Purinergic P2X(7) Receptors Mediate ATP-induced Saliva Secretion by the Mouse Submandibular Gland

Center for Oral Biology, University of Rochester Medical Center, Rochester, New York 14642, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 12/2008; 284(8):4815-22. DOI: 10.1074/jbc.M808597200
Source: PubMed


Salivary glands express multiple isoforms of P2X and P2Y nucleotide receptors, but their in vivo physiological roles are unclear. P2 receptor agonists induced salivation in an ex vivo submandibular gland preparation. The nucleotide selectivity sequence of the secretion response was BzATP > ATP > ADP > UTP, and removal of external Ca(2+) dramatically suppressed the initial ATP-induced fluid secretion ( approximately 85%). Together, these results suggested that P2X receptors are the major purinergic receptor subfamily involved in the fluid secretion process. Mice with targeted disruption of the P2X(7) gene were used to evaluate the role of the P2X(7) receptor in nucleotide-evoked fluid secretion. P2X(7) receptor protein and BzATP-activated inward cation currents were absent, and importantly, purinergic receptor agonist-stimulated salivation was suppressed by more than 70% in submandibular glands from P2X(7)-null mice. Consistent with these observations, the ATP-induced increases in [Ca(2+)](i) were nearly abolished in P2X(7)(-/-) submandibular acinar and duct cells. ATP appeared to also act through the P2X(7) receptor to inhibit muscarinic-induced fluid secretion. These results demonstrate that the ATP-sensitive P2X(7) receptor regulates fluid secretion in the mouse submandibular gland.

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Available from: Mireya Gonzalez, Apr 13, 2015
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    • "In contrast, other studies have shown an apparent antagonistic relationship between muscarinic and purinergic signaling. For example one study demonstrated enhanced muscarinic receptor-evoked [Ca 2+ ] i rises in parotid acini isolated from P2X7R knockout mice compared to the background strain [48], and another study on submandibular gland acini showed that ATP and CCh co-stimulation decreased fluid secretion in wild type mice [16]. The differences in findings between groups are not immediately clear but may be related to differences in the agonist concentrations, mouse strains or tissues used. "
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    ABSTRACT: Isolated clusters of mouse parotid acinar cells in combination with live cell imaging were used to explore the crosstalk in molecular signaling between purinergic, cholinergic and adrenergic pathways that integrate to control fluid and protein secretion. This crosstalk was manifested by 1) β-adrenergic receptor activation and amplification of P2X4R evoked Ca2+ signals, 2) β-adrenergic-induced amplification of P2X7R-evoked Ca2+ signals and 3) muscarinic receptor induced activation of P2X7Rs via exocytotic activity. The findings from our study reveal that purinoceptor-mediated Ca2+ signaling is modulated by crosstalk with canonical signaling pathways in parotid acinar cells. Integration of these signals are likely important for dynamic control of saliva secretion to match physiological demand in the parotid gland.Keywordscalcium signaling; salivary gland; purinergic receptor; Epac; cAMP; parotid; exocytosis; crosstalk
    Cell Calcium 09/2015; DOI:10.1016/j.ceca.2015.09.006 · 3.51 Impact Factor
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    • "ARC channels rely on the generation of arachidonic acid and are store-independent, since their activation does not depend on the loss of Ca2+ from the endoplasmic reticulum [5], [6]. In addition to Ca2+-sensitive ion movements that occur in response to Ca2+ entry via the SOCE pathway in salivary gland cells, the entry of extracellular Ca2+ via the P2X7 receptor/ion channel also activates Ca2+-sensitive ion channels and initiates fluid secretion and saliva formation [7], [8], [9]. SOCE can be activated in a receptor-independent manner using agents such as TG that block the Ca2+-ATPase on the endoplasmic reticulum membrane, which depletes endoplasmic reticulum Ca2+ stores and thereby promotes Ca2+ entry. "
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    ABSTRACT: The regulation of intracellular Ca(2+) concentration ([Ca(2+)]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 Ca(2+) stores, followed by the sustained elevation of [Ca(2+)]i from Ca(2+) entry across the plasma membrane via store-operated Ca(2+) entry (SOCE). Ca(2+) entry is also increased in a store-independent manner by arachidonate-regulated Ca(2+) (ARC) channels. Using rat parotid salivary gland cells, we examined multiple pathways of Ca(2+) 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 Ca(2+) stores using the endoplasmic reticulum Ca(2+)-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 [Ca(2+)]i by Ca(2+) ionophore or by Ca(2+) entry via ARC channels in native cells, although ERK1/2 phosphorylation was increased by Ca(2+) 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 Ca(2+), 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 Ca(2+) entry (SOCE) in these cells, and suggest that ERK1/2 may contribute to events downstream of SOCE.
    PLoS ONE 08/2013; 8(8):e72881. DOI:10.1371/journal.pone.0072881 · 3.23 Impact Factor
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    • "Aside from its pro-inflammatory effects, ATP activation of the P2X7 receptor has also emerged as an important regulator of autonomic function, including salivary gland secretion. While P2X7 receptor activation can stimulate secretion in mouse submandibular bands, activation also results in the inhibition of muscarinic receptor-induced fluid secretion [19]. Therefore, activation of P2X7 in the milieu of salivary gland inflammation in pSS may contribute to the failure of fluid secretion irrespective of the extent of glandular destruction, through interference of autonomic signalling, a well-documented phenomenon in pSS. "
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    ABSTRACT: The aim of this study was to examine the association between functional polymorphisms in the pro-inflammatory P2X7 receptor and the Ro/La autoantibody response in primary Sjogren's syndrome (pSS). Twelve functional P2RX7 polymorphisms were genotyped in 114 pSS patients fulfilling the Revised American-European Consensus Criteria for pSS and 136 controls. Genotyping of the A1405G (rs2230912) polymorphism was performed on a replication cohort consisting of 281 pSS patients and 534 controls. P2X7 receptor function in lymphocytes and monocytes was assessed by measurement of ATP-induced ethidium+ uptake. Serum IL-18 levels were determined by ELISA. The minor allele of P2RX7 A1405G is a tag for a common haplotype associated with gain in receptor function, as assessed by ATP-induced ethidium+ uptake. A positive association between 1405G and anti-Ro+/-La seropositive pSS patients was observed in Cohort 1. Although not replicated in Cohort 2, there was a consistent, significant, negative epistatic interaction effect with HLA-DR3 in seropositive pSS patients from both cohorts, thereby implicating this gain of function variant in the pathogenesis of pSS. Serum IL-18 was elevated in seropositive pSS patients, but was not influenced by P2RX7 A1405G. The P2RX7 1405G gain-of-function haplotype may be a risk factor for seropositive pSS in a subset of subjects who do not carry HLA risk alleles, but has no effect in subjects who do (epistasis). Potential mechanisms relate to autoantigen exposure and inflammatory cytokine expression. The observed elevation of IL-18 levels are consistent with P2X7 receptor activation in seropositive pSS patients. Collectively these findings implicate P2X7 receptor function in the pathogenesis of pSS.
    Arthritis research & therapy 07/2013; 15(4):R71. DOI:10.1186/ar4248 · 3.75 Impact Factor
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