Neurokinin A in the parotid and submandibular glands of the rat: immunohistochemical localization and effect on protein and peroxidase secretion

An indirect immunofluorescence technique was used to study the distribution of neurokinin A immunoreactive (NKA-IR) nerve fibres in submandibular and parotid glands of the rat. The functional role of neurokinin A on protein and peroxidase secretion in these glands was evaluated by using in vitro methods. In the parotid gland neurokinin A immunoreactive fibres were mainly distributed around the secretory acini, but some were also in evidence around the stromal blood vessels and ducts. The number of the neurokinin A immunoreactive nerve fibres was lower in the submandibular gland than in the parotid gland. They were mainly distributed around the secretory acini and stromal blood vessels and ducts. In vitro, neurokinin A significantly stimulated the release of total amount of released proteins and peroxidase from parotid gland fragments, while in the submandibular gland only the release of peroxidase was increased. By using SDS polyacrylamide gel electrophoresis (SDS-PAGE) specific changes were found in the release of proteins after neurokinin A stimulation. The results of the present study demonstrate that neurokinin A immunoreactive nerve fibres are present in the rat parotid and submandibular glands. Their localization around the secretory elements of the glands and the effect of neurokinin A in vitro experiments indicates that neurokinin A might have a significant role in the regulation of salivary secretion.     

Fluid and protein secretion from ferret submandibular and parotid glands in response to sympathetic nerve stimulation or administration of sympathomimetics.

Electrical stimulation of the sympathetic innervation evoked secretion of submandibular and parotid saliva. By changing the mode of stimulation from a continuous to an intermittent one the fluid response increased and glandular blood flow improved. The volumes from the submandibular glands were larger than those from the parotid glands and further, the protein concentration of submandibular saliva was higher than that of parotid saliva. Adrenaline, isoprenaline and phenylephrine evoked larger fluid responses from submandibular than from parotid glands. However, the fluid response was small compared to the parasympathetic one. Substance P-evoked saliva was used as carrier for protein released by sympathetic nerve stimulation or administration of adrenaline and isoprenaline. In vitro tissues of submandibular and parotid glands responded to adrenaline with a dose-dependent release of protein. Taken together, the analytical pharmacology performed in vivo and in vitro, and including the antagonists phentolamine, dihydroergotamine, propranolol and metoprolol, showed that in submandibular glands, α(α₁)adrenoceptors were predominantly involved in fluid secretion and β(β₁)-adrenoceptors predominantly involved in protein secretion. In parotid glands, fluid secretion seemed solely to depend on α(α₁)-adrenoceptors, while β(β₁)-adrenoceptors seemed almost solely involved in protein secretion.     

The effects of antidepressants and pilocarpine on rat parotid glands: an immunohistochemical study

OBJECTIVES:

To evaluate the effects of antidepressants and pilocarpine on the quantity of myoepithelial cells and on the proliferation index of the epithelial cells of rat parotid glands.

INTRODUCTION:

Hyposalivation, xerostomia, and alterations in saliva composition are important clinical side effects related to the use of antidepressants.

METHODS:

Ninety male Wistar rats were allocated to nine groups. The control groups received saline for 30 (group C30) or 60 days (group C60) or pilocarpine for 60 days (group Pilo). The experimental groups were administered fluoxetine (group F30) or venlafaxine for 30 days (group V30); fluoxetine (group FS60) or venlafaxine (group VS60) with saline for 60 days; or fluoxetine (group FP60) or venlafaxine (group VP60) with pilocarpine for 60 days. Parotid gland specimens were processed, and the immunohistochemical expression of calponin and proliferating cell nuclear anti-antigen on the myoepithelial and parenchymal cells, respectively, was evaluated. Analysis of variance (ANOVA), Tukey HSD and Games-Howell tests were applied to detect differences among groups (p<0.05).

RESULTS:

Compared with the controls, chronic exposure to antidepressants was associated with an increase in the number of positively stained cells for calponin. In addition, venlafaxine administration for 30 days was associated with an increase in the number of positively stained cells for proliferating cell nuclear anti-antigen. Fluoxetine and pilocarpine (group FP60) induced a significant decrease in the number of positively stained cells for calponin compared with all other groups.

CONCLUSIONS:

The number of positively stained cells for calponin increased after chronic administration of antidepressants. The proliferation index of the epithelial cells of rat parotid glands was not altered by the use of antidepressants for 60 days.     

Further evidence for AQP8 expression in the myoepithelium of rat submandibular and parotid glands

Previously (Wellner et al., Pflugers Arch 441:49–56, 2000) we suggested that the localization of the aquaporins (AQPs) AQP5 and AQP8 in the apical and basolateral membranes of rat submandibular gland (SMG) acinar cells, respectively, provides for transcellular water flow during saliva formation. While the localization of AQP5 in this gland has been verified in several laboratories, there have been differing reports regarding AQP8 localization. Other investigators subsequently reported that AQP8 is not expressed in the acinar or ductal cells of the major salivary glands of the rat, but in the myoepithelium of each gland. Thus, we have carried out additional studies: (1) to reassess the localization of AQP8 in the rat SMG and (2) to assess the localization of AQP8 in the rat parotid gland (PG). Initially, we compared the localizations of AQP8 with recognized basolateral markers in acinar cells [the Na⁺,K⁺-ATPase and the Na⁺–K⁺–2Cl^– cotransporter (NKCC1)]. Our results indicated that Na⁺,K⁺-ATPase localized in both the basal and lateral membranes of rat SMG acinar cells, whereas AQP8 was detected only in the basal regions of the acini. In the rat PG, AQP8 was invested near intercalated ducts and adjacent acini, whereas NKCC1 localized in the basolateral membranes of acinar cells. As these results were suggestive of myoepithelial localization in both glands, we compared AQP8 localization with the localization of smooth muscle actin, a myoepithelial marker. We found that AQP8 and smooth muscle actin colocalized in both the rat SMG and PG, providing additional strong support for a myoepithelial localization of AQP8. Thus, in agreement with an earlier report by other investigators (Elkjaer et al., Am J Physiol Renal Physiol 281:F1047–F1057, 2001), we report that AQP8 is expressed in the myoepithelial cells, but not in the acinar cells, of both the rat SMG and PG.     

A granular cell in the proximal intercalated duct of human parotid and submandibular glands

In human parotid and submandibular gland unusual granulated cells are observed at the acinar-intercalated duct junction. These cells, which show a well developed Golgi apparatus, greatly differ from the typical elements of the intercalated ducts mainly due to the presence of abundant secretory granules of unknown nature. A complex substructure distinguishes these granules from those of conventional ductal cells and from those of acinar cells as well. In addition, some differences exist in the morphology of the granules observed in parotid with respect to those of submandibular gland.     

The fine structural localization of acetylcholinesterase activity in the rat parotid and sublingual glands

The fine structural localization of acetylcholinesterase activity was studied in the rat parotid and sublingual glands. In both glands, reaction product was found in association with the axolemmae of stromal axons invested by Schwann cells and between the contact with effector cells. In the sublingual gland, reaction product was also found in association with surface vesicles and pits and the rough endoplasmic reticulum of myoepithelial cells.     

Menkes protein localization in rat parotid acinar cells

The aim was to study the subcellular localization of the Menkes protein (MNK; ATP7A) in the rat parotid acinar cell. MNK protein is a copper transporting P-type ATPase whose absence or dysfunction causes a fatal neurodegenerative disorder, MNK disease. Rat parotid glands were fixed and low-temperature embedded in Lowicryl K4M resin, and ultrathin sections were prepared for immunocytochemical analysis. Immunolocalization of MNK was demonstrated mainly over the trans Golgi network (TGN) area. Immature and mature secretory granules were also labelled, indicating that MNK protein could be involved here in copper secretion from acinar cells into saliva, consistent with a proposed cariostatic role for copper.     

Histological analysis of parotid and submandibular glands in chronic alcohol abuse: a necropsy study.

A quantitative histological analysis of the major salivary glands was carried out at necropsy in 28 alcoholics and in a series of age and sex matched controls. The findings were related to the different types of histologically diagnosed liver disease present. Significant quantitative changes of salivary gland structure were noted in cirrhosis but not in other forms of alcoholic liver disease. In cirrhotic subjects the parotid contained proportionally more adipose but less acinar tissues than in controls. The submandibular gland showed a proportional increase in adiposity and reduction in fibrovascular tissues but no noticeable reduction in its acinar proportional volume. Neither grossly detectable parotid enlargement nor acinar hypertrophy, a feature which has previously been noted as characteristic of alcoholic sialadenosis, were evident in this series. These findings provide little structural support for the reportedly increased secretory capacity of salivary glands in chronic alcohol abuse.     

Cellular localization of kallikreins in rat submandibular and sublingual salivary glands: immunofluorescence tracing related to histological characteristics.

Immunohistochemical localization of rat salivary gland kallikrein was related to glandular structures in tissue processed and stained by various methods. In the submandibular gland, most of the kallikrein was located to cytoplasmic granules of the granular tubules. Cells of the striated ducts showed a faint cytoplasmic staining with a bright luminal rim that occasionally was seen also in the excretory ducts. Minor amounts of kallikrein was found in the interstitial tissue. In the sublingual gland, kallikrein was found in the cytoplasm of the striated duct cells and as a luminal rim in the main ducts. Acini were negative in both glands. Fixation in Helly's fluid preserved cytoplasmic granules and was thus superior for intracellular localization of kallikrein, whereas ethanol fixation, due to absence of non-specific background staining, afforded the most sensitive method for detection of small amounts of antigen. In the submandibular gland, best identification of granular tubules and striated ducts was achieved with DMAB-nitrite staining for tryptophan and counterstaining with Mayer's haemalum on sections of tissue fixed in Helly's fluid. In the sublingual gland, the duct system was best demonstrated by haematoxylin-eosin staining.     

Immunoglobulin A secretion into saliva during dual sympathetic and parasympathetic nerve stimulation of rat submandibular glands

Salivary secretion of proteins from rat submandibular glands was studied using graded stimulation of the parasympathetic nerve in isolation, and then at a fixed rate in combination with graded sympathetic nerve stimulation. Increasing the frequency of parasympathetic nerve stimulation per se caused a gradual increase in the secretion of peroxidase (from acini) but only small increases in proteinase (from ductal cells) and IgA outputs. Dual stimulations, with an increasing frequency of sympathetic nerve stimulation on a background of low frequency parasympathetic nerve stimulation, showed that maximal acinar secretion of peroxidase required only a low frequency of additional sympathetic stimulation, whereas ductal secretion of kallikrein was greatest with the highest frequency of additional sympathetic stimulation (20 Hz in bursts). IgA secretion also required high frequency additional sympathetic stimulation in bursts for greatest output. Although a synergism occurred with parasympathetic plus sympathetic nerve stimulation for the secretion of both peroxidase and kallikrein it was not evident for the secretion of IgA. This presumably reflects a difference for exocytosis of proteins stored in granules (e.g. peroxidase and kallikrein) compared to those proteins continuously transported across the plasma membrane in vesicles by transcytosis. This work confirms that vesicular movement of secretory IgA can be increased by both parasympathetic and sympathetic nerve stimulation, but the frequency parameters differ for each nerve.     

Nerve-evoked secretion of immunoglobulin A in relation to other proteins by parotid glands in anaesthetized rat

Secretion of fluid and proteins by salivary cells is under the control of parasympathetic and sympathetic autonomic nerves. In a recent study we have shown that, in the rat submandibular gland, autonomic nerves can also increase the secretion of IgA, a product of plasma cells secreted into saliva as SIgA (IgA bound to Secretory Component, the cleaved poly-immunoglobulin receptor). The present study aimed to determine if parotid secretion of SIgA is increased by autonomic nerves and to compare SIgA secretion with other parotid proteins stored and secreted by acinar and ductal cells. Assay of IgA in saliva evoked by parasympathetic nerve stimulation immediately following an extended rest period under anaesthesia indicated that it had been secreted into intraductal saliva in the absence of stimulation during the rest period. The mean rate of unstimulated IgA secretion (2.77+/-0.28 microg min(-1) g(-1)) and the 2.5-fold increase in IgA secretion evoked by parasympathetic stimulation were similar to results found previously in the rat submandibular gland. Sympathetic nerve stimulation increased SIgA secretion 2.7-fold, much less than in the submandibular gland. SDS-PAGE and Western blot analysis with anti-IgA and anti-Secretory Component antibodies confirmed that SIgA was the predominant form of IgA in saliva. Acinar-derived amylase and ductal-derived tissue kallikrein were more profoundly increased by parasympathetic and particularly sympathetic stimulation than SIgA. Overall, the results of the present study indicate that SIgA forms a prominent component of unstimulated parotid salivary protein secretion and that its secretion is similarly increased by stimulation of either autonomic nerve supply. The secretion of other parotid salivary proteins that are synthesized and stored by acinar or ductal cells is upregulated to a much greater extent by parasympathetic and particularly sympathetic stimulation.     

Long‐term dexamethasone treatment alters the histomorphology of acinar cells in rat parotid and submandibular glands

Glucocorticoids (GCs) induce insulin resistance (IR), a condition known to alter oral homeostasis. This study investigated the effects of long‐term dexamethasone administration on morphofunctional aspects of salivary glands. Male Wistar rats received daily injections of dexamethasone [0.1 mg/kg body weight (b.w.), intraperitoneally] for 10 days (DEX), whereas control rats received saline. Subsequently, glycaemia, insulinaemia, insulin secretion and salivary flow were analysed. The parotid and submandibular glands were collected for histomorphometric evaluation and Western blot experiments. The DEX rats were found to be normoglycaemic, hyperinsulinaemic, insulin resistant and glucose intolerant (P < 0.05). DEX rat islets secreted more insulin in response to glucose (P < 0.05). DEX rats had significant reductions in the masses of the parotid (29%) and submandibular (16%) glands (P < 0.05) that was associated with reduced salivary flux rate. The hypotrophy in both glands observed in the DEX group was associated with marked reduction in the volume of the acinar cells in these glands of 50% and 26% respectively (P < 0.05). The total number of acinar cells was increased in the submandibular glands of the DEX rats (P < 0.05) but not in the parotid glands. The levels of proteins related to insulin and survival signalling in both glands did not differ between the groups. In conclusion, the long‐term administration of dexamethasone caused IR, which was associated with significant reductions in both mass and flux rate of the salivary glands. The parotid and submandibular glands exhibited reduced acinar cell volume; however, the submandibular glands displayed acinar hyperplasia, indicating a gland‐specific response to GCs. Our data emphasize that GC‐based therapies and insulin‐resistant states have a negative impact on salivary gland homeostasis.     

Quantitative immunohistochemistry of immunoglobulin- and J-chain-producing cells in human parotid and submandibular salivary glands.

Ig-producing cells were quantified by paired immunohistochemical staining in saline-extracted and paraffin-embedded normal tissue specimens from human parotid (ten) and submandibular (seven) salivary glands. The density of such cells (number/mm2 of 6 micron thick tissue section) was significantly higher in the submandibular than in the parotid gland (P less than 0.005), but the Ig-class distribution was fairly similar. The mean percentage class ratios for IgG, IgA, IgM and IgD cells in the parotid were 4.5:86.5:5.9:3.1, and in the submandibular gland 3.7:86.9:7.9:1.6. In the parotid gland of a patient with selective IgA deficiency the same class ratios were 27:0:20:53. Thus, the IgA cells were especially replaced by IgD cells. In normal glands most of the IgA (80-93%), IgM (99-100%) and IgD cells (81-95%) were J-chain-positive; this was likewise true for a substantial proportion of the IgG cells (32-46%). Of additional interest was the finding that in the IgA-deficient parotid gland, 99% of the numerous IgD cells and 86% of the increased number of IgG cells contained cytoplasmic J chain. IgE-producing cells were virtually absent from the IgA-deficient as well as from the normal salivary glands.     

Radioprotective effect of heat shock protein 25 on submandibular glands of rats

Irradiation (IR) is a fundamental treatment modality for head and neck malignancies. However, a significant drawback of IR treatment is irreversible damage of salivary gland in the IR field. In the present study, we investigated whether heat shock protein (HSP) 25 could be used as a radioprotective molecule for radiation-induced salivary gland damage in rats. HSP25 as well as inducible HSP70 (HSP70i) that were delivered to the salivary gland via an adenoviral vector significantly ameliorated radiation-induced salivary fluid loss. Radiation-induced apoptosis, caspase-3 activation, and poly(ADP-ribose) polymerase cleavage in acinar cells, granular convoluted cells, and intercalated ductal cells were also inhibited by HSP25 or HSP70i transfer. The alteration of salivary contents, including amylase, protein, Ca+, Cl-, and Na+, was also attenuated by HSP25 transfer. Histological analysis revealed almost no radiation-induced damage in salivary gland when HSP25 was transferred. Aquaporin 5 expression in salivary gland was inhibited by radiation; and HSP25 transfer to salivary gland prevented this alteration. The protective effect of HSP70i on radiation-induced salivary gland damage was less or delayed than that of HSP25. These results indicate that HSP25 is a good candidate molecule to protect salivary gland from the toxicity of IR.     

Polyamines and long-term disuse of rat parotid glands.

A decrease in nerve reflex activation for 7-14 days, induced by a liquid diet, caused the rat parotid gland to lose weight, involving reduction in both cell size and number. In the atrophied glands, the activity of ornithine decarboxylase, the key enzyme in polyamine formation, and the levels of the polyamines putrescine, spermidine and spermine were found to be lowered. The present results are compatible with a role for polyamines in cellular growth.     

Nitric oxide-dependent protein synthesis in parotid and submandibular glands of anaesthetized rats upon sympathetic stimulation or isoprenaline administration

In anaesthetized female rats, the beta-adrenoceptor agonist isoprenaline was intravenously infused (20 microg kg(-1) min(-1)) for 30 min or the ascending cervical sympathetic nerve trunk was intermittently stimulated (50 Hz, 1 s every tenth second) on one side for 30 min. The incorporation of [3H]leucine into trichloroacetic acid (TCA)-insoluble material was used as an index of protein synthesis. In response to isoprenaline, the [3H]leucine incorporation increased by 79% in the parotid glands and by 82% in the submandibular glands. The neuronal type NO-synthase inhibitor N-PLA, reduced (P < 0.001) this response to 26% and 20%, respectively. Sympathetic stimulation under alpha-adrenoceptor blockade increased the [3H]leucine incorporation by 192% in the parotid glands and by 35% in the submandibular glands. N-PLA reduced the corresponding percentage figures to 86% (P < 0.01) and 8% (P < 0.05). When tested in the parotid glands, the non-selective NO-synthase inhibitor L-NAME reduced (P < 0.01) the nerve-evoked response to 91%. The increase in [3H]leucine incorporation in response to sympathetic stimulation under beta-adrenoceptor blockade was not affected by N-PLA in the parotid (139% versus 144%) and submandibular glands (39% versus 34%). In non-stimulated glands, the [3H]leucine incorporation was not influenced by the NO-synthase inhibitors. In conclusion, beta-adrenoceptor mediated salivary gland protein synthesis is largely dependent on NO generation by neuronal type NO-synthase, most likely of parenchymal origin.     

Ultrastructure of the main excretory duct epithelia of the rat parotid and submandibular glands with a review of the literature

The fine structure of the main excretory duct (MED) epithelia of the rat parotid and submandibular glands was investigated by scanning and transmission electron microscopy. The structure of these two MED epithelia was compared. Five principal cell types were observed in the MED epithelium of the parotid gland--cuboidal, basal, light (types I and II), dark, and tuft cells. The cuboidal cells, which were stratified in two or three layers, were the most numerous. Basal cells were situated adjacent to the basal lamina of the epithelium. The type I light cells had well-developed basal plasma membrane infoldings. The type II light cells had poorly developed basal plasma membrane infoldings. The dark and tuft cells resembled those of the MED epithelium of the rat submandibular gland. Four principal cell types were observed in the MED epithelium of the submandibular gland--light cells (types I and II), dark, tuft, and basal cells. The two types of light cells were the most numerous. Dark cells with regular and dense microvilli were relatively narrow in shape and had complicated basal plasma membrane infoldings. There were numerous vesicles or channel-like vesicles in their apical cytoplasm. Tuft cells were characterized by prominent microvilli, many vesicles of various sizes in the apical cytoplasm, and no basal infoldings. Peroxisomes were present in the MED epithelia of the rat parotid and submandibular glands, especially in light and cuboidal cells.