Effects of carbamylcholine and isoproterenol on electrolyte fluxes in perfused main duct of submandibular gland of reserpinized rat

Administration of reserpine (RES) at a dosage of 0.5 mg/kg body wt, ip daily for 7 days was found to lower the dose of carbamylcholine and isoproterenol that alters sodium and potassium transport by cells of the main duct of rat submandibular gland. In the perfused main excretory duct of the submandibular gland of the RES rat, administration of carbamylcholine at a dosage of 1 microgram/kg body wt, inhibited net efflux of sodium (17%) and administration of isoproterenol at a dosage of 2 micrograms/kg body wt increased net efflux of sodium (20%); these drugs, at the same dosages, did not induce significant change in electrolyte flux of normal rat. At a dosage of 5 micrograms/kg body wt, carbamylcholine decreased net influx of potassium (15%) in the RES rat but was without effect on normal rat. Isoproterenol at the dosage of 5 micrograms/kg body wt significantly inhibited net influx of potassium in both the RES rat and normal rat. The data suggested that the duct cells developed supersensitivity to sympathomimetic and parasympathomimetic stimulation after chronic RES treatment.     

Expression of transforming growth factor beta 1 in the submandibular gland of the rat

We employed immunocytochemical and in situ hybridization techniques to study the expression of transforming growth factor beta 1 (TGF-beta 1) in rat submandibular gland. Immunoreactivity for TGF-beta 1 was observed in the cells of granular convoluted tubules (GCTs), striated ducts, and excretory ducts, whereas it was absent in the intercalated ducts and secretory acini in both male and female rats. Immunoelectron microscopy revealed the ultrastructural localization of TGF-beta 1 in the secretory granules of GCT cells. On the other hand, signals for rat TGF-beta 1 mRNA were abundant in the GCT and striated duct cells but were lacking in the excretory duct cells. These results provided evidence for the production of TGF-beta 1 in the GCTs and striated ducts of rat submandibular gland.     

Differential localization of "brain-specific" S-100 and its subunits in rat salivary glands

In the rat, the S-100 antigens in the submandibular gland were found to be immunochemically identical with those in the brain (glial cells) when compared using crossed immunoelectrophoresis. Specific antibodies against the S-100a non-beta and against the S-100 beta subunit were prepared from antibodies against crude S-100 protein and from S-100 components (S-100a and b) by affinity chromatography. In the rat salivary glands a differential distribution of subunit immunoreactivity was clearly evidenced using indirect immunofluorescence. Certain intercalated duct cells of the submandibular gland as well as Schwann cells contained the S-100 beta subunit immunoreactivity exclusively, while other duct cells in parotid, submandibular, and sublingual glands contained S-100a non-beta subunit immunoreactivity. Both subunits were present in astrocytes and ependymal cells. The immunocytochemical localization of alpha and beta subunits is a promising technique for the classification of various types of S-100-containing cells.     

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde--2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H₂O₂, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaral dehyde and incubation in a DAB reaction medium containing a high concentration of H₂O₂ inhibits the peroxidase activity of acinar cells in the hamster submandibular gland     

Cytochemical detection of endogenous peroxidase in the acinar cells of the hamster submandibular gland

The presence of endogenous peroxidase activity in the hamster submandibular gland was investigated cytochemically by light and electron microscopy using diaminobenzidine methods. After fixation of tissue with 2% paraformaldehyde--2.5% glutaraldehyde and incubation in a DAB reaction medium containing 0.01% H₂O₂, the peroxidase reaction product was localized in the nuclear envelope, the cisternae of the endoplasmic reticulum, secretory granules and the Golgi apparatus in both the acinar and granular duct cells of the submandibular gland. This is in contrast to earlier investigators who failed to detect peroxidase activity in acinar cells of the hamster submandibular gland and reported that peroxidase is localized only in the granular duct cells. The discrepancy may be caused by differences in experimental procedures. It is suggested that fixation of tissue with a high concentration of glutaral dehyde and incubation in a DAB reaction medium containing a high concentration of H₂O₂ inhibits the peroxidase activity of acinar cells in the hamster submandibular gland This revised version was published online in November 2006 with corrections to the Cover Date.     

The immunohistolocalization of carbonic anhydrase III in the submandibular gland of rats and hamsters

Summary Carbonic anhydrase III has been localized using the avidin-biotin-glucose oxidase complex (ABC) method in the submandibular gland of the rat and hamster. This isozyme, which is predominant in skeletal muscle, was observed in intercalated duct, striated duct and excretory duct cells in the rat submandibular glands. In contrast, only some striated duct cells in hamster submandibular glands were stained.     

Submandibular gland resection and bilateral parotid duct ligation as a management for chronic drooling in cerebral palsy

The control of chronic drooling in cerebral palsy has been difficult in the past. Previous methods to control drooling include the Wilkie procedure, which diverts the salivary flow from the parotid glands by means of surgically created tunnels to the tonsillar fossa, along with submandibular gland resection. An alternative is submandibular gland resection with bilateral parotid duct ligation. Previous published studies using this method have resulted in good to excellent results, but population sizes were felt to be too small to be conclusive. Since 1979, a total of 58 patients have been treated by parotid duct ligation with submandibular gland resection, and 86 percent have shown good to excellent results. These results compare favorably with those published by Wilkie. Also, parotid duct ligation is technically easier, associated with less postoperative morbidity, and has shown a decreased duration of hospitalization compared to parotid duct transposition.     

Apoptosis and proliferation of myoepithelial cells in atrophic rat submandibular glands.

This study was designed to determine whether apoptosis and proliferation of myoepithelial cells occur in atrophic rat submandibular glands. The excretory duct of the right submandibular gland was doubly ligated with metal clips. The atrophic right submandibular glands removed after 1-28 days of duct ligation were investigated using immunohistochemical double staining for actin as a marker for myoepithelial cells and proliferating cell nuclear antigen (PCNA) as a marker for proliferating cells, double staining for actin immunohistochemistry, nick end-labeling (TUNEL) as a marker for apoptotic cells, and transmission electron microscopy (TEM). A few PCNA- and no TUNEL-positive myoepithelial cells were found in the control submandibular glands taken from animals with no operation. In the experimental glands, PCNA-positive myoepithelial cells were common 2 and 3 days after duct ligation and then decreased in number. TUNEL-positive myoepithelial cells appeared at 2 days and were observed most frequently at 5 days. Apoptotic myoepithelial cells were also identified by TEM. These observations suggest that both apoptosis and proliferation of myoepithelial cells occur, especially in the early phase of atrophy, in the rat submandibular gland.     

Immunohistochemistry of carbonic anhydrase isozymes VI and II during development of the rat salivary glands

 Secreted carbonic anhydrase (isozyme VI; CA VI) was localized by immunohistochemistry in the developing postnatal rat submandibular and parotid glands using a specific monoclonal antibody to the rat enzyme. CA VI immunostaining was not detectable in the glands before birth. In the submandibular gland, granular immunostaining for CA VI was detectable in several terminal tubule cells of 1-day-old rats. At 1 week, the CA VI-positive cells were located at the periphery of the terminal tubules and appeared to be budding off the tubules. These cellular buds gradually increased, and, by 4 weeks, formed acini. CA VI was also detected in the duct lumen from day 1. The immunostaining in the parotid gland was detected sporadically in the acinar cells at 2 or 3 weeks. By 4 weeks, when the gland was almost indistinguishable from the adult one, the number of positive acinar cells had increased. Their number, however, was far smaller than in the adult gland, and the enzyme could not be detected in the duct lumen. CA II was also localized using specific antibodies to the rat isozyme. CA II was detectable in the inter- and intralobular striated ducts at 2 weeks after birth in the submandibular gland and at 3 weeks in the parotid gland. These results suggset that CA VI is secreted into saliva from soon after birth and that CA II appears in parallel with the functional maturation of the ducts. In addition, CA II was transiently expressed by the cellular buds of the submandibular gland at 2 and 3 weeks. Accepted: 7 January 1998     

Effects of substance P on Na and K transport in perfused main submandibular duct of rat

Substance P (2 and 4 micrograms/kg . min, iv) caused an inhibition of net efflux of Na and net influx of K in perfused main excretory duct of rat submandibular gland. These effects could not be blocked by atropine sulfate. The data suggest that substance P receptors are present in the duct cells and play a role in the regulation of transductal electrolyte transport.     

Excision of the submandibular gland by an intraoral approach

To improve the outcome in patients with benign diseases of the submandibular gland, we have developed an entirely intraoral technique for excision of the submandibular gland. This procedure is anatomically safe and can be performed with minimal morbidity. We believe the essential surgical steps are as follows: (1) infiltration with Xylocaine plus epinephrine with an adequate waiting period for hemostasis; (2) careful identification of the submandibular duct/lingual nerve relationship; (3) anterior retraction of the mylohyoid muscle to expose the superficial lobe; (4) superiorly directed, extraoral, manipulation of the submandibular gland; and (5) close and blunt dissection to the gland laterally to avoid injury to the facial artery and vein.     

Evaluation of a reservoir in the main excretory duct of rat submaxillary gland.

Cannulation of salivary gland main excretory duct at its oral opening is routinely used for collecting fluid, in situ, from the luminally perfused duct, or saliva from the stimulated gland. For perfusion of the main excretory duct, in situ, or for saliva collection, rat submaxillary gland is often the organ of choice, since electrolyte transport occurs at high rates both in the whole gland and in the main excretory duct. Recently, it has been reported that there is a pouchlike dilatation of the main excretory duct at its oral end, and that this dilatation may serve as a fluid reservoir. Because of possible effects of such a reservoir on measurements of electrolyte transport by the whole gland or the main duct segment, the size and form of the reservoir have now been examined. For this, techniques of histology, radiography, and microcatherization were employed. It was found that, while the functional volume of the reservoir exceeds that of the main duct proper, the time needed for displacement of reservoir fluid by perfusate or saliva would probably be only on the order of 1-3 min at higher rates of saliva or perfusate flow. Therefore, if adequate allowance is made for equilibration time, collection of saliva or luminal perfusate by oral cannula seems justified.     

Androgenic actions of 5 alpha-androstane-3 alpha,17 beta-diol in rat submandibular gland

To evaluate the action of 5 alpha-androstane-3 alpha,17 beta-diol(3 alpha-diol) in rat submandibular gland, 5 alpha-reductase, 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSD) and oxidative 3 alpha-hydroxysteroid dehydrogenase (3 alpha-HSDO) activities, and trypsin-like protease activities, were assayed in control, castrated and 3 alpha-diol injected rats. 3 alpha-Diol (1 mg/kg) was injected subcutaneously in castrated male rats daily for 7 days. A 47% decrease of 5 alpha-reductase activity in the nuclei and a 30% decrease of 3 alpha-HSD(O) activities in the cytosol were shown after castration. 3 alpha-Diol restored the 5 alpha-reductase and 3 alpha-HSD(O) activities to 82 and 140% of the control submandibular gland, respectively. 3 alpha-Diol raised the trypsin-like protease activity to near control values in the submandibular gland of castrated rats. Morphological observations also revealed a distinct effect of 3 alpha-diol on the number of granules of granular duct cells. It is concluded that 3 alpha-diol has an androgenic action in the rat submandibular gland. It stimulates the 3 alpha-HSD(O). The 3 alpha-HSD(O) in its turn may be responsible for DHT accumulation in the cells.     

The postnatal development of the submandibular gland of the mouse

Summary The postnatal development of the submandibular gland was investigated in male mice of the Swiss-Webster strain, which were killed at 1, 2, 3, 4, 5, 6, 8, 10, 12, 16 and 20 weeks of age, while the older mice had been weaned at 3 weeks of age. The mean weight of the submandibular gland increases from 9.5 mg at 1 week to 232.9 mg at 20 weeks of age, and the rate of increase is rapid between 3 and 10 weeks of age. The gland's contents of DNA, RNA and protein increase in a similar manner.The changes in the constituent cell types of the gland were studied in radioautographs prepared from Epon-embedded sections of mice given ³H-thymidine and stained with toluidine blue. At 1 week of age, the gland consists of acinar cells (36%), intercalated duct cells (26%), juxta-acinar cells (13%), striated duct cells (12%) and others. The cellular composition of the gland changes little before weaning, but the absolute number of all types of cells increases with age. Between 3 and 4 weeks, juxta-acinar cells disappear and granular convoluted tubule cells appear and increase rapidly in number with age. The rapid expansion of the population size of granular convoluted tubule cells after weaning coincides with the second peak of increased proliferative activity of intercalated duct cells, whereas all the other cell types show a progressive decrease in their proliferative activity with age. In spite of the burst in proliferative activity, there is no corresponding increase in the absolute number of intercalated duct cells. The number of striated duct cells peak at 5 weeks of age and then declines. These findings indicate that the mitoses of intercalated duct cells give rise to granular convoluted tubule cells through a stage of striated duct cells. At 20 weeks of age, the gland consists of granular convoluted tubule cells (47%), acinar cells (28%), intercalated duct cells (12%), striated duct cells (1%) and others.Supported by Public Health Service Research Grant AMDE 19753 from the National Institute of Health. The authors are indebted to Mr. I. Borcsanyi for technical assistance     

Comparison of effects of surgical and chemical sympathectomy on beta adrenergic and muscarinic receptors of parotid gland of young and adult rats

Surgical (removal of a superior cervical ganglion) or chemical [(administration of a single dose of 6 hydroxydopamine (6 OHDA) (50 mg/kg dose body wt)] sympathectomy of rats at 2 or 8 days of age resulted in an increase in [3H]DHA binding of membranes of parotid gland of young rats (age range 21 days to 48 days). The increase progressed with postnatal age; at 21 days of age (surgical sympathectomy), it was 13%; at 32 days of age with 6 OHDA, it was as much as 34%, but only 26% at 42 days of age with surgical sympathectomy. No change in [3H]QNB binding was observed at any postnatal ages following neonatal sympathectomy. Conversely, surgical sympathectomy of the parotid of adult rat resulted in little or no change in [3H]DHA binding at 1, 2, 3, or 4 weeks postdenervation, but [3H]QNB binding was reduced at all periods, with the reduction from control values at 2 weeks being 34%, and at the subsequent intervals, 24-26%. The increase in number of beta adrenoceptors of the parotid gland was not related to the kind of sympathectomy (chemical or surgical) or neonatal age at which it was done; however, duration of the denervation for 2-3 weeks was necessary for the receptor increase to occur. In the adult, however, the duration of the denervation was of no importance since change in number of beta adrenoceptors did not occur at 1, 2, 3, or 4 weeks after surgical denervation but did occur after only 1 week after of reserpine-induced denervation. QNB binding was decreased with surgical sympathectomy as well as reserpine-induced sympathectomy of adult parotid gland; norepinephrine concentration was decreased to levels of a few percent of innervated glands. The relation between development of glandular supersensitivity and increase in beta adrenoceptors is discussed.     

Apical maxi-K (KCa1.1) channels mediate K+ secretion by the mouse submandibular exocrine gland

The exocrine salivary glands of mammals secrete K+ by an unknown pathway that has been associated with HCO3(-) efflux. However, the present studies found that K+ secretion in the mouse submandibular gland did not require HCO3(-), demonstrating that neither K+/HCO3(-) cotransport nor K+/H+ exchange mechanisms were involved. Because HCO3(-) did not appear to participate in this process, we tested whether a K channel is required. Indeed, K+ secretion was inhibited >75% in mice with a null mutation in the maxi-K, Ca2+-activated K channel (KCa1.1) but was unchanged in mice lacking the intermediate-conductance IKCa1 channel (KCa3.1). Moreover, paxilline, a specific maxi-K channel blocker, dramatically reduced the K+ concentration in submandibular saliva. The K+ concentration of saliva is well known to be flow rate dependent, the K+ concentration increasing as the flow decreases. The flow rate dependence of K+ secretion was nearly eliminated in KCa1.1 null mice, suggesting an important role for KCa1.1 channels in this process as well. Importantly, a maxi-K-like current had not been previously detected in duct cells, the theoretical site of K+ secretion, but we found that KCa1.1 channels localized to the apical membranes of both striated and excretory duct cells, but not granular duct cells, using immunohistochemistry. Consistent with this latter observation, maxi-K currents were not detected in granular duct cells. Taken together, these results demonstrate that the secretion of K+ requires and is likely mediated by KCa1.1 potassium channels localized to the apical membranes of striated and excretory duct cells in the mouse submandibular exocrine gland.     

Rate of protein synthesis in rat salivary gland cells after pilocarpine or feeding

The effect of pilocarpine and food uptake on the rate of incorporation of [3H]-leucine in vivo was measured by means of quantitative radioautography in three exocrine cells of the rat: the acinar and the granular duct cells of the submandibular and the acinar cells of the parotid gland. The three cell types react differently. The submandibular acinar cells showed a decrease in incorporation rate after pilocarpine administration but not after feeding. The incorporation rate of the granular duct cells of the submandibular gland remains constant after both stimulations. The acinar cells of the parotid gland show an increase in incorporation rate of [3H]-leucine in response to both. The contrast between the submandibular and the parotid gland could also be demonstrated radiobiochemically, the results reflecting the incorporation rates of the acinar cells of both glands, giving no information on the contribution of other cell types. The decrease in incorporation rate of the submandibular gland acinar cells is accompained by a shift of polyribosomes towards monomers.     

Comparative histochemical study of glycoconjugates of the major salivary glands and pancreas in humans

The human parotid, submandibular, sublingual salivary glands and pancreas have been studied with lectin--horseradish peroxidase conjugates (con A, PNA, SBA, WGA, LAL), aldehyde fuchsin and Bismark brown. Intercalated duct cells produce a specific aldehyde-fuchsin-reactive substance. These cells are found only in the submandibular and parotid, but not in the sublingual glands. Similar reactivity is found in B-insulocytes of the pancreas. Aldehyde-fuchsin marks cytoplasmic granularity of the striated duct cells of all large salivary glands. This specific granularity is also selectively stained with Bismark brown and con A. Using fucose-specific lectin from Laburum anagyroides bark (LAL), granularity in serocytes of the submandibular gland is demonstrated. Some individual variations are observed in PNA binding to serocytes of the submandibular gland. It reveals that thyroglobulin-peroxidase conjugate (previously reported as an available second-step reagent for indirect lectin histochemical methods) non-specifically binds to the striated duct cells of the submandibular gland. During control staining it is also found, that DAB-reaction for endogenous peroxidase can be used as a test-system for a selective histochemical exposure of nuclear regions of endotheliocytes, pericytes and striated duct epitheliocytes of the human salivary glands. Possible significance of the phenomena observed is discussed.     

Regeneration of acinar cells following ligation of rat submandibular gland retraces the embryonic-perinatal pathway of cytodifferentiation

Rat submandibular gland can regenerate following ligation-induced atrophy, eventually recovering its normal morphology and function. Previous studies have suggested that the regeneration process implies both self-proliferation of existing acini and formation of new acinar cells. One hypothesis is that new acinar cells may differentiate from the ductal cells in a similar fashion to the process of cytodifferentiation occurring during submandibular glandular development. In this study atrophy was induced, under recovery anaesthesia, by applying a metal clip on the main duct of the submandibular gland without including the chorda lingual nerve. After 2 weeks the duct was deligated for 3, 5 or 7 days or 8 weeks and the glands collected. Tissue was prepared for immunohistochemstry, biochemical analysis and RNA extraction. The histology of the regenerated glands shows several normal-looking acini, which have regained their glycoprotein content (AB/PAS positive), data also confirmed by biochemical analysis (SDS-PAGE/PAS). Regenerating tissue was characterized by the presence of embryonic-like branched structures ending with AB/PAS positive acinar cells. The proteins SMG-B and PSP are normally expressed in acinar cell precursors during development but only by intercalated ductal cells in the adult stage. In the adult regenerating gland mRNA levels of both SMG-B and PSP were found to be up-regulated compared to ligated glands and SMG-B expression localized to acinar cells whilst the ductal cells were negative. This study of rat submandibular gland regeneration suggests new acinar cells have differentiated from ducts and express markers of acinar cell precursors in a similar manner to the cytodifferentiation process occurring during glandular development.