Parotid Acinar Cell Research Articles

Role of NAADP for calcium signaling in the salivary gland

Coordination of intracellular Ca2+ signaling in parotid acini is crucial for controlling the secretion of primary saliva. Previous work from our lab has demonstrated acidic-organelle Ca2+ release as a participant in agonist-evoked signaling dynamics of the parotid acinar cell. Furthermore, results implicated a potential role for the potent Ca2+ releasing second messenger NAADP in these events. The current study interrogated a direct role of NAADP for Ca2+ signaling in the parotid salivary gland acinar cell. Use of live-cell Ca2+ imaging, patch-clamp methods, and confocal microscopy revealed for the first time NAADP can evoke or enhance Ca2+ dynamics in parotid acini. These results were compared with pancreatic acini, a morphologically similar cell type previously shown to display NAADP-dependent Ca2+ signals. Findings presented here may be relevant in establishing new therapeutic targets for those suffering from xerostomia produced by hypofunctioning salivary glands.

Undiagnosed paraganglioma; A challenge during laparoscopic retroperitoneal resection

Report our experience of the management of a patient with undiagnosed retroperitoneal paraganglioma and the intraoperative complications that the theatre team faced. We present a case of a 36-year-old patient who during oncological follow-up for a previous diagnosis of parotid acinar cell carcinoma was incidentally identified as having an interaortocaval tumour. Following routine preoperative assessment the patient was arranged to undergo a laparoscopic retroperitoneal tumour resection. After minimal tumour manipulation the patient developed cardiac rhythm abnormalities and became hypertensive. The tumour was successfully removed laparoscopically after a cautious inter- aortocaval dissection. Abruptly, prior to extraction of the tumour containing endobag, the patient developed cardiac arrest. Following 35 minutes of life support measures there was a return of spontaneous circulation. The endobag was laparoscopically removed from the abdominal cavity 24 hours later using the initial operative port sites. The patient´s progression was satisfactory and he could be discharged six days postoperatively. Asymptomatic undiagnosed paragangliomas represent a real challenge during laparoscopic operations. Haemodynamic changes and life-threatening events can arise acutely intraoperatively, where an immediate and coordinated response of the whole theatre team may be required to avoid fatal outcome.

A Model of [Formula: see text] Dynamics in an Accurate Reconstruction of Parotid Acinar Cells.

We have constructed a spatiotemporal model of [Formula: see text] dynamics in parotid acinar cells, based on new data about the distribution of inositol trisphophate receptors (IPR). The model is solved numerically on a mesh reconstructed from images of a cluster of parotid acinar cells. In contrast to our earlier model (Sneyd et al. in J Theor Biol 419:383-393. https://doi.org/10.1016/j.jtbi.2016.04.030 , 2017b), which cannot generate realistic [Formula: see text] oscillations with the new data on IPR distribution, our new model reproduces the [Formula: see text] dynamics observed in parotid acinar cells. This model is then coupled with a fluid secretion model described in detail in a companion paper: A mathematical model of fluid transport in an accurate reconstruction of a parotid acinar cell (Vera-Sigüenza etal. in Bull Math Biol. https://doi.org/10.1007/s11538-018-0534-z , 2018b). Based on the new measurements of IPR distribution, we show that Class I models (where [Formula: see text] oscillations can occur at constant [[Formula: see text]]) can produce [Formula: see text] oscillations in parotid acinar cells, whereas Class II models (where [[Formula: see text]] needs to oscillate in order to produce [Formula: see text] oscillations) are unlikely to do so. In addition, we demonstrate that coupling fluid flow secretion with the [Formula: see text] signalling model changes the dynamics of the [Formula: see text] oscillations significantly, which indicates that [Formula: see text] dynamics and fluid flow cannot be accurately modelled independently. Further, we determine that an active propagation mechanism based on calcium-induced calcium release channels is needed to propagate the [Formula: see text] wave from the apical region to the basal region of the acinar cell.

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.

Modeling calcium waves in an anatomically accurate three-dimensional parotid acinar cell

We construct a model of calcium waves in a three-dimensional anatomically accurate parotid acinar cell, constructed from experimental data. Gradients of inositol trisphosphate receptor (IPR) density are imposed, with the IPR density being greater closer to the lumen, which has a branched structure, and inositol trisphosphate (IP3) is produced only at the basal membrane. We show (1) that IP3 equilibrates so quickly across the cell that it can be assumed to be spatially homogeneous; (2) spatial separation of the sites of IP3 action and IP3 production does not preclude the formation of stable oscillatory Ca2+ waves. However, these waves are not waves in the mathematical sense of a traveling wave with fixed profile. They result instead from a time delay between the Ca2+ rise in the apical and basal regions; (3) the ryanodine receptors serve to reinforce the Ca2+ wave, but are not necessary for the wave to exist; (4) a spatially independent model is not sufficient to study saliva secretion, although a one-dimensional model might be sufficient. Our results here form the first stages of the construction of a multiscale and multicellular model of saliva secretion in an entire acinus.

A systems biology approach identifies a regulatory network in parotid acinar cell terminal differentiation.

ObjectiveThe transcription factor networks that drive parotid salivary gland progenitor cells to terminally differentiate, remain largely unknown and are vital to understanding the regeneration process.MethodologyA systems biology approach was taken to measure mRNA and microRNA expression in vivo across acinar cell terminal differentiation in the rat parotid salivary gland. Laser capture microdissection (LCM) was used to specifically isolate acinar cell RNA at times spanning the month-long period of parotid differentiation.ResultsClustering of microarray measurements suggests that expression occurs in four stages. mRNA expression patterns suggest a novel role for Pparg which is transiently increased during mid postnatal differentiation in concert with several target gene mRNAs. 79 microRNAs are significantly differentially expressed across time. Profiles of statistically significant changes of mRNA expression, combined with reciprocal correlations of microRNAs and their target mRNAs, suggest a putative network involving Klf4, a differentiation inhibiting transcription factor, which decreases as several targeting microRNAs increase late in differentiation. The network suggests a molecular switch (involving Prdm1, Sox11, Pax5, miR-200a, and miR-30a) progressively decreases repression of Xbp1 gene transcription, in concert with decreased translational repression by miR-214. The transcription factor Xbp1 mRNA is initially low, increases progressively, and may be maintained by a positive feedback loop with Atf6. Transfection studies show that Xbp1Mist1 promoter. In addition, Xbp1 and Mist1 each activate the parotid secretory protein (Psp) gene, which encodes an abundant salivary protein, and is a marker of terminal differentiation.ConclusionThis study identifies novel expression patterns of Pparg, Klf4, and Sox11 during parotid acinar cell differentiation, as well as numerous differentially expressed microRNAs. Network analysis identifies a novel stemness arm, a genetic switch involving transcription factors and microRNAs, and transition to an Xbp1 driven differentiation network. This proposed network suggests key regulatory interactions in parotid gland terminal differentiation.

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.

Acinic cell carcinoma of parotid gland metastasis to left cavernous sinus: a case report and review of literature

Objective To investigate the clinical manifestations and pathological features of parotid gland papillary acinic cell carcinoma metastasis to left cavernous sinus. Methods The clinical manifestations, pathological features and differential diagnosis were studied in one case of parotid papillary acinic cell carcinoma metastasis to left cavernous sinus. Related literatures were also reviewed. Results The patient was a 50-year-old female who presented paroxysmal dizziness for 5 months and blurred vision in her left eye for 10 months. The MRI examination showed left parasellar space-occupying mass in the cavernous sinus. In operation, the tumor was located in the superior wall of left cavernous sinus, soft and red-grey in color, with abundant blood supply. The histomorphological examination revealed the tumor cells were arranged in solid, acinar or papillary pattern. The tumor cells were large, with eosinophilic cytoplasm, round or oval nuclei and small nucleoli. Immunohistochemical staining found that the tumor cells expressed cytokeratin (CK), epithelial membrane antigen (EMA), vimentin (Vim) and S-100 protein (S-100), and showed weak positive expression of glial fibrillary acidic protein (GFAP) and focal positive expression of P53 protein. Ki-67 labeling index was about 5%-10% . The tumor cells were negative for neuroendocrine markers and pituitary hormone protein markers. This case was difficult to differentiate from other primary intracalvarium tumors, including papillary meningioma, papillary tumor of choroid plexus, papillary ependymoma, papillary glioneuronal tumors as well as chordoma. According to the medical history and the comparison of histomorphology and immunophenotyping between parotid gland tumor cells and left cavernous sinus tumor cells, the final diagnosis was metastatic papillary parotid acinar cell carcinoma of the left cavernous sinus. The patient was followed for 21 months and no recurrence was seen. Conclusion It is very rare and to our knowlege it is the second case with metastasic papillary parotid acinar cell carcinoma in the cavernous sinus. Understanding the patient's history and differentiating from other primary intracalvarium tumors is one of the key steps to give right pathological diagnosis and clinical therapy. doi：10.3969/j.issn.1672-6731.2013.12.008

Apical Ca2+ activated K+ Channels in Mouse Lacrimal Acinar Cells

Fluid secretion of salivary glands relies on intracellular calcium transients activating calcium dependent Cl− conductances in the luminal membrane of acinar cells. Older parotid acinar cell models place Ca2+-activated K+ channels into the basolateral membrane because early studies found Ca2+-activated K+ channel activity in cell-attached patches targeting this membrane domain. Recently we have used the combination of whole cell patch clamp electrophysiology, spatially localized flash photolysis of caged Ca2+ and temporally resolved digital Ca2+ imaging to show that Ca2+ -activated K+ channels colocalize with Cl− channels and occur in much higher density in the luminal membrane of parotid acinar cells compared to the basal. This finding provided evidence that supported a new mathematical model in which optimal fluid secretion requires significant K+ conductance inserted into the luminal membrane. The aim of the present study was to test the localization of Ca2+ -dependent K+ channels in acinar cells from a different tissue, the lacrimal gland. Our initial experiments have shown both intermediate (IK) and big conductance (BK) Ca2+-activated K+ currents in these cells, although the abundance of IK conductance is less when compared to parotid. Flash-induced localized Ca2+ elevations at the luminal membrane resulted in a robust increase in K+ and Cl− currents. The K+ current evoked by apical photolysis was abolished by 10 μM paxilline, a specific inhibitor of BK channels. Our failure to activate IK in these experiments suggests that the role of IK in lacrimal acinar cells is smaller compared to parotid. In contrast, when the [Ca2+] was increased at the basal plasma membrane, no increase in either K+ or Cl− currents was evoked. These data indicate that the density of large conductance Ca2+ activated K+ channels appears higher in the apical versus basal plasma membrane.

A dynamic model of saliva secretion

We construct a mathematical model of the parotid acinar cell with the aim of investigating how the distribution of K + and Cl − channels affects saliva production. Secretion of fluid is initiated by Ca 2+ signals acting on Ca 2+ dependent K + and Cl − channels. The opening of these channels facilitates the movement of Cl − ions into the lumen which water follows by osmosis. We use recent results into both the release of Ca 2+ from internal stores via the inositol (1,4,5)-trisphosphate receptor (IP 3R) and IP 3 dynamics to create a physiologically realistic Ca 2+ model which is able to recreate important experimentally observed behaviours seen in parotid acinar cells. We formulate an equivalent electrical circuit diagram for the movement of ions responsible for water flow which enables us to calculate and include distinct apical and basal membrane potentials to the model. We show that maximum saliva production occurs when a small amount of K + conductance is located at the apical membrane, with the majority in the basal membrane. The maximum fluid output is found to coincide with a minimum in the apical membrane potential. The traditional model whereby all Cl − channels are located in the apical membrane is shown to be the most efficient Cl − channel distribution.

The contribution of AKAP5 in amylase secretion from mouse parotid acini

A-kinase (PKA) anchoring proteins (AKAPs) are essential for targeting type II PKA to specific locales in the cell to control function. In the present study, AKAP5 (formerly AKAP150) and AKAP6 were identified in mouse parotid acini by type II PKA regulatory subunit (RII) overlay assay and Western blot analysis of mouse parotid cellular fractions, and the role of AKAP5 in mouse parotid acinar cell secretion was determined. Mice were euthanized with CO(2). Immunofluorescence staining of acinar cells localized AKAP5 to the basolateral membrane, whereas AKAP6 was associated with the perinuclear region. In functional studies, amylase secretion from acinar cells of AKAP5 mutant [knockout (KO)] mice treated with the beta-adrenergic agonist, isoproterenol, was reduced overall by 30-40% compared with wild-type (WT) mice. In contrast, amylase secretion in response to the adenylyl cyclase (AC) activator, forskolin, and the cAMP-dependent protein kinase (PKA) activator, N(6)-phenyl-cAMP, was not statistically different in acini from WT and AKAP5 KO mice. Treatment of acini with isoproterenol mimicked the effect of the Epac activator, 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (8-pMeOPT-2'-O-Me-cAMP), in stimulating Rap1. However, in contrast to isoproterenol, treatment of acini with 8-pMeOPT-2'-O-Me-cAMP resulted in stimulation of amylase secretion from both AKAP5 KO and WT acinar cells. As a scaffolding protein, AKAP5 was found to coimmunoprecipitate with AC6, but not AC8. Data suggest that isoproterenol-stimulated amylase secretion occurs via both an AKAP5/AC6/PKA complex and a PKA-independent, Epac pathway in mouse parotid acini.

Early events of secretory granule formation in the rat parotid acinar cell under the influence of isoproterenol. An ultrastructural and lectin cytochemical study.

The events involved in the maturation process of acinar secretory granules of rat parotid gland were investigated ultrastructurally and cytochemically by using a battery of four lectins [Triticum vulgaris agglutinin (WGA), Ulex europaeus agglutinin I (UEA-I), Glycine max agglutinin (SBA), Arachys hypogaea agglutinin (PNA)]. In order to facilitate the study, parotid glands were chronically stimulated with isoproterenol to induce secretion. Specimens were embedded in the Lowicryl K4M resin. The trans-Golgi network (TGN) derived secretory granules, which we refer to as immature secretory granules, were found to be intermediate structures in the biogenesis process of the secretory granules in the rat parotid acinar cell. These early structures do not seem to be the immediate precursor of the mature secretory granules: in fact, a subsequent interaction process between these early immature granule forms and TGN elements seems to occur, leading, finally, to the mature granules. These findings could explain the origin of the polymorphic subpopulations of the secretory granules in the normal acinar cells of the rat parotid gland. The lectin staining patterns were characteristic of each lectin. Immature and mature secretory granules were labelled with WGA, SBA, PNA, and lightly with UEA-I. Cis and intermediate cisternae of the Golgi apparatus were labelled with WGA, and trans cisternae with WGA and SBA.

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.

Visualizing form and function in organotypic slices of the adult mouse parotid gland

An organotypic slice preparation of the adult mouse parotid salivary gland amenable to a variety of optical assessments of fluid and protein secretion dynamics is described. The semi-intact preparation rendered without the use of enzymatic treatment permitted live-cell imaging and multiphoton analysis of cellular and supracellular signals. Toward this end we demonstrated that the parotid slice is a significant addition to the repertoire of tools available to investigators to probe exocrine structure and function since there is currently no cell culture system that fully recapitulates parotid acinar cell biology. Importantly, we show that a subpopulation of the acinar cells of parotid slices can be maintained in short-term culture and retain their morphology and function for up to 2 days. This in vitro model system is a significant step forward compared with enzymatically dispersed acini that rapidly lose their morphological and functional characteristics over several hours, and it was shown to be long enough for the expression and trafficking of exogenous protein following adenoviral infection. This system is compatible with a variety of genetic and physiological approaches used to study secretory function.

A mathematical model of fluid secretion from a parotid acinar cell

Salivary fluid secretion is crucial for preventing problems such as dryness of mouth, difficulty with mastication and swallowing, as well as oral pain and dental cavities. Fluid flow is driven primarily by the transepithelial movement of chloride and sodium ions into the parotid acinus lumen. The activation of Cl - channels is calcium dependent, with the average elevated calcium concentration during calcium oscillations increasing the conductance of the channels, leading to an outflow of Cl - . The accumulation of NaCl in the lumen drives water flow by osmosis. We construct a mathematical model of the calcium concentration oscillations and couple this to a model for Cl - efflux. We also construct a model governing fluid flow in an isolated parotid acinar cell, which includes a description of the rate of change of intracellular ion concentrations, cell volume, membrane potential and water flow rate. We find that [ Ca 2 + ] oscillations lead to oscillations in fluid flow, and that the rate of fluid flow is regulated by the average calcium concentration and not the frequency of the oscillations.

Water permeability of parotid acinar cell membrane measured by H-1 NMR

Water permeability of parotid acinar cell membrane measured by H-1 NMR

Molecular Identification and Physiological Roles of Parotid Acinar Cell Maxi-K Channels

The physiological success of fluid-secreting tissues relies on a regulated interplay between Ca(2+)-activated Cl(-) and K(+) channels. Parotid acinar cells express two types of Ca(2+)-activated K(+) channels: intermediate conductance IK1 channels and maxi-K channels. The IK1 channel is encoded by the K(Ca)3.1 gene, and the K(Ca)1.1 gene is a likely candidate for the maxi-K channel. To confirm the genetic identity of the maxi-K channel and to probe its specific roles, we studied parotid glands in mice with the K(Ca)1.1 gene ablated. Parotid acinar cells from these animals lacked maxi-K channels, confirming their genetic identity. The stimulated parotid gland fluid secretion rate was normal, but the sodium and potassium content of the secreted fluid was altered. In addition, we found that the regulatory volume decrease in acinar cells was substantially impaired in K(Ca)1.1-null animals. We examined fluid secretion from animals with both K(+) channel genes deleted. The secretion rate was severely reduced, and the ion content of the secreted fluid was significantly changed. We measured the membrane potentials of acinar cells from wild-type mice and from animals with either or both K(+) channel genes ablated. They revealed that the observed functional effects on fluid secretion reflected alterations in cell membrane voltage. Our findings show that the maxi-K channels are critical for the regulatory volume decrease in these cells and that they play an important role in the sodium uptake and potassium secretion process in the ducts of these fluid-secreting salivary glands.

Endotoxin can decrease isolated rat parotid acinar cell amylase secretion in a nitric oxide-independent manner

Salivary mucus and amylase have an anti-bacterial nature. Bacterial endotoxin is considered to decrease mucus secreting cell activity by nitric oxide-dependent mechanisms. In this study, the actions of endotoxin on amylase secreting cell activity have been studied. Endotoxin ( Escherichia coli lipopolysaccharide; 3 mg/kg, i.v., 5 h) evoked nitric oxide synthase 2 (NOS2) induction in the rat whole parotid tissue (assessed by Western blot and the citrulline assay) and in rat isolated parotid acinar cells (assessed by Western blot and immunohistochemistry), and reduced basal and acetylcholine-stimulated amylase secretion from these isolated cells. However, N G-nitro- l-arginine methyl ester (0.1 mg/ml, 4 days in drinking water, yielding a dose of 25 mg/kg/day) did not affect amylase release under basal or acetylcholine-stimulated conditions, either in control acinar cells or those from endotoxin challenged rats. Thus, basal, acetylcholine-evoked or endotoxin-decreased cellular amylase secretion from rat isolated parotid acinar cells does not appear to be modulated by endogenous nitric oxide.

Menkes protein localization in rat parotid acinar cells

The aim was to study the subcellular localization of the Menkes protein (MNK; ATP7A) in the rat parotid acinar cell. MNK protein is a copper transporting P-type ATPase whose absence or dysfunction causes a fatal neurodegenerative disorder, MNK disease. Rat parotid glands were fixed and low-temperature embedded in Lowicryl K4M resin, and ultrathin sections were prepared for immunocytochemical analysis. Immunolocalization of MNK was demonstrated mainly over the trans Golgi network (TGN) area. Immature and mature secretory granules were also labelled, indicating that MNK protein could be involved here in copper secretion from acinar cells into saliva, consistent with a proposed cariostatic role for copper.

A primary culture of parotid acinar cells retaining capacity for agonists-induced amylase secretion and generation of new secretory granules

Exocrine acinar cells, like parotid cells, have difficulty in maintaining their functions in cell lines or in primary cultures. For this reason, molecular studies on exocrine cell functions are unsatisfactory. To examine the mechanisms whereby the functions of parotid acinar cells are maintained, we attempted to establish a system for primary culture and transfection of exogenous genes. Acinar cells were dispersed from rat parotid glands by digestion with enzymes and were cultured in a medium containing rat serum. Most of the cultured cells had secretory granules that contained amylase, suggesting that they were derived from acinar cells, although they spread on the dish surface and formed filopodia. The cultured cells retained both granules and the ability to release amylase in response to beta-adrenergic and cholinergic agonists, even 48 h after dispersion. However, the total amount of amylase in the cells decreased rapidly from 24 to 48 h after dispersion. These results suggested that amylase synthesis was more damaged than the machinery for exocytosis during culture in vitro. VAMP2 gene fused with enhanced green fluorescence protein was transfected into the dispersed acinar cells, and VAMP2 protein was expressed and localized to amylase-containing granules, as normally seen for endogenous VAMP2 protein. This indicated that new granules were generated, and that protein sorting was functional. The cells cultured by this method maintained their functions for at least 48 h. They can be used for examining the effects of exogenous genes on parotid acinar cell functions, such as regulated exocytosis and the maturation of secretory granules.