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MS 7709 Received 16 December 1997; accepted after revision 28 May 1998.
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ABSTRACT |
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INTRODUCTION |
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The intracellular pH (pHi) level is thought to play an important role in a variety of cellular processes, including cellular metabolism, protein synthesis, contractility, ion channel conductivity, and cell cycle control (Madshus, 1988). Therefore, pHi needs to be regulated in a narrow range to maintain cellular functions. In the enterocytes of the small intestine, the uptake of such nutrients as oligopeptides (Thwaites et al. 1993a; Fei et al. 1994), amino acids (Kanai & Hediger, 1992; Thwaites et al. 1995), fatty acids (Harig et al. 1991) and carboxylic acids (Tamai et al. 1995) are mediated by cotransport with H+ or by exchange with OH- or HCO3- across the microvillus membrane. Thus, in the enterocytes, the acid-base regulatory mechanisms are important not only for stabilizing the pHi level, but also for supporting the intestinal absorption of those nutrients. Several relevant membrane acid-base transporter molecules, including the Na+-H+ exchanger (Tse et al. 1991; Bookstein et al. 1994; Yun et al. 1995; Hoogerwerf et al. 1996; Wakabayashi et al. 1997), the Na+-HCO3- cotransporter (Osypiw et al. 1994; Peral et al. 1995; Bernardo et al. 1996; MacLeod et al. 1996), and the Cl--HCO3- exchanger (Sundaram et al. 1991; Chow et al. 1992) have been demonstrated or suggested to be involved in the enterocytes. However, the role of most of these acid-base transporters in pHi regulation during intestinal absorption of nutrients has yet to be elucidated.
In respect of oligopeptide transport in the intestine, it is generally assumed that the sequential action of Na+,K+-ATPase in the basolateral membrane and of the Na+-H+ exchanger in the apical membrane creates an acidic extracellular microclimate, providing an inward electrochemical gradient of H+ which is necessary for H+-coupled oligopeptide absorption (Ganapathy et al. 1994; Adibi, 1997). In support of this, it has been demonstrated in the Caco-2 colonic tumour cell line that the apical membrane Na+-H+ exchanger is mainly responsible for discharging H+ that enters the cells through the H+-oligopeptide cotransporter (Thwaites et al. 1993b, 1994). On the other hand, in native intestinal epithelia, this hypothesis has never been verified by experimental evidence. In addition, it is conceivable that several acid extrusion mechanisms in the basolateral membrane could also be involved in oligopeptide absorption, although this currently remains conjecture. The aim of this study was to identify the acid-base transporters in the ileal enterocytes which are responsible for extruding H+ that enters cells through the H+-coupled oligopeptide transporter. To achieve this, pHi was measured by microfluorometry in a villus isolated from the guinea-pig ileum that had been loaded with pH-sensitive fluorescent dye. The dipeptide-induced short-circuit current (Isc) was also determined in an isolated ileal sheet in Ussing chambers to identify the localization, apical vs. basolateral membrane, of these transporters.
Parts of this work have been reported previously (Hayashi & Suzuki, 1996, 1997).
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METHODS |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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Solutions
The Hepes-buffered standard solution contained (mM): NaCl, 100; mannitol, 80; CaCl2, 1·5; MgCl2, 1·0; KCl, 5; and Hepes, 10; pH adjusted to 7·4 with Tris at 37°C, and was gassed with 100 % O2. The CO2/HCO3--buffered standard solution contained (mM): NaCl, 75; KCl, 5; NaHCO3, 25; CaCl2, 1·5; MgCl2, 1·0; and mannitol, 80; gassed with 95 % O2-5 % CO2 (pH 7·4 at 37°C). The Na+-free solution was prepared by equimolar substitution with N-methyl-D-glucamine or choline. In the ammonium-containing solution for NH4Cl pulsing, 40 mM NH4Cl was added by substitution of 80 mM mannitol. As a metabolic substrate in the Ussing chamber experiments, 10 mM D-glucose and 2·5 mM L-glutamine were added to the serosal solution.
Microfluorometric recording of pHi level
Male guinea-pigs (Hartley strain, Japan SLC, Shizuoka, Japan) weighing between 300-700 g were acclimatized on a standard diet, with food and water provided ad libitum. The animals were anaesthetized with urethane (1 g (kg body weight)-1, intraperitoneal injection). A segment of distal ileum was excised, opened along the mesenteric border and kept in an oxygenated Hepes-buffered solution. The animals were killed by intracardially injecting a lethal dose of a saturated KCl solution.
Several samples containing the top one-third of the villus were cut from each ileal segment by microdissection scissors under a stereomicroscope while the intestinal segment was pinned with the mucosal side upward. The villus tips were incubated in an oxygenated Hepes-buffered solution containing Dispase (2000 proteinase units ml-1) for 5 min at 37°C while gently shaking to increase the efficiency of a subsequent adhesion to the perfusion vessel. The villus tip preparations were then washed several times by centrifugation, before being resuspended in a Hepes-buffered solution containing 8 µM of 2'7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein, acetoxymethyl ester (BCECF AM) for 5 min in the dark at room temperature (20-25°C). The BCECF AM had first been dissolved to a concentration of 8 mM in dimethyl sulphoxide. After dye loading, the villus tip preparations were washed with the Hepes-buffered solution and used for the subsequent experiments. The suspension of dye-loaded villus tips was seeded on to a specially designed glass perfusion vessel, which had been coated with the cell adhesive, Cell-Tak, and the vessel was centrifuged at 200 g for 5 min at room temperature to fix the villus tips. The vessel was then placed on the stage of an inverted microscope (TMD, Nikon, Tokyo, Japan) equipped with a microscopic dual-wavelength fluorometer system (CAM-230, Japan Spectroscopic, Tokyo, Japan), perfused at 6 ml min-1 with the gas-equilibrated solution and maintained at 35-37°C. The whole area of a selected villus tip was illuminated alternately at 440 and 500 nm for 50 ms by a chopper at 100 Hz, and the fluorescence was measured at 520-570 nm through a band-pass filter. The pHi value was calculated from the mean fluorescence ratio (F500/F440) every 2 or 3 s. All these procedures were controlled by a computer (Macintosh LC) which was equipped with a data acquisition and analysis system (Lab View 2, National Instruments, Houston, TX, USA). Any autofluorescence from the villus tips was found to be negligible.
The pHi level was calibrated in situ by the nigericin-K+ method (Thomas et al. 1979). Dye-loaded villus tips were incubated with nigericin (20 µM) for 5 min, and then with a high-K+ perfusion solution of various pH values (KCl, 130 mM; NaCl, 10 mM; CaCl2, 1·5 mM; MgCl2, 1·0 mM; Hepes or Mes, 10 mM; pH was adjusted with Tris at 37°C) to determine the relationship between the fluorescence ratio (F500/F440) and pHi. Calibration curves were obtained over a pH range of 6-8.
The rate of H+ efflux or intracellular H+ accumulation was calculated from the initial rate of pHi recovery or from the pHi decrease, respectively, and the cellular total buffer capacity (
t). t can be expressed as follows:
t = i + 2·303 [HCO3-]i,
where i is the intrinsic buffer capacity and [HCO3-]i is the intracellular HCO3- concentration. The intrinsic buffer capacity was determined by the ammonia withdrawal method in a Na+- and Cl--free solution (Cl- was replaced with gluconate in a Na+-free, Hepes-buffered solution; Boyarsky et al. 1988). Although this solution would prevent any undesirable contribution from an acid-base transport mechanism depending upon Na+ or Cl-, which may be the major influence on pHi regulation, a mechanism depending upon K+ (e.g. H+,K+-ATPase) and/or other mechanisms such as proton conductance and H+-ATPase would still be operative. The relationship between pHi and i was estimated by regression of the data to a fourth-order polynomial. [HCO3-]i was calculated by the Henderson-Hasselbach equation at the measured pHi value.
To assess the effects of inhibitors on pHi recovery, three experiments were performed consecutively on every tissue sample, first in the absence of an inhibitor, second in the presence of the inhibitor, and then finally in the absence of the inhibitor again. The mean value of pHi recovery rate from the first and final measurements was taken as the control value and compared with the rate in the presence of the inhibitor for the same tissue sample. The initial rate of pHi recovery after acid loading was determined by a linear least-squares fit to data collected during the initial linear phase of pHi recovery.
Measurements of the dipeptide-induced short-circuit current
A segment of the guinea-pig distal ileum, about 10 cm long, was excised and opened along the mesenteric border. A mucosal preparation, consisting of the mucosa and part of the submucosa, was made by removing the tunica muscularis with fine forceps. The mucosal preparation was then mounted vertically between Lucite chambers that provided an exposed area of 0·5 cm2. The volume of the bathing solution on each side was 10 ml, and the solution temperature was maintained at 37°C by a water jacket.
The short-circuit current (Isc) was measured by using an automatic voltage-clamping device that compensated for the solution resistance (CEZ-9000, Nihon Kohden, Tokyo, Japan). The transepithelial potential was recorded through 1 M KCl salt-agar bridges connected to a pair of calomel half-cells, and the transepithelial current was applied across the tissue via a pair of Ag-AgCl electrodes that were kept in contact with the luminal and serosal bathing solutions by using a pair of 1 M N-methyl-D-glucamine Cl- salt-agar bridges. The Isc value is referred to as positive when current flowed from the lumen to the serosa. We measured the dipeptide-induced Isc value three times in the same preparation, first under control conditions, second under experimental conditions and finally under control conditions again. The Isc increase from the second measurement is presented relative to the mean Isc increase from the first and final measurements.
Chemicals
We obtained amiloride and nigericin from Sigma, 4,4'-diisothiocyanostilbene-2,2'-disulphonic acid (DIDS) from Wako (Tokyo, Japan), BCECF AM from Molecular Probes, Dispase from Godo Susei (Tokyo, Japan), and Cell-Tak from Becton Dickinson Labware (Bedford, MA, USA).
Data and statistical analyses
The relationship between the amiloride concentration and inhibition of pHi recovery rate was fitted to the Michaelis-Menten equation by using a non-linear least-squares algorithm (Ultra Fit, Biosoft, Cambridge, UK). Each experimental value is given as the mean ±
As is shown in Fig. 1, the villus tip isolated from the guinea-pig ileum was covered with polygonal absorptive enterocytes, except for the cut end. The fluorescent dye was accumulated at a higher level in this surface enterocyte layer than in the villus core region. Figure 2 shows the relationship between intracellular pH (pHi) and buffer capacity. The total buffer capacity did not differ much in the pHi range of 7·2 to 6·6, but it increased markedly at pHi values lower than this. The thick continuous line in this figure was used to calculate the rate of H+ efflux or intracellular H+ accumulation in some of the following results.
A, transmitted light micrograph of an isolated villus tip. B, epifluorescent micrograph of the same sample. Fluorescence is present at a higher level in the surface epithelial layer than in the villus core. The bar represents 100 µm.
The intrinsic buffer capacity (
In the resting condition, the pHi level of villus tips was 7·34 ± 0·07 pH units (n = 9) in the CO2/HCO3--free, Hepes-buffered solution. When the villus tips were perfused with the CO2/HCO3--buffered solution, the resting pHi value was 7·04 ± 0·02 pH units (n = 33), a value significantly lower than that in the Hepes-buffered solution.
Na+-dependent and amiloride-sensitive pHi recovery from acid loading by pulsed NH4+
We first examined the recovery of pHi from acid loading in the CO2/HCO3--free, Hepes-buffered solution so as to minimize the activity of the HCO3--dependent pHi regulatory mechanism. An acid load was imposed on the cells by pulsed perfusion with 40 mM NH4Cl. As shown in Fig. 3, upon replacement of the perfusate with a Na+-free solution, the pHi level rapidly decreased with little recovery (pHi = 6·54 ± 0·03, n = 24). However, when the perfusate was replaced with the Na+-containing solution, the pHi level rapidly returned to the resting level. Amiloride, a known inhibitor of Na+-H+ exchange, inhibited the Na+-dependent pHi recovery rate (Fig. 3, middle part). The dose-response curve for the inhibition of Na+-dependent pHi recovery by amiloride indicated a half-maximal concentration (IC50) of 20 ± 2 µM and maximum inhibition of 92 ± 2 % (Fig. 4, open circles). At the concentration of 0·6 mM amiloride, the initial rate of pHi recovery was strongly, but not completely, inhibited (87 ± 3 %, n = 6).
The villus tip was acid-loaded 3 times by being exposed to 40 mM NH4Cl for 90 s. Dashed lines indicate the initial rate of pHi recovery. A representative trace from 6 experiments is presented.
Effect of amiloride on pHi recovery from acid loading by glycylglycine
The activation of H+-coupled oligopeptide transport in the enterocytes by a superfusion of 10 mM glycylglycine (Gly-Gly) decreased the pHi level by 0·29 ± 0·01 pH units (final pHi = 6·74 ± 0·02, n = 33) in the CO2/HCO3--buffered solution (Fig. 5). The pHi decrease reached its nadir within 3 min. Recovery of the pHi level was observed upon replacing the perfusate with the control solution. The superfusion of another dipeptide glycylproline (Gly-Pro) also decreased the pHi level by 0·18 ± 0·03 pH units (n = 4). On the other hand, the superfusion of 20 mM glycine (Gly) caused a transient increase in the pHi level by 0·08 ± 0·03 pH units followed by a decrease to slightly below the basal pHi level by 0·03 ± 0·01 pH units (n = 5). These results suggest that oligopeptide-induced acidification was mainly due to H+-coupled oligopeptide transport. While the hydrolysis of oligopeptides may take place, the released amino acids, which have demonstrated H+-coupled transport under certain conditions (Thwaites et al. 1995), may not be mainly responsible for the oligopeptide-induced acidification.
Representative traces in the CO2/HCO3--buffered solution are presented. Each trace was obtained from 3 separate experiments. Substrates were added to the perfusate by replacing equimolar mannitol.
We next examined a contribution of the amiloride-sensitive acid extruder just described to the pHi recovery from acid loading by Gly-Gly. In the CO2/HCO3--buffered solution, the initial rate of pHi recovery was partially inhibited by 0·3 mM amiloride (Fig. 6A and C). The dose-response relationship for inhibition of the initial rate of pHi recovery by amiloride was examined and the result is illustrated in Fig. 7. The IC50 for amiloride was 41 ± 19 µM and maximum inhibition was 63 ± 5 %. This IC50 value for amiloride is not markedly different from that for the inhibition by amiloride of pHi recovery from acid loading imposed by pulsed NH4+ in the Hepes-buffered solution (Fig. 4, open circles). Thus, both the amiloride-sensitive and the amiloride-insensitive acid extruders (or base loaders) contribute to the pHi recovery from acid loading by Gly-Gly in the CO2/HCO3--buffered solution. In the CO2/HCO3--free, Hepes-buffered solution, the superfusion of Gly-Gly caused a decrease in the pHi level by 0·60 ± 0·05 pH units (final pHi = 6·74 ± 0·05, n = 9) (Fig. 6B). In the Hepes-buffered solution, the initial rate of pHi recovery upon removing Gly-Gly was completely abolished in the presence of 0·3 mM amiloride (Fig. 6B and C). In the presence of 30 µM amiloride, the pHi recovery rate was inhibited by 62 ± 5 % (n = 4), suggesting that the amiloride dose-inhibition relationship agrees with the relationship when acid loading was imposed by pulsed NH4+ (Fig. 4). The efflux of H+-equivalent as estimated from the initial rate of pHi recovery was significantly less in the Hepes-buffered solution than in the CO2/HCO3--buffered solution (Fig. 6C).
These findings indicate that, in the absence of CO2 and HCO3-, the amiloride-sensitive acid extruder is totally responsible for the pHi recovery, whereas, in the presence of CO2 and HCO3-, the amiloride-insensitive acid extruder is also activated and contributes to the pHi recovery.
A, representative trace in the CO2/HCO3--buffered solution. Gly-Gly was added to the perfusate at a concentration of 10 mM by replacing equimolar mannitol. Dashed lines show the initial rates of pHi recovery. B, representative trace in the Hepes-buffered solution. C, effect of 0·3 mM amiloride on H+ efflux after removing Gly-Gly in the presence and absence of CO2 and HCO3-. The control value is the mean value of H+ efflux from the first measurement without an inhibitor and the final measurement once the inhibitor had been removed. Each H+-equivalent efflux was estimated from the initial rate of pHi recovery and buffer capacity. n = 4 for the Hepes- and n = 8 for the CO2/HCO3--buffered solution. *P < 0·05 compared with the CO2/HCO3--buffered solution.
An acid load was imposed on cells by pulsed perfusion with 10 mM Gly-Gly, and the initial rate of pHi recovery was determined in the presence of various concentrations of amiloride. n = 4-6 for each concentration of amiloride. The curve was determined by fitting the data to the Michaelis-Menten equation.
Na+ dependency of pHi recovery from acid loading by Gly-Gly
The Na+ dependency of pHi recovery from acid loading by Gly-Gly was tested in the CO2/HCO3--buffered solution (Fig. 8). Replacement of the perfusate with a Na+-free solution in the presence of Gly-Gly caused a marked decrease in the pHi value (pHi = 6·35 ± 0·01, n = 24). Under Na+-free conditions, pHi recovery was hardly apparent after Gly-Gly had been removed, except for an occasional small change. However, the pHi level rapidly recovered when Na+ was introduced into the perfusate again. These results suggest that the main pHi recovery mechanisms from acid loading induced by Gly-Gly are all dependent upon extracellular Na+.
We next examined the effect of amiloride on this Na+-dependent pHi recovery from acid loading by Gly-Gly. In the presence 0·3 mM amiloride, the initial rate of pHi recovery activated by Na+ was inhibited by 74 % (Fig. 8A and C). This degree of inhibition was not enhanced when 2 mM amiloride was used (Fig. 8C). We next tested whether the component of pHi recovery that was insensitive to amiloride would be inhibited by the stilbene derivative DIDS. In the presence of both DIDS (0·6 mM) and amiloride (0·3 mM), the initial rate of pHi recovery induced by Na+ was inhibited more, although not completely abolished, than in the presence of amiloride alone (Fig. 8B and C). Therefore, the amiloride-insensitive pHi recovery mechanism that is active in the presence of CO2 and HCO3- is at least partly sensitive to DIDS.
A, the villus tip was acid loaded by pulsed perfusion with 10 mM Gly-Gly in the absence of extracellular Na+. The initial rates of pHi recovery upon the reintroduction of Na+ (dashed lines) were compared in the absence and presence of 0·3 mM amiloride. A representative trace from 6 experiments is shown. B, initial rate of Na+-dependent pHi recovery (dashed lines) in the absence and presence of 0·3 mM amiloride + 0·6 mM DIDS. A representative trace from 6 experiments is shown. C, initial slopes of Na+-dependent pHi recovery in the presence of various inhibitors were obtained from experiments similar to those shown in A and B. The slope under Na+-free conditions was obtained from the series of inhibitor experiments. Results are given as a percentage of the control value. Columns not sharing the same lower-case letters are significantly different by ANOVA. n = 6 for each column except for the Na+-free column where n = 18.
Activation of the amiloride-sensitive and DIDS-sensitive acid extrusion mechanism during superfusion with Gly-Gly
To determine whether the amiloride-sensitive acid extruder and DIDS-sensitive acid extruder (or base loader) just described would be activated or not during the absorption of Gly-Gly, we measured the pHi decreases induced by amiloride and DIDS both under resting conditions and during 10 mM Gly-Gly superfusion in the presence of CO2 and HCO3- (Table 1). The application of 0·3 mM amiloride caused a decrease in pHi under resting conditions, which was enhanced in the presence of Gly-Gly. We determined the net intracellular H+ accumulation rather than H+ influx because it was difficult to determine the very small initial rate of pHi change. The intracellular H+ accumulation induced by 0·3 mM amiloride was two times greater during Gly-Gly superfusion than under resting conditions. An increase in the dose of amiloride from 0·3 to 2 mM resulted in a further pHi decrease. However, the increase in net H+ accumulation induced by increasing to 2 mM amiloride was not significantly different in either the presence or absence of Gly-Gly. The application of 0·6 mM DIDS under resting conditions resulted in a small alkalinization, whereas the application of DIDS during Gly-Gly superfusion caused a marked decrease in pHi. Thus, the net intracellular H+ accumulation induced by DIDS was significantly greater during Gly-Gly superfusion than under resting conditions. These results indicate that acid extrusion (or base loading) by the amiloride-sensitive mechanism and the DIDS-sensitive mechanism were both enhanced during Gly-Gly superfusion. It was also observed that the pHi decrease and intracellular H+ accumulation induced by 0·3 mM amiloride during Gly-Gly superfusion were significantly enhanced when applied in the presence of DIDS (-0·15 ± 0·003 pH units and 14·0 ± 5·5 mM for
Effects of Na+ removal, amiloride and DIDS on oligopeptide-induced Isc
We next measured oligopeptide-induced Isc to investigate whether the amiloride-sensitive and the DIDS-sensitive acid extruders that were implied by the results of pHi measurements were located on the apical or basolateral membrane. We used Gly-Pro, a dipeptide presumably more resistant than Gly-Gly to hydrolysis by brush-border amino peptidases, to minimize the increment in Isc that would be induced by amino acids released due to hydrolysis of the dipeptide. As shown in Fig. 9, the increase in Isc induced by luminal dipeptides attained a plateau within a few minutes and remained at that level for at least the following 15 min. We thus determined the maximum increase in Isc from the baseline level. In the CO2/HCO3--buffered solution, the luminal application of 10 mM Gly-Pro induced an increase in Isc with a mean value of 48 ± 3 µA cm-2 (n = 29). In the Hepes-buffered solution, Gly-Pro induced an increase in Isc with a mean value of 56 ± 3 µA cm-2 (n = 43).
Representative traces are shown. The increase in Isc induced by a luminal application of 10 mM Gly-Pro was determined 3 times, each being preceded by washing several times and with a stabilizing period of at least 15 min as indicated by W. A, time control. B, the second measurement was done in the absence of serosal Na+, while the first and final measurements were under control conditions.
Table 1. Comparison of the effects of inhibitors on pHi between resting conditions and during Gly-Gly superfusion
The first series of experiments was performed in the CO2/HCO3--buffered solution. As shown in Fig. 9B, Gly-Pro-induced Isc appeared to be markedly reduced by removing Na+ from the serosal side. Since the Gly-Pro-induced Isc values gradually decreased when the measurements were repeated (Fig. 9A), the Isc increase from the second test measurements is presented relative to the mean Isc increase from the first and final control experiments (Table 2). Removing Na+ from the serosal solution significantly decreased Gly-Pro-induced Isc. While removing Na+ from the luminal solution also seemed to decrease Gly-Pro-induced Isc, this decrease was not significant. Removing Na+ from both sides did not inhibit further Gly-Pro-induced Isc than did serosal Na+ removal alone. We then examined the effects of amiloride and DIDS. As shown in Table 2, a pre-application of 0·5 mM amiloride to either the luminal or serosal side had little effect on Gly-Pro-induced Isc. However, when 0·5 mM amiloride was applied to both sides, Gly-Pro-induced Isc was significantly decreased. An increase in luminal amiloride concentration to 2 mM did not further inhibit Gly-Pro-induced Isc in either the presence or absence of 0·5 mM amiloride on the serosal side. As shown in Table 2, serosal DIDS also had an inhibitory effect on Gly-Pro-induced Isc: a pre-application of 0·6 mM DIDS simultaneously with 0·5 mM amiloride to the serosal side significantly decreased Gly-Pro-induced Isc. In addition, when amiloride was present on both the luminal (2 mM) and the serosal (0·5 mM) sides, the application of 0·6 mM DIDS to the serosal side markedly reduced Gly-Pro-induced Isc when compared with the absence of DIDS. On the other hand, the application of 0·6 mM DIDS to the luminal side did not further inhibit Gly-Pro-induced Isc either in the presence of 0·5 mM amiloride on the serosal side or in the presence of 0·5 mM amiloride on both sides (Table 2).
Thus, the application of serosal amiloride and DIDS, as well as the removal of serosal Na+ each had an inhibitory effect on Gly-Pro-induced Isc, at least under certain conditions.
Table 2. Effects of added amiloride and DIDS and of Na+ replacement on the glycylproline-induced Isc value in a CO2/HCO3--buffered solution
Gly-Pro-induced Isc was also measured in the Hepes-buffered solution to confirm the inhibitory effects of serosal amiloride and Na+ removal (Table 3). The increment in Isc induced by the luminal application of 10 mM Gly-Pro was significantly decreased by the removal of Na+ from either the serosal side or the luminal side. When Na+ was simultaneously removed from both sides, Gly-Pro-induced Isc was reduced more than by serosal Na+ removal alone. Pre-application of 0·5 mM amiloride to the serosal side caused subsequent Gly-Pro-induced Isc to be significantly decreased. However, luminal 0·5 mM amiloride had little effect on Gly-Pro-induced Isc. The inhibition of dipeptide-induced Isc by amiloride on both sides was not significantly different from that by serosal amiloride alone.
Table 3. Effects of added amiloride and Na+ replacement on the glycylproline-induced Isc value in a Hepes-buffered solution
Our aim was to identify the acid-base transporters in the ileal enterocyte membrane that are responsible for extruding H+ that enters the cells through the H+-coupled oligopeptide transporter. The results of pHi measurements show that at least two acid extrusion (or base loading) mechanisms, both being dependent on extracellular Na+, were activated and were responsible for extruding H+ loaded with superfusing Gly-Gly: (1) an amiloride-sensitive acid extruder; and (2) a DIDS-sensitive, HCO3--dependent acid extruder. The findings that Gly-Pro-induced Isc was attenuated by either removing serosal Na+ or by adding amiloride or DIDS to the serosal side imply that both the amiloride-sensitive acid extruder and the DIDS-sensitive, HCO3--dependent acid extruder were present at least in the basolateral membrane of the enterocytes. It is generally thought, and has actually been demonstrated in a Caco-2 colonic tumour cell line, that a Na+-H+ exchanger in the apical membrane is mainly responsible for the extrusion of H+ that enters through the H+-coupled oligopeptide transporter (Thwaites et al. 1993b, 1994; Ganapathy et al. 1994; Adibi, 1997). From the present results, however, we propose that at least in guinea-pig ileum the amiloride-sensitive and the DIDS-sensitive acid extrusion mechanisms in the basolateral membrane would also probably play an important role in the H+ extrusion in combination with the apical membrane mechanism.
We used a villus tip preparation in this study on which to take microfluorometric pHi measurements, because this preparation responded to acid loading by pulsed NH4+ or Gly-Gly at least several times with reasonable reproducibility (see e.g. Figs 3 and 6). This preparation contained not only epithelial enterocytes but also non-epithelial cells in the villus core region and, therefore, interpreting the results of pHi measurements would be difficult if the fluorescence signal was derived mainly from the latter cells. However, fluorescent dye was accumulated at a higher level in the surface layer than in the villus core region (Fig. 1). In addition, superfusion of Gly-Gly could decrease pHi to a considerable degree. The expression of mRNA and protein for the oligopeptide transporter are restricted to epithelial enterocytes with no detectable expression in the lamina propria (Freeman et al. 1995; Walker et al. 1998). Consequently, it is reasonable to assume that the major part of the fluorescence signal was derived from enterocytes.
Amiloride-sensitive acid extruder
We have demonstrated here a Na+-dependent acid extrusion mechanism that was inhibited by amiloride with an IC50 value of 20 µM by acid loading with pulsed NH4+ in a CO2/ HCO3--free solution (Fig. 4). This acid extrusion mechanism was presumably activated and partially responsible for extruding H+ loaded with superfusion of Gly-Gly because: (1) pHi recovery after Gly-Gly superfusion was dependent on Na+ and partially inhibited by amiloride with an IC50 value of 41 µM (Figs 7 and 8), and (2) intracellular H+ accumulation induced by 0·3 mM amiloride was greater during superfusion of Gly-Gly than under resting conditions (Table 1). The membrane transporters responsible for this Na+-dependent, amiloride-sensitive acid extrusion are probably Na+-H+ exchangers. Three Na+-H+ exchanger isoforms, NHE1, 2 and 3, have been demonstrated in rat, rabbit and human enterocytes (Yun et al. 1995; Wakabayashi et al. 1997). In addition, the presence of another isoform, NHE4, has been reported in rat intestine (Orlowski et al. 1992). Of these, NHE1 and NHE2 have been reported to be inhibited by a relatively low concentration of amiloride. In the guinea-pig ventricular myocyte, amiloride has been reported to inhibit NHE1 with an IC50 value of 87 µM in the presence of 140 mM Na+ (Loh et al. 1996), a value similar to the present findings. Unfortunately, the IC50 value for the inhibitory effect of amiloride on NHE2 in the presence of a physiological concentration of Na+ has not been reported. On the other hand, the IC50 value for the inhibitory effect of amiloride on NHE3 and NHE4 has been suggested to be more than 300 µM in the presence of a physiological concentration of Na+ (Chambrey et al 1997). Consequently, either NHE1 or NHE2 (or both) is probably responsible for the amiloride-sensitive pHi recovery after acid loading by a superfusion of Gly-Gly.
The results of Gly-Pro-induced Isc measurements imply that the amiloride-sensitive Na+-H+ exchanger is present in the basolateral membrane of the enterocytes. Peptide-induced Isc was inhibited by removing serosal Na+ or adding amiloride to the serosal side, although, in the CO2/HCO3--buffered solution, inhibition by serosal amiloride was only observed when amiloride was simultaneously present in the luminal solution (Tables 2 and 3). Previous functional and morphological studies on the intestines have demonstrated that NHE1 is localized in the basolateral membrane, whereas NHE2 and NHE3 are localized in the apical membrane of the enterocytes (Tse et al. 1991; Bookstein et al. 1994; Hoogerwerf et al. 1996), although any functional activity of NHE2 in the apical membrane remains to be demonstrated. The membrane localization of NHE4 has not been reported in the intestine. Consequently, we propose that an amiloride-sensitive NHE1 in the basolateral membrane is involved in extruding H+ loaded into the enterocytes through the oligopeptide-H+ cotransport in the apical membrane, and would thereby play a supporting role in intestinal oligopeptide absorption.
HCO3--dependent, DIDS-sensitive acid extruder
The present results suggest that, besides the amiloride-sensitive acid extruder, pHi recovery from acid loading by Gly-Gly was also mediated by another Na+-dependent acid extruder (or base loader) that was only active in a CO2/HCO3--solution and inhibited by DIDS (Fig. 8). It was also shown that the activity of the DIDS-sensitive acid extrusion was probably enhanced during Gly-Gly superfusion (Table 1). The HCO3--dependent, DIDS-sensitive acid extrusion mechanism is probably also present in the basolateral membrane because Gly-Pro-induced Isc was significantly reduced by serosal DIDS (when amiloride was present in the serosal, or in both the serosal and luminal solution) (Table 2). The membrane protein responsible for this DIDS-sensitive acid extrusion could be a Na+-HCO3- cotransporter that works in the inward direction. Operation of the putative Na+-HCO3- cotransporter in the inward direction may facilitate H+-coupled, electrogenic oligopeptide influx not only due to the neutralization of accumulated H+, but possibly also due to the hyperpolarization of membrane potential, because most subtypes of the Na+-HCO3- cotransporter are known to transport more than one HCO3- with each Na+ (Boron et al. 1997). The presence of a Na+-HCO3- cotransporter has been suggested in intestinal cells from several species (Osypiw et al. 1994; Peral et al. 1995; Bernardo et al. 1996; MacLeod et al. 1996), although the basolateral membrane localization of a Na+-HCO3- cotransporter has not been described (Hagenbuch et al. 1987; Tosco et al. 1995). In many cell types, Na+-HCO3- cotransporters have been shown to be inhibited by DIDS (Boron et al. 1997), although the Na+-HCO3- cotransporter in the crypt cells of the guinea-pig has been suggested to be resistant to DIDS (MacLeod et al. 1996).
Apical membrane acid extruder
Our results support the current idea that a Na+-dependent mechanism in the apical membrane plays a role in intestinal oligopeptide absorption. Thus, Gly-Pro-induced Isc was probably reduced by removing Na+ from the luminal side (Tables 2 and 3). In addition, luminal 0·5 mM amiloride significantly attenuated Gly-Pro-induced Isc when amiloride was simultaneously present on the serosal side in the CO2/HCO3--buffered solution, but not in the Hepes-buffered solution (Tables 2 and 3). These findings suggest the possibility of two different Na+-dependent acid extrusion mechanisms in the apical membrane: one is inhibited by 0·5 mM amiloride and operates mainly in the CO2/HCO3--buffered solution, while the other is not inhibited by 0·5 mM amiloride and operates mainly in the Hepes-buffered solution. Previous studies have reported that intestinal oligopeptide absorption was reduced by luminal amiloride (Hu et al. 1995; Westphal et al. 1995) or by preincubating the luminal side with a Na+-free solution (Rubino et al. 1971). In addition, Na+ absorption has been reported to increase in association with oligopeptide absorption (Hellier et al. 1973; Himukai et al. 1983). Furthermore, pHi recovery from an oligopeptide-induced acid load has been shown to be markedly increased by luminal Na+ in Caco-2 cells (Thwaites et al. 1993b, 1994). These and the present results can be explained by the operation of the apical membrane Na+-H+ exchanger, probably NHE3 or NHE2 (Hoogerwerf et al. 1996). However, perhaps surprisingly, the results of our pHi measurements seem to exclude the involvement of NHE3 in pHi recovery from Gly-Gly superfusion: the pHi recovery was mostly abolished by 0·3 mM amiloride in the Hepes-buffered solution and, in the CO2/HCO3--buffered solution, the degree of inhibition of pHi recovery by amiloride was no different at concentrations between 0·3 and 5 mM. In addition, the H+ accumulation observed with 2 mM amiloride (in the presence of 0·3 mM amiloride) was no different in the presence or absence of Gly-Gly (Table 1). These results suggest that an acid extrusion mechanism requiring more than 0·3 mM amiloride to be inhibited would not contribute substantially to pHi recovery from acid loading by Gly-Gly. Since NHE3 has been reported to be inhibited by amiloride with an IC50 value of at least 300 µM in the presence of a physiological concentration of Na+ (Chambrey et al. 1997), it is unlikely, though cannot be excluded, that apical membrane NHE3 played a substantial role in extruding H+ during oligopeptide absorption. Further study is necessary to elucidate the role of the NHE3 and other acid extruders in the apical membrane in maintaining intestinal oligopeptide absorption.
In summary, we have proposed that, in addition to the acid extruder in the apical membrane, a basolateral amiloride-sensitive acid extruder, probably NHE1, and a basolateral DIDS-sensitive, HCO3--dependent acid extruder (base loader), possibly the Na+-HCO3- cotransporter, play an important role in maintaining intestinal oligopeptide absorption. Our proposition of the basolateral presence of these acid extruders is based on the inhibition of Gly-Pro-induced Isc by serosal amiloride, DIDS or Na+ removal. Oligopeptide-induced Isc has been demonstrated to reflect closely the rate of intestinal oligopeptide absorption at least under certain conditions (Hoshi 1986; Abe et al. 1987). This notion has been reinforced by the evidence that H+-coupled oligopeptide transporter is electrogenic (Boyd & Ward 1982; Abe et al. 1987; Thwaites et al. 1993c; Fei et al. 1994). To confirm our proposition of the role by basolateral acid extruders, however, it should be directly demonstrated by, for example, tracer uptake measurements that oligopeptide absorption across the apical membrane is actually reduced by serosal amiloride, DIDS or Na+ removal. Our hypothesis predicts that H+ entering with oligopeptide across the apical membrane would not be totally removed back to the luminal side, but that a substantial amount of accumulated H+ may be released to the serosal side across the basolateral membrane. Although this prediction should be verified by measuring the H+ flux across the intestinal epithelium, the previous observation that dipeptides actually increased the microclimate pH level (decreased H+ at the luminal surface) is consistent with this prediction (Shimada, 1987; Ikuma et al. 1996).
Acknowledgements
We would like to thank Drs T. Hoshi (University of Shizuoka, Japan) and M. Ikuma (Yale University, USA) for helpful discussions and for commenting on the manuscript. This study was supported by a Grant-in-Aid for Scientific Research on Priority Areas of 'Channel-Transporter Correlation' from the Ministry of Education, Science and Culture, Japan (07276101).
Corresponding author
H. Hayashi: Laboratory of Physiology, School of Food and Nutritional Sciences, University of Shizuoka, 52-1 Yada, Shizuoka 422-8526, Japan.
Email: hayashih{at}smail.u-shizuoka-ken.ac.jp
RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References
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Figure 1. Top view of an isolated villus tip from the guinea-pig ileum placed in the perfusion vessel after being loaded with BCECF fluorescent dye
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Figure 2. Relationship between intracellular pH and buffer capacity
i) was determined by the ammonia-withdrawal method. The thin continuous line is a fourth-order polynomial of best fit to the data points by the least-squares method. Data were obtained from 6 animals. The thick continuous line represents the total buffer capacity (t) when cells were bathed in the CO2/HCO3--buffered solution, which includes additional buffer capacity that is expressed as 2·303 [HCO3-]i (see Methods for a detailed explanation).
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Figure 3. Na+-dependent pHi recovery from acid loading imposed by pulsed NH4Cl and the effect of amiloride on pHi recovery in the Hepes-buffered solution
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Figure 4. Dose-response relationship for the inhibition of pHi recovery by amiloride in the Hepes-buffered solution
, an acid load was imposed on cells by pulsed perfusion with 40 mM NH4Cl. The effects of various concentrations of amiloride on the initial rate of Na+-dependent pHi recovery were determined from the series of experiments shown in Fig. 3. The mean value of pHi recovery rate from the first and final measurements was taken as the control value and compared with the rate in the presence of amiloride. The continuous curve was determined by fitting the data to the Michaelis-Menten equation. The number of animals was 4-6 for each concentration of amiloride. 
, an acid load was imposed on cells by pulsed perfusion with 10 mM Gly-Gly. The effects of two different concentrations of amiloride on the initial rate of pHi recovery were determined from the experiments shown in Fig. 6B. The dashed curve was drawn arbitrarily. n = 4 for each concentration of amiloride.
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Figure 5. Effects of glycylglycine (Gly-Gly), glycylproline (Gly-Pro) and glycine (Gly) on the resting pHi level
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Figure 6. Effect of amiloride on pHi recovery from acid loading imposed by Gly-Gly
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Figure 7. Dose-response relationship for the inhibition of pHi recovery from Gly-Gly-imposed acid loading by amiloride in the CO2/HCO3--buffered solution
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Figure 8. Effects of amiloride and DIDS on the rate of Na+-dependent pHi recovery from acid loading imposed by Gly-Gly in the CO2/HCO3--buffered solution
pHi and H+ accumulation, respectively, n = 3, P < 0·05 compared with the absence of DIDS by the unpaired t test).
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Figure 9. Glycylproline-induced short-circuit current (Isc) in the CO2/HCO3--buffered solution
Resting Peptide superfusion Inhibitor Initial pHi pHiH+ accumulation (mM) Initial pHi pHiH+ accumulation (mM) n 0·3 mM amiloride 6·89 ± 0·04 -0·04 ± 0·01 1·4 ± 0·4 6·75 ± 0·04 -0·07 ± 0·01 * 3·5 ± 0·3 * (5) 2 mM amiloride1 6·85 ± 0·04 -0·06 ± 0·01 2·7 ± 0·4 6·68 ± 0·03 -0·05 ± 0·01(n.s.) 2·9 ± 0·4 (n.s.) (5) 0·6 mM DIDS 6·86 ± 0·08 0·03 ± 0·003 -1·2 ± 0·2 6·81 ± 0·08 -0·13 ± 0·04 * 6·4 ± 1·1 * (5) pHi) are shown. H+ accumulation was evaluated by integrating the pHi-buffer capacity curve (Fig. 2). Each value represents the mean ±
Serosal treatment Luminal treatment None 0·6 mM DIDS 0·5 mM amiloride 0·5 mM amiloride + 0·6 mM DIDS 2 mM amiloride Na+ replacement None 0·88 ± 0·05a - 0·80 ± 0·04a,b - 0·77 ± 0·07a,b,c 0·74 ± 0·08a,b,c 0·5 mM amiloride 0·80 ± 0·04a,b,c 0·76 ± 0·07a,b,c 0·65 ± 0·05b,c 0·63 ± 0·04c 0·67 ± 0·06b,c - 0·5 mM amiloride + 0·6 mM DIDS 0·67 ± 0·06b,c - - - 0·39 ± 0·02d - Na+ replacement 0·36 ± 0·03d - - - - 0·28 ± 0·05d
Serosal treatment Luminal treatment None 0·5 mM amiloride Na+ replacement None 0·95 ± 0·04a 0·90 ± 0·04a 0·50 ± 0·06b 0·5 mM amiloride 0·32 ± 0·05c,d 0·32 ± 0·06c,d - Na+ replacement 0·39 ± 0·04b,c - 0·21 ± 0·02d
DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References
REFERENCES
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Abstract
Introduction
Methods
Results
Discussion
References
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