Introduction

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NO has been shown to be a ubiquitous intracellular messenger and is thought to play an important role in pathways that involve the regulation of [Ca²⁺]_i. NOS, which synthesizes NO, has been discovered in nerve endings from where it diffuses to the surrounding tissue (Alm et al., 1997). Recently, it was reported that NOS is also expressed in acinar and duct cells of pancreatic, submandibular (Xu et al., 1997), and parotid salivary (Tritsaris et al., 2000) glands as well as macrophages and endothelial cells (Forstermann et al., 1995). These findings suggest that NO has an important role in secretory processes.

NO activates the soluble GC (GC-S) and leads to an increase in cGMP synthesis. NO-induced cGMP production initiates a cascade of signaling events. The first step is activation of PKG. This kinase activates ADP-ribosyl cyclase by the phosphorylation of the enzyme, which, in turn, synthesizes the Ca²⁺-mobilizing nucleotide, cADP-ribose, thereby leading to the release of Ca²⁺ from ryanodine-sensitive Ca²⁺ stores in lacrimal acinar cells (Gromada et al., 1995).

The interaction of M₃ muscarinic (M₃) receptors in rat parotid cells with their respective agonists such as acetylcholine, CCh, or MCh results in the activation of PLC followed by the generation of IP₃, which, in turn, leads to the rapid release of Ca²⁺ from intracellular stores and subsequent influx of Ca²⁺ from extracellular sites. [Ca²⁺]_i plays an important role in the regulation of exocytosis induced by muscarinic agonists.

An increase in [Ca²⁺]_i activates Ca²⁺- and CaM-dependent proteins such as CaM kinases and NOS. CaM kinase II is a multifunctional enzyme which, in pancreatic  cells, is required for both granule mobilization under stimulated conditions and the maintenance of secretory capacity under control conditions (Gromada et al., 1999). However, the role of CaM kinase II in amylase secretion from parotid acinar cells has not been clear. The action of NO generated by NOS in signaling pathways is mainly mediated by cGMP and PKG (Clementi, 1998; Watson et al., 1999). Three isoforms of NOS, namely endothelial NOS (eNOS), inducible NOS (iNOS), and neuronal NOS (nNOS), are present in many kinds of mammalian tissue cells. However, it has not been clear which type of isoform of NOS is expressed in rat parotid acinar cells, whether NO is produced by NOS within rat parotid acinar cells in response to the stimulation of M₃ receptors with MCh, and whether the M₃ receptor regulates NOS-PKG signaling in rat parotid acinar cells. The aim of this study is to identify the isoform of NOS present in rat parotid acinar cells and to investigate the possible role of CaM kinase II, NOS, and PKG in exocytosis induced by MCh, an M₃ agonist, in these cells. We indicated in this study that nNOS is endogenously present in rat parotid acinar cells and that the activation of this enzyme, together with CaM kinase II and PKG, is coupled with MCh-induced amylase secretion.

	Experimental Procedures

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Materials. Aprotinin, 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethylester) (BAPTA-AM), 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (carboxy-PTIO), CCh, 4,5-diaminofluorescein diacetate (DAF-2/DA), forskolin, hyaluronidase, leupeptin, MCh, and RPMI 1640 medium were from Sigma-Aldrich (St. Louis, MO). Phorbol 12-myristate 13-acetate (PMA), 1-[6[(17)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]1H-pyrrole2,5 dione (U-73122), hexahydro-sila-difenidol hydrochloride, p-fluoro analog (p-F-HHSiD), 3,4,5-trimethoxybenzoic acid 8-(diethylamino)- octyl ester (TMB-8), 1H-(1,2,4)-oxadiazolo[4,3-a] quinoxaline-1-one (ODQ), N-[2-(N-(4-chlorocinnamyl)-N-methylaminomethyl) phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide (KN-93), and N^G-nitro-L-arginine methylester (L-NAME) were from Funakoshi Co. (Tokyo, Japan). BPDEtide, KT5720, and KT5823 were from Calbiochem-Novabiochem Co. (Darmstadt, Germany). Collagenase was from Worthington Biochemicals (Freehold, NJ). The CaM kinase II assay system was obtained from Invitrogen (Carlsbad, CA). The PKG assay system was from Promega (Madison, WI). L-[³H]Arginine (2.0 TBq/mmol) and [-³²P]ATP (0.37 TBq/mmol) were obtained from PerkinElmer Life Sciences (Boston, MA). Antibrain NOS (or nNOS) antibodies and their respective immunizing peptides were obtained from BIOMOL Research Laboratories, Inc. (Plymouth Meeting, PA).

Animals and Diet. Male Wistar rats (8 weeks old) were used for the experiments. They were provided with a standard laboratory diet (MF; Oriental Yeast, Tokyo, Japan) and water ad libitum and were maintained in a temperature-controlled environment (22 ± 2°C) with a 12-h light/dark cycle (lights on at 6:00 AM) for at least 2 weeks before the experiments. All procedures were approved by the Animal Care Committee of Tokushima University.

Preparation and Incubation of Rat Parotid Tissue Slices and Isolation of Acinar Cells from Rat Parotid Glands. Rat parotid glands were transferred to ice-cold, oxygenated Krebs-Ringer-Tris (KRT) solution consisting of 120 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 1.2 mM MgSO₄, 1.0 mM CaCl₂, 16 mM Tris-HCl (pH 7.4), and 5 mM glucose. Tissue slices (0.4 mm thick) were prepared from the glands with a McIlwain Tissue Chopper (Mickle Laboratory Engineering, Surrey, UK) and were equilibrated with KRT solution for 15 min at 37°C with shaking before the various experimental incubations, which were performed with ~50 mg of tissue slices in a final volume of 10 ml of KRT solution. Pretreatment and treatment of the tissue slices with the various agents were carried out by using KRT solution containing 1.0 mM CaCl₂, except as otherwise indicated in the legends for figures and tables. In some experiments, rat parotid acinar cells were isolated by collagenase and hyaluronidase digestion by a method described previously (Ishikawa et al., 1988) and used for Western blot analysis to identify the isoform of NOS in the isolated parotid acinar cells of rats and for fluorescence study to measure NOS activity in the cells.

Western Blotting for nNOS. Acinar cells isolated from rat parotid glands by the method described above were homogenized on ice in 10 mM Tris-HCl buffer (pH 7.4) containing 255 mM sucrose, 2 mM EDTA, 12 µM leupeptin, 1 µM pepstatin A, 0.3 µM aprotinin, and 1 mM phenylmethylsulfonyl fluoride to perform Western blot analysis for nNOS by the method of Resta et al. (1999). In brief, the homogenate was centrifuged at 1500g at 4°C for 10 min to remove insoluble debris. The supernatant was dissolved and subjected to SDS-polyacrylamide gel electrophoresis in 7.5% linear polyacrylamide gel. After electrophoresis, the protein was electrophoretically transferred from the unstained gel to a nitrocellulose transfer membrane (Hybond ECL; Amersham Biosciences UK, Ltd., Little Chalfont, Buckinghamshire, UK) using a Trans-Blot apparatus (Bio-Rad, Hercules, CA). The blots were probed with anti-nNOS antibody diluted 1:1500 or anti-nNOS antibody preadsorbed with the excess immunizing peptide, followed by incubation for 3 h at room temperature with a horseradish peroxidase-conjugated secondary antibody. Immunodetection was performed according to the enhanced chemiluminescence method (Amersham).

Determination of nNOS Activity in Rat Parotid Tissue Slices and Rat Parotid Acinar Cells. Rat parotid acinar cells isolated from rat parotid glands were incubated in RPMI 1640 medium with 10 µM DAF-2/DA for 30 min at 37°C, which was aerated with 95% O₂/5% CO₂ at pH 7.4. The acinar cells were washed and resuspended in a HEPES-buffered Krebs-Ringer-bicarbonate medium containing 118.46 mM NaCl, 4.74 mM KCl, 1.18 mM KH₂PO₄, 1.00 mM CaCl₂, 1.18 mM MgSO₄, 24.88 mM NaHCO₃, and 5 mM HEPES, pH 7.4, and then suspended to measure NOS activity according to a fluorescence study with DAF-2/DA as described by Tritsaris et al. (2000). The cells were gently stirred in a cuvette maintained at 37°C with or without MCh and the other agents. Changes in the fluorescence, which are generated by the reaction of DAF-2 with NO, were monitored with a fluorescence spectrometer (CF-4000; Hitachi, Tokyo, Japan). The experiments were done with excitation wavelength at 495 nm (5-nm bandwidth) and emission wavelength at 515 nm (5-nm bandwidth). Agents were added to the cuvette to give the final concentration given in the figure.

Assay of CaM Kinase II and PKG Activities in Rat Parotid Tissue Slices. After experimental incubations, the parotid tissue slices were rapidly frozen at 80°C. For measurement of CaM kinase II activity, the frozen slices were homogenized in a solution containing 20 mM Tris-HCl (pH 8.0), 2 mM EDTA, 2 mM EGTA, 20 µg/ml soybean trypsin inhibitor, 10 µg/ml aprotinin, 5 µg/ml leupeptin, 2 mM DTT, 25 mM benzamidine, and 1 mM phenylmethylsulfonylfluoride. The homogenate was centrifuged at 350g for 5 min, and the resulting supernatant was diluted with a solution containing 50 mM Tris-HCl (pH 7.5), 10 mM MgCl_2, 0.1 mM DTT, and 0.1 mg/ml of bovine serum albumin before assay of CaM kinase II activity with a specific assay kit. For measurement of PKG activity, the frozen tissue slice was homogenized in a solution comprising 20 mM HEPES-NaOH (pH 7.5), 10 mM EGTA, 40 mM -glycerophosphate, 1% Nonidet P-40, 25 mM MgCl₂, 2 mM sodium orthovanadate, 140 mM NaCl, 1 mM DTT, and a mixture of protease inhibitors [1 mM Pefabloc, 10 µg/ml aprotinin, and 10 µg/ml leupeptin]. The homogenate was centrifuged for 15 min at 15,000g, and the resultant supernatant was assayed for PKG activity with a specific kit. In brief, 100 µl of the supernatant were added to 50 µl of assay mixture containing 20 mM Tris-HCl (pH 7.4), 200 µM ATP, 100 µM BPDEtide, 20 mM MgCl₂, 100 µM 1-methyl-3-isobutylxanthine, 1 µM 6-22amide, and 0.5 µCi [-³²P]ATP. After incubation for 10 min at 30°C, the reaction was terminated by the addition of 140 µl of ice-cold 10% trichloroacetic acid. The mixture was centrifuged for 5 min at 15,000g to separate proteins, and the resulting supernatant was spotted onto phosphocellulose filters. After removal of unreacted [-³²P]ATP, the filter-associated radioactivity was measured with a liquid scintillation spectrometer to determine the incorporation of ³²P into BPDEtide.

Other Methods. The amount of cGMP in a parotid tissue slice was measured with a radioimmunoassay kit (Yamasa Shoyu, Tokyo, Japan). The amylase activity of the incubation medium was measured as described by Bernfeld (1955) with amylose as the substrate and was expressed as milligrams of maltose produced during incubation for 5 min at 20°C.

Statistical Analysis. Data are expressed as means ± S.E. and unless indicated otherwise were tested for statistical significance with Student's t test. A P <0.05 was considered statistically significant.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Effects of U-73122, TMB-8, Dantrolene, and BAPTA-AM on MCh-Induced Amylase Secretion in Rat Parotid Tissue Slices. MCh induced amylase secretion in a concentration-dependent manner from rat parotid tissue slices in the presence of extracellular calcium; this effect was significant at 100 nM and maximal at 100 µM, with a median effective concentration (1.59 ± 0.1 µM) similar to that of CCh (2.16 ± 0.1 µM) (Fig. 1, left). This secretory response to MCh was rapid, being detectable at 1 min after the addition of agonist (Fig. 1, right). On the basis of these results, a submaximal MCh concentration of 1 or 10 µM was selected for subsequent experiments. As reported by Dai et al. (1991), MCh-induced amylase secretion was completely inhibited by p-F-HHSiD, an M₃ receptor antagonist, but not by methoctramine, an M₂ receptor antagonist, showing that the effect of MCh was mediated by M₃ receptor (data not shown). Exposure of parotid acinar cells to M₃ receptor agonists such as CCh is well established to result in the PLC-mediated generation of IP₃ and the consequent mobilization of Ca²⁺ from intracellular stores (Merritt and Rink, 1987). To investigate the possible role of PLC in the stimulatory effect of MCh on amylase secretion, we exposed rat parotid tissue for 10 min to 10 µM U-73122, a selective inhibitor of PLC (Jørgensen et al., 1995), before incubation with 1 µM MCh. This inhibitor prevented the stimulation of amylase secretion by MCh (Table 1), suggesting that activation of PLC by MCh contributes to Ca²⁺ mobilization from intracellular stores and then to amylase secretion. Incubation of tissue cells with 15 µM TMB-8, a muscarinic receptor antagonist (Ellis and Seidenberg, 2000), also inhibited amylase secretion induced by 1 µM MCh (Table 1). The increase in [Ca²⁺]_i induced by the release of Ca²⁺ through IP₃-gated channels may in turn result in Ca²⁺ release through ryanodine receptors. It was reported that ryanodine and IP₃ receptors were differentially distributed and expressed in rat parotid gland (Zhang et al., 1999) and that dantrolene bound the ryanodine receptors and inhibited Ca²⁺ release through ryanodine receptor channels in skeletal muscle (Zhao et al., 2001). We also reported previously that dantrolene (15 µM) inhibited the increase in the amount of aquaporin 5 water channel in the apical membranes caused by M₃ agonist-induced Ca²⁺ release through ryanodine receptors in rat parotid glands (Ishikawa et al., 1998, 2000). These findings show that dantrolene inhibits Ca²⁺ release through ryanodine receptors. Treatment of rat parotid tissue slices with 15 µM dantrolene inhibited MCh-induced amylase secretion (Table 1). Treatment of tissue with 100 µM BAPTA-AM, a cell-permeable Ca²⁺ chelator, also prevented MCh-induced amylase secretion (Table 1), demonstrating the importance of Ca²⁺ in MCh-induced exocytosis in parotid tissues. Preincubation of tissue slices for 10 min in KRT lacking CaCl₂, before the addition of 1 µM MCh and incubation for 10 min, prevented the effect of MCh on amylase secretion (Table 1), suggesting that increase in [Ca²⁺]_i caused by Ca²⁺ entry from extracellular sites plays an important role in M₃ receptor-mediated stimulation of amylase secretion.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Concentration and time dependence of the effects of MCh and CCh on amylase secretion from rat parotid tissue slices. Tissue slices were incubated in the presence of 1.0 mM CaCl2 for 10 min with the indicated concentrations of MCh or CCh (left), or for the indicated times in the absence or presence of 1 µM MCh or 1 µM CCh (right), after which the activity of amylase released into the incubation medium was measured. Each point represents the mean ± S.E. of values from three to four experiments. , P < 0.05, , P < 0.01, and , P < 0.001 versus corresponding control value.

View this table: [in this window] [in a new window]   TABLE 2 Effects of KN-93, L-NAME, and ODQ on MCh, PMA, or A23187-induced amylase secretion from rat parotid tissue slices Tissue slices were pretreated for 10 min with or without KN-93, L-NIL, or ODQ, and then incubated for 10 min in the additional absence or presence of MCh, PMA, or A23187. The amylase activity of the medium was then determined. Data are means ± S.E. of values from three experiments.

Effects of KN-93, L-NAME, and ODQ on MCh-, PMA-, or A23187-Induced Amylase Secretion in Rat Parotid Tissue Slices. To investigate the mechanisms by which Ca²⁺ promotes amylase secretion, we first examined the possible role of CaM kinase II. Treatment of parotid tissue slices for 10 min with 10 µM KN-93, a selective inhibitor of CaM kinase II, completely blocked amylase secretion induced by either 1 µM MCh or 10 µM A23187 (Table 2). CaM kinase II activity was also assayed by using CaM kinase II biotinylated peptide substrate. As shown in Table 3, addition of MCh (1 µM) induced a 40% increase in this enzyme activity, which was apparent after 10 min. The treatment of the tissue slices with 10 µM KN-93 did not increase CaM kinase II activity (0.111 ± 0.012, 0.152 ± 0.016, and 0.105 ± 0.011 pmol/min/mg protein in the control and MCh-treated tissues in the absence and presence of 10 µM KN-93, respectively). These results thus suggest that the activation of CaM kinase II contributes to the Ca²⁺-mediated secretory responses to MCh and A23187.

Identification of nNOS in Isolated Parotid Acinar Cells of Rats and Effect of MCh on nNOS Protein Level. Rat parotid tissue slices consist of acinar cells, duct cells, epithelial cells, nerve endings, blood vessels, and so on, in which different kinds of isoform of NOS are known to be present. To identify the isoform of NOS in rat parotid acinar cells by Western blot analysis, we prepared enzymatically isolated acinar cells from rat parotid tissues.

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View larger version (17K): [in this window] [in a new window]   Fig. 2.   Western blots for nNOS in isolated parotid acinar cells of rats. Rat parotid acinar cells were incubated without (lane 1) or with MCh at the concentrations of 106 M (lane 2) and 105 M (lane 3) at 37°C for 10 min. After removal of insoluble debris, the proteins were separated on SDS-polyacrylamide gels and then transferred to a nitrocellulose membrane and immunoblotted with anti-nNOS antibody. A typical Western blot of nNOS in parotid acinar cells is shown.

Effects of MCh on NOS Activities in Isolated Parotid Acinar Cells of Rats. To measure NO production by nNOS in rat parotid acinar cells in response to the stimulation with MCh in real time, we used the fluorescent NO indicator DAF-2/DA. The isolated parotid acinar cells of rats were preloaded with 10 µM DAF-2/DA and then challenged with 1 µM MCh. Stimulation with MCh of the parotid acinar cells preloaded with DAF-2/DA induced the generation of increase in DAF-2 fluorescence corresponding to an increase in the NO synthesis in the cells (Fig. 3). To investigate whether the activation of nNOS in the parotid acinar cells is dependent on [Ca²⁺]_i elevated by MCh, the cells were treated with 50 µM BAPTA-AM for 10 min as described in Table 1. Stimulation of the cells with 1 µM MCh in the presence of 50 µM BAPTA-AM did not induce an increase in DAF-2 fluorescence (Fig. 3). Treatment with MCh of the isolated parotid acinar cells under the condition of the absence of extracellular calcium did not induce the generation of increase in DAF-2 fluorescence (data not shown), suggesting that the increase in [Ca²⁺]_i caused by Ca²⁺ entry from extracellular sites upon stimulation with MCh plays an important role in the induction of the increase in DAF-2 fluorescence. When the L-NAME (300 µM)-pretreated acinar cells were treated simultaneously with 1 µM MCh, the increase in DAF-2 fluorescence was not observed (data not shown). These findings demonstrate that NO that was produced by stimulation with MCh reacted with DAF-2 and resulted in a production of triazolofluorescein. In some experiments, NOS activity was also assayed by the method of Bredt and Snyder (1989). MCh (1 µM) induced a rapid increase in NOS activity of parotid tissue slices with an approximately 40% increase apparent after 10 min of the incubation with the agonist (Table 3). This result was consistent with the result described above.

View larger version (21K): [in this window] [in a new window]   Fig. 3.   Effect of MCh on nNOS activity in isolated parotid acinar cells of rats. NOS activity was measured as changes in fluorescence intensity in isolated parotid acinar cells of rat that were preincubated with 10 µM DAF-2/DA for 30 min. Cells were incubated with (b) or without (a) 100 µM BAPTA/AM for 10 min and then with 1 µM MCh in the presence of 1.0 mM CaCl2. Cells that had been incubated with Ca2+-free KRT solution for 10 min were incubated with 1 µM MCh in the continuous presence of Ca2+-free KRT solution (c). Trace is a representative of four to five separate experiments. MCh was applied as indicated by the arrow.

Effect of MCh on cGMP Concentration in Rat Parotid Tissue Slices and the Effects of KT5823, KT5720, or Carboxy-PTIO on MCh- and A23187-Induced Amylase Secretion. Activation of M₃ receptors with MCh on rat parotid acinar cells was shown to lead to activations of nNOS (Figs. 2 and 3) and of GC resulting in a rise in the amount of cGMP (Table 3), suggesting that NO is the link between M₃ receptor activation and cGMP production. To test whether NO can activate GC, we investigated the effect of MCh on cGMP concentration in rat parotid tissue slices. We showed that MCh stimulated cGMP production (Table 3) and amylase secretion (Table 4). MCh-induced amylase secretion was inhibited by 10 µM KT5823, a selective inhibitor of PKG (Cataldi et al., 1999), but not by 10 µM KT5720, a selective inhibitor of cAMP-dependent protein kinase (Cataldi et al., 1999) (Table 4). KT5823 (10 µM) also inhibited A23187-induced amylase secretion (Table 4). The effect of carboxy-PTIO, an NO scavenger, on MCh-induced amylase secretion was also examined. Pretreatment of parotid tissue for 10 min with 0.3 mM carboxy-PTIO completely inhibited MCh- or A23187-induced amylase secretion (Table 5). This NO scavenger did not inhibit isoprenaline-induced amylase secretion (data not shown), showing that it does not affect exocytosis per se. These results show that production of NO induced by MCh stimulated the increase in the accumulation of cGMP and then caused the activation of PKG and resulted in the induction of amylase secretion.

Effects of Atriopeptin and Sodium Azide on Amylase Secretion in Rat Parotid Tissue Slices. To examine further the role of cGMP in modulation of amylase secretion, we investigated the effect of atriopeptin, which binds to a cell surface receptor and stimulates cGMP production by particulate GC, as well as that of sodium azide, which releases NO that stimulates cGMP production by GC-S. Atriopeptin (50 and 100 pmol) and sodium azide (10 and 20 nM) each caused amylase secretion in a concentration-dependent manner (Table 6), demonstrating that the increase in cGMP concentration induced amylase secretion in rat parotid tissues. This finding was supported by the result reported by Watson et al. (1982).

Time Course of Amylase Secretion in Rat Parotid Tissue Slices Induced by Dibutyryl cGMP and MCh. We next compared the time courses of amylase secretion induced by dibutyryl cGMP and MCh to show the sequential transduction of signal in MCh-induced amylase secretion (Fig. 4). The times required to secrete half the amount of amylase released during incubation for 10 min with the respective agent at 20°C were 3.5 and 5.7 min for dibutyryl cGMP and MCh, respectively. These results are consistent with the notion that the signal of the activation of M₃ receptors by MCh is transduced to the activation of nNOS via Ca²⁺-CaM complex and then to the activation of GC-S and finally induced amylase secretion.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Time courses of amylase secretion from rat parotid tissue slices induced by dibutyryl-cGMP or MCh. Tissue slices were incubated with 0.5 mM dibutyryl-cGMP, 1 mM SIN-1, or 1 µM MCh for the indicated times at 20°C, after which the amylase activity in the incubation medium was determined. Data are expressed as a percentage of the amylase secreted after incubation for 10 min with the respective agent and are the means ± S.E. of values from three experiments. Statistical significance was assessed by analysis of variance: F ratio = 119.8, P < 0.01 (at 3 min); and F ratio = 150.4, P < 0.001 (at 6 min).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

We have shown that MCh acts at M₃ receptors on rat parotid tissue slices to induce rapidly amylase secretion in a concentration-dependent manner (Fig. 1). MCh-induced amylase secretion was completely blocked by U-73122 and TMB-8 (Table 1). The activation of M₃ receptors on parotid tissue slices also induces the mobilization of Ca²⁺ from intracellular stores as a result of the generation of IP₃ by PLC (Wang et al., 1994; Ishikawa et al., 1998). In the present study, the Ca²⁺ ionophore A23187 stimulated amylase secretion in the presence of extracellular Ca²⁺ (Tables 2 and 4). Exposure of rat parotid tissue slices to dantrolene or BAPTA-AM completely inhibited MCh-induced amylase secretion (Table 1). Incubation with MCh of the parotid tissue slices in the absence of extracellular Ca²⁺ did not induce amylase secretion (Table 1). These observations suggest that the stimulatory effect of MCh on amylase secretion from the rat parotid tissue slices depends on the increase in [Ca²⁺]_i caused by the release of Ca²⁺ from intracellular storage sites and Ca²⁺ entry from extracellular sites.

CaM is an important effector of Ca²⁺ signaling in many mammalian cell types, and the Ca²⁺-CaM complex activates various protein kinases (Babb et al., 1996; Möhling et al., 1997; Gromada et al., 1999) including the multifunctional enzyme CaM kinase II. This enzyme is expressed in many mammalian cell types, and it is activated on exposure of insulinoma to muscarinic agonists (Babb et al., 1996). We have now shown that MCh induces the activation of CaM kinase II in rat parotid tissue slices (Table 3). The selective CaM kinase II inhibitor KN-93 also inhibited completely amylase secretion induced by MCh or A23187 but had no effect on that induced by PMA (Table 2), suggesting that activation of CaM kinase II contributes to the stimulatory action of MCh and A23187 on exocytosis. In mouse pancreatic  cells, CaM kinase II is thought to be required for both granule mobilization by acetylcholine and the maintenance of secretory capacity under control conditions (Gromada et al., 1999). Treatment of parotid tissue slices with L-NAME, a selective inhibitor of nNOS, or with ODQ, a selective inhibitor of GC-S, completely inhibited MCh- or A23187-induced amylase secretion (Table 2). We performed Western blot analysis by using anti-nNOS antibody and showed that nNOS is localized in isolated parotid acinar cells of rats (Fig. 2). nNOS was also detected in abundance in the parotid acinar cells incubated with 10⁶ and 10⁵ M MCh for 10 min. This finding suggests that nNOS is expressed in rat parotid acinar cells. To measure NO production in the acinar cells in response to the stimulation with MCh in real time, the isolated rat parotid acinar cells were incubated with DAF-2/DA. As shown in Fig. 3, it was revealed that stimulation with MCh of isolated parotid acinar cells of rats induced a fast increase in DAF-2 fluorescence corresponding to an increase in the NO synthesis in parotid acinar cells. These findings demonstrate the presence of endogenous nNOS in rat parotid acinar cells and a large and rapid increase in nNOS activity upon stimulation of M₃ receptors with MCh in the acinar cells. This addresses not only the potential role of NO release in the parotid acinar cells for fluid formation, but also its function as a signaling element for the tissue surrounding the glands under in vivo conditions. As shown in Fig. 3, incubation with MCh of the isolated parotid acinar cells in the presence of BAPTA-AM did not induce an increase in DAF-2 fluorescence. Incubation of the isolated parotid acinar cells with MCh in the absence of extracellular Ca²⁺ did not also induce an increase in DAF-2 fluorescence, showing the importance of Ca²⁺ entry from extracellular sites on MCh-induced increase in nNOS activity in rat parotid acinar cells. Exposure of parotid tissue slices to MCh also induced an increase in the conversion of L-[³H]arginine to L-[³H]citrulline (Table 3). Both atriopeptin, which activates particulate GC, and sodium azide, which releases NO to stimulate GC-S (Shahidullah and Wilson, 1999), induced amylase secretion from parotid tissue (Table 6), supported by the result that cGMP mediates amylase secretion from mouse parotid acini (Watson et al., 1982). We also showed that MCh induced the activation of PKG (Table 3) and that KT5823, a selective inhibitor of PKG, abolished the stimulatory effects of MCh and A23187 on amylase secretion, but not KT5720, a selective inhibitor of cAMP-dependent protein kinase (Table 4).

It is of importance and relevance to show the sequential transduction of signal in the induction of amylase secretion by MCh in parotid acinar cells. We reported previously that short-term treatment of rat parotid tissue slices with IPR for less than 10 min resulted in supersensitivity of amylase secretion from the tissue slices, but such treatment for more than 20 min resulted in desensitization (Hata et al., 1983), and that such short-term treatments of rat submandibular tissue slices with IPR (Ishikawa et al., 1995) and of rat parotid tissue slices with histamine (Eguchi et al., 1998) induced only desensitization of mucin and amylase secretion from the tissue slices, respectively. These phenomena were accompanied by alterations in the number of -adrenoceptors or histamine H₂ receptors in the tissue slices and in the affinity of these receptors for their agonists but no changes in adenylate cyclase activity. For the purpose of studying the mechanisms responsible for the regulation of signal transduction coupled with the exocytosis from the tissue slices, we compared the time course of the phosphorylation of Gi2 and amylase secretion in the tissues and demonstrated that the onset of the IPR-induced changes in the phosphorylation level of Gi2 preceded temporally the initiation of amylase secretion (Amano et al., 1996). By using the same method, we compared the time required to secrete half the amount of amylase from rat parotid tissue slices during the incubation for 10 min with MCh and dibutyryl cGMP. The time required for dibutyryl cGMP was shorter than that for MCh (Fig. 3), suggesting that the signal of the activation of M₃ receptors by MCh in rat parotid acinar cells was transduced to the activation of nNOS via Ca²⁺-CaM complex and then to the activation of GC-S and finally induced amylase secretion.

It is most interesting to identify possible substrates for CaM kinase II and PKG. CaM kinase II phosphorylates the synapsin I-like protein identified in MIV6 insulinoma cells (Matsumoto et al., 1995). Phosphorylation of synapsin I by CaM kinase II in neurons results in the dissociation of synaptic vesicles from the cytoskeleton and thereby facilitates vesicle translocation and fusion with the plasma membrane (Llinäs et al., 1991). Hormones that increase in the intracellular concentration of cAMP also induce phosphorylation of synapsin-like protein in pancreatic  cells (Gromada et al., 1998). Organized granule movement toward a specific region of pancreatic  cells was also shown to require Ca²⁺- and CaM-dependent phosphorylation of myosin light chain (Niwa et al., 1998). CaM kinase II associates with secretory granules in insulinoma cells (Möhling et al., 1997), and substrates for this enzyme include a subunit of tubulin (Colca et al., 1983), microtubule-associated protein 2 (Krueger et al., 1997), and myosin light chain (Niki et al., 1993), suggesting the possibility that CaM kinase II regulates various aspects of the interaction between secretory granules and the cytoskeleton. As to the substrate for PKG, it was recently reported that norepinephrine bound to a novel adrenergic receptor in the chick ciliary ganglion activated GC and that the increase in accumulation of cGMP activated PKG, which might phosphorylate a target protein involved in the exocytosis of synaptic vesicles (Yawo, 1999). The septins are known to be a family of GTPase, some of which are required for the cytokinesis stage of cell division and others of which are associated with exocytosis. A new class of brain-specific septin was purified and cloned and was a substrate for PKG (Xue et al., 2000). Further experiments are necessary to identify the substrates for CaM kinase II and PKG that associate with the exocytosis in rat parotid acinar cells.

In summary, we indicated here that endogenous nNOS was present in rat parotid acinar cells and that rapid increases in CaM kinase II and nNOS activities contributed to amylase secretion induced by MCh.