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Different rate-limiting activities of intracellular pH regulators for HCO3 secretion stimulated by forskolin and carbachol in rat parotid intralobular ducts

Abstract

Intracellular pH (pHi) regulation fundamentally participates in maintaining HCO3 release from HCO3 -secreting epithelia. We used parotid intralobular ducts loaded with BCECF to investigate the contributions of a carbonic anhydrase (CA), anion channels and a Na+–H+ exchanger (NHE) to pHi regulation for HCO3 secretion by cAMP and Ca2+ signals. Resting pHi was dispersed between 7.4 and 7.9. Forskolin consistently decreased pHi showing the dominance of pHi-lowering activities, but carbachol gathered pHi around 7.6. CA inhibition suppressed the forskolin-induced decrease in pHi, while it allowed carbachol to consistently increase pHi by revealing that carbachol prominently activated NHE via Ca2+-calmodulin. Under NHE inhibition, forskolin and carbachol induced the remarkable decreases in pHi, which were slowed predominantly by CA inhibition and by CA or anion channel inhibition, respectively. Our results suggest that forskolin and carbachol primarily activate the pHi-lowering CA and pHi-raising NHE, respectively, to regulate pHi for HCO3 secretion.

Introduction

Salivary glands are formed by acinar and ductal parts. Acini secrete the plasma-like, isotonic fluid “primary saliva”, whereas ducts modify the saliva components by secretion of HCO3 and K+ as well as reabsorption of Na+ and Cl [13]. The volume of saliva induced by the Ca2+ signal is much larger than that by the cAMP signal in vivo and ex vivo [4, 5]. Most of the salivary fluid is secreted by acinar parts. However, it is suggested that both signals also induce fluid secretion from ductal parts, which accompanies HCO3 release [6, 7]. Secretion of the appropriate concentration of HCO3 is an important function of the ducts to keep oral health by means of the pH buffering effect of HCO3 [2, 3]. Several models were proposed for mechanisms of HCO3 secretion from salivary ducts, corresponding to divergence among animal species, types of salivary glands, and parts of the salivary ducts [2, 6, 810]. The relative amount of secreted HCO3 also depends on the divergence. A parasympathomimetic Ca2+-mobilizing agonist (carbachol; CCh) and a sympathomimetic cAMP-increasing agonist (isoproterenol) stimulate HCO3 secretion in the main excretory duct of rat submandibular glands [11]. In rat parotid glands, there is no report about HCO3 secretion in the main excretory duct and expression levels of factors involved in HCO3 secretion in each part of the duct is unknown. However, HCO3 secretion evoked by forskolin, a cAMP-increasing agent, and CCh in the rat parotid intralobular ducts is clearly demonstrated in the cell and tissue levels [6, 7, 12]. The intracellular pH (pHi) regulation plays an important role in keeping HCO3 secretion. Therefore, in this paper, we focused on the mechanism of pHi regulation during HCO3 secretion in the rat parotid intralobular ducts. In a proposed model for the rat parotid intralobular duct, intracellular HCO3 is generated by the cooperative activities of carbonic anhydrases (CA) and Na+–H+ exchangers (NHE) [6, 12]. HCO3 is released through Cl channels in the model. One of the channels is a CFTR Cl channel mainly dependent on the cAMP signal [7, 12], as reported in the submandibular ducts [13, 14]. The other is a diphenylamine-2-carboxylate (DPC)-sensitive Cl channel apparently activated by the Ca2+ signal [6, 15] and this channel has not yet been identified. The amplitude of inward currents evoked by the cAMP signal is around 75 pA at the membrane potential of −80 mV in the single cells of rat parotid intralobular ducts [12]. Removal of external HCO3 and CO2 reduces these currents, indicating that the currents reflect HCO3 secretion [12]. The Ca2+ signal also elicits the HCO3 - and CO2-dependent currents with the amplitude similar to those induced by the cAMP signal (unpublished data). The rate of HCO3 secretion is determined by the activities of the HCO3 -releasing Cl channels, NHE and CA, which appear to cooperatively maintain and regulate pHi. However, the relative contribution of those activities to the pHi regulation and HCO3 secretion induced by cAMP and Ca2+ signals remains elusive.

Among the NHE isoforms expressed in salivary glands, NHE1 located in the basolateral membrane mainly regulates the pHi in mouse parotid and sublingual acinar cells [16, 17] and in rat parotid acinar cells [18]. In rat parotid ductal cells, the basolaterally-located NHE1 and apically-located NHE3 are concerned with regulation of pHi [18]. Nevertheless, NHE3 located in the apical membrane of mouse parotid ductal cells does not play a major role in Na+ absorption [19]. Moreover, physiological function of NHE3 that is sensitive to cAMP-mediated inhibition [20] remains unclear in rat submandibular gland ducts [21]. It is very likely that NHE1 is the functionally relevant NHE isoform in parotid ductal cells.

There are many reports about pHi regulation by NHE and the mechanisms of NHE activation in various organs. Osmotic cell shrinkage causes an alkaline shift in the pHi set point of NHE in resident alveolar macrophages [22]. NHE1 is active during metabolic inhibition in rabbit ventricular myocytes if pHi is driven more acidic than NHE1 set-point pHi [23]. Ca2+-calmodulin activates NHE1, and the calmodulin-binding autoinhibitory domain controls “pH-sensing” in NHE1 expressed in the exchanger-deficient cell PS120 [24, 25]. On the other hand, MAP kinase-dependent phosphorylation is involved in the regulation of NHE in the rat myocardium [26]. In parotid acinar cells, CCh induces phosphorylation-independent activation of NHE1 [27]. There may exist some processes, such as association of Ca2+-calmodulin complexes to the cytosolic domain of NHE1 during CCh stimulation [27], as reported for the cultured cells. However, the detailed mechanism of NHE activation in salivary glands, especially in ductal cells, is still unknown.

Carbonic anhydrase isozymes I, II and VI (CAI, CAII and CAVI) are localized in rat parotid glands [28, 29], whereas CAVI is secreted into saliva [29]. It is reported that an increase in the intracellular cAMP concentration activates CAIX through phosphorylation at the intracellular domain by the protein kinase A in hypoxia in cultured cell lines [30]. However, the activation mechanism of the CA isozymes expressed in salivary glands during the cAMP signaling has not yet been reported.

In this paper, we characterized the regulatory mechanisms of pHi in rat parotid intralobular ductal cells stimulated by forskolin and CCh, and investigated which factor, the Cl channel, NHE or CA, has the rate-limiting activity for pHi regulation and HCO3 secretion induced by each of cAMP and Ca2+ signals. Although there may be a cross-talk between cAMP and Ca2+ signals, the results obtained suggest that forskolin-induced cAMP signals and CCh-induced Ca2+ signals have different primary ways of pHi regulation controlling mainly CA and NHE activities, respectively, during HCO3 secretion.

Materials and methods

Preparation of intralobular duct segments from rat parotid glands

Male Wistar rats were purchased from Charles River Laboratories Japan (Yokohama, Japan). The animal use protocol was approved by the Committee of Animal Experimentation, Hiroshima University. Parotid glands were removed from the rats (250–400 g) anesthetized with sodium pentobarbital (somnopentyl, 70–100 mg/kg, i.p.). The minced glands were digested for 45 min at 37 °C with collagenase S-1 (1 mg/ml) dissolved in a modified Krebs–Henseleit Ringer solution (KHR: 103 mM NaCl, 4.7 mM KCl, 2.56 mM CaCl2, 1.13 mM MgCl2, 25 mM NaHCO3, 1.15 mM NaH2PO4, 2.8 mM glucose, 4.9 mM Na-pyruvate, 2.7 mM Na2-fumarate, 4.9 mM Na-glutamate, 12.5 mM HEPES, pH 7.4). KHR was gassed with 95 % O2 + 5 % CO2 before each experiment. The digestives were dispersed by pipetting to separate intralobular duct segments from acini and washed with KHR twice.

Chemicals

Carbamylcholine chloride (carbachol, CCh), EDTA and trypsin were obtained from Nacalai Tesque (Kyoto, Japan), and collagenase (type S-1) and sodium pentobarbital (somnopentyl) were from Nitta Gelatin (Osaka, Japan) and Kyoritsu Seiyaku (Tokyo, Japan), respectively. Diphenylamine-2-carboxylate (DPC) and forskolin were purchased from Wako Pure Chemical Industries (Osaka, Japan). Bovine serum albumin fraction V (BSA), 5(N,N)-dimethyl amiloride (DMA), gramicidin d, 3-isobutyl-methylxantine (IBMX), methazolamide, nigericin, and poly-l-lysin were obtained from Sigma-Aldrich (St. Louis, MO, USA). 1,2-Bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid, tetraacetoxymethyl ester (BAPTA-AM) and 3′-o-acetyl-2′,7′-bis(carboxyethyl)-4 or 5-carboxyfluorescein, diacetoxymethyl ester (BCECF-AM) were obtained from Dojindo Laboratories (Kumamoto, Japan). CFTR inhibitor 172 and W-7 hydrochloride were purchased from Calbiochem (EMD Biosciences, La Jolla, CA, USA, and EMD Chemicals, San Diego, CA, USA).

Measurement of intracellular pH in the duct segments

The duct segments were incubated for 15 min at 37 °C in KHR containing the pH-sensitive fluorescence dye BCECF-AM (0.3 μM) and 0.1 % BSA. In the experiments for intracellular Ca2+ chelation, 50 μM BAPTA-AM was added to the incubation medium. Then, the duct segments were washed with KHR and allowed to adhere on cover slips coated with poly-l-lysin. The ducts placed in the chamber were continuously perfused with bathing solutions, basically KHR, gassed with 95 % O2 + 5 % CO2. The intracellular pH (pHi) measurement was performed by using the image analysis equipment ARGUS-HiSCA system (Hamamatsu Photonics, Hamamatsu, Japan) with the inverted microscope TE300 (Nikon, Tokyo, Japan) at room temperature (23–26 °C). The ratio of fluorescence excited at 490 nm to that at 450 nm (F490/F450) was calculated at 10-s intervals. pHi calibration was performed based on the linear relationship between F490/F450 and pHi in the ducts perfused with pH standard solutions (5 mM NaCl, 55 mM KCl, 85 mM K-gluconate, 0.8 mM MgSO4, 10 mM HEPES, and 5 μM nigericin, pH 6.8, 7.2 or 7.6). pHi at the 10–30 regions with the size similar to a cell in each duct was averaged and then expressed as a mean ± SE at each measurement time. These data were used for presenting the time course of pHi change. For statistical analyses, the average pHi was determined in the final 1 min before the addition of the agents for the resting state and from 4 to 5 min after the agent addition (or from 9 to 10 min after the DMA addition), and then expressed as mean ± SE of at least 5 experiments. The statistical difference was regarded as significant when P < 0.05 by using paired or unpaired Student’s t test.

Ionic current measurements in the ductal cells

Dispersed parotid glands, obtained by the collagenase treatment and pipetting, were poured into a 90-mm dish to allow the duct segments to adhere tightly to the dish. Acini in the dish were roughly washed out with KHR. The ducts still attaching on the dish were taken off by pipetting in 0.05 % trypsin + 0.016 % EDTA dissolved in phosphate-buffered saline, immediately collected and washed with KHR. The ducts obtained were digested again with 0.16 % collagenase (type S-1) at 37 °C for 25 min and dispersed by pipetting. The ductal cells were washed with KHR, placed on poly-l-lysine-coated cover slips and used for patch clamp experiments.

The gramicidin-perforated patch techniques were used for measuring the ionic currents from the ductal cells with a patch/whole-cell clamp amplifier CEZ-2400 (Nihon Kohden, Tokyo, Japan). Patch pipettes were pulled from borosilicate glass capillaries. The pipette solution containing 150 mM KCl, 10 mM HEPES and 0.1 mg/ml gramicidin d was adjusted to pH 7.4 using KOH. The bathing solution (KHR solution) was constantly perfused in a chamber. KHR was gassed with 5 % CO2 + 95 % O2. To eliminate K+ currents, the membrane potential was held at −80 mV, which was approximately equal to the equilibrium potential of K+. All measurements were performed at room temperature (23–27 °C). Other details of the gramicidin-perforated patch techniques were similar to those in the previous papers [12, 31].

Results

Forskolin + IBMX-induced pHi changes and effects of methazolamide and CFTR inhibitor 172 on the responses

We previously reported that forskolin + IBMX (F+I) induces HCO3 generation by the carbonic anhydrase (CA) and Na+–H+ exchanger (NHE) system, and HCO3 release through the CFTR Cl channel in rat parotid intralobular ductal cells [7, 12]. To sustain the HCO3 secretion via Cl channels, the intracellular HCO3 concentration above its electrochemical equilibrium value are maintained by extrusion of H+ by NHE, and generation of H+ and HCO3 by CA. Thus, the pHi are determined by the pHi-raising NHE activity and the pHi-lowering activities of CA and Cl channels. In the present study, we examined which factors were dominantly involved in the pHi regulation during HCO3 secretion. The resting pHi level in 9 ducts tested was distributed between 7.44 and 7.74 (7.62 ± 0.05, n = 9). Although variability in the resting pHi was observed, application of F+I decreased pHi from the resting level in all the ducts tested (Fig. 1a). F+I-induced pHi changes (ΔpH) were correlated with the resting pHi, showing the tendency toward gradually increasing the pHi changes as the resting pHi became higher (Fig. 1b). There was a statistically marked difference between pHi levels in the resting state and during the F+I stimulation (Fig. 1c; P < 0.001 in the paired Student’s t test). The F+I-induced decrease in pHi was significantly suppressed by pre-addition of the CA inhibitor, methazolamide (Fig. 1d, e), comparing the data shown in Fig. 1e (Met + F+I; pHi 7.56 ± 0.03, n = 10) with those in Fig. 1c (F+I; pHi 7.42 ± 0.02, n = 9) (P < 0.001 in the unpaired Student’s t test). This indicates that H+ production by some isozymes of CA contributes to the F+I-induced decrease in pHi. F+I induces HCO3 release via CFTR Cl channels [12], which would be expected to reduce the pHi level. However, the F+I-induced pHi change was not affected by the presence of CFTR inhibitor 172, a potent inhibitor of the CFTR Cl channel [32] (Fig. 1f, g; pHi 7.45 ± 0.06, n = 5). We confirmed the effectiveness of CFTR inhibitor 172 in the ductal cells by measuring F+I-induced inward currents. The inhibitor almost completely blocked the currents (Fig. 1h). These results suggest that the F+I-induced decrease in pHi may be due to the CA activity, which is functionally dominant in pHi regulation, rather than the CFTR Cl channel, and that the pHi-lowering CA activity may dominate over the pHi-raising NHE during F+I stimulation.

Fig. 1
figure1

Forskolin + IBMX-induced decrease in the pHi. a Time courses of the pHi changes in rat parotid intralobular ducts by the addition of 10 μM forskolin + 100 μM IBMX (F+I). Each point indicates the mean ± SE of pHi in 19–28 regions of the ducts. The pHi level was reduced by the addition of F+I in all the ducts tested. b Relationships between the F+I-induced pHi changes averaged during 4–5 min after F+I addition (ΔpH) and the resting pHi levels averaged for 1 min just before F+I addition. Each point was obtained from the data in a (n = 9). c The pHi averaged for 1 min just before F+I addition (control) and 4–5 min after F+I application (F+I). Data shown are mean ± SE calculated from the traces in a (n = 9). d A time course of pHi change in the duct by F+I addition of under the inhibition of carbonic anhydrases by 1 mM methazolamide (Met). Each point indicates the mean ± SE of pHi in 20 regions of the duct. e The effect of 1 mM methazolamide on the F+I-induced pHi change. The pHi averages for 1 min just before (control, pHi; 7.57–7.83) and 4–5 min after (Met) the addition of methazolamide, and 4–5 min after the subsequent addition of F+I (Met + F+I) are denoted. Data shown are mean ± SE (n = 10). f A time course of pHi change in the duct by F+I addition under the blocking of CFTR Cl channels by 10 μM CFTR inhibitor 172 (CFTR172). Each point indicates the mean ± SE of pHi in 17 regions of the duct. g The pHi averages for 1 min just before (control, pHi; 7.39–7.91) and 4–5 min after the addition of the CFTR inhibitor (CFTR172), and 4–5 min after the subsequent addition of F+I (CFTR172 + F+I) are shown. Data shown are mean ± SE (n = 5). h Addition of 10 μM CFTR inhibitor 172 (CFTR172) almost completely blocked the inward current induced by 10 μM forskolin + 100 μM IBMX (F+I). The current was measured by using the gramicidin-perforated patch techniques in a single ductal cell. Small and brief voltage pulses (5 mV, 0.2 s) were superimposed on the holding potential (−80 mV) in every 10 s. The broken line indicates the zero level of the current. Similar results were obtained in the other 4 experiments

F+I-induced pHi changes under the inhibition of NHE

To further characterize the contributions of NHE, CA, and CFTR Cl channels to pHi regulation during F+I stimulation, we deduced and analyzed the pHi changes mediated by the CA and CFTR Cl channel activities under NHE inhibition by 5(N,N)-dimethyl amiloride (DMA). Application of DMA alone significantly decreased the pHi (Fig. 2a, b), suggesting that NHE may be activated in the resting state. Addition of F+I in the presence of DMA induced a more remarkable decrease in pHi (Fig. 2a, b; ΔpHi 0.61) than did F+I alone (Fig. 1a, c; ΔpHi 0.20). Application of DMA that inhibits the pHi-raising NHE activity allows the pHi-lowering activities to be profoundly unveiled. The difference in the F+I-induced ΔpHi between the presence and absence of DMA implies that NHE is further activated during F+I stimulation, compared with that in the resting state. However, the pHi-raising NHE might be passively activated following the F+I-induced activation of the pHi-lowering CA and CFTR Cl channels, since F+I alone decreased the pHi, and the pHi value during F+I stimulation in the presence of methazolamide was not greater than that in the initial resting level. In the presence of CFTR inhibitor 172 or methazolamide with DMA, F+I induced a decrease in pHi to the same level as that during F+I stimulation with DMA alone [Fig. 2a, b (DMA + F+I); pHi 6.97 ± 0.05 (n = 6); Fig. 2c, d (DMA + CFTR172 + F+I); pHi 7.02 ± 0.05 (n = 7); and Fig. 2e, f (DMA + Met + F+I); pHi 7.00 ± 0.04 (n = 6)]. Judging from the F+I-induced ΔpHi, both CFTR inhibitor 172 and methazolamide seem to inhibit the F+I-induced ΔpHi in the presence of DMA in appearance. However, no difference among those three pHi levels reached during F+I stimulation could be due to the presence of the lowest limit of pHi in those conditions. Therefore, in the case in which the pHi values just before the application of F+I are varied because of any effects of pre-added inhibitors, it is considered that the values of F+I-induced ΔpHi are not suitable to evaluate and compare the effects of inhibitors on F+I-induced pHi regulation. The pHi level in the presence of DMA and methazolamide (Fig. 2f; pHi 7.31 ± 0.05, n = 6) was lower than that in the presence of DMA only (Fig. 2b; pHi 7.58 ± 0.04, n = 6), or DMA and CFTR inhibitor 172 (Fig. 2d; pHi 7.59 ± 0.07, n = 7). Therefore, to evaluate the contributions of CA and Cl channels to the pHi changes during F+I stimulation, here we calculated the rate of pHi change per 10 s, −ΔpH/10 s. This rate should reflect the combined influences of HCO3 release through Cl channels, and generation of H+ and HCO3 by CA under the inhibition of NHE-mediated H+ extrusion. The maximal rate of F+I-induced pHi change in the presence of DMA was 0.105 ± 0.009/10 s, n = 6, and slowed by the pre-addition of methazolamide (0.039 ± 0.004/10 s, n = 6) more prominently than that by the CFTR inhibitor 172 (0.069 ± 0.012/10 s, n = 7) (Fig. 2g). Nevertheless, since the maximal rate of F+I-induced pHi change may be affected by pHi levels, we also calculated the rate at the constant pHi of 7.27 in each condition to evaluate the contribution of CA and CFTR Cl channels to pHi regulation. The pHi value, 7.27, was determined, considering the data during F+I stimulation in this section and those during CCh stimulation in a later section. pHi and −ΔpH/10 s were averaged, respectively, at each sampling time during F+I stimulation in the ducts (Fig. 3a–c). Average of −ΔpH/10 s at pHi of 7.27 was calculated by linear interpolation between two data points, as is indicated by an arrow in each figure, and shown in Fig. 3d. Relative amplitude of the rate in each condition was similar to that of the maximal rate in Fig. 2g. These results (Figs. 2g, 3d) suggest a dominant involvement of CA-mediated H+ generation rather than CFTR-mediated HCO3 release in the F+I-induced decrease in pHi.

Fig. 2
figure2

Effects of 5(N,N)-dimethyl amiloride (DMA) and its combination with CFTR inhibitor 172 (CFTR172) or methazolamide on forskolin + IBMX (F+I)-induced pHi changes. a A time course of pHi change during the applications of DMA and DMA + forskolin + IBMX. The pHi was decreased by the addition of 20 μM DMA and further decreased by the subsequent addition of 10 μM forskolin + 100 μM IBMX (F+I). Each point indicates the mean ± SE of pHi in 19 regions of the duct. b The pHi averages for 1 min just before (control) and 9–10 min after DMA addition (DMA), and 4–5 min after the subsequent addition of F+I (DMA + F+I). Data shown are mean ± SE (n = 6). c A representative trace of pHi change during the applications of DMA + CFTR172 (10 μM) and DMA + CFTR172 + F+I. Each point indicates the mean ± SE of pHi in 13 regions of the duct. d The pHi averages for 1 min just before (control) and 9–10 min after (DMA + CFTR172) the addition of DMA + CFTR inhibitor 172, and 4–5 min after the subsequent F+I addition (DMA + CFTR172 + F+I). Data shown are mean ± SE (n = 7). e A time course of pHi change during the applications of DMA + 1 mM methazolamide (DMA + Met) and DMA + Met + F+I. Each point indicates the mean ± SE of pHi in 14 regions of the duct. f The pHi averages for 1 min just before (control) and 9–10 min after (DMA + Met) the addition of DMA + methazolamide, and 4–5 min after the subsequent F+I addition (DMA + Met + F+I). Data shown are mean ± SE (n = 6). g The maximal rates of F+I-induced decreases in pHi (−ΔpH per 10 s) in the presence of DMA. Simultaneous addition of CFTR172 with DMA significantly reduced the rate, and the addition of methazolamide reduced the rate more effectively. *P < 0.05, ***P < 0.001 in the unpaired Student’s t test. Data shown are mean ± SE (n = 6, 7, and 6, respectively)

Fig. 3
figure3

Evaluation of the contribution of carbonic anhydrases and CFTR Cl channels to forskolin + IBMX-induced pHi changes. ac Relationships between the averaged rates of pHi decreases (−ΔpH per 10 s) versus the corresponding, averaged pHi during the pHi-decreasing phase by 10 μM forskolin + 100 μM IBMX (F+I) stimulation in the presence of 5(N,N)-dimethyl amiloride (DMA; 20 μM), and its combination with CFTR inhibitor 172 (CFTR172; 10 μM) or methazolamide (Met; 1 mM). The averaged rates of pHi decreases were plotted at 10 s intervals as the pHi was decreased by F+I stimulation with the starting point indicated as F+I added. Data shown are mean ± SE (n = 6, 7, and 6, respectively). d The rates of F+I-induced decreases in pHi (−ΔpH per 10 s) at pHi of 7.27 in the presence of DMA. Simultaneous addition of CFTR172 with DMA reduced the rate, and the addition of methazolamide reduced the rate more effectively (n = 6, 7, and 6, respectively). The rate at pHi 7.27 was obtained by using linear interpolation between two data points of the means, as indicated by an arrow, in (ac) of each condition

CCh-induced pHi changes

To study the mechanisms underlying pHi regulation during HCO3 secretion induced by Ca2+ signaling, we applied CCh, a Ca2+-mobilizing muscarinic agonist [15, 33]. Figure 4a shows time courses of pHi changes elicited by the addition of CCh in rat parotid intralobular ducts. Resting pHi was dispersed between 7.40 and 7.85 (n = 30), and the dispersion of pHi at the steady state of CCh stimulation became smaller mostly to a range between 7.49 and 7.68 (n = 28) except two high pHi data during the stimulation. Altogether, pHi in 30 ducts (Fig. 4a) changed from 7.63 ± 0.02 to 7.59 ± 0.01 by CCh stimulation on average (mean ± SE, n = 30) although CCh mainly lowered pHi at the steady state (70 % of 30 ducts). Interestingly, some ducts showed two phases of the pHi change during CCh stimulation: pHi was transiently increased and then decreased to be lower (n = 11) or higher (n = 5) than the initial resting level, presumably dependent on faster activation of the pHi-raising NHE and slower activation of the pHi-lowering CA and/or Cl channels. There was a correlation between CCh-induced ΔpH and the resting pHi (Fig. 4b). These results indicate that CCh tends to decrease pHi in the ducts with the high resting pHi and increase pHi with the low resting pHi, in contrast to the F+I-induced responses showing the decreased pHi in all the ducts. Variability in the resting pHi of the ducts may be derived from differences in relative expression levels of NHE, CA and Cl channels, and in the intracellular Ca2+ concentrations in the resting state. CCh stimulation gathered the pHi level together and maintained it at a higher level in comparison with that during F+I stimulation, suggesting the existence of a certain activity that was stimulated by CCh, but not by F+I, to elevate the pHi level.

Fig. 4
figure4

pHi changes during the stimulation by 10 μM carbachol (CCh) in rat parotid ducts. a Time courses of pHi changes induced by CCh addition. pHi dispersion in the resting state became smaller after the addition of CCh. pHi traces for 30 ducts are shown. b Relationships between the CCh-induced pHi changes (ΔpH) and the resting pHi levels. Each point was obtained from the data in (a) (n = 30)

Effects of CA inhibition by methazolamide on the CCh-induced pHi change

To characterize the contributions of NHE, CA, and Cl channels to the pHi regulation during CCh stimulation, we first addressed whether the CCh-induced changes in pHi were due to H+ and HCO3 generation by CA. Under the CA inhibition by methazolamide, CCh increased pHi in all the ducts tested (Fig. 5a, b; P < 0.01 in the paired Student’s t test). This is in contrast with the effect of methazolamide on F+I-induced pH changes (Fig. 1d, e). These results suggest not only the presence of CA (some isozymes) activity during CCh stimulation but also that CCh remarkably activates NHE-mediated H+ extrusion to maintain the higher pHi, which has been unveiled by inhibiting CA.

Fig. 5
figure5

Effects of blocking H+ generation by methazolamide, Ca2+ chelation and calmodulin inhibition on 10 μM carbachol (CCh)-induced pHi changes. a A time course of CCh-induced pHi change in the presence of 1 mM methazolamide (Met). CCh evoked an increase in pHi in all the ducts tested in the presence of methazolamide. Each point indicates the mean ± SE of pHi in 20 regions of the duct. b The pHi averaged for 1 min just before (control) and 4–5 min after (Met) the addition of 1 mM methazolamide and 4–5 min after 10 μM CCh addition (Met + CCh). The pHi was increased clearly by CCh in the presence of methazolamide. Data shown are mean ± SE (n = 5). c A representative trace of CCh-induced pHi change in the absence of the external Ca2+ (Ca free) and the presence of 1 mM methazolamide (Met) in the duct loaded with the Ca2+-chelating agent, BAPTA (50 μM BAPTA-AM for 15 min at 37 °C). Each point indicates the mean ± SE of pHi in 18 regions of the duct. d Loading BAPTA into the ducts and external Ca2+ removal significantly reduced the CCh-induced increase in pHi, comparing the data shown in b (Met + CCh) and d (Ca free + Met + CCh) (P < 0.05 in the unpaired Student’s t test, n = 5 and 7). The data in d are shown as mean ± SE. e A time course of CCh-induced pHi change in the presence of the calmodulin inhibitor, W-7 (W7, 100 μM). Each point indicates the mean ± SE in 24 regions of the ducts. f W-7 suppressed the CCh-induced increase in pHi in the presence of 1 mM methazolamide (Met). Data shown are mean ± SE (n = 5)

Effects of intracellular Ca2+ chelation by BAPTA and calmodulin inhibition by W-7 on CCh-induced pHi changes

To understand the regulatory mechanisms underlying NHE activation during CCh stimulation, we examined whether CCh activated NHE via Ca2+-calmodulin by monitoring the pHi in the ductal cells under the inhibition of CA. The CCh-induced increase in pHi in the presence of methazolamide was reduced by extracellular Ca2+ removal after intracellular Ca2+ chelation by BAPTA (Fig. 5c, d). The effects of Ca2+ removal and chelation were significant, comparing the data shown in Fig. 5d (Ca free + Met + CCh) with those in Fig. 5b (Met + CCh) (P < 0.05 in the unpaired Student’s t test, n = 7 and 5). The CCh-induced increase in pHi in the presence of methazolamide was also significantly suppressed by the calmodulin inhibitor W-7 (Fig. 5e, f). Importantly, our data show that the pHi level of the experimental condition of Met + W7 + CCh (Fig. 5f) was lower than that of Met + CCh (Fig. 5b) (P < 0.05 in the unpaired Student’s t test, n = 5 and 5). These results suggest that Ca2+-calmodulin is involved in the CCh-induced activation of NHE.

CCh-induced pHi changes under the inhibition of NHE by DMA

To further characterize the contributions of NHE, CA and Cl channels to the pHi regulation during CCh stimulation, we deduced and analyzed the pHi changes mediated by CA and Cl channel activities under the inhibition of NHE by DMA. DMA significantly decreased pHi, and the addition of CCh markedly reduced pHi in the presence of DMA (Fig. 6a, b; pHi at DMA + CCh was 7.11 ± 0.05, n = 7). Thus, inhibition of NHE activity unveiled the pHi-lowering activities of CA and/or Cl channels, enhanced during CCh stimulation. In the presence of the Cl channel blocker DPC or methazolamide with DMA, CCh induced a decrease in pHi to the same level as that during CCh stimulation with DMA alone [Fig. 6c, d (DMA + DPC + CCh); pHi 7.20 ± 0.08 (n = 6), and Fig. 6e, f (DMA + Met + CCh); pHi 7.15 ± 0.03 (n = 5)]. No difference among these three pHi levels could be due to the lowest limit of pHi changes during CCh stimulation. Therefore, to evaluate the contributions of CA and Cl channel activities to the pHi changes, we calculated the rate of pHi change per 10 s, −ΔpH/10 s. The maximal rate of CCh-induced decrease in pHi in the presence of DMA was shown in Fig. 6g [DMA + CCh; max(−ΔpH/10 s) = 0.071 ± 0.003, n = 7]. Addition of DPC in the presence of DMA diminished the maximal rate of the CCh-induced pHi reduction (Fig. 6g), indicating the involvement of Cl channel activation in lowering pHi. Methazolamide also significantly slowed the rate (Fig. 6g), suggesting the contribution of CA activity to the pHi decrease. Average of −ΔpH/10 s at pHi of 7.27 was also calculated as is described above: pHi and −ΔpH/10 s were averaged respectively at each sampling time during CCh stimulation in the ducts (Fig. 7a–c). Figure 7d shows that relative amplitude of the rate in each condition was similar to that of the maximal rate in Fig. 6g. These results (Figs. 6g, 7d) and the CCh-induced pHi changes in the absence and presence of methazolamide (Figs. 4a, 5a, b) reveal that CCh remarkably activates NHE which keeps the pHi at around 7.6 against the pHi-lowering activities, and that the CCh-induced decrease in pHi is associated with both the HCO3 release through DPC-sensitive Cl channels and H+ generation by CA.

Fig. 6
figure6

Effects of 5(N,N)-dimethyl amiloride (DMA) and its combination with diphenylamine-2-carboxylate (DPC) or methazolamide on carbachol (CCh)-induced pHi changes. a A time course of pHi change during the applications of DMA and DMA + CCh. The pHi was decreased by the addition of 20 μM DMA and further decreased by the subsequent addition of 10 μM CCh. Each point indicates the mean ± SE of pHi in 16 regions of the duct. b The pHi averages for 1 min just before (control) and 9–10 min after DMA addition (DMA), and 4–5 min after the subsequent addition of CCh (DMA + CCh). Data shown are mean ± SE (n = 7). c A representative trace of pHi change during the applications of DMA + DPC (200 μM) and DMA + DPC + CCh. Each point indicates the mean ± SE of pHi in 17 regions of the duct. d The pHi averages for 1 min just before (control) and 9–10 min after (DMA + DPC) the addition of DMA + DPC, and 4–5 min after the subsequent CCh addition (DMA + DPC + CCh). Data shown are mean ± SE (n = 6). e A time course of pHi change during the applications of DMA + 1 mM methazolamide (DMA + Met) and DMA + Met + CCh. Each point indicates the mean ± SE of pHi in 18 regions of the duct. f The pHi averages for 1 min just before (control) and 9–10 min after (DMA + Met) the addition of DMA + methazolamide, and 4–5 min after the subsequent CCh addition (DMA + Met + CCh). Data shown are mean ± SE (n = 5). g The maximal rates of CCh-induced decreases in pHi (−ΔpH per 10 s) in the presence of DMA. Simultaneous addition of either DPC or methazolamide with DMA reduced the rate. ***P < 0.001 in the unpaired Student’s t test. Data shown are mean ± SE (n = 7, 6, and 5, respectively)

Fig. 7
figure7

Evaluation of the contribution of carbonic anhydrases and diphenylamine-2-carboxylate (DPC)-sensitive Cl channels to carbachol (CCh)-induced pHi changes. ac Relationships between the averaged rates of pHi decreases (−ΔpH per 10 s) versus the corresponding, averaged pHi during the pHi-decreasing phase by 10 μM CCh stimulation in the presence of 5(N,N)-dimethyl amiloride (DMA; 20 μM), and its combination with 200 μM DPC or methazolamide (Met; 1 mM). The averaged rates of pHi decreases were plotted at 10-s intervals as the pHi was decreased by CCh stimulation with the starting point indicated as CCh added. Data shown are mean ± SE (n = 7, 6, and 5, respectively). d The rates of CCh-induced decreases in pHi (−ΔpH per 10 s) at pHi of 7.27 in the presence of DMA. Simultaneous addition of either DPC or methazolamide with DMA reduced the rate (n = 7, 6, and 5, respectively). The rate at pHi of 7.27 was obtained by using linear interpolation between two data points of the means, as is indicated by an arrow, in the figure (ac) of each condition

Discussion

F+I-induced pHi changes and activities of CA, CFTR and NHE in parotid intralobular ducts

F+I consistently decreased the pHi level despite variability in the resting pHi observed among the ducts (Fig. 1a–c), indicating the dominance of the pHi-lowering activities of CA and/or CFTR Cl channels over the pHi-raising NHE activity during F+I stimulation. Since the F+I-induced change was suppressed by methazolamide (Fig. 1d, e) as reported in the previous paper [7], we expected that the F+I-induced decrease in pHi could be due to facilitation of H+ and HCO3 generation by CA and HCO3 release through the CFTR Cl channel [12]. Unexpectedly, CFTR inhibitor 172 did not significantly affect the F+I-induced pHi change (Fig. 1c, g). In addition, CFTR inhibitor 172 moderately reduced the rate of the F+I-induced pHi change in the presence of DMA, while methazolamide more effectively slowed the rate (Figs. 2g, 3d). These suggest that activation of CFTR Cl channels during F+I stimulation is not a major rate-limiting step for pHi regulation, and that some isozymes of CA in salivary ducts would be activated by F+I (Fig. 8) as CAIX in cultured cells is [30]. F+I-induced CA activation may primarily contribute to the pHi regulation via generation of H+ and HCO3 , while being followed by and concerting with NHE-mediated H+ extrusion and CFTR-mediated HCO3 release to sustain HCO3 secretion by the cAMP signal.

Fig. 8
figure8

Schematic illustration for pHi regulation during HCO3 secretion in rat parotid intralobular ducts. Sympathetic β-adrenergic stimulation induces activation of the carbonic anhydrase (CA) rather than the Na+–H+ exchanger (NHE), whereas parasympathetic muscarinic stimulation activates NHE on the basolateral membrane at least partially via Ca2+-calmodulin. Both mechanisms facilitate HCO3 secretion through the Cl channels activated by the stimulations. Thick arrows indicate the rate-limiting activation

Involvement of the pHi-lowering activities of CA and Cl channels in pHi regulation during CCh stimulation

The CCh-induced decrease in pHi, observed in the ducts with high resting pHi, suggests that the pHi-lowering CA and/or Cl channels would be activated during the stimulation. First, we examined the effects of CA inhibition on the CCh-induced pHi change. In the presence of methazolamide, CCh increased the pHi level in all the ducts tested (Fig. 5a, b). It is very likely that the inhibition of CA by methazolamide reduced the cytosolic H+ generation, and unveiled the activity of the NHE-mediated H+ extrusion during CCh stimulation. This effect and the methazolamide-induced reduction in CCh-induced pHi change in the presence of DMA (Figs. 6g, 7d) suggest that some isozymes of CA participate in lowering pHi by generating H+ and HCO3 during CCh stimulation in order to effectively elicit HCO3 secretion.

We also examined the effects of Cl channel blockage on the pHi change. Since the molecular identity of Cl channels activated by the Ca2+ signal has not been clarified yet in rat parotid intralobular ducts [2], we used the non-specific Cl channel inhibitor DPC to block CCh or Ca2+-activated Cl channels in the present study as reported in the previous papers [6, 15, 34]. Accordingly, DPC slowed the rate of CCh-induced pHi reduction in the presence of DMA (Figs. 6g, 7d). These results suggest that the CCh-induced decrease in pHi is associated with HCO3 release via DPC-sensitive Cl channels [6] in addition to H+ generation by CA.

Involvement of CCh-induced NHE activation in pHi regulation

Although both pHi-lowering activities of CA and DPC-sensitive Cl channels participate in pHi regulation during CCh stimulation, the prominent features in the pHi regulation are derived from pHi-raising activity. First, the pHi-raising activity of NHE, which was enhanced during CCh stimulation, maintained the pHi at a higher level, compared with that during F+I stimulation. Second, CA inhibition suppressed the F+I-induced decrease in pHi to a level close to, but lower than, the resting level, whereas it caused pHi to increase during CCh stimulation. Thus, the pHi-raising NHE is prominently activated by CCh.

The two-phase pHi change in some ducts during CCh stimulation (Fig. 4a) suggests that NHE activation by the agent may be faster than its Cl channel activation. In fact, the time to half-maximal responses of CCh-induced increase in pHi in the presence of methazolamide (0.36 ± 0.05 min, n = 5) (Fig. 5a; and other 4 similar data) was much shorter than that of CCh-induced decrease in pHi in the presence of DMA (1.25 ± 0.07 min, n = 7) (Fig. 6a; and other 6 similar data). The NHE activity may promote an initial increase in pHi via H+ extrusion, and the Cl channel activity may contribute to a delayed decrease by releasing HCO3 through the channel in association with H+ and HCO3 generation by CA. The two exceptional data in Fig. 4a may be due to the much higher NHE activity than those of pHi-lowering CA and Cl channels in the CCh-stimulated states. Since pHi is regulated by cooperative activities of CA, NHE and Cl channels, variability in the expression level of each factor may elicit some pHi differences among the ducts (Fig. 4).

The resting pHi was decreased after the addition of DMA (Figs. 2a, b, 6a, b), suggesting that NHE would be partially activated before the stimulation, dependent on the resting NHE phosphorylation or resting intracellular Ca2+ concentration ([Ca2+]i) [27]. Importantly, CCh more remarkably decreased pHi in the presence of DMA than it did in the absence of DMA, suggesting that NHE may be more activated by CCh than in the resting level to maintain the pHi at around 7.6 against the pHi-lowering activities of CA and Cl channels.

Ca2+-calmodulin-mediated activation of NHE

The Ca2+ chelator BAPTA and the calmodulin inhibitor W-7 suppressed the NHE-mediated increase in pHi, induced by CCh under the inhibition of CA by methazolamide. The effect of Ca2+ chelation suggests that the CCh-induced increase in [Ca2+]i may be a primary trigger of NHE activation by CCh, although CCh also activates other signals and the Ca2+ signal has a cross-talk with the cAMP signal in salivary gland cells [2, 3]. Removal of external Ca2+ also suppresses HCO3 secretion from the duct segments in our previous paper [6]. These results indicate that CCh is most likely to activate the NHE at least partially via Ca2+-calmodulin in the parotid ducts (Fig. 8). This is in accord with the previous reports on parotid acinar cells [27] and cultured cells [24, 25]. To keep pHi at a rather high level (around 7.6) in a steady state during CCh stimulation, the pHi-raising NHE may be activated the most among the three pHi regulators. In other words, activation of NHE may be the rate-limiting step for pHi regulation and HCO3 secretion during the CCh stimulation. In the parotid ducts, the high pHi level maintained by Ca2+-calmodulin-mediated activation of NHE would advance efficient generation of HCO3 from CO2 and H2O by CA, resulting in an effective increase in HCO3 secretion via DPC-sensitive Cl channels. From the viewpoint of pHi regulation, parasympathetic muscarinic stimulation may be more beneficial to HCO3 secretion than sympathetic β-adrenergic stimulation.

Conclusion

Taken together, we conclude that F+I may accelerate HCO3 secretion by increasing CA activity for HCO3 and H+ generation, rather than activating NHE for H+ extrusion and the CFTR Cl channel for HCO3 release, whereas CCh facilitates HCO3 secretion mainly through an increase in H+ extrusion by the Ca2+-calmodulin-mediated activation of NHE, in addition to activation of DPC-sensitive Cl channels and CA (Fig. 8). In this paper, we have revealed that different rate-limiting activities of pHi regulation for HCO3 secretion stimulated primarily by cAMP and Ca2+ signals exist in the intralobular ducts of rat parotid glands.

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Acknowledgments

This work was carried out partially at the Analysis Center of Life Science, Natural Science Center for Basic Research and Development, Hiroshima University.

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Correspondence to Chikara Hirono.

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All procedures performed in studies involving animals were in accordance with the ethical standards of the institution at which the studies were conducted.

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Ueno, K., Hirono, C., Kitagawa, M. et al. Different rate-limiting activities of intracellular pH regulators for HCO3 secretion stimulated by forskolin and carbachol in rat parotid intralobular ducts. J Physiol Sci 66, 477–490 (2016). https://doi.org/10.1007/s12576-016-0443-6

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Keywords

  • Intracellular pH
  • Rat parotid intralobular ducts
  • Bicarbonate secretion
  • Na+–H+ exchanger
  • Carbonic anhydorase
  • Cl channels