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Background: Radiotherapy to the head and neck area damages the salivary glands. As a consequence hyposalivation may occur, but also the protein composition of saliva may be affected possibly compromising oral health. The aim of our study was to compare the relative abundance of proteins and peptides in parotid saliva of irradiated patients to that of healthy controls., Methods: Using Lashley cups and citric acid, saliva from the parotid glands was collected from nine irradiated patients and ten healthy controls. The samples were analyzed with SELDI-TOF-MS using a NP20 and IMAC-30 chip in the molecular weight range of 1-30 kDa., Results: On the NP20 chip 61 (out of 217) and on the IMAC-30 chip 32 (out of 218) peaks differed significantly in intensity between the saliva of the irradiated patients and healthy controls. 55 % of the significant peaks showed higher intensity and 45 % showed lower intensity in the saliva of irradiated patients. The peaks may represent, amongst others, the salivary proteins lysozyme, histatins, cystatin, protein S100 and PRP's., Conclusions: Large differences were found in the relative abundance of a wide range of proteins and peptides in the parotid saliva of irradiated patients compared to healthy controls.

The diagnosis of primary Sjögren's syndrome (pSS) is difficult due to the lack of specific laboratory and clinical tests. As an initial step for the global discovery of changes in the abundance of parotid salivary proteins in pSS, a pooled sample was compared to that from healthy control subjects by multidimensional protein identification technology (MudPIT). A total of 1246 proteins were identified by MudPIT. The abundance of 477 of these proteins did not change, 529 were only detected in either the pSS or HC sample, while 206 of these proteins were significantly upregulated ≥ twofold and 34 were downregulated ≤ 0.5. Ingenuity Pathway Analyses of differentially expressed proteins identified by MudPIT resulted in the identification of 100 significant pathways. The same samples were quantified in parallel using RP MS. Fifty-eight of 71 proteins identified by RP overlapped with MudPIT results. Five proteins were further analyzed by targeted label-free quantification to confirm the similar relative differential expression observed by RP and MudPIT approaches. The present study supports the use of MS for global discovery and validation of marker proteins for improved and early diagnosis of pSS., (© 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Background and Objective: Alteration of salivary gland secretion is one of the consequences of diabetes. In a recent study on the submandibular gland of diabetic subjects, we found changed expression of statherin, a salivary protein of fundamental importance in preserving tooth integrity, whose reduction was related with the high incidence of oral diseases in patients with diabetes. The goal of this report is to extend the study to human parotid gland and to compare the effects of diabetes on statherin expression with those previously described in submandibular gland., Materials and Methods: Fragments of parotid glands obtained from diabetic and non-diabetic patients were fixed, dehydrated, embedded in Epon Resin and processed for the immunogold histochemistry. The staining density was expressed as number of gold particles per μm(2) and statistically evaluated., Results and Conclusions: In all samples, statherin reactivity was specifically localized in secretory granules of acinar cells. The statistical analysis showed that labelling density was significantly lower in diabetic than in non-diabetic parotid glands and that diabetes affects protein expression at identical extent in parotid and submandibular glands. The results strengthen the hypothesis that a reduced statherin secretion may be responsible for the higher incidence of oral disorders in diabetic subjects., (© 2011 John Wiley & Sons A/S.)

Objectives: Published studies of gene transfer to mouse salivary glands have not employed the parotid glands. Parotid glands are the likely target tissue for most clinical applications of salivary gene transfer. The purpose of the present study was to develop a convenient and reproducible method of retroductal gene transfer to mouse parotid glands., Methods: The volume for vector delivery was assessed by infusion of Toluidine Blue into Stensen's ducts of Balb/c mice after direct intraoral cannulation. Recombinant, serotype 5 adenoviral vectors, encoding either firefly luciferase or human erythropoietin (hEpo), were constructed and then administered to parotid glands (10(7) vector particles/gland). Transgene expression in vivo was measured by enzyme activity (luciferase) or an enzyme-linked immunosorbent assay (hEpo). Vector biodistribution was measured by real-time quantitative (Q) PCR., Results: The chosen volume for mouse parotid vector delivery was 20μL. Little vector was detected outside of the targeted glands, with both QPCR and luciferase assays. Transgene expression was readily detected in glands (luciferase, hEpo), and serum and saliva (hEpo). Most secreted hEpo was detected in saliva., Conclusion: These studies show that mouse parotid glands can be conveniently and reproducibly targeted for gene transfer, and should be useful for pre-clinical studies with many murine disease models., (© 2010 John Wiley & Sons A/S.)

Background: Although radiation therapy (RT) causes permanent xerostomia, parotid-sparing radiation therapy (PSRT) ensures recovery of saliva quantity over time. Salivary epidermal growth factor (EGF) is produced primarily by parotid glands., Objectives: The aim of this study was to determine whether salivary EGF can be detected in parotid saliva after PSRT and whether protein secretion is time dependent., Study Design: Salivary EGF concentration (pg/mL) was determined by enzyme-linked immunosorbent assay in stimulated parotid saliva before RT and at 3, 6, and 12 months after RT from 22 patients with head and neck cancer treated with PSRT., Results: Saliva samples were from 17 men and 5 women (age ranges 23-70 years and 46-71 years, respectively). At 6 months after RT, EGF concentration was 407 pg/mL lower than at baseline (P = .045). Twelve months after PSRT, parotid glands produce substantial amounts of EGF and other proteins, eventually approximating pre-RT levels, with recovery of salivary function., Conclusions: This proof-of-principle study shows that even proteins in picogram quantities, such as EGF, can be detected in saliva after PSRT., (Copyright © 2011 Mosby, Inc. All rights reserved.)

Objective: To evaluate the effect of astaxanthin on antioxidant parameters of salivary gland from diabetic rats. The hypothesis of the study was whether the supplementation of diabetic rats with astaxanthin might antagonize, or at least prevent, the defect in their antioxidative status., Design: Wistar rats (n=32) were divided in 4 groups: untreated control, treated control, untreated diabetic and treated diabetic rats. Astaxanthin (20mg/kg body weight) was administered daily by gavage for 30 days. On day 23, diabetes was induced by injection of alloxan (60 mg/kg body weight). After 7 days of diabetes induction, the rats were killed and submandibular and parotid removed. Superoxide dismutase (SOD), catalase, glutathione peroxidase and reductase activities and the content of thiol groups were determined. Data were compared by ANOVA and the Tukey test (p<0.05)., Results: Diabetes caused a reduction of SOD, and thiol content and increase of catalase and glutathione peroxidase activities of submandibular gland whilst in the parotid gland diabetes caused an increase of thiol content and no effect in the antioxidant system. The astaxanthin restores the enzymatic activities in the salivary gland, however does not prevent its oxidative damage., Conclusion: The submandibular gland presented more susceptibility to oxidative alterations induced by diabetes. Astaxanthin presented a positive effect on the oxidative protection of the salivary gland from diabetic rats., (2010 Elsevier Ltd. All rights reserved.)

Background: Handheld mobile phones (MPHs) have become a 'cultural' accessory device, no less so than a wrist watch. Nevertheless, the use of MPHs has given rise to great concern because of possible adverse health effects from exposure to the radiofrequency radiation (RFR) emitted by the device. Previous studies suggested correlation between MPH and salivary gland tumors., Objective: To evaluate whether MPH induces physiologic changes in the adjacent parotid gland, located on the dominant side, in terms of secretion rates and protein levels in the secreted saliva., Materials and Method: Stimulated parotid saliva was collected simultaneously from both glands in 50 healthy volunteers whose MPH use was on a dominant side of the head., Results: A significantly higher saliva secretion rate was noticed in the dominant MPH side compared with that in the non-dominant side. Lower total protein concentration was obtained in the dominant compared with the non-dominant MPH side among the right dominant MPH users., Conclusions: Parotid glands adjacent to handheld MPH in use respond by elevated salivary rates and decreased protein secretion reflecting the continuous insult to the glands. This phenomenon should be revealed to the worldwide population and further exploration by means of large-scale longitudinal studies is warranted.

Human saliva is a protein-rich, easily accessible source of potential local and systemic biomarkers to monitor changes that occur under pathological conditions; however, little is known about the changes in abundance associated with normal aging. In this study, we performed a comprehensive proteomic profiling of pooled saliva collected from the parotid glands of healthy female subjects, divided into two age groups 1 and 2 (20-30 and 55-65 years old, respectively). Hydrophobic charge interaction chromatography was used to separate high- from low-abundance proteins prior to characterization of the parotid saliva using multidimensional protein identification technology (MudPIT). Collectively, 532 proteins were identified in the two age groups. Of these proteins, 266 were identified exclusively in one age group, while 266 proteins were common to both groups. The majority of the proteins identified in the two age groups belonged to the defense and immune response category. Of note, several defense related proteins (e.g., lysozyme, lactoferrin and histatin-1) were significantly more abundant in group 2 as determined by G-test. Selected representative mass spectrometric findings were validated by Western blot analysis. Our study reports the first quantitative analysis of differentially regulated proteins in ductal saliva collected from young and older female subjects. This study supports the use of high-throughput proteomics as a robust discovery tool. Such results provide a foundation for future studies to identify specific salivary proteins which may be linked to age-related diseases specific to women.

Amphetamine induces xerogenic effects, but its mechanism of action and xerogenic potency are unknown. In the current in vivo study on the rat parotid gland, the effects of amphetamine on reflex-evoked and acetylcholine-evoked salivation were examined in the absence and presence of adrenergic and dopaminergic antagonists. Under anaesthesia, amphetamine increased the secretion of salivary fluid and the amount of protein therein in response to acetylcholine. Phentolamine abolished the increase in salivary flow and had no effect on the salivary protein concentration, whereas propranolol only reduced the salivary protein concentration. Reflex activation of the secretion evoked a well-maintained level of secretion that was reduced by amphetamine [50% inhibitory dose (ID(50)) 1.9 +/- 0.1 mg kg(-1) intravenously); the salivary protein concentration was increased in the presence of amphetamine. Phentolamine and haloperidol reduced the amphetamine-inhibitory effect on the reflex-evoked fluid response, whereas propranolol had no effect on the fluid response. The xerogenic effect of amphetamine is mainly exerted by central mechanisms involving alpha-adrenoceptors, while, indirectly, amphetamine causes secretion of protein by inducing the release of noradrenaline from glandular nerve terminals.

Objective: Parotid saliva flow is increased by mastication and its composition is also modified. The aim of this work was to clarify the relationships between flow rate, pH and protein concentration, during resting and short-duration simulated chewing, using continuous and fractional saliva collections., Design: Parotid saliva flow rate, pH and protein concentration, as it exits the Stensen's duct, were determined on seven subjects in response to one and 30 ipsilateral jaw clenches. To achieve this, we have developed a system able to collect parotid saliva and to measure continuously flow rate, pH and protein concentration and synchronised to the values at the exit of the duct., Results: With increase in flow rate, pH increased linearly and its protein concentration decreased linearly with the logarithm of the flow rate. With an increase in flow rate from 50 to 500 microl/min, the pH increased from 5.8 to 7.0 and the protein concentration decreased from 1.0 to 0.7 g/l. Measurements made on parotid saliva fractions confirmed the variations in pH and protein concentration with flow rate and showed that alpha-amylase concentration was significantly related to both salivary conductivity and protein concentration., Conclusions: Continuous measurements of salivary flow and composition offer a simple and convenient way to determine the precise relationships between different types of oral stimulation and parotid salivary flow and composition.

Objective: Saliva is important for oral health, including the prevention of dental decay. The purpose of the present work was to indicate whether the parotid gland has altered function in the elderly, particularly in terms of proteins secreted, and whether its functional status could be associated with the presence of root caries., Material and Methods: Ductal parotid gland secretion was obtained from 51 individuals: 21 elderly with carious roots (Patients), 20 elderly (Controls), and 10 adults (Young) without root caries. Pooled aliquots were analyzed by liquid chromatography/tandem mass spectrometry to yield lists of major proteins present in the three groups., Results: Approximately 200 unique proteins were detected, of which 73 were identified repeatedly with high confidence and therefore included in the comparison. Some of the differences observed, when comparing Patients with respectively elderly Controls and the Young, resembled changes found in patients with Sjogren's syndrome, a condition associated with dental decay. Other changes involved proteins that are likely to impact on the oral microbiota, such as the absence of dermcidin and the presence of collagen in Patients. Cystatin S, a putative indicator of caries, was present at a higher level in Patients., Conclusions: Parotid function tends to change upon aging, with possible consequences as to caries activity. However, analyses of individual samples revealed considerable variations in protein patterns.

Saliva is a body fluid with important functions in oral and general health. A consortium of three research groups catalogued the proteins in human saliva collected as the ductal secretions: 1166 identifications--914 in parotid and 917 in submandibular/sublingual saliva--were made. The results showed that a high proportion of proteins that are found in plasma and/or tears are also present in saliva along with unique components. The proteins identified are involved in numerous molecular processes ranging from structural functions to enzymatic/catalytic activities. As expected, the majority mapped to the extracellular and secretory compartments. An immunoblot approach was used to validate the presence in saliva of a subset of the proteins identified by mass spectrometric approaches. These experiments focused on novel constituents and proteins for which the peptide evidence was relatively weak. Ultimately, information derived from the work reported here and related published studies can be used to translate blood-based clinical laboratory tests into a format that utilizes saliva. Additionally, a catalogue of the salivary proteome of healthy individuals allows future analyses of salivary samples from individuals with oral and systemic diseases, with the goal of identifying biomarkers with diagnostic and/or prognostic value for these conditions; another possibility is the discovery of therapeutic targets.

To elucidate the localization of post-translational modifications of different classes of human salivary proteins and peptides (acidic and basic proline-rich proteins (PRPs), Histatins, Statherin, P-B peptide, and "S type" Cystatins) a comparative reversed phase HPLC-ESI-MS analysis on intact proteins of enriched granule preparations from parotid and submandibular glands as well as parotid, submandibular/sublingual (Sm/Sl), and whole saliva was performed. The main results of this study indicate the following. (i) Phosphorylation of all salivary peptides, sulfation of Histatin 1, proteolytic cleavages of acidic and precursor basic PRPs occur before granule storage. (ii) In agreement with previous studies, basic PRPs are secreted by the parotid gland only, whereas all isoforms of acidic PRPs (aPRPs) are secreted by both parotid and Sm/Sl glands. (iii) Phosphorylation levels of aPRPs, Histatin 1, and Statherin are higher in the parotid gland, whereas the extent of cleavage of aPRP is higher in Sm/Sl glands. (iv) O-Sulfation of tyrosines of Histatin 1 is a post-translational modification specific for the submandibular gland. (v) The concentration of Histatin 3, Histatin 5, and Histatin 6, but not Histatin 1, is higher in parotid saliva. (vi) Histatin 3 is submitted to the first proteolytic cleavage (generating Histatins 6 and 5) during granule maturation, and it occurs to the same relative extent in both glands. (vii) The proteolytic cleavages of Histatin 5 and 6, generating a cascade of Histatin 3 fragments, take place after granule secretion and are more extensive in parotid secretion. (viii) Basic PRPs are cleaved in the oral cavity by unknown peptidases, generating various small proline-rich peptides. (ix) C-terminal removal from Statherin is more extensive in parotid saliva. (x) P-B peptide is secreted by both glands, and its relative quantity is higher in submandibular/sublingual secretion. (xi) In agreement with previous studies, S type Cystatins are mainly the product of Sm/Sl glands.

Parotid saliva placed in 35-mm-diameter tissue culture dishes developed increasing surface viscoelasticity at the interface with air. A surface layer became visible with time, and was collected and analysed by protein electrophoresis which indicated that a single protein (pI 4.2; molecular mass approx. 6 kDa) predominated. Western blot analysis demonstrated that the major protein band reacted with an antiserum directed against the C-terminal of the calcium-binding salivary protein statherin. Matrix-assisted laser-desorption ionization-time-of-flight MS analysis gave a molecular mass of 5380 Da for the protein, corresponding to the molecular mass of statherin. Staining of film protein in electrophoresis gels was compared with statherin synthesized on a solid phase, and the mean statherin content of film formed from 1 ml of parotid saliva was measured as 7 nmol. The mean calcium content of the surface layer was 250 nmol. Surface rheology was greatly decreased in the presence of EDTA, whereas surface tension of saliva was unaffected by calcium chelation, suggesting that protein accumulated at the surface was unaffected. The results suggest that a layer rich in statherin forms at the interface of saliva and air, and that the surface rheology developed is dependent upon protein interactions mediated by calcium. The surface layer may enhance the function of saliva as a protective layer on the mucosal surfaces and teeth.

Background: The purpose of this study was to compare the sensitivity of parotid saliva to that of serum in detecting anti-SSA/Ro and anti-SSB/La autoantibodies in patients with Sjögren's syndrome., Methods: Forty patients and 20 controls participated in the study; all patients met the 1993 European Community criteria for the diagnosis of Sjögren's syndrome. Healthy controls were age- and sex-matched individuals with no signs or symptoms of Sjögren's syndrome. Serum and saliva samples were evaluated using AffiniTech SSA/Ro and SSB/La antibodies kits (AffiniTech, Ltd. Bentonville, AR, USA). The results were also compared with serological status of SS-A and SS-B as reported by an independent clinical laboratory., Results: Serum was significantly more sensitive than saliva in detecting SSA/Ro and SSB/La antibodies (P = 0.001). There was high agreement between the results with the AffiniTech kits and the independent laboratory (kappa = 0.80; P < 0.001). However, there was poor agreement between saliva and serum results (kappa = 0.174; P = 0.168)., Conclusions: The overall results appear to support that serum analysis is effective method for evaluating the presence of SS-A and SS-B autoantibodies.

Ectodermal dysplasias (EDs) constitute a group of conditions comprising developmental defects in two or more of the following tissues: hair, teeth, nails, and sweat glands. The aim of the present study was to contribute to a better understanding of salivary gland involvement in EDs. An ED group (n = 39, median age 12 yr; 24 males, 15 females) and a healthy age- and sex-matched control group were studied. Citric acid stimulated submandibular and parotid salivary flow rates and salivary concentrations, and output of total protein, acidic proline-rich proteins and histatins were analysed. The associations between quantitative and qualitative salivary parameters were also studied. In the ED group, 13 persons (33%) demonstrated a significantly reduced secretion of submandibular and/or parotid saliva, in addition to a low unstimulated and/or chewing-stimulated whole salivary flow. In the ED group as a whole, a reduced median secretory rate of submandibular saliva was found, whereas the median concentrations of some protein parameters were increased. However, the overall output of proteins was normal or reduced. Submandibular glands seemed to be more affected than parotid glands in EDs. In conclusion, salivary secretory tests are recommended in persons with known or suspected EDs.

Histatins, a family of small-molecular-weight, histidine-rich cationic salivary proteins, have been difficult to isolate in an efficient way by conventional procedures due to their anomalous interactions with chromatographic resins. In the present study we explored the possibility of developing a new isolation procedure based on recent observations that histatins associate with various metal ions, including zinc. Since solubility studies showed that histatin 5 forms precipitates with zinc under alkaline conditions, we investigated whether this characteristic could be exploited for the preparative isolation of histatins from salivary secretions. A fast and efficient two-step procedure was developed using zinc precipitation of histatins from human parotid secretion followed by final purification using reversed-phase high-performance liquid chromatography (HPLC). Analysis of zinc precipitates by Tricine-SDS-PAGE, cationic PAGE, HPLC, and mass spectrometry revealed the presence of the three major histatins, 1, 3, and 5, as well as statherin. The histatin yield obtained by the precipitation step was approximately 90%. Therefore, zinc precipitation of histatins from glandular salivary secretions is a novel, rapid, and effective means for the isolation of these proteins., (Copyright 2001 Academic Press.)

While fluoroquinolones are widely used in the treatment of various infectious disease, not enough attention has been paid to their adverse effects on salivary glands functions. In the present study, the effects of ofloxacin, a fluoroquinolone antibiotic, on rat parotid and submandibular gland functions, were examined in an acute experiment. Ofloxacin (OFLX) was administered intraperitoneally at various doses (20, 40, and 80 mg/kg). Pure parotid and submandibular saliva were collected intraorally by microployethylene tubes under anaesthesia using a dissecting microscope. Flow rate, amylase activity, total protein, and calcium concentrations were reduced by all doses of OFLX (20, 40, 80 mg/kg, P < 0.01) in parotid saliva. In parotid saliva, sodium and potassium were increased by doses of 20 and 40 mg/kg (P < 0.01) and 20, 40, 80 mg/kg (P < 0.01) respectively. In submandibular saliva, flow rate, total protein and calcium concentrations were significantly reduced by all doses (20, 40, 80 mg/kg, P < 0.01). Sodium and potassium concentrations were also increased by a dose of 80 mg/kg, (P < 0.05) in submandibular saliva. It is concluded that ofloxacin inhibits rat salivary gland functions, which might be observed as a side-effect in humans. Properties of fluoroquinolones to alter intracellular cAMP and calcium levels and their ability to suppress DNA, RNA and protein synthesis of acinar cells might be possible reasons for the observed changes.

Salivary tissue kallikrein is stored in an active form in human salivary glands. Pre-kallikrein has been demonstrated in mixed saliva, but it is not clear if the various salivary glands contribute equally. This study set out to determine if pre-kallikrein is present in human parotid and submandibular salivas at rest, whether levels change during stimulation, and to compare the pattern of pre-kallikrein and kallikrein secretion with that of total protein. Resting and citric acid-stimulated parotid and submandibular, and gum-stimulated parotid saliva samples were collected from 6 healthy subjects. Salivary flows were determined gravimetrically. Total protein concentration and kallikrein enzymic activity were assayed using standard techniques. Pre-kallikrein was assayed following trypsinisation of duplicate samples. Pre-kallikrein was present in parotid and submandibular ductal saliva. Proportions of pre-kallikrein and active kallikrein were similar in salivas secreted at rest and during stimulation, and both outputs mirrored protein output in both major glands. Gum-stimulated parotid saliva showed lower activity than resting, and no differences were seen between resting and stimulated submandibular samples.

Secretion of fluid and proteins by salivary cells is under the control of parasympathetic and sympathetic autonomic nerves. In a recent study we have shown that, in the rat submandibular gland, autonomic nerves can also increase the secretion of IgA, a product of plasma cells secreted into saliva as SIgA (IgA bound to Secretory Component, the cleaved poly-immunoglobulin receptor). The present study aimed to determine if parotid secretion of SIgA is increased by autonomic nerves and to compare SIgA secretion with other parotid proteins stored and secreted by acinar and ductal cells. Assay of IgA in saliva evoked by parasympathetic nerve stimulation immediately following an extended rest period under anaesthesia indicated that it had been secreted into intraductal saliva in the absence of stimulation during the rest period. The mean rate of unstimulated IgA secretion (2.77+/-0.28 microg min(-1) g(-1)) and the 2.5-fold increase in IgA secretion evoked by parasympathetic stimulation were similar to results found previously in the rat submandibular gland. Sympathetic nerve stimulation increased SIgA secretion 2.7-fold, much less than in the submandibular gland. SDS-PAGE and Western blot analysis with anti-IgA and anti-Secretory Component antibodies confirmed that SIgA was the predominant form of IgA in saliva. Acinar-derived amylase and ductal-derived tissue kallikrein were more profoundly increased by parasympathetic and particularly sympathetic stimulation than SIgA. Overall, the results of the present study indicate that SIgA forms a prominent component of unstimulated parotid salivary protein secretion and that its secretion is similarly increased by stimulation of either autonomic nerve supply. The secretion of other parotid salivary proteins that are synthesized and stored by acinar or ductal cells is upregulated to a much greater extent by parasympathetic and particularly sympathetic stimulation.

The protein-protein interaction between [soluble NSF attachment protein (SNAP) receptor] (SNARE) proteins found in the lysate of parotid acinar cells was investigated. Immunoblotting analysis showed that parotid acini contain both syntaxin-4 and SNAP-23, plausible candidates of target membranes (t-) SNAREs in non-neuronal cells. However, when vesicle-associated membrane protein (VAMP)-2 was immunoprecipitated from lysates of parotid acinar cells, syntaxin-4 and SNAP-23 were not coprecipitated with VAMP-2, although syntaxin-1 and SNAP-25, t-SNAREs in neuronal cells, were clearly coprecipitated with VAMP-2 from brain lysates. Inversely, when syntaxin-4 was immunoprecipitated from parotid lysates, SNAP-23, Munc18c, and N-ethylmaleimide-sensitive fusion protein (NSF) were coprecipitated, but VAMP-2 was again undetectable. When proteins in the crude secretory-granule fraction were biotinylated and then immunoprecipitated with anti-VAMP-2, 35- and 80-kDa proteins were coprecipitated along with VAMP-2. These results suggest that the interaction between syntaxin-4, SNAP-23 and VAMP-2 is fairly weak and their concentrations in the cell lysate are insufficient to make a readily detectable complex, and that bindings between these proteins are hindered by other proteins in parotid acinar cells.

To address whether there are associations between the peptide composition of human parotid saliva and dental decay (caries) experience, we have characterized the peptides from parotid ductal saliva collected from nine adults who have remained free from dental caries (mean age = 59.2; Decayed Missing Filled Surfaces index [DMFS] = 0) and nine individuals who have experienced caries (mean age = 51.2; mean DMFS = 38.4). Ethanol-soluble peptides were size-fractionated on columns of Bio-Gel P-2; the salivary peptides derived from caries-susceptible subjects appeared larger than those found in the saliva of caries-free subjects. Peptides were then resolved into 19 species by cation exchange HPLC. Sequence analysis identified 18 peptides that appear to be proteolytic cleavage products of the basic proline-rich proteins IB-4, IB-5, IB-7, IB-8b, and P-B. The peptides that were more abundant in saliva obtained from the caries-free group differed from those isolated from the caries-susceptible group. The median peptide concentration of one possible precursor protein, IB-7, was found to be higher in saliva collected from caries-free individuals than in that from caries-susceptible individuals. Although differences were found in the phenotypes of proline-rich proteins expressed by these groups of caries-free and caries-susceptible subjects, no statistically significant associations were observed among proline-rich phenotypes and the level of any peptide. Collectively, our results indicate that proteolytic processing of parotid salivary proteins differs among individuals who have remained caries-free and those who have experienced dental decay.

Ferret parotid saliva has been analysed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and two-dimensional electrophoresis (2-DE) to determine, for the first time, its protein composition. SDS-PAGE, in combination with Coomassie Brilliant Blue (CBB) staining, revealed up to 20 bands and the patterns were characterised by major protein constituents of Mr 105000, 51000, 47000, 33000, 22000 and 16 400 common to all samples from all animals. Sequential samples collected from the same animal during prolonged stimulation of the parasympathetic nerve (40 min at 40 Hz) showed subtle but reproducible protein changes. Saliva collected from different animals varied widely in the amount of a protein Mr 66000. 2-DE, in combination with silver staining, revealed up to 300 spots and the patterns were characterised by major protein constituents of Mr 105000 (pI 6.3-7.2), Mr 66000 (pI 4.7-5.3), Mr 51000 (pI 5.0-5.7), Mr 47000 (pI 6.0-7.5), and Mr 33000 (pI 4.7-6.0). Many of the polypeptide spot clusters consisted of one or more horizontal strings of spots suggesting extensive microheterogeneity. Both SDS-PAGE and 2-DE indicated that the protein patterns of ferret parotid saliva evoked by electrical stimulation of the parasympathetic nerve in the absence or presence of atropine are similar, i.e., the protein composition of the atropine-resistant nonadrenergic, noncholinergic (NANC) secretion is similar to that of saliva evoked in the absence of muscarinic receptor blockade.

The rat parotid gland produces a number of well-characterized secretory proteins. Relatively little is known, however, about the onset of their synthesis and cellular localization during gland development. Secretory protein expression was studied in parotid glands of fetal and postnatal rats using light and electron microscopic immunocytochemistry and Northern blotting. Amylase, parotid secretory protein (PSP), common salivary protein-1 (CSP-1), and SMGB were first detected by immunofluorescence in parotid glands of 18 day fetuses. By 5 days after birth, light and electron microscopic immunolabeling localized all of these proteins to the secretory granules of developing acinar cells. Labeling of acinar cells for DNAse I, however, was not observed until 18 days after birth. Between 9 and 25 days, CSP-1 and SMGB reactivity of acinar cells declined, but increased in intercalated duct cells. After 25 days, CSP-1 and SMGB were found only in intercalated ducts, and amylase, PSP, and DNAse I were restricted to acinar cells. Levels of CSP-1 and SMGB mRNA were relatively constant through 21 postnatal days, but declined significantly after that. Amylase and PSP mRNA increased rapidly and continuously from five days after birth to the adult stage. In contrast, DNAse I mRNA was not detectable until 18 days after birth. The immunocytochemical and molecular analyses define three basic patterns of protein expression in the rat parotid gland: proteins whose synthesis is initiated early in development and is maintained in the acinar cells, such as amylase and PSP; proteins that are initially synthesized by immature acinar cells but are restricted to intercalated ducts in the adult gland, such as CSP-1 and SMGB; and proteins that are synthesized only by mature acinar cells and first appear during the third postnatal week, such as DNAse I. The parotid gland exhibits four distinct developmental stages: prenatal, from initiation of the gland rudiment until birth; neonatal, from 1 day up to about 9 days postnatal; transitional, from 9 days to 25 days of age; and adult, from 25 days on. Although differences exist in timing and in the specific proteins expressed, these developmental stages are similar to those seen in the rat submandibular gland. Additionally, the results support the suggestion that intercalated ducts may differentiate from the neonatal acini.

Parotid and mandibular saliva was obtained from red kangaroos by concurrent acetylcholine isoprenaline stimulation. Salivary proteins were separated by horizontal electrophoresis on either cellulose acetate or starch gels and assessed by specific staining techniques for 23 enzymes commonly found in mammalian tissues and body fluids. Parotid saliva was positive for acid phosphatase, alpha-amylase, carbonic anhydrase, glucose-6-phosphate dehydrogenase, sorbitol dehydrogenase and superoxide dismutase activities. Mandibular saliva was positive for alcohol dehydrogenase in addition to the above six enzymes. The kangaroo salivas lacked activity for alkaline phosphatase, beta-galactosidase and non-specific esterase which occur in saliva from some mammalian species.

Previous studies of experimental diabetes have demonstrated changes in the levels of specific salivary proteins. The present study is part of a larger effort aimed at elucidating the mechanism(s) by which insulin regulates salivary protein expression in the rat parotid gland. Diabetes was induced in 2-3-month-old male Fischer 344 rats by injection of streptozotocin (STZ). After 30 days one group of rats was given insulin for 7 days. Untreated rats served as controls. As previously observed, parotid acinar cells from diabetic rats accumulated lipid and contained occasional crystalloid lysosomes. Quantitative immunogold labeling of secretory granules in diabetic glands revealed decreases of 30-60% for proline-rich-proteins (PRPs), amylase and parotid secretory protein (PSP), but labeling for acidic epididymal glycoprotein (AEG) was unchanged. The response to insulin treatment was variable: amylase and PSP labeling were partly restored, but PRP and AEG labeling showed little change. Photoaffinity labeling of cyclic AMP receptor proteins (cARP) showed changes in several tissues including a consistent increase in the diabetic parotid gland. Immunogold labeling of secretory granules with antibody to cARP was similar in control and diabetic parotids, but nuclear and cytoplasmic label was decreased in diabetic acinar cells. These results indicate that STZ-diabetes and insulin reconstitution cause variable changes in the expression of parotid secretory proteins. Changes in cARP levels suggest that the insulin and cyclic AMP pathways may interact in regulating expression of salivary secretory proteins.

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.

The objective was to determine the percentage contribution of parotid saliva to whole saliva and to the saliva at 11 sites in the mouth, when flow rate was unstimulated or stimulated with chewing-gum. The marker substance used was alpha-amylase, as this is in much higher concentration in parotid saliva than in secretions from other salivary glands. Formulae were derived for calculation of the minimum, maximum, and mean percentage contributions of parotid saliva to saliva in different areas of the mouth. The results, from 10 individuals, showed that the contributions of parotid to unstimulated and stimulated whole saliva averaged 30.1% and 35.6%, respectively, whereas the corresponding values for samples from the region vestibular to the upper molars were 56.1% and 61.4%, but only 2.8% and 6.8% for samples from an area vestibular to the upper incisors. Thus parotid saliva was not evenly distributed throughout the mouth. Stimulated samples mostly contained significantly higher proportions of parotid saliva, but the distribution of the parotid saliva was still extremely variable. Because the different regions of the mouth are not exposed to the same fluid environment, this may influence the site-specificity of supragingival calculus deposition and of various diseases such as dental caries.

Objective: The objective of this study was to determine if there were any differences in the parotid saliva output and composition related to caries activity., Study Design: Stimulated parotid saliva samples were collected from 85 healthy young adults, caries-active or caries-free. Flow rates were determined, and samples were analyzed for pH and buffer capacity, total protein, electrolytes, proteins with a high performance liquid chromatography method, and histatins., Results: There were no differences in flow rates or pH, but buffer capacity was higher in women than in men, and K+ and Cl- were both slightly higher in the caries-active group. The women had a significantly higher total protein concentration, as well as higher concentrations of each of the individual protein components assayed. There were no differences attributable to caries activity., Conclusions: Significant sex differences in salivary protein concentrations exist. Caries activity may be related to some salivary electrolyte alterations, but not to protein composition.

Detection of human parotid salivary proteins by dansylation and UV-transillumination after sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) has been compared with Coomassie Blue R-250 and silver staining procedures. Dansylation gives superior results in terms of both resolution and sensitivity, especially with basic proline-rich proteins (PRPs).

Bacterial colonisation of oral surfaces by microorganisms may be dependent on their interaction with specific host receptor molecules. Primary oral colonisers are known to remove specific proteins from parotid saliva. The aim of this study was to determine whether these interactions facilitate microbial attachment to a surface and hence identify specific salivary components as putative host receptor molecules. Parotid saliva was resolved by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto nitrocellulose membranes. Suspensions of fluorescently labelled microorganisms were incubated with the blots and salivary components with adherent bacteria identified as fluorescent bands under ultraviolet (UV) transillumination. Species of streptococci known to be early colonisers of the clean tooth surface were found to adhere specifically to certain salivary proteins, especially to basic proline-rich proteins (PRPs). Polymorphic variations in these patterns could form the basis of differences in oral microflora, susceptibility to oral infections and consequent disease.

Protein deficiency results in an increased susceptibility to dental caries, suggesting that oral host-defense properties are compromised. An important component of oral host defense is salivary gland function, which is affected by both protein deficiency and diet consistency. This study describes the effects on rat parotid gland growth and secretory function induced by feeding rats diets of normal (20%) or moderately low (7%) protein content, provided in either a powdered or solid form. In addition, since protein deficiency may result in a secondary zinc deficiency which, in turn, may affect salivary gland function, the effects of these diets on liver zinc concentration were also measured. From 22 to 47 days of age, rats (18/group) were fed the following diets: normal protein, powdered; normal protein, solid; low protein, powdered; and low protein, solid. With each diet consistency, liver zinc was higher for the normal protein group. Within each protein level, liver zinc was higher for the solid diet. This latter observation suggests that food mastication and the resultant stimulation of salivary gland function may also play a role in zinc metabolism. With the normal-protein diet, parotid gland weight was higher for the solid diet; with the low-protein diet, parotid gland weight was similar for both consistencies and did not differ from that of the group fed the "normal protein, powdered" diet. For both consistencies, parotid saliva protein concentration was greater for malnourished rats.(ABSTRACT TRUNCATED AT 250 WORDS)

A specific radioimmunoassay for human carbonic anhydrase (CA) VI has been developed and used to determine the concentrations of the enzyme in saliva. The assay detected as little as 200 pg of CA VI and the antibody used did not cross-react with CA II or other salivary proteins. The method showed an intra-assay variation of 8.5% and an inter-assay variation of 16.9%. The concentration in parotid saliva varied over a wide range (from 9.7 micrograms/ml to 121 micrograms/ml) with an average value of 47.0 +/- 39.2 (SD) micrograms/ml (n = 50). The mean secretion rate of CA VI from the combined parotid glands was 42.8 +/- 37.9 micrograms/min. CA VI represented about 3% of the total protein in parotid saliva.

The flow rate of extra-parotid and parotid saliva was compared in patients with Bell's palsy and in healthy volunteers. Samples were analysed for the concentration of total protein and seven acute-phase proteins. There was no difference between younger and older patients with regard to oral status, salivary flow rate, total protein or acute-phase proteins, in either extra-parotid or parotid saliva. No significant difference in flow rate for both extra-parotid and parotid saliva was found in the Bell's palsy patients in comparison with the controls. In the patients the salivary flow rate from the parotid gland on the paralysed side was slightly lower than on the healthy side, but not to a significant extent. The quantity of total protein was lower in the extra-parotid saliva in the patient group; there were no differences between the two groups with regard to parotid saliva. We were able to demonstrate small amounts of various acute-phase proteins in the control group. In the patients we found higher quantities per minute of acute-phase proteins in both extra-parotid saliva and parotid saliva than in the controls. In extra-parotid saliva there were significant differences in haptoglobulin, alpha 2-macroglobulin, C3-complement factor and ceruloplasmin; in parotid saliva the differences in haptoglobulin and ceruloplasmin were significant. However, there was a large inter individual variation in both groups studied. In the patient group no significant difference in the secretion of acute-phase proteins from the parotid gland could be demonstrated between the paralysed side and the healthy side.(ABSTRACT TRUNCATED AT 250 WORDS)

The protein constituents of cat parotid saliva evoked by parasympathetic stimulation or the infusion of bethanechol have been compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The SDS-PAGE patterns were highly reproducible for an individual cat and revealed up to 30 polypeptide bands in microlitre volumes of unconcentrated saliva. Only minor and predominantly quantitative differences were detected in the salivary protein composition of different cats. SDS-PAGE demonstrates quantitative rather than qualitative changes in the protein composition of saliva evoked in response to different stimuli. The latter included preliminary observations on the effect of vasoactive intestinal peptide (VIP) on the bethanechol-evoked response.

Stimulated parotid salivary flow rates were compared in elderly normotensive, hypertensive, and controlled hypertensive African-Americans, the latter group taking hydrochlorothiazide (HCTZ). The normotensive group consisted of 15 healthy unmedicated subjects with systolic blood pressures of less than 150 mm Hg and diastolic pressures less than 90 mm Hg. The hypertensive group consisted of 10 unmedicated subjects with systolic pressures greater than 160 mm Hg and diastolic pressures greater than 100 mm Hg. The controlled hypertensive group consisted of 20 subjects taking HCTZ (50 mg, daily) with controlled blood pressures similar to the normotensive control group. Stimulated parotid salivary samples were collected from each subject. A 2% citrate solution applied to the dorsum of the tongue was used for stimulation. The results showed no significant differences in stimulated parotid flow rates between normotensive and uncontrolled hypertensive subjects. However, the medicated, controlled hypertensive subjects had a significant reduction of stimulated parotid salivary flow rates compared to both the normotensive and hypertensive groups.

This study examined parotid salivary flow rate and composition in three groups of differently treated diabetics and a control group of non-diabetics. The study population was composed of edentulous African-Americans at least 65 years of age. Group A was the control, Group B insulin-dependent (Regular Iletin, U-100 qd.), Group C controlled by oral medication (Tolbutamide, 500 mg qd.), and Group D was diet controlled. All diabetic patients had serum glucose values under 250 mg/dl. Salivary flow rates, secretory IgA, electrolytes (Na+, Cl-, K+, Ca++) and total protein concentrations were evaluated. The results showed no significant differences between groups with respect to salivary flow rates, electrolytes and IgA concentrations. Additionally, all diabetic groups demonstrated a significantly lower salivary total protein concentration when compared to the controls. There appears to be no evident decrease in salivary flow rate in these three differently controlled diabetic groups compared with healthy non-diabetics.

C57BL mice were injected intraperitoneally with 300,000 Cetus units/day of human recombinant interleukin-2 (rIL-2) for 2, 4, and 5 days to study its effect on salivary gland function and morphology. The pilocarpine-stimulated parotid salivary flow was collected via cannulation of the glandular duct. Total salivary protein was assayed spectrophotometrically, salivary electrolytes were determined by atomic absorption, and glandular lymphoid cell infiltration was evaluated histologically. After 5 days of rIL-2 administration salivary output and total salivary protein concentrations were reduced significantly. Similar changes, albeit to a lesser extent, were observed following 2 and 4 days of rIL-2 treatment. Increased lymphoid infiltration of the salivary glands was observed, and was directly related to the duration of rIL-2 administration. The effect of the lymphokine on the parotid gland gradually dwindled after cessation of treatment: 4 days posttreatment this salivary gland showed signs of recovery, which at 10 days proved to be complete. The possible use of this animal model in the study of lymphocyte-induced salivary gland diseases is discussed.

Parotid salivation was stimulated by infusion of bethanechol chloride into anaesthetized brushtail possums to ascertain maximal flow rates, salivary composition and dietary adaptations of salivary function. Secretion rates for one gland ranged from 5.3 +/- 0.16 to 84.3 +/- 3.20 microliters/min (2.4 +/- 0.07 to 37.8 +/- 1.43 microliters/min per kg body weight). Salivary osmolality (160.8 +/- 15.39 to 248.2 +/- 8.70 mosmol/kg) and the concentrations of Na (63.1 +/- 10.93 to 124.1 +/- 5.52 mmol/l) and HCO3 (19.5 +/- 3.41 to 89.0 +/- 3.19 mmol/l) were positively correlated with flow rate. The concentrations of urea (7.8 +/- 0.64 to 4.6 +/- 0.33 mmol/l), PO4 (2.6 +/- 0.25 to 0.96 +/- 0.10 mmol/l), H+ (46.2 +/- 16.30 to 9.0 +/- 1.51 nequiv/l), K (21.4 +/- 3.73 to 13.7 +/- 2.33 mmol/l), Ca (3.7 +/- 0.53 to 2.2 +/- 0.27 mmol/l) and Mg (0.3 +/- 0.07 to 0.04 +/- 0.007 mmol/l) fell with increasing flow rate. The relations between flow rate and amylase activity (22.6 +/- 10.47 to 10.5 +/- 3.68 mu kat/l), protein concentration (2.7 +/- 0.61 to 1.3 +/- 0.28 g/l), and Cl concentration (62.1 +/- 6.88 to 50.6 +/- 6.37 mmol/l) were inconsistent between experiments. Salivary Na/K ratios were not decreased by infusion of aldosterone (2.2-22.2 nmol/h for 5 h), showing that the gland of Na-replete possums is unresponsive to short-term increases in mineralocorticoids. The low salivary amylase activity, less than that of kangaroos, presumably reflects the interaction of evolutionary history of the possum with its natural low-starch diet.(ABSTRACT TRUNCATED AT 250 WORDS)

This study examined the effect of pilocarpine on caries and saliva composition in rats with compromised salivary gland function. Eight litters of specific-pathogen-free female Sprague-Dawley rats were divided into five groups for surgery. Partial desalivation was performed in either of three ways: (1) Both parotid ducts were ligated; (2) both submandibular/sublingual (SM/SL) glands were excised, or (3) one parotid duct was ligated, and submandibular/sublingual glands were removed unilaterally. Completely desalivated animals and unoperated animals served as positive and negative controls, respectively. One week following surgery, groups were subdivided so that half of each group had osmotic pumps implanted which delivered pilocarpine HCl (24 mg/kg/day). All animals were infected with Streptococcus sobrinus 6715 and fed cariogenic diet ad libitum for 28 days. Caries scores, microbiological data, and saliva flow rate and composition were determined for all animals. Animals which had both SM/SL glands removed and received pilocarpine developed significantly lower sulcal caries (p < or = 0.05) compared with the animals that had both SM/SL removed but did not receive pilocarpine. The concentration of protein in parotid saliva in these pilocarpine-treated animals was unaffected, and no differences were observed in the electrophoretic profiles on SDS-PAGE. Pilocarpine appears to exert its greatest caries-protective effect when the parotid glands remain intact.

Electron microscopic immunocytochemistry was used to investigate the distribution of four secretory proteins within the granules of human parotid and submandibular gland acinar cells. The results demonstrate that the distribution of the individual proteins differs significantly. Amylase is concentrated within the electron-dense regions of the granules, whereas agglutinin is present mainly in the regions of lower density. In contrast, the proline-rich proteins and histatins are distributed throughout the content of the granules and do not appear to be concentrated in either the high- or low-density regions. The results also suggest that different mechanisms, such as self-aggregation or ionic complexes with metal ions and charged macromolecules, may be involved in the packaging of individual proteins within the same granule. Further, they also show that among the salivary glands of various species, the intragranular distribution of similar proteins (e.g., amylase and proline-rich proteins) may differ.

The present investigation was undertaken to study the effect of adrenoreceptor modulators on the expression of salivary proteins. Sprague-Dawley rats were treated for 10 consecutive days with adrenergic agonists isoproterenol, dobutamine, terbutaline, salbutamol, methoxyphenamine, or methoxamine. Antiserum to selected salivary proteins was used to compare the concentration of these proteins in the submandibular and parotid glands of treated animals. Chronic treatments of rats (50 mumol/kg body weight for 10 d) with either isoproterenol or dobutamine induced synthesis of a cysteine-proteinase inhibitor (cystatin) in the submandibular glands. When isoproterenol was injected concomitantly with the mixed beta-antagonist propranolol or the beta 1-adrenergic antagonists metaprolol, protocol, or atenolol, the induction of cystatin was totally suppressed. However, the beta 2-antagonist, ICI-118551, produced only partial reduction in cystatin induction elicited by isoproterenol. On the contrary, rats treated with either isoproterenol or beta 1-agonists demonstrated a significantly reduced concentration of serine-proteinase kallikrein in submandibular glands. The decrease observed in submandibular kallikrein of rats treated with isoproterenol was prevented by concomitant treatment with beta 1-antagonists but not with beta 2-antagonists. Because kallikreins are produced by ductal cells and cystatins are produced by acinar cells of submandibular glands, these observations suggest that there may be differential control of expression of proteins synthesized by ductal and acinar cells. Chronic treatment of rats with nonselective beta-agonist isoproterenol or beta 1-selective agonists increased markedly the proline-rich proteins (PRP) in parotid glands, but the parotid amylase concentration was not significantly affected by beta-adrenergic agonists.(ABSTRACT TRUNCATED AT 250 WORDS)

Rabbit polyclonal antibodies against isoproterenol-induced mouse proline-rich proteins (PRPs) were used to localize PRPs in the parotid salivary glands of normal adult BALB/c mice. The antibodies recognized both acidic-type and basic-type PRPs. Immunoblotting experiments revealed that the glands contained an acidic-type and a basic-type PRP. Parotid gland tissue was fixed with Karnosky's fixative and embedded in Lowicryl resin at low temperature. PRPs were localized at the electron microscope level using an indirect post-embedding staining technique with protein A-gold. The secretion granules of the acinar cells were strongly labelled. Pre-absorption of the antibody with purified acidic-type and basic-type PRPs indicated that the basic-type PRP is mainly located at the periphery of the granules but that the acidic-type PRP is more evenly distributed within the granules. Pre-absorption of the antibody with alpha-amylase did not affect the staining pattern, suggesting minimal cross-reactivity. PRPs were also detected within the rough endoplasmic reticulum and the Golgi apparatus of acinar cells, within the granules of the proacinar cells and in the lumena of the ducts, but not within the intercalated or striated duct cell granules.

These three neuropeptides were measured at daily baseline values by radioimmunoassay. Stimulated parotid saliva was collected from 31 subjects using a modified Carlson-Crittenden device affixed over Stenson's duct. Methionine enkephalin-like immunoreactivity ranged from 6.6 to 11.7 fmol/ml, with a mean of 9.3 fmol/ml. Substance P-like immunoreactivity ranged from 6.1 to 12.6 fmol/ml, with a mean of 9.3 fmol/ml. beta-Endorphin-like immunoreactivity ranged from 1.2 to 3.6 fmol/ml, with a mean of 2.6 fmol/ml. This is believed to be the first documentation of methionine enkephalin- and substance P-like activities in human parotid saliva and the first demonstration of beta-endorphin-like activity in any type of human saliva. Substance P-like activity was significantly higher in morning than evening samples; beta-endorphin-like activity also tended to be higher in the morning samples. Substance P and beta-endorphin-like immunoreactivities covaried in a significant positive manner, suggesting either common control mechanisms or similar responses to physiological variables.

This analysis of physiological, biochemical and molecular changes related to aging was made in 3-, 12- and 24-month-old rats. The salivary gland weight/body weight ratio and the structural membrane proteins did not change with age for either gland, but a significant age-related decline in DNA synthesis for both glands was detected, unrelated to the hormonal responsiveness at the level of the plasma membrane. There was a marked increase in the concentration of soluble proteins in adolescent parotid gland and, for the two older age groups, in submandibular gland. The saliva flow rate was different when expressed as volume per time, as volume per time and g glandular wet weight, and/or kg body weight. The concentration of secreted proteins was not affected by age in either gland. The total amount of proteins secreted over 30 min revealed no age-related perturbation for the parotid gland, but showed a significant age-related increase in submandibular saliva. Sodium dodecyl sulphate-polyacrylamide gel analysis revealed changes in the protein bands between 39 and 50 kDa in the Coomassie blue-stained gels from 12-month-old animals. Amylase showed an initial increase (12 months), followed by a marked decline in its activity in parotid saliva. The glandular supernatant had low residual cellular amylase activity after stimulation. Therefore, secretory impairment with age after pilocarpine-isoproterenol stimulation was excluded. Analysis of total RNA showed a pronounced decrease of amylase mRNA in the parotid gland between 12 and 24 months of age. No amylase mRNA was expressed in any of the submandibular samples. For epidermal growth factor, total saliva showed a decrease with age. It seemed that the submandibular gland followed the same picture with age as the parotid gland, with a specific decline in the biosynthesis of single secretory proteins.

Parotid salivation was investigated in two species of potoroine marsupial, Aepyprymnus rufescens and Potorous tridactylus to ascertain flow rates and composition, the buffer capacity of the saliva with respect to possible dependence of these animals on foregut fermentation, and the similarity of anion excretion patterns to those of the kangaroo parotid. Under anaesthesia neither species secreted spontaneously and secretion was stimulated by intravenous infusion of carbachol, bethanechol and isoprenaline. Under cholinergic stimulation in Aepyprymnus, the concentrations of Na, Cl, HCO3 and osmolality were positively correlated with flow rate, whereas K, Mg, PO4, H+ and urea were negatively correlated with flow. Amylase activity and the concentrations of protein and Ca showed no consistent relation to flow. Relative to Aepyprymnus, saliva of Potorous had much lower amylase activity and amylase activity per gram protein, lower concentrations of urea and Ca, and higher Na. Protein, K and HCO3 concentrations were similar in both species. The plasma of both species had similar electrolyte concentrations, but Potorous had lower protein, urea, osmolality and amylase activity. Plasma amylase activity in Aepyprymnus rose during cholinergic stimulation to levels in excess of rodent plasma. Isoprenaline infusion in Aepyprymnus increased salivary amylase activity and concentrations of protein, Ca, HCO3 and PO4, and reduced the concentrations of Cl and H+. The patterns of anion excretion in the two potoroine marsupials were dissimilar to those of the kangaroo parotid suggesting that parotid fluid secretion is not HCO3 driven to the same extent as that of Kangaroos. Buffer anion concentrations and secretion rates were similar to koalas and low relative to kangaroos, indicating that these potoroines do not rely on foregut fermentation.

Isoelectric focusing of human salivary proteins with carrier ampholyte-isoelectric focusing systems requires prior desalting and concentration of samples, a procedure which is time-consuming and requires relatively large volumes of samples. By contrast, immobilized pH gradient gels are more tolerant to salt loads. Thus pretreatment of samples consists only of centrifugation prior to isoelectric focusing. If larger loads (greater than 50 micrograms) are required, the samples may be concentrated by lyophilization and reconstitution in a smaller volume of water or by dialysis against 30% w/v polyethylene glycol. Immobilized pH gradient polyacrylamide gels (incorporating a hybrid carrier ampholyte system) of two pH ranges (pH 4-9 and pH 3.5-5.0) have been used to separate the proteins in human parotid saliva. The effects of urea on focused patterns were studied; in pH 4-9 gels it gave improved resolution of protein bands, whereas in pH 3.5-5.0 gels it prevented protein precipitation. The salivary proteins were then visualized by staining with Coomassie Brilliant Blue G-250 or a silver procedure. Using the latter, 25-30 well-resolved bands were formed on a pH 4-9 gel loaded with 20 micrograms of proteins. The method offers considerable advantages compared with carrier ampholyte-isoelectric focusing.

Changes in the concentrations of ions (K+, Cl-, Na+), total protein, amylase activity and flow rate of parotid saliva were observed by tongue stimulation with 3% tartaric acid various intervals in a subject after about one hour rest. The results may be summarized as follows: 1) After about one hour rest state, the values of each item were K+: 42-56 mEq/l, Cl-: 8-28 mEq/l, Na+: 1-10 mEq/l, total protein: 5.7-11 mg/ml, amylase activity: 2-6.5 x 10(3)U/ml and flow rate: 0.03-0.05 ml/min. 2) The changes were classified into two groups based on the recovery from peak to rest values. One group showed short recovery times (Cl-: 20 min., Na+: 8 min. and flow rate: 2 min.) and the other group had rather longer recovery times (K+: 40 min., total protein: 55 min. and amylase activity: 40 min.). The differences between these two groups were clear when the stimulus intervals were 30 sec. to 5 min. 3) When the tongue was stimulated every 15 sec. all items showed first responses, after that, the all values except the flow rate remained unchanged. 4) Different values in ions and protein concentrations and amylase activity were observed when the flow rate was similar before and after stimulation. 5) The recovery time of the responses of protein concentration and amylase activity to stimulation was about one hour, and a rest period of more than one hour is necessary to analyze the concentration of total protein and amylase activity in parotid saliva.

Male rats were maintained on a regimen of twice daily intragastric administration of ethanol or a calorifically equivalent sucrose solution for thirty days. A second control group received no intragastric solution and all groups received chow and water ad libitum. Parotid saliva elicited by pilocarpine was collected by unilateral duct cannulation. The parotid flow rate over the initial post-stimulatory five minute period was raised by 44% in ethanol-dosed rats and the salivary sodium concentration was also raised, in line with higher flow rate. There were no histopathological changes related to ethanol or sucrose dosing, but stereological analysis showed a 64% increase in the proportional volume of intralobular vascular tissue in ethanol-dosed rats. These quantified histological findings suggest that parotid intralobular haemodynamics may be altered after chronic ethanol-dosing and this may contribute to the hypersecretory response exhibited by the ethanol-dosed rats.