The effects of electrical and pharmacological stimulation on the types of proteins secreted by rat parotid and submandibular glands.

In an attempt to determine whether the types of proteins in rat parotid and submandibular saliva could be influenced by the nature of the stimulus, ductal secretions were collected from anaesthetized, adult male Sprague-Dawley rats in response to a variety of stimuli. The different types of proteins were quantified after separation by anionic and cationic disc gel electrophoresis on acrylamide gels. The relative proportions of the different proteins in either secretion were not influenced by the age of the rats above 2 months. For parotid saliva, no differences in the types or proportions of the different proteins were detected in saliva elicited by electrical stimulation of the parasympathetic nerve to the glands, by cholinergic or by α- or β-adrenergic agonists, despite large differences in salivary flow rates and protein concentrations in response to those different stimuli. The relative proportions of proteins were the same in the secretions collected at both 7:00 and 18:00h, the times of expected minima and maxima, respectively, in the secretory protein content of the glands. For submandibular saliva an α-adrenergic agonist, methoxamine, and electrical stimulation of the chordalingual nerve elicited the secretion of additional types of proteins to those released in response to methacholine, pilocarpine or isoprenaline. Secretion of these additional proteins was abolished by the α-adrenergic blocker, phentolamine, but not the β-adrenergic blocker, propranolol.It is postulated that in rat submandibular glands the acinar cells secrete protein in response to cholinergic, α- and β-adrenergic stimulation, with β-adrenergic stimuli being most effective, whereas the granular tubules secrete proteins different from those secreted by acinar cells and only in response to α-adrenergic stimulation.     

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.     

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.     

Production of microliths and sialadenitis in rats by a short combined course of isoprenaline and calcium gluconate.

The relationship between microliths and sialadenitis in man is unclear, so an attempt was made to investigate it experimentally in rats with the use of isoprenaline and calcium gluconate either alone or combined. The acini of the submandibular and parotid glands of rats that were given isoprenaline were enlarged, and degenerate acinar cells were seen, with extravasated secretions in the submandibular gland. Similar changes were seen in the submandibular and parotid glands of rats that were given isoprenaline combined with calcium gluconate; in addition, ductal microliths with regions of atrophic sialadenitis were observed. The results suggest that there is temporary obstruction to the salivary flow after isoprenaline is injected, and in the rats that were also given calcium gluconate some of the stagnant saliva calcified to form microliths, which produced a lasting obstruction and obstructive sialadenitis. This supports the possibility that microliths, which are present in normal salivary glands of man, are a primary etiologic factor in sialadenitis.     

Developmental changes in the volumes, protein, and some electrolyte concentrations of male and female rat submandibular saliva secreted in response to methoxamine and pilocarpine

Saliva secreted in response to methoxamine and pilocarpine was collected from the cannulated ducts of the submandibular glands of male and female rats at weekly intervals from two to 10 weeks of age. It was analyzed for volume and for concentrations of protein, potassium, calcium, and inorganic phosphate. Following the collection of saliva, the submandibular glands were removed and weighted. The wet weights of the glands increased substantially up to seven weeks of age and then reached almost plateau values in both sexes. The salivary volumes secreted in response to both agents in both sexes were positively correlated with the gland weights, except that after five to six weeks of age there was no correlation between gland weight and methoxamine-stimulated salivary volume. The concentrations of protein, potassium, and inorganic phosphate were inversely related to the flow rates only at relatively low rates of flow. The concentration of calcium was positively correlated with the protein concentration and was independent of the nature of the stimulus and of sex differences during postnatal development.     

Dopamine-induced secretion of protein and of some electrolytes by rat submandibular and parotid glands

The secretory effects of dopamine were tested in Nembutal-anaesthetized adult male Sprague-Dawley rats injected either intraperitoneally (i.p.; 5–100 mg/kg) or intravenously (i.v.; 250 μg-3 mg/kg) and with or without α- and β-adrenergic and cholinergic blockers and haloperidol. Polyacrylamide-disc gel electrophoresis was used to separate the different proteins in parotid and submandibular duct saliva and the latter was also analysed for Na, K, Ca and P. The proteins secreted in parotid saliva were similar to those elicited by α- and β-adrenergic and cholinergic agonists and the effects of dopamine appeared to be similar to those produced by β-adrenergic stimulation. Parotid secretion was partially blocked by both phentolamine and haloperidol, suggesting that dopamine is also acting on α-adrenergic and on specific dopamine receptors. The submandibular gland is able to secrete different types of proteins in response to α-adrenergic agonists (α-type proteins) as opposed to β-adrenergic or cholinergic agonists (β-type proteins). With i.v. dopamine at all doses and i.p. dopamine at doses of 75 mg/kg or above, the proteins secreted by the submandibular glands were of the α-type. However, at i.p. doses of 40 mg/kg or less, proteins of the β-type were secreted. Thus the types of proteins secreted were dose-dependent. Administration of each of six α-adrenergic blockers prior to the higher doses of dopamine caused secretion only of proteins of the β-type, but this change did not occur after administration of atropine, propranolol or haloperidol, although haloperidol partially blocked the secretory effects of dopamine. Despite the difference in types of proteins secreted in response to i.p. dopamine at 40 and 75 mg/kg, the salivary electrolyte and total protein concentrations were not significantly changed.     

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.     

Secretory responses to autonomic stimulation of rat salivary glands following reserpine treatment

The response to stimulation of the parasympathetic innervation to parotid or submandibular glands of reserpinized rats was altered from that of untreated rats. Thus, acute reserpinization, like other types of sympathectomy, resulted in increase in volume of parasympathetically-evoked parotid or submandibular saliva when comparison was made with evoked saliva from untreated glands. As norepinephrine is depleted by reserpine, there was no response to stimulation of sympathetic nerves to these reserpinized glands. Adrenergic receptors were normally activated by administration of autonomic agonists. Thus a single high dose of reserpine can cause the same effects as those induced by chronic administration of low doses of reserpine, i.e. a 3-fold increase in calcium (Ca) concentration of submandibular gland but no change in Ca concentration of parotid gland. Although sympathetic stimulation caused no change in Ca concentration of submandibular or parotid glands of reserpine (acute)-treated rats, stimulation with isoproterenol (25 mg/kg, i.p., 60 min) produced a 32-35 per cent decrease in glandular Ca concentration from that of unstimulated reserpinized glands. Glands of untreated rats showed a 52 per cent depletion after 60 min of isoproterenol stimulation; however, Ca output in parotid saliva from reserpinized rat for 60 min of stimulation was not changed from that of untreated rats, but that of submandibular saliva was two times greater. Ca concentration of submandibular saliva was unchanged during 60 min-stimulation of reserpine-treated rats, but that of untreated rats decreased.     

Epidermal growth factor concentration in hyperplastic and hypertrophic submandibular salivary glands of mice.

Submandibular salivary glands of mice contain high concentrations of epidermal growth factor (EGF). The EGF content of mouse submandibular salivary glands undergoing hyperplastic and hypertrophic changes was measured and compared to that in the glands of control mice. Salivary gland hyperplasia was induced by giving mice a single injection of isoprenaline and hypertrophy was produced either by repeated, daily injections of isoprenaline, repeated amputation of a lower incisor tooth or by removing the right submandibular salivary gland and thus producing a compensatory hypertrophy of the left submandibular gland. The EGF content of the hyperplastic submandibular salivary glands was not different from that of the control glands. While the EGF content of the hypertrophied glands resulting from either repeated isoprenaline injections or partial sialoadenectomy did not differ from that of the control glands, the concentration of EGF was significantly lower. This reduced concentration is probably a reflection of acinar hypertrophy with a resultant smaller proportionate contribution of the granular tubules to the mass of the gland. Incisor-amputation-induced hypertrophy did not result in a reduced concentration of EGF in the submandibular salivary glands, but the reason for the different response is unknown.The findings provide no evidence for the involvement of EGF in the induced changes of submandibular salivary gland hyperplasia or hypertrophy resulting from either isoprenaline treatment or partial sialoadenectomy. The reason for the higher concentration of EGF in hypertrophied submandibular salivary glands resulting from incisor amputation compared to that measured in hypertrophied glands resulting from the other stimuli used remains unresolved.     

Volume and composition of pilocarpine- and isoproterenol-stimulated submandibular saliva of early postnatal rats

Submandibular saliva was collected from early postnatal rats by cannulation of the main excretory duct of individual glands after i.p. injections of pilocarpine (10 mg/kg body weight) or isoproterenol (10 mg/kg body weight). With this method of saliva collection, a secretory response to pilocarpine was observed at two wk of age. The average weight of 40 glands was 35.6 +/- 1.6 mg, and the average volume of saliva secreted in 60 min was 32 +/- 2.2 microliters. By three wk of age, the gland had approximately doubled in size (average weight of 39 glands = 61.9 +/- 3.1 mg), and the average total volume of saliva secreted in 60 min was more than three times larger (120.4 +/- 10.5 microliters) than that secreted by two-week-old rats. The relationship between electrolyte (Na+, K+, Ca++) and protein concentrations and rate of flow was similar to that observed in pilocarpine-stimulated adult saliva, and did not differ appreciably in the saliva of two- and three-week-old animals. A measurable secretory response to isoproterenol was observed at three wk of age when saliva was collected by duct cannulation. The average total volume of saliva secreted in 60 min was 48 +/- 3.1 microliters, and salivary Na+ and K+ concentrations, and their relationship to flow rate, were similar to those of isoproterenol-stimulated adult saliva. Saliva Ca++ and protein concentrations were also generally similar to those of isoproterenol-stimulated adult saliva. Total protein output (in 60 min) was 2 1/2 times greater in three-week-old rats with isoproterenol stimulation (compared to pilocarpine stimulation), but was significantly smaller than that of isoproterenol-stimulated adult glands. It is concluded that the submandibular gland of early postnatal rats is capable of secreting saliva in vivo following cholinergic and beta-adrenergic stimulation, and that this ability corresponds with the appearance of the corresponding autonomic receptors, but precedes cytodifferentiation. Ductal transport of electrolytes is well-developed at this stage of postnatal development, but fluid and protein output is smaller than in adult glands and requires full morphological maturation of acinar cells.     

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.     

The effects of upper incisor separation on the submandibular and sublingual glands of rats

The effects of upper incisor separation on the submandibular and sublingual glands of rats were examined biochemically, immunohistologically, and radio-immunologically during 28 days of treatment. Lateral separation of the upper incisors by application of force from an orthodontic appliance caused significant enlargement of the sublingual and submandibular glands of rats by three and seven days, respectively, after the beginning of the orthodontic treatment. This enlargement was followed by a significant increase of both RNA and DNA content, with some evidence of hyperplasia and hypertrophy. The enlargement was also associated with a significant increase of substance P at early stages after treatment, suggesting the involvement of the sensory nerves. These changes were largely inhibited by phenoxybenzamine, an alpha-adrenergic blocker, but not by atropine or morphine. Wet weights and RNA contents of the sublingual glands were markedly reduced by atropine. In comparison with control animals, the enlarged submandibular glands of rats subjected to orthodontic treatment secreted additional proteins identical with those secreted by glands enlarged by chronic administration of isoproterenol. In addition, chemical sympathectomy with 6-hydroxydopamine and phenoxybenzamine stimulated the synthesis of these abnormal proteins, but atropine and morphine did not. In contrast, protease activities in the convoluted granular tubule cells in the submandibular glands were increasingly reduced after treatment, as seen in rats subjected to chronic treatment with isoproterenol. However, the submandibular and sublingual glands completely recovered after removal of the orthodontic appliance.     

Functional and proteomic analysis of submandibular saliva in rats exposed to chronic stress by immobilization or constant light

ObjectiveIn this study, we have evaluated the effects of stress on functional and proteomic changes in submandibular saliva of rats.DesignMale adult rats were divided in three groups: IMO (2 h/day of immobilization for 7 days), LL (constant light during 20 days), C (unstressed controls submitted to 14 h light–10 h dark cycle). Body weight, food intake and the dry weight of submandibular gland were recorded. Saliva samples, collected under anaesthesia following i.p. administration of isoproterenol and pilocarpine (5 mg/kg), were assayed for total proteins (TP), amylase activity and SDS-PAGE electrophoresis.ResultsBody weight, food intake and the dry weight of submandibular gland of IMO rats were lower than those of C and LL groups. The salivary volumes secreted in IMO and LL rats, were significantly higher than in controls. The TP output (μg protein/μg saliva/mg of dry tissue) and amylase activity output (AU/μg of saliva/mg of dry tissue) in IMO were significantly higher than in C and LL animals. The electrophoretic pattern of saliva proteins of LL rats, revealed the absence of a protein band of approximately 25 kDa. This band was composed by the common salivary protein-1 and a prolactin-induced protein as identified by peptide mass fingerprinting.ConclusionsDifferences in body weight and food intake between IMO and LL might be attributed to the sort and intensity of stressors stimuli. The changes in the volume of secreted saliva could be a compensatory mechanism in response to stressors. The increase of total protein in IMO rats and the absence of 25 kDa proteins in LL, would suggest that the submandibular glands respond to the sympathetic nervous system stimuli induced by the stress with an increase of activity of the sympathetic nerves in IMO and a reduction in LL rats.     

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.     

Age changes in secretory function of male and female rat parotid glands in response to methoxamine and pilocarpine

Saliva secreted in response to methoxamine and pilocarpine was collected from the cannulated ducts of both parotid glands of male and female rats at weekly age intervals from three to 10 weeks, and at 3.5, 8, and 15 months of age. It was analyzed for the concentrations of protein, potassium, calcium, inorganic phosphate, and for amylase activity. The type of protein were determined electrophoretically, and an amino acid analysis of the total protein was also carried out. The wet weights of the glands increased substantially up to eight weeks of age, then reached almost plateau values, and finally tended to decrease at 15 months of age in both sexes. The salivary volumes secreted in response to methoxamine and pilocarpine were positively correlated with the parotid gland weights in both sexes. The concentrations of protein, potassium, and inorganic phosphate were inversely related to the salivary flow rates only at relatively low rates of flow. The amylase activity was positively correlated with the concentration of protein, independent of the nature of the stimulus, age, and sex. With methoxamine as a stimulus, the amylase activity was positively correlated with the concentration of calcium, independent of age and sex. The types of protein and amino acid concentrations were independent of the nature of the stimulus, age, and sex up to 15 months of age. However, in parotid saliva of several rats at 8 and 15 months of age, unusual proteins were observed electrophoretically, independent of the nature of the stimulus and sex.     

Effects of thyroxine and dexamethasone on rat submandibular glands

Glucocorticoids and thyroxine are known to have a marked effect on the flow rate and protein composition of rat parotid saliva in hormonally intact animals. In the present study, the effects of a one-week treatment of male rats with dexamethasone and thyroxine were studied by electron microscopy and x-ray micro-analysis, and by measurement of the flow rate and determination of the chemical composition of pilocarpine-induced submandibular saliva. Thyroxine had the most extensive effects on the submandibular gland. The acinar cells were enlarged and filled with mucus; the cellular calcium concentration was significantly increased. The flow rate of the submandibular saliva was significantly reduced compared with that in saline-injected control animals. Thyroxine caused an increase in the concentrations of protein, total calcium, and potassium in the saliva. Dexamethasone had no significant effects on gland ultrastructure or on the elemental composition of the acinar cells; flow rate was not affected, but the concentrations of protein, calcium, and potassium were significantly increased. The effects of dexamethasone and thyroxine on the flow rate and protein composition of pilocarpine-induced rat submandibular saliva differ from those reported earlier for rat parotid saliva after simultaneous stimulation with pilocarpine and isoproterenol.     

Enlargement of rat submandibular salivary gland induced by single amputation of lower incisor teeth

Histological, histometric and ultrastructural studies of rat submandibular salivary glands (SMG) following single amputation of lower incisor teeth were made. The SMG enlargement occurred after the tooth amputation, and its maximal increase of weight was reached 7 days later. The results of histological and histometric observations showed that the enlargement of SMG was due to both hypertrophy and hyperplasia of the acinar cells, and hypertrophic acinar cells were ultrastructurally characterized by well-developed organelles such as rough-surfaced endoplasmic reticulum and Golgi apparatus which filled the entire cytoplasm. Hydropically swollen nerve endings with disappearance of vesicles were frequently found. In later stages, degenerative or necrotic cells were scattered in the acini, and the size and the structure of the remaining acinar cells became gradually similar to those of non-treated glands. The morphological changes seen in the enlarged SMG induced by single amputation of the lower incisor teeth in rat were similar in many ways to those of sialadenosis induced experimentally by active sympathomimetic drugs.     

The effect of food consistency and dehydration on reflex parotid and submandibular salivary secretion in conscious rats

Changes in salivary secretion with different consistency of diet and dehydration were studied in male Wistar rats under unrestricted conditions. To measure the salivary secretion, a stop-flow method was used. There was little unstimulated salivary secretion from the parotid and submandibular glands, but eating solid, powdered, and liquid diets induced parotid and submandibular saliva. There was no significant change in the volume and flow rate of saliva in bilateral parotid glands during the eating of solid diets. The solid and powdered diets induced significantly more salivary secretion from the parotid gland than did the liquid. The salivary flow rate with solid diets was significantly greater from the parotid gland than from the submandibular. On the other hand, the salivary flow rate with the liquid diet was significantly smaller from the parotid gland than from the submandibular. Appreciable amounts of submandibular saliva, but little parotid saliva were secreted during grooming. Clearly, parotid and submandibular saliva have different roles in the rat. When injected intraperitoneally with 1.5 M NaCl solution or water-deprived for 24 h, rats took longer to eat the solid diets, and had increased salivary volume and decreased flow rate from the parotid gland. These results indicate that the moisture content of the diet and the dryness of the mouth alters the volume of parotid saliva secreted in rats and show that parotid saliva plays an important part in mastication and swallowing.     

Enlargement of rat submandibular salivary gland induced by single amputation of lower incisor teeth. Histological, histometric and ultrastructural studies

Histological, histometric and ultrastructural studies of rat submandibular salivary glands (SMG) following single amputation of lower incisor teeth were made. The SMG enlargement occurred after the tooth amputation, and its maximal increase of weight was reached 7 days later. The results of histological and histometric observations showed that the enlargement of SMG was due to both hypertrophy and hyperplasia of the acinar cells, and hypertrophic acinar cells were ultrastructurally characterized by well-developed organelles such as rough-surfaced endoplasmic reticulum and Golgi apparatus which filled the entire cytoplasm. Hydropically swollen nerve endings with disappearance of vesicles were frequently found. In later stages, degenerative or necrotic cells were scattered in the acini, and the size and the structure of the remaining acinar cells became gradually similar to those of non-treated glands. The morphological changes seen in the enlarged SMG induced by single amputation of the lower incisor teeth in rat were similar in many ways to those of sialadenosis induced experimentally by active sympathomimetic drugs.     

Levels of pre-kallikrein in resting and stimulated human parotid and submandibular saliva

Salivary tissue kallikrein is stored in an active form in human salivary glands. Pre-kallikrein has been demonstrated in mixed saliva, but it is not clear if the various salivary glands contribute equally. This study set out to determine if pre-kallikrein is present in human parotid and submandibular salivas at rest, whether levels change during stimulation, and to compare the pattern of pre-kallikrein and kallikrein secretion with that of total protein. Resting and citric acid-stimulated parotid and submandibular, and gum-stimulated parotid saliva samples were collected from 6 healthy subjects. Salivary flows were determined gravimetrically. Total protein concentration and kallikrein enzymic activity were assayed using standard techniques. Pre-kallikrein was assayed following trypsinisation of duplicate samples. Pre-kallikrein was present in parotid and submandibular ductal saliva. Proportions of pre-kallikrein and active kallikrein were similar in salivas secreted at rest and during stimulation, and both outputs mirrored protein output in both major glands. Gum-stimulated parotid saliva showed lower activity than resting, and no differences were seen between resting and stimulated submandibular samples.