P2X7 receptors induce degranulation in human mast cells.

Mast cells play important roles in host defence against pathogens, as well as being a key effector cell in diseases with an allergic basis such as asthma and an increasing list of other chronic inflammatory conditions. Mast cells initiate immune responses through the release of newly synthesised eicosanoids and the secretion of pre-formed mediators such as histamine which they store in specialised granules. Calcium plays a key role in regulating both the synthesis and secretion of mast-cell-derived mediators, with influx across the membrane, in particular, being necessary for degranulation. This raises the possibility that calcium influx through P2X receptors may lead to antigen-independent secretion of histamine and other granule-derived mediators from human mast cells. Here we show that activation of P2X7 receptors with both ATP and BzATP induces robust calcium rises in human mast cells and triggers their degranulation; both effects are blocked by the P2X7 antagonist AZ11645373, or the removal of calcium from the extracellular medium. Activation of P2X1 receptors with αβmeATP also induces calcium influx in human mast cells, which is significantly reduced by both PPADS and NF 449. P2X1 receptor activation, however, does not trigger degranulation. The results indicate that P2X7 receptors may play a significant role in contributing to the unwanted activation of mast cells in chronic inflammatory conditions where extracellular ATP levels are elevated

Local Ca2+ Entry Via Orai1 Regulates Plasma Membrane Recruitment of TRPC1 and Controls Cytosolic Ca2+ Signals Required for Specific Cell Functions

Store-operated Ca2+ entry (SOCE) has been associated with two types of channels: CRAC channels that require Orai1 and STIM1 and SOC channels that involve TRPC1, Orai1, and STIM1. While TRPC1 significantly contributes to SOCE and SOC channel activity, abrogation of Orai1 function eliminates SOCE and activation of TRPC1. The critical role of Orai1 in activation of TRPC1-SOC channels following Ca2+ store depletion has not yet been established. Herein we report that TRPC1 and Orai1 are components of distinct channels. We show that TRPC1/Orai1/STIM1-dependent ISOC, activated in response to Ca2+ store depletion, is composed of TRPC1/STIM1-mediated non-selective cation current and Orai1/STIM1-mediated ICRAC; the latter is detected when TRPC1 function is suppressed by expression of shTRPC1 or a STIM1 mutant that lacks TRPC1 gating, STIM1(684EE685). In addition to gating TRPC1 and Orai1, STIM1 mediates the recruitment and association of the channels within ER/PM junctional domains, a critical step in TRPC1 activation. Importantly, we show that Ca2+ entry via Orai1 triggers plasma membrane insertion of TRPC1, which is prevented by blocking SOCE with 1 µM Gd3+, removal of extracellular Ca2+, knockdown of Orai1, or expression of dominant negative mutant Orai1 lacking a functional pore, Orai1-E106Q. In cells expressing another pore mutant of Orai1, Orai1-E106D, TRPC1 trafficking is supported in Ca2+-containing, but not Ca2+-free, medium. Consistent with this, ICRAC is activated in cells pretreated with thapsigargin in Ca2+-free medium while ISOC is activated in cells pretreated in Ca2+-containing medium. Significantly, TRPC1 function is required for sustained KCa activity and contributes to NFκB activation while Orai1 is sufficient for NFAT activation. Together, these findings reveal an as-yet unidentified function for Orai1 that explains the critical requirement of the channel in the activation of TRPC1 following Ca2+ store depletion. We suggest that coordinated regulation of the surface expression of TRPC1 by Orai1 and gating by STIM1 provides a mechanism for rapidly modulating and maintaining SOCE-generated Ca2+ signals. By recruiting ion channels and other signaling pathways, Orai1 and STIM1 concertedly impact a variety of critical cell functions that are initiated by SOCE

Calcium Homeostasis and Cone Signaling Are Regulated by Interactions between Calcium Stores and Plasma Membrane Ion Channels

Calcium is a messenger ion that controls all aspects of cone photoreceptor function, including synaptic release. The dynamic range of the cone output extends beyond the activation threshold for voltage-operated calcium entry, suggesting another calcium influx mechanism operates in cones hyperpolarized by light. We have used optical imaging and whole-cell voltage clamp to measure the contribution of store-operated Ca2+ entry (SOCE) to Ca2+ homeostasis and its role in regulation of neurotransmission at cone synapses. Mn2+ quenching of Fura-2 revealed sustained divalent cation entry in hyperpolarized cones. Ca2+ influx into cone inner segments was potentiated by hyperpolarization, facilitated by depletion of intracellular Ca2+ stores, unaffected by pharmacological manipulation of voltage-operated or cyclic nucleotide-gated Ca2+ channels and suppressed by lanthanides, 2-APB, MRS 1845 and SKF 96365. However, cation influx through store-operated channels crossed the threshold for activation of voltage-operated Ca2+ entry in a subset of cones, indicating that the operating range of inner segment signals is set by interactions between store- and voltage-operated Ca2+ channels. Exposure to MRS 1845 resulted in ∼40% reduction of light-evoked postsynaptic currents in photopic horizontal cells without affecting the light responses or voltage-operated Ca2+ currents in simultaneously recorded cones. The spatial pattern of store-operated calcium entry in cones matched immunolocalization of the store-operated sensor STIM1. These findings show that store-operated channels regulate spatial and temporal properties of Ca2+ homeostasis in vertebrate cones and demonstrate their role in generation of sustained excitatory signals across the first retinal synapse

International Consensus Statement on Rhinology and Allergy: Rhinosinusitis

Background: The 5 years since the publication of the first International Consensus Statement on Allergy and Rhinology: Rhinosinusitis (ICAR‐RS) has witnessed foundational progress in our understanding and treatment of rhinologic disease. These advances are reflected within the more than 40 new topics covered within the ICAR‐RS‐2021 as well as updates to the original 140 topics. This executive summary consolidates the evidence‐based findings of the document. Methods: ICAR‐RS presents over 180 topics in the forms of evidence‐based reviews with recommendations (EBRRs), evidence‐based reviews, and literature reviews. The highest grade structured recommendations of the EBRR sections are summarized in this executive summary. Results: ICAR‐RS‐2021 covers 22 topics regarding the medical management of RS, which are grade A/B and are presented in the executive summary. Additionally, 4 topics regarding the surgical management of RS are grade A/B and are presented in the executive summary. Finally, a comprehensive evidence‐based management algorithm is provided. Conclusion: This ICAR‐RS‐2021 executive summary provides a compilation of the evidence‐based recommendations for medical and surgical treatment of the most common forms of RS

Sustained activation of the tyrosine kinase Syk by antigen in mast cells requires local Ca2+ influx through Ca2+ release-activated Ca2+ channels.

Mast cell activation involves cross-linking of IgE receptors followed by phosphorylation of the non-receptor tyrosine kinase Syk. This results in activation of the plasma membrane-bound enzyme phospholipase Cgamma1, which hydrolyzes the minor membrane phospholipid phosphatidylinositol 4,5-bisphosphate to generate diacylglycerol and inositol trisphosphate. Inositol trisphosphate raises cytoplasmic Ca2+ concentration by releasing Ca2+ from intracellular stores. This Ca2+ release phase is accompanied by sustained Ca2+ influx through store-operated Ca2+ release-activated Ca2+ (CRAC) channels. Here, we find that engagement of IgE receptors activates Syk, and this leads to Ca2+ release from stores followed by Ca2+ influx. The Ca2+ influx phase then sustains Syk activity. The Ca2+ influx pathway activated by these receptors was identified as the CRAC channel, because pharmacological block of the channels with either a low concentration of Gd3+ or exposure to the novel CRAC channel blocker 3-fluoropyridine-4-carboxylic acid (2',5'-dimethoxybiphenyl-4-yl)amide or RNA interference knockdown of Orai1, which encodes the CRAC channel pore, all prevented the increase in Syk activity triggered by Ca2+ entry. CRAC channels and Syk are spatially close together, because increasing cytoplasmic Ca2+ buffering with the fast Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis failed to prevent activation of Syk by Ca2+ entry. Our results reveal a positive feedback step in mast cell activation where receptor-triggered Syk activation and subsequent Ca2+ release opens CRAC channels, and the ensuing local Ca2+ entry then maintains Syk activity. Ca2+ entry through CRAC channels therefore provides a means whereby the Ca2+ and tyrosine kinase signaling pathways can interact with one another

Targeting Ca2+ release-activated Ca2+ channel channels and leukotriene receptors provides a novel combination strategy for treating nasal polyposis.

BACKGROUND: Nasal polyposis is a chronic inflammatory disease of the upper respiratory tract that affects around 2% of the population and almost 67% of patients with aspirin-intolerant asthma. Polyps are rich in mast cells and eosinophils, resulting in high levels of the proinflammatory cysteinyl leukotrienes. OBJECTIVES: To better understand the role of the proinflammatory leukotrienes in nasal polyposis, we asked the following questions: (1) How do nasal polyps produce leukotriene C(4) (LTC(4))? (2) Can LTC(4) feed back in a paracrine way to maintain mast cell activation? (3) Could a combination therapy targeting the elements of this feed-forward loop provide a novel therapy for allergic disease? METHODS: We have used immunohistochemistry, enzyme immunoassay, and cytoplasmic calcium ion (Ca(2+)) imaging to address these questions on cultured and acutely isolated human mast cells from patients with polyposis. RESULTS: Ca(2+) entry through store-operated Ca(2+) release-activated Ca(2+) (CRAC) channels in polyps produced LTC(4) in a manner dependent on protein kinase C. LTC(4) thus generated activated mast cells through cysteinyl leukotriene type I receptors. Hence Ca(2+) influx into mast cells stimulates LTC(4) production, which then acts as a paracrine signal to activate further Ca(2+) influx. A combination of a low concentration of both a CRAC channel blocker and a leukotriene receptor antagonist was as effective at suppressing mast cell activation as a high concentration of either antagonist alone. CONCLUSION: A drug combination directed against CRAC channels and leukotriene receptor antagonist suppresses the feed-forward loop that leads to aberrant mast cell activation. Hence our results identify a new potential strategy for combating polyposis and mast cell-dependent allergies

Intercellular Ca2+ wave propagation involving positive feedback between CRAC channels and cysteinyl leukotrienes.

Mast cells are key components of the immune system, where they help orchestrate the inflammatory response. Aberrant mast cell activation is linked to a variety of allergic diseases, including asthma, eczema, rhinitis, and nasal polyposis, which in combination affect up to 20% of the population in industrialized countries. On activation, mast cells release a variety of signals that target the bronchi and vasculature and recruit other immune cells to the inflammatory site. Prominent among such signals are the cysteinyl leukotrienes, a family of potent proinflammatory lipid mediators comprising leukotriene C(4) (LTC(4)), LTD(4), and LTE(4). LTC(4), the parent compound, is secreted from mast cells following Ca(2+) influx through store-operated calcium release-activated calcium (CRAC) channels. Here, we show that activated mast cells release a paracrine signal that evokes Ca(2+) signals in spatially separate resting mast cells. The paracrine signal was identified as a cysteinyl leukotriene because 1) RNAi knockdown or pharmacological block of the 5-lipoxygenase enzyme prevented activated mast cells from stimulating resting cells. 2) Block of cysteinyl leukotriene type I receptors on resting mast cells with the clinically prescribed receptor antagonist montelukast prevented their activation by active mast cells. 3) RNAi knockdown of cysteinyl leukotriene type I receptors on resting cells prevented them from responding to the paracrine signal derived from activated mast cells. 4) Purified LTC(4) evoked Ca(2+) signals in mast cells that were identical to those triggered by the paracrine signal. Low levels of stimulus intensity released sufficient levels of leukotriene to activate resting cells. Leukotriene secretion still occurred tens of minutes after stimulation, suggesting a role as a long-lasting trigger in mast cell activation. Stimulation of the cysteinyl leukotriene receptor activated CRAC channels and evoked prominent store-operated Ca(2+) entry. This resulted in further cysteinyl leukotriene production, triggering a positive feedback cascade. Acutely isolated mast cells from patients with allergic rhinitis exhibited store-operated Ca(2+) influx through CRAC channels and responded to cysteinyl leukotrienes. Histological analysis of samples taken from patients revealed clustering of mast cells, often located within 20 microm of each other, a distance sufficient for paracrine signaling by leukotrienes to operate effectively. We conclude that a positive-feedback cascade involving CRAC channels and cysteinyl leukotrienes constitute a novel mechanism for sustaining mast cell activation

Different agonists recruit different stromal interaction molecule proteins to support cytoplasmic Ca2+ oscillations and gene expression.

Stimulation of cells with physiological concentrations of calcium-mobilizing agonists often results in the generation of repetitive cytoplasmic Ca(2+) oscillations. Although oscillations arise from regenerative Ca(2+) release, they are sustained by store-operated Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels. Here, we show that following stimulation of cysteinyl leukotriene type I receptors in rat basophilic leukemia (RBL)-1 cells, large amplitude Ca(2+) oscillations, CRAC channel activity, and downstream Ca(2+)-dependent nuclear factor of activated T cells (NFAT)-driven gene expression are all exclusively maintained by the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule (STIM) 1. However, stimulation of tyrosine kinase-coupled FCεRI receptors evoked Ca(2+) oscillations and NFAT-dependent gene expression through recruitment of both STIM2 and STIM1. We conclude that different agonists activate different STIM proteins to sustain Ca(2+) signals and downstream responses