Irradiation-induced damage to the salivary glands: the role of redox-active iron and copper

The mechanism of irradiation-induced hypofunction of the salivary glands is a process that is not fully understood. Here we examine the hypothesis that intracellular and redox-active ions of iron and copper, which are associated with the secretion granules, play a catalytic role in the irradiation-induced damage. Rats were subjected to head and neck irradiation (15 Gy X rays) and allowed to recover for 2 months. The function of the parotid and submandibular glands was then determined by pilocarpine-stimulated salivary secretion. A 45% decrease in the function of both glands was obtained when compared to nonirradiated controls. Treatment prior to irradiation (90 min) with cyclocytidine (200 mg/kg) led to a massive degranulation of the parotid gland and yielded nearly complete protection from radiation-induced damage. In contrast, pilocarpine stimulation prior to irradiation led to a marginal degranulation of the parotid gland and yielded only 13% protection. Neither agent caused degranulation of the submandibular gland mucous cells or yielded functional protection of this gland. Treatment with both agents yielded a marked increase in iron, copper and manganese levels in the parotid gland saliva. An analogous marked increase in the redox activity of iron and copper ions was recorded for the parotid saliva stimulated by pilocarpine and cyclocytidine. Pilocarpine-stimulated submandibular gland saliva contained metal levels similar to those of the parotid gland saliva. However, no redox activity and no increase in metal mobilization could be demonstrated in the submandibular gland saliva stimulated by both agents. The correlation between the patterns of gland degranulation, mobilization of redoxactive metals and the protection of gland function, for both parotid and submandibular glands, focuses attention on the catalytic roles played by transition metal ions in promoting free radical reactions, which likely participate in the process of injury to the tissue.     

Protein diffusion patterns in human saliva

BACKGROUND: There are two modes of saliva protein diffusion on a cellulose flat matrix. The monophasic mode of diffusion consists in a homogeneous distribution of salivary protein components on the matrix. On the contrary, in the biphasic mode, an area occupied by nondiffusible proteins is surrounded by an area occupied by diffusible proteins. AIM: To study protein diffusion patterns of saliva obtained from normal human volunteers. MATERIAL AND METHODS: Saliva was obtained from 33 subjects aged 67.5 +/- 12 years old (29 female). Forty microliters of saliva were deposited in the center of cellulose disks for protein diffusion assays. RESULTS: Thirty three whole saliva samples and 31 submandibular/sublingual samples showed a biphasic diffusion pattern. On the contrary, 62 out of 66 samples of parotid saliva displayed a monophasic diffusion pattern. In one parotid saliva samples and 2 submandibular/sublingual samples, the diffusion pattern could not be established. Patterns of salivary protein diffusion were highly consistent within the same individual. Eighty percent of subjects had an unequivocal pattern of saliva protein diffusion. CONCLUSIONS: The monophasic mode of saliva protein diffusion is a feature of parotid saliva, whereas the biphasic mode is characteristic of submandibular/sublingual and whole saliva.     

Value of quantitative salivary gland scintigraphy in the early stage of Sj?gren's syndrome

The aim of this study was to test the impact of quantitative salivary gland scintigraphy in patients with suspected Sj?gren's syndrome. Thirteen patients with suspected Sj?gren's syndrome were investigated. During clinical work-up, three had severe and four had mild Sj?gren's syndrome, while six were normal. Quantitative salivary gland scintigraphy was performed using a standardized method. The normal data-base consisted of 172 patients without any evidence of salivary gland malfunction. Visual and quantitative comparisons of the patients' scintigrams were made. In the patients with severe Sj?gren's syndrome, uptake was 0.10 +/- 0.04% and 0.09 +/- 0.03% in the parotid and submandibular glands respectively, confirming the visual diagnosis. In the patients without Sj?gren's syndrome, concordance between the visual and quantitative evaluations could also be shown. In contrast, among the patients with mild Sj?gren's syndrome, uptake was diminished (P < 0.05), amounting to 0.21 +/- 0.05% and 0.16 +/- 0.02% in the parotid and submandibular glands respectively, while visual analysis indicated normal parenchymatous function. In conclusion, quantitative salivary gland scintigraphy is essential for the reliable detection of parenchymatous malfunction at an early stage of Sj?gren's syndrome, which may be missed by visual analysis alone.     

Expression of gross cystic disease fluid protein-15/Prolactin-inducible protein in rat salivary glands

Gross cystic disease fluid protein-15 (GCDFP-15)/prolactin-inducible protein (PIP) is present at moderate levels in human submandibular and sublingual glands and is barely detectable in human parotid gland. The rodent homologue, PIP, has previously been identified in adult submandibular and lacrimal glands. Here we present the molecular characterization of rat PIP and show that this protein is a product of neonatal and adult rat submandibular, sublingual, and parotid glands. cDNA clones encoding rat PIP were isolated and sequenced. The deduced amino acid sequence of rat PIP shows 56% overall identity and 80% similarity with mouse PIP. By SDS-PAGE, secreted rat PIP has an apparent Mr of 17,000, with a minor proportion present as Mr 20-22,000 N-glycosylated forms. PIP was localized in rat salivary glands by immunogold silver staining. PIP was identified in acinar cells of developing and mature submandibular and parotid glands and at very low levels in sublingual gland serous demilunes. Typically, rat submandibular gland secretory proteins are produced by either acinar cell progenitors (Type III cells) or mature acinar cells. The expression pattern observed for PIP is similar to that previously reported for salivary peroxidase, an important component of nonimmune mucosal defense.     

Devices for saliva collection from the major salivary glands. Results in normal subjects

BACKGROUND: Collection of saliva produced by the major salivary glands may be accomplished either by cannulation of the glandular ducts or by the application of specific collecting devices to the emergence area of the glandular ducts. Those procedures are complex, slow, invasive and require skilled personnel. AIM: To report the design and application of a device to collect parotid saliva (snail collector) and another device to collect saliva from the submandibular/sublingual complex. MATERIAL AND METHODS: The saliva collection devices were tested in 40 healthy volunteers (20 male) aged 18 to 22 years old. Saliva was collected using conventional conditions, during 5 to 15 min. RESULTS: An average of 1 to 1.5 ml of saliva was collected in the 10-15 min period from both parotid and submandibular/sublingual glands. Flow rates from parotid glands were 80 microliters/min and 180 microliters/min from submandibular/sublingual glands. Parotid saliva had a protein and organic material concentration twice as high than saliva from submandibular/sublingual glands. The presence of human alpha-amylase duplet (Mr 55 kD and 58 kD) predominated in parotid saliva, whereas saliva from submandibular/sublingual glands had other molecular markers such as the lysozyme duplet (Mr 18.5 kD and 17 kD). CONCLUSIONS: The tested devices were easily applicable, comfortable and allowed the collection of both parotid saliva and submandibular/sublingual saliva from various subjects at once, under the supervision of a single professional.     

Differences in thermal salivation between the FOK rat (a model of genotypic heat adaptation) and three other rat strains

Rats secrete saliva in response to heat. In the present study, details of thermal salivation were investigated using the FOK rat in comparison with Sprague-Dawley (SD), Donryu, and ACI rats. The FOK rat is a strain inbred for genotypic heat adaptation and endures heat for long periods. Conscious rats of all four strains were exposed to 42.5 degrees C. The order of heat endurance times at this temperature was FOK > SD > Donryu = ACI. FOK rats spread their saliva over their entire ventral surface, their faces, and their outside legs. This saliva area was wider than those made by the other three strains. SD rats spread in an area wider than those of the Donryu and ACI rats. Saliva spreading in the FOK rats continued for 4.0-4.5 h, far longer than in the other strains. Under ketamine anesthesia and exposure to 40 degrees C, the FOK rats secreted saliva at 1390+/-235 microL/100 g of body weight during a 60-min observation period. This was the highest rate among the four rat strains (p < 0.0001). The body temperature increase rate in anesthetized FOK and SD rats was lower than in the other two strains, suggesting a minor contribution of unknown factors. Ligation of the submandibular gland ducts abolished the thermal salivation of the FOK rats, whereas ligation of the parotid duct had no effect. The submandibular, sublingual, and lachrymal glands in the FOK rats were 1.3-1.5, 1.25-1.4, and 1.3-1.5 times heavier, respectively, than those in the other three strains, whereas the parotid gland of the FOK rats was not enlarged. These findings indicate that the rats' saliva spreading and ET values are significantly correlated. A potentiated and long-lasting salivation from the submandibular gland was acquired during development of genotypic heat adaptation. This salivation is actuated in response to heat. The pronounced thermal salivation is probably attributable to adaptive changes in the superior salivatory nucleus-chorda tympani-submandibular gland pathway.     

Protein binding effects of salivary excretion of phenobarbital in dogs

The salivary excretion of phenobarbital was investigated by collecting parotid saliva (Pr) and mandibular-sublingual saliva (MS) separately after intravenous administration in beagle dogs. (1) The alterations in the proportions of saliva secreted by the different glands were produced by salivation stimulants such as citric acid, ascorbic acid, sodium chloride and sodium glutamate. (2) The phenobarbital concentrations in both Pr amd MS were lower than those in plasma. The drug concentrations in MS were significantly lower than in Pr with stimulus of 10% citric acid of 15% sodium chloride (p less than 0.05). There was a significant correlation between phenobarbital concentration in each saliva and plasma specimen ( p less than 0.05). (3) The stimulation with 10% citric acid produced higher saliva /plasma drug concentration ratios (S/P ratios: 0.923 +/- 0.175 for Pr, 0.633 +/- 0.073 for MS) than that with 15% sodium chloride (S/P ratios: 0.597 +/- 0.071 for Pr, 0.509 +/- 0.067 or MS). (4) The S/P ratios were hardly influenced by salivary flow rates, at least under the experimental conditions examined in this study. (5) The increased S/P ratios were observed with higher salivary pH and then the equation of Matin et al. 3) seemed to hold for the average values of salivary pH and S/P ratio. (6) The stimulation with 10% citric acid produced higher protein concentration in saliva and higher S/P ratio than that with 15% sodium chloride following alternate stimulations in the same dog.     

Radiation-induced progressive decrease in fluid secretion in rat submandibular glands is related to decreased acinar volume and not impaired calcium signaling

The mechanism(s) of radiation-induced salivary gland dysfunction is poorly understood. In the present study, we have assessed the secretory function (muscarinic agonist-stimulated saliva flow, intracellular calcium mobilization, Na+/K+/2Cl- cotransport activity) in rat submandibular glands 12 months postirradiation (single dose, 10 Gy). The morphological status of glands from control and irradiated rats was also determined. Pilocarpine-stimulated salivary flow was decreased by 67% at 12 months (but not at 3 months) after irradiation. This was associated with a 47% decrease in the wet weight of the irradiated glands. Histological and morphometric analysis demonstrated that acinar cells were smaller and occupied relatively less volume and convoluted granular tubules were smaller but occupied the same relative volume, while intercalated and striated ducts maintained their size but occupied a greater relative volume in submandibular glands from irradiated compared to control animals. In addition, no inflammation or fibrosis was observed in the irradiated tissues. Carbachol- or thapsigargin-stimulated mobilization of Ca2+ was similar in dispersed submandibular gland cells from control and irradiated animals. Further, [Ca2+]i imaging of individual ducts and acini from control and irradiated groups showed, for the first time, that mobilization of Ca2+ in either cell type was not altered by the radiation treatment. The carbachol-stimulated, bumetanide-sensitive component of the Na+/K+/ 2Cl- cotransport activity was also similar in submandibular gland cells from control and irradiated animals. These data demonstrate that a single dose of gamma radiation induces a progressive loss of submandibular gland tissue and function. This loss of salivary flow is not due to chronic inflammation or fibrosis of the gland or an alteration in the neurotransmitter signaling mechanism in the acinar or ductal cells. The radiation-induced decrease in fluid secretion appears to be related to a change in either the water-handling capacity of the acini or the number of acinar cells in the gland.     

Fine structure of the medullary lateral line area of Chelon labrosus (order perciformes), a nonelectroreceptive teleost

Changes in serum levels of rat tissue kallikrein (rK1) in venous blood were measured, using a newly developed radioimmunoassay, before and after autonomic nerve stimulations of submandibular salivary secretion. rK1 secreted into saliva under these conditions was measured by radioimmunoassay and by enzymic activity assay, using the fluorogenic peptide substrate D-Val-Leu-Arg-7-amino-4-trifluoromethylcoumarin (AFC). Following an overnight fast, serum rK1 concentration was 30-40 ng ml-1. Unilateral electrical stimulation of the submandibular sympathetic nerve supply (at 50 Hz in bursts of 1 s every 10 s for 60 min) evoked a small flow of saliva with a very high rK1 concentration, resulting in a large output of rK1 of 2104.4 +/- 603.5 micrograms (n = 6). Such stimulation caused a large degranulation of granular duct cells and a corresponding reduction in glandular rK1 content. Unilateral electrical stimulation of the parasympathetic nerve supply (at 5 Hz continuously for 60 min) evoked a copious flow of saliva with a very low rK1 concentration, resulting in a low output of rK1 (18.1 +/- 4.9 micrograms; n = 6). Despite these large differences in salivary outputs of rK1, serum concentrations of rK1 were increased similarly following either sympathetic or parasympathetic stimulation by 48 and 46%, respectively. If the submandibular duct was briefly obstructed during sympathetic stimulation, inducing leakage and glandular oedema, then serum rK1 increased greatly (40-fold); a similar increase to that seen by others in previous studies without deliberate obstruction. Four days after bilateral submandibular-sublingual sialadenectomy serum rK1 concentration was reduced by approximately 50%. The results indicate that submandibular glands normally contribute to circulating levels of rK1 in rats, but this contribution is independent of the amounts of rK1 secreted into saliva by sympathetically induced exocytosis, and is likely to arise from basal vesicular transport. However, if glandular leakage occurs during sympathetic stimulation of submandibular secretion this then causes increases in the circulating levels of rK1 that correlate with the large amounts being secreted into saliva.     

Observations on two cases of apparent submandibular gland cysts in HIV positive patients: MR and CT findings

OBJECTIVE: To present two cases of probable lymphoepithelial cysts of the submandibular glands in patients who were human immunodeficiency virus (HIV) positive and who also had lymphoepithelial cysts of the parotid glands. MATERIALS AND METHODS: Computed tomography and MRI of two HIV positive patients with lymphoepithelial cysts of the parotid glands and cysts in the submandibular glands were correlated with the histories and the possible presence of other known causes of submandibular gland multiple cysts. RESULTS: Because of the present treatment philosophy regarding HIV positive patients with major salivary gland cysts, surgical resection of these glands was not performed. All other known causes of multiple submandibular gland cysts were excluded by either history or laboratory data. CONCLUSION: Computed tomography and MRI on two patients with known HIV infection and bilateral parotid lymphoepithelial cysts are presented. Both patients also had bilateral multiple submandibular gland cysts and no evidence of obstructive glandular disease, autoimmune disease, or other organ system cysts. These cases of presumed submandibular gland lymphoepithelial cysts are rare in the literature. They are presented in the hope that other radiologists will be stimulated to document the occurrence of this entity.     

Infant survival after orthotopic liver transplantation

Of all head and neck neoplasms, 3% are malignant salivary neoplasms. Only 20% of them affect submandibular glands. These tumours vary histologically, which results from the complex embryogenesis of the glands. Malignant submandibular gland tumours are twice as frequent as parotid gland tumours. Simultaneous occurrence of quite different malignant tumours in the same salivary gland is extremely rare. The age range of patients affected with salivary gland neoplasms is wide. However, the occurrence of these neoplasms in children is exceptionally rare. The authors describe a case of a 13-year-old girl with acinose adenoid carcinoma and cystiscarcinoma coexisting in one submandibular salivary gland.     

The effect of local radiotherapy on kallikrein activity in saliva secreted by parotid gland in patients with head and neck cancers

The samples of parotid gland saliva, collected from control human subjects and those taken from patients with head and neck cancers were submitted to the assay of protein concentration and kininogenase and amidolytic kallikrein activities. No patients with parotid gland tumours were included. The effect of pilocarpine stimulation on these parameters was studied. It was found that the saliva secreted by parotid gland of the investigated patients contains less protein and lower kininogenase activity in comparison to control subjects. Pilocarpine administration resulted in an increase of protein concentration and a decrease of kallikrein activity both in control and investigated subjects. Radiotherapy did not evoke any significant changes in spontaneously secreted saliva. The radiotherapy resulted in a progressive decrease of protein concentration and kallikrein activity in saliva secreted by pilocarpine stimulated glands. The kallikrein activity per mg of protein contained in spontaneously secreted saliva increased significantly during radiotherapy but it distinctly decreased in the saliva of pilocarpine-treated patients. It allows to conclude that the parotid glands do not lose their ability to synthesize and secrete kallikrein during radiotherapy of head and neck cancers.     

Ultrasonographic biometry in salivary glands

Specifications about the size of healthy salivary glands are not available to date. Therefore, we determined the size of the submandibular and the parotid glands by ultrasonography in 50 subjects (25 men, 25 women, mean age 45 y, range 20-68) with no history of disease affecting the salivary glands. The subjects were equally distributed concerning gender and age. Body weight did not differ more than 20% from the ideal weight following Broca's formula (mean body weight 71 kg, range 46-95 kg). In the submandibular glands we found an anterior-posterior length of 35 mm +/- 5.7 mm, a paramandibular dimension to the depth of 14.3 mm +/- 2.9 mm and a dimension in frontal scanning of 33.7 mm +/- 5.4 mm. The parotid glands were measured 46.3 mm +/- 7.7 mm in the axis parallel to the mandibular ramus and 37.4 mm +/- 5.6 mm in the transversel axis. The dimension of the parotid parenchyma was measured with 7.4 mm +/- 1.7 mm lateral to the mandible and 22.8 mm +/- 3.6 mm dorsal to the mandible. No statistically significant difference to the 5%-level was found concerning gender. The dimension of the parotid glands correlated statistically significantly with body weight (p = 0.03). This correlation was not found in the dimension of the submandibular glands. Age did not correlate with the dimension of salivary glands. Results of the submandibular glands were compared with volume measurements of submandibular glands from cadavers.     

Breast cancer diagnosis using N2 laser excited autofluorescence spectroscopy

Carbonic anhydrase VI (CA VI) is a secreted enzyme produced predominantly by serous acinar cells of submandibular and parotid glands. We have investigated the developmental pattern of CA VI production by these glands in the sheep, from fetal life to adulthood, using immunohistochemistry. Also, a specific radioimmunoassay for CA VI was used to measure changes in enzyme expression in the parotid gland postnatally. CA VI is detectable by immunohistochemistry in parotid excretory ducts from 106 days gestation (term is 145 days), in striated ducts from 138 days and in acinar cells from 1 day postnatal. The duct cell content of CA VI declined as the acinar cell population increased, a feature also of CA VI immunoreactivity in the submandibular gland. Production of CA VI by submandibular duct cells was detectable initially at 125 days gestation, and acinar production was not seen before 29 days post-natal. Apart from the differing ontogeny of CA VI production in ducts and acini of parotid and submandibular glands, there was a parallel pattern of CA VI expression during the development of these major salivary glands. With the development of the acinar tissues in the postnatal lamb, there was a dramatic increase (about 600-fold) in the level of expression of CA VI in the parotid gland between days 7 and 59 as measured by radioimmunoassay.     

Submandibular secretion of immunoglobulins IgA and IgG in peripheral paralysis of the facial nerve

INTRODUCTION: In all lesions of the facial nerve suprachoroidally localized, and due to disturbance of parasympathetic and sympathetic component, there comes to qualitative and quantitative disorders of the secretion of submandibular salivary gland. Glandular immunoglobulins IgA and IgG are the secretion of the specific plasma cells in the interstice of this gland. The mechanism of the secretion of immunoglobulins is not sufficiently clear, but it is certainly under the direct neurogenic control, since the disorders of the secretion emerge after the denervation of the submandibular salivary gland. The aim of the study was to prove the direct relation between the degree of submandibular immunoglobulin secretion IgA and IgG, and the degree of the lesion of the facial nerve U which is vitally important in the clinical estimation of the peripheral paralysis of this nerve. MATERIAL AND METHODS: In 35 patients with peripheral idiopathic facial nerve paralysis, the quantity of the secreted immunoglobulins IgA and IgG was examined by laser nephelometar BLN, Module 3. The quantity of the secreted immunoglobulins IgA and IgG (mg lit) in the saliva of the paralysed side was indirectly compared to the secreted immunoglobulins of the healthy, i.e. control side. The examination was performed three times: a) after the appearance of the disease, in the first 30 days; b) two to three months later; c) after six to twelve months. RESULTS: The quantity of the secreted immunoglobulins is significantly higher in the saliva samples taken from the paralysed side (9.50U204.77 mg lit), in comparison with the samples taken from the healthy side (9.50U70.36 mg lit). In the group of patients with favourable results and significantly higher secretion (p 0.01) normalization occurred in the final period of observation. In patients with unfavourable results the difference in secretion was continuously present (p 0.05) (table 1). DISCUSSION: In the lesions of the facial nerve suprachoroidally localized, there comes to disorder concerning the secretion of immunoglobulins IgA and IgG by submandibular salivary gland, which can be applied in the estimation of the degree of paralysis and the prognosis of the final result. CONCLUSION: The results of the research show that in the peripheral idiopathic facial nerve paralysis, there comes to increased secretion of immunoglobulins IgA and IgG in submandibular gland, at the paralysed side. In the patients who, during the paralysis, show quicker fall and normalization of the previously increased quantities of immunoglobulins, the recovery of the motor function of the facial nerve comes more successfully and more certainly. The degree of the secretion of immunoglobulins IgA and IgG can be used for the estimation of the severely of the pathological process in the suprachoroidal part of the nerve, and it can be used as a reliable parameter for the prognosis of the paralysis outcome.     

Mucoepidermoid carcinoma of the major salivary glands: clinical and histopathologic analysis of 234 cases with evaluation of grading criteria

BACKGROUND: The authors had previously conducted an investigation of minor salivary gland mucoepidermoid carcinoma, in which they demonstrated that certain clinical and histopathologic features were useful in predicting biologic outcome. The current study investigated the usefulness of these features in determining the prognoses of patients with mucoepidermoid carcinomas of the major salivary glands. METHODS: Clinical data and 15 histopathologic features were compared in 4 patient groups based on outcome after initial treatment. The outcome groups were 1) survival without disease, 2) survival with tumor recurrence only, 3) survival with metastasis, and 4) death related to tumor. A numeric score was assigned to each unfavorable histopathologic feature. Low grade tumors had scores of 0-4. Intermediate grade tumors scored 5 or 6. High grade tumors had scores higher than 6. RESULTS: Most patients (75%) were tumor free after the initial treatment. Twenty-one patients (9%) had local recurrence only, 12 (5%) demonstrated metastasis and survived, and 25 patients (11%) died of their disease. CONCLUSIONS: Clinical features associated with metastasis or death were more advanced age, tumor size, and preoperative symptoms. Histopathologic features that correlated with poor outcome were cystic component less than 20%, 4 or more mitotic figures per 10 high-power fields, neural involvement, necrosis, and anaplasia. All five of these histopathologic features demonstrated statistical prognostic significance when parotid gland tumors from Groups 1 and 4 were compared (P < 0.001). The point-based grading system demonstrated a statistically significant correlation with outcome for parotid tumors but not for submandibular tumors. The authors' findings indicate that patients with tumors of equal histopathologic grade have a better prognosis when their tumors are in the parotid gland than when their tumors are in the submandibular gland. Six of eight submandibular tumors that metastasized or resulted in death were low grade lesions, and none were high grade.     

Submandibular salivary proteases: lack of a role in anti-HIV activity

Whole human saliva contains a number of proteolytic enzymes, mostly derived from white blood cells and bacteria in the oral cavity. However, less information is available regarding proteases produced by salivary glands and present in salivary secretions. In the present study, we have analyzed submandibular saliva, collected without contaminating cells, and identified multiple proteolytic activities. These have been characterized in terms of their susceptibility to a series of protease inhibitors. The submandibular saliva proteases were shown to be sensitive to both serine and acidic protease inhibitors. We also used protease inhibitors to determine if salivary proteolytic activity was involved in the inhibition of HIV infectivity seen when the virus is incubated with human saliva. This anti-HIV activity has been reported to occur in whole saliva and in ductal saliva obtained from both the parotid and submandibular glands, with highest levels of activity present in the latter fluid. Protease inhibitors, at concentrations sufficient to block salivary proteolytic activity in an in vitro infectivity assay, did not block the anti-HIV effects of saliva, suggesting that the salivary proteases are not responsible for the inhibition of HIV-1 infectivity.     

The effect of transport-blocking drugs on secretion of fluid and electrolytes by the mandibular gland of red kangaroos, Macropus rufus

Mechanisms of primary fluid formation by macropodine mandibular glands were investigated in anaesthetized red kangaroos using ion-transport and carbonic anhydrase inhibitors. Bumetanide at carotid plasma concentrations of 0.005-0.1 mmol/l progressively reduced a stable, acetylcholine-evoked flow rate of 1.02 +/- 0.024 ml/min to 0.16 +/- 0.016 ml/min (mean +/- SEM). Concurrently, saliva [Na], [Cl] and osmolality decreased, [K] and [HCO3] increased and HCO3 excretion was unaffected. High-rate cholinergic stimulation was unable to increase salivary flow above 12 +/- 1.5% of that for equivalent pre-bumetanide stimulation. Furosemide (1.0 mmol/l) and ethacrynate (0.5 mmol/l) caused depression of salivary flow and qualitatively similar effects on ion concentrations to those of bumetanide. Amiloride (up to 0.5 mmol/l) caused no reduction in salivary flow rates or [Na] but decreased [K] and [Cl] and increased [HCO3]. When compared with bumetanide alone, amiloride combined with bumetanide further augmented [K] and [HCO3] and lowered [Cl], but had no additional effects on Na or flow. At the higher level, 4-acetamido-4'- isothiocyanatostilbene-2,2'disulphonic acid (SITS) (0.05 and 0.5 mmol/l) stimulated fluid output, increased [HCO3] and [protein], and depressed [Na], [K] and [Cl]. Relative to bumetanide alone, SITS given with bumetanide had no additional effects on salivary flow or electrolytes. Methazolamide (0.5 mmol/l) in combination with bumetanide curtailed the decrease in [Cl] and the increases in [K] and [HCO3] associated with bumetanide. The residual methazolamide-resistant HCO3 excretion was sufficient to support 2-6% of primary fluid secretion. It was concluded that secretion of primary fluid by the kangaroo mandibular gland is initiated mainly (> 90%) by Cl transport resulting from Na-K-2Cl symport activity. A small proportion of the fluid secretion (up to 6%) appears to be supported by HCO3 secretion. No evidence was found for fluid secretion being dependent on Cl transport involving Na/H and Cl/HCO3 antiports or on HCO3 synthesis involving carbonic anhydrase.     

Abnormally high levels of cystatin S in submandibular glands, saliva, and gingiva of plaque-resistant rats

To identify salivary biomarkers of periodontal diseases, we used plaque-resistant and -susceptible rats as animal models. The levels of salivary cystatin S in saliva, salivary glands, and gingiva were tested in Nembutal-anesthetized young and adult plaque-resistant and -susceptible rats of both sexes with and without chronic treatment with isoproterenol. Isoproterenol was injected i.p. once a day for 4 or 6 consecutive days. Isoelectric focusing electrophoresis by the PhastSystem and the western blotting method were used to separate different proteins and to identify a salivary cystatin S band in these samples. The expression of salivary cystatin S mRNA was also determined by the northern blotting method. Depending upon the types of agonists, a few differences were observed in secretory functions between both strains of rats in both sexes, but the levels of salivary cystatin S in saliva elicited from the submandibular gland and in the extracts of the submandibular glands and gingiva were significantly higher in plaque-resistant rats when compared with those of plaque-susceptible rats in both sexes. However, no significant difference was seen between the strains after chronic treatment with isoproterenol. The N-terminal 26-amino-acid sequence of salivary cystatin S purified from submandibular saliva of plaque-resistant rats was identical with that purified from submandibular saliva of Sprague-Dawley rats subjected to chronic treatment with isoproterenol. The expression of salivary cystatin S mRNA was dramatic in the submandibular glands of the plaque-resistant rats and in the submandibular glands of Wistar rats subjected to chronic treatment with isoproterenol, but not in those of plaque-susceptible rats. These results suggest that salivary cystatin S might be a good biomarker in distinguishing between the two strains of rats and that its concentration is correlated with plaque resistance.     

Hepatitis C virus infection of salivary gland epithelial cells. Lack of evidence

BACKGROUND: Hepatitis C virus genome (HCV-RNA) has been detected in whole salivary gland tissue of chronically infected patients. However, contamination of the tissue by plasma or blood cells was not excluded by the previous reports. AIMS: To assess whether HCV infects the salivary gland epithelial cells in patients with chronic HCV liver disease. METHODS: Twenty unselected patients with chronic active hepatitis (11 cases) or active cirrhosis (nine cases) were examined. Serum and saliva samples were obtained from all patients, 12 of whom (seven, chronic active hepatitis; five, active cirrhosis) underwent salivary gland biopsy. PCR for HCV-RNA was performed on RNA extracted from serum, saliva and salivary gland epithelial cells collected by isokinetic gradient separation after trypsin digestion of whole salivary gland tissue. Saliva samples were also examined for the presence of secretory IgA anti-HCV by gel chromatography and ELISA testing. RESULTS: HCV-RNA was detected in all sera with titers ranging from 5.42 x 10(5) genome equivalents/ml to 123.2 x 10(5) genome equivalents/ml. Thirteen patients were infected with genotype 1b, four patients had genotype 1a, two patients had genotype 2a and one patient was unclassifiable. Low titer HCV-RNA (<2 x 10(5) genome equivalents/ml) was detected in 3/20 saliva samples (15%) from highly viremic patients infected with 1b genotype. RNA extracted from salivary gland epithelial cells consistently tested negative for HCV-RNA. In addition, all saliva specimens tested negative for secretory-IgA (S-IgA) anti-HCV, even after a 10-fold concentration of the samples. CONCLUSIONS: There was no evidence that HCV infects the salivary gland epithelial cells in our viremic patients with HCV chronic liver disease. Low level HCV-RNA in saliva is most probably due to virus spillover from blood.