M Diana

Morphological evidence that pentagastrin regulates secretion in the human parotid gland

Salivary secretion is principally regulated by autonomic nerves. However, recent evidence from in vivo animal experiments suggests that gastrointestinal peptide hormones can also influence saliva production. The aim of the present study was to define the secretagogue activity of the gastrin‐analogue pentagastrin in human salivary glands. For this purpose, parotid tissues were exposed to pentagastrin in vitro. Morphological techniques were used to evaluate modifications to serous acinar cells associated with secretion. Using a variant of the osmium maceration method, high resolution scanning electron microscopy allowed assessment of the morphology of the cytoplasmic aspect of the plasmalemma to demonstrate secretory activity. To quantify responses to pentagastrin, we recorded morphometric data on microvilli, microbuds, and protrusions. Dose‐dependent morphological changes were observed, whereas protein concentration increased in the incubate. The use of selective receptor antagonists showed pentagastrin to act principally via cholecystokinin‐A receptors. The morphological responses observed following exposure to pentagastrin differed from those elicited following exposure to the pan‐muscarinic agonist carbachol. This study provides the first demonstration of a direct secretory action of gastrointestinal peptides on salivary glands in humans.

Diabetes reduces statherin in human parotid: immunogold study and comparison with submandibular gland.

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

Diabetes affects statherin expression in human labial glands.

BACKGROUND AND OBJECTIVE Salivary statherin, which plays a special role in the defense of tooth integrity, is secreted by both major and minor salivary glands. A significantly reduced expression of this was recently found in human major salivary glands removed from diabetic subjects and was correlated with the high incidence of dental diseases occurring in patients with diabetes. In this study, we measured the density of gold particles indicating statherin immunoreactivity in labial glands to reveal a significant difference between diabetic and non-diabetic patients. MATERIALS AND METHODS Surgical samples of labial glands obtained from both diabetic and non-diabetic patients were fixed with a glutaraldehyde and paraformaldehyde mixture, embedded in Epon, and treated for immunogold histochemistry using a polyclonal antibody specific for statherin. RESULTS Statherin immunoreactivity was detected onto small vesicles diffused throughout the cytoplasm of serous cells. Statistical analysis revealed that the number of stained particles was significantly lower in the samples from diabetic subjects than from non-diabetic subjects. CONCLUSIONS The results indicate that diabetes affects statherin secretion in labial glands and support the hypothesis that the increased susceptibility to oral diseases associated with diabetes could be related with a reduced statherin secretion.

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.

Mitochondrial metabolism and morphology in a rat model of NAFLD

NAFLD (non-alcoholic fatty liver disease) is a condition, characterized by fatty liver, that, if untreated, can progress to NASH (non-alcoholic steatohepatitis) and, finally, to cirrhosis. Mitochondrial dysfunction is a crucial element for the progression of NAFLD to NASH. Our objective was studying both morphological and physiological mitochondrial alterations in a rat model of NAFLD and NASH. Sprague Dawley rats were fed either by a standard diet (35% of energy from fat) or by a high fat diet (HFD) (71% of energy from fat). Diets were given ad libitum for a maximum of four weeks and then rats were killed by decapitation after 1, 2, 3, and 4 weeks. A portion of liver samples was processed for light and transmission electron microscopy (TEM) for steatosis assessment, a part for high resolution scanning electron microscopy (HRSEM) to evaluate 3D mitochondria morphology, the remaining part for oxidative phosphorylation experiments (OXPHOS). Measurements of OXPHOS were performed on freshly isolated mitochondria in a Clark-type oxygen electrode. Several substrates were added to mitochondria to test the efficiency of electron transport chain complexes (ETC) and to test the efficiency of several enzymes involved in fatty acid oxidation (FAO). At three and four weeks of HFD, signs of hepatic steatosis were evident (as an increased amount of intracellular lipid droplets) on histological sections and TEM images. OXPHOS measurements indicated that, compared to controls, in the first week, HFD rats had an increased oxygen consumption with each substrate, in the second, third and fourth week OXPHOS efficiency had a tendency to decline with substrates inherent to glycolitic metabolism, but it was higher with substrates stimulating FAO. There were not notable differences in Respiratory Control Ratio and ADP/O ratio between HFD and controls, proving that the integrity of mitochondria was preserved and coupled to phosphorylation. HRSEM observations indicated that variations on inner mitochondrial morphology in HFD rats, appeared in the third and fourth week, when mitochondrial cristae were less, and cristal shape was often lamellar rather than tubular. Concluding, following HFD mitochondria exhibited first an increase in oxidative metabolism (with normal ultrastructural morphology) and later an alteration in the oxidative phosphorylation apparatus associated to mitochondrial cristae injury. These preliminary results suggest that in order to develop a consistent mitochondrial impairment associated to NASH is necessary to extend HFD treatment for longer. This study was supported by a grant by RAS (Regione Autonoma della Sardegna).

Immunolocalization of AQP5 in human parotid salivary glands

Background . Aquaporins (AQPs) are channel protein essential in the transport of water across the biological cell membranes. Thirteen different AQPs have been identified in different tissues of mammals. In rat parotid, the AQP-5 is principally localized in the luminal membrane of the acinar serous cells (1). In mouse parotid a weak but distinct labeling has also observed in lateral, facing the neighboring cells, and basal plasma membranes (2). The AQP seems to be directly involved in transepithelial pathway for osmotic water flow, and in salivary fluid secretion. To date, in human only a report (3) testifies the presence of AQP-5 in luminal surface of secretory cells of parotid glands. So, in order to extend the knowledge regard to aquaporins, our intention was to define the sublocalization of AQP-5 in human parotid glands by immunogold post embedding method. Methods . Surgical samples of human parotid glands were cut into small fragments, fixed in a mixture of paraformaldehyde and glutaraldehyde and embedded in Epon resin without previous osmication. Ultrathin sections were incubated overnight at 4°C with primary antibody (1:25) against human AQP-5. Labeled sections were examined by transmission electron microscope (TEM). Results . The AQP-5 labeled was observed to the apical membrane of the serous cells. Gold particles were, occasionaly, found in the surface of intercellular canaliculi. Few small vesicles exhibited reactivity for AQP-5. Conclusions . Our preliminary studies show that the apical membrane of serous cells of human parotid glands is a specific site of AQP-5. Because the water-rich fluid is secreted in the luminal membrane where aqp5 was present, it is reasonable to speculate that aqp5 plays an important role in the secretion in the human parotid salivary gland. The reactivity of labeled vesicles could mean that in serous cells of human parotid gland occurs a translocation of aqp5 from the intracytoplasmatic compartment to the apical membrane (4). This study is supported by a grant by Regione autonoma della Sardegna. 1. Nielsen et al., Am J Physiol Cell Physiol, 1997. 2. Larsen et all., J Mol Hist, 2001. 3. Gresz et al., Am J Physiol Gastrointest Liver Physiol, 2001. 4. Hishikawa et all., biochemical and biophysical research communications, 1998.

Human submandibular glands treated in vitro with amisulpride. An HRSEM morphological and morphometrical study

Amisulpride, alike sulpiride, is a benzamide substitute used in treating schizophrenia and dysthymia in a few European countries (1). Authors describe amisulpride as D 2 and D 3 receptor antagonist, and recently as 5-HT 7a serotonin receptors antagonist (2). Moreover, a few case reports and clinical trials indicate amisulpride as a drug to reduce atypical antipsychotic-induced sialorrhea (3). Aim of this study is to investigate, by high resolution scanning electron microscopy (HRSEM), morphological changes induced in vitro by amisulpride in serous cells of human submandibular glands. Samples of human non-pathological submandibular glands obtained at surgery, were immersed in an oxygenated inorganic medium, according to the procedure described in our previous works (4), stimulated in vitro with amisulpride, and treated by our modification of the osmium maceration technique (5). By removing from serous cells all cytoplasmic organelles, we are able to visualize, by HRSEM, and quantify, with statistical method, the morphological changes on the surfaces of the plasmalemma involved in secretory processes. In particular, we calculated the density of microvilli, that of microbuds, and that of protrusions per µm 2 of the intercellular canaliculi luminal membrane. Our results show that amisulpride acts on secretory serous cells of human submandibular glands, promoting a reduction of microvilli and an increase of microbuds density. In particular, microbuds increased density indicate the presence of microexocytosis profiles that allow secretion of protein into lumen. Whereas the clinical treatment of sialorrhea with amisulpride (3) demonstrates a reduction of saliva production, our data illustrate that this drug has peculiar effects on secretory mechanisms involved in protein secretion. 1) Mortimer AM.: Neuropsychiatr Dis Treat , 5:267-77. 2009 2) Abbas AI. et al.: Psychopharmacology , 205:E-pub. 2009 3) Kreinin A. et al.: Int Clin Psychopharmacol , 21:99-103. 2006 4) Testa Riva F. et al.: Cell Tissue Res , 324:347-52. 2006 5) Riva A. et al.: Scanning Microscopy 13:111-22. 1999

A HRSEM morphometrical study on the secretory response of human submandibular serous cells treated in vitro by pentagastrin

In human submandibular glands, saliva production is principally regulated by parasympathetic and sympathetic nerves. Recently, however, Aras & Ekström (2006) and Loy et al. (2008) demonstrated, in rats and human parotid glands respectively, that secretion of parotid glands is induced by gastrointestinal hormones as well. Aim of this work is to study, in human submandibular serous cells, the morphological changes due to the gastrin-analogue pentagastrin with our in vitro technique, and to compare the results with findings obtained in response to autonomic receptors stimulation, as isoproterenol, a β-adrenergic agonist that induces a proteic secretion with little fluid, and carbachol, the pan-muscarinic receptors agonist that stimulates an abundant fluid saliva. Samples of normal human submandibular glands, obtained at surgery, were stimulated in an oxygenated inorganic medium both with pentagastrin and with other secretagogue drugs. Specimens incubated in the same medium without the drugs served as controls. Samples were then processed for light (LM) and high resolution scanning electron microscopy (HRSEM) according to our modification of the OsO4 maceration method (Riva et al., 1998). After pentagastrin treatment, serous acini show, by LM, a few dilated intercellular canaliculi with sign of exocytosis, while cells are full of secretory granules. To quantify the response we have measured, in HRSEM images, the density of the sign of exocytosis (protrusions), that of microvilli and that of microbuds located in 1 μm2 of the cytoplasmic surface of the canaliculi lumenal membrane, exposed after removal of cellular organelles. Pentagastrin induced a significative reduction of microvilli and an increase of protrusions and microbuds with respect to controls. Since as results from our previous works, these findings are indicative of a secretory stimulation, we conclude that they demonstrate the activity of pentagastrin on salivary secretion. Moreover the fact that no large vacuoles are present, whereas a large number of microbuds possibly related to microexocytosis are seen, suggests that pentagastrin provokes a protein secretion with little fluid.