Differentiation of rat salivary glands by thermoanalytical analysis.

The water release from the sublingual, parotid and submandibular glands of male and female rats was analyzed by thermal analysis in order to detect the total water content and types. Different types of water, which are increasing from the sublingual to the parotid gland, were found and the relative distribution appeared to be a function of the bond energy of water to glandular components. In addition, evidence of a sexual dimorphism in the rat sublingual gland was demonstrated.

The parotid gland is the main source of human salivary epidermal growth factor.

To clarify the production of human epidermal growth factor (EGF) by different salivary glands, we measured its concentration by radioimmunoassay separately in whole saliva, in parotid gland (PG) saliva and in mixed submandibular (SMG) and sublingual gland (SLG) saliva. Also, we studied the presence of EGF in PG and SMG by immunohistochemistry. The mean (geometric) concentrations of EGF in PG saliva (2704 pg/ml, +/- SEM interval 2393-3056 pg/ml, n = 20) was higher (p less than 0.001) than in whole saliva (864 pg/ml, +/- 733-1019 pg/ml, n = 29), which in turn was higher (p less than 0.001) than in mixed SMG + SLG saliva (357 pg/ml, +/- 296-430 pg/ml, n = 16). No sex difference existed in any salivary gland EGF. Immunohistochemistry revealed EGF in the acinar cells of both PG and SMG, but only in PG there were prominent EGF deposits in luminal spaces. Our data suggest that EGF is produced by both PG and SMG, but that more of it is secreted from the PG. This result is new and challenges the general view that human salivary EGF is mainly from SMG. In mouse almost all salivary EGF comes from SMG and its amount is androgen dependent. Thus there are great differences in sources and regulation of salivary EGF between man and mouse.

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.

Salivary glands in long-term alloxan-diabetic rats. A quantitative light and electron-microscopic study.

Fifty untreated diabetic animals were compared with 58 age-matched non-diabetic controls. Reduced salivary gland weight was evident after one month's diabetes and this was unchanged after 12 months of diabetes. Submandibular/sublingual gland weight was proportional to the reduced body weight in the diabetic rats. Parotid gland weight, however, was proportionally more reduced. Only diabetic rats had lipid inclusions in the acinar cells of their submandibular glands and the morphometrically estimated amount of inclusions was positively correlated to the blood glucose level. Acinar cell size was significantly increased in long-term diabetic rats as compared with short-term diabetic rats and controls. Capillary basement membrane width was significantly increased in long-term diabetic rats compared with age-matched controls and with short-term diabetic rats. Thus, both the degree and duration of diabetes have a major effect on salivary gland morphology in alloxan diabetic rats.

Immunocytochemical localization of insulin- and glucagonlike peptides in rat salivary glands.

An avidin-biotin immunocytochemical technique was used to localize cells containing an insulin- or glucagon-like peptide in the major salivary glands of Sprague-Dawley rats. Cells with insulin-like staining were observed in the intercalated ducts of both the parotid and submandibular glands, but none were found in the sublingual gland. A discrete population of cells with intense glucagon-like immunostaining was associated with the acini of all three major salivary glands. This immunostaining only followed use of a glucagon antiserum with N-terminal specificity and not after incubation of tissues with an anti-glucagon serum having C-terminal specificity. These results suggest that rat salivary glands may contain peptides potentially capable of influencing substrate metabolism. In addition, the present findings indicate that the glucagon-like peptide found in salivary glands has a greater immunocytochemical similarity to glicentin (gut-type glucagon) and/or glucagon precursors than to the 3500 molecular weight pancreatic glucagon.

Two-dimensional electrophoresis in the analysis of a mixture of human sublingual and submandibular salivary proteins.

Complex protein mixtures of unstimulated human sublingual and submandibular saliva were fractionated by two-dimensional (2-D) gel electrophoresis, visualized by silver staining and then analysed by immunostaining. Specific proteins were identified by incubation with specific antibody and peroxidase-conjugated second antibody (Western blot). Electrophoresis and silver staining revealed over 50 protein components in 2 microliter of unconcentrated mixture. The Western-blot technique allowed detection of protein spots of plasma origin when an antibody against whole serum was used, but only the albumin spot could be found. Albumin, secretory IgA, acid phosphatase and alpha-amylase were identified with specific antibodies.

Study on membrane fragments released from the sublingual glands of the mouse during secretion in vitro and in vivo.

During in vitro secretion membrane fragments are released by the sublingual glands (SL) of the mouse. After stimulation of saliva with 1 microM carbamylcholine, these membranes have been isolated by centrifugation at 100,000 X g for 1 h. The release of 0.56% of the total tissue content of DNA during a 3 h period denotes that the amount of damaged tissue is very low. After 3 h, based on the determination of sialic acid, 25.4% of the secretory product, the sublingual mucin, has been released. The amount of membrane-bound alkaline phosphatase, released during a period of 3 h is 0.63%. As at least three quarters of this amount is due to broken cells, the amount of membrane-bound alkaline phosphatase released during the secretory process (0.16%) is very low compared to the amount of secretory product (25.4%). The low amount of alkaline phosphatase is in accordance with EM observations, which show that secretory granule membranes lack alkaline phosphatase activity. However, at those locations, where luminal membranes fuse with the granule membranes, alkaline phosphatase has been detected. So, the low alkaline phosphatase activity may be due to the presence of some luminal membranes in the secretory product. The involvement of alkaline phosphatase in the secretory process is also indicated by the complete inhibition of the secretory process by tetramisole (5 mM). The SL membrane preparation, isolated from the incubation fluid, had a relatively simple electrophoretic pattern and some antigenic determinants in common with the granular membranes from the Par and SM glands. The phospholipid composition of the released SL membranes differed strongly from the Par and SM granule membranes, especially in their relatively low amount of sphingomyelin (6.8%) and their high amount of phosphatidylethanolamine (26.4%). The lysophosphatidylcholine was only 3.5%. Among others, the phospholipid composition of the SL membranes may be responsible for the specific properties and behaviour of the sublingual granular membranes during the secretory process.

S-100 alpha-like immunoreactivity in tubules of rat kidney. A clue to the function of a "brain-specific" protein.

The localization of the alpha and beta subunits of S-100 protein was studied in normal tissue where the identification of three subclasses of S-100 containing cells was derived: i) cells that contain both alpha and beta subunits; ii) cells that contain only the alpha subunit; and iii) cells that contain only the beta subunit. In this study monospecific antibodies against the S-100 alpha and beta subunits were used to characterize the S-100-like immunoreactivity in the rat kidney: Certain cells in the distal nephron, i.e., the connecting piece, collecting ducts, and the thin limb of Henle's loop, contained S-100 alpha immunoreactivity. Proximal tubules, the thick ascending limb of Henle's loop, the distal tubules, and the juxtaglomerular apparatus were negative. No S-100 beta immunoreactivity was found in kidney tubules. However, Schwann cells of renal pelvic nerves contained S-100 beta immunoreactivity. The presence of S-100 alpha antigen in certain cells of the kidney gives further support to the assumption that the alpha subunit of S-100a is related to cells that are highly involved in pH, electrolyte, and water regulation.

Influence of fixation on the lectin binding sites in the rabbit salivary glands.

In order to verify the influence of fixative on the formation of any nonspecific lectin bindings, the authors have carried out an investigation on rabbit salivary glands. The results obtained applying peanut, soybean, wheat germ, and winged pea lectins to unfixed samples of rabbit submandibular and sublingual glands agreed almost completely with the results of our previous research effected on the same samples fixed with aldehydes. The most important differences between the fixed samples and the unfixed ones consisted in the lack of reactivity of the material inside the secretion lumina to all the lectins used, and in the lack of peanut binding to the submandibular gland. No significant differences in intensity and location were found for soybean, wheat germ, and winged pea lectins.

Immunohistological studies on ABH-activities in secretory cells of human major salivary glands--correlation between ABH-activities in the secretory cells and secretor-nonsecretor.

The activities of A, B and H in serous cells (S-cells), mucous cells (M-cells) and excretory duct cells were examined in a large number of paraffin sections of three major salivary glands obtained from 91 corpses, using the immunofluorescence technique. The results are: By taking H activity in S-cells of the submandibular gland or A, B and H activity in M-cells of the sublingual gland as an indicator, the salivary glands were classified as Type I showing activity and Type II showing no activity. No glands corresponding to the intermediate type, as seen in the case of saliva, were noted at all. Among 91 corpses, 70 cases were classified as Type I and 21 as Type II. The results matched well with those of Lewis type tested on blood. The frequencies of the typing (Type I; 76.9%, Type II; 23.1%) were approximately in concordance with those of secretor and nonsecretor in Japanese saliva. From these results, it was assessed that the former corresponded to the secretor type in the case of saliva, and the latter to the nonsecretor type. Even in the same individual, both S-cells and M-cells exhibited different productivities of substances, depending on the glands to which they belonged. Namely, only S-cells in the submandibular gland belonging to Type I showed only H activity independent of the blood group of the individual, but the other S-cells in the other major glands did not show any activity for A, B and H. M-cells exhibited strong activity for H and/or A and/or B in the sublingual and submandibular gland and belonged to Type I, but little activity in the sublingual gland belonged to Type II. In the submandibular gland of Type II, some M-cells showed activity and others did not. On the basis of the above results, we discuss the applicability of the present genetic theory concerning the secretor and nonsecretor type in saliva to salivary glands and cells, and further refer to the reasons for appearance of the weak secretor type or intermediate type in saliva.

Immunochemical study and acinar localization of AM1, a murine submandibular glycoprotein with esterolytic activity.

Glycoprotein AM1, a glycoprotein from the submandibular glands of the mouse was isolated from the 100 000 X g tissue extract by polyacrylamide gel electrophoresis. An antiserum to purified glycoprotein AM1 was prepared, and its specificity was tested by immunodiffusion and immunoelectrophoresis. Glycoprotein AM1 could be detected in large quantity only in the submandibular glands of the mouse and in very small amounts in the parotid and sublingual glands and in serum. No glycoprotein AM1 was found in the murine brain, heart, lung, liver, spleen, kidney, pancreas, spinal cord and testis. In addition, glycoprotein AM1 was not detectable in the submandibular glands of the rat and rabbit, and in whole human saliva. No cross-reactivity was found with murine submandibular proteinase A and porcine pancreatic kallikrein. The cellular localization of glycoprotein AM1 was determined by the indirect immunofluorescence technique. In the submandibular glands bright fluorescence was only present in the acinar cells, throughout the whole gland. In the sublingual glands faint fluorescence was detectable as a diffuse network around the acini and possibly in the serous acinar demilune cells. On a subcellular level, glycoprotein AM1 could be demonstrated in the extract of the SMC secretory granular fraction, which originates largely from the acinar cells. On the other hand, glycoprotein AM1 was hardly detectable in the SMB secretory granular fraction, which originates predominantly from the granular convoluted tubular cells. Concomitantly, glycoprotein AM1 was secreted in vivo and could be detected in whole saliva, particularly after stimulation with isoproterenol and carbamylcholine, and also with phenylephrine, but to a much lesser extent.

Reactivity of peroxidase-labeled lectins in rabbit submandibular and sublingual glands.

The carbohydrate histochemistry of rabbit submandibular and sublingual glands has been examined by the use of 4 peroxidase-labeled lectins at the light microscopic level. In the submandibular gland, the striated ducts appeared to be the formations which were more reactive to all lectins. In the sublingual gland, the terminal tracts were the most reactive secreting portions, because they bound all the lectins used. The material contained in the lumen of the ducts of submandibular and sublingual glands always reacted fairly intensely. The binding of lectins to salivary glands indicated the possibility to use lectins for the explanation of the transport properties both of the striated ducts and of the terminal tracts.

Variability and ultrastructural histochemical localization of sulphates in the salivary glands of rodents and Lagomorpha.

Stainings were effected on an ultrastructural histochemical level to localize sulphomucins in the submandibular and sublingual glands of growing mice and rabbits. Sulphates behave in an entirely different way in the salivary glands of Lagomorpha and in those of Rodents. In growing rabbits sulphates can be demonstrated in both glands; at maturity they can be demonstrated only in the sublingual gland and no longer in the submandibular gland. In the salivary glands of Rodents, sulphates cannot be demonstrated histochemically in new born subjects or in adults. The histochemical results are compared to the biochemical ones, and discrepancies, where present, are discussed.

Histochemical application of mild alkaline hydrolysis for selective elimination of O-glycosidically linked glycoproteins.

A new technique to eliminate O-glycosidically linked glycoprotein (mucin-type glycoprotein) selectively has been developed. Composite paraffin sections were collodionized before and after alkaline treatment with 0.5 M NaOH in 70% ethanol; the effect of this procedure on mucosubstances was examined using the periodic acid-Schiff reaction. Exposure to alkaline hydrolysis for 72 to 144 hours at 4 C led to a complete loss of periodic acid-Schiff reactivity of epithelial mucins in rat sublingual gland, stomach and small intestine, but that of fuzzy coat, thyroid colloid, collagen fibers and tracheal cartilage was well preserved. These results agreed fairly well with biochemical findings. The present study also revealed that materials prepared by freeze-substitution provided the most satisfactory results.

Epidermal growth factor, renin, and protease in hormonally responsive duct cells of the mouse sublingual gland.

In the striated ducts of the sublingual glands of normal adult male, but not female, Swiss-Webster mice a few scattered cells have apical secretion granules. These sublingual duct cells resemble the granular convoluted tubule (GCT) cells of the submandibular glands of adult female mice, in that they are smaller than submandibular GCT cells of adult males, and contain fewer apical granules, and prominent basal striations. These cells stain immunocytochemically for epidermal growth factor (EGF), renin, and protease A. Such granular striated duct cells could be induced in the sublingual glands of adult female mice by treatment with either testosterone propionate or thyroxine; the two hormones given simultaneously acted synergistically in this induction.

Ultrastructural localization of complex carbohydrates present in the sublingual gland of rabbits.

Stainings with dialized iron (DI), high-iron diamine-thiocarbohydrazide-silver proteinate HID-TCH-SP), tannic acid-uranylacetate (TA-U), tannic acid-ferric chloride (TA-F), and periodate-thiocarbohydrazide-silver proteinate (PA-TCH-SP) were applied to samples of sublingual glands of rabbits. Comparing the results obtained with what had been previously demonstrated biochemically, it is possible to assert that the faintly electron-opaque, granules of the cells of the preterminal tracts contain sulphated glycoconjugates because of the presence of sulphated hexosamines and hexoses, acid glycoconjugates because of the presence of sialic acid and uronic acids, and neutral glycoconjugates because of the presence of hexoses.