Water Channels and Zymogen Granules in Salivary Glands

Yasuko Ishikawa1,*, Gota Cho2, Zhenfang Yuan3, Mariusz T. Skowronski4, Yan Pan5, and Hajime Ishida1,# Departments of 1Pharmacology and 2Dental Anesthesiology, The University of Tokushima School of Dentistry, 3-18-15 Kuramoto-cho, Tokushima 770-8504, Japan Departments of 3Internal Medicine and 5Pharmacology, Peking University First Hospital, Xishiku Street, Xicheng District, Beijing, 100034, China 4Department of Animal Physiology, Faculty of Biology, University of Warmia and Mazury, Oczapowskiego 1A 10-718 Olsztyn, Poland

Received April 5, 2006

Abstract. Salivary secretion occurs in response to stimulation by neurotransmitters released from autonomic nerve endings. The molecular mechanisms underlying the secretion of water, a main component of saliva, from salivary glands are not known; the plasma membrane is a major barrier to water transport. A 28-kDa integral membrane protein, distributed in highly water- permeable tissues, was identified as a water channel protein, aquaporin (AQP). Thirteen AQPs (AQP0 – AQP12) have been identified in mammals. AQP5 is localized in lipid rafts under unstimulated conditions and translocates to the apical plasma membrane in rat parotid glands upon stimulation by muscarinic agonists. The importance of increases in intracellular calcium 2+ concentration [Ca ]i and the nitric oxide synthase and protein kinase G signaling pathway in the translocation of AQP5 is reviewed in section I. Signals generated by the activation of Ca2+ mobilizing receptors simultaneously trigger and regulate exocytosis. Zymogen granule exocytosis occurs under the control of essential process, stimulus-secretion coupling, in salivary glands. Ca2+ signaling is a principal signal in both protein and water secretion from salivary glands induced by cholinergic stimulation. On the other hand, the cyclic adenosine mono- phosphate (cAMP)/cAMP-dependent protein kinase system has a major role in zymogen granule 2+ exocytosis without significant increases in [Ca ]i. In section II, the mechanisms underlying the control of salivary protein secretion and its dysfunction are reviewed.

Keywords: aquaporin, lipid raft, zymogen granule, supersensitivity, desensitization

Introduction...... 496 4. Disorders of AQP5 in parotid glands: age- or I. Water secretion from salivary glands ...... 496 diabetes-related xerostomia...... 500 1. Aquaporin (AQP) water channel proteins in salivary II. Protein secretion from salivary glands ...... 500 glands ...... 496 1. Role of salivary gland membrane receptors in 1) Structure of AQP5 water channel protein secretion ...... 500 2) Localization of AQP5 with lipid rafts in rat 1) Exocytosis regulated by a membrane receptor parotid gland cells system in salivary glands 2. Translocation of AQP5 with lipid rafts to the apical 2) Exocytotic machinery in salivary glands plasma membrane (APM) and its dissociation to 3) Intracellular signals in stimulation-secretion non-rafts within the APM ...... 498 coupling in salivary glands 3. Intracellular signals for the translocation of AQP5 2. Altered protein secretion in salivary glands...... 506 toward the APM...... 498 1) Functional alterations of membrane receptors in salivary glands 2) Post-receptor mechanisms underlying *Corresponding author. [email protected] # the supersensitivity and desensitization of protein Professor Emeritus at the University of Tokushima secretion in salivary glands Published online in J-STAGE Concluding remarks ...... 507 DOI: 10.1254/jphs.CRJ06007X

Invited article

495 496 Y Ishikawa et al

Introduction delivered to the plasma membranes for the secretion of various substances. Mammalian salivary glands are composed of the Saliva has important roles in the function of the oral major paired glands, parotid, submandibular, and sub- cavity and the protection of the oral mucosa and lingual glands, and hundreds of minor salivary glands gastrointestinal epithelium. Saliva in the oral cavity scattered over most of the oral mucosa. All these functions in mastication, digestion, swallowing, speech, salivary glands are innervated by the autonomic nervous taste, denture holding, and it has anticaries activity, system. Parasympathetic nerves innervate all of the antihalitosis activity, and anti-infective activity. Based major and minor salivary glands, demonstrating that on the presence of secretory immunoglobulin (Ig) A, parasympathetic innervation has a role in the control of IgG, IgM, lysozyme, peroxidase, lactoferrin, histatin, the salivary gland function. Parasympathetic nerves that epidermal growth factor, fibroblast growth factor, nerve innervate the submandibular and sublingual glands are growth factor, and endorphin in saliva, saliva also derived from the superior salivary nucleus, whereas functions in protection of the oral mucosa and gastro- parasympathetic nerves that innervate the parotid glands intestinal epithelium. On the other hand, the molecular are derived from the inferior salivary nucleus. Sympa- mechanisms underlying the secretion of water from the thetic nerves innervate the parotid and submandibular salivary gland cells are not clarified; the presence of glands and are derived from the superior cervical plasma membranes is a major barrier to water transport. ganglion. Activation of M3 muscarinic acetylcholine Recently, aquaporins (AQPs) were discovered as water receptors (M3 mAChRs) by ACh released from para- channel proteins (12 – 16). There have been many sympathetic nerve endings and pilocarpine and cevime- efforts to study the molecular basis of water movement. line produces the largest increase in fluid flow rates in The various roles of saliva are obvious in the clinical salivary glands and a modest increase in salivary protein problems of patients with reduced salivary flow rate, secretion (1). Norepinephrine released from sympathetic xerostomia. In this review, we focus on studies demon- nerve endings acts at both α- and β-adrenoceptors (β- strating the molecular mechanisms underlying the ADRs) in parotid and submandibular glands. Activation secretion of water and proteins from salivary glands. For of β-ADRs induces the secretion of the salivary proteins descriptions of the molecular mechanisms underlying contained in zymogen granules by exocytosis, whereas the secretion of electrolytes from the glands, see the activation of α-ADRs increases modest fluid secretion reviews by Nauntofte (10) and Turner (11). (2). Salivary glands also secrete saliva as resting saliva even under the resting conditions. Dopamine (3), I. Water secretion from salivary glands substance P (4), vasoactive intestinal peptide (5), histamine (6), bradykinin (7), γ-aminobutyric acid (8), and 1. Aquaporin water channel proteins in salivary benzodiazepine (9) act at their respective receptors and glands contribute to regulate salivary secretion. Acinar cells in Water is the main component of saliva, but the the salivary glands generate and secrete primary saliva molecular mechanisms by which water is secreted from containing isotonic plasma-like electrolyte concentra- the salivary gland cells are unknown; the plasma tions, proteins in secretory granules, and water. The membrane is a major barrier to water transport. In 1988, primary saliva is subsequently modified by reabsorption a 28-kDa integral protein was discovered as a water and secretion of electrolytes and water as it passes channel (12, 13). This protein is now known as AQP1. along the duct, resulting in the generation of a hypotonic A number of AQPs that selectively transport water solution, saliva (10, 11). Amylase and mucin, major across the plasma membrane have been cloned in a salivary proteins, are synthesized in the parotid and variety of mammalian cells. AQPs have homology with sublingual glands, respectively, and both the proteins are the major intrinsic proteins of the lens (14) and thirteen synthesized in the submandibular glands. Salivary pro- members of the AQP family, AQP0 – AQP12, have teins are stored in zymogen granules in the acinar cells been identified from many mammalian cells (15, 16). of these salivary glands and secreted by exocytosis, the The AQP family consists of three subsets: aquaporins fusion of zymogen granules with the apical plasma (AQP0, AQP1, AQP2, AQP4, AQP5, AQP6, and membranes (APMs) of the salivary glands. Zymogen AQP8); aquaglyceroporins (AQP3, AQP7, AQP9, and granule exocytosis in the salivary glands is the called AQP10); and superaquaporins (AQP11 and AQP12) regulated exocytosis, because it is tightly regulated by (15, 16). Among these members of the AQP family, intracellular signals produced via plasma membrane AQP1, AQP3, AQP4, AQP5 (17), and AQP8 (18, 19) receptors, and can be distinguished from the unregulated are present in mammalian salivary glands. Hybridization exocytosis in which integral membrane proteins are studies indicate that AQP1, AQP3, and AQP5 mRNA Salivary Water and Protein Secretion 497 are present at significant levels, but AQP4 is not salivary glands results in a 2- and 3-fold increase in detected (17). AQP homologues are localized in salivary secretion compared with secretion from glands different cell membranes including various intracellular exposed to control virus (27). AQP4, a 28-kDa protein, organelles in many kinds of cells. For example, AQP5 is is expressed in brain and parotid and submandibular expressed in the APM, the lateral membrane, and glands (17). AQP4 is not detected, however, in immuno- intracellular organelles, but not in the basilar membrane fluorescence microscopy images of salivary gland cells of salivary gland cells (20, 21). AQP5 is also present in (17). AQP8 is also a 28-kDa protein, and it is abundant zymogen granules in rat parotid glands (22). AQP5 in the testis (28), pancreas, and liver (19) and is mRNA is abundantly present in rat serous acinar cells expressed in the myoepithelial cells of rat submandi- of exocrine glands such as the parotid and submandibular and parotid glands (18). These findings indicate bular glands, lacrimal glands, and subepithelial glands of that AQP5 has a significant role in water secretion from the upper airway, and eye, but not in kidney, brain, or serous acinar cells and interlobular duct cells of parotid intestine (23). Confocal immunofluorescence micro- and submandibular glands. scopy revealed AQP5 fluorescence not only in the acinar cells, but also in the interlobular duct cells in 1) Structure of AQP5 water channel rat parotid glands (21). The AQP5 cDNA, which Hydropathy analysis of the deduced amino acid encodes a 265-residue polypeptide, was isolated from sequence of AQP5 predicted a protein with a six trans- rat submandibular glands using a homology-based membrane domain (12, 23, 25, 29, 30) (Fig. 1). The cloning approach by Raina et al. (23). In vitro transcrip- amino acid sequence contains two tandem repeats tion and translation of this cDNA yielded a 27-kDa corresponding approximately to the NH2-terminal and polypeptide. Expression of the corresponding cRNA in COOH-terminal halves of a single polypeptide. There Xenopus oocytes resulted in a 20-fold increase in water are three extracellular loops (loop A, loop C, and loop E) permeability. The increase in water permeability was and two intracellular loops (loop B and loop D). The reversibly inhibited by HgCl2, but did not enhance two halves of each AQP share substantial sequence membrane transport of urea or glycerol. In AQP5 similarity, but are oriented in the plasma membrane in knockout mice, although protein secretion from salivary such a manner that corresponding regions are located on glands and amylase activity in the saliva were not opposite sides of the membrane. Both loop B and loop E, affected, the body growth rate was reduced by 20% and which contain the Asp-Pro-Ala (NPA) sequence that is pilocarpine-stimulated saliva production was reduced characteristic of AQPs and are intracellular and extra- by more than 60% compared with those in wild-type cellular, respectively, are critical for the formation of mice (24). AQP1 is a 28-kDa protein that was first functional water-selective pores. In polarized mamma- isolated from human red blood cells (25). Antibodies to lian cells, proteins anchored to the cell membrane by AQP1 react with endothelial cells and erythrocytes in glycophospholipids are generally located at the APM capillaries and venules within the salivary glands, but (31). Glycosylation might, therefore, have an important not with the gland cells themselves (26). Adenovirus- role in the function of integral membrane proteins. mediated transfer of AQP1 cDNA to rat irradiated AQP5, AQP2, AQP3, and AQP4 each contain an N-

Fig. 1. Proposed membrane topology of AQP5. The topology is based on the sequence analysis and the hourglass model proposed by Raina et al. (23). The AQP molecule is thought to consist of six transmembrane domains and five connecting loops (loop-A, -B, -C, -D, and -E). The aqueous pore is thought to form when loop- B and -E fold together into an hourglass. There are characteristic Asp-Pro-Ala (NPA) motifs in loop-B and -E. Loop-C contains N-glycosylation site (N) and loop-E does mercury-sensitive site (C). This figure is reproduction with permission from ref. 1. 498 Y Ishikawa et al glycosylation site in loop C (23), whereas AQP1 con- conditions. tains an N-glycosylation site in loop A. Elimination of the N-glycosylation site in AQP1, however, has no effect 2. Translocation of AQP5 with lipid rafts to the APM on the trafficking of this protein when expressed in and its dissociation to non-rafts within the APM Xenopus oocytes (25). AQP5 contains a mercury-sensi- Treatment of rat parotid tissues with the M3 agonists tive cysteine residue at position 182, which is located ACh for 15 s (44) and SNI-2011 (cevimeline) for 10 min just upstream of the NPA sequence in loop E (23, 32, (45) or the α1 agonists epinephrine and phenylephrine for 33). Treatment of Xenopus oocytes expressing AQP5 1 min (46) increases the amount of AQP5 in the APM with 1 mM HgCl2 results in a marked decrease in water and decreases in the amount of AQP5 in the intracellular permeability, which is reversed by incubation with β- membranes (ICM) in the tissues, indicating that M3 mercaptoethanol (23, 32, 33). The AQP5 amino acid agonists and α1 agonists induce the translocation of sequence also contains potential phosphorylation sites AQP5 from the ICM to the APM. Immunoelectron for cAMP-dependent protein kinase (PKA) (23, 32, 33), microscopy of parotid glands of rats with cevimeline but not protein kinase C (PKC) and casein kinase II (34). injected into the tail vein revealed that the number of gold particles marking AQP5 markedly increases in the 2) Localization of AQP5 with lipid rafts in rat parotid APM and decreases in the cytoplasm in the interlobular gland cells duct cells (21). Confoal immunofluorescence micro- Cholesterol- and glycolipid-enriched microdomains, scopy also revealed that intravenous injection of commonly known as lipid rafts, have been suggested to cevimeline induces trafficking of the AQP5 immuno- be involved in a number of cell functions such as fluorescence together with flotillin-2 and GM1 between membrane sorting and trafficking (35 – 39), receptor the APM and the cytoplasm in interlobular duct cells of signaling (40), and cholesterol homeostasis (41). For rat parotid glands (21). The distribution of AQP5 was example, transcytosis of IgA and exocytosis of newly- compared with that of F-actin which is known to locate made brush-border proteins in enterocytes occur through beneath the APM (Fig. 2). Under control conditions, an apical lipid raft-containing compartment (42). In AQP5 was predominantly located outside of F-actin. addition, the involvement of several other protein Within 10 min of the injection of cevimeline, AQP5 families is implicated in structural and functional translocated towards the luminal side. After 10-min modifications of lipid rafts (43). These reports suggest treatment of rat parotid tissues with cevimeline, AQP5 that lipid rafts are involved in sorting some apical levels decrease in the 1% Triton X-100-insoluble resident proteins. Whether AQP5 is located in lipid rafts fraction and increase in the Triton X-100-soluble in rat parotid gland cells was examined using flotillin-2, fraction (21), but AQP levels in the tissues treated a lipid raft-associated integral membrane protein, and without cevimeline did not show these changes in GM1 ganglioside, a glycosphingolipid, as markers of detergent solubility (21), showing the dissociation of lipid rafts (38). Confocal immunofluorescence micro- AQP5 from lipid rafts to non-rafts within the APM. scope images revealed that under resting conditions, These results revealed that cevimeline induced the AQP5 colocalizes with flotillin-2 and GM1 in the translocation of AQP5 with lipid rafts to the APM and cytoplasm of interlobular duct cells of rat parotid glands, the dissociation of AQP5 from lipid rafts within the indicating that AQP5 is located in lipid rafts (21). This APM. These findings are also supported by the results finding was further supported by the fact that under from fractionation studies using sucrose or Opti-Prep resting conditions, AQP5 in rat parotid gland tissues is density gradients (21). fractionated in sucrose density gradients to the same fractions containing flotillin-2 and GM1 (21). Lipid rafts 3. Intracellular signals for the translocation of AQP5 float to the lighter density fractions in an Opti-Prep towards the APM discontinuous density gradient (21). After separation of The M3 agonist- or α1 agonist-induced increases in the the homogenate of parotid tissues of control rats on the amount of AQP5 in the APM in rat parotid tissue cells is Opti-Prep gradient, the amount of AQP5 was the largest inhibited by TMB-8 and dantrolene, calcium release in the lighter fractions, supporting the finding from inhibitors from intracellular stores (44 – 47); BAPTA- unstimulated conditions, in which AQP5 is located with AM, a cell permeable calcium chelator (45); or U73122, lipid rafts in rat parotid gland cells. This result was also an inhibitor of phospholipase C (PLC) (46), but not by confirmed by the finding that AQP5 in unstimulated H-7 or GF 109203X (46), PKC inhibitors; or phorbol parotid tissues of rats fractionates to the 1% Triton X- 12-myristate13-acetate, an activator of PKC (45). 100-insoluble fraction. Thus, AQP5 localizes in lipid Conversely, A23187, a calcium ionophore (44), alone rafts in the cytoplasm of parotid gland cells under resting increases the amount of AQP5 in the APM and decreases Salivary Water and Protein Secretion 499

Fig. 2. Changes in confocal immunofluorescence microscope images of tissue slices showing AQP5 and F-actin in interlobular ducts of parotid glands of rat injected with cevimeline. Parotid glands were obtained from rats injected with physiological saline (A) and cevimeline (5.0 mg/kg, B and C). At 10 (B) and 60 (C) min after injection, the glands were embedded. The section was immunostained to detect AQP5 with Alexa Fluor 488 (green) and stained with rhodamine-phalloidin (red). Bars, 10 µm. the amount of AQP5 in the Triton X-100-insoluble in the APM in parotid glands. Intravenous injection of fraction (21). These findings indicate that an increase A23187 into rats increases AQP5 levels in the APM 2+ in the intracellular calcium concentration ([Ca ]i) by the and, moreover, decreases in the amount of AQP5 in the activation of inositol-1,4,5-trisphosphate (IP3) receptors 1% Triton X-100-insoluble fraction (21). These findings 2+ and ryanodine receptors with IP3 and cADP ribose, indicate that the increase in [Ca ]i mediates the effect of respectively, via M3 mChRs or α1-ADRs in rat parotid M3 agonists on the translocation of AQP5 with lipid rafts gland cells causes the translocation of AQP5 towards the from the cytoplasm to the APM and subsequently the APM. Exposure of isolated rat parotid acinar cells to dissociation of AQP5 from lipid rafts to non-rafts within 2+ 2+ ACh or cevimeline induces a rapid increase in [Ca ]i the APM. The site of action of Ca for the translocation with marked oscillations (45, 47). This increase and dissociation of AQP5 in parotid gland cells, coincides with the increased AQP5 levels in the APM in however, is not clear. rat parotid gland cells. ML-9, an inhibitor of myosin In Ca2+-mediated intracellular signal transduction, an 2+ light chain kinase (MLCK), which is identified in increase in [Ca ]i has an important role in the activation parotid glands and regulates capacitative Ca2+ entry, of Ca2+/calmodulin (CaM)-dependent proteins such as inhibits the ACh- and pilocarpine-induced increase in CaM kinases, MLCK, and nitric oxide (NO) synthase the AQP5 levels in the APM in rat parotid tissue cells (NOS). Cevimeline increases CaM kinase II activity in 2+ (47), suggesting that the increase in [Ca ]i caused by the rat parotid glands (48). CaM kinase II is a multifunc- release of Ca2+ from intracellular stores and by the tional enzyme that is required for both granule mobili- capacitative Ca2+ entry into the cells is necessary for zation under stimulating conditions and maintenance of the maximum effect of ACh on the increase of AQP5 secretory capacity under resting conditions in pancreatic 500 Y Ishikawa et al

β cells (48). MLCK regulates capacitative Ca2+ entry parotid acinar cells from young adult (10-week-old) and (49) and is involved in Ca2+-dependent secretion of senescent (110-week-old) rats treated with ACh or insulin (50) and rennin (51). NO increases cGMP forma- epinephrine using DAF-2/DA are coincident with those tion through the stimulation of soluble guanylate cyclase in the responsiveness of AQP5 in the cells of these (GC-s) (52, 53). The possible roles of CaM kinase II, rats to the agonists (55). Confocal immunofluorescence NOS, MLCK, and PKG in the regulation of AQP5 microscopy images revealed that under unstimulated function were investigated to clarify the molecular basis conditions, AQP5 fluorescence localizes in a diffuse of water movement across biologic membranes in pattern in the cytoplasm parotid interlobular duct cells of parotid gland cells (47). Neuronal NOS (nNOS) is both young adult and senescent rats. Under stimulated expressed in isolated parotid acinar cells, and endothelial conditions with cevimeline, there is an increase in the and inducible NOS are not (54). M3 agonist-induced fluorescence for AQP5 in the cells of young adult rats, increases in the amount of AQP5 in the APM in rat but not senescent rats (55, 61). These findings indicate parotid gland cells is inhibited by ML-9, an inhibitor of that the age-related impairment in the responsiveness of MLCK; carboxy-PTIO, an NO scavenger; or KN-62, an AQP5 in rat parotid gland cells to muscarinic stimula- inhibitor of CaM kinase II (47). The NO donors, SIN-1 tion might account for the concomitant changes in NOS or SNAP, increase the amount of AQP5 in the APM; activity in the cells, and might induce age-related and the NO donor-induced increase is inhibited by xerostomia. To study the mechanisms underlying KT5823, an inhibitor of PKG, and L-Nil, an nNOS diabetic xerostomia, changes in the distribution of AQP5 inhibitor (47). The increase in the amount of AQP5 in in parotid gland cells of control and streptozocin- the APM is induced by treating rat parotid tissues induced diabetic (diabetic) rats were investigated after with dibutyryl cGMP, but not with dibutyryl cAMP (47). intravenous injection of cevimeline. Confocal immuno- Studies using the NO probe, 4,5,-diaminofluorescein fluorescence microscopy revealed that cevimeline- (DAF-2)/diacetate (DA), revealed that NOS activity induced translocation of AQP5 with lipid rafts to the increases rapidly after treating isolated rat parotid acinar APM from the cytoplasm in the parotid interlobular duct cells with an M3 agonist (54, 55). These findings suggest cells of control rats is not observed in diabetic rats (61). that NO/cGMP signal transduction has a crucial role in The decreased solubility of AQP5 by 1% Triton X-100 2+ Ca homeostasis in the M3 agonist-stimulated increase observed in parotid gland cells of control rats is also not in AQP5 in the APM of rat parotid glands. AQP1 has a observed in diabetic rats. These findings indicate that cyclic nucleotide-binding domain in the C-terminus cevimeline-induced translocation of AQP5 with lipid (56), and PKG phosphorylates the C-terminal residue in rafts to the APM and the dissociation of AQP5 from AQP2 and increases the insertion of AQP2 into renal lipid rafts to non-rafts within the APM in parotid gland epithelial cells (57). The site of action of PKG on AQP5 cells is impaired in diabetic rats. The amount of AQP5 in parotid glands, however, remains unknown. in parotid glands in diabetic rats does not decrease in comparison with control rats. In contrast, the amount of 4. Disorders of AQP5 in parotid glands: age- or M3 receptors decreases 50% in parotid glands of diabetic diabetes-related xerostomia rats. NOS activity, as measured using DAF-2/DA, in Great importance has been attached to AQPs as a isolated parotid acinar cells from diabetic rats is not cause of human adult diseases. Diabetic patients and increased compared to that in control rats, suggesting a aged people often complain of xerostomia, which is decrease in the release of Ca2+ from intracellular Ca2+ characterized by oral dryness and difficulty in perform- stores by IP3 synthesized through the activation of M3 ing oral functions (58 – 60). The mechanisms underlying mAChRs with their respective agonists. Finally, there is 2+ xerostomia are not known. The stimulatory effect of a decline in Ca signaling via M3 mAChRs in salivary ACh on AQP5 levels in the APM of parotid tissue glands in diabetic xerostomia (61). cells of rats decreases markedly during aging, but this decrease is not observed in the effect of epinephrine on II. Protein secretion from salivary glands AQP5 levels in the APM in the glands (55). The amounts of AQP5, M3 mAChRs, IP3, and Gq/11α protein do not 1. Role of salivary gland membrane receptors in decrease in rat parotid glands during aging (55). This protein secretion finding indicates a marked decrease in the responsive- 1) Exocytosis regulated by a membrane receptor system ness of AQP5 in parotid gland cells of senescent rats to in salivary glands cholinergic stimulation, but not to adrenergic stimula- The salivary proteins amylase and mucin are synthe- tion. sized in acinar cells and stored in zymogen granules in The age-related changes in NOS activities in isolated the cells of parotid and sublingual glands, respectively. Salivary Water and Protein Secretion 501

Submandibular gland acinar cells synthesize both these proteins and store them in the granules. Activation of M3 mAChRs (1, 49, 54), β2-ADRs (2, 62 – 66), H2 histamine receptors (6), NK1 (4), and vasoactive intestinal peptide (VIP) (5) receptors on the basolateral plasma membrane (BLM) in salivary glands with their respective agonists induces marked secretion of proteins in zymogen granules by exocytosis. The fusion of zymogen granules, which are mostly present in the apical region in acinar cells of salivary glands, with the APM as well as the other exocrine gland cells is tightly regulated by intracellular signals produced in response to secretagogues, ACh (1), norepinephrine (2), histamine (6), substance P (4), and VIP (5), in a dose-dependent manner. Regulated exocytosis differs from unregulated exocytosis, which delivers integral membrane proteins to the plasma membrane for the secretion of various substances from the cells (67). Stimulation of salivary gland β2-ADRs induces the largest increase in protein secretion from the glands, while stimulation of M3 mAChRs induces a modest increase in protein secretion (1, 66). Both β- ADR and mAChR are present in parotid tissues of 3- day-old rats, as indicated by binding studies with radiolabeled ligands that specifically bind their respective receptors (66). Thereafter, the number of β-ADRs increases steadily to the tissue level at 28 days of age, when it levels off and this level is maintained till 730 days of age (66). These changes are coupled with those of the affinity of β-ADR for the respective agonists, but not for the antagonists. On the other hand, the number of mAChR also increases steadily to the tissue level at 56 days of age, at which point it levels off and is maintained till 730 days of age. The affinities Fig. 3. Conformational changes in zymogen granules by ATP-Mg of mAChR, for both the agonists and antagonists do not and a low concentration of Ca2+ in the presence of cytosolic proteins change after birth, which differs from the affinities of β- from parotid glands. Panel A: Zymogen granules in rat parotid ADR (66). The ability of the cholera toxin to catalyze tissues. AC: acinar cells. Panel B: Zymogen granules after incubation ADP-ribosylation of Gs proteins increases steadily in with ATP-Mg and EGTA in the presence of cytosolic proteins. Panel C: Zymogen granules after incubation with ATP-Mg and a the tissues at 14 to 56 days of age and thereafter this low concentration of Ca2+ in the presence of cytosolic proteins. level is maintained until 365 days of age, in association Bars, 1 µm. This figure is a reproduction with permission from with the responsiveness of adenylate cyclase (AC) to ref. 63. isoproterenol (IPR) in the tissues (66). However, the ability of pertussis toxin (IAP) to catalyze ADP-ribosylation is recognized in the tissues at 14 days of age and stimulated by ATP-Mg, showing that this process is a this affinity does not change thereafter (66). These priming step (Fig. 3) (62). ATP was hydrolyzed in this findings support the superior efficacy of β-ADR stimula- step, suggesting the presence ATPase in the zymogen tion for the induction of amylase secretion in rat parotid granules. The ATP-dependent release of amylase was tissues (66). inhibited by glycoletheldiamine tetraacetic acid (EGTA), a calcium chelator (Fig. 3) (62), suggesting the impor- 2) Exocytotic machinery in salivary glands tance of Ca2+ in this step. The release of amylase from The mechanisms underlying zymogen granule exo- zymogen granules was, however, not caused by ATP- cytosis have been studied using the granules isolated Mg and a low concentration of Ca2+ (62). The stimula- from rat parotid glands (62, 63, 67 – 70). The release tory effect of Ca2+ was observed in the presence of of amylase from the isolated zymogen granules was ATP-Mg and the cytosolic proteins from parotid glands 502 Y Ishikawa et al

(62). Changes of the turbidity of the reaction medium granules in this step (63). This finding was confirmed containing zymogen granules in the presence of by the electron microscope images (63). It is known that cytosolic proteins from parotid glands was caused by the priming step itself is stimulated by lower levels of the addition of ATP-Mg and a low concentration of Ca2+ and is required by ATP hydrolysis (67). Priming Ca2+, showing the conformational changes of zymogen and fusion are shown to be stimulated by cytosolic

Fig. 4. Schematic representation of signal transduction for regulated exocytosis of zymogen granules. The events in nor- adrenalin-induced exocytosis in parotid gland cells are represented. Priming step is ATP-dependent and zymogen granules move physically to subplasmalemmal region of the cells in this step. Tethering step and docking step are also ATP-dependent. Fusion of zymogen granules with the APM and release of amylase are triggered by Ca2+ without ATP.

Table 1. Major proteins that function in zymogen granule exocytosis Class of protein Protein Function

SNARE Syntaxin 1 Formation of SNARE complex which mediates membrane fusion. SNAP-25 VAMP

SNARE cofactor αSNAP αSNAP/ATPase NSF acts in priming step and in disassembly of SNARE complex. NSF Munc 18/nSec 1 Prevention of formation of SNARE complex. Munc 13 Promotion of syntaxin 1 for formation of SNARE complex.

Rab Rab 3D Regulation of granule maturation. Rab 27B Reguration of formation of SNARE complex.

Ca2+-binding protein Synaptotagmin Ca2+ sensor

Others cAMP-GEF/Epac Mediation of cAMP dependent, PKA-independent exocytosis.

VAMP (synaptobrevin): vesicle associated membrane protein, SNAP-25: 25-kDa synaptosomal-associated protein, SNARE: soluble N-ethylmaleimide-sensitive factor attachment protein (SNAP) receptor, NSF: N-ethylmaleimide-sensitive fusion protein, GEF: guanine nucleotide exchange factor, Epac: exchange proteins directly activated by cAMP. Salivary Water and Protein Secretion 503 proteins (67 – 69). Requirement of the cytosolic proteins involved in the Ras superfamily of small G proteins in the Ca2+-dependent step in zymogen granule exo- that function in vesicle transport (81). Rab proteins cytosis suggests the importance of the modification of localize in the cytosol as the GDP-bound form, an the proteins that function in the zymogen granule exo- inactive state, and in the membrane as the GTP-bound cytosis, such as soluble N-ethylmaleimide-sensitive form, an active state. Mammals have many Rab protein factor (NSF) attachment protein receptors (SNAREs). isoforms. Rab3 is involved in secretory granule exo- The priming, tethering, docking, fusion, and release cytosis (82, 83) and has several potential effectors, steps are sequential steps in zymogen granule exocytosis Rabphilin3 (84), Rims (Rim1 and Rim2) (85), Noc2 (Fig. 4). Recently, the idea of “kiss and run” exocytosis (86), and Granuphilin (87). SNAREs, Rabs, syntaxins is proposed (67, 70). In this phenomenon, the fusion (except for syntaxin 1), VAMPs (except for VAMP-7), pore is rapidly reclosed after formation and expansion of SNAP-23, α-SNAP, and NSF are expressed in parotid a fusion pore and release of vesicle contents. Evidences acinar cells and are involved in cAMP- and Ca2+- of “kiss and run” exocytosis have been shown in triggered exocytosis (88). Syntaxin 1 and SNAP25 are synaptic vesicles and chromaffin granules exocytosis, not expressed in parotid acinar cells (71, 88). Rab3D, but not have been shown in zymogen granule exocytosis one of four structurally related Rab3 isoforms (Rab3A, (67). The exocytotic machinery in salivary acinar cells 3B, 3C, and 3D), and Rab27B are present in parotid (Table 1) as well as in neurons and endocrine cells acinar cells (89, 90). Rab3D is not essential for the includes SNAREs, ATPase, NSF, and its cofactor (α- regulation of exocytosis, but Rab27B regulates the synaptosomal-associated protein (α-SNAP)), Munc18 formation of the SNARE complex with its effector /Sec1, Munc13, synaptotagmins, Rab3, and its effectors protein (90, 91). Salivary glands of Noc2 knockout mice (67, 70, 71). SNAREs are membrane proteins in parotid have a marked accumulation of secretory granules (92). acinar cells and are involved in basic components of exocytosis. SNAREs are classified as arginine (R)- 3) Intracellular signals in stimulation-secretion SNAREs and glutamine (Q)-SNAREs based on the coupling in salivary glands presence of arginine or glutamine in the center of the Ca2+ signaling: ACh stimulates PLCβ via the acti- SNARE motif (72). SNARE complex formation is vation of Gq/11α protein coupled to M3 mAChRs and proposed to mediate the fusion of zymogen granules generates phospholipids-derived messengers, DG and with the APM. The SNARE complex comprises three IP3. DG activates PKC and induces salivary protein proteins, vesicle-associated membrane protein-2 secretion by regulating the exocytotic process (Fig. 4). (VAMP-2, synaptobrevin), syntaxin 1, and 25-kDa Norepinephrine acts both at α1-ADRs and β2-ADRs in SNAP (SNAP-25). SNAP-25 is a Q-SNARE and salivary glands. Activation of α1-ADRs stimulates PLCβ contains two SNARE motifs. VAMP-2, an R-SNARE and generates IP3. Norepinephrine also acts at β2-ADRs that is an integral membrane protein, and syntaxin 1, a in salivary glands and activates AC via the activation of Q-SNARE, each contain a single SNARE motif. These Gsα protein, thereby increasing the amount of cAMP four SNARE motifs form a stable SNARE complex (Fig. 4). Increases in the amount of cAMP activate PKA and mediate the zymogen granule fusion (73, 74). For in the cells and induce zymogen granule exocytosis 2+ 2+ the SNARE complexes to be recycled, they must be without a significant increase in [Ca ]i (2). Ca signal- 2+ disassembled after exocytosis (75). NSF, a chaperone- ing IP3 mobilizes Ca from intracellular stores via the 2+ like ATPase, and αSNAP, an adaptor protein, interact activation of IP3 receptors. A rise in [Ca ]i is the primary with the assembled SNARE motifs and disassemble signal that triggers zymogen granule exocytosis as well the complexes. The complex is dissociated into its as AQP5 translocation to the APM in salivary glands individual components by NSF ATPase. Munc18/Sec 1 (1, 21, 44 – 47). The mode of Ca2+ release depends on was discovered in C. elegans in the UNC-18 mutant the concentration of IP3-generated secretagogues. Low (Munc18) and in yeast in a secretory mutant (Sec 1) (76, concentrations of cevimeline (21, 45 – 47) and metha- 77). Both Sec 1 and Munc18 were also discovered in choline (MCh) (93), cholinergic agonists, evoke Ca2+ mammals (78). Munc18 binds to syntaxin 1 and prevents release in parotid isolated acinar cells with oscillation. the formation of the SNARE complex (79). A recent High concentrations of the agonists evoke Ca2+ release study, however, demonstrated that Munc18 might with an initial rapid rise followed by a gradual decline to facilitate the SNARE complex formation by regulating the plateau level observed under unstimulated conditions the conformation of syntaxin 1 (70). Munc13 promotes (94). Cevimeline- and MCh-induced exocytosis in rat the activation of syntaxin 1 for SNARE complex parotid glands are completely inhibited by BAPTA-AM, formation by disturbing the interaction between KN-93 (CaM kinase II inhibitor), ML-9 (MLCK syntaxin 1 and Munc18 (79, 80). Rab proteins are inhibitor), L-NAME (nNOS inhibitor), ODQ (GC-s 504 Y Ishikawa et al inhibitor), or KT5823 (PKG inhibitor), but not KT5720 logue of SNAP-25, is phosphorylated by PKC in plate- (PKA inhibitor) (21, 54). Exocytosis induced by cevime- lets and mast cells (102, 103). The effect of phosphory- line or MCh is inhibited by dantrolene, which prevents lated SNAP-23 on SNARE proteins, however, is not the release of Ca2+ from ryanodine-sensitive stores (45, known (102). In Munc-18 knockout mice, vesicle 54, 93). nNOS is expressed in rat isolated parotid acinar exocytosis is completely blocked (104). Munc18-1, 18- cells, and cevimeline-induced amylase secretion from 2, and 18-3 are involved in exocytosis in mammals, and parotid tissues in nNOS knockout mice has not been Munc18-1 and 18-3 are phosphorylated in the intact observed in spite of the expression of M3 receptors and cells in response to secretory stimuli and phorbol esters the maintenance of the IPR-induced secretory response (105 – 108). Munc18-1 is phosphorylated by PKC and in the tissues (54). These findings suggest the activation reduces the binding ability of PKC to syntaxin 1A (109). of Ca2+- and CaM-dependent enzymes and the NOS- Thus, further studies are required to understand the PKG signaling pathway in cevimeline- and MCh- molecular mechanisms by which PKC phosphorylates induced salivary protein secretion. The changes in SNAP-25/23 and Munc18, and alters the protein secre- 2+ [Ca ]i in salivary glands are coupled with the initial tion from exocrine cells. 2+ Ca release from intracellular stores by IP3 and both cAMP/PKA: cAMP regulates exocytosis in various the subsequent Ca2+ extrusion mediated by Ca2+-ATPase secretory cells. In synapses (110), adrenal chromaffin in the APM (95) and Ca2+ influx into the cells through cells (111, 112), PC12 cells (113), and pancreatic β cells receptor-operated Ca2+ channels (capacitative Ca2+ influx) (114), cAMP enhances exocytosis, but an increase in the (95) and Ca2+ release from intracellular stores by amount of cAMP in the absence of Ca2+ elevation is not cADPribose (96). sufficient to trigger exocytosis. In these cells, increases DG/PKC: DG generated by the activation of PLCβ in the amount of cAMP activate PKA and regulate Ca2+- activates PKC. Although Ca2+ is the key intracellular triggered exocytosis, suggesting that cAMP-dependent trigger in zymogen granule exocytosis in salivary pathways exist with Ca2+-dependent pathways for exo- glands, PKC together with PKA regulates exocytosis cytosis and SNARE proteins have an important role (97). PKC is a family of isoenzymes classified into in this exocytosis. In contrast, in parotid and sub- classical (α, β, and γ) PKC activated both by Ca2+ and mandibular glands, cAMP directly triggers zymogen DG; novel (δ, ε, η, and θ) PKC activated by DG; and granule exocytosis without significant increases in 2+ 2+ atypical (ξ, and ι/λ) PKC activated by neither Ca nor [Ca ]i (115 – 117). In parotid glands, the amount of 2+ DG (98). Because phorbol esters act mainly to regulate amylase secretion due to an increase in [Ca ]i is less exocytosis via PKC, PKC is suggested to have a role than that induced by an increase in cAMP (66). modulating regulated exocytosis. There is another The cAMP-triggered exocytosis is also observed in process in exocytosis, however, that is stimulated in a glucagon-induced insulin release in pancreatic β cells PKC-independent manner (99). The secretion of (114) or parathyroid hormone secretion in parathyroid amylase from rat parotid tissues induced by phorbol 12- cells (118). Botulinum toxin B inhibits IPR-induced myristate 13-acetate (PKC activator) is not inhibited amylase secretion from parotid glands, which is cAMP- by KN-93, L-NAME, or ODQ (93), suggesting that triggered exocytosis (88). This toxin cleaves VAMP-2, PKC-mediated exocytosis is independent of Ca2+- which is present on the membrane of zymogen granules mediated exocytosis in rat parotid glands. The site at and mediates fusion with syntaxin2 on the plasma which PKC affects the exocytosis process, that is the membrane (119). The inhibition of cAMP exocytosis by PKC substrate, is not known (100). Recently, the botulinum toxin B indicates the requirement of VAMP- characteristics of proteins phosphorylated by PKC in the 2, a SNARE protein, in cAMP- and Ca2+-triggered exocytosis process were studied. Phosphorylation of exocytosis (88). The involvement of other SNARE SNAP-25 occurs in pancreatic β cells in response to proteins or other components of fusion machinery in phorbol ester treatment and stimulation with physiologic cAMP-triggered exocytosis has not been reported. It is stimuli (101). SNAP-25 contributes to form SNARE proposed that cAMP signaling and Ca2+ signaling act at complexes with VAMP and syntaxin 1. The formation different steps in amylase secretion from pancreatic cells of SNARE complexes is thought to mediate vesicle (120). The involvement of cAMP in Ca2+-triggered docking/fusion at the plasma membrane (98). The exocytosis might be due to the protein of the exocytotic phosphorylation of SNAP-25 on Ser187 is induced by machinery, which is a PKA substrate (121). Cysteine phorbol esters, but PKC does not phosphorylate string protein (Csp) (122) phosphorylated by PKA recombinant SNAP-25 on Ser187. This finding indicates reduces the affinity to bind syntaxin and synaptotagmin, that the effect of PKC on the phosphorylation of and thus, Csp might be a candidate substrate (122, 123). SNAP-25 is not clear. SNAP23, a non-neuronal homo- Another candidate is cAMP-guanine nucleotide Salivary Water and Protein Secretion 505 exchange factor (GEF), which is a cAMP-binding pro- with PKC (130). DG stimulates the activity of types I, tein (124). This protein is part of a family of novel II, III, and VII by PKC-mediated phosphorylation (131). cAMP-binding proteins and is suggested to have many Conversely, PKA phosphorylates types V and VI and roles in cAMP-regulated, PKA-independent exocytosis inhibits their activities (132, 133). PDEs, enzymes (123 – 127). These findings suggest the involvement of a that hydrolyze the phosphodiester bond of cyclic cAMP regulated, PKA-independent mechanism in nucleotides, comprise more than 50 different PDE amylase secretion from parotid glands. It is, however, proteins. PDE families have different responses to unknown whether cAMP-induced amylase secretion in inhibitors. PDE1s are stimulated by Ca2+/CaM activity parotid glands depends only on a PKA-dependent (134, 135), and PDE4 regulates cAMP signaling by its mechanism. cAMP is synthesized from ATP by adeny- PKA-mediated phosphorylation (136 – 138). PKA is a late cyclase. The addition of dibutyry cAMP alone major target of cAMP-regulated exocytosis in exocrine induces amylase secretion from rat parotid gland tissues cells. Various ligands bind to GPCR and activate AC, (66). The intracellular concentration is regulated by resulting in the activation of PKA. In the salivary glands, four components, heterotrimetric GTP-binding protein β2 agonists (2, 64) and H2 agonists (6) activate AC and (G protein) coupled receptors (GPCR), G protein, AC, PKA, resulting in the induction of protein secretion. and cyclic nucleotide specific phosphodiesterase (PDE). PKA is composed of two regulatory subunits, type I and AC comprises a large superfamily and its activation is type II, and two catalytic subunits, RI and RII. Each induced by Gsα protein. Nine membrane-bound forms regulatory subunit comprises R Iα, R Iβ, and R IIα and R (type I – IX) are expressed in mammals and one soluble IIβ, respectively (139, 140). In parotid glands, there are form is found in sperm (128). Types I, III, and VIII are two PKA isozymes, type I and II (64). PKA type I is the expressed in neurons and in nonneuronal secretory cells membrane-bound form that is activated during β-ADR and are regulated by Ca2+/CaM. Types II, IV, VI, VII, stimulation and is accompanied by an increase in and IX are expressed in various tissues, and type V is specific membrane phosphorylation during amylase expressed in heart. Gsα protein stimulates the activity of secretion induced by β-ADR agonists (64, 141). These all forms of AC; Giα protein inhibits the activity of findings suggest that Gi2α protein is selectively phos- types I, V, VI, and VIII (129). Ca2+/CaM stimulates the phorylated by PKA type I, resulting in decreased func- activities of types I and III by binding to the CaM- tion and supersensitivity of amylase secretion (6, 64, binding site in the cytosolic domain, and Ca2+ also 65). stimulates the activity of type V by its phosphorylation

Fig. 5. Schematic representation of mechanism of isoproterenol-induced supersensitivity and desensitization of amylase secretion in rat parotid glands. Regulation of phosphorylation of Gi2α by protein kinase A (PKA) and protein phosphatase 2A (PP2A) is coupled with isoproterenolol (IPR)-induced supersensitivity and desensitization of amylase secretion. βAR: β-adrenergic receptor. 506 Y Ishikawa et al

2. Altered protein secretion in salivary glands hand, histamine acts at H2 receptors and induces amylase 1) Functional alterations of membrane receptors in sal- secretion from rat parotid gland tissues (6). Short-term ivary glands treatment of rat parotid tissues with histamine for less Plasma membrane receptors not only relay physio- than 10 min does not result in supersensitivity of logic information to effector cells, but also control amylase secretion during further treatment with hista- peripheral responses by functional alterations due to mine, but rather in desensitization (6). These changes in interactions with their agonists. Supersensitivty and the secretory response are accompanied by both desensitization of protein secretion from salivary glands alterations in the numbers of β2-ADRs (2, 64, 65) and H2 mediated plasma membrane receptors is induced by the histamine receptors (6) and in the affinity of the interaction with their agonists (2, 6, 64, 65) (Fig. 5). receptors for the agonists, as assessed by measurement Desensitization is a phenomenon in which short-term of the specific binding of radiolabeled ligands. The exposure of plasma membrane receptors coupled with increases and decreases in the number of β2-ADRs or H2 G proteins to an agonist results in a reduction of the histamine receptors in salivary gland tissues are coupled physiologic response. This phenomenon commonly with supersensitivity and desensitization, respectively. occurs in response to a variety of stimuli and is found in The affinity of these receptors for their agonists, but many organisms, indicating the importance of the not their antagonists, increases and decreases in the plasma membrane receptors in cell signaling. Desensiti- supersensitivity and desensitization, respectively (2, 6, zation of salivary protein secretion in response to IPR is 64, 65). also induced by repeated administration of a tricyclic antidepressant (142). Regarding the mechanisms under- 2) Post-receptor mechanisms underlying the super- lying the rapid β2-ADR desensitization, the sequestration sensitivity and desensitization of protein secretion in of β2-ADR quickly follows the rapid uncoupling of the salivary glands β2-ADR from Gs proteins (143 – 147). This uncoupling The cAMP concentration in the tissues that induces phenomenon involves the phosphorylation of β2-ADRs the supersensitivity of amylase secretion is lower than by PKA and β-ADR kinase, which are activated under that after pre-treatment with IPR (2). The cAMP concen- different conditions. Receptor phosphorylation, presum- tration in the parotid or submandibular tissues desensi- ably caused by any hormone or drug that increases tized by IPR (2, 64, 65) or histamine (6) treatment is cAMP levels in the cell, reduces the ability of a receptor also significantly lower than that after pre-treatment occupied by the agonist to stimulate the GTPase activity with IPR or histamine. G proteins have a key role in of the Gs proteins with which it couples, resulting in the transmembrane signal transduction as coupling proteins, induction of heterologous desensitization. The mecha- and in transducing information from a variety of nisms underlying homologous desensitization of β2- membrane receptors to their respective effector proteins. ADR involve receptor phosphorylation by β-ADR Gs, Gi, and Gq have been identified in rat parotid glands kinase (143 – 147). (151, 152). β-ADRs are coupled with the carboxyl- On the other hand, supersensitivity is observed in terminus of Gsα protein, and the binding of β-agonists exocrine glands, skeletal and smooth muscles, and with the β-ADRs promotes activation of Gs proteins neurons after chronic interruption of the neuronal input (153) and results in the activation of AC. Functional by denervation in mammals. Supersensitivity of salivary modifications and decreases in Gs protein levels are fluid secretion and salivary protein secretion develop involved in agonist-induced heterologous desensitiza- after parasympathetic (148) and sympathetic (149) tion of AC stimulation in erythrocytes (154) and hepato- denervation, respectively. Supersensitivity of salivary cytes (155). Activation of Gs proteins leads to the secretion also occurs following sympathetic nerve activation of PKA via the stimulation of AC, suggesting stimulation after partial sympathetic ganglionectomy the importance of PKA in the regulation of G protein (150). These findings suggest that only β-ADRs- function. In rat salivary gland, however, changes in the mediated amylase secretion becomes supersensitive levels and function of Gs proteins, measured as cholera after chronic denervation. Short-term treatment (<10 toxin-catalyzed ADP-ribosylation and GTP-binding min) of rat parotid tissues with IPR results in supersensi- capacities of these proteins, are not involved in the tivity of amylase secretion during further treatment with mechanism underlying IPR-induced supersensitivity, or the same agonist, but longer (>20 min) IPR treatment in IPR- and histamine-induced desensitization of protein results in desensitization (2, 64). In submandibular gland secretion from rat parotid (2, 6, 64) and submandibular tissues, however, IPR treatment for various periods of glands (65). IAP-sensitive G proteins, termed Gi1, Gi2, time only desensitizes mucin secretion from the tissues, and Gi3, comprise a family of Gi proteins and Gi2 but does not induce supersensitivity (65). On the other inhibits adenylate cyclase activity (156). Giα protein is Salivary Water and Protein Secretion 507 phosphorylated by PKA (64, 157, 158). The phosphory- transduction via plasma membrane receptors. The lation causes some conformational changes in Gi protein studies have revealed the characteristics of physiologic 2+ trimers and decreases in both the IAP-catalyzed ADP- regulation of salivary secretion. A rise in [Ca ]i is an ribosylation of Gi protein and their dissociation into α- important trigger in the stimulation-secretion coupling in and βγ-subunits (157, 158). In parotid tissues super- salivary gland cells as with any other secretory cells. In sensitized with IPR, there is a 60% decrease in IAP- most secretory cells, an increase in cAMP levels in the catalyzed ADP-ribosylation of Giα (64). Conversely, cells triggers exocytosis under conditions of increased 2+ 2+ however, a 40% increase in IAP-catalyzed ADP- [Ca ]i, and in the absence of Ca , an increase in cAMP ribosylation of Giα is coupled with the desensitization levels acting through PKA alone is not sufficient to of amylase and mucin secretion from rat parotid and trigger exocytosis. In the parotid and submandibular submandibular tissues induced by IPR and histamine, gland cells, however, an increase in cAMP levels in the respectively (6, 64, 65). In agonist-induced desensitiza- cells alone triggers exocytosis through a PKA-dependent 2+ tion of AC stimulation in MDCK cells (159) and rat pathway without an increase in [Ca ]i. On the other heart muscle cells (160), there are also increases in the hand, salivary protein is also secreted from these gland level and function of Gi proteins, as measured by IAP- cells by the PKC- and PKG-dependent pathways. The catalyzed ADP-ribosylation and GTP-binding capacities amount of salivary protein secreted by cAMP alone is 2+ of these proteins. Treatment of rat parotid tissue larger than that by increased [Ca ]i, indicating that membranes with the PKA catalytic subunit or alkaline cAMP is the principal signal for exocytosis in salivary phosphatase induces a decrease or increase in IAP- gland cells. Recently, PKA-independent, cAMP-regu- catalyzed ADP-ribosylation of Giα protein, respectively. lated exocytosis was reported to be present in certain A 40% increase and a 50% decrease in the phosphoryla- neurons and endocrine cells, but not in salivary gland tion of Gi2α immunoprecipitated with AS/7 (anti-G cells. There is quite a difference in the mechanisms protein antiserum) from [32P]Pi-labeled parotid cells underlying regulated exocytosis between salivary gland is coupled with supersensitivity and desensitization, cells and other secretory cells. Salivary fluid secretion 2+ respectively (64). Pretreatment with okadaic acid at a from parotid gland cells is regulated by [Ca ]i acting concentration that inhibits the activities of protein through a NO/PKG-dependent pathway. These findings phosphatase (PP) I and IIA increases the phosphoryla- demonstrate that PKA-, PKC-, and PKG-dependent tion level of Gi2α protein and results in enhanced pathways for exocytosis, and PKG-dependent pathways supersensitivity of salivary protein secretion and the for water secretion, coexist in salivary gland cells. One disappearance of the desensitization (6, 64). Phosphory- or more of the proteins of the fundamental machinery for lation by PKA of a specific serine residue on the G- exocytosis and water secretion, however, are not known. subunit of PP I inhibits the activity by dissociation of the To better understand the universality of the mechanisms catalytic subunit from the G-subunit (161). Inhibitor I, underlying exocytosis and water secretion, various an inhibitor of PP I, is phosphorylated through the substances, including the substrates for PKA, PKC, and activation of PKA by adrenaline and results in an PKG, must be identified. inhibition of PP I activity (161), indicating that PP I activity is inhibited through the activation of PKA by Acknowledgments cAMP elevation. PP IIA is suggested to be involved in dephosphorylation of Gi2α. In histamine-induced We thank the many colleagues who have studied with desensitization of amylase secretion from rat parotid us. This work was supported in part by Grant-in-Aid tissues, there is a 50% increase in PP IIA activity, but for Scientific Research and that for Knowledge Cluster PP I levels do not change (6). These findings indicate Initiative from Ministry of Education, Culture, Sports, that the function of Gi2α protein is dynamically Science, and Technology of Japan and a Research-Grant regulated by phosphorylation and dephosphorylation from the Daiichi Pharmaceutical Company (Tokyo, systems involving PKA and PP IIA, and its phosphory- Japan). lation is directly involved in the regulation of cellular responses in rat salivary glands. References

Concluding remarks 1 Ishikawa Y, Ishida H. Aquaporin water channel in salivary glands. Jpn J Pharmacol. 2000;83:95–101. 2 Hata F, Ishida H, Kagawa K, Kondo E, Kondo S, Noguchi Y. The secretory mechanisms of water and protein from β-Adrenoceptor alterations coupled with secretory response in salivary glands have been studied as models to investi- rat parotid tissue. J Physiol. 1983;341;185–196. gate water transport and exocytosis regulated by signal 3 Hata F, Ishida H, Kondo E. Dopamine-induced amylase secre- 508 Y Ishikawa et al

tion from rat parotid salivary gland in vitro: an effect mediated Shono M, et al. Identification of AQP5 in lipid rafts and its via noradrenergic and cholinergic nerve. Br J Pharmacol. translocation to apical membranes by activation of M3 mAChRs 1986;89:473–483. in intracellular ducts of rat parotid gland. Am J Physiol Cell 4 Guillemain B, Rollandy I, Imhoff V, Rossignol B. The NK-1 Physiol. 2005;289:C1303–C1311. receptor and a calcium-phospholipid pathway: inositoltrisphos- 22 Matsuki M, Hashimoto S, Shimono M, Murakami M, Fujita- phate production and calcium movement by selective agonists of Yoshinaga J, Furuyama S, et al. Involvement of aquaporin-5 neurokinin receptors in rat parotid glands. J Neurochem. 1992; water channel in osmoregulation in parotid secretory granules. 58:2321–2325. J Mem Biol. 2005;203:119–126. 5 Turner JT, Camden JM. Regulation of secretion by vasoactive 23 Raina S, Preston GM, Guggino WB, Agre P. Molecular cloning intestinal polypeptide in isolated perfused rat submandibular and characterization of an aquaporin cDNA from salivary, glands. Arch Oral Biol. 1992;37:281–287. lacrimal, and respiratory tissues. J Biol Chem. 1995;270:1908– 6 Eguchi T, Ishikawa Y, Ishida H. Mechanism underlying hista- 1912. mine-induced desensitization of amylase secretion in rat parotid 24 Ma T, Song Y, Gillespie A, Carlson EJ, Epstein CJ, Verkman glands. Br J Pharmacol. 1998;124:1523–1533. AS. Defective secretion of saliva in transgenic mice lacking 7 Gallacher DV. Are there purinergic receptors on parotid acinar aquaporin-5 water channels. J Biol Chem. 1999;274:20071– cells. Nature. 1982;296:83–86. 20074. 8 Sawaki K, Ouchi K, Sato T, Kawaguchi M. Existence of 25 King LS, Agre P. Pathophysiology of the aquaporin water gamma-aminobutyric acid and its biosynthetic and metabolic channels. Ann Rev Physiol. 1996;58:619–648. enzymes in rat salivary glands. Jpn J Pharmacol. 1995;67:359– 26 He X, Tse CM, Donowitz M, Alper SL, Gabriel S, Baum BJ. 363. Polarization distribution of key membrane transport proteins in 9 Kujirai M, Sawaki K, Kawaguchi M. Inhibitory effect of rat submandibular gland. Pflugers Arch Eur J Physiol. 1997; diazepam on muscarinic receptor-stimulated inositol 1,4,5- 433:260–268. trisphosphate production in rat parotid acinar cells. Br J 27 Delporte C, Hoque ATNMS, Kulakusky JA, Braddon VR, Pharmacol. 2002;137:945–952. Goldsmith CM, Wellner RB, et al. Relationship between 10 Nauntofte B. Regulation of electrolyte and fluid secretion in adenovirus-mediated aquaporin-1 expression and fluid move- salivary acinar cells. Am J Physiol Gastrointest Liver Physiol. ment across epithelial cells. Biochem Biophys Res Commun. 1992;263:G823–G837. 1998;246:584–588. 11 Turner RJ. Mechanisms of fluid secretion by salivary glands. 28 Ishibashi K, Kuwahara M, Kageyama Y, Tohsaka A, Marumo F, Ann N Y Acad Sci. 1993;694:24–35. Sasaki S. Cloning and functional expression of a second new 12 Preston GM, Agre P. Isolation of the cDNA for erythrocyte aquaporin abundantly expressed in testis. Biochem Biophys Res integral membrane protein of 28 kilodaltons: member of an Commun. 1997;237:714–718. ancient channel family. Proc Natl Acad Sci U S A. 1991;88: 29 Smith BL, Agre P. Erythrocyte Mr28,000 transmembrane 11110–11114. protein exists as multisubunit similar to channel proteins. J Biol 13 Preston GM, Carroll TP, Guggino WB, Agre P. Appearance of Chem. 1991;266:6407–6415. water channels in Xenopus oocytes expressing red cell CHIP28 30 Agre P, Kozono D. Aquaporin water channels: molecular protein. Science. 1992;256:385–387. mechanisms for human diseases. FEBS Lett. 2003;555:72–78. 14 Knepper MA. The aquaporin family of molecular water 31 Ramamoorthy S, Melikian H, Qian Y, Blakely RD. Bio- channels. Proc Natl Acad Sci U S A. 1994;91:6255–6258. synthesis, N-glycosylation and surface trafficking of biogenic 15 King LS, Kozono D, Agre P. From structure to disease; the amine transporter protein. Method Enzymol. 1998;296:347–370. evolving tale of aquaporin biology. Nat Rev Mol Cell Biol. 32 Lee MD, Bhakta KY, Raina S, Yanescu R, Griffin CA, 2004;5:687–698. Copeland NG, et al. The human aquaporin-5 gene. J Biol Chem. 16 Morishita Y, Sakube Y, Sasaki S, Ishibashi K. Aquaporin 1995;271:8500–8604. superfamily (superaquaporins) expansion of aquaporins 33 Krane CM, Towne JE, Menon AG. Cloning and characterization restricted to multicellular organisms. J Pharmacol Sci. of murine AQP5: evidence for a conserved aquaporin gene 2004;96:276–279. cluster. Mamm Genome. 1999;10:498–505. 17 Gresz V, Kwon TH, Hurley PT, Varga G, Zelles T, Nielsen S, 34 Sidhaye V, Hoffert JD, King LS. cAMP has distinct acute and et al. Identification and localization of aquaporin water channels chronic effects on aquaporin-5 in lung epithelial cells. J Biol in human salivary glands. Am J Physiol Gastrointest Liver Chem. 2005;280:3590–3596. Physiol. 2001;281:G247–G257. 35 Simons K, Ikonen E. Functional rafts in cell membranes. Nature. 18 Wellner RB, Redman RS, Swain WD, Baum BJ. Further 1997;387:569–572. evidence for AQP8 expression in the myoepithelium of rat 36 Simons K, Meer G. Lipid sorting in epithelial cells. Bio- submandibular and parotid glands. Pflugers Arch Eur J Physiol. chemistry. 1988;27:6197–6202. 2006;451:642–645. 37 Brown DA, Rose JK. Sorting of GPI-anchored proteins to 19 Koyama Y, Yamamoto T, Kondo D, Funaki H, Yaoita E, glycolipid-enriched membrane subdomains during transport to Kawasaki K, et al. Molecular cloning of a new aquaporin from the apical cell surface. Cell. 1992;68:533–544. rat pancreas and liver. J Biol Chem. 1997;272:30329–30333. 38 Paladino S, Sarnataro D, Pillich R, Tivodar S, Nitsch L, Zurzolo 20 Takata K, Matsuzaki T, Tajika Y. Aquaporins; water channel C. Protein oligomerization modulates raft partitioning and proteins of the cell membrane. Progr Histochem Cytochem. apical sorting of GPI-anchored proteins. J Cell Biol. 2004;39:1–84. 2004;167:699–709. 21 Ishikawa Y, Yuan Z, Inoue N, Skowronski MT, Nakae Y, 39 Li X, Galli S, Leu JB, Wade EV, Weinman MD, Donowitz M. Salivary Water and Protein Secretion 509

Na+-K+ exchanger3 (NHE3) is present in lipid rafts in the rabbit parotid glands. J Dent Res. 2003;82:476–480. ileal brush border: a role for rafts in trafficking and rapid 56 Anthony TI, Brooks HL, Boasse D, Reonov S, Yanohko GM, stimulations of NHE3. J Physiol. 2001;541:537–552. Regan JW, et al. Cloned human aquaporin-1 is a cyclic GMP- 40 Simons K, Toomre D. Lipid rafts and signal transduction. Natl gated ion channel. Mol Pharmacol. 2000;57;576–588. Rev Mol Cell Biol. 2000;1:31–39. 57 Bouley R, Breton S, Sun T-X, McLaughlin M, Nsumu NN, Lin 41 Simons K, Ehehalt R. Cholesterol, lipid rafts, and disease. J Clin HY, et al. Nitric oxide and atrial natriuretic factor stimulation Invest. 2002;110:597–603. cGMP-dependent membrane insertion of aquaporin-2 in renal 42 Hansen GH, Pedersen J, Niels-Christensen LL, Immerdal, L, epithelial cells. J Clin Invest. 2000;106:1115–1126. Danielsen EM. Deep-apical tubules: dynamic lipid-raft micro- 58 Mandel ID. The functions of saliva. J Dent Res. 1987;66:623– domains in the brush-border region of enterocytes. Biochem J. 627. 2003;373:125–132. 59 Ship JA, Baum BJ. Is reduced salivary flow normal in old 43 Galbianti FI, Razani B, Lisanti MP. Emerging themes in lipid people? Lancet. 1990;336:1507. rafts and caveolae. Cell. 2001;106:403–411. 60 Mata AD, Marques D, Rocha S, Francisco H, Santos C, 44 Ishikawa Y, Eguchi T, Skowronski MT, Ishida H. Acetylcholine Mesquita MF, et al. Effect of diabetes mellitus on salivary acts on M3 muscarinic receptors and induces the translocation of secretion and its composition in the human. Mol Cell Biochem. aquaporin-5 water channel via cytosolic Ca2+ elevation in rat 2004;261:137–142. parotid glands. Biochem Biophys Res Commun. 1998;245:835– 61 Ishikawa Y, Inoue N, Zhenfang Y, Nakae Y. Molecular 840. mechanisms and drug development in aquaporin water channel 45 Ishikawa Y, Skowronski MT, Ishida H. Persistent increase in the diseases: the translocation of aquaporin-5 from lipid rafts to the amount of aquaporin-5 in the apical membrane of rat parotid apical plasma membranes of parotid glands of normal rats and acinar cells induced a muscarinic agonist SNI-2011. FEBS Lett. impairment of it in diabetic or aged rats. J Pharmacol Sci. 2000;477:253–257. 2004;96:271–273. 46 Ishikawa Y, Skowronski MT, Inoue N, Ishida H. α1-Adreno- 62 Ishida H, Miki N, Yoshida H. Role of Ca2+ in the secretion of ceptor-induced trafficking of aquaporin-5 to the apical plasma amylase from the parotid glands. Jpn J Pharmacol. 1971;21:227– membrane of rat parotid cells. Biochem Biophys Res Commun. 238. 1999;245:835–840. 63 Ishida H, Miki N, Hata F, Yoshida H. Conformational changes 47 Ishikawa Y, Iida H, Ishida H. The muscarinic acetylcholine in zymogen granules from the parotid glands induced by ATP receptor-stimulated increase in aquaporin-5 levels in the apical and a low concentration of Ca2+. Jpn J Pharmacol. 1971;21:239– plasma membrane in rat parotid acinar cells is coupled with 251. activation of nitric oxide/cGMP signal transduction. Mol 64 Amano I, Ishikawa Y, Eguchi T, Ishida H. Regulation of Pharmacol. 2002;61:1423–1434. phosphorylation of Gi2α protein controls the secretory response 48 Gromada J, Hog M, Renstrom E, Bokvist E, Eliasson L, to isoproterenol in rat parotid tissues. Biochim Biophys Acta. Noguchi Y. CaM kinase II-dependent mobilization of secretory 1996;1313:146–156. granules underlies acetylcholine-induced stimulation of exo- 65 Ishikawa Y, Amano I, Eguchi T, Ishida H. Mechanism of cytosis in mouse pancreatic β-cells. J Physiol. 1999;518:745– isoproterenol-induced heterologous desensitization of mucin 759. secretion: Regulation of phosphorylation of Gi proteins controls 49 Watanabe H, Takahashi R, Zhang X, Hayashi H, Ando J, Ishiki the cell response to the subsequent stimulation. Biochim M, et al. An essential role of myosin light chain kinase in the Biophys Acta. 1995;1265:173–180. regulation of agonist- and fluid flow-stimulated Ca2+ influx in 66 Ishikawa Y, Chen C, Eguchi T, Skowronski MT, Ishida H. endothelial cells. FASEB J. 1998;12:341–348. Developmental enhancement of secretory response to iso- 50 Niwa T, Matsukawa Y, Senda T, Nimura Y, Hidaka H, Niki I. proterenol coupled with increases in β-adrenoceptor density of Acetylcholine activates intracellular movements of insulin Gs protein function in rat parotid tissues. Mech Aging Develop. granules in pancreatic β-cells via inositol triphosphate- 1998;104:75–90. dependent mobilization of Ca2+. Diabetes. 1998;47:1699–1706. 67 Burgoyne RD, Morgan A. Secretory granule exocytosis. Physiol 51 Kim M, Kim S, Kim HS, Chang JW, Kim HW, Park CS. Rev. 2003;83:581–632. Regulation of rennin secretion through reversible phosphoryla- 68 Hay JC, Martin TFJ. Resolution of regulated secretion into tion of myosin light chain kinase and protein phosphatase type I. sequential MgATP-dependent and calcium dependent stages J Pharmacol Exp Ther. 1998;285:968–974. mediated by distinct cytosolic proteins. J Cell Biol. 1992;119: 52 Moncade S, Palmer RMJ, Higgs EA. Nitric oxide: physiology, 139–151. pathology, and pharmacology. Pharmacol Rev. 1991;517:317– 69 Walent JH, Porter BW, Martin TFJ. A novel 145Kd brain 326. cytosolic protein reconstitutes Ca2+-regulated secretion in 53 Lucas KA, Pitari GM, Kazerounian S, Ruiz-Stwat I, Schulz S, permeable neuroendocrine cells. Cell. 1992;70:765–775. Chepenik KP, et al. Guanyl cyclases and signaling by cGMP. 70 Seino S, Shibasaki T. PKA-dependent and PKA-independent Pharmacol Rev. 2000;52:375–413. pathway for cAMP-regulated exocytosis. Physiol Rev. 2005;85: 54 Yuan Z, Iida H, Inoue N, Ishikawa Y, Ishida H. Effect of SNI- 1303–1342. 2011 on amylase secretion from parotid tissue in rats and in 71 Imai A, Nashida T, Shimomura H. mRNA expression of neuronal nitric oxide synthase knockout mice. Eur J Pharmacol. membrane-fusion-related proteins in rat parotid gland. Arch 2003;464:197–216. Oral Biol. 2001;46:955–962. 55 Inoue N, Iida H, Yuan Z, Ishikawa Y, Ishida H. Age-related 72 Fasshauser HC, Sutton RB, Brunger AT, Jahn R. Conserved decreases in the response of aquaporin-5 to acetylcholine in rat structural features of the synaptic fusion complex: SNARE 510 Y Ishikawa et al

proteins reclassified as Q- and R-SNAREs. Proc Natl Acad Sci small GTPase Rab27B regulates amylase release fro m rat U S A. 1998;95:15781–15786. parotid acinar cells. J Cell Sci. 2004;117:1945–1953. 73 Bennett MK, Scheller RH. The molecular machinery for secre- 91 El-Amraoui A, Schonn JS, Kussel-Andermann P, Blanchard S, tion is conserved yeast to neurons. Proc Natl Acad Sci U S A. Desnos C, Henry JP, et al. MyRIP, a novel Rab effector, enables 1993;90:2559–2563. myosin VIIa recruitment to retinal melanosomes. EMBO Rep. 74 Rothman JE, Warren G. Implication of the SNARE hypothesis 2002;3:463–470. for intracellular membrane topology and dynamics. Curr Biol. 92 Wang Y, Sugita S, Dsudhof TC. The RIM/NIM family of 1994;4:220–233. neuronal C 2 domain proteins. Interaction with Rab3 and a new 75 Barnard RJ, Morgan A, Burgoyne RD. Stimulation of NSF class of Src homology 3 domain proteins. J Biol Chem. ATPase activity by αSNAP is required for SNARE complex 2000;275:20033–20044. disassembly and exocytosis. J Cell Biol. 1997;139:875–883. 93 Ishikawa Y, Iida H, Skowronski MT, Ishida H. Activation of 76 Hosono R, Hekimi S, Kamiya Y, Sassa T, Murakami S, endogenous nitric oxide synthase coupled with methacholin- Nishiwaki S, et al. The unc-18 gene encodes a novel protein induced exocytosis in rat parotid acinar cells. J Pharmacol Exp affecting the kinetics of acetylcholine metabolism in nematode Ther. 2002;301:355–363. Caenorhabditis elegans. J Neurochem. 1992;58:1517–1525. 94 Campos-Toimil M, Edwardson JM, Thomas P. Real-time studies 77 Novick P, Schekman R. Secretion and cell-surface growth are of zymogen granule exocytosis in intact rat pancreatic acinar blocked in a temperature sensitive mutant of Saccharomyces cells. J Physiol. 2000;528:317–326. cerevisiae sites. Proc Natl Acad Sci U S A. 1979;76:1858–1862. 95 Belan PV, Gerasimenko O, Tepikin A, Petersen OH. Localiza- 78 Halachmi N, Lev Z. The Sec-1 family: a novel family of proteins tion of Ca2+ extrusion sites in pancreatic acinar cells. J Biol involved in synaptic transmission and general secretion. J Chem. 1996;271:7615–7619. Neurochem. 1996;66:889–897. 96 Putney JW Jr. TRP, inositol 1,4,5,-trisphosphate receptors, and 79 Dububova I, Sugita S, Hill S, Hosaka M, Fernandez I, Sudhof capacitative calcium entry. Proc Natl Acad Sci U S A. 1999;96: TC, Rizo J. A conformational switch in syntaxin during 14669–14671. exocytosis: role of munc18. EMBO J. 1999;18:4372–4382. 97 Thorn P, Gerasimenko O, Petersen OH. Cyclic ADP-ribose 80 Yang B, Steegmaier M, Gonzalez LC Jr, Scheller RH. nSec1 regulation of ryanodine receptors involved in agonist evoked binds a closed conformation of syntaxin 1A. J Cell Biol. cytosolic Ca2+ oscillation in pancreatic acinar cells. EMBO J. 2000;148:247–252. 1994;13:2038–2043. 81 Pfeffer SR. Rab GTPase: specifying and deciphering organelle 98 Morgan RD, Burgoyne JW, Barclay TJ, Craig GR, Prescott LF, identify and function. Trends Cell Biol. 2001;11:487–491. Giufo GJO, et al. Regulation of exocytosis by protein kinase C. 82 Geppert M, Bolshakov VY, Siegelbaum SA, Takei K, DeCamilli Biochem Soc Trans. 2005;33:1341–1344. P, Hammer RE, et al. The role of Rab3A in neurotransmitter 99 Newton AC. Protein kinase C: structural and spatial regulation release. Nature. 1994;369:493–497. by phosphorylation, cofactors, and macromolecular interactions. 83 Darchen F, Goud B. Multiple aspects of Rab protein action in the Chem Rev. 2001;101:2353–2364. secretory pathway: focus on Rab 3 and Rab 6. Biochimie. 100 Rhee JS, Betz A, Pyott S, Reim K, Varoqueaux FIA, Hesse D, 2000;82:375–384. et al. Beta phorbol ester- and diacylglycerol-induced augmenta- 84 Shirataki H, Kaibuchi K, Sakuda T, Kishida S, Yamaguchi T, tion of transmitter release is mediated by Munc13s and not by Wada K, et al. Rabphillin-3A, a putative target protein for PKCs. Cell. 2002;108:121–133. smg p25A/rab3A p25 small GTP binding protein related to 101 Gonelle-Gispert C, Costa M, Takahashi M, Sadoul K, Halban P. synaptotagmin. Mol Cell Biol. 1993;13:2061–2068. Phosphorylation of SNAP-25 on serine-187 is induced by 85 Wang Y, Okamoto M, Schmitz F, Hoffman K, Sudhof TC. Rim secretagogues in insulin-secreting cells, but is not correlated is a putative Rab3 effector in regulating synaptic vesicle fusion. with insulin secretion. Biochem J. 2002;368:223–232. Nature. 1997;388:593–598. 102 Risinger C, Bennett MK. Differential phosphorylation of 86 Matsumoto M, Miki T, Shibasaki T, Kawaguchi M, Shinozaki syntaxin and synaptosome-associated protein of 25 kDa (SNAP- H, Nio J, et al. Noc2 is essential in normal regulation of exo- 25) isoforms. J Neurochem. 1999;72:614–624. cytosis in endocrine and exocrine cells. Proc Natl Acad Sci 103 Hepp R, Puri N, Hohenstein AC, Crawford GL, Whiteheart SW, U S A. 2004;101:8313–8318. Roche PA. Phosphorylation of SNAP-23 regulates exocytosis 87 Coppola T, Frantz C, Perret-Menoud V, Gattesco S, Hirling H, from mast cells. J Biol Chem. 2005;280:6610–6620. Regazzi R. Pancreatic β cell protein granuphilin binds Rab3 and 104 Polgar J, Lane WS, Chung SH, Houng AK, Reed GL. Phos- Munc-18 and control exocytosis. Mol Biol Cell. 2002;13:1906– phorylation of SNAP-23 in activated human platelets. J Biol 1915. Chem. 2003;278:44369–44376. 88 Fuguta-Yoshigaki J, Dohke Y, Hara-Yokoyama M, Kamata Y, 105 Toonen RF, Verhage M. Vesicle trafficking: pleasure and pain Kozaki S, Furuyama S, et al. Vesicle-associated membrane from SM genes. Trends Cell Biol. 2003;13:177–186. protein2 is essential for cAMP-regulated exocytosis in rat 106 Barclay JW, Craig TJ, Fisher RJ, Ciufo Lf, Evans GJO, Morgan parotid acinar cells. The inhibition of cAMP-dependent amylase A, et al. Phosphorylation of Munc18 by protein kinase C release by botulinum neurotoxin B. J Biol Chem. 1996;271: regulates the kinetics of exocytosis. J Biol Chem. 2003;278: 13130–13134. 10538–10545. 89 Raffaniello RD, Lin J, Schwimmer R, Ojakian GK. Expression 107 Craig TJ, Evans GJ, Morgan A. Physiological regulation of and localization of Rab3D in rat parotid gland. Biochim Biophys Munc18/nSec1 phosphorylation on serine-313. J Neurochem. Acta. 1990;1450:352–363. 2003;86:1450–1457. 90 Imai A, Yoshie S, Nashida T, Shimomura R, Fukuda M. The 108 Reed GI, Houng AK, Fitzgerald ML. Human platelets contain Salivary Water and Protein Secretion 511

SNARE proteins and a Sec1p homologue that interacts with nucleotide-activated channel activation, and actin temporal syntaxin 4 and is phosphorylated after thrombin activation: synaptic tagging. J Neurosci. 2004;24:4205–4212. implications for platelet secretion. Blood. 1999;93:2617–2626. 126 Kaneko M, Takahashi T. Presynaptic mechanism underlying 109 Fujita Y, Sasaki T, Fukai K, Kotani H, Kimura T, Hata Y, et al. cAMP-dependent synaptic potentiation. J Neurosci. 2004;24: Phosphorylation of Munc-18/n-Sec1/rbSec1 by protein kinase 5204–5208. C: its implication in regulating the interaction of Munc-18/n- 127 Renstrom E, Elliasson I, Rorsman P. Protein kinase A-dependent Sec1/rbSec1 with syntaxin. J Biol Chem. 1996;271:7265–7268. and -independent stimulation of exocytosis by cAMP in mouse 110 Chavez-Noriega LE, Stevens CF. Increased transmitter release pancreatic B-cells. J Physiol. 1997;502:105–118. at excitatory synapses produced by direct activation of adenylate 128 Kripenski J, Coussen F, Bakalyar Ha, Tang WJ, Feinstein PG, cyclase in rat hippocampal slices. J Neurosci. 1974;4:310–317. Orth K, et al. Adenylyl cyclase amino acid sequence: possible 111 Burgoyne RD. Mechanisms of secretion from adrenal channel- or transporter-like structure. Science. 1989;244:1558– chromaffin cells. Biochim Biophys Acta. 1984;779:201–216. 15564. 112 Morgan A, Wilkinson M, Burgoyne RD. Identification of Exo2 129 Taussig R, Tang WJ, Hepler JR, Gilman AG. Distinct patterns of as the catalytic subunit of protein kinase A reveals a role for bidirectional regulation of mammalian adenylyl cyclases. J Biol cyclic AMP in Ca2+-dependent exocytosis in chromaffin cells. Chem. 1994;269:6093–6100. EMBO J. 1993;12:3747–3752. 130 Kawabe J, Ebina T, Toya Y, Oka N, Schwencke C, Duzic E, 113 Rabe CS, Schneider J, McGee R: Enhancement of depolariza- et al. Regulation of type V adenylyl cyclase by PMA-sensitive tion-dependent neurosecretion from PC12 cells by forskolin- and -insensitive protein kinase C isoenzymes in intact cells. induced elevation of cAMP. J Cyclic Nucleotide Res. 1982;8: FEBS Lett. 19965;384:273–276. 371–384. 131 Yoshimura M, Cooper DM. Type-specific stimulation of 114 Ammala C, Ashcroft FM, Rorsman P. Calcium-independent adenylyl cyclase by protein kinase C. J Biol Chem. 1993;268: potentiation of insulin release by cyclic AMP in single B-cells. 4604–4607. Nature. 1993;363:356–358. 132 Chen Y, Harry A, Li J, Smit MJ, Bai X, Magnusse NR, et al. 115 Baldys-Waligorska A, Pour A, Moriarty CM, Dowd F. The Adenylyl cyclase VI is selectively regulated by protein kinase A effect of calcium and cyclic AMP on amylase release in phosphorylation in a region involved in Gαs stimulation. Proc digitonin-permeabilized parotid gland cells. Biochim Biophy Natl Acad Sci U S A. 1997;94:14100–14104. Acta. 1987;929:190–196. 133 Iwami G, Kawabe J, Ebina T, Cannon PJ, Homcy CJ, Ishikawa 116 Quissell DO, Barzen KA, Deishen LM. Rat submandibular and Y. Regulation of adenylyl cyclase by protein kinase A. J Biol parotid protein phosphorylation and exocytosis: effect of site Chem. 1995;270:12481–12484. selective cAMP analogs. Crit Rev Oral Biol Med. 1993;4:443– 134 Charbonneau H, Kumar S, Novack JP, Blumenthal DK, Griffn 448. PR, Shabanowitz J, et al. Evidence for domain organization 117 Takuma T, Ichida T. Cyclic AMP antagonist Rp-cAMPS inhibits within the 61-kDa calmodulin-dependent cyclic nucleotide amylase exocytosis from saponin-permeabilized parotid acini. phosphodiesterase from bovine brain. Biochemistry. 1991;30: J Biochem. 1991;110:292–294. 7931–7940. 118 Muff R, Fisher JA. Parathyroid hormone secretion does not 135 Yan C, Zhao AZ, Rentley JK, Loughney K, Ferguson K, Veavo respond to changes of free calcium in electropermeabilized JA. Molecular cloning and characterization of a calmodulin- bovine parathyroid cells, but is stimulated with phorbol ester and dependent phosphodiesterase enriched in olfactory sensory cyclic AMP. Biochem Biophys Res Commun. 1986;139:1233– neuron. Proc Natl Acad Sci U S A. 1995;92:9677–9681. 1238. 136 Conti M, Richter W, Mehats C, Livera G, Park JY, Jin C. Cyclic 119 Gaisano HY, Shen L, Grondin G, Ghai M, Bouquillon A, Lowe AMP- specific PDE4 phosphodiesterases as critical components A, et al. The vesicle-associated membrane protein family of of cyclic AMP signaling. J Biol Chem. 2003;278:5493–5496. proteins in rat pancreatic and parotid acinar cells. Gastro- 137 Houslay MD, Adams DR. PDE4 cAMP phosphodiesterases: enterology. 1997;111:1661–1669. modular enzymes that orchestrate signaling cross-talk, desensiti- 120 Hansen J, Antonin W, Edwardson JM. Identification of SNAREs zation and compartmentalization. Biochem J. 2003;370:1–18. involved in regulated exocytosis in the pancreatic acinar cells. 138 Oki N, Takahashi SI, Hidaka H, Conti M. Short term feedback J Biol Chem. 1999;274:22871–22876. regulation of cAMP in FRTL-5 thyroid cells. Role of PDE4D3 121 Turner KM, Burgoyne RD, Morgan A. Protein phosphorylation phosphodiesterase activation. J Biol Chem. 2000;275:10831– and the regulation of synaptic membrane traffic. Trends 10837. Neurosci. 1999;22:459–464. 139 Taylor SS, Yang JU, Wu J, Baste NM, Radzio-Andzelm E, 122 Evans GJO, Wilkinson MC, Graham ME, Turner KM, Anand G. PKA: a portrait of protein kinases dynamics. Biochim Chamberlain LH, Burgoyne RD, et al. Phosphorylation of Biophys Acta. 2004;1697:259–269. cysteine string protein by PKA: Implication for the modulation 140 Zimmermann B, Chiorini JA, Ma Y, Kotin RM, Herberg FW. of exocytosis. J Biol Chem. 2001;276:47878–47885. PrKX is a novel catalytic subunit of the cAMP-dependent 123 Evans GJ, Morgan A. Phosphorylation-dependent interaction of protein kinase regulated by the regulatory subunit type I. J Biol the synaptic vesicle proteins cysteine string protein and Chem. 1999;274:5370–5378. synaptotagmin I. Biochem J. 2002;364:343–347. 141 Quissell DO, Deisher LM, Barzen KA. Activation and distri- 124 Ozaki N, Shibzaki T, Kashima Y, Miki T, Takahashi K, Ueno H, bution of rat parotid cAMP-dependent protein kinase following et al. cAMP, GEF a direct target of cAMP in regulated exo- beta-adrenergic receptor stimulation in vitro. Arch Oral Biol. cytosis. Nat Cell Biol. 2000;2:805–811. 1989;34:23–26. 125 Zong N, Zucker RS. Roles of Ca2+, hyperpolarization and cyclic 142 Scarpace PJ, Koller MM, Rajakumar G. Desipramine 512 Y Ishikawa et al

desensitizes beta-adrenergic signal transduction in salivary in rat parotid gland membranes. Arch Biochem Biophys. glands: differential regulation with age. Eur J Pharmacol. 1993;305:546–550. 1993;247:65–72. 153 Palm D, Munch G, Malek D, Hekman K. Identification of a 143 Benovic JL, Pike LJ, Cerione RA, Staniszewski C, Yoshimasa Gs-protein coupling domain to the beta-adrenoceptor using T, Codina J, et al. Phosphorylation of the mammalian beta- site-specific synthetic peptides. Carboxyl terminus of Gs alpha is adrenergic receptor by cyclic AMP-dependent protein kinase. involved in coupling to beta-adrenoceptors. FEBS Lett. 1990; Regulation of the rate of receptor phosphorylation and dephos- 261:294–298. phorylation by agonist occupancy and effects on coupling of the 154 Briggs MM, Stadel JM, Iyengar R, Lefkowitz RJ. Functional receptor to the stimulatory guanine nucleotide regulatory modification of the guanine nucleotide regulatory protein after protein. J Biol Chem. 1985;269:7094–7101. desensitization of turkey erythrocytes by catecholamines. Arch 144 Clark RB, Kunkel MW, Friedman J, Goka TJ, Johnson JA. Biochem Biophys. 1983;224:142–151. Activation of cAMP-dependent protein kinase is required for 155 Garrity MJ, Andreasen TJ, Storm DR, Robertson RP. Prosta- heterologous desensitization of adenylyl cyclase in S49 wild- glandin E-induced heterologous desensitization of hepatic type lymphoma cells. Proc Natl Acad Sci U S A. 1988;85:1442– adenylate cyclase. Consequences on the guanyl nucleotide 1446. regulatory complex. J Biol Chem. 1983;258:8692–8697. 145 Hausdorff WP, Bouvier M, O’ Dowd BE, Iros GP, Caron MG, 156 Simonds WF, Goldsmith PK, Codina J, Unson C, Spiegel AM. Lefkowitz RJ. Phosphorylation sites on two domains of the beta Gi2 mediates alpha 2-adrenergic inhibition of adenylyl cyclase 2-adrenergic receptor are involved in distinct pathways of in platelet membranes: in situ identification with G alpha C- receptor desensitization. J Biol Chem. 1989;264:12657–12665. terminal antibodies. Proc Natl Acad Sci U S A. 1989;86:7809– 146 Krasel C, Vilardaga JP, Bunemann M, Lohse MJ. Kinetics of 7813. G-protein-coupled receptor signalling and desensitization. 157 Katada T, Gilman AG, Watanabe Y, Bauer S, Jakobs KH. Biochem Soc Trans. 2004;32:1029–1031. Protein kinase C phosphorylates the inhibitory guanine- 147 Lohse MJ, Benovic CJ, Caron MG, Lefkowitz RJ. Multiple nucleotide-binding regulatory component and apparently pathways of rapid beta 2-adrenergic receptor desensitization. suppresses its function in hormonal inhibition of adenylate Delineation with specific inhibitors. J Biol Chem. 1990;265: cyclase. Eur J Biochem. 1985;151:431–437. 3202–3209. 158 Watanabe Y, Imaizumi T, Misaki N, Iwakura K, Yoshida H. 148 Ekstrom J. Sensitization of the rat parotid gland to secretagogues Effects of phosphorylation of inhibitory GTP-binding protein by following either parasympathetic denervation or sympathetic cyclic AMP-dependent protein kinase on its ADP-ribosylation denervation or decentralization. Acta Physiol Scand. 1980;108: by pertussis toxin, islet-activating protein. FEBS Lett. 1988;236: 253–261. 372–374. 149 De Peusner ICW, Perec CJ, Stefano FJE. Effects of sym- 159 Rich KA, Codina J, Floyd G, Sekura R, Hildebrandt JD, Iyengar pathectomy on the in vitro and β-responses of the parotid glands. R. Glucagon-induced heterologous desensitization of the MDCK Naunynn Schmiedeberg Arch Pharmacol. 1979;308:217–221. cell adenylyl cyclase. Increases in the apparent levels of the 150 Asking B, Emmelin N. Amylase-secretory responses to inhibitory regulator (Ni). J Biol Chem. 1984;259:7893–7901. sympathetic nerve impulses in the rat parotid gland following 160 Reithmann C, Gierschik P Sidiropoulos D, Werdan K, partial sympathetic ganglionectomy. Arch Oral Biol. 1987;32: Jakob KH. Mechanism of noradrenaline-induced heterologous 469–472. desensitization of adenylate cyclase stimulation in rat heart 151 Watson EL, DiJulio D, Kauffman D, Iversen J, Robinovitch MR, muscle cells: increase in the level of inhibitory G-protein Izutsu KT. Evidence for G proteins in rat parotid plasma alpha-subunits. Eur J Pharmacol. 1989;172:211–221. membranes and secretory granule membranes. Biochem J. 161 Hubbard MJ, Cohen P. Regulation of protein phosphatase-1G 1992;285:441–449. from rabbit skeletal muscle. 1. Phosphorylation by cAMP- 152 Sawaki K, Hiramatsu Y, Baum BJ, Ambudkar IS. Involvement dependent protein kinase at site 2 releases catalytic subunit of G alpha q/11 in m3-muscarinic receptor stimulation of from the glycogen-bound holoenzyme. Eur J Biochem. 1989; phosphatidylinositol 4,5 bisphosphate-specific phospholipase C 186:701–709.