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Chronic intrauterine pulmonary hypertension increases capacitative calcium entry in fetal pulmonary artery smooth muscle cells

³Center of Excellence in Pulmonary Biology, Division of Pediatric Pulmonary, Allergy and Critical Care Medicine, Department of Pediatrics, Stanford University Medical School, Palo Alto, California; ¹Division of Pediatric Pulmonary and Critical Care Medicine, Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota; and ²Department of Physiology, College of Medicine, Biomedical Engineering Program, Genomics Research Laboratory, University of Arizona, Tucson, Arizona

Submitted 24 August 2006 ; accepted in final form 1 December 2006

	ABSTRACT

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Oxygen causes perinatal pulmonary dilatation. Although fetal pulmonary artery smooth muscle cells (PA SMC) normally respond to an acute increase in oxygen (O₂) tension with a decrease in cytosolic calcium ([Ca²⁺]_i), an acute increase in O₂ tension has no net effect on [Ca²⁺]_i in PA SMC derived from lambs with chronic intrauterine pulmonary hypertension (PHTN). The present experimental series tests the hypothesis that an acute increase in O₂ tension decreases capacitative calcium entry (CCE) in normal, but not hypertensive, fetal PA SMC. PA SMC were isolated from late-gestation fetal lambs after either ligation of the ductus arteriosus (PHTN) or sham (control) operation at 127 days gestation. PA SMC were isolated from the distal PA (4th generation) and maintained under hypoxic conditions (25 Torr) in primary culture. After fura 2 loading, apparent [Ca²⁺]_i in PA SMC was determined as the ratio of 340- to 380-nm fluorescence intensity. Under both hypoxic and normoxic conditions, cyclopiazonic acid (CPA) increased [Ca²⁺]_i more in PHTN than in control PA SMC. CCE was determined in PA SMC under hypoxic and normoxic conditions, after superfusion with zero extracellular Ca²⁺ and intracellular store depletion with CPA, followed by superfusion with Ca²⁺-containing solution, in the presence of the voltage-operated calcium channel blockade. CCE was increased in PHTN compared with control PA SMC under conditions of both acute and sustained normoxia. Transient receptor potential channel gene expression was greater in control compared with PHTN PA SMC. PHTN may compromise perinatal pulmonary vasodilation, in part, by modulating PA SMC CCE.

cytosolic calcium; oxygen sensing; persistent pulmonary hypertension of the newborn

Fetal stress can compromise neonatal pulmonary vasodilation. Intrauterine hypoxemia, infection, hyperglycemia, and drug exposure can result in an incomplete response to perinatal vasodilator stimuli and persistently elevated pulmonary arterial blood pressure, limited pulmonary blood flow, and severe central hypoxemia. Persistent pulmonary hypertension of the newborn (PPHN) is characterized by extrapulmonary shunting of blood across the patent foreman ovale and through the patent ductus arteriosus (DA), severe hypoxemia, high levels of circulating endothelin-1, decreased nitric oxide production, and vascular remodeling due to increased cell proliferation (1, 3, 13, 14, 29).

Previous work from our laboratory demonstrated that pulmonary artery smooth muscle cells (PA SMC) derived from late-gestation fetal lambs directly sense an acute increase in oxygen tension and respond with activation of a calcium-sensitive K⁺ (K_Ca) channel, membrane hyperpolarization, and a decrease in intracellular Ca²⁺ ([Ca²⁺]_i) (23). In contrast, in PA SMC derived from an ovine model of PPHN, K_Ca channel expression is diminished, and an acute increase in oxygen tension has no effect on [Ca²⁺]_i (18). The mechanism that accounts for the persistent elevation of PA SMC [Ca²⁺]_i derived from animals with PPHN remains unknown. Given the biologically imperative nature of perinatal pulmonary vasodilation, it is likely that more than a single mechanism accounts for the pulmonary vascular response to the acute increase in oxygen tension that occurs at birth.

Recent data suggest that capacitative Ca²⁺ entry (CCE) plays a key role in intracellular calcium homeostasis in a wide variety of excitable cells. CCE, or store-operated Ca²⁺ entry, is activated by depletion of intracellular Ca²⁺ stores (21). Although CCE occurs through the TRP (transient receptor potential) family of channel proteins (6, 12), whether CCE is operative in fetal PA SMC has not been previously addressed. Golovina et al. (10) demonstrated that in primary pulmonary hypertension, a disease state characterized by increased pulmonary vascular resistance and smooth muscle cell hypertrophy (10, 26), CCE and TRP channel expression is increased.

Since PA SMC derived from fetal lambs demonstrate a decrease in cytosolic calcium in response to an acute increase in oxygen tension (22, 23), we hypothesized that acute normoxia attenuates PA SMC CCE. Moreover, since PA SMC derived from fetal lambs with chronic intrauterine pulmonary hypertension are insensitive to an acute increase in oxygen tension, we hypothesized that sustained elevation of PA SMC [Ca²⁺]_i, despite an acute increase in oxygen tension, results, in part, from increased CCE. To test these hypotheses, the role of CCE and TRP channel expression was determined in PA SMC derived from late-gestation fetal lambs with and without chronic intrauterine pulmonary hypertension (6, 12, 16, 27). The present experimental series provides data that in fetal PA SMC: 1) acute normoxia normally diminishes CCE; 2) chronic intrauterine pulmonary hypertension increases CCE; and 3) specific TRP channels are expressed.

	METHODS

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Animals. The procedures used in these studies were reviewed and approved by the Animal Care and Use Committee at the University of Minnesota Medical School.

Cell culture. Techniques used for cell isolation and culture have been previously described (18). Late-gestation fetal sheep (term = 147 days) from ewes with time-dated pregnancies were used in this study. Ewes were fasted for 24 h and sedated with pentobarbital sodium. Fetal lambs were partially delivered through an hysterotomy incision, with the head remaining inside the womb to prevent spontaneous breathing, and intracardiac pentobarbital sodium was administered. After thoracotomy, the lung and heart block was isolated.

Distal (4th generation) PA were quickly excised and placed in MEM (0.2 mM Ca²⁺). Loose connective tissue and adventitia were removed, and the vessels were liberally rinsed with MEM. Vessels were cut into small pieces and placed into 50-ml conical flasks containing 5.0 ml of enzymatic dissociation mixture, which consisted of 0.125 mg/ml elastase (Elastin Products, Owensville, MO), 1 mg/ml collagenase (Worthington Biochemical, Lakewood, NJ), 2.0 mg/ml bovine serum albumin (Sigma), 0.375 mg/ml soybean trypsin inhibitor (Sigma), and 4.8 ml of MEM. After incubation at 37°C for 60 min in a shaking bath, the tissue suspension was triturated six to seven times every 15 min in a glass pipette for a total incubation period of 120 min. The tissue suspension was then passed through a 100-µm nylon mesh (Nitex; Tetka, Elmsford, NJ) to separate dispersed SMC from undigested vessel wall fragments and debris. The filtered suspension was centrifuged (300 g for 15 min), and the cell pellet was resuspended in MEM supplemented with 10% fetal bovine serum. The dispersed cell suspension was aliquoted onto 25-mm² glass coverslips. Cells were incubated at 37°C in a humidified 10% O₂, 5% CO₂, balance N₂ (hypoxia) atmosphere. After 18–24 h, medium was removed to remove nonadherent cells and debris, and cells were refed with fresh medium. Medium was routinely exchanged at 48-h intervals. Cells were studied between day 5 and day 14 of culture at a cell density of subconfluent monolayers. Identity of cells was confirmed using SMC-specific antibodies for -SMC actin, calponin, and caldesmin.

Chronic intrauterine pulmonary hypertension model. Surgical ligation of the DA was performed as previously described (1). Pregnant ewes between 126 and 128 days gestation were fasted for 24 h before surgery. Ewes were sedated with intravenous pentobarbital sodium (total dose 2–4 g) and anesthetized with 1% tetracaine hydrochloride (3 mg) by lumbar puncture. Throughout surgery, the ewes were sedated but breathed spontaneously. Under sterile conditions, the gravid uterus was delivered through a midline laparotomy. The fetal lamb's left forelimb was withdrawn through a small hysterotomy. A skin incision was made under the left forelimb after local infiltration with lidocaine (3 ml, 1% solution). A left thoracotomy exposed the heart and great vessels. The DA was isolated with blunt dissection, and a 2–0 silk suture was placed around the DA and tied. The ribs and skin were reapproximated, the hysterotomy incision was closed, and the uterus was returned into the maternal abdominal cavity. The ewes recovered rapidly from surgery and were generally standing in their pens within 6 h. Food and water were provided ad libitum. After 7–10 days, animals were euthanized rapidly after high-dose maternal and fetal infusions of pentobarbital sodium, and the PA SMC were harvested as described above. In this paper, cells derived from animals with the chronic intrauterine pulmonary hypertension model are referred to as hypertensive; cells derived from normal animals are referred to as normotensive or control.

Ca²⁺ imaging. To assess dynamic changes in [Ca²⁺]_i in individual PA SMC, the Ca²⁺-sensitive fluorophore fura 2-AM (Molecular Probes) was used. Subconfluent fetal PA SMC on 25-mm² glass coverslips were placed on the stage of an inverted microscope (Nikon Diaphot). Cells were loaded with 10 nM fura 2-AM and 2.5 µg/ml Pluronic acid (Molecular Probes) for 20 min, followed by a 20-min wash in Ca²⁺-containing solution before the start of the experiment. Ratiometric imaging was performed with the excitation wavelengths of 340 and 380 nm and an emission wavelength of 510 nm. Imaging was performed with an ICCD camera (Photonic Science; Robertsbridge, United Kingdom) using Axon Instruments (Foster City, CA) or Metafluor (Universal Imaging, Downingtown, PA) image capture and analysis software. Ca²⁺ calibration was achieved by measuring a maximum (with 1 mM ionomycin) and a minimum (with 10 mM EGTA). PO₂ was controlled by aerating the recording solution reservoir and the stage microincubator with either 21% O₂ with balance N₂ (normoxia) or 100% N₂ (hypoxia). pH was 7.40 ± 0.05 and did not change during the experiments. For each experiment, 10–30 cells were visualized, and ratiometric data was acquired from individual cells. Fluorescent intensity measurements were made after stable baseline values were obtained in either hypoxic (PO₂ 25 Torr) or normoxic (PO₂ 120 mmHg) recording solution. The recording solution was superfused onto the cells at a rate of 2 ml/min in all experiments. Peak levels of fluorescence ratios reached following an intervention were used for data analysis. KCl (60 mM) was superfused onto the cells at the conclusion of the experiment to ensure cellular viability.

Mn²⁺ quenching. To assess changes in the rate of extracellular Ca²⁺ entry in individual PA SMC, Mn²⁺ quenching was used. The protocol has been previously described (6). Mn²⁺ containing buffer (0.5 mM) was superfused over the cells 1 min before the beginning of the experiment and throughout the entire experiment. Imaging was performed with an excitation wavelength of 360 nm. The emission wavelength remained 510 nm. Fura 2 fluorescence is quenched when bound to Mn²⁺. As Mn²⁺ enters the cell, it quenches the fura 2 signal. The rate of quenching is proportional to the rate of Mn²⁺ entry. In the absence of Ca²⁺, Mn²⁺ acts as a surrogate divalent cation, entering cells through Ca²⁺ channels. The rate of quenching of the fura 2 dye by Mn²⁺ in a zero-Ca²⁺ buffer is proportional to the rate of Ca²⁺ entry.

Solutions. Recording solutions consisted of (in mM) 10 HEPES, 10 glucose, 135 NaCl, 5.6 KCl, 1.8 CaCl₂, and 1.2 MgCl₂. Zero-Ca²⁺ solution was identical except it contained no CaCl₂. Mn²⁺ buffer was identical to zero-Ca²⁺ buffer except it contained 0.5 mM MnCl₂. All solutions were made with nanopure distilled water. pH was adjusted to 7.4.

Drugs. Cyclopiazonic acid (CPA), a Ca²⁺-ATPase inhibitor that passively empties intracellular Ca²⁺ stores (Alexis Biochemical, San Diego, CA), and nifedipine, a blocker of L-type voltage-operated Ca²⁺ channels (Sigma, St. Louis, MO), were used in separate experiments.

Real-time quantitative RT-PCR. The expression of mRNAs was analyzed by real-time quantitative RT-PCR (qPCR). Total RNA was isolated from SMC, as described earlier. cDNA was synthesized (SuperScript III, Invitrogen) and subsequently amplified on the Mx3000P instrument (Stratagene, La Jolla, CA) using Brilliant SYBR Green QPCR master mix, which contains SureStart Taq DNA polymerase, SYBR Green-I dye, dNTPs, and the reference dye (Stratagene). The PCR reaction was run under the following conditions: denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 30 s. Amplified products were incubated at 95°C for 1 min and 55°C for 30 s to plot dissociation curves. Primers were designed from matching sequences of known mammalian (human, cow, mice) genes, and their use was optimized for qPCR analysis as suggested by the manufacturer. PCR quality and specificity was verified by dissociation curve analysis. The primer sets used are shown in Table 1.

Experimental conditions. PA SMC were maintained in two distinct culture experimental conditions. In chronic hypoxia experiments, cells were maintained in primary culture and studied under low-oxygen tension conditions (25 Torr). In experiments examining the effects of acute normoxia, PA SMC were maintained in primary culture under low-oxygen tension conditions (25 Torr). Oxygen tension was increased (120 Torr) 5 min after the start of the experiments. In chronically normoxic PA SMC, cells were maintained in primary culture under normoxic conditions and studied under the same oxygen tension (120 Torr).

Experimental protocols: release of Ca²⁺ from CPA-sensitive intracellular Ca²⁺ stores. After obtaining stable baseline values in hypoxic recording solution, hypoxic (chronic hypoxia) or normoxic (acute normoxia) zero-Ca²⁺ buffer was superfused over the cells for 5 min, the stage microincubator was aerated with hypoxic or normoxic air, and zero-Ca²⁺ buffer containing CPA (10 µM) was superfused over the cells while fluorescence measurements continued.

CCE. After obtaining stable baseline values in hypoxic recording solution, hypoxic (chronic hypoxia) or normoxic (acute normoxia) zero-Ca²⁺ buffer was superfused over the cells for 5 min, and the stage microincubator was aerated with hypoxic or normoxic air. To assess CCE, cells were first superfused with zero-Ca²⁺ buffer containing CPA (10 µM) followed by superfusion with Ca²⁺-containing buffer with nifedipine (5 µM) while fluorescence measurements continued.

To address the potential that sustained normoxia modulates release of calcium from intracellular stores, the protocols outlined above were performed using cells maintained in primary culture under conditions of normoxia.

Rate of CCE. Cells were superfused with hypoxic Mn²⁺-containing buffer for 5 min, normoxic zero-Ca²⁺ buffer was superfused over the cells, and the stage microincubator was aerated with normoxic air. To deplete intracellular stores, zero-Ca²⁺ buffer containing CPA (10 µM) was superfused over the cells. Finally, Mn²⁺-containing buffer was superfused over the cells while the rate of fura 2 fluorescence signal intensity was measured.

For real-time PCR experiments, total RNA was isolated from six control and six pulmonary hypertension animals. Analysis was performed on PA SMC from either control or hypertensive animals after being under normoxic conditions for 7 days. Each experiment was repeated a minimum of three times. Student's t-test was used to assess differences between experimental groups. P values <0.05 were considered significant.

Western analysis. PA SMC maintained in primary culture were rinsed twice in cold PBS then lysed in RIPA buffer with protease inhibitor cocktail and 0.1% Triton X-100 (200 µl/flask). The homogenate was passed 10 times through a needle and syringe and then centrifuged at 1,000 g for 5 min. The supernatant protein concentration was determined with the BCA protein assay (Pierce). Seventy-five micrograms of protein were combined with SDS-PAGE reducing sample buffer and electrophoresed in a 4–20% gradient gel (Bio-Rad). The proteins were electroblotted onto polyvinylidene difluoride membrane (Bio-Rad) for 1 h. Six percent skim milk in 20 mM Tris-buffered saline was used for blocking and washing the membranes. Antibody against TRP6 channel (Alomone, Jerusalem, Israel) was diluted in milk-TBS, and membrane rotated in the solution overnight at 4°C. In TRP6 blocking experiments, the antibody was incubated 1 h with the specific peptide antigen provided with the antibody and then diluted 1:200. The second antibody was anti-rabbit IgG horseradish peroxidase (HRP) conjugate (Stressgen) 1:20,000 in milk-TBS with 0.01% Tween 20, rocked for 2 h at room temperature. After being washed, the membranes were incubated for 10 min at room temperature with SuperSignal West Femto chemiluminescent reagent (Pierce). Kodak x-omat fs-1 film was exposed. The blots were washed, reblocked, and incubated with anti-SMC actin monoclonal antibody (Sigma), followed by anti-mouse IgG-HRP conjugate (Sigma) and processed as above.

Statistics. For fluorescence microscopy experiments, an average baseline value was obtained for each cell, and all changes were calculated for each cell compared with its own baseline. P values of 0.01 were considered significant. A two-way ANOVA with repeated measures and a Student-Newman-Keuls post hoc test were used to assess the differences between and among groups in each experimental protocol. Values are expressed as means ± SE.

	RESULTS

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Release of Ca²⁺ from CPA-sensitive intracellular Ca²⁺ stores. Under hypoxic conditions, CPA increased fluorescence intensity by 20.9 ± 2.5% (P < 0.01, vs. baseline) in control PA SMC and by 69.6% ± 1.7% in hypertensive PA SMC (P < 0.01, vs. baseline; P < 0.01, vs. control). With acute normoxia, CPA increased fluorescence intensity by 47.4 ± 1.1% (P < 0.01, vs. baseline) in control PA SMC and by 114.0 ± 5.3% in hypertensive PA SMC (P < 0.01, vs. baseline; P < 0.01, vs. control; Fig. 1, A and B).

View larger version (23K): [in this window] [in a new window]   Fig. 1. Effect of cyclopiazonic acid (CPA) on cytosolic calcium in control and hypertensive fetal pulmonary artery smooth muscle cells (PA SMC). A: under conditions of chronic hypoxia (control, n = 92; hypertensive, n = 62), acute normoxia (control, n = 163; hypertensive, n = 112), or sustained normoxia (control, n = 67; hypertensive, n = 81), CPA caused a greater increase in cytosolic calcium in hypertensive, compared with control, fetal PA SMC. The results are presented as percent change ± SE in fluorescence emission intensity in response to CPA. *P < 0.001, vs. control cells. B: physiological tracings of experiments performed in each experimental condition.

View larger version (23K): [in this window] [in a new window]   Fig. 2. Capacitative calcium entry (CCE) in control and hypertensive fetal PA SMC. A: under hypoxic conditions, there is no difference in CCE between control (n = 67) and hypertensive (n = 34) fetal PA SMC. With acute normoxia, CCE decreases in control cells (n = 90) and increases in hypertensive (n = 56) fetal PA SMC. Sustained normoxia increases CCE in control (n = 59) cells. Despite sustained normoxia, CCE in hypertensive (n = 59 cells) cells remains greater than in control PA SMC. The results are presented as percent change ± SE in fluorescence emission intensity. *P < 0.001, vs. control cells. B: representative physiological tracings of individual experiments under experimental conditions described in A.

Rate of CCE. To evaluate the rate of CCE, the rate of Mn²⁺ quenching of the fura 2 fluorescence intensity was studied in control and hypertensive cells under acutely normoxic conditions following CPA-sensitive store depletion. The rate of Mn²⁺ quenching of the fura 2 signal was –1.8419 in control cells compared with –3.1036 in hypertensive cells. The rate of Mn²⁺ quench following CPA-sensitive store depletion was 71% higher in hypertensive compared with control cells (P < 0.01; Fig. 3). A similar pattern was present when the calcium channel blocker nifedipine was included in the perfusate.

View larger version (25K): [in this window] [in a new window]   Fig. 3. A: rate of quenching of fura 2 fluorescence intensity in control and hypertensive PA SMC after depletion of CPA-sensitive stores. Manganese acts as a surrogate cation for calcium and upon entry into the cell quenches fura 2 fluorescence. The rate of fura 2 fluorescence intensity decrease is directly proportional to the rate of extracellular calcium entry. The rate of fura 2 fluorescence signal intensity quenching is greater in hypertensive (slope = –3.1036; P < 0.001, vs. control) compared with control (slope = –1.8149) PA SMC. B: rate of quenching of fura 2 fluorescence intensity in control and hypertensive PA SMC after depletion of CPA-sensitive stores and in the presence of nifedipine. The rate of fura 2 fluorescence signal intensity quenching is greater in hypertensive (slope = –2.6293; P < 0.001, vs. control) compared with control (slope = –0.9146) PA SMC.

View larger version (30K): [in this window] [in a new window]   Fig. 4. Western Blot analysis of transient receptor potential channel 6 (TRPC6) protein expression from fetal PA SMC derived from control or hypertensive late-gestation fetal lambs. Representative immunoblot of PA SMC lysates (25 µg protein/lane) from control and hypertensive fetal PA SMC is shown.

	DISCUSSION

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Our laboratory has reported previously that an acute increase in oxygen tension results in a decrease in PA SMC cytosolic calcium (23). The present report provides data that an acute increase in oxygen tension normally decreases CCE in PA SMC. Thus, not only does an acute increase in oxygen tension decrease cytosolic calcium via K_Ca channel activation, membrane hyperpolarization, and closure of voltage-operated calcium channels (20), but it diminishes CCE, thereby contributing to the overall decrease in cytosolic calcium in normal fetal PA SMC.

Previous studies have demonstrated that despite an acute increase in oxygen tension, cytosolic calcium in PA SMC derived from animals with chronic intrauterine pulmonary hypertension remains elevated (18). In PA SMC derived from animals with chronic intrauterine pulmonary hypertension, K_Ca channel expression is decreased (7), the ion channel that determines resting membrane potential changes to a voltage-sensitive K⁺ channel (19), and the ryanodine-sensitive intracellular calcium store is more replete (18). The present data suggests that augmented loading of intracellular stores and CCE are additional mechanisms whereby cytosolic calcium remains persistently elevated in hypertensive PA SMC. In specific, while an acute increase in oxygen tension normally decreases CCE in fetal PA SMC, CCE does not decrease in response to an increase in oxygen tension in hypertensive PA SMC. Despite sustained normoxia, stores remain relatively more loaded, and CCE is greater in hypertensive, compared with normotensive, PA SMC. Further evidence for a role for CCE in the incomplete response to vasodilator stimuli that characterizes PPHN is the observation that in normoxic conditions, the rate of Ca²⁺ entry after store depletion is greater in hypertensive cells than in control cells.

The present report is the first to demonstrate the presence of these channels in the perinatal pulmonary circulation. Although the observation that TRP channel expression is greater in control compared with hypertensive cells may be considered inconsistent with the physiology, the divergent expression pattern may represent an effort to compensate for elevated cytosolic calcium that has been previously reported in hypertensive PA SMC (18). TRP6 protein expression is, however, consistent with the physiology data in that expression is greater in hypertensive compared with normotensive PA SMC. The present report does not represent a comprehensive survey of all TRP channels. Thus more complete molecular data may be more consistent with the physiology data included in the manuscript. Furthermore, the molecular data included in the present report supports the proposition that TRP channel expression in PA SMC is oxygen sensitive. Whereas previous investigators have demonstrated that hypoxia increases TRP channel expression (28), the present study was undertaken in PA SMC derived from the neonatal pulmonary circulation. Developmental differences in oxygen sensing may inform the response of neonatal compared with more mature PA SMC. The highly divergent responses to normoxia in control compared with hypertensive PA SMC suggest that TRP channels normally play a role in the postnatal adaptation of the pulmonary circulation. How chronic intrauterine hypertension effects this response remains unknown. On one level, the molecular data may be interpreted as at odds with the physiology data presented in the study. However, the physiologic data clearly demonstrates that CCE is increased in PA SMC derived from hypertensive compared with control PA SMC. One explanation may be that molecular expression of TRP channels decreases to compensate for the increase in calcium loading. Although this explanation represents speculation, there is precedent for this construct (17). Similarly, expression of TRP channels has been altered in response to injury (4).

Thus CCE may play a role in the persistently elevated pulmonary vascular tone that characterizes PPHN. In other experimental models, CCE plays a role in elevated pulmonary vascular tone (8, 15, 25). The physiological data included in the present manuscript in combination with the molecular data indicating a divergent response in the molecular expression of TRP 1, 3, 5, and 6 between control and hypertensive PA SMC suggests that CCE plays a role in maintaining elevated high pulmonary vascular tone in the normal fetus and the oxygen-induced perinatal pulmonary vasodilation.

Previous reports indicate that the amount of Ca²⁺ stored in the intracellular Ca²⁺ stores is relatively more filled in proliferating than nonproliferating human PA SMC (9). The observation that even after long-term exposure to normoxia, CPA-sensitive stores are more replete in hypertensive, compared with normal, PA SMC, is consistent with the notion that in chronic intrauterine pulmonary hypertension, CCE might play a role in increased PA SMC proliferation. Increased loading of intracellular stores may contribute to the increased cell proliferation that characterizes the histology of PPHN. Even after sustained normoxia, CCE is increased in hypertensive PA SMC. The fetal environment may increase the sensitivity of PA SMC to a postnatal insult that would otherwise have no effect on pulmonary vascular tone. An alternate interpretation is that elevated loading of intracellular stores serves to mitigate the increased rate of extracellular Ca²⁺ entry that results from augmented CCE (2). Putatively, the relatively greater loading of the intracellular stores may derive from diminished K_Ca channel expression and thereby compromise the ability of calcium release from intracellular stores to cause membrane hyperpolarization and closure of voltage-operated calcium channels.

Whether all TRP channels are store-operated remains unknown. Recent evidence supports a role for at least some TRP channels in CCE in PA SMC (6, 12, 27). In human PA SMC, TRP1 inhibition decreased CCE (26), and TRP1 overexpression increased CCE-induced constriction of rat pulmonary artery rings (15). The present study was unable to identify an alteration in molecular expression that mirrors the physiological changes reported in the present manuscript. However, the present results do indicate that normal postnatal adaptation of the pulmonary circulation entails an increase in TRP molecular expression in neonatal PA SMC. These molecules are expressed in adult PA SMC (6, 12, 16, 27). Hence, the response of fetal PA SMC to sustained normoxia may normally involve diminished TRP channel expression, thereby promoting a decrease in cytosolic calcium and diminished pulmonary vascular tone.

In conclusion, the present report provides data that TRP channels 1, 3, 5,and 6 are expressed and CCE is involved in calcium homeostasis in normal PA SMC. PA SMC normally respond to acute normoxia with decreased CCE. Chronic intrauterine pulmonary hypertension results in sustained increased CCE in PA SMC. The exaggerated CCE in hypertensive, compared with normal, PA SMC may play an etiological role in both the vasoconstriction and increased cellular proliferation that characterizes PPHN. Given these observations, CCE may represent an additional target for therapeutic intervention in a disease for which neither prevention nor definitive treatment is currently available.

	GRANTS

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This work was supported by National Heart, Lung, and Blood Institute Grants RO1-HL-60784 (D. N. Cornfield) and RO1-HL-70628 (D. N. Cornfield), American Heart Association Established Investigator Award (D. N. Cornfield), and Viking Children's Fund (E. R. Resnik).

	ACKNOWLEDGMENTS

 
These findings were presented in part at the Society for Pediatric Research Meeting on May 5, 2004. D. N. Cornfield is an Established Investigator of the American Heart Association.

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. N. Cornfield, Center of Excellence in Pulmonary Biology, Division of Pediatric Pulmonary, Allergy and Critical Care Medicine, Stanford Univ. Medical School, 300 Pasteur Drive, Palo Alto, CA (e-mail: cornfield{at}stanford.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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