, an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. M 3 muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and M 3 mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular Ca 2ϩ signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands.translocation; aquaporin-5 AQUAPORINS (AQPs) form water channels that selectively transport water across the plasma membrane (19). Thirteen mammalian AQPs, AQP0 -AQP12, have been identified (1, 27). AQP5, initially cloned from rat submandibular glands (32), is an apical membrane water channel that is distributed to epithelial cells in several secretory glands, such as salivary glands (10). Salivary fluid secretion is defective in transgenic mice lacking AQP5, indicating that AQP5 has an important role in fluid secretion (24).The parotid glands are innervated by both sympathetic and parasympathetic nerves (2). The activation of M 3 muscarinic acetylcholine receptors (mAChRs) and ␣ 1 -adrenoceptors increases intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) and induces salivary fluid secretion (2). In vitro experiments using rat parotid slices demonstrated that ACh and epinephrine acting at M 3 mAChRs and ␣ 1 -adrenoceptors, respectively, induce a rapid increase in the AQP5 levels in the apical plasma membrane (APM) by increasing [Ca 2ϩ ] i (14, 15). We previously investigated (16) the possible role of Ca 2ϩ -mediated intracellular signal transduction in the M 3 mAChR agonist-induced increase in AQP5 levels in the APM and demonstrated that activation of endogenous nitric oxide synthase and protein kinase G in the cells is coupled with t...

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 waterpermeable 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 concentration [Ca 2+ ] 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 Ca 2+ mobilizing receptors simultaneously trigger and regulate exocytosis. Zymogen granule exocytosis occurs under the control of essential process, stimulus-secretion coupling, in salivary glands. Ca 2+ 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 monophosphate (cAMP)/ cAMP-dependent protein kinase system has a major role in zymogen granule exocytosis without significant increases in [Ca 2+ ] i . In section II, the mechanisms underlying the control of salivary protein secretion and its dysfunction are reviewed.

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. The activation of M3 muscarinic acetylcholine receptors (mAChRs) or alpha1-adrenoceptors on the salivary glands induces salivary fluid secretion. AQP5 localizes in lipid rafts and activation of the M3 mAChRs or alpha1-adrenoceptors induced its translocation together with the lipid rafts to the APM in the interlobular ducts of rat parotid glands. This review focuses on the mechanisms of AQP5 translocation together with lipid rafts to the APM in the interlobular duct cells of parotid glands of normal rats and the impairment of AQP5 translocation in diabetes and senescence.

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