Rat Parotid Tissue Research Articles

Effects of ageing on morphology, amylase release, cytosolic Ca2+signals and acyl lipids in isolated rat parotid gland tissue

Xerostomia (oral dryness sensation) is due to dryness of the oral cavity and it is more prevalent in the elderly. This study investigated the effect of ageing on parotid gland structure and function of control (2-6 months) and aged (12, 16-18 and 22-24 months) rats employing light microscopic, colorimetric, gas chromatographic and microspectrofluorimetric methods to investigate the morphological changes of the parotid glands, amylase release, endogenous lipid distribution and cytosolic free calcium levels, respectively. When compared to controls, age-related changes were apparent in glands obtained from rats aged 16-18 and 22-24 months, which included reduced acinar cell distribution, enlarged parotid ducts with fatty and connective tissue and mast cell infiltrations. Parotid acini from 12, 16-18 and 22-24-month-old glands showed significant (p < 0.05) age-related decreases in amylase release, compared to controls when challenged with acetylcholine (ACh). No change in basal calcium signals was observed in parotid acini from 2-6 to 16-18-month-old-animals. However, stimulation of 16-18-month-old parotid acini with 10(-5)M ACh resulted in a significant (p < 0.001) decrease in both peak and plateau phases of the cytosolic Ca2+ signal when compared to control. Gas chromatography of de novo and essential acyl lipids revealed no changes in the amount of either acyl lipid group in glands obtained from 2-6 to 22-24-month-old animals. Lipid analysis of phospholipid associated acyl chains showed a higher relative proportion of linoleic acid in older glands. The results reveal that ageing is associated with marked and distinct morphological changes including infiltrations of lipids and mast cells of the parotid gland and decreases in amylase release and cytosolic Ca2+ signals.

The muscarinic acetylcholine receptor-stimulated increase in aquaporin-5 levels in the apical plasma membrane in rat parotid acinar cells is coupled with activation of nitric oxide/cGMP signal transduction.

The present study investigated the role of nitric oxide (NO)/cGMP signal transduction in the M(3) muscarinic acetylcholine receptor (mAChR)-stimulated increase in aquaporin-5 (AQP5) levels in the apical plasma membrane (APM) of rat parotid glands. Pretreatment of rat parotid tissue with the NO scavenger 2-(4carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide potassium inhibited both acetylcholine (ACh)- and pilocarpine-induced increases in AQP5 in the APM. NO donors [3-morpholinosydnonimine (SIN-1) and (S)-nitroso-N-acetylpenicillamine (SNAP)] mimicked the effects of mAChR agonists. A selective protein kinase G inhibitor [(9S,10R,12R)-2,3,9,10,11,12-hexahydro-10-methoxy-2,9-dimethyl-1-oxo-9,12-epoxy-1H-diindolo-[1,2,3-fg-3',2',1'-kl]pyrrolo[3,4-i][1,6]benzodiazocine-10-carboxylic acid methyl ester (KT5823)] and an NO synthase inhibitor (N(6)-imminoethyl-L-lysine) blocked SIN-1- and SNAP-induced increases in AQP5 in the APM. A calmodulin kinase II inhibitor [(8)-5-isoquinolinesulfonic acid, 4-[2-(5-isoquinolinyl-sulfonyl)methylamino]-3-oxo-(4-phenyl-1-piperazinyl)-propyl]phenyl ester (KN-62)] decreased the pilocarpine-induced increase of AQP5 in the APM. Using diaminofluorescinein-2 diacetate, enhanced NO synthase activity was detected in isolated parotid acinar cells after ACh-treatment. Treatment with dibutyryl cGMP, but not dibutyryl cAMP, induced an increase in AQP5 levels in the APM. BAPTA-AM inhibited the cGMP-induced increase in AQP5 in the APM. Pretreatment of the tissues with a myosin light chain kinase inhibitor [(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9)] inhibited a mAChR-stimulated increase in AQP5 levels in the APM. Although there was a significant ACh-induced increase in AQP5 in the APM in the absence of extracellular Ca(2+), the maximal effect of ACh on the AQP5 levels in the APM occurred in the presence of extracellular Ca(2+). These results suggest that NO/cGMP signal transduction has a crucial role in Ca(2+) homeostasis in the mAChR-stimulated increase in AQP5 levels in the APM of rat parotid glands.

Activation of endogenous nitric oxide synthase coupled with methacholine-induced exocytosis in rat parotid acinar cells.

Methacholine (MCh) interacted with M(3) muscarinic receptors in rat parotid tissue slices and induced amylase secretion. MCh- and calcimycin-induced exocytosis was completely inhibited by N-[2-(N-(4-chlorocinnamyl)-N-methylaminomethyl)phenyl]-N-[2-hydroxyethyl]-4-methoxybenzenesulfonamide, N(G)-nitro-L-arginine methylester (L-NAME), 1H-(1,2,4)-oxadiazolo[4,3-a]quinoxaline-1-one, and 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide, suggesting that activations of calmodulin (CaM) kinase II, nitric oxide synthase (NOS), and cGMP-dependent protein kinase (PKG) were coupled with the exocytosis. These suggestions were supported by the results that exposure of the slices to MCh induced a rapid increase in these enzyme activities. Western blot analysis showed that neuronal NOS (nNOS) was expressed in isolated parotid acinar cells of rats. To measure nitric oxide (NO) production in response to the stimulation with MCh in real time, the isolated parotid acinar cells had been preloaded with 4,5-diaminofluorescein diacetate and incubated with the agonist. MCh (1 microM) induced a fast increase in 4,5-diaminofluorescein fluorescence, corresponding to an increase in the NO synthesis in the presence of extracellular Ca(2+) but not in the absence of it. When the isolated parotid acinar cells preloaded with L-NAME or 2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethylester) were treated simultaneously with MCh, the increase in the fluorescence also was not observed. The MCh-induced increase in the fluorescence was not observed in the cells incubated in the absence of extracellular calcium, showing the importance of Ca(2+) entry from extracellular sites for MCh-induced NOS activation. These results indicate that nNOS is endogenously present in rat parotid acinar cells and that the rapid activation of this enzyme together with those of CaM kinase II and PKG contributes to MCh-induced amylase secretion.

α1-Adrenoceptor-Induced Trafficking of Aquaporin-5 to the Apical Plasma Membrane of Rat Parotid Cells

Incubation of rat parotid tissue with 10 μM epinephrine resulted in a transient and marked trafficking of aquaporin-5 (AQP5) from intracellular membranes to the apical plasma membrane (APM) that was maximal at 1 min. This effect of epinephrine was mimicked by phenylephrine, but not by clonidine, dobutamine, or salbutamol, and it was inhibited by phentolamine, but not by propranolol. Furthermore, the epinephrine-induced trafficking of AQP5 was inhibited by phospholipase C inhibitor U73122 as well as dantrolene and TMB-8, both of which inhibit the release of Ca2+ from intracellular stores. Cytochalasin D and tubulozole-C also inhibited this action of epinephrine. These results indicate that epinephrine, acting at α1-adrenoceptors, induces the trafficking of AQP5 to the APM by triggering the release of Ca2+ from intracellular stores through inositol 1,4,5-trisphosphate and ryanodine receptors. In addition, the potent involvement of the cytoskeleton was shown in the epinephrine-induced trafficking of AQP5.

O–229 - Changes in the distribution of histamine H2 receptors coupled with secretory response in rat parotid tissues

O–229 - Changes in the distribution of histamine H2 receptors coupled with secretory response in rat parotid tissues

Developmental enhancement of secretory response to isoproterenol coupled with increases in β-adrenoceptor density and Gs protein function in rat parotid tissues

The β-adrenergic agonist, isoproterenol (IPR), stimulated more significantly and sensitively amylase secretion from both the tissues of 7- and 56-day-old rats than a cholinergic agonist, carbachol, at the same concentration. The EC 50 value of amylase secretion with IPR decreased significantly during development but that with carbachol did not change. Estimation by measuring bindings of [ 3H]dihydroalprenolol and [ 3H]quinuclidynylbenzylate indicated the marked increases in the numbers of both β-adrenoceptors and muscarinic receptors in the tissues during development. The affinity of β-adrenoceptors for the agonist was also enhanced during development, but that of muscarinic receptors for the agonist was not. These developmental changes in the number and affinity of β-adrenoceptors and muscarinic receptors paralleled those in amylase secretory response of the tissues to their agonists. The response of adenylate cyclase (AC) of the tissues to 1 μM IPR was steadily enhanced after birth. In contrast, the response of AC to 1 μM forskolin was high until 14 days old, but markedly decreased at 28 days old and thereafter maintained this level. The increase in cholera toxin-catalyzed ADP-ribosylation (AR) of stimulatory GTP binding proteins (Gs proteins) in the tissues was apparent at 14 days old, reaching a maximum at 56 days old and thereafter decreasing with age. On the other hand, pertussis toxin-catalyzed AR of inhibitory GTP binding proteins (Gi proteins) did not change after birth. Thus, the ratio of apparent levels of Gs to Gi proteins increased significantly after birth, reaching a maximum at 56 days old, but decreased rapidly till 84 days old and thereafter maintained this level. These changes in the ratio paralleled those in the response of AC to IPR. These results showed that the rapid and marked increases in the number and affinity of β-adrenoceptors and the ratio of apparent levels of Gs to Gi proteins in rat parotid tissues during development had a key role in the enhancement of the secretory response of the tissues to β-agonists.

Mechanism underlying histamine-induced desensitization of amylase secretion in rat parotid glands.

1. Histamine acted on H2 receptors in rat parotid tissues and induced the amylase secretion. Immunoblot analysis by using anti-H2 receptor protein antiserum demonstrated that histamine induced the increase and decrease in the amounts of H2 receptor proteins in basolateral and intracellular membranes, respectively. 2. Short-term treatment with histamine resulted in decreases in amylase secretion, the density of H2 receptors and their affinity for the agonists during further incubation with histamine, but showed an unaltered secretory response to isoproterenol, indicating that the histamine-induced desensitization was confined to H2 receptors. 3. This treatment triggered a 20% decrease in the histamine-stimulated adenylate cyclase activity and a 40% decrease in the phosphorylation level of Gi2alpha protein in the tissues, resulting in an increase in pertussis toxin (IAP)-catalyzed ADP-ribosylation of the protein. An enhancement of cholera toxin-catalyzed ADP-ribosylation of Gs protein was observed only during the first incubation with histamine. 4. This treatment triggered a 30% decrease and a 60% increase in the histamine-stimulated activities of protein kinase A and protein phosphatase 2A in the tissues, respectively. 5. Pretreatment with okadaic acid completely blocked the histamine-induced decrease in amylase secretion and increase in IAP-catalyzed ADP-ribosylation of Gi protein. The levels of Gi2alpha and Gs alpha proteins in the tissues were not modified by histamine treatment and the level of Gi2alpha protein was not affected by pretreatment with okadaic acid, as assessed by immunoblot analyses with anti-Gi2alpha and anti-Gs alpha protein antiserum. 6. The regulation of Gi2alpha protein phosphorylation in parotid tissues plays an important role in the histamine-induced desensitization of amylase secretion.

Atrial Natriuretic Factor-Induced Amylase Output in the Rat Parotid Gland Appears to be Mediated by the Inositol Phosphate Pathway

In previousin vivostudies we have reported that atrial natriuretic factor enhanced induced salivary secretion and increased isoproterenol-induced amylase release in the rat suggesting that, ANF effect could be mediated by phosphatidylinositol hydrolysis. In the present work, the effect of ANF on rat parotid tissue incubatedin vitrowas investigated with the aim to assess whether the phosphoinositol pathway was involved in ANF intracellular signaling in the parotid gland. Results showed that ANF induced a dose dependent increase in amylase fractional release, which was lower than that evoked by any concentration of isoproterenol. Furthermore 100 nM ANF enhanced isoproterenol-evoked amylase release. The effect of ANF was not affected in the presence of propranolol suggesting the noninvolvement of the β adrenergic receptor, which is the main stimulus for the output of the enzyme in the parotid gland. However, ANF increased phosphatidylinositol hydrolysis, which implies an increase in intracellular calcium, which is necessary for the achievement of maximal response in amylase release. This effect was abolished in the presence of neomycin supporting ANF direct stimulation of phospholipase C. These results suggest the involvement of the C type natriuretic peptide receptor coupled to phospholipase C in ANF evoked amylase release and ANF enhancement of the isoproterenol-induced output of the enzyme.

Acetylcholine Acts on M3Muscarinic Receptors and Induces the Translocation of Aquaporin5 Water Channel via Cytosolic Ca2+Elevation in Rat Parotid Glands

To evaluate the role of aquaporin5 (AQP5) in salivary secretion induced by cholinergic stimulation, the alteration of the distribution of AQP5 in rat parotid tissues induced by acetylcholine (ACh) was studied by immunoblot analysis. The treatment of the tissues with ACh within 1min induced the translocation of AQP5 from intracellular membranes (ICM) to apical membranes (APM), but that for more than 5min resulted in the converse translocation from APM to ICM. The ACh-induced increase in the amount of AQP5 in APM was inhibited by atropine,p-F-HHSiD and TMB-8, but not by methoctramine, staurosporine or H-7. The calcium ionophore A-23187 alone stimulated the translocation of AQP5 between APM and ICM. These results indicated that ACh acted on M3muscarinic receptors and induced the translocation of AQP5 between ICM and APM, and that the cytosolic Ca2+elevation by ACh may play a key role in this translocation in rat parotid glands.

S19-5 - Regulation of phosphorylation of Gi2a protein controls the secretory responponse to isoproterenol in rat parotid tissues

S19-5 - Regulation of phosphorylation of Gi2a protein controls the secretory responponse to isoproterenol in rat parotid tissues

P-438 - Effect of trifluoperazine on the secretory response of rat parotid tissues to histamine

P-438 - Effect of trifluoperazine on the secretory response of rat parotid tissues to histamine

O-57 - Histamine H2 receptor alterations coupled with secretory response of rat parotid tissues

O-57 - Histamine H2 receptor alterations coupled with secretory response of rat parotid tissues

Regulation of phosphorylation of Gi2α protein controls the secretory response to isoproterenol in rat parotid tissues

Treatment of rat parotid tissues with 1 μM isoproterenol (IPR) for 10 min caused a 60% decrease in pertussis toxin (IAP)-catalyzed ADP-ribosylation of Giα and resulted in supersensitivity of amylase secretion from the tissues. However, conversely, IPR treatment for 30 min caused a 40% increase in IAP-catalyzed ADP-ribosylation of Giα, coupled with desensitization of amylase secretion. No changes in Gs function were observed in IPR-induced phenomena. Pretreatment with okadaic acid induced enhancement of the supersensitivity of amylase secretion and disappearance of the desensitization. These phenomena were accompanied with decreases in IAP-catalyzed ADP-ribosylation of Giα. IPR treatment for 30 min caused a 50% decrease in phosphorylation of Gi2α immunoprecipitated with anti-G protein antiserum (AS/7) from [ 32P]P i-labeled cells, but such treatment for 10 min caused a 40% increase in phosphorylation in the cells pretreated with okadaic acid. Phosphorylation and dephosphorylation of immunoprecipitates with AS/7 by protein kinase A (PKA) and alkaline phosphatase caused decreases and increases in IAP-catalyzed ADP-ribosylation, respectively, indicating the presence of PKA-mediated phosphorylation sites on Gi2α. Thus, the control of the phosphorylation of Gi2α is of importance and relevance in the regulation of biological processes and cellular responses.

S29-5 - Regulation of phosphorylation of Gi2 α controls amylase secretion from rat parotid tissues to subsequent stimulation by isoproterenol.

S29-5 - Regulation of phosphorylation of Gi2 α controls amylase secretion from rat parotid tissues to subsequent stimulation by isoproterenol.

P-416 - The relation between regulation of phosphorylation of Gi2 α protein and secretory response of rat parotid tissues to the subsequent stimulation by isoproterenol

P-416 - The relation between regulation of phosphorylation of Gi2 α protein and secretory response of rat parotid tissues to the subsequent stimulation by isoproterenol

Effects of dopamine on adenylyl cyclase activity and amylase secretion in rat parotid tissue.

Several previous studies have shown that dopamine causes amylase secretion from rat parotid tissue. However, the mechanism of this dopamine action is still unclear. The present study was designed to characterize dopamine action in rat parotid gland tissue by examining the effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release. Dopamine significantly enhanced accumulation of cyclic AMP in parotid slices and stimulated adenylyl cyclase activity in parotid membrane preparations. It also significantly stimulated amylase release from parotid slices. The stimulatory effects of dopamine on cyclic AMP accumulation, adenylyl cyclase activity, and amylase release were effectively blocked with propranolol, a beta-adrenergic antagonist, but not by either SCH 23390, a preferential D1 antagonist, or butaclamol, a preferential D2 antagonist. No substantial specific binding sites for D1 receptors were detectable by [3H]SCH 23390 binding in parotid membranes. These results suggest that the stimulatory effect of dopamine on amylase secretion in rat parotid tissue is not mediated through specific D1 dopamine receptors but rather through beta-adrenergic receptors.

P3-48 - Regulation of phosphorylation of Gi proteins controls the secretory response of rat parotid tissues to the subsequent stimulation by isoproterenol

P3-48 - Regulation of phosphorylation of Gi proteins controls the secretory response of rat parotid tissues to the subsequent stimulation by isoproterenol

O-419 - Role of PLA2 on fusion of secretory granules and membranes of rat parotid tissue.

O-419 - Role of PLA2 on fusion of secretory granules and membranes of rat parotid tissue.

Identification and localization of G proteins in exocrine glands.

GTP-binding proteins were identified in rat parotid acinar plasma-enriched membranes (PM) by immunoblot analysis and localized immunohistochemically in the parotid gland as well as in other exocrine glands by using affinity-purified antisera specific for alpha subunits of the G proteins. Isolated rat parotid acinar PM immunoreacted strongly to antisera directed against Gs alpha, Gi alpha 1/alpha 2, Gi alpha 3, and Go alpha; the signal for Go alpha, however, was weak with crude Go antisera. Immunohistochemical studies to identify and localize Go in rat parotid tissue revealed that antisera to Go alpha immunoreacted with ductal cells. In addition, strong immunoreactivity to Go alpha antisera was noted in ductal cells of other salivary glands including rat submandibular, mouse parotid, and mouse submandibular glands. Light labeling of rat parotid and submandibular gland acinar cells was also noted. In contrast, in the rat and mouse pancreas, Go antisera immunoreacted primarily with islet cells. Ductal cells were negative, but there was light labeling of rat pancreatic acinar cells. The apparent ductal specificity of Go alpha staining was further verified by demonstrating that Go alpha antisera immunoreacted strongly with HSG-PA cells, a human transformed salivary ductal cell line. The results demonstrate that rat parotid acinar plasma membranes express a number of G proteins including Go and that Go appears to be selectively expressed in the ductal cells of rat parotid gland and other salivary glands. The selective enrichment of Go in ductal cells suggests that this G protein may play an important role in ductal cell physiology.

P-291 - Studies on proteins secreted by isoproterenol from rat parotid tissues briefly with agonist.

P-291 - Studies on proteins secreted by isoproterenol from rat parotid tissues briefly with agonist.