Top Banner

of 12

Calcium Sensing Receptor Antagonists Abrogate Airway Hyperresponsiveness and Inflammation in Allergic Asthma

Feb 25, 2018

Download

Documents

Welcome message from author
This document is posted to help you gain knowledge. Please leave a comment to let me know what you think about it! Share it to your friends and learn new things together.
Transcript
  • 7/25/2019 Calcium Sensing Receptor Antagonists Abrogate Airway Hyperresponsiveness and Inflammation in Allergic Asthma

    1/12

    A S T H M A

    Calcium-sensing receptor antagonists abrogateairway hyperresponsiveness and inflammationin allergic asthma

    Polina L. Yarova,1* Alecia L. Stewart,2* Venkatachalem Sathish,2* Rodney D. Britt Jr.,2*

    Michael A. Thompson,2 Alexander P. P. Lowe,4 Michelle Freeman,2 Bharathi Aravamudan,2

    Hirohito Kita,3 Sarah C. Brennan,1 Martin Schepelmann,1 Thomas Davies,1 Sun Yung,1

    Zakky Cholisoh,4 Emma J. Kidd,4 William R. Ford,4 Kenneth J. Broadley,4 Katja Rietdorf,5

    Wenhan Chang,6 Mohd E. Bin Khayat,7 Donald T. Ward,7 Christopher J. Corrigan,8

    Jeremy P. T. Ward,8 Paul J. Kemp,1 Christina M. Pabelick,2 Y. S. Prakash,2 Daniela Riccardi1

    Airway hyperresponsivenessand inflammationare fundamental hallmarks of allergicasthmathat are accompanied by

    increases in certain polycations, such as eosinophil cationic protein. Levels of these cations in body fluids correlate

    with asthma severity. We show that polycations and elevated extracellular calcium activate the human recombinant

    and native calcium-sensing receptor (CaSR), leading to intracellular calcium mobilization, cyclic adenosine monophos-

    phate breakdown, and p38 mitogen-activated protein kinase phosphorylation in airway smooth muscle (ASM) cells.

    These effects can be prevented by CaSR antagonists, termed calcilytics. Moreover, asthmatic patients and allergen-

    sensitized mice expressed more CaSR in ASMs than did their healthy counterparts. Indeed, polycations induced hyper-reactivity in mouse bronchi, andthis effectwas preventedby calcilytics and absentin mice with CaSR ablationfrom ASM.

    Calcilytics also reduced airway hyperresponsiveness and inflammation in allergen-sensitized mice in vivo. These data

    show that a functional CaSR is up-regulated in asthmatic ASM and targeted by locally produced polycations to induce

    hyperresponsiveness and inflammation. Thus, calcilytics may represent effective asthma therapeutics.

    INTRODUCTION

    Despite substantial advances in our understanding of its pathophys-iology and improved therapeutic regimens, asthma remains a tremen-dous worldwide health care burden with around 300 million individualsufferers. Although the symptomsof asthma are potentially controllablein most asthma sufferers using conventional therapy such as topicalbronchodilators and corticosteroids, these are troublesome to adminis-

    ter efficiently and present unwanted side effects. There remains a signif-icant minority of patients whose symptoms fail to be controlled withthese approaches and who face chronically impaired quality of life withincreased risk of hospital admission and even death, although in aminority such patients account for the major share of asthma healthcare costs. Accordingly, there is an urgent unmet need for identificationof novel asthma therapies that target the root cause of the disease ratherthan its clinical sequelae.

    Asthma is characterized by inflammation-driven exaggeration ofairway narrowing in response to specificand nonspecific environmentalstimuli [nonspecific airway hyperresponsiveness (AHR)], as well aschronic remodeling of the conducting airways (1). A number of mech-anisms, many driven by inflammation, have been hypothesized to con-tribute to AHR and/or remodeling. Among these, there is increasingrecognition that airway inflammation results in augmented local con-

    centrations of polycations (27). The polycations eosinophil catioprotein (ECP) and major basic protein are well-established markforasthmaseverity andstability, with some evidence that they may cotribute directly to the pathogenesis of asthma (6, 810). Furthermoreasthma, increased arginase activity diverts L-arginine toward increasproduction of the polycations spermine, spermidine, and putresci(4, 5,11). Although in human peripheral blood monocytes spermi

    exhibits anti-inflammatory properties (12), associations between creases in polycations in the asthmatic airway mucosa and AHR/airwremodeling and inflammation (4,5,13) have long been apparent aascribed to their positive charge (9). However, the cause-effect relationsremains hitherto unexplained. Here, we provide evidence that activationthe cell surface, G protein (heterotrimeric guanine nucleotidebindprotein)coupled calcium-sensing receptor (CaSR) by polycations driAHR and inflammation in allergic asthma.

    The CaSR is the master controller of extracellular free ionized calum ion (Ca2+o) concentration via the regulation of parathyroid homone (PTH) secretion (14). Accordingly, CaSR-based therapeuticsused for the treatment of systemic disorders of mineral ion metabolisPharmacological activators of the CaSR (calcimimetics) are usedto trhyperparathyroidism, and negative allosteric modulators of the Ca(calcilytics) are in clinical development for treating autosomal domnant hypocalcemia (15).

    In addition to its pivotal role in divalent cation homeostasis, tCaSR is expressed in tissues not involved in mineral ion metabolissuch as the blood vessels, breast, andplacenta, where the CaSR regulamany fundamental processes including gene expression, ion chanactivity, and cell fate (16). Furthermore, altered CaSR expression halso been associated with several pathological conditions including flammation, vascular calcification, and certain cancers (1619). In thnoncalciotropictissues, the CaSR responds to a range of stimuli includ

    1School of Biosciences, Cardiff University, Cardiff CF10 3AX, UK. 2Department of Anes-thesiology, Mayo Clinic, Rochester, MN 55905, USA. 3Department of Medicine, MayoClinic, Rochester, MN 55905, USA. 4Division of Pharmacology, Cardiff University, Schoolof Pharmacy and Pharmaceutical Sciences, Cardiff University, Cardiff CF10 3XF, UK.5Department of Life, Healthand Chemical Sciences, TheOpen University, Milton KeynesMK7 6AA, UK. 6Department of Medicine, UCSF School of Medicine, San Francisco, CA 94143,USA. 7Facultyof Life Sciences, University of Manchester, Manchester M13 9PT,UK. 8Division ofAsthma, Allergy and Lung Biology, Kings College London, London SE1 9RT, UK.*These authors contributed equally to this work.Corresponding author. E-mail: [email protected] (D.R.); [email protected] (Y.S.P.)

    R E S E A R C H A R T I C L E

    www.ScienceTranslationalMedicine.org 22 April 2015 Vol 7 Issue 284 284ra60

  • 7/25/2019 Calcium Sensing Receptor Antagonists Abrogate Airway Hyperresponsiveness and Inflammation in Allergic Asthma

    2/12

    not only Ca2+obut also polyvalent cations,amino acids,ionic strength, andpH, makingthis receptor uniquely capable of integratingmultiple environmental signals. Owing toits ability to act as a multimodal chemo-sensor, the potential relevance of CaSRto asthma pathophysiology is manifold,

    yet there is currently no evidence regard-ing CaSR expression or function in asth-ma. In this regard, a fundamental aspectof asthma pathophysiology is elevatedintracellular calcium ion concentration([Ca2+]i) in airway smooth muscle (ASM)cells that is not only critical to theenhancedbronchoconstriction of nonspecific AHRbut also implicated in longer-term, likelygenomic effects that result in airway re-modeling such as increased ASM cellproliferation(leading to airway wall thick-ening) and deposition of extracellularmatrix components (20,21). There is cur-rently no information as to whether theCaSR can regulate [Ca2+]iin the asthmaticairways, even though a polycation sensorsuch as the CaSR, whose activation leadsto anincrease in [Ca2+]i, seems a likely can-didate. Therefore, we hypothesized thatif a CaSR was to be found in the airways,it would sense and respond not only toinflammation-enhanced Ca2+obut alsoto polycations such as the ECPs andL-argininederived polyaminesputrescine,spermidine, and spermine, whose produc-tion is markedly increased during asth-

    ma (37, 11) or by many RNArespiratoryviruses that exacerbate asthma, such as in-fluenza A and Newcastle disease virus,which either contain polyamines in the vi-ral envelope or produce them as part oftheir requirement for replication (22,23).To test our hypothesis, we examined hu-man ASM samples from nonasthmaticand asthmatic subjects, and used two mod-els of allergen-induced airway inflammation,together with a mouse model of targetedCaSR gene ablation from ASM.

    RESULTS

    CaSR expression in human andmouse airways is increasedduring asthmaIn human bronchialbiopsies and in mouseinterlobular bronchi, CaSR was immu-nolocalized within the SM22a-positivesmooth muscle layer, with additional ex-pression in bronchial epithelium (Fig. 1A).

    Fig. 1. CaSR immunolocalizes to human and mouse airways and is overexpressed in asthma. (A) Hman airway biopsy (upper panels) or mouse intralobular bronchi (lower panels) stained with CaSR antibo(red) and SM22a(green) show immunoreactivity in both smooth muscle and epithelium. Scale bars,10 m(B) Human and mouse ASM cells stained with anti-SM22aantibody and showing CaSR immunoreactivScale bars,100 mm. (C) qRT-PCR shows higher CaSR expression in moderate asthmatics than in healtsubjects [n = 4 patients per group, fold change versus healthy, mean (line) SD (box)]. (D) Western analyof CaSR protein shows substantially elevated CaSR expression in moderate asthmatics ( n= 5 patients pgroup). (E) Exposure of healthy human ASM cells to TNF-a(20 ng/ml) or IL-13 (50 ng/ml) for 48 hours signcantly increased CaSR protein expression (n= 5 patients per group) compared to vehicle control for either tokine. (F) CaSR mRNA expression was significantly greater in mice after induction of airway inflammation wMAs in comparison to unsensitized mice [n= 4 mice per group; 10 airways per mouse, fold change versunsensitized, mean (line) SD (box)]. Statistical comparisons were performed [on DDCtvalues for (C) a(F)] by two-tailed, unpaired Studentsttests (C, D, and F) and one-way analysis of variance (ANOVA) wBonferroni post hoctest (E). *P< 0.05, **P< 0.01, ***P< 0.001 versushealthy, control, or unsensitized, as shoSource data, details of the statistical analysis, and Pvalues are given in the Supplementary Materials.

    R E S E A R C H A R T I C L E

    www.ScienceTranslationalMedicine.org 22 April 2015 Vol 7 Issue 284 284ra60

  • 7/25/2019 Calcium Sensing Receptor Antagonists Abrogate Airway Hyperresponsiveness and Inflammation in Allergic Asthma

    3/12

    Isolated human and mouse ASM cells retained CaSR expression(Fig. 1B).

    Quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western analysis of human ASM cells demonstrated that bothCaSR mRNA (Fig. 1C) and protein (Fig. 1D) expression were increasedabout threefold in moderate asthmatics compared to nonasthmatics(healthy). Furthermore, in human ASM cells from healthy indivi-

    duals, 48 hours of exposure to the asthma-associated proinflammatorycytokines, tumor necrosis factora (TNF-a) and interleukin-13 (IL-13),significantly increased CaSR protein expression (Fig. 1E and fig. S1C).qRT-PCR of laser capture microdissected ASM layers of intralobularbronchi in lung sections showed an about threefold increase in CaSRmRNA expression in mixed allergen (MA)sensitized mice comparedto ASM from unsensitized mice (Fig. 1F). Together, these results indi-cate that the CaSR is present in human and mouse ASM and its expres-sion is increased in asthma. Furthermore, in vitroeffects of cytokines onhuman ASM, and the effects of MA sensitization in a mouse model ofallergic asthma, provide an evidence for the role of inflammation in up-regulation of CaSR expression.

    Polycations implicated in asthma pathogenesis activatethe human CaSRIn many cell types, CaSR activation results in an increase in intracellularCa2+ concentration([Ca2+]i) arising from mobilization of Ca

    2+

    i (14).Totest the hypothesis that polycations that are up-regulated during asthmaactivate theCaSR,we measuredchangesin[Ca2+]i in human embryonic kid-ney (HEK) 293 cells stably expressing the human CaSR (HEK-CaSR),or HEK293 cells stably expressing an empty vector (HEK-0). A repre-sentative Western blot of HEK-CaSR and HEK-0 is shown in fig. S2A.Consistent with this hypothesis, HEK-CaSR, but not HEK-0, cells ex-hibited significantincreases in [Ca2+]i after exposure to (i)ECP[10mg/ml,a concentration well below the cytotoxic levels (24) and comparable tothose measured in the sputum of some asthmatic subjects (6,7)], (ii)the polycationic peptide poly-L-arginine [PLA; 300 nM; a mimetic of ma-

    jor basic protein (8)], and (iii) the polycation spermine (1 mM) (Fig. 2Aand fig. S2, B to D, for single traces). For each of these agonists, the in-crease in [Ca2+]i was inhibited by the calcilytic NPS89636 (100 nM) (Fig.2A and fig. S2, B to D, for single traces). Additional calcilytics, NPS2143(1mM) or Calhex 231 (1 mM), also prevented spermine-induced CaSRactivation (Fig. 2A and fig. S2D).

    Calcilytics prevent increases in Ca2+i in ASM fromasthmatic patientsIn human ASM, several endogenously produced agents such as acetyl-choline (ACh) and histamine evoke increases in [Ca2+]i, which driveAHR, remodeling, and production of a range of inflammatory cytokinesand other mediators in asthma (20,25). Accordingly, we hypothesizedthat activation of CaSR in ASM also leads to an increase in [Ca2+]i, andpredicted that this effect would be enhanced in asthmatics.

    We found that the sensitivity of human asthmatic ASM in the ab-sence of agentsthat increase [Ca2+]i wassignificantly higher than that ofASM from nonasthmatics in the presence of 2 mM [Ca2+]o (Fig. 2B, leftpanel, and fig. S3, A and B, forsingletraces).Inhibitionof theCaSR withthe calcilytic NPS2143 (1 mM) blunted the [Ca2+]o hyperresponsivenessof asthmatic ASM cells, highlighting the functional role of CaSR in thissetting (Fig. 2B, right panel, and fig. S3, A and B, for single traces).

    Having determined the sensitivity of human ASM to [Ca 2+]o, wethen tested the ability of the CaSR to alter [Ca2+]iresponses to ACh

    in human asthmatic and healthy ASM. In the presence of physlogical [Ca2+]o[that is, 1 mM, at which the CaSR is half-maximaactive (14)], exposure to 1mM ACh produced the expected increase[Ca2+]i, which was significantly greater in ASM from asthmatic ptients (Fig. 2C). Inhibiting CaSR with NPS2143 reduced the [Ca 2

    response to ACh to levels that were not significantly different frothose measured in healthy ASM (Fig. 2C and fig. S3C). These effe

    were even more pronounced in the presence of 2 mM Ca2+

    o, a cocentration at which the CaSR is fully active, whereas they could notobserved in the presence of 0.5 mM [Ca 2+]o, which is below tthreshold for CaSR activation (fig. S3C).

    Histamine (1 mM) also evoked an increase in [Ca2+]i in both healtand asthmatic ASM, which was significantly greater in asthmatic AS(Fig. 2D). Preexposure of asthmatic ASM to calcilytic also reduced [Ca2

    responses to histamine so that they did not differ statistically frothose in healthy ASM (Fig. 2D). Furthermore, an alternative, membranimpermeant CaSR agonist, Gd3+ (0.1 mM), evoked a further increase[Ca2+]i in human ASM in the absence (fig. S3D) or presence (fig. S3E)histamine, effects that were greater in asthmatic than in nonasthmaASM cells. Together, these results demonstrate that, in ASM cells, tCaSR is functional and contributes to the regulation of baseline AS[Ca2+]i. Accordingly, in asthmatic ASM cells, the CaSR may contributo a higher baseline [Ca2+]i, a leading cause of AHR, whereas calcilytrestore baseline [Ca2+]i.

    Calcilytics abrogate signaling pathways characteristic ofairway contractility and asthma in human ASMTo determine potential mechanisms by which CaSR modulates cotractility relevant to asthma, we explored two mechanisms in noasthmatic and asthmatic ASM: cyclic adenosine monophospha(cAMP), which should induce bronchodilation, and phospholipaC (PLC)/inositol 1,4,5-trisphosphate (IP3), an important contributo bronchoconstriction, with the idea that CaSR activation shousuppress cAMP but elevate IP3(16). Indeed, in the presence of 2 m

    Ca2+

    o, cAMP levels were low, and calcilytics increased cAMP, partularly in asthmatic ASM (fig. S3F). Measurements of cellular IP3cotent showed that, particularly in asthmatic ASM, CaSR antagonsuppressed the elevated levels of IP3 in the presence of 2 mM Ca(fig. S3G).

    In addition to targeting phosphodiesterases to inhibit cAMbreakdown, many of the pipeline or existing drugs for asthma targactivation of signaling pathways dependent on extracellular signaregulated kinase 1/2 (ERK1/2), p38 mitogen-activated protein kina(MAPK), and phosphatidylinositol 3-kinase/Akt phosphorylation (26,2Therefore, we examined the effect of activation of the ASM CaSR these pathways in human ASM cells. In healthy ASM, CaSR activatiwith5mMCa2+o induced a significant increasein p38 MAPKphosphrylation, an effect that was prevented by co-incubation with a calcilyCalcilytic treatment reduced ERK1/2 and Akt phosphorylation at 5 mCa2+o (Fig. 2, E and F, and fig. S4 for technical replicates). Overall, thdata highlight the ability of CaSR to modulate signaling pathways ac

    vated during asthma, which may contribute to altered ASM functibeyond [Ca2+]i.

    SM22aCaSRflox/flox mice are protected frompolycation-induced bronchoconstrictionTo determine whether activation of the CaSR in ASM leads to AHin vivo, we generated mice with targeted CaSR ablation from visce

    R E S E A R C H A R T I C L E

    www.ScienceTranslationalMedicine.org 22 April 2015 Vol 7 Issue 284 284ra60

  • 7/25/2019 Calcium Sensing Receptor Antagonists Abrogate Airway Hyperresponsiveness and Inflammation in Allergic Asthma

    4/12

    SM cells by breeding LoxP-CaSR mice wSM22a-Cre mice (28, 29). The resultiSM22aCaSRflox/flox mice [knockout (KOfig. S5] were comparable to Cre-negat[wild-type (WT)] littermates in appeance, reproductive abilities, body weigand life expectancy (fig. S5, B and C

    Fluorescence-activated cell sorting analyshows that molecular CaSR ablation froASM resulted in a significant reductionCaSR immunoreactivity in KO cells, whwas 27% of that seen in WT cells. In cotrast, CaSR ablation from ASM cells dnot significantly alter the expressionthe smooth muscle marker SM22a (fS5D). Successful ASM CaSR ablation wdemonstrated functionally by the evidenthat Ca2+o(1 to 5 mM) and an alternatimembrane-impermeant CaSR agonGd3+ (100mM to 1 mM), evoked an crease in [Ca2+]iin WT ASM cells, whwas significantly greater than that mesured in cells from KO mice (fig. S5upper panels for single traces and lowpanels for biological replicates). Nevtheless, lungs from KO mice appeared htomorphologically comparable to thoseWT mice and did not exhibit fibrosis, flammation, or impaired alveolarizati(fig. S5F). Intralobular bronchi from Wand KO mice also had comparable lumidiameters (fig. S5G).

    The intrinsic baseline contractilityintralobular bronchi was not affected

    CaSR ablation from ASM cells, as showby exposure either to high K+ (40 mfig. S5H) or to increasing concentratioof ACh (1 nM to 30 mM), both of whevoked bronchoconstriction of comprable magnitude in WT and KO moubronchi (Fig. 3A). In intralobular brochi from WT mice, treatment with eithspermine (300 mM, Fig. 3B) or 2.5 m[Ca2+]o(Fig. 3E) enhanced the bronchconstrictor response to ACh. CaSR ablatfrom ASM blunted both spermine-induc(Fig. 3C) and [Ca2+]o-induced (Fig. 3sensitization of the ACh response. Furthmore, spermine (10mM to 3 mM) inducsensitization of the ACh response in pcontracted WT, but notin KO mouse brochi (Fig. 3D). Consistentwith these findinspermine also enhanced the responseACh (0.5mM) in precision-cut lung slifrom WT animals (Fig. 3G, and summain Fig. 3H). This effect was prevented calcilytic treatment (NPS89636, 300 nFig. 3H). However, the effects of eith

    Fig. 2. Polycations activate the human CaSR in recombinant systems and human ASM cells, partic-ularly those from asthmatics.(A) ECPs (n= 7), PLA (n= 6), or spermine (n= 17) each increased [Ca2+]iinHEK-CaSR, but not in HEK-0 cells (ECP, n = 3; PLA,n = 6; spermine,n = 6). In HEK-CaSR cells, the calcilyticNPS89636 prevented these increases (ECP, n = 3;PLA, n = 3; and spermine, n = 4). Two alternative calcilytics,NPS2143 (n = 4)and Calhex231 (n = 5), alsopreventedspermine-induced CaSRactivation. (B) InhumanASMcells, exposure to 2 mM [Ca2+]oincreased [Ca

    2+]iin asthmatic but not in healthy ASM cells (left), an effectpreventedby the calcilytic NPS2143 (right) (n =3each).(C and D) Inthe presenceof 1 mMCa2+o, exposure toACh (C) (n = 4 healthy, n = 4 asthmatic) or histamine (D) (n = 5 healthy, n = 4 asthmatic) resulted in increasesin [Ca2+]i, which was greater in asthmatic ASM cells. This effect was prevented by NPS2143. ns, not signif-icant. (E and F) Western analysis [exemplar gel (E) and summary data (F)] of healthy ASM cell lysates showsthe effects of 5 mM Ca2+oin the absence or presence of NPS2143 on Akt, p38 MAPK, and ERK phosphoryl-ation (n = 17 to 19 independentexperimentsfrom cells isolated from n = 2 nonasthmaticpatients). Statisticalsignificance was determined by one-way ANOVA with Bonferroni post hoc test (A), two-way ANOVA withBonferroniposthoc test (B to D), or one-way ANOVA with Dunn post hoc test(F).*P< 0.05, **P< 0.01, ***P 0.05; n =3 per experimental group) and up to 24 hours (1.0 0.1 mM and 1.0 0.1 mM, 4 and 24 hours after calcilytic treatment, respectively;P> 0.05;n = 3 perexperimentalgroup). These results suggest that theobserved effectsof the calcilytics are not ascribed to systemic changes in Ca2+o homeostasis.

    DISCUSSION

    Ourstudy highlights the expression of the CaSR in ASMand identifies afundamental pathophysiological role for this receptor in the context of

    asthma. The fact that inflammatory cationic proteins known to corlate with asthma severity can activate the CaSR expressed by ASM ceat physiologically relevant concentrations to elevate [Ca2+]i and increthe contractility of the ASM nonspecifically provides both a rationalplanation for the genesis of nonspecific AHR in asthma and a basis the direct mechanistic link between this phenomenon and airway flammation. These findings raise the possibility that the CaSR direc

    influences mechanisms involved in inflammatory cell recruitment aactivation. In turn, production of asthma-relevant cytokines can furthincrease CaSR expression, thereby generating a positive feedback looThus, locally delivered calcilytics would have the advantage of breakthis cycle by reducing inflammation and by blunting ASM hypersponsiveness. Indeed, in two different in vivo models of allergic asthminterfering with CaSR signaling positively affects multiple aspectsairway disease, benefits not achieved by single-drug therapies. In tsense, the CaSR represents a truly novel potential therapeutic targin asthma.

    [Ca2+]ois known to be increased at inflammation sites (18, 3therefore activating the CaSR, leading to an increase in [Ca 2+]i ap38 MAPK activation and a decrease in the intracellular cAMP poIn addition, the CaSR is activated by a plethora of molecules, particlarly polyamines, whichact orthosterically (independently of [Ca2+]o)help stabilize the unique conformations of the receptor. This leato preferential coupling to different G proteins, a process definedligand-directed targeting of receptor stimulus (33). The relevance of tCaSR to local and systemic symptoms in asthma and other airway deases is potentially immense, extending beyond its innate expressionASM and any local regulation of [Ca2+]o. In asthma, airway inflammtionleads to increased release of polycations, which are accepted markof asthmaseverity, locally andinto the systemic circulation (27). SputuECP concentrations in asthmatics have been reported to attain abo10 mg/ml (6, 7), which here we show to be well within the concentratirange sufficient to activate the CaSR. Added to this is arginase-drivproduction of spermine, spermidine, and putrescine, which are incre

    ingly implicated in asthma pathophysiology (4,5,11). From an enronmental perspective, CaSR agonists may also be presented to tairways in the form of smoke (Ni 2+) or car fumes (Pb2+ and Cd(34) and bacterial/viral infections (polyamines) (22,23).

    In addition to elevating [Ca2+]i and, therefore, priming ASM cellsrespond with a lower threshold to pathophysiological stimuli, Camay also enhance sensitization of airways to Ca2+, for example, via copling to protein kinase C and Rho kinase, as demonstrated in other csystems, namely, HEK-CaSR (35), a topic that is currently unexplorin the lung but is highly relevant to the increasing interest in targetisensitization mechanisms (36). Beyond contributions to AHR aairway remodeling (25), calcilytics prevent activation of intracellupathways, which are currently being targeted by pipeline asthma druspecifically p38 MAPK and phosphodiesterase inhibitors (26). Indeboth classes of inhibitors target various inflammatory cells, which lease key mediators responsible for the remodeling and inflammaticharacteristic of these diseases. For this reason, local delivery of calcitics has the potential to target not only one of the key possible causesasthma but also the production of proinflammatory cytokines that cotribute to its exacerbations. Consistent with this hypothesis is the abiof the calcilytics to reduce inflammatory cell infiltration in the BALFOVA-sensitized mice.

    Our ex vivo experiments show that activation of the airway Caincreases responses to bronchoconstrictors by about 20 to 25%. Alb

    Fig. 4. Activation of the airway CaSR exacerbates AHR in vivo. (A) In me-chanically ventilated, unsensitized mice, acute exposure to the calcimimeticR568 increased bronchoconstriction to MCh challenge, measured as increasedairway resistance (RL). The calcilytic prevented the AHR induced by R568, butevinced little effect on its own. (B) MA-sensitized mice showed enhanced re-sponse to MCh. Preexposure to the calcimimetic resulted in a further increasein RL , whereas thecalcilytic NPS2143reduced AHR. Statistical comparisons be-tween thecurveswere made by two-way ANOVA, Bonferroni post hoctest. n = 5per condition. **P< 0.01, ***P< 0.001 statistically different from control, ###P