Morphological and biochemical changes in the rat parotid gland after compensatory and isoproterenol-induced enlargement

Enlargement of parotid glands can be induced in rats by treatment with isoproterenol (ISP) or by removal of the submandibular and sublingual glands. In this study, morphological changes in the enlarged parotid glands and qualitative changes in secreted proteins were examined in rats that had been treated with ISP for 10 days or that had been partially sialoadenectomized by removal of the submandibular/sublingual glands 2 weeks prior to killing. After ISP treatment or salivary gland ablation, secretory cells were enlarged and contained enlarged secretory granules that stained differently from granules in normal glands. Isoproterenol treatment induced the greatest enlargement of cells and granules. Even though gland structure was altered in both experimental groups, electrophoresis of saliva showed that submandibular/sublingual gland ablation did not lead to significant qualitative changes in secreted proteins, while ISP treatment induced major changes in the pattern of secreted protein. The results suggest that compensatory enlargement of the parotid glands and changes after ISP treatment are induced by stimulation of different regulatory pathways.     

Changes in protein secretion by rat submandibular salivary glands after enlargement caused by repeated amputation of the lower incisor teeth.

The lower incisors of male rats were repeatedly amputated at 3- or 4-day intervals for about 1 month. Four days after the last amputation, submandibular saliva was collected from the anaesthetized rats by intra-oral cannulation, using methoxamine (6 mg/kg), an α-adrenomimetic drug, as an acute secretory stimulus. The rats showed enlarged submandibular and sublingual glands and the protein concentration was significantly less in submandibular saliva than in controls. The types of protein secreted by the rat submandibular gland in response to methoxamine normally differ from those secreted in response to isoprenaline. In the enlarged glands, methoxamine stimulation caused the secretion of proteins similar, by polyacrylamide gel electrophoresis, to those normally secreted in response to isoprenaline, suggesting that repeated lower incisor amputation had caused the glands to lose their normal responsiveness to α-adrenergic agonists. An abnormal protein was also secreted, identical electrophoretically and immunologically with one secreted by the enlarged submandibular glands of rats exposed to chronic isoprenaline administration.     

The effects of an incisal bite plane on rat submandibular glands

The submandibular glands of rats subjected to application of an incisal bite plane became slightly, but not significantly, enlarged. However, in comparison with control animals, they secreted additional proteins identical with those secreted by glands enlarged by periodic incisor amputation or chronic isoproterenol treatment.     

Quantitative changes in amylase activity in the salivary glands, pancreas, saliva, and serum after administration of isoproterenol, pilocarpine, and acetylcholine

Amylase activity in various tissues--i.e., submandibular/sublingual and parotid glands, the pancreas, saliva, and serum--in rats was measured after injection of isoproterenol, pilocarpine, and acetylcholine. All agents reduced amylase activity in the parotid gland and increased the enzyme activity in the submandibular/sublingual glands, in saliva and serum.     

Glucose-6-phosphate dehydrogenase activity in the salivary glands of incisor-amputated rats.

The activity of glucose-6-phosphate dehydrogenase (G6PD) was studied in the hypertrophied salivary glands of rats submitted to periodic amputation of the lower incisors. Hypertrophy of submandibular and sublingual salivary glands became evident after the second amputation, as shown by the relative glandular weights. The increased specific activity of G6PD was statistically significant only after the first amputation, reaching 1.9 times the control value for the submandibular glands and 1.8 times the control value for the sublingual glands 24 h after the stimulus. An increase in G6PD activity suggests that there is an activation of the pentose phosphate cycle in order to prepare the cells for biosynthetic processes.     

Studies on saliva in ODU plaque-susceptible rats having experimental gingivitis

Saliva was collected from the submandibular, parotid, and sublingual glands at to compare the amount of saliva between ODU plaque-susceptible (Sus) and plaque-resistant (Res) rats. The amount of saliva secreted was less in the Sus rats than in the Res rats. This difference was mainly due to a decrease in salivation from the submandibular glands, which resulted from the decrease in the weight of these glands. This reduction in salivation may be associated with the rapid rate of plaque formation seen around the mandibular teeth of these animals and the development of periodontal diseases.     

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目的    观察糖尿病大鼠腮腺、颌下腺、舌下腺超微结构变化。方法    雄性Wistar大鼠20只,随机分对照组和实验组,各10只。实验组：腹腔注射四氧嘧啶制作糖尿病大鼠模型;对照组：正常饲养。动物模型建成后第8周时称重并测定各组大鼠空腹血糖,然后取材制片,应用透射电镜观察大鼠腮腺、颌下腺、舌下腺超微结构变化。结果    建模后第8周时对照组大鼠体重（282.40±14.00）g,血糖（5.50±0.40）mmol／L;实验组大鼠体重 （148.45±8.45）g,血糖（25.20±4.10）mmol／L,组间比较差异有统计学意义（P < 0.05）。实验组大鼠腮腺细胞细胞核轮廓不规则,线粒体肿胀,粗面内质网扩张;颌下腺细胞细胞核固缩,线粒体出现空泡变性、嵴断裂;舌下腺细胞未见明显改变。结论    糖尿病可导致腮腺和颌下腺分泌功能受损而出现口干症状,但未影响舌下腺功能,舌下腺可能起到一定的代偿作用。     

A comparative quantitative histological investigation of atrophic changes in the major salivary glands of liquid-fed rats.

Adult male rats were maintained on a wholly liquid diet for 9 days. The three pairs of major glands were removed, weighed, histologically sectioned and examined by stereological and morphometric techniques. The results were compared with the same glands from control rats maintained on normal hard diet and water, and the extent of the differences was compared between the three different types of gland. Up to 50% of the serous acinar volume in the parotid glands, but only 15% in the submandibular glands, was lost after liquid feeding. There was no loss of the mucous acinar tissue in the sublingual gland. Mean acinar diameters were reduced by 33% in parotids and 15% in submandibular glands after liquid feeding, whereas the mucous acini of the sublingual gland remained unaltered. The results point to varying levels of susceptibility to the loss of masticatory reflexes between the parotid serous acini and the submandibular serous acini, and show that the morphology of the sublingual mucous acini is independent of masticatory reflex stimulation. The reductions in acinar diameters suggest that most of the glandular atrophy after liquid feeding is due to acinar cell shrinkage rather than to losses of acinar cell numbers in both parotid and submandibular glands.     

Relative secretory contributions of the three major salivary glands of the rat in response to substance P and super-sensitivity

In vivo salivation in the rat in response to a range of intravenous doses of substance P was studied. The ducts of the parotid, submandibular and sublingual glands were cannulated. The secretory threshold dose of substance P, in microgram/kg, was 0.05-0.1 in the submandibular glands, 0.2 in the parotid glands and 0.2-0.5 in the sublingual glands. The maximal secretory response in all three types of glands was obtained at a dose level of 5-10 micrograms/kg. The total amount of saliva secreted at this dose level from the three pairs of glands was calculated to about 300 mg; the submandibular glands were responsible for 65 per cent, the parotid glands for 32 per cent and the sublingual glands for 3 per cent. Parasympathetic decentralization but not sympathetic denervation caused the sublingual glands to develop a super-sensitivity to substance P. The secretory effect of substance P was not exerted via cholinergic, alpha-adrenergic or beta-adrenergic receptors.     

Binding of wheat-germ agglutinin to glycoconjugates in the salivary glands of reserpinized rats

Salivary glands from adult rats that had received reserpine for 1, 3 or 7 days and from saline-treated controls were treated with rhodamine-labelled wheat-germ agglutinin conjugates (WGA-TRITC) to localize and characterize the distribution of glycoconjugates. Fluorescent and morphometric analysis of the parotid, submandibular and sublingual glands indicated that each gland responded differently to reserpine treatment. Parotid gland acinar cells and ducts showed no change in pattern or intensity of WGA-TRITC staining after reserpine. Mucous acinar cells of the submandibular gland had increased WGA binding and an accumulation of WGA-positive material in duct lumina after 3-7 days of reserpine. Morphometric analysis showed that the maximal increase in submandibular acinar-cell size occurred after 1 day of reserpine treatment. In sublingual glands, there was no detectable increase in mucous acinar-cell staining, but progressive accumulation of WGA-positive material was seen in duct lumina after 7 days of reserpine. As WGA binds to N-acetyl-glucosamine and N-acetyl-neuraminic acid residues, it may be that the eventual blockage of the duct system is related to increased production and secretion of glycoconjugates that contain these carbohydrates.     

Impaired induction of cystatin S gene expression by isoproterenol in the submandibular gland of hypophysectomized rats

Cystatin S, an inhibitor of cysteine proteases, is produced and secreted by acinar cells of the rat submandibular gland. Expression of the cystatin S gene is known to be induced at high levels by the beta-adrenergic agonist isoproterenol. In the present study, we revealed that in the submandibular gland of hypophysectomized adult male rats, the levels of induced cystatin S mRNA 24 h after a single administration of isoproterenol are strikingly lower than those in the gland of normal rats. Administration of one of the pituitary-dependent hormones testosterone, estradiol, dexamethasone and thyroxine, together with isoproterenol resulted in marked enhancement of the isoproterenol-induced cystatin S mRNA expression in hypophysectomized rats, whereas administration of any of these hormones alone had no significant effect. These results suggested the existence of cross-talk between the signaling pathways of steroid hormones and isoproterenol in inducing cystatin S gene expression in the rat submandibular gland.     

放疗对小型猪腮腺颌下腺舌下腺微血管损伤影响的实验研究

目的:观察放疗对小型猪腮腺、颌下腺、舌下腺的微血管损伤状况。方法:将6只实验用小型猪分为2组。2组动物进行放疗,将双侧腮腺、颌下腺、舌下腺加入放射野中,放疗组20Gy/每侧,对照组0Gy/每侧。放疗结束后4 h处死两组动物,取两组动物腮腺、颌下腺、舌下腺标本行HE染色与CD31免疫组化染色,观察放疗早期3种腺体微血管密度变化。结果:两组小型猪腮腺、颌下腺、舌下腺CD31阳性染色颗粒数有显著差异(P＜0.05);3个腺体间统计结果有显著性差异(P＜0.05);腮腺与颌下腺及舌下腺有显著性差异(P＜0.05),颌下腺与舌下腺无显著性差异。结论:放疗可导致小型猪腮腺、颌下腺、舌下腺微血管密度明显减低,且各腺体间微血管密度减低有差异,腮腺的损伤程度大于颌下腺和舌下腺。     

Effects of the selective beta 2-adrenergic agonists, procaterole and terbutaline, on protein secretion by rat submandibular glands

We examined the secretory effects of two beta 2-adrenergic agonists, procaterole and terbutaline, on the submandibular glands of anesthetized rats. After stimulation with these agents with and without a range of antagonists (non-specific alpha- and beta-adrenergic blockers), submandibular saliva was collected. The flow rate, protein concentration, the electrophoretic patterns, and amino acid composition of saliva were examined. These parameters were compared with their counterparts in saliva stimulated with isoproterenol (IPR), with and without antagonists. Assessed by these criteria, secreted proteins were classified as the alpha- or beta-type. In addition, IPR-stimulated proteins were compared in submandibular saliva of rats chronically treated with IPR or procaterole. Both beta 2-agonists were potent secretagogues for the submandibular glands of rats. All beta-antagonists completely abolished the secretory effects elicited by both beta 2-agonists, with the exceptions of carteolol and propranolol. However, no blocking agent abolished the secretory effects of IPR (60 mg/kg). The types of proteins in all submandibular saliva samples elicited by both beta 2-agonists with and without antagonists were the beta-type. Enlargement of the submandibular glands was not observed in rats subjected to chronic administration of procaterole, nor were abnormal and additional proteins observed, as confirmed by electrophoresis and by the amino acid analyses.     

Mechanisms of increase in amylase activity in rat submandibular and sublingual glands after administration of pilocarpine

The increase in parotid rather than pancreatic-type amylase activity in the submandibular and sublingual glands of rats caused by administration of pilocarpine was abolished or diminished when pilocarpine was injected into rats which had been parotidectomized, sympathectomized by superior cervical ganglionectomy or pretreated with reserpine. These results suggest that the increases in amylase activity in the submandibular and sublingual glands by pilocarpine are not due to increase in enzyme synthesis, but to uptake of enzyme released into the blood in large quantities from the parotid gland and that the release from the parotid gland by pilocarpine is primarily mediated by sympathetic nerves.     

Physostigmine-induced salivary secretion in the rat

The purpose of this study was to see if physostigmine, a reversible cholinesterase inhibitor, affects the secretion and composition of saliva of the major salivary glands of the rat. Low doses of physostigmine did not elicit secretion. At higher doses there was significant flow from the parotid and submandibular glands within 5 min; however, no sublingual secretion was observed. The submandibular flow rate was highest for the first 5 min, then declined rapidly. The parotid flow rate initially was one-fifth of the maximum submandibular rate and then gradually decreased. The concentrations of Ca, Na and K of physostigmine-induced parotid saliva, and the Na of submandibular saliva, were similar to those with carbachol stimulation. The Ca and K concentrations of submandibular saliva were significantly higher than with carbachol or parasympathetic stimulation, and resembled those of alpha-adrenergic stimulation. The protein concentrations of physostigmine-evoked saliva from both glands were similar. The amylase activity of physostigmine-evoked parotid saliva was much higher than that of carbachol or parasympathetic stimulation. Physostigmine-evoked secretion was completely blocked by atropine, a cholinergic antagonist, and by reserpine, partially blocked by phentolamine, an alpha-adrenergic antagonist and not affected by surgical sympathectomy. Morphologically, physostigmine resulted in a moderate decrease in the number of acinar, but not ductal, secretory granules of both the parotid and submandibular glands, while the sublingual gland was unaffected. Numerous patches of parotid acini also developed vacuoles or vesicles. These results suggest that physostigmine-induced salivary secretion is mediated primarily by direct effects on cholinergic and alpha-adrenergic receptors.     

Changes in protein secretion by rat submandibular glands in response to isoproterenol, alpha-methylnoradrenaline, and clonidine during post-natal development

We studied developmental changes in salivary volumes and proteins secreted by the submandibular glands of male rats at weekly intervals from two to ten weeks of age in response to the beta 1-, alpha 1-, and alpha 2-adrenoceptor agonists, isoproterenol (IPR), alpha-methylnoradrenaline (alpha-mNA), and clonidine (Clonid). The types of proteins in saliva samples were determined and compared by isoelectric-focusing electrophoresis with the Phast system in both the gradient pH -3.5-to-5 and pH-3.5-to-9 gels by means of silver staining. Salivary volume and protein concentration in saliva samples elicited by IPR and alpha-mNA were positively related to the weight of the submandibular glands up to six or seven weeks of age, whereas in saliva elicited by Clonid, no relation was found in the protein concentration [corrected]. The isoelectric-focusing electrophoretic patterns of proteins secreted by the glands in response to three stimuli were different from each other during post-natal development. Within one stimulation, differences were also observed at two and three weeks of age for Clonid, and from seven weeks of age for the three stimuli, respectively. The alpha-type proteins, but not the beta-type proteins, were very similar to those in extracts from glands of rats at seven weeks of age. Almost all of the alpha-type proteins, but not the beta-type proteins, reacted with antibodies to two proteases. We conclude that functional maturation precedes morphological maturation in the submandibular glands of rats.     

Phenotypic characterization of resident macrophages in submandibular salivary glands of normal and isoproterenol-treated rats.

Macrophages exert a major effect in the stimulation of lymphocytes and the modulation of immunological responses. To determine the presence and phenotypic distribution of the resident cells of the mononuclear phagocyte system in submandibular glands, frozen sections were prepared from five normal rats, and from six rats treated with 20 mg/kg isoproterenol/day for 10 days. A panel of six monoclonal antibodies was used to identify membrane markers associated primarily with circulatory monocytes (ED1), mature tissue macrophages (ED2), lymphoid macrophages (ED3), Ia antigen (OX6), CD5-positive T lymphocytes (OX19) and rat B lymphocytes (OX33). Cells identified by each monoclonal antibody were quantified by averaging the number of positive cells in 10 consecutive random high-power fields. ED2 cells (165 cells/field) were predominant in normal rat submandibular gland, followed by lower numbers of OX6-positive cells (18 cells/field). Cells positive for the remaining markers were also present in smaller amounts. In submandibular glands, treatment of rats with isoproterenol resulted in an increase in ED1-positive cells (from 2 to 39 cells/field), but also in substantial decreases in the number of cells positive for the remaining cell markers. B cells were not detected in any of the submandibular glands examined. These data suggest that isoproterenol induces a mild inflammatory response within rat submandibular glands that is not observed in normal glands. This results in an increase in the relative number of infiltrating monocytes compared to the number of more mature tissue macrophages.     

Secretion from minor salivary glands following ablation of the major salivary glands in rats

Since minor salivary glands are tiny and dispersed, ductal cannulation cannot be used when studying their function. The present study was devised to develop a method of measuring minor salivary gland function by excision of the major glands. Female rats (230–280 g) were anaesthetized with sodium pentobarbital. Ablation of the submandibular, sublingual and parotid glands was performed through a sagittal neck incision. Sham-operated rats served as controls. Groups of sialadenectomized animals were investigated immediately and after 1 week, 2 weeks and 3 months. To study secretory function, the mouth was rinsed with 250 μl water in every 5 min and protein and amylase concentrations were measured. After an initial 50 min of basal secretion pilocarpine (1 mg/kg, i.p.) was given. Bilateral ablation of both submandibular, sublingual and parotid glands led to a moderate loss of body weight and a considerable increase in water intake. No other obvious abnormality was observed for periods up to 90 days following surgery. We deduce that the minor glands secrete approximately 14% of protein and 1% of amylase in whole saliva. Secretion is maintained even after 90 days following removal of the major glands. Surgical removal of the major salivary glands allows the secretory function of the minor glands in rats to be studied in vivo.     

The contribution of oral minor mucous gland secretions to the volume of whole saliva in man

To determine the contribution of minor mucous gland secretions to total saliva by a direct method, flow rates of both unstimulated and sour lemon drop (SLD)-stimulated saliva were initially determined in 15 subjects. The right and left lingual nerves were then anaesthetized to halt submandibular and sublingual secretion, and both parotid ducts were cannulated. The only remaining saliva in the mouth was that secreted by minor salivary glands. Unstimulated and SLD-stimulated minor mucous gland secretions were then collected and the median percentage contributions to whole saliva were calculated to be 8 and 7 per cent, respectively. Comparable results were obtained on 3 subjects using an indirect method similar to that of Schneyer (1956). With the left parotid duct cannulated, subjects maintained a constant, SLD-stimulated, left parotid flow rate of 1 ml/min and the remaining mixed saliva was collected to determine its flow rate. The right parotid and the submandibular and sublingual glands were then also cannulated and the flow rate from these glands determined whilst that from the left parotid was maintained at 1 ml/min. The contribution from minor mucous glands was the difference between the flow rate of mixed saliva and the combined flow rate from the right parotid, submandibular and sublingual glands.