Rat Parotid Acinar Cells Research Articles

Increased GLUT1 expression and localization to Golgi apparatus of acinar cells in the parotid gland of Goto-Kakizaki diabetic rats

ObjectivePatients with diabetes are known to have high salivary glucose levels. But the mechanisms are still unclear. We hypothesized that the topological changes of glucose transporters affect the salivary glucose level. MethodsWe used adult Goto-Kakizaki (GK) rats, an animal model of advanced diabetes, and Wistar rats as a control, with or without glucose load. The sections of salivary glands from the animals were processed for standard histological, immunohistochemical, and immunofluorescent staining. ResultsParotid acinar cells of GK rats appeared like mucous filled with low-eosin-stained granules and possessing a flat nucleus located basally, whereas those of Wistar rats appeared as a typical serous gland with eosin-rich cytoplasm and a spherical nucleus. Cytoplasmic granules of GK rat parotid acinar cells showed no reaction of polysaccharide staining. In acinar cell cytoplasm of GK rats, intense GLUT1 immunoreactivity was observed compared to Wistar rats. By double immunostaining for GLUT1 and Golgi apparatus-specific markers, it was determined that GLUT1 was localized to the Golgi apparatus. By glucose loading in starved GK rats, the distribution of GLUT1-immunoreactive signals was spread out clearly from the apical side of the nucleus to the basolateral side. ConclusionsIn rat model of diabetes, highly localized GLUT1 at Golgi apparatus in acinar cells seems to increase taking up cytoplasmic glucose to form exocytotic vesicles. This phenomenon may transform parotid glands from serous to mucous-like and result in saccharide-rich saliva.

Transdifferentiation of periodontal ligament stem cells into acinar cells using an indirect co-culture system.

Serous Acinar Cells (ACs) are mature and functional secretory epithelial cells that develop and complete through other stem cells at the end of the ductal system. So, the regeneration of the salivary gland damaged by radiation does not occur without cell therapy. Todays, an accessible tissue like the Periodontal Ligament (PDL) of the tooth was considered to easily extract the Mesenchymal Stem Cells (MSCs). In-vitro differentiation of stem cells before transplantation to damaged tissue reduces the risk of tumorigenesis. This study was conducted to evaluate the feasibility of differentiation of PDLSCs into salivary acinar cells by a co-culture system. PDLSCs were isolated from adult human PDL tissue and co-cultured with rat parotid ACs using an indirect co-culture system. The transdifferentiation of PDLSCs was evaluated by PCR of Aquaporin 5 (AQP5) and Carbonic anhydrase 6 (CA6) genes, then quantitative real-time PCR was used to measure the gene expression levels. The data were analyzed by ANOVA. Specific bond with the correct size on 6% acrylamide gel and TBE5X buffer showed the expression of AQP5 and CA6 in PDLSCs co-cultured with acinar cells. RT-PCR revealed co-cultured PDLSCs with or without KGF (Keratinocyte Growth Factor) showed significantly increased expression of AQP5 genes in compared to the initial PDLSCs. Expression of AQP5 and CA6, indicating successful transdifferentiation of PDLSCs into ACs, in co-culture system for 3weeks.

Mechanisms Underlying Activation of α₁-Adrenergic Receptor-Induced Trafficking of AQP5 in Rat Parotid Acinar Cells under Isotonic or Hypotonic Conditions.

Defective cellular trafficking of aquaporin-5 (AQP5) to the apical plasma membrane (APM) in salivary glands is associated with the loss of salivary fluid secretion. To examine mechanisms of α1-adrenoceptor (AR)-induced trafficking of AQP5, immunoconfocal microscopy and Western blot analysis were used to analyze AQP5 localization in parotid tissues stimulated with phenylephrine under different osmolality. Phenylephrine-induced trafficking of AQP5 to the APM and lateral plasma membrane (LPM) was mediated via the α1A-AR subtype, but not the α1B- and α1D-AR subtypes. Phenylephrine-induced trafficking of AQP5 was inhibited by ODQ and KT5823, inhibitors of nitric oxide (NO)-stimulated guanylcyclase (GC) and protein kinase (PK) G, respectively, indicating the involvement of the NO/ soluble (c) GC/PKG signaling pathway. Under isotonic conditions, phenylephrine-induced trafficking was inhibited by La3+, implying the participation of store-operated Ca2+ channel. Under hypotonic conditions, phenylephrine-induced trafficking of AQP5 to the APM was higher than that under isotonic conditions. Under non-stimulated conditions, hypotonicity-induced trafficking of AQP5 to the APM was inhibited by ruthenium red and La3+, suggesting the involvement of extracellular Ca2+ entry. Thus, α1A-AR activation induced the trafficking of AQP5 to the APM and LPM via the Ca2+/ cyclic guanosine monophosphate (cGMP)/PKG signaling pathway, which is associated with store-operated Ca2+ entry.

Hydrogen sulfide: a novel gaseous signaling molecule and intracellular Ca2+ regulator in rat parotid acinar cells.

In addition to nitric oxide (NO), hydrogen sulfide (H2S) is recognized as a crucial gaseous messenger that exerts many biological actions in various tissues. An attempt was made to assess the roles and underlying mechanisms of both gases in isolated rat parotid acinar cells. Ductal cells and some acinar cells were found to express NO and H2S synthases. Cevimeline, a muscarinic receptor agonist upregulated endothelial NO synthase in parotid tissue. NO and H2S donors increased the intracellular Ca(2+) concentration ([Ca(2+)]i). This was not affected by inhibitors of phospholipase C and inositol 1,4,5-trisphosphate receptors, but was decreased by blockers of ryanodine receptors (RyRs), soluble guanylyl cyclase, and protein kinase G. The H2S donor evoked NO production, which was decreased by blockade of NO synthases or phosphoinositide 3-kinase or by hypotaurine, an H2S scavenger. The H2S donor-induced [Ca(2+)]i increase was diminished by a NO scavenger or the NO synthases blocker. These results suggest that NO and H2S play important roles in regulating [Ca(2+)]i via soluble guanylyl cyclase-cGMP-protein kinase G-RyRs, but not via inositol 1,4,5-trisphosphate receptors. The effect of H2S may be partially through NO produced via phosphoinositide 3-kinase-Akt-endothelial NO synthase. It was concluded that both gases regulate [Ca(2+)]i in a synergistic way, mainly via RyRs in rat parotid acinar cells.

Activation of muscarinic receptors in rat parotid acinar cells induces AQP5 trafficking to nuclei and apical plasma membrane

BackgroundThe subcellular distribution of aquaporin-5 (AQP5) in rat parotid acinar cells in response to muscarinic acetylcholine receptor (mAChR) activation remains unclear. MethodsImmunoconfocal and immunoelectron microscopy were used to visualize the distribution of AQP5 in parotid acinar cells. Western blotting was used to analyze AQP5 levels in membranes. To clarify the characteristics of membrane domains associated with AQP5, detergent solubility and sucrose-density flotation experiments were performed. ResultsUnder control conditions, AQP5 was diffusely distributed on the apical plasma membrane (APM) and apical plasmalemmal region and throughout the cytoplasm. Upon mAChR activation, AQP5 was predominantly located in the nucleus, APM and lateral plasma membrane (LPM). Subsequently, localization of AQP5 in the nucleus, APM and LPM was decreased. Prolonged atropine treatment inhibited mAChR agonist-induced translocation of AQP5 to the nucleus, APM and LPM. AQP5 levels were enhanced in isolated nuclei and nuclear membranes prepared from parotid tissues incubated with mAChR agonist. mAChR agonist induced AQP5 levels in both soluble and insoluble nuclear fractions solubilized with Triton X-100 or Lubrol WX. Small amounts of AQP5 in nuclei were detected using low-density sucrose gradient. When AQP5 was present in the nuclear membrane, nuclear size decreased. ConclusionThe activation of mAChR induced AQP5 translocation to the nucleus, APM and LPM, and AQP5 may trigger water transport across the nuclear membrane and plasma membrane in rat parotid acinar cells. General significanceAQP5 translocates to the nuclear membrane and may trigger the movement of water, inducing shrinkage of the nucleus and the start of nuclear functions.

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

ObjectiveThe 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. DesignTo induce internalization of the luminal membrane after exocytosis, we injected the β-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. ResultsThe 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. ConclusionsAlthough 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.

P2Y2 nucleotide receptor activation enhances the aggregation and self-organization of dispersed salivary epithelial cells

Hyposalivation resulting from salivary gland dysfunction leads to poor oral health and greatly reduces the quality of life of patients. Current treatments for hyposalivation are limited. However, regenerative medicine to replace dysfunctional salivary glands represents a revolutionary approach. The ability of dispersed salivary epithelial cells or salivary gland-derived progenitor cells to self-organize into acinar-like spheres or branching structures that mimic the native tissue holds promise for cell-based reconstitution of a functional salivary gland. However, the mechanisms involved in salivary epithelial cell aggregation and tissue reconstitution are not fully understood. This study investigated the role of the P2Y2 nucleotide receptor (P2Y2R), a G protein-coupled receptor that is upregulated following salivary gland damage and disease, in salivary gland reconstitution. In vitro results with the rat parotid acinar Par-C10 cell line indicate that P2Y2R activation with the selective agonist UTP enhances the self-organization of dispersed salivary epithelial cells into acinar-like spheres. Other results indicate that the P2Y2R-mediated response is dependent on epidermal growth factor receptor activation via the metalloproteases ADAM10/ADAM17 or the α5β1 integrin/Cdc42 signaling pathway, which leads to activation of the MAPKs JNK and ERK1/2. Ex vivo data using primary submandibular gland cells from wild-type and P2Y2R(-/-) mice confirmed that UTP-induced migratory responses required for acinar cell self-organization are mediated by the P2Y2R. Overall, this study suggests that the P2Y2R is a promising target for salivary gland reconstitution and identifies the involvement of two novel components of the P2Y2R signaling cascade in salivary epithelial cells, the α5β1 integrin and the Rho GTPase Cdc42.

Activation of ERK1/2 by Store-Operated Calcium Entry in Rat Parotid Acinar Cells

The regulation of intracellular Ca2+ concentration ([Ca2+]i) plays a critical role in a variety of cellular processes, including transcription, protein activation, vesicle trafficking, and ion movement across epithelial cells. In many cells, the activation of phospholipase C-coupled receptors hydrolyzes membrane phosphoinositides and produces the depletion of endoplasmic reticulum Ca2+ stores, followed by the sustained elevation of [Ca2+]i from Ca2+ entry across the plasma membrane via store-operated Ca2+ entry (SOCE). Ca2+ entry is also increased in a store-independent manner by arachidonate-regulated Ca2+ (ARC) channels. Using rat parotid salivary gland cells, we examined multiple pathways of Ca2+ entry/elevation to determine if they activated cell signaling proteins and whether this occurred in a pathway-dependent manner. We observed that SOCE activates extracellular signal-related kinases 1 and 2 (ERK1/2) to ∼3-times basal levels via a receptor-independent mechanism when SOCE was initiated by depleting Ca2+ stores using the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (TG). TG-initiated ERK1/2 phosphorylation increased as rapidly as that initiated by the muscarinic receptor agonist carbachol, which promoted an increase to ∼5-times basal levels. Notably, ERK1/2 phosphorylation was not increased by the global elevation of [Ca2+]i by Ca2+ ionophore or by Ca2+ entry via ARC channels in native cells, although ERK1/2 phosphorylation was increased by Ca2+ ionophore in Par-C10 and HSY salivary cell lines. Agents and conditions that blocked SOCE in native cells, including 2-aminoethyldiphenyl borate (2-APB), SKF96363, and removal of extracellular Ca2+, also reduced TG- and carbachol-stimulated ERK1/2 phosphorylation. TG-promoted ERK1/2 phosphorylation was blocked when SRC and Protein Kinases C (PKC) were inhibited, and it was blocked in cells pretreated with β-adrenergic agonist isoproterenol. These observations demonstrate that ERK1/2 is activated by a selective mechanism of Ca2+ entry (SOCE) in these cells, and suggest that ERK1/2 may contribute to events downstream of SOCE.

Characterization of cysteine string protein in rat parotid acinar cells

Cysteine string proteins (CSPs) are secretory vesicle chaperone proteins that contain: (i) a heavily palmitoylated cysteine string (comprised of 14 cysteine residues, responsible for the localization of CSP to secretory vesicle membranes), (ii) an N-terminal J-domain (DnaJ domain of Hsc70, 70kDa heat-shock cognate protein family of co-chaperones), and (iii) a linker domain (important in mediating CSP effects on secretion). In this study, we investigated the localization of CSP1 in rat parotid acinar cells and evaluated the role of CSP1 in parotid secretion. RT-PCR and western blotting revealed that CSP1 was expressed and associated with Hsc70 in rat parotid acinar cells. Further, CSP1 associated with syntaxin 4, but not with syntaxin 3, on the apical plasma membrane. Introduction of anti-CSP1 antibody into SLO-permeabilized acinar cells enhanced isoproterenol (IPR)-induced amylase release. Introduction of GST-CSP11–112, containing both the J-domain and the adjacent linker region, enhanced IPR-induced amylase release, whereas neither GST-CSP11–82, containing the J-domain only, nor GST-CSP183–112, containing the linker region only, did produce detectable enhancement. These results indicated that both the J-domain and the linker domain of CSP1 are necessary to function an important role in acinar cell exocytosis.

MADD/DENN/Rab3GEP functions as a guanine nucleotide exchange factor for Rab27 during granule exocytosis of rat parotid acinar cells

We previously reported that the small GTPase Rab27 and its effectors regulate isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. Although activation of Rab27 by a specific guanine nucleotide exchange factor (GEF) is thought to be required for amylase release, its activation mechanism is poorly understood, because GEF for Rab27 has not been reported in parotid acinar cells. In the present study, we investigated the possible involvement of MADD/DENN/Rab3GEP, which was recently described as a Rab27-GEF in melanocytes, in amylase release from rat parotid acinar cells. Reverse transcription-PCR analyses indicated that mRNA of DENND family members, including MADD, was expressed in parotid acinar cells. MADD protein was also expressed in the cytosolic fraction of parotid acinar cells. Incubation of an antibody against the C-terminal 150 amino acids of MADD (anti-MADD-C antibody) with streptolysin O-permeabilized parotid acinar cells caused not only inhibition of IPR-induced amylase release but also reduction in the amount of GTP-Rab27. Our findings indicated that MADD functions as a GEF for Rab27 in parotid acinar cells and that its GEF activity for Rab27, i.e., GDP/GTP cycling, is required for IPR-induced amylase release.

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.

Enhancing CCL28 expression through the gene transfer to salivary glands for controlling cariogenic microbe

The chemokine CCL28 participates in direct antimicrobial activities as well as homing of certain types of lymphocytes. The present study was conducted to harness these properties of the chemokine for the prevention of dental caries. The gene encoding CCL28 was transferred to salivary glands to enhance the production of this chemokine locally. First, a recombinant eukaryotic plasmid expressing CCL28 was constructed. Then, the CCL28 protein from 293 cells transfected with the recombinant plasmid was verified to inhibit the caries pathogen Streptococcus mutans (S. mutans) in a biofilm. Finally, the recombinant plasmid was retrogradely administered to the parotid glands of rats through the secretory ducts. The successful transfer of the gene encoding CCL28 to rat parotid acinar cells was confirmed by immunofluorescence and real-time PCR. Increases in both CCL28 and secretory IgA (SIgA) in the rat saliva were tested by ELISA. It was revealed that the CCL28 protein obtained from the study was able to strongly inhibit S. mutans living in biofilm in vitro. The delivery of the recombinant plasmid to the rat parotid glands was able to induce high levels of CCL28 and SIgA in saliva, and the increased levels of CCL28 and SIgA in saliva were maintained for 2weeks. Notably, the dental plaque from the rats treated with the delivery of the recombinant plasmid in the study harbored significantly less S. mutans. These data indicated that the present strategy may hold hope for the effective prevention of dental caries.

Exocyst subunits are involved in isoproterenol‐induced amylase release from rat parotid acinar cells

Exocytosis of secretory granules in parotid acinar cells requires multiple events: tethering, docking, priming, and fusion with a luminal plasma membrane. The exocyst complex, which is composed of eight subunits (Sec3, Sec5, Sec6, Sec8, Sec10, Sec15, Exo70, and Exo84) that are conserved in yeast and mammalian cells, is thought to participate in the exocytotic pathway. However, to date, no exocyst subunit has been identified in salivary glands. In the present study, we investigated the expression and function of exocyst subunits in rat parotid acinar cells. The expression of mRNA for all eight exocyst subunits was detected in parotid acinar cells by RT-PCR, and Sec6 and Sec8 proteins were localized on the luminal plasma membrane. Sec6 interacted with Sec8 after 5 min of stimulation with isoproterenol. In addition, antibodies to-Sec6 and Sec8 inhibited isoproterenol-induced amylase release from streptolysin O-permeabilized parotid acinar cells. These results suggest that an exocyst complex of eight subunits is required for amylase release from parotid acinar cells.

The Human Submandibular Gland

Submandibular acinar glands secrete numerous proteins such as digestive enzymes and defense proteins on the basis of the exocrine secretion mode. Exocytosis is a complex process, including a soluble NSF attachment protein receptor (SNARE)-mediated membrane fusion of vesicles and target membrane and the additional activation of cytoskeletal proteins. Relevant data are available predominantly for animal salivary glands, especially of the rat parotid acinar cells. The authors investigated the secretory molecular machinery of acinar (serous) cells in the human submandibular gland by immunohistochemistry and immunofluorescence and found diverse proteins associated with exocytosis for the first time. SNAP-23, syntaxin-2, syntaxin-4, and VAMP-2 were localized at the luminal plasma membrane; syntaxin-2 and septin-2 were expressed in vesicles in the cytoplasm. Double staining of syntaxin-2 and septin-2 revealed a colocalization on the same vesicles. Lactoferrin and α-amylase served as a marker for secretory vesicles and were labeled positively together with syntaxin-2 and septin-2 in double-staining procedures. Cytoskeletal components such as actin, myosin II, cofilin, and profilin are concentrated at the apical plasma membrane of acinar submandibular glands. These observations complement the understanding of the complex exocytosis mechanisms.

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 β-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.

EPI64 Protein Functions as a Physiological GTPase-activating Protein for Rab27 Protein and Regulates Amylase Release in Rat Parotid Acinar Cells

Rab27, a small GTPase, is generally recognized as an important regulator of secretion that interacts with Rab27-specific effectors to regulate events in a wide variety of cells, including endocrine and exocrine cells. However, the mechanisms governing the spatio-temporal regulation of GTPase activity of Rab27 are not firmly established, and no GTPase-activating protein (GAP) specific for Rab27 has been identified in secretory cells. We previously showed that expression of EPI64, a Tre-2/Bub2/Cdc16 (TBC)-domain-containing protein, in melanocytes inactivates endogenous Rab27A on melanosomes (Itoh, T., and Fukuda, M. (2006) J. Biol. Chem. 281, 31823-31831), but the EPI64 role in secretory cells has never been investigated. In this study, we investigated the effect of EPI64 on Rab27 in isoproterenol (IPR)-stimulated amylase release from rat parotid acinar cells. Subcellular fractionation and immunohistochemical analyses indicated that EPI64 was enriched on the apical plasma membrane of parotid acinar cells. We found that an antibody against the TBC/Rab-GAP domain of EPI64 inhibited the reduction in levels of the endogenous GTP-Rab27 in streptolysin-O-permeabilized parotid acinar cells and suppressed amylase release in a dose-dependent manner. We also found that the levels of EPI64 mRNA and EPI64 protein increased after IPR stimulation, and that treatment with actinomycin D or antisense-EPI64 oligonucleotides suppressed the increase of EPI64 mRNA/EPI64 protein and the amount of amylase released. Our findings indicated that EPI64 acted as a physiological Rab27-GAP that enhanced GTPase activity of Rab27 in response to IPR stimulation, and that this activity is required for IPR-induced amylase release.

P2X 7 receptor antagonists display agonist-like effects on cell signaling proteins

Background The activation of various P2 receptors (P2R) by extracellular nucleotides promotes diverse cellular events, including the stimulation of cell signaling protein and increases in [Ca 2+] i. We report that some agents that can block P2X 7R receptors also promote diverse P2X 7R-independent effects on cell signaling. Methods We exposed native rat parotid acinar cells, salivary gland cell lines (Par-C10, HSY, HSG), and PC12 cells to suramin, DIDS (4,4′-diisothiocyano stilbene-2,2′-disulfonic acid), Cibacron Blue 3GA, Brilliant Blue G, and the P2X 7R-selective antagonist A438079, and examined the activation/phosphorylation of ERK1/2, PKCδ, Src, CDCP1, and other signaling proteins. Results With the exception of suramin, these agents blocked the phosphorylation of ERK1/2 by BzATP in rat parotid acinar cells; but higher concentrations of suramin blocked ATP-stimulated 45Ca 2+ entry. Aside from A438079, these agents increased the phosphorylation of ERK1/2, Src, PKCδ, and other proteins (including Dok-1) within minutes in an agent- and cell type-specific manner in the absence of a P2X 7R ligand. The stimulatory effect of these compounds on the tyrosine phosphorylation of CDCP1 and its Src-dependent association with PKCδ was blocked by knockdown of CDCP1, which also blocked Src and PKCδ phosphorylation. Conclusions Several agents used as P2X 7R blockers promote the activation of various signaling proteins and thereby act more like receptor agonists than antagonists. General significance Some compounds used to block P2 receptors have complicated effects that may confound their use in blocking receptor activation and other biological processes for which they are employed, including their use as blockers of various ion transport proteins.

Evidence for amylase release by cyclin-dependent kinase 5 in the rat parotid

Cyclin-dependent kinase 5 (Cdk5) plays no apparent role in cell cycle regulation, and Cdk5 is not activated by cyclins but only p35 or p39. Although the enzymatic activity of Cdk5 is highest in the central nervous system, recent reports indicate that it also has important functions in non-neuronal cells. In the present study, we investigated whether Cdk5 and its activators are expressed in rat parotid acinar cells, whether a β-adrenergic agonist enhances the expression of Cdk5, and whether Cdk5 mediates amylase release. We found that Cdk5 and its activator, cyclin I, were expressed in rat parotid acinar cells, and that the expression of Cdk5 was enhanced by treatment of the cells with isoproterenol. Amylase release stimulated by isoproterenol was depressed by the addition of olomoucine, a Cdk5 inhibitor, or by the introduction of an anti-Cdk5 antibody. Cdk5 activity was enhanced by treatment with isoproterenol and this enhanced activity was attenuated by the addition of olomoucine. Olomoucine also attenuated both phosphorylation of Munc18c and translocation of Munc18c from the plasma membrane induced by isoproterenol. These results indicated that β-stimulation of rat parotid acinar cells enhanced the expression of Cdk5, and that this Cdk5 activation may mediate amylase release through phosphorylation of Munc18c.

Regulation and Identification of Na,K-ATPase α1 Subunit Phosphorylation in Rat Parotid Acinar Cells

The stimulation of fluid and electrolyte secretion in salivary cells results in ionic changes that promote rapid increases in the activity of the Na,K-ATPase. In many cell systems, there are conflicting findings concerning the regulation of the phosphorylation of the Na,K-ATPase α subunit, which is the catalytic moiety. Initially, we investigated the phosphorylation sites on the α1 subunit in native rat parotid acinar cells using tandem mass spectrometry and identified two new phosphorylation sites (Ser(222), Ser(407)), three sites (Ser(217), Tyr(260), Ser(47)) previously found from large scale proteomic screens, and two sites (Ser(23), Ser(16)) known to be phosphorylated by PKC. Subsequently, we used phospho-specific antibodies to examine the regulation of phosphorylation on Ser(23) and Ser(16) and measured changes in ERK phosphorylation in parallel. The G-protein-coupled muscarinic receptor mimetic carbachol, the phorbol ester phorbol 12-myristate 13-acetate, the Ca(2+) ionophore ionomycin, and the serine/threonine phosphatase inhibitor calyculin A increased Ser(23) α1 phosphorylation. Inhibition of classical PKC proteins blocked carbachol-stimulated Ser(23) α1 subunit phosphorylation but not ERK phosphorylation, which was blocked by an inhibitor of novel PKC proteins. The carbachol-initiated phosphorylation of Ser(23) α1 subunit was not modified by ERK or PKA activity. The Na,K-ATPase inhibitor ouabain reduced and enhanced the carbachol-promoted phosphorylation of Ser(23) and Ser(16), respectively, the latter because ouabain itself increased Ser(16) phosphorylation; thus, both sites display conformational-dependent phosphorylation changes. Ouabain-initiated phosphorylation of Ser(16) α1 was not blocked by PKC inhibitors, unlike carbachol- or phorbol 12-myristate 13-acetate-initiated phosphorylations, suggesting that this site was also a substrate for a kinase other than PKC.

Isoproterenol and cAMP Block ERK Phosphorylation and Enhance [Ca2+]i Increases and Oxygen Consumption by Muscarinic Receptor Stimulation in Rat Parotid and Submandibular Acinar Cells

Salivary glands are innervated by sympathetic and parasympathetic neurons, which release neurotransmitters that promote fluid secretion and exocytosis when they bind to muscarinic and beta-adrenergic receptors, respectively. Signaling pathways downstream of these receptors are mainly distinct, but there is cross-talk that affects receptor-dependent events. Here we report that the beta-adrenergic ligand isoproterenol blocks increases in extracellular signal-related kinase (ERK) phosphorylation, a protein kinase C-dependent event promoted by the muscarinic receptor ligand carbachol in freshly dispersed rat parotid acinar cells. The inhibitory action of isoproterenol was reproduced by cAMP stimuli (forskolin) and mimetics (dibutyryl-cAMP, 8-(4-chlorophenylthio)-cAMP), including one highly selective for protein kinase A (N(6)-benzoyl-cAMP). In contrast, Epac (exchange proteins directly activated by cAMP)-selective activators did not mimic the blockade of ERK by isoproterenol, suggesting that inhibition involved protein kinase A. Isoproterenol also blocked ERK downstream of phorbol 12-myristate 13-acetate and the P2X(7) and epidermal growth factor receptors. Isoproterenol and forskolin blocked MEK phosphorylation, reduced RAF phosphorylation on a stimulatory site (Ser-338), and increased RAF phosphorylation on an inhibitory site (Ser-259). Inhibitory effects on ERK were also observed in freshly dispersed rat submandibular acinar cells but not in three immortalized/cancer salivary cell lines (Par-C10, HSY, HSG), indicating significant differences between native cells and cell lines. Notably, in native parotid cells isoproterenol enhanced the carbachol-promoted increases in [Ca(2+)](i) and oxygen consumption, events that initiate and accompany, respectively, the stimulation of fluid secretion by muscarinic ligands. Thus, isoproterenol produces opposite effects on prominent events downstream of the muscarinic receptor second messengers diacylglycerol (decrease in ERK phosphorylation) and inositol trisphosphate (increase in [Ca(2+)](i) and fluid secretion).