functional NHE3 activity is required to allow optimal absorption of dipeptides across the human intestinal epithelium.

Expression and role of sodium, potassium, chloride cotransport (NKCC1) in mouse inner medullary collecting duct (mIMCD-K2) epithelial cells.

Loop-diuretic-sensitive 86Rb+(K+) transmembrane fluxes were determined in cells of a mouse inner medullary collecting duct cell line (mIMCD-K2). The furosemide-sensitive (0.1 mM) influx was a substantial fraction of the total influx (0.39+/-0.04 or 0.42+/-0.03, n=5 in the presence or absence of ouabain, respectively). Furosemide also reduced 86Rb+(K+) efflux by a similar fraction (0.46). RT-PCR analysis revealed expression of mRNA for the Na+-K+-2Cl- cortransporter-1 (NKCC1), but not NKCC2. Loop-diuretic-sensitive 86Rb+(K+) influx was confined to the basolateral membrane, confirming its localisation there. The physiological properties of NKCC1 expressed in mIMCD-K2 cells, including the dependence upon medium Na+, K+ and Cl- and the relative sensitivity to loop diuretics as assessed by the concentration required for half-maximal inhibition (IC50) (bumetanide 3.3+/-1.4x10-7 M>piretanide 2.5+/-0.15x10-6 M>furosemide 2.3+/-1.2x10-5 M) were typical for NKCC1. Possible functions of NKCC1 were tested; furosemide did not inhibit the majority of forskolin-stimulated secretory short-circuit current (Isc) (83.5+/-5.3% of the maintained response at 5 min). Secondly, total 86Rb+(K+) influx was stimulated markedly when external osmolarity was increased to 600 mosmol/l by mannitol due to an increase via NKCC1 from 55+/-11 to 191+/-2 nmol/106 cells per 15 min, (both n=4, P<0.01). In contrast, 10-5 M forskolin did not stimulate total 86Rb+(K+) influx. Finally, the ability of both K+ and NH4+ to compete for ouabain-insensitive 86Rb+(K+) influx via NKCC1 was confirmed with similar concentrations for half-maximal influx reduction (K0.5). Apical exposure to NH4+ elicited rapid cytosolic alkalinisation in 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein (BCECF)-loaded epithelial layers, consistent with selective permeability of the apical membrane to NH3. Conversely, NH4+ (5 mM) at the basal cell surface resulted in progressive acidification, the initial rate being reduced by 43% by furosemide. We conclude that NKCC1 participates in selective uptake of NH4+ at the basal surface, and that IMCD may function in direct NH4+ deposition to urine.