©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Na in Resting and Stimulated Submandibular Salivary Ducts (*)

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

Xin Xu Hong Zhao 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

In the preceding manuscript (Zhao, H., Xu, X., Diaz, J., and Muallem, S.(1995) J. Biol. Chem. 270, 19599-19605), we described a K-dependent H/HCO(3) and Na influx pathway in the luminal membrane of salivary duct cells. In the present studies, we further characterized this pathway to show that the K-dependent Na influx was not mediated by the luminal amiloride-sensitive Na channel, the Na/H exchangers, or any electroneutral or conductive Cl-dependent transport pathway. Thus, K efflux probably maintained electroneutrality during Na influx induced by removal of K. Accordingly, Na influx was largely inhibited by 2.5 mM external Ba. The K site of the K-dependent Na influx showed the selectivity sequence Cs > K > NH(4) > Li which is different from that of several known K channels. More importantly, Na influx is 50% inhibited at about 20 mM K, and significant Na influx occurred even at 80 mM K. This is a critical property for the pathway to play a role in Na reabsorption and K secretion by the duct. The large Na influx in resting duct cells is matched by high activity of the ductal Na pump which is about 8-fold faster than that of acinar cells. Stimulation of submandibular ducts with various agonists increased [Na] in an agonist-specific manner. The parasympathetic agonist epinephrine was more effective than isoproterenol and the sympathetic agonist carbachol. The use of various inhibitors of Na and K transporters suggests that different pathways mediate Na influx in stimulated acinar and duct cells of the gland. In duct cells, Na influx was inhibited only by extracellular Cs and Ba. The overall findings support a significant role for the K-dependent pathway(s) in Na reabsorption and K and HCO(3) secretion and explain several features of transepithelial electrolyte transport by salivary ducts.


INTRODUCTION

The main function of the salivary gland ductal system is the reabsorption of Na and Cl and the secretion of K and HCO(3)(1) . Cl absorption is coupled in part to HCO(3) secretion and in part to Na absorption(2, 3, 4) . A significant portion of HCO(3) secretion is coupled to K secretion(2, 5) . However, the bulk of K secretion is coupled to Na absorption(5, 6, 7) . These findings together with measurements of inter-relationships among ions transported during transepithelial electrolyte transport (2) have led to a model in which most Na reabsorption and K and HCO(3) secretion are mediated by a luminal Na/H and K/H exchangers(1, 2) .

In the preceding manuscript (8) we measured pH(^1)and [Na] in isolated submandibular ducts and the perfused main duct to show the localization and inter-relationships between several anticipated (1, 2) and several newly discovered (8) ion transporters. We confirmed the existence of luminal Na/H and Cl/HCO(3) exchangers and demonstrated the presence of similar transporters in the basolateral membrane of duct cells. However, we could not obtain evidence for a coupled K/H exchanger, similar to that described in other epithelial cells(9, 10, 11) . Instead, we identified a luminal, duct-specific, K-sensitive ion transport pathway(s) which transports H/HCO(3) and Na. We postulate that the K-sensitive pathway(s) mediates most of the K secretion and some of the Na reabsorption by salivary ducts. To perform these tasks, the pathway should be regulated by ductal secretagogues and allow the transport of H/HCO(3) and Na in the presence of high K. Thus, the concentration of K in the primary saliva produced by acinar cells is about 5 mM whereas that measured in the secondary saliva is about 70-80 mM(3, 7, 12, 13, 14, 15) .

In the present studies we show that Na transport initiated by changes in K is not mediated by any of the previously described Na transporters of duct cells and that the pathway can mediate Na influx in the presence of 40-60 mM K. Stimulation of duct cells with various ductal secretagogues increases [Na], which may be mediated by the K-dependent pathway.


EXPERIMENTAL PROCEDURES

All materials and experimental procedures were the same as in the preceding manuscript(8) . In the present studies we used SBFI or Na-Green to estimate [Na]. It was necessary to use Na-Green to evaluate the effect of amiloride on ductal Na transport because amiloride and all analogues tested have strong fluorescence when excited at the 340-380 nm range. A disadvantage of using Na-Green is that this dye has no isosbestic point and in many experiments a large fraction of the dye behaved as if it were extracellular. This reduces the signal/noise ratio, prevents proper calibration, and precludes testing the effect of various Na concentrations on Na transport.

Na-Green was loaded by incubating the cells with 2.5 µM Na-Green/AM for 15 min at room temperature. The cells were then washed once, resuspended in about 5 ml of PSA, and kept on ice until plating on coverslips. Na-Green fluorescence was measured by photon counting at an excitation wavelength of 490 nm. The emitted light was passed through a dichroic mirror and filter set DM510 from Nikon and directed to a photomultiplier tube. The operational, storage, and data analysis programs were from PTI.


RESULTS

Characterization of Na Influx

Several amiloride-sensitive Na transporters were reported in duct cells(8, 16, 17) . Fig. 1a shows that they make minimal contributions to the K-induced Na influx, since 0.5 mM amiloride, which is sufficient to inhibit the luminal Na channel and the Na/H exchanger, had no effect on Na influx. Fig. 1b shows that DMA (and amiloride) had no apparent effect on the rate of Na efflux induced by addition of K.


Figure 1: Effect of amiloride on K-dependent Na influx. Intralobular ducts from the submandibular salivary gland loaded with Na-Green were perfused with solution A (NaCl, 5 mM K) or solution B (K-free) (a and b). As indicated, 0.5 mM amiloride (a) or 25 µM DMA (b) was included in the perfusate.



These cells have a luminal Cl channel (18) which may affect electrogenic cation transport. The ubiquitous Na-K-2Cl cotransporter (19) can potentially mediate some of the Na influx. Fig. 2a shows that removal of Cl slightly reduced the rate of Na influx (32 ± 8%, n = 4). However, this may be secondary to the acidification of the cytosol caused by Cl removal (see Fig. 2in the preceding manuscript(8) ). Indeed, in the presence of HCO(3), Na influx was not affected by Cl removal (within 8 ± 11% of control) (Fig. 2c). Inhibition of the cotransporter with bumetanide had no apparent effect on the rates of Na influx or efflux induced by changes in K (Fig. 2b). We therefore conclude that Cl fluxes are not required for Na influx and/or K efflux induced by removal of K.


Figure 2: Na influx is independent of Cl transport in duct cells. In experiment a, SBFI-loaded ducts were perfused with solution A (NaCl) containing 5 mM K, solution B (K-free), and then solution A (control period), before perfusion with solution D (Cl-free, 5 mM K), K-free solution D, and then solution D. In experiment b, ducts were perfused with solution A or solution B and with or without 0.1 mM bumetanide as indicated. In experiment c, the ducts were equilibrated in solution E (NaCl, HCO(3)) before perfusion with solution F (Cl-free, HCO(3)) and K-free solution F.



To test the possible involvement of ion channels in Na influx, we measured the effect of known inhibitors of K and nonselective cation channels on Na influx and the ionic selectivity of the K site. Fig. 3a shows that 10 mM tetraethylammonium had no effect on Na fluxes. Similarly, Na influx was not inhibited by tetrabutylammonium, quinine, quinidine, and apamin (not shown). On the other hand, 2.5 mM Ba reduced the steady-state [Na] of resting duct cells by about 4.6 ± 0.6 mM (n = 5) (Fig. 3b). Ba also inhibited the Na influx induced by K removal in a reversible manner. Fig. 3c shows that addition of Ba to cells incubated in K-free medium caused only a small reduction in [Na]. However, when K was added to the medium, the cells rapidly reduced [Na], in the presence (Fig. 2c) or absence (not shown) of Ba. The effects of Ba on Na influx and efflux are identical with those on H fluxes shown in the preceding manuscript (8) .


Figure 3: Effect of channel blockers on Na influx. In all experiments (a-c), the ducts were alternately perfused with solution A (NaCl, 5 mM K) or solution B (K-free). Where indicated, these solutions also contained 10 mM tetraethylammonium (a) or 2.5 mM Ba (b and c).



The ionic selectivity of the K site is shown in Fig. 4. Fig. 4a shows that Li up to 10 mM could not substitute K either in preventing Na influx or in activating the Na pump-mediated Na efflux from Na-loaded cells. NH(4) at 5 mM partially substituted K in inhibiting Na influx but was a poor substitute for the Na pump (Fig. 4b). Higher concentrations of NH(4) were not tested because of its effect on pH. Fig. 4c shows that Cs substituted well for K in inhibiting Na influx and as a substrate for the Na pump. Interestingly, unlike K, the second removal of Cs from the medium failed to cause rapid and large Na influx. This is probably because Cs cannot be transported by the K-dependent pathway to mediate Cs- Na exchange. Fig. 4d shows the apparent affinity for Cs in inhibiting Na influx. In this experiment, Na influx was inhibited by Cs and the Na pump was inhibited by ouabain. Stepwise reduction in Cs caused a stepwise increase in [Na]. Cs inhibited the K-dependent Na influx pathway with an apparent IC of about 2.5 mM.


Figure 4: Effect of K congeners on Na influx in duct cells. In experiment a, ducts in solution A (NaCl, 5 mM K) were perfused with solution B (K-free) in which 10 mM Li replaced 10 mM Na before perfusion with solution B, solution B containing 10 mM Li, and then solution A. In experiments b and c, the same protocol was followed except that 5 mM NH(4) (b) or 10 mM Cs (c) replaced equivalent amounts of Na in solution B. In experiment d, the duct was perfused with solution B containing 10 mM Cs and then the same solution containing 0.5 mM ouabain. Subsequently, the concentration of Cs was reduced by replacement with an appropriate concentration of Na.



Fig. 5shows the K dependence of Na influx. In the absence of ouabain, reducing K from 5 to 2 mM had a small effect on [Na] (Fig. 5a). Further reduction in K to 1, 0.5, 0.25, and 0 mM resulted in a stepwise increase and stabilization of [Na] at different levels. A subsequent stepwise increase in K caused a proportional reduction in [Na]. Notably, [Na] stabilized at different levels at the same K concentration during Na influx and efflux. This was observed in all experiments tested (n = 7). This probably reflects the effect of [Na] on the apparent affinity of the pump for K(20) . The behavior of Na influx in Fig. 5a shows the marked impact and activity of the Na pump in intralobular ducts.


Figure 5: Dependence of Na influx on K concentration. In experiment a, ducts were perfused with solution A containing the indicated K concentrations. In experiment b(1), the duct was perfused with solution A in which 55 mM Na was replaced with 55 mM K and then with the high K solution containing 1 mM ouabain. Trace b(2) shows the control experiment, in which NMG replaced Na. The experiment in c shows the protocol used to measure the effect of K on Na influx independent of the Na pump. The ducts were perfused with solution A in which different concentrations of K replaced equivalent concentrations of Na. All solutions also contained 1 mM ouabain to inhibit the Na pump. Na influx independent of the K-dependent channel and due to inhibition of the Na pump was estimated from Na influx in the presence of ouabain and 60 mM K (broken line, taken from experiment b(2)). The contribution of the K-dependent channel to Na influx was calculated as the differences between the [Na] in experiments c and b(1) (panel d).



Fig. 5b shows how the contribution of the K-sensitive pathway and the Na pump to Na influx were separated. Fig. 5b(1) shows that increasing K from 5 to 60 mM reduced [Na] by about 6.7 ± 1.2 mM (n = 3). This was due to inhibition of Na influx and not stimulation of the Na pump, since pump activity was maximal at 5 mM K. Fig. 5b(2) provides a control experiment showing that the reduced Na influx at 60 mM K was not due to the reduction in Na. Hence, at 90 mM Na and 5 mM K, ouabain caused substantial Na influx.

The conditions of Fig. 5b were used to determine the dependence of Na influx on K, and the results are illustrated in Fig. 5c. Na influx was inhibited by 60 mM K, and the Na pump was inhibited by 1 mM ouabain. Remarkably, reducing K from 60 to 40 mM was sufficient to cause Na influx, and the influx was almost maximal at 5 mM K. Fig. 5d displays the summary of 4 similar experiments and shows that the dependence of Na influx on K followed simple saturation kinetics with an apparent affinity for K of about 18.2 ± 3.4 mM (n = 4). Furthermore, extrapolation of the results to higher K indicates that the pathway will allow some Na influx even at 80 mM K. This property is a key feature of the luminal K-dependent pathway with respect to its physiological role.

Regulation of [Na]in Stimulated Duct Cells

The use of an image acquisition system allowed us to simultaneously measure [Na] of acini and ducts present in the same recording field and, when informative, compare regulation of [Na] in the two cell types. [Na] of resting submandibular acinar cells was about 8.9 ± 0.78 mM (n = 43) which is in the same range reported in sublingual (21) and parotid acinar cells(22) . In the same experiments, [Na] of resting duct cells was somewhat higher and averaged 14.4 ± 1.03 mM (n = 43).

Fig. 6a shows that stimulation of duct cells with Epi caused a rapid (27.8 ± 1.6 mM/min) and sustained increase in [Na] by about 7.4 ± 0.4 mM (n = 21). Carbachol stimulation increased [Na] of duct cells at a rate of 12.5 ± 0.8 mM/min to an average of 4.7 ± 0.3 mM (n = 11) above resting (Fig. 6b). In the same experiments, carbachol rapidly and strongly increased [Na] of acinar cells by 62 ± 4.7 mM (not shown). Isoproterenol had the smallest effect on [Na]. Isoproterenol stimulation increased ductal [Na] (Fig. 6c) at a rate of 5.6 ± 0.4 mM/min, which stabilized at 2.8 ± 0.55 mM (n = 6) above control.


Figure 6: Effect of agonist stimulation on [Na] of submandibular duct cells. The cells were stimulated by perfusion with solution A containing 10 µM Epi (a), 100 µM carbachol (b), or 1 µM isoproterenol (c). In experiment b, cell stimulation was terminated by perfusion with solution A containing 10 µM atropine.



In an attempt to evaluate the contribution of the different Na influx pathways to the agonist-dependent [Na] increase, we tested the effect of several transport inhibitors on Na influx. Results with the submandibular acinar cells are presented as a positive control and to contrast the behaviors of the two cell types. Carbachol was the most active agonist in acinar cells, and, therefore, results obtained with this agonist are shown in Fig. 7, a-d. Inhibition of the Na-K-2Cl cotransporter with bumetanide reduced the rate of Na influx into carbachol-stimulated acinar cells by about 24 ± 9% (n = 4), but had a minimal effect on the extent of [Na] increase. Removal of bumetanide slightly increased [Na] (Fig. 7b). Ba reduced the rate of Na influx by 46 ± 11% (n = 3) (Fig. 7c). However, most of the effect of Ba is probably indirect since Ba is likely to interfere with the initial KCl efflux and cell shrinkage, which is critical for activation of Na influx in stimulated acinar cells(22) . DMA inhibited Na influx rate by about 72 ± 17% (n = 4) (Fig. 7a). Since Epi is the most active agonist in granular intralobular ducts, the results obtained with this agonist are shown in Fig. 7, e-h. Fig. 7f shows that bumetanide had no apparent effect on the Epi-stimulated [Na] increase (n = 6). Notably, 25 µM DMA had no apparent effect on the agonist-mediated [Na] increase in the presence or absence of K (Fig. 7g), excluding both the amiloride-sensitive Na channel and the Na/H exchangers as major contributors to this Na influx. Fig. 7h shows that 2.5 mM Ba nearly abolished the Epi-stimulated [Na]increase and the large Na influx observed after removal of K.


Figure 7: Effect of various inhibitors on agonist-induced [Na] increase in duct and acinar cells. In experiments a-c, SBFI-loaded acinar cells were stimulated with 100 µM carbachol after perfusion in the absence (a) or presence of 0.1 mM bumetanide (b) or 2.5 mM Ba (c). In experiment d, a Na-Green-loaded acinus was perfused with solution A containing 25 µM DMA before stimulation with 100 µM carbachol. In experiments e, f, and h, SBFI-loaded ducts were stimulated with 10 µM Epi in the absence (e) or presence of 0.1 mM bumetanide (f) or 2.5 mM Ba (h). Where indicated in experiment h, the stimulated duct was also perfused with solution B containing 2.5 mM Ba and then solution B. In experiment g, a Na-Green-loaded submandibular duct was incubated with 25 µM DMA before stimulation with 10 µM Epi. Removal of K in the continuous presence of DMA rapidly increased [Na].



Additional evidence that different pathways mediate the major portion of the Na influx in duct and acinar cells is presented in Fig. 8. Fig. 8a shows that replacing K with 5 mM Cs inhibited the Epi-evoked [Na] increase in duct cells. Removal of Cs from the incubation medium resulted in a rapid increase in [Na]. Replacing K with 5 mM Cs had a minimal effect on the carbachol-induced [Na] increase in acinar cells (not shown). Fig. 8, b and c, shows that increasing K from 5 to 40 mM reduced [Na] in Epi-stimulated ducts by 4.7 ± 0.6 mM (n = 3), whereas it increased acinar [Na] by 6.2 ± 0.8 mM (n = 3).


Figure 8: Inhibition of agonist-induced [Na] increase by Cs and high K in duct but not acinar cells. In experiment a, the duct was incubated in solution B (K-free) containing 5 mM Cs before stimulation with 10 µM Epi and then with solution B and again with solution B containing 5 mM Cs. In experiments b and c, acinar and duct cells present in the same recording field were stimulated with 10 µM Epi before exposure to solution A in which 35 mM K replaced 35 mM Na.



The lack of effect of DMA and the inhibition by Cs and Ba of the agonist-dependent [Na] increase in duct cells suggests that the K-dependent pathway may mediate a significant part of Na influx in stimulated duct cells. To further test this possibility, we measured the effect of agonist stimulation on Na influx evoked by removal of K. Fig. 9a shows that Epi stimulation increased the rate of Na influx due to K removal by 1.84 ± 0.06-fold, and [Na] stabilized at about 67 ± 5.1 mM (n = 8). In addition, Epi stimulation of cells incubated in the absence of K further increased [Na] by about 9.6 ± 1.7 mM (Fig. 9b).


Figure 9: Stimulation by Epi of K-dependent Na influx in duct cells. In experiment a, the duct was perfused with solution A (NaCl, 5 mM K), solution B (K-free), and then solution A (control period), before stimulation with 10 µM Epi and perfusion with solution B. In experiment b, the duct was perfused with solution B before stimulation with 10 µM Epi and perfusion with solution A.



To evaluate the effect of cell stimulation on Na influx independent of Na pump activity, we measured the effect of ouabain on Na influx in stimulated cells. Fig. 10a shows that stimulation of cells incubated with ouabain increased the rate of Na influx from 9.8 ± 0.6 to 34.7 ± 4.2 mM/min, and [Na] stabilized at about 58 ± 4 mM (n = 3), which was about 10-12 mM lower than that caused by removal of K. Removal of K increased [Na] in duct cells treated with ouabain and Epi (Fig. 10b) by about 13 mM. The reciprocal protocol is shown in Fig. 10b. Addition of ouabain to Epi-stimulated cells caused [Na] to increase at a rate of 28.4 ± 2.6 mM/min, which was about 2.9-fold faster than in unstimulated cells (Fig. 10a).


Figure 10: Effect of ouabain on [Na] of Epi-stimulated duct cells. Ducts were treated with 0.5 mM ouabain before (a) or after (b) stimulation with 10 µM Epi. In experiment b, where indicated, the duct was exposed to solution B (K-free) containing 0.5 mM ouabain.




DISCUSSION

The major function of the salivary ductal system is the modification of electrolyte composition of the primary saliva(1) . This is achieved by HCO(3) and K secretion and Na reabsorption, processes that are coupled to each other(1, 2) . It has been difficult to fully explain the coupling of these activities under various physiological and experimental conditions(2) . The most commonly accepted model assumes that luminal Na/H and K/H exchangers are responsible for the cotransport of these ions in duct cells(1, 2) . The model, however, does not account well for HCO(3) secretion and does not explain several properties of ductal electrolyte transport. For example it was shown that 10 µM amiloride, which blocks the luminal Na channel (16) but is not sufficient to inhibit the epithelial type Na/H exchanger(8, 17) , largely blocked Na reabsorption(5, 23, 24, 25) . It is not clear why Na concentration of the luminal solution has to be reduced well below that needed for Na reabsorption (2, 5) in order to stimulate HCO(3) secretion(2, 15) . In addition, a K/H exchange activity cannot be demonstrated in mildly (8, 17) or strongly acidified duct cells(8) . Hence, alternative mechanisms have to be invoked for HCO(3) and K fluxes at the luminal membrane.

In the preceding manuscript(8) , we reported the presence of a luminal, K-dependent pathway which allows the transport of H/HCO(3) and Na(8) . In the present studies, we show that this pathway is different from any amiloride-sensitive Na pathway present in duct cells. Neither low nor high concentrations of amiloride inhibited Na influx induced by removal of K. Low concentrations of amiloride (up to 20 µM) had no effect on [Na]. Amiloride reduced resting [Na] only at high concentrations. This indicates that the K-dependent Na influx is not mediated by the amiloride-sensitive Na channel and that this channel has minimal contribution to Na influx in resting duct cells.

Another critical implication of the experiments with amiloride is that a coupling between K/H and Na/H exchangers cannot possibly mediate Na influx and K efflux from duct cells. Coupling between the exchangers requires that when Na influx is initiated by removal of K, H influx induced by K/H exchange is matched by Na/H exchange to maintain relatively stable pH and mediate large Na influx. In this case, a high concentration of amiloride was expected to completely inhibit Na influx. In fact, as judged from measurements with Na-Green, amiloride reduced Na influx by only about 13%. This corresponds to about 5.7 mM Na influx based on measurements of [Na] with SBFI. This value agrees reasonably well with measurement of pH shown in the preceding manuscript (8) in which amiloride increased the K-dependent acidification by about 0.16 pH unit. Measurement of H buffer capacity of duct cells using established procedures(26, 27) showed it to be about 35 mM/pH unit at a pH of 7.1. This translates to the transport of 5.4 mM H, and, therefore Na, by the Na/H exchanger. Hence, Na/H exchange mediates a small fraction of Na influx in duct cells and thus coupled Na/H and K/H exchange cannot account for K and HCO(3) secretion and Na reabsorption by the duct.

Other pathways that do not contribute to, or are essential for, the Na influx induced by removal of K are the Cl-dependent pathways. These include Na-K-2Cl and NaCl cotransport and the luminal and basolateral Cl channels present in duct cells from various species(1, 18, 28) . An important implication of this conclusion is that another ion must be transported together with Na to maintain electroneutrality. This ion must be K. Therefore, although we did not measure K transport directly, K efflux must occur and account for the bulk of Na influx induced by removal of K.

A potential transporter that can mediate the K-dependent Na influx is the nonselective cation channel found in many epithelial cells, including pancreatic and salivary acinar cells(29) . We could not obtain evidence for the involvement of this channel in Na influx into resting or stimulated submandibular duct cells. Although the K-dependent Na influx was completely inhibited by Ba, it was not sensitive to other channel blockers such as tetraethylammonium, quinidine, or apamin. Furthermore, the K-dependent Na influx was active at resting levels of [Ca] in duct cells, whereas no such activity was found in submandibular acinar cells present in the same recording field. Finally, replacing K with Cs inhibited Na influx even when the Na pump was inhibited, indicated that Cs directly inhibited the K-dependent pathway. Cs should not inhibit Na influx by the nonselective channel, as replacing K with Cs in the absence of Cs influx should have hyperpolarized the cells and augmented Na influx mediated by a nonselective channel. Although these observations do not completely exclude that the K-dependent Na influx is mediated by the nonselective cation channel, they suggest that such is unlikely.

An important property of the K-dependent pathway is that it allows Na influx in the presence of very high luminal K (60-80 mM). Of course, the high concentration of K in saliva(3, 7, 12, 13, 14, 15) dictates that if the pathway is to play a role in salivary electrolyte transport it must allow Na influx at high luminal K. Nevertheless finding such a property was important in establishing a physiological role for this transporter. Since the K-sensitive pathway dominates Na influx in submandibular duct cells, it is possible that the main function of the amiloride-sensitive Na channel is to regulate the potential across the luminal membrane and thus control fluxes through the K-dependent pathway. In this case, the activity of both transporters is required for electrolyte transport by the duct. The major role of the luminal Na/H exchanger may be to prevent accumulations of H next to the apical membrane during HCO(3) secretion and thus facilitate Cl reabsorption by the luminal Cl/HCO(3) exchanger. Indeed, 100 µM amiloride inhibits the ductal Na/H exchanger by less than 50% (17) , whereas 10 µM amiloride inhibits Na absorption by more than 90%(5, 23, 25) . If a major role of the luminal Na/H exchanger was Na reabsorption, then 10 µM amiloride should have reduced the reabsorption by about 10-15%.

It is clear that the K-dependent pathway is active in resting cells when luminal K is 5 mM. This is concluded from the unmasking of significant Na influx by ouabain. The effect of ouabain on [Na] also indicates a tight coupling between Na influx by the luminal K-dependent pathway and the basolateral Na pump. This finding was also demonstrated in the main perfused duct when the luminal and basolateral transporters and solutions are separated (Fig. 10, (8) ). Therefore, unlike most cells, in salivary duct cells the major Na influx mechanism that fuels the Na pump is the K-dependent pathway and not the Na/H exchanger. The physiological consequence of this coupling is the maintenance of low [Na] during Na reabsorption. Low [Na] prevents inhibition of the Na/H exchangers, which probably function to remove the excess cytosolic H accumulating during HCO(3) secretion.

Another interesting finding of the present studies was the properties of the agonist-dependent [Na]increase in submandibular duct cells. Bumetanide and amiloride had no effect on the agonist-dependent [Na] increase. On the other hand, this activity was blocked by Cs and Ba, and agonist stimulation increased the rate of K-dependent Na influx by 2-3-fold. This would suggest that most Na influx in stimulated submandibular duct cells is mediated by the K-dependent pathway. Hence, independent of its identity, it is quite remarkable that the K-dependent pathway dominates Na influx in resting and stimulated submandibular duct cells, whereas the amiloride-sensitive pathways make minor contributions to Na influx. It is therefore possible that the K-dependent transporter is the major Na influx pathway responsible for Na reabsorption. If it can also mediate K efflux during Na influx, it might also be responsible for the secretion of K into the duct lumen.

The findings presented in the preceding (8) and present manuscripts explain several features of electrolyte transport by duct cells which could not be explained by previous models. The K-dependent pathway provides a suitable mechanism for ductal secretion of HCO(3) and for the coupling between HCO(3) and K secretion. Our results also explain why the secretion of K and HCO(3) is augmented and becomes increasingly coupled in the absence of luminal Na(2, 3, 5, 6) . Under these conditions, the K-dependent pathway is expected to mostly extrude HCO(3) rather than absorb Na. In the absence of luminal Cl, Na reabsorption is reduced by 25%, and Na influx becomes equal to K efflux(2, 3) . Under these conditions, Na reabsorption can occur through the K-sensitive pathway in exchange for intracellular K. HCO(3) secretion by the rat submandibular duct is low compared to the active transport of Na and K(1, 2) . In the presence of 5 mM luminal K, removal of luminal Na increases HCO(3) secretion about 3-fold(2) . The lack of luminal K/H exchange (8) requires alternative pathways that allow the K and HCO(3) uptake under these conditions. The K-dependent pathway(s) has the required properties to mediate such fluxes. Finally, our results explain why agonist stimulation reduces the transepithelial potential difference of the ducts(14, 15) . The agonists activate the luminal K-dependent, Na- and K-permeable pathways and increase [Na] and probably reduce [K]. Both of these effects reduce the transepithelial potential difference. The maintained high [Na] in stimulated cells (Fig. 6) is expected to reduce the rate of Na influx at the luminal membrane and at least partially inhibit the Na/H exchangers(30, 31) . As a consequence, agonist stimulation is expected to reduce the rate of Na reabsorption. This was indeed found in many studies using the perfused main duct(1) .


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK 38938 and DK 36591. 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: 214-648-8585.

(^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; Epi, epinephrine; DMA, dimethylamiloride.


ACKNOWLEDGEMENTS

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


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