Populations of Mast Cells Agonist Elicits Graded Responses in All-Or
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All-or-None Activation of CRAC Channels by Agonist Elicits Graded Responses in Populations of Mast Cells This information is current as Wei-Chiao Chang, Joseph Di Capite, Charmaine Nelson and of October 1, 2021. Anant B. Parekh J Immunol 2007; 179:5255-5263; ; doi: 10.4049/jimmunol.179.8.5255 http://www.jimmunol.org/content/179/8/5255 Downloaded from References This article cites 44 articles, 12 of which you can access for free at: http://www.jimmunol.org/content/179/8/5255.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average by guest on October 1, 2021 Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2007 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology All-or-None Activation of CRAC Channels by Agonist Elicits Graded Responses in Populations of Mast Cells1 Wei-Chiao Chang, Joseph Di Capite, Charmaine Nelson, and Anant B. Parekh2 In nonexcitable cells, receptor stimulation evokes Ca2؉ release from the endoplasmic reticulum stores followed by Ca2؉ influx -through store-operated Ca2؉ channels in the plasma membrane. In mast cells, store-operated entry is mediated via Ca2؉ release activated Ca2؉ (CRAC) channels. In this study, we find that stimulation of muscarinic receptors in cultured mast cells results in Ca2؉-dependent activation of protein kinase C␣ and the mitogen activated protein kinases ERK1/2 and this is required for the 2؉ subsequent stimulation of the enzymes Ca -dependent phospholipase A2 and 5-lipoxygenase, generating the intracellular mes- senger arachidonic acid and the proinflammatory intercellular messenger leukotriene C4. In cell population studies, ERK acti- vation, arachidonic acid release, and leukotriene C4 secretion were all graded with stimulus intensity. However, at a single cell level, Ca2؉ influx was related to agonist concentration in an essentially all-or-none manner. This paradox of all-or-none CRAC channel activation in single cells with graded responses in cell populations was resolved by the finding that increasing agonist Downloaded from concentration recruited more mast cells but each cell responded by generating all-or-none Ca2؉ influx. These findings were extended to acutely isolated rat peritoneal mast cells where muscarinic or P2Y receptor stimulation evoked all-or-none activation of Ca2؉entry but graded responses in cell populations. Our results identify a novel way for grading responses to agonists in immune cells and highlight the importance of CRAC channels as a key pharmacological target to control mast cell activation. The Journal of Immunology, 2007, 179: 5255–5263. http://www.jimmunol.org/ 2ϩ n eukaryotic cells, Ca influx is a critical trigger for a di- acid liberated by cPLA2 is then converted to the proinflammatory verse array of cellular responses including exocytosis, mus- paracrine signal leukotriene C4 (LTC4) by the 5-lipoxygenase en- cle contraction, gene transcription, and cell growth (1). In zyme (12, 13). I 2ϩ nonexcitable cells, Ca entry occurs through store-operated and The physiological trigger for CRAC channel activation is an second messenger-gated Ca2ϩ channels in the plasma membrane increase in the levels of the ubiquitous second messenger inositol 2ϩ (2, 3). Store-operated Ca channels are activated following the 1,4,5-trisphosphate (InsP3), which occurs following stimulation of emptying of intracellular Ca2ϩ stores, are regulated by a diverse cell surface receptors that couple to phospholipase C (2, 14). The range of agonists, and are found in a variety of nonexcitable cells. extent of I activation is steeply related to the cytoplasmic CRAC by guest on October 1, 2021 Electrophysiological evidence indicates that store-operated InsP3 concentration with a Hill coefficient of 12, and this essen- channels represent a heterogeneous family (4). The best charac- tially all-or-none relationship is seen either by infusing cells with 2ϩ 2ϩ 3 terized member is the Ca release-activated Ca (CRAC) chan- InsP3 or following stimulation of plasma membrane receptors that nel and the whole cell current flowing through CRAC channels is activate phospholipase C (15). Such nonlinear behavior arises from called ICRAC (5, 6). Defective CRAC channel activity has been the metabolism of InsP3 by inositol 5-phosphatase because the causally linked to inherited primary immunodeficiencies (7) and nonmetabolisable InsP3 analog Ins2,4,5P3 generates a graded re- 2ϩ Ca entry through CRAC channels regulates exocytosis (8), gene sponse whereas InsP3-F, another analog that is only broken down transcription (9), and cytoplasmic enzymes such as NO synthase by the 5-phosphatase, activates ICRAC in a highly supralinear man- (10), adenylate cyclase (11), and Ca2ϩ-dependent phospholipase ner (15, 16). Consistent with this is the finding that inhibition of 2ϩ A2 (cPLA2; Ref. 12). Ca influx through CRAC channels acti- inositol 5-phosphatase with InsP4 reduces the steepness of the re- vates cPLA2 indirectly, via recruitment of conventional protein lationship between InsP3 concentration and activation of ICRAC ␣  2ϩ kinase C and I isozymes and subsequent stimulation of the mi- and enables lower concentrations of InsP3 to evoke robust Ca togen activated protein kinases ERK1 and ERK2 (13). Arachidonic entry (17). Hence, following stimulation of receptors that elevate intracellular InsP3 levels, ICRAC appears to activate in an essen- Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, tially all-or-none manner. Although several negative feedback United Kingdom mechanisms can subsequently inhibit ICRAC and hence grade the 2ϩ Received for publication June 19, 2007. Accepted for publication August 12, 2007. overall extent of Ca influx (2, 18), the fact that ICRAC activates The costs of publication of this article were defrayed in part by the payment of page in an all-or-none manner following receptor stimulation neverthe- charges. This article must therefore be hereby marked advertisement in accordance less raises an intriguing paradox. Weak stimuli (presented as con- with 18 U.S.C. Section 1734 solely to indicate this fact. centrations of agonist that are below the affinity of the receptor for 1 This work was supported by an Medical Research Council Programme Grant (to its cognate ligand) often produce weak cellular responses whereas A.B.P.). W.-C.C. was a recipient of an Overseas Research Studentship award and J.D.C. held a Christopher Welch Scholarship. higher concentrations elicit stronger responses. If ICRAC is essen- 2 Address correspondence and reprint requests to Prof. Anant Parekh, Oxford Uni- tially an all-or-none process, how can responses be graded with versity, Parks Road, Oxford, U.K. E-mail address: [email protected] stimulus intensity? 3 2ϩ 2ϩ Abbreviations used in this paper: CRAC, Ca release-activated Ca ; cPLA2, We have addressed this issue by examining the effect of different 2ϩ 2ϩ Ca -dependent phospholipase A2; LTC4, leukotriene C4; InsP3, inositol 1,4,5- levels of receptor activation on ICRAC,Ca entry, protein kinase trisphosphate; 2-APB, 2-aminoethyldiphenyl borate. ␣ C , and ERK stimulation and subsequent activation of the cPLA2 Copyright © 2007 by The American Association of Immunologists, Inc. 0022-1767/07/$2.00 and 5-lipoxygenase enzymes in a mast cell line as well as in www.jimmunol.org 5256 ALL-OR-NONE CALCIUM INFLUX IN MAST CELLS acutely isolated rat peritoneal mast cells. Our new results demon- from a holding potential of 0 mV and normalized to cell size (capacitance). strate that increasing agonist concentration increases protein kinase Currents were filtered using an 8-pole Bessel filter at 2.5 kHz and digitized ␣ at 100 s. Capacitative currents were compensated before each ramp. Leak C and ERK activation, cPLA2 stimulation, and LTC4 secretion in 2ϩ currents were obtained by averaging two ramp currents taken just before a graded manner. However, activation of ICRAC and Ca influx application of carbachol and subtracting this from all subsequent record- remain essentially all-or-none processes. Responses appear graded ings. Series resistance was Ͻ10 MOhms. with stimulus intensity because increasing agonist concentration Preparation of cell lysates recruits more cells in the population but each cell that responds does so by activating Ca2ϩ influx in an essentially all-or-none Attached cells from 10-cm plastic dishes were washed twice with PBS and manner. Our results therefore identify a mechanism for eliciting lysed with PBS buffer containing 0.5% Triton X-100 and protease mixture inhibitor (Sigma-Aldrich), as described (12). Lysates were centrifuged at graded responses in a multicellular system despite all-or-none ac- 8000 rpm for 5 min, and the supernatants were collected and stored at tivation of CRAC channels by receptor stimulation. Ϫ70°C until used. Materials and Methods Western blotting Cell culture Total