Highly localized intracellular Ca2+ signals promote optimal salivary gland fluid secretion.
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ABSTRACT:
SUBMITTER: Takano T
PROVIDER: S-EPMC8352588 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Agonist-induced Ca(2+) entry via store-operated Ca(2+) (SOC) channels is suggested to regulate a wide variety of cellular functions, including salivary gland fluid secretion. However, the molecular components of these channels and their physiological function(s) are largely unknown. Here we report that attenuation of SOC current underlies salivary gland dysfunction in mice lacking transient receptor potential 1 (TRPC1). Neurotransmitter-regulated salivary gland fluid secretion in TRPC1-deficient TRPC1(-/-) mice was severely decreased (by 70%). Further, agonist- and thapsigargin-stimulated SOC channel activity was significantly reduced in salivary gland acinar cells isolated from TRPC1(-/-) mice. Deletion of TRPC1 also eliminated sustained Ca(2+)-dependent potassium channel activity, which depends on Ca(2+) entry and is required for fluid secretion. Expression of key proteins involved in fluid secretion and Ca(2+) signaling, including STIM1 and other TRPC channels, was not altered. Together, these data demonstrate that reduced SOC entry accounts for the severe loss of salivary gland fluid secretion in TRPC1(-/-) mice. Thus, TRPC1 is a critical component of the SOC channel in salivary gland acinar cells and is essential for neurotransmitter-regulation of fluid secretion.
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Project description:Airway submucosal glands contribute to airway surface liquid (ASL) composition and volume, both important for lung mucociliary clearance. Serous acini generate most of the fluid secreted by glands, but the molecular mechanisms remain poorly characterized. We previously described cholinergic-regulated fluid secretion driven by Ca(2+)-activated Cl(-) secretion in primary murine serous acinar cells revealed by simultaneous differential interference contrast (DIC) and fluorescence microscopy. Here, we evaluated whether Ca(2+)-activated Cl(-) secretion was accompanied by secretion of HCO(3)(-), possibly a critical ASL component, by simultaneous measurements of intracellular pH (pH(i)) and cell volume. Resting pH(i) was 7.17 +/- 0.01 in physiological medium (5% CO(2)-25 mM HCO(3)(-)). During carbachol (CCh) stimulation, pH(i) fell transiently by 0.08 +/- 0.01 U concomitantly with a fall in Cl(-) content revealed by cell shrinkage, reflecting Cl(-) secretion. A subsequent alkalinization elevated pH(i) to above resting levels until agonist removal, whereupon it returned to prestimulation values. In nominally CO(2)-HCO(3)(-)-free media, the CCh-induced acidification was reduced, whereas the alkalinization remained intact. Elimination of driving forces for conductive HCO(3)(-) efflux by ion substitution or exposure to the Cl(-) channel inhibitor niflumic acid (100 microM) strongly inhibited agonist-induced acidification by >80% and >70%, respectively. The Na(+)/H(+) exchanger (NHE) inhibitor dimethylamiloride (DMA) increased the magnitude (greater than twofold) and duration of the CCh-induced acidification. Gene expression profiling suggested that serous cells express NHE isoforms 1-4 and 6-9, but pharmacological sensitivities demonstrated that alkalinization observed during both CCh stimulation and pH(i) recovery from agonist-induced acidification was primarily due to NHE1, localized to the basolateral membrane. These results suggest that serous acinar cells secrete HCO(3)(-) during Ca(2+)-evoked fluid secretion by a mechanism that involves the apical membrane secretory Cl(-) channel, with HCO(3)(-) secretion sustained by activation of NHE1 in the basolateral membrane. In addition, other Na(+)-dependent pH(i) regulatory mechanisms exist, as evidenced by stronger inhibition of alkalinization in Na(+)-free media.
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Project description:Reactive oxygen species (ROS) can have divergent effects in cerebral and peripheral circulations. We found that Ca(2+)-permeable transient receptor potential ankyrin 1 (TRPA1) channels were present and colocalized with NADPH (reduced form of nicotinamide adenine dinucleotide phosphate) oxidase 2 (NOX2), a major source of ROS, in the endothelium of cerebral arteries but not in other vascular beds. We recorded and characterized ROS-triggered Ca(2+) signals representing Ca(2+) influx through single TRPA1 channels, which we called "TRPA1 sparklets." TRPA1 sparklet activity was low under basal conditions but was stimulated by NOX-generated ROS. Ca(2+) entry during a single TRPA1 sparklet was twice that of a TRPV4 sparklet and ~200 times that of an L-type Ca(2+) channel sparklet. TRPA1 sparklets representing the simultaneous opening of two TRPA1 channels were more common in endothelial cells than in human embryonic kidney (HEK) 293 cells expressing TRPA1. The NOX-induced TRPA1 sparklets activated intermediate-conductance, Ca(2+)-sensitive K(+) channels, resulting in smooth muscle hyperpolarization and vasodilation. NOX-induced activation of TRPA1 sparklets and vasodilation required generation of hydrogen peroxide and lipid-peroxidizing hydroxyl radicals as intermediates. 4-Hydroxy-nonenal, a metabolite of lipid peroxidation, also increased TRPA1 sparklet frequency and dilated cerebral arteries. These data suggest that in the cerebral circulation, lipid peroxidation metabolites generated by ROS activate Ca(2+) influx through TRPA1 channels in the endothelium of cerebral arteries to cause dilation.
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Project description:Radiotherapy for head and neck cancers commonly causes damage to salivary gland tissue, resulting in xerostomia (dry mouth) and numerous adverse medical and quality-of-life issues. Amifostine is the only Food and Drug Administration-approved radioprotective drug used clinically to prevent xerostomia. However, systemic administration of amifostine is limited by severe side effects, including rapid decrease in blood pressure (hypotension), nausea, and a narrow therapeutic window. In this study, we demonstrate that retroductal delivery of amifostine and its active metabolite, WR-1065, to murine submandibular glands prior to a single radiation dose of 15 Gy maintained gland function and significantly increased acinar cell survival. Furthermore, in vivo stimulated saliva secretion was maintained in retrograde-treated groups at levels significantly higher than irradiated-only and systemically treated groups. In contrast to intravenous injections, retroductal delivery of WR-1065 or amifostine significantly attenuated hypotension. We conclude that localized delivery to salivary glands markedly improves radioprotection at the cellular level, as well as mitigates the adverse side effects associated with systemic administration. These results support the further development of a localized delivery system that would be compatible with the fractionated dose regimen used clinically.
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Project description:Recent research efforts in oral biology have resulted in elucidation of the proteomes of major human salivary secretions and whole saliva. One might hypothesize that the proteome of minor gland secretions may show significantly different characteristics when compared with the proteomes of parotid or submandibular/sublingual secretions. To test this hypothesis, we conducted the first exploration into the proteome of minor salivary gland secretion. Minor gland secretion was obtained from healthy volunteers, and its components were subjected to liquid-chromatography-electrospray-ionization-tandem-mass-spectrometry. This led to the identification of 56 proteins, 12 of which had never been identified in any salivary secretion. The unique characteristics of the minor salivary gland secretion proteome are related to the types as well as the numbers of components present. The differences between salivary proteomes may be important with respect to specific oral functions.
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Project description:Activation of an apical Ca(2+)-activated Cl(-) channel (CaCC) triggers the secretion of saliva. It was previously demonstrated that CaCC-mediated Cl(-) current and Cl(-) efflux are absent in the acinar cells of systemic Tmem16A (Tmem16A Cl(-) channel) null mice, but salivation was not assessed in fully developed glands because Tmem16A null mice die within a few days after birth. To test the role of Tmem16A in adult salivary glands, we generated conditional knockout mice lacking Tmem16A in acinar cells (Tmem16A(-/-)). Ca(2+)-dependent salivation was abolished in Tmem16A(-/-) mice, demonstrating that Tmem16A is obligatory for Ca(2+)-mediated fluid secretion. However, the amount of saliva secreted by Tmem16A(-/-) mice in response to the ?-adrenergic receptor agonist isoproterenol (IPR) was comparable to that seen in controls, indicating that Tmem16A does not significantly contribute to cAMP-induced secretion. Furthermore, IPR-stimulated secretion was unaffected in mice lacking Cftr (Cftr(?F508/?F508)) or ClC-2 (Clcn2(-/-)) Cl(-) channels. The time course for activation of IPR-stimulated fluid secretion closely correlated with that of the IPR-induced cell volume increase, suggesting that acinar swelling may activate a volume-sensitive Cl(-) channel. Indeed, Cl(-) channel blockers abolished fluid secretion, indicating that Cl(-) channel activity is critical for IPR-stimulated secretion. These data suggest that ?-adrenergic-induced, cAMP-dependent fluid secretion involves a volume-regulated anion channel. In summary, our results using acinar-specific Tmem16A(-/-) mice identify Tmem16A as the Cl(-) channel essential for muscarinic, Ca(2+)-dependent fluid secretion in adult mouse salivary glands.
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Project description:New strategies for tissue engineering have great potential for restoring and revitalizing impaired tissues and organs, including the use of smart hydrogels that can be modified to enhance organization and functionality of the salivary glands. For instance, monomers of laminin-111 peptides chemically conjugated to fibrin hydrogel (L1pM-FH) promote cell cluster formation in vitro and salivary gland regeneration in vivo when compared with fibrin hydrogel (FH) alone; however, L1pM-FH produce only weak expression of acinar differentiation markers in vivo (e.g., aquaporin-5 and transmembrane protein 16). Since previous studies demonstrated that a greater impact can be achieved when trimeric forms were used as compared with monomeric or dimeric forms, we investigated the extent to which trimers of laminin-111 chemically conjugated to FH (L1pT-FH) can increase the expression of acinar differentiation markers and elevate saliva secretion. In vitro studies using Par-C10 acinar cells demonstrated that when compared with L1pM-FH, L1pT-FH induced similar levels of acinar-like cell clustering, polarization, lumen formation, and calcium signaling. To assess the performance of the trimeric complex in vivo, we compared the ability of L1pM-FH and L1pT-FH to increase acinar differentiation markers and restore saliva flow rate in a salivary gland wound model of C57BL/6 mice. Our results show that L1pT-FH applied to wounded mice significantly improved the expression of the acinar differentiation markers and saliva secretion when compared with the monomeric form. Together, these positive effects of L1pT-FH warrant its future testing in additional models of hyposalivation with the ultimate goal of applying this technology in humans.
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Project description:Hyposalivation contributes to dental caries, periodontitis, and microbial infections. Additionally, it impairs activities of daily living (e.g., speaking, chewing, and swallowing). Treatments for hyposalivation are currently limited to medications (e.g., the muscarinic receptor agonists pilocarpine and cevimeline) that induce saliva secretion from residual acinar cells and the use of saliva substitutes. However, given that these therapies provide only temporary relief, the development of alternative treatments to restore gland function is essential. Previous studies demonstrated that laminin 1 (L1) is critical for intact salivary cell cluster formation and organization. However, the full L1 sequence is not suitable for clinical applications, as each protein domain may contribute to unwanted effects, such as degradation, tumorigenesis, and immune responses that, when compounded, outweigh the potential benefits provided by their sum. Although the L1 peptides YIGSR and A99 linked to fibrin hydrogels (FHs) promote intact salivary epithelial formation in vitro, little is known about their role during salivary gland regeneration in vivo. Therefore, the goal of this study was to demonstrate whether L1 peptides conjugated to FHs promote tissue regeneration in a wound-healing model of mouse submandibular glands (mSMGs). Our results suggest that YIGSR-A99 peptides, chemically conjugated to FHs and applied to wounded mSMGs in vivo, formed new organized salivary tissue. In contrast, wounded mSMGs treated with FHs alone or in the absence of a scaffold showed disorganized collagen formation and poor tissue healing. Together these studies indicate that damaged salivary gland tissue can grow and differentiate when treated with FHs containing L1 peptides.
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Project description:The addition of 5-hydroxytryptamine to the isolated blowfly salivary gland stimulates fluid secretion, transepithelial calcium transport and the breakdown of 32P- or 3H-labelled phosphatidylinositol The breakdown of [32P]phosphatidylcholine and [32P]-phosphatidylethanolamine was not stimulated by 5-hydroxytryptamine. In salivary glands incubated with myo-[2-3H]inositol for 1--3 h, more than 95% of the label retained by the tissue was in the form of phosphatidylinositol. The addition of 5-hydroxytryptamine resulted in an increase in the accumulation of label in intracellular inositol 1:2-cyclic phosphate, inositol 1-phosphate and free inositol along with an increase in the release of [3H]inositol to the medium and saliva. The release of [3H]inositol to the medium served as a sensitive indicator of phosphatidylinositol breakdown. The release of [3H]inositol was not increased by cyclic AMP or the bivalent-cation ionophore A23187 under conditions in which salivary secretion was accelerated. The stimulation of fluid secretion by low concentrations of 5-hydroxytryptamine was potentiated by 3-isobutyl-1-methylxanthine, which had no effect on inositol release. The stimulation of fluid secretion by 5-hydroxytryptamine was greatly reduced in calcium-free buffer, but the breakdown of phosphatidylinositol continued at the same rate in the absence of calcium. These results support the hypothesis that breakdown of phosphatidylinositol by 5-hydroxytryptamine is involved in the gating of calcium.
| S-EPMC1186479 | biostudies-other
Project description:PurposeThe role of adrenergic innervation in the regulation of lacrimal gland (LG) ductal fluid secretion is unknown. The Aim of the present study was to investigate the effect of adrenergic stimulation on fluid secretion in isolated LG duct segments and to study the underlying intracellular mechanisms.MethodsFluid secretion of isolated mouse LG ducts was measured using video-microscopy. Effect of various adrenergic agonists (norepinephrine, phenylephrine, and isoproterenol) on fluid secretion as well as inhibitory effects of specific antagonists on adrenergic agonist-stimulated secretory response were analyzed. Changes in intracellular Ca2+ level [Ca2+i] were investigated with microfluorometry.ResultsBoth norepinephrine and phenylephrine initiated a rapid and robust fluid secretory response, whereas isoproterenol did not cause any secretion. Phenylephrine-induced secretion was completely blocked by α1D-adrenergic receptor blocker BMY-7378. The endothelial nitric oxide synthase (eNOS) inhibitor L-NAME or guanylyl cyclase inhibitor ODQ reduced but not completely abolished the phenylephrine-induced fluid secretion, whereas co-administration of Ca2+-chelator BAPTA-AM resulted in a complete blockade. Phenylephrine stimulation induced a small, but statistically significant elevation in [\(Ca_i^{2 + }\)].ConclusionsOur results prove the direct role of α1-adrenergic stimulation on LG ductal fluid secretion. Lack of isoproterenol-induced fluid secretory response suggests the absence of β-receptor mediated pathway in mouse LG ducts. Complete blockade of phenylephrine-induced fluid secretion by BMY-7378 and predominant inhibition of the secretory response either by L-NAME or ODQ suggest that α-adrenergic agonists use the NO/cGMP pathway through α1D receptor. Ca2+ signaling independent from NO/cGMP pathway may also play an at least partial role in α-adrenergic induced ductal fluid secretion.
| S-EPMC7718821 | biostudies-literature