Rat Parotid Gland Research Articles

Expression and biological effects of CB 1 cannabinoid receptor in rat parotid gland

Experiments were designed to determine whether cannabinoids affect salivary gland function. For this purpose, the effect of anandamide on cAMP accumulation, amylase release and Na +-K +-ATPase activity was studied in rat parotid glands. Anandamide induced a concentration-dependent increase in cAMP and led to amylase release but inhibited Na +-K +-ATPase activity. These effects were blocked by the CB 1 cannabinoid receptor antagonist, AM281. The inhibition of adenylyl cyclase activity by SQ 22536 impaired amylase release and Na +-K +-ATPase inhibition. The effect of anandamide on cAMP accumulation significantly correlated with its action either on amylase release or on Na +-K +-ATPase activity. Such correlation strongly supports the view that the effect of anandamide on amylase release and Na +-K +-ATPase activity is the result of cAMP accumulation. The relative potencies of the CB 1 cannabinoid receptor antagonist, AM281, to block these three functional responses were similar, supporting the view that anandamide actions in parotid glands were achieved through a single receptor subtype, the CB 1. Binding studies using the selective cannabinoid CB 1 receptor antagonist, [ 3H]SR141716A, indicated the presence of the specific binding site. It may be concluded that in parotid glands the endogenous cannabinoid anandamide, bound to the CB 1 cannabinoid receptor subtype, induces cAMP accumulation which in turn leads to amylase release and Na +-K +-ATPase inhibition.

Stimulation of guanylate cyclase in rat parotid membranes by phosphate

The natriuretic peptide receptor guanylate cyclases are members of the membrane-bound guanylate cyclase family. Atrial natriuretic pepticle (ANP) stimulates guanylate cyclase A, which is predominantly expressed in the rat parotid gland. ATP is well known to increase ANP-stimulated guanylate cyclase, with Mg2+ as a cofactor. We investigated the regulation of guanylate cyclase activity in rat parotid membranes with Mn2+ as a cofactor, because enzyme activity was much higher with Mn2+ than with Mg2+. ANP (10(-7)M) stimulated guanylate cyclase activity to 120%-130% of the control level, and ATP (0.1-1.0 mM) depressed its activity, with or without ANP, to 70%-80% of the control. Enzyme activity was increased by the addition of phosphate (5-20 mM). In the presence of phosphate (5 mM), guanylate cyclase with and without ANP was maximally stimulated to 5- and 6.6-fold of the control, respectively. The net stimulation of guanylate cyclase by ANP was increased at ATP concentrations between 0.2 and 0.5 mM in the presence of phosphate (5 mM or 10 mM), but no change was observed in the absence of phosphate. Phosphate not only stimulated guanylate cyclase in the absence of ATP but altered the ATP regulation of ANP-stimulated guanylate cyclase. The stimulation of guanylate cyclase by phosphate may depend on a peculiarity of rat parotid membranes.

Evidence for the involvement of cAMP-GEF (Epac) pathway in amylase release from the rat parotid gland

Amylase release from the rat parotid gland is mainly mediated in a cAMP-dependent protein kinase (PKA)-dependent manner. In the present study, amylase release mediated in cAMP-dependent and PKA-independent manners was investigated with a cAMP-regulated guanine nucleotide exchange factor (cAMP-GEF: Epac)-selective cAMP analogue, 8CPT-2Me-cAMP. The Epac was localized in the intracellular and the plasma membrane fractions. PKA activation by 8CPT-2Me-cAMP was 100-fold lower than that by cAMP. The amylase release (% of the total) from the intact parotid acinar cells was 16 and 3.6% by isoproterenol (1 μM) and 8CPT-2Me-cAMP (200 μM), respectively, and that from the saponin-permeabilized cells was 15 and 3% by cAMP (100 μM) and 8CTP-2Me-cAMP (10 μM), respectively. H-89 inhibited cAMP-induced amylase release, but did not inhibit 8CPT-2Me-cAMP-induced amylase release. These results indicated that amylase release by β-adrenergic stimulation is mediated through both the cAMP/PKA and cAMP/Epac signal pathways.

Streptozotocin-induced type 1 diabetes mellitus alters the morphology, secretory function and acyl lipid contents in the isolated rat parotid salivary gland.

Diabetes mellitus (DM) is associated with numerous conditions including hypo-secretion of digestive enzymes. This study investigated the morphology, secretory function (alpha-amylase release) and acyl lipid contents in the isolated parotid gland of STZ-induced diabetic and age-matched control rats in order to provide insights into diabetes-induced salivary insufficiency. The techniques employed included light microscopy, colourimetric and gas chromatography (GC) analysis, respectively. Diabetes mellitus was induced in adult male Wistar rats by a single intraperitoneal (IP) injection of streptozotocin (STZ) (60 mg per kg body weight). Control animals were injected with a similar volume of citrate buffer. The animals were tested for DM 4 days after STZ injection and 2 months later when they were humanely killed for the experiment. The morphological results showed diabetic parotid glands to be extensively infiltrated with lipid droplets of various magnitudes, whereas glands from control animals display normal structure with the absence of lipid droplets. The analysis of parotid secretory function revealed a significant (p < 0.05) dose-dependent decrease in alpha-amylase release in response to noradrenaline (NA) in STZ-treated glands when compared to age-match control parotid glands. Furthermore, the levels of acyl lipids (16:0, 16:1, 18:0 and 18:1) in diabetic parotid glands was significantly (p < 0.01) reduced compared to control glands, along with a reduced ratio of 16:1/16:0. The results indicate DM can elicit changes in the morphology, secretory function and acyl fatty acid quantity in the isolated rat parotid gland.

Soluble guanylyl cyclase is localised in the acinar cells and participates in amylase secretion in rat parotid gland

It is well known that the muscarinic cholinergic agonists, carbachol and methacholine, enhance nitric oxide synthase (NOS) activity, and also stimulate salivary secretion. In the present study, we investigated whether salivary secretion by muscarinic cholinergic stimulation is mediated through the NO/cGMP signaling pathway in rat salivary glands. Since NO activates soluble guanylyl cyclase (sGC) and cGMP may function as a mediator, the localisation of sGC was investigated in the salivary glands. sGC was localized in both the acinar and duct cells of the rat parotid and sublingual glands, and localized only in the acinar cells of the submandibular glands. S-Nitroso-glutathione (NO generator; GSNO) and YC-1 (NO-independent sGC activator) stimulated sGC in the cytosol to synthesise cGMP. The combination of GSNO and YC-1 stimulated sGC synergistically. Carbachol, GSNO and YC-1 enhanced amylase release from the rat parotid glands. Amylase release stimulated by carbachol and GSNO was inhibited by addition of the sGC inhibitor, ODQ, and cGMP-dependent protein kinase inhibitor, KT-5823. These results indicate that amylase release may be mediated through the NO/cGMP signaling pathway.

The binding of VIP36 and alpha-amylase in the secretory vesicles via high-mannose type glycans.

Vesicular integral protein of 36 kDa (VIP36) is an intracellular lectin recognizing high-mannose type glycans and is highly expressed in salivary glands, especially the parotid gland, which secretes alpha-amylase in large quantities. Here immunoelectron microscopy demonstrated that VIP36 was primarily localized to secretory vesicles in the glandula parotis of the rat, where alpha-amylase also resided. A secretory vesicle fraction, prepared by Percoll density gradient centrifugation, contained both VIP36 and alpha-amylase. Moreover, alpha-amylase that was localized to these secretory vesicles contained high-mannose type glycans. In addition, VIP36 coprecipitated with alpha-amylase in an endo H treatment-sensitive manner. These results suggest that VIP36 is involved in the secretion of alpha-amylase in the rat parotid gland.

The small GTPase Rab27B regulates amylase release from rat parotid acinar cells.

Small GTPase Rab is a large family of putative membrane trafficking proteins, and each member is thought to regulate a specific type(s) of membrane trafficking. However, little is known about the involvement of Rab protein(s) in secretory granule exocytosis in exocrine cells or the molecular mechanism underlying this process. We show that Rab27B, a closely related isoform of Rab27A that regulates lysosome-related granule exocytosis in cytotoxic T lymphocytes, is abundantly expressed on amylase-containing secretory granules in rat parotid gland acinar cells. We also identify the putative Rab27B effector protein, Slac2-c (Slp homologue lacking C2 domains-c)/MyRIP, which was originally described as a myosin Va/VIIa and actin binding protein, in rat parotid glands. The results of subcellular fractionation, immunoprecipitation and immunohistochemical studies indicate that the Rab27B-Slac2-c complex is formed on secretory granules in vivo. The introduction of either a specific Rab27 binding domain (i.e. a recombinant Slp homology domain of Slac2-b that specifically binds Rab27A/B but not other Rabs) or functionally blocking antibodies that specifically disrupt Rab27B-Slac2-c complex in vitro strongly inhibited isoproterenol-stimulated amylase release from streptolysin O-permeabilized parotid acinar cells. Our results indicate that the Rab27B-Slac2-c complex is an important constituent of secretory granule exocytosis in parotid acinar cells.

Immunolocalization of AQP-5 in rat parotid and submandibular salivary glands after stimulation or inhibition of secretion in vivo

In vitro studies of cultured salivary gland cells and gland slices have indicated that there may be regulated translocation of aquaporin (AQP)-5 between the apical plasma membrane and intracellular compartments of the secretory cells. However, it remains unknown whether AQP-5 in salivary glands is subject to regulated trafficking in vivo. To examine this possibility, we have investigated the subcellular localization of AQP-5 in rat parotid and submandibular glands fixed in vivo under conditions of stimulated or inhibited salivary secretion. Immunofluorescence and immunoelectron microscopy was used to determine the subcellular distribution of AQP-5 in control conditions following the stimulation of secretion with pilocarpine (a muscarinic agonist) or epinephrine (an alpha-adrenoceptor agonist) or during inhibition of basal secretion with atropine (a muscarinic antagonist) or phentolamine (an alpha-adrenoceptor antagonist). Under control conditions, >90% of AQP-5 was associated with the apical plasma membrane of acinar and intercalated duct cells, with only rare gold particles associated with intracellular membrane domains. Pilocarpine treatment dramatically increased saliva production but had no discernible effect on AQP-5 distribution. However, the increased salivary secretion was associated with luminal dilation and the appearance of a markedly punctate AQP-5 labeling pattern due to clustering of AQP-5 at the microvilli (especially evident in the parotid gland) after 10 min of drug injection. No changes in the subcellular localization of AQP-5 were seen in response to epinephrine, atropine, or phentolamine treatment compared with control tissues. Thus AQP-5 is localized predominantly in the apical plasma membrane under control conditions, and neither the onset nor the cessation of secretion is associated in vivo with any significant short-term translocation of AQP-5 between intracellular structures and the apical plasma membrane.

Expressions of CCAAT-enhancer-binding proteins and c-Myc in the parotid gland of the rat: in vivo effects of isoprenaline, bethanechol, vasoactive intestinal peptide and food intake

Parotid glands of adult female rats were exposed to agonists mimicking sympathetic (isoprenaline, 1 mg/kg, I.P.) or parasympathetic activity (bethanechol, 10 μg/kg/min i.v. for 30 min, and vasoactive intestinal peptide, VIP, 0.2 μg/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 1 h 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/EBPα of 42, 38 and 30 kDa increased by 45–50% above control value, C/EBPβ LAP (38/35 kDa) by 80% and C/EBPδ (35 kDa) by 230%, while C/EBPβ LIP (20 kDa) decreased by 45%. In response to VIP, C/EBPα of 42 kDa increased by 75% and C/EBPα of 30 kDa by 10%, C/EBPβ LAP by 65% and C/EBPδ by 410%, while C/EBPα of 38 kDa as well as C/EBPβ LIP were not changed. In response to bethanechol, C/EBPα of 42 kDa increased by 105%, C/EBPβ LAP by 40% and C/EBPδ by 170%, while C/EBPα of 30 kDa decreased by 30% and C/EBPα of 38 kDa and C/EBPβ 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/EBPα of 42 kDa by 105%, of 30 kDa by 40% and of 38 kDa, by 10%, in C/EBPβ LAP by 65% and in C/EBPδ by 215%, whereas C/EBPβ 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.

Cholinergic regulation of Na +-K +-ATPase activity in rat parotid gland: changes after castration

In this study, we investigated the different signalling pathways involved in muscarinic acetylcholine M 3 receptor-dependent modulation of Na +-K +-ATPase in parotid glands from normal and castrated rats. Carbachol inhibited the enzyme activity in parotid glands from control rats while it stimulated the enzyme activity in castrated rats. The inhibition of Ca 2+ calmodulin by trifluoperazine abolished the inhibitory effect of carbachol in control rats, while the inhibition of protein kinase C by staurosporine stimulated Na +-K +-ATPase. In castrated rats, trifluoperazine inhibited the carbachol-stimulant effect while staurosporine had no effect. Results indicate that in control glands the activation of a phospholipid-Ca 2+ calmodulin-dependent protein kinase C is responsible for the inhibitory effect of carbachol on Na +-K +-ATPase activity. In castrated rats, the activation of the enzyme by carbachol is regulated by its Ca 2+ calmodulin-stimulating action, and not by activation of protein kinase C. The activation of the Na +-K +-ATPase observed in castrated rats resulted in a decrease in carbachol-induced net K + efflux and thereby could decrease salivary fluid production.

Distribution and properties of arginase in the salivary glands of four species of laboratory mammals.

Important progress in arginine metabolism includes the discovery of widespread expression of two isoforms of arginase, arginase I and II, not only in hepatic cells but also in non-hepatic cells, and the formation of nitric oxide, a widely distributed signal-transducing molecule, from arginine by nitric oxide synthase. Possible physiological roles of arginase may therefore include regulation of nitric oxide synthesis through arginine availability for nitric oxide synthase. In this paper, arginase was investigated in the submandibular, sublingual, and parotid glands of rat, mouse, guinea pig, and rabbit. From their arginase contents, the salivary glands of these species were divided into two groups. Variable levels of arginase activity were detected in the salivary glands of mouse and rat. However, salivary glands of rabbit and guinea pig had almost no arginase activity. The presence of nitric oxide synthase has been reported in all the salivary glands used in this study. Therefore, one of the important findings was the presence of species specificity in the co-localization of arginase and nitric oxide synthase in the salivary glands of the four species. The highest specific activity of arginase was found in mouse parotid gland. In rat, considerable arginase activity was detected in all three glands, at 3.6-7.3% of that in rat liver. In rat submandibular gland, arginase was detected in both cytosolic and particulate fractions. In addition, arginase was detected in isolated acinar cells, but not in duct cells. Experiments on the intracellular distribution and the effects of the arginase inhibitors ornithine and N(omega)-hydroxy-L-arginine (NOHA), suggested the presence of both arginase I and arginase II in rat submandibular gland.

Molecular Mechanisms and Drug Development in Aquaporin Water Channel Diseases: The Translocation of Aquaporin-5 From Lipid Rafts to the Apical Plasma Membranes of Parotid Glands of Normal Rats and the Impairment of It in Diabetic or Aged Rats

Salivary secretion from rat salivary glands occurs in response to stimulation by acetylcholine and norepinephrine released from nerve endings. Aquaporin-5 (AQP5) localizes in lipid rafts under control conditions and is induced to traffic to the apical plasma membrane in interlobular ducts of rat parotid glands by the activation of M3 muscarinic acetylcholine receptors or α1-adrenoceptors. This review will focus on the mechanisms of the translocation of AQP5 from lipid rafts to the apical plasma membrane in the interlobular duct cells of parotid glands of normal rats and the impairment of its translocation in diabetic or senescent rats.

Relationship between apoptosis and menstrual cycle on the rat parotid gland

Relationship between apoptosis and menstrual cycle on the rat parotid gland

Initial activation processes of the Na+-K+-2Cl− cotransporter and the Cl− channel in carbachol-stimulated acinar cells of rat parotid glands

Initial activation processes of the Na+-K+-2Cl− cotransporter and the Cl− channel in carbachol-stimulated acinar cells of rat parotid glands

ラット耳下腺と顎下腺におけるL系アミノ酸トランスポーターの発現と局在に関する研究(博士(医学))(杏林大学学位論文要旨および審査要旨)

ラット耳下腺と顎下腺におけるL系アミノ酸トランスポーターの発現と局在に関する研究(博士(医学))(杏林大学学位論文要旨および審査要旨)

Circulating antibodies against rat parotid gland M3 muscarinic receptors in primary Sjögren's syndrome.

In this study we demonstrate that IgG present in the sera of patients with primary Sjögren's syndrome (PSS) could bind and activate muscarinic acetylcholine receptors (mAChRs) of rat parotid gland. These antibodies were able to inhibit in a non-competitive manner the binding of 3H-quinuclidinyl benzilate (QNB) to mAChRs of purified rat parotid gland membranes. Moreover, IgG from PSS could modify biological effects mediated by mAChR activation; i.e. decrease cAMP, increase phosphoinositide turnover without affecting cGMP. Atropine and 4-DAMP blocked all of these effects, and carbachol mimicked them, confirming the M3 subtype mAChRs mediated PSS IgG action. Neither binding nor biological effect were obtained with IgG from sera of normal women. The prevalence of cholinergic antibody was 100% in PSS, and was independent of Ro/SS-A and La/SS-B antibodies. It could be concluded that antibody against mAChRs may be another serum factor to be considered in the pathophysiology of the development of PSS.

Expression and anti-bacterial activity of human parotid secretory protein (PSP).

Parotid secretory protein (PSP) is an abundant protein in mouse and rat parotid glands. A related sequence (C20orf70) was identified on human chromosome 20. The goal of this study was to determine if PSP is expressed in the human parotid gland. The cDNA for human PSP was amplified from a human parotid cDNA sample. A peptide antibody, raised to the C-terminal peptide of PSP, identified the protein in human parotid tissue by immunofluorescence microscopy. Immunoaffinity chromatography suggested that PSP was expressed in human saliva. PSP is related to bactericidal/permeability-increasing protein (BPI). To test if PSP exhibits anti-bacterial activity, epitope-tagged PSP was expressed in rat GH4C1 cells. The secretion medium exhibited bacteristatic or bactericidal effects on Pseudomonas aeruginosa in a colony-forming assay when compared with secretion medium from GH4C1 cells that did not express PSP. These results suggest that PSP is expressed in the human parotid gland and saliva, where it functions as a BPI-like anti-bacterial protein.

Localization of VIP36 in the post-Golgi secretory pathway also of rat parotid acinar cells.

VIP36 (36-kD vesicular integral membrane protein), originally purified from Madin-Darby canine kidney (MDCK) epithelial cells, belongs to a family of animal lectins and may act as a cargo receptor. To understand its role in secretory processes, we performed morphological analysis of the rat parotid gland. Immunoelectron microscopy provided evidence that endogenous VIP36 is localized in the trans-Golgi network, on immature granules, and on mature secretory granules in acinar cells. Double-staining immunofluorescence experiments confirmed that VIP36 and amylase co-localized in the apical regions of the acinar cells. This is the first study to demonstrate that endogenous VIP36 is involved in the post-Golgi secretory pathway, suggesting that VIP36 plays a role in trafficking and sorting of secretory and/or membrane proteins during granule formation.

Detection and characterization of a rat parotid gland protein kinase that catalyzes phosphorylation of matured destrin at Ser-2

Destrin, an actin-binding protein, is partly phosphorylated at Ser-2 (numbering on the matured form) in the resting rat parotid gland, and β-adrenergic or cholinergic stimulation of this gland induces its dephosphorylation. In this study, we searched for the protein kinase involved in phosphorylation of destrin. We developed an assay method for the kinase, using an antibody specific to destrin phosphorylated at Ser-2, and detected the kinase in the rat parotid homogenate. This enzyme was predominantly (93%) present in the soluble fraction, and the enzyme in this fraction was characterized. It had an optimum pH at 6.8 and required 3–5 mM Mg 2+ for its maximum activity. Ca 2+ (1 mM) had no effect whereas Mn 2+ (5 mM) inhibited the enzyme activity by 75%. The apparent K m values for destrin and ATP were 92 μg/ml and 170 μM, respectively. GTP was an inefficient phosphate donor, and cAMP had no effect. Heat-denatured destrin was poorly phosphorylated. Two-dimensional PAGE analysis of destrin phosphorylated with the soluble fraction indicated that it was exclusively phosphorylated at Ser-2. None of the protein kinase inhibitors tested here was specific to this enzyme. At 1 mM, ML-7, Y-27632, KN-93, HA-1077, H-7, and H-8 inhibited the activity by 88, 61, 58, 49, 46, and 42%, respectively. Staurosporine (2 μM) and H-89 (50 μM) inhibited the activity by 48 and 33%, respectively. Heparin (30 μg/ml) had no effect. These results suggest that the rat parotid gland contains a novel, constitutively active, soluble protein kinase catalyzing specific phosphorylation of destrin at Ser-2.

Age-related decreases in the response of aquaporin-5 to acetylcholine in rat parotid glands.

Aquaporin-5 (AQP5) is important in salivary fluid secretion in response to cholinergic and adrenergic stimuli in rat parotid glands. We hypothesized that expression and function of AQP5 might change with age. Acetylcholine and epinephrine induced increases in AQP5 levels in the apical plasma membranes of both young adult and senescent rats. The stimulatory effect of acetylcholine, but not that of epinephrine, on AQP5 levels in the apical plasma membranes of the cells decreased markedly during aging. The quinuclidine derivative, SNI-2011, induced a persistent increase in AQP5 levels in the apical plasma membrane in the cells of both these rats. The amounts of M(3)-muscarinic receptor and Gq proteins did not decrease during aging. The age-related alteration in the responsiveness of AQP5 in the cells to these stimuli might account for the concomitant changes in nitric oxide synthase activity. These results suggest that SNI-2011 might have therapeutic benefit for the treatment of age-related xerostomia.