©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Na, K, and H/HCO Transport in Submandibular Salivary Ducts
MEMBRANE LOCALIZATION OF TRANSPORTERS (*)

(Received for publication, March 30, 1995; and in revised form, May 31, 1995)

Hong Zhao Xin Xu Julie Diaz Shmuel Muallem (§)

From the Department of Physiology, University of Texas Southwestern Medical Center, Dallas, Texas 75235

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

The mechanisms mediating transepithelial ion transport in salivary ducts were characterized and localized by studying the regulation of [Na], [K], and pH in isolated intralobular ducts and perfused main ducts of the submandibular salivary gland. A new procedure was developed for the rapid preparation of intralobular ducts. Measurements of pH revealed the presence of Na/H and Cl/HCO(3) exchange activities in intralobular duct cells. We could not obtain evidence for a coupled K/H exchange activity which was postulated to exist in the luminal membrane of duct cells. Rather, a K-dependent pathway which mediates the transport of H/HCO(3) and Na was found. This pathway was absent from acinar cells of the same gland and was active in unstimulated duct cells incubated in 5 mM K. Accordingly, inhibition of the Na pump with ouabain resulted in rapid and large Na influx in duct but not acinar cells. Perfusion experiments with the experimentally accessible main duct and measurements of pH were used to provide the first direct localization of ion transporters in salivary ducts. The luminal and basolateral membranes of the duct express separate Na/H and Cl/HCO(3) exchangers. Na/H exchange activity in both membranes was similar, whereas the luminal Cl/HCO(3) exchange activity was higher than that in the basolateral membrane. The perfused main duct was also used to localize the newly discovered K-dependent H/HCO(3) and Na transport pathway to the luminal membrane, which suggests that this pathway may play an important role in Na reabsorption and K and HCO(3) secretion by the salivary ductal system.


INTRODUCTION

Fluid and electrolyte secretion by the submandibular salivary gland occurs in two steps(1, 2) . First, a plasma like fluid is secreted by the acinar cells and cells in the acinar-intercalated duct region. Subsequently, the electrolyte composition of the primary fluid is modified by the ductal system. The ducts reabsorb Na and Cl, secrete K and HCO(3), and reduce the osmolarity of the fluid, apparently without a significant change in the volume of the secreted fluid(3) .

The majority of the studies to reveal the mechanism of vectorial ion transport in duct cells was performed with the experimentally available perfused main duct(3) . Ion substitution and the use of blockers led to a model in which luminal K secretion is mediated by a coupled, electroneutral K/H exchanger in parallel with an epithelial type Na/H exchanger(3, 4) . Na absorption in the luminal membrane is mediated by the Na/H exchanger and an amiloride-sensitive Na channel(5, 6) . Na exit and K entry at the basolateral membrane are mediated by the Na pump. Salivary secretion is under autonomic control. Both cholinergic and alpha-adrenergic stimulation inhibit Na reabsorption and reduce the transepithelial potential (7, 8, 9, 10, 11, 12) . beta-Adrenergic stimulation has variable effects depending on agonist concentration(8, 10, 13) . Several gastrointestinal hormones, including substance P, also modulate ductal secretion similar to cholinergic stimulation(3, 14) .

In recent years techniques were developed to examine the mechanisms by which agonists modulate salivary electrolyte secretion on the cellular level. Patch clamp of intralobular duct cells was used to demonstrate the presence of an amiloride-sensitive Na channel (6) and a Cl channel in the luminal membrane (15) . Measurement of pH(^1)by microspectrofluorometry demonstrated the presence of a Na/H exchange in intralobular striated duct cells(16) , but the membrane localization of the exchanger has not been determined. No evidence for Cl/HCO(3) or coupled K/H exchange was found(16) . Measurements of [Ca] showed that cholinergic, alpha-adrenergic(17, 18) , and beta-adrenergic (19) stimulation caused an increase in [Ca].

In the present study we identified the major mechanisms of Na, K and H/HCO(3) transport in submandibular duct cells. For the purpose of these studies we simplified the technique of Dehaye and Turner (20) for preparation of intralobular granular duct cells. We also used measurements of pH in the perfused main duct to provide the first direct membrane localization of the transporters participating in regulation of pH, [Na], and transepithelial electrolyte transport. The subsequent manuscript (38) describes the properties and regulation of this pathway during agonist stimulation.


EXPERIMENTAL PROCEDURES

Materials and Solutions

All fluorescent probes were from Molecular Probes. Dimethylamiloride (DMA) was from RBI, Natick, MA. Collagenase P was from Boehringer Mannheim. The standard perfusion solution A (NaCl, Hepes) contained (in mM): 140 NaCl, 5 KCl, 1 MgCl(2), 1 CaCl(2), 10 glucose, and 10 Hepes (pH 7.4 with NaOH). K-free, Hepes-buffered solution B was prepared by replacing 5 mM K with 5 mMN-methyl-D-glucamine (NMG). The composition of all other solutions was identical with that detailed in (21) . In general Na-free solutions were prepared by replacing Na with NMG, and Cl-free solutions were prepared by replacing Cl with gluconate (Glu). HCO(3)-buffered solutions contained 2.5 mM Hepes, and 25 mM HCO(3) replaced 25 mM Cl. The solutions used in the present studies were labeled as follows: Na-free, Hepes solution C; Cl-free, Hepes solution D; NaCl, HCO(3) solution E; Cl-free, HCO(3) solution F; Na-free, HCO(3) solution G. The osmolarity of all solutions was adjusted to 310 mosM with the major salt.

Preparation of Isolated Intralobular Ducts

A new procedure was developed for the preparation of intralobular ducts from the rat submandibular salivary gland. The submandibular glands were removed from male rats (75-100 g) to an ice-cold solution A in which 10 mM Na-pyrovate replaced 10 mM NaCl and which contained 0.02% soybean trypsin inhibitor and 0.1% bovine serum albumin (PSA). Each gland was cleaned by injection of 5-10 ml of PSA and removal of the capsule and finely minced. The minced tissue was transferred to 8 ml of PSA containing 2.5 mg of collagenase P, the flask was gassed with 100% O(2), and capped. After 2 min of digestion at 37 °C, the tissue was partially dispersed by pipetteting 5 times with a 5-ml plastic pipette tip, and the digestion continued for an additional 8 min. The digest was washed twice with PSA by a 10-s centrifugation at 100 g, resuspended in about 6 ml of PSA and kept on ice until use. Fig. 1A shows the mixture of ducts and acini obtained by this procedure.


Figure 1: Light micrographs of isolated acini, intralobular ducts and the perfused main submandibular ducts. A shows the mixture of acini and ducts obtained after collagenase digestion of submandibular glands (magnification 80). B shows the cannula perfusing a main duct (left side) held in place by a polyethylene tubing (magnification 16).



Microdissection and Perfusion of the Main Submandibular Duct

The procedure used to prepare the main duct for perfusion was similar to that described before(7) , while the perfusion set up was similar to the one we used to perfuse the main pancreatic duct (21) . Male rats (250-300 g) were anesthetized and tracheotomized. The submandibular glands were exposed and the connective tissue was cleared in the region of the gland hilum. The submandibular duct was opened close to the gland hilum, and a cannula was inserted and ligated. The duct was cut, transferred to a Petri dish containing PSA, and perfused luminally with a PSA containing 2 µM BCECF/AM. After a 10-15-min incubation at room temperature, the duct lumen was flushed with 0.2 ml of PSA, and the duct was transferred to a perfusion chamber. The open end of the duct was cut and pulled to allow attachment of the holding cannula as shown in Fig. 1B, right. Then the chamber was placed on a stage of an inverted microscope and the cannula connected to the lumen perfusion line. The lumen was perfused at a rate of 25 µl/min, and the bath was perfused in an anterograde direction at a rate of 10-12 ml/min.

Fluorescence Measurements

Intralobular ducts and the acini were loaded with BCECF by a 15-min incubation at room temperature in PSA containing 2 µM BCECF/AM. The cells were then washed once, resuspended in 5 ml of PSA, and kept on ice until use. Loading with SBFI was by a 30-min incubation at room temperature with 5 µM SBFI/AM. These cells were also washed once with PSA and kept on ice until use. The fluorescence was recorded either by photon counting or image acquisition and analysis using the systems described before(21, 22) . For photon counting, the fluorescence was recorded from 6-12 cells. In the case of the main duct, the fluorescence was recorded from an area equivalent to 8-12 cells and as close as possible to the lumen perfusing cannula. BCECF fluorescence was recorded at excitation wavelengths of 490 and 450 nm at a resolution of 2/s. The fluorescence ratios of 490/450 were calibrated intracellularly by perfusing the cells with solutions containing 145 mM KCl, 10 mM Hepes, 5 µM nigericin with pH adjusted to 6.2-7.6, as described previously(21) . In the case of image acquisition, images were recorded at a resolution of 4 s for each set of images, and the fluorescence of single cells or cell clusters was analyzed as desired(21) .

SBFI fluorescence was recorded at excitation wavelengths of 340 and 370 nm. The image ratios of 340/370 were calibrated essentially as described before(23) . To clamp Na, the cells were perfused with solutions containing several concentrations of NaCl (between 0 and 90 mM), 10 mM Hepes (pH 7.4), 5 µM gramicidin, and 2.5 µM monensin. The dye behaved similarly in duct and acinar cells, and similar changes in fluorescence ratio were obtained by clamping Na at different concentrations. Therefore, the calibration curves from both cells were pooled. The apparent K for Na in the cells was about 48 ± 6.3 mM (n = 4). The calibration curve and the ratios were used to calculate [Na].


RESULTS

H/HCO Transport in Intralobular Ducts

The only H/HCO(3) transport mechanism described before in submandibular duct is a Na/H exchange(16, 24) . Fig. 2, a and b, shows the presence of a Cl/HCO(3) exchange mechanism in duct and acinar cells of the submandibular gland. In the absence of HCO(3), removal of Cl acidified the cells, whereas in the presence of HCO(3), removal of Cl caused cytosolic alkalinization. Treatment of the ducts with DIDS inhibited the effect of Cl removal and readdition on pH. Similar results were obtained in the absence of Na (not shown). Hence, a Na-independent, DIDS-sensitive Cl/HCO(3) exchange similar to that found in acinar cells is present in submandibular intralobular duct cells.


Figure 2: Cl/HCO(3) and Na/H exchange in intralobular ducts. Ducts and acini loaded with BCECF were used to measure pH by image acquisition and analysis (a and b). Cells incubated in solution A (NaCl, Hepes) were perfused in sequence with solutions D (Cl-free, Hepes), A, E (NaCl, HCO(3)), F (Cl-free, HCO(3)), and E. After treatment with 0.5 mM DIDS for 8 min, the cells were perfused with solution F containing 0.5 mM DIDS. The figure shows 1 of 4 experiments with similar results. In c, ductal pH was measured by photon counting of BCECF fluorescence from 8 cells. The duct was perfused with solution E in which 20 mM NH(4)Cl replaced 20 mM NaCl and then solution G (Na-free, HCO(3)). After stabilization of pH, the cells were perfused with solution G in which 5 or 140 mM K replaced similar [NMG] and then with solution E containing 20 µM DMA before perfusion with solution E. This experiment is similar to 2 others with the same conditions and to 6 others in Hepes-buffered solutions.



In an attempt to identify the proposed(3, 4) K/H exchange activity, we tested the effect of external K on H efflux from acid-loaded cells (Fig. 2c). Addition of 5 mM K to acidified cells caused a small increase in pH. However, increasing K up to 140 mM had no further effect on pH. Exposing these cells to 5 mM K and 140 mM Na in the presence of 20 µM dimethylamiloride (DMA) increased pH similar to that observed with K alone. Removal of DMA resulted in recovery of resting pH due to Na/H exchange. Hence, even in strongly acidified cells and in the presence of HCO(3), external K had minimal effect on pH.

Another protocol to test for K/H exchange is shown in Fig. 3. Removal of external K caused cytosolic acidification at an initial rate of about 0.13 ± 0.02 pH unit/min (n = 12). Up to 20 µM amiloride, which is sufficient to block the luminal Na channel(6) , had no effect on the rate or extent of the acidification. Inhibition of the Na/H exchanger with 0.5 mM amiloride caused a further reduction of pH by about 0.16 ± 0.03 pH unit (n = 4) (Fig. 3a). Inhibition of the Na/H exchanger prior to removal of K showed that the maximal rate of acidification due to removal of K was 0.34 ± 0.02 pH unit/min (n = 5) with stabilization of pH within 128 ± 5.7 s (Fig. 3b). Fig. 3c shows that HCO(3) somewhat reduced the rate and significantly blunted the extent of the acidification. However, a similar set point of pH was attained under the various conditions. In Hepes-buffered medium, pH stabilized at 6.98 ± 0.03 (n = 4), whereas in HCO(3)-buffered medium, pH stabilized at 6.93 ± 0.04 (n = 3).


Figure 3: Effect of K on pH(i) in intralobular ducts. In experiment a, ducts loaded with BCECF were perfused with solution B (K-free, Na containing, Hepes), solution B containing 0.5 mM amiloride, solution B, and then solution A (containing 5 mM K). In experiment b, ducts were incubated in solution A containing 25 µM DMA before exposure to solution B containing DMA. In experiment c, the ducts were equilibrated with HCO(3)-buffered solution E (NaCl, HCO(3)) before exposure to 25 µM DMA and then solution E in which 5 mM NMG-Cl replaced 5 mM KCl (K-free).



In the absence of amiloride or DMA, addition of K rapidly increased pH back to resting levels (Fig. 3a). DMA (Fig. 3, b and c) reduced, but did not prevent, the rapid alkalinization observed on addition of K. Hence, the initial rapid increase in pH induced by K did not require functioning Na/H exchanger.

Fig. 4, a and b, illustrates the dependence of H influx and efflux on [K]. Gradual reduction of K from 5 to 1 mM had a small effect on pH (Fig. 4a). The same effect was observed if K was reduced in one step from 5 to 1 mM. Reducing K from 1 to 0 mM resulted in the typical acidification. Fig. 4b shows that in duct cells acidified by reducing K from 5 to 0 mM, the addition of 1 mM K was sufficient to rapidly restore normal pH. In an attempt to determine the specificity of the extracellular K site, we found that substituting Li for K had no effect on the K-dependent acidification (not shown). Fig. 4c shows that Cs at 5 mM largely prevented the acidification. On the other hand, when 5 mM Cs was added to acidified cells incubated in the absence of K, resting pH was recovered.


Figure 4: Dependence of pH on K concentration. Ducts incubated in solution A were perfused with the same solutions in which increasing concentrations of NMG substituted for K (a and b). In trace c, the duct was perfused with solution A in which 5 mM CsCl replaced 5 mM KCl before perfusion with solution B (K-free) and then solution A.



The characteristics of H fluxes demonstrated in Fig. 2-4 are not compatible with a coupled K/H exchange. Another possibility is transport of the ions through conductive pathways as was suggested for the striated duct(16) . However, K and H fluxes did not require anion transport since incubation of the cells in Cl-free medium in the presence or absence of HCO(3), to deplete intracellular and extracellular Cl, had no effect on H fluxes due to K removal or addition (not shown, but see Na fluxes in accompanying manuscript (38) ). The K-dependent acidification was not affected by up to 10 mM triethylammonium (not shown), but was inhibited by Ba. Fig. 5a shows that incubating the cells with 2.5 mM Ba increased steady-state pH by 0.067 ± 0.015 (n = 3) unit and inhibited the acidification by about 84 ± 7% (n = 3). The inhibition by Ba was reversible. A somewhat unexpected finding is illustrated in Fig. 5b. Inhibition of the pathway required for H fluxes by Ba did not result in recovery of pH due to Na/H exchange. This did not occur until K was added to the incubation medium in the absence (not shown) or presence (Fig. 5b) of Ba. Hence, Ba inhibits H efflux but not H influx induced by changes in K.


Figure 5: Inhibition by Ba of K-dependent acidification. In experiment a, the duct was perfused with solution A containing 2.5 mM Ba, solution B (K-free) containing 2.5 mM Ba, and then solution B. In experiment b, the duct was perfused with solution B and then solution B and A containing 2.5 mM Ba.



A clue to what might be limiting the activity of the Na/H exchanger to set pH at about 7.1-7.2 in the absence of K (Fig. 3-5) was obtained by testing the effect of Na on pH. Fig. 6a shows that removal of Na reduced pHto 6.86 ± 0.03 (n = 3). Removal of K in the absence of Naincreased, rather than decreased, pH to about 6.96 ± 0.04 (n = 3). On the other hand, removal of Na after incubation in the absence of K resulted in a rapid and large reduction in pH to about 6.34 ± 0.03 (n = 5) (Fig. 6b). The same behavior was observed in the presence of HCO(3) (Fig. 6c).


Figure 6: Effect of Na on pH of ducts incubated in the presence or absence of K. In experiment a (dashed line), the duct was perfused with solution C (Na-free, Hepes), K-free solution C and finally with solution A. In experiment b (solid line), the same duct was subsequently perfused with solution B (K-free, Hepes) and then with the Na- and K-free solution. The same results were obtained when only part b of the experiment was performed. In experiment c, a duct incubated in solution E (NaCl, HCO(3)) was perfused with solution E and solution G (Na-free, HCO(3)) in which 5 mM NMG replaced the K and finally with solution E.



Intracellular Na

One explanation of the results in Fig. 6is that removal of K caused rapid and large increases in [Na]. This was directly confirmed by measuring [Na] with SBFI. Fig. 7a shows that reducing K from 5 to 0 mM caused a rapid increase in duct cells [Na] from 13.6 ± 0.4 (n = 59) to 45.3 ± 2.7 mM (n = 33). Increasing K from 0 to 5 mM resulted in a rapid Na efflux to restore resting [Na] within 183 ± 16 s at 37 °C. HCO(3) had minimal effect on the [Na] increase due to removal of K. Fig. 7b shows that this behavior was unique to duct cells.


Figure 7: Effect of K on [Na] of intralobular ducts and acini. SBFI-loaded submandibular ducts and acini present in the same recording field were used to measure [Na] by image acquisition and analysis. Cells in solution A (NaCl, Hepes) were perfused with solution B (K-free, Hepes) and then solution A. Subsequently, the cells were perfused sequentially with solution E (NaCl, HCO(3)), K-free solution E, and then solution E. Note that removal of K in the presence or absence of HCO(3) increased Na in ducts but initially reduced Na in acini.



To evaluate the Na permeability of resting duct cells and demonstrate that removal of K increased [Na] beyond that expected from Na pump inhibition, we tested the effect of ouabain on [Na]. Fig. 8, a-d, shows that ouabain inhibited the Na pump of duct cells at resting (before removal of K) and at high [Na] with an apparent affinity of about 100 mM, typical of the alpha1 isoform found in rat tissue(25) . Further, 1 mM ouabain inhibited the pump even in the presence of 40 mM K (Fig. 8e). Yet, ouabain at 2 mM increased [Na] less and slower than removal of K (Fig. 8a). Thus, removal of K increased Na influx through a K-dependent pathway.


Figure 8: Effect of ouabain on [Na] of duct cells. SBFI-loaded ducts were perfused with solution A containing 0.02 (a), 0.1 (b), 0.5 (c), or 2 (d) mM ouabain and then solution B (K-free, Hepes). After stabilization of [Na], the ducts were perfused with solution B and solution A containing the above concentrations of ouabain. Subsequent perfusion with solution A caused rapid reversal of the inhibition by ouabain as shown in experiment d. Resting [Na] of all ducts was similar to that in trace d. Traces a-c were separated vertically to improve visualization of the effect of ouabain. In experiment e, a duct incubated in solution B was perfused with solution B containing 1 mM ouabain, solution A in which 35 mM K replaced 35 mM Na and also containing 1 mM ouabain, the high K solution, and then solution A. Results similar to those in a-e were observed in at least 3 separate experiments.



The combined experiments in Fig. 7and Fig. 8show that at 5 mM K duct cells have significant permeability to Na which is countered by the Na pump. Removal of K inhibits the Na pump but also increases Na permeability by 4.2-fold (4.56 ± 0.6 mM/min (n = 4) in the presence of ouabain and 19.2 ± 0.8 mM/min (n = 33) due to removal of K). The accompanying manuscript describes the characterization and regulation of this duct cell specific ion transport pathway.

Localization of Ion Transporters

In the initial phase of this study we attempted to microperfuse the intralobular ducts of the submandibular gland, similar to our previous studies with pancreatic ducts(21) . Because of the large amount of connective tissue and the fragility of the granular duct, we were unable to microdissect and perfuse intact intralobular ducts. The only reliable technique was to perfuse the main duct (see Fig. 1). The problem in this case was that a significant amount of the dye was extracellular. In fact it was not possible to obtain sufficient signal/noise with SBFI or Na-Green to measure [Na]. However, this problem was less acute with BCECF. Therefore, we used this dye to localize the transporters.

Fig. 9shows the presence of a Na/H exchange activity in the luminal side and provides the first demonstration of similar activity in the basolateral side of the duct. Fig. 8a shows that removal of Na from the lumen had a small effect on pH. Removal of Na from the bath in the absence of luminal Na caused rapid acidification. Addition of Na to the lumen in the absence of Na in the bath rapidly increased pH. However, addition of Na to the bath was required to restore resting pH. The second part of Fig. 8a shows a reverse sequence of Na removal and addition. In this case, addition of Na to the bath in the absence of luminal Na partially restored pH, and addition of Na to the lumen further increased pH. Fig. 8b shows that DMA inhibited the Na-dependent H efflux in both membranes. Removal of DMA was followed by recovery of normal pH.


Figure 9: Localization of Na/H exchangers in the luminal and basolateral membranes of the main submandibular duct. The main duct was cannulated, loaded with BCECF, and perfused with separate luminal and bath solutions. In experiment a, the luminal and bath perfusion solutions were switched from solution A (NaCl, Hepes) to solution C (Na-free, Hepes) as indicated. In experiment b, the duct was perfused with Na-free solution C and acidified by an NH(4) pulse before perfusing the lumen with solution A (140 mM Na) containing 20 µM DMA and then solution A. In the second part of the experiment, the bath of the acidified duct was perfused with solution A containing 20 µM DMA and then solution A. The experiments were performed with separate ducts and represent 5 (a) and 3 (b) others with the same results whether both experiments were performed with the same or separate ducts.



Fig. 10provides evidence for separate luminal and basolateral Cl/HCO(3) exchangers in submandibular ducts. Removal of Cl from the bath in the presence of luminal Cl caused a small, transient (in 5 out of 5 experiments) increase in pH (Fig. 10a). Removal of Cl from the lumen caused a rapid, large, and sustained increase in pH. Addition of Cl to the bath in the absence of luminal Cl partially reduced pH, and addition of Cl to the lumen restored normal pH. The reverse experiment is shown in Fig. 10b. In this case, removal of Cl from the lumen in the presence of Cl in the bath resulted in a large and sustained increase in pH. Addition of Cl to the lumen in the absence of Cl in the bath was nearly sufficient to restore resting pH. All effects of Cl removal were largely inhibited by incubation with 0.5 mM DIDS.


Figure 10: Localization of Cl/HCO(3) exchange activity in the luminal and basolateral membranes of the main submandibular duct. The lumen and bath of a BCECF-loaded duct in solution E (NaCl, HCO(3)) were perfused with solution F (Cl-free, HCO(3)) and then solution E (a). The same duct was used to perform the experiment in trace b in which the lumen and then the bath were perfused with solution F and then solution E. Finally, the lumen and then the bath were perfused with solution F containing 0.5 mM DIDS (c). All the experiments were performed with the same duct and represent another two with similar results. Similar results were also obtained in two experiments in which part b was performed before part a.



Fig. 11shows the localization of the K-sensitive pathway in the luminal membrane. Removal of K from the luminal solution caused small acidification. Removal of K from the bath was required to obtain the maximal acidification (Fig. 10a) (n = 3). On the other hand, removal of K from the bath in the presence of luminal K had a small effect on pH, and removal of K from the lumen caused significant acidification (Fig. 10b) (n = 3). Addition of K to the lumen in the absence of K in the bath had a small effect on pH. Addition of K to the bath was required for the rapid H efflux (Fig. 10a). The simplest explanation for the need to remove bath K to observe the maximal acidification is the effect of bath K on the Na pump. In the perfused duct, large intracellular Na accumulation due to removal of luminal K was prevented because of Na efflux across the basolateral membrane by the Na/K pump. This prevented partial inhibition of the Na/H exchangers which removed some of the H entering the cytosol. That this is likely the case is shown by the experiments in the second part of Fig. 10, a and b. Fig. 10a shows that when K was removed from the lumen and then the bath to allow accumulation of intracellular Na as in Fig. 6b, removal of Na from the bath and then the lumen caused large and rapid cytosolic acidification. On the other hand, Fig. 10b shows that maintaining luminal K at 5 mM throughout the experiment reduced the acidification observed on removal of Na from both sides. Thus, removal of luminal K increased the accumulation of intracellular Na and the acidification. Unfortunately we were unable to confirm this finding by measuring [Na] with SBFI or Na-Green due to small signal/noise.


Figure 11: Localization of the K-dependent H and Na influx pathway. In experiment a, the lumen and bath of the duct were alternately perfused with solution B (K-free, Hepes) and then solution A. In the second part of the experiment after perfusing the lumen and then bath with solution B, the bath and then the lumen were perfused with a Na- and K-free solution C. This caused large acidification of the cytosol. In experiment b, the reverse protocol was performed in which the bath and then the lumen were perfused with solution B. In the second part of the experiment, the lumen was continuously perfused with a solution containing 5 mM K. When the lumen was not exposed to a K-free solution, removal of Na from the bath and then the lumen caused a small reduction in pH.




DISCUSSION

Ductal systems of various exocrine glands control the final electrolyte composition of the secreted fluid. In the gastrointestinal tract, the ducts alkalize the fluid by secreting HCO(3) and/or absorbing H. HCO(3) concentration in the secreted fluid of some species can be as high as 140 mM(3, 26) . The submandibular salivary gland has been extensively used as a model system to study electrolyte secretion by duct cells(3) . Salivary duct cells reabsorb Na and Cl and secrete K and HCO(3). The concentrations of Na and K in the salivary fluid varies depending on secretory rate but, in general, they change in a reciprocal manner(3) . Cl absorption parallels that and accounts for about 25% of Na absorption (4, 27) and appears to be only partially coupled to HCO(3) secretion(3) . HCO(3) and K secretion partially correlates, but usually K secretion significantly exceeds HCO(3) secretion(5, 7, 8, 11, 12) . In the extreme cases of total removal of luminal Na(4, 5, 28) or in the presence of luminal amiloride(16) , when secretion is slow but measurable, K and HCO(3) secretion (or H absorption) become increasingly coupled(4) . Based on these findings and their elegant studies on the dependence of ion transport on the luminal concentrations of Na and K, Knauf et al.(4) suggested that luminal K and HCO(3) secretion are mediated largely by separate, but functionally coupled, Na/H and K/H exchangers. With few modifications, the model proposed by Knauf et al.(4) becomes the accepted mechanism of ion transport by the salivary duct.

Partial validation of the model was obtained with the findings of an amiloride-sensitive Na channel (6) and a Cl conductance (15) in the luminal membrane of salivary duct cells. Measurement of pH in the present studies confirmed the presence of a Na/H exchange activity in intralobular duct cells(16, 24) . Unlike a previous report using similar cells and techniques, we also found a Cl/HCO(3) exchange activity in these cells (Fig. 2). The simplified cell preparation technique used for the present studies may have allowed the demonstration of this activity in intralobular ducts. As a control for these experiments, we measured pH in ducts and acini in the same recording field since the acini were shown to have a Cl/HCO(3) exchanger(29) . In both cell types, removal of Cl in the absence of HCO(3) acidified, rather than alkalinized, the cytosol. These findings are similar to those in pancreatic acini (30) in which the exchanger transports HCO(3) but not OH. In the presence of HCO(3), removal of Cl caused a DIDS-sensitive, Na-independent alkalinization, all indicative of a Cl/HCO(3) exchange.

The perfusion experiments with the main submandibular ducts provide the first direct localization and the first evidence for the presence of Na/H and Cl/HCO(3) exchange activities on both sides of the duct. Thus, the luminal and basolateral sides of the ducts displayed similar rates of Na/H exchange. The rate and extent of pH changes due to Cl removal suggests that luminal Cl/HCO(3) exchange activity is much higher than the basolateral activity. The influence of the ions in one side on the activity of the exchanger present in the opposite side indicates that the exchangers share common pools of intracellular Na, Cl, and H/HCO(3). This would imply that the luminal and basolateral exchangers are present in the same cells or in cells coupled with respect to Na, Cl, and H/HCO(3). This is particularly relevant since the submandibular duct contains at least two morphologically distinct cell types(3) . In the present studies, we were unable to address this question directly since the thick layer of connective tissue of the main duct precluded identification of single cells (see Fig. 1B). However, pancreatic duct cells were shown to be electrically coupled through gap junctions(31) , which are permeable to a variety of small charged molecules(32) . It is thus possible that the exchangers exist in the same or separate submandibular duct cells coupled by gap junction.

The presence of more than one type of a similar transporter in selective membranes of the same epithelial cell has been extensively studied in the kidney(33, 34) . It is believed that in these cases one of the transporters is involved in transepithelial electrolyte transport, whereas the other maintains the normal cytosolic concentration of the respective ion(34) . It is likely that the basolateral Na/H and Cl/HCO(3) exchangers in the salivary duct regulate pH. The luminal Na/H exchanger probably mediates some of the nonelectrogenic Na reabsorption(3, 4) . On the other hand, the luminal Cl/HCO(3) exchanger is likely to play only a minor role in Na absorption and K and HCO(3) secretion since removal of luminal Cl reduces Na reabsorption by only 25%(4, 27) .

The most significant finding of the present studies is a K-dependent pathway permeable to Na and/or H (HCO(3)) in submandibular duct, but not acinar cells. We believe that this can be a major pathway of K and HCO(3) secretion in the luminal membrane of the duct.

Our results do not support the existence of a coupled K/H exchange or a K/HCO(3) cotransport mechanism in submandibular duct cells. The most compelling evidence is the inability of K to cause substantial H efflux from acid-loaded cells (Fig. 2). In separate experiments we used the protocol in Fig. 6b to first deplete the cells from K and load them with Na and then acidify the cells by incubation in Na-free medium. Even in these acid-loaded and K-depleted cells K was not effective in causing H efflux. In duct cells incubated in the absence of Na, removal of K alkalinized, rather than acidified, the cytosol (Fig. 6a). All previously reported K/H exchange mechanisms had markedly different properties (35, 36, 37) from the K-dependent H fluxes in submandibular ducts. These include saturation at a 100-150 mM K(35, 37) , H transport only against the concentration gradient for K(35, 36, 37) insensitivity to Ba(36, 37) and transport of Cs and Li(37) . Since Ba and 5 mM Cs inhibited the K-dependent H/HCO(3) and Na(38) fluxes, it is possible that the pathway is sensitive to the transepithelial potential and K and H (HCO(3)) transport is mediated by conductive pathways. Obviously, additional studies are needed to identify the nature of this pathway(s) with certainty. However, irrespective of the exact mechanism, this transporter is likely to play a major role in ductal electrolyte secretion.

At this point it is useful to indicate the similarities and inter-relationship between the K-induced H and Na fluxes which are described in the accompanying manuscript(38) . This suggests that H/HCO(3) and Na and maybe K are transported by the same pathway or by separate but tightly coupled pathways. The physiological significance of these observations is highlighted by our ability to localize this pathway to the luminal membrane of submandibular duct cells (Fig. 11). Thus, the luminal membrane of the submandibular duct has an ion transport pathway(s) which is sensitive to the extraluminal concentrations of K and mediates the concomitant transport of Na and H and/or HCO(3) into the cells and K out of the cells. Such a pathway is ideal in mediating K and HCO(3) efflux to the lumen and some of the Na reabsorption during salivary secretion.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK38938 and DK36591. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence and reprint requests should be addressed: Dept. of Physiology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235. Tel.: 214-648-2593; Fax: 214-648-8685.

(^1)
The abbreviations used are: pH, intracellular pH; [Na], intracellular Na concentration; Na, intracellular Na; [K], intracellular K concentration; [Ca], intracellular Ca concentration; K, extracellular K concentration; DMA, dimethylamiloride; NMG, N-methyl-D-glucamine; DIDS, 4,4`-diisothiocyanostilbene-2,2`-disulfonic acid; BCECF, 2`,7`-bis(2-carboxyethyl)-5(6)-carboxyfluorescein.


ACKNOWLEDGEMENTS

We thank Daniel Mlcoch for technical assistance and Mary Vaughn for expert administrative support.


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