Circulation Research 2012 Yamamura 469 81

Integrative Physiology

Enhanced Ca2�-Sensing Receptor Function in IdiopathicPulmonary Arterial Hypertension

Aya Yamamura,* Qiang Guo,* Hisao Yamamura,* Adriana M. Zimnicka, Nicole M. Pohl,Kimberly A. Smith, Ruby A. Fernandez, Amy Zeifman, Ayako Makino, Hui Dong, Jason X.-J. Yuan

Rationale: A rise in cytosolic Ca2� concentration ([Ca2�]cyt) in pulmonary arterial smooth muscle cells (PASMC)is an important stimulus for pulmonary vasoconstriction and vascular remodeling. Increased resting [Ca2�]cyt

and enhanced Ca2� influx have been implicated in PASMC from patients with idiopathic pulmonary arterialhypertension (IPAH).

Objective: We examined whether the extracellular Ca2�-sensing receptor (CaSR) is involved in the enhanced Ca2�

influx and proliferation in IPAH-PASMC and whether blockade of CaSR inhibits experimental pulmonary hypertension.Methods and Results: In normal PASMC superfused with Ca2�-free solution, addition of 2.2 mmol/L Ca2� to

the perfusate had little effect on [Ca2�]cyt. In IPAH-PASMC, however, restoration of extracellular Ca2� induceda significant increase in [Ca2�]cyt. Extracellular application of spermine also markedly raised [Ca2�]cyt inIPAH-PASMC but not in normal PASMC. The calcimimetic R568 enhanced, whereas the calcilytic NPS 2143attenuated, the extracellular Ca2�-induced [Ca2�]cyt rise in IPAH-PASMC. Furthermore, the protein expression levelof CaSR in IPAH-PASMC was greater than in normal PASMC; knockdown of CaSR in IPAH-PASMC with siRNAattenuated the extracellular Ca2�-mediated [Ca2�]cyt increase and inhibited IPAH-PASMC proliferation. Usinganimal models of pulmonary hypertension, our data showed that CaSR expression and function were both enhancedin PASMC, whereas intraperitoneal injection of the calcilytic NPS 2143 prevented the development of pulmonaryhypertension and right ventricular hypertrophy in rats injected with monocrotaline and mice exposed to hypoxia.

Conclusions: The extracellular Ca2�-induced increase in [Ca2�]cyt due to upregulated CaSR is a novel pathogenicmechanism contributing to the augmented Ca2� influx and excessive PASMC proliferation in patients andanimals with pulmonary arterial hypertension. (Circ Res. 2012;111:469-481.)

Key Words: pulmonary artery � smooth muscle cell � proliferation � G-protein–coupled receptor� pulmonary hypertension � receptors

Idiopathic pulmonary arterial hypertension (IPAH) is a fataland progressive disease with unidentified etiologic causes.

Pulmonary vascular remodeling and sustained pulmonary vaso-constriction are 2 major causes for the elevated pulmonaryvascular resistance and pulmonary arterial pressure in patientswith IPAH. A central aspect of pulmonary vascular remodelingis intimal and medial hypertrophy caused by enhanced prolifer-ation and inhibited apoptosis of pulmonary arterial smoothmuscle cells (PASMC).1 An increase in cytosolic free Ca2�

concentration ([Ca2�]cyt) in PASMC is a major trigger for pulmo-nary vasoconstriction and an important stimulus for PASMCproliferation leading to pulmonary vascular remodeling.2–4

In PASMC, [Ca2�]cyt can be increased by Ca2� releasefrom the intracellular stores and Ca2� influx through plasma-lemmal Ca2� channels.1,5 PASMC express various Ca2�-permeable channels including voltage-dependent Ca2� chan-nels (VDCC), receptor-operated Ca2� channels (ROC), andstore-operated Ca2� channels (SOC). VDCC are activated bymembrane depolarization. ROC channels are mainly openedby vasoconstrictors (eg, endothelin-1 and serotonin) via anintracellular second messenger, diacylglycerol (DAG), andby growth factors (eg, epidermal growth factor and platelet-derived growth factor). Activation of ROC by interactionwith ligands greatly contributes to the increase in [Ca2�]cyt

Original received February 2, 2012; revision received June 14, 2012; accepted June 22, 2012. In May 2012, the average time from submission to firstdecision for all original research papers submitted to Circulation Research was 12.0 days.

From the Kinjo Gakuin University School of Pharmacy, Nagoya, Japan (A.Y.); First Affiliated Hospital of Soochow University, Suzhou, China (Q.G.);Nagoya City University Graduate School of Pharmaceutical Sciences, Nagoya, Japan (H.Y.); Department of Medicine, Institute for PersonalizedRespiratory Medicine, Center for Cardiovascular Research, Department of Pharmacology, University of Illinois at Chicago (A.M.Z., N.M.P., K.A.S.,R.A.F., A.Z., A.M., J.X.-J.Y.); and the Department of Medicine, University of California, San Diego, La Jolla, CA (H.D.).

*These authors contributed equally to this workThe online-only Data Supplement is available with this article at http://circres.ahajournals.org/lookup/suppl/doi:10.1161/CIRCRESAHA.112.

266361/-/DC1.Correspondence to Jason X.-J. Yuan, MD, PhD, Department of Medicine, University of Illinois at Chicago, COMRB, Room 3131 (MC 719), 909 S

Wolcott Ave, Chicago, IL 60612. E-mail [email protected]© 2012 American Heart Association, Inc.

Circulation Research is available at http://circres.ahajournals.org DOI: 10.1161/CIRCRESAHA.112.266361

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due to receptor-operated Ca2� entry (ROCE) in PASMC. Onactivation of membrane receptors and subsequent increase ininositol 1,4,5-trisphosphate (IP3) synthesis, SOC are opened bythe depletion of Ca2� from the sarcoplasmic reticulum (SR), animportant intracellular store in PASMC, to elicit store-operatedCa2� entry (SOCE) or capacitative Ca2� entry. SOCE is animportant mechanism involved in maintaining a sustained ele-vation of [Ca2�]cyt and refilling Ca2� into the SR.

We have previously demonstrated that the resting [Ca2�]cyt

is increased, whereas SOCE and ROCE are both enhanced inPASMC isolated from IPAH patients in comparison to PASMCisolated from normal subjects and patients without pulmonaryhypertension.6,7 We have also shown that increased Ca2� influxduring PASMC proliferation is due largely to the enhancementof SOCE; downregulation and blockade of SOC significantlyinhibits PASMC proliferation. These results indicate that SOCEplays an important role in regulating cell proliferation in vascularsmooth muscle cells.2–4,8 Furthermore, expression and activityof ROC and SOC are both upregulated in PASMC isolated frompatients with IPAH and animals with hypoxia-induced pulmo-nary hypertension.2,9–11

Opening of ROC and SOC is caused initially by activationof various membrane receptors including G protein-coupledreceptors (GPCR) and receptor tyrosine kinases. The extra-cellular Ca2�-sensing receptor (CaSR) is a member of GPCRsubfamily C (also known as GPRC2A),12–14 which wasoriginally identified in the parathyroid glands and is activatedby its ligands including Ca2�, Gd3�, Mg2�, polyamines,antibiotics, and amino acids.12,15 The CaSR is involved inmultiple cellular processes of the parathyroid glands inresponse to changes in serum Ca2� concentrations such asproliferation, differentiation, and apoptosis.12,13,15 In additionto parathyroid glands, the CaSR is also expressed in kidney,bone, smooth muscle, endothelium, gastrointestinal tract, andbrain.13,16–19 The physiological and pathological significance

of CaSR in the development and progression of pulmonaryarterial hypertension, however, remains unknown.

In this study, we examined whether CaSR was involved in theenhanced Ca2� signaling and augmented proliferation inPASMC from patients with IPAH and whether inhibition ofCaSR attenuates IPAH-PASMC proliferation and prevents thedevelopment of experimental pulmonary hypertension in animalmodels.

MethodsPreparation of Human and Animal PASMCHuman PASMC were isolated from normal control subjects andpatients with IPAH and chronic thromboembolic pulmonary hyper-tension (CTEPH).8,20,21 Approval to use the human lung tissues andcells was granted by the UIC Institutional Review Board. HumanPASMC were cultured in Medium 199 supplemented with 10% fetalbovine serum at 37°C. The cells at passages 5 to 8 were used for theexperiments. In some experiments, we also used freshly-dissociatedPASMC from rats22 and PASMC from mice.23

[Ca2�]cyt Measurement[Ca2�]cyt was measured in PASMC using fura-2 and a Nikon digitalfluorescent imaging system.24 Cells were loaded with 4 �mol/Lfura-2 acetoxymethyl ester (fura-2/AM) for 60 minutes at 25°C and[Ca2�]cyt was measured using a ratiometric method at 32°C.

Western BlotSolubilized protein isolated from PASMC, pulmonary arteries, andlung tissues was loaded on an 8% acrylamide gel, transferred to anImmobilon-P transfer membrane (Millipore, Bedford, MA), andimmunoblotted with anti-CaSR monoclonal antibody (MA1–934,1:200; Thermo Scientific, Rockford, IL). To isolate the fraction ofcellular membrane, the cell lysate was centrifuged at 100 000g andthen the pellet was resuspended to use for Western blot analysis.Signals were detected using a Super Signal West Pico Chemilumi-nescent Substrate (Thermo Scientific). The protein levels werenormalized to �-tubulin (sc-9104, 1:200; Santa Cruz Biotechnology)and expressed in arbitrary units.

Transfection of cDNA and Small Interfering RNAPASMC were transiently transfected with vector cDNA (2 �g,pcDNA3.1[�]), human CaSR cDNA (2 �g), control small interfer-ing RNA (siRNA) (50 nmol/L, sc-37007; Santa Cruz Biotechnol-ogy), or CaSR siRNA (50 nmol/L, s2440; Applied Biosystems,Austin, TX) using an Amaxa Basic Nucleofector kit for primarysmooth muscle cells (Lonza). [Ca2�]cyt measurements and Westernblot using cDNA- and siRNA-transfected cells were preformed 48 to72 hours after electroporation.

Proliferation AssayProliferation of PASMC was determined using an automated cellcounter (TC10; Bio-Rad Laboratories, Hercules, CA). At 48 hoursafter subculture, PASMC were counted and replated (0 hours) into8-well multidishes (Nunclon �, 10.5 cm2 of culture area per well;Thermo Scientific) with 1�105 cells/well (0.95�104 cells/cm2).

Preparation of MCT-PH Rats andHypoxia-Induced PH MiceAll experiments were approved by the Ethics/Animal Care Commit-tee of University of Illinois at Chicago. For MCT-PH rat experi-ments, male Sprague-Dawley rats (190–200 g) were treated withsingle subcutaneous (sc) injection of vehicle (dimethyl sulfoxide,DMSO) or 60 mg/kg MCT. For NPS 2143-treated group, rats wereintraperitoneally injected (ip) with NPS 2143 at a dose of 4.5 mg/kgper day (from day 1–10). Fourteen days after injection, rats wereanesthetized with ketamine/xylazine and then RVSP was measuredusing an MPVS Ultra system (Millar Instruments). For hypoxia-

Non-standard Abbreviations and Acronyms

[Ca2�]cyt cytosolic free Ca2� concentration

CaSR Ca2�-sensing receptor

CTEPH chronic thromboembolic pulmonary hypertension

DAG diacylglycerol

GPCR G-protein–coupled receptor

HPH hypoxia-induced pulmonary hypertension

IP3 inositol 1,4,5-trisphosphate

IPAH idiopathic pulmonary arterial hypertension

MCT monocrotaline

PASMC pulmonary arterial smooth muscle cell

ROC receptor-operated Ca2� channels

ROCE receptor-operated Ca2� entry

RVSP right ventricular systolic pressure

SOC store-operated Ca2� channels

SOCE store-operated Ca2� entry

SR sarcoplasmic reticulum

TB trypan blue

VDCC voltage-dependent Ca2� channels

470 Circulation Research August 3, 2012

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induced pulmonary hypertension (HPH) mouse experiments, malemice (8-week-old C57BL/6) were exposed to hypoxia (10% O2) in aventilated chamber to develop pulmonary hypertension. For NPS2143-treated group, mice were injected (ip) with NPS 2143 (1.0mg/kg per day; from day 1–22). Four weeks after exposure tonormobaric hypoxia, mice were anesthetized with ketamine/xyla-zine, and then RVSP was measured by right heart catheterization.

Reverse Transcription-PCRThe extraction of total RNA from rat PASMC and the reverse transcrip-tion were performed using TRIzol Reagent (Invitrogen) and High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, FosterCity, CA), respectively. For semiquantitative analysis of mRNA, Plat-inum PCR SuperMix (Invitrogen) and specific primers for rat CaSR andGAPDH were used. Quantitative real-time PCR analysis was performedbased on the SYBR assay (SYBR Green Master Mix; Roche AppliedScience, Indianapolis, IN) using gene-specific primers for rat CaSR andGAPDH on a Bio-Rad CFX384 Real-Time System C1000 ThermalCycler system (Bio-Rad Laboratories, Hercules, CA).

Immunohistochemical and Hematoxylin andEosin StainingImmunohistochemistry and hematoxylin and eosin staining wereperformed using formalin-fixed and paraffin-embedded sections(3 �m) from left lung lobes of rats. The tissue sections were treatedwith PBS containing 5% normal goat serum and either CaSR (1:100dilution) or SM �-actin (1:100; EMD Millipore) antibody for 12hours at 4°C. After washing repeatedly in PBS, the sections werecovered with PBS containing Alexa Fluor 488-labeled (1:500 dilu-tion) or Cy3-labeled (1:500) secondary antibody for 1 hour at roomtemperature and then rinsed with PBS. Then sections were mountedin VECTASHIELD hard-set mounting medium with DAPI (1.5�g/mL). To measure the external diameter of pulmonary arteriesstained with hematoxylin and eosin, the microscopic images wereanalyzed using ImageScope software (Ver.11).

DrugsPharmacological reagents were obtained from Sigma-Aldrich exceptfor KB-R7943, NPS 2143, and R568 (Tocris, Ellisville, MO). Allhydrophobic compounds were dissolved in DMSO at the concentra-tion of 10 or 100 mmol/L as a stock solution.

Statistical AnalysisComposite data are shown as the mean�SE. The statistical signifi-cance between two groups was determined by Student t test. Thestatistical significance among groups was determined by Scheffe testafter 1-way analysis of variance. Significant difference is expressedas P�0.05 or P�0.01.

ResultsExtracellular Ca2� Induces a Significant Increasein [Ca2�]cyt in IPAH-PASMC But Not inNormal PASMCWe first examined and compared the effects of extracellularCa2� restoration on changes in [Ca2�]cyt in PASMC fromnormal subjects, IPAH patients and CTEPH patients. Innormal PASMC superfused with Ca2�-free solution (plus1 mmol/L EGTA; for 10 minutes), restoration of extracellularCa2� (2.2 mmol/L) into the bath solution had no effect on[Ca2�]cyt (Figure 1A through 1C). Even after long exposure(30 and 60 minutes) of normal PASMC to Ca2�-free solution,there was no change in [Ca2�]cyt after restoration of extra-cellular Ca2�. In IPAH-PASMC superfused with Ca2�-freesolution (for 10 minutes); however, restoration of extracellu-lar Ca2� resulted in a significant increase in [Ca2�]cyt in 96%of the cells tested. The extracellular Ca2�-induced increase in

[Ca2�]cyt in IPAH-PASMC was independent of the exposuretime (5–30 minutes) to Ca2�-free solution. Interestingly, asshown in Figure 1A through 1C (IPAH1 versus IPAH2), thekinetics of the extracellular Ca2�-induced increases in [Ca2�]cyt

were different in IPAH-PASMC. Approximately 48% of IPAH-PASMC tested (IPAH1) exhibited a rapid transient increase in[Ca2�]cyt, whereas more than 50% of the cells (IPAH2) had asustained plateau phase of [Ca2�]cyt increase after the initialtransient (Figure 1A, middle panels, and Figure 1C). In PASMCisolated from CTEPH patients superfused with Ca2�-free solu-tion (for 10 minutes), restoration of extracellular Ca2� had noeffect on [Ca2�]cyt (Figure 1A through 1C).

The extracellular Ca2�-induced [Ca2�]cyt increase inIPAH-PASMC was dependent on Ca2� concentration (Figure1D); the EC50 was approximately 1.22 mmol/L (n�57–183cells), which resulted an increase of [Ca2�]cyt by 1,250 nmol/L(Figure 1D, right panel). In contrast, we were unable to detect asignificant increase in [Ca2�]cyt in normal PASMC with resto-ration of 10 mmol/L extracellular Ca2� (Figure 1D).

Protein Expression of CaSR in IPAH-PASMC IsGreater Than in Normal PASMCTo investigate the potential mechanism of extracellular Ca2�-mediated increase in [Ca2�]cyt, we compared the proteinexpression level of CaSR in normal and IPAH PASMC. Asshown in Figure 2, the protein expression of CaSR inIPAH-PASMC (Figure 2A and 2B) and lung tissues fromIPAH patients (Figure 2C and Online Figure I) was signifi-cantly higher than in normal PASMC and lung tissues in bothtotal protein lysates and in the membrane bound fraction(n�5, P�0.01). These data indicate that upregulation ofCaSR may be involved in the enhanced Ca2�-induced[Ca2�]cyt increase in IPAH-PASMC.

The Extracellular Ca2�-Induced [Ca2�]cyt Rise inIPAH-PASMC Is Not Due Simply toCa2� LeakageTo rule out the possibility that the extracellular Ca2�-induced[Ca2�]cyt increase in IPAH-PASMC was caused by mem-brane leakage to Ca2�, we first compared the resting[Ca2�]cyt in normal and IPAH-PASMC. In cells superfusedwith 2.2 mmol/L Ca2�-containing solution, the resting[Ca2�]cyt in IPAH-PASMC (n�370 cells) was significantlyhigher than in normal PASMC (n�96 cells). Removal ofextracellular Ca2� negligibly affected the resting [Ca2�]cyt innormal PASMC but significantly decreased the resting[Ca2�]cyt in IPAH-PASMC (Online Figure IIA). To examinewhether IPAH-PASMC had leaky membranes, we incubatednormal and IPAH PASMC with trypan blue (TB). No blue(TB-stained) cells were detected in normal and IPAH PASMCincubated in TB-containing solution, whereas after treatment ofthe cells with 10 �mol/L ionomycin (for 10 minutes), a Ca2�

ionophore, all normal and IPAH PASMC were TB-stained; therewas no difference between normal and IPAH PASMC (OnlineFigure IIB). Furthermore, we were unable to detect any fluores-cent signals in normal and IPAH PASMC incubated with themembrane-impermeable fura-2 (Online Figure IIC). All theseexperiments indicate that the extracellular Ca2�-induced

Yamamura et al Ca2�-Sensing Receptor in Pulmonary Hypertension 471



[Ca2�]cyt increase in IPAH-PASMC was not due to Ca2�

leakage through the plasma membrane.

Effects of Specific CaSR Modulators on theExtracellular Ca2�-Induced [Ca2�]cyt Increase inNormal and IPAH-PASMCTo further confirm that the upregulated CaSR in IPAH-PASMC is involved in the enhanced extracellular Ca2�-induced [Ca2�]cyt rise, we examined whether the polyaminespermine and the calcimimetic R568 induced the same effecton [Ca2�]cyt as did restoration of extracellular Ca2�, andwhether the calcilytic NPS 2143 inhibited the extracellularCa2�-induced [Ca2�]cyt increase. As shown in Figure 3A,extracellular application of 3 mmol/L spermine, a CaSRagonist, induced a slight increase in [Ca2�]cyt (52�24nmol/L, n�13) in normal PASMC in the presence of2.2 mmol/L extracellular Ca2�. In IPAH-PASMC; however,extracellular application of spermine caused a huge [Ca2�]cyt

increase (1308�123 nmol/L, n�49; P�0.01 versus normalPASMC) in the presence of 2.2 mmol/L extracellular Ca2�. InIPAH-PASMC, short-time treatment with the positive allostericmodulator of CaSR, R568 (1 �mol/L) significantly enhanced the

extracellular Ca2�-mediated increase in [Ca2�]cyt (Figure 3B),whereas the negative allosteric modulator of CaSR, NPS 2143(10 �mol/L), significantly inhibited extracellular Ca2�-mediatedincrease in [Ca2�]cyt (Figure 3C). Collectively, these resultsdemonstrate that the upregulated CaSR (Figure 2) is involved inthe enhancement of the extracellular Ca2�-induced [Ca2�]cyt

increase in PASMC from IPAH patients.

Extracellular Ca2�-Induced [Ca2�]cyt Increase inIPAH-PASMC Is Dependent of Phospholipase Cand the Inositol-2,4,5-Trisphosphate ReceptorTo examine the potential signaling pathway involved in theCaSR-mediated increase in [Ca2�]cyt in IPAH-PASMC, weperformed pharmacological experiments on the extracellularCa2�-mediated increase in [Ca2�]cyt. As shown in Figure 4,short-time pretreatment of IPAH-PASMC with the inhibitorof phospholipase C (PLC), U73122 (1 �mol/L), or thespecific blocker of Inositol-2,4,5-Trisphosphate receptor(IP3R), xestospongin C (3 �mol/L), significantly inhibited theextracellular Ca2�-induced increase in [Ca2�]cyt (OnlineFigure IIIA and C). The inactive form of U73122, U73343(1 �mol/L), however, had no effect on the extracellular

Figure 1. Enhancement of extracellularCa2�-induced increase in [Ca2�]cyt inPASMC from IPAH patients. A throughC, Representative records (A), pseudo-color images (B), and summarized data(mean�SE) (C) showing extracellularCa2�-mediated changes in [Ca2�]cyt inPASMC from normal subjects (n�45cells), IPAH patients (n�160 cells), andCTEPH patients (n�27 cells). Two kineti-cally different responses of [Ca2�]cyt toextracellular Ca2� in PASMC from 2IPAH patients are shown in the middle.D, Representative traces of [Ca2�]cytchanges in response to extracellularapplication of 0.1, 0.5, 1.1, 2.2, and10 mmol/L Ca2� (left) and the dose-response curves (right) in normal PASMC(top) and IPAH-PASMC (bottom). TheEC50 for extracellular Ca2�-induced[Ca2�]cyt increase in IPAH-PASMC is1.22 mmol/L.




Ca2�-induced increase in [Ca2�]cyt (Online Figure IIIB).Pretreatment of IPAH-PASMC with the blocker of VDCC,diltiazem (10 �mol/L) or the inhibitor of Na�/Ca2� ex-changer KB-R7943 (10 �mol/L), however, had no effect onthe extracellular Ca2�-induced increase in [Ca2�]cyt (OnlineFigure IIID and E). These results indicate that activation ofPLC and IP3R is involved in the CaSR-mediated [Ca2�]cyt

increase in IPAH-PASMC, whereas VDCC and the reversemode of Na�/Ca2� exchanger are not involved in the Ca2R-mediated Ca2� influx or inward transportation.

Downregulation of CaSR in IPAH-PASMCInhibits Extracellular Ca2�-Induced [Ca2�]cyt

Increase and Attenuates Cell ProliferationTo obtain direct evidence for the involvement of CaSR inextracellular Ca2�-induced [Ca2�]cyt increase and cell prolifer-ation, we used siRNA to knockdown CaSR expression andexamined whether CaSR was necessary for the extracellularCa2�-induced [Ca2�]cyt increase in IPAH-PASMC. Treatmentof IPAH-PASMC with 50 nmol/L siRNA significantly de-creased protein level of CaSR (Figure 4A) and markedlyinhibited the extracellular Ca2�-induced increase in [Ca2�]cyt

(Figure 4B). In comparison to normal PASMC, the proliferaterate of IPAH-PASMC, determined by a change in cell number,was much faster (Figure 4C), whereas downregulation of CaSRin IPAH-PASMC by transiently transfecting 50 nmol/L siRNAfor CaSR significantly attenuated cell proliferation (Figure 4D).These experiments provide compelling evidence that CaSR isnecessary for the augmented extracellular Ca2�-induced

[Ca2�]cyt increase and enhanced cell proliferation inIPAH-PASMC.

Overexpression of CaSR in Normal PASMCAugments the Extracellular Ca2�-Induced[Ca2�]cyt Increase and Enhances Cell ProliferationExtracellular application of either 2.2 mmol/L Ca2� or3 mmol/L spermine failed to induce a significant increase in[Ca2�]cyt in normal PASMC because of a low proteinexpression level of CaSR (Figures 2 and 3). We examinedwhether CaSR was sufficient to mediate extracellular Ca2�-induced [Ca2�]cyt increase in both human and rat PASMC bytransiently transfecting the human CaSR into normalPASMC. As shown in Figure 5, overexpression of CaSR(with 2 �g of the human CaSR cDNA) in normal (human andrat) PASMC significantly augmented the extracellular Ca2�-mediated increase in [Ca2�]cyt (Figure 5A and 5B) andenhanced cell proliferation (Figure 5C) in comparison tonormal PASMC transiently transfected with an empty vector.Taken together with the data showed earlier (Figure 4), theseresults indicate that (1) CaSR is sufficient to mediate extra-cellular Ca2�-induced [Ca2�]cyt increase and cell prolifera-tion in normal PASMC, and (2) CaSR is necessary for theaugmented extracellular Ca2�-induced [Ca2�]cyt increase andenhanced cell proliferation in IPAH-PASMC.

CaSR Is Functionally Upregulated in PASMCfrom Animal Models of Experimental PulmonaryHypertension, and Blockade of CaSR Prevents theDevelopment of PHThe presented in vitro experimental data show that theupregulated CaSR and augmented extracellular Ca2�-mediated [Ca2�]cyt increase in PASMC are involved in theenhanced PASMC proliferation in patients with IPAH. Toinvestigate whether CaSR can be a target for treatment ofpulmonary arterial hypertension, we used the rat model ofmonocrotaline (MCT)-induced pulmonary hypertension(MCT-PH) and the mouse model of HPH to test the potentialtherapeutic effect of the calcilytic NPS 2143. We firstexamined and compared the mRNA and protein expressionlevel of CaSR in PASMC from control and MCT-treated rats.As shown in Figure 6A and 6B, the mRNA level of CaSR inPASMC isolated from rats (rPASMC) with MCT-PH wasmuch greater than in PASMC isolated from normotensivecontrol rats injected with vehicle. The immunohistochemistryand immunoblotting experimental data indicate that the pro-tein expression level of CaSR in the small pulmonary artery(Figure 6C) and PASMC (Figure 6D) of MCT-rats wassignificantly higher than in the small pulmonary artery andPASMC of control rats. Furthermore, the basal or resting[Ca2�]cyt and the extracellular Ca2�-induced increase in[Ca2�]cyt were both enhanced in freshly-dissociated PASMCfrom MCT-PH rats compared with freshly-dissociatedPASMC from normotensive control rats (Figure 6E). Treat-ment with NPS 2143 not only decreased the basal [Ca2�]cyt,but also inhibited the extracellular Ca2�-induced increase in[Ca2�]cyt in PASMC isolated from MCT-PH rats (Figure 6E).These results imply that upregulation of CaSR and subse-quent enhancement of extracellular Ca2�-induced [Ca2�]cyt

Figure 2. Upregulation of CaSR in PASMC from IPAHpatients. A, Western blot analysis on CaSR in total proteins(top) and membrane proteins (bottom) isolated from normalPASMC (Nor) and IPAH-PASMC (IPAH). B, Summarized data(mean�SE), bottom showing CaSR protein levels that were nor-malized to the �-tubulin level. **P�0.01 versus normal PASMC.C, Western blot analysis on CaSR in total and membrane pro-teins isolated from lung tissues of normal subjects and IPAHpatients.




increase in PASMC contribute to the development of pulmo-nary hypertension in rats injected with MCT.

To test the in vivo therapeutic effect of the CaSR antago-nist, we examined and compared the right ventricular systolicpressure (RVSP), the Fulton index [ie, the ratio of rightventricle/left ventricle�septum, RV/(LV�S)], and muscular-ization of distal pulmonary arteries in normotensive control(Norm) rats and MCT-injected rats with and without treat-ment with NPS 2143, a CaSR antagonist. Injection of MCT(60 mg/kg) in rats significantly increased RVSP and causedright ventricular (RV) hypertrophy compared with the nor-motensive control (Norm) rats injected with vehicle (DMSO)(Figure 7A through 7C). Intraperitoneal injection of NPS2143 (4.5 mg/kg per day) had little effect on RVSP andRV/(LV�S) ratio in Norm rats but significantly attenuatedthe increase in RVSP and the Fulton index in MCT-PH rats(Figure 7A through 7C). There were no significant changes in

heart rate in Norm rats with (412�17 bpm, n�6) or without(411�21 bpm, n�6) NPS treatment and MCT-injected ratswith (414�23 bpm, n�6) or without (413�21 bpm, n�6)NPS treatment. The MCT-induced increases in RVSP and RVhypertrophy were associated with significant pulmonary vas-cular remodeling; the vascular medial wall thickness of smallpulmonary arteries with the outer diameter less than 100 �mwas significantly greater in MCT-injected rats than in Normrats (Figure 7D and 7E). Treatment with the CaSR antagonist(NPS 2143) significantly inhibited the muscularization ofsmall pulmonary arteries (Figure 7D and 7E). The in vivoanimal experiments are consistent with the in vitro experi-ments using normal and IPAH PASMC.

To further validate the pathogenic role of upregulated CaSRin the development of pulmonary hypertension and the thera-peutic effect of CaSR antagonists on experimental pulmonaryhypertension, we repeated the experiments mentioned above in

Figure 3. Effects of CaSR modulators on[Ca2�]cyt in IPAH-PASMC. A, Representativetraces (left) and summarized data (mean�SE),right showing the effect of spermine (3 mmol/L), a CaSR agonist, on [Ca2�]cyt in the absence(0 Ca) and presence (2.2 Ca) of extracellularCa2� (2.2 mmol/L) in normal (n�30) and IPAHPASMC (n�52). **P�0.01 versus normalPASMC (spermine/0Ca); ##P�0.01 versus nor-mal PASMC (spermine/2.2 Ca). B and C, Rep-resentative records of [Ca2�]cyt changes (left)and summarized data (mean�SE) (right) of thestimulatory effect of R568 (1 �mol/L, n�63, B),a positive allosteric modulator of CaSR, andthe inhibitory effect of NPS-2143 (10 �mol/L,n�120 (C), a negative allosteric modulator ofCaSR, on the 0.5 mmol/L extracellular Ca2�-induced [Ca2�]cyt increases in IPAH-PASMC.**P�0.01 versus control IPAH-PASMC (withouttreatment with vehicle, R568 or NPS 2143).




the HPH mouse model. As shown in Figure 8, the mRNA andprotein expression of CaSR was significantly higher in pulmo-nary arteries and lung tissues in HPH mice than in normoxiccontrol mice (Figure 8A and 8B), whereas the extracellularCa2�-induced increase in [Ca2�]cyt in freshly isolated PASMCfrom HPH mice was significantly enhanced in comparison tocells from normoxic mice (Figure 8C). These data indicate thatCaSR is functionally upregulated in PASMC from HPH mice.

Intraperitoneal injection of NPS 2143 (1 mg/kg per day fromday 1–10) had little effect on RVSP and RV/(LV�S) ratio innormoxic control mice (Nor) but significantly inhibited theincrease in RVSP and RV/(LV�S) ratio in hypoxic mice (Hyp)(Figure 8D through 8F). Furthermore, inhibition of CaSR alsosignificantly inhibited the hypoxia-mediated pulmonary arterialwall thickening (Figure 8G) and reversed the hypoxia-induceddecrease in branch and junction numbers (and total length) ofsmall pulmonary arterial trees (Figure 8H and 8I). There were nosignificant changes in heart rate in normoxic mice with (411�17bpm, n�6) or without (410�20 bpm, n�6) NPS treatment andhypoxic mice with (412�18 bpm, n�6) or without (413�18bpm, n�6) NPS treatment.

These data imply that intraperitoneal injection of the CaSRantagonist is an efficient therapeutic approach to inhibit thedevelopment and progression of pulmonary vascular remod-eling and right ventricular hypertrophy in animal models withexperimental pulmonary hypertension induced by injection ofmonocrotaline and exposure to hypoxia. The observationsfrom this study strongly indicate that increased expressionand function of CaSR may play a pathogenic role in thedevelopment of pulmonary vascular remodeling and antago-nists of CaSR, or calcilytics, may have great therapeuticpotential for patients with pulmonary arterial hypertension.

DiscussionIn this study, we found that (1) extracellular application of Ca2�

(0.5–10 mmol/L) and spermine (3 mmol/L) induced a largeincrease in [Ca2�]cyt in IPAH-PASMC but not in normalPASMC; (2) the protein expression level of CaSR in IPAH-PASMC was greater than in normal PASMC; (3) downregula-tion of CaSR in IPAH-PASMC (with siRNA) inhibited theextracellular Ca2�-induced increase in [Ca2�]cyt and attenuatedcell proliferation, whereas overexpression of CaSR in normalPASMC augmented the extracellular Ca2�-induced rise in[Ca2�]cyt and enhanced cell proliferation; (4) the expression andfunction of CaSR were also increased in PASMC from rats withMCT-PH and mice with HPH, and intraperitoneal injection of aCaSR antagonist (NPS 2143) significantly inhibited pulmonaryvascular remodeling and attenuated the development and pro-gression of the experimental pulmonary hypertension. Collec-tively, the observations from this study indicate that (1) func-tionally upregulated CaSR and subsequently augmentedextracellular Ca2�-induced rise in [Ca2�]cyt in PASMC contrib-ute to the enhanced Ca2� signaling and excessive cell prolifer-ation in IPAH patients; and (2) blockade of the upregulatedCaSR with calcilytics may be a novel therapeutic approach forpulmonary arterial hypertension.

[Ca2�]cyt plays an important role in the regulation ofcontraction, proliferation, and migration of PASMC. Anincrease in [Ca2�]cyt in PASMC is a major trigger forpulmonary vasoconstriction and an important stimulus forPASMC proliferation and pulmonary vascular remodelingunder pathological conditions. Elevation of [Ca2�]cyt inPASMC results from Ca2� release from intracellular storesand Ca2� influx through plasmalemmal Ca2� channels.1,5 We

Figure 4. Downregulation of CaSR with siRNAattenuates extracellular Ca2�-induced [Ca2�]cytincreases in IPAH-PASMC and inhibits IPAH-PASMC proliferation. A, Western blot analysis onCaSR in IPAH-PASMC treated with a control (orscrambled) siRNA (control) and siRNA for CaSR (at25 and 50 nmol/L, n�3 for each group). B, Repre-sentative records of [Ca2�]cyt changes (left) andsummarized data (mean�SE) (right) showing theextracellular Ca2�-induced [Ca2�]cyt increases inIPAH-PASMC transfected with control siRNA(n�57) and CaSR-siRNA (n�57). **P�0.01 versuscontrol-siRNA. C, Summarized data (mean�SE)showing the total cell numbers of normal PASMC(open circles) and IPAH-PASMC (solid circles) aftercultured in growth media for 8, 24, 48, 72, and 96hours. The growth curves for normal and IPAHPASMC are significantly different (P�0.001, n�3experiments). D, Summarized data (mean�SE,n�3 experiments) showing inhibitory effects ofCaSR-siRNA on the cell proliferation in IPAH-PASMCs. **P�0.01 versus control-siRNA.




previously showed that the resting [Ca2�]cyt was increased,whereas ROCE and SOCE were enhanced in PASMC fromIPAH patients compared with PASMC from normal subjectsand patients without pulmonary hypertension.6,7

CaSR is a GPCR (belongs to the Family C GPCR) with 1085amino acids, which is present constitutively in a homodimericconfiguration formed by covalent and noncovalent linkages25–27

and is able to form heterodimers with the metabotropic gluta-mate receptors (mGLuR1 and mGluR5).27 One of the hallmarksof CaSR is the cysteine-rich large N-terminal extracellulardomain (ECD, approximately 600 amino acids). The regionbetween alanine 116 and proline 136 in the ECD is important formaintaining CaSR in an inactive conformation28 and is associ-ated with the activating mutations or single nucleotide poly-morphism (SNPs) identified in the human CaSR gene. Theligands or activators of CaSR include polyvalent cations (eg,Ca2�, Mg2�, Gd3�), polypeptides (eg, amyloid-� peptide),polyamines (eg, spermine, spermidine, putrescine), aminoglyco-side antibiotics (eg, neomycin, kanamycin), and amino acids (eg,phenylalanine, tyrosine, tryptophan, glutamate). In addition,there are synthetic CaSR activators, or calcimimetics (eg, NPS-R-568, NPS-R-467), and CaSR antagonists, or calcilytics (eg,NPS 2143), that affect CaSR function.12–14,29–31 The cysteine-rich domain in the ECD also sensitizes the receptor to redoxchanges and hypoxia/hyperoxia. In Sprague-Dawley rats, treat-ment with the CaSR activator or the calcimimetic R-568 atten-

uates aortic wall thickening induced by uremia.32 Our data fromthis study, however, indicate that the extracellular Ca2�-mediated [Ca2�]cyt increase in IPAH-PASMC was potentiatedby NPS-R-568, an allosteric agonist of CaSR, and inhibited byNPS 2143, an allosteric antagonist of CaSR. Extracellularapplication of spermine significantly increased [Ca2�]cyt inIPAH-PASMC superfused in Ca2�-containing solution. Thesedata imply that multiple ligands can activate the upregulatedCaSR in IPAH-PASMC leading to cell proliferation, contractionand migration via Ca2� signaling and other signal transductioncascades.

Extracellular Ca2� binding to CaSR is a highly cooperativeprocess. The EC50 for extracellular Ca2�-induced [Ca2�]cyt

increase in IPAH-PASMC is approximately 1.2 mmol/L (Figure1D), whereas the EC50 has been reported to be 1.7 mmol/L inparathyroid cells,33 1.5 mmol/L in cardiomyocytes,34 and5.6 mmol/L in bronchial epithelial cells.35 However, in recon-stituted systems with CaSR clones isolated from parathyroidglands, the EC50 for CaSR activation (or the Ca2� sensitivity ofCaSR) is 3.0 to 3.5 mmol/L.12,13,15,36–38 The lower EC50 forCa2�-induced [Ca2�]cyt rise in IPAH-PASMC and native vas-cular smooth muscle cells (compared with the EC50 for therecombinant CaSR) is presumably due to the high degree ofcooperativity in the interaction between the upregulated CaSR(eg, in IPAH-PASMC) and ligands. CaSR is always exposed tovarious coagonists physiologically (eg, Mg2�, polyamines, and

Figure 5. Overexpression of CaSR in normalPASMC enhances extracellular Ca2�-induced[Ca2�]cyt increase and promotes cell prolifera-tion. A and B, Representative records of [Ca2�]cytchanges (left) and summarized data (mean�SE)(right) extracellular Ca2�-induced [Ca2�]cytincreases in human (A) and rat (B) PASMC trans-fected with an empty vector (vector, n�12) andthe human CaSR cDNA (CaSR, n�8). C, Summa-rized data (mean�SE) showing the total numbersof normal human PASMC transfected with thevector (solid circles) and CaSR gene (gray circles)after incubation in growth media for 24, 48, 72, 96,and 120 hours. The growth curves for vector con-trol PASMC and IPAH PASMC are significantlydifferent (P�0.01, n�3 experiments). **P�0.01 ver-sus vector. D, Western blot analysis on CaSR inhuman PASMC transfected with an empty vector(Vector) or CaSR.




amino acids) and other activators pathologically (eg, antibiotics,heavy metals, and amyloid-� peptide). Therefore, the sensitivityof CaSR to extracellular Ca2� is expected to be higher inpathologically conditions than in physiological conditions.

As mentioned earlier, the CaSR senses extracellular Ca2�

concentration and transduces it to intracellular space thoughmultiple signal pathways.12–14,18 CaSR interacts directly with G

proteins (Gq� and G11�). Activation of CaSR by extracellularCa2� (or calcimimetics) induces increases in [Ca2�]cyt throughPLC-mediated hydrolysis of phosphatidylinositol-4,5-bisphosphate (PIP2) to inositol-1,4,5- trisphosphate (IP3) anddiacylglycerol (DAG). IP3 binds to the IP3 receptor (IP3R) on theSR membrane and induces Ca2� release from the SR to thecytosol. Depletion (or reduction) of Ca2� from the intracellularstores (ie, SR) via activated IP3R (or opened Ca2� releasechannels) then causes SOCE through SOC in the plasmamembrane. Furthermore, DAG causes ROCE by activatingROC in the plasma membrane. The IP3-mediated Ca2� mobili-zation, the store depletion-mediated SOCE and the DAG-mediated ROCE all contribute to the increase in [Ca2�]cyt onactivation of CaSR in the plasma membrane by extracellularCa2� and CaSR activators. In IPAH-PASMC, the extracellularCa2�-mediated [Ca2�]cyt increases were significantly attenuatedby the PLC inhibitor (U73122) and the IP3R blocker (xesto-spongin C) but not affected by the voltage-dependent Ca2�

channel blocker (diltiazem) and the Na�/Ca2� exchanger inhib-itor (KB-R7943) (Online Figure III). These data further confirmthe important role of the PLC-IP3 signaling cascade in CaSR-mediated increase [Ca2�]cyt and its proliferative effect onPASMC isolated from patients with IPAH. Most membranereceptors, including many GPCRs, become desensitized withprolonged exposure to agonists. However, CaSR desensitizesvery slowly,39 indicating that CaSR signals for long periods oftime through the regulation of intracellular signaling cascadesand other signal transduction pathways.

In addition to increasing [Ca2�]cyt, extracellular Ca2�-mediated activation of CaSR has been linked to several signaltransduction cascades. CaSR activates the mitogen-activatedprotein kinase (MAPK) cascade, for example, extracellularsignal-regulated kinase 1/2 (ERK1/2), p38, and c-jun N-terminalkinase (JNK), potentially through the interaction with filamin Ascaffolding protein and caveolin-1 in cholesterol-rich invagina-tions in the plasma membrane known as caveolae.16,17,40–44 InIPAH-PASMC, the number of caveoli and the protein expres-sion level of caveolin-1/-2 are both increased, which are asso-ciated with increased SOCE.45 Downregulation of caveolin-1with siRNA and treatment with methyl-�-cyclodextrin (M�CD)significantly diminish the number of caveoli on the surfacemembrane of IPAH-PASMC, significantly attenuate SOCE, andmarkedly inhibit cell proliferation.6,45 It is unknown, however,whether upregulated CaSR in IPAH-PASMC is exclusively orpredominantly localized in caveoli and functionally interactswith upregulated TRPC3/C6 channels.7,8

CaSR is widely distributed in the parathyroid glands,15

kidney,46 bone,47 gastrointestinal tract,48 skin,49 brain,50 im-mune cells,51 and heart.34 In addition, CaSR is expressed invascular smooth muscle cells from rat subcutaneous artery,52

aorta,53 pulmonary artery,16,17 gerbil spiral modiolar artery,54

human renal artery,55 aorta,55,56 and internal mammary ar-tery,57 as well as in endothelial cells from rat mesenteric artery,porcine coronary artery,58 and human aorta.19 Activation ofCaSR in vascular smooth muscle cells increases [Ca2�]cyt andinduces vasoconstriction17,54,56; therefore, CaSR is involved inregulating myogenic tone, peripheral vascular resistance,52 andarterial blood pressure.59,60 Recent observations demonstrate thatthe CaSR in vascular smooth muscle cells contributes to regu-

Figure 6. Upregulation of CaSR in PASMC from rats withMCT-induced pulmonary hypertension. A and B, Regular (A)and real-timer (B) RT-PCR analyses on CaSR in PASMC iso-lated from normal rats (Norm, n�6) and rats with MCT-inducedpulmonary hypertension (MCT, n�5). **P�0.01 versus Norm. C,Immunohistochemistry data showing CaSR expression level(green fluorescence intensity) in normal and MCT-treated rats.D, Western blot analysis on CaSR in PASMC isolated from nor-mal and MCT-PH rats. *P�0.05, **P�0.01 versus Norm. E,Summarized data (mean�SE) showing the basal [Ca2�]cyt (left,n�12) and the amplitude of extracellular Ca2�-induced [Ca2�]cytincreases (right, n�11) in PASMC freshly isolated from normalrats and MCT-injected rats that are treated with vehicle (�NPS)or 10 �mol/L NPS 2143 (�NPS), a synthetic calcilytic. **P�0.01versus norm (�/�NPS).




lating cell proliferation and apoptosis through the MEK1/ERK1/2 and PLC signaling pathways17,53,55 and expression ofCaSR is involved in the regulation of mouse lung develop-ment.61 Our study indicated that in normal human PASMC,CaSR protein was expressed at a low level and extracellularapplication of Ca2� (from 0.5–10 mmol/L) failed to cause asignificant increase in [Ca2�]cyt (Figure 1D), whereas extracel-lular application of spermine caused a small increase in [Ca2�]cyt

(Figure 3A). These observations imply that CaSR is expressed atvery low level in normal human PASMC and is not a majorcontributor to the regulation of pulmonary vascular tone underphysiological conditions.

In patients with IPAH and animals with experimental pulmo-nary hypertension, CaSR is functionally upregulated in PASMCand thus becomes an important GPCR involved in the initiationand progression of pulmonary vascular remodeling and pulmo-nary hypertension. Knockdown of CaSR with siRNA inPASMC from IPAH patients not only diminished extracellularCa2�-induced increase in [Ca2�]cyt but also inhibited cell pro-liferation (Figure 4). Overexpression of CaSR in PASMC fromnormal subjects conferred an extracellular Ca2�-induced in-crease in [Ca2�]cyt and enhanced cell proliferation (Figure 5).

These experimental data provide compelling evidence that CaSRis necessary and sufficient for the augmented Ca2� signaling andexcessive PASMC proliferation in IPAH patients. In this sce-nario, Ca2� is an extracellular ligand and an intracellularsignaling element involved in the development and progressionof sustained pulmonary vasoconstriction and vascular remodel-ing in patients with IPAH. The pathogenic role of upregulatedCaSR in pulmonary hypertension is further confirmed by thetherapeutic effect of CaSR antagonists on experimental pulmo-nary hypertension in rats injected with MCT and in miceexposed to chronic hypoxia.

The gain-of-function (or activating) mutations or SNP inthe human CaSR gene causes autosomal dominant hypocal-cemia62 and Bartter syndrome type V.12,63 CaSR antagonists(calcilytics), that is, negative allosteric modulators that indi-rectly stimulate parathyroid hormone secretion through adecrease in CaSR activity, are potential drug candidates forthe treatment of osteoporosis and other bone metabolismdiseases.64,65 Using 2 well-established animal models ofpulmonary hypertension (MCT-injected rats and chronichypoxic mice), we found that increased RVSP and pulmonaryvascular medial hypertrophy, as well as right ventricular

Figure 7. Blockade of CaSR by NPS2143 inhibits the development of pul-monary vascular remodeling and pul-monary hypertension in rats injectedwith MCT. A and B, Representative rec-ord of right ventricular pressure (RVP, A)and summarized data (mean�SE) show-ing the peak value of right ventricular sys-tolic pressure (RVSP, B) in normal controlrats (norm, n�6) and MCT-injected rats(MCT, n�6) that are treated with vehicle(�NPS) or NPS 2143 (�NPS, 4.5 mg/kgonce a day). C, Averaged Fulton index[RV/(LV�S) ratio, mean�SE] showing thatRV hypertrophy is significantly inhibited inMCT-rats treated with NPS. *P�0.05 ver-sus MCT along. D and E, Representativehematoxylin and eosin images of smallpulmonary arteries (D) and summarizeddata of the medial thickness of pulmonaryarteries with a diameter (Ø) less than50 �m, between 50 and 100 �m, andgreater than 100 �m (E) in normal controlrats (norm, n�6) and MCT-injected rats(MCT, n�6) that are treated with vehicleor NPS 2143. *P�0.05 versus MCT-treated rats (red bars).




hypertrophy [determined by the Fulton index, RV/(LV�S)]were associated with upregulated CaSR expression and en-hanced extracellular Ca2�-induced [Ca2�]cyt rise (and basal[Ca2�]cyt) in PASMC (Figures 6 through 8). Intraperitonealinjection of the CaSR antagonist NPS 2143 (4.5 mg/kg once aday) had little effect on the pulmonary hemodynamics and theFulton index in control rats or normoxic mice, but significantlydecreased RVSP, RV/(LV�S) ratio and small pulmonary vas-cular wall thickening in rats with MCT-PH (Figure 7) and micewith HPH (Figure 8). These results strongly suggest that CaSRis involved in the development of experimental pulmonaryhypertension and a potential target for developing therapeuticapproach for pulmonary arterial hypertension.

In conclusion, upregulated expression of CaSR in PASMCand augmented function of CaSR through intracellular Ca2�

signaling (and other signal transduction cascades) are new

pathogenic mechanisms involved in the initiation and pro-gression of pulmonary vascular remodeling in patients withpulmonary arterial hypertension. Pharmacological blockadeof CaSR in the pulmonary vasculature by synthetic calcilyticsand downregulation of CaSR by siRNA (and/or specificmicroRNA) may be a novel therapeutic approach for IPAHpatients who do not respond to the conventional drug therapy.

Sources of FundingThis work was supported in part by grants from the National Heart,Lung, and Blood Institute of the National Institutes of Health(HL066012 and HL098053 to J.X.-J.Y.).

DisclosuresNone.

Figure 8. Upregulation of CaSR in PASMC from HPH mice and blockade of CaSR by NPS 2143 inhibits the development of HPHin mice. A and B, Real-time RT-PCR (A) and Western blot (B) analyses on CaSR in PA and lung tissues isolated from normoxic (Nor)and hypoxic (Hyp) mice. C, Representative record (left) and summarized data (mean�SE, right) showing the extracellular Ca2�-inducedincrease in [Ca2�]cyt in PASMC isolated from normoxic and hypoxic mice. **P�0.01, ***P�0.001 versus Nor. D and E, Representativerecord of right ventricular pressure (RVP, D) and summarized data (mean�SE) showing the peak value of right ventricular systolic pres-sure (RVSP, E) in normoxic (Nor, n�6) and hypoxic (Hyp, n�6) mice that are treated with vehicle or NPS 2143 (�NPS, 1 mg/kg once aday). F, Averaged Fulton index [RV/(LV�S) ratio, mean�SE] in normoxic and hypoxic mice treated with or without NPS 2143. *P�0.05versus Hyp along. G, Representative hematoxylin and eosin images of small pulmonary arteries showing that the medial thickness issignificantly increased in Hyp mice and the hypoxia-induced medial hypertrophy is inhibited by NPS 2143. H, Representative angiogra-phy of the whole lung (top) and enlarged area of the whole lung (bottom, indicated by the box in the top) in normoxic and hypoxicmice treated with vehicle or NPS (�NPS). The vertical bars denote 2 mm (top) and 0.5 mm (bottom). I, Summarized data (mean�SE)showing the number of branches, the number of junctions, and the total length of vascular segments per square millimeter. **P�0.01versus other bars.




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Novelty and Significance

What Is Known?

● Pulmonary vascular remodeling and sustained pulmonary vasocon-striction contribute to the development of idiopathic pulmonaryarterial hypertension (IPAH).

● Increased cytosolic free Ca2� concentration ([Ca2�]cyt) stimulatespulmonary arterial smooth muscle (PASMC) proliferation leading topulmonary vascular remodeling.

● Ca2�-sensing receptor (CaSR) is a G-protein–coupled receptorimportant for multiple cellular processes of the parathyroid glandssuch as proliferation, differentiation, and apoptosis.

What New Information Does This Article Contribute?

● CaSR is functionally upregulated in IPAH-PASMC contributing toenhanced Ca2� signaling and excessive cell proliferation in IPAHpatients.

● Blockade of the CaSR with an antagonist inhibits the development ofpulmonary hypertension in animal models.

● Targeting the CaSR may be a novel therapeutic approach for IPAHpatients.

Idiopathic pulmonary arterial hypertension is a rare, progressiveand fatal disease that predominantly affects women. The

pathogenic mechanisms involved in the pulmonary vascularabnormalities (eg, arterial remodeling and sustained vasocon-striction) in IPAH patients remain unclear. An increase incytosolic Ca2� concentration ([Ca2�]cyt) in PASMC is a majortrigger for pulmonary vasoconstriction and an important stimu-lus for PASMC migration and proliferation (which subsequentlycause pulmonary vascular wall thickening leading to the in-crease in pulmonary vascular resistance). In this study, wereport that a unique G-protein–coupled receptor (GPCR), CaSR,is significantly upregulated in PASMC isolated from patients withIPAH and animals with experimental pulmonary hypertension.The upregulated CaSR is necessary for the enhanced extracel-lular Ca2�-induced increase in [Ca2�]cyt and the augmented cellproliferation in IPAH-PASMC. Pharmacological blockade of CaSRwith a calcilytic, NPS 2143, markedly inhibits the extracellularCa2�-induced rise in [Ca2�]cyt and attenuates the developmentof experimental pulmonary hypertension in animal models.These data indicate that functionally upregulated CaSR inPASMC may play an important role in causing sustainedpulmonary vasoconstriction and excessive pulmonary vascularremodeling in IPAH patients. Targeting CaSR in PASMC may helpdevelop novel therapeutic approach for pulmonary hypertension.




Online Supplementary METHODS Preparation of human and animal PASMC. Human PASMC were isolated from explanted lung tissues of normal control subjects (2 unsuitable organ donors and 2 COPD patients without pulmonary hypertension) and patients with IPAH (3 patients diagnosed on the basis of NIH IPAH Registry with an averaged mean PAP of 56±5 mmHg).1, 2 In addition, PASMC were isolated and prepared from endarterectomized pulmonary arterial tissues of patients with chronic thromboembolic pulmonary hypertension (CTEPH, 4 patients with an averaged mean PAP of 39±5 mmHg)3. Approval to use the human lung tissues and cells was granted by the UIC Institutional Review Board. Human PASMC were cultured in Medium 199 supplemented with 10% fetal bovine serum (Invitrogen, Grand Island, NY), 100 U/ml penicillin plus 100 g/ml streptomycin, 50 g/ml D-valine (Sigma-Aldrich, St. Louis, MO), and 20 g/ml endothelial cell growth supplement (BD Biosciences, Franklin Lakes, NJ) at 37ºC. The cells at passages 5 to 8 were used for the experiments. In some experiments, we also used freshly-dissociated PASMC from rats (by a modified protocol based on the methods published previously).4 In the modified protocol, cell suspensions were plated onto cover slips for a short time (2-3 hrs) before experiments. We also used PASMC from mice by a modified method previously published by Marshall et al.5. [Ca2+]cyt measurement. The [Ca2+]cyt measurement was performed as described previously6. In brief, cultured PASMC on cover slips were placed in a recording chamber on the stage of an invert fluorescent microscope (Eclipse Ti-E; Nikon, Tokyo, Japan) equipped with an objective lens (S Plan Fluor 200.45 ELWD; Nikon), an EM-CCD camera (Evolve; Photometrics, Tucson, AZ), and NIS Elements 3.2 software (Nikon). The cells were loaded with 4 M fura-2 acetoxymethyl ester (fura-2/AM; Invitrogen/Molecular Probes, Eugene, OR) for 60 min at room temperature (25C). The HEPES-buffered external solution contained an ionic composition of 137 mM NaCl, 5.9 mM KCl, 2.2 mM CaCl2, 1.2 mM MgCl2, 14 mM glucose, and 10 mM HEPES (pH 7.4 with NaOH). In the Ca2+-free solution, 2.2 mM CaCl2 was replaced by equimolar MgCl2 and 1 mM EGTA was added to chelate the residual Ca2+. The fura-2-loaded cells in the recording chamber were continuously perfused with HEPES-buffered solution at 32C for 30 min to wash out extracellular fura-2/AM and allow sufficient time for the intracellular esterase to cleave acetoxymethyl ester from fura-2 so it can stay in the cytosol. The cells were then excited with 340-nm and 380-nm wavelength (D340×v2 and D380×v2 filters, respectively; Chroma Technology, Bellows Falls, VT) by a xenon arc lamp (Lambda LS; Sutter Instrument, Novato, CA) and an optical filter changer (Lambda 10-B; Sutter Instrument). Fura-2 fluorescence (or emission of fura-2) from a region of interest (5×5 μm) of PASMC, as well as background fluorescence, was collected through a dichroic mirror (400DCLP; Chroma Technology) and a wide band emission filter (D510/80m; Chroma Technology). The fluorescence signals emitted from the region of interest in cells were monitored and recorded continuously on a Nikon digital imaging fluorescence microscopy system and recorded in a PC. The ratio (R) of 340-nm and 380-nm fluorescence intensities (F340/F380), recorded every 2 seconds, was then used to calculate [Ca2+]cyt using the following equation: [Ca2+]cyt = Kd × (Sf2/Sb2) × (R-Rmin)/(Rmax-R), where Sf2 and Sb2 are the emission fluorescence values at 380-nm excitation in the presence of EGTA and triton X-100, respectively; Kd (225 nM) is the dissociation constant of the Ca2+-fura-2 complex; and Rmax and Rmin were calculated according to the standard protocol.7, 8

Pharmacological Experiments in Rats with MCT-PH. Adult male Sprague-Dawley rats (190-200 g in body weight; Charles River Laboratories, Wilmington, MA) were randomly divided into two groups: 1) normal control group which was injected subcutaneously of vehicle (DMSO); 2) MCT-PH group which was given a subcutaneous injection of MCT (60 mg/kg, Sigma-Aldrich) to induce pulmonary hypertension. The normal control group was further divided into two subgroups (n=6 for each): one subgroup received the intraperitoneal injection of NPS2143 (Tocris, solubilized into DMSO) at a dose of 4.5 mg/kg per day (from day 1 to day 10) and the other subgroup was injected intraperitoneally with the vehicle (DMSO) (from day 1 to day 10). The MCT-PH group was also further divided into two subgroups (n=6 for each): one subgroup received the intraperitoneal injection of NPS2143 at a dose of 4.5 mg/kg per day (from day 1 to day 10) and the other subgroup was injected intraperitoneally with the vehicle (DMSO) (from day 1 to day 10). Two weeks (14 days) later, the rats were anesthetized with ketamine (100 mg/kg, i.p.) and xylazine (26 mg/kg, i.p.) and then right ventricular pressure (RVP) was measured by a pressure-transducer catheter (Millar Instruments: SPF869 and SPF1030) inserted into the right ventricle through the right jugular vein. The hemodynamic data were recorded using an MPVS Ultra® data acquisition system and analyzed with an AD Instruments Lab Chart Pro7.0 software. The protocol used for the rat experiments was approved by the Animal Care Committee of the University of Illinois at Chicago. Pharmacological Experiments in Mice with HPH. Male C57BL/6N mice (18-22 g in body weight and 6 weeks of age, Charles River Laboratories, Wilmington, MA) were randomly divided into two groups: normoxic control group and hypoxic group which were placed into a normobaric hypoxic chamber (10% O2) for 4 weeks to establish pulmonary hypertension. The normoxic group was further divided into two subgroups (n=6 for each): one subgroup received the intraperitoneal injection of NPS2143 at a dose of 1 mg/kg per day (from day 1 to day 22) and the other subgroup was injected intraperitoneally with the vehicle (DMSO) (from day 1 to day 22). The hypoxic group was also further divided into two subgroups (n=6 for each): one subgroup received the intraperitoneal injection of NPS2143 at a dose of 1 mg/kg per day (from day 1 to day 22) and the other subgroup was injected intraperitoneally with the vehicle (DMSO) (from day 1 to day 22). Four weeks (28 days) later, the mice were anesthetized with ketamine/xylazine (i.p.) and then right ventricular pressure (RVP) was measured by the right heart catheterization similar to that protocol for rats. The hemodynamic data were recorded using the MPVS Ultra® data acquisition system and analyzed with the AD Instruments Lab Chart Pro7.0 software. The protocol used for the mouse experiments was approved by the Animal Care Committee of the University of Illinois at Chicago. Assessment of Pulmonary Artery Thickness. The left lung lobes (from rats or mice) were fixed in a 3% paraformaldehyde solution and the lobes were dissected in the middle for paraffin embedding. Sectioning at 3 µm was performed for all paraffin-embedded tissue blocks. H&E staining was performed according to common histopathological procedures. Histological analysis was performed in a blinded way: the person who dissected the lobes coded the tissue blocks with numbers and gave to another person to analyze the pulmonary vascular wall thickness. Microscopic images were analyzed using a computerized morphometric system (Aperio Imagescope v11.1.2.752 software) to assess the pulmonary arterial wall thickness. Pulmonary arteries were categorized according to their external diameter: category 1 are the arteries with an

external diameter between 25 and 50 µm; category II are the arteries with an external diameter between 51 and 100 µm; and category III are the arteries with an external diameter greater than 100 µm. Thickness of an artery was determined by the average values obtained in multiple areas of the artery. Angiography in Rats and Mice. Rats and mice were anesthetized to dissect out the whole lungs. A PE10 (BD) tube was inserted into the main pulmonary artery and superfused with warm PBS solution (36-38°C) via a syringe pump (Syringepump.com NE-300) at a rate of 0.25 ml/min for 4 min. Then MICROFIL® Silicone Rubber Injection Compounds (Flow-Tech, Inc., Carver, MA), a liquid silicon polymer, was infused into the main pulmonary artery at 0.25 ml/min for 4 min using the syringe infusion pump. The MICROFIL-infused lung tissues were stored at 4°C for 12 hrs before the lungs were sequentially bathed in ethanol at gradually increased concentrations. The tissues were then placed in methyl-salicylate and photographed with a digital camera (MD600E, Amscope, USA) through a dissecting microscope (WILD M651, Leica, Switzerland). The ImageJ software was used to evaluate the number of arterial branches, the number of arterial junctions and the total length of arterial segments in a given area (usually a square mm area, i.e., 1 mm2). We selected the areas (in 1 mm2 unit) of the lung vascular images with Photoshop CS software, and made binary images using ImageJ software. The pictures were then skeletonized for analyzing the vessel branches, junctions and total length. These data were normalized by the area selected in each lung. Immunofluorescence Staining of Human and Rat Lung Sections. Immunohistochemistry for CaSR and SM-α-actin was performed on formalin-fixed, paraffin-embedded (human, rat and mouse) lung tissue sections. Briefly, sections were first deparaffinized in xylene, rehydrated in graded ethanol solutions, and rinsed in deionized water. Then, sections were incubated (for 30 min) in a Tris-EDTA Buffer (10 mM Tris Base,1 mM EDTA solution, with 0.05% Tween20; pH 9.0) for heat-induced epitope retrieval. After washing in PBS, the sections were blocked by incubating with 5% bovine serum albumin (Sigma) for 1 hr. The slides were rinsed in PBS and incubated first with a CaSR polyclonal antibody (rabbit anti-mouse IgG, Abcam) at a dilution of 1:50 for 1 day at 4°C. Then the slides were incubated with a monkey anti-rabbit IgG antibody conjugated with Alexa488 (Invitrogen) as the secondary antibody for 1 hr and a monoclonal antibody against the SM-α-actin clone 1A4 (Cy3 conjugate) (Purified Mouse Immunoglobulin) at a dilution of 1:200. After washing in PBS, sections were mounted in Vectashield hard-set mounting medium with DAPI (Vector Laboratories) and viewed under an Axiovert 200M fluorescence microscope. The expression of CaSR was quantified by grey value using the of Image J software.

Online Supplementary FIGURES and FIGURE LEGENDS

Normal IPAH

CaS

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Online Figure I. Upregulated CaSR in pulmonary arteries from IPAH patients. A. Immunohistochemistry data showing CaSR expression level (green fluorescence intensity) in pulmonary arteries of normal subjects and IPAH patients. The lung tissue sections were stained with antibodies against CaSR (green) and smooth muscle α-actin (SM-α-Actin, red) and with DAPI (blue). The overlay images are shown in the bottom. B. Summarized data (means±SE, n=3 for each group) showing CaSR expression level (green fluorescence intensity) in pulmonary arteries of normal subjects (Norm) and IPAH patients. **P<0.01 vs. Normal.

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Online Figure II. Extracellular Ca2+-induced increase [Ca2+]cyt in IPAH-PASMCs is not due to Ca2+ leakage. A. Representative traces showing change in resting [Ca2+]cyt before and during application of Ca2+-free solution in normal and IPAH PASMC. Summarized data (right panel, n=96-370 cells) showing the resting [Ca2+]cyt in normal and IPAH PASMC superfused with 2.2 mM Ca2+-containing solution (black bars) or Ca2+-free solution (grey bars). **P<0.001 vs. normal PASMC. B. Representative images (left panels) of trypan (TB) blue staining (0.4%, 1 min) before (-) and during (+) treatment with 10 M ionomycin for 10 min in normal (upper panels) and IPAH (lower panels) PASMC. Summarized data (right panel) showing no correlation between the extracellular Ca2+-induced increase in [Ca2+]cyt and the Ca2+ leakage through the plasma membrane. C. Representative images showing [Ca2+]cyt (or Fura-2 fluorescence intensity) in normal (left panels) and IPAH (right panels) PASMC in the absence and presence of 10 M CPA, an inhibitor of Ca2+-ATPase in the SR. The cells were loaded with the membrane-permeable fura-2/AM (4 M, upper panels) or the membrane-impermeable fura-2 (4 and 40 M, middle and lower panels). Data were obtained from 21 to 64 cells.

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Online Figure III. Inhibition of PLC and IP3R attenuates extracellular Ca2+-induced [Ca2+]cyt increases in IPAH-PASMC Representative records of [Ca2+]cyt changes (left panels), pseudo images (middle panels) and summarized data (means±SE, right panels) showing extracellular Ca2+-induced increase in [Ca2+]cyt before and during treatment with 1 M U73122 (a PLC inhibitor; n=57, A), 1 M U73343 (an inactive form of U73122; n=45, B), 3 M xestospongin C (an IP3R blocker; n=34, C), 10 M diltiazem (a VDCC blocker; n=46, D), and KB-R7943 (an Na+/Ca2+ exchanger inhibitor; n=22, E) in IPAH-PASMC. **P<0.01 vs. control.

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Aya Yamamura, Qiang Guo, Hisao Yamamura, Adriana M. Zimnicka, Nicole M. Pohl,Hypertension

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