Autonomic effects on protein secretion by rat submandibular salivary glands

1. Following treatment with cholinergic and beta-adrenergic drugs, the beta-type protein, associated with cAMP, was secreted regardless of the doses used. 2. Following treatment with alpha 1-adrenergic drugs, both the beta-type and alpha-type proteins were secreted depending on the doses used and the alpha-type protein was completely converted to the beta-type with alpha-blockers. 3. Following treatment with alpha 2-adrenergic drugs, the gamma-type protein, associated with cGMP, was secreted independent of the doses used.     

Depletion of neuropeptides in rat parotid glands and declining atropine-resistant salivary secretion upon continuous parasympathetic nerve stimulation

In rats the parasympathetic auriculo-temporal nerve on one side was continuously stimulated at 40 Hz for 20-80 min in the presence of adrenergic blockers (dihydroergotamine and propranolol) +/- atropine. During the first 10 min this gave rise to a flow of saliva from the parotid gland that in the atropinized rats amounted to 35% of that found in rats not treated with atropine, while the protein and amylase outputs were 75% of those in non-atropinized rats. The atropine-resistant secretion of fluid and proteins declined to 5-10% of the initial value within 40 min but did not cease completely even after 80 min. The marked reduction in secretory responses was not due to desensitization or exhaustion of the gland cells. The nerve stimulation reduced the parotid gland content of vasoactive intestinal peptide (VIP) and substance P (SP) to approximately 60 and 25% of that of contralateral glands after 20 and 60 min, respectively. The probable explanation for the decline in secretory response seems to be depletion of non-adrenergic, non-cholinergic transmitter(s). The present results suggest that neuropeptides are involved in the regulation of salivary secretion but provide no direct evidence that either VIP or SP is responsible for the atropine-resistant salivary secretion.     

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.     

Guanylin and uroguanylin in the parotid and submandibular glands: potential intrinsic regulators of electrolyte secretion in salivary glands

The intestinal peptides guanylin and uroguanylin regulate the electrolyte/water transport in the gastrointestinal epithelium via activation of cystic fibrosis transmembrane conductance regulator (CFTR), the cystic fibrosis gene product. Because a major but incompletely understood function of the salivary glands is the CFTR-mediated secretion of an electrolyte-rich fluid, we investigated the rat and guinea pig parotid and submandibular glands for expression, cellular distribution, and subcellular localization of guanylin and uroguanylin. RT-PCR analyses with guanylin and uroguanylin-specific primers revealed that both peptides are highly expressed in the parotid and submandibular glands. At the translational level, western blotting analyses with peptide-specific guanylin and uroguanylin antibodies identified the expected 12.5-kDa immunoreactive peptides in these organs. At the cellular level, guanylin and uroguanylin were exclusively confined to epithelial cells of the intralobular and interlobular ducts. At the subcellular level, the immunoreactivities were localized by preembedding immunoelectron microscopy to small vesicles which were concentrated at the apical part of the secretory epithelial cells. The expression and cell-specific localization of guanylin and uroguanylin in the salivary glands indicate that these peptides may be specifically involved in the regulation of CFTR-mediated electrolyte/water secretion in the salivary gland ductal system.     

Ultrastructural phosphatase histochemistry of submandibular and parotid salivary glands of man

Summary Thiamine pyrophosphatase was demonstrated in the Golgi complex and acid phosphatase in the GERL of acinar cells of submandibular and parotid glands and were previously demonstrated in cells of intercalary ducts. Thiamine pyrophosphatase was also demonstrated in the Golgi complex of cells of striated and excretory ducts and myoepithelial cells. Acid phosphatase was also demonstrated in lysosomes. Alkaline phosphatase was rarely demonstrated light microscopically at luminal surfaces of striated and excretory ducts and electron microscopically in luminal vesicles in cells of striated ducts. The demonstration of the phosphatases in Golgi complexes and GERLs indicates that investigations on these structures in experimental animals are relevant to human salivary glands and supports the opinion that ductal cells as well as acinar cells secrete organic material. The presence of alkaline phosphatase at luminal surfaces of striated and excretory ducts suggests that resorption as well as secretion may occur in them.     

Tyrosylprotein sulfotransferase in rat submandibular salivary glands.

1. The transfer of sulfate ester group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to poly-(Glu6, Ala3, Tyr1) (EAY; Mr 47 kDa) in rat submandibular salivary gland has been investigated. The highest tyrosylprotein sulfotransferase activity was obtained in the Golgi-enriched fraction in the presence of 2 mM 5'AMP, 20 mM MnCl2 and 50 mM NaF at pH 6.2. 2. The apparent Km values for EAY and PAPS were 1.6 x 10(-6) and 1.9 x 10(-6) M, respectively. 3. Inclusion of NaCl, EDTA, NEM and DTT was inhibitory for the enzyme activity. The enzyme was 28 times less susceptible to 2,6-dichloro-4-nitrophenol inhibition than to phenol sulfotransferase inhibition. 4. This study is the first report characterizing a sulfotransferase activity specific for tyrosylprotein in rat submandibular salivary glands.     

The effects of alpha-methylnoradrenaline on protein and electrolyte secretion by rat submandibular and parotid glands

alpha-Methylnoradrenaline (alpha-mNA) is a potent secretagogue for the parotid and submandibular glands of rats. With regard to the parotid glands, alpha-mNA activates mainly beta-adrenoceptors. In the submandibular glands, alpha-mNA activates alpha-adrenoceptors at higher doses whereas at relatively lower doses it activates beta-adrenoceptors. alpha-mNA may not stimulate the specific alpha 2-adrenoceptors of the salivary glands of rats.     

The effects of m-octopamine on salivary flow rates and protein secretion by rat submandibular glands

1. m-Octopamine given i.v. or i.p. was a potent sialogogue for rat salivary glands.2. Salivation in response to i.v. m-octopamine was completely abolished by prazosin and phenoxybenzamine.3. The α-type of proteins were secreted in response to all doses of i.v. and i.p. m-octopamine and these were converted into the β-type with prazosin, but not with yohimbine.4. m-Octopamine stimulated both α- and β-adrenoceptors and was a much more selective α₁-agonist than was the p-isomer.     

The effects of oxymetazoline on secretion of protein and some electrolytes by rat submandibular and parotid glands

Oxymetazoline is a potent secretagogue for the salivary glands of rats. In the parotid gland, it activates preferentially alpha-adrenoceptors. As for the submandibular glands, it activates alpha-adrenoceptors at relatively low doses but at higher doses it allows secretion of new types of proteins.     

Fatty acid acylation of salivary mucin in rat submandibular glands

The acylation of salivary mucin with fatty acids and its biosynthesis was investigated by incubating rat submandibular salivary gland cells with [3H]palmitic acid and [3H]proline. The elaborated extracellular and intracellular mucus glycoproteins following delipidation, Bio-Gel P-100 chromatography, and CsCl equilibrium density gradient centrifugation were analyzed for the distribution of the labeled tracers. Both preparations gave single bands at the CsCl density of 1.48, in which carbohydrate peaks coincided with that of the labels. The [3H]palmitic acid in these glycoproteins was susceptible to cleavage by alkali and hydroxylamine, thus indicating the ester nature of the bond. With both intracellular and extracellular glycoproteins deacylation caused the glycoproteins to band in the CsCl gradient at a density of 1.55. The incorporation of both markers into mucus glycoprotein increased steadily with time up to 4 h, at which time about 65% of [3H]palmitate and [3H]proline were found in the extracellular glycoprotein and 35% in the intracellular glycoprotein. The incorporation ratio of proline/palmitate, while showing an increase with incubation time in the extracellular glycoprotein, remained essentially unchanged with time in the intracellular glycoprotein and at 4 h reached respective values of 0.14 and 1.12. The fact that the proline/palmitate incorporation ratio in the intracellular glycoprotein at 1 h of incubation was 22 times higher than in the extracellular and 8 times higher after 4 h suggests that acylation occurs intracellularly and that fatty acids are added after apomucin polypeptide synthesis. As the incorporation of palmitate within the intracellular mucin was greater in the mucus glycoprotein subunit, it would appear that fatty acid acylation of mucin subunits preceeds their assembly into the mucus glycoprotein polymer.     

Mucin histochemistry of submandibular and parotid salivary glands of man: light and electron microscopy

Light-microscopy showed parotid serous acinar cells to contain neutral mucin, serous and mucous acinar cells of submandibular gland and intercalary ductal cells of both glands to contain acid and neutral mucins, and cells of striated ducts and excretory ducts to contain neutral mucin. Mucins were demonstrated ultrastructurally in a portion of the components of secretory granules of acinar cells and intercalary ductal cells, and in secretory granules of striated and excretory ductal cells. The mucins were all stained by techniques that reveal 1,2-glycols. Secretory granules of submandibular mucous and serous acinar cells and intercalary ductal cells were stained variably by the low iron-diamine technique for acid mucin, and those of mucous acinar cells by the high iron-diamine technique for sulphomucins mucin and possibly consisted of protein. The results suggest that one type of cell may be able to produce a range of secretory products and to package them variously into secretory granules.     

Immunolocalization of electroneutral Na(+)-HCO cotransporters in human and rat salivary glands

Patterns of salivary HCO secretion vary widely among species and among individual glands. In particular, virtually nothing is known about the molecular identity of the HCO transporters involved in human salivary secretion. We have therefore examined the distribution of several known members of the Na(+)-HCO cotransporter (NBC) family in the parotid and submandibular glands. By use of a combination of RT-PCR and immunoblotting analyses, the electroneutral cotransporters NBC3 and NBCn1 mRNA and protein expression were detected in both human and rat tissues. Immunohistochemistry demonstrated that NBC3 was present at the apical membranes of acinar and duct cells in both human and rat parotid and submandibular glands. NBCn1 was strongly expressed at the basolateral membrane of striated duct cells but not in the acinar cells in the human salivary glands, whereas little or no NBCn1 labeling was observed in the rat salivary glands. The presence of NBCn1 at the basolateral membrane of human striated duct cells suggests that it may contribute to ductal HCO secretion. In contrast, the expression of NBC3 at the apical membranes of acinar and duct cells in both human and rat salivary glands indicates a possible role of this isoform in HCO salvage under resting conditions.     

Fluid secretion rates from mouse and rat parotid glands are markedly different following pilocarpine stimulation

1. Parotid saliva production in two commonly employed laboratory animals, mouse and rat, was studied following pilocarpine stimulation. 2. When normalized to body wt, average parotid saliva output rates in mice were 3-4-fold greater than those observed in rats. When parotid salivary flow rates were normalized to gland weight, mice still displayed 2-3-fold higher values than rats. 3. The Na+ and K+ content of parotid saliva showed small differences between the two species, while saliva from rats contained 3-fold higher protein levels than observed with mice.     

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.     

Changes in parotid acinar cells accompanying salivary secretion in rats on sympathetic or parasympathetic nerve stimulation

Morphological and secretory effects of stimulating autonomic nerves have been studied in parotid glands of rats. Sympathetic stimulation evoked a slow flow of saliva which had a high concentration of amylase. After long term sympathetic stimulation secretory granules were heavily depleted from the parotid acinar cells. Parasympathetic stimulation evoked a copious flow of saliva with a low concentration of amylase. However, at high frequency stimulation the total amount of amylase secreted on parasympathetic stimulation was as great or even greater than on symphatetic stimulation, nevertheless, any loss of secretory granules from the acinar cells was very small. It is concluded that secretion of parotid acinar granules in the rat is prinicipally a sympathetic function. Secretion of fluid is more effectively produced by parasympathetic stimulation and much of the amylase in such saliva appears to have arisen from sources other than the secretory granules.     

Effect of translocator protein (18 kDa)-ligand binding on neurotransmitter-induced salivary secretion in rat submandibular glands

Background information. TSPO (translocator protein), previously known as PBR (peripheral‐type benzodiazepine receptor), is a ubiquitous 18 kDa transmembrane protein that participates in diverse cell functions. High‐affinity TSPO ligands are best known for their ability to stimulate cholesterol transport in organs synthesizing steroids and bile salts, although they modulate other physiological functions, including cell proliferation, apoptosis and calcium‐dependent transepithelial ion secretion. In present study, we investigated the localization and function of TSPO in salivary glands. Results. Immunohistochemical analysis of TSPO in rat salivary glands revealed that TSPO and its endogenous ligand, DBI (diazepam‐binding inhibitor), were present in duct and mucous acinar cells. TSPO was localized to the mitochondria of these cells, whereas DBI was cytosolic. As expected, mitochondrial membrane preparations, which were enriched in TSPO, exhibited a high affinity for the TSPO drug ligand, ³H‐labelled PK 11195, as shown by B_max and K_d values of 10.0±0.5 pmol/mg and 4.0±1.0 nM respectively. Intravenous perfusion of PK 11195 increased the salivary flow rate that was induced by muscarinic and α‐adrenergic agonists, whereas it had no effect when administered alone. Addition of PK 11195 also increased the K⁺, Na⁺, Cl⁻ and protein content of saliva, indicating that this ligand modulated secretion by acini and duct cells. Conclusions. High‐affinity ligand binding to mitochondrial TSPO modulates neurotransmitter‐induced salivary secretion by duct and mucous acinar cells of rat submandibular glands.