Oxygen increases ductus arteriosus smooth muscle cytosolic calcium via release of calcium from inositol triphosphate-sensitive stores
Maggie Keck,1
Ernesto Resnik,1
Bradley Linden,2
Franklin Anderson,1
David J. Sukovich,1
Jean Herron,1 and
David N. Cornfield1,2,3
Division of Pediatric Pulmonary and Critical Care Medicine, Departments of 1Pediatrics, 2Physiology, and 3Surgery, University of Minnesota, Minneapolis, Minnesota
Submitted 29 October 2004
; accepted in final form 4 January 2005
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ABSTRACT
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In utero, blood shunts away from the lungs via the ductus arteriosus (DA) and the foramen ovale. After birth, the DA closes concomitant with increased oxygen tension. The present experimental series tests the hypothesis that oxygen directly increases DA smooth muscle cell (SMC) cytosolic calcium ([Ca2+]i) through inactivation of a K+ channel, membrane depolarization, and entry of extracellular calcium. To test the hypothesis, DA SMC were isolated from late-gestation fetal lambs and grown to subconfluence in primary culture in low oxygen tension (25 Torr). DA SMC were loaded with the calcium-sensitive fluorophore fura-2 under low oxygen tension conditions and studied using microfluorimetry while oxygen tension was acutely increased (120 Torr). An acute increase in oxygen tension progressively increased DA SMC [Ca2+]i by 11.7 ± 1.4% over 40 min. The effect of acute normoxia on DA SMC [Ca2+]i was mimicked by pharmacological blockade of the voltage-sensitive K+ channel. Neither removal of extracellular calcium nor voltage-operated calcium channel blockade prevented the initial increase in DA SMC [Ca2+]i. Manganese quenching experiments demonstrated that acute normoxia initially decreases the rate of extracellular calcium entry. Pharmacological blockade of inositol triphosphate-sensitive, but not ryanodine-sensitive, intracellular calcium stores prevented the oxygen-induced increase in [Ca2+]i. Endothelin increased [Ca2+]i in acutely normoxic, but not hypoxic, DA SMC. Thus acute normoxia 1) increases DA SMC [Ca2+]i via release of calcium from intracellular calcium stores, and subsequent entry of extracellular calcium, and 2) potentiates the effect of contractile agonists. Prolonged patency of the DA may result from disordered intracellular calcium homeostasis.
oxygen sensing; pulmonary hypertension potassium channels; vascular biology
IN THE FETAL PULMONARY CIRCULATION oxygen tension and nitric oxide production are low (13). Fetal pulmonary blood flow is limited, and blood pressure is high. Blood is shunted away from the lungs through the foramen ovale and the ductus arteriosus (DA), a structure that connects the pulmonary artery to the aorta in utero. At birth, pulmonary blood flow immediately increases by 8- to 10-fold, and pulmonary blood pressure decreases by 50% within the initial 24 h of life (24). An acute increase in oxygen tension causes perinatal pulmonary vasodilation (3, 26), even while it results in constriction of the DA (1, 5). Initial closure of the DA in response to an increase in PO2 is caused by vessel constriction (14), whereas long-term closure is accomplished through cell migration, apoptosis (25), and cell proliferation (4, 15, 18).
In the fetal state, elevation of pulmonary vascular tone and patency of the DA is of critical importance. How pulmonary vascular tone remains elevated while DA tone remains low despite being adjacent vascular structures is unknown. In the fetal pulmonary circulation, endothelin, a powerful vasoconstrictor (29) produced by the endothelial cell (20), plays a key role in maintaining elevated tone, as endothelin inhibition results in sustained fetal pulmonary vasodilation without affecting either pulmonary or aortic pressures (17). This observation suggests that endothelin possesses site-specific properties in the fetal circulation. Whether the low oxygen tension environment of the normal fetus enables endothelin to modulate perinatal pulmonary vascular tone without affecting DA tone is unknown.
In pulmonary artery smooth muscle cells (PA SMC), an acute increase in oxygen tension (PO2) activates calcium-sensitive K+ (KCa) channels, causing membrane hyperpolarization, closure of voltage-operated Ca2+ channels (VOCC), a decrease in cytosolic Ca2+ concentration ([Ca2+]i), and vasodilation (9, 23). In contrast, current data indicate that in DA SMC oxygen causes inactivation of a voltage-sensitive K+ (Kv) channel, membrane depolarization, and entry of calcium via VOCC. The data for this construct derive, in large measure, from studies performed in rabbit DA rings wherein an acute increase in PO2 caused constriction concomitant with an increase in [Ca2+]i. 4-Aminopyridine (4-AP), an inhibitor of the KV channel, mimics the effect of acute normoxia (27). Evidence for the role of calcium entry via VOCC is the observation that nifedipine, a VOCC blocker, causes dilation of rabbit DA rings constricted by oxygen exposure (19, 28).
Despite this data, a direct relationship between normoxia and an increase in [Ca2+]i in DA SMC has not been demonstrated. Whether release of calcium from intracellular stores plays a role in the response to normoxia is unknown. Thus the present experimental series tests the hypotheses that, in DA SMC, an increase in O2 tension 1) results in calcium release from intracellular stores, 2) increases cytosolic calcium via Kv channel inhibition and entry of extracellular Ca2+ through L-type Ca2+ channels, and 3) potentiates the effect of endothelin, resulting in DA constriction only after birth. To test these hypotheses, DA SMC were isolated from late-gestation fetal lambs maintained in primary culture and studied by microfluorimetry.
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METHODS
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Animals.
The procedures used in these studies were previously 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 (10). Late-gestation fetal lambs (135140 days gestation, term = 147 days) obtained from time-dated pregnant ewes were used in this study. Ewes were fasted for 24 h and were sedated with pentobarbital sodium. Fetal lambs were rapidly partially delivered through hysterotomy, with the head remaining inside the womb to prevent spontaneous breathing, and intracardiac pentobarbital sodium was administered. After fetal thoracotomy, the lung and heart block was isolated.
The DA was quickly excised and placed in physiological saline solution composed of (in mM) 120 NaCl, 5.9 KCl, 11.5 dextrose, 25 NaHCO3, 1.2 NaH2PO4, 1.2 MgCl2, and 1.5 CaCl2. Loose connective tissue and adventitia were removed, and the vessels were liberally rinsed with minimal essential media (MEM: 0.2 mM Ca2+). Vessels were carefully 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 5 ml MEM. After incubation at 37°C for 60 min in a shaking bath, the tissue suspension was triturated six or 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 cells 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 aliquotted onto 25-mm2 glass coverslips at a density of 510 x 103 cells/cm2. Cells were incubated at 37°C in a humidified 10% O2, 5% CO2, balance N2 (hypoxia) or humidified 95% air, 5% CO2 (normoxia) atmosphere. After 1824 h, medium, nonadherent cells, and debris were removed, 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.
Ca2+ imaging.
To assess dynamic changes in [Ca2+]i in individual DA SMC, the Ca2+-sensitive fluorophore fura-2 AM (Molecular Probes) was used. Subconfluent fetal DA 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 20 min in Ca2+-containing solution to allow for deesterification before 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 intensified charge-coupled device camera (Photonic Science, Robertsbridge, UK) using Axon Instruments (Foster City, CA) or Metafluor (Fryer, Bloomington, MN) image capture and analysis software. Ca2+ calibration was achieved by measuring a maximum (with 1 mM ionomycin) and a minimum (with 10 mM EGTA). Oxygen tension 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. Intracellular free Ca2+ was calculated assuming a dissociation constant of 220 (12). For each experiment 1020 cells were visualized and ratiometric data was acquired from individual cells.
Mn2+ quenching.
To assess changes in the rate of entry of extracellular Ca2+ in individual DA SMC, Mn2+ (0.5 mM) was substituted for calcium in the recording solution, as previously described (6). Mn2+-containing solution was superfused over the cells 1 min before the beginning of the experiment and throughout the experiment. Cells were excited with an excitation wavelength of 360 nm. The emission wavelength remained 510 nm. Because Mn2+ quenches the fura-2 signal, the rate of decrease of the fura-2 fluorescence signal is proportional to the rate of entry of Mn2+ into the cell. As Mn2+ acts as a surrogate divalent cation, entering cells through Ca2+ channels, the rate of fura-2 quenching is proportional to the rate of Ca2+ entry.
Statistical analysis.
Throughout, results are presented as means ± SE. Statistical significance was tested with Student's t-test (paired or unpaired as appropriate). P < 0.05 was taken as the threshold level for statistical significance. Experiments were designed to have a statistical power of at least 90% at a probability level of P < 0.05. 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 manganese quenching experimental protocol.
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 contained no CaCl2. Mn2+ buffer was identical to zero Ca2+ buffer except contained 0.5 mM MnCl2. All solutions were made with nanopure distilled water. Osmolality was adjusted to
300 mosM, and pH adjusted to 7.4.
Drugs and concentrations.
4-AP (103 M), nifedipine (5 µM), tetraethylammonium (TEA, 103 M), endothelin (106 M), caffeine (5 x 104 M), and glibenclamide (103 M) were obtained from Sigma (St. Louis, MO). Ryanodine (50 µM) was obtained from Molecular Probes (Eugene, OR). 2-Aminoethoxydiphenyl borate (2-APB, 50 µM), ionomycin, and angiotensin II (107 M) were obtained from Calbiochem (San Diego, CA).
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RESULTS
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Effects of an acute increase in PO2 on [Ca2+]i in chronically hypoxic DA SMC.
To determine the effect of an acute increase in PO2 on [Ca2+]i, baseline [Ca2+]i measurements were obtained under hypoxic conditions for 5 min, then PO2 was acutely increased to normoxic conditions. Acute normoxia increased DA SMC [Ca2+]i (Fig. 1) within 12 min (P = 0.016). Forty minutes after the switch to normoxia, [Ca2+]i increased by 11.7% ± 1.4% (P < 0.005; n = 193 cells, 7 animals).

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Fig. 1. Effect of acute normoxia on cytosolic calcium in ductus arteriosus smooth muscle cells (DA SMC). DA SMC were loaded with fura-2 under hypoxic conditions. Over 40 min, acute normoxia caused a progressive increase in fura-2 fluorescence. Results are presented as percent change in fluorescence emission intensity in response to acute normoxia over time. After 12 min of normoxia, fura-2 fluorescence increased compared with baseline (P = 0.016 vs. baseline; n = 193 cells, 7 animals).
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Effects of K+ channel blockers on [Ca2+]i.
Under hypoxic conditions, the effect of K+ channel blockers on DA SMC [Ca2+]i was studied. Cells were treated with glibenclamide, 4-AP, or TEA. Cells were washed with basic recording solution between each drug. In a separate set of experiments only 4-AP (103 M) was superfused over the cells. Neither TEA, a KCa channel blocker in mM concentrations (11), nor glibenclamide, a blocker of ATP-sensitive K+ channels, had an effect on DA SMC [Ca2+]i. 4-AP, a KV channel blocker, increased DA SMC [Ca2+]i by 4.85 ± 0.56% (P < 0.01; n = 102 cells, 3 animals). The increase in DA SMC [Ca2+]i was the same as after 12 min of acute normoxia (Fig. 2).

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Fig. 2. Voltage-sensitive K+ channel inhibition increases DA SMC cytosolic calcium. In DA SMC, 4-aminopyridine (4-AP, 103 M) increased fluorescence by the same degree (4.85 ± 0.56%; n = 107 cells, 3 animals; P < 0.001 vs. baseline) as that caused by acute normoxia (4.89 ± 0.96%) for 12 min (P = 0.7, 4-AP vs. normoxia for 12 min).
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Contribution of extracellular Ca2+ entry to oxygen-induced increase in DA SMC [Ca2+]i.
To determine the role of extracellular Ca2+ entry on the O2-induced increase in [Ca2+]i DA SMC, cells were superfused with zero-Ca2+ buffer or nifedipine, a blocker of L-type VOCC, concomitant with an acute increase in PO2. In the presence of nifedipine, DA SMC [Ca2+]i initially increased to a greater degree than the increase associated with acute normoxia alone. For the initial 12 min after addition of nifedipine and acute normoxia, [Ca2+]i was greater in cells treated with nifedipine than cells treated with acute normoxia alone. However, the increase was not sustained in nifedipine-treated DA SMC, as during minutes 12-40, the increase in [Ca2+]i was greater in control cells compared with DA SMC treated with nifedipine (P
0.001 vs. control; n = 93 cells, 3 animals). DA SMC exposed to an acute increase in oxygen tension in the presence of zero extracellular calcium behaved similarly, as the initial increase in [Ca2+]i was greater than control cells but was less than control cells for the duration of the study (P
0.01 vs. control; n = 41 cells, 3 animals; Fig. 3).
Effects of an acute increase in PO2 on the rate of extracellular Ca2+ entry in DA SMC.
To address the issue of whether entry of extracellular Ca2+ is necessary for the initial increase in [Ca2+]i, Mn2+ quenching was used. Mn2+-containing solution was superfused over DA SMC while the rate of fura-2 signal quenching was measured before and after an acute increase in PO2. Under hypoxic conditions, the rate of fura-2 quenching was 2.17 (fluorescence units over time, R2 = 0.9803). During the initial 5 min after an increase in oxygen tension, the rate of fura-2 quenching decreased by 38% to 1.35 (R2 = 0.9789) (Fig. 4; n = 115 cells, 3 animals; P < 0.001 hypoxia vs. normoxia).

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Fig. 4. Rate of manganese quenching of fura-2 fluorescence intensity in DA SMC under hypoxic (solid line) and normoxic (dashed line) conditions. Manganese acts as a surrogate cation for calcium and upon entry into the cell quenches fura-2 fluorescence. The rate of the fura-2 fluorescence intensity decrease is directly proportional to the rate of extracellular calcium entry. In response to acute normoxia (slope = 1.35, R2 = 0.9789), the rate of quenching of the fura-2 fluorescence intensity decreases by 38% compared with the rate of quenching under hypoxic conditions (slope = 2.17, R2 = 0.9803; P < 0.001 hypoxia vs. normoxia; n = 115 cells, 3 animals).
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Role of intracellular Ca2+ release on O2-induced increase in [Ca2+]i.
To examine the role of intracellular Ca2+ release on the O2-induced increase in DA SMC [Ca2+]i, either 2-APB, a blocker of Ca2+ release from inositol 1,4,5-triphosphate (IP3)-sensitive stores, or ryanodine at a dose that blocks release of Ca2+ from ryanodine-sensitive stores, was superfused over the cells concurrent with an acute increase in PO2. As illustrated in Fig. 5A, 2-APB, an antagonist of IP3-induced calcium release (2), blocked the acute normoxia-induced increase in [Ca2+]i (n = 62 cells, 4 animals; P
0.001 vs. control). To determine whether release of Ca2+ from intracellular stores is necessary for the sustained increase in [Ca2+]i, 2-APB was superfused over the cells 25 min after exposure to acute normoxia. When added after 25 min of acute normoxia, 2-APB had no effect on the oxygen-induced increase in DA SMC [Ca2+]i (n = 50 cells, 2 animals; Fig. 5B). The addition of ryanodine either at the onset of or after 25 min of acute normoxia had no effect on the oxygen-induced increase in DA SMC.

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Fig. 5. Effect of 2-aminoethoxydiphenyl borate (2-APB), a pharmacologic antagonist of calcium release from the inositol-triphosphate-sensitive stores, on normoxia-induced increase in DA SMC fura-2 fluorescence intensity. A: application of 2-APB upon onset of acute normoxia blocked the normoxia-induced increase in fura-2 fluorescence (n = 62 cells, 4 animals). B: normoxia-induced increase in DA SMC fura-2 fluorescence is unaffected by the application of 2-APB (n = 50 cells, 2 animals).
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Effect of endothelin on hypoxic or normoxic DA SMC [Ca2+]i.
In hypoxic DA SMC endothelin had no effect on [Ca2+]i (n = 78 cells, 4 animals). However, in DA SMC rendered acutely normoxic, endothelin increased fluorescence by almost threefold, from 0.61 ± 0.04 to 1.55 ± 0.17 (n = 34; P < 0.01 vs. hypoxia, baseline; Fig. 6). The initial increase was transient, with calcium fluorescence returning to baseline levels within 167 ± 35 s of endothelin exposure. The initial increase was followed by a more sustained, but less pronounced, increase in DA SMC [Ca2+]i, wherein DA SMC [Ca2+]i increased from 0.58 ± 0.01 to 0.89 ± 0.02 (P < 0.01 vs. hypoxia, baseline).

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Fig. 6. Effect of endothelin (ET-1) on hypoxic and normoxic DA SMC. DA SMC are treated with ET-1. Application of ET-1 caused a marked and sustained increase in DA membrane fluorescence in normoxic (n = 78 cells, 3 animals), but not hypoxic (n = 65 cells, 3 animals), DA SMC.
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To determine whether the dramatically increased response to endothelin in normoxia is specific, DA SMC were treated with angiotensin or caffeine either under hypoxic conditions or after acute normoxia. In hypoxia, endothelin had no effect on DA SMC fluorescence ratio, whereas in normoxia, endothelin increased DA SMC fluorescence ratio by 254 ± 26%. Angiotensin increased DA SMC fluorescence ratio by 8.05 ± 1.10% in hypoxia and 7.20 ± 0.33% in normoxia (P < 0.01 vs. baseline, P = not significant hypoxia vs. normoxia; Fig. 7). Caffeine increased DA SMC fluorescence ratio by 5.28 ± 0.34% in hypoxia and 3.25 ± 0.30% in normoxia (P < 0.01 vs. baseline, P < 0.01 hypoxia vs. normoxia).

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Fig. 7. Response of DA SMC to ET-1 (A), angiotensin (B), or caffeine (C) under conditions of either chronic hypoxia or acute normoxia. Although the response to ET-1 was dramatically increased in acute normoxia (n = 78 cells, 3 animals) compared with hypoxia (n = 65 cells), acute normoxia had little effect on the response of DA SMC to either angiotensin (hypoxia n = 47, normoxia n = 43) or caffeine (hypoxia n = 54, normoxia n = 25).
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DISCUSSION
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The present data demonstrate that an acute increase in PO2 directly increases [Ca2+]i in a subconfluent monolayer of DA SMC (Fig. 1). Pharmacological inactivation of voltage-sensitive, but not ATP- or calcium-sensitive, K+ channels mimics the effect of an acute increase in oxygen tension (Fig. 2), providing support for the proposition that KV channel inactivation mediates the O2-induced increase in DA SMC [Ca2+]i (19, 27). Contrary to previous reports (28), an acute increase in oxygen tension initially decreases the rate of calcium entry into DA SMC. Three separate experiments demonstrate that entry of extracellular calcium is not necessary for the initial increase in DA SMC [Ca2+]i. Rather, release of calcium from IP3-sensitive stores accounts for the initial increase in DA SMC [Ca2+]i. The sustained and progressive increase in DA SMC [Ca2+]i entails subsequent extracellular calcium entry (Fig. 3). Consistent with previous reports (6, 7), the present data suggest a role for endothelin in the normal transition of the pulmonary circulation, as the combination of acute normoxia and endothelin cause a marked calcium transient in DA SMC. The observation that acute normoxia has no effect on either angiotensin- or caffeine-induced increases in DA SMC [Ca2+]i implies a distinct role for the interaction between endothelin and oxygen tension in the DA.
Data supporting the notion that the initial increase in DA SMC [Ca2+]i is not dependent on the entry of extracellular calcium includes the observation that acute normoxia increased [Ca2+]i in the presence of either zero extracellular calcium or nifedipine. These results were further validated by experiments wherein the rate at which manganese quenched the fura-2 signal decreased with acute normoxia, consistent with a diminished rate of entry of extracellular calcium. Interestingly, after 12 min the normoxia-induced increase in DA SMC [Ca2+]i was no longer potentiated by prevention of extracellular calcium entry. However, the sustained and progressive increase in DA SMC [Ca2+]i associated with normoxia was prevented by the blockade of extracellular calcium entry.
Although the initial component of the normoxia-induced increase in DA SMC [Ca2+]i is not mediated by entry of extracellular calcium, the present data demonstrate that release of Ca2+ from IP3-sensitive stores accounts for the initial increase in DA SMC [Ca2+]i. Pharmacological blockade of calcium release from IP3-sensitive stores prevented normoxia-induced increase in DA SMC [Ca2+]i. However, blockade of calcium release from IP3-sensitive stores had no effect on normoxia-induced increase in DA SMC [Ca2+]i when administered 25 min after increasing oxygen tension. Together, these data suggest a biphasic response to an acute increase in oxygen tension in DA SMC. Initially, Ca2+ is released from IP3-sensitive intracellular stores as entry of extracellular Ca2+ slows. Subsequently, extracellular Ca2+ enters DA SMC, leading to a sustained and progressive increase in [Ca2+]i. The initial release of Ca2+ from intracellular stores is necessary to trigger the second phase of extracellular Ca2+ entry, as the addition of 2-APB concomitant with the introduction of acute normoxia prevents a rise in DA SMC [Ca2+]i. The observation that blockade of calcium release from ryanodine-sensitive stores has no effect on the normoxia-induced increase in DA SMC offers further support for the notion that release of calcium from an IP3-sensitive store plays a pivotal role in the normoxia-induced increase in DA SMC [Ca2+]i.
Closure of the DA at birth is a necessary step for the normal transition from fetal to air-breathing life (4). Endothelin, in combination with an acute increase in oxygen tension, causes a calcium transient that is sufficient to prompt DA SMC contraction. Previous studies demonstrated that an acute increase in PO2 causes an increase in tension of DA rings (8). In rabbit DA SMC acute normoxia inhibits K+ current, leading to membrane depolarization (19, 21, 28). Pharmacological blockade of the voltage-gated K+ channels mimics the effect of acute normoxia, whereas overexpression of the Kv channel confers oxygen sensitivity (27). Consistent with such data, the present results show that 4-AP mimics an acute increase in PO2 in DA SMC. In contrast, the present results demonstrate that preventing extracellular Ca2+ entry accentuates the initial increase in [Ca2+]i in DA SMC exposed to acute normoxia.
There are several possible explanations for the seemingly divergent results. First, prior studies that addressed the ability of L-type VOCC blockers to cause DA dilation were performed in preconstricted DA rings, whereas the present study addresses the effect of VOCC blockade on the normoxia-induced increase in DA SMC [Ca2+]i. Second, our data indicate a biphasic role of extracellular Ca2+ entry, with the first phase completed within 12 min after an increase in PO2. Previous studies have examined the effects of L-type VOCC blockers after DA rings or DA SMC have been normoxic for an unspecified amount of time and thus may not have observed the first phase of the response. The present data do not address the underlying mechanism whereby closure of KV channels decreases the rate of Ca2+ entry through VOCC. Potentially, sufficient KV channel closure to cause membrane depolarization does not occur until after 12 min of acute normoxia. Alternatively, there is precedent for intracellular Ca2+ modulating VOCC activity (16). Thus Ca2+ released from IP3-sensitive stores by an increase in PO2 may inhibit L-type Ca2+ channels.
The present study is limited by the absence of a mechanistic link among an increase in PO2, release of Ca2+ from IP3-sensitive stores, and subsequent extracellular Ca2+ entry. How augmented calcium release from intracellular stores slows the rate of extracellular calcium entry remains unknown. Given that the DA SMC maintained in primary culture respond to an acute increase in oxygen tension with an increase in cytosolic calcium, in direct contrast to the response of fetal PA SMC maintained in primary culture (22, 23), it is unlikely that the present findings are an artifact of culture conditions. Rigorous measures were undertaken to ensure that DA SMC were not subject to dedifferentiation while maintained in culture. DA SMC stained positively with SMC-specific markers and did not stain positively for endothelial cell-specific (factor VIII) markers. However, the response of the DA SMC is likely influenced by the absence of DA endothelial cells in the experimental design. An additional limitation of the present studies is that the conclusions are contingent upon the specificity of the pharmacological probes.
In conclusion, the present data indicate that DA SMC respond directly to an acute increase in PO2 with an increase in [Ca2+]i. Although the normoxia-induced increase is mimicked by pharmacological blockade of a voltage-sensitive K+ channel, release of Ca2+ from IP3-sensitive stores is necessary for the O2-induced increase in [Ca2+]i. With the initial increase in oxygen tension, the rate of extracellular Ca2+ entry slows. The sustained increase in DA SMC [Ca2+]i requires entry of extracellular Ca2+. The combination of an acute increase in oxygen tension and endothelin results in a sustained and marked elevation of DA SMC, thereby facilitating DA closure. The present results suggest a previously undescribed, yet centrally important, role for intracellular calcium homeostasis in mediating the response of DA SMC to an acute increase in oxygen. The present data may provide insight into the mechanisms responsible for pathological patency of the DA.
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GRANTS
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This work was supported by National Heart, Lung, and Blood Institute Grants RO1 HL-60784 and RO1 HL-70628 (D. N. Cornfield), an American Heart Association Established Investigator Award (D. N. Cornfield), and by the Viking Children's Fund (E. Resnik).
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ACKNOWLEDGMENTS
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The findings were presented in part at the Society for Pediatric Research Meeting (May 5, 2004; San Francisco, CA). Dr. Cornfield is an Established Investigator of the American Heart Association.
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FOOTNOTES
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Address for reprint requests and other correspondence: D. N. Cornfield, Div. of Pediatric Pulmonary and Critical Care Medicine, Univ. of Minnesota Medical School, 420 Delaware St. SE, MMC 742, Minneapolis, MN 55455 (E-mail: cornf001{at}umn.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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