A.P. Vreugdenhil

Comparison of the secretory process in the parotid and sublingual glands of the mouse: I. Regulation of the secretory processes

1. Secretion from the mucous sublingual gland of the mouse has been investigated and compared with the serous parotid gland. The influence of acetylcholine, noradrenalin and adrenalin on the secretion of glycoproteins (e.g. mucins) and proteins (e.g. amylase) from these glands in vitro, and the involvement of cyclic AMP and Ca2+ has been studied. 2. Secretion from the parotid gland could be stimulated by both acetylcholine and the catecholamines. It appears that cyclic AMP plays an important role in the adrenergic secretory process, but not in the cholinergic-induced secretion. In the latter case, exogenous Ca2+ strongly increased the secretion. 3. Mucin secretion from the sublingual gland could be affected by acetylcholine in the presence of exogenous Ca2+. Noradrenalin and adrenalin induced only a slow mucin secretion and, for this secretory process, exogenous Ca2+ is also required. Though cyclic AMP is present in the sublingual gland, no influence on its level could be detected in this gland after stimulation of the adrenergic beta-receptor, whereas, in contrast to the parotid gland, dibutyryl cyclic AMP induced only a slow secretion. Because it was observed that the sublingual gland of the mouse is not innervated sympathetically, it seems reasonable to suppose that the catecholamines stimulate the mucin secretion from this gland via hormonal receptors and not via the adrenergic beta-receptor. 4. The protein secretion from the sublingual gland could be stimulated by both acetylcholine and the catecholamines. An involvement of cyclic AMP in this process was not observed. Addition of exogenous Ca2+ is less important, as was found for the mucin secretion. So it has been concluded that protein and mucin secretion from the sublingual gland are regulated via different pathways.

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

SummaryAntibodies 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.

Biochemical and immunochemical studies of α-amylase from the salivary glands of the mouse

Abstract α-Amylase was purified 8-fold from the parotid glands by complexing the enzyme with glycogen, followed by precipitation of the complex with ethanol. For the submandibular α-amylase the specific activity increased by a factor 112. With acrylamide gel electrophoresis, one major and 3 minor parotid isoenzymes were detected. By sodium dodecyl sulphate-acrylamide gel electrophoresis, one band was obtained, mol. wt about 65,000. Isoelectric focusing separated 2 major and 7 minor parotid isoenzymes. The isoelectric points of the major components were 6.9 and 7.1; those of the minor isoenzymes ranged from 5.4 to 7.4. The submandibular α-amylase preparation gave a diffuse protein pattern on electrophoresis with an isoelectric point of 6.9. The native submandibular and the sublingual α-amylase were identical immunochemically, but dissimilar from the parotid, the serum and the pancreatic α-amylase of the mouse. In contrast to native submandibular α-amylase, the submandibular amylase-glycogen complex was immunochemically identical to the parotid α-amylase. The parotid α-amylase was partially identical to the serum and the pancreatic α-amylase of the mouse.

Electrophoretic isolation and partial characterization of a major secretory glycoprotein from the submandibular glands of the mouse

After either cholinergic or adrenergic stimulation of the submandibular glands of the mouse, a major protein of the incubation medium could be isolated by electrophoresis, designated the AM2 protein. About 5 per cent of the secreted proteins and 2.4 per cent of the secreted protein-bound sialic acid was recovered as the purified AM2 protein. The AM2 protein appeared to be electrophoretically pure in 7.5% polyacrylamide gel both at pH 8.9 and at pH 4.3. In sodium dodecyl sulfate-electrophoresis the molecular weight was estimated to be about 80 000 for the major component and about 40 000 for the minor component. By isoelectric focusing the isoelectric point has been determined to be 4.7. The amino acid analysis indicated Glx, Asx, Leu and Ala as the major amino acids, comprising 15.0, 10.6, 9.2 and 9.1 per cent of the amino acid residues, respectively. The ratio of the acidic amino acids and their amides (Glx plus Asx) to the basic amino acids (Lys plus Arg) was 2.2. The sugar analysis showed that the AM2 glycoprotein consists of 17.3 per cent of carbohydrate, with as major carbohydrate component glucosamine. The molar ratio of the sugars was Man : Gal : Glc : GlcNH2 : sialic acid = 2.3 : 1.0 : 4.7 : 9.8 : 2.9. Galactosamine could be detected as a trace component and fucose was not detectable.

Electrophoretic isolation and partial characterization of a glycoprotein of the submandibular glands of the mouse.

A protein has been isolated from the water-extract of the submandibular glands of the mouse, after Biogel P-300 column passage, followed by preparative polyacrylamide gel electrophoresis at pH 4.3 and subsequently at pH 8.9, designated the AM1 protein. The isolated protein was electrophoretically pure in 7.5, 10 and 15% polyacrylamide gels both at pH 4.3 and at pH 8.9. Likewise, by electrophoresis in 15% sodium dodecyl sulfate-polyacrylamide gel only one protein band could be detected. Of the total amount of the water-extractable submandibular proteins the recovery of this protein component comprised 3 to 5 per cent. The molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 28 000, both in 7.5 and 15% gel. The isoelectric point was determined by isoelectric focusing in 4.8% polyacrylamide slabgel to be 4.85. The amino acid analysis showed that the ratio of acidic amino acids (Glx plus Asx) to basic amino acids (Lys plus Arg) is 2.3. The glycoprotein consists of protein for 77.4 per cent and of carbohydrate for 22.6 per cent. The molar ratio of the carbohydrates was GlcNH2:GalNH2:Man:Gal:Glc:Fuc:sialic acid = 22.0:1.3:3.0:1.7:10.0:2.6:0.3. The glycoprotein was not secreted from the submandibular glands by stimulation with cholinergic (acetylcholin) or adrenergic (noradrenalin) drugs both in vitro and in vivo. So, it appeared that this glycoprotein could be characterized as a cellular, non-secretory component of these salivary glands.

Comparison in vitro of the incorporation of [3H]-leucine and N-acetyl-[14C]-mannosamine into proteins and glycoproteins of the parotid, submandibular and sublingual glands of the mouse

Abstract During incubation for 10 h, the glands remained active in both protein and glycoprotein secretion and incorporation of the radioactive precursors. The incorporation of [3H]-Leu was completely due to de-novo biosynthesis of protein. At least 80 per cent of the incorporated [14C]-ManNAc could be recovered as sialic acid. The incorporation of [3H]-Leu could be detected as early as 10 min after the onset of incubation and reached the maximum level in all glands after 4 h of incubation. The sublingual glands were more active in incorporating [3H]-Leu than the other two types of salivary glands per mg of glandular wet wt. The pattern of incorporation of [14C]-ManNAc differed from that of [3H]-Leu, in having a time lag in the parotid and submandibular glands of at least 1 h, and an acceleration of the incorporation after 6 h. In spite of their high content of sialomucin, [14C]-ManNAc was hardly incorporated by the sublingual glands. This was apparently not due to a slow cellular uptake of ManNAc, but possibly because the conversion to sialic acid was slow in the sublingual glands of the mouse.

Comparison of adenylate cyclase activity and in vitro secretion in the parotid and sublingual glands of the mouse.

1. Adenylate cyclase (EC 4.6.1.1) activity has been determined in the parotid and sublingual glands of the mouse. Optimal activity of the enzyme was obtained at a Mg2+‐concentration of 8 mM at pH 8.2, using AMP‐PNP as the substrate. 2. Cyclic AMP degradation during the adenylate cyclase assay was relatively high in both the homogenate and the 40,000 g pellet‐fraction of the glands. Theophylline was effective in inhibiting this degradation only in the parotid hemogenate, whereas isobutylmethylxanthine inhibited the cyclic AMP degradation in both salivary glands. Using the latter phosphodiesterase inhibitor, we observed a higher adenylate cyclase activity in the sublingual glands than in the parotid glands. 3. Various receptor‐selective sympathetic and parasympathetic agonists and antagonists have been tested for their capacity to influence the adenylate cyclase activity and the glycoprotein secretion in the parotid and sublingual glands of the mouse, in vitro. (a) The parotid glycoprotein secretion was increased by beta‐adrenergic agonists, which stimulate adenylate cyclase, and by cholinergic muscarinic drugs, which do not activate this enzyme. The adrenergic alpha‐agonist phenylephrine appeared to be involved neither in the glycoprotein secretion nor in the direct regulation of the adenylate cyclase activity. (b) The sublingual protein and mucin secretion was increased by cholinergic muscarinic agents. The over‐all protein secretion was stimulated also by phenylephrine, but this effect could be blocked by propranolol. The adenylate cyclase activity in membrane preparations was not stimulated by these secretogogues.

REGULATION OF SUBLINGUAL (GLYCO)PROTEIN SECRETION IN THE MOUSE

Publisher Summary This chapter presents an experiment to examine how secretion of sublingual protein can be regulated in a mouse. The sublingual glands (SL) of the mouse contain mixed acini with large mucin-containing cells and small serous demilune cells. The chapter describes an in vitro investigation that showed how neuroreceptors are involved in the regulation of the SL-secretion. In addition, 10 μM phenylephhne, an α-adrenergic agonist, exerted a significant influence on the overall protein secretion but did not induce mucin secretion. Both the α-adrenergic agonist phenylephhne and the β-adrenergic agonist isoproterenol stimulated only the demilune cells to exocytosis, suggesting that the significant protein secretion from SL under the influence of phenylephhne in vitro originated from these cells. So, it is possible that the adrenergic nerve fibers, which were observed in SL, are involved in the regulation of the demilune cells.

MORPHOLOGICAL CHANGES OF ACINAR CELLS IN THE SUBMANDIBULAR GLANDS OF MICE AFTER STIMULATION WITH AUTONOMIC DRUGS

Publisher Summary This chapter presents an experiment concerning the acinar cells of submandibular glands of mice. In the experiment, female Swiss mice of 20–22 g were used. They were kept with free access to water and food and under constant conditions of temperature and changes of light and darkness. The mice were starved overnight to synchronize the secretory state of the cells as much as possible. The perfusion with drugs was continued for periods varying from 15 to 90 min. The intraperitoneal-injected mice were sacrified 90-120 min after administration of the agonist. The control mice were treated in the same way. After fixation, the tissues were dehydrated and embedded in Epon–Araldite. After perfusion during periods varying from 15–60 min and subsequent perfusion fixation, the morphology of the control glands did not differ from that after immediate perfusion fixation.

CHARACTERISTICS OF SALIVARY α-AMYLASE OF THE MOUSE

Publisher Summary This chapter describes the characteristics of salivary alpha-amylase of the mouse. This enzyme is involved in the hydrolysis of complex carbohydrates. It is derived from the major salivary glands and is present in saliva in high concentrations. Murine salivary amylase is predominantly localized in the parotid glands (Par), whereas a minor fraction is present in the submandibular (SM) and sublingual (SL) glands. This chapter presents a study in which this enzyme was isolated and compared with Par-amylase by biochemical, immunochemical, and immune histochemical techniques. In addition, different types of SM secretory granules were isolated and identified by their protein composition. The native SM-amylase appeared to be immunochemically nonidentical to Par-amylase, and the localization of SM-amylase was apparently dependent on the method applied. In a few studies, various types of secretory granules were isolated and studied by electron microscopy and by biochemical and immunochemical methods.