Development of the rat salivary glands. II. The identification of a trypsinlike protease in the submaxillary gland

Trypsinlike protease activity at pH 9.2 was measured in tissue extracts of adult rat salivary glands by using a fluorometric assay in which β-naphthylamine is released by the hydrolysis of benzylarginine β-naphthylamide. The submaxillary gland contains high levels of this activity, and the parotid and sublingual glands have a maximum of 2000-fold and 200-fold less. After polyacrylamide disc gel electrophoresis at pH 8.3, the protease activity of submaxillary extracts is associated with a major protein band. Neither this protein band nor its protease activity is detectable in extracts of parotid or sublingual glands. Homogenates of newborn submaxillary gland do not have this protease activity at detectable levels, suggesting that its major accumulation is postnatal.     

Vasoactive intestinal peptide and substance P in salivary glands of the rat following denervation or duct ligation

Immunoreactive vasoactive intestinal peptide (VIP) and substance P (SP) were studied in parotid, submaxillary and sublingual glands of the rat. The concentration of VIP was highest in the submaxillary gland and lowest in the parotid gland. The concentration of SP was highest in the parotid gland; it was at, or below the limit of detection in the sublingual gland. In the parotid gland the total amounts of VIP and SP were reduced by 95% after parasympathetic denervation (section of the auriculo-temporal nerve). In the submaxillary gland the total amounts of the peptides were unchanged after parasympathetic decentralization (section of the chorda-lingual nerve). In this gland the total amount of SP was reduced by 92% and that of VIP by 50%, when the chorda tympani nerve fibres were cut deep into the hilum. Cutting the nerve fibres at the hilum left the total amounts of the peptides unchanged in the submaxillary gland, whereas in the sublingual gland the total amount of VIP was reduced by 70%. Sympathetic denervation did not reduce the total amounts of the peptides. Duct ligation caused gland atrophy. In the parotid gland the total amounts of VIP and SP were reduced by 40%. In the submaxillary gland the same percentage reduction occurred with regard to SP; however, the total amount of VIP was reduced by 99%. The VIP- and SP-containing nerve fibres reach the salivary glands by the parasympathetic nerves. In both submaxillary and sublingual glands a certain fraction of VIP originates within the glands.     

Expression and localization of immunoreactive-sialomucin complex (Muc4) in salivary glands

Sialomucin Complex (SMC; Muc4) is a heterodimeric glycoprotein consisting of two subunits, the mucin component ASGP-1 and the transmembrane subunit ASGP-2. Northern blot and immunoblot analyses demonstrated the presence of SMC/Muc4 in submaxillary, sublingual and parotid salivary glands of the rat. Immunocytochemical staining of SMC using monoclonal antisera raised against ASGP-2 and glycosylated ASGP-1 on paraffin-embedded sections of parotid, submaxillary and sublingual tissues was performed to examine the localization of the mucin in the major rat salivary glands. Histological and immunocytochemical staining of cell markers showed that the salivary glands consisted of varying numbers of serous and mucous acini which are drained by ducts. Parotid glands were composed almost entirely of serous acini, sublingual glands were mainly mucous in composition and a mixture of serous and mucous acini were present in submaxillary glands. Since immunoreactive (ir)-SMC was specifically localized to the serous cells, staining was most abundant in parotid glands, intermediate levels in submaxillary glands and least in sublingual glands. Ir-SMC in sublingual glands was localized to caps of cells around mucous acini, known as serous demilunes, which are also present in submaxillary glands. Immunocytochemical staining of SMC in human parotid glands was localized to epithelial cells of serous acini and ducts. However, the staining pattern of epithelial cells was heterogeneous, with ir-SMC present in some acinar and ductal epithelial cells but not in others. This report provides a map of normal ir-SMC/Muc4 distribution in parotid, submaxillary and sublingual glands which can be used for the study of SMC/Muc4 expression in salivary gland tumors.     

Developmental changes in the activities of prolinase and prolidase in rat salivary glands,and the effect of thyroxine administration

Summary As the salivary glands are interesting tissues to study proliferation, we studied the activities of prolinase and prolidase using Pro-Ala and Pro-Hyp as substrates, respectively, in developing rat salivary glands between day 1 and week 10 after birth. Developmental changes of prolinase activity in the submandibular and sublingual glands were similar to those in the parotid gland, which steadily increased and reached the adult level by 20–25 days after birth. However, the changes in the activity of prolidase in the submandibular and sublingual glands were different from those in the parotid gland: the activity in the parotid gland slowly increased with maturation and reached a maximum level on day 30, but the activity in the submandibular and sublingual glands continuously increased with maturation. When thyroxine was injected every two days from day 1 to day 19, both enzyme activities were induced precociously in the parotid gland but not in the submandibular and sublingual glands. On the study of regional distribution in rat tissues, the correlation coefficient between prolinase and prolidase activities was high in the peripheral but not high in the brain regions.These results indicate that the physiological roles of prolinase and prolidase are very similar but not the same.     

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     

Comparative developmental analysis of the parotid, submandibular and sublingual glands in the neonatal rat.

Analysis of the soluble protein fractions from the rat parotid, submandibular and sublingual glands by polyacrylamide-gel electrophoresis reveals similarities in overall patterns of protein synthesis at birth. Tissue-specific changes in protein and glycoprotein synthesis occur shortly after birth and again at the time of weaning, 21--28 days later. Incorporation of [3H]thymidine into DNA was at its highest after birth and gradually decreased in both the parotid and submandibular gland, whereas [3H]thymidine incorporation in the sublingual gland was low throughout the time of neonatal development. [14C]Leucine incorporation into total protein increased in all glands with age after birth, showing an accelerated rate 21--28 days later. Trichloroacetic acid/phosphotungstic acid-precipitable [3H]fucose in glycoproteins declined over the time of neonatal development in the parotid and submandibular gland, but its incorporation remained higher in the sublingual gland. alpha-Amylase (EC 3.2.1.1) in the salivary glands increased at the time of weaning, as judged by detectability in sodium dodecyl sulphate/polyacrylamide gels and by immune precipitation. Two membrane-bound enzymes, UDP-galactose:2-acetamido-2-deoxy-D-glucosamine 4 beta-galactosyltransferase (EC 2.4.1.22) and UDP-galactose:2-acetamido-2-deoxy-D-galactosaminyl-protein 3 beta-galactosyltransferase (no EC number), undergo tissue-specific change rather than changes induced by physiological stimulation of the salivary glands.     

Effect of Neural Stimulation on Acinar Cell Membrane Potentials in Isolated Pancreas and Salivary Gland Segments

The effect of cholinergic neural excitation by field stimulation on the acinar cell membrane potential was investigated in superfused segments of mouse pancreas and salivary glands (sublingual, submaxillary, and parotid glands).

Responses of acinar cells in both exocrine pancreas and salivary glands to the neural excitation obtained by field stimulation were similar to responses previously described in each gland to the externally applied acetylcholine.

In the pancreatic acinar cell, electrical field stimulation induced depolarization with a latency of 0.3 to 1.2 sec. This depolarization was accompanied by a marked decrease in membrane resistance. The equilibrium potential of the depolarization induced by stimulation was between -10 and -20 mV. In the sublingual gland, field stimulation induced depolarization of the acinar cell with a latency of 0.2 to 0.3 sec. The stimulus induced depolarization was blocked by the addition of atropine. In the submaxillary and parotid glands, field stimulation induced depolarization in some acinar cell and hyper-polarization in other cells.

The results support evidence previously presented by Petersen and his colleagues that acetylcholine acts to increase Na⁺ and K⁺ or Na⁺, K⁺, and Cl^- permeabilities in the pancreatic acinar cell and to increase K⁺ and Na⁺ permeabilities in the salivary gland [11,24].     

Acinar cell response to liquid diet during rats’ growth period differs in submandibular and sublingual glands from that in parotid glands

Continuously feeding a liquid diet to growing rodents strongly inhibits parotid gland growth, due to suppressed growth of acinar cells. This study investigated whether a liquid diet had a similar effect on submandibular and sublingual glands of growing rats. Rats were weaned on day 21 after birth and then fed a pellet diet in the control group and a liquid diet in the experimental group for 0, 1, 2, 4, and 8 weeks. Their submandibular and sublingual glands were excised, weighed, and examined histologically, immunohistochemically (using antibodies to 5′-bromo-2-deoxyuridine and cleaved caspase 3), and ultrastructurally. The submandibular glands did not significantly differ between the control and experimental groups at all tested points. Only at Week 8, acinar cell area and 5′-bromo-2-deoxyuridine-labeling index of acinar cells in sublingual glands were significantly lower in the experimental group than in the control group. These results show that a liquid diet during rats’ growth period had no effect on acinar cells in their submandibular glands, and only a slight effect on acinar cells in their sublingual glands of growing rats, in contrast to the marked effect of a liquid diet on parotid glands.     

Carbohydrate histochemistry of the opossum submandibular and major sublingual glands.

Submandibular and major sublingual salivary glands of the opossum contain histochemically demonstrable neutral mucosubstances, nonsulfated acid musosubstances and sulfomucins. Sialomucins could not be demonstrated conclusively with the methods used in this study. Special serous cells of the opossum submandibular gland contained low concentrations of acidic mucosubstances but no appreciable concentration of neutral mucosubstances was seen. Sulfomucins were not observed in special serous cells. The mucous tubules of the submandibular gland contained high concentrations of neutral mucosubstances. No appreciable acidic mucosubstance was demonstrated in the submandibular gland mucous tubules. Unlike the mucous tubules of the submandibular gland, the major sublingual gland mucous tubules contained high concentrations of both neutral and acidic mucosubstances. The mucous tubules often contained sulfomucin-positive cells interspersed among cells that contained high concentrations of non-sulfated acidic mucosubstance. Marked staining of sulfated acidic mucosubstance was seen only in the major sublingual gland, in both the mucous tubules and in the seromucous demilunes. The seromucous demilunes contained both sulfated and non-sulfated acidic mucosubstances.     

Histology and histochemistry of the parotid and the principal and accessory submandibular glands of the little brown bat

The parotid and the principal and accessory submandibular glands of the little brown bat. Myotis lucifugus (Vespertilionidae), were examined using light microscopy and staining methods for mucosubstances. The parotid gland is a compound tubuloacinar seromucous gland. Parotid gland secretory cells contain both neutral and nonsulfated acidic mucosubstances. The principal and accessory submandibular glands are compound tubuloacinar mucus-secreting glands. They contain somewhat atypical mucus-secreting demilunar cells that often appear to be interspersed between mucous tubule cells. The mucous tubule cells in both the principal and accessory submandibular glands contain sulfonmucins. Demilunar cells of the principal submandibular gland contain moderate amounts of nonsulfated acidic mucosubstances, but the corresponding cells of the accessory submandibular gland contain considerable neutral mucosubstance with very little acid mucosubstance. Intercalated ducts composed of cuboidal or low columnar epithelial cells are present in all three glands. Striated ducts in all glands are composed of columnar cells whose apices bulge into the ductal lumina. Excretory ducts are composed of simple columnar epithelium, with occasional basal cells that suggest a possible pseudostratified nature. The cells of the excretory ducts also have bulging apices. All duct types contain apical cytoplasmic secretory material that is a periodic acid-Schiff positive, neutral mucosubstance. Ductal apical secretory material is more evident in intercalated and striated ducts than in excretory ducts.     

Characterization of mouse salivary glands by water content and type

The thermoanalytical analysis was applied to samples of sublingual, submandibular and parotid glands from sexually mature mice of both sexes. Findings indicated that the three salivary glands show a behaviour of water release characteristic for each type of gland. Derivative thermogravimetry curves concerned with the sublingual and parotid glands belonging to male and female subjects exhibited overlapped results. As regards submandibular gland, instead, some differences emerged between subjects of different sex. Water content and types in sublingual, submandibular and parotid glands were discussed and related to the different morphological expression, histochemical reactivity and chemical composition of these organ tissues.     

Effects of sialoadenectomy and castration in female rats

Previous work on the role of the submaxillary glands in the control of the oestrous cycle in rats has been extended to castrated rats in order to avoid the overlapping between sexual and salivary hormones. Animals were sacrificed 30 days after sialadectomy or pseudosialadectomy. The data show that simultaneous castration and sialadectomy increases significantly the glucaemia level and decreases the weight of the adrenal glands. Non-simultaneous castration and removal of the submaxillary glands decreases the weight of the parotid glands. This effect decreases when both actions are simultaneous. On the other hand, castration produces an important decrease in QO2 uptake in tested structures. Removal of submaxillary glands produces a significant increase of QO2 in hypothalamus and thyroid glands. Simultaneous castration and sialadectomy at the anterior cortex, posterior cortex and parotid gland level shows similar results with respect to desalivated rats; other structures show results similar to the castrated group values. From these results, the role played by submaxillary glands in the control of the sexual cycle of the rat and the possible relation to other structures is discussed.     

Characterization of primary translation products from ovine and rat salivary gland mRNAs

Ovine and rat salivary gland mRNAs have been prepared and their translation products characterized. A 60 kD translation product from ovine submaxillary and sublingual gland mRNAs is identical in mass to the ovine apomucin. Two additional ovine translation products, 25 and 40 kD, are specific to mucin-producing salivary glands. Four rat mRNA translation products are encoded by mucin-producing salivary glands (38, 44, 67, 69 kD). These polypeptides were not detected in the parotid gland mRNAs, a serous gland. Each of these products has a high level of [3H]serine incorporation, a characteristic of mucins. The nature of these products suggests that they are mucins or mucin-like and that their molecular weights should approximate that of the corresponding apomucins.     

Expression and distribution of osteopontin and matrix metalloproteinase (MMP)-3 and -7 in mouse salivary glands

We investigated the expression and distribution of osteopontin in mouse salivary glands. Western blot analysis showed intense positive bands at the predicted molecular mass (about 60 kDa) in mouse parotid and sublingual glands. However, a cross-reacted band around 30 kDa was strongly detected in submandibular glands. Indirect immunofluorescent analysis showed that osteopontin was localized at the luminal (apical) membranes of the acinar cells in parotid and sublingual glands. However, it was not detected in acinar cells of submandibular glands. No expression was found in ductal cells of any glands. We also examined the expression of matrix metalloproteinase (MMP)-3 and -7. In parotid gland, MMP-3 was observed at 57 kDa, indicating a latent form, but MMP-7 was not detected. In contrast, MMP-7 definitely was observed at 28 kDa area in submandibular gland, whereas MMP-3 was not detected. These results suggest that osteopontin localizes at luminal sites of acinar cells and may be associated with saliva secretion in mouse salivary gland. It is also suggested that osteopontin may be cleaved by MMP-7 in mouse submandibular gland.     

Microstereological and histochemical studies of the salivary glands of the giant rat (Cricetomys gambianus, waterhouse)

The histology and histochemistry of the parotid, submandibular and sublingual glands were studied. The submandibular gland contained only serous acini as in the guinea pig, but unlike in many other mammals. The parotid gland contained only serous acini while the sublingual gland was mixed, mucous acini being the predominant secretory tissue interspersed by a few serous acini. Serous demilunes also commonly formed caps on the mucous acini. The ducts of the gland contributed over 30% of the volume of the submandibular gland, while those of the parotid and sublingual glands formed about 12 and 10% of the gland, respectively. The secretions of the parotid gland, as judged by histochemical methods, contained neutral mucins and some sialomucins. Neutral mucins, sulphomucins and sialomucins were detected in both the submandibular gland and sublingual gland.     

Adrenergic innervation of the salivary glands in the rat

Summary The adrenergic innervation of the major salivary glands in the rat has been studied by a specific histochemical method for the visualization of the adrenergic transmitter. Adrenergic varicose nerve fibres were found, located in a typical adrenergic ground plexus closely surrounding the serous acini of the submaxillary and parotid glands, but not the acini of the mainly mucous sublingual gland. The ducts were found to be completely devoid of adrenergic innervation. Arterioles and venules in the stroma of all three glands and certain very small vessels, possibly the sphincters of arterio-venous anastomoses, were also richly innervated by adrenergic vasomotor fibres. The relationship of the adrenergic nerve fibres to the different functional units of the gland parenchyma is discussed.The investigation has been supported by a research grant (B 66–257) from the Swedish Medical Research Council and by a Public Health Service Research Grant (NB 05236-01) from the National Institute of Neurological Diseases and Blindness.     

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

In untreated, fasting animals the cells of the serous demilunes of the sublingual gland incorporate [3H]-leucine at a higher rate than any other of the 5 main cell types of the 3 major salivary glands. The acinar cells of the submandibular and the mucous cells of the sublingual gland show intermediate values, while the cells of the granular ducts of the submandibular and the acini of the parotid gland have a low rate of incorporation. In fasting animals extrusion of newly synthesized protein starts early in the cells of the serous demilunes. It starts between 4 and 7 hrs after [3H]-leucine injection in the acinar cells of the submandibular gland, while the other cell types did not lose substantial amounts of labelled (glyco)protein within 7 hrs. The secretion of protein is stimulated by the cholinergic drug pilocarpine in all but one of the 5 types of salivary gland cells studied. The acinar cells of the submandibular gland react strongly, the granular duct cells less strongly. Still less are the reactions of the acinar cells of the parotid and of the nucous cells of the sublingual gland. The cells of the serous demilunes of the latter appear to be insensible to pilocarpine. The effect of food uptake on secretion does not differ from pilocarpine stimulation, with one exception: the acinar cells of the parotid gland react more strongly on food uptake than on cholenergic stimulation.