Strain-specific differences in the proline-rich proteins and glycoproteins induced in rat salivary glands by chronic isoprenaline treatment.

Parotid and submandibular glands were isolated from five strains of rat after chronic injection of the beta-adrenergic receptor agonist isoprenaline (isoproterenol). The glands were observed to have undergone a marked increase in wet weight, owing to hypertrophy and hyperplasia. The 100 000 g soluble fraction of gland cell lysates were extracted with 10% (w/v) trichloroacetic acid, and the soluble material subsequently analysed by SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis. By this procedure, evidence was obtained for the induction, in isoprenaline-treated parotid and submandibular glands, of proline-rich proteins with apparent Mr values ranging from 20 000 to 40 000. Heterogeneity was evident in the proteins produced for a specific gland between the rat strains, although the amino acid compositions were the same. Products from induced mRNAs translated in vitro had similar mobilities in SDS/polyacrylamide gels, despite the apparent difference in mobility of trichloracetic acid-extracted proline-rich proteins from the various strains. Strain-specific differences were noted for the proline-rich glycoproteins from control salivary glands as well as those induced as a consequence of isoprenaline treatment. Although the glycoproteins had similar amino acid compositions, there was considerable heterogeneity in the carbohydrate compositions for these proteins, suggesting that the differences were the result of post-translational modifications during glycosylation. Induction of the increased activity of the Golgi membrane marker enzyme UDP-galactose:2-acetamido-2-deoxy-D-glucosamine 4 beta-galactosyltransferase (EC 2.4.1.22) occurred to the same extent in the parotid glands of all strains examined. There was no change in the specific activity of a second enzyme, UDP-galactose:N-acetylgalactosaminyl-protein 3 beta-galactosyltransferase (no EC designation).     

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.     

Acinar cell proliferation in the parotid and submandibular salivary glands of the neonatal rat

Abstract. Although the rat salivary glands are deficient in acini at birth, acinar cells proliferate rapidly during the early post-natal period. The pattern of acinar cell proliferation was analysed in the parotid and submandibular glands of neonatal rats from day of birth until day 34. Mitotic and [³H]thymidine ([³H]TdR) labelling indices of the two glands show distinctly different patterns. Analysis of cell division in the rat parotid gland demonstrated a peak of mitotic index at 14 days (2.9 ± 0.4%) and labelling index at 16 days (25.2 ± 2.1%). Submandibular gland acinar cell proliferation reaches a zenith between 7–8 days; labelling index (14.2 ± 1.1%) and mitotic index (2.3 ± 0.3%). Cell proliferation decreases rapidly in both glands after reaching a peak in activity. Gland size increases more rapidly in the submandibular gland which correlates with the earlier shift from cell proliferation to differentiation which occurs in this organ. Circadian rhythms of [³H]TdR incorporation were also investigated in this study. A circadian rhythm of [³H]TdR incorporation into DNA occurs at 15 days after birth with a peak at 06.00 hours in both glands and a trough occurring at 15.00 hours in parotid gland and 18.00 hours in the submandibular gland. Determination of specific activity of DNA (ct/min per μg DNA) on days 8, 10, 12, 13, 14, 15, and 16 after birth at 06.00 and 15.00 hours indicated that a circadian rhythm in [³H]TdR incorporation into DNA began on day 14. The developmental switch from suckling to solid food may be an initiating factor in the sychronization of the circadian rhythm in cell proliferation.     

Isoprenaline-induced changes in rat parotid and submandibular glands are age- and dosage-dependent.

Neonatal rats treated with chronic injections of isoprenaline (isoproterenol) for 10 days revealed differential induction of proline-rich proteins and glycoprotein synthesis between the parotid and submandibular glands. Biosynthesis of proline-rich proteins (Mr 17000-35000) and a Mr-220000 glycoprotein were detectable by solubilization in 10%-trichloroacetic acid extracts from parotid glands 14 days after birth. The enzyme lactose synthase (UDP-galactose: 2-acetamido-2-deoxy-D-glucosamine 4 beta-galactosyltransferase) (EC 2.4.1.22) is also induced 4-7-fold in specific activity compared with control neonatal rats, but again only after 14 days post partum, with isoprenaline treatment. This is in accord with the ability of the parotid gland to respond to beta-receptor stimulation and subsequent increases in intracellular cyclic AMP necessary for induction of protein synthesis [Grand, Chong & Ryan (1975) Am. J. Physiol. 228, 608-612]. Induction of the proline-rich proteins and a Mr-190000 glycoprotein in the soluble fraction from the submandibular gland were not detected until 49 days after birth under identical conditions in the same animal. Cyclic AMP in the submandibular gland undergoes increases on beta-receptor stimulation similar to those achieved in the adult animal, 1 day after birth (Grand et al., 1975). This same differential induction between parotid and submandibular gland was obtained with a range of isoprenaline dosages in adult animals. Trichloroacetic acid-soluble proline-rich proteins were isolated from parotid glands at a dosage of 4.0 mg of isoprenaline/kg body wt., but 7.0 mg/kg was required to induce also biosynthesis of these proteins in the submandibular gland. Gland hypertrophy showed the same differential dosage kinetics, based on gland weight, between the two glands; however, hypertrophy could be accomplished at a lower dosage of isoprenaline than that used to induce proline-rich-protein biosynthesis.     

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.     

Isoproterenol-mediated parotid gland hypertrophy is inhibited by effectors of 4 beta-galactosyltransferase

Chronic administration of the beta-adrenergic receptor agonist isoproterenol results in parotid gland hypertrophy and hyperplasia. This physiological change in the gland has recently been reported to be accompanied by a dramatic increase in the rate of synthesis and accumulation of the enzyme 4 beta-galactosyltransferase. Here, isoproterenol-induced hypertrophy of the parotid gland is shown to be blocked by the introduction of the 4 beta-galactosyltransferase modifier protein alpha-lactalbumin and also by specific antiserum to bovine 4 beta-galactosyltransferase. Partial inhibition of gland enlargement was observed stereospecifically with UDP-galactose and with the competitive acceptor glycoprotein, ovalbumin, as well. These agents did not affect the induction by isoproterenol of the biosynthesis of the proline-rich proteins nor of 4 beta-galactosyltransferase. These agents also inhibited the incorporation of thymidine by isoproterenol-stimulated parotid cells in culture.     

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.     

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.     

Inhibition of isoproterenol-induced DNA and RNA synthesis in rat parotid and pancreas following removal of submandibular-sublingual glands

Summary [³H] thymidine incorporation into DNA of the parotid (PA) gland of adult and 20-day-old rats and into DNA of the pancreas (PANC) of 20-day-old rats was increased markedly following a 2-day regimen of isoproterenol (ISO) administration. However, when the submandibular-sublingual (SM-SL) glands had been removed just prior to initiation of the ISO injections, the [³H] thymidine incorporation into PA and PANC was inhibited, and cpm/mg protein of these organs was even lower than that of organs of untreated rats with SM-SL glands present. Removal of the PA glands just prior to initiation of the ISO regimen had no effect on the ISO-induced [³H] thymidine incorporation into DNA of PANC but partially inhibited that of the submandibular (SM) gland. It is suggested that the inhibitory effects on DNA and RNA synthesis that follow removal of SM-SL glands are attributable to the growth factors (epidermal growth factor and nerve growth factor) found in the rat SM gland. These factors appear to regulate normal DNA synthetic activity of exocrine glands as well as ₁-adrenoceptor mediated DNA synthesis. Cellular hypertrophy induced by the ISO was less markedly affected by absence of the SM glands, but a partial inhibition of [³H] uridine incorporation into RNA of PA of adult rats also occurred when SM-SL glands were removed prior to initiation of the ISO-regimen.     

Purification of inorganic pyrophosphatase from rat salivary glands

Inorganic pyrophosphatase [EC 3.6.1.1] isolated from rat sublingual and submandibular glands was purified 2300-fold and 2600-fold, respectively. The purified enzymatic preparations separated on electrophoresis into two protein bands, of which only one showed the pyrophosphatase activity. Inorganic pyrophosphatase from rat salivary glands is a monomeric anionic protein, its isoelectric point is 5.42 and 4.90 for the sublingual and submandibular glands, respectively.     

Mapping Relative Differences in Human Salivary Gland Secretions by Dried Saliva Spot Sampling and nanoLC–MS/MS

Dried saliva spot sampling is a minimally invasive technique for the spatial mapping of salivary protein distribution in the oral cavity. In conjunction with untargeted nano‐flow liquid chromatography tandem mass spectrometry (nanoLC–MS/MS) analysis, DSS is used to compare the proteomes secreted by unstimulated parotid and submandibular/sublingual salivary glands. Two hundred and twenty proteins show a statistically significant association with parotid gland secretion, while 30 proteins are at least tenfold more abundant in the submandibular/sublingual glands. Protein identifications and label‐free quantifications are highly reproducible across the paired glands on three consecutive days, enabling to establish the core proteome of glandular secretions categorized into eight salivary protein groups according to their biological functions. The data suggest that the relative contributions of the salivary glands fine‐tune the biological activity of human saliva via medium‐abundant proteins. A number of biomarker candidates for Sjögren's syndrome are observed among the gland‐specifically expressed proteins, which indicates that glandular origin is an important factor to consider in salivary biomarker discovery.     

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.     

Use of Biometric Characteristics of Major Salivary Glands as a Baseline to Develop a Formula for the Quantitative Estimation of Posttraumatic Regeneration of Glandular Tissue

In experiments conducted on 903 rats, we studied the biometric characteristics of the major salivary glands (parotid, submandibular, and sublingual glands) during ontogenesis. We calculated the indices of nondirectional fluctuation asymmetry for the submandibular and sublingual glands and determined correlation coefficients and the coefficients of linear regression between salivary glands, rat body weight, and the weight of the femoral bone (the largest bone in the rat). The strongest correlation was found between the dry weight of the submandibular gland and the rat body weight. Mathematical analysis of the growth of the submandibular gland after sialotomy allowed us to derive a formula for the quantitative estimation of regeneration, taking into account the natural growth of the rat.     

Use of biometric characteristics of major salivary glands as a base line to develop a formula for the quantitative estimation of posttraumatic regeneration of glandular tissue

In experiments conducted on 903 rats, we have studied biometric characteristics of the major salivary glands (parotid, submandibular, and sublingual glands) during ontogenesis. We have calculated the indices of non-directional fluctuation asymmetry for the submandibular and sublingual glands and determined correlation coefficients and coefficients of linear regression between salivary glands, rat body weight, and the weight of the femoral bone (the largest bone in the rat). The strongest correlation was found between the dry weight of the submandibular gland and the rat body weight. Mathematical analysis of the growth of the submandibular gland after sialotomy allowed us to derive a formula for the quantitative estimation of regeneration, taking into account the natural growth of the rat.     

Modification of cyclocytidine-induced changes in [3H]thymidine incorporation and weight of parotid gland by partial sialoadenectomy.

Cyclocytidine (CC), a potent antitumor agent, caused a 2.3- to 5.0-fold increase in [3H]thymidine uptake of rat parotid gland after 3 days of daily administration of 500 mg/kg body wt. Gland weight also showed a 47-67% increase from that of controls. Ablation of the submandibular-sublingual glands prior to initiation of the CC regimen prevented the usual CC-induced increase in [3H]thymidine uptake, but did not inhibit the increase in gland size. It is postulated that CC-induced parotid hyperplasia requires an initial release of growth factors from the submandibular gland; however, enlargement of the parotid gland by CC is independent of such factors.     

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.     

Differential expression of proline-rich proteins in rabbit salivary glands.

Salivary glands synthesize and secrete an unusual family of proline-rich proteins (PRPs) that can be broadly divided into acidic and basic PRPs. We studied the tissue-specific expression of these proteins in rabbits, using antibodies to rabbit acidic and basic PRPs as well as antibodies and cDNA probes to human PRPs. By immunoblotting, in vitro translation, and Northern blotting, basic PRPs could be readily detected in the parotid gland but were absent in other salivary glands. In contrast, synthesis in vitro of acidic PRPs was detected in parotid, sublingual, and submandibular glands. Ultrastructural localization with immunogold showed heavy labeling with antibodies to acidic PRPs of secretory granules of parotid acinar cells and sublingual serous demilune cells. Less intense labeling occurred in the seromucous acinar cells of the submandibular gland. With antibodies to basic PRPs, the labeling of the parotid gland was similar to that observed with antibodies to acidic PRPs, but there was only weak labeling of granules of a few sublingual demilune cells, and no labeling of the submandibular gland. These results demonstrate a variable pattern of distribution of acidic and basic PRPs in rabbit salivary glands. These animals are therefore well suited for study of differential tissue expression of PRPs.     

Localization of amylase and mucins in the major salivary glands of the mouse

Summary Antibodies against murine submandibular and sublingual mucins have been raised in rabbits. Both antisera appeared to be specific. Using these antibodies, the mucins were localized in the acinar cells of the submandibular and sublingual glands respectively.The dyed amylopectin method was used to estimate the activity of amylase in the salivary glands. The enzyme was localized either by a starch-substrate film method or with antibodies against purified parotid amylase. The activity of amylase in parotid homogenates is about 1000-fold higher than that in homogenates of either submandibular or sublingual glands, in which the activity was comparable. Amylase was localized in the acinar cells of the parotid gland with both localization techniques. In the sublingual gland, amylase was found predominantly in the stroma around the acini, and there was some evidence that amylase was present in the demilune cells as well. In the submandibular gland, contradictory results were obtained with both techniques. With the starch-substrate film method, amylase activity was found in the granular convoluted tubular cells, whereas immuno-reactive amylase could only be demonstrated in the acinar cells of this gland. It is concluded that in the submandibular gland amylase and mucin are present in the same cell type.     

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.