Ontogenetic development of the water channel protein AQP5 in mouse salivary gland tissue.

Aquaporins (AQP) are a family of channel proteins expressed in the cell membranes of many tissue types. As water channels, they enable the selective permeation of water molecules and thus play an important role in water transport through the plasma membrane. There are numerous AQP sub-types, among which AQP5 is expressed in the salivary glands. The expression and localization of AQP5 in different salivary gland cells of animal models during fetal development and after birth have enabled the physiological functions of AQP5 to be elucidated, but subsequent changes in the adult phase are unknown. It is known that saliva production tends to decrease with age, but it is unclear how AQP5 activity and function changes developmentally, from young to old including gender differences. In the present study, we sampled the parotid, submandibular, and sublingual glands from young (8 weeks old) and aged (12 months old) mice of both sexes to study the effects of age- and sex-related differences in AQP5 expression. Positive fluorescence immunostaining was detected in the membranes of cells from all gland types, and this was enhanced in juvenile mice from both sexes. Western blot analyses revealed that AQP5 expression levels tended to decrease with age in both male and female animals. Conversely, AQP5 gene expression levels did not change significantly with aging, but were found to be high in submandibular gland cells of both sexes, in parotid gland cells of older female mice, and in the sublingual gland cells of young male mice.

Expression and localization of CD63 in the intracellular vesicles of odontoblasts.

We hypothesized that odontoblasts release exosomes as well as dental pulp cells and focused on the exosome membrane marker CD63. Odontoblasts are well-differentiated mesenchymal cells that produce dentin. Dental pulp, a tissue complex formed with odontoblasts, releases exosomes to epithelial cells and stimulates their differentiation to ameloblasts. However, the localization of CD63 in differentiated odontoblasts is poorly understood. Therefore, herein, we aimed to reveal the expression of CD63 in odontoblasts during tooth development. We first investigated the localization of CD63 in mouse incisors and molars using immunofluorescence. In adult mouse incisors, the anti-CD63 antibody was positive in mature odontoblasts and dental pulp cells but not in pre-odontoblasts along the ameloblasts in the apical bud. Additionally, the anti-CD63 antibody was observed as a vesicular shape in the apical area of odontoblast cytosol and inside Tomes' fibers. The anti-CD63 antibody-positive vesicles were also observed using immunoelectron microscopy. Moreover, during mouse mandibular molar tooth morphogenesis (E16 to postnatal 6 weeks), labeling of anti-CD63 antibody was positive in the odontoblasts at E18. In contrast, the anti-CD63 antibody was positive in the dental pulp after postnatal day 10. Furthermore, anti-CD63 antibody was merged with the multivesicular body marker Rab7 in dental pulp tissues but not with the lysosome marker Lamp1. Finally, we determined the effect of a ceramide-generation inhibitor GW4869 on the mouse organ culture of tooth germ in vitro. After 28 days of GW4869 treatment, both CD63 and Rab7 were negative in Tomes' fibers, but were positive in control odontoblasts. These results suggest that CD63-positive vesicular organelles are important for mouse tooth morphogenesis.

Effect of ovariectomy induced osteoporosis on metaphysis and diaphysis repair process.

The fracture repair process is known to be delayed in postmenopausal women, under estrogen-deficient status. Osteoporotic fracture mainly occurs in the metaphyseal region of the long bone; however, most studies on fracture healing have focused on the diaphyseal region. In this study, we compared the repair process between metaphysis and diaphysis of ovariectomized (OVX) and Sham mice, and analyzed the effects of short-term estrogen administration in OVX mice. Mice were divided into four experimental groups, including Sham, OVX, OVX + vehicle, and OVX + 17ß-estradiol (E2). Bone apertures were formed in the tibial metaphysis and diaphysis. The samples were collected and examined by micro-computed tomography, and using histological, histochemical, and immunohistochemical analysis at different time points after the surgery. The cartilaginous callus was formed at the diaphysis site of both the groups, which was sequentially replaced by bone on the periosteum side. Medullary callus was formed in all the groups; however, the volume of the callus in OVX mice was significantly lesser (˜30%) than that in Sham mice. Furthermore, in the metaphysis, no differences were observed in the medullary callus and bone mineral density between the two groups from day 21 to 28. The diaphysis of OVX group was not completely repaired even by day 28. In both the sites of OVX mice, ALP activity and disappearance of Gr-1 positive cells were delayed compared to that of Sham. Estrogen administration improved medullary callus formation in the diaphysis, however not in the metaphysis. The effect of ovariectomy on the repair process in diaphysis was greater than that in metaphysis. Our findings clarify the differences between the metaphysis and diaphysis repair process using OVX mouse model and suggest that the estrogen sensitivities differ between the sites during the bone repair process.

Trabecular structure and composition analysis of human autogenous bone donor sites using micro-computed tomography.

OBJECTIVES: To evaluate the bone microstructure of autogenous graft bone in elderly people (mean age, 66 years), we compared the bone volume/total volume and bone mineral density of four donor sites that are commonly harvested for maxillofacial surgery and dental implant treatments, using X-ray micro-computed tomography. METHODS: Eighteen Japanese cadavers were included in this study. Overall, 66 harvested bones (mandibular symphysis, mandibular ramus, ilium, and tibia) were studied. Micro-computed tomography scans of four sites were performed to analyze the trabecular structures, bone mineral density, and bone volume/total volume in these bones. RESULTS: The mandibular symphysis bones showed the highest bone volume/total volume and bone mineral density at the four sites. There was a significant difference in the bone volume/total volume between the mandibular symphysis and tibia groups. There was also a significant difference in bone mineral density between the mandibular symphysis group and the ilium and tibia groups. In the three-dimensional observations, the structures of the mandibular trabecular were plate-type. The structures of the tibial bone were mixtures of plate- and rod-types. In the ilium, most trabecula were rod-shaped. CONCLUSIONS: Mandibular symphysis and ramus had a higher bone volume/total volume and bone mineral density of the four sites and did not show regressive changes in our findings. Mandibular bone is the most suitable source of autogenous graft bone material because of its superior bone quality and quantity.

Role of Innate Inflammation in the Regulation of Tissue Remodeling during Tooth Eruption.

Tooth eruption is characterized by a coordinated complex cascade of cellular and molecular events that promote tooth movement through the eruptive pathway. During tooth eruption, the stratum intermedium structurally changes to the papillary layer with tooth organ development. We previously reported intercellular adhesion molecule-1 (ICAM-1) expression on the papillary layer, which is the origin of the ICAM-1-positive junctional epithelium. ICAM-1 expression is induced by proinflammatory cytokines, including interleukin-1 and tumor necrosis factor. Inflammatory reactions induce tissue degradation. Therefore, this study aimed to examine whether inflammatory reactions are involved in tooth eruption. Reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed sequential expression of hypoxia-induced factor-1α, interleukin-1ß, and chemotactic factors, including keratinocyte-derived chemokine (KC) and macrophage inflammatory protein-2 (MIP-2), during tooth eruption. Consistent with the RT-PCR results, immunohistochemical analysis revealed KC and MIP-2 expression in the papillary layer cells of the enamel organ from the ameloblast maturation stage. Moreover, there was massive macrophage and neutrophil infiltration in the connective tissue between the tooth organ and oral epithelium during tooth eruption. These findings suggest that inflammatory reactions might be involved in the degradation of tissue overlying the tooth organ. Further, these reactions might be induced by hypoxia in the tissue overlying the tooth organ, which results from decreased capillaries in the tissue. Our findings indicate that bacterial infections are not associated with the eruption process. Therefore, tooth eruption might be regulated by innate inflammatory mechanisms.

Effect of nitrogen-containing bisphosphonates on osteoclasts and osteoclastogenesis: an ultrastructural study.

We have previously indicated that a single injection of alendronate, one of the nitrogen-containing bisphosphonates (NBPs), affects murine hematopoietic processes, such as the shift of erythropoiesis from bone marrow (BM) to spleen, disappearance of BM-resident macrophages, the increase of granulopoiesis in BM and an increase in the number of osteoclasts. NBPs induce apoptosis and the formation of giant osteoclasts in vitro and/or in patients undergoing long-term NBP treatment. Therefore, the time-kinetic effect of NBPs on osteoclasts needs to be clarified. In this study, we examined the effect of alendronate on mouse osteoclasts and osteoclastogenesis. One day after the treatment, osteoclasts lost the clear zone and ruffled borders, and the cell size decreased. After 2 days, the cytoplasm of osteoclasts became electron dense and the nuclei became pyknotic. Some of the cells had fragmented nuclei. After 4 days, osteoclasts had euchromatic nuclei attached to the bone surface. Osteoclasts had no clear zones or ruffled borders. After 7 days, osteoclasts formed giant osteoclasts via the fusion of multinuclear and mononuclear osteoclasts. These results indicate that NBPs affect osteoclasts and osteoclastogenesis via two different mechanisms.

Repair processes of flat bones formed via intramembranous versus endochondral ossification.

OBJECTIVES: Although fractures occur in various bones, including long, short, and flat bones, fracture repair investigations focus on the diaphysis of the long bone. The cell composition, osteogenic capacity, and bone matrix differ among osteogenesis patterns. However, the differences in the bone repair process have not been studied. Here, we compared the bone repair processes in the parietal bone and scapula of adolescent mice. METHODS: Bone apertures were created in the parietal bone and scapula. Samples were collected at indicated times after surgery, and the repair process was analyzed using micro-computed tomography, histological, immunohistochemical, and mRNA expression analyses. RESULTS: In both repair processes, cartilage formation was not detected on the periosteum side. The parietal bone aperture was gradually filled with newly formed bone produced from the edge of the aperture by day 14 but was not completely repaired even by day 49. In the scapula, a bony callus was detected on the periosteum at day 7, and the aperture was bridged by day 14. Subsequently, the bony callus was remodeled to the original bone architecture. Alkaline phosphatase activity and osteocalcin synthesis occurred earlier in the repair region of the scapular periosteum, compared with that in the parietal periosteum. The mRNA expression of osteogenic markers in the periosteum was markedly upregulated in the scapula versus the parietal bone. CONCLUSION: Our study findings clarify the differences between parietal bone and scapula repair and suggest that the bone repair process differs among ossification patterns.

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.

Expression and localization of aqua-glyceroporins AQP3 and AQP9 in rat oral epithelia.

Aquaporins (AQPs) are a family of small integral membrane proteins made up of 6 hydrophobic, a-helical, membrane-spanning domains surrounding a highly selective aqueous pore. AQP3, AQP7, and AQP9, termed aqua-glyceroporins, are known to be involved in the transport of water, glycerol, and other small molecules. In this study, we investigated the expression and localization of aqua-glyceroporins in rat oral stratified squamous epithelia of the palate, the buccal mucosa, the inferior aspect of the tongue, and the oral floor by using RT-PCR, immunofluorescence, and immunogold electron microscopy. AQP3 and AQP9 mRNAs were expressed in whole oral epithelium. Immunostaining for AQP3 was recognized in each type of epithelium. The results suggest that AQP3 synthesis begins predominantly in the cytoplasm of the basal cells. During the process of epithelial cell differentiation, AQP3 protein appears to accumulate and be transported to the plasma membrane, from where it is incorporated into the cornified or surface layers. The intracellular localization of AQP3 appears to correlate with the differentiation of keratinocytes, suggesting that it acts as an enhancer of the physiological permeability barrier together with membrane coating granules. The distribution pattern of AQP9 was limited to the marginal areas of the basal and suprabasal layers, which was different from that of AQP3. This difference in distribution between AQP3 and AQP9 suggests that AQP9 in rat oral epithelia acts as a channel by facilitating glycerol uptake from the blood through the endothelial cells of the capillary vessels to the oral stratified squamous epithelium. AQP3 and AQP9 facilitate both transcellular osmotic water flow and glycerol transport as pore-like passive transporters in the keratinocytes of oral epithelia, and may play a key role in not only hydration and the permeability barrier, but also cell proliferation, differentiation, migration, development, and wound healing by generating ATP.

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.

E2f1-deficient NOD/SCID mice have dry mouth due to a change of acinar/duct structure and the down-regulation of AQP5 in the salivary gland.

Non-obese diabetic (NOD) mice have been used as a model for dry mouth. NOD mice lacking the gene encoding E2f1, a transcription factor, develop hyposalivation more rapidly progressively than control NOD mice. However, the model mice are associated with an underlying disease such as diabetes. We have now established E2f1-deficient NOD/severe combined immunodeficiency disease (NOD/SCID.E2f1(-/-)) mice to avoid the development of diabetes (Matsui-Inohara et al., Exp Biol Med (Maywood) 234(12):1525-1536, 2009). In this study, we investigated the pathophysiological features of dry mouth using NOD/SCID.E2f1(-/-) mice. In NOD/SCID.E2f1(-/-) mice, the volume of secreted saliva stimulated with pilocarpine is about one third that of control NOD/SCID mice. In behavioral analysis, NOD/SCID.E2f1(-/-) mice drank plenty of water when they ate dry food, and the frequency and time of water intake were almost double compared with control NOD/SCID mice. Histological analysis of submandibular glands with hematoxylin-eosin stain revealed that NOD/SCID.E2f1(-/-) mice have more ducts than NOD/SCID mice. In western blot analysis, the expression of aquaporin 5 (AQP5), a marker of acinar cells, in parotid and in submandibular glands of NOD/SCID.E2f1(-/-) mice was lower than in NOD/SCID mice. Immunohistochemical analysis of parotid and submandibular acini revealed that the localization of AQP5 in NOD/SCID.E2f1(-/-) mice differs from that in NOD/SCID mice; AQP5 was leaky and diffusively localized from the apical membrane to the cytosol in NOD/SCID.E2f1(-/-) mice. The ubiquitination of AQP5 was detected in submandibular glands of NOD/SCID.E2f1(-/-) mice. These findings suggest that the change of acinar/duct structure and the down-regulation of AQP5 in the salivary gland cause the pathogenesis of hyposalivation in NOD/SCID.E2f1(-/-) mice.

Roles of AQP5/AQP5-G103D in carbamylcholine-induced volume decrease and in reduction of the activation energy for water transport by rat parotid acinar cells.

In order to assess the contribution of the water channel aquaporin-5 (AQP5) to water transport by salivary gland acinar cells, we measured the cell volume and activation energy (E (a)) of diffusive water permeability in isolated parotid acinar cells obtained from AQP5-G103D mutant and their wild-type rats. Immunohistochemistry showed that there was no change induced by carbamylcholine (CCh; 1 µM) in the AQP5 detected in the acinar cells in the wild-type rat. Acinar cells from mutant rats, producing low levels of AQP5 in the apical membrane, showed a minimal increase in the AQP5 due to the CCh. In the wild-type rat, CCh caused a transient swelling of the acinus, followed by a rapid agonist-induced cell shrinkage, reaching a plateau at 30 s. In the mutant rat, the acinus did not swell by CCh challenge, and the agonist-induced cell shrinkage was delayed by 8 s, reaching a transient minimum at around 1 min, and recovered spontaneously even though CCh was persistently present. In the unstimulated wild-type acinar cells, E (a) was 3.4 ± 0.6 kcal mol(-1) and showed no detectable change after CCh stimulation. In the unstimulated mutant acinar cells, high E (a) value (5.9 ± 0.1 kcal mol(-1)) was detected and showed a minimal decrease after CCh stimulation (5.0 ± 0.3 kcal mol(-1)). These results suggested that AQP5 was the main pathway for water transport in the acinar cells and that it was responsible for the rapid agonist-induced acinar cell shrinkage and also necessary to keep the acinar cell volume reduced during the steady secretion in the wild-type rat.

The actin-specific reagent jasplakinolide induces apoptosis in primary rat parotid acinar cells.

Jasplakinolide is a reagent that stabilizes and polymerizes actin filaments and is a commonly used tool in cell biology. In primary rat parotid acinar cells, jasplakinolide partially inhibited the release of amylase induced by ß-adrenergic receptor activation, as previously reported. However, in confocal microscopic observation with fluorescence conjugated anti-actin antibody, the jasplakinolide-treated cells not only showed decreased fluorescence intensity and aggregation of cortical F-actin but also revealed events characteristic of apoptosis such as cell shrinkage, membrane blebbing and apoptotic body formation. Such characteristic events of apoptosis were confirmed by transmission electron microscopy. The occurrence of apoptosis in jasplakinolide-treated cells was further confirmed by biochemical analysis: a DNA ladder was detected by electrophoresis, and DNA fragmentation was revealed using ELISA with an antibody to single-stranded DNA. Moreover, the degradation of fodrin was detected in jasplakinolide-treated cells by Western blotting, and the K(+) release induced by the fluid secretagogue carbachol was impaired. Taken together, these results demonstrate that jasplakinolide induces apoptosis and suppresses the secretory functions of rat parotid acinar cells.

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.

Phosphorylation of myristoylated alanine-rich C kinase substrate is involved in the cAMP-dependent amylase release in parotid acinar cells.

Myristoylated alanine-rich C kinase substrate (MARCKS) is known as a major cellular substrate for protein kinase C (PKC). MARCKS has been implicated in the regulation of brain development and postnatal survival, cellular migration and adhesion, as well as phagocytosis, endocytosis, and exocytosis. The involvement of MARCKS phosphorylation in secretory function has been reported in Ca(2+)-mediated exocytosis. In rat parotid acinar cells, the activation of beta-adrenergic receptors provokes exocytotic amylase release via accumulation of intracellular cAMP levels. Here, we studied the involvement of MARCKS phosphorylation in the cAMP-dependent amylase release in rat parotid acinar cells. MARCKS protein was detected in rat parotid acinar cells by Western blotting. The beta-adrenergic agonist isoproterenol (IPR) induced MARCKS phosphorylation in a time-dependent manner. Translocation of a part of phosphorylated MARCKS from the membrane to the cytosol and enhancement of MARCKS phosphorylation at the apical membrane site induced by IPR were observed by immunohistochemistry. H89, a cAMP-dependent protein kinase (PKA) inhibitor, inhibited the IPR-induced MARCKS phosphorylation. The PKCdelta inhibitor rottlerin inhibited the IPR-induced MARCKS phosphorylation and amylase release. IPR activated PKCdelta, and the effects of IPR were inhibited by the PKA inhibitors. A MARCKS-related peptide partially inhibited the IPR-induced amylase release. These findings suggest that MARCKS phosphorylation via the activation of PKCdelta, which is downstream of PKA activation, is involved in the cAMP-dependent amylase release in parotid acinar cells.

Characterization of neurokinin A-evoked salivary secretion in the perfused rat submandibular gland.

Neurokinin A (NKA) evokes salivary secretion. Despite such reports, the direct effect of NKA on salivary secretion in submandibular gland has not been clarified. Here we studied characterization of salivary fluid secretion induced by NKA in the perfused submandibular grand (SMG) of the rat. NKA (3-100 nM) stimulated salivary fluid secretion in a dose-dependent manner. The profile of secretion induced by NKA consisted of two phases, transient and sustained phases. When the gland was perfused with Lucifer yellow (LY)-containing perfusate buffer and stimulated by NKA, concentration of LY in saliva was increased. In the absence of Ca(2+) in the perfusate, NKA induced only a transient salivary fluid and a transient LY secretion. When the gland was treated with BAPTA, NKA failed to induce both salivary fluid secretion and LY secretion. These results suggest that NKA induces salivary secretion via both transcellular and paracellular pathways, which depends on intracellular Ca(2+) mobilization.

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.

Role of protein kinase C-delta in isoproterenol-induced amylase release in rat parotid acinar cells.

In parotid acinar cells, beta-adrenergic receptor activation results in accumulation of intracellular cAMP. Subsequently, cAMP-dependent protein kinase (PKA) is activated and consequently amylase release is provoked. In this paper, we investigated involvement of protein kinase C-delta (PKC delta), a novel isoform of PKC, in amylase release induced by beta-adrenergic receptor stimulation. Amylase release stimulated with the beta-agonist isoproterenol (IPR) was inhibited by rottlerin, an inhibitor of PKC delta. IPR activated PKC delta and the effect of IPR were inhibited by a PKA inhibitor, H89. Myristoylated alanine-rich C kinase substrate (MARCKS), a major cellular substrate for PKC, was detected in rat parotid acinar cells, and a MARCKS inhibitor, MARCKS-related peptide, inhibited the IPR-induced amylase release. IPR stimulated MARCKS phosphorylation, which was found to be inhibited by H89 and rottlerin. These observations suggest that PKC delta activation is a downstream pathway of PKA activation and is involved in amylase release via MARCKS phosphorylation in rat parotid acinar cells stimulated with beta-adrenergic agonist.