Parotid Acini Research Articles

Salivary Gland Tissue Recombination Can Modify Cell Fate

Although mesenchyme is essential for inducing the epithelium of ectodermal organs, its precise role in organ-specific epithelial fate determination remains poorly understood. To elucidate the roles of tissue interactions in cellular differentiation, we performed single-cell RNA sequencing and imaging analyses on recombined tissues, where mesenchyme and epithelium were switched ex vivo between two types of embryonic mouse salivary glands: the parotid gland (a serous gland) and the submandibular gland (a predominantly mucous gland). We found partial induction of molecules that define gland-specific acinar and myoepithelial cells in recombined salivary epithelium. The parotid epithelium recombined with submandibular mesenchyme began to express mucous acinar genes not intrinsic to the parotid gland. While myoepithelial cells do not normally line parotid acini, newly induced myoepithelial cells densely populated recombined parotid acini. However, mucous acinar and myoepithelial markers continued to be expressed in submandibular epithelial cells recombined with parotid mesenchyme. Consequently, some epithelial cells appeared to be plastic, such that their fate could still be modified in response to mesenchymal signaling, whereas other epithelial cells appeared to be already committed to a specific fate. We also discovered evidence for bidirectional induction: transcriptional changes were observed not only in the epithelium but also in the mesenchyme after heterotypic tissue recombination. For example, parotid epithelium induced the expression of muscle-related genes in submandibular fibroblasts that began to mimic parotid fibroblast gene expression. These studies provide the first comprehensive unbiased molecular characterization of tissue recombination approaches exploring the regulation of cell fate.

Structure of the parotid gland in natural infection by Leishmania infantum in Canis familiaris

ObjectiveThe objective of this study was to perform a histopathological, morphometric and proteomic study of the parotid gland of dogs naturally infected with Leishmania infantum treated and not treated with Allopurinol. DesignParotid glands from 14 dogs were used, divided into two groups: untreated and treated with oral allopurinol (20 mg / kg, once daily for 90 days). After adequate dissection, the organs were submitted to histopathological, histomorphometric and immunohistochemical techniques, using the monoclonal anti-β-catenin antibody. ResultsHistopathological evaluation of treated and untreated groups showed acinar hypertrophy, structural disorganization of the nucleus and cytoplasm. There was an increase in the area and perimeter of the parotid acini in the experimental groups. The immunostaining of the β-catenin protein in the membrane was severely reduced in the treated and untreated groups. ConclusionsThese findings suggest that Leishmania infantum infection and treatment with Allopurinol alter the tissue structure of the parotid gland in dogs, promoting an increase in the acinar volume and a decrease in the expression of β-catenin in cell membranes.

Outcomes of Gallic Acid on Alternariol Induced Cyto-Morphic and Genotoxic In Vivo Changes in Parotid Gland: 4-HNE Incorporated.

Alternaria toxins are emerging mycotoxins that gained considerable interest with increasing evidence of their existence and toxicological properties. There is limited research and insufficient data about their in vivo hazardous effects. We designed this study to evaluate histopathological and genotoxic in vivo impacts of alternariol (AOH) on the parotid gland as well as to assess the competency of gallic acid (GA) in reversing these effects. Forty healthy adult male Wister rats were utilized and assigned equally on control, GA, alternariol and AOH+ gallic treated groups. Parotid gland samples from experimental groups were collected and then examined for histopathological, ultrastructural and immunohistochemical examination for 4-hydroxynonenal “4-HNE as lipid peroxidation marker” as well as Comet assay for DNA damage. Additionally, parotid tissue homogenates were tested for catalase “CAT”, superoxide dismutase “SOD” and malondialdehyde “MDA” levels. Our data proved that alternariol produced various histopathological and ultrastructural alterations of parotid acini as well as significant DNA damage, significant reduction of CAT and SOD enzymatic activity and significant boosting of 4-HNE immunohistochemical expression and MDA levels as compared to control group. On the other hand, gallic acid administration almost restored histological and ultrastructural parotid architecture, 4-HNE immune-expression and biochemical levels. Ultimately, we demonstrated alternariol-induced histopathological and genotoxic alterations on parotid gland as well as the competency of gallic acid in reversing these effects.

Oxidative Stress Changes Induced by Methotrexate on Parotid Gland Structure of Adult Male Albino Rat: Can Vitamin C Ameliorate These Changes?

AbstractBackground: Methotrexate (MTX) is a cytotoxic chemo-therapeutic agent for several malignancies and various inflam-matory diseases. Salivary dysfunction is one of MTX muco-toxic effects. Vitamin C (vit. C) scavenges the oxidative molecules and free radicals since it is a potent hydrophilic antioxidant.Aim of Study: Is to throw light on the structural alternation in the rat parotid gland after administration of MTX and the probable antioxidant role of vit. C on these structure alterations.Material and Methods: Fifty male albino rats were equally divided into 5 groups; Group I: Rats had received a balanced diet for 9 days; Group II: Rats had received vit. C daily by oral gavage (500mg/kg body weight) dissolved in 5cm distilled water for 9 days; Group III (MTX-treated group): Animals had received MTX intraperitoneally as one single dose (20mg/ kg B.w) on the third day of the experiment; Group IV (MTX-withdrawal group): Rats had received MTX intraperitoneally as one single dose (20mg/kg B.w) on the third day of the experiment then left without treatment for another 15 days; Group V (MTX + vit. C-treated group): Rats had received MTX intraperitoneally as one single dose on the third day of the experiment and vit. C by oral gavage 2 days before and 6 days after MTX administration (at same doses mentioned before). The animals of Group I, II, III and V were anesthetized and scarified after 9 days of experiment, while rats of Group IV were anesthetized and scarified after 18 days of experiment. At the end of experiment, parotid tissues were collected for both light and electron microscopy and biochemical investi-gations.Results: MTX caused parotid histopathological and ultra-structural alterations. The MTX-treated group revealed loss of normal architecture of parotid tissue. Some parotid acini were completely degenerated leaving large spaces. Other acini appeared with numerous intracellular vacuoles. The ducts appeared dilated with stagnation of secretion. Collagen fibers, inflammatory cells infiltration, congested blood vessels and hemorrhage in between the acini were also detected. Biochem-ically, the Malondialdehyde (MDA) level of the MTX- treated group was significantly increased while the Glutathione (GSH) level was significantly reduced in comparison with other groups. The MTX-withdrawal group showed little improvement of biochemical parameters, while structural examination still showed signs of degenerations. However, the MTX + vit. C-treated group demonstrated some improvement of histological features and biochemical parameters as compared to the MTX-treated group.Conclusions: Methotrexate produces marked degenerative changes in the parotid structure while vitamin C has the ability to ameliorate these parotid injuries.

Carboxyl Ester Lipase May Not Mediate Lipotoxic Injury during Severe Acute Pancreatitis

Acute lipolysis of visceral fat or circulating triglycerides may worsen acute pancreatitis (AP)-associated local and systemic injury. The pancreas expresses pancreatic triacylglycerol lipase (PNLIP), pancreatic lipase-related protein 2 (PNLIPRP2), and carboxyl ester lipase (CEL), which may leak into the visceral fat or systemic circulation during pancreatitis. We, thus, aimed to determine the pancreatic lipase(s) regulating lipotoxicity during AP. For this AP, associated fat necrosis was analyzed using Western blot analysis. Bile acid (using liquid chromatography-tandem mass spectrometry) and fatty acid (using gas chromatography) concentrations were measured in human fat necrosis. The fat necrosis milieu was simulated invitro using glyceryl trilinoleate because linoleic acid is increased in fat necrosis. Bile acid requirements to effectively hydrolyze glyceryl trilinoleate were studied using exogenous or overexpressed lipases. The renal cell line (HEK 293) was used to study lipotoxic injury. Because dual pancreatic lipase knockouts are lethal, exocrine parotid acini lacking lipases were used to verify the results. PNLIP, PNLIPRP2, and CEL were increased in fat necrosis. Although PNLIP and PNLIPRP2 were equipotent in inducing lipolysis and lipotoxic injury, CEL required bile acid concentrations higher than in human fat necrosis. The high bile acid requirements for effective lipolysis make CEL an unlikely mediator of lipotoxic injury in AP. It remains to be explored whether PNLIP or PNLIPRP2 worsens AP severity invivo.

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.

Role of NAADP for Calcium Signaling in the Salivary Gland

The secretion of saliva in response to physiological demand is under control of the autonomic nervous system. Coordination and regulation of intracellular Ca2+ signaling following autonomic activation is crucial for proper salivary secretion. Previous work from our lab has demonstrated parotid acinar cells contain an abundant and polarized distribution of acidic organelles. Furthermore, acidic organelles are recruited for agonist-induced Ca2+ signaling following cAMP elevation. The current study interrogated a role for NAADP, a potent second messenger known to release Ca2+ from acidic organelles. In parotid acinar cells, initial results demonstrated NED19, an inherently fluorescent NAADP receptor inhibitor, was localized to acidic endosomes. Additionally, using a whole-cell patch clamp method to dialyze NAADP into parotid acini resulted in robust Ca2+ -activated Cl-currents. Peak activation occurred at low nanomolar concentrations of NAADP and higher concentrations showed an inhibitory effect. These observations suggest a role for NAADP in producing the fluid component of saliva. Thus, this work identifies a NAADP signaling pathway as a potential therapeutic target for people suffering from salivary hypofunction.

CAMP-dependent recruitment of acidic organelles for Ca2+ signaling in the salivary gland.

Autonomic neural activation of intracellular Ca2+ release in parotid acinar cells induces the secretion of the fluid and protein components of primary saliva critical for maintaining overall oral homeostasis. In the current study, we profiled the role of acidic organelles in shaping the Ca2+ signals of parotid acini using a variety of imaging and pharmacological approaches. Results demonstrate that zymogen granules predominate as an apically polarized population of acidic organelles that contributes to the initial Ca2+ release. Moreover, we provide evidence that indicates a role for the intracellular messenger NAADP in the release of Ca2+ from acidic organelles following elevation of cAMP. Our data are consistent with the "trigger" hypothesis where localized release of Ca2+ sensitizes canonical intracellular Ca2+ channels to enhance signals from the endoplasmic reticulum. Release from acidic stores may be important for initiating saliva secretion at low levels of stimulation and a potential therapeutic target to augment secretory activity in hypofunctioning salivary glands.

Redistribution of Gαs in mouse salivary glands following β-adrenergic stimulation

ObjectiveSignalling via β-adrenergic receptors activates heterotrimeric G-proteins, which dissociate into α and βγ subunits. In salivary glands, the α subunit of Gs stimulates adenylate cyclase, increasing cyclic AMP levels and promoting exocytosis. The goals of this study were to determine Gαs localization in salivary glands and whether it undergoes redistribution upon activation. MethodsMouse parotid and submandibular (SMG) glands were fixed with paraformaldehyde and prepared for immunofluorescence labelling with anti-Gαs. ResultsIn unstimulated parotid and SMG acinar cells, Gαs was localized mainly to basolateral membranes. Some parotid acinar cells also exhibited cytoplasmic fluorescence. Isoproterenol (IPR) stimulation resulted in decreased membrane fluorescence and increased cytoplasmic fluorescence, which appeared relatively uniform by 30min. Beginning about 2h after IPR, cytoplasmic fluorescence decreased and membrane fluorescence increased, approaching unstimulated levels in SMG acini by 4h. Some parotid acini exhibited cytoplasmic fluorescence up to 8h after IPR. The IPR-induced redistribution of Gαs was prevented (SMG) or reduced (parotid) by prior injection of propranolol. Striated duct cells of unstimulated mice exhibited general cytoplasmic fluorescence, which was unchanged after IPR. ConclusionsGαs is localized to basolateral membranes of unstimulated salivary acinar cells. Activation of Gαs causes its release from the cell membrane and movement into the cytoplasm. Reassociation of Gαs with the membrane begins about 2h after stimulation in the SMG, but complete reassociation takes several hours in the parotid gland. The presence of Gαs in striated duct cells suggests a role in signal transduction of secretion and/or electrolyte transport processes.

Apical Ca2+-activated potassium channels in mouse parotid acinar cells

Ca2+ activation of Cl and K channels is a key event underlying stimulated fluid secretion from parotid salivary glands. Cl channels are exclusively present on the apical plasma membrane (PM), whereas the localization of K channels has not been established. Mathematical models have suggested that localization of some K channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca2+] was used to investigate if Ca2+-activated K channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca2+-buffering conditions that produced brief, localized increases in [Ca2+] after focal laser photolysis of caged Ca2+. Conditions were used to isolate K+ and Cl− conductances. Photolysis at the apical PM resulted in a robust increase in K+ and Cl− currents. A localized reduction in [Ca2+] at the apical PM after photolysis of Diazo-2, a caged Ca2+ chelator, resulted in a decrease in both K+ and Cl− currents. The K+ currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of large-conductance “maxi-K” (BK) and intermediate K (IK), respectively, and almost abolished by incubation with both antagonists. Apical TRAM-34–sensitive K+ currents were also observed in BK-null parotid acini. In contrast, when the [Ca2+] was increased at the basal or lateral PM, no increase in either K+ or Cl− currents was evoked. These data provide strong evidence that K and Cl channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain, and the density of these channels appears higher in the apical versus basolateral PM. Collectively, this study provides support for a model in which fluid secretion is optimized after expression of K channels specifically in the apical PM.

Effects of Low Magnetic Irradiation on Morphology and Ultrastructure of Parotid Glands in Rats and Amelioration by Vitamin E

Background: Human is daily exposed to electromagnetic field from different source in modern societies with associated health impacts. This experiment aims to study the histological structure of parotid gland after exposure to extremely low frequency magnetic field ELF-MF associated with utility grid 50/60 Hertz (Hz) and to detect the possible role of Vitamin E supplementation. Materials and methods: Thirty adult male albino rats were classified into three groups: Group (I) was control animals, group (II) and (III) were exposed to ELF-MF (50 Hz and 100 – 300 mili Tesla (mT)). Animals of group III were supplemented with daily doses of vitamin E directly before exposure to ELF-MF. Light and electron microscope examination of parotid glands were done. Results: Degeneration of parotid gland acini and ducts together with cellular infiltrations, fibrosis and mucous transformation of the acinar cells were detected after exposure to ELF-MF. These changes were limited with vitamin E supplementation in group III. So, exposure to ELF-MF leads to parotid gland affection which could be tolerated with vitamin E supplementation.

Apical Ca2+-Activated K+-Channels in Mouse Parotid Acinar Cells

Ca2+ activation of Cl-- and K+-channels are key events underlying stimulated fluid secretion from parotid salivary glands. Cl--channels are exclusively present on the apical plasma membrane (PM) while the localization of K+-channels has not been established. Mathematical models have suggested that localization of some K+-channels to the apical PM is optimum for fluid secretion. A combination of whole cell electrophysiology and temporally resolved digital imaging with local manipulation of intracellular [Ca2+] was used to investigate if Ca2+-activated K+-channels are present in the apical PM of parotid acinar cells. Initial experiments established Ca2+ buffering conditions that produced brief, localized increases in [Ca2+] following focal laser photolysis of caged-Ca2+. Conditions were employed to isolate K+ and Cl- conductances. Photolysis at the apical PM resulted in a robust increase in K+ and Cl- currents. A localized reduction in [Ca2+] at the apical PM following photolysis of Diazo-2, a caged-Ca2+ chelator, resulted in a decrease in both K+ and Cl- currents. The K+ currents evoked by apical photolysis were partially blocked by both paxilline and TRAM-34, specific blockers of BK and IK respectively and almost abolished by incubation with both antagonists. Apical TRAM-34 sensitive K+ currents were also observed in BK null parotid acini. In contrast, when the [Ca2+] was increased at the basal PM no increase in either K+ or Cl- currents was evoked. These data provide strong evidence that K+- and Cl--channels are similarly distributed in the apical PM. Furthermore, both IK and BK channels are present in this domain and that the density of these channels appears higher in the apical vs. basal PM. In total, this study provides support for a model in which fluid secretion is optimized following expression of K+-channels specifically in the apical PM.

Nicotinic receptor agonist-induced salivation and its cellular mechanism in parotid acini of rats

Cigarette smoking and nicotine enhance parotid saliva secretion, however, the underlying mechanism is unclear. To address the mechanism of nicotine-induced salivation and to explore the possibility that nicotinic receptor agonists act as sialogogues, we investigated the effects of nicotinic receptor agonists on salivary secretion in vivo and on intracellular Ca 2+ concentration in digested parotid acini in vitro in rats. In urethane-anesthetized rats, intravenous administration of nicotinic receptor agonists, nicotine and cytisine, at 3 μmol/kg increased whole saliva output accompanied by a pressor response with nicotine, but not with cytisine. Using Ca 2+-imaging system on digested parotid acini in which autonomic nerve terminals were kept intact, nicotine and cytisine dose-dependently increased intracellular Ca 2+ concentration at μM level. This was not observed in single acinar cells containing no nerve terminal. The nicotine-induced Ca 2+ response was largely blocked by a muscarinic receptor antagonist and partly blocked by an adrenergic receptor antagonist. Furthermore, the same nicotine-induced Ca 2+ response was blocked by mecamylamine, a relatively selective nicotinic antagonist for α3β4 subtype receptor, but not by other selective antagonists, dihydro-β-erythroidine for α4-containing receptor and methyllycaconitine for α7 nicotinic receptors. These results suggest that nicotinic agonists-induced salivation is due to a release of acetylcholine and noradrenaline from autonomic nerve terminals through activation of α3β4 nicotinic receptor subtype. In addition, considering the blood pressure response and development of addiction with nicotine, cytisine may be a better therapeutic candidate to serve as a sialogogue for xerostomia patients.

Immunohistochemical and Immunocytochemical Localization of Amylase in Rat Parotid Glands and von Ebner's Glands by Ion Etching-Immunoscanning Electron Microscopy

The distribution of amylase in rat parotid glands and von Ebner’s glands was examined using ion etching-immunoscanning electron microscopy, which enables both light and electron microscopic observations of identical semi-thin resin sections immunolabeled with anti-α-amylase and immunogold in association with silver enhancement. At the light microscopic level, most acinar secretory granules (SG) and striated duct secretions of parotid glands were strongly stained dark brown. In von Ebner’s glands, acinar SG and duct secretions were weakly to strongly stained light to dark brown. At the electron microscopic level, labeling was observed as bright gold-silver particles. The labeling intensity of acinar SG of parotid glands was higher than that of von Ebner’s glands. In parotid glands, weak labeling of SG in transitional cells between acini and intercalated ducts, very weak labeling of SG in intercalated ducts, and strong labeling of striated duct secretions were observed. In von Ebner’s glands, the secretions and some SG of interlobular ducts were strongly labeled compared to those of intralobular ducts and SG of acini. Less amylase was synthesized in von Ebner’s acini compared to parotid acini, whereas von Ebner’s ducts may secrete significantly more amylase to modify saliva than parotid ducts.

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.

Differences in the Ca 2+ response resulting from neurotransmitter stimulations of rat parotid acini and ducts

There are few data available regarding the differences in intracellular Ca 2+ responses of parotid acinar and ductal cells. This study investigated the Ca 2+ mobilization that was induced by the chemical stimulation of acinar and ductal cells from rat parotid glands. In fura-2 loaded parotid cells, carbachol increased the intracellular Ca 2+ concentration ([Ca 2+] i) to a greater extent in the acinar cells than in the ductal cells, but noradrenaline increased the [Ca 2+] i in the ductal cells more than in the acinar cells. Although there was no difference in the α1-adrenergic receptor agonist phenylephrine-induced Ca 2+ mobilization between acini and ducts, the β-adrenergic receptor agonist isoproterenol increased the [Ca 2+] i in only the ductal cells. Additionally, the effects of non-adrenergic, non-cholinergic neurotransmitters were investigated. Substance P and ATP increased the [Ca 2+] i in parotid acini and/or ducts. A substance P-induced Ca 2+ response was observed in only acini, while the ATP-induced Ca 2+ response was significantly higher in ducts than in acini. These results suggest that parotid acini have a greater sensitivity to cholinergic and substance P stimulation and a lesser sensitivity to β-adrenergic and ATP stimulation than the ductal cells. In light of these results, substance P and isoproterenol will be useful for identifying parotid acini and ducts, respectively.

Ca2+ mobilization by nicotine through synaptic activation in rat parotid acini

Nicotine has been reported to increase the intracellular Ca(2+) concentration ([Ca(2+)](i)) in sublingual acini due to neurotransmitter release from nerve terminals associated with the cell preparation (1). However, it is unclear whether or not the same reaction exists in parotid cells. Therefore, we investigated effect of nicotine on Ca(2+) mobilization in digested parotid acini from rats. After removing the parotid gland from Wistar rats, the tissues were minced and digested with collagenase. Then, the intracellular Ca(2+) indicator fura-2 was added to the preparation, and the change in [Ca(2+)](i) was monitored using fluorescent microscope. In many but not all parotid acini, K(+) stimulation induced transient increases in [Ca(2+)](i). The K(+)-induced Ca(2+) response in parotid acini was completely blocked by Cd(2+)-containing solution. These results suggest that the parotid cell preparation has nerve terminals. In all high-K(+)-sensitive parotid acini, over 3 microM of nicotine increased [Ca(2+)](i), and the response was blocked by a Cd(2+)-containing solution and nicotinic receptor antagonists. All high-K(+)-insensitive acinar cells were resistant to the effect of nicotine on Ca(2+) mobilization. These results suggest that nicotine induces increases in [Ca(2+)](i) in parotid acini due to neurotransmitter release from associated nerve terminals.

Inhibition of serine/threonine phosphatase enhances arachidonic acid-induced [Ca2+]i via protein kinase A

Arachidonic acid (AA) regulates intracellular calcium concentration ([Ca2+]i) in a variety of cell types including salivary cells. In the present study, the effects of serine/threonine phosphatases on AA-induced Ca(2+) signaling in mouse parotid acini were determined. Mice were euthanized with CO2. Treatment of acini with the serine/threonine phosphatase inhibitor calyculin A blocked both thapsigargin- and carbachol-induced Ca2+ entry but resulted in an enhancement of AA-induced Ca2+ release and entry. Effects were mimicked by the protein phosphatase-1 (PP1) inhibitor tautomycin but were inhibited by the PP2A inhibitor okadaic acid. The protein kinase A (PKA) inhibitor PKI(14-22) significantly attenuated AA-induced enhancement of Ca2+ release and entry in the presence of calyculin A, whereas it had no effect on calyculin A-induced inhibition of thapsigargin-induced Ca2+ responses. The ryanodine receptor (RyR) inhibitor, tetracaine, and StHt-31, a peptide known to competitively inhibit type II PKA regulatory subunit binding to PKA-anchoring protein (AKAP), abolished calyculin A enhancement of AA-induced Ca2+ release and entry. StHt-31 also abolished forskolin potentiation of 4-chloro-3-ethylphenol (4-CEP) and AA on Ca2+ release but had no effect on 8-(4-methoxyphenylthio)-2'-O-methyladenosine-3',5'-cAMP potentiation of 4-CEP responses. Results suggest that inhibition of PP1 results in an enhancement of AA-induced [Ca2+]i via PKA, AKAP, and RyRs.

ATP Potentiates Insitol‐1,4,5‐triphosphate‐induced Calcium Release in Permeabilized Parotid and Pancreatic Acini

A pivotal event underlying exocytosis from exocrine cells is Ca2+ mobilization from intracellular stores initiated by Insitol‐1,4,5‐triphosphate (InsP3) binding to InsP3 receptors (InsP3R). InsP3R gating is regulated by a diverse array of regulatory inputs. We aimed to investigate the effects of ATP on InsP3‐induced Ca2+ release in permeabilized parotid and pancreatic acini. ATP dependency is known to differ between InsP3R subtypes. Type II InsP3R is predominantly present in parotid gland, and both Type II and III InsP3R are expressed in pancreas. Following the SERCA driven filling of Ca2+ stores, 0.1 μM to 10 μM InsP3 was infused with or without ATP in β‐escin permeabilized acini. InsP3 increased the Ca2+ release rate in a concentration‐dependent manner in both parotid and pancreatic acini. ATP (5 mM) significantly enhanced Ca2+ release at sub‐maximal [InsP3]. Next, the ATP dependency of the InsP3‐induced Ca2+ release was tested. ATP dose‐dependently potentiated the 0.3 μM InsP3‐induced Ca2+ release rate in both parotid and pancreatic acini. However, the ATP concentration dependency was shifted to the left in parotid acini. These results provide evidence that ATP can enhance InsP3‐induced Ca2+ release in both parotid and pancreatic acini and that the complement of InsP3R in parotid acini is more sensitive to this form of regulation.

Enhanced Formation of a HCO−3 Transport Metabolon in Exocrine Cells of Nhe1–/– Mice

Cl(-) influx across the basolateral membrane is a limiting step in fluid production in exocrine cells and often involves functionally linked Cl(-)/HCO(3)(-) (Ae) and Na(+)/H(+) (Nhe) exchange mechanisms. The dependence of this major Cl(-) uptake pathway on Na(+)/H(+) exchanger expression was examined in the parotid acinar cells of Nhe1(-/-) and Nhe2(-/-) mice, both of which exhibited impaired fluid secretion. No change in Cl(-)/HCO(3)(-) exchanger activity was detected in Nhe2-deficient mice. Conversely, Cl(-)/HCO(3)(-) exchanger activity increased nearly 4-fold in Nhe1-deficient mice, despite only minimal or any change in mRNA and protein levels of the anion exchanger Ae2. Acetazolamide completely blocked the increase in Cl(-)/HCO(3)(-) exchanger activity in Nhe1-null mice suggesting that increased anion exchange required carbonic anhydrase activity. Indeed, the parotid glands of Nhe1(-/-) mice expressed higher levels of carbonic anhydrase 2 (Car2) polypeptide. Moreover, the enhanced Cl(-)/HCO(3)(-) exchange activity was accompanied by an increased abundance of Car2.Ae2 complexes in the parotid plasma membranes of Nhe1(-/-) mice. Anion exchanger activity was also significantly reduced in Car2-deficient mice, consistent with an important role of a putative Car2.Ae2 HCO(3)(-) transport metabolon in parotid exocrine cell function. Increased abundance of this HCO(3)(-) transport metabolon is likely one of the multiple compensatory changes in the exocrine parotid gland of Nhe1(-/-) mice that together attenuate the severity of in vivo electrolyte and acid-base balance perturbations.