Stimulation of mucin release from rat submandibular salivary-gland cells by NaF

The effect of NaF on cAMP accumulation, cAMP-dependent protein kinase activity (cAMP-dPK) ratios and [14C]-glucosamine-labelled mucin release from these isolated cells was investigated. NaF (0.01-5 mM) increased significantly the cellular cAMP concentration and cAMP-dPK activity ratios in a dose- and time-dependent manner. NaF (5.0 mM) increased [14C]-glucosamine-labelled mucin release in a time-dependent manner. Thus the stimulation of prelabelled mucin secretion by NaF is mediated by an increase in the cAMP concentration, which exerts its effect, at least partly, via the activation of cAMP-dPK activity.     

Differences in the regulatory mechanism of amylase release by rat parotid and submandibular glands

It is not known whether the mechanisms involved in amylase release in submandibular and parotid glands are similar. Here, the participation of different signalling pathways in amylase release by the parotid and submandibular glands of the male rat was compared by studying the secretory response after beta-adrenergic stimulation. The beta-adrenergic agonist isoproterenol induced an increase of cAMP in both salivary glands, but while in the parotid it triggered amylase release, in the submandibular it was unable to increase amylase secretion. Parotid amylase release was dependent on adenylate cyclase activation, as SQ-22536 inhibited the secretory effect. In contrast, submandibular amylase secretion did not depend on the intracellular concentration of cAMP, as SQ-22536 did not modify its secretory response. Moreover, other activators of adenylate cyclase, such as forskolin and prostaglandin E2, also failed to modify amylase release by the submandibular gland. Neither ionophores nor calcium-blocking agents, as well as calcium-calmodulin and nitric oxide synthase inhibitors, were effective in modifying basal amylase release by the submandibular gland. However, the disruption of microfilaments with cytochalasin B, but not the disruption of microtubules with colchicine, prevented amylase release in that gland. It is concluded that amylase exocytosis in the submandibular gland is a constitutive non-regulated phenomenon, as it is independent of extracellular or intracellular signals. It depends only on the integrity of the microfilaments, probably used by the vesicles to travel from the Golgi apparatus to the plasma membrane.     

Evidence for amylase release by cGMP via cAMP-dependent protein kinase in rat parotid acinar cells

Amylase release from the rat parotid gland is primarily mediated by a cAMP-dependent protein kinase (PKA). We previously reported that cGMP/cGMP-dependent protein kinase (PKG) signaling evokes amylase release. In the present study, we investigated whether cGMP-mediated amylase release might be due to cGMP/PKA signaling, as well as cGMP/PKG pathway. Activation of PKA by cGMP was required 100-1000-fold greater concentration than activation by cAMP in a parotid cytosol fraction. Synergistic activation of PKA by the combination of physiological cAMP and low concentration of cGMP was observed. Amylase release from intact acinar cells was synergistically stimulated by the combination of diBu-cAMP and 8-pCPT-cGMP. cGMP dose-dependently stimulated amylase release from saponin-permeabilized parotid acinar cells. Phosphorylation by cGMP produced phosphorylated proteins of the same size as those produced by cAMP. Phosphorylation by cGMP was inhibited by the addition of PKA inhibitor, H-89. These results suggest that cGMP activates both PKG and PKA. Thus, it appears that both cGMP/PKG and cGMP/PKA pathways mediate amylase release from rat parotid acinar cells.     

Stimulation of rat parotid secretion by cAMP analogues that synergistically activate the type II isoenzyme of the cAMP-dependent protein kinase.

Beta-adrenergic-stimulated parotid secretion is believed to be mediated by activation of the cAMP-dependent protein kinase (PK-A). However, the relative roles of the type I and II PK-A isoenzymes are still unclear. Combinations of site-selective, lipophilic cAMP analogues that synergistically activate each PK-A were used to investigate this problem. The selectivity of synergistic activation with these combinations was verified with the partially purified parotid PK-A isoenzymes, using kemptide as a substrate. Synergism in activation of PK-AII was only seen with 8-thiomethyl cAMP (8-TM) and N-6-benzoyl cyclic AMP (N6B), while PK-AI was only synergistically activated by 8-(6-aminohexyl) amino cyclic AMP (AHA) and N6B. Additive activation of each isoenzyme was observed for the combination of 8-TM and AHA. Rates of amylase secretion from dispersed parotid acini in response to secretagogues were determined with a coupled enzyme assay for amylase activity, which was adapted for use in a microplate reader. Cells were stimulated to secrete during 30 min with different doses (0.1-1.0 mM) and combinations of the cyclic nucleotide analogues. Alone, N6B was most effective in stimulating amylase secretion. The basal amylase secretory rate was stimulated by these secretagogues (0.44 mM) to the following extent: 53-fold (N6B), 8-fold (8-AHA), 2-fold (8-TM). In combination at a series of concentrations, only 8-TM + N6B produced synergistic stimulation of secretion, while AHA + N6B and 8-TM + AHA did not.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of streptozotocin diabetes on amylase release and cAMP accumulation in rat parotid acinar cells.

Rat parotid responses to sympathetic nerve stimulation in vivo are impaired 2-4 weeks after the induction of streptozotocin diabetes. In this study, the effects of experimental diabetes of similar duration and severity on noradrenaline-stimulated amylase release and cAMP accumulation were examined in vitro. Amylase levels were significantly lower in acinar cells isolated from diabetic animals than in controls, and cellular amylase increased after treatment of the diabetic animals with either thyroxine (T4) or insulin. Diabetes and T4 had no apparent affect on amylase release measured as a percentage of the total. In contrast, giving insulin resulted in a significant reduction in maximal secretion (20.4 +/- 2.4% compared with 43.6 +/- 7.6%). Similar results were observed when amylase release was stimulated with forskolin. Basal cAMP levels were unaffected by diabetes or T4 (7.8 +/- 2.3 pmol/mg protein), but stimulated cAMP levels were significantly greater in diabetic acinar cells than in controls. Insulin reversed the effects of diabetes on cAMP accumulation, whereas T4 had no effect. Thus, diabetes (2-4 weeks) and insulin in vivo appear to have paradoxical effects on parotid amylase release and cAMP accumulation in vitro. Further, the effects of diabetes appear to be unrelated to thyroid status.     

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.     

Increase nitric oxide synthase activity in parotid glands from rats with experimental periodontitis

Oral Diseases (2010) 16 , 801–806 Objective: In this study we investigated the activity of the nitric oxide synthase (NOS) in parotid glands from rats with experimental periodontitis and controls. Methods: Periodontitis was produced by a ligature placed around the cervix of the two lower first molar. Experiments were carried out 22 days after the ligature. Results: Ligation caused an increase in parotid NOS activity. The selective blocker of the inducible isoform of the enzyme partially inhibited its activity in parotid glands from rat with ligature. In controls, the activity was partially inhibited by the antagonists of the selective neural and endothelial isoforms. NOS activity in rats with ligature was cyclic adenosine monophosphate (cAMP)‐dependent while in controls it was calcium‐dependent. Prostaglandin E₂ concentration was increased in parotid gland from rats with ligature. The inhibitor of prostaglandin production, FR 122047, diminished both, prostaglandin production and NOS activity. In rats with ligature unstimulated amylase released is increased. Both, prostaglandin and NOS were involved in the increment of amylase release. Conclusion: It can be concluded that in parotid glands from ligated rats, prostaglandin E₂ production is increased and, through cAMP accumulation, activates the inducible NOS isoform. The increment of nitric oxide production participates in the increase in basal amylase release.     

A biochemical analysis of parotid and submandibular salivary gland function with age after simultaneous stimulation with pilocarpine and isoproterenol in female NIA Fischer 344 rats.

This analysis of physiological, biochemical and molecular changes related to aging was made in 3-, 12- and 24-month-old rats. The salivary gland weight/body weight ratio and the structural membrane proteins did not change with age for either gland, but a significant age-related decline in DNA synthesis for both glands was detected, unrelated to the hormonal responsiveness at the level of the plasma membrane. There was a marked increase in the concentration of soluble proteins in adolescent parotid gland and, for the two older age groups, in submandibular gland. The saliva flow rate was different when expressed as volume per time, as volume per time and g glandular wet weight, and/or kg body weight. The concentration of secreted proteins was not affected by age in either gland. The total amount of proteins secreted over 30 min revealed no age-related perturbation for the parotid gland, but showed a significant age-related increase in submandibular saliva. Sodium dodecyl sulphate-polyacrylamide gel analysis revealed changes in the protein bands between 39 and 50 kDa in the Coomassie blue-stained gels from 12-month-old animals. Amylase showed an initial increase (12 months), followed by a marked decline in its activity in parotid saliva. The glandular supernatant had low residual cellular amylase activity after stimulation. Therefore, secretory impairment with age after pilocarpine-isoproterenol stimulation was excluded. Analysis of total RNA showed a pronounced decrease of amylase mRNA in the parotid gland between 12 and 24 months of age. No amylase mRNA was expressed in any of the submandibular samples. For epidermal growth factor, total saliva showed a decrease with age. It seemed that the submandibular gland followed the same picture with age as the parotid gland, with a specific decline in the biosynthesis of single secretory proteins.     

Exocytosis in rat parotid acini after in vitro treatment with forskolin is accompanied by cellular redistribution of regulatory subunits of cyclic AMP-dependent protein kinase

Incubation of rat parotid acinar tissue with 1 mumol/L forskolin resulted in progressive exocytosis which was virtually complete after a 30-minute incubation period. Cyclic AMP binding to protein kinase (cA-PK) regulatory (R) subunits, determined by photo-affinity labeling with [32P]-8-azido cyclic AMP, was found to increase in a time-dependent manner in the 10,000-g supernatant fraction of a broken cell preparation. An apparent redistribution of protein kinase R subunits took place in the 600-g supernatant after in vitro treatment of cells with forskolin. In control cells, RI and RII subunits and a 35-to-40-kdal fragment were present in approximately equal amounts throughout the incubation. Azido labeling of RII appeared either to increase or to remain unchanged, while that of RI decreased in the 600-g pellet. Only type I isozyme R subunits were found in the 600-g pellet in either the absence or presence of forskolin. These finding indicate that a temporal relationship exists between stimulated protein exocytosis and cyclic AMP-dependent protein kinase activation. Forskolin stimulation of adenylate cyclase in salivary gland cells therefore provides a defined system for the study of cyclic AMP-mediated protein secretion.     

Transport to the bloodstream of amylase following retrograde infusion of amylase into the parotid glands in the rat

Amylase secretion into parotid saliva was increased more by isoprenaline whereas the secretion rate of parotid saliva was increased more by pilocarpine. Retrograde infusion of parotid-amylase solution into excretory ducts of the parotid gland elevated the activity of the enzyme in the serum. Direct evidence that retrogradely-infused amylase reaches the blood from the parotid gland was obtained using amylase prepared from Bacillus subtilis. Pretreatment with pilocarpine accelerated the release of amylase into the blood. The elevation of intraductal pressure may be the primary mechanism for the release of amylase into the blood from the parotid glands.     

Mechanisms of increase in amylase activity in rat submandibular and sublingual glands after administration of pilocarpine

The increase in parotid rather than pancreatic-type amylase activity in the submandibular and sublingual glands of rats caused by administration of pilocarpine was abolished or diminished when pilocarpine was injected into rats which had been parotidectomized, sympathectomized by superior cervical ganglionectomy or pretreated with reserpine. These results suggest that the increases in amylase activity in the submandibular and sublingual glands by pilocarpine are not due to increase in enzyme synthesis, but to uptake of enzyme released into the blood in large quantities from the parotid gland and that the release from the parotid gland by pilocarpine is primarily mediated by sympathetic nerves.     

Parasympathetic nerve-evoked protein synthesis, mitotic activity and salivary secretion in the rat parotid gland and the dependence on NO-generation

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.     

Response of rat parotid acini to barium.

The effect of barium on isolated acini was tested. Barium in the 0.1-10 mM concentration range non-competitively inhibited the efflux of 86Rb+ stimulated by carbamylcholine or substance P. This inhibition was independent of the presence of calcium in the extracellular medium. In the same preparation, barium did not affect the efflux of 45Ca2+ but, at a 10 mM concentration, it increased amylase release by 70%. Removal of extracellular calcium decreased basal amylase release and the response to carbamylcholine. Adding back calcium or barium to the incubation medium increased basal and carbamylcholine-stimulated amylase secretion, but calcium was more effective than barium. These results suggest that barium has two opposite effects on calcium-regulated processes in rat parotid gland: (1) it is an inhibitor of calcium-activated potassium channels; (2) it is a partial agonist of calcium-activated amylase secretion.     

Effects of dietary consistency and water content on parotid amylase secretion and gastric starch digestion in rats

The aim was to estimate the significance of oral sensation and mastication in inducing amylase secretion from the parotid gland and subsequent starch digestion in the stomach. Rats were fed three diets of similar chemical composition but different physical presentations. Two were solid, either pellets or powder, and one was liquid. Oral sensory activity would be greatest with the pellets and least with the liquid. Only the pellets would require significant mastication. Three criteria were used to estimate amylase secretion, amylase activity in the stomach, the depletion of glandular amylase activity and plasma amylase concentrations. Gastric starch digestion was estimated by measuring the concentration of reducing-sugars in the stomach contents. Parotid amylase secretion and gastric starch digestion were similar whether rats were fed pelleted or powdered solid food but much lower in rats fed a liquid diet. These findings support the view that it is the contact of dry food with the oral mucosa rather than the jaw movements involved in mastication that stimulates parotid amylase secretion.     

Translocation of the α-isozyme of protein kinase C during stimulation of rat parotid acinar cells by phorbol ester and carbachol

Protein kinase C activity was detected in the cytosolic fraction of quiescent parotid acinar cells; the particulate fraction contained a much smaller proportion of the enzyme. Protein kinase C activity was increased in the membrane fraction and decreased in the cytosol after exposure of intact cells to phorbol 12-myristate 13-acetate (PMA) or the muscarinic-receptor agonist carbachol. The effect of PMA was potentiated by a subthreshold concentration of ionomycin. Immunoblot analysis with anti-protein kinase C antibodies revealed that the protein kinase C-α isoform is expressed in rat parotid cells. Other Ca²⁺dependent isoforms were not detected. Further, agonist stimulation caused the redistribution of protein kinase C-α from cytosol to a membrane fraction. Agonists may promote parotid acinar cell activity, including amylase secretion, by increasing the affinity of protein kinase C-α for the membrane fraction, presumably via a rise in Ca²⁺ and diacylglycerol derived from polyphosphoinositide hydrolysis.     

Effects of prolonged parotid duct ligation, parotidectomy and acute hepatitis of rats on amylase activity.

Amylase activity in the parotid gland was extremely reduced (to 2.4 per cent) by prolonged duct ligation but that in the pancreas and serum as similar to controls. The serum amylase activity of parotidectomized rats was remarkably reduced within a week of surgery, but recovered to approximately the previous level in 4 weeks. The amylase activities in the pancreas and liver were not different between control and parotidectomized rats. The serum amylase activity of rats with acute hepatitis (1 day after administration of D-galactosamine) decreased to 18.5 per cent compared with controls; however, the activity recovered to its original level after 4-7 days. There was no pancreatic amylase in the serum either of parotidectomized rats or those with acute hepatitis. These observations show that the parotid glands may not be the only source of serum amylase. Serum amylase is mainly derived from the parotid gland, but liver may contribute to serum amylase in compensation for the parotid glands.     

Sialagogue-stimulated protein phosphorylation related to ornithine decarboxylase induction in cultured rat parotid explants.

Both beta-adrenergic (isoproterenol) and cholinergic (carbachol) sialagogues increase amylase secretion, ornithine decarboxylase activity and DNA synthesis in murine parotid gland in vivo and in vitro. These agonists enhanced the incorporation of labelled inorganic orthophosphate into parotid proteins in rat parotid explants cultured on siliconized lens paper floating on serum-free 199 medium. Analysis of the labelled proteins by SDS-PAGE and autoradiography revealed that isoproterenol enhanced the phosphorylation of four proteins with apparent molecular weights of 17, 20, 31 and 32 kDa and carbachol stimulated the phosphorylation of 31 and 32 K proteins. Isoproterenol-dependent ornithine decarboxylase induction and phosphorylation of the proteins were selectively suppressed by monensin but not by polymyxin B, whereas carbachol-dependent ornithine decarboxylase induction and protein phosphorylation were inhibited by polymyxin B but not by monensin. Neither monensin nor polymyxin B suppressed isoproterenol- or carbachol-stimulated amylase secretion. Time course experiments showed that sialagogue-stimulated protein phosphorylation preceded the increase of ornithine decarboxylase activity and had almost disappeared when it was maximal. Propranolol and atropine, antagonists of isoproterenol and carbachol, respectively, completely inhibited not only amylase secretion and ornithine decarboxylase induction but also protein phosphorylation stimulated by the corresponding agonists. These findings suggest that increased phosphorylation of specific proteins is associated with sialagogue-stimulated ornithine decarboxylase induction but not amylase secretion.     

Effect of methotrexate on protein and amylase secretion by rat parotid and submandibular salivary glands

Saliva was collected from anaesthetized, methotrexate (MTX)-treated, pair-fed and control rats using either acetylcholine or bethanechol as secretagogue. Parotid saliva was analysed for protein and amylase content, and submandibular saliva for protein. Acetylcholine stimulation after MTX increased parotid protein output by 151 per cent, amylase output by 125 per cent and submandibular protein output by 229 per cent when compared to control values. There were no significant differences in either protein or amylase output between the control and pair-fed animals. With bethanechol, salivary amylase and protein secretion did not differ between MTX-treated and control animals. The significant increase in parotid protein and amylase output with acetylcholine was reduced by pretreatment with hexamethonium (5 mg/kg) or propranolol (5 mg/kg), but not by phenoxybenzamine (1 mg/kg). MTX did not increase the number or the dissociation constant of parotid beta-adrenergic receptors but did increase their number in the submandibular gland. Thus increases in protein and amylase output caused by acetylcholine MTX-treated rats may be the result of increased beta-adrenergic activity.     

Biochemical changes in rat parotid gland lysosomal enzyme activities after isoprenaline or starvation.

The content of several lysosomal enzymes was measured in the rat parotid gland when the normal secretory cycle was altered by either reducing or enhancing secretion. The findings support the concept that lysosomes play a role in regulating the gland content of secretory products. The secretory protein, amylase, began to accumulate when secretion was reduced. This was immediately followed by an increase in lysosomal enzymes (cathepsin D and acid phosphatase). Thereafter, amylase activity decreased rapidly followed by a smaller reduction in lysosomal enzymes. When secretion was enhanced by an injection of isoprenaline, amylase was reduced by 95 per cent within two hours; the lysosomal enzymes were decreased by 15–30 per cent. Within 24 h, amylase was restored and increased by 50 per cent whereas the lysosomal enzymes were increased only 10–30 per cent.The activity of acid phosphatase was measured with two different substrates. Activity measured with β-glycerophosphate paralleled that of cathepsin D whereas that measured with p-nitrophenylphosphate did not when secretion was reduced but did when secretion was enhanced. The results support the hypothesis that acid phosphatase activity measured with β-glycerophosphate is lysosomal whereas that measured with p-nitrophenylphosphate may have another cellular location in addition to lysosomes.     

Effects of secretory nerve stimulation on the movement of rat parotid amylase into the circulation

Stimulation of the nerves to the rat parotid gland demonstrated that increases in serum amylase levels can originate exclusively from this gland. However, whilst parasympathetic nerve stimulation caused a significant movement of parotid amylase into the blood, sympathetic nerve stimulation did not, despite the higher concentration of amylase in the saliva evoked. Experiments in which conscious animals were fed for a short period showed that parotid amylase reaches the blood during normal activity. The mechanisms underlying the movement of parotid amylase into the circulation are still unknown but there appears to be some dependence on the volume of saliva being produced.