Prostate-specific antigen: An unfamiliar protein in the human salivary glands.

OBJECTIVES: The presence of prostate-specific antigen (PSA) in saliva and salivary glands has been reported. Nevertheless, its release pathway in these glands remains to be elucidated. Here, we showed PSA subcellular distribution focusing on its plausible route in human salivary parenchyma. MATERIALS AND METHODS: Sections of parotid and submandibular glands were subjected to the immunohistochemical demonstration of PSA by the streptavidin-biotin method revealed by alkaline phosphatase. Moreover, ultrathin sections were collected on nickel grids and processed for immunocytochemical analysis, to visualize the intracellular distribution pattern of PSA through the observation by transmission electron microscopy. RESULTS: By immunohistochemistry, in both parotid and submandibular glands PSA expression was detected in serous secretory acini and striated ducts. By immunocytochemistry, immunoreactivity was retrieved in the cytoplasmic compartment of acinar and ductal cells, often associated with small cytoplasmic vesicles. PSA labeling appeared also on rough endoplasmic reticulum and in the acini's lumen. A negligible PSA labeling appeared in most of the secretory granules of both glands. CONCLUSIONS: Our findings clearly support that human parotid and submandibular glands are involved in PSA secretion. Moreover, based on the immunoreactivity pattern, its release in oral cavity would probably occur by minor regulated secretory or constitutive-like secretory pathways.

Melatonin ultrastructural localization in mitochondria of human salivary glands.

The hormone melatonin was initially believed to be synthesized exclusively by the pineal gland and the enterochromaffin cells, but nowadays its production and distribution were observed in several other tissues and organs. Among others, the ultrastructural localization of melatonin and its receptors has been reported in human salivary glands. In these glands, the fine localization of melatonin in intracellular organelles, above all in mitochondria, remains to be explored comprehensively. Bioptic samples of parotid and submandibular glands were treated to search for melatonin using the immunogold staining method by transmission electron microscopy. Morphometric analysis was applied to micrographs. The results indicated that, both in parotid and submandibular glands mitochondria, a certain melatonin positivity was present. Within glandular cells, melatonin was less retrieved in mitochondria than in secretory granules; however, its presence in this organelle was clearly evident. Inside striated duct cells, melatonin staining in mitochondria was more prominent than in glandular cells. Our data provide an ultrastructural report on the presence of melatonin in mitochondria of human major salivary glands and represent a fundamental prerequisite for a better understanding of the melatonin role in this organelle.

Histopathological and biochemical alterations of the parotid gland induced by experimental hypothyroidism in adult male rats and the possible therapeutic effect of Nigella sativa oil.

Thyroid hormones are essential for metabolic rate regulation and play a role on the integrity of the salivary glands. Nigella sativa is a widely used plant in medicine. This study aimed to evaluate the effect of Nigella sativa oil (NSO) on the hypothyroidism-induced parotid gland pathological alterations. Rats were divided into four groups: control group, hypothyroid group: received daily oral carbimazole for 3 weeks, hypothyroid-NSO group: NSO was orally given for 4 weeks after hypothyroidism induction and NSO group: administrated NSO only for 4 weeks. After 7 weeks, all rats were sacrificed, serum thyroid hormones were estimated, and parotid glands were assessed by histopathological, immunohistochemical, ultrastructural and morphometric analyses. Hypothyroid group showed a significant decrease in thyroid hormones with increase in thyroid-stimulating hormone (TSH) levels and decrease in body and parotid weights compared to the control rats that were improved with NSO treatment. Sections of the hypothyroid group showed fibrosis, acinar cytoplasmic vacuolations, vascular congestion, ductal dilatation, wide intercellular canaliculi, nuclear pyknosis and decreased number of secretory granules. Also, there were decreased B-cell lymphoma 2 (Bcl-2) and increased p53, Bcl-2 Associated X (Bax) and alpha-smooth muscle actin (α-SMA) immune-expressions; with decreased Bax/ Bcl-2 ratio that all were attenuated by NSO. NSO ameliorates hypothyroidism-induced parotid changes by altering p53, Bax and Bcl-2 pathway.

Distribution, nature, and origin of CXCL14-immunoreactive fibers in rat parotid gland.

The distribution and nature of CXCL14-immunoreactive nerve fibers in salivary glands, especially the parotid gland was immunohistochemically investigated. Furthermore, the origin of parotid CXCL14-immunoreactive nerve fibers was determined by retrograde tracing experiments. CXCL14-immunoreactive nerve fibers were localized in the parotid, submandibular, and sublingual glands, particularly in the parotid gland. Double staining using identical sections revealed that a subpopulation of cells neuropeptide Y (NPY)-containing fibers was immunopositive for CXCL14 in the parotid gland. In the peripheral regions of acinar cells, CXCL14-immunoreactive fibers tended to coexist with NPY; however, perivascular NPY-immunoreactive fibers tended to be immunonegative for CXCL14. Parotid CXCL14-immunoreactive fibers were immunopositive for tyrosine hydroxylase (TH) but immunonegative for choline acetyltransferase and vasoactive intestinal peptide (VIP). After injection of horseradish peroxidase-labeled wheat germ agglutinin (WGA-HRP) in the parotid gland, retrogradely labeled neurons were seen in the superior cervical ganglion (SCG) and otic ganglion. Some of the WGA-immunoreactive somata in the SCG were immunopositive for CXCL14; however, no doubly-labeled somata were noted in the otic ganglion. These results indicate that CXCL14-immunoreactive nerve fibers originate in the SCG, and are sympathetic in nature. The coexistence of CXCL14 with NPY/TH suggests that CXCL14 may be associated with NPY/TH functions as a neuromodulatory chemokine in the parotid gland. The localization of CXCL14 nerve fibers around the acinar cells of the parotid gland indicates its involvement in acinar cell function, but not vasoconstriction.

Localization of phospholipase C ß3 in the major salivary glands of adult mice.

Phospholipase C (PLC)ß has a role in saliva secretion by controlling intracellular Ca2+via its product, IP3. The present study was attempted to localize PLCß isoforms in mouse salivary glands in situ. A single major band was detected for PLCß3 in immunoblots of the parotid and sublingual glands (PG, SLG), while no such band was seen in the submandibular gland (SMG). No bands were detected for PLCß1 or 4 in the three glands. In immuno-light microscopy of PG and SLG, substantial immunoreactivity for PLCß3 was seen in the cytoplasm including the plasmalemma of almost all ductal cells, while no distinct immunoreactivity was discerned in most acinar cells except for sublingual demilune cells. Numerous ductal cells exhibited higher immunoreactivity for PLCß3 in their apical/supranuclear cell domain including the plasmalemma than in the basal/infranuclear domain, indicating an apico-basal polarity. In immuno-gold electron microscopy of PG ducts and SLG ducts and demilunes, most gold particles were found in association with plasma membranes as well as various intracellular membranes, most of which formed small oblong or flattened vesicles and vacuoles. A few particles were seen without association with any membranous structures. The present finding supports the previous physio-pharmacological result that Ca2+-signaling proteins as well as initial intracellular Ca2+ changes occur in the apical cell domain including the plasma membranes of the exocrine cells.

Localization of phosphatidylinositol 4-phosphate 5-kinase (PIP5K) α, ß, γ in the three major salivary glands in situ of mice and their response to ß-adrenoceptor stimulation.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), which is composed of three isozymes (α, ß and γ), catalyzes the production of phosphatidylinositol bisphosphate (PIP2). This phospholipid functions in membrane trafficking, as an anchor for actin cytoskeletons and as a regulator of intramembranous channels/transporters. It is also a precursor of such second messengers as diacylglycerol, inositol triphosphate and phosphatidylinositol (3,4,5)-triphosphate. In the present study, the expression and localization of endogenous PIP5Ks were examined in the three major salivary glands of young adult mice in situ. In western blotting of normal control glands, immunoreactive bands for individual PIP5Ks were detectable, with the highest density in the parotid gland and the weakest density in the submandibular gland. In immuno-light microscopy under non-stimulated condition, weak immunoreactivity for PIP5Kα was confined to the apical plasmalemma in parotid, but not sublingual or submandibular, acinar cells. Immunoreactivity for PIP5Kß was weak to moderate and confined to ductal cells but not acinar cells, whereas that for PIP5Kγ was selectively and intensely detected in myoepithelial cells but not acinar cells, and it was weak in ductal cells in the three glands. In western blot of the parotid gland stimulated by isoproterenol, a ß-adrenoceptor agonist, no changes were seen in the intensity of immunoreactive bands for any of the PIP5Ks. In contrast, in immuno-light microscopy, the apical immunoreactivity for PIP5Kα in parotid acinar cells was transiently and distinctly increased after the stimulation. The increased immunoreactivity was ultrastructurally localized on most apical microvilli and along contiguous plasma membrane, where membranous invaginations of various shapes and small vesicles were frequently found. It was thus suggested that PIP5Kα is involved in post-exocytotic membrane dynamics via microvillous membranes. The present finding further suggests that each of the three isoforms of PIP5K functions through its product PIP2 discretely in different cells of the glands to regulate saliva secretion.

Melatonin release by exocytosis in the rat parotid gland.

Several beneficial effects on oral health are ascribed to melatonin. Due to its lipophilic nature, non-protein-bound circulating melatonin is usually thought to enter the saliva by passive diffusion through salivary acinar gland cells. Recently, however, using transmission electron microscopy (TEM), melatonin was found in acinar secretory granules of human salivary glands. To test the hypothesis that granular located melatonin is actively discharged into the saliva by exocytosis, i.e. contrary to the general belief, the ß-adrenergic receptor agonist isoprenaline, which causes the degranulation of acinar parotid serous cells, was administered to anaesthetised rats. Sixty minutes after an intravenous bolus injection of isoprenaline (5 mg kg-1 ), the right parotid gland was removed; pre-administration, the left control gland had been removed. Samples were processed to demonstrate melatonin reactivity using the immunogold staining method. Morphometric assessment was made using TEM. Gold particles labelling melatonin appeared to be preferentially associated with secretory granules, occurring in their matrix and at membrane level but, notably, it was also associated with vesicles, mitochondria and nuclei. Twenty-six per cent of the total granular population (per 100 µm2 per cell area) displayed melatonin labelling in the matrix; three-quarters of this fraction disappeared (P < 0.01) in response to isoprenaline, and melatonin reactivity appeared in dilated lumina. Thus, evidence is provided of an alternative route for melatonin to reach the gland lumen and the oral cavity by active release through exocytosis, a process which is under the influence of parasympathetic and sympathetic nervous activity and is the final event along the so-called regulated secretory pathway. During its stay in granules, anti-oxidant melatonin may protect their protein/peptide constituents from damage.

Bone marrow-derived mesenchymal stem cells ameliorate parotid injury in ovariectomized rats.

BACKGROUND AIMS: Parotid hypofunction causes life-disrupting effects, and there are no effective medications for xerostomia. We hypothesized that mesenchymal stem cells (MSCs) have repairing effects on parotid glands of ovariectomized (OVX) rats. METHODS: Forty-five adult female rats were divided into three equal groups: group I (Control group), group II (OVX-group) and group III (OVX rats that received MSCs at 4 and 8 weeks post-ovariectomy). At 12 weeks post-ovariectomy, histological (Masson's trichrome and periodic acid-Schiff with alcian blue stains), immunohistochemical (caspase-3 and CD44) and morphometric studies and salivary flow rate and saliva pH determination were carried out. RESULTS: Histologically, the OVX group displayed numerous irregular vacuolated acini, thickened septa with marked cellular infiltration and vascular congestion. Degenerated organelles and few or irregular secretory granules with a different density were observed. Caspase-3-positive cells were highly expressed. MSC-treated glands exhibited a considerable degree of preservation of glandular architecture with numerous CD44-expressing and few caspase-3-expressing cells. Significant decrease of the salivary flow rate in the OVX group was detected, which reverted to normal levels in group III. CONCLUSIONS: MSCs ameliorated the damaging effects of ovariectomy on the parotid glands.

Developmental changes in the parotid salivary gland of prenatal buffalo: an ultrastructural study / Cambios en el desarrollo de la glándula salival de la parótida de búfalo prenatal: un estudio ultraestructural

SUMMARY: The present study was undertaken to elucidate ultrastructural changes in development of parotid salivary gland of buffalo during different stages of prenatal life. The ultrastructural studies revealed that the cytoplasm of acinar cells was filled with mitochondria, rough endoplasmic reticulum and Golgi complex in mid and late foetal age groups. Medium electron dense secretory granules first appeared in the acinar cells of parotid gland at 30 cm CVRL (141st day). However, at 49.5 cm CVRL (185th day) two types of electron dense granules were identified on basis of granule density viz., dark and light. The dark electron dense granules were more in number, whereas light granules were comparatively less having electron lucent content within them was identified. The mean diameter of dark and light granules was measured about 0.45±0.1 µm and 0.30±0.1 µm, respectively, which showed that the dark granules were comparatively larger in size. The secretory granules were increased in number during the late foetal age group. The myoepithelial cells were located at the base of the acinar cells as well as intercalated and striated ducts, and were stellate in shape. The ultrastructure of myoepithelial cell revealed parallel stream of myofilaments in the cytoplasm and its processes. Lipofuscin pigments were also observed in between the acinar cells of parotid gland.

RESUMEN: El estudio se realizó para elucidar los cambios ultraestructurales en el desarrollo de la parótida del búfalo durante las diferentes etapas de la vida prenatal. Los estudios ultraestructurales revelaron que el citoplasma de las células acinares estaba saturado de mitocondrias, de retículo endoplasmático rugoso y Complexo golgiensis en las edades fetal media y tardía. Se observó un número mayor de gránulos oscuros densos de electrones, mientras que los gránulos ligeros fueron comparativamente menor en número con contenido de electrones. El diámetro medio de gránulos oscuros y ligeros se midió aproximadamente 0,45 / pm 0,1 / mu m y 0,30 / pm 0,1 / mu m, respectivamente, lo que mostró que los gránulos oscuros eran comparativamente mayores en tamaño. Los gránulos secretores aumentaron en número durante el último grupo de edad fetal. Las células mioepiteliales se localizaron en la base de las células acinares, así como en conductos intercalados y estriados, y tenían una forma estrellada. La ultraestructura de las células mioepiteliales reveló una corriente paralela de miofilamentos en el citoplasma y sus procesos. También se observaron pigmentos de lipofuscina entre las células acinares de la glándula parótida.

Low-frequency noise effects on the rat parotid gland: A transmission electron microscopy study.

INTRODUCTION: Low-frequency noise (LFN) is a ubiquitous physical stressor known to cause degenerative cellular changes and organ alterations with functional repercussions both in humans and animals. MATERIALS AND METHODS: After acceptance of the study protocol by a local ethics committee, 20 Wistar rats were randomly divided into two equal groups. One group was kept in silence and the other continuously exposed to LFN during 13 weeks. The rats had unlimited access to water and were fed standard rat chow. After exposure, the animals were sacrificed and the parotid glands were excised and prepared for transmission electron microscopy. RESULTS: The acinar cells showed marked ultrastructural alterations, such as intracellular vacuolization, loss of cell polarity, increased heterochromatin, cytoplasmic inclusions, and oncocytic transformation. CONCLUSIONS: LFN induces ultrastructural changes in the rat parotid gland that correlate with previously described functional changes.

Acinar cell response to liquid diet during rats' growth period differs in submandibular and sublingual glands from that in parotid glands.

Continuously feeding a liquid diet to growing rodents strongly inhibits parotid gland growth, due to suppressed growth of acinar cells. This study investigated whether a liquid diet had a similar effect on submandibular and sublingual glands of growing rats. Rats were weaned on day 21 after birth and then fed a pellet diet in the control group and a liquid diet in the experimental group for 0, 1, 2, 4, and 8 weeks. Their submandibular and sublingual glands were excised, weighed, and examined histologically, immunohistochemically (using antibodies to 5'-bromo-2-deoxyuridine and cleaved caspase 3), and ultrastructurally. The submandibular glands did not significantly differ between the control and experimental groups at all tested points. Only at Week 8, acinar cell area and 5'-bromo-2-deoxyuridine-labeling index of acinar cells in sublingual glands were significantly lower in the experimental group than in the control group. These results show that a liquid diet during rats' growth period had no effect on acinar cells in their submandibular glands, and only a slight effect on acinar cells in their sublingual glands of growing rats, in contrast to the marked effect of a liquid diet on parotid glands.

Parotoid, radial, and tibial macroglands of the frog Odontophrynus cultripes: Differences and similarities with toads.

Anuran integument is characterized by the presence of glands, some of which are responsible for toxin production. In some species these glands accumulate in parts of the body strategically located against predators, forming structures known as macroglands. This is the case for parotoid macroglands, on the dorsum of the head, tibial macroglands, on the rear limbs, and radial macroglands, on the forelimbs of toads and some other anurans. The toad Rhinella jimi, for example, simultaneously displays all three types of macroglands, which is unusual even among bufonids. Interestingly, considering the phylogenetic distance, the frog Odontophrynus cultripes (Odontophrynidae) also presents these three macroglandular types. In this study we analyze the morphology of O. cultripes macroglands and the chemical composition of their poison using an interdisciplinary approach. In this species, the parotoid, tibial, and radial macroglands consist of aggregates of elongated and juxtaposed poison glands, arranged in a honeycomb style, very similar to that of toads. Comparative analysis of these three macrogland types shows significant differences in both the morphology of secretory granules and biochemical composition. The present work on O. cultripes contributes to the evidence that amphibians, or at least anurans, share a basic design for all cutaneous glandular accumulations. The determinant factor for macroglandular formation may be the selective pressure for defense against predators.

[STUDYING OF MORPHOLOGICAL PECULIARITIES OF SUBMANDIBULAR SALIVARY GLANDS AFTER GASTRIC RESECTION IN EXPERIMENT].

The impact of gastric resection on the submandibular salivary gland (SSG) state, using histological and histochemical methods of investigation in experiment, was studied up. A relative mass of a SSG after gastric resection conduction have had reduced, and the accompanying changes in stroma were revealed with the gland's secretion enhancement. Essential dystrophic changes in the SSG parenchyma and stroma after gastric resection conduction may cause a pronounced disorders of their function.

Step-by-step protocol to perfuse and dissect the mouse parotid gland and isolation of high-quality RNA from murine and human parotid tissue.

Macroscopic identification and surgical removal of the mouse parotid gland is demanding because of its anatomic location and size. Moreover, the mouse parotid gland contains high concentrations of RNases, making it difficult to isolate high-quality RNA. So far, appropriate methods for optimal perfusion-fixation and dissection of mouse parotid glands, as well as the isolation of high quality RNA from this tissue, are not available. Here we present a simple, optimized, step-by-step surgical method to perfuse and isolate murine parotid glands. We also compared two common RNA extraction methods (RNeasy Mini Kit versus TRIzol) for their yields of high-quality, intact RNA from human and murine parotid gland tissues that were either snap-frozen or immersed in RNAlater stabilization solution. Mouse parotid tissue that was perfused and immersed in RNAlater and human samples immersed in RNAlater exhibited the best RNA quality, independent of the isolation method.

Morphometric Study of Diabetes Related Alterations in Human Parotid Gland and Comparison with Submandibular Gland.

Type 2 diabetes mellitus represents one of the principal diseases that afflict the world population and is often associated with malfunction of salivary glands and consequent oral diseases. We recently described significant ultrastructural alterations in the human submandibular gland in diabetic patients without evident oral pathologies. Herein, an analogs morphometrical investigation was focused on the parotid gland in order to evaluate if one of the two glands is more affected by diabetes. Parotid fragments from diabetic and nondiabetic patients were fixed, dehydrated, and processed for light and electron microscopy. Serous cells were randomly photographed and the density and size of several structures involved in the secretory process were examined by morphometry. Scanning electron microscopy images revealed significant changes in the number of apically docked granules and vesicles, suggesting that the last steps in exocytosis are somehow altered in diabetic cells. Other variables analyzed by light and transmission electron microscopy such as the size of acini and secretory granules did not show significant changes, but comparison with previous data obtained with submandibular gland cells demonstrated that the two glands are affected differently.

Ultrastructural evidence of a secretory role for melatonin in the human parotid gland.

In vivo animal studies show that pentagastrin, cholecystokinin and melatonin cause the secretion and synthesis of salivary proteins. Melatonin occurs in large amounts in the gut and is released into the blood on food intake. In vitro experiments suggest that pentagastrin exerts secretory activity in human salivary glands, as judged by ultrastructural changes, reflecting secretion, and an actual protein output. Currently, it is hypothesised that melatonin induces secretory exocytotic events in the human parotid gland. Human parotid tissues were exposed to a high single concentration of melatonin in vitro, processed for high resolution scanning electron microscopy and then assessed morphometrically with the emphasis on the membrane of the intercellular canaliculi, a site of protein secretion. Compared with controls and in terms of density, the melatonin-exposed parotid tissues displayed increases in protrusions (signalling anchored granules) and microbuds (signalling membrane recycling and/or vesicle secretion) and decreases in microvilli (signalling cytoskeletal re-arrangement related to exocytosis), phenomena abolished or very largely reduced by the melatonin receptor blocker, luzindole. In conclusion, acinar serous cells of parotid tissue displayed in vitro exocytotic activity to melatonin, signalling protein secretion. Whether, under physiological conditions, melatonin influences the secretion of human parotid glands remains to be explored, however.

Análisis morfométrico ultraestructural de células normales de glándula parótida de rata / Ultrastructural morphometric analysis from normal parotid gland cells of rat

A partir de 10 ratas hembras con un peso aproximado de 250 g y 4 meses de vida, fueron obtenidas quirúrgicamente muestras de glándula parótida las que se trataron con técnicas de microscopía electrónica de transmisión para posteriormente obtener microfotografías de células parotideas con aumentos finales de hasta 21300 X. En las citadas microfotografías se aplicaron técnicas morfométricas con el objetivo de cuantificar las fracciones volumétricas que los distintos componentes ocupan en estas células normales, describiendo de esta manera sus volúmenes y relacionándolos con la funcionalidad que desempeñan en esta célula normal. Se evaluaron las fracciones volumétricas pertenecientes a: citoplasma, núcleo, mitocondrias, retículo endoplasmático rugoso (RER), gránulos de zimógeno, eu y heterocromatina. De igual forma, se cuantificó las áreas celulares y nucleares. Contando con los datos numéricos producto de la evaluación morfométrica de sus componentes se podrá determinar el patrón de distribución de sus organelos y de funcionalidad de esta célula activa en la síntesis y secreción de proteínas representada por los gránulos de zimógeno de diastasa y diversas proteínas salivales.

From 10 female rats weighing approximately 250 g and aged 4 months, samples of parotid gland were obtained surgically which were treated with transmission electronic microscopy in order to obtain microphotographs with final increases of up to 21,300 X. Morphometric techniques were applied to these microphotographs to quantify the volumetric fractions that the different components occupy in these normal cells, thus describing their volumes and relating them to their functionality in this normal cell. Volumetric fractions were evaluated pertaining to: cytoplasm, nucleus, mitochondria, rough endoplasmic reticulum (RER), zymogen granules, eu and heterochromatin. Likewise, cell and nuclear areas were quantified. With the numerical data from the morphometric evaluation of its components, it was possible to determine the distribution pattern of the organelles and functionality of this cell active in protein synthesis and secretion represented by diastase zymogen granules and various salivary proteins.

Subcellular distribution of melatonin receptors in human parotid glands.

The hormone melatonin influences oral health through a variety of actions, such as anti-inflammatory, anti-oxidant, immunomodulatory and antitumour. Many of these melatonin functions are mediated by a family of membrane receptors expressed in the oral epithelium and salivary glands. Using immunoblotting and immunohistochemistry, recent studies have shown that the melatonin membrane receptors, MT1 and MT2, are present in rat and human salivary glands. To date, no investigation has dealt with the ultrastructural distribution of the melatonin receptors. This was the aim of the present study, using the immunogold method applied to the human parotid gland. Reactivity to MT1 and, with less intensity, to MT2 appeared in the secretory granules of acinar cells and in the cytoplasmic vesicles of both acinar and ductal cells. Plasma membranes were also stained, albeit slightly. The peculiar intracytoplasmic distribution of these receptors may indicate that there is an uptake/transport system for melatonin from the circulation into the saliva.

Study of human adult parotid duct in the area of penetration through buccinator muscle and their functional relationship as a sphincter.

The adult human parotid duct is roughly 6-8 cm long. From the parotid gland, parotid duct traverses through masseter muscle, penetrates through buccinator muscle, and opens into the oral cavity. This unique form of the parotid duct is likely correlated with the function of the duct, directly affected by the movement of the buccinator muscle during mastication and swallowing. Histological structure of the duct is known to be different in each region, and details of smooth muscle present in the parotid duct are mostly unclear. In this study, we conducted SEM and histological observations of the area where the parotid duct penetrates the buccinator muscle, and an observation of smooth muscle to investigate its existence using alpha-smooth muscle antibody. We confirmed the presence of an abundance of skeletal muscle bundles likely originating from the buccinator muscle under the epithelium of the parotid duct wall in the region where it penetrates the buccinator muscle. We also observed that some of the muscle fibers were completely attached to the epithelium. We observed a lack of smooth muscle in this region of the duct wall. From these results, we suggest that the area of the duct penetrating buccinator muscle plays a role in regulating the salivary passage through the contraction of the surrounding buccinator muscle fibers.