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Molecular Mechanisms and Drug Development in Aquaporin Water Channel Diseases: The Translocation of Aquaporin-5 From Lipid Rafts to the Apical Plasma Membranes of Parotid Glands of Normal Rats and the Impairment of It in Diabetic or Aged Rats
Journal of Pharmacological Sciences
J Pharmacol Sci 96, 271 – 275 (2004)
©2004 The Japanese Pharmacological Society
Forum Minireview
Molecular Mechanisms and Drug Development in Aquaporin Water Channel Diseases: The Translocation of Aquaporin-5 From Lipid Rafts to the Apical Plasma Membranes of Parotid Glands of Normal Rats and the Impairment of It in Diabetic or Aged Rats Yasuko Ishikawa1,*, Noriko Inoue1, Yuan Zhenfang1, and Yoshiko Nakae2 Departments of 1Medical Pharmacology and 2Oral and Maxillofacial Anatomy, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15, Kuramoto-cho, Tokushima 770-8504, Japan Received September 29, 2004; Accepted October 13, 2004
Abstract. Salivary secretion from rat salivary glands occurs in response to stimulation by acetylcholine and norepinephrine released from nerve endings. Aquaporin-5 (AQP5) localizes in lipid rafts under control conditions and is induced to traffic to the apical plasma membrane in interlobular ducts of rat parotid glands by the activation of M3 muscarinic acetylcholine receptors or a 1-adrenoceptors. This review will focus on the mechanisms of the translocation of AQP5 from lipid rafts to the apical plasma membrane in the interlobular duct cells of parotid glands of normal rats and the impairment of its translocation in diabetic or senescent rats. Keywords: aquaporin-5, [Ca2+]i, lipid raft, interlobular duct, parotid gland
thetic nerve that innervates the submandibular and sublingual glands is derived from the superior salivary nucleus, whereas that which innervates the parotid glands is derived from the inferior salivary nucleus. The sympathetic nerve that innervates the major salivary glands is derived from the superior cervical ganglion. In general, parasympathetic stimulation produces a high flow rate of saliva as a result of the activation of M3 muscarinic acetylcholine receptors (mAChRs) on the salivary gland cells. In contrast, norepinephrine released from sympathetic nerve endings acts at both b- and a adrenoceptors on the salivary gland cells. Activation of b 2-adrenoceptors induces the secretion by exocytosis of salivary proteins contained in zymogen and mucinogen granules in acinar cells of salivary glands, whereas activation of a 1-adrenoceptors induces the fluid and ion secretion (3 – 5). The aquaporin-5 (AQP5) water channel has been cloned from the salivary glands of rats (6), humans (7), and mice (8). This review will focus on the mechanisms of the translocation of AQP5 in the interlobular duct cells of parotid glands of normal, diabetic, and aged rats after the activation of M3 mAChs.
Introduction Saliva has manifold functions in maintaining the integrity of the oral tissues, for example, in protecting teeth from caries; in tasting, mastication, and swallowing of food; in speech; and in the tolerance of dentures. Xerostomia, or dry mouth, is caused by a reduction of salivary secretion associated with the malfunction of salivary glands. This condition often accompanies diseases of maturity such as diabetes insipidus and cardiac failure, psychological emotional states such as fear and depression, autoimmune diseases, and the response to therapeutic irradiation of salivary glands. The use of anticholinergic drugs, anorectics, antihistamines, antidepressants, antihypertensives, and calcium antagonists also often induces xerostomia (1). This drug-induced side effect is a problem mostly in the elderly who are the principal consumers of such medications. The salivary glands are innervated by both sympathetic and parasympathetic nerves (2). The parasympa*Corresponding author. FAX: 088-633-7332 E-mail: [email protected]
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Characteristics of AQP5 in rat parotid glands The AQP5 cDNA, which encodes a 265-residue polypeptide, was isolated from rat submandibular glands by using a homology-based cloning approach as described by Raina et al. (6). Northern blot analysis revealed that AQP5 mRNA is expressed in submandibular, parotid, and sublingual glands, lacrimal gland, trachea; eye, and distal lung, but not in kidney, brain, or intestine (6). In vitro transcription and translation of this cDNA yielded a 27-kDa polypeptide, and expression of the corresponding cRNA in Xenopus oocytes resulted in a 20-fold increase in Pf that was reversibly inhibited by HgCl2, but which was not accompanied by increases in the membrane transport of urea or glycerol. Hydropathy analysis of the deduced amino acid sequence of AQP5 predicted a protein with six transmembrane domains (6, 9). The amino acid sequence contains two tandem repeats corresponding approximately to the NH2-terminal and COOH-terminal halves of the polypeptide (7, 8). In AQP5 knockout mice, although protein secretion by salivary glands and amylase activity in saliva were not affected, the body growth rate was reduced by 20% and pilocarpinestimulated salivary fluid secretion was reduced by more than 60% compared with those in wild-type mice. On the basis of these observations, Ma et al. concluded that AQP5 plays an important role in salivary fluid secretion (10). AQP5 located in lipid rafts was translocated to the apical plasma membranes in interlobular duct cells of rat parotid glands Lipid rafts have been found to be involved in various
cellular events such as signal transduction (11), membrane sorting and trafficking (12), cell polarization (13), and the allergic response (14). Especially, it is noteworthy that lipid rafts are implicated in the sorting of some membrane proteins to the apical plasma membrane domain of epithelial cells (15). Lipid rafts are defined as glycosphingolipid- and cholesterol-enriched microdomains that are characterized by their insolubility in the non-ionic detergent Triton X-100 in the cold (16 – 18) or are detected in the high buoyant fraction when separating membranes of differing densities using discontinuous sucrose-density centrifugation (19). Under unstimulated conditions, AQP5 in rat parotid gland cells was present in the Triton X-100-insoluble fraction and float to the light density fraction (20). AQP5 fluorescence was localized in both the apical plasma membranes and intracellular structures with a raft marker, flotillin-2 or GM1, in interlobular duct cells under unstimulated conditions. At 10 min after the injection of cevimeline, AQP5 was located predominantly on the apical plasma membrane in interlobular duct cells. Conversely, at 60 min after the injection, AQP5 fluorescence showed diffuse pattern of staining with a raft marker in the cytoplasm (Fig. 1). These findings demonstrated that AQP5 was located in lipid rafts, and the activation of M3 mAChRs with cevimeline induced the translocation of AQP5 from lipid rafts to the apical plasma membrane in interlobular duct cells of rat parotid glands. These results are supported by the report that AQP5 was detected in the Rab4-containing intracellular membrane fraction located in the subapical region of rat parotid glands (21). Although it was reported that AQP5 is not located in lipid rafts of lung (22), AQP1 is located in a particular subtype of lipid rafts called caveolae, which are plasmalemmal vesicles in lung (23) and
Fig. 1. Effect of cevimeline on the distribution of AQP5 in the intralobular duct of rat parotid glands. Cevimeline (B and C) or saline (A) was injected intravenously into rats. At 0 (A), 10 (B), and 60 (C) min after the injection, parotid glands were prepared and then examined by immunofluorescence microscopy. The section was exposed to anti-AQP5 antibody. Reactivity was then detected by the use of Alexa-488 fluorescent secondary antibody.
Aquaporin in Parotid Glands
cardiomyocytes (24), and appear to shuttle molecules into and out of cells. Exposure to hypertonic solution triggers a reversible translocation of AQP1 in cardiac myocytes (24). Our biochemical studies also showed that the activation of M3 mAChRs (25) or a 1-adrenoceptors (26) induces the translocation of AQP5 from intracellular membranes to the apical plasma membrane in rat parotid gland acinar cells. AQP5 is not present in the basolateral membrane (27). Taken together, it was demonstrated that the activation of M3 mAChRs induces the translocation of AQP5 from lipid rafts to the apical plasma membrane in the interlobular duct cells of rat parotid glands. The amount of AQP5 on the apical plasma membrane in rat parotid glands Immunoblot analysis with antibodies to AQP5 revealed that exposure of rat parotid tissues to ACh or epinephrine induced the rapid increases in the amount of AQP5 on the apical plasma membrane. The effects of these agents were maximal at 15 s and 1 min, respectively (25, 26), resulting in a rapid and transient increase in AQP5 levels on the apical plasma membrane by increasing the cytosolic Ca2+ concentration ([Ca2+]i) (25, 26). In contrast, muscarinic receptor agonists, (±)-cis2-methylspilo(1,3-oxathiolane-5,3')quinuclidine hydrochloride hemihydrate (SNI-2011, cevimeline) and pilocarpine induce a long-lasting increase in AQP5 levels in the apical plasma membrane of rat parotid
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glands (28). The effect of cevimeline on an increase in AQP5 levels on the apical plasma membrane of rat parotid gland acinar cells is longer than that of pilocarpine (29). Regulation of the function of AQP5 by [Ca2+]i In parotid glands, the stimulation of M3 mAChRs with their agonists generates inositol trisphosphate and diacylglycerol through the stimulation of Gq/ 11 protein and phospholipase Cb. Inositol trisphosphate is then involved in the subsequent elevation of [Ca2+]i (30). The rise in [Ca2+]i has a key role in M3 mAChR agonistinduced increases in AQP5 levels on the apical plasma membrane (30). Recently, generation and release of NO was recognized as an important second messenger pathway when elevated [Ca2+]i induced the activation of the calmodulin-dependent enzyme, NO synthase (Fig. 2). NO increases cGMP formation via stimulation of soluble guanylate cyclase. The inhibition of calmodulin kinase II decreased the M3 mAChR agonist-induced increases in AQP5 levels on the apical plasma membrane (30). The exposure of dbcGMP induced the translocation of AQP5 in the presence of Ca2+. In the presence of a Ca2+ chelator, dbcGMP failed to induce the translocatopn of AQP5 to the apical plasma membrane (30), suggesting that enhancement of [Ca2+]i fulfills a crucial role in M3 mAChRs agonist-induced the translocation of AQP5 to the apical plasma membrane (30).
Fig. 2. Schematic representation of signal transduction in M3 muscarinic receptorinduced the translocation of AQP5 in the intralobular duct of rat parotid glands. M3R, M3 muscarinic receptor; Gq, G protein that stimulates phospholipase C (PLC); PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-triphosphate; IP3R, IP3 receptor; RyR, Ryanodine receptor; Arg, arginine; CaM, calmodulin; CaMKII, calmodulin-dependent kinaseII; nNOS, neuronal nitric oxide synthase; sGC, soluble guanylate cyclase; PKG, cGMPdependent protein kinase; cADPr, cADPribose; APM, apical plasma membrane; BLM, basolateral plasma membrane.
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Impairment in the translocation of AQP5 to the apical plasma membranes in the interlobular duct cells of rat parotid glands during aging ACh increased the amount of AQP5 on the apical plasma membrane by 3.6-fold in parotid tissue cells of young adult rats, but 1.8-fold in those of senescent rats. In contrast, epinephrine induced smaller increases in the amount of AQP5 on the plasma membranes in the tissue cells of young and senescent rats, which were 2.3- and 1.8-fold, respectively (31). The changes in the responsiveness of AQP5 in the tissues to nervous stimuli during aging corresponded to Ca2+-dependent signal transduction activity. Cevimeline induced a persistent increase in AQP5 levels on the apical plasma membrane, even in senescent rats (31). M3 mAChRs and Gq/ 11 protein levels in parotid glands did not decrease during aging. These results suggest that the stimulatory effect of ACh on the translocation of AQP5 to the apical plasma membrane in rat parotid cells is markedly reduced during aging and that cevimeline is effective for treatment of age-related xerostomia. Defect of the translocation of AQP5 to the apical plasma membranes in interlobular duct cells of parotid glands of diabetic rats Parotid secretory response to ACh is reduced in streptozotosin (STZ)-induced diabetic rats. Lowered susceptibility of mAChRs was recognized (32). Although mRNA levels for AQP5 were increased, the amount of AQP5 in parotid homogenatre was decreased in STZ rats. Cevimeline induced the translocation of AQP5 from lipid rafts to the apical plasma membrane in interlobular duct cells of parotid glands of control rats, but did not in those of STZ-induced diabetic rats (Y. Ishikawa et al., unpublished data). Conclusion AQP5 localized in interlobular duct cells of rat parotid glands was translocated from lipid rafts to the apical plasma membrane and plays an important role in salivary secretion in response to the activation of M3 mAChRs. The implication of the activation of Ca2+ and calmodulin-dependent enzymes and the NOS-PKG signaling pathway was demonstrated in this translocation of AQP5. The impairment of the translocation of AQP5 was also shown in parotid glands of senescent rats and diabetic rats and suggested to cause xerostomia.
Acknowledgment This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. References 1 FDI Working Group 10. Saliva: its role in health and disease. Int Dent J. 1992;42:291–304. 2 Bradley RM. Salivary secretion. In: Bradley RM, editor. Essentials of oral physiology. St. Louis: Mosby-Year Book, Inc; 1995; p. 161–186. 3 Baum BJ. Principles of saliva secretion. Ann NY Acad Sci. 1993;694:17–23. 4 Hata F, Ishida H, Kagawa K, Kondo E, Kondo S, Noguchi Y. b -Adrenoceptor alterations coupled with secretory response in rat parotid tissue. J Physiol (Lond). 1983;341:185–196. 5 Ishikawa Y, Amano I, Eguchi T, Ishida H. Mechanism of isoproterenol-induced heterologous desensitization of mucin secretion from rat submandibular glands; Regulation of phosphorylation of Gi proteins controls the cell response to the subsequent stimulation. Biochim Biophys Acta. 1995;1265:173– 180. 6 Raina S, Preston GM, Guggino WB, Agre P. Molecular cloning and characterization of an aquaporin cDNA from salivary, lacrimal, and respiratory tissues. J Biol Chem. 1995;270:1908– 1912. 7 Lee MD, Bhakta KY, Raina S, Yanescu R, Griffin CA, Copeland NG, et al. The human aquaporin-5 gene. J Biol Chem. 1995;271:8599–8604. 8 Krane CM, Towne JE, Menon AG. Cloning and characterization of murine Aqp5: evidence for a conserved aquaporin gene cluster. Mammalian Genome. 1999;10:498–505. 9 Lee MD, King LS, Agre P. The aquaporin family of water channel proteins in clinical medicine. Medicine. 1997;76:141– 156. 10 Ma T, Song Y, Gillespie A, Carlson EJ, Epstein CJ, Verkman AS. Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels. J Biol Chem. 1999;274:20071– 20074. 11 Simons K, Toomre D. Lipid rafts and signal transduction. Nat Rev Mol Cell Biol. 2000;1:31–39. 12 Jacobson K, Dietrich C. Looking at lipid rafts. Trends Cell Biol. 2002;9:87–91. 13 Simons K, Ehehalt R. Cholesterol, lipid rafts, and disease. J Clin Invest. 2002;110:597–603. 14 Brown DA, Rose JK. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992;68:533–544. 15 Polishchuk R, Di Pentima A, Lippincott-Schwartz J. Delivery of raft-associated, GPI-anchored proteins to the apical surface of polarized MDCK cells by a transcytotic pathway. Nat Cell Biol. 2004;6:297–307. 16 Lafont F, Lecat S, Verkade P, Simons K. Annexin XIIIb associates with lipid microdomains to function in apical delivery. J Cell Biol. 1998;142:1413–1427. 17 Simons K, Ikonen E. Functional rafts in cell membranes. Nature. 1997;387:569–572.
Aquaporin in Parotid Glands 18 Keller P, Simons K. Cholesterol is required for surface transport of influenza virus hemagglutinin. J Cell Biol. 1998;140:1357– 1367. 19 Igarashi J, Michel T. Agonist-modulated targeting of the EDG-1 receptor to plasmalemmal caveolae. J Biol Chem. 2000;275: 32363–32370. 20 Ishikawa Y, Zhenfang Y, Inoue N, Shono M, Nakae Y. Translocation of aquaporin-5 from rafts to apical membrane in rat parotid glands stimulated by muscarinic agonists. J Histochem Cytochem. 2004;52:Suppl I S56. 21 Nashida T, Yoshie S, Imai A, Shimomura H. Co-localization of Rab4 with endocytosis-related proteins in the rat parotid glands. Arch Histol Cytol. 2003;66:45–52. 22 Palestini P, Calvi C, Conforti E, Daffara R, Botto L, Miserocchi G. Compositional changes in lipid microdomains of air-blood barrier plasma membranes in pulmonary intestinal edema. J Appl Physiol. 2003;95:1446–1452. 23 Schenitzer JE, Oh P. Aquaporin-1 in plasma membrane and caveolae provides mercury-sensitive water channels across lung endothelium. Am J Physiol. 1996;270:H416–H422. 24 Page E, Winterfield J, Goings G, Bastawrous A, Upshaw-Earley J, Doyle D. Water channel proteins in rat cardiac myocyte caveolae: osmolarity-dependent reversible internalization. Am J Physiol. 1998;274:H1998–H2000. 25 Ishikawa Y, Eguchi T, Skowronski MT, Ishida H. Acetylcholine acts on M3 muscarinic receptors and induces the translocation of aquaporin5 water channel via cytosolic Ca2+ elevation in rat
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parotid glands. Biochem Biophys Res Commun. 1998;245:835– 840. Ishikawa Y, Skowronski MT, Inoue N, Ishida H. a 1-Adrenoceptor-induced trafficking of aquaporin-5 to the apical plasma membrane of rat parotid cells. Biochem Biophys Res Commun. 1999;265:94–100. He X, Tse CM, Donowitz M, Alper SL, Gabriel SE, Baum BJ. Polarized distribution of key membrane transport proteins in rat submandibular gland. Pflugers Arch. 1997;433:260–268. Ishikawa Y, Skowronski MT, Ishida H. Persistent increase in the amount of aquaporin-5 in the apical plasma membrane of rat parotid acinar cells induced by a muscarinic agonist SNI-2011. FEBS Lett. 2000;477:253–257. Ishikawa Y, Ishida H. Aquaporin water channel in salivary glands. Jpn J Pharmacol. 2000;83:95–101. Ishikawa Y, Iida H, Ishida H. The muscarinic acetylcholine receptor-stimulated increase in aquaporin-5 levels in the apical plasma membrane in rat parotid acinar cells is coupled with activation of nitric oxide /cGMP signal transduction. Mol Pharmacol. 2002;61:1423–1434. Inoue N, Iida H, Yuan Z, Ishikawa Y, Ishida H. Age-related decrease in the response of aquaporin-5 to acetylcholine in rat parotid glands. J Dent Res. 2003;82:416–480. Watanabe M, Yamagishi-Wang H, Kawaguchi M. Lowered susceptibility of muscarinic receptor involved in salivary secretion of streptozotocin-induced diabetic rats. Jpn J Pharmacol. 2001;87:117–124.
J Pharmacol Sci 96, 271 – 275 (2004)
©2004 The Japanese Pharmacological Society
Forum Minireview
Molecular Mechanisms and Drug Development in Aquaporin Water Channel Diseases: The Translocation of Aquaporin-5 From Lipid Rafts to the Apical Plasma Membranes of Parotid Glands of Normal Rats and the Impairment of It in Diabetic or Aged Rats Yasuko Ishikawa1,*, Noriko Inoue1, Yuan Zhenfang1, and Yoshiko Nakae2 Departments of 1Medical Pharmacology and 2Oral and Maxillofacial Anatomy, Institute of Health Biosciences, The University of Tokushima Graduate School, 3-18-15, Kuramoto-cho, Tokushima 770-8504, Japan Received September 29, 2004; Accepted October 13, 2004
Abstract. Salivary secretion from rat salivary glands occurs in response to stimulation by acetylcholine and norepinephrine released from nerve endings. Aquaporin-5 (AQP5) localizes in lipid rafts under control conditions and is induced to traffic to the apical plasma membrane in interlobular ducts of rat parotid glands by the activation of M3 muscarinic acetylcholine receptors or a 1-adrenoceptors. This review will focus on the mechanisms of the translocation of AQP5 from lipid rafts to the apical plasma membrane in the interlobular duct cells of parotid glands of normal rats and the impairment of its translocation in diabetic or senescent rats. Keywords: aquaporin-5, [Ca2+]i, lipid raft, interlobular duct, parotid gland
thetic nerve that innervates the submandibular and sublingual glands is derived from the superior salivary nucleus, whereas that which innervates the parotid glands is derived from the inferior salivary nucleus. The sympathetic nerve that innervates the major salivary glands is derived from the superior cervical ganglion. In general, parasympathetic stimulation produces a high flow rate of saliva as a result of the activation of M3 muscarinic acetylcholine receptors (mAChRs) on the salivary gland cells. In contrast, norepinephrine released from sympathetic nerve endings acts at both b- and a adrenoceptors on the salivary gland cells. Activation of b 2-adrenoceptors induces the secretion by exocytosis of salivary proteins contained in zymogen and mucinogen granules in acinar cells of salivary glands, whereas activation of a 1-adrenoceptors induces the fluid and ion secretion (3 – 5). The aquaporin-5 (AQP5) water channel has been cloned from the salivary glands of rats (6), humans (7), and mice (8). This review will focus on the mechanisms of the translocation of AQP5 in the interlobular duct cells of parotid glands of normal, diabetic, and aged rats after the activation of M3 mAChs.
Introduction Saliva has manifold functions in maintaining the integrity of the oral tissues, for example, in protecting teeth from caries; in tasting, mastication, and swallowing of food; in speech; and in the tolerance of dentures. Xerostomia, or dry mouth, is caused by a reduction of salivary secretion associated with the malfunction of salivary glands. This condition often accompanies diseases of maturity such as diabetes insipidus and cardiac failure, psychological emotional states such as fear and depression, autoimmune diseases, and the response to therapeutic irradiation of salivary glands. The use of anticholinergic drugs, anorectics, antihistamines, antidepressants, antihypertensives, and calcium antagonists also often induces xerostomia (1). This drug-induced side effect is a problem mostly in the elderly who are the principal consumers of such medications. The salivary glands are innervated by both sympathetic and parasympathetic nerves (2). The parasympa*Corresponding author. FAX: 088-633-7332 E-mail: [email protected]
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Characteristics of AQP5 in rat parotid glands The AQP5 cDNA, which encodes a 265-residue polypeptide, was isolated from rat submandibular glands by using a homology-based cloning approach as described by Raina et al. (6). Northern blot analysis revealed that AQP5 mRNA is expressed in submandibular, parotid, and sublingual glands, lacrimal gland, trachea; eye, and distal lung, but not in kidney, brain, or intestine (6). In vitro transcription and translation of this cDNA yielded a 27-kDa polypeptide, and expression of the corresponding cRNA in Xenopus oocytes resulted in a 20-fold increase in Pf that was reversibly inhibited by HgCl2, but which was not accompanied by increases in the membrane transport of urea or glycerol. Hydropathy analysis of the deduced amino acid sequence of AQP5 predicted a protein with six transmembrane domains (6, 9). The amino acid sequence contains two tandem repeats corresponding approximately to the NH2-terminal and COOH-terminal halves of the polypeptide (7, 8). In AQP5 knockout mice, although protein secretion by salivary glands and amylase activity in saliva were not affected, the body growth rate was reduced by 20% and pilocarpinestimulated salivary fluid secretion was reduced by more than 60% compared with those in wild-type mice. On the basis of these observations, Ma et al. concluded that AQP5 plays an important role in salivary fluid secretion (10). AQP5 located in lipid rafts was translocated to the apical plasma membranes in interlobular duct cells of rat parotid glands Lipid rafts have been found to be involved in various
cellular events such as signal transduction (11), membrane sorting and trafficking (12), cell polarization (13), and the allergic response (14). Especially, it is noteworthy that lipid rafts are implicated in the sorting of some membrane proteins to the apical plasma membrane domain of epithelial cells (15). Lipid rafts are defined as glycosphingolipid- and cholesterol-enriched microdomains that are characterized by their insolubility in the non-ionic detergent Triton X-100 in the cold (16 – 18) or are detected in the high buoyant fraction when separating membranes of differing densities using discontinuous sucrose-density centrifugation (19). Under unstimulated conditions, AQP5 in rat parotid gland cells was present in the Triton X-100-insoluble fraction and float to the light density fraction (20). AQP5 fluorescence was localized in both the apical plasma membranes and intracellular structures with a raft marker, flotillin-2 or GM1, in interlobular duct cells under unstimulated conditions. At 10 min after the injection of cevimeline, AQP5 was located predominantly on the apical plasma membrane in interlobular duct cells. Conversely, at 60 min after the injection, AQP5 fluorescence showed diffuse pattern of staining with a raft marker in the cytoplasm (Fig. 1). These findings demonstrated that AQP5 was located in lipid rafts, and the activation of M3 mAChRs with cevimeline induced the translocation of AQP5 from lipid rafts to the apical plasma membrane in interlobular duct cells of rat parotid glands. These results are supported by the report that AQP5 was detected in the Rab4-containing intracellular membrane fraction located in the subapical region of rat parotid glands (21). Although it was reported that AQP5 is not located in lipid rafts of lung (22), AQP1 is located in a particular subtype of lipid rafts called caveolae, which are plasmalemmal vesicles in lung (23) and
Fig. 1. Effect of cevimeline on the distribution of AQP5 in the intralobular duct of rat parotid glands. Cevimeline (B and C) or saline (A) was injected intravenously into rats. At 0 (A), 10 (B), and 60 (C) min after the injection, parotid glands were prepared and then examined by immunofluorescence microscopy. The section was exposed to anti-AQP5 antibody. Reactivity was then detected by the use of Alexa-488 fluorescent secondary antibody.
Aquaporin in Parotid Glands
cardiomyocytes (24), and appear to shuttle molecules into and out of cells. Exposure to hypertonic solution triggers a reversible translocation of AQP1 in cardiac myocytes (24). Our biochemical studies also showed that the activation of M3 mAChRs (25) or a 1-adrenoceptors (26) induces the translocation of AQP5 from intracellular membranes to the apical plasma membrane in rat parotid gland acinar cells. AQP5 is not present in the basolateral membrane (27). Taken together, it was demonstrated that the activation of M3 mAChRs induces the translocation of AQP5 from lipid rafts to the apical plasma membrane in the interlobular duct cells of rat parotid glands. The amount of AQP5 on the apical plasma membrane in rat parotid glands Immunoblot analysis with antibodies to AQP5 revealed that exposure of rat parotid tissues to ACh or epinephrine induced the rapid increases in the amount of AQP5 on the apical plasma membrane. The effects of these agents were maximal at 15 s and 1 min, respectively (25, 26), resulting in a rapid and transient increase in AQP5 levels on the apical plasma membrane by increasing the cytosolic Ca2+ concentration ([Ca2+]i) (25, 26). In contrast, muscarinic receptor agonists, (±)-cis2-methylspilo(1,3-oxathiolane-5,3')quinuclidine hydrochloride hemihydrate (SNI-2011, cevimeline) and pilocarpine induce a long-lasting increase in AQP5 levels in the apical plasma membrane of rat parotid
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glands (28). The effect of cevimeline on an increase in AQP5 levels on the apical plasma membrane of rat parotid gland acinar cells is longer than that of pilocarpine (29). Regulation of the function of AQP5 by [Ca2+]i In parotid glands, the stimulation of M3 mAChRs with their agonists generates inositol trisphosphate and diacylglycerol through the stimulation of Gq/ 11 protein and phospholipase Cb. Inositol trisphosphate is then involved in the subsequent elevation of [Ca2+]i (30). The rise in [Ca2+]i has a key role in M3 mAChR agonistinduced increases in AQP5 levels on the apical plasma membrane (30). Recently, generation and release of NO was recognized as an important second messenger pathway when elevated [Ca2+]i induced the activation of the calmodulin-dependent enzyme, NO synthase (Fig. 2). NO increases cGMP formation via stimulation of soluble guanylate cyclase. The inhibition of calmodulin kinase II decreased the M3 mAChR agonist-induced increases in AQP5 levels on the apical plasma membrane (30). The exposure of dbcGMP induced the translocation of AQP5 in the presence of Ca2+. In the presence of a Ca2+ chelator, dbcGMP failed to induce the translocatopn of AQP5 to the apical plasma membrane (30), suggesting that enhancement of [Ca2+]i fulfills a crucial role in M3 mAChRs agonist-induced the translocation of AQP5 to the apical plasma membrane (30).
Fig. 2. Schematic representation of signal transduction in M3 muscarinic receptorinduced the translocation of AQP5 in the intralobular duct of rat parotid glands. M3R, M3 muscarinic receptor; Gq, G protein that stimulates phospholipase C (PLC); PIP2, phosphatidylinositol 4,5-bisphosphate; IP3, inositol 1,4,5-triphosphate; IP3R, IP3 receptor; RyR, Ryanodine receptor; Arg, arginine; CaM, calmodulin; CaMKII, calmodulin-dependent kinaseII; nNOS, neuronal nitric oxide synthase; sGC, soluble guanylate cyclase; PKG, cGMPdependent protein kinase; cADPr, cADPribose; APM, apical plasma membrane; BLM, basolateral plasma membrane.
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Impairment in the translocation of AQP5 to the apical plasma membranes in the interlobular duct cells of rat parotid glands during aging ACh increased the amount of AQP5 on the apical plasma membrane by 3.6-fold in parotid tissue cells of young adult rats, but 1.8-fold in those of senescent rats. In contrast, epinephrine induced smaller increases in the amount of AQP5 on the plasma membranes in the tissue cells of young and senescent rats, which were 2.3- and 1.8-fold, respectively (31). The changes in the responsiveness of AQP5 in the tissues to nervous stimuli during aging corresponded to Ca2+-dependent signal transduction activity. Cevimeline induced a persistent increase in AQP5 levels on the apical plasma membrane, even in senescent rats (31). M3 mAChRs and Gq/ 11 protein levels in parotid glands did not decrease during aging. These results suggest that the stimulatory effect of ACh on the translocation of AQP5 to the apical plasma membrane in rat parotid cells is markedly reduced during aging and that cevimeline is effective for treatment of age-related xerostomia. Defect of the translocation of AQP5 to the apical plasma membranes in interlobular duct cells of parotid glands of diabetic rats Parotid secretory response to ACh is reduced in streptozotosin (STZ)-induced diabetic rats. Lowered susceptibility of mAChRs was recognized (32). Although mRNA levels for AQP5 were increased, the amount of AQP5 in parotid homogenatre was decreased in STZ rats. Cevimeline induced the translocation of AQP5 from lipid rafts to the apical plasma membrane in interlobular duct cells of parotid glands of control rats, but did not in those of STZ-induced diabetic rats (Y. Ishikawa et al., unpublished data). Conclusion AQP5 localized in interlobular duct cells of rat parotid glands was translocated from lipid rafts to the apical plasma membrane and plays an important role in salivary secretion in response to the activation of M3 mAChRs. The implication of the activation of Ca2+ and calmodulin-dependent enzymes and the NOS-PKG signaling pathway was demonstrated in this translocation of AQP5. The impairment of the translocation of AQP5 was also shown in parotid glands of senescent rats and diabetic rats and suggested to cause xerostomia.
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