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Purification and characterization of an inducible cysteine proteinase inhibitor from submandibular glands of isoproterenol-treated rats.

A low-molecular-weight protein, induced in rats following prolonged isoproterenol treatment, has been purified from rat submandibular glands by chromatography on columns of Sepharose CL-6B, DEAE-Sepharose CL-6B, and Sephadex G-75. The purified protein is homogeneous based on gel electrophoresis and Ouchterlony double diffusion. The molecular weight of the purified protein was 14,000 and 15,500 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration on a Superose 12 column, respectively. This protein contains 31% glutamic acid/glutamine and aspartic acid/asparagine, 3.6% cysteine, and 2.5% proline. This protein is shown to be an inhibitor of several cysteine proteinases, papain and ficin being inhibited very strongly in approximately 1:1 molar ratio of enzyme to inhibitor. The protein is not detected in normal rat tissues but is induced in submandibular and sublingual glands even after 1 day of isoproterenol treatment of rats as early as 7 days after birth. Based on cysteine proteinase inhibitor activity, molecular size, and chemical composition this protein appears to belong to the cystatin superfamily.

Amino Acids↗

L-arginine/nitric oxide pathway and interaction with lead acetate on rat submandibular gland function.

The effects of lead acetate, L-arginine (nitric oxide precursor) and L-NAME (nitric oxide synthesis inhibitor) on rat submandibular secretory function were studied. Pure submandibular saliva was collected intraorally from anaesthetized rats by a micro polyethylene cannula using pilocarpine as secretagogue. Treatment for twenty-eight days with three doses of lead acetate (0.01%, 0.04%, 0.05% w/v) in drinking water caused significant alterations on salivary function. Salivary flow rate was decreased by lead at all doses used. The total protein concentration and amylase activity of saliva were both decreased by lead (0.04% and 0.05%). All doses of lead decreased saliva calcium concentrations. Two weeks' treatment of rats by L-arginine (2.25% w/v) and L-NAME (0.7% w/v) in drinking water also affected the saliva secretory function. L-Arginine caused increase in submandibular gland weight. The saliva flow rate was reduced by L-NAME. The total protein concentration of saliva was increased by L-arginine and decreased by L-NAME. Amylase activity was reduced by L-arginine treatment. Calcium concentration was reduced by L-arginine and increased by L-NAME. Concurrent L-arginine treatment with lead acetate recovered lead-induced reduction of flow rate but L-NAME potentiated it. Concurrent therapy of lead and L-NAME resulted in greater reduction of protein concentration when compared to that of lead. L-Arginine showed a preventive effect on lead-induced decrease of protein concentration. Both L-arginine and L-NAME prevented lead-induced reduction in calcium concentration. It is concluded that nitric oxide plays a role in salivary gland function. Also lead acetate inhibitory effect on submandibular function is somewhat diminished by L-arginine and partially increased by L-NAME. It seems that lead acetate interacts with nitric oxide modulatory role in salivary gland.

Amylases↗

Submandibular gland mucocele of the extravasation type. Report of two cases.

The clinical and radiological differential diagnosis of cystic lesions of the head and neck region can be difficult. We report two cases of submandibular gland mucoceles presenting as cystic masses in the submandibular region. The imaging characteristics and differential diagnosis are discussed.

Adolescent↗

Delayed gastric ulcer healing after extirpation of submandibular glands is sex-dependent.

This study examines the effect of excision of the submandibular salivary glands, the main source of epidermal growth factor (EGF), and the role of gender on the healing of acetic acid-induced gastric ulcers in rats. In male rats excision of the submandibular glands delayed ulcer healing. At 15 and 25 days the unhealed ulcer areas were significantly larger in the sialoadenectomy group than in control animals, and fewer completely healed ulcers were seen in this group at 25 days. Ulcer healing in female rats was slower. At day 25 ulcers were healed in 12% of female rats with intact salivary glands, compared with 68% in males. Female rats also showed larger unhealed ulcer areas after sialoadenectomy than controls. We conclude that removal of the main source of EGF in the gastrointestinal tract is associated with a delay in healing of gastric ulcers. The significant difference in healing observed between female and male rats may be influenced by the known androgenic regulation of EGF production in the salivary glands.

Animals↗

Histopathological study of the human submandibular gland in graft versus host disease.

Major salivary gland dysfunction and severe xerostomia is one of the manifestations of graft versus host disease (GVHD). The histopathological evaluation of the major salivary gland in patients with GVHD has never been reported. The pathological findings of the submandibular glands in a GVHD patient who succumbed to the disease are described. Lymphocytic infiltration, parenchymal destruction, and fibrosis were observed, which may provide the pathophysiological mechanism for the xerostomia and hyposalivation observed in GVHD.

Adult↗

Osmotically inactive space during hyperosmotic stress in the perfused submandibular gland of the rat.

Cell volume changes were measured by an impedance method during hyperosmotic stress in the perfused rat submandibular gland. When the perfusate osmolarity was raised to 484 mosmol, the cell volume decreased and remained at a plateau level (79%). The decrease was smaller than expected from the extracellular fluid osmolarity change. Furthermore, the potassium content of the gland increased by 7% during the hyperosmotic stress. These results suggest that the cell volume changes observed during hyperosmotic stress are affected by the existence of an osmotically inactive space and by an increase in the intracellular solute content.

Animals↗

Characterization and localization of two forms of active Ca2+ transport in vesicles derived from rat submandibular glands.

Energy-dependent Ca2+ uptake was characterized in vesicles derived from rat submandibular salivary glands. Ca2+ transport was stimulated by submicromolar levels of Ca2+, reached a plateau at 1-20 microM Ca2+ then again increased as the Ca2+ concentration rose to millimolar levels. Ruthenium red (2.5 microM) was used to resolve this pattern of uptake into two components: ruthenium red-insensitive Ca2+ transport occurs in the presence of the dye, is stimulated by submicromolar Ca2+ concentrations and reaches a maximum steady state at about 1 microM Ca2+. The distribution of ruthenium red-insensitive Ca2+ uptake in membrane subfractions obtained by differential centrifugation is positively (r = 0.717) and significantly (p = 0.001) correlated with the distribution of membrane-bound RNA in the same subfractions. Ca2+ uptake which is abolished by ruthenium red is greatest at millimolar Ca2+ concentrations. Its distribution is positively (r = 0.828) and significantly (p = 0.0001) correlated with the cytochrome-c oxidase activity of the membrane subfractions but is unrelated to the distribution of particulate RNA and is negatively correlated with Na+-K+ ATPase activity. We conclude that vesicles derived from the endoplasmic reticulum of rat submandibular glands actively transport Ca2+ by a ruthenium red-insensitive mechanism which is stimulated at Ca2+ concentrations typical of the cytosol. Membranes derived from mitochondria also sequester Ca2+ but by a mechanism which is inhibited by ruthenium red and which reaches its maximum steady state capacity at relatively high Ca2+ concentrations.

Adenosine Triphosphate↗

Immunochemical analysis of laminin during postnatal development of the rat submandibular gland.

Anti-laminin serum was used to investigate distribution patterns and chain composition of laminin during postnatal development of rat submandibular gland. Immunohistochemical analysis showed that in glands of newborn rats laminin was not uniformly present around growing acini. Staining was frequently weak or absent in clefts formed between adjacent cells. This irregular staining pattern decreased progressively over the subsequent periods, and in 30-day-old animals immunoreactivity was observed only at the periphery of glands. Immunoblot analysis showed that laminin was composed of bands corresponding to the alpha 1, beta 1 and gamma 1 polypeptides. The correlation between the pattern of laminin expression and gland maturation suggests a role of laminin in the functional maturation of acinar cells.

Age Factors↗

The prevalence of consolidated salivary deposits in the small ducts of human submandibular glands.

Deposits of apparently solid material were observed in the small salivary ducts in a series of non-diseased submandibular glands obtained from necropsies evenly divided by sex and adult age. The deposits were of granular or laminated structure. They were of mixed staining, the majority being predominantly eosinophilic. A small number of deposits were essentially wholly hematoxylinophilic and some of these were probably calcified. The prevalence of deposits, expressed as the number per unit sectional area, increased steadily with increasing age but was unrelated to sex. An apparently close correlation between intraductal salivary deposits and foci of obstructive adenitis in the same series of glands was shown to be mainly due to the common influence of age. The deposits probably form by precipitation of salivary glycoprotein, possibly because of parenchymal age changes. They may be related to early salivary lithogenesis but the statistical evidence of this study suggests that they are unlikely to be a major factor in the etiology of focal obstructive adenitis.

Adolescent↗

Relation between lingual nerve, submandibular gland duct and mandibular body in the sublingual space.

It was shown that the crossing of the duct of the submandibular gland and of the lingual nerve appeared constantly in the close vicinity of the upper surface of the posterior portion of the mylohyoid muscle closely to the inner surface of the mandible within the range of the third permanent molar. In the lateral view the nerve/duct crossing is projected on the external mandibular surface distally from the retrotrigonal triangle into the rounded region where the mandibular body and mandibular branch are fused.

Humans↗

Purification and properties of guinea-pig submandibular-gland kallikrein.

Guinea-pig submandibular kallikrein has been purified from the glands to electrophoretic homogeneity by conventional procedures. The enzyme is active as a kininogenase, releasing kallidin at a rate of 462 micrograms/min per mg of protein from bovine kininogen, and proved potently hypotensive in the guinea pig and in the dog, properties which indicate its tissue kallikrein nature. The specific activity determined on the substrate N-alpha-benzoyl-L-arginine ethyl ester (11.1 mumol/min per mg of protein) is much lower than that measured with N-acetyl-L-phenylalanyl-L-arginine ethyl ester (483 mumol/min per mg of protein). The latter value is of an order of magnitude comparable with the specific activities of other tissue kallikreins determined with this sensitive kallikrein substrate. The enzyme is a glycoprotein consisting of 237 amino acid residues and containing three to four glucosamine molecules. Its amino acid composition is not identical with that reported for guinea-pig coagulating-gland kallikrein, but is remarkably similar to that of the porcine tissue kallikreins. Apparent Mr values are 29000 (sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) or 34000 (gel filtration). The amino acid sequence of the first 31 N-terminal residues was determined and was found to be closely homologous with that of other tissue kallikreins.

Amino Acid Sequence↗

Mechanism of isoproterenol-induced heterologous desensitization of mucin secretion from rat submandibular glands. Regulation of phosphorylation of Gi proteins controls the cell response to the subsequent stimulation.

Short-term treatment of rat submandibular tissues with 10 microM isoproterenol (IPR) resulted in reduction of mucin secretion in response to the agonist during further incubation, and in increases in EC50 values. This IPR-induced reduction of secretion was coupled with selective decreases in the number of beta-adrenoceptors in the tissues and in their affinity for agonists, as assessed by measurement of the specific binding of [3H]dihydroalprenolol. Treatment of the tissues with IPR caused a 30% decrease in IPR-stimulated adenylate cyclase activity and a 25% increase in the GTP binding capacity of inhibitory G proteins (Gi proteins). This IPR treatment triggered a 60% increase in the ability of pertussis toxin (IAP) to catalyze ADP-ribosylation of Gi proteins in the tissue membranes. Enhanced function of stimulatory G proteins (Gs proteins) was observed only during the first incubation of the tissues with IPR. The IAP-catalyzed ADP-ribosylation of Gi proteins in tissues treated with IPR was decreased by prior treatment with cyclic AMP dependent protein kinase, but was increased markedly by prior treatment with alkaline phosphatase. Neither IPR-induced desensitization of protein secretion nor increase in the IAP-catalyzed ADP-ribosylation of Gi proteins was observed in the tissues pretreated with 0.25 microM okadaic acid. These findings suggest that the regulation of Gi protein phosphorylation plays an important role in the IPR-induced heterologous desensitization of mucin secretion from rat submandibular glands.

Adrenergic beta-Agonists↗

Supporting effects of myoepithelial cells in submandibular glands of dogs when acting against increased intraluminal pressure.

1. In dogs under chloralose-urethane anaesthesia secretion from the submandibular gland was recorded with the outflow at gland level and at heights of up to 50 cm above the gland.2. With the outflow level increased, secretion elicited by sympathetic nerve stimulation was far better maintained before than after injection of the alpha-adrenoceptor blocking agent dihydroergotamine.3. When the outflow level was raised while no secretion occurred, fluid flowed into the gland. Part of this amount was returned on lowering the outflow to gland level. This volume was assumed to have been accommodated in the distended luminal system, whereas some fluid was obviously lost into the gland tissues.4. Both these fractions of the fluid flowing into the gland when the outflow level was high could be reduced by injecting the alpha-receptor agonist phenylephrine. Bradykinin, which like phenylephrine is known to contract salivary myoepithelial cells, had the same effect on the two inflow volumes.5. It is concluded that myoepithelial contraction reduced the distensibility of the luminal system and in addition supported the acini, thereby diminishing backflow into the glandular tissues and enabling the gland to discharge saliva against a high pressure.6. Morphologically it was found that in resting glands PAS-positive saliva was displaced from the ductal system when the outflow cannula was raised, but it was preserved in the lumina under similar conditions when the myoepithelial cells were being stimulated by phenylephrine or bradykinin.7. Although sympathetic secretion could be maintained against a head of pressure, so long as it was accompanied by myoepithelial contraction, the increased force caused by the secretion led to disruptive damage of striated ducts which are the first part of the luminal system not supported by myoepithelial cells.8. The morphological findings reinforce the belief that contraction of myoepithelial cells gives active support to the underlying parenchyma.

Animals↗

Crystalloids in the excretory ducts of the accessory submandibular gland of the long-winged bat, Miniopterus magnator.

Cytoplasmic crystalloids are abundant in the excretory ducts of the accessory submandibular gland of the long-winged bat, Miniopterus magnator. The crystalloids, which always lack a membranous enclosure, may have an intricate silhouette. They consist of parallel linear densities with a 12.5 nm periodicity. These densities actually may be thin-walled tubules. In some crystalloids, intersecting subcrystalloids produce a complex pattern of decussate densities. In a few rare instances, continuities were detected between a crystalloid and a smooth-surfaced cisternal element. In other mammalian species, similar crystalloids connected to smooth endoplasmic reticulum play a role in steroid metabolism. We postulate that the ductular crystalloids in M. magnator might be involved in production of a factor that influences mating behavior.

Animals↗

Binding protein for 5 alpha-dihydrotestosterone in mouse submandibular gland.

The changes in the levels of the binding protein for 5 alpha-dihydrotestosterone in cytoplasmic extract of the submandibular glands during development were compared in male and female mice using a DEAE-cellulose filter assay. The binding protein was first detectable 5 days after birth in both sexes, at a time coincident with androgen-independent cytodifferentiation of the convoluted tubular cells in the submadibular gland. The level of the binding protein in female mice was maintained at 5 pmol/mg protein after birth, whereas in males it began to decrease from 3 weeks after birth with inccrease in serum testosterone, becoming much less than a quarter of the level in females or immature mice by 4 weeks after birth. However, after castration, the level of detectable binding protein in mature male mice increased within 7 days to the same level as that in females or immature mice. This suggests that the low binding capacity for exogenous hormone in mature male mice is due to occupancy of the binding sites by endogenous hormone.

Age Factors↗

Homeobox protein, Hmx3, in postnatally developing rat submandibular glands.

Homeobox-containing (Hox) genes play important roles in development, particularly in the development of neurons and sensory organs, and in specification of body plan. The Hmx gene family is a new class of homeobox-containing genes defined by a conserved homeobox region and a characteristic pattern of expression in the central nervous system that is more rostral than that of the Hox genes. To date, three closely related members of the Hmx family, Hmx1, Hmx2, and Hmx3, have been described. All three Hmx genes are expressed in the craniofacial region of developing embryos. Here we show, for the first time, the expression of the transcription factor Hmx3 in postnatally developing salivary glands. Hmx3 protein is expressed in a cell type-specific manner in rat salivary glands. Hmx3 is present in both the nuclei and cytoplasm of specific groups of duct cells of the submandibular, parotid, and sublingual glands. Hmx3 expression increases during postnatal development of the submandibular gland. The duct cells show increasing concentrations of Hmx3 protein with progressive development of the submandibular gland. In contrast, the acinar cells of the three salivary glands do not exhibit detectable levels of Hmx3 protein.

Animals↗

Biosynthesis of a low-molecular-mass rat submandibular gland mucin glycoprotein in COS7 cells.

We have examined the biosynthesis of a low-molecular-mass mucin from rat submandibular gland (RSMG) expressed recombinantly in COS7 tissue culture cells, focusing primarily on the addition of carbohydrate to the protein core of the mucin. We find evidence for N-linked glycosylation, but this modification is not required for secretion of the mucin. Similarly, although the recombinant RSMG mucin, like its native counterpart, contains large amounts of O-linked carbohydrate, chain extension beyond the initial O-linked GalNAc moiety is not required for secretion. We have identified partially glycosylated mucin by a combination of metabolic pulse-chase and lectin precipitations of the biosynthetic intermediates. Our results suggest that the addition of GalNAc to threonine and serine in the RSMG mucin does not occur simultaneously, as has been described for other O-glycosylated proteins.

Acetylgalactosamine↗