Physiological role of aquaporin 5 in salivary glands.

Regarding the 13 known mammalian aquaporins (AQPs), their functions in their expressing tissues, effects of their mutation/polymorphisms in humans, and effects of knockout of their genes are summarized in this review article. The roles of AQP5, an exocrine gland-type water channel, in the salivary gland under normal and pathophysiological conditions are reviewed in detail. First, the involvement of AQP5 in water secretion from acinar cells was demonstrated by measuring volume changes of acini/acinar cells, as well as activation energy (E a) in transepithelial water movement by NMR spectrometry, and a functional linkage between AQP5 and TRPV4 was suggested. Next, involvement of the parasympathetic nervous system on the AQP5 levels in the acinar cells of the submandibular and that of a ß-adrenergic agonist on those in the parotid gland are described. That is, chorda tympani denervation induces autophagy of the submandibular gland, causing AQP5 degradation/metabolism, whereas isoproterenol, a ß-adrenergic agonist, causes first an increase then decrease in AQP5 levels in the parotid gland, which action is coupled with the secretory-restoration cycle of amylase-containing secretory granules. The PG also responded to endotoxin, a lipopolysaccharide that activates NF-κB and MAPK pathways. Elevated NF-κB and AP-1 (c-Fos/c-Jun) form a complex that can bind to the NF-κB-responsive element on the AQP5 promoter and thus potentially downregulate AQP5 transcription. Salivary gland pathologies and conditions involving AQP5 and possible treatments are described as well.

Reduced salivary gland size and increased presence of epithelial progenitor cells in DLK1-deficient mice.

DLK1 (PREF1, pG2, or FA1) is a transmembrane and secreted protein containing epidermal growth factor-like repeats. Dlk1 expression is abundant in many tissues during embryonic and fetal development and is believed to play an important role in the regulation of tissue differentiation and fetal growth. After birth, Dlk1 expression is abolished in most tissues but is possibly reactivated to regulate stem cell activation and responses to injury. We have recently reported that DLK1 regulates many aspects of salivary gland organogenesis. Here, we have extended our studies of the salivary gland phenotype of Dlk1 knock-out mice. We have observed that salivary glands are smaller and weigh significantly less in both Dlk1 knock-out males and females compared with gender and age-matched wild-type mice and regardless of the natural sexual dimorphism in rodent salivary glands. This reduced size correlates with a reduced capacity of Dlk1-deficient mice to secrete saliva after stimulation with pilocarpine. However, histological and ultrastructural analyses of both adult and developing salivary gland tissues have revealed no defects in Dlk1 ((-/-)) mice, indicating that genetic compensation accounts for the relatively mild salivary phenotype in these animals. Finally, despite their lack of severe anomalies, we have found that salivary glands from Dlk1-deficient mice present a higher amount of CK14-positive epithelial progenitors at various developmental stages, suggesting a role for DLK1 in the regulation of salivary epithelial stem cell balance.

Serotonergic Innervation of the Salivary Glands and Central Nervous System of Adult Glossina pallidipes Austen (Diptera: Glossinidae), and the Impact of the Salivary Gland Hypertrophy Virus (GpSGHV) on the Host.

Using a serotonin antibody and confocal microscopy, this study reports for the first time direct serotonergic innervation of the muscle sheath covering the secretory region of the salivary glands of adult tsetse fly, Glossina pallidipes Austen. Reports to date, however, note that up until this finding, dipteran species previously studied lack a muscle sheath covering of the secretory region of the salivary glands. Direct innervation of the salivary gland muscle sheath of tsetse would facilitate rapid deployment of saliva into the host, thus delaying a host response. Our results also suggest that the neuronal and abnormal pattern seen in viral infected glands by the Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) is due to a compensatory increased branching of the neurons of the salivary glands, which is associated with the increased size of the salivary glands in viral infected flies. This study shows for the first time serotonin in the cell bodies of the brain and thoracico-abdominal ganglion in adult tsetse, G. pallidipes Austen (Diptera: Glossinidae). A hypothesis is proposed as to whether innervation of the muscle sheath covering of the secretory region of the salivary glands is present in brachyceran compared with nematoceran dipterans; and, a plea is made that more research is needed to develop a blood feeding model, similar to that in the blow flies, for elucidating the various mechanisms involved in production and deployment of saliva.

Orcokinin-like immunoreactivity in central neurons innervating the salivary glands and hindgut of ixodid ticks.

Orcokinins are conserved neuropeptides within the Arthropoda but their cellular distribution and functions in ticks are unknown. We use an antibody against the highly conserved N-terminal (NFDEIDR) of mature orcokinin peptides to examine their distribution in six ixodid species: Amblyomma variegatum, Dermacentor reticulatus, Hyalomma anatolicum, Ixodes scapularis, Ixodes ricinus and Rhipicephalus appendiculatus. Numerous immunoreactive neurons (~100) were detected in various regions of the synganglion (central nervous system) in all examined tick species. Immunoreactive projections of two prominent groups of efferent neurons in the post-oesophageal region were examined in detail: (1) neurons innervating the salivary glands; (2) neurons innervating the hindgut. Using matrix-assisted laser desorption/ionisation-time-of-flight (MALDI-TOF), we detected orcokinin peaks in extracts of the synganglia and hindguts but not in the salivary glands of I. scapularis females. Our data provide further evidence of the presence of orcokinin in ixodid ticks and establish a morphological basis for functional studies of identified peptidergic neuronal networks.

Dopamine, vesicular transporters, and dopamine receptor expression in rat major salivary glands.

The localization of dopamine stores and the expression and localization of dopamine (DAT) and vesicular monoamine transporters (VMAT) type-1 and -2 and of dopamine D1-like and D2-like receptor subtypes were investigated in rat submandibular, sublingual, and parotid salivary glands by HPLC with electrochemical detection, as well as immunochemical and immunohistochemical techniques. Male Wistar rats of 2 mo of age were used. The highest dopamine levels were measured in the parotid gland, followed by the submandibular and sublingual glands. Western blot analysis revealed DAT, VMAT-1, VMAT-2, and dopamine receptors immunoreactivity in membrane preparations obtained from the three glands investigated. Immunostaining for dopamine and transporters was developed within striated ducts. Salivary glands processed for dopamine receptors immunohistochemistry developed an immunoreaction primarily in striated and excretory ducts. In the submandibular gland, acinar cells displayed strong immunoreactivity for the D2 receptor, while cells of the convoluted granular tubules were negative for both D1-like and D2-like receptors. Parotid glands acinar cells displayed the highest immunoreactivity for both D1 and D2 receptors compared with other salivary glands. The above localization of dopamine and dopaminergic markers investigated did not correspond closely with neuron-specific enolase (NSE) localization. This indicates that at least in part, catecholamine stores and dopaminergic markers are independent from glandular innervation. These findings suggest that rat major salivary glands express a dopaminergic system probably involved in salivary secretion. The stronger immunoreactivity for dopamine transporters and receptors in striated duct cells suggests that the dopaminergic system could regulate not only quality, but also volume and ionic concentration of saliva.

A human capsaicin model to quantitatively assess salivary CGRP secretion.

BACKGROUND: Capsaicin induces the release of calcitonin gene-related peptide (CGRP) via the transient receptor potential channel V1 (TRPV1). The CGRP response after capsaicin application on the tongue might reflect the "activation state" of the trigeminal nerve, since trigeminal CGRP-containing vesicles are depleted on capsaicin application. We tested (i) the quantitative CGRP response after oral capsaicin application; (ii) the optimal concentration of red chili homogenate; and (iii) the day-to-day variability in this response. METHODS: Saliva was collected for two consecutive days after oral application of eight capsaicin dilutions (red chili homogenates) of increasing concentrations in 13 healthy individuals. Effects of homogenate concentration were assessed. Consecutively, saliva was sampled after application of vehicle and undiluted homogenates. RESULTS: CGRP secretion (pg/ml) increased dose-dependently with homogenate concentration (p < 0.001). CGRP levels were highest after application of nondiluted homogenate (vs. baseline: 13.3 (5.0) vs. 9.7 (2.9); p = 0.003, as was total CGRP secretion in five minutes (pg) with undiluted (vs. baseline): 89.2 (44.1) vs. 14.1 (2.8); p < 0.001. The dose-dependent response in CGRP was not affected by day (p = 0.14) or day*concentration (p = 0.60). Increase in CGRP (undiluted - baseline; pg/ml) did not differ between measurements on dose-finding (p = 0.67) and follow-up days (p = 0.46). CONCLUSION: Oral application of red chili homogenate is well tolerated and causes a dose-dependent CGRP release in saliva, without day-to-day effects in this response. This model could be used to noninvasively study the activation state of the trigeminal nerve innervating salivary glands.

Roles of innervation in developing and regenerating orofacial tissues.

The head is innervated by 12 cranial nerves (I-XII) that regulate its sensory and motor functions. Cranial nerves are composed of sensory, motor, or mixed neuronal populations. Sensory neurons perceive generally somatic sensations such as pressure, pain, and temperature. These neurons are also involved in smell, vision, taste, and hearing. Motor neurons ensure the motility of all muscles and glands. Innervation plays an essential role in the development of the various orofacial structures during embryogenesis. Hypoplastic cranial nerves often lead to abnormal development of their target organs and tissues. For example, Möbius syndrome is a congenital disease characterized by defective innervation (i.e., abducens (VI) and facial (VII) nerves), deafness, tooth anomalies, and cleft palate. Hence, it is obvious that the peripheral nervous system is needed for both development and function of orofacial structures. Nerves have a limited capacity to regenerate. However, neural stem cells, which could be used as sources for neural tissue maintenance and repair, have been found in adult neuronal tissues. Similarly, various adult stem cell populations have been isolated from almost all organs of the human body. Stem cells are tightly regulated by their microenvironment, the stem cell niche. Deregulation of adult stem cell behavior results in the development of pathologies such as tumor formation or early tissue senescence. It is thus essential to understand the factors that regulate the functions and maintenance of stem cells. Yet, the potential importance of innervation in the regulation of stem cells and/or their niches in most organs and tissues is largely unexplored. This review focuses on the potential role of innervation in the development and homeostasis of orofacial structures and discusses its possible association with stem cell populations during tissue repair.

Effect of the neuropeptides vasoactive intestinal peptide, peptide histidine methionine and substance P on human major salivary gland secretion.

OBJECTIVE: The parasympathetic transmitters vasoactive intestinal peptide (VIP) and substance P (SP) are secretagogues in salivary glands of animals. Currently, we hypothesise that in human salivary glands, these neuropeptides and the VIP-related peptide histidine methionine (PHM) also exert secretory actions, reflected morphologically by exocytosis of acinar protein/glycoprotein-storing granules. MATERIALS AND METHODS: Submandibular and parotid gland tissues, exposed in vitro to VIP and PHM, and SP, respectively, were examined by light and transmission electron microscopy. For comparison, the response to in vitro stimulation of isoproterenol, phenylephrine and carbachol was examined. Moreover, the peptidergic innervation of the glands was examined by immunohistochemistry. RESULTS: Vasoactive intestinal peptide- and PHM-immunoreactive nerves were in close proximity to acini and ducts in the two glands, while these elements lacked a SP-positive innervation. While no morphological changes occurred in response to SP (parotid glands), VIP and PHM administration (submandibular glands) caused conspicuous acinar degranulation accompanied by luminal space broadening. In the two glands, both α1 - and ß-adrenergic receptor stimulation and muscarinic receptor stimulation caused similar changes as to VIP/PHM, although to varying extent. CONCLUSIONS: Vasoactive intestinal peptide and PHM, but not SP, are likely transmitters in the parasympathetic control of salivary (protein) secretion in humans.

[Changes of the innervation in the mucous membrane and glands of the tongue in early and late experimental diabetes mellitus]. / A nyelv nyálkahártya és mirigyek innervációjának változása korai és késoi kisérletes diabetes mellitusban.

The number of the different neuropeptides-containing nerve fibres and immunocompetent cells was changed in diabetes mellitus (DM) in different organs. In this work we investigated the effect of DM on quantitation of the nerve fibres using immunhistochemistry. After two weeks of the DM the quantitiy of the different nerve fibres increased significantly both in the mucous membrane and glands of the tongue. The number of the immunocompetent cells (lymphocytes, plasma cells, mast cells) increased as well significantly. Some of these cells showed also immunoreactivity for substance P and neuropeptide Y. A few substance P cells were in very close relation to the SP immunoreactive nerve fibres. After four weeks of DM the number of the nerve fibres was decreased compared to the 2 weeks treatment, however, the number of them was higher compared to the control. The close correlation between the nerve fibres and immune cells might play a crucial role in maintaining the homeostasis in the mucous membrane and glands of the tongue as well as in the increasing inflammation and elimination of it.

Endothelins are vascular-derived axonal guidance cues for developing sympathetic neurons.

During development, sympathetic neurons extend axons along a myriad of distinct trajectories, often consisting of arteries, to innervate one of a large variety of distinct final target tissues. Whether or not subsets of neurons within complex sympathetic ganglia are predetermined to innervate select end-organs is unknown. Here we demonstrate in mouse embryos that the endothelin family member Edn3 (ref. 1), acting through the endothelin receptor EdnrA (refs 2, 3), directs extension of axons of a subset of sympathetic neurons from the superior cervical ganglion to a preferred intermediate target, the external carotid artery, which serves as the gateway to select targets, including the salivary glands. These findings establish a previously unknown mechanism of axonal pathfinding involving vascular-derived endothelins, and have broad implications for endothelins as general mediators of axonal growth and guidance in the developing nervous system. Moreover, they suggest a model in which newborn sympathetic neurons distinguish and choose between distinct vascular trajectories to innervate their appropriate end organs.

Distribution of alpha-synuclein in rat salivary glands.

Expression of alpha-synuclein (Syn), a presynaptic neuronal protein, was immunohistochemically examined in intact rat submandibular, sublingual, and lingual glands. The submandibular gland contained abundant periductal Syn-immunoreactive (-ir) nerve fibers. Abundant Syn-ir varicosities were present in acini of the sublingual and serous lingual glands. By confocal laser scanning microscopy, Syn-ir nerve fibers around smooth muscle actin (SMA)-ir cells alone were infrequent; however, those around aquaporin-5 (AQP5)-ir cells alone and both SMA- and AQP5-ir cells were abundant in the sublingual and serous lingual glands. SMA-ir cells were occasionally immunoreactive for toll-like receptor 4, a Syn receptor. Syn-ir nerve fibers contained tyrosine hydroxylase (TH) in the submandibular gland and choline acetyltransferase (ChAT) in all examined salivary glands. In the superior cervical (SCG), submandibular, and intralingual ganglia, sympathetic and parasympathetic neurons co-expressed Syn with TH and ChAT, respectively. SCG neurons innervating the submandibular gland contained mostly Syn. In the thoracic spinal cord, 14.7% of ChAT-ir preganglionic sympathetic neurons co-expressed Syn. In the superior salivatory nucleus, preganglionic parasympathetic neurons projecting to the lingual nerve co-expressed Syn and ChAT. The present findings indicate that released Syn acts on myoepithelial cells. Syn in pre- and post-ganglionic neurons may regulate neurotransmitter release and salivary volume and composition.

Microinjection of NMDA-neurotoxin into the superior salivatory nucleus of the rat: Short-term secretory and long-term drinking behavior effects.

The anatomical location of the superior salivatory nucleus (SSN), the site of origin of the parasympathetic preganglionic cell bodies that innervate the submandibular-sublingual salivary glands, is well established in rats. However, as of yet there is no functional data that convincingly shows the secretory nature of this region. Previous studies have not been able to differentiate between interventions on efferent or afferent fibers connected to the SSN versus interventions on the salivatory nucleus itself. Taking advantage of the fact that salivatory neurons express NMDA-receptors on their somas, in the present study SSN cell bodies were activated and lesioned sequentially by means of intracerebral application of NMDA-neurotoxin. In exp. 1 two effects, a short- and a long-term effect, were observed following NMDA administration. The first effect was high submandibular-sublingual saliva secretion during the hour following administration of the neurotoxin and the second was a profound change in drinking behavior once the animals recovered from the lesion. Thus, on post-surgery days 16, 17 and 18, the rats exhibited hyperdipsia in the presence of dry food but not in the presence of wet food. In expt. 2 results showed that saliva hypersecretion observed after NMDA-microinjection was completely blocked by the administration of atropine (a cholinergic blocker) but not after the administration of dihydroergotamine plus propranolol (α and ß-adrenergic blockers, respectively). From a functional perspective, these data suggest that the somata of the parvocellular reticular formation control the secretory activity of the submandibular-sublingual salivary glands and thus constitute the SSN.

Inhibition of pilocarpine-induced saliva secretion by adrenergic agonists in ICR mice.

The aim of the present study was to clarify the effects of the adrenoceptor agonist isoproterenol (IPR) on saliva secretion stimulated by the muscarinic receptor agonist pilocarpine (PILO) in mice. Mice were injected with either 0.5 mg/kg, i.p. PILO alone or simultaneously with 2 mg/kg, i.p., IPR to evaluate the inhibitory effects of adrenoceptor agonists on saliva secretion. The mechanisms underlying changes in saliva flow rate were evaluated by histological examination of aquaporin 5 (AQP5) and saliva flow rate using the adenylate cyclase (AC) inhibitor SQ22536 (0.25 mg per mouse, s.c.), which was administered 30 min prior to PILO and/or IPR. Saliva volume decreased significantly in the mice treated simultaneously with PILO + IPR compared with that in mice treated with PILO alone. Changes in the intracellular localization of AQP5 were seen in PILO + IPR-treated mice, and those changes were reversed by SQ22536 pretreatment. In addition, the decreased salivary flow rate in the PILO + IPR-treated mice was partially restored by SQ22536 pretreatment. There were no significant changes in intracellular calcium or ATP levels among the groups. The results of the present study suggest the existence of an inhibitory effect of the sympathetic nervous system on parasympathetic-stimulated salivary secretion from the salivary gland.

The neuropeptide SMYamide, a SIFamide paralog, is expressed by salivary gland innervating neurons in the American cockroach and likely functions as a hormone.

The SMYamide genes are paralogs of the SIFamide genes and code for neuropeptides that are structurally similar to SIFamide. In the American cockroach, Periplanea americana, the SMYamide gene is specifically expressed in the SN2 neurons that innervate the salivary glands and are known to produce action potentials during feeding. The SN2 axon terminals surround rather than directly innervate the salivary gland acini. Therefore one may expect that on activation of these neurons significant amounts of SMYamide will be released into the hemolymph, thus suggesting that SMYamide may also have a hormonal function. In the Periplaneta genome there are two putative SIFamide receptors and these are both expressed not only in the central nervous system and the salivary gland, but also in the gonads and other peripheral tissues. This reinforces the hypothesis that SMYamide also has an endocrine function in this species.

Botulinum toxin assessment, intervention and aftercare for paediatric and adult drooling: international consensus statement.

Many individuals with neurological problems or anatomical abnormalities of the jaw, lips or oral cavity may drool, which can impact on health and quality of life. A thorough evaluation of the patient's history, examination of the oral region by a speech pathologist and, in individuals over 3 years, a dental examination is warranted. Questionnaires with established validity such as the Drooling Impact Scale are useful assessment tools. A hierarchical approach to treatment is taken from least invasive therapies, such as speech pathology, to more invasive, such as injection of botulinum neurotoxin type-A (BoNT-A) into the salivary glands (parotid and submandibular). The wishes of the individual and their carer are crucial considerations in determining the suitability of the intervention for the patient. In the presence of dysphagia and cerebral palsy (CP), careful assessment is required prior to the injection of BoNT-A. Favourable responses to intervention include a reduction in the secretion of saliva and in drooling, as well as psychosocial improvements. BoNT-A is usually well tolerated, although potential side effects should be discussed with the patient and carer.

Psychological stress and its influence on salivary flow rate, total protein concentration and IgA, IgG and IgM titers.

UNLABELLED: The hypothalamic-pituitary-adrenal and sympathetic-adrenomedullary axes are the main systems activated in response to stress. Alterations in salivary components and flow rate have been associated with oral health problems and psychological stress. OBJECTIVES: The aim of the present study was to investigate the influence of psychological stress on salivary flow, total protein concentration and IgG, IgM and IgA concentrations. METHODS: Thirty-eight medical students, average age of 21.4 +/- 2.1 years and enrolled in the 2nd to 5th years of their course, took part voluntarily in the study which involved two different periods: the first after vacations and the second during the final exams (a gap of 4 months). An Oral Health Questionnaire and the Lipp Inventory of Stress Symptoms for Adults (ISSL) were applied during both these periods. The flow rate, total protein concentration and immunoglobulin titers of saliva samples, collected after stimulation and stored in a container with protease inhibitor, were measured. RESULTS: Analysis of the ISSL showed that 42.1% (n = 16) of the students had stress during the post-vacation period, and 44.7% (n = 17) during the final exams. The students' salivary flow rate was significantly lower during the latter period than during the post-vacation period (p < 0.0001), regardless of the presence or absence of psychological stress as measured by the ISSL. There was a reduction in salivary flow rate and a consequent reduction in total protein concentration during the exam period (p = 0.0058). However, during both periods of the study there was no significant difference in total salivary protein concentration between the groups of students with or without psychological stress according to the ISSL (p > 0.05). IgG predominated over IgA and IgM (p < 0.001) during both study periods, regardless of the presence or absence of psychological stress. The study period and the presence of stress influenced the secretion of salivary immunoglobulins. IgM titers during the post-vacation period (p = 0.0044), and IgA (p = 0.028), IgG (p = 0.022) and IgM (p = 0.0075) titers during the final exams were higher in students with symptoms of psychological stress. CONCLUSIONS: Although the immunoglobulin titers were high, there was a reduction in the students' salivary flow rates and a consequent reduction in total protein concentrations.

Saliva and other taste stimuli are important for gustatory processing of linoleic acid.

Paradoxically, bilateral transection of the chorda tympani nerve (CTX) raises the taste discrimination threshold for the free fatty acid, linoleic acid (LA), yet the chorda tympani nerve (CT) is unresponsive to lingual application of LA alone. LA may require a background of saliva to activate taste cells, since CTX decreases saliva production through denervation of the submaxillary and sublingual salivary glands. To assess the role of saliva, we measured LA taste discrimination thresholds for animals whose submaxillary and sublingual salivary glands were removed and also recorded CT responses to LA mixed in artificial saliva. Partial desalivation shifted LA discrimination thresholds from between 5.5 and 11 microM to between 11 and 22 microM. However, this effect was not as pronounced as previously seen with CTX animals. Surprisingly, the CT was unresponsive to LA mixed with artificial saliva, suggesting that artificial saliva may lack components necessary for LA taste. Additionally, fats may primarily enhance other tastes. We previously reported that LA increases CT responses to monosodium glutamate (MSG). Thus we also recorded CT whole nerve responses to taste mixtures of LA and sodium chloride (NaCl), sucrose (SUC), citric acid (CA), or quinine hydrochloride (QHCl) in anesthetized rats. We found that LA increased CT responses to NaCl but did not alter CT responses to SUC, CA, and QHCl. Thus CT recordings either lack the sensitivity to detect small changes to SUC, CA, and QHCl or LA may affect CT responses to MSG and NaCl only, perhaps by specifically modulating gustatory processing of Na(+).

Dopamine-beta-hydroxylase: evidence for increased activity in sympathetic neurons during psychotic states.

Tritiated dopamine was infused into psychiatric patients during acute psychotic episodes and in remission. An index of the activity of dopamine-beta-hydroxylase of salivary gland sympathetic neurons was determined by measuring the distribution of tritiated metabolites in salivary fluid. Increased synthesis of norepinephrine occurred in acute schizophrenia and in the manic state of manic-depressive psychosis but not in the depressed phase.

Vasoactive intestinal polypeptide and muscarinic receptors: supersensitivity induced by long-term atropine treatment.

Long-term treatment of rats with atropine induced large increases in the numbers of muscarinic receptors and receptors for vasoactive intestinal polypeptide in the salivary glands. Since receptors for vasoactive intestinal polypeptide coexist with muscarinic receptors on the same neurons in this preparation, the results suggest that a drug that alters the sensitivity of one receptor may also affect the sensitivity of the receptor for a costored transmitter and in this way contribute to the therapeutic or side effects of the drugs.