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Am J Physiol Gastrointest Liver Physiol 294: G738-G747, 2008. First published January 10, 2008; doi:10.1152/ajpgi.00239.2007
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HORMONES AND SIGNALING

Suppression of carbachol-induced oscillatory Cl– secretion by forskolin in rat parotid and submandibular acinar cells

Takahide Shintani, Chikara Hirono, Makoto Sugita, Yoshiko Iwasa, and Yoshiki Shiba

Department of Oral Physiology, Hiroshima University Graduate School of Biomedical Sciences, Minami-ku, Hiroshima, Japan

Submitted 27 May 2007 ; accepted in final form 8 January 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sympathetic stimulation induces weak salivation compared with parasympathetic stimulation. To clarify this phenomenon in salivary glands, we investigated cAMP-induced modulation of Ca2+-activated Cl– secretion from rat parotid and submandibular acinar cells because fluid secretion from salivary glands depends on the Cl– secretion. Carbachol (Cch), a Ca2+-increasing agent, induced hyperpolarization of the cells with oscillatory depolarization in the current clamp mode of the gramicidin-perforated patch recording. In the voltage clamp mode at –80 mV, Cch induced a bumetanide-sensitive oscillatory inward current, which was larger in rat submandibular acinar cells than in parotid acinar cells. Forskolin and IBMX, cAMP-increasing agents, did not induce any marked current, but they evoked a small nonoscillatory inward current in the presence of Cch and suppressed the Cch-induced oscillatory inward current in all parotid acinar cells and half (56%) of submandibular acinar cells. In the current clamp mode, forskolin + IBMX evoked a small nonoscillatory depolarization in the presence of Cch and reduced the amplitude of Cch-induced oscillatory depolarization in both acinar cells. The oscillatory inward current estimated at the depolarized membrane potential was suppressed by forskolin + IBMX. These results indicate that cAMP suppresses Ca2+-activated oscillatory Cl– secretion of parotid and submandibular acinar cells at –80 mV and possibly at the membrane potential during Cch stimulation. The suppression may result in the weak salivation induced by sympathetic stimulation.

Na+-K+-2Cl– cotransporter; Cl– channel; cAMP; gramicidin-perforated patch recording; bumetanide

SYMPATHETIC NERVE STIMULATION, via the Ca2+-increasing {alpha}-adrenergic and cAMP-increasing β-adrenergic actions, induces smaller saliva than parasympathetic nerve stimulation via the muscarinic cholinergic action. cAMP-increasing β-adrenergic agents themselves do not induce fluid secretion from salivary glands but modulate the fluid secretion induced by Ca2+-increasing muscarinic agents (22, 29, 40). However, the cellular mechanism of these modulations of fluid secretion is still unclear.

The transcellular Cl– secretion from salivary gland acinar cells plays a key role in fluid secretion from the acini. It consists of two processes, Cl– release through channels on the luminal membranes (27, 30, 32, 35) and Cl– replenishment through cotransporters and exchangers on the basolateral membranes (17, 19, 38, 43). Muscarinic agonists induce Cl– release through the Ca2+-dependent Cl– channel from the luminal side and K+ release through K+ channels from the basolateral side in salivary acinar cells (9, 30, 35). The Cl– release (secretion) results in a decrease in the intracellular Cl– level (13, 31, 42), and this activates Cl– uptake via the Na+-K+-2Cl– cotransporter of which activity is regulated by Ca2+ elevation (12, 26, 37). Secretory Cl– is transported into the cells mainly via the Na+-K+-2Cl– cotransporter on the basolateral side and partially through the Cl–-HCO3– exchanger (9, 11, 28, 30, 36, 44), although HCO3– partially contributes to anion secretion by direct HCO3– efflux (31). Therefore, it is important to investigate the regulatory mechanism of the Cl– secretion via the Ca2+-activated Cl– channel and the Na+-K+-2Cl– cotransporter for understanding the regulation of fluid secretion from salivary glands.

In this paper, we investigated effects of cAMP-increasing agents on Cl– secretion, which is induced by the Ca2+-increasing agonist carbachol (Cch) in the acinar cells to clarify the cellular mechanism of the modulation of Ca2+-activated fluid secretion by cAMP-increasing agents, by using the gramicidin-perforated patch recording method. The conventional whole-cell patch clamp recording method reveals the Cl– channel activity as an agonist-induced Cl– current in salivary acinar cells, since the current-carrying anion Cl– is supplied by the pipette solution. On the other hand, the gramicidin-perforated patch recording method, in which the current-carrying anion is supplied by the cells, reveals the transcellular anion movement via anion transporters and anion channels (for the details, see MATERIALS AND METHODS). Using this technique in salivary acinar cells, we demonstrated the suppression of Cch-induced transcellular anion current by cAMP-increasing agents in this paper.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell preparation. Cells were prepared by almost the same method described in the previous paper (22). Briefly, parotid and submandibular glands were removed from male Wistar rats (310–460 g), which were anesthetized by pentobarbital sodium (Nembutal, 70 mg/kg ip). The animal use protocol was approved by Research Facilities for Laboratory Animal Science, Hiroshima University. After 7–10 min of pretreatment by 0.05% trypsin (Nacalai Tesque, Kyoto, Japan) dissolved in phosphate-buffered saline containing 0.016% EDTA, rat parotid and submandibular glands were digested with 0.16% collagenase (type S-1; Nitta Gelatin, Osaka, Japan) to disperse the cells. They were further dispersed by pipetting, and undigested large fragments of tissues were removed by gravity. Obtained cells were washed and placed on poly-L-lysin-coated cover glasses with a diameter of 5 mm.

Patch clamp. The gramicidin-perforated patch recording was originally developed to measure ionic currents, while avoiding disturbance of intracellular Cl– concentration ([Cl–]i) in central nervous system cells (1, 2, 10). Since gramicidin pores are permeable only to small monovalent cations, anions are not introduced by the gramicidin-perforated patch electrode but rather controlled by the cell itself. Accordingly, intracellular anions are not replaced with those in the pipette solution, and the cytoplasmic anion concentration is determined by anion-supplying and anion-releasing activities of the cell itself. Therefore, the method has been used for measuring intact Cl– currents and calculating the intact [Cl–]i from the reversal potential of the current response induced by inhibitory amino acids in central nervous system cells. We applied this method to anion-secreting cells in salivary glands and were able to confirm the secreted anion and secretion mechanism in anion-secreting individual cells because the current response measured with this method reflects both anion supply, such as Cl– uptake by transporters, and anion release, such as Cl– release through ion channels (20, 39).

In the gramicidin-perforated patch recording experiments, the pipette solution contained 150 mM KCl and 10 mM HEPES buffer (pH 7.2). For a gramicidin stock solution, 10 mg gramicidin D or gramicidin (Sigma, St. Louis, MO) was dissolved in 1 ml methanol and stored at –20°C. The gramicidin stock was diluted by 100 times with the pipette solution, and this solution was then sonicated until gramicidin was dissolved. Patch pipettes were pulled from borosilicate glass capillaries. Pipette tips were placed for a few seconds in the gramicidin-free pipette solution, and the pipette was then back-filled with the gramicidin-containing pipette solution. The access resistance was 9–30 M{Omega}. The pipette potential was held at –80 mV (approximately equal to the potassium equilibrium potential) to prevent the potassium current from passing through the potassium channels (22) and instead make the K+ flux pass through the electrode (21). Contribution of other cations such as Na+ was negligibly small. Thus the transcellular anion current was indirectly measured as a cation current flowing into the electrode with the same amplitude as the anion current (21). Ionic currents were measured with a patch/whole-cell clamp amplifier CEZ-2400 (Nihon Kohden, Tokyo, Japan) and recorded with a data acquisition system, MacLab/200 (AD Instruments, New South Wales, Australia) and Macintosh Performa 5260 (Apple Computer, Cupertino, CA). The external solution used through the experiments was basically the modified Krebs-Henseleit-Ringer (KHR) solution containing (in mM) 103 NaCl, 4.7 KCl, 2.56 CaCl2, 1.13 MgCl2, 2.8 glucose, 4.9 sodium pyruvate, 2.7 fumaric acid disodium salt, 4.9 L-glutamic acid monosodium salt, 12.5 HEPES-NaOH (pH 7.4), 25 NaHCO3, and 1.15 NaH2PO4. It was gassed with 5% CO2-95% O2. All experiments were made at the room temperature of 24–28°C.

In the conventional whole-cell recording experiments, cells were obtained by the same preparation as in the gramicidin-perforated patch experiments. The whole-cell pipette solution contained (in mM): 140 KCl, 1 MgCl2, 0.5 EGTA, 10 glucose, 1 ATP, and 10 HEPES (pH 7.4). The access resistance was 7–26 M{Omega}.

Cch-induced oscillatory anion current was measured from the base line in the period. After addition of forskolin + IBMX, a nonoscillatory current was measured from the stable base line just before the addition of forskolin + IBMX, and the oscillatory anion current was measured from the nonoscillatory current in the period (see Fig. 2, A, D, and E and Fig. 4, A, D, and E). Similarly, Cch-induced oscillatory depolarization and nonoscillatory depolarization after addition of forskolin + IBMX were measured (see Fig. 2, B, F, and G and Fig. 4, B, F, and G). Current amplitude of the oscillatory anion current was obtained by averaging the current for the time (20 or 30 s) described in the figure legend (integrating the current for the time and dividing the integral by the time). The effects of drugs on the anion current were analyzed quantitatively and shown in bar graphs. Bumetanide, forskolin, gramicidin, and IBMX were purchased from Sigma. Carbamylcholine chloride (carbachol, Cch) was obtained from Nacalai Tesque. Experimental values are means ± SE for the number of determinations indicated. P < 0.05 (the paired or unpaired Student's t-test) was taken to represent statistically significant differences.


Figure 2
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  		Fig. 2. A: typical suppression of Cch (0.25 µM)-induced oscillatory anion current and induction of a nonoscillatory current by 10 µM forskolin + 100 µM IBMX (F + I) in the presence of Cch at the holding potential of –80 mV in the gramicidin-perforated patch configuration of the rat parotid acinar cell. The currents were normalized by membrane capacitance to avoid the factor of cell size. Other 6 cells showed the similar time course of the currents. The cell was voltage clamped at –80 mV for 20 s to minimize Cch-induced K+ current and then at –87 mV for 20 s every minute. Parts of the current trace [and the membrane potential (Vm) trace in B] during holding at –87 mV were omitted for simplicity. B: Cch-induced hyperpolarization and oscillation of membrane potential Vm. Forskolin + IBMX suppressed Vm oscillation and evoked a nonoscillatory Vm. Vm was measured by changing the clamp mode from voltage clamp to current clamp for 20 s every minute. Time axis is common in A and B. C: time course of changing the clamp mode between voltage clamp at –80 mV (V-clamp) and current clamp at 0 A/F (I-clamp). D–G: time scale enlargement of traces indicated by short bars (D–G) in A and B reveals that the frequency of the current oscillation and Vm oscillation did not change after addition of forskolin + IBMX.

 

Figure 4
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  		Fig. 4. A: typical suppression of 0.25 µM Cch-induced oscillatory anion current and induction of a nonoscillatory current by 10 µM forskolin + 100 µM IBMX in the presence of Cch at the holding potential of –80 mV in the gramicidin-perforated patch configuration of the rat submandibular acinar cell. The currents were normalized by membrane capacitance. Other 4 cells showed the similar time course of the response. B: Cch-induced hyperpolarization and depolarizing oscillation of membrane potential Vm. Forskolin + IBMX suppressed Vm oscillation and evoked a nonoscillatory Vm. Vm was measured by changing the clamp mode from voltage clamp to current clamp for 15 s every 2 min. Time axis is common in A and B. C: time course of changing the clamp mode between voltage clamp at –80 mV and current clamp at 0 A/F. D–G: time scale enlargement of the traces indicated by short bars (D–G) in A and B reveals that the frequency of the current oscillation and Vm oscillation did not change after the addition of forskolin + IBMX.

 

   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Cch-induced oscillatory anion current in acinar cells. In the gramicidin-perforated patch recording at the holding potential of –80 mV, Cch (0.25 µM) evoked a long-lasting oscillatory anion current both in the parotid and submandibular acinar cells as shown in Fig. 1A and reported previously (39). The amplitude of the oscillatory current in acinar cells of both glands became a steady-state level in 5 min from the addition of 0.25 µM Cch. The oscillation continued at least for another 7 min without marked change in the amplitude. Therefore, we studied effects of cAMP-increasing agents on the oscillatory current comparing the current amplitude just before their addition with that 3–5 min after the addition.


Figure 1
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  		Fig. 1. A: carbachol (Cch, 0.25 µM)-induced anion current measured with the gramicidin-perforated patch recording method at the holding potential of –80 mV to minimize Cch-induced K+ current in the rat parotid acinar cell. The current was normalized by membrane capacitance to avoid the factor of cell size. Current amplitude did not markedly change during the steady state (t > 5 min after Cch addition). Other 5 cells showed the similar time course of the current. B and C: suppression of 0.25 µM Cch-induced oscillatory anion current by 0.5 mM bumetanide at the holding potential of –80 mV in the gramicidin-perforated patch configuration of the rat parotid acinar cells (means ± SE, *P < 0.05, paired Student's t-test, n = 4 in C). Current amplitude was obtained by averaging the oscillatory current for 2 min from 10–12 min after the addition of Cch (from 3–5 min after the addition of bumetanide), whereas the control current was averaged for 1 min from 5–6 min after the addition of Cch before the addition of bumetanide in the same cells. D: Cch (0.25 µM)-induced anion current measured by the conventional whole-cell recording method at the holding potential of –80 mV in the rat parotid acinar cell. The current was normalized by membrane capacitance. E and F: no effect of 0.5 mM bumetanide on Cch-induced oscillatory anion current in the conventional whole-cell configuration (means ± SE, n = 4 in F). Current amplitude was obtained by averaging the oscillatory current for 1 min from 10–11 min after the addition of Cch (from 4–5 min after the addition of bumetanide), whereas the control current was averaged for 1 min from 5–6 min after the addition of Cch before the addition of bumetanide in the same cells.

 
To examine whether Cch (0.25 µM)-induced oscillatory anion current at –80 mV is actually carried by Cl– in rat parotid acinar cells, we investigated effects of the Na+-K+-2Cl– cotransporter inhibitor, bumetanide, on the current amplitude in the steady-state phase of the current oscillation. Bumetanide (0.5 mM) markedly (by 75%) suppressed the oscillatory current in the gramicidin-perforated patch configuration (Fig. 1, B and C). Cch also induced an oscillatory anion current, which reflected the Cl– channel activity, in the conventional whole-cell configuration (Fig. 1D), but bumetanide did not affect the Cch-induced oscillatory current (Fig. 1, E and F). These results suggest that bumetanide suppresses the oscillatory current in the gramicidin-perforated patch configuration through the inhibition of the Na+-K+-2Cl– cotransporter, but not the Ca2+-activated Cl– channel activity in the parotid acinar cells, and that the major carrier of the oscillatory current in the gramicidin-perforated patch configuration is very likely Cl– taken up via the Na+-K+-2Cl– cotransporter.

Effect of forskolin + 3-isobutyl-methylxantine on current amplitude. Cch (0.25 µM)-induced oscillatory anion current was suppressed by cAMP-increasing agents [forskolin (10 µM) + IBMX (100 µM)] in the parotid acinar cell (Fig. 2, A, D, and E). Time course of the Cch-induced oscillatory current, averaged for 20 s every 1 min, is shown in Fig. 3A. The oscillatory current 5–7 min after the Cch addition was then averaged for 1–1.5 min in parotid acinar cells. It was –2.39 ± 0.58 A/F (mean ± SE, n = 7) (Fig. 3B). The addition of forskolin (10 µM) + IBMX (100 µM) for 3 min to the external solution did not induce any marked current response in the absence of Cch [only very small nonoscillatory current was induced; –0.23 ± 0.21 A/F (mean ± SE, n = 5)]. Their addition for 3 min during Cch stimulation reduced the Cch-induced oscillatory current to –0.89 ± 0.22 A/F (mean ± SE, n = 7) in the averaged amplitude in addition to the induction of a nonoscillatory inward current (–1.46 ± 0.31 A/F, mean ± SE, n = 7) (Fig. 2, A, D, and E and Fig. 3). The suppression of Cch-induced oscillatory anion current by forskolin + IBMX was marked (by 63% in average) and significant (P < 0.01, paired Student's t-test, n = 7) in parotid acinar cells (Fig. 3B).


Figure 3
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  		Fig. 3. A: time course of the suppression of 0.25 µM Cch-induced oscillatory anion current by 10 µM forskolin + 100 µM IBMX at the holding potential of –80 mV in the gramicidin-perforated patch configuration of the rat parotid acinar cell. Each point was obtained by averaging of Cch-induced oscillatory current (Iosc) for 20 s in voltage clamp at –80 mV. Other 6 cells showed the similar time course of the response. B: effects of forskolin + IBMX on the current amplitude and the frequency of the oscillation. Three points just before addition of forskolin + IBMX in A were averaged (solid bar), and similar averaging was performed for 3–5 min after addition of forskolin + IBMX (open bar). Then data were statistically analyzed for 7 cells. The current was significantly suppressed by forskolin + IBMX (means ± SE, **P < 0.01, paired Student's t-test, n = 7), but the frequency of the current oscillation was not affected by the drugs (n = 7).

 
Forskolin + IBMX also suppressed Cch-induced oscillatory anion current in the submandibular acinar cell (Figs. 4, A, D, and E). Time course of the Cch-induced oscillatory current, averaged for 30 s in every 1 min, is shown in Fig. 5A. The Cch-induced oscillatory current 5–7 min after the Cch addition was averaged for 1–1.5 min in submandibular acinar cells. It was –5.89 ± 0.45 A/F (mean ± SE, n = 9) and significantly larger than that of parotid cells (P < 0.001, unpaired Student's t-test). The Cch-induced oscillatory current per unit membrane capacitance in submandibular acinar cells is two and a half times as large as that in parotid acinar cells at –80 mV. The addition of forskolin + IBMX for 3 min in the absence of Cch induced a small nonoscillatory current response in submandibular acinar cells (–0.22 ± 0.18 A/F, mean ± SE, n = 4). Their addition for 3 min significantly reduced the Cch-induced oscillatory current from –6.63 ± 0.54 A/F to –4.41 ± 0.76 A/F (means ± SE, n = 5; P < 0.05, paired Student's t-test) and induced a nonoscillatory inward current (–1.31 ± 0.70 A/F, mean ± SE, n = 5) in the 5 of 9 submandibular acinar cells in the presence of Cch (Fig. 4, A, D, and E and Fig. 5). No significant effect on the Cch-induced oscillatory current was observed in the other 4 cells (from –4.98 ± 0.60 A/F to –5.29 ± 0.91 A/F, means ± SE). The nonoscillatory current in the 4 cells was –0.76 ± 0.40 A/F (mean ± SE).


Figure 5
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  		Fig. 5. A: time course of the suppression of 0.25 µM Cch-induced oscillatory anion current by 10 µM forskolin + 100 µM IBMX at the holding potential of –80 mV in the gramicidin-perforated patch configuration of the rat submandibular acinar cell. Each point was obtained by averaging Cch-induced oscillatory current for 30 s in voltage clamp at –80 mV. B: three points just before the addition of forskolin + IBMX in A were averaged (solid bar), and similar averaging was performed for 3–5 min after the addition of forskolin + IBMX (open bar). Then data were statistically analyzed for 5 cells. The current was significantly suppressed by forskolin + IBMX (means ± SE, *P < 0.05, paired Student's t-test, n = 5), but the frequency of the current oscillation was not affected by the drugs (n = 5).

 
The frequency of current oscillation did not change by the addition of forskolin + IBMX both in parotid (Fig. 2, D and E and Fig. 3B) and submandibular acinar cells (Fig. 4, D and E and Fig. 5B), indicating that the frequency of Ca2+ oscillation was not affected by the cAMP-increasing agents because the current oscillation reflects the Ca2+ oscillation in salivary acinar cells (16, 18).

Effect of forskolin + IBMX on membrane potential. Membrane potential gives us somehow physiological secretory information, such as the driving force of secretory ions. Therefore, we measured membrane potential of the same acinar cells by changing the patch-clamp mode from voltage clamp to current clamp (0 A/F) for a short period (e.g., for 15 or 20 s in every 1 or 2 min as shown in Figs. 2C and 4C). We analyzed the membrane potential in the 7 parotid cells and the 5 submandibular cells sensitive to forskolin + IBMX. Cch induced a nonoscillatory hyperpolarization through sustained activation of K+ channels and an oscillatory depolarization through oscillatory activation of Cl– channels in the acinar cells. The depolarizing oscillatory component of membrane potential was superimposed on the nonoscillatory membrane potential (Fig. 2, B, F, and G and Fig. 4, B, F, and G).

Addition of forskolin + IBMX did not significantly affect the depolarizing peak value of the oscillation both in parotid (from –66.3 ± 2.9 mV to –68.5 ± 3.4 mV; means ± SE, n = 7) and submandibular acinar cells (from –36.2 ± 2.2 mV to –38.5 ± 3.0 mV; means ± SE, n = 5) but induced a nonoscillatory depolarization in parotid (from –84.3 ± 1.3 mV to –79.6 ± 2.3 mV; means ± SE, P < 0.05, paired Student's t-test, n = 7) and submandibular cells (–85.6 ± 10.5 mV to –71.7 ± 10.9 mV; means ± SE, P < 0.01, paired Student's t-test, n = 5). Therefore, the amplitude of the oscillation was reduced from 18.0 ± 3.1 mV to 11.1 ± 3.8 mV (means ± SE, P < 0.05, paired Student's t-test, n = 7) in parotid and from 49.4 ± 11.5 mV to 33.3 ± 12.6 mV (means ± SE, P < 0.05, paired Student's t-test, n = 5) in submandibular acinar cells. In the other 4 submandibular acinar cells, the changes in membrane potential were comparable to those in the forskolin + IBMX-sensitive acinar cells.

Estimation of Cch-induced Cl– secretion in the physiological condition and its suppression by forskolin + IBMX. Cch-induced oscillatory anion current in the gramicidin-perforated patch recording reflects rather physiological Cl– secretion because the current depends on the Na+-K+-2Cl– cotransporter activity and the Cl– channel activity, unlike the current measured with the conventional whole-cell recording and other perforated patch recordings. However, since the membrane potential was held at –80 mV during voltage clamp, the secretion is not completely physiological. Therefore, to take a better estimation of the physiological Cl– secretion, we compensated the current amplitude by multiplying the ratio (r); r = (Vm – VCl–)/(–80 – VCl–), where Vm – VCl– was the physiological driving force between membrane potential (Vm) and the Cl– equilibrium potential (VCl–), and (–80 – VCl–) was the experimental driving force between the holding potential (–80 mV) and VCl– at the voltage clamp mode. VCl– (–21 mV) was calculated from the intracellular Cl– concentration ([Cl–]i; ~50 mM), which was based on data shown in Figs. 2B and 4 of a paper by Foskett (13) and also reported by Tanimura et al. (42) for rat parotid acinar cells during 10 µM Cch stimulation. We hypothesized that rat submandibular acinar cells have the same [Cl–]i at a steady state during Cch stimulation and that [Cl–]i does not change by addition of forskolin + IBMX in the presence of Cch in the acinar cells of both glands. Vm was obtained from oscillatory membrane potential, averaged for the period (about 15 s) from one peak to the other peak of the oscillation, in the current clamp mode during the stimulation. The averaged membrane potential was –75.1 ± 3.4 mV (mean ± SE, n = 7) in parotid and –59.9 ± 4.3 mV (mean ± SE, n = 5) in submandibular acinar cells at 5 min after the Cch addition (Fig. 6, A and B). They depolarized to –73.2 ± 2.5 mV (mean ± SE, n = 7) in parotid and –56.0 ± 3.9 mV (mean ± SE, n = 5) in submandibular acinar cells at 5 min after the addition of forskolin + IBMX. On the basis of these data, we calculated r; 0.92 ± 0.06 (mean ± SE, n = 7) and 0.66 ± 0.07 (mean ± SE, n = 5) in parotid and submandibular acinar cells, respectively, during Cch stimulation, and 0.89 ± 0.04 (mean ± SE, n = 7) and 0.59 ± 0.07 (mean ± SE, n = 5) in parotid and submandibular acinar cells, respectively, during Cch + forskolin + IBMX stimulation (Fig. 6, C and D). Vm during the stimulations in parotid acinar cells was similar to the holding potential, but Vm in submandibular acinar cells depolarized more than the holding potential; r in the submandibular cells was smaller than that of the parotid cells. Finally, the physiological Cl– secretion as Cl– current was estimated as shown in Fig. 6, E and F. The estimated Cl– current was –2.05 ± 0.44 A/F (mean ± SE, n = 7) in parotid acinar cells and –4.48 ± 0.78 A/F (mean ± SE, n = 5) in submandibular acinar cells. It was similar to that measured in voltage clamp at –80 mV in parotid acinar cells and smaller than that measured in voltage clamp at –80 mV in submandibular acinar cells. The estimated current during Cch stimulation in the submandibular glands was significantly larger than that in the parotid gland acinar cells (Fig. 6, E and F; P < 0.05, unpaired Student's t-test). The estimated current was significantly reduced by addition of forskolin + IBMX to –0.75 ± 0.18 A/F (mean ± SE, n = 7) in parotid and to –2.79 ± 0.76 A/F (mean ± SE, n = 5) in submandibular acinar cells (Fig. 6, E and F). We have demonstrated that forskolin + IBMX suppressed the estimated Cch-induced Cl– secretion from both parotid and submandibular acinar cells in the physiological condition.


Figure 6
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  		Fig. 6. Estimation of the physiological Cch-induced Cl– secretory current from the current data obtained in the voltage clamp at –80 mV and the membrane potential data in the current clamp and the effect of forskolin + IBMX on the physiological Cch-induced secretory Cl– current. Solid bars show the data under the voltage clamp at –80 mV, and open bars show the estimated values under the physiological condition. A and B: membrane potential Vm in parotid (A) and submandibular (B) acinar cells. C and D: ratio of Cl– driving force under the physiological condition to that under the voltage clamp at –80 mV in parotid (C) and submandibular (D) acinar cells. E and F: Cl– secretory current normalized by membrane capacitance in parotid (E) and submandibular (F) acinar cells. Estimate of Cch-induced Cl– secretion in the physiological condition is suppressed by forskolin + IBMX in both parotid and submandibular acinar cells (means ± SE, *P < 0.05, **P < 0.01 in paired Student's t-test).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Using the gramicidin-perforated patch recording techniques, we clarified that bumetanide-sensitive oscillatory anion current was mediated by the transcellular Cl– flux that passed through the Na+-K+-2Cl– cotransporter and the Cch-activated Cl– channel connected in series in single exocrine cells. The amplitude of the Cch-induced oscillatory Cl– current in submandibular acinar cells was significantly larger than that in parotid acinar cells even after normalizing the current by membrane capacitance to remove the factor of cell size. These suggest that the amount of secreted Cl– per unit cell membrane area of submandibular acinar cells is larger than that of parotid acinar cells and that it may at least partially contribute to the large amount of stimulated fluid secretion from submandibular glands (40) compared with that from parotid glands (22, 29).

Membrane potential measured in the current clamp mode during Cch stimulation was at the more hyperpolarizing level in parotid acinar cells than that in submandibular acinar cells. Membrane potential of parotid acinar cells during Cch stimulation is very likely closer to the K+ equilibrium potential (about –80 mV) than that of submandibular acinar cells during the stimulation. This is probably due to the larger Cch-induced K+ conductance in parotid acinar than that in submandibular acinar cells (22, 40) since the K+ channel is activated by Ca2+ at a low dose of acetylcholine, which evokes Cl– current, in parotid acinar cells but not in submandibular acinar cells (14, 18). Ca2+-activated K+ channels of parotid acinar cells may contribute to Cl– secretion from the cells more than the channels of submandibular acinar cells by increasing the driving force of Cl–. However, Cl– secretion in submandibular acinar cells per unit membrane area was larger than that in parotid acinar cells under the hyperpolarized membrane potential. The capacity to secrete Cl– via the Na+-K+-2Cl– cotransporter and the Ca2+-activated Cl– channel may be larger in submandibular acinar cells than in parotid acinar cells.

Effects of forskolin + IBMX on Cl– current. In rat parotid and submandibular acinar cells, forskolin + IBMX during Cch stimulation induced two kinds of response: one of the responses to forskolin + IBMX was the induction of nonoscillatory anion current in the voltage clamp mode, which was corresponding to the nonoscillatory depolarization in the current clamp mode, and the other was suppression of Cch-induced oscillatory anion current. AMP-increasing agents in the presence of Cch evoke the nonoscillatory Cl– channel activity in rat submandibular acinar cells (40) but not in rat parotid acinar cells (22). The nonoscillatory Cl– channel activity is suppressed in the hypertonic bathing solution in the conventional whole-cell configuration of rat submandibular acinar cells (40), which suggests that the current may pass through the volume-sensitive Cl– channel (3, 5). This component of Cl– current does not contribute to the salivary secretion in the submandibular glands (40). The presence of functional epithelial Na+ channels has not been reported in the acinar cells, and K+ channels make almost no contribution to ionic current at the holding potential of –80 mV. However, there remains a possibility that some part of the nonoscillatory current may be a cation current through nonselective cation channels, such as P2X4R, a purinergic receptor subtype (7). In this study, cAMP-increasing agents induced the nonoscillatory currents in the presence of Cch at the holding potential, but their physiological nature is not determined.

The other response to forskolin + IBMX was the suppression of Cch-induced oscillatory anion current in the voltage clamp mode, which was corresponding to the suppression of oscillatory depolarization amplitude in the current clamp mode. The consistency in both clamp mode measurements indicates that cAMP-increasing agents modulate the Cch-induced Cl– secretion in the salivary acinar cells. Since the addition of forskolin + IBMX did not change the frequency of the current oscillation, a decrease in the amplitude and/or the duration of each current pulse probably contributed to the suppression.

If the nonoscillatory current was an anion current as discussed above, averaged amplitude of total anion current, the nonoscillatory current and the oscillatory current in the presence of Cch and forskolin + IBMX (–2.34 ± 0.33 A/F, mean ± SE, n = 7) was almost the same as that of total anion current, the oscillatory current only, in the presence of Cch (–2.39 ± 0.58 A/F, mean ± SE, n = 7) in parotid acinar cells. In submandibular acinar cells, the former (–5.72 ± 0.67 A/F, mean ± SE, n = 5) was slightly but significantly (P < 0.05, paired Student's t-test) smaller than the latter (–6.63 ± 0.54 A/F, mean ± SE, n = 5). In the gramicidin-perforated patch recording, Cl– is supplied not by the electrode but by the cells themselves through Cl– transporters. Therefore, in the steady state of Cl– current responses, where the amount of intracellular Cl– does not markedly change, total release of Cl– from the cells through the two kinds of Cl– channels equals uptake of Cl– through Cl– transporters, mainly the Na+-K+-2Cl– cotransporter. Ineffectiveness of forskolin + IBMX on the total current amplitude in parotid acinar cells suggests that the Na+-K+-2Cl– cotransporter activity did not markedly change after the addition of the agents. However, Cl– release from the Ca2+-dependent Cl– channel on the luminal membrane (Cl– secretion), corresponding to the oscillatory anion current, was probably reduced by the agents because the Cl– efflux was divided into the two pathways, nonoscillatory current pathway in the basolateral membrane and the Ca2+-dependent Cl– channel on the luminal membrane. These may be involved in the mechanism of suppression of Cch-induced oscillatory anion current by cAMP-increasing agents in parotid acinar cells. In submandibular acinar cells, the small decrease in total anion current by addition of forskolin + IBMX suggests that Cl– uptake through the Cl– transporter may have been slightly reduced: if the transporter activity was not reduced under the suppression of the total anion current, net Cl– influx would increase the [Cl–]i. This would evoke further depolarization at the peak of Cch-induced oscillatory membrane potential, which reflects the Cl– equilibrium potential rather than that the K+ equilibrium potential due to small K+ currents in submandibular acinar cells (18, 40), but the further depolarization was not observed. The induction of the nonoscillatory current may contribute to the suppression of Cch-induced oscillatory anion current by forskolin + IBMX in submandibular acinar cells, under the condition of the small decrease in total anion current (the reduction of Cl– uptake). Moreover, the conventional whole-cell data in submandibular acinar cells suggest suppression of Cch-induced oscillatory Cl– channel activity by cAMP (40), which also may contribute to the suppression of the oscillatory current. On the other hand, if the nonoscillatory current was a cation current, the total anion current was equal to the oscillatory anion current, which was suppressed by forskolin + IBMX in both parotid and submandibular acinar cells. Then, Cl– uptake through the Cl– transporter may have been reduced.

Suppression of Cch-induced oscillatory anion current by forskolin + IBMX apparently argues against the reports that cAMP-increasing agents potentiate Cch-induced increase of the [Ca2+]i markedly in the parotid acinar cells (7, 8, 22, 41) and slightly (but not significantly) in submandibular acinar cells (40); since the rise of [Ca2+]i activates the Ca2+-dependent Cl– channel (4, 5, 14, 23), the further increase in [Ca2+]i would facilitate Cch-induced Cl– current. In fact, Cch-induced channel activity is often potentiated by cAMP-increasing agents at the negative membrane potential in the conventional whole-cell configuration of parotid acinar cells (22), though the Cl– channel activity of high Ca2+-buffered parotid acinar cells is inhibited by cAMP only at the positive membrane potential through a CFTR-mediated mechanism (34). In the gramicidin-perforated patch recording, Cch-induced oscillatory anion current was suppressed by the agents at the negative membrane potential (the present article). This apparent inconsistency may be explained by following reasons. First, the potentiated increase in [Ca2+]i is rather sustained, whereas an oscillatory increase in [Ca2+]i is suppressed (7, 8), and our present results of current responses in the gramicidin-perforated patch configuration are rather consistent with the [Ca2+]i responses but not with the previous whole-cell recording data (22), possibly due to the difference of intracellular conditions between the two patch clamp methods. Under the condition of the sustained increase in [Ca2+]i, cAMP may activate the volume-sensitive Cl– channel. In submandibular acinar cells, the conventional whole-cell data indicate suppression of Cch-induced oscillatory Cl– channel activity and induction of nonoscillatory Cl– channel activity by cAMP during Cch stimulation (40), which are consistent with our present results in the gramicidin-perforated patch configuration. Second, potentiation of Cch-induced increase in [Ca2+]i by cAMP-increasing agents is often analyzed at the initial transient phase (8), whereas the suppression of Cch-induced oscillatory anion current by the agents was at the steady state in the latter phase; the potentiation of [Ca2+]i response is dramatic in the initial transient phase and it decreases in a few minutes to a steady-state level, which is slightly larger than or not very different from the control level induced by Cch alone (7, 8). We analyzed the current suppression at the steady state 3–5 min after the addition of forskolin + IBMX and therefore, the nonoscillatory (sustained) increase in current responses was not very marked compared with that in the [Ca2+]i responses but was similar to that in the steady-state [Ca2+]i level. Last, the oscillatory anion current measured with the gramicidin-perforated patch recording does not directly reflect the [Ca2+]i; the Cl– current in the gramicidin-perforated patch configuration depends on both the Ca2+-dependent Cl– channel activity and the Cl– transporter activity and is modulated by the [Cl–]i, whereas the Cl–current in the conventional whole-cell configuration reflects the Ca2+-dependent Cl– channel activity but not the Cl– transporter activity, and the [Cl–]i is determined by the Cl– content of the pipette solution. Therefore, the Cl– current in the gramicidin-perforated patch configuration could not keep facilitated without an increase in the Cl– transporter activity, even if the [Ca2+]i and the Cl– channel activity increased.

Ca2+ and cAMP-increasing agents individually facilitate the Na+-K+-2Cl– cotransporter activity in parotid acinar cells (12, 33). However, the effect of the interaction between Ca2+ and cAMP signals on the Cl– transporter activity is unknown. Our present results suggest that the Cl– cotransporter activity may not be affected in parotid acinar cells and may be slightly reduced in submandibular acinar cells by cAMP under the rather high [Ca2+]i in the gramicidin-perforated patch configuration.

Using the gramicidin-perforated patch recording, we have shown that cAMP may exert their modulatory action mainly on Cl– release through Cl– channels in both the acinar cells and partially on Cl– uptake through the Cl– transporter in submandibular acinar cells, though our results suggest the existence of the submandibular acinar cells with different sensitivity to cAMP. It is likely that the Na+-K+-2Cl– cotransporter activity may be suppressed in the cAMP-sensitive submandibular acinar cells and not affected in parotid acinar cells by cAMP under the high [Ca2+]i conditions.

Cch-induced Cl– secretion in the physiological condition and its inhibition by forskolin + IBMX. Our results in the suppression of the oscillatory anion current (Cl– secretion) by forskolin were obtained at –80 mV in the gramicidin-perforated patch recording method, and the membrane potential influences the Cl– secretion as the driving force. The membrane potential in the Cch-stimulated condition somehow depolarized compared with –80 mV in submandibular glands and was almost near to the holding level of –80 mV in parotid glands. Forskolin + IBMX furthermore depolarized the membrane potential in both the gland acinar cells. Under the compensation of the Cl– current in the physiological membrane potential, the Cl– secretion decreased, compared with that measured at –80 mV, but it was still suppressed by forskolin + IBMX in both the acinar cells.

In this study, we clarified the Cl– secretion nature in the salivary acinar cell level, using the gramicidin-perforated patch clamp technique. The salivary secretion is marked in the submandibular glands compared with that in the parotid glands. This is due to the difference in the Cl– secretion capacity in the acinar cell level; larger Cch-induced Cl– secretion capacity in the submandibular acinar cells than in the parotid acinar cells. Our present results on reduction of Cl– secretion are consistent with the previous reports that Cch-induced fluid secretion from perfused salivary glands is suppressed by cAMP-increasing agents (29, 40). Agonist-induced fluid secretion from salivary glands in vivo is potentiated by cAMP-increasing agents (6, 22, 25), though the fluid secretion is reduced by the agents when doses of a muscarinic agonist and/or a cAMP-increasing agent are high (6). In addition, intravenous injection of cAMP-increasing agents induce a transient increase in glandular blood flow (25), whereas the vascular modulation around the salivary glands by parasympathetic nerve is postulated to be involved in the mechanism of the prominent salivation in vivo (15, 24). Taken together, fluid secretion from salivary glands may basically depend on Cl– secretion, and other factors such as the increase in glandular blood flow also may affect the fluid secretion in in vivo experiments, especially at low doses. We clarified that the Cch-induced Cl– secretion is suppressed by cAMP-increasing agents in the acinar cell levels in both parotid and submandibular glands. It is likely that the interaction between Ca2+ and cAMP signals in the acinar cells leads to the suppression of the fluid secretion through the decrease in the Cl– secretion from the salivary gland acini.

Conclusions. We have demonstrated inhibitory regulation of Ca2+-dependent Cl– secretion from rat parotid and submandibular acinar cells possibly through the modulation of the Cl– channel activity and partially the Na+-K+-2Cl– cotransporter activity via the interaction between Ca2+ and cAMP signalings. Suppression of Cl– secretion in the acinar cells is also consistent with the suppression of salivary secretion in the perfused glands (29, 40). It is strongly suggested that the inhibition of Cl– secretion in the acinar cells may contribute to the weaker fluid secretion induced by sympathetic ({alpha}- and β-adrenergic) stimulations than that by parasympathetic stimulation in salivary glands.


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This research was partially supported by Grants-in-Aid for Scientific Research (C) (No. 16591857 and No. 17591939) from Japan Society for the Promotion of Science.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Hirono, Dept. of Oral Physiology, Hiroshima Univ. Graduate School of Biomedical Sciences, 2-3 Kasumi 1-Chome, Minami-ku, Hiroshima 734-8553, Japan (e-mail: chikara{at}hiroshima-u.ac.jp)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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