Store-operated channels in the pulmonary circulation of high- and low-altitude neonatal lambs.

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Store-operated channels in the pulmonary circulation of high- and low-altitude neonatal lambs.
  doi: 10.1152/ajplung.00024.2012304:L540-L548, 2013. First published 15 February 2013;  Am J Physiol Lung Cell Mol Physiol V. ReyesFerrada, Marcela Diaz, Julian T. Parer, Gertrudis Cabello, Aníbal J. Llanos and RobertoRiquelme, César E. Ulloa, Rodrigo T. Rojas, Pablo Silva, Ismael Hernandez, Javiera Daniela Parrau, Germán Ebensperger, Emilio A. Herrera, Fernando Moraga, Raquel A. high- and low-altitude neonatal lambsStore-operated channels in the pulmonary circulation of  You might find this additional info useful...  60 articles, 33 of which you can access for free at: This article cites including high resolution figures, can be found at: Updated information and services can be found at:  Molecular Physiology American Journal of Physiology - Lung Cellular and  about Additional material and information information is current as of April 19, 2013. ESSN: 1522-1504. Visit our website at Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright © 2013 the American Physiological Society. components of the respiratory system. It is published 24 times a year (twice monthly) by the American Physiologicalthe broad scope of molecular, cellular, and integrative aspects of normal and abnormal function of cells and publishes srcinal research covering  American Journal of Physiology - Lung Cellular and Molecular Physiology   a t   U N I  V E R  S I  D A D D E  C H I  L E  onA  pr i  l  1  9  ,2  0 1  3 h  t   t   p:  /   /   a j   pl   un g. ph  y  s i   ol   o g y . or  g /  D  ownl   o a d  e d f  r  om   Store-operated channels in the pulmonary circulation of high- and low-altitudeneonatal lambs Daniela Parrau, 1,8 * Germán Ebensperger, 1 * Emilio A. Herrera, 1,2 Fernando Moraga, 9 Raquel A. Riquelme, 3 César E. Ulloa, 1 Rodrigo T. Rojas, 1 Pablo Silva, 1,4 Ismael Hernandez, 1 Javiera Ferrada, 1 Marcela Diaz, 1,6 Julian T. Parer, 5 Gertrudis Cabello, 7 Aníbal J. Llanos, 1,2 and Roberto V. Reyes 1 1  Laboratorios de Fisiología y Fisiopatología del Desarrollo, y de Bioquímica y Biología Molecular de la Hipoxia, Programade Fisiopatología, Instituto de Ciencias Biomédicas (ICBM), Facultad de Medicina, Universidad de Chile, Santiago, Chile; 2  International Center for Andean Studies (INCAS), Universidad de Chile, Santiago, Chile;  3  Departamento de Bioquímica y Biología Molecular, Facultad de Ciencias Químicas y Farmacéuticas, Universidad de Chile, Santiago, Chile;  4 Universidad Cayetano Heredia, Lima, Perú;  5  Department of Obstetrics, Gynecology and Reproductive Sciences, University of CaliforniaSan Francisco, San Francisco, California;  6  Escuela de Obstetricia, Universidad de Chile, Santiago, Chile;  7 Facultad deCiencias, Universidad de Tarapacá, Arica, Chile;  8 Facultad de Medicina, Universidad Diego Portales, Santiago, Chile; and  9 Facultad de Medicina, Universidad Católica del Norte, Coquimbo, Chile Submitted 17 January 2012; accepted in final form 12 February 2013 ParrauD,EbenspergerG,HerreraEA,MoragaF,RiquelmeRA,UlloaCE,RojasRT,SilvaP,HernandezI,FerradaJ,DiazM,ParerJT, Cabello G, Llanos AJ, Reyes RV.  Store-operated channels in thepulmonary circulation of high- and low-altitude neonatal lambs.  Am J Physiol Lung Cell Mol Physiol  304: L540–L548, 2013. First publishedFebruary 15, 2013; doi:10.1152/ajplung.00024.2012.—We determinedwhether store-operated channels (SOC) are involved in neonatalpulmonary artery function under conditions of acute and chronichypoxia, using newborn sheep gestated and born either at high altitude(HA, 3,600 m) or low altitude (LA, 520 m). Cardiopulmonary vari-ables were recorded in vivo, with and without SOC blockade by2-aminoethyldiphenylborinate (2-APB), during basal or acute hypoxicconditions. 2-APB did not have effects on basal mean pulmonaryarterial pressure (mPAP), cardiac output, systemic arterial bloodpressure, or systemic vascular resistance in both groups of neonates.During acute hypoxia 2-APB reduced mPAP and pulmonary vascularresistance in LA and HA, but this reduction was greater in HA. Inaddition, isolated pulmonary arteries mounted in a wire myographwere assessed for vascular reactivity. HA arteries showed a greaterrelaxation and sensitivity to SOC blockers than LA arteries. Thepulmonary expression of two SOC-forming subunits, TRPC4 and STIM1,was upregulated in HA. Taken together, our results show that SOCcontribute to hypoxic pulmonary vasoconstriction in newborn sheepand that SOC are upregulated by chronic hypoxia. Therefore, SOCmay contribute to the development of neonatal pulmonary hyperten-sion. We propose SOC channels could be potential targets to treatneonatal pulmonary hypertension.hypoxia; pulmonary vasoconstriction; pulmonary hypertension;2-aminoethydiphenylborinate; pulmonary vascular reactivity PULMONARY ARTERIES HAVE AN  intrinsic vasoconstrictor responseto low oxygen levels when exposed to acute hypoxia. This is areversible, rapid and physiological response, known as hypoxicpulmonary vasoconstriction (HPV), that redirects blood flowfrom poorly oxygenated to better oxygenated alveoli. Thus,when total lung is exposed to hypoxia, HPV results in anincrease in pulmonary artery pressure (PAP) that reverseswhen normoxia is reestablished (31). However, exposure tochronic hypoxia produces an imbalance between vasodilatorand vasoconstrictor mechanisms, and there is pulmonary vas-cular remodeling that includes proliferation of pulmonary ar-tery myocytes among other cellular processes (46). The resultis a pathological and persistent increase in pulmonary arterycontractile tone and pulmonary arterial hypertension, which inmany cases leads to right ventricular hypertrophy, right heartfailure, and eventually death (13).In pulmonary artery smooth muscle cells, an increase inintracellular calcium concentration ([Ca 2  ] i ) is essential forHPV, proliferation, and remodeling (13, 19, 20, 42). Thisincrease in [Ca 2  ] i  greatly depends on an influx of extracellularcalcium (13, 59), which may enter the smooth muscle cellthrough store-operated channels (SOC) among other pathways(5, 8, 22, 40). These are channels physiologically activated byinternal calcium store depletion induced by agonists of inosi-tol-(1,4,5)-triphosphate receptor and/or the ryanodine receptorsuch as endothelin-1 (ET-1), among other effects of the latter.They can also be experimentally stimulated by agents that block the sarcoplasmic reticulum calcium pump like cyclopiazonic acidor thapsigargin (7, 40) and blocked by 2-aminodiphenylborinate(2-APB) or 1-[2-(4-methoxyphenyl)  2-[3-(4-methoxyphenyl)propoxy]ethyl]imidazole (SKF-96365) (1, 22, 57).SOC-forming molecules of the TRPC, ORAI, and STIMfamilies are expressed in lung, isolated pulmonary arteries, andcultured smooth muscle from pulmonary arteries (9, 25, 27, 30,35, 36, 50, 62, 63). Pharmacological inhibition and genesuppression experiments have shown that SOC are involved inthe pulmonary vascular [Ca 2  ] i  increase and contractile re-sponse to acute hypoxia (27, 28, 48, 55, 57, 58).On the other hand, chronic hypoxia upregulates SOC inpulmonary artery myocytes from rat, mouse, and human (25,52, 54). SOC upregulation is also observed in myocytes stim-ulated to proliferate as happens in pulmonary artery remodel-ing (12, 22, 53, 61, 62, 63).Despite the substantial advances in the SOC-related mech-anisms regulating pulmonary vascular function, most of thesedata have been obtained from ex vivo and in vitro approaches * D. Parrau and G. Ebensperger contributed equally.Address for reprint requests and other correspondence: R. V. Reyes, Pro-grama de Fisiopatología, Instituto de Ciencias Biomédicas, Facultad de Me-dicina, Universidad de Chile. Avda. Salvador 486, Providencia, CP 6640871,Santiago, Chile (e-mail:  Am J Physiol Lung Cell Mol Physiol  304: L540–L548, 2013.First published February 15, 2013; doi:10.1152/ajplung.00024.2012.1040-0605/13 Copyright  ©  2013 the American Physiological Society http://www.ajplung.orgL540   a t   U N I  V E R  S I  D A D D E  C H I  L E  onA  pr i  l  1  9  ,2  0 1  3 h  t   t   p:  /   /   a j   pl   un g. ph  y  s i   ol   o g y . or  g /  D  ownl   o a d  e d f  r  om   using isolated cells or organs from adult individuals. Never-theless, studies covering the in vivo role of SOC on theneonatal pulmonary circulation are lacking.Neonatal pulmonary hypertension in humans is associatedwith developmental chronic hypoxia and results in high mor-tality, decreased postnatal growth and long lasting neurologi-cal, respiratory, and cardiac complications (4, 10, 14, 21, 45).We have developed a model of neonatal pulmonary hyperten-sion in sheep, gestated and born at high altitude in the AndeanAltiplano, characterized by an increased PAP, impaired vascu-lar reactivity, and altered arterial structure in the pulmonarycirculation (14, 15, 16, 18).In the present work, we tested the hypotheses that SOCcontribute to the control of PAP in low-altitude and high-altitude newborn sheep and that SOC function is enhanced athigh altitude. We used an integrative approach at the wholeanimal, isolated organ, and molecular levels in comparing thetwo groups of animals. Our studies include  1 ) in vivo cardio-pulmonary function under normoxia and acute hypoxia, with orwithout SOC blockade with 2-APB;  2 ) ex vivo relaxationinduced by SOC blockade with 2-APB, in isolated smallpulmonary arteries; and  3 ) the pulmonary expression of puta-tive SOC-forming molecules TRPC1, TRPC3, TRPC4,TRPC5, TRPC6, ORAI1, and STIM1. MATERIALS AND METHODS The Faculty of Medicine Ethics Committee of the University of Chile approved all experimental procedures (protocols CBA N° 040and CBA 0232 FMUCH). The studies on animals were performedaccording with the Guide for the Care and Use of Laboratory Animalspublished by the US National Institutes of Health (NIH publicationno. 85-23, revised 1996) and adhere to the American PhysiologicalSociety’s Guiding Principles in the Care and Use of Animals.  Animals.  The animals used in the present study consisted of 10newborn sheep gestated, born, and raised at the University of Chilefarm, Rinconada de Maipú, at 580 m altitude (LA, 13.9    0.5 daysold; 7.4  0.3 kg) and 10 newborn sheep gestated, born, and raised atPutre Research Station, International Center for Andean Studies(INCAS), at 3,600 m altitude (HA, 13.7  0.7 days old; 5.1  0.3 kg; P  0.05, HA vs. LA for body weight). Surgical preparation and in vivo experiments.  Five lambs pergroup were surgically prepared between 6 and 8 days of age for invivo experimentation as described previously (16). In brief, theanimals were anesthetized with a ketamine (10 mg/kg im) and diaz-epam (0.1–0.5 mg/kg im) association with additional local infiltrationof 2% lidocaine. Polyvinyl catheters were placed into the descendingaorta and inferior vena cava and a Swan-Ganz catheter was placedinto the pulmonary artery. Following 3–4 days of postsurgical recov-ery, the animals were subjected to a 3-h experimental protocol,consisting of 1 h of basal recording (breathing room air), 1 h of acuteisocapnic hypoxia, and 1 h of recovery during which they werereturned to room air breathing. This protocol was performed twice onseparate days on each animal in random order either with vehicleinfusion (DMSO-0.9% NaCl, 1:10) or under SOC blockade with2-APB (8 mg/kg bolus in vehicle). Infusion of vehicle or 2-APBstarted 15 min prior to hypoxia, ran continuously for 15 min, andfinished before the beginning of the hypoxic challenge. Acute isocap-nic hypoxia was induced via a transparent, loosely tied polyethylenebag placed over the animal’s head into which a known mixture of air,N 2  and CO 2  (  14% O 2  in Santiago and 17.5% O 2  in Putre; 2–3% CO 2 in N 2 ) was passed at a rate of 20 l/min to reach an arterial P O 2  of   30mmHg. Arterial blood samples were taken during the experimentalprotocol to determine arterial pH, P CO 2 , P O 2 , hemoglobin concentra-tion and percentage saturation of hemoglobin (SO 2 ) (IL-Synthesis 25,Instrumentation Laboratories measurements corrected to 39°C). PAPand systemic arterial pressure (SAP) were recorded continuously viaa data-acquisition system (PowerLab/8SP System and LabChart v7.0Software; ADInstruments) connected to a computer. Cardiac output(CO) was determined at set intervals with the thermodilution methodby the injection of 3 ml of chilled (0°C) 0.9% NaCl into thepulmonary artery through the Swan-Ganz catheter connected to acardiac output computer (COM-2 model, Baxter). Heart rate (HR),mean PAP (mPAP), mean SAP, pulmonary vascular resistance (PVR),and systemic vascular resistance (SVR) were calculated as describedpreviously (14).  Ex vivo and in vitro experiments.  Five uninstrumented lambs pergroup underwent euthanasia with an overdose of sodium thiopentone(100 mg/kg iv) for collection of their lungs. WIRE MYOGRAPHY.  The lungs were removed by dissection andimmediately immersed in cold saline. Parenchymal pulmonary arter-ies (200–300   m) were dissected, isolated, and mounted on a wiremyograph and maintained at 37°C aerated with 95% O 2 -5% CO 2  inKrebs buffer. The resting tension, defined as transmural pressureexerted in the vessels (at the pulmonary arterial circulation thispressure is  17–25 mmHg, as seen on our in vivo experiments), wascalculated by stretching the vessel in a stepwise manner to a stan-dardized tension equivalent to the physiological transmural pressure(32). This was done to simulate conditions in vivo for two mainreasons: first, because the stimulated vascular response is dependenton the degree of stretch; and secondly, because this degree of stretchgives the maximal vascular response (32, 44, 56).Concentration-response curves (CRCs) were constructed for con-traction induced by potassium chloride (KCl, 6.25 to 125 mM) andET-1 (10  12 to 10  7 M) to assess contractile function. The ex vivorelaxant effect of SOC blockade with 2-APB and SKF-96365 wasdetermined as follows: tension of pulmonary arteries was previouslyrecorded in a calcium-free Krebs buffer (calcium was omitted andtraces were removed with 2    10  4 M EGTA) under voltage-dependent calcium channel blockade with nifedipine (10  5 M). Next,ET-1 (10  7 M) was added to promote depletion of internal calciumstores and to evaluate contraction under these conditions, followed byexternal calcium restitution (2    10  3 M) to promote additionalcontraction. When maximal contraction was reached under theseconditions, the relaxation promoted by increasing cumulative concen-trations of 2-APB (10  7 to 10  4 M) or SKF-96365 (10  7 to 10  4 M)was recorded to have a CRC for SOC blockade. The response to everyCRC dose was recorded 3 min after each condition. Contractileresponses were expressed in terms of tension (N/m) or percentagerelative to a submaximal dose of KCl (62.5 mM). Relaxation re-sponses to 2-APB and SKF-96365 were expressed as a percentage of decrease of the tension reached after external calcium restitution.CRCs were fitted to a nonlinear equation (Prism 5.0, GraphPadSoftware, La Jolla, CA), and differences between groups were com-pared by calculating the area under the curve and the sensitivity aspD2, where pD2    log[EC 50 ], EC 50  being the concentration atwhich 50% of the maximal response was achieved (14, 17). RT-PCR.  Total RNA purification from lung tissue, cDNA synthesis,and PCR amplification were performed as described previously (6, 17).Primers for amplification of partial DNA sequences from TRPC1 (for-ward 5 = -ATGGGACAGATGTTACAAGATTTTGGG-3 =  and reverse5 = -AGCAAACTTCCATTCTTTATCCTCATG-3 = , accession numberNM_053558), TRPC3 (forward 5 = -TGACCTCTGTTGTGCT-CAAATATG-3 =  and reverse 5 = -CCACTCTACATCACTGTAATCC-3 = ,accession number NM_021771), TRPC4 (forward 5 = -TCTGCA-GATATCTCTGGGAAGGATGC-3 =  and reverse 5 = -AAGCTTTGTTC-GAGCAAATTTCCATTC-3 = , accession number NM_080396), TRPC5(forward 5 = -AACTCCCTCTACCTGGCAACTA-3 =  and reverse 5 = -GGATATGAGACGCCACGAACTT-3 = , accession number NM_080898),TRPC6 (forward 5 = -AAAGATATCTTCAAATTCATGGTCATA-3 = and reverse 5 = -ATCCGCATCATCCTCAATTTC-3 = , accession number L541 STORE-OPERATED CHANNELS IN NEONATAL PULMONARY ARTERIES  AJP-Lung Cell Mol Physiol  •  doi:10.1152/ajplung.00024.2012  •   a t   U N I  V E R  S I  D A D D E  C H I  L E  onA  pr i  l  1  9  ,2  0 1  3 h  t   t   p:  /   /   a j   pl   un g. ph  y  s i   ol   o g y . or  g /  D  ownl   o a d  e d f  r  om   NM_053559), ORAI1 (forward 5 = -AGGTGATGAGCCTCAAC-GAG-3 =  and reverse 5 = -CTGATCATGAGCGCAAACAG-3 = , accessionnumber NM_001013982), and STIM1 (forward 5 = -GGCCAGAGTCT-CAGCCATAG-3 =  and reverse 5 = -CATAGGTCCTCCACGCTGAT-3 = ,accessionnumberNM_001108496)werederivedfromthecorrespondingrat genes after alignment and identification of conserved sequences fromrat, mouse, bovine, and human orthologs, whereas the 18S-rRNA (for-ward 5 = -GTAACCCGTTGAACCCCATT-3 =  and reverse 5 = -CCATC-CAATCGGTAGTAGCG-3 = , the housekeeping gene, accession numberDQ013885) was derived from the corresponding ovine sequence. All thePCR products were sequenced to verify their identity. The PCR productswere visualized under UV light and the signals obtained on RT-PCRdeterminations were quantified by densitometric analysis using the ScionImage Software (Scion Image Beta 4.02 Win; Scion, Frederick, MD). Statistical analysis.  Data are expressed as means    SE. Groupswere compared by two-way ANOVA and the post hoc Newman-Keuls test or by Student’s  t  -test for unpaired data, as appropriate. Forall comparisons, differences were considered statistically significantwhen  P  0.05 (11). RESULTS  In vivo cardiopulmonary function with and without 2-APB. Arterial blood gases and acid-base status. Basal P O 2 , SO 2 , andP CO 2  were lower, whereas pH was higher in HA than LA lambs(Table 1). During acute hypoxia with either vehicle or 2-APBinfusion, a fall to similar values of P O 2  and SO 2  occurred inboth groups of lambs, without any changes of P CO 2  relative tobasal period (Table 1). During recovery, all variables returnedtoward basal values in both groups (Table 1). Treatment with2-APB had no significant effect on arterial blood gas andacid-base state either during basal and acute hypoxic condi-tions relative to controls (Table 1). Cardiovascular and pulmonary functions.  HA lambsshowed higher mPAP than LA lambs throughout the experi-mental protocol. Furthermore, during acute superimposed hyp-oxia, mPAP increased in both LA and HA animals infused withvehicle (Fig. 1,  A  and  B ). SOC blockade with 2-APB did nothave any effects during basal, but significantly attenuated theincrease of mPAP induced by acute hypoxia in LA lambs (Fig.1  A ). SOC blockade with 2-APB reduced mPAP under hypoxiaand recovery in HA lambs (Fig. 1  B ). The net decrease of mPAP elicited by SOC blockade, expressed as the differ-ence between mPAP under vehicle infusion and 2-APBinfusion during acute hypoxia was greater in HA than in LAlambs (Fig. 1 C  ). Consequently, PVR was higher in HArelative to LA newborns during all of the experimentalTable 1.  Arterial pH and blood gases in LA and HA lambs Basal Basal  Infusion Hypoxemia Recovery pHLAVehicle 7.432  0.014 7.432  0.009 7.419  0.014 7.419  0.0132-APB 7.400  0.009 7.411  0.013 7.393  0.028 7.412  0.012HAVehicle 7.496  0.016* 7.489  0.020* 7.469  0.014* 7.464  0.017*2-APB 7.484  0.014* 7.489  0.015* 7.477  0.021* 7.475  0.017*P CO 2 , mmHgLAVehicle 40.1  1.1 40.6  1.3 39.3  1.5 39.3  1.32-APB 41.1  1.2 39.4  1.5 38.8  1.5 37.5  1.1HAVehicle 29.2  0.8* 28.8  1.1* 29.1  0.9* 28.6  1.6*2-APB 27.8  0.8* 26.8  0.8* 27.6  0.7* 26.7  0.9*P O 2 , mmHgLAVehicle 79.6  1.8 81.9  2.6 31.3  0.3‡ 81.9  1.52-APB 79.5  1.8 82.0  2.1 30.4  0.5‡ 84.8  3.8HAVehicle 42.7  1.9* 43.0  2.2* 29.3  0.6‡ 44.3  1.6*2-APB 44.0  1.9* 47.2  1.7* 29.6  0.4‡ 46.8  1.3*SO 2 , %LAVehicle 100.2  1.0 100.9  1.0 56.7  2.0‡ 100.4  0.92-APB 94.3  0.9 100.1  0.7 52.9  2.2‡ 100.8  1.0HAVehicle 74.4  2.0* 75.2  2.8* 49.2  2.3‡ 75.1  2.3*2-APB 74.6  1.9* 78.4  1.2* 49.6  3.9‡ 77.6  2.5*Hb, g/dlLAVehicle 9.4  0.3 8.9  0.3 9.6  0.2 9.2  0.22-APB 9.9  0.4 9.8  0.5 10.2  0.4 9.7  0.5HAVehicle 11.0  0.7 10.6  0.6 12.5  1.5* ‡ 10.5  0.52-APB 10.6  0.5 10.5  0.4 11.3  0.5‡ 10.7  0.6Values are the means  SE for arterial pH (pH), partial pressure of carbon dioxide (P CO 2 ), partial pressure of oxygen (P O 2 ), saturation of hemoglobin withoxygen (SO 2 ) and hemoglobin concentration ([Hb]). LA, conception pregnancy and delivery at low altitude (580 m,  n    5); HA, conception pregnancy anddelivery at high altitude (3,600 m,  n  5). Vehicle, dimethylsulfoxide: NaCl 0.9% (1:10); 2-APB, 2-aminoethyldiphenylborinate. Blood samples were taken andmeasured during preinfusion (Basal), during infusion with vehicle or 2-APB (Basal  Infusion), during acute hypoxia, and during recovery. Significant differences( P  0.05): *LA vs. HA; ‡vs. all in the same group. L542  STORE-OPERATED CHANNELS IN NEONATAL PULMONARY ARTERIES  AJP-Lung Cell Mol Physiol  •  doi:10.1152/ajplung.00024.2012  •   a t   U N I  V E R  S I  D A D D E  C H I  L E  onA  pr i  l  1  9  ,2  0 1  3 h  t   t   p:  /   /   a j   pl   un g. ph  y  s i   ol   o g y . or  g /  D  ownl   o a d  e d f  r  om   protocol. LA and HA lambs showed an increased PVRduring acute hypoxia and exhibited a partial attenuation of this response by 2-APB (Fig. 2,  C   and  D ).CO was similar in LA and HA lambs under basal conditions.During acute hypoxia, CO increased and returned to basalvalues during recovery in both LA and HA lambs. The infusionof 2-APB did not have any effect on CO throughout theexperimental protocol (Fig. 2,  A  and  B ).Basal HR was similar in both vehicle-infused LA and HAgroups; it increased during acute superimposed hypoxia butreturned to basal values during recovery. Infusion of 2-APBdid not modify HR in basal or acute hypoxic conditions, but itmaintained an elevated HR during recovery in both groups(Table 2).SAP remained stable during all of the experimental proto-cols, either with vehicle or 2-APB in LA and HA lambs (Table2). In contrast, SVR was similar during baseline but diminishedduring acute hypoxia and returned to basal values duringrecovery in LA animals. Infusion of 2-APB did not induce anychanges of SVR relative to the vehicle in this group (Table 2). 30 60 90 120 150 18001020304050 Hypoxemia 2-APB ‡† ‡ † * Time (min) 30 60 90 120 150 18001020304050 Hypoxemia † ‡‡§§   m   P   A   P   (  m  m   H  g   ) A B C LAHA 0246810 *    m   P   A   P   (  m  m   H  g   ) Vehicle Time (min) Fig. 1. Mean pulmonary arterial pressure (mPAP) during in vivo acute hypoxia protocol in LA (  A ) or HA (  B ) lambs; LA, conception pregnancy and deliveryat low altitude (580 m,  n  5); HA, conception pregnancy and delivery at high altitude (3,600 m,  n  5). Acute hypoxia was induced following a vehicle infusion( Œ ) or with the store-operated channel (SOC) blocker 2-aminoethyldiphenylborinate (2-APB;   ). The horizontal gray bar indicates the infusion period. Valuesare means  SE. calculated every minute during the experimental protocol. The sensitivity of the acute hypoxic response to 2-APB, was calculated accordingto the following formula:  mPAP  mPAP vehicle- mPAP 2-APB , where mPAP vehicle  and mPAP 2-APB  represents the mean pulmonary artery pressure (mPAP) during1 h of hypoxia, in the presence of 2-APB or its vehicle, respectively ( C  ). Significant differences ( P  0.05): *LA vs. HA; †vs. vehicle; ‡vs. all in the same group;§vs. Basal, Basal  Infusion. 0200400600Vehicle2-APB ‡§ A B C D    B  a  s  a   l   B  a  s  a   l  +   I  n  f  u  s   i  o  n   H  y  p  o  x  e  m   i  a    R  e  c  o  v  e  r  y † § §§    P   V   R   (  m  m   H  g .  m   l   -   1  .  m   i  n .   K  g   )    B  a  s  a   l   B  a  s  a   l  +   I  n  f  u  s   i  o  n   H  y  p  o  x  e  m   i  a    R  e  c  o  v  e  r  y † * ‡ 0200400600‡‡    C   O   (  m   l  m   i  n   -   1     K  g   -   1    ) Fig. 2. Cardiac output (CO) and pulmonaryvascular resistance (PVR) in LA (  A  and  C  )and HA (  B  and  D ) newborn sheep. Values areexpressed as means    SE. for the differentexperimental periods under vehicle (openbars) or 2-APB administration (solid bars).Significant differences ( P    0.05): *LA vs.HA; †vs. vehicle; ‡vs. all in the same group;§vs. Basal, Basal  Infusion. L543 STORE-OPERATED CHANNELS IN NEONATAL PULMONARY ARTERIES  AJP-Lung Cell Mol Physiol  •  doi:10.1152/ajplung.00024.2012  •   a t   U N I  V E R  S I  D A D D E  C H I  L E  onA  pr i  l  1  9  ,2  0 1  3 h  t   t   p:  /   /   a j   pl   un g. ph  y  s i   ol   o g y . or  g /  D  ownl   o a d  e d f  r  om 
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