Postprandial transformations of enzymatic and hormonal properties of saliva and blood

In 14 volunteers, saliva from both parotid, submandibular and sublingual glands were collected by capsules under stimulation of sialosis with citric acid or alimentary trial breakfast. It was taken immediately and on the 1st and 3rd hours of postprandial response. In saliva and the blood serum, alpha-amylases, trypsin, common protein, thyrotropin, thyroxine, triiodthyronin, luteinizing hormone, follicle-stimulating hormone, prolactin, progesterone, oestradiol and hydrocortisone were assessed by means of immuno-assay technique. All but oestradiol hormones had a lower concentration in the saliva than in the blood serum. The concentration and deficits of hormones and trypsin in saliva of submandibular and sublingual glands is higher, than in saliva of parotid glands, the latter having a higher alpha-amylolytic activity. The share of p-amylase in comparison with s-amylase in saliva of parotid glands is lesser than in saliva of submandibular and sublingual glands. In alimentary stimulation of sialosis, the saliva with higher amylolytic and tryptic activity, higher concentration of thyrotropin and thyroxine was found than under a non-alimentary stimulation. After the 1st and the 3rd hours following a trial breakfast, in response to a non-alimentary stimulation of sialosis the saliva was found to preserve properties of a postprandial saliva.     

Modulating effects of prostaglandins on parasympathetic-mediated secretory activities of rat salivary glands

Exogenously administered PGE1 or PGE2, like atropine, markedly decreased both the flow and calcium concentration of parasympathetically evoked rat parotid saliva; PGF2 alpha was less effective. Despite the fact that prostaglandins greatly reduced the Ca concentration of nerve-evoked saliva, they did not change the glandular Ca concentration of either control or parasympathetically stimulated parotid glands. Prostaglandins (20 micrograms/kg, i.a.) decreased the Na or K concentration of nerve-evoked parotid saliva, but at lower doses had no significant effect. PGE1, PGE2, PGF2 alpha or atropine markedly decreased flow rates of similarly evoked rat submandibular saliva. Prostaglandins and atropine, however, decreased the Na concentration and increased the K concentration of parasympathetically evoked submandibular saliva. PGF2 alpha, like atropine, increased the Ca concentration of such saliva. Drug vehicle, ethanol, slightly decreased the flow of both parotid and submandibular saliva but not the ion secretion, Endogenous prostaglandins themselves may not play a role in secretory activities during parasympathetic nerve stimulation of rat salivary glands, since administration of indomethacin, and inhibitor of prostaglandin biosynthesis, prior to or during nerve stimulation did not significantly alter nerve-evoked salivary secretion, The mechanisms by which prostaglandins modulate secretory responses of salivary glands during parasympathetic stimulation are not understood.     

Parasympathetic NANC-secretion of saliva in the mink, and effects of substance P and VIP.

In both parotid and submandibular glands a parasympathetic non-adrenergic, non-cholinergic (NANC) nerve-evoked secretion of saliva was demonstrated. Saliva evoked by exogenous substance P was poor in protein, while saliva evoked by VIP was protein-rich. In a subthreshold dose for fluid secretion VIP released protein and potentiated the responses elicited by substance P, particularly regarding the output of protein. The two neuropeptides may contribute to the parasympathetic NANC secretion of saliva in the mink. Further, agonists responsible for the secretory NANC response are also likely to contribute to the secretory response of the glands to parasympathetic stimulation in the absence of autonomic receptor blockade in this species.     

Modulating effects of prostaglandins on parasympathetic-mediated secretory activities of rat salivary glands

Exogenously administered PGE₁ or PGE₂, like atropine, markedly decreased both the flow and calcium concentration of parasympathetically evoked rat parotid saliva: PGF_2α was less effective. Despite the fact that prostaglandins greatly reduced the Ca concentration of nerve-evoked saliva, they did not change the glandular Ca concentration of either control or parasympathetically stimulated parotid glands. Prostaglandins (20 μg/kg, i.a.) decreased the Na or K concentration of nerve-evoked parotid saliva, but at lower doses had no significant effect. PGE₁, PGE₂, PGF_2α or atropine markedly decreased flow rates of similarly evoked rat submandibular saliva. Prostaglandins and atropine, however, decreased the Na concentration and increased the K concentration of parasympathetically evoked submandibular saliva. PGF_2α, like atropine, increased the Ca concentration of such saliva. Drug vehicle, ethanol, slightly decreased the flow of both parotid and submandibular saliva but not the ion secretion. Endogenous prostaglandins themselves may not play a role in a secretory activities during parasympathetic nerve stimulation of rat salivary glands, since administration of indomethacin, an inhibitor of prostaglandins biosynthesis, prior to or during nerve stimulation did not significantly alter nerve-evoked salivary secretion. The mechanisms by which prostaglandins modulate secretory responses of salivary glands during parasympathetic stimulation are not understood.     

Radioisotopic and biochemical determination of salivary secretion after long term psychotropic therapy

The aim of this work was to investigate the effect of prolonged psychotropic therapy (neuroleptics and antidepressants over 5 yr on salivary secretion. The flow rate in the parotid and submandibular glands were measured separately by scintigraphy. Flow rates, total protein concentration and total IgA level were determined in the unstimulated saliva in 30 control subjects and 73 patients treated with psychotropic drugs. As evidenced by measurement of flow rates and scintigraphy, psychotropic therapy reduced the unstimulated salivary secretion from parotid glands and to a lesser extent from submandibular gland. The scintigraphic study showed a lower response to stimulation in patients than controls.     

Effects of saliva from chronically reserpinized rat on Na and K transport in perfused main excretory duct of submandibular gland of normal rat

Reserpine (RES) (0.5 mg/kg body wt, ip) was administered to rats for 7 days. On Day 8 saliva was evoked from these animals by intraperitoneal injection of pilocarpine nitrate (10 mg/kg body wt) and saliva from submandibular and parotid glands was collected separately. These collected salivas were used to perfuse through the main ducts of the submandibular glands of normal rats. After a control period of perfusion of the main duct with bicarbonate saline solution, parotid saliva from RES rats was perfused through the duct followed by regular perfusion. There was inhibition of Na absorption (22%) and K secretion (23%). Moreover, when submandibular saliva from treated rat was perfused through the main duct prior to regular perfusion, there was a decrease in Na absorption (31%) and K secretion (28%). In contrast, perfusion of the main duct with either parotid or submandibular saliva from normal rats caused no significant changes in Na and K transport. The present experiments confirm previous studies that there is some Na-inhibitory factor(s) present in saliva of the chronically RES-treated rat.     

Trafficking and postsecretory events responsible for the formation of secreted human salivary peptides: a proteomics approach

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.     

Altered secretion and processing of epidermal growth factor in adrenergic-induced growth of the rat submandibular gland

The granular convoluted tubule (GCT) cells of the submandibular glands represent a major production site for epidermal growth factor (EGF). This study investigates EGF production in the submandibular glands in relation to beta-adrenergic stimulation. Rats were treated with isoproterenol (beta-agonist), which caused up to a 400% increase in submandibular tissue weight after 3 weeks. The weight increase coincided with marked morphologic changes, with degranulation and an apparent decrement in the number of the GCT cells. Immunostaining against EGF revealed a reduction in the number of EGF-immunoreactive cells. Concomitantly, the glandular contents of 6-kDa EGF decreased from 12.86+/-3.42 nmol/gland (mean+/-S.E.M.) in controls to 0.26+/-0.03 nmol/gland. EGF mRNA levels, expressed relative to total RNA levels, only tended to be reduced after 3 weeks as judged from RT-PCR and in situ hybridization (ISH). The isoproterenol-treated rats had increased output of EGF in the saliva, but the salivary secretion of protein was also increased. In both glandular tissue and saliva, gel filtration revealed partially processed high molecular weight forms of EGF in the isoproterenol-treated rats. These data indicate that isoproterenol treatment leads to a hyperstimulatory state of the GCT cells, which then causes depletion of the cellular stores of mature EGF, and most likely due to a shortened posttranslational transit, incomplete peptide processing.     

Effects of nutritional conditions around weaning on carbonic anhydrase activity in the parotid gland and ruminal and abomasal epithelia of Holstein calves

Thirty-two male Holstein calves were used to investigate the effects of nutritional conditions around weaning and aging on carbonic anhydrase (CA) activity in the parotid gland and epithelium from the rumen and abomasum. We fed calf starter and lucerne hay as well as milk replacer (group N) or fed milk replacer either with (group S) or without (group M) administration of short-chain fatty acids (SCFA) through polypropylene tubing into the forestomach until 13 weeks of age. The diets were fed at 1000 hours and 1600 hours, and SCFA were administrated after milk replacer feeding at 1600 hours. Slaughter and tissue sampling were carried out between 1300 hours and 1430 hours at 1, 3, 7, 13, and 18 weeks of age. Tissue samples from five adult (1.5-2.0 years-old) Holstein steers were obtained from a local abattoir. In group N, CA activity in the parotid gland gradually and significantly increased toward the adult value, whilst in the epithelium from the rumen and abomasum, adult values were reached at 3 and 7 weeks of age, respectively. At 13 weeks, the activity for group N was significantly higher than that for the other two groups in the parotid gland, but there was no significant difference in the epithelium from the rumen and abomasum. The concentration of the carbonic isozyme VI in the parotid gland also changed with age but, in contrast to CA activity, had not reached adult levels by 13 weeks of age. In groups M and S, parotid saliva did not show any change toward an alkaline pH or toward a reciprocal change in the concentrations between Cl(-) and HCO(3)(-), even at 13 weeks of age. From these results we conclude that a concentrate-hay based diet around weaning has a crucial role in CA development in the parotid gland, but not in the epithelium of the rumen and abomasum.     

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.     

An immunohistochemical study of secretagogue-induced secretion of epidermal growth factor in the submandibular salivary glands of mice

Routine histological and indirect immunofluorescence techniques were used to examine the histological details of changes in the distribution of epidermal growth factor (EGF) in the submandibular salivary glands of mice during secretion. Comparisons were made bewteen glands of normal mice and those of mice given one of a number of secretagogues at various times prior to sampling. Normal submandibular salivary glands in male mice had an extensive system of convoluted granular tubules (CGT), the cells of which contained EGF. When adrenaline of alpha-phenylephrine was administered, the CGT cells degranulated, and there was a concomitant loss of intracellular EGF-positive immunofluorescence. The excretory ducts were engorged with immunofluorescent material, indicating secretion of EGF into saliva, while the ductal cells themselves remained EGF-negative. The degranulation response could be blocked by phentolamine, but not by propranolol, and no changes in EGF distribution followed the administration of pilocarpine. It was concluded that EGF is secreted, at least partly into the saliva, following an alpha-adrenergic response, and that this occurs with degranulation of the cells of the CGT.     

Clinicopathological characteristics of immunoglobulin G4-related sialadenitis

IntroductionImmunoglobulin G4-related disease (IgG4-RD) is a newly recognized fibro-inflammatory condition. Forty-two cases with immunoglobulin G4-related sialadenitis (IgG4-RS) confirmed by histopathological and immunohistochemical assessment were studied to clarify the clinicopathologic characteristics of the salivary glands involved in IgG4-RS, especially the relationship between the histopathologic features and function of salivary glands or serum levels of IgG4.MethodsClinical, serologic, imaging and histopathological data of these cases were analyzed. CT volumes of submandibular, parotid, and lacrimal glands were calculated. The saliva flow rate was measured. Scintigraphy with 99mTc-pertechnetate was undertaken in 31 cases, and the concentration index (CI) and secretion index (SI) was calculated. Relationships between fibrosis severity and salivary gland function or serum IgG4 levels were analyzed.ResultsThe first symptom was swelling of bilateral submandibular or lacrimal glands. Physical examination showed multiple bilateral major salivary glands (including sublingual and accessory parotid glands) and lacrimal glands were enlarged in IgG4 RS. Multiple enlarged cervical lymph nodes were noted in 30 patients. Saliva flow at rest was lower than normal in 34 cases; stimulated saliva flow was lower than normal in 15 cases. Secretory function was reduced more severely in the submandibular glands than in the parotid glands. Serum levels of IgG4 were elevated in 95.2% of cases and 78.6% patients had increased IgE levels. Serum IgG4 level was higher and saliva secretion lower as glandular fibrosis increased.ConclusionsProminent changes in the morphology, histology, immunohistochemistry and secretion of the major salivary glands of IgG4-RS patients were accompanied by involvement of the lacrimal glands and cervical lymph nodes. Elevated IgE, allergic history, eosinophil infiltration suggest allergic reactions as a potential pathogenesis of IgG4-RS. Severity of glandular fibrosis correlated with salivary function and serum levels of IgG4.     

Expression and distribution of osteopontin and matrix metalloproteinase (MMP)-3 and -7 in mouse salivary glands

We investigated the expression and distribution of osteopontin in mouse salivary glands. Western blot analysis showed intense positive bands at the predicted molecular mass (about 60 kDa) in mouse parotid and sublingual glands. However, a cross-reacted band around 30 kDa was strongly detected in submandibular glands. Indirect immunofluorescent analysis showed that osteopontin was localized at the luminal (apical) membranes of the acinar cells in parotid and sublingual glands. However, it was not detected in acinar cells of submandibular glands. No expression was found in ductal cells of any glands. We also examined the expression of matrix metalloproteinase (MMP)-3 and -7. In parotid gland, MMP-3 was observed at 57 kDa, indicating a latent form, but MMP-7 was not detected. In contrast, MMP-7 definitely was observed at 28 kDa area in submandibular gland, whereas MMP-3 was not detected. These results suggest that osteopontin localizes at luminal sites of acinar cells and may be associated with saliva secretion in mouse salivary gland. It is also suggested that osteopontin may be cleaved by MMP-7 in mouse submandibular gland.     

Tissue and species distribution of the secreted carbonic anhydrase isoenzyme.

The secreted carbonic anhydrases, CA VI, are high molecular mass, oligomeric enzymes originally found in the sheep parotid gland and saliva. The enzymes have been purified from the saliva or parotid glands of several different species. All the CA VI enzymes studied have an apparent subunit Mr of about 45,000 as previously reported for the sheep enzyme. By Western analysis, CA VI from human, cow and dog cross-reacted with antibody raised against the purified sheep enzyme whereas that of the mouse did not. The N-terminal sequences of the sheep, human, cow and mouse enzymes are reported. The sheep, cow and human N-terminal sequences are similar to one another while the mouse sequence is substantially different. Nevertheless, the amino acids in the aromatic cluster I (Trp-5, Tyr-7, Trp-16 and Tyr/Phe-20) have all been conserved, as is the case with the cytoplasmic carbonic anhydrases. Eighteen tissues from the sheep have been examined for the presence of CA VI by Western analysis but it has been found only in the salivary glands. Northern analysis and hybridization histochemistry show that the mRNA for CA VI in sheep is expressed specifically in the acinar cells of the parotid and submandibular glands.     

Decreased saliva secretion and down-regulation of AQP5 in submandibular gland in irradiated rats

The molecular mechanisms of radiation-induced xerostomia remain unclear. The purpose of this study was to investigate the alterations of aquaporins (AQPs) and Na(+)/K(+)-ATPase in irradiated rat submandibular glands and to test the hypothesis that down-regulation of AQP5 expression in irradiated salivary glands is one of the mechanisms of radiation-induced xerostomia. Saliva from control and irradiated rat submandibular glands was analyzed. The mRNA level of AQP5 in the submandibular glands was assessed by semi-quantitative RT-PCR and in situ hybridization. The protein expression of AQP5, AQP1 and Na(+)/K(+)-ATPase was determined by Western blotting and immunohistochemistry. The body weight, submandibular gland weight, and saliva secretion of irradiated rats significantly decreased by 12, 24 and 32% on day 3 and 24, 16 and 38% on day 30 postirradiation, respectively. There was a significant increase in the protein concentration and osmolality of saliva in irradiated rats on days 3 and 30 postirradiation. However, there was no significant difference between irradiated and control rats in total saliva protein secretion. RT-PCR analysis showed that mRNA expression of AQP5 was significantly down-regulated by 37 and 51% in irradiated rats on days 3 and 30 postirradiation, respectively. Immunoblotting showed that the AQP5 protein level was decreased by 40 and 60% in irradiated glands, in contrast to the slight reductions of AQP1 and Na(+)/K(+)-ATPase proteins. Immunohistochemical analysis demonstrated that loss of AQP5 protein occurred throughout the irradiated glands, while no significant reduction was detected in AQP1 and Na(+)/ K(+)-ATPase labeling density. These results suggest that the preferential down-regulation of AQP5 with minor effects on AQP1 and Na(+)/K(+)-ATPase may contribute to radiation-induced salivary dysfunction.     

Nervous control of mammalian salivary glands

In the production and flow of saliva, sympathetic and parasympathetic nerves generally cooperate, although variations between the different salivary glands are considerable, particularly in the sympathetic innervation. In the submandibular gland of the dog, sympathetic impulses cause secretion via beta-adrenoceptors, and since sympathetic motor effects are elicited via alpha-adrenoceptors it is possible to study separately motor and secretory effects in this gland. Such experiments indicate that myoepithelial contractions serve to accelerate the salivary flow and to support the secreting acinar cells and prevent back-flow of fluid from the luminal system into the glandular tissues. The contractions are elicited reflexly from the oral mucosa together with secretion. A potentiation interaction between sympathetic and parasympathetic nerves occurs in the formation of the primary saliva. In parotid glands of rabbits and rats such an interaction has been demonstrated in the secretion of amylase.     

The origin of immunoglobulins in salivary secretion of sheep.

A comparative study was made on the secretion of IgG1 and IgA into parotid and submaxillary saliva of sheep. The purpose of the study was to examine the suggestion that in the external secretory organs of ruminants there is an inverse relationship between the capacity to selectively transfer IgG1 and the development of the IgA secretory system. Despite a marked difference in the secretion of IgA between the parotid and submaxillary salivary glands, the magnitude of selective transfer of IgG1 was similar. Thus, there appears to be no relationship between the selective transfer of IgG1 and the secretion of IgA into the saliva of sheep.     

Exocrine and endocrine secretion of renin and epidermal growth factor from the mouse submandibular glands

Renin and epidermal growth factor (EGF) are synthesized in large amounts by the male mouse submandibular glands. We report the peptides to be secreted mainly in an exocrine manner. The highest values in saliva are obtained upon stimulation with the alpha-adrenergic agonist phenylephrine. The median value for renin is 54 700 nmol/l and the median value for EGF is 211 800 nmol/l. Aggressive behaviour and beta-adrenergic stimulation also increase salivary output of both peptides, while vasoactive intestinal polypeptide (VIP) plus pilocarpine selectively stimulate the secretion of renin. The pattern of increase in plasma is comparable to that in saliva though the substance concentration is lower by a factor of 2 to 70 for renin and a factor of 280 to 12 000 for EGF.     

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.