Intracellular pH regulation in bombesin-stimulated secretion in isolated bile duct units from rat liver

Won Kyoo Cho1, Albert Mennone2, and James L. Boyer2

1 Division of Gastroenterology/Hepatology, Indiana University School of Medicine, Indianapolis, Indiana 46202; and 2 Department of Internal Medicine and Liver Center, Yale University School of Medicine, New Haven, Connecticut 06520

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Bombesin, a neuropeptide, stimulates fluid and HCO-3 secretion from cholangiocytes, but the underlying mechanisms are poorly understood. In this study, we aimed to examine the effects of bombesin on ion transport processes involved in the regulation of intracellular pH (pHi) and HCO-3 secretion in polarized cholangiocytes. Isolated bile duct units from normal rat liver were used to measure pHi by 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein 495 nm-to-440 nm dual ratio methods. Bombesin increased Cl--HCO-3 exchange activity but did not affect basal pHi or the activities of Na+/H+ exchange or Na+-HCO-3 symport. Depolarization of cholangiocytes increased basal pHi and the activity of Cl-/HCO-3 exchange, suggesting that an electrogenic Na+-HCO-3 symport might function as a counterregulatory pHi mechanism. Na+-independent acid-extruding mechanisms were not observed. We conclude that bombesin stimulates biliary secretion from cholangiocytes by activating luminal Cl-/HCO-3 exchange, which may be coupled to basolateral electrogenic Na+-HCO-3 symport.

bicarbonate secretion; ion transport; chloride-bicarbonate exchange; electrogenic sodium-bicarbonate symport

    INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

BOMBESIN IS A tetradecapeptide isolated from the skin of the European frog (2, 12) and chemically and biologically homologous to gastrin-releasing peptide (GRP) (25, 35). In the gastrointestinal tracts of various mammals (4, 6, 33, 42), bombesin and GRP are exclusively found in nervous tissue (10, 11, 24) and mediate diverse secretory and motor functions and release numerous peptides, such as neurotensin, motilin, insulin, CCK, secretin, and glucagon (13, 16, 22, 37). The presence of bombesin and GRP in enteric nerves and their potent effects on various gastrointestinal functions strongly suggest a potential role for endogenous GRP (bombesin) in gastrointestinal and liver physiology.

HCO-3 secretion from cholangiocytes plays an important physiological role through counteracting the increase in acid loads resulting from the cephalic phase of gastric acid secretion or during food digestion. The ion transport processes responsible for this basal or hormone-stimulated HCO-3 secretion have been characterized previously in cholangiocytes isolated from normal and bile duct-obstructed rats, as well as a cholangiocyte cell line (1, 14, 39). These studies (1, 14, 39) demonstrated the presence of two acid extruders in the bile duct epithelium (BDE), an Na+/H+ exchanger and an electrogenic Na+-HCO-3 symporter, and one acid loader, an Na+-independent Cl-/HCO-3 exchanger, which is located in the luminal membrane and is functionally coupled to Cl- channels. It was also shown (1) that secretin induces an HCO-3-rich choleresis by stimulating the activity of this Cl-/HCO-3 exchanger in cholangiocytes indirectly via activation of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB)-sensitive Cl- channels.

Bombesin and GRP are also known to increase an HCO-3-rich bile secretion in dogs and pigs (16, 22, 23, 27), but the underlying mechanism or site of action is poorly understood, partly because of the lack of adequate in vitro models and complex interactions with other secretagogues. Recently, we have shown (9) that bombesin stimulates biliary fluid and HCO-3 secretion by acting at the level of cholangiocytes, but not hepatocytes, and may mediate the neural regulation of bile secretion. A dose-dependent fluid secretion in isolated bile duct units (IBDU) was demonstrated over a wide range of physiologically relevant bombesin concentrations from 0.1 to 100 nM. Bombesin (10 nM)-stimulated fluid secretion in IBDU was mediated by specific bombesin receptors and was almost completely inhibited after omitting HCO-3 or Cl- from the perfusate, suggesting a significant dependence on HCO-3 and Cl- (9). Moreover, this stimulated secretion was associated with an increase in luminal pH in IBDU and biliary HCO-3 secretion in isolated perfused rat livers, consistent with an increased HCO-3 secretion at the level of the bile ducts (9).

In this study, we have examined the effect of bombesin on the H+/HCO-3 transport processes involved in intracellular pH (pHi) regulation, using a novel functional polarized IBDU. IBDU have been used previously to study the effects of secretin and bombesin on cholangiocytes and are an ideal functional polarized cholangiocyte preparation to study bile ductular secretion (9, 26). The present study is the first to show that the major underlying ion transport mechanism mediating the HCO-3-rich choleresis observed in isolated perfused rat liver and IBDU during bombesin stimulation is the Cl-/HCO-3 exchanger in cholangiocytes. Bombesin-stimulated increases in Cl-/HCO-3 exchanger activity result in increased HCO-3 secretion coupled to basolateral electrogenic Na+-HCO-3 symport. Unlike pig BDE (19, 44), there is no significant Na+-independent counterregulatory mechanism present in rat BDE.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials

BSA, penicillin/streptomycin, EDTA, heparin, HEPES, D-(+)-glucose, insulin, soybean trypsin inhibitor (type I-s), DMSO, hyaluronidase, DNase (DN-25), nigericin, amiloride, valinomycin, sodium gluconate, potassium gluconate, and hemicalcium gluconate were purchased from Sigma Chemical (St. Louis, MO). 2',7'-Bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM (BCECF-AM) was obtained from Molecular Probes (Eugene, OR). Bombesin was purchased from Bachem (Torrance, CA). Matrigel was from Collaborative Biomedical (Bedford, MA), collagenase D was from Boehringer Mannheim Biochemicals (Indianapolis, IN), and Pronase was from Calbiochem (San Diego, CA). L-15 medium Leibovitz, MEM, alpha -MEM, L-glutamine, gentamicin, and FCS were from GIBCO (Grand Island, NY). All other chemicals were of the highest purity available commercially. Monoclonal H+-ATPase antibody was a generous gift from Dr. Gluck at the Barnes-Jewish Hospital (St. Louis, MO).

Solutions

The compositions of the Krebs-Ringer bicarbonate (KRB), HEPES, and gluconate buffer solutions have been described previously (1, 39). All peptides were made up in perfusion buffer with 1% (wt/vol) BSA as a carrier.

Isolation of Bile Duct Units

Male Sprague-Dawley rats (Camm Laboratory Animals, Wayne, NJ) weighing 200-250 g were housed and allowed free access to water and Purina rodent chow (St. Louis, MO). Intrahepatic bile duct units (IBDU) were isolated as previously described by our laboratory and have been extensively characterized as polarized biliary epithelial cells with a central lumen (26).

IBDU were plated on Matrigel-coated small glass coverslips in modified alpha -MEM medium as previously described (26), incubated at 37°C in an air-5% CO2 equilibrated incubator, and used between 18 and 30 h after plating. Viability by trypan blue exclusion was consistently >95% and was not affected by preincubations with KRB, HEPES, gluconate, NH4Cl buffer solutions, and valinomycin in KRB solution.

pHi Determination

pHi of IBDU was measured as previously described (26), using a SPEX-AR-CM Microsystem microfluorometric method (Spex Industries, Edison, NJ). Previously, a heterogeneous response was observed with respect to the initial luminal size of IBDU (9). IBDU with an initial luminal area <170 µm2 did not show secretory responses to bombesin, possibly due to heterogeneity in receptor distribution; thus these smaller IBDU were excluded from the present study. IBDU, incubated overnight on Matrigel-coated glass coverslips, were loaded with 12 µM BCECF-AM for 20-30 min, washed for 10-20 min with BCECF-free medium, and then transferred into a 37°C thermostated perfusion chamber on the stage of an inverted microscope (IM 35; Carl Zeiss, Thornwood, NY). IBDU easily took up BCECF with a uniform distribution throughout the cytoplasm, and leakage and photobleaching of the dye were negligible. To assess the activities of Na+/H+ exchange, Na+-HCO-3 symport, and Cl-/HCO-3 exchange, we performed two sequential pH maneuvers with and without bombesin in alternating order to minimize intercellular variability. A 15- to 20-min reequilibration period between each maneuver was allowed for pHi to return to baseline. The fluorescence signal was >50 times the background autofluorescence. The 440 nm-to-490 nm fluorescence intensity ratio data were converted to pHi values using a previously described nigericin calibration technique (17, 26, 41). Fluorescence ratios over pHi 6.4-7.6 were linear for IBDU, and the intrinsic buffering power (beta i) was determined previously (26). Total buffering power (beta t) was calculated from the formula beta t = beta i + 2.303 × [HCO-3], where intracellular HCO-3 concentration ([HCO-3]) is derived from the Henderson-Hasselbalch equation.

Immunofluorescent Staining of H+-ATPase in Rat Liver and IBDU

Rat liver and kidney cryosections were fixed for 10 min in 4% formaldehyde and 6% HgCl2 in 140 mM sodium acetate buffer. After a wash in 0.1 M PBS, the sections were blocked with 20% calf serum and 1% polyethylene glycol in PBS for 30 min. The primary antibody (anti-H+-ATPase) was used undiluted for 2 h at room temperature. The section were next washed in PBS and incubated in a goat anti-mouse secondary antibody for 1 h at room temperature, washed in PBS, and mounted in Vectashield (Vector, Burlingame, CA). Sections were viewed and photographed with a Nikon epifluorescence microscope.

Statistical Analysis

All data from pH measurements are given as arithmetic means ± SD. Statistical differences were assessed by the unpaired or paired Student's t-test using the INSTAT statistical computer program (GraphPad Software, San Diego, CA).

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of Bombesin on Basal pHi

In a HEPES, nominally HCO-3-free medium, infusion of 10 nM bombesin for ~10 min had no effect on basal pHi (7.10 ± 0.08, n = 11) in IBDU compared with the albumin carrier controls (7.09 ± 0.09, n = 11). These findings suggest that bombesin does not influence the activity of the Na+/H+ exchanger, which is the main ion transporter maintaining basal pHi in a nominally HCO-3-free medium. Similarly, bombesin did not affect basal pHi (7.19 ± 0.07, n = 17) of IBDU in HCO-3-enriched medium compared with albumin carrier controls (7.18 ± 0.07, n = 17).

Effect of Bombesin on Recovery of pHi From an Acute Acid Load in HEPES

When IBDU were exposed to 20 mM NH4Cl, pHi increased promptly (Fig. 1), as the uncharged NH3 diffused into the cell and combined with intracellular H+. However, this process reversed when NH4Cl was withdrawn from the perfusate and NH3 leaves the cell after releasing H+, causing a rapid fall in pHi to 6.81 ± 0.05 (n = 11). In the absence of HCO-3, the recovery rates from this acid load reflect the activity of Na+/H+ exchangers (1, 26, 39). Bombesin (10 nM) induced a slight but statistically insignificant (P = 0.15) increase in the recovery rates [0.14 ± 0.04 pH units (pHU)/min, n = 11] from this acid load, compared with albumin carrier controls (0.12 ± 0.03 pHU/min, n = 11) (Table 1). Bombesin did not affect the nadir pHi (6.82 ± 0.08, n = 8), compared with albumin controls (6.81 ± 0.05, n = 8; P = 0.44). These findings suggest that bombesin (10 nM) has no significant effect on the activity of Na+/H+ exchangers in IBDU.


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Fig. 1.   Effect of bombesin (Bom; 10 nM) on Na+/H+ exchange activity in isolated bile duct units (IBDU). Intracellular pH (pHi) of IBDU was studied by SPEX microfluorospectrophotometry using a BCECF 440/490 ratio method. Two sequential 20 mM NH4Cl acid-loading and recovery maneuvers in HEPES with and without bombesin were performed with a 15- to 20-min reequilibration period between each maneuver. Activities of Na+/H+ exchange of IBDU were assessed by measuring pHi recovery rate (Delta pHi/Delta t) after 20 mM NH4Cl acid loading in HEPES. Combined data are summarized in Table 1 (means ± SD, n = 11). pHi recovery rates with bombesin administration were not different from those of the albumin (Alb) controls.

                              
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Table 1.   Effect of 10 nM bombesin on activity of Na+/H+ exchange

Effect of Bombesin on Recovery of pHi from an Acute Acid Load in KRB

When isolated cholangiocytes from normal rat livers are maintained in an HCO-3-enriched medium, the Na+/H+ exchanger and Na+-HCO-3 symporter are the main acid extruders that restore pHi back to baseline pHi from an acute acid load with NH4Cl (1, 26). To evaluate more specifically the effect of bombesin on Na+-HCO-3 symport activity in HCO-3-enriched medium, we superfused 1 mM amiloride at the moment of 20 mM NH4Cl withdrawal (Fig. 2) to inhibit the Na+/H+ exchanger (1, 39). In the presence of 1 mM amiloride in KRB, the recovery rates from an NH4Cl acid load reflect the activity of the Na+-HCO-3 symporter (1, 26, 39). As shown in Table 2, bombesin did not affect the recovery rates (0.12 ± 0.05 pHU/min, n = 8; P = 0.60) compared with the albumin controls (0.12 ± 0.05 pHU/min, n = 8). This finding suggests that bombesin had no significant effect on Na+-HCO-3 symport activity as assessed by the recovery rates from an acute acid load with NH4Cl.


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Fig. 2.   Effect of bombesin (10 nM) on Na+-HCO-3 symport activity in IBDU. Two sequential 20 mM NH4Cl acid-loading and recovery maneuvers in Krebs-Ringer bicarbonate (KRB) with and without bombesin in the presence of 1 mM amiloride (Ami), a specific inhibitor of Na+/H+ exchanger, were performed with a 15- to 20-min reequilibration period between each maneuver. Activities of Na+-HCO-3 symport in IBDU were assessed by measuring pHi recovery rate (Delta pHi/Delta t) from 20 mM NH4Cl acid load and are summarized in Table 2 (means ± SD, n = 8). pHi recovery rates with bombesin administration were not different from those of albumin controls.

                              
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Table 2.   Effect of 10 nM bombesin on activity of Na+-HCO-3 symport

Effect of Bombesin on Activity of Cl-/HCO-3 Exchange

To measure Cl-/HCO-3 exchanger activity, IBDU were subjected to the acute removal and readmission of Cl- by substitution with equimolar amounts of gluconate. As shown in Fig. 3, Cl- removal resulted in a rapid alkalinization of pHi from 7.19 ± 0.06 to 7.42 ± 0.10 pHU, which was reversed after Cl- readmission. The Cl-/HCO-3 exchange activity is reflected by the rate of pHi change during Cl- removal and readmission. The administration of 10 nM bombesin resulted in a significant increase in this Cl-/HCO-3 exchange activity as measured in 12 experiments (Table 3). Alkalinization rate and proton flux during Cl- removal increased from 0.10 ± 0.04 pHU/min and 5.66 ± 2.27 mM/min for the albumin control to 0.16 ± 0.09 pHU/min (n = 12, P = 0.04) and 9.24 ± 5.51 mM/min (n = 12, P = 0.03), respectively, during bombesin (10 nM) administration. Similarly, the acidification rate and proton flux during Cl- readmission increased from 0.15 ± 0.04 pHU/min and 11.15 ± 4.09 mM/min for the albumin control to 0.25 ± 0.09 pHU/min (n = 12, P = 0.004) and 21.54 ± 10.06 mM/min during bombesin administration (n = 12, P = 0.005), respectively. Moreover, the peak pHi also increased from 7.42 ± 0.10 pHU for the albumin control to 7.47 ± 0.12 pHU (n = 12, P = 0.015) after bombesin (10 nM), although no change in baseline pHi was observed. These results strongly suggest that bombesin-stimulated HCO-3 secretion in cholangiocytes is mediated by the increase in the Cl-/HCO-3 exchange activity in this epithelial tissue.


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Fig. 3.   Effect of bombesin (10 nM) on Cl-/HCO-3 exchange activity in IBDU. Two sequential Cl- removal and readmission maneuvers with and without bombesin were performed with a 15- to 20-min reequilibration period between each maneuver (A). Bombesin (10 nM) stimulated maximal rates of alkalinization after Cl- removal [0.16 ± 0.09 pH units (pHU)/min, P < 0.05] and pHi recovery after Cl- readmission (0.25 ± 0.09 pHU/min, P < 0.01) compared with control values (0.10 ± 0.04 and 0.15 ± 0.05 pHU/min, respectively; n = 12), indicating stimulation of Cl-/HCO-3 exchange activity (B) (means ± SD).

                              
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Table 3.   Effect of 10 nM bombesin on activity of Cl-/HCO-3 exchange

Study of Counterregulatory pH Mechanism for Cl-/HCO-3 Exchange Activation

Although an acidification of pHi is expected from increased acid loads from the stimulated Cl-/HCO-3 exchange during bombesin-stimulated HCO-3-rich choleresis, there is no significant change in pHi of cholangiocytes, suggesting an activation of acid extruder(s) as a counterregulatory pH mechanism(s). The Na+-HCO-3 symporter and Na+/H+ exchanger, which were previously characterized in rat cholangiocytes, are such potential acid extruders (1, 39). In addition, H+-ATPase is another possible candidate, since it was shown to be a major acid-extruding mechanism during secretin-stimulated HCO-3-rich choleresis in pig BDE (19, 44). Each of these Na+-dependent and -independent ion transporters was examined.

Na+/H+ exchanger. As shown in Fig. 1 and Table 1, bombesin did not have any significant effect on the Na+/H+ exchanger, because neither basal pHi nor recovery rate of pHi from an acute acid load in HEPES medium was altered during bombesin stimulation. We also studied the effect of a specific Na+/H+ exchanger inhibitor, amiloride (1 mM), on basal pHi with and without bombesin stimulation in an HCO-3-enriched medium. As shown in Fig. 4, there were no significant changes in basal pHi with administration of amiloride in KRB. In addition, the pHi of IBDU in the presence of amiloride also remained unaffected during superfusion with bombesin, which was expected to increase the intracellular acid load from stimulation of the Cl-/HCO-3 exchanger activity. Thus, as observed previously with secretin in rat cholangiocytes and pig BDE (20, 43), these results, in addition to the findings from Table 1, suggest that the Na+/H+ exchanger has no significant role in maintaining the pHi of cholangiocytes either in the basal state or after bombesin stimulation.


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Fig. 4.   Effect of amiloride (1 mM) on basal and bombesin-stimulated pHi of IBDU. No significant change in basal pHi was observed with or without bombesin coadministration in KRB in the presence of amiloride (n = 5), suggesting minimal involvement of Na+/H+ exchange in maintaining pHi during basal or bombesin-stimulated secretion.

Na+-HCO-3 symporter. This symporter is a major acid extruder in HCO-3-containing medium, but, as shown in Fig. 2 and Table 2, bombesin had no effect on Na+-HCO-3 symporter activity as assessed by the recovery rates from an acute acid load with NH4Cl. However, unlike other pH regulatory mechanisms, this electrogenic symport can also be activated secondarily by changes in the membrane potential, as occurs with Cl- channel activation. Unfortunately, specific inhibitors for the Na+-HCO-3 symporter are not available. Thus the role of this symporter as a counterregulatory pHi mechanism was studied indirectly by examining the effects of membrane depolarization on basal pHi and Cl-/HCO-3 exchange activities using valinomycin (0.5 µM) in KRB solution with 30 mM KCl substituted for equimolar NaCl (1, 46). As shown in Fig. 5 and Table 4, when the membrane was depolarized by administration of valinomycin and high K+, the basal pHi increased by 0.05 ± 0.04 pHU (n = 9, P < 0.05), suggesting stimulation of an electrogenic Na+-HCO-3 symport resulting in an increased HCO-3 influx into the cholangiocytes. Furthermore, the increase in HCO-3 influx that results from this electrogenic Na+-HCO-3 symport, in turn, increases Cl-/HCO-3 exchange activity, as shown in Fig. 5 and Table 4. Together these findings indicate that a basolateral electrogenic Na+-HCO-3 symport is most likely coupled to the luminal Cl-/HCO-3 exchange by changes in the membrane potential and/or HCO-3 concentrations and functions to counteract the increased intracellular acid loads resulting from bombesin-stimulated biliary HCO-3 secretion. In addition, these findings also offer a plausible explanation for the apparent absence of change in the recovery rates from NH4Cl acid loading during bombesin stimulation, since this pH maneuver, which involves the same amount of acid loading, may not adequately account for secondary effects such as changes in membrane potential and/or HCO-3 concentration gradients.


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Fig. 5.   Effect of depolarization by valinomycin (0.5 µM) and 30 mM KCl on basal pHi and Cl-/HCO-3 exchange activity. Membrane potential of IBDU was depolarized by perfusing valinomycin (0.5 µM) in KRB solution with 30 mM KCl substituted for equimolar NaCl and followed basal pHi. In addition, to assess changes in Cl-/HCO-3 exchange activities with depolarization, 2 sequential Cl- removal and readmission maneuvers before and after depolarization were performed with a 15- to 20-min reequilibration period between each maneuver. Basal pHi and Cl-/HCO-3 exchange activities increased with depolarization, and results are summarized in Table 4.

                              
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Table 4.   Effect of depolarization by 0.5 µM valinomycin and 30 mM KCl on basal pHi and activity of Cl-/HCO-3 exchange

Na+-independent acid-extruding mechanism(s). A vesicular H+-ATPase has been proposed as another potential counterregulatory pH mechanism after secretin-stimulated HCO-3 secretion in pig BDE (19, 44). However, its presence in rat or human cholangiocytes has not been previously demonstrated (8, 40). As shown in Fig. 6, a rapid acidification of the pHi of IBDU, which was observed by substituting Na+ with choline either in HCO-3-enriched or -free medium (data not shown) for 30-60 min, could not be overcome by administration of bombesin and is only reversed with readmission of Na+. Moreover, as shown in Fig. 7, immunofluorescent staining of rat liver and IBDU with H+-ATPase antibody was also negative in contrast to the positive control in rat kidney, in which H+-ATPase has been previously demonstrated (50). These findings strongly suggest that an Na+-independent acid-extruding mechanism such as H+-ATPase is not present in rat cholangiocytes.


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Fig. 6.   Effect of Na+ withdrawal on pHi recovery after acidification in IBDU. Substitution of Na+ with choline in KRB or HEPES medium with or without bombesin coadministration results in acidification of basal pHi, which was not reversed until Na+ was resubstituted (n = 6).


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Fig. 7.   Immunofluorescent staining of anti-H+-ATPase. A: kidney, note positive labeling (arrowheads) in tubules. B: liver, note lack of positive staining in bile ducts (arrowhead). Magnification, ×250.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Although secretin has been thought to be the major regulator of biliary HCO-3 secretion, early physiological studies in dogs showed that an HCO-3-rich choleresis increased not only with feeding (28) but also with sham feeding (34), suggesting that neural regulation may be involved in postprandial bile formation. Recently, we have shown (9) that the neuropeptide bombesin can stimulate biliary HCO-3 secretion in the rat. These studies (9, 34) as a whole suggested that neuropeptides such as bombesin stimulate biliary HCO-3 secretion at physiological concentrations and thus may play a significant role in a neurally mediated HCO-3-rich choleresis.

The present study demonstrates for the first time that the bombesin-stimulated HCO-3 and fluid secretion in BDE is mediated by an increase in Cl-/HCO-3 exchanger activity, as assessed by rates of pHi recovery in IBDU after established Cl- removal and readmission protocols (1, 26, 39). This increase in the Cl-/HCO-3 exchanger activity occurred without affecting basal pHi. In cholangiocytes (1, 39) and in many other cell types, including hepatocytes (3, 47) and pancreatic ducts (18, 30), Cl-/HCO-3 exchange is the major acid-loading mechanism, and it mediates HCO-3 secretion/absorption. Thus stimulation of this exchanger by bombesin can account for the increases in biliary HCO-3 secretion previously demonstrated in isolated perfused rat livers and IBDU (9). Together, these findings suggest that Cl-/HCO-3 exchange is most likely to be the primary mechanism for bombesin-stimulated HCO-3 and fluid secretion in cholangiocytes (7). This conclusion is further supported by previous ion substitution studies demonstrating near complete dependence of the bombesin response on the presence of both Cl- and HCO-3 in the medium (9).

In pancreatic duct cells (18, 30) and isolated cholangiocytes (1), an increased HCO-3 secretion via the Cl-/HCO-3 exchanger is thought to be coupled to an activation of Cl- channels. Thus the bombesin-stimulated secretion in cholangiocytes also likely involves an activation of Cl- channels that provides a favorable Cl- gradient and electrical membrane potential changes necessary for the stimulation of Cl-/HCO-3 exchange and electrogenic Na+-HCO-3 symport, respectively. Furthermore, this coupling of Cl- channels with the Cl-/HCO-3 exchanger at the luminal membrane, in turn, can provide a "Cl- recycling" mechanism whereby Cl- exits cholangiocytes via Cl- channels on the apical side and recycles via the Cl-/HCO-3 exchanger after bombesin stimulation. This mechanism leads to an increased net vectoral HCO-3 secretion accompanied by a passive net H2O movement into bile during the bombesin-stimulated HCO-3-rich choleresis, while Cl- shuttles in and out of the cholangiocytes at the luminal membrane via this recycling pathway. Detailed electrophysiological studies would be needed to better elucidate this mechanism. At present, it is not clear whether Cl- fluxes also occur at the basolateral membrane during this process.

Although an intracellular acid load should follow the bombesin-stimulated increase in HCO-3 secretion into bile from increased Cl-/HCO-3 exchange activity, bombesin had no measurable effect on the basal pHi of IBDU, suggesting the presence of a counterregulatory pH mechanism(s) in cholangiocytes. Several acid-extruding mechanisms have been described in cholangiocytes, including Na+/H+ exchange, H+-ATPase, and Na+-HCO-3 symport (1, 39, 44). However, Na+/H+ exchange is unlikely to play any significant role, since bombesin did not affect either basal pHi in KRB in the presence of amiloride or in HEPES or the recovery rates after an NH4Cl acid load in HEPES medium. This conclusion is consistent with previous findings (1) that the Na+/H+ exchanger is minimally involved in basal pHi maintenance in physiological HCO-3-containing medium and is only active at low pHi. Moreover, Na+/H+ exchange also does not play a major role in secretin-stimulated biliary HCO-3 secretion in pigs (19) and in the isolated guinea pig liver (5).

The present study also presents the first convincing functional evidence that Na+-independent acid-extruding mechanisms, such as H+-ATPase or the K+/H+ exchanger, are unlikely to be counterregulatory mechanisms in rat cholangiocytes. As shown in Fig. 6, pHi did not recover after acidification when Na+ is excluded from the medium and infusion of bombesin also could not reverse this acidification until Na+ is replaced, strongly suggesting the functional absence of Na+-independent acid-extruding mechanisms. Furthermore, immunostaining of rat bile ducts with specific antibodies to H+-ATPase (Fig. 7) also failed to detect this exchanger, providing additional evidence that rat cholangiocytes lack H+-ATPase. In addition, the absence of pHi recovery in HCO-3-containing medium when Na+ is withdrawn also suggests that reversal of the Cl-/HCO-3 exchanger by admitting HCO-3 in exchange for Cl- or nonspecific HCO-3 influx through certain ion channels, such as the cystic fibrosis transmembrane conductance regulator, does not occur (or is not enough) to compensate for the acidification of pHi. Thus, unlike cholangiocytes in pigs but analogous to those in humans (40), rat cholangiocytes do not have Na+-independent acid-extruding mechanisms.

Having excluded Na+/H+ exchange and Na+-independent acid extrusion as counterregulatory mechanisms, our findings are instead most consistent with HCO-3 entry at the basolateral membrane via Na+-HCO-3 symport after bombesin-stimulated HCO-3 secretion. A similar mechanism has been previously proposed for secretin-stimulated HCO-3 secretion in cholangiocytes (1) and in rabbit and guinea pig gallbladder epithelium (32, 48). Unlike the Na+/H+ exchanger, the Na+-HCO-3 symporter is active in maintaining basal pHi of cholangiocytes in HCO-3-containing medium (1, 39) and is much more active at basal than at lower or higher pHi in the basolateral membrane of rabbit renal proximal tubules (38). Moreover, the electrogenic Na+-HCO-3 symporter also can be activated secondarily by changes in membrane potential (15, 36, 49). Interestingly, measurements of the primary activity of this acid extruder, as assessed by the recovery rates from NH4Cl acid loading in KRB medium in the presence of amiloride, suggest that Na+-HCO-3 symport is not directly stimulated by bombesin. There are two plausible explanations for this absence of stimulatory effects of bombesin on Na+-HCO-3 symport activity assessed by this method. First, this NH4Cl pH maneuver, which causes intracellular acidification/alkalization by shifting H+-HCO-3 balance inside the cell, can also alter membrane potentials as shown in hepatocytes (21). Therefore, this maneuver cannot directly evaluate the effects of membrane potential changes resulting from bombesin stimulation on the Na+-HCO-3 symport activity. Alternatively, the known dependence of certain Cl- channels on pHi (31, 45) can result in Cl- channel inactivation at low pHi used to test the symport activity. Such an inhibition of Cl- channel activity at low pHi, in turn, would prevent the membrane depolarization expected with bombesin stimulation that would be necessary to increase electrogenic Na+-HCO-3 symport activity.

To circumvent these problems, we assessed indirectly the role of membrane depolarization as a secondary activator of the electrogenic Na+-HCO-3 symport activity. As shown in Fig. 5, this study was performed near basal pHi so that changes in pHi should not significantly affect the activities of various ion transporters and channels with possible pHi dependence. Such secondary activation of the electrogenic Na+-HCO-3 symport is evident by an increase in basal pHi from HCO-3 influx (Fig. 5 and Table 4) after depolarization of cholangiocytes by valinomycin and high K+. Although less likely, this increase in pHi could also be from an activation of K+-dependent transporters such as K+/H+ exchanger due to shifts in the K+ gradient. However, as discussed above, no significant Na+-independent acid-extruding mechanisms are present in cholangiocytes. Thus it is unlikely that this increase in pHi is secondary to transporters such as K+/H+ exchanger.

In addition, as we have shown previously in isolated cholangiocytes (1), the depolarization of cholangiocytes in IBDU by valinomycin and high K+ also increased Cl-/HCO-3 exchange activity (Fig. 5 and Table 4), providing evidence for coupling of the electrogenic Na+-HCO-3 symport with Cl-/HCO-3 exchange. Thus, when bombesin stimulates biliary HCO-3 secretion, the electrogenic Na+-HCO-3 symporter is secondarily activated, not only from the increased HCO-3 concentration gradient due to an increase in HCO-3 efflux as luminal Cl-/HCO-3 exchange is stimulated, but also from the membrane depolarization that presumably occurs from Cl- channel activation. This secondary activation of the Na+-HCO-3 symporter then, in turn, may function to maintain the pHi of the cholangiocytes.

Although the underlying ion transport mechanisms mediating bombesin-stimulated biliary secretion are quite similar to secretin, some important differences appear to exist. Unlike secretin, preliminary studies (7) suggest that the bombesin response is not dependent on the function of microtubules or on cAMP as a secondary messenger. In addition, previous Ca2+ measurements (29) indicate that bombesin does not change intracelluar Ca2+ in isolated cholangiocytes or IBDU, suggesting that bombesin-stimulated biliary secretion also does not involve the Ca2+ messenger system. Although preliminary, these findings suggest a unique signal transduction pathway for this bombesin response in cholangiocytes.

In summary (Fig. 8), the present study demonstrates for the first time that bombesin stimulates luminal HCO-3 secretion by increasing Cl-/HCO-3 exchange, presumably coupled to basolateral HCO-3 entry via electrogenic Na+-HCO-3 symport mediated by changes in the HCO-3 gradient and/or in the membrane potential. In addition, we also present the first functional evidence that, unlike pig cholangiocytes but analogous to those in human BDE, rat cholangiocytes have no Na+-independent acid-extruding mechanisms.


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Fig. 8.   Bombesin-stimulated secretion in rat bile duct epithelium. Bombesin-stimulated fluid and HCO-3 secretion in cholangiocytes is mediated by an increase in Cl-/HCO-3 exchanger activity, coupled to Cl- channels, which is counterbalanced by the electrogenic Na+-HCO-3 symporter to maintain pHi. PKA, protein kinase A. CFTR, cystic fibrosis transmembrane conductance regulator; CA carbonic anhydrase.

    ACKNOWLEDGEMENTS

We thank Drs. W. F. Boron and J. Geibel for helpful discussions.

    FOOTNOTES

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-25636 and DK-34989 to J. L. Boyer and to the cell isolation, culture, organ perfusion, and morphology cores of the Yale Liver Center, respectively. W. K. Cho was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-07356 and an American Gastroenterology Association Advanced Research Award.

Part of this work was presented at the American Gastroenterology Association meeting in San Diego, CA, May 1995, and AASLD meeting in Chicago, IL, November 1995, and published in abstract form (Gastroenterology 108: A1049, 1995; Hepatology 22: 315A, 1995).

Address for reprint requests: W. K. Cho, Division of GI/Hepatology, Indiana Univ. School of Medicine, 975 West Walnut St., IB 424, Indianapolis, IN 46202.

Received 30 December 1997; accepted in final form 22 July 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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