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PubMed1984

Release of 86Rb from rat submandibular gland slices: relation to sodium pump activity.

Slices of rat submandibular gland were preloaded with 86Rb, an isotope that can substitute for K+ in the K+ release process. The efflux of 86Rb was monitored in a superfusion apparatus that efficiently removed the 86Rb as it exited from the tissue slices. Carbachol and the calcium ionophore A23187 activated a calcium-dependent increase in 86Rb efflux. Dibutyryl cGMP had no detectable effect on 86Rb efflux in contrast to its activation of ouabain-sensitive uptake of 86Rb observed in an earlier study. The stimulated release of 86Rb was not dependent on the presence of either sodium or chloride ions. When 86Rb efflux was stimulated by carbachol, the efflux rate returned toward the basal rate after a few minutes of exposure to carbachol in the medium. If ouabain was then introduced into the superfusate, a large increase in efflux was stimulated. In the absence of carbachol, only a small increase in 86Rb efflux was stimulated by ouabain. The effect of ouabain indicates that there was a substantial recycling of 86Rb between the release and uptake processes in the extracellular space of the tissue slice. The significance of this observation is discussed.

Animals↗

PubMed1975

The development of the granular convoluted duct in the rat submandibular gland.

The development of the granular convoluted duct in the submandibular gland of male rats, 4 to 12 weeks of age, was investigated. During this period, the average weight of the gland increased from 213 to 526 mg, the total DNA and RNA contents doubled, and the protein content tripled. Radioautographs were prepared from Epon embedded sections of the gland of the rats given 3-H-thymidine and stained with toluidine blue. The glands of 4-week-old rats consisted mainly of acinar cells (45%), intercalated ductal cells (20%) and striated ductal cells (16%). A few granular convoluted ductal cells were seen in the striated duct close to the intercalated duct. The frequency (and absolute number) of granular convoluted ductal cells increased linearly from 1% (3 X 10-6) at four weeks to 26% (68 X 10-6) at eight weeks, while the calculated number of striated ductal cells remained stationary. The absolute number of acinar cells and intercalated ductal cells nearly doubled between four to eight weeks of age. The proliferative activity of all cell types declined with age but between six and ten weeks of age the rate of proliferation of ductal cells was relatively higher than the rate of proliferation of the acinar cells. Morphologically the size and number of granules in the granular convoluted ductal cells increased with age. Based on the above data it is concluded that the granular convoluted ductal cells developed from that segment of the striated ductal cells which is in close proximity with the intercalated ductal cells. The heterogeneity of the granules in the granular convoluted ductal cells observed from six weeks of age might denote the functional diversity of the cells.

Age Factors↗

PubMed1971

The functional organization of the parasympathetic secretory innervation of the submandibular gland.

1. In cats the hypoglossal and chorda-lingual nerves were cut and their central ends sewn to the peripheral end of the latter nerve. At monthly intervals the sensitivity of the submandibular gland to a secretory agent was then estimated.2 When the supersensitivity resulting from the decentralization of the gland had been lowered to a constant level acute experiments were made. Electrical stimulation of both nerves caused secretion, which was abolished by ganglion blocking drugs and atropine.3. Simultaneous stimulation of the nerves at frequencies subliminal for each nerve did not cause any secretion.4. Stimulation of one nerve at a subliminal frequency did not increase a flow of saliva evoked by stimulation of the other nerve. However, as soon as the secretory threshold was reached, the flow was markedly accelerated, provided it was submaximal.5. In many cases stimulation of the regenerated chorda fibres at a high frequency caused a flow corresponding to the maximal secretory capacity of the gland. In some experiments this was true for the hypoglossal fibres also.6. The functional organization of the parasympathetic secretory innervation of the submandibular gland of the cat is discussed in the light of these observations.

Animals↗

PubMed1997

Age-related changes in collagen, laminin and tenascin in the infant rat submandibular gland.

We investigated biochemically and immunohistochemically the age-related changes in collagen (types I, III and IV), laminin and tenascin of the extracellular matrix in the submandibular gland of infant rats. The hydroxyproline content markedly increased on day 14 after birth. Electrophoretic profile revealed alpha chains of only type I collagen up to day six after birth. Type III collagen, which was diffusely distributed in interlobular connective tissue, was gradually expressed from day seven after birth. Type IV collagen content increased from day seven. Laminin content gradually increased with age. Tenascin immunohistochemically showed weak expression only around the vessel walls up to day seven. In addition, this expression markedly increased around acinar and ductal walls as basement membrane components on day 14 after birth. These findings suggest that rat submandibular gland types I and IV collagen, and laminin are already present during the fetal stage and relate to construction of tissue, and that type III collagen and tenascin, which increased from day seven after birth, relate to maturation.

Aging↗

PubMed2004

Co-expression of tyrosine hydroxylase, dopamine beta-hydroxylase and neuropeptide Y in the sympathetic neurons projecting to the submandibular gland in the sheep.

Sympathetic neurons projecting to the ovine submandibular gland (SMG) from the superior cervical ganglion (SCG) and middle cervical ganglion (MCG) were identified using retrograde tracing with fluorescent dye (Fast Blue). Antibodies to tyrosine hydroxylase (TH), dopamine beta-hydroxylase (DbetaH) and neuropeptide Y (NPY) were used to determine the immunochemical characteristics of SMG-innervating sympathetic neurons. Immunohistochemistry combined with the retrograde tracing revealed that the population of SMG-projecting neurons consist of four distinct sub-populations, but taking into account their possible different physiological properties only three major sub-populations can be distinguished. The vast majority of neurons in both ganglia are noradrenergic in nature (co-express TH and DbetaH). All examined TH-immunoreactive (IR) neurons also show immunoreactivity to DbetaH. Sub-population of noradrenergic neurons can be divided into NPY-IR and non-NPY-IR. Noradrenergic neurons expressing NPY may act as vasoconstrictors. The second sub-population of SMG projecting neurons in the ganglia studied consists of non-noradrenergic neurons (containing NPY, but not TH). It is known that these kinds of neurons may play a vasodilatory role. In both examined ganglia the third sub-population consists of non-TH-IR and non-NPY-IR neurons of unknown physiological function. Since no DbetaH immunoreactivity was found in any of TH- neurons these nerve cells can also be regarded as non-noradrenergic. It is possible that some neurons of the second as well as the third sub-population are cholinergic and some of them are non-noradrenergic/non-cholinergic in character.

Animals↗

PubMed2001

An alternatively spliced Muc10 glycoprotein ligand for putative L-selectin binding during mouse embryonic submandibular gland morphogenesis.

Late-gestation (embryonic day 18; E18) mouse submandibular glands (SMG) comprise a network of large and small ducts that terminate in lumen-containing, presumptive acini (terminal buds) expressing unique, cell membrane-associated embryonic mucin. The objective here was to clone and sequence embryonic low molecular-weight SMG mucin, predict its secondary structure, and begin to investigate its possible role in SMG development. Evidence was found that: (1) embryonic low molecular-weight mucin is an alternatively spliced Muc10 gene product, 220 amino acids in size (approximately 25 kDa), rich in potential O-glycosylation sites, and variably glycosylated (approximately 40 and 68 kDa); (2) consensus secondary-structure prediction for embryonic low molecular-weight mucin is consistent with a molecule that is anchored to the plasma membrane, directly or indirectly (via a glycolipid), and has a protein core that serves as a scaffold for carbohydrate presentation; (3) embryonic L-selectin is immunolocalized to the plasma membrane region of terminal-bud epithelial cells in a pattern similar to that seen for embryonic mucin; (4) embryonic, but not adult, mucin is able to bind L-selectin and does so endogenously in E18 SMG. As the primary role of L-selectin is to mediate cell adhesion and its ligands are mucin-like glycoproteins, it is suggested that this embryonic low molecular-weight mucin be termed MucCAM.

Age Factors↗

PubMed1992

Thyroid hormone and androgen regulation of nerve growth factor gene expression in the mouse submandibular gland.

The nerve growth factor (NGF) content of the mouse submandibular gland (SMG) is under hormonal control and is modulated by both thyroid hormones (TH) and androgens. The sexual dimorphism of the gland is well documented. In the adult male mouse, the SMG contains 10 times more NGF compared to the female. Conversely, castration of male mice reduces the SMG NGF levels to those found in control females. In order to determine the locus at which androgens and TH exert their effect on NGF gene expression in the SMG, steady-state NGF mRNA levels were determined. Daily treatment of adult female mice with TH for 1 week increased NGF mRNA levels 6-fold. Androgen treatment produced a 20-fold increase in SMG NGF mRNA, which was comparable to levels detected in the control adult male SMG. The effect of TH on NGF mRNA levels was time-dependent and coincided with the increase in NGF protein concentrations. At 48 h after a single TH injection, NGF mRNA levels (measured in SMG total RNA) increased 2-4-fold, while heteronuclear (hn) RNA levels were increased 1.5-2-fold. The NGF gene transcription rate was determined by run-on assay following TH treatment. A small but significant 2-fold induction by TH of NGF gene transcription was found at 24-48 h. Cytoplasmic RNA prepared from the same SMGs used in the run-on experiments was tested by S1 nuclease protection; NGF cytoplasmic RNA was increased 7-fold in the SMGs of females treated with TH 48 h previously. These results demonstrate that the effect of TH on NGF gene expression is due in part to an induction of NGF gene transcription. The discrepancies observed between transcription rate and mRNA levels suggest that the major effect of TH is at the post-transcriptional level, possibly mRNA stabilization. The time required to observe an induction of TH on NGF gene transcription is suggestive of an indirect effect, possibly through the induction by TH of another protein which in turn activates the NGF gene.

Anabolic Agents↗

PubMed1991

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.

Animals↗

PubMed1996

Testosterone metabolism in new squamous cell carcinoma cell line (RSS18) from 7,12-dimethylbenz[a]anthracene-induced submandibular gland of female rat.

We established a new squamous cell carcinoma cell line, designated RSS18, from a 7,12-dimethyl-benz[a]anthracene (DMBA)-induced submandibular gland of the female rat, and investigated a testosterone metabolism in the cells. During 6 h incubation of RSS18 cells with testosterone as a substrate, the cells produced a significant amount of 5alpha-dihydrotestosterone (DHT) and three kinds of minor metabolites, and their percentages metabolized against total metabolites were in descending order of DHT (89 %) > 5alpha-androstane-3alpha,17beta-diol (9.0 %) > 5alpha-androstanedione(1.6%) > 4-androstene-3,17-dione (0.69%). Therefore, testosterone in RSS18 cells was predominantly converted to DHT by 5alpha-reductase. Growth of RSS18 cells was stimulated by DHT (10(-11)-10(-9) M) to around 170%. By reverse transcription-polymerase chain reaction, the androgen receptor mRNA was significantly detected in RSS18 cells. As a result of these findings, DHT production from testosterone and expression of androgen receptor mRNA, we concluded that RSS18 proliferation may be stimulated by DHT through 5alpha-reductase from testosterone.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1998

Effects of calcitonin and calcitonin gene-related peptide on the substance P-mediated secretion of fluid from the rat submandibular gland.

1. The effects of rat calcitonin gene-related peptide (rCGRP), rat calcitonin (rCT) and salmon calcitonin (sCT) on the substance P-mediated (SP-mediated) secretion of fluid from the rat submandibular gland were investigated. 2. Rat CGRP potentiated and prolonged the SP-mediated secretion of fluid from the rat submandibular gland in a dose-dependent manner. CGRP also enhanced methacholine- and phenylephrine-mediated secretion of fluid. 3. The potentiating effect of the combination of rCGRP and SP was somewhat reduced by pretreatment with spantide, human CGRP8-37 and atropine but not by pretreatment with phentolamine or with propranolol. 4. Salmon CT at a low dose mimicked the effect of rCGRP on the SP-mediated secretion of fluid, but its potency was lower than that of rCGRP. However, rCT had no effect on the SP-mediated secretion of fluid. 5. These results suggest that the potentiating effects of rCGRP and SP might involve cholinergic receptors, as well as CGRP and tachykinin receptors, and that sCT, but not rCT, is able to mimic rCGRP in potentiating the secretion of fluid induced by SP.

Animals↗

PubMed1993

Tissue kallikrein and tonin levels in submandibular glands of STZ-induced diabetic rats and the effects of insulin.

The levels of tissue kallikrein, tonin, and other kallikrein-like proteinases were determined in extracts of rat submandibular glands 3 mo after the induction of diabetes with STZ (65 mg/kg i.v.). Total kallikrein-like proteinase activity was assayed catalytically with the fluorogenic substrate DVLR-AFC. Tissue kallikrein was assayed by using the same substrate in the presence of SBTI. Activity of other kallikrein-like proteinases was defined as the difference between the total kallikrein-like activity and that of tissue kallikrein. Tonin was assayed by using the substrate ZVKKR-AFC in the presence of aprotinin. Results were compared with age-matched controls and with diabetic rats that had received daily insulin injections for the last week of the test period. The results showed that all activities were significantly reduced in diabetic glands compared with controls. Insulin treatment restored concentrations of tissue kallikrein activity, whereas the activities of tonin and other kallikrein-like proteinases were unchanged. RIA supported these findings. The results indicate that in rat submandibular glands, insulin affects the synthesis of kallikrein-like proteinases in different ways and, allowing for the slowness of the processes involved, insulin may exert a direct influence on the regulation of tissue kallikrein synthesis but only have indirect effects on the synthesis of tonin and the closely related kallikrein-like proteinases.

Amino Acid Sequence↗

PubMed1991

Immunohistochemical localization of [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 in sympathetic and parasympathetic neurons and nerve fibers projecting to the rat submandibular gland.

The localization of [Met5]enkephalin, [Met5]enkephalin-Arg6-Gly7-Leu8, vasoactive intestinal polypeptide and tyrosine hydroxylase immunoreactivities was studied in the submandibular gland of adult Sprague-Dawley and Wistar rats using the indirect immunofluorescence technique. Immunoreactivities for [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8, a proenkephalin A-derived octapeptide, showed identical distributions. A large number of enkephalin-immunoreactive nerve fibers were detected around secretory acini, along intercalated ducts, convoluted granular tubules, intra- and interlobular ducts, as well as in close contact with blood vessels. The submandibular ganglia contained several enkephalin-immunoreactive neurons and nerve fibers. In the superior cervical ganglion numerous enkephalin-immunoreactive neurons and nerve fibers were also detected. Immunohistochemical co-localization studies indicated that [Met5]enkephalin and [Met5]enkephalin-Arg6-Gly7-Leu8 immunoreactivities co-exist with vasoactive intestinal polypeptide in a subpopulation of neurons of the rat submandibular ganglia, in nerve trunks along the salivary ducts of the gland, and in nerve fibers around the acini. Uni- or bilateral superior cervical ganglionectomies for 1-4 weeks resulted in a complete disappearance of tyrosine hydroxylase immunoreactivity in the glandular parenchyma, while moderate tyrosine hydroxylase immunoreactivity was seen in some neurons of the submandibular ganglia. Abundant [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive nerve fibers were still seen around the acini and blood vessels, as well as close to salivary ducts. These operations did not affect the [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive neurons in the submandibular ganglia. Many principal neurons in the superior cervical ganglion contained both [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase immunoreactivity. Nerve ligation experiments indicated that [Met5]enkephalin-Arg6-Gly7-Leu8-immunoreactive sympathetic fibers project along the external carotid nerve. Accordingly, nerve fibers were found around the acini and blood vessels as well as in nerve trunks along the salivary ducts of the submandibular gland, showing co-localization of [Met5]enkephalin-Arg6-Gly7-Leu8 and tyrosine hydroxylase. Taken together, these observations suggest that the nerve fibers of the rat submandibular gland containing proenkephalin A-derived peptides are of both sympathetic and parasympathetic origin.

Animals↗

PubMed1993

Age-dependent changes in the phosphorylation of nuclear proteins of submandibular glands in isoproterenol-treated rats.

Age-dependent changes in the regulation of the phosphorylation of nuclear proteins were investigated in relation to DNA synthesis in rat submandibular glands after injection of isoproterenol (IPR). The level of phosphorylation of nuclear proteins in the tissue increased rapidly after birth, reaching a maximum at 4 weeks, and then decreased to the level of 52-week-old rats. The level of protein kinase activity in nuclei varied in parallel with that of phosphorylation of nuclear non-histone proteins after birth. The time after the injection of IPR required to initiate the phosphorylation of nuclear non-histone proteins of rat submandibular glands, which occurs prior to the onset of RNA synthesis that precedes the replication of DNA, increased with age. These results suggest that this delayed onset of phosphorylation plays a regulatory role in cell proliferation and cell function during aging.

Aging↗

PubMed1989

Effect of protein malnutrition on the composition of submandibular glands of aged rats.

Fifty 22-month-old male Sprague-Dawley rats were fed for 8 weeks ad libitum with either a 20% protein diet (control) or a 5% protein diet (protein malnourished group). Submandibular glands were removed, weighed and homogenate prepared. After the low protein diet, there was a slight but significant decrease in body weight, glandular content of total protein (mg/g tissue), kallikrein activity/mg protein and glutamine-rich peptide. In contrast, the gland weight, DNA and mucin content were not changed by feeding a low protein diet. Thus in aged rats submandibular glands are only modestly affected by protein malnutrition and the effects on gland-specific proteins vary.

Aging↗

PubMed1993

Giant mitochondria in the seromucous secretory cells of the accessory submandibular gland of the long-haired fruit bat, Stenonycteris lanosus.

Giant mitochondria, measuring up to 6.4 microns in diameter, are present in the seromucous secretory cells of the accessory submandibular gland of the long-haired fruit bat, Stenonycteris lanosus. These mitochondria, as well as all of the smaller ones in the same cells, contain in their matrix compartment an abundance of 33 nm threads that probably consist of protein. Some mitochondria, regardless of size, contain 5 nm helical filaments within an expanded crista. Despite their altered morphology, the enlarged mitochondria in the accessory submandibular gland of S. lanosus must be able to function normally in energy metabolism, since the secretory cells in which they are found elaborate numerous secretory granules.

Animals↗

PubMed2006

Effects of low-intensity focused ultrasound on the mouse submandibular gland.

Ultrasound is expected to make a considerable contribution to drug delivery systems (DDSs). We tested the hypothesis that low-intensity focused ultrasound (LIFU) increases vessel permeability in the mouse submandibular gland without causing parenchymal damage. In a preliminary study, LIFU at 3 W/cm2 with a 50% duty cycle for 2 minutes did not cause histologic damage. We therefore applied LIFU to mouse submandibular gland at these conditions before and after injecting horseradish peroxidase. Single labeling laser scanning confocal microscopy revealed positive horseradish peroxidase staining around the excretory ducts in the mucous-producing part of the gland, but absence of staining in control glands. Immunostaining for fibrinogen was positive in the same region. Fibrinogen is an intravascular protein that does not pass through intact vessels. These findings suggest that LIFU increases vessel permeability and disruption without destruction. It is anticipated that this process will be useful in establishing a DDS that uses LIFU.

Animals↗

PubMed1979

Denervation-induced changes in alpha and beta adrenergic receptors of the rat submandibular gland.

Denervation resulted in a marked increase in the beta adrenergic response and isoproterenol-stimulated cyclic adenosine 3':5'-monophosphate accumulation of dispersed cells prepared from a rat submandibular gland. This increase in beta adrenergic response was paralleled by an increase in the density of beta adrenergic receptors in membranes prepared from these glands. Denervation also produced a moderate increase in alpha adrenergic receptor density in membranes prepared from whole glands. However, the alpha adrenergic response in cells, epinephrine-induced release of potassium, appeared unaltered by denervation. Furthermore, membranes prepared from denervated dispersed cells did not show the increase in alpha adrenergic receptor density seen in membranes from an intact denervated gland. These data demonstrate that removal of noradrenergic nerve terminals affects alpha and beta adrenergic receptors differently. While it is clear that beta adrenergic membrane receptors participate in denervation-induced supersensitivity, the changes in alpha adrenergic membrane receptors are more complex and may not contribute to the supersensitivity seen after denervation. On the basis of competitive binding studies, the adrenergic receptors in membranes from intact submandibular glands were subclassified as beta-1 and alpha-2. Denervation did not alter the binding characteristics of the alpha-2 receptors in this gland, demonstrating that alpha-2 adrenergic membrane receptors can be postsynaptic in this adrenergically innervated tissue.

Animals↗

PubMed1990

Three-dimensional localization of DNA synthesis in secretory elements of adult female mouse submandibular gland.

A system based in part on three-dimensional structural relationships is described for precisely characterizing the location of cells within secretory complexes of the adult female mouse submandibular gland. The pattern of DNA synthesis during a 90-minute pulse with 3H-thymidine was characterized based upon the above system. Seventy-eight percent of all radiolabeled nuclei were found in the intercalated duct system. One-half of these were in second-order intercalated ducts. DNA synthesis was also observed in acinar cells, granular intercalated duct cells, striated granular duct cells, and granular duct cells. Some secretory complexes contained multiple radiolabeled nuclei, with some of these nuclei in a side-by-side configuration. Approximately one-half of all secretory complexes contained radiolabeled nuclei. A second survey of the frequency of complexes containing radiolabeled nuclei was conducted following four pulses at eight-hour intervals over a 26-hour period. Only about 30% of all complexes contained radiolabeled nuclei. This reduction in the frequency of radiolabeled nuclei when compared with the single pulse suggests the possibility of individual variation. However, a more prolonged period of daily injections for nine days with 3H-thymidine resulted in all but one of the secretory complexes containing radiolabeled nuclei. This latter observation suggests that cell addition in adult submandibular glands is widespread.

Animals↗