Polyamines and long-term disuse of rat parotid glands

Nilsson , B.-O., Rosengren , E. & Ekström , J. 1990. Polyamines and long-term disuse of rat parotid glands. Acta Physiol Scand 140 , 105–109. Received 1 February 1990, accepted 7 April 1990. ISSN 0001–6772. Department of Physiology and Biophysics, University of Lund and Department of Pharmacology, University of Gothenburg, Sweden. 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.     

Neuropeptides and disuse of the rat parotid gland.

Rats were fed a liquid diet with the aim of decreasing nervous reflex activity in the parotid glands. In rats killed after 21 days on this diet the glands were atrophied and the total amounts of the neuropeptides, substance P, vasoactive intestinal peptide and calcitonin gene-related peptide, were lower than in control glands.     

Changing myoepithelial cell distribution during regeneration of rat parotid glands

The distribution of the myoepithelial cells during regeneration of the rat parotid gland after atrophy induced by one week of parotid duct ligation was investigated by immunohistochemistry for actin and transmission electron microscopy (TEM). Immunohistochemically, residual ducts were surrounded by actin-positive cells when clips were removed from the duct. Three days later, most of the newly formed acini originating from the residual ducts were also embraced by actin-positive cells. After 10 days, actin-positivity tended to be seen as dots around acini that decreased in number day by day. On day 21 actin-positive cells mainly surrounded intercalated ducts with only a few positive reactions identified at the acinar periphery. Electron microscopically, residual ducts and newly formed acini were peripherally embraced by myoepithelial cells before day 5. After day 7, shift of myoepithelial cells from the periphery of acini to the duct-acinar junctional region was identified. Then few myoepithelial cells were identified at the periphery of acini. These observations indicate that myoepithelial cells migrate from the acinar periphery to the duct-acinar junctional region during rat parotid regeneration, and that such behaviour is closely related to that seen during rat parotid development.     

Ultrastructural study on nuclear bodies of the rat parotid glands

Nuclear bodies of types I, II, III and IV are constantly present in rat parotid glands. They become apparent on the 21st day and afterwards. Type IV nuclear bodies were chiefly found in the striated ducts. Nuclei containing 3 and more nuclear bodies were found as early as the 3rd month, although only in the striated ducts. Nuclear bodies are not related to glycogen or DNA replication but probably to rRNA metabolism. This study leads to a better understanding of nuclear bodies modifications which can be observed ultrastructurally in human salivary glands pathology. Their increased number in some nuclei may be the expression of an alteration of the cell metabolism.     

Studies on hypoxia. 8. Ultrastructural and biochemical effects of prolonged exposure on rat parotid glands

The effect of chronic hypoxia upon the rat parotid gland is studied by electron microscopy and biochemistry. Male Sprague-Dawley rats are subjected to 88% N₂ and 12% O₂ at less than 2 psi pressure for 7 days. For electron microscopy, the tissues are perfusion-fixed, embedded and sectioned in a routine manner. In the biochemical studies, rats are injected with ³H-phenylalanine (2 μCi/g; s.i. ～ 5 Ci/mM) 60 min before sacrifice. Fresh glands are then prepared for analysis of amylase (Cibachrome-amylase substrate method), DNA (diphenylamine reaction), and total protein (Lowry method). The radioactivity in the acid precipitable and soluble fractions is determined by liquid scintillation spectrometry. The control animals are pair-fed and handled identically except that they are maintained in an ambient atmosphere. The ultrastructure of the hypoxic cells is altered in several areas. The Golgi apparatus demonstrates a decrease in organization and contains fewer transport vesicles. The rough-surfaced endoplasmic reticulum is broken and presents many vesicular and concentric profiles. The mitochondria have undergone several changes including swelling, clumping of intramitochondrial matrix, and fragmentation of cristase. The nucleus demonstrates a fibrillar pattern and contains a reduced amount of heterochromatin. The biochemical results indicate that, compared with controls, the hypoxic cells contain only 55% of amylase and 84% of the DNA, and thus suggest a drastic reduction of exportable protein production and an increase in cellular size, respectively. On the contrary, the incorporation of ³H-phenylalanine demonstrates an increase in the amount of radioactivity in the acid precipitable fraction of the hypoxic cells. These results lead to the conclusion that hypoxic stress causes a suppression of exportable protein synthesis, but may induce other cytoplasmic protein production, reflecting the biochemical and morphologic adjustment of the cell to a recovery phase.     

Co-localization of rab4 with endocytosis-related proteins in the rat parotid glands

Small GTP-binding proteins have been implicated in the regulation of vesicular traffic. We investigated the localization of Rab4 in the rat parotid glands by Western blotting and light-microscopic immunohistochemistry. Rab4 was localized mainly on the intracellular membranes in the subapical-actin terminal web, but was not present in the basolateral region both in acinar and ductal cells. Actin, alpha-adaptin, Rab5A and aquaporin5 were detected in the Rab4-containing intracellular membrane fraction prepared using anti-Rab4 antibody covalently coupled to magnetic beads. Detection of actin indicated that the Rab4-containing intracellular membranes were attached to the actin filaments. Although alpha-adaptin was immunohistochemically distributed along the plasma membrane, this protein coexisted with Rab4 only at the apical region. Rab5A immunoreactivity was distributed all around the cytoplasm. These findings suggested that Rab4 participates in endocytosis at the apical membrane of parotid glands.     

Purification of zymogen granules from monkey parotid glands.

A method giving highly purified zymogen granules from Macaca irus and Cercopithecus aethiops parotid glands in reported. A 0.3 M sucrose medium for homogenization was supplemented with 10 mM tris/HCl, pH 7.3, and 0.1 mM lauric acid to stabilize the fragile monkey zymogen granules. Nuclei and cell debris were sedimented at 150 times g. A "crude" zymogen granule fraction was trapped in the 1.0 M sucrose layer of a discontinuous sucrose gradient at 1000 times g. Equilibrium centrifugation in a continuous sucrose gradient gave a fraction of zymogen granules at 1.85 M sucrose. Compared to the homogenate, this fraction exhibited about two-fold increase in zymogen granule marker, whereas mitochondrial marker was reduced to 1/4 and lysosomal marker to 1/2. A low level of contamination from other cell organelles was confirmed by electron microscopic investigation.     

Parasympathetic non-adrenergic, non-cholinergic-induced protein synthesis and mitogenic activity in rat parotid glands

Electrical stimulation of the parasympathetic auriculo-temporal nerve (40 Hz, 30 min), in the anaesthetized rat under - and -adrenoceptor blockade, increased [3H]thymidine and [3H]leucine uptake into the parotid glands by 80 and 263 %, respectively. The increase in response to parasympathetic stimulation was almost the same ([3H]thymidine 82 % and [3H]leucine 283 %) when atropine (2 mg kg-1 I.P. or I.V.) was included in the pretreatment. Neither intravenous infusion of vasoactive intestinal peptide (0.5-20 mg kg-1 min-1, 30 min) nor of bethanechol (10 mg kg-1 min-1, 30 min), under adrenoceptor blockade, increased the uptake of [3H]thymidine into the glands. However, these drugs increased [3H]leucine uptake, and in combination they interacted positively. Whereas vasoactive intestinal peptide is likely to be involved in the parasympathetic nerve-evoked protein synthesis, the nature of the non-adrenergic, non-cholinergic component(s) involved in the mitogenic response is presently unknown.     

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.     

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.     

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.     

Incidence and histology of human accessory parotid glands

The parotid glands of 228 Japanese human cadavers were examined to determine the incidence and histological features of accessory parotid glands. The incidence was found to be 56% with no differences between right and left sides or between sexes. Thirty parotid glands and their associated accessory glands were examined histologically: eight of these accessory glands were found to be mixed secretory glands (i.e., containing both serous and mucous acini). Thus, the pattern of differentiation of a significant fraction of accessory glands differs from that of the main parotid gland: it appears that mixed acini, present in the early stages of development, persist into later life, and their presence may be related to tumors developing at these sites. © 1993 Wiley-Liss, Inc.     

Dissociation of rat parotid gland.

Rat parotid gland was dissociated by sequential collagenase and hyaluronidase digestions, chelation with ethylenediaminetetraacetic acid, and mild shearing force to yield predominantly single cells. The isolated acinar cells retained their morphologic characteristics and their amylase activity. The functional integrity of the isolated cells was assessed by measuring their secretory response to isoproterenol, epinephrine, and carbamylcholine and by their ability to incorporate radioactively labeled leucine and thymidine. The discharge of amylase from the dissociated cells was not effected by isoproterenol or norepinephrine and the response to carbamylcholine was minimal. The data indicate a destruction or perturbation of hormone receptors during the dissociation procedure. The maintenance of the cells in culture for up to 18 hours failed to restore the responsiveness of the isolated parotid gland acinar cells to isoproterenol. The isolated cells incorporated 14C-leucine into proteins at a linear rate between 30 and 180 minutes. Chromatographic and electrophoretic profiles of newly synthesized proteins indicated that all major proteins synthesized in vivo were also synthesized by the isolated cells. The isolated cells incorporated tritiated thymidine into DNA. Furthermore, stimulation of DNA synthesis by isoproterenol in vivo was reflected by a higher rate of thymidine incorporation by the isolated cells as compared with controls. The dissociated parotid gland cells offer a convenient system for studying various cellular processes, particularly the synthesis of macromolecules with high specific activity. However, some functions, notably the response to beta- adrenergic agonists, are lost during the dissociation procedure.