Muscarinic agonists induce phosphorylation-independent activation of the NHE-1 isoform of the Na+/H+ antiporter in salivary acinar cells.

Cholinergic agonists stimulate isotonic fluid secretion in the parotid gland. This process is driven by the apical exit of Cl-, which enters the cells partly via Cl-/HCO-3 exchange across the basolateral membrane. Acidification of the cytosol by the extrusion of HCO-3 is prevented by the concomitant activation of the Na+/H+ exchanger (NHE), which is directly activated by cholinergic stimulation. Multiple isoforms of the NHE have been described in mammalian cells, but the particular isoform(s) present in salivary glands and their mechanism of activation have not been defined. Reverse transcriptase-polymerase chain reaction with isoform-specific primers was used to establish that NHE-1 and NHE-2, but not NHE-3 or NHE-4, are expressed in parotid glands. The presence of NHE-1 was confirmed by immunoblotting and immunofluorescence, which additionally demonstrated that this isoform is abundant in the basolateral membrane of acinar cells. The predominant role of NHE-1 in carbachol-induced Na+/H+ exchange was established pharmacologically using HOE694, an inhibitor with differential potency toward the individual isoforms. Because muscarinic agonists induce stimulation of protein kinases in acinar cells, we assessed the role of phosphorylation in the activation of the antiport. Immunoprecipitation experiments revealed that, although NHE-1 was phosphorylated in the resting state, no further phosphorylation occurred upon treatment with carbachol. Similar phosphopeptide patterns were observed in control and carbachol-treated samples. Together, these findings indicate that NHE-1, the predominant isoform of the antiporter in the basolateral membrane of acinar cells, is activated during muscarinic stimulation by a phosphorylation-independent event. Other processes, such as association of Ca2+-calmodulin complexes to the cytosolic domain of the antiporter, may be responsible for the activation of Na+/H+ exchange.

Stimulation of salivary acinar cells induces rapid and abun-dant secretion of isotonic fluid, a process that is driven primarily by efflux of Cl Ϫ and HCO 3 Ϫ across the apical membrane (1). The stimuli, such as cholinergic agonists, increase secretion by elevating the anion permeability of the apical membrane, while promoting accumulation of Cl Ϫ in the cytosol. The latter is accomplished in part by activation of a loop diuretic-sensitive Na ϩ -K ϩ -2Cl Ϫ co-transporter, but also by parallel operation of the Cl Ϫ /HCO 3 Ϫ and Na ϩ /H ϩ antiporters in the basolateral membrane (1,2). Accordingly, fluid secretion in perfused salivary glands was found to be sensitive to inhibitors of the Na ϩ /H ϩ antiporter (3). Moreover, in isolated rat parotid acinar cells carbachol markedly activated Na ϩ influx, and the initial rate of influx was inhibited by 75% in the presence of dimethylamiloride (DMA), 1 a relatively specific inhibitor of Na ϩ /H ϩ exchange (4,5).
Stimulation of salivary cells is accompanied by a tendency of the cytosol to become acidic. This is attributable in part to generation of acid equivalents by the metabolic pathways supplying energy to the secretory process, but mainly to the electrodiffusional exit of HCO 3 Ϫ across the apical membrane (6,7). Because the changes are caused by HCO 3 Ϫ itself, the cells are unable to buffer the cytosolic pH (pH i ) using HCO 3 Ϫ /CO 2 and must resort to other regulatory mechanisms. Na ϩ /H ϩ exchange fulfills this role as well, extruding the excess cytosolic acid across the basolateral membrane (5,8,9). The alkalinization that accompanies activation of the antiporter not only regulates pH i , but also promotes the intracellular accumulation of HCO 3 Ϫ , facilitating secretion of this anion (10). Jointly, these observations indicate that activation of the basolateral Na ϩ /H ϩ antiporter plays an essential role in salivary fluid secretion. Despite its importance, however, neither the identity nor the molecular mechanism of activation of the antiporter have been elucidated.
Five distinct isoforms of the Na ϩ /H ϩ exchanger (NHE) which differ in their kinetic and pharmacological properties have been identified in mammalian cells (11,12). They are differentially expressed in various tissues, suggesting distinct functions for the individual isoforms. NHE-1 is ubiquitously expressed and is involved in the regulation of pH i and cell volume in both epithelial and non-polarized cells (12). NHE-2 and NHE-3 are prominent in intestinal and renal tissues where they ostensibly participate in transepithelial NaCl transport. NHE-3 is located on the apical membrane, while the specific location of NHE-2 is still controversial (11)(12)(13)(14). The two other isoforms are poorly characterized. NHE-4 is abundant in the stomach (14), but its precise cellular location and function remain obscure. Similarly, the distribution and function of NHE-5 (15) are still unknown, and even its full sequence remains to be defined. Little is known about the distribution of these isoforms in the salivary gland.
The purpose of the experiments described in this article was to identify the isoform(s) of the Na ϩ /H ϩ exchanger present in acinar cells of the rat parotid gland, to explore their individual contribution to the uptake of Na ϩ , and the regulation of pH i , and to define their mechanisms of activation during cholinergic stimulation. Ϫ replaced Cl Ϫ and the solution was gassed with 95% O 2 , 5% CO 2 (solution B). Acid loading was accomplished by pre-pulsing the cells for the indicated time in a medium where 40 mM NH 4 ϩ replaced Na ϩ (solution C). In the Na ϩ -free medium (solution D) all Na ϩ was iso-osmotically replaced with N-methyl-D-glucammonium ϩ .

Materials
Preparation of Acinar Cells-Parotid acinar cells from male Wistar rats were isolated by sequential treatment of the glands with trypsin (Life Technologies, Inc.) and purified collagenase (Worthington, type CLSPA), as described previously (16). The fraction used for optical studies, which consisted of single cells, doublets, triplets, and "strings" of cells, was kept at room temperature with periodic top gassing with 100% O 2 .
Microscopy and Fluorescence Measurements-Approximately 200 l of the cell suspension was layered onto a poly-L-lysine (0.8 mg/ml) coated coverslip. Cells adhering within 2 min were covered with solution A and loaded with the dye by incubation with either 1 M BCECFacetoxymethyl ester for 5 min at 37°C or with 7 M SBFI-acetoxymethyl ester for 60 min at room temperature under 100% O 2 gassing. After loading, cells were allowed to recover for 30 -60 min in solution A at 37°C under 100% O 2 gassing, to minimize possible toxic effects of dye loading (4). The coverslip was next mounted in a chamber and perfused continuously with solution A on the stage of an inverted microscope (Zeiss Axiovert). BCECF was excited sequentially at 440 and 490 nm (10 nm band pass) and emission was detected at 530 nm (10-nm band pass). Fluorescence was quantified by averaging pixel intensities throughout the cell and pH i was determined by in situ calibration of the excitation ratio using the K ϩ /nigericin technique. SBFI was excited at 340 and 380 nm (10-nm band pass) and emission was measured at 500 nm (40-nm band pass). Additional details of the optical setup were described previously (4, 16 -18). To convert the ratio of SBFI fluorescence to [Na ϩ ] i , cells were exposed to various extracellular concentrations of Na ϩ (substitution for K ϩ ) in the presence of 10 M gramicidin. Ionophore-induced cell swelling was prevented by replacing 60 mM Cl Ϫ with gluconate Ϫ . The data fitted the equation where R 0 and R max were the ratios measured in the absence and presence of saturating (150 mM) [Na ϩ ] i , respectively. K D was 20.8 Ϯ 1.4 mM (n ϭ 6). During all experiments, cells were viewed simultaneously by differential interference contrast while measuring low light-level fluorescence, using red illumination and the dichronic mirrors and filter sets described earlier (18). This enabled us to simultaneously estimate cell volume, as described (16 -17).
Calculation of Net Proton Flux-Net proton flux, J H ϩ (in mM/min), was calculated as the product of the rate of pH i recovery (dpH/dt) and the intrinsic buffering capacity of the cells. The latter was measured using weak electrolyte pulses, as described (19), while dpH/dt over a discrete pH i interval was determined by fitting a straight line to 3 or more consecutive data points. Lines were fitted by least squares using Cricket Graph 1.3.2 and consistently yielded r 2 Ͼ 0.95. The slope of this line was considered to be the rate of pH i change at the mean pH i of the interval analyzed. An alternative method of calculating the slope involved fitting all the data of a pH i recovery curve to an exponential function (pH i ϭ k 0 ϩ K 1 *e Ϫk 2 t) using the IGOR curve-fitting software. Similar results were obtained with both approaches. Where indicated (e.g. Fig. 2B) the rate of acid accumulation induced by the stimulus was added to the rate of extrusion, to calculate total Na ϩ -dependent H ϩ efflux. Acid accumulation was calculated by measuring the pH changes upon removal of Na ϩ in stimulated cells. No spontaneous acid loading was detected in control cells when Na ϩ was removed (n ϭ 25).
Isolation of RNA, Reverse Transcription, and Polymerase Chain Reaction (RT-PCR)-Total RNA was isolated from partially purified acinar cells by guanidinium thiocyanate-phenol-chloroform extraction (Trizol; Life Technologies, Inc.), based on the method of Chomczynski and Sacchi (20). Poly(A ϩ ) RNA was purified by affinity chromatography with an oligo(dT)-cellulose column (Pharmacia). Parotid mRNA was then reverse-transcribed and the complementary DNA amplified by the polymerase chain reaction, using the GeneAmp RNA PCR kit (Perkin-Elmer) and a Perkin-Elmer DNA thermal cycler Model 480. After completion of the PCR reaction (35 cycles), a 10-l sample of the PCR tube was analyzed by electrophoresis on a 0.8% agarose gel pre-stained with 0.5 g/ml ethidium bromide and the gel was photographed under UV illumination. Four isoform-specific sets of primers were used, which hybridized to unique regions of the rat NHE-1, NHE-2, NHE-3, and NHE-4. Primers were as follows: NHE-1, 5Ј primer: CCT ACG TGG AGG CCA AC, 3Ј primer: CAG CCA ACA GGT CTA CC, size of the PCR product: 429 base pairs (bp); NHE-2, 5Ј primer: GCT GTC TCT GCA GGT GG, 3Ј primer: CGT TGA GCA GAG ACT CG, size of PCR product: 680 bp; NHE-3, 5Ј primer: CTT CTT CTA CCT GCT GC, 3Ј primer: CAA GGA CAG CAT CTC GG, size of PCR product: 574 bp; NHE-4, 5Ј primer: CTG AGC TCT GTG GCT TC, 3Ј primer: C GAG GAA ATG CAG CAG C, size of PCR product: 381 bp. All four sets of primers yielded the expected PCR products when pCMV plasmids containing the full-length clone of the corresponding isoform were used as a template, but did not yield discernible products when any of the other isoforms was used as template.
Immunoblotting and Immunoprecipitation-The preparation and purification of anti-NHE-1 antibodies and the method used for immunoblotting of membranes have been described in detail elsewhere (21). For immunoprecipitation, acinar cells were labeled for 2 h at 37°C in nominally phosphate-free medium containing [ 32 P]orthophosphate (500 Ci/ml). Cells were then treated with or without carbachol in medium A for 2 min at 37°C. The reaction was stopped by sedimentation, followed by resuspension in immunoprecipitation buffer. The samples were extracted for 30 min at 4°C and sedimented for 30 min at 100,000 ϫ g at 0°C. Immunoprecipitation proceeded as described previously (21).
Peptide Mapping-Samples were immunoprecipitated as above and eluted from the beads by boiling for 5 min in 125 mM Tris-HCl, pH 6.8, 0.5% SDS, 10% glycerol, and 0.0001% bromphenol blue. After cooling to room temperature, 25 g/ml chymotrypsin was added to the eluate. Digestion was stopped at the indicated times by addition of mercaptoethanol and SDS to final concentrations of 10 and 2%, respectively, and boiling for 5 min. The peptides were resolved by SDS-PAGE using 15% acrylamide, the gels were dried and autoradiograms obtained using Kodak X-Omat AR film.
Immunofluorescence-Cells attached to coverslips or frozen tissue sections were rinsed twice with PBS and then fixed by incubation with 3% paraformaldehyde for 10 min. Fixation was terminated by rinsing and incubating with 100 mM glycine in PBS, pH 7.4, for 15 min. Cells were then permeabilized by incubation with a solution of 0.1% Triton-X and 0.1% (w/v) bovine serum albumin in PBS (TA-PBS solution) for 15 min, followed by three washes with the same solution. Blocking was then performed by incubation for 20 min in TA-PBS containing 5% goat serum. The coverslips were then washed three more times with TA-PBS. All the preceding steps were at room temperature. The cells were next incubated overnight with a 1:100 dilution of anti-NHE-1 antibodies in TA-PBS at 4°C. Where indicated, the primary antibody was omitted to control for specificity of staining. After three more washes in TA-PBS, the samples were incubated with a 1:200 dilution of fluorescently labeled donkey anti-rabbit antibody in TA-PBS for 50 min at room temperature. The cells were finally washed three times with PBS and mounted in 50% glycerol containing 1% n-propyl gallate.
Other Methods-Protein was determined using the Pierce BCA Assay Reagent. All experiments were performed at least three times. Representative radiograms or confocal images are illustrated. Quanti-tative data are presented as mean Ϯ S.E. of the number of experiments (n) in parentheses. Fig. 1, A and B, illustrate measurements of pH i in isolated acinar cells using quantitative imaging of the fluorescence of intracellular BCECF. In agreement with earlier observations (4, 5), we found that muscarinic stimulation of parotid acinar cells in the presence of HCO 3 Ϫ induces a rapid and transient cytosolic acidification (Fig. 1A). Exposure of cells to 10 M carbachol in HCO 3 Ϫ -containing medium (solution B), resulted in an drop in pH i averaging 0.20 Ϯ 0.02 pH units (n ϭ 40). The transient acidification was superseded by a secondary alkalinization, which exceeded the base-line pH i by an average of 0.21 Ϯ 0.01 (n ϭ 38). As shown in Fig. 1B, the initial acidifi-cation was absent when the cells were stimulated in nominally HCO 3 Ϫ -free medium (solution A). Instead, the cells immediately became alkaline, reaching a final pH i of 7.48 Ϯ 0.08 (n ϭ 66). The occurrence of a transient acidification in the presence, but not in the absence of HCO 3 Ϫ , is consistent with the notion that this pH change reflects loss of intracellular HCO 3 Ϫ through Ca 2ϩ -activated apical anion channels (22,23).

Effect of Carbachol on pH i and on Na ϩ /H ϩ Exchange
The alkalinization that follows the transient acidosis in solution A (Fig. 1A), as well as that elicited immediately by carbachol in solution B (Fig. 1B) are attributable to activation of Na ϩ /H ϩ exchange. This view is supported by the following observations. First, omission of Na ϩ following stimulation with carbachol induced a pronounced cytosolic acidification (Fig.  1B), suggestive of accumulation of metabolic acid and/or reversal of the antiport. This acidification was rapidly reversed upon reintroduction of Na ϩ (Fig. 1B). Second, the alkalinization induced by the muscarinic agonist was not observed in the presence of DMA, a relatively specific inhibitor of Na ϩ /H ϩ exchange (data not shown). Third, the net extrusion of H ϩ (equivalents) after exposure to carbachol was accompanied by Na ϩ influx, readily detectable as an increase in [Na ϩ ] in cells treated with ouabain to preclude extrusion by the Na ϩ /K ϩ pump. As shown in Fig. 1C, where intracellular [Na ϩ ] was measured using SBFI, addition of the glycoside alone increased [Na ϩ ] at a marginal rate prior to addition of carbachol, and DMA had little effect. Upon muscarinic stimulation, however, intracellular [Na ϩ ] increased drastically, at a rate of approximately 40 mM/min. Importantly, the influx was greatly reduced in the presence of DMA, implying that at least 75% of the Na ϩ enters the cell via the antiporter in HCO 3 Ϫ -containing medium (Ϸ55% in nominally HCO 3 Ϫ -free solution A). Assuming a 1:1 stoichiometry, the amount of Na ϩ that enters the cell through the DMA-sensitive pathway (22 mM/min) suffices to account for the net H ϩ extrusion induced by carbachol (24 mM/min), calculated from the rate of change of pH i and considering the buffering power of the cytosol (approximately 8 mM/pH in the pH 7.3-7.5 range). Together, these results indicate that Na ϩ /H ϩ exchange is stimulated markedly by treatment of acinar cells with carbachol. Of note, the failure of ouabain-treated cells to gain Na ϩ prior to muscarinic activation (in the presence and absence of DMA) implies that the antiporter is virtually quiescent in resting (unstimulated) cells.
Effect of Carbachol on Na ϩ /H ϩ Exchange pH i Dependence-The preceding results suggest that treatment with carbachol converts the antiporter from a quiescent to an active mode. Further insight into this transition was gained by analyzing the properties of Na ϩ /H ϩ exchange in resting and stimulated cells. Because the antiporter is not detectable in untreated cells at normal pH, its activity was unmasked by acid-loading the cytosol, using an NH 4 ϩ pre-pulse. A representative experiment is shown in Fig. 2A. Acinar cells were pulsed with the weak base, which was then removed while simultaneously replacing Na ϩ with N-methyl-D-glucammonium ϩ (solution D). Under these conditions, the cells underwent rapid acidification and failed to recover within the period studied, due to the absence of Na ϩ . Upon readdition of Na ϩ , however, a rapid alkalinization ensued. In otherwise untreated cells, pH i recovered to near the original basal level. By contrast, if the cells were treated with carbachol prior to Na ϩ readdition (open circles in Fig. 2A), the recovery surpassed the resting pH i level, resulting in a net cytosolic alkalinization, reminiscent of that recorded in Fig. 1. Calculation of the rates of Na ϩ -induced H ϩ (equivalent) extrusion in cells with or without muscarinic stimulation are summarized in Fig. 2B. Two features are noteworthy: first, that following acid loading the rates of recovery are very large (upwards of 100 mM/min), comparing very favorably with other cell types where absolute antiport rates have been reported (e.g. Ref. 24). This likely reflects the specialized function of these secretory cells. Second, it is apparent that the pH i sensitivity of the antiporter is increased following exposure to carbachol. Although the rates of both control and carbacholtreated cells were similar at more acidic pH, exchange is clearly noticeable in the stimulated cells at H ϩ concentrations where the basal antiporter is essentially inactive (i.e. between 7.3 and 7.45). Similar shifts in the activation threshold or "set point" of the antiport have been reported in other systems (see Ref. 25 for review). That the antiport is truly quiescent in unstimulated cells is suggested by several observations: (i) the absence of a DMA-sensitive Na ϩ gain in cells treated with ouabain (Fig.  1C); (ii) the failure of DMA and other amiloride analogs to alter baseline pH i (not shown), and (iii) the absence of a cytosolic acidification upon removal of external Na ϩ in unstimulated cells, a finding that contrasts sharply with the large pH i drop noted when Na ϩ is removed after stimulation (Fig. 1B).
Isoforms of NHE in Acinar Cells-To better understand the mechanism underlying NHE activation by muscarinic agonists, it was important to establish which isoform(s) of the antiport operate in acinar cells. To this end, we extracted mRNA from parotid glands and assessed the expression of the four well known isoforms of the antiporter (NHE-1 to 4) by RT-PCR (Fig.  3). Isoform-specific primers which hybridized to unique regions of the rat NHE-1, NHE-2, NHE-3, and NHE-4 were used. All four sets of primers yielded the expected PCR products when linearized pCMV plasmids containing the full-length cDNA clone of the corresponding isoform were used as template (Fig.  3, lanes 1, 4, 7, and 10). No discernible products were detected when a specific primer set was used with any of the noncorresponding isoforms as template (not shown). When cDNA obtained by reverse transcription of rat parotid mRNA was used as a template, the NHE-1 primers yielded a product of Ϸ500 bp (Fig. 3, lane 2), while a smaller yield of the expected product (Ϸ700 bp) was also observed for NHE-2. The NHE-3 and NHE-4 primers did not yield discernible products in repeated trials (e.g. Fig. 3, lanes 8 and 11). Omission of reverse transcriptase prevented appearance of the 500-and 700-bp products, ruling out contamination with genomic DNA. Thus, the predominant isoforms expressed in parotid glands are NHE-1 and NHE-2, with no detectable NHE-3 and NHE-4.
The presence of NHE-1 was further documented immunochemically. Acinar cell membranes were probed with an antibody raised against the C-terminal 157 amino acids of NHE-1. The specificity of the antibody was first ascertained comparing Chinese hamster ovary cells transfected with NHE-1 with their untransfected, antiport-deficient counterparts (Fig. 4). The an- ϩ (solution C) and then perfused with Na ϩ -free medium (solution D) with (open circles) or without (solid circles) carbachol. pH i recovery was next induced by reintroducing extracellular Na ϩ . Traces are data from single cells representative of at least cells 30 cells from six different animals. B, pH i dependence of the Na ϩ -induced net H ϩ (equivalent) flux (J H ϩ). This was calculated as the sum of the Na ϩ -dependent rate of pH i recovery and the rate of acid loading observed when Na ϩ was removed, multiplied by the buffering power, which was determined independently throughout the pH range of interest as described under "Experimental Procedures." No spontaneous acid loading was detected in control cells when Na ϩ was removed (n ϭ 25). Straight lines were fitted by least squares using Cricket Graph 3.1.2. and data were analyzed using Statworks. Control cells: closed circles (fitted by the equation y ϭ 1072-146ϫ; r ϭ 0.76, p Ͻ 0.001, n ϭ 36). Carbachol-stimulated cells: open circles (fitted by y ϭ 2125-283x; r ϭ 0.88, p Ͻ 0.001, n ϭ 28). tibody recognized a major band of 110 -115 kDa, the expected size of mature NHE-1, in the transfectants but not in the deficient precursor cells. A smaller and sharper band also present in the transfectants but missing in the controls is in all likelihood the incompletely (core) glycosylated form of NHE-1, a biosynthetic precursor. A third polypeptide, present in both samples, is likely nonspecific. As shown in the leftmost lanes of Fig. 4, one major and one minor polypeptide were also recognized by the antibody in acinar cell membranes. Both polypeptides remained associated with the membranes following alkaline extraction of extrinsic components, suggesting that they are transmembrane proteins. The predominant immunoreactive band of acinar cells likely represents the mature form of NHE-1, which is known to be heterogenously glycosylated (12), accounting for its diffuse mobility on SDS-PAGE. The smaller, sharper band may be the core-glycosylated biosynthetic precursor.
Parotid glands are composed of acini and ducts. Because these were not separated for preparation of RNA or for membrane isolation, it cannot be definitively stated that NHE-1 is present in the acinar cells. To verify this point, the distribution of NHE-1 in parotid slices was assessed immunocytochemically, using the polyclonal antibody described above. Representative confocal fluorescence images are shown in Fig. 5. The low power image of panel A demonstrates that NHE-1 is present in both ductal and acinar cells. In both instances, the staining is observed predominantly on the basolateral membrane, which can be more clearly discerned in panels B and C. Plasmalemmal immunoreactivity was also evident in isolated acinar cells (Fig. 5D). Such staining likely reflects the presence of NHE-1 on the basolateral membrane, inasmuch as this occupies by far the largest fraction of surface area in acinar cells (1). Whether NHE-1 is present also in the apical membranes cannot be defined unambiguously, but in several instances staining appeared to be minimal in the region of the membrane facing the lumen of the acinus (e.g. arrow in Fig. 5B), suggesting that the apical membranes are largely devoid of NHE-1. Discontinuities in the staining of the luminal membrane are also apparent in ductal cells, suggesting preferential distribution of NHE-1 on the basolateral side.
The preceding results indicate that NHE-1 is present in acinar cells, but do not clarify the contribution of this isoform to the Na ϩ /H ϩ exchange activity across the basolateral mem-brane. The fraction of the exchange mediated by NHE-1 was assessed pharmacologically in isolated acinar cells using HOE694. This compound inhibits NHE-1, NHE-2, and NHE-3 at widely differing concentrations (26), thus providing a means of discerning between the isoforms. As shown in Fig. 6A, the antiport activity of acinar cells, measured as the Na ϩ -dependent recovery of pH i from an acid load, could be effectively inhibited by low doses of HOE694. The concentration required for half-maximal inhibition was Ϸ0.06 M (Fig. 6B), similar to that reported to inhibit NHE-1 (26), and much lower than that needed to inhibit either NHE-2 or NHE-3 (K 0.5 of 5 and 650 M, respectively). Together, the biochemical and functional findings indicate that NHE-1 is the primary Na ϩ /H ϩ antiporter of rat acinar cells. Of note, low doses of HOE694 inhibited the pH i recovery effectively both before and after treatment with carbachol. In carbachol-stimulated cells the recovery was inhibited by Ͼ95% by 3 M HOE694, from 4.6 Ϯ 0.53 to 0.2 Ϯ 0.02 pH/min (n ϭ 10; measured at pH i 6.8). These data imply that NHE-1 is the isoform mediating muscarinic activation of the antiport.

Mechanism of Activation of Na ϩ /H ϩ Exchange
Having established that NHE-1 is the isoform activated by muscarinic agonists in acinar cells, we proceeded to explore the mechanism(s) underlying this form of regulation. Phosphorylation of serine residues within the cytoplasmic (C-terminal) domain has been postulated to mediate receptor-mediated activation of NHE-1 in cultured cells and in platelets (see Refs. 12 and 25, for reviews). Because muscarinic stimulation triggers protein kinase activity in parotid cells, we compared the phosphorylation of NHE-1 before and after challenge with carbachol (Fig. 7A). Freshly isolated cells were labeled with [ 32 P]orthophosphate for 2 h and, after washing, they were incubated for 2 min with or without 10 M carbachol at 37°C. Finally, the cells were solubilized and NHE-1 was immunoprecipitated and analyzed by SDS-PAGE and radiography. Immunoblotting with anti-NHE-1 antibody was used to ensure that comparable amounts of the protein were precipitated from control and treated cells. 2 One of four similar experiments is FIG. 5. Localization of NHE-1 in parotid glands. Frozen sections obtained from parotid glands (A-C) and isolated acinar cells (D) were fixed, permeabilized, and stained with polyclonal anti-NHE-1 antibody, followed by fluorescently labeled secondary (donkey anti-rabbit) antibody, as detailed under "Experimental Procedures." Samples were analyzed using a Bio-Rad 600 laser scanning confocal imaging system mounted on a Leitz Metallux-3 microscope using ϫ 63 (1.3 NA) and ϫ 100 (1.32 NA) oil-immersion objectives (Leitz). The arrow in B points to the apical membrane. illustrated in Fig. 7 (top panel). As reported for other cell types (11,12,21) NHE-1 was found to be phosphorylated in resting acinar cells. The absence of radiolabel in samples treated with preimmune serum confirmed the specificity of the immunoprecipitation protocol (not shown). Importantly, the extent of phosphorylation was indistinguishable before and after muscarinic stimulation. Densitometric integration of radiograms from four independent experiments indicated that phosphate incorporation in carbachol-treated cells was 102 Ϯ 19% (mean Ϯ S.E.) of the control level (Fig. 7, botton panel). Multiple exposures were performed to ensure that differences in intensity were not obscured by film saturation. Moreover, direct quantification by PhosphorImaging similarly showed no significant difference between untreated and stimulated cells.
The above experiments indicate that net phosphorylation of NHE-1 does not change when cells are acutely stimulated by muscarinic agonists. While the overall phosphate content of NHE-1 appears to remain constant, it is conceivable that phosphorylation of one site occurs and is accompanied by dephosphorylation of a different site. Alternatively, multiple phosphorylation sites may exist in the resting state. In this case phosphorylation or dephosphorylation of a single residue may affect the total 32 P content only moderately and could go undetected when the total radioactivity is compared. To test these possibilities, we carried out phosphopeptide mapping of radio-labeled immunoprecipitates from untreated and stimulated cells. The precipitates were eluted from the beads and denatured with SDS, then hydrolyzed using chymotrypsin. The protease yielded 4 phosphopeptides of molecular mass between 4 and 6 kDa. This pattern was essentially identical whether proteolysis was carried out for 1 or 5 min, suggesting that the reaction had reached completion. Importantly, the phosphopeptide composition and relative intensity of the bands were identical in control and carbachol-treated samples (Fig.  8). These findings indicate that dephosphorylation of one site with concomitant phosphorylation of another is unlikely. In addition, no evidence was found for preferential dephosphorylation of any one of the phosphopeptides resolved by chymotryptic cleavage of NHE-1.

DISCUSSION
Primary fluid secretion by rat salivary glands is driven osmotically by transepithelial salt gradients. As in most secretory epithelia, these gradients are generated by Na ϩ -coupled anion uptake across the basolateral membrane. In salivary glands, such secondary active uptake of anions is mediated not only by Na ϩ -K ϩ -2Cl Ϫ cotransport, but also by Cl Ϫ /HCO 3 Ϫ exchange coupled to Na ϩ /H ϩ exchange via the intracellular pH. By alkalinizing the cytosol, NHE is important also in driving HCO 3 Ϫ secretion and in guarding the cells against metabolic acidosis. Despite recognition of the important role of the antiporter in fluid secretion in salivary glands, the identity of the isoform activated by muscarinic stimulation and the molecular details of this activation had not been elucidated.
The present studies provide evidence that NHE-1 is the predominant isoform in the parotid gland and that it is prefer- entially localized to the basolateral membrane of acinar cells. This site is in agreement with the reported location of NHE-1 in other epithelia and is consistent with a role for the antiporter in promoting secondary active, pH i -coupled Cl Ϫ entry into the cell. That this isoform is the major contributor to Na ϩ uptake in carbachol-stimulated cells was shown using HOE694. At low micromolar or submicromolar concentrations, this benzoylguanidine derivative greatly inhibits NHE-1, while leaving the other isoforms unaffected. In our studies, low doses of HOE694 markedly inhibited Na ϩ -induced H ϩ extrusion, implicating NHE-1 in the process.
Transcripts encoding NHE-2 have been detected in kidney medulla and cortex, jejunum, ileum, duodenum, stomach, and adrenal gland (11,14,27,28), although the subcellular distribution of this isoform remains controversial. NHE-2 was also detected in the parotid, using RT-PCR. Its location and regulation were not pursued here partly because of unavailability of effective antibodies, but mainly because it seems to contribute little to the muscarinic response of the acinar cells, as judged by the effects of HOE694. NHE-2 may be located on the apical surface of the ductal cells, which failed to stain for NHE-1.
Activation of parotid cells by carbachol resulted in an apparent alkaline shift of the pH dependence of NHE, suggesting increased affinity for intracellular H ϩ . A similar mode of activation was reported earlier in submandibular glands and in a variety of systems where NHE-1 mediates transport. In contrast, there is evidence that activation of NHE-2 entails primarily an increase in maximal velocity, at constant affinity (11). Albeit indirect, this evidence further supports the notion that NHE-1 is the isoform activated in acinar basolateral membranes.
Despite earlier controversy, it is now generally accepted that NHE-1 can be activated by elevation of intracellular [Ca 2ϩ ]. In the parotid acinar cells, increased [Ca 2ϩ ] i was sufficient to mimic the stimulation effected by muscarinic agonists: treatment with thapsigargin induced a Na ϩ -dependent cytosolic alkalinization (not illustrated). Interestingly, the apical isoforms of epithelia (NHE-2 and/or NHE-3) have been suggested to become inhibited when [Ca 2ϩ ] increases (29,30). This would further argue for a predominant role of NHE-1 in parotid cells.
NHE-1 has been shown to be constitutively phosphorylated in other cells and additional phosphate groups are acquired upon stimulation by growth factors, phorbol esters, or okadaic acid (31,32). It has therefore been postulated that phosphorylation mediates at least part of the biological responses of NHE-1. It was conceivable that the muscarinic activation, and that elicited by thapsigargin, were similarly mediated by activation of Ca 2ϩ -dependent kinases and/or inhibition of phosphatases. However, no change in phosphorylation of NHE-1 was detectable when the cells were stimulated. This observa-tion is not without precedent, since the osmotic activation of the antiporter had been demonstrated earlier to occur in the absence of phosphorylation (21) and ionomycin-induced elevation of [Ca 2ϩ ] similarly activated the exchanger without altering the phosphorylation state of NHE-1 (33,34).
The simplest hypothesis available to explain our observations is that muscarinic stimulation of the antiporter results, at least in part, from the elevation of [Ca 2ϩ ] i and formation of Ca 2ϩ -calmodulin complexes that bind and activate the antiporter directly. Wakabayashi and colleagues (33,34) reported the existence of two calmodulin-binding domains in NHE-1, which upon binding Ca 2ϩ -calmodulin are believed to induce a conformational change in the protein that displaces an autoinhibitory domain, thereby stimulating the exchanger. Indeed, deletion of the putative autoinhibitory segment resulted in constitutively activated exchangers. We attempted to demonstrate the applicability of this model to acinar cells. At the concentrations required to block calmodulin, compound W7 completely inhibited the alkalinization of carbachol-stimulated cells. However, recovery of unstimulated cells from an acid load was also impaired. This may reflect a constitutive effect of calmodulin on NHE-1 of unstimulated acinar cells, and/or a direct (nonspecific?) effect of W7 on the antiport, which complicates the assessment of the role of calmodulin. Another calmodulin inhibitor, calmidazolium, induced a variable acidification of the cells, particularly after stimulation with carbachol. The confounding nature of this acidification again precluded evaluation of the role of calmodulin in muscarinic stimulation. Other approaches to evaluate the role of calmodulin are currently being considered.
In summary, carbachol stimulation of salivary acinar cells results in marked activation of NHE-1, which resides predominantly, if not exclusively, in the basolateral membrane. Such activation is independent of phosphorylation and can be at least partially mimicked by simply raising the concentration of [Ca 2ϩ ] i to levels similar to those observed upon muscarinic stimulation. Although direct proof is as yet unavailable, we speculate that interaction of the antiporter with Ca 2ϩ -calmodulin is largely responsible for the stimulation. FIG. 8. Phosphopeptide composition of NHE-1 isolated from control and carbachol-treated cells. Untreated (Con) and carbachol-stimulated (Car) cells were extracted and immunoprecipitated with NHE-1 antibodies. The radiolabeled immunoprecipitates were subjected to proteolytic degradation by chymotrypsin, as described under "Experimental Procedures." The resulting phosphopeptides were resolved by SDS-PAGE using 15% acrylamide. The position of major phosphopeptides is indicated by arrows. The location of molecular mass markers is also noted. Representative of four similar experiments.