Effects of chronic clonidine administration on parasympathetic-evoked rat saliva.

Effects of chronic administration of clonidine on parasympathetic-evoked saliva from both parotid and submandibular glands were investigated. Clonidine at 1 mg/kg/day for 5 or 7 days caused a significant reduction in the salivary secretion (flow rate and total volume) evoked by parasympathetic nerve stimulation of parotid but not submandibular glands. Ion concentrations (Na, K and Ca) of parasympathetically nerve-evoked parotid saliva were not altered. However, the total protein concentration as well as output, amylase activity, and output of such saliva were markedly increased. Possible mechanisms for clonidine-induced increase in nerve-elicited salivary protein concentration include release of neuropeptides, and changes in adrenergic receptor binding which need further study.     

Electrophoretic behavior of amylase in mouse: the parotid gland is major source of serum amylase

Amylase activity in the saliva, salivary glands, serum, liver (perfused and non perfused) and pancreas was assayed and isoamylases were separated by electrophoresis in these organs using C57BR/cdJ and M. m. molossinus (Kor) mice. Amylase isozymes in the saliva, parotid gland, serum and liver were identical in both strains, respectively. Amylase activity in the liver was lower than that in the serum and liver amylase disappeared almost by perfusion. Major serum amylase was released from the parotid gland in intact animals.     

Isolation and characterization of alpha-amylase messenger RNA from bank vole parotid glands. Evidence for two separate messenger RNAs coding for amylase and an amylase-related protein

Bank vole saliva contains two glycogen-precipitable proteins, both of which show affinity for the alpha-amylase inhibitor cycloheptaamylose. One of these proteins, amylase, has a molecular weight of 55,000, judged from dodecylsulphate/acrylamide gel electrophoresis. The other has an apparent molecular weight of 59,000 and has no amylase activity. We report here that tryptic peptide maps as well as amino-acid composition analyses indicate extensive homology between the two proteins. We have also isolated total poly(A)-containing mRNA from amylase-rich bank vole parotid glands. These mRNAs were translated in the presence of [35S]methionine in an mRNA-dependent cell-free translation system from rabbit reticulocyte lysate. The radioactive translation products were examined by dodecylsulphate/polyacrylamide gel electrophoresis. Two major translation products with apparent molecular weights of approximately 56,500 and 60,500, respectively, were further characterized by tryptic peptide analyses. Our data indicate that the 56,500-Mr product is the biosynthetic precursor of amylase, whereas the 60,500-Mr translation product is a precursor of the 59,000-Mr amylase-like protein. Both precursors appear to contain extra peptide material, presumably as amino-terminal 'pre' or 'signal' peptides, in analogy with that found for other precursors of secretory proteins. Thus, amylase and the 59,000-Mr protein, although very similar, are translated from two separate mRNAs. These two messengers sediment in a sucrose gradient at about 17-S, corresponding to lengths of about 1,800 nucleotides.     

Response of the parotid gland of red kangaroos,Macropus rufus,to phenylephrine stimulation

Intracarotid infusions of l-phenylephrine at 1.0 or 10 nmol kg(-1) min(-1) were accompanied by increases in salivary amylase activity, protein, potassium, magnesium and chloride relative to cholinergically-stimulated saliva. Intravenous infusions of phenylephrine at the same dose rates had a lesser effect on salivary composition particularly protein. Propranolol administered with phenylephrine via the carotid artery, at an antagonist/agonist ratio of 10:1, was much more effective in blocking the phenylephrine-induced changes in salivary composition than equimolar infusion of phentolamine with phenylephrine. It was concluded that alpha(1)-adrenoreceptors were not present in functionally significant numbers in the gland and that the effect of phenylephrine on the kangaroo parotid was mediated by beta-adrenoreceptors. As the phenylephrine dose rates in the kangaroos were comparable with those used to determine alpha-adrenergic responses of eutherian salivary glands and as phentolamine appeared to have minor beta-sympathomimetic activity, at least one subtype of beta-adrenoreceptors in macropods may not be identical to its eutherian counterpart.     

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.     

VAMP8/endobrevin as a general vesicular SNARE for regulated exocytosis of the exocrine system

The molecular mechanism governing the regulated secretion of most exocrine tissues remains elusive, although VAMP8/endobrevin has recently been shown to be the major vesicular SNARE (v-SNARE) of zymogen granules of pancreatic exocrine acinar cells. In this article, we have characterized the role of VAMP8 in the entire exocrine system. Immunohistochemical studies showed that VAMP8 is expressed in all examined exocrine tissues such as salivary glands, lacrimal (tear) glands, sweat glands, sebaceous glands, mammary glands, and the prostate. Severe anomalies were observed in the salivary and lacrimal glands of VAMP8-null mice. Mutant salivary glands accumulated amylase and carbonic anhydrase VI. Electron microscopy revealed an accumulation of secretory granules in the acinar cells of mutant parotid and lacrimal glands. Pilocarpine-stimulated secretion of saliva proteins was compromised in the absence of VAMP8. Protein aggregates were observed in mutant lacrimal glands. VAMP8 may interact with syntaxin 4 and SNAP-23. These results suggest that VAMP8 may act as a v-SNARE for regulated secretion of the entire exocrine system.     

Tissue-specific expression in the salivary glands of transgenic mice.

Using a DNA construct, named Lama, derived from the murine parotid secretory protein (PSP) gene, we have obtained salivary gland specific gene expression in transgenic mice. Lama is a PSP minigene and allows analysis of the PSP gene 5' regulatory region by transgenesis. We show here that the regulatory region included in Lama with 4.6 kb of 5' flanking sequence is sufficient to direct expression specifically to the salivary glands. The expression level in the parotid gland is only about one percent of the PSP mRNA level, while that of the sublingual gland is near the PSP mRNA level. This suggests significant differences in the PSP gene regulation in the two glands. In addition, Lama is a secretory expression vector in which cDNAs or genomic fragments can be inserted. We demonstrate that the Lama construct can direct the expression of a heterologous cDNA encoding the C-terminal peptide of human factor VIII to salivary glands and that the corresponding peptide is secreted into saliva.     

Salivary secretion induced by substance P

Secretion of fluid, ions, and amylase from parotid and submaxillary glands of rat, induced by intravenous injection of substance P (SP), was examined. The action of SP on salivary glands, like physalaemin, resembled that of cholinergic stimulation. While SP-evoked salivary flow from both glands was blocked by atropine, atropine did not modify composition of SP-evoked saliva. The present study suggests that salivary secretion and secretion of ions and amylase evoked by SP are mediated via SP-sensitive cholinergic receptors and specific SP receptors, respectively.     

The salivary flow rate and composition of whole and parotid resting and stimulated saliva in young and old healthy subjects

Resting and stimulated whole and parotid salivary composition and flow rate were examined in 63 healthy volunteers. No significant differences were found between the young and old in secretion rates and salivary concentrations of sodium, potassium, calcium, magnesium, and total protein. The activity of amylase in the resting and stimulated parotid saliva was significantly lower in the old.     

Na+-Glucose Cotransporter SGLT1 Protein in Salivary Glands: Potential Involvement in the Diabetes-Induced Decrease in Salivary Flow

Oral health complications in diabetes include decreased salivary secretion. The SLC5A1 gene encodes the Na⁺-glucose cotransporter SGLT1 protein, which not only transports glucose, but also acts as a water channel. Since SLC5A1 expression is altered in kidneys of diabetic subjects, we hypothesize that it could also be altered in salivary glands, contributing to diabetic dysfunction. The present study shows a diabetes-induced decrease (p < 0.001) in salivary secretion, which was accompanied by enhanced (p < 0.05) SGLT1 mRNA expression in parotid (50%) and submandibular (30%) glands. Immunohistochemical analysis of parotid gland of diabetic rats revealed that SGLT1 protein expression increased in the luminal membrane of ductal cells, which can stimulate water reabsorption from primary saliva. Furthermore, SGLT1 protein was reduced in myoepithelial cells of the parotid from diabetic animals, and that, by reducing cellular contractile activity, might also be related to reduced salivary flux. Six-day insulin-treated diabetic rats reversed all alterations. In conclusion, diabetes increases SLC5A1 gene expression in salivary glands, increasing the SGLT1 protein content in the luminal membrane of ductal cells, which, by increasing water reabsorption, might explain the diabetes-induced decrease in salivary secretion.     

Amylase levels in the tissues and body fluids of the domestic cat (Felis catus).

1. The amylase activities of serum, liver, pancreas, submaxillary and parotid glands, saliva, duodenum, colon, lung, heart, spleen, kidney and skeletal muscle of the domestic cat, were determined. 2. As in most mammals, the highest amylase content was found in the pancreas. Levels in saliva and salivary glands were very low. 3. Amylase activities of the serum, liver and pancreas have an optimum pH 7.0-7.5. 4. Amylases of serum, liver and pancreas were inactivated by removal of chloride and these activities mainly restored by addition of chloride.     

Inheritance of a Parotid Secretory Protein in Mice and Its Use in Determining Salivary Amylase Quantitative Variants

Among inbred strains of mice, a major protein, PSP, produced and secreted by the parotid glands, shows variation in electrophoretic mobility and in the peptides produced by cyanogen bromide treatment. This variation is inherited as a single Mendelian factor with two alleles showing co-dominant expression. In genetic crosses, it segregates independently from the amylase complex and is closely linked to the agouti locus on chromosome 2. The protein ratios between amylase and PSP in saliva, obtained by scanning of electrophoretic gel separations, were found to reflect genetic differences in salivary amylase production in strains YBR/Cv and C3H/As.     

Cl(-)/HCO(3)(-) exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca(2+)](i) increase in salivary acinar cells

Large volumes of saliva are generated by transepithelial Cl(-) movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl(-) uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na(+) independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an approximately 170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive Cl(-)/HCO(3)(-) exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the Cl(-)/HCO(3)(-) exchanger activity in acinar cells from both glands. Intracellular Ca(2+) chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca(2+) concentration with the Ca(2+)-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating Cl(-)/HCO(3)(-) exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca(2+)-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.     

Analysis of parotid and mixed saliva in Roe deer (Capreolus capreolus L.)

In ruminants, different functions have been ascribed to the different salivary glands according to the feeding type. In this context, possible adaptations of salivary functions were investigated regarding the secretion of various proteins by different types of salivary glands. To yield uncontaminated parotid saliva in large quantities, a non-surgical method has been developed. Parotid gland secretions were collected via endoscopic placement of guide wires into each parotid duct, which were subsequently used for placement of collection catheters. Salivary flow was stimulated by intra-glandular administration of the parasympathomimetic compound pilocarpine-hydrochloride into the parotid gland. Mixed saliva (excluding parotid saliva) was collected into sterile tubes by normal outflow during the sampling of parotid saliva. The total flow volume, flow rate and the content of proteins as well as of several ions (Na⁺, K⁺, Ca²⁺, inorganic phosphate) of both types of saliva were measured in sheep, fallow deer and roe deer. Roe deer secreted the highest amount of total salivary proteins relative to body mass [mg/kg body mass] and the highest relative volume [ml/10 min/kg body mass], both in parotid and mixed saliva, of all ruminant species examined. Additionally, the protein profile and the tannin-binding properties of parotid and mixed saliva in roe deer were investigated. Parotid saliva bound almost twice as much tannin as mixed saliva, underlining the importance of yielding uncontaminated parotid saliva for tannin-binding studies. Accepted: 6 January 1998     

Sorting of transgenic secretory proteins in miniature pig parotid glands following adenoviral-mediated gene transfer

BACKGROUND: Gene transfer to salivary glands for use in treating both systemic and upper gastrointestinal tract diseases shows considerable potential. Numerous studies in rodents demonstrate that salivary glands can secrete transgenic secretory proteins either into saliva, primarily via the regulated secretory pathway (RSP), or into the bloodstream, primarily by the constitutive secretory pathway (CSP). The purpose of the present study was to assess the sorting characteristics of human growth hormone (hGH), a RSP protein, and human erythropoietin (hEpo), a CSP protein, in a large animal model of salivary gland gene transfer, the miniature pig. METHODS: Recombinant serotype 5 adenoviral (Ad5; 10(11) particles/gland) vectors encoding either hGH (AdCMVhGH) or hEpo (AdCMVhEpo) were administered to both parotid glands of male miniature pigs by intraductal cannulation. The secretion of hGH or hEpo was measured in both saliva and serum on days 3, 7 and 14 following administration. Detailed serum chemistry and hematological analyses were performed, and the presence of serum antibodies to hGH and hEpo was measured. For AdCMVhEpo-treated minipigs vector distribution in multiple tissues was determined by quantitative polymerase chain reaction (QPCR). RESULTS: The RSP protein hGH was secreted entirely into saliva, while the CSP protein hEpo was secreted into both saliva and serum. Most hEpo was found in saliva, but serum hEpo levels were sufficient to significantly increase hematocrit levels in treated animals by approximately 10%. Expression of both transgenes was maximal on day 3 and declined to near background by day 14. The amount of vector found in the targeted glands was 100 x more than in other tissues. CONCLUSIONS: Secretion of transgenic hGH from minipig parotid glands occurred principally into saliva via the RSP, as seen in rodents, while hEpo was secreted into both saliva and serum, the latter presumably via the CSP. Even though hEpo secretion into the bloodstream was not to the extent previously observed in rodents, serum hEpo levels were considerable and the hEpo was biologically active. Ad5 vector distribution was highly restricted to the parotid glands with little vector detected elsewhere. While the results in this large animal model support the established notion that salivary gland gene transfer can be used for treating systemic single protein deficiency disorders, they also highlight differences in transgenic CSP protein sorting between rodents and miniature pigs.     

Variation in the Number of Genes Coding for Salivary Amylase in the Bank Vole, CLETHRIONOMYS GLAREOLA

A Danish population of bank voles is polymorphic for three electrophoretically different salivary amylases; A, H and S, of which A is the most common. Both single-, double- and triple banded phenotypes were observed, and in several crosses two electrophoretic forms cosegregated. In addition to the qualitative variation, some individuals show consistent quantitative variation in the relative activities of their amylase bands. This variation has been qualified by spectrophotometrical measurements of the relative amounts of amylase protein in the various bands. --Seventy wild chromosomes were analyzed by determining the amounts of amylase they produced when heterozygous with a laboratory stock chromosome known to carry two closely linked amylase genes, both coding for a fourth electrophoretic variant, B. The amount of A-protein divided by half the amount of B-protein was used as an estimate of the number of A-genes on the tested chromosomes. The wild chromosomes fell into three clearly distinguishable classes: 9 clustered around a gene number estimate of one, 45 chromosomes yielded estimates around two genes, and the gene number estimate of the remaining 16 was close to three. The integer values of the gene number estimates and the cosegregation of electrophoretically different salivary amylases are consistent with the model that the population is polymorphic for chromosomes with either one, two, or three closely linked amylase genes. It is suggested that such gene number variation may be more common than generally recognized, and some other reported cases of quantitative enzyme variation, for instance that of human red cell acid phosphatase, are interpreted in terms of variation in the number of genes involved.     

Unexpected beta adrenergic effects of the antitumor agent, cyclocytidine, on rat salivary glands.

In addition to its potent antileukemic properties, cyclocytidine has a sialogogue action that depends on stimulation of beta adrenergic ereceptors of salivary glands. Furthermore, when chronically administered (for 3 days), cyclocytidine caused enlargement of parotid and submaxillary glands and heart that resembled the hypertrophy caused by chronic isoproterenol administration. The salivas evoked by cyclocytidine also closely resembled those evoked by isoproterenol, and were extremely viscous, and high in K+, (121 plus or minus 5.6, for submaxillary, and 42 plus or minus 2.9, for parotid), low in flow rate (0.007 mg/min times mg) and parotid saliva contained high concentrations of amylase (805 plus or minus 33 mg/mg gland). Cyclocytidine also caused marked emptying of parotid gland amylase. The cyclocytidine-induced salivary flow and gland emptying of amylase were prevented for 90 min when propranolol (but not dibenzyline or atropine) was administered prior to injection of the cyclocytidine. In addition, when the superior cervical ganglion was acutely removed, administration of cyclocytidine elicited salivary flow from the denervated as well as the innervated glands. These findings suggest that cyclocytidine does not affect salivary glands through indirect central or ganglionic actions. Cyclocytidine action does not exclusively involve beta receptors, since even in the presence of propranolol, secretory flow was evident after 90 min but when dibenzyline was given with the propranolol, complete blockade of cyclocytidine-stimulated saliva was effected. The dominant effect is, however, a beta adrenergic one. The undesirable side effects of cyclocytidine (parotid pain, postural hypotension, and cardiac hypertrophy) probably stem chiefly from its beta adrenergic properties and might be eliminated (or at least modified) by administration of propranolol with the cyclocytidine.     

Transgenic mice expressing bacterial phytase as a model for phosphorus pollution control

We have developed transgenic mouse models to determine whether endogenous expression of phytase transgenes in the digestive tract of monogastric animals can increase the bioavailability of dietary phytate, a major but indigestible form of dietary phosphorus. We constructed phytase transgenes composed of the appA phytase gene from Escherichia coli regulated for expression in salivary glands by the rat R15 proline-rich protein promoter or by the mouse parotid secretory protein promoter. Transgenic phytase is highly expressed in the parotid salivary glands and secreted in saliva as an enzymatically active 55 kDa glycosylated protein. Expression of salivary phytase reduces fecal phosphorus by 11%. These results suggest that the introduction of salivary phytase transgenes into monogastric farm animals offers a promising biological approach to relieving the requirement for dietary phosphate supplements and to reducing phosphorus pollution from animal agriculture.     

High levels of GM1-ganglioside beta-galactosidase in the salivary glands and GM1-like-ganglioside storage in parotids of deficient mice.

We have previously demonstrated high levels of GM1-ganglioside beta-galactosidase (beta-gal) in the salivary glands of Swiss-Webster mice (Nowroozi et al., J Craniofac Genet Dev Biol 18:51, 1998), and suggested that this activity reflects an important role for the lysosome in catabolism of salivary glycoconjugates. Here, we characterized and compared activities of lysosomal glycosidases among the salivary glands, spleen, and muscle of C57BL/6 mice, beta-gal hexosaminidase, and beta-glucuronidase activities are high in all three glands relative to muscle. Enzyme activities in the sublingual gland were substantially higher than in the submandibular and parotid glands. Spleen displays levels of activity that are comparable or higher (for beta-glucuronidase) than those in the salivary glands, whereas muscle displays substantially lower levels of these lysosomal glycosidases. In order to investigate the role of beta-gal in the salivary glands, we further characterized the salivary phenotype of knock-out mice deficient in this enzyme, mimicking human GM1-gangliosidosis. In contrast with the relative levels of beta-gal specific-activity among the salivary glands, only the parotid developed severe, generalized, degenerative histopathological changes in beta-gal-deficient knock-out mice. GM1-like-ganglioside, typically found at high levels only in the nerve tissue, where its exact function is still not clear, was demonstrated in storage vacuoles of the parotid glands of the deficient mice by binding of cholera toxin subunit B. Thus, beta-gal activity observed in the parotid gland most likely reflects its role in GM1-ganglioside catabolism, and this ganglioside, never previously reported in the salivary glands, may have a role in parotid exocrine secretory functions. beta-gal may also serve in secretory glycoprotein catabolism in other salivary glands, but this function may be non-essential for these glands.     

Identification of aquaporin-5 and lipid rafts in human resting saliva and their release into cevimeline-stimulated saliva

BACKGROUND: It is unknown whether AQP5 and lipid rafts are released into human unstimulated (resting) saliva and saliva in response to secretagogues. METHODS: In order to quantitate the salivary concentration of AQP5, we produced a polyclonal antibody for human AQP5 and developed an enzyme-like immunosorbent assay (ELISA). RESULTS: AQP5 and lipid rafts were identified in human resting saliva. The amount of AQP5 in resting saliva showed a diurnal variation with high levels during waking hours, and an age-related decrease in AQP5 was coincident with the volume of resting saliva. Cevimeline, a muscarinic acetylcholine receptor (mAChR) agonist, induced the release of AQP5 with lipid rafts, amylase, mucin, and lysozyme. Changes in saliva AQP5 levels after cevimeline administration occurred simultaneously with changes in saliva flow rates. Confocal microscopy revealed that AQP5 was located in the apical plasma membrane and showed a diffuse pattern in parotid glands under resting conditions. Following cevimeline administration, AQP5 was predominantly associated with the APM and was localized in the lumen. GENERAL SIGNIFICANCE: AQP5 and lipid rafts were released with salivary proteins from human salivary glands by the stimulation of M3 mAChRs, and that changes in saliva AQP5 levels can be used as an indicator of salivary flow rate and also as a useful index of M3 mAChR agonist's action on human salivary glands.