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Glutamate receptor (GluR) subunit composition of inferior salivatory nucleus (ISN) neurons was studied by immunohistochemical staining of retrogradely labeled neurons. Preganglionic ISN neurons innervating the von Ebner or parotid salivary glands were labeled by application of a fluorescent tracer to the lingual-tonsilar branch of the glossopharyngeal nerve or the otic ganglion respectively. We used polyclonal antibodies to glutamate receptor subunits NR1, NR2A, NR2B, (NMDA receptor subunits) GluR1, GluR2, GluR3, GluR4 (AMPA receptor subunits), and GluR5-7, KA2 (kainate receptor subunits) to determine their expression in ISN neurons. The distribution of the NMDA, AMPA and kainate receptor subunits in retrogradely labeled ISN neurons innervating the von Ebner and parotid glands was qualitatively similar. The percentage of retrogradley labeled ISN neurons innervating the parotid gland expressing the GluR subunits was always greater than those innervating the von Ebner gland. For both von Ebner and parotid ISN neurons, NR2A subunit staining had the highest expression and the lowest expression of GluR subunit staining was NR2B for von Ebner ISN neurons and GluR1 for parotid ISN neurons. The percentage of NR2B and GluR4 expressing ISN neurons was significantly different between the two glands. The percentage of ISN neurons that expressed GluR receptor subunits ranged widely indicating that the distribution of GluR subunit expression differs amongst the ISN neurons. While ISN preganglionic neurons express all the GluR subunits, differences in the percentage of ISN neurons expression between neurons innervating the von Ebner and parotid glands may relate to the different functional roles of these glands.

Purpose: To evaluate the function of the parotid glands before and during gustatory stimulation, using an intrinsic susceptibility-weighted MRI method (blood oxygenation level dependent, BOLD-MRI) at 1.5T and 3T., Materials and Methods: A total of 10 and 13 volunteers were investigated at 1.5T and 3T, respectively. Measurements were performed before and during gustatory stimulation using ascorbate. Circular regions of interest (ROIs) were delineated in the left and right parotid glands, and in the masseter muscle for comparison. The effects of stimulation were evaluated by calculating the difference between the relaxation rates, DeltaR(2)*. Baseline and stimulation were statistically compared (Student's t-tests), merging both parotid glands., Results: The averaged DeltaR(2)* values prestimulation obtained in all parotid glands were stable (-0.61 to 0.38 x 10(-3) seconds(-1)). At 3T, these values were characterized by an initial drop (to -2.7 x 10(-3) seconds(-1)) followed by a progressive increase toward the baseline. No significant difference was observed between baseline and parotid gland stimulation at 1.5T, neither for the masseter muscle at both field strengths. A considerable interindividual variability (over 76%) was noticed at both magnetic fields., Conclusion: BOLD-MRI at 3T was able to detect DeltaR(2)* changes in the parotid glands during gustatory stimulation, consistent with an increase in oxygen consumption during saliva production.

Parotid masses present both functional and aesthetic consequences due to the proximity of the facial nerve and the glands' alignment with the contours of the mandible. As the largest salivary glands, the parotids participate in providing moisture to the mouth through the production of saliva. This paper presents an overview of the anatomy and physiology of the parotid glands, signs and symptoms of parotid conditions, relevant diagnostic testing, medical and surgical treatment of parotid masses, postoperative recovery, and the role of the otorhinolaryngology (ORL) nurse in the continuum of care and patient education.

Among the chemical moieties present in human parotid saliva, some, such as gustin or carbonic anhydrase VI, have been useful to distinguish patients with taste and smell dysfunction from normal subjects. To continue these studies we compared levels of salivary cAMP and cGMP in patients with taste and smell dysfunction with those in normal subjects. We were also interested in exploring physiological characteristics of salivary cAMP and cGMP including changes with gender and age because previous studies had not clearly defined these issues. To perform these studies parotid saliva was collected from 61 normal volunteers and 253 patients with taste and smell dysfunction. cAMP and cGMP were measured by a spectrophotometric 96 plate ELISA technique; parotid salivary protein and flow rate were also measured. Both cAMP and cGMP were found in saliva of normal subjects and patients in the detection range of the assay used. In patients mean concentrations of both cAMP and cGMP were lower than in normal subjects; for cAMP levels were lower among both men and women patients. cAMP was 7 to 10 times higher than cGMP in both normal subjects and patients. Concentrations of cAMP were consistently higher in normal women than in normal men. cAMP levels were generally lower and cGMP levels were generally higher than in previously reported studies. There was a complex pattern of change for both cAMP and cGMP with age with concentrations increasing to about age 50, then decreasing, then increasing again at age >70 years.

The aim of the study was to analyse the appearance of the superficial muscolo-aponeurotic system (SMAS) in radiological images (Magnetic Resonance -MR- and Computed tomography -CT- scans, 10M, 10F randomly selected) in the three regions of the face (the parotid and cheek regions and the nasolabial fold). In axial CT images, the SMAS appears as a relatively hyperdense tortuous line between the hypodense superficial fibroadipose tissue (SAT) and the hypodense deep adipose tissue (DAT). In parotid region SAT is well represented (mean thickness 4.32 +/- 2.9 mm), whereas DAT is very thin (0.33 +/- 0.48 mm); SMAS appears as a thin hyperdense line, close to the parotid gland (0.76 +/- 0.43 mm). In cheek region, SAT is well represented (5.57 +/- 1.17 mm), whereas DAT is thinner (2.94 +/- 0.62 mm), and SMAS is well recognisable (1.69 +/- 0.52 mm). At the level of the nasolabial fold, the SAT is poorly represented (0.37 +/- 0.06 mm); the SMAS continues in the mimic muscles (2.41 +/- 0.05 mm), and DAT shows a mean thickness of 2.15 +/- 0.63 mm. In the MR examination, the SMAS appears as a thin continuous line hypointense in the T1-and T2-weighted sequence, from parotid region to nasolabial fold, comprising mimic muscles in the anterior region of the cheek and at the level of the nasolabial fold. No significative differences in thickness between CT and MR were found. Our anatomo-radiological study confirms that the subcutaneous architecture of the face consists of multiple layers of tissues that connect facial muscles with the dermis. This pattern of arrangement shows a progressive centrifugal thinning towards the adjacent regions.

Background and Purpose: Our aim was to compare different field strengths monitoring physiologic changes due to oral stimulation of parotid glands by using diffusion-weighted (DW) echo-planar imaging (EPI)., Materials and Methods: Twenty-seven healthy volunteers were examined with a DW-EPI sequence at 1.5T and 3T before and after oral stimulation with commercially available lemon juice. The b factors used were 0, 500, and 1000 s/mm(2). Apparent diffusion coefficient (ADC) maps were evaluated with a manually placed region of interest including the entire parotid gland. For comparison of results, a Student t test was used on the basis of the mean of the volunteer median values. To compare both field strengths, we calculated the Pearson correlation coefficient (r)., Results: DW-EPI MR imaging visualized the parotid glands of all volunteers. With 1.5T, the mean ADC before stimulation was 1.12 x 10(-3) mm(2)/s +/- 0.08 x 10(-3) mm(2)/s. After stimulation with lemon juice, the ADC increased to 1.18 x 10(-3) mm(2)/s +/- 0.09 x 10(-3) mm(2)/s. For 3T, the ADC before stimulation was 1.14 x 10(-3) mm(2)/s +/- 0.04 x 10(-3) mm(2)/s, with an increase to 1.17 x 10(-3) mm(2)/s +/- 0.05 x 10(-3) mm(2)/s after stimulation. For both field strengths, the increase in ADC after stimulation was significant (P < .001). High correlations between both field strengths were found pre- and poststimulation (r = 0.955, and 0.936, respectively)., Conclusion: DW-EPI MR imaging allows monitoring of physiologic changes due to oral stimulation of parotid glands by using DW imaging with high correlation between 1.5T and 3T.

Selective deep lobe parotidectomy is a demanding technique, but it preserves healthy glandular tissue, improves cosmetic results and minimises the incidence of Frey's syndrome. We have evaluated postoperative function of the superficial lobe of the parotid after selective resection of the deep lobe. Fourteen patients who each had a mass involving the deep lobe of the parotid were selected from 127 patients with tumours of the parotid gland who were seen and treated between January 2001 and March 2004. Of the 14, 12 matched the study criteria. The preoperative diagnosis was made using both computed tomography (CT) and ultrasound or fine needle aspiration cytology, and the diagnosis was confirmed by histological analysis. All cases were treated by the same surgeon. At 6 months follow-up all patients had a House-Brackmann test, iodine starch test, and scintigraphy of both parotid glands. After scintigraphy the maximum uptake value and function of the gland were evaluated with the concentration index (CI) and the CI percentage ratio. The concentration function of the gland in the resected side of the study group had a mean (S.D.) CI index of 5.5 (3.6) and a CI percentage ratio of 84%. Selective deep lobe parotidectomy has the following advantages: it minimises the impact of treatment on the facial contour, it does not increase postoperative morbidity and it preserves the function of the gland.

Objective: Intraperitoneal injection of a sialogogue, pilocarpine, at high concentrations induces salivation via peripheral pathways and thirst sensation via central pathways. In this study, we report that the effects of another sialagogue, cevimeline, on salivation and water intake in conscious rats differ from those of pilocarpine., Design: We investigated that effects of peripherally and centrally injected cevimeline on parotid saliva flow rate and water intake in conscious rats. The results were compared with those of pilocarpine., Results: The intraperitoneal injection of cevimeline induced salivation from the parotid gland, but not water intake. In contrast, the intracerebroventricular injection of cevimeline induced water intake without salivation. The concentration of cevimeline needed to induce salivation by intraperitoneal injection was several 10 times that of pilocarpine, but that needed to induce water intake by intracerebroventricular injection was over a 1000 times greater., Conclusions: The finding that intraperitoneally injected cevimeline induces salivation without inducing water intake, suggests that the effects on the thirst center in the brain are weaker than those of pilocarpine.

Objective: A gender difference in the unstimulated whole saliva flow rate (UWSFR) may be due to a difference in the sizes of the salivary glands. In this study, we investigated the relationships among the UWSFR, gland sizes and body sizes of healthy young adult males and females., Design: Unstimulated whole saliva was collected for 5 min by the spitting method in 50 healthy young adults, and the flow rate of the saliva was measured. Heights and weights were measured, and body mass indices (BMI) were calculated. The sizes of the salivary glands were measured by use of a magnetic resonance imaging technique., Results: Parotid and submandibular gland sizes and flow rates in females were significantly smaller than those in males, as were also the weights, heights and BMI. In both males and females, there were significant positive correlations between gland sizes and the flow rates, weights and BMI. The variations of the flow rates were reduced by standardizing them with gland sizes, weights and BMI., Conclusions: These results suggest that the lower UWSFR in females as compared with males is due to the smaller gland sizes due to the smaller body sizes.

Purpose: The purpose of this study was to evaluate function of major salivary glands, subjective xerostomia, and correlation between subjective and objective xerostomia scoring after precision-oriented radiotherapy for nasopharyngeal carcinoma., Materials and Methods: From 2000 to 2002, 34 patients with histologically proven non-metastatic nasopharyngeal carcinoma received definitive therapy by parotid-sparing radiotherapy, which included intensity-modulated radiotherapy (33 patients>60 Gy), 3D-conformal radiotherapy, and brachytherapy boost. Salivary function was assessed by sialoscintigraphy pre-irradiation and post-irradiation at 1, 6, 12, and 18 months. The salivary stimulated secretion ratio (SSR) was used to evaluate function of submandibular and parotid glands. Subjective and objective xerostomia was monitored by the LENT/SOMA system., Results: The median dose to parotid gland was 34.6 Gy (interquartile range, 32.9-36.5 Gy). The median dose to submandibular gland was 60.5 Gy (interquartile range, 58.1-61.5 Gy). Parotid-gland post-irradiation median SSR at 1 (0.01, P=0.000) and 6 (0.08, P=0.002) months showed significant reduction compared with pre-irradiation data (0.30). After 12 months, parotid-gland median SSR (12 months, 0.22, P = 0.734; 18 months, 0.16, P=0.885) lost significance compared with pre-irradiation data. Submandibular-gland post-irradiation median SSR at 1 (P=0.000), 6 (P=0.000), 12 (P=0.000), and 18 (P=0.000) months all showed significant reduction compared with pre-irradiation data. There were significant correlations between LENT/SOMA subjective and objective xerostomia scores at 6 months (r=0.657, P=0.000), 12 months (r=0.480, P=0.013), and 18 months (r=0.591, P=0.002)., Conclusions: With parotid-sparing radiotherapy for nasopharyngeal carcinoma, gland function can recover significantly 12 months after radiotherapy. There were significant rank-order correlations between LENT/SOMA subjective and objective (analytic) grading scores at 6 to 18 months' follow-up.

There is no satisfactory conventional treatment for patients who experience irreversible salivary gland damage after therapeutic radiation for head and neck cancer or because of Sjögren's syndrome. Additionally, if most parenchyma is lost, these patients also are not candidates for evolving gene transfer strategies. To help such patients, several years ago we began to develop an artificial salivary gland. In the present study, we used a non-human primate tissue source, parotid glands from rhesus monkeys, to obtain potential autologous graft cells for development of a prototype device for in situ testing. Herein, we present 3 major findings. First, we show that primary cultures of rhesus parotid gland (RPG) cells are capable of attaining a polarized orientation, with Na(+)/K(+)-adenosine triphosphatase, zonula occludens-1, and claudin-1 distributed in specific domains appropriate for epithelial cells. Second, we show that RPG cells exhibit 2 essential epithelial functions required for graft cells in an artificial salivary gland device (i.e., an effective barrier to paracellular water flow and the generation of a moderate transepithelial electrical resistance). Third, we show that RPG cells can express functional water channels, capable of mediating directional fluid movement, after transduction by adenoviral and adeno-associated virus type 2 vectors. Together these results demonstrate that it is feasible to individually prepare RPG cells for eventual use in a prototype artificial salivary gland.

Objective: To study the preservation of parotid glands function and relationship between parotid glands function and dose-volume histogram (DVH) in nasopharyngeal carcinoma (NPC) patients treated by intensity modulated radiation therapy (IMRT)., Methods: From August 2002 to December 2004, the excretion index (EI) and uptake index (UI) of parotids in 48 NPC patients underwent radical IMRT was examined by ECT at the beginning, the end of and the 3 months after radiotherapy. The relationship between parotid function (EI and UI) and DVH were analyzed., Results: The mean doses to the contralateral parotid and ipsilateral parotid were 22.8 +/- 4.5 Gy and 31.9 +/- 4.1 Gy, respectively. The symptom of xerostomia was mild at the end of radiotherapy. ECT showed EI of contralateral parotid were 0.35 +/- 0.25, 0.31 +/- 0.24 and 0.33 +/- 0.22 at the beginning, the end of and 3 months after radiotherapy (RT), respectively. UI were 7.12 +/- 3.56, 5.81 +/- 2.25 and 5.72 +/- 2.81 at the same intervals. This shows no statistical difference. The EI and UI of ipsilateral parotid at the completion of radiotherapy declined significantly (0.21 +/- 0.16 and 4.87 +/- 2.45, respectively) compared with those of pre-treatment (0.36 +/- 0.27 and 8.02 +/- 3.89, respectively) (P < 0.05). DVH showed: at the end of RT, the EI was significant difference between mean dose < 26 Gy and > or = 26 Gy group (P = 0.009) and decreased significantly in the group of V25 (the percentages of parotid volume irradiated with < 25 Gy) > or = 50% compared with the group of V25 < 50% (P < 0.01). The UIs were no significant difference in two groups (P > 0.05)., Conclusion: 26 Gy is a threshold dose for the preservation of parotid glands function. There is also a threshold volume irradiated for the preservation of the parotid glands function. Based on the precondition of assuring significant dose to the target volume (PTV), we should reduce the irradiated volume and dose to parotid glands as possible as we can so as to preserve its function.

Objective: To evaluate the clinical utility of dynamic magnetic resonance (MR) sialographic images as a diagnostic tool for patients with Sjögren's syndrome., Methods: The morphological findings and various kinds of functional parameters in volunteers on dynamic MR sialographic images were compared with those in five patients with definite Sjögren's syndrome., Results: On the MR sialographs of all five patients with Sjögren's syndrome, the so-called 'apple-tree appearance' was seen. The difference in two functional parameters using the dynamic MR sialographic data was elucidated between the two groups. The maximum area of the detectable ducts in the group of patients was significantly smaller (P < 0.001) than that in the group of volunteers. The ratio of change in the detectable ducts in the group of patients was significantly lower (P = 0.011) than that in the group of volunteers., Conclusions: Our study suggests that dynamic MR sialographic data in addition to MR sialographic images might be useful for the diagnosis of Sjögren's syndrome.

Incorporation of radiolabelled leucine and thymidine into trichloroacetic acid-insoluble material of the parotid gland was used as indices of protein synthesis and mitotic activity, respectively, following electrical stimulation of the parasympathetic auriculo-temporal nerve for 30 min in pentobarbitone-anaesthetized rats under adrenoceptor blockade (phentolamine and propranolol, 2mg/kg intravenous of each) in the absence or presence of atropine (2mg/kg intravenous) and without or with nitric oxide synthase inhibitors. In atropinized rats, the parasympathetic non-adrenergic, non-cholinergic (NANC) nerve-evoked mean increases in protein synthesis at a frequency of 10 Hz (142%) and 40 Hz (200%) were not affected in a statistically significant way (124 and 275%, respectively) by the neuronal type NO-synthase inhibitor N(w)propyl-l-arginine (N-PLA) (30 mg/kg intravenous). Neither were the increase (175%) in protein synthesis at 10 Hz in non-atropinized animals affected by N-PLA (180%). The increase (65%) in mitotic activity, 19 h after the end of stimulation at 40 Hz, in the presence of atropine, was not affected by N-PLA (55%). Neither were the increase (95%) in gland content of amylase at this point of observation statistically significant affected by N-PLA (144%). The secretion of fluid and output of amylase from the parotid gland upon nerve stimulation was not affected by N-PLA. When examining the non-selective NO-synthase inhibitor l-NAME (30 mg/kg intravenous) in atropinized rats subjected to stimulation at 10 Hz, neither the increase in protein synthesis nor the evoked fluid response or amylase outputs were affected. Hence, in contrast to an NO-dependent sympathetic-induced protein synthesis and mitosis in the parotid gland, involving the activity of the neuronal type NO-synthase, no support for a parasympathetic-induced protein synthesis (and gain in gland amylase) and mitosis, depending on NO-generation, was found. Likewise, the present findings provide no evidence for a role of NO in the parasym pathetic nerve-evoked fluid secretion and amylase output.

We evaluated the role of VAMP-2/synaptobrevin, VAMP-7/TI-VAMP, and VAMP-8/endobrevin in exocytic pathways of HSY cells, a human parotid epithelial cell line, by coexpressing these VAMP proteins tagged with green fluorescent protein (GFP) and human growth hormone (hGH) as a secretory cargo. Exocytosis of hGH was constitutive and the fluorescent signal of hGH-GFP was observed in the Golgi area and small vesicles quickly moving throughout the cytoplasm. The cytoplasmic vesicles containing hGH overlapped well with VAMP-7-GFP, but did so scarcely with VAMP-2-GFP or VAMP-8-GFP. However, when the vesicle transport from the trans-Golgi network to the plasma membrane was arrested by incubation at 20 degrees C for 2 h and then released by warming up to 37 degrees C; VAMP-2-GFP and hGH were clearly colocalized together in small cytoplasmic vesicles. Neither VAMP-7-GFP nor hGH-GFP was colocalized with LAMP-1, a marker for lysosomes and late endosomes. These results suggest that (1) VAMP-2 can be one of the v-SNAREs for constitutive exocytosis; (2) VAMP-7 is involved in the constitutive exocytosis as a slow, minor v-SNARE, but not in the lysosomal transport; and (3) VAMP-8 is unlikely to be a v-SNARE for constitutive exocytosis in HSY cells.

The complexity of mammalian physiology requires a diverse array of ion channel proteins. This diversity extends even to a single family of channels. For example, the family of Ca2+-activated K channels contains three structural subfamilies characterized by small, intermediate, and large single channel conductances. Many cells and tissues, including neurons, vascular smooth muscle, endothelial cells, macrophages, and salivary glands express more than a single class of these channels, raising questions about their specific physiological roles. We demonstrate here a novel interaction between two types of Ca2+-activated K channels: maxi-K channels, encoded by the KCa1.1 gene, and IK1 channels (KCa3.1). In both native parotid acinar cells and in a heterologous expression system, activation of IK1 channels inhibits maxi-K activity. This interaction was independent of the mode of activation of the IK1 channels: direct application of Ca2+, muscarinic receptor stimulation, or by direct chemical activation of the IK1 channels. The IK1-induced inhibition of maxi-K activity occurred in small, cell-free membrane patches and was due to a reduction in the maxi-K channel open probability and not to a change in the single channel current level. These data suggest that IK1 channels inhibit maxi-K channel activity via a direct, membrane-delimited interaction between the channel proteins. A quantitative analysis indicates that each maxi-K channel may be surrounded by four IK1 channels and will be inhibited if any one of these IK1 channels opens. This novel, regulated inhibition of maxi-K channels by activation of IK1 adds to the complexity of the properties of these Ca2+-activated K channels and likely contributes to the diversity of their functional roles.

Aim: In this study, we have determined signalling pathways involved in adenosine A(1) receptor (A(1) receptor)-dependent stimulation of amylase release in rat parotid gland., Methods: Amylase release, binding and cyclic adenosine monophosphate (cAMP) assays, inositol phosphates (IPs) production and nitric oxide synthase (NOS) activity in the presence of cyclopentyl-1,3-dipropylxanthine (CPA) alone or in the presence of different inhibitory drugs were performed., Results: The binding parameters of specific A(1) antagonist [(3)H]-cyclopentyl 1,3-dipropilxanthine ([(3)H]-DPCPX) in parotid gland membranes show a population of high affinity sites with K(d) (nm) 0.53 +/- 0.06 and B(max) (fmol mg(-1) protein) 122.6 +/- 10.2. CPA stimulation of A(1) receptor exerts an increase in amylase release, IPs accumulation, cAMP production and NOS activity. All these A(1) agonist effects were blocked by the A(1) receptor antagonist DPCPX. Inhibitors of phospholipase C (PLC), calcium/calmodulin (CaM), protein kinase C (PKC), and adenylate cyclase, but not NOS, activities attenuated the CPA stimulatory effect on amylase release. The effect of CPA on amylase release significantly correlated with its action either on cAMP or on IPs accumulation., Conclusion: These results suggest that CPA activation of parotid gland A(1) receptor induces a stimulatory effect on amylase release associated with increased production of cAMP and IPs accumulation. The mechanism appears to occur secondarily to stimulation of phosphoinositide turnover via PLC activation. This, in turn, triggers cascade reactions involving CaM and PKC. The CPA stimulation of NOS does not appear to participate in amylase release.

Various ClC-type voltage-gated chloride channel isoforms display a double barrel topology, and their gating mechanisms are thought to be similar. However, we demonstrate in this work that the nearly ubiquitous ClC-2 shows significant differences in gating when compared with ClC-0 and ClC-1. To delineate the gating of ClC-2 in quantitative terms, we have determined the voltage (V(m)) and time dependence of the protopore (P(f)) and common (P(s)) gates that control the opening and closing of the double barrel. mClC-2 was cloned from mouse salivary glands, expressed in HEK 293 cells, and the resulting chloride currents (I(Cl)) were measured using whole cell patch clamp. WT channels had I(Cl) that showed inward rectification and biexponential time course. Time constants of fast and slow components were approximately 10-fold different at negative V(m) and corresponded to P(f) and P(s), respectively. P(f) and P(s) were approximately 1 at -200 mV, while at V(m) > or = 0 mV, P(f) approximately 0 and P(s) approximately 0.6. Hence, P(f) dominated open kinetics at moderately negative V(m), while at very negative V(m) both gates contributed to gating. At V(m) > or = 0 mV, mClC-2 closes by shutting off P(f). Three- and two-state models described the open-to-closed transitions of P(f) and P(s), respectively. To test these models, we mutated conserved residues that had been previously shown to eliminate or alter P(f) or P(s) in other ClC channels. Based on the time and V(m) dependence of the two gates in WT and mutant channels, we constructed a model to explain the gating of mClC-2. In this model the E213 residue contributes to P(f), the dominant regulator of gating, while the C258 residue alters the V(m) dependence of P(f), probably by interacting with residue E213. These data provide a new perspective on ClC-2 gating, suggesting that the protopore gate contributes to both fast and slow gating and that gating relies strongly on the E213 residue.

Aquaporin-5 (AQP5), an apical plasma membrane (APM) water channel in salivary glands, lacrimal glands, and airway epithelium, has an important role in fluid secretion. M(3) muscarinic acetylcholine receptor (mAChR)-induced changes in AQP5 localization in rat parotid glands were investigated with immunofluorescence or immunoelectron microscopy, detergent solubility, and gradient density floatation assays. Confocal microscopy revealed AQP5 localization in intracellular vesicles of interlobular duct cells in rat parotid glands and AQP5 trafficking to the APM 10 min after injection of the mAChR agonist cevimeline. Conversely, 60 min after injection, there was a diffuse pattern of AQP5 staining in the cell cytoplasm. The calcium ionophore A-23187 mimicked the effects of cevimeline. Immunoelectron microscopic studies confirmed that cevimeline induced AQP5 trafficking from intracellular structures to APMs in the interlobular duct cells of rat parotid glands. Lipid raft markers flotillin-2 and GM1 colocalized with AQP5 and moved with AQP5 in response to cevimeline. Under control conditions, the majority of AQP5 localized in the Triton X-100-insoluble fraction and floated to the light-density fraction on discontinuous density gradients. After 10-min incubation of parotid tissue slices with cevimeline or A-23187, AQP5 levels decreased in the Triton X-100-insoluble fraction and increased in the Triton X-100-soluble fraction. Thus AQP5 localizes in the intracellular lipid rafts, and M(3) mAChR activation induces AQP5 trafficking to the APM with lipid rafts via intracellular Ca(2+) signaling and induces AQP5 dissociation from lipid rafts to nonrafts on the APM in the interlobular duct cells of rat parotid glands.

Exocrine cells of the digestive system are specialized to secrete protein and fluid in response to neuronal and/or hormonal input. Although morphologically similar, parotid and pancreatic acinar cells exhibit important functional divergence in Ca(2+) signaling properties. To address whether there are fundamental differences in exocytotic release of digestive enzyme from exocrine cells of salivary gland versus pancreas, we applied electrophysiological and optical methods to investigate spatial and temporal characteristics of zymogen-containing secretory granule fusion at the single-acinar cell level by direct or agonist-induced Ca(2+) and cAMP elevation. Temporally resolved membrane capacitance measurements revealed that two apparent phases of exocytosis were induced by Ca(2+) elevation: a rapidly activated initial phase that could not be resolved as individual fusion events and a second phase that was activated after a delay, increased in a staircaselike fashion, was augmented by cAMP elevation, and likely reflected both sequential compound and multivesicular fusion of zymogen-containing granules. Optical measurements of exocytosis with time-differential imaging analysis revealed that zymogen granule fusion was induced after a minimum delay of approximately 200 ms, occurred initially at apical and basolateral borders of acinar cells, and under strong stimulation proceeded from apical pole to deeper regions of the cell interior. Zymogen granule fusions appeared to coordinate subsequent fusions and produced persistent structures that generally lasted several minutes. In addition, parotid gland slices were used to assess secretory dynamics in a more physiological context. Parotid acinar cells were shown to exhibit both similar and divergent properties compared with the better-studied pancreatic acinar cell regarding spatial organization and kinetics of exocytotic fusion of zymogen granules.

Using patch-clamp and molecular biological techniques, we identified and characterized membrane currents most likely generated by an electrogenic Na+-HCO3- cotransporter (NBCe) in acutely dissociated bovine parotid acinar (BPA) cells. When BPA cells were dialysed with a N-methyl-D-glucamine (NMDG)-glutamate-rich pipette solution, switching a Na-glutamate-rich, nominally HCO3--free bath solution to the one containing 25 mM HCO3-, but not Cl-, elicited a whole-cell current with a linear current-voltage relation. The HCO3- evoked current was abolished by total replacement of extracellular Na+ (Na+o) with NMDG or by 0.5 mM 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS), and was only partially supported by Li+o, but not by K+o, Cs+o, and cholineo. The reversal potential shift of DIDS (0.5 mM)-sensitive current induced by a change of [Na+]o corresponded to an apparent coupling ratio of HCO3- to Na+ of 2. RT-PCR analysis showed the presence of transcripts of NBCe1-B, but not NBCe1-A in BPA cells. Electrophysiological and pharmacological properties of whole-cell currents recorded from HEK293 cells transfected with the NBCe1-B, which was cloned from BPA cells resembled those of the native currents. Non-invasive measurements of membrane potential changes in the cell-attached patch configuration indicated that an NBCe activity is present in intact unstimulated BPA cells bathed in a 25 mM HCO3--containing solution. Collectively, these results not only suggest that an NBCe is present, functional and may be mediated, at least in part, by NBCe1-B in BPA cells, but also provide the first electrophysiological characterization of transport properties of NBCe expressed in native exocrine glands.

Objective: We introduce a new technique for the functional evaluation of the salivary glands using continuous magnetic resonance (MR) sialography before and after citric acid stimulation., Methods: In 10 volunteers, the time-dependent changes in the maximum area of the detectable parotid gland ducts on MR sialographic images taken every 30 seconds before and after citric acid stimulation were analyzed. The time period to the occurrence of the maximum duct area poststimulation was noted, and then the time for the area to return to its 50% value pre-citric acid stimulation was also observed. This new technique was clinically applied in 1 patient with an excessive supply impression of saliva and in 1 patient with a short supply impression with saliva., Results: In all volunteers after citric acid stimulation, the maximum area of the detectable salivary gland ducts first increased and then decreased. A strong relationship was found between the maximum area of the detectable salivary gland ducts before citric acid stimulation and total saliva volume (Pearson r = 0.672, P = .031). Compared with all the volunteers, the ratio of change in the detectable ducts was the highest in the patient with an excessive supply impression of saliva, but lowest in the patient with a short supply impression with saliva., Conclusions: This initial study suggests that dynamic MR sialography allows for functional and morphological evaluation of the salivary glands. This technique appears to have many possible applications and further investigation in this field is necessary.

Background and Purpose: Radiation treatment of head-and-neck tumors mostly leads to a damage to the salivary glands and a consequential permanent loss of saliva. The aim of this investigation was to establish a modern three-dimensional conformal radiotherapy (3D-CRT) to show a decrease in severe xerostomia in contrast to the proven conventional technique (K-RT) with photons and electrons., Patients and Methods: Between April 2002 and September 2003, 32 patients (25 male, seven female, mean age: 58 years) with malignant tumors of the head and neck were included-after surgery or in case of inoperability with curative intent-in a prospective, nonrandomized study. 10/32 patients (31%) received K-RT with photons and electrons, and 22/32 patients (69%) 3D-CRT (six to eight photon portals). The quantity of saliva was measured as stimulated saliva flow rate (ml/5 min) prior to treatment, at the end, and 1, 6, and 12 months after termination of treatment. To find out the resulting mean dose of both parotid glands for every patient in Gray (D(mean) doses), the D(mean) doses of the ipsilateral and the contralateral parotid gland, determined by dose-volume histograms (DVHs), were averaged over. For calculation of the NTCP (normal tissue complication probability), the logistic model was used., Results: In the trend the stimulated salivary flow rates were higher in the group with 3D-CRT than in the group with K-RT during the whole observation period (at 10 weeks after the start of radiotherapy 3D-CRT vs. K-RT with 1.56 +/- 1.6 vs. 0.82 +/- 1.2 ml/5 min; p < 0.1). The patients treated with the K-RT had, on average, significantly higher averaged D(mean) values than those irradiated with 3D-CRT (p < 0.012). Patients, who were irradiated with 3D-CRT for tumors of the larynx or hypopharynx, showed, on average, significantly lower D(mean) values than patients, who were treated with 3D-DRT because of oral cavity or oropharynx carcinomas or with K-RT irrespective of the primary tumor site (p < 0,003). The resulting dose for 50% complication probability (TD(50)) of the salivary glands was 36.9 Gy (30.9-43.5 Gy; 95% confidence interval). The gradient k of the curve located in point TD(50) was 7.7 (4.8-15.8; 95% confidence interval)., Conclusion: Basically, 3D-CRT seems to be suitable as a standard for all patients with carcinomas of the oral cavity, oro- and hypopharynx. Especially in patients with tumors located in the larynx and hypopharynx, averaged D(mean) doses of both parotids during irradiation can be reached, to conserve salivary flow rates, which are similar to baseline flow rates.

Upon continuous stimulation, the pore of the monovalent cation-selective P2X7 receptor (P2X7R) expands to accommodate large molecules such as N-methyl-D-glucamine (NMDG+). How the change in P2X7R permeability is regulated is not known. Here we report that extracellular Cl- (Cl-(o)) regulates the outward current, whereas extracellular Na+ (Na+(o)) regulates the inward current of large molecules by P2X7Rs. The P2X7R-mediated current was measured in parotid acinar and duct cells of wild type and P2X7R-/- mice and in HEK293 cells expressing the human or mouse P2X7R isoforms. In symmetrical NaCl, triethylammonium chloride, and NMDG+ chloride solutions, the P2X7R current followed a linear current/voltage relationship. In symmetrical NaCl, removal of Cl-(o) reduced the inward Na+ current by approximately 35% and the outward Na+ current by only 10%. By contrast, in the absence of Na+(i) and the presence of Na+(o) or NMDG+(o), the removal of Cl-(o) reduced the inward Na+ or NMDG+ currents by 35% but the outward NMDG+ current by >95%. The effect of Cl-(o) was half-maximal at approximately 60 mm. Reducing Cl-(i) from 150 to 10 mm did not reproduce the effects of Cl-(o). All currents were eliminated in P2X7R-/- cells and reproduced by expressing the P2X7Rs in HEK cells. These findings suggest that Cl-(o) primarily regulates the outward P2X7R current of large molecules. When cells dialyzed with NMDG+ were stimulated in the presence of Na+(o), subsequent removal of Na+(o) resulted in a strongly outward rectifying NMDG+ current, indicating maintained high selectivity for Na+ over NMDG+. During continuous incubation in Na+-free medium, the permeability of the P2X7Rs to NMDG+ gradually increased. On the other hand, when the cells were incubated in symmetrical NMDG+ and only then stimulated with ATP, the NMDG+ current by P2X7Rs followed a linear current/voltage relationship and did not change with time. These findings suggest that the P2X7R has a "Na+(o) memory" and that Na+(o) regulates the inward permeability of P2X7Rs to large molecules. The novel regulation of P2X7R outward and inward permeability to large molecules by Cl-(o) and Na+(o), respectively, may have an important protective function, particularly in secretory epithelial cells.

Purpose: We investigated the factors that affect salivary function after head-and-neck radiotherapy (RT), including parotid gland dose-volume effects, potential compensation by less-irradiated gland tissue, and functional recovery over time., Methods and Materials: Sixty-five patients with head-and-neck tumors were enrolled in a prospective salivary function study. RT was delivered using intensity-modulated RT (n = 45), forward-planning three-dimensional conformal RT (n = 14), or three-dimensional conformal RT with an intensity-modulated RT boost (n = 6). Whole salivary flow was measured before therapy and at 6 months (n = 61) and 12 months (n = 31) after RT. A wide variety of dose-volume models to predict post-RT salivary function were tested. Xerostomia was defined according to the subjective, objective, management, analytic (SOMA) criteria as occurring when posttreatment salivary function was < 25% of the pretreatment function. Multivariate logistic regression analysis was used to assess the combined effect of dose-volume, patient-, and treatment-related factors., Results: A significant correlation was observed between the relative quality-of-life scores and relative stimulated saliva values at 6 months after RT (Spearman's correlation coefficient [R(s)] = 0.46, p < 0.001). The dose-volume factors were by far the strongest correlates with stimulated saliva flow, although other factors showed modest significance in multimetric models (chemotherapy, gender, and Karnofsky performance status). Several fitted dose-volume models provided a good mathematical description of the data. Significant noise in the salivary measurements (repeated measurement coefficient of variation was 27% in normal subjects) precluded selection of any one of the models presented solely on the basis of the objective fit criteria. Nevertheless, the mean dose-exponential model, in which each parotid gland's relative salivary gland function equaled exp(-A x mean gland dose), with A equal to 0.054/Gy (68% confidence interval 0.052-0.059), provided a good representation of the data and was incorporated into our multimetric analysis. Using that model, we estimated that a mean parotid dose of 25.8 Gy, on average, was likely to reduce a single parotid gland's flow to 25% of its pretreatment value, regardless of the treatment delivery method. Significant correlations were observed between a logistic multivariate model (incorporating the mean dose-exponential equation, gender, and Karnofsky performance status) and stimulated saliva flow at 6 months (R(s) = 0.73), stimulated saliva flow at 12 months (R(s) = 0.54), and quality-of-life score at 6 months (R(s) = 0.35) after RT., Conclusion: Stimulated parotid salivary gland dose-volume models strongly correlated with both stimulated salivary function and quality-of-life scores at 6 months after RT. The mean stimulated saliva flow rates improved from 6 to 12 months after RT. Salivary function, in each gland, appeared to be lost exponentially at a rate of approximately 5%/1 Gy of mean dose. Additional research is necessary to distinguish among the models for use in treatment planning. The incidence of xerostomia was significantly decreased when the mean dose of at least one parotid gland was kept to < 25.8 Gy with conventional fractionation. However, even lower mean doses imply increased late salivary function.

Echo-planar diffusion-weighted (DW) magnetic resonance (MR) imaging was used to evaluate changes in the parotid glands during gustatory stimulation. The study protocol was approved by the local ethics committee, and informed consent was obtained from all volunteers. Twelve healthy volunteers (five women, seven men) with a median age of 25 years (range, 22-30 years) were examined with a 1.5-T MR unit. A DW MR imaging sequence was performed once at rest and continuously repeated over a mean period of 26 minutes (range, 24-28 minutes) during salivary stimulation with a tablet of ascorbic acid given orally. During the first 5 minutes (range, 1 minute 30 seconds--7 minutes 30 seconds) of salivary stimulation, a decrease in apparent diffusion coefficient (ADC) was observed in both the parotid (P = .0001) and the submandibular (P = .0004) glands in all volunteers. During the following 15 minutes, a steady increase in ADC from the baseline value was noted for the parotid glands (P = .0022), and peak ADC was reached a median of 21 minutes (range, 14-21 minutes) after the start of gustatory stimulation. The ADC of the submandibular glands did not increase significantly after the start of gustatory stimulation compared with the ADC at baseline. In conclusion, DW MR imaging allows physicians to noninvasively demonstrate functional changes in the salivary glands., ((c) RSNA, 2005)

Objective: Saliva is a critical fluid necessary for oral health. Medications, radiation therapy, and systemic conditions can decrease salivary function and increase a patient's risk for caries and other oral infections. Palliative management of xerostomia includes wetting agents such as ice chips and saliva substitutes. Systemic agents stimulate salivary flow but often have unfavorable side effects. All have met with limited success. The purpose of this study is to assess the effectiveness of transcutaneous electric nerve stimulation (TENS) as a means of stimulating salivary function in healthy adult subjects., Study Design: Twenty-two healthy, adult subjects with no history of salivary gland disorder enrolled in the protocol. The TENS electrode pads were placed externally on the skin overlying the parotid glands. Unstimulated saliva was collected for 5 minutes via the Carlson-Crittenden cup into preweighed vials using standardized collection techniques. The TENS unit was then activated and stimulated saliva collected for an additional 5 minutes., Results: Fifteen of 22 subjects demonstrated increased parotid salivary flow when stimulated via the TENS unit. Five experienced no increase and 2 experienced a decrease. The mean unstimulated salivary flow rate was 0.02418 mL/min (SD 0.03432) and mean stimulated salivary flow rate was 0.04946 mL/min (SD 0.04328). Statistical analysis of flow rates utilizing the paired t test demonstrated the difference to be statistically significant, P < .001. In 7 subjects with 0 baseline flow, 5 continued to have no flow., Conclusions: The TENS unit was effective in increasing parotid gland salivary flow in two-thirds of healthy adult subjects. A further study in a cohort of patients with salivary gland disorders is warranted.

The purpose of this study is to develop an initial step in salivary gland tissue engineering through proliferation and phenotypic preservation of rat parotid acinar cells in vitro. By using the explant outgrowth technique and M199 medium with the addition of sialic acid, acinar cells not only survived for more than 30 days in the absence of basement membrane substrates but also proliferated to yield cells with acinar phenotypic expression. Furthermore, we tested whether chitosan can be used as a synthetic extracellular matrix to culture salivary acinar cells. Chitosan is a deacetylated product of chitin, which is a plentiful polysaccharide found in nature and is safe for the human body, but little is known about the utility of chitosan in culturing salivary acinar cells. It was found that coating fibronectin on chitosan membrane improved the attachment of acinar cells in the initial stage. However, the poor attachment of acinar cells on pure chitosan membrane did not affect cell growth after longer culture times, indicating that chitosan is potentially useful as a tissue-engineering scaffold of the salivary gland. These in vitro results are encouraging because such a culture system may serve as an artificial salivary gland for future use in the treatment of patients with salivary hypofunction.

The inferior salivatory nucleus (ISN) contains parasympathetic neurons controlling the parotid and von Ebner salivary glands. To characterize the neurophysiological and morphological properties of these neurons, intracellular recordings were made from anatomically identified ISN neurons in rat brain slices. Neurons were also filled with Lucifer yellow and morphometrically analyzed. Based on responses to membrane hyperpolarization followed by depolarization, three types of repetitive discharge patterns were defined for neurons innervating the parotid gland. The regular, repetitive discharge response to membrane depolarization was changed by hyperpolarization resulting either in a delay in the occurrence of the first spike or to an increase in the length of the first interspike interval in the action potential train. Membrane hyperpolarization had little effect on the discharge pattern of some neurons. Similar response discharge patterns were found for neurons innervating the von Ebner salivary gland, which also included a further group of neurons that responded with a short burst of action potentials. Neurons innervating the parotid salivary glands differed morphologically from the von Ebner salivary glands having significantly larger soma and more and longer dendrites than von Ebner gland neurons. In addition, the mean membrane input resistance, time constant, and spike half-width of parotid gland neurons was significantly lower than in von Ebner gland neurons. These differences in intrinsic membrane properties and morphology may relate to the functions of the von Ebner and parotid glands. von Ebner glands are involved in taste stimulus delivery and removal from posterior tongue papillae while the parotid glands contribute saliva to the entire mouth.

The aim was to study the subcellular localization of the Menkes protein (MNK; ATP7A) in the rat parotid acinar cell. MNK protein is a copper transporting P-type ATPase whose absence or dysfunction causes a fatal neurodegenerative disorder, MNK disease. Rat parotid glands were fixed and low-temperature embedded in Lowicryl K4M resin, and ultrathin sections were prepared for immunocytochemical analysis. Immunolocalization of MNK was demonstrated mainly over the trans Golgi network (TGN) area. Immature and mature secretory granules were also labelled, indicating that MNK protein could be involved here in copper secretion from acinar cells into saliva, consistent with a proposed cariostatic role for copper.

Background and Purpose: To determine the salivary function, after parotid-sparing radiotherapy (RT), of different regions within the parotid gland and to evaluate dose-function relationships within the parotid glands and between patients., Patients and Methods: Sixteen head and neck cancer patients, irradiated between September 1999 and November 2000 using a conformal parotid-sparing technique, were included in this study. Before RT and 7 months after RT (range 6-10 months), a salivary gland scintigraphy was performed in all patients combined with a single photon emission computed tomography (SPECT). The salivary excretion fraction (SEF) was measured, after stimulation, in 8-12 transverse 5mm SPECT slices of each parotid. Loss of salivary excretion fraction (dSEF %) of these slices was calculated as the proportion of SEF after RT as compared to SEF before RT. Since the planning CT-scan and the SPECT-scintigraphy were performed in the same treatment position, the dose to a transverse slice within the parotid gland could be matched to the loss of salivary excretion fraction of that respective slice. A non-linear model was fitted to the dose-loss of function data and the dose resulting in 50% loss of salivary excretion fraction (D50) was calculated., Results: Before RT, all but one patient presented with normal salivary excretion fractions (SEF) of both parotid glands. Within the same parotid gland, the SEF's of the different slices were almost equal. Seven months after RT, the reduction in SEF was statistically significant (P-value<0.0001). A significant difference in loss of salivary excretion fraction (dSEF) was also observed between both parotid glands (P<0.0001) as a result of the parotid-sparing technique. When plotting the dSEF of a slice versus the dose given to that slice, doses as low as 10-15 Gy could result in a serious loss of function (dSEF>50%). After fitting a non-linear model to these plots, the mean dose resulting in 50% loss of salivary excretion fraction (D50) 7 months after RT was 22.5 Gy. A large inter-patient variability was found in D50., Conclusions: Salivary SPECT is a useful tool for the evaluation of the salivary function of different slices within the parotid gland. Before irradiation, the different slices within one parotid gland act as functional sub-units contributing equally to the function of the entire gland. Seven months after an average dose of 22.5 Gy (D50) the functional sub-unit has lost 50% of its excretion fraction. The high inter-patient variability in D50 and the observation that low doses (10-15 Gy) can induce serious loss of function should prompt us in the clinic to reduce the dose to the parotids even lower than the threshold of 22.5 Gy.

Background: Xerostomia is a uniform complication after radiotherapy (RT) for nasopharyngeal carcinoma (NPC). Dosimetric studies suggested that intensity-modulated RT (IMRT) can spare part of the parotid glands from high-dose radiation. Disease control and salivary function after IMRT for early-stage NPC was studied prospectively., Methods: Thirty-three patients with T1,N0-N1,M0 NPC were treated with IMRT from 2000 to 2002. The prescribed dose was 68-70 grays (Gy) in 34 fractions to gross tumor volume, 64-68 Gy to the planning target volume, and 70 Gy to enlarged cervical lymph nodes. Nineteen patients had stimulated whole salivary (SWS) flow assessment and stimulated parotid salivary (SPS) flow assessment at baseline and at 2 months, 6 months, 12 months, 18 months, and 24 months after the completion of IMRT., Results: At a median follow-up of 2 years, only 1 neck failure was observed. The 2-year and 3-year local control, distant metastases-free, and overall survival rates all were 100%. The lymph node control and progression-free survival rates were 100% at 2 years and 92.3% at 3 years, respectively. The average mean dose to the parotid gland was 38.8 Gy. The SWS and SPS flow showed continuous recovery: 60% and 47.1% of patients recovered at least 25% of their baseline SPS flow and SWS flow, respectively, at 1 year after completion of IMRT, and the proportions rose to 85.7% and 71.4%, respectively, by 2 years. The pH and buffering capacity of saliva also improved with time., Conclusions: Parotid-sparing IMRT achieved good locoregional control, and there was continuous recovery of salivary flow, pH, and buffering capacity in the first 2 years after IMRT in patients with NPC., ((c) 2004 American Cancer Society.)

Purpose: To investigate the feasibility of diffusion-weighted (DW) echo-planar imaging (EPI) for measuring different functional conditions of the parotid gland and to compare different measurement approaches., Materials and Methods: Parotid glands of 27 healthy volunteers were examined with a DW EPI sequence (TR 1,500 msec, TE 77 msec, field-of-view 250 x 250 mm, pixel size 2.10 x 1.95 mm, section thickness 5 mm) before and after oral stimulation with commercially available lemon juice. The b factors used were 0, 500, and 1,000 sec/mm (2). Apparent diffusion coefficient (ADC) maps were digitally transferred to MRIcro (Chris Rorden, University of Nottingham, Great Britain) and evaluated with a manually placed circular region of interest (ROI) containing 100 - 200 pixel. Additional ROIs including the entire parotid gland were placed on either side. The results of both measurements were compared, using the Student's t test based on the median ADC values for each person. A two-tailed p-value of less than.05 was determined to indicate statistical significance. To compare both measurement approaches, the Pearson's correlation coefficient (r) was calculated., Results: Diffusion-weighted echo-planar MR imaging successfully visualized the parotid glands of all volunteers. In a first step, the median ADC value per person was computed. Using ROIs of 100 - 200 pixels, the mean was calculated to be 1.08 x 10 (- 3) mm (2)/sec +/- 0.12 x 10 (- 3) mm (2)/sec for both parotid glands prior to stimulation. After stimulation, the mean ADC was measured at 1.15 x 10 (- 3) mm (2)/sec +/- 0.11 x 10 (- 3) mm (2)/sec for both parotid glands. Evaluating the entire parotid gland, the ADC was 1.12 x 10 (- 3) mm (2)/sec +/- 0.08 x 10 (- 3) mm (2)/sec prior to stimulation, whereas the ADC increased to 1.18 x 10 (- 3) mm (2)/sec +/- 0.09 x 10 (- 3) mm (2)/sec after stimulation with lemon juice. For both types of measurements, the increase in ADC after stimulation proved to be significant (p < 0.001). High correlations between both measurement types were found (r >.83)., Conclusion: Diffusion-weighted echo-planar MR imaging allows non-invasive quantification of functional changes in the parotid glands.

Background: The purpose of the present study was to elucidate whether myoepithelial cells proliferate mitotically during regeneration of rat submandibular glands after atrophy., Methods: The excretory duct of the right submandibular gland of rats was doubly ligated near the hilum with metal clips, which were removed after 7 days of ligation (day 0). The regenerating right submandibular glands were removed from 0 to 14 days after removal of the clips. The removed tissue was examined with immunohistochemical double staining for proliferating cell nuclear antigen (PCNA) as a marker of proliferating cells and actin as a marker of myoepithelial cells, as well as with transmission electron microscopy (TEM)., Results: The PCNA-positive myoepithelial cells were observed at the periphery of transitional duct-acinar structures, ducts and acini in the regenerating glands at every time-point, and the PCNA-labeling index of myoepithelial cells increased greatly especially between day 2 and 4. The mitosis of myoepithelial cell was also identified by TEM at day 4., Conclusion: These findings suggest that myoepithelial cells are able to proliferate mitotically during regeneration of rat submandibular gland.

Store-operated Ca(2+) influx, suggested to be mediated via store-operated cation channel (SOC), is present in all cells. The molecular basis of SOC, and possible heterogeneity of these channels, are still a matter of controversy. Here we have compared the properties of SOC currents ( I(SOC)) in human submandibular glands cells (HSG) and human parotid gland cells (HSY) with I(CRAC) (Ca(2+) release-activated Ca(2+) current) in RBL cells. Internal Ca(2+) store-depletion with IP(3) or thapsigargin activated cation channels in all three cell types. 1 muM Gd(3+) blocked channel activity in all cells. Washout of Gd(3+) induced partial recovery in HSY and HSG but not RBL cells. 2-APB reversibly inhibited the channels in all cells. I(CRAC )in RBL cells displayed strong inward rectification with E(rev)(Ca) = >+90 mV and E(rev) (Na) = +60 mV. I(SOC) in HSG cells showed weaker rectification with E(rev)(Ca) = +25 mV and E(rev)(Na) = +10 mV. HSY cells displayed a linear current with E(rev) = +5 mV, which was similar in Ca(2+)- or Na(+)-containing medium. pCa/ pNa was >500, 40, and 4.6 while pCs / pNa was 0.1,1, and 1.3, for RBL, HSG, and HSY cells, respectively. Evidence for anomalous mole fraction behavior of Ca(2+)/Na(+) permeation was obtained with RBL and HSG cells but not HSY cells. Additionally, channel inactivation with Ca(2+) + Na(+) or Na(+) in the bath was different in the three cell types. In aggregate, these data demonstrate that distinct store-dependent cation currents are stimulated in RBL, HSG, and HSY cells. Importantly, these data suggest a molecular heterogeneity, and possibly cell-specific differences in the function, of these channels.

Ca2+-induced Ca2+ release (CICR) is a well characterized activity in skeletal and cardiac muscles mediated by the ryanodine receptors. The present study demonstrates CICR in the non-excitable parotid acinar cells, which resembles the mechanism described in cardiac myocytes. Partial depletion of internal Ca2+ stores leads to a minimal activation of Ca2+ influx. Ca2+ influx through this pathway results in an explosive mobilization of Ca2+ from the majority of the stores by CICR. Thus, stimulation of parotid acinar cells in Ca2+ -free medium with 0.5 microm carbachol releases approximately 5% of the Ca2+ mobilizable by 1 mm carbachol. Addition of external Ca2+ induced the same Ca2+ release observed in maximally stimulated cells. Similar results were obtained by a short treatment with 2.5-10 microm cyclopiazonic acid, an inhibitor of the sarco/endoplasmic reticulum Ca2+ ATPase pump. The Ca2+ release induced by the addition of external Ca2+ was largely independent of IP(3)Rs because it was reduced by only approximately 30% by the inhibition of the inositol 1,4,5-trisphosphate receptors with caffeine or heparin. Measurements of Ca2+ -activated outward current and [Ca2+](i) suggested that most CICR triggered by Ca2+ influx occurred away from the plasma membrane. Measurement of the response to several concentrations of cyclopiazonic acid revealed that Ca2+ influx that regulates CICR is associated with a selective portion of the internal Ca2+ pool. The minimal activation of Ca2+ influx by partial store depletion was confirmed by the measurement of Mn2+ influx. Inhibition of Ca2+ influx with SKF96365 or 2-aminoethoxydiphenyl borate prevented activation of CICR observed on addition of external Ca2+. These findings provide evidence for activation of CICR by Ca2+ influx in non-excitable cells, demonstrate a previously unrecognized role for Ca2+ influx in triggering CICR, and indicate that CICR in non-excitable cells resembles CICR in cardiac myocytes with the exception that in cardiac cells Ca2+ influx is mediated by voltage-regulated Ca2+ channels whereas in non-excitable cells Ca2+ influx is mediated by store-operated channels.

Parotid glands of adult female rats were exposed to agonists mimicking sympathetic (isoprenaline, 1mg/kg, I.P.) or parasympathetic activity (bethanechol, 10 microg/kg/min i.v. for 30 min, and vasoactive intestinal peptide, VIP, 0.2 microg/kg/min, i.v. for 30 min) or they were reflexly activated by a meal demanding chewing. The stimulated glands were removed at varying times (15(30)-360 min) following the onset of the agonist administration or 75-300 min after the start of a 1h long feeding period, and a number of transcription factors was studied using Western blot. The protein bands were semi-quantitatively measured by densitometry. In response to isoprenaline, C/EBPalpha of 42, 38 and 30 kDa increased by 45-50% above control value, C/EBPbeta LAP (38/35 kDa) by 80% and C/EBPdelta (35 kDa) by 230%, while C/EBPbeta LIP (20 kDa) decreased by 45%. In response to VIP, C/EBPalpha of 42 kDa increased by 75% and C/EBPalpha of 30 kDa by 10%, C/EBPbeta LAP by 65% and C/EBPdelta by 410%, while C/EBPalpha of 38 kDa as well as C/EBPbeta LIP were not changed. In response to bethanechol, C/EBPalpha of 42 kDa increased by 105%, C/EBPbeta LAP by 40% and C/EBPdelta by 170%, while C/EBPalpha of 30 kDa decreased by 30% and C/EBPalpha of 38 kDa and C/EBPbeta LIP remained unchanged. c-Myc increased in response to isoprenaline and VIP by 40-55%, but not to bethanechol. In rats offered a pelleted diet, the parotid glands displayed increases in C/EBPalpha of 42 kDa by 105%, of 30 kDa by 40% and of 38 kDa, by 10%, in C/EBPbeta LAP by 65% and in C/EBPdelta by 215%, whereas C/EBPbeta LIP decreased by 25%. Thus, in parotid glands transcription factors of importance for growth and metabolism were shown to be influenced by autonomimetics as well as by nervous activity.

The effects of external anions (SCN(-), NO3-, I(-), Br(-), F(-), glutamate, and aspartate) on gating of Ca(2+)-dependent Cl(-) channels from rat parotid acinar cells were studied using the whole-cell configuration of the patch-clamp technique. Shifts in the reversal potential of the current induced by replacement of external Cl(-) with foreign anions, gave the following selectivity sequence based on permeability ratios ( P(x)/ P(Cl)): SCN(-)>I(-)>NO3->Br(-)>Cl(-)>F(-)>aspartate>glutamate. Using a continuum electrostatic model we calculated that this lyotropic sequence resulted from the interaction between anions and a polarizable tunnel with an effective dielectric constant of approximately 23. Our data revealed that anions with P(x)/P(Cl) > 1 accelerated activation kinetics in a voltage-independent manner and slowed deactivation kinetics. Moreover, permeant anions enhanced whole-cell conductance ( g, an index of the apparent open probability) in a voltage-dependent manner, and shifted leftward the membrane potential- g curves. All of these effects were produced by the anions with an effectiveness that followed the selectivity sequence. To explain the effects of permeant anions on activation kinetics and g(Cl) we propose that there are 2 different anion-binding sites in the channel. One site is located outside the electrical field and controls channel activation kinetics, while a second site is located within the pore and controls whole-cell conductance. Thus, interactions of permeant anions with these two sites hinder the closing mechanism and stabilize the channel in the open state.

Objective: To determine the efficacy of botulinum toxin A in the management of drooling (sialorrhea) in children and young adults with cerebral palsy., Design: Twenty-one children were enrolled in an open-label, nonblinded prospective study. Subjective and objective measures were used to determine the effect of botulinum toxin A on drooling and saliva production. Subjective measures included visual scales to document the child's severity and frequency of drooling. Objective measures included the number of bibs used per day and salivary secretion. At the initial visit, subjective and objective measures established the child's baseline drooling and saliva production. Fifteen units of botulinum toxin A was injected into each parotid glans. At each fellow-up visit of telephone survey, subjective and objective measures were recorded to monitor the child's drooling and saliva production. A postinjection questionnaire evaluated overall effect and caregiver satisfaction., Results: The visual analog scales and number of bibs used per day demonstrated statistically significant reduction in severity and frequency of drooling at 2 wks, 1 mo and 2 mos. Salivary production was significantly reduced at 1-mo fellow-up. Eighty-nine percent of the caregivers reported and improvement of their child's drooling after botulinum toxin A injection. Severity-nine percent of caregivers were satisfied with the treatment and would perform the treatment again., Conclusion: Intraparotid injections of botulinum toxin A are efficacious in decreasing severity and frequency of drooling, the number of bibs used per day, and the production of saliva in children with cerebral palsy. The injections are relatively safe and adverse effects were observed in this study.

The duct of the parotid gland had been indiscriminately named Stenon's duct or Stensen's duct. In order to establish its origin we carried out a biographic search and a brief revision of the life of the great anatomist, geologist, palaeontologist, bishop and finally Saint Nicolaus Stensen.

Rab3D is a low molecular weight GTP-binding protein believed to be involved with regulated secretion in many cell types. In parotid, Rab3D is localized to secretory granule membranes or present in the cytosol as a complex with Rab escort protein. In the present study, we examined the redistribution of membrane-associated Rab3D during secretion in permeabilized parotid acini. When permeabilized acini were stimulated with calcium and cAMP, amylase release increased greater than twofold over basal. Quantitative immunoblotting of subcellular fractions revealed that Rab3D did not dissociate from parotid membranes during secretion. Immunohistochemical staining demonstrated that Rab3D co-localizes with amylase containing granules that are found in the apical pole of the cell. Upon stimulation with calcium and cAMP, Rab3D and amylase immunostaining of granules appeared to be more dispersed. However, Rab3D immunostaining was not observed on the plasma membrane and appeared to reside in the apical cytoplasm. To examine the role of Rab3D in amylase release, cytosolic extracts containing myc-tagged Rab3D and Rab3DQ81L, a GTP-binding mutant, were prepared and incubated with streptolysin O-permeabilized acini. Rab3D, but not Rab3DQ81L, bound to parotid membranes suggesting that Rab3D-binding to parotid membranes is guanine nucleotide-dependent. Moreover, wild-type and mutant Rab3D inhibited agonist-induced amylase release from permeabilized parotid acini. These observations indicate that in parotid acini, Rab3D does not dissociate from parotid membranes or redistribute to the plasma membrane during secretion, and may play an inhibitory role in regulated secretion. The fact that both wild-type Rab3D and the GTP-binding mutant inhibit amylase release suggests that binding of Rab3D to the membrane is not essential for secretory inhibition.

Objective: The first clinical studies indicate that Botox provides effective treatment for hyperhidrosis and sialorrhea. The aim of this work is to sum up current evaluation of this use., Method: A systematic literature search was conducted on the Pub Med database, along with on chapters in other publications. The most interesting articles in relation to our own personal experience were chosen., Results: Despite recent use of BT to treat focal hyperhidrosis, there have been numerous publications since 1997. However, the injected areas have not been listed so frequently. Axillary hyperhidrosis has been studied most; it is also in this case and in the case of gustatory sweating that the best results have been obtained. Publications about palmar and especially plantar hyperhidrosis are much rarer, almost anecdotic. It has been demonstrated to a lesser extent that BT injections are effective in these cases. Literature about sialorrhea is just beginning. However, the reduction of the production of saliva following intra parenchymatic injection of toxin into the parotid and submandibular glands, thus rarifying drooling, has been demonstrated. For each of the pathological indications, both the injection techniques and the optimal doses remain to be determined., Discussion: Because BT blocks all cholinergic transmission, including the autonomous nervous system, it was plausible to expect a reduction in sweating and salivation on local injection of the product. In fact, the first publications indicated such efficiency without serious side effects. For hyperhidrosis, there has developed a consensus for making intracutaneous injections only. Of the injections in axillary areas, the palms of the hands, the plantar regions, the face or other cutaneous areas, palmoplantar hyperhidrosis is the least accessible, in any case causes the most technical problems, because of difficulty in pain management. For sialorrhea and the drooling that accompanies certain chronical neurological diseases, BT seems to have very promising effects. However, it has not been precisely determined whether to inject the parotid gland, the submandibular gland, or both. Necessary and sufficient means of targeting are still imprecise. It also remains to be determined the number of sites per gland and the doses to be injected.

Apoptosis is a highly organized cellular process that is critical for maintaining glandular homeostasis. We have used primary rat salivary acinar cells from the parotid and submandibular glands to investigate the critical regulatory events involved in apoptosis. Caspase-3 activity, cleavage of caspase substrates, and deoxyribonucleic acid (DNA) fragmentation were assayed in cells treated with etoposide, a DNA-damaging agent, or brefeldin A (BFA), a Golgi toxin. Dose-response studies showed that the sensitivity of both cell types to etoposide and BFA was similar, with 150 microM etoposide or 1.5 microM BFA inducing maximal caspase activation. However, BFA induced a more robust activation of caspase and DNA fragmentation in both cell types. Similar results were observed when the caspase cleavage of poly(adenosine 5'-diphosphate ribose) polymerase and protein kinase C delta were analyzed by Western blot. Analysis of the kinetics of apoptosis showed that caspase-3 activation was maximal at 8 h of etoposide or BFA treatment in the parotid cells and at 8-18 h in the submandibular cells. A similar time course was observed when DNA fragmentation was assayed, although maximal DNA fragmentation in BFA-treated cells was two- to threefold higher than that observed in etoposide-treated cells. Despite slight kinetic differences, it would appear that the apoptotic cascade is very similar in both primary parotid and submandibular acinar cells. Although limited in their long-term stability in culture, the use of primary, nonimmortalized salivary acinar cultures will also permit the use of specific transgenic animals to further characterize the molecular events involved in the regulation of salivary gland acinar cell apoptosis.

Using electrophysiological and molecular techniques, we investigated the molecular nature of an inwardly rectifying K+ channel in bovine parotid acinar (BPA) cells and examined its role in setting resting membrane potential. In whole-cell recordings from freshly isolated BPA cells, a predominant current was a K+ current rectified strongly in the inward direction. An inward conductance of the inwardly rectifying K+ (Kir) current was proportional to [K+]o(0.57). The selectivity sequence based on permeability ratios was K+ (1.00) > Rb+ (0.63) >> Li+ (0.04) = Na+ (0.02) and the sequence based on conductance ratios was K+ (1.00) >> Rb+ (0.03) = Li+ (0.03) = Na+ (0.02). The current was blocked by extracellular Ba2+ and Cs+ in a voltage- and a concentration-dependent manner, with a Kd at 0 mV of 11.6 microM and 121 mM, respectively. Cell-attached patch measurements identified 27 pS K+ channels as being the most likely to mediate whole-cell Kir currents. Addition of Ba2+ (100 microM) to the bathing solution reversibly depolarized the resting membrane potential in intact unstimulated cells. RT-PCR of RNA from bovine parotid cells revealed transcripts of bovine Kir2.1 (bKir2.1). HEK293 cells stably expressing bKir2.1 cloned from bovine parotid exhibited whole-cell and single channel Kir currents, of which electrophysiological characteristics were quantitatively similar to those of native Kir currents. Immunohistochemical studies showed a bKir2.1 immunoreactivity in BPA cells. Collectively, these results suggest that Kir2.1 may mediate native Kir currents responsible for setting resting membrane potential in BPA cells and might be, at least in part, involved in spontaneous secretion in ruminant parotid glands.

The role of salivary glands in nitrate and nitrite metabolism is poorly understood. The aim of the present study was to investigate the effect of parotid gland ablation on dynamic metabolism of nitrate and nitrite in miniature pigs. The parotid glands of 5 healthy miniature pigs were bilaterally ablated by methyl violet. Concentrations of nitrate and nitrite of whole saliva, serum, and urine samples were analyzed by high-performance liquid chromatography. Results showed that bilateral ablation of the parotid glands led to a significant decrease of nitrate secretion from blood to saliva (P < 0.05) and thus low nitrite levels. Dysfunction of the parotid glands temporarily increased the systemic level of nitrate in miniature pigs after nitrate loading. This study suggests that the parotid glands play an important role in the balance of nitrate and nitrite levels in both whole saliva and the body.

The role of adrenoceptors in the control of parotid salivary function has been investigated in anaesthetized sheep. The enhancement of parotid protein output that occurs when the parasympathetic and sympathetic innervations to the gland are stimulated simultaneously in bursts at a low frequency (20 Hz for 1 s at 10-s intervals) was effectively abolished by pretreatment with propranolol (> or = 1.0 mg kg(-1), i.v., P < 0.001), without a comparable reduction in the flow of saliva or in the output of sodium or potassium. Secretion of protein was similarly augmented by simultaneous stimulation of the sympathetic innervation and an intracarotid infusion of acetylcholine (0.4-0.6 microg min(-1) g gland(-1)). This effect was also abolished by pretreatment with propranolol. Pretreatment with phentolamine (>1.0 mg kg(-1), i.v.) had no effect on the output of protein that occurred during combined stimulation of the parasympathetic and sympathetic innervations but increased the flow of saliva and the output of electrolytes. Stimulation of the parasympathetic innervation to the parotid gland caused a substantial fall in vascular resistance, which was reduced by the administration of atropine (0.5 mg kg(-1)). Stimulation of the sympathetic innervation caused a substantial rise in parotid vascular resistance in atropinized sheep. This effect was greater during continuous stimulation than during intermittent stimulation and enhanced by pretreatment with propranolol. It was virtually eliminated by pretreatment with phentolamine. It is concluded that the enhancement of protein output from the ovine parotid gland, that occurs during combined stimulation of the parasympathetic and sympathetic innervations at relatively low frequencies, depends upon interaction between cholinergic muscarinic and beta-adrenergic receptors. The vasoconstriction that occurs during sympathetic stimulation alone can be accounted for by activation of alpha-adrenoceptors., (Copyright 2002 Elsevier Science B.V.)

Although Ca(2+)-activated K(+) (K(Ca)) channels distinct from maxi-K(+) channels have been suggested to contribute to muscarinically stimulated K(+) currents in salivary acinar cells, the molecular nature of the channels is unclear. Using electrophysiological and RT-PCR techniques, we have now investigated the involvement of SK4/IK1-like channels in native K(Ca) currents in bovine parotid acinar (BPA) cells. Ca(2+)-dependent K(+) efflux from perfused bovine parotid tissues was not inhibited by a maxi-K(+) channel blocker, tetraethylammonium (TEA). Whole cell recordings from BPA cells showed a TEA-insensitive K(Ca) conductance, which was highly permeable to Rb(+). In inside-out macropatches, TEA-insensitive Rb(+) currents were activated by Ca(2+) with half-maximal values of 0.4 microM. 1-Ethyl-2-benzimidazolinone (1-EBIO) increased the Ca(2+) sensitivity of the currents. The calmodulin antagonists trifluoperazine, calmidazolium, and W-7 inhibited the Ca(2+)-activated Rb(+) currents. In outside-out macropatches, Ca(2+)-activated Rb(+) currents were inhibited by Ba(2+), quinine, clotrimazole, and charybdotoxin but not by d-tubocrarine or apamin. RT-PCR analysis showed transcripts of SK4/IK1 in BPA cells. These results collectively suggest that SK4/IK1-like channels mediate the native K(Ca) currents in BPA cells.

Arginase in salivary glands is potentially involved in the synthesis of proline, glutamate, and polyamines that play specific physiological roles in the glands, and also in depletion of arginine in the oral cavity to protect teeth from microorganisms. We detected protein and mRNA for the type I isoform of arginase in mouse salivary glands. Enzymes of the arginine-biosynthetic pathway were also detected. Immunohistochemical analysis revealed that arginase I was enriched in the striated duct, and was also present in the acinus, demilune and granulated duct. Mice with targeted disruption of the gene for C/EBPalpha, which is a transcription factor essential for expression of the arginase I gene in the liver, showed dramatically reduced immunoreactivity for arginase I in the parotid gland but not in the submandibular and sublingual glands. Therefore, C/EBPalpha is specifically required for expression of the arginase I gene in the parotid gland.