1 Department of Anesthesiology, Justus Liebig University, 35392 Giessen, Germany; 2 Veterans Affairs Medical Center, Minneapolis 55417; and 4 Department of Pediatrics, Division of Pediatric Pulmonology and Critical Care, Departments of 3 Surgery, 6 Physiology, and 5 Medicine, University of Minnesota Medical School, Minneapolis, Minnesota 55455
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ABSTRACT |
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Ca2+-sensitive K+
(KCa) channels play an important role in mediating
perinatal pulmonary vasodilation. We hypothesized that lung KCa channel function may be decreased in persistent
pulmonary hypertension of the newborn (PPHN). To test this hypothesis,
pulmonary artery smooth muscle cells (PASMC) were isolated from fetal
lambs with severe pulmonary hypertension induced by ligation of the ductus arteriosus in fetal lambs at 125-128 days gestation. Fetal lambs were killed after pulmonary hypertension had been maintained for
at least 7 days. Age-matched, sham-operated animals were used as
controls. PASMC K+ currents and membrane potentials were
recorded using amphotericin B-perforated patch-clamp techniques. The
increase in whole cell current normally seen in response to normoxia
was decreased (333.9 ± 63.6% in control vs. 133.1 ± 16.0%
in hypertensive fetuses). The contribution of the KCa
channel to the whole cell current was diminished in hypertensive,
compared with control, fetal PASMC. In PASMC from hypertensive fetuses,
a change from hypoxia to normoxia caused no change in membrane
potential compared with a 14.6 ± 2.8 mV decrease in membrane
potential in PASMC from control animals. In PASMC from animals with
pulmonary hypertension, 4-aminopyridine (4-AP) caused a larger
depolarization than iberiotoxin, whereas in PASMC from control animals,
iberiotoxin caused a larger depolarization than 4-AP. These data
confirm the hypothesis that the contribution of the KCa
channel to membrane potential and O2 sensitivity is decreased in an ovine model of PPHN, and this may contribute to the
abnormal perinatal pulmonary vasoreactivity associated with PPHN.
pulmonary hypertension; fetus; oxygen sensing; persistent pulmonary hypertension of the newborn; Ca2+-sensitive K+ channel
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INTRODUCTION |
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THE MECHANISMS RESPONSIBLE for the maintenance of the high-tone, low-flow fetal pulmonary vasculature and the rapid increase in pulmonary blood flow that occurs at birth remain incompletely understood. At birth, pulmonary blood flow increases 8- to 10-fold, and pulmonary artery pressure declines steadily over the first several hours of life (18). Although physical factors and vasoactive products elaborated by the pulmonary vascular endothelium are involved in the regulation of perinatal pulmonary vascular tone (1, 7), recent data indicate that Ca2+-sensitive K+ channel (KCa) activation plays a key role in mediating perinatal pulmonary vasodilation. Perinatal pulmonary vasodilator stimuli, such as an increase in shear stress (23), ventilation (24), an acute increase in O2 tension (8), and nitric oxide (NO) (3, 5, 21), act, at least in part, through KCa channel activation. In pulmonary artery smooth muscle cells (PASMC), K+ activation causes membrane hyperpolarization, closure of voltage-operated Ca2+ channels, and a decrease in cytosolic Ca2+ (19) that correlates with vasodilation. Data from our laboratory demonstrate that KCa channel expression and activity are developmentally regulated, greatest in the late-gestation fetus, and decreasing with maturation (17). Thus, when the biological response to pulmonary vasodilator stimuli is imperative, KCa channel expression and activity are greatest.
In some newborn infants, pulmonary vascular resistance remains elevated after birth, resulting in a clinical syndrome termed persistent pulmonary hypertension of the newborn (PPHN), characterized by extrapulmonary right-to-left shunting of blood across the ductus arteriosus (DA) or patent foramen ovule, causing severe hypoxemia (11). Evidence suggests that adverse intrauterine stimuli such as chronic hypoxia or hypertension (12) can decrease endothelial nitric oxide synthase (NOS) gene and protein expression (22, 26) and NOS activity, and limit perinatal NO production, thereby contributing to the pathophysiology of PPHN.
Recent data demonstrated that KCa channel gene expression is decreased in whole lung tissue from animals with experimental chronic intrauterine pulmonary hypertension caused by ligation of the DA (9). The physiology and histology that characterize this experimental model of perinatal pulmonary hypertension in fetal lambs resemble the pathophysiology of PPHN in humans (2, 13, 20). The implications of that study were that, in PPHN, decreases in KCa channel activity may contribute to failure of the pulmonary circulation to achieve and sustain low pulmonary vascular resistance after birth. Whether the alterations in whole lung K+ channel mRNA expression correlate with alterations in K+ channel physiology in PASMC remains unknown. Thus the present study was undertaken to test the hypotheses that chronic intrauterine pulmonary hypertension directly affects fetal PASMC 1) O2 sensing; 2) ion channel currents; and 3) K+ channel that determines resting membrane potential.
To test these hypotheses, PASMC were isolated from late-gestation fetal lambs with and without chronic intrauterine pulmonary hypertension. Electrophysiological studies performed in freshly isolated PASMC demonstrated that chronic intrauterine pulmonary hypertension compromises PASMC O2 sensing, changes the ion channel currents, and decreases the contribution of the KCa channel to the whole cell current.
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MATERIALS AND METHODS |
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Animals. Procedures used in these studies were reviewed and approved by the Animal Care and Use Committee at the University of Minnesota.
Experimental model of chronic intrauterine pulmonary hypertension. Surgical ligation of the DA was performed as previously described (13, 20). Eighteen mixed-breed pregnant ewes between 124 and 128 days gestation (term = 147 days) 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. Ewes were kept sedated but breathed spontaneously throughout the operation. The gravid uterus was delivered through a midline laparotomy. The fetus' left forelimb was withdrawn through a small hysterotomy. A skin incision was made under the left forelimb after subcutaneous infiltration with lidocaine (2-3 ml, 1% solution). A left thoracotomy exposed the heart and great vessels. The DA was visualized. A 2-0 silk suture was placed around the DA and tied. The ribs and skin were reapproximated. The hysterotomy was closed, and the uterus was returned to 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 7-12 days, both control (n = 7) and hypertensive (n = 11) fetal lambs were rapidly delivered through a hysterotomy incision after injection of pentobarbital sodium in the umbilical artery to prevent spontaneous breathing. After thoracotomy, lungs were isolated, lung tissue was placed on ice and rinsed with sterile HBSS, and the distal pulmonary arteries were isolated.Electrophysiology studies of freshly isolated cells. Distal (4th and 5th order, intralobar) pulmonary arteries were dissected from fetal hypertensive and normotensive sheep and placed immediately in hypoxic solution. To maintain the low O2 state of the fetal environment, cells were prepared and stored in a hypoxic Ca2+-free Hanks' solution (see Solutions and drugs). Single cells were enzymatically dispersed using a papain digestion protocol. Briefly, arteries were incubated for 30 min at 4°C in Hanks' solution containing 0.5 mg/ml papain, 1 mg/ml albumin, and 1 mg/ml dithiothreitol, without EGTA, and then incubated at 37°C for 15-20 min. The arteries were washed thoroughly in enzyme-free Hanks' solution for at least 10 min and then maintained at 4°C. Several digestions were done each day to ensure cell viability. All cells were studied in identical conditions within 2 h of preparation.
Gentle trituration produced a suspension of single cells, which was then separated into aliquots in a perfusion chamber on the stage of the inverted microscope (Diaphot 200; Nikon) for electrophysiological studies. After a brief period to allow a partial adherence to the bottom of the recording chamber, cells were superfused via gravity with an experimental solution (see Solutions and drugs) at a rate of 2-3 ml/min for the recording of K+ current (Ik) and resting membrane potential. Whole cell recordings were performed using the amphotericin-perforated patch-clamp technique (15). Patch pipettes were pulled from glass tubes (PG 150T; Warner Instruments). The pipettes were fire-polished directly before the experiments and had a resistance of 2-3 MSolutions and drugs. The Hanks' solution contained (in mM) 145 NaCl, 4.2 KCl, 1 MgCl2, 1.2 KH2PO4, 10 HEPES, 10 glucose, and 0.1 EGTA (pH was adjusted to 7.4 by KOH). The extracellular or experimental solution contained (in mM) 115 NaCl, 5.4 KCl, 1 MgCl2, 1.8 CaCl2, 25 NaHCO3, 10 HEPES, and 10 glucose (pH 7.4 with NaOH). The standard intracellular pipette solution contained (in mM) 145 KCl, 1 MgCl2, 1 EGTA, 10 HEPES, and 120 µg/ml amphotericin B (pH was adjusted to 7.3 by KOH).
The effect of hypoxia was studied by switching between normoxic and hypoxic perfusate reservoirs. Normoxic solutions were equilibrated with 21% O2, 5% CO2, and 74% N2. Hypoxic solutions were achieved by bubbling with 0% O2 (plus 5% CO2-balance N2) for at least 20 min before cell perfusion. These procedures produced PO2 values in the cell chamber of 130-150 mmHg (21% O2) and 24-30 mmHg (0% O2). PCO2 was 36-42 mmHg, and pH was 7.37-7.42 under these conditions. O2 levels were measured with a Rapidlab Chiron blood gas analyzer from samples taken directly from the experimental chamber containing the PASMC during perfusion, which allows an exact measurement of PO2. By using a small recording chamber (400 µl), high perfusion rate (2-3 ml/min), and short dead space, bath exchange could be achieved in <30 s. Iberiotoxin was obtained from Alomone Laboratories (Jerusalem, Israel). All other compounds were purchased from Sigma Chemical (St. Louis, MO). Iberiotoxin was solubilized in distilled water. 4-Aminopyridine (4-AP) was dissolved in the extracellular solution. The drug solutions were adjusted to pH 7.4 before use.Statistical analysis.
Intergroup differences were assessed by ANOVA with post hoc analysis
with Fischer's least-significant difference test. P values <0.05 were considered significant. Numerical values are given as
means ± SE of n cells. In Figs. 1-4, the SE is
indicated when it exceeds the symbol size.
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RESULTS |
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Response of whole cell currents to normoxia in PASMC from normotensive and hypertensive animals. Ik recorded in PASMC under hypoxic conditions was small in both groups, as shown in Fig. 1 (284.3 ± 71.2 pA at +50 mV; n = 9 for control vs. 336.7 ± 38.3 pA at +50 mV; n = 11 for PASMC from hypertensive animals, P > 0.05), with no significant difference in cell capacitance (15.7 ± 1.1 pF, n = 17; 17.4 ± 0.8 pF, n = 18 for normotensive and hypertensive PASMC, respectively, P > 0.05). Figure 1C shows Ik recorded at more negative membrane potential in both groups and clearly demonstrates a lack of significant difference at all investigated potentials.
PASMC isolated from control animals demonstrated spiking, oscillatory outward Ik, consistent with the appearance of spontaneous outward transient currents (STOC; see Refs. 4 and 6 and Fig. 1A), whereas in PASMC from hypertensive animals the STOC pattern was completely absent (Fig. 1B). As previously described (8), PASMC from normal fetuses respond to an acute increase in O2 tension with an increase in Ik (Fig. 1A). In PASMC from control animals, the O2-induced increase in whole cell current was 333.9 ± 63.6% (n = 8) compared with an increase of 133.1 ± 16.1% (n = 8) in PASMC from hypertensive fetuses (P < 0.05 control vs. hypertensive; Fig. 1, A and B).Pharmacology of whole cell currents. In the present experiments, in PASMC from normal fetuses, iberiotoxin decreased whole cell current by 58.4 ± 10.4% (n = 5) under normoxic conditions (Fig. 2A), whereas the 4-AP-induced inhibition was 32.7 ± 8.1% at +50 mV (n = 4; Fig. 2B). In contrast, in PASMC isolated from fetal animals with pulmonary hypertension, the contribution of the KCa channel to the whole cell current was decreased, since iberiotoxin inhibited just 30.2 ± 9.7% (n = 5; +50 mV; Fig. 3A) of the whole cell current, whereas the voltage-gated K+ (Kv) channel blocker 4-AP inhibited 62.2 ± 9.8% (n = 5; +50 mV; Fig. 3B) of the current.
Membrane potential.
In PASMC from normal fetuses, switching from hypoxia to normoxia caused
membrane hyperpolarization of 14.6 ± 2.8 mV, a degree of
membrane hyperpolarization that is consistent with previous reports
(Fig. 4; Refs. 8 and 10).
Iberiotoxin (100 nM) caused a similar change in membrane potential
(14.2 ± 1.8 mV; n = 5), whereas 5 mM 4-AP had no
significant effect on membrane potential (n = 5). In
PASMC isolated from fetal animals with pulmonary hypertension, a change
in O2 tension from hypoxia to normoxia had no effect on
membrane potential change. 4-AP caused a larger depolarization than
iberiotoxin (13.7 ± 1.1 mV; n = 5 vs.
8.8 ± 1.2 mV; n = 5).
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DISCUSSION |
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The present study demonstrates that chronic intrauterine pulmonary hypertension directly affects PASMC ion channel currents. Specifically, chronic intrauterine pulmonary hypertension attenuated the increase in K+ channel current associated with an acute increase in O2 tension. Resting membrane potential did not change with an increase in O2 tension. Furthermore, in PASMC derived from animals with chronic intrauterine pulmonary hypertension, the contribution of Kv channels to the whole cell current was greater than in PASMC from control animals. Finally, the STOC that characterizes whole cell current in PASMC from normal fetal animals (8, 16) was not appreciated in PASMC from fetal animals with chronic intrauterine pulmonary hypertension.
The present study demonstrates that chronic elevation of intrauterine pulmonary artery blood pressure directly affects PASMC O2 sensing. Although in normal fetal PASMC the KCa channel predominates and is sensitive to an increase in O2 tension (16), in PASMC from animals with pulmonary hypertension the Kv channel predominates and does not respond to an acute increase in O2 tension. Membrane potential in PASMC obtained from hypertensive animals does not change in response to an acute increase in O2 tension.
Given data that point to a critically important role for pulmonary vascular KCa channel activation in perinatal pulmonary vasodilation, the incomplete response of the perinatal pulmonary vasculature to vasodilator stimuli in PPHN may result from effects on the PASMC KCa channel. O2 (8), NO (3, 5, 21), ventilation (24), shear stress (23), and physiological stimuli that cause perinatal pulmonary vasodilation act through KCa channel activation. Thus, if intrauterine events compromise PASMC KCa channel activity, then essential perinatal vasodilator stimuli may be unable to prompt the sustained and progressive pulmonary vasodilation that characterizes the normal transition of the pulmonary circulation (18).
The observed changes in K+ channel current seen in this study correlate well with the changes in K+ channel mRNA expression previously observed with this model (9). However, the present study examines currents through functional channels in isolated smooth muscle cells. This is the first evidence that there is a change in the balance of the ion channel currents that control smooth muscle membrane potential and O2 sensitivity in an animal model of PPHN. The present findings suggest that changes in Ik could underlie the vascular reactivity and attenuated perinatal pulmonary vasodilation that characterizes this model (2, 13) and perhaps PPHN.
Although the contribution of the KCa channel to the whole cell Ik was dramatically reduced in this model, it was not completely eliminated. The present finding is consistent with the previous studies that demonstrated a 30% reduction in KCa channel expression in whole lung tissue of fetal lambs with a ligated DA (9). The present data imply that the decrease in KCa channel mRNA expression is sufficient to compromise the O2 sensitivity of the current and may be responsible for the impaired vascular reactivity seen with this model. In human infants, intrauterine closure of the DA likely leads to an increase in PPHN (25). The present data suggest that pulmonary vascular KCa channel activity may be affected in human infants with intrauterine closure of the DA.
The electrophysiology data reported in the present study address effects of ductal ligation and chronic intrauterine pulmonary hypertension on plasmalemmal K+ channels. However, the effects of chronic intrauterine pulmonary hypertension may extend beyond ion channels in the membrane. For example, an acute increase in O2 tension causes a decrease in PASMC intracellular Ca2+ concentration through quantal release of intracellular Ca2+ from ryanodine-sensitive stores (14), thereby causing activation of the KCa channel. Chronic intrauterine hypertension may affect the spatial relationship of the ryanodine-sensitive stores in a manner that precludes KCa channel activation. The experimental approach in the present studies does not permit ready separation of membrane events from cytosolic events. Further studies are necessary to more fully elucidate the subcellular events that may be affected by chronic intrauterine hypertension and thereby compromise O2 sensing in the pulmonary vasculature. Moreover, additional experiments that use an integrative physiology model to confirm the findings outlined in this study are essential to more fully characterize the significance and physiological implications of the present electrophysiological findings.
It is interesting to note that the switch from a KCa-dominated current to a Kv current seen in hypertensive fetuses parallels the change that is associated with maturation (16). In fetal PASMC, the KCa channel determines resting membrane potential (8), whereas in the adult PASMC, the Kv channel is the major determinant of resting membrane potential (27). The KCa channel predominance in the fetal PASMC renders the fetal PASMC uniquely well adapted and ready to respond to an acute increase in O2 tension at a critical point in development. In contrast, the Kv channel predominance in the adult PASMC enables the adult pulmonary circulation to respond to an acute decrease in O2 tension (28), thereby optimizing the match between ventilation and perfusion and minimizing intrapulmonary shunting. Chronic intrauterine pulmonary hypertension may decrease KCa channel expression and activity while concomitantly increasing Kv channel expression and activity. The molecular signal responsible for the change in PASMC expression is unknown but may have significant therapeutic implications.
In summary, the present data provide evidence that chronic intrauterine pulmonary hypertension directly affects O2 sensing in fetal PASMCs. The ion channel responsible for determining resting membrane potential changes from the Ca2+-sensitive to a voltage-sensitive K+ channel. The membrane hyperpolarization that normally occurs in fetal PASMC in response to an acute increase in O2 tension is absent in PASMC from animals with chronic intrauterine pulmonary hypertension. Whether the findings reported in the present study derive entirely from effects on plasma membrane K+ channel expression and activity remains unknown. The present study suggests that the attenuated O2-induced pulmonary vasodilation that characterizes PPHN may result from compromised PASMC KCa channel activity. Further studies are necessary to determine the molecular signal responsible for the alterations in PASMC K+ channel expression and activity.
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ACKNOWLEDGEMENTS |
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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-65322 (E. K. Weir), the National Research Service Award (B. C. Linden), an American Heart Association Established Investigator Award (D. N. Cornfield), Veterans Affairs Merit Review Funding (E. K. Weir), and Deutsche Forschungsgemeinschaft Grant Ol 127/1-1 (A. Olschewski).
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FOOTNOTES |
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Address for reprint requests and other correspondence: D. N. Cornfield, Box 742, Div. of Pediatric Pulmonology and Critical Care, Univ. of Minnesota School of Medicine, 420 Delaware St. S.E., Minneapolis, MN 55455 (E-mail: cornf001{at}tc.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.
August 2, 2002;10.1152/ajplung.00100.2002
Received 4 April 2002; accepted in final form 8 July 2002.
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