The secretory ability of newly formed secretory granules is regulated by pro-cathepsin B and amylase in parotid glands.

Parotid glands are exocrine glands that release saliva into the oral cavity. Acinar cells of parotid glands produce many secretory granules (SGs) that contain the digestion enzyme amylase. After the generation of SGs in the Golgi apparatus, they mature by enlarging and membrane remodeling. VAMP2, which is involved in exocytosis, accumulates in the membrane of mature SGs. The remodeling of SG membranes is regarded as a preparation process for exocytosis but its detailed mechanism remains unknown. To address that subject, we investigated the secretory ability of newly formed SGs. Although amylase is a useful indicator of secretion, the cell leakage of amylase might affect the measurement of secretion. Thus, in this study, we focused on cathepsin B (CTSB), a lysosomal protease, as an indicator of secretion. It has been reported that some procathepsin B (pro-CTSB), which is a precursor of CTSB, is initially sorted to SGs after which it is transported to lysosomes by clathrin-coated vesicles. Because pro-CTSB is processed to mature CTSB after its arrival in lysosomes, we can distinguish between the secretion of SGs and cell leakage by measuring the secretion of pro-CTSB and mature CTSB, respectively. When acinar cells isolated from parotid glands were stimulated with isoproterenol (Iso), a ß-adrenergic agonist, the secretion of pro-CTSB was increased. In contrast, mature CTSB was not detected in the medium although it was abundant in the cell lysates. To prepare parotid glands rich in newly formed SGs, the depletion of per-existing SGs was induced by an intraperitoneal injection of Iso into rats. At 5 h after that injection, newly formed SGs were observed in parotid acinar cells and the secretion of pro-CTSB was also detected. We confirmed that the purified newly formed SGs contained pro-CTSB, but not mature CTSB. At 2 h after Iso injection, few SGs were observed in the parotid glands and the secretion of pro-CTSB was not detected, which proved that the Iso injection depleted pre-existing SGs and the SGs observed at 5 h were newly formed after the Iso injection. These results suggest that newly formed SGs have a secretory ability prior to membrane remodeling.

Acinar Cell Proliferation Promoted by BMP2 in Injured Mouse Parotid Gland: BMP2 Promotes Cell Proliferation in Parotid Gland.

Objective: To identify factors that affect salivary gland recovery, we investigated the expression and function of bone morphogenetic protein 2 (BMP2) in mice. Materials and Methods: Using a micro clip, mice parotid glands were removed 7 days after the ligation of the unilateral parotid excretory duct. Thereafter, they were weighed and stained with hematoxylin and eosin, and BMP2 expression was examined via real-time reverse transcription-polymerase chain reaction. Primary cultures of parotid glands were prepared, and BMP2 protein was added to the culture medium for 48 hr to examine its effect on cell proliferation. E-cadherin and vimentin expression was examined using western blotting. Finally, immunohistochemical staining using an anti-Ki67 antibody was performed. Results: Duct-ligated parotid glands weighed less than those that were collected after sham surgery and showed acinar cell atrophy. They also showed higher BMP2 expression than control glands. Primary-cultured parotid acinar cells supplemented with BMP2 showed higher proliferative potential than control cells. Furthermore, they showed E-cadherin, but not vimentin, expression, and their percentage of Ki67-positive cells were higher than that corresponding to the controls. Conclusions: Injury to salivary glands by excretory duct ligation increased BMP2 expression, which may be involved in maintaining salivary gland function by inducing acinar cell proliferation.

Switching of cargo sorting from the constitutive to regulated secretory pathway by the addition of cystatin D sequence in salivary acinar cells.

The mechanism for segregation of cargo proteins into the regulated and constitutive secretory pathways in exocrine cells remains to be elucidated. We examined the transport of HaloTag proteins fused with full-length cystatin D (fCst5-Halo) or only its signal peptide (ssCst5-Halo) in parotid acinar cells. Although both fusion proteins were observed to be colocalized with amylase in the secretory granules, the coefficients for overlapping and correlation of fCst5-Halo with amylase were higher than those of ssCst5-Halo. The secretion of both the proteins was enhanced by the addition of the ß-adrenergic receptor agonist isoproterenol as well as endogenous amylase. In contrast, unstimulated secretion of ssCst5-Halo without isoproterenol was significantly higher than that of fCst5-Halo and amylase. Simulation analysis using a mathematical model revealed that a large proportion of ssCst5-Halo was secreted through the constitutive pathway, whereas fCst5-Halo was transported into the secretory granules more efficiently. Precipitation of fCst5-Halo from cell lysates was increased at a low pH, which may mimic the milieu of the trans-Golgi networks. These data suggest that the addition of a full-length sequence of cystatin D facilitates efficient selective transport into the regulated pathway by aggregation at low pH in the trans-Golgi network.NEW & NOTEWORTHY The mechanism underlying the segregation of cargo proteins to the regulated and constitutive secretory pathways in exocrine cells remains to be solved. We analyzed unstimulated secretion in salivary acinar cells by performing double-labeling experiments using HaloTag technology and computer simulation. It revealed that the majority of HaloTag with only signal peptide sequence was secreted through the constitutive pathway and that the addition of a full-length cystatin D sequence changed its sorting to the regulated pathway.

Suppression of parotid acinar cell dysfunction by the free radical scavenger 3-methyl-1-phenyl-2-pyrazolin-5-one.

Salivary gland atrophy and consequent hyposalivation are serious problems in clinical dentistry, as saliva regulates the environment of the oral cavity. To clarify the mechanisms underlying salivary gland dysfunction, a system for primary culture of parotid acinar cells has been established. It has been reported previously that the process of cell isolation from parotid glands triggers stress signaling mediated by Src and p38 mitogen-activated protein (MAP) kinase (p38), leading to dedifferentiation of acinar cells, and that an nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor suppresses this activation of Src and p38, suggesting that reactive oxygen species initiate the dedifferentiation signal. The present study examined the effect of a free radical scavenger, 3-methyl-1-phenyl-2-pyrazolin-5-one (also termed MCI-186 or edaravone), on activation of the stress signal and the secretory function of parotid acinar cells. Activation of p38 during cell isolation was suppressed by addition of MCI-186. The retention of the activity of amylase, a major salivary protein, and the number of amylase-containing secretory granules were improved by isolation and culture in the presence of MCI-186. In addition, calcium elevation upon stimulation with a muscarinic agonist was higher in MCI-186-treated cells than in untreated cells. These results suggest that MCI-186 (edaravone) is a promising agent for prevention of salivary gland dysfunction.

Maintenance of claudin-3 expression and the barrier functions of intercellular junctions in parotid acinar cells via the inhibition of Src signaling.

OBJECTIVES: Salivary acinar and duct cells show different expression patterns of claudins, which may reflect their different functions. To study the role of claudins in saliva secretion, we examined alterations in the expression patterns of cell adhesion molecules in parotid glands of γ-irradiated rats and analyzed the influence of those changes on intercellular barrier function using primary cultures of parotid acinar cells. DESIGN: Rats were γ-irradiated with doses of 5, 15 or 20Gy, and expression levels of cell adhesion molecules were examined by immunoblotting analysis. Acinar cells were isolated from parotid glands and were cultured in the absence or presence of the Src kinase inhibitor PP1. Changes in protein and mRNA expression patterns were determined by immunoblotting and by RT-PCR analyses, respectively. Intercellular barrier function was examined by measuring transepithelial electrical resistance and the paracellular flux of FITC-dextran. RESULTS: In irradiated parotid glands, the expression of claudin-4 was enhanced at 15Gy or higher, levels that induce the hyposecretion of saliva, although that increase was transient. At 30days after irradiation, expression levels of cell adhesion molecules were decreased. In primary cultures, the expression of claudin-4 was also increased transiently but the expression of claudin-3 and E-cadherin was decreased. The barrier function of tight junctions was disrupted although the localization of occludin was maintained. The Src kinase inhibitor PP1 suppressed those changes in gene expression and retained the intercellular barrier function. CONCLUSIONS: These results suggest that the inhibition of Src signaling maintains the barrier functions of intercellular junctions in salivary glands, which can be lost due to tissue injury.

Involvement of myristoylated alanine-rich C kinase substrate phosphorylation and translocation in cholecystokinin-induced amylase release in rat pancreatic acini.

Cholecystokinin (CCK) is a gastrointestinal hormone that induces exocytotic amylase release in pancreatic acinar cells. The activation of protein kinase C (PKC) is involved in the CCK-induced pancreatic amylase release. Myristoylated alanine-rich C kinase substrate (MARCKS) is a ubiquitously expressed substrate of PKC. MARCKS has been implicated in membrane trafficking in several cell types. The phosphorylation of MARCKS by PKC results in the translocation of MARCKS from the membrane to the cytosol. Here, we studied the involvement of MARCKS in the CCK-induced amylase release in rat pancreatic acini. Employing Western blotting, we detected MARCKS protein in the rat pancreatic acini. CCK induced MARCKS phosphorylation. A PKC-δ inhibitor, rottlerin, inhibited the CCK-induced MARCKS phosphorylation and amylase release. In the translocation assay, we also observed CCK-induced PKC-δ activation. An immunohistochemistry study showed that CCK induced MARCKS translocation from the membrane to the cytosol. When acini were lysed by a detergent, Triton X-100, CCK partially induced displacement of the MARCKS from the GM1a-rich detergent-resistant membrane fractions (DRMs) in which Syntaxin2 is distributed. A MARCKS-related peptide inhibited the CCK-induced amylase release. These findings suggest that MARCKS phosphorylation by PKC-δ and then MARCKS translocation from the GM1a-rich DRMs to the cytosol are involved in the CCK-induced amylase release in pancreatic acinar cells.

Secretory proteins without a transport signal are retained in secretory granules during maturation in rat parotid acinar cells.

OBJECTIVE: The acinar cells of the parotid gland are filled with numerous secretory granules (SGs), which accumulate the digestion enzyme amylase. SGs mature accompanied with membrane remodelling such as fusion and budding of small vesicles. However, little is understood about the mechanism of the condensation of SG contents during maturation. In this study, we examined whether secretory proteins need a specific signal to be retained in SGs. DESIGN: To induce internalization of the luminal membrane after exocytosis, we injected the ß-adrenergic agonist isoproterenol into rats. Acinar cells were then incubated with Lucifer Yellow (LY) dye as a tracer for 3h for uptake into immature secretory granules (ISGs). To observe whether LY was retained in SGs after maturation, we continued incubating the cultured acinar cells for 2 days. RESULTS: The localization of LY into ISGs was confirmed by the following four methods: (1) co-localization of the fluorescence of LY and amylase by confocal laser microscopy, (2) detection of the fluorescence from purified ISGs, (3) secretion of the fluorescence together with amylase upon stimulation, and (4) observation of the intracellular localization of LY by electron microscopy. Moreover, we observed co-localization of some of the SGs with the fluorescence of LY after cell culture. CONCLUSIONS: Although the fusion and budding of small vesicles may contribute to the process of granule maturation, LY remained in the SGs even after maturation. These results suggest that secretory proteins that have no transport signal are not excluded from SGs, and they are retained in SGs during granule maturation in exocrine parotid glands.

Sorting of a HaloTag protein that has only a signal peptide sequence into exocrine secretory granules without protein aggregation.

The mechanism involved in the sorting and accumulation of secretory cargo proteins, such as amylase, into secretory granules of exocrine cells remains to be solved. To clarify that sorting mechanism, we expressed a reporter protein HaloTag fused with partial sequences of salivary amylase protein in primary cultured parotid acinar cells. We found that a HaloTag protein fused with only the signal peptide sequence (Met(1)-Ala(25)) of amylase, termed SS25H, colocalized well with endogenous amylase, which was confirmed by immunofluorescence microscopy. Percoll-density gradient centrifugation of secretory granule fractions shows that the distributions of amylase and SS25H were similar. These results suggest that SS25H is transported to secretory granules and is not discriminated from endogenous amylase by the machinery that functions to remove proteins other than granule cargo from immature granules. Another reporter protein, DsRed2, that has the same signal peptide sequence also colocalized with amylase, suggesting that the sorting to secretory granules is not dependent on a characteristic of the HaloTag protein. Whereas Blue Native PAGE demonstrates that endogenous amylase forms a high-molecular-weight complex, SS25H does not participate in the complex and does not form self-aggregates. Nevertheless, SS25H was released from cells by the addition of a ß-adrenergic agonist, isoproterenol, which also induces amylase secretion. These results indicate that addition of the signal peptide sequence, which is necessary for the translocation in the endoplasmic reticulum, is sufficient for the transportation and storage of cargo proteins in secretory granules of exocrine cells.

Involvement of AQP6 in the Mercury-sensitive osmotic lysis of rat parotid secretory granules.

In secretory granules and vesicles, membrane transporters have been predicted to permeate water molecules, ions and/or small solutes to swell the granules and promote membrane fusion. We have previously demonstrated that aquaporin-6 (AQP6), a water channel protein, which permeates anions, is localized in rat parotid secretory granules (Matsuki-Fukushima et al., Cell Tissue Res 332:73-80, 2008). Because the localization of AQP6 in other organs is restricted to cytosolic vesicles, the native function or functions of AQP6 in vivo has not been well determined. To characterize the channel property in granule membranes, the solute permeation-induced lysis of purified secretory granules is a useful marker. To analyze the role of AQP6 in secretory granule membranes, we used Hg²âº, which is known to activate AQP6, and investigated the characteristics of solute permeability in rat parotid secretory granule lysis induced by Hg²âº (Hg lysis). The kinetics of osmotic secretory granule lysis in an iso-osmotic KCl solution was monitored by the decay of optical density at 540 nm using a spectrophotometer. Osmotic secretory granule lysis was markedly facilitated in the presence of 0.5-2.0 µM Hg²âº, concentrations that activate AQP6. The Hg lysis was completely blocked by ß-mercaptoethanol which disrupts Hg²âº-binding, or by removal of chloride ions from the reaction medium. An anion channel blocker, DIDS, which does not affect AQP6, discriminated between DIDS-insensitive and sensitive components in Hg lysis. These results suggest that Hg lysis is required for anion permeability through the protein transporter. Hg lysis depended on anion conductance with a sequence of NO(3) (-) > Br⁻ > I⁻ > Cl⁻ and was facilitated by acidic pH. The anion selectivity for NO(3) (-) and the acidic pH sensitivity were similar to the channel properties of AQP6. Taken together, it is likely that AQP6 permeates halide group anions as a Hg²âº-sensitive anion channel in rat parotid secretory granules.

Thiol-oxidation reduces the release of amylase induced by ß-adrenergic receptor activation in rat parotid acinar cells.

In parotid acinar cells, the activation of ß-adrenergic receptors induces the accumulation of intracellular cAMP, and consequently provokes the exocytotic release of amylase, a digestive enzyme. The cellular redox status plays a pivotal role in regulating various cellular functions. Cellular redox imbalance caused by the oxidation of cellular antioxidants, as a result of oxidative stress, induces significant biological damage. In this study, we examined the effects of diamide, a thiol-oxidizing reagent, on amylase release by rat parotid acinar cells. In cells treated with diamide, the formation of cAMP and the release of amylase induced by the ß-agonist isoproterenol (IPR) were partially reduced. The inhibitory effect of diamide on the IPR-induced release of amylase could be abrogated by reduced glutathione or dithiothreitol. Diamide had no effect on the amylase release induced by forskolin, an adenylate cyclase activator, or by mastoparan, a heterotrimeric GTPbinding protein activator. In cells treated with diamide, the binding affinity for [(3)H]DHA, but not the number of binding sites, was reduced. These results suggest that ß-adrenergic receptor function is reduced by thiol-oxidation, which inhibits amylase secretion by parotid acinar cells.

Maintenance of paracellular barrier function by insulin-like growth factor-I in submandibular gland cells.

Insulin-like growth factor-I (IGF-I) is expressed in salivary glands. We examined the effects of IGF-I on cell number, the expression and distribution of tight junction proteins and the paracellular barrier function in cells derived from rat submandibular glands. When those cells were cultured in medium containing 10% foetal bovine serum (FBS) or IGF-I, the number of cells was comparable at 10 days. However, in the presence of inhibitor of IGF-I receptors, the number of cells cultured with FBS only was clearly reduced. The tight junction proteins occludin and claudin-3 were similarly detected by Western blotting in cells cultured with IGF-I or FBS. Immunostaining revealed that occludin and another tight junction protein (ZO-1) were similarly localized at intracellular junctions of cells cultured with IGF-I or FBS. The barrier functions were evaluated by transepithelial resistance (TER) and by FITC-dextran permeability. The TER values and FITC-dextran permeability of cells cultured with IGF-I or FBS were comparable. These observations suggest that IGF-I contributes to the maintenance not only of the cell number of salivary gland cells but also of their paracellular barrier function via the expression and distribution of tight junction proteins.

Characteristics of neurokinin A-induced salivary fluid secretion in perfused rat submandibular gland.

Tachykinins such as neurokinin A (NKA) and substance P have been demonstrated to induce salivary fluid secretion in vivo. However, characteristics of salivary fluid secretion induced by tachykinins in salivary glands have not been well elucidated. In this study, the effects of the tachykinin NKA on salivary fluid secretion were investigated in isolated, perfused rat submandibular gland. NKA provoked salivary fluid secretion, which consisted of transient and sustained phases, in a dose-dependent manner. In fura-2-loaded dispersed cells of the rat submandibular gland, the doses of NKA in which induced salivary fluid secretion caused an increase in intracellular Ca(2+) concentration. When Ca(2+) was removed from the perfusate to examine the effect of Ca(2+) mobilization on NKA-induced fluid secretion, only the transient salivary fluid secretion occurred. When the gland was perfused with the Ca(2+)-free perfusate containing the intracellular Ca(2+) chelator BAPTA-AM, NKA failed to induce salivary fluid secretion. NKA also induced an increase in oxygen consumption, but which was reduced by the removal of Ca(2+) from perfusate. Salivary fluid is secreted via transcellular and paracellular pathways in acinar cells of salivary glands. To examine the contribution of paracellular pathway to NKA-induced salivary fluid secretion, the glands were perfused with a perfusate containing Lucifer yellow (LY), a cellular impermeable substance, and then were stimulated with NKA, which provoked secretion of LY in the saliva. These results suggest that the NKA-induced salivary fluid secretion is Ca(2+)-dependent and that the paracellular pathway contributes to the secretion.

The thiol-oxidizing agent diamide reduces isoproterenol-stimulated amylase release in rat parotid acinar cells.

In parotid acinar cells, activation of beta-adrenergic receptors provokes exocytotic amylase release via the accumulation of intracellular cAMP. Cellular redox status plays a pivotal role in the regulation of various cellular functions. Cellular redox imbalance caused by the oxidation of cellular antioxidants, as a result of oxidative stress, induces significant biological damages. In this study, we examined effect of diamide, a thiol-oxidizing reagent, on amylase release in rat parotid acinar cells. In the presence of diamide, isoproterenol (IPR)-induced cAMP formation and amylase release were partially reduced. Diamide had no effect on amylase release induced by forskolin and mastoparan, an adenylate cyclase activator and heterotrimeric GTP binding protein activator, respectively. In the cells pretreated with diamide, the binding affinity of [(3)H]dihydroalprenolol to beta-receptors was reduced. These results suggest that oxidative stress results in reduction of binding affinity of ligand on beta-receptor and consequently reduces protein secretory function in rat parotid acinar cells.

Separation of immature granules containing color dye from the rat parotid gland.

Parotid acinar cell contains many secretory granules. Most of granules are mature, but only little immature granules are included. These immature granules are not enough for investigation of granule maturation. In this study, we show an easy method of separation of immature granules from the rat parotid gland. In addition, we succeeded in detection of color dye in the granules. These results suggest that secretory granules can be visualized through endocytosis.