Departments of 2 Medicine,
5 Pediatrics, and
1 Physiology, The mechanism responsible for the abrupt
decrease in resistance of the pulmonary circulation at birth may
include changes in the activity of
O2-sensitive
K+ channels. We characterized the
electrophysiological properties of fetal and adult ovine pulmonary
arterial (PA) smooth muscle cells (SMCs) using conventional and
amphotericin B-perforated patch-clamp techniques. Whole cell
K+ currents of fetal PASMCs in
hypoxia were small and characteristic of spontaneously transient
outward currents. The average resting membrane potential (RMP) was
ion channels; hypoxia; pulmonary circulation; fetus; adult
THE MECHANISMS RESPONSIBLE for 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 (28). Closely regulated and limited pulmonary blood flow is
necessary for normal pulmonary vascular development (7, 22, 34). At
birth, pulmonary blood flow increases 8- to 10-fold, whereas pulmonary
arterial (PA) pressure declines by >50% over the first 24 h of life
(9, 13, 14). Although physical factors and vasoactive products
elaborated by the pulmonary vascular endothelium have been shown to be
involved in the regulation of perinatal vascular tone (3, 10, 11, 15,
30), evidence also suggests an important role for PA smooth muscle
cells (SMCs) (11, 12).
The low PO2 environment
of the normal fetus may contribute to the high fetal pulmonary vascular
resistance by direct effects on the fetal PASMC
K+ channels. In adult animals,
acute (6, 26, 35) and chronic (29) hypoxia cause inhibition of
K+-channel activity and membrane
depolarization of PASMCs. The observation that
O2-induced fetal pulmonary
vasodilation is prevented by blockade of
Ca2+-dependent
K+
(K Ca) channels suggests a
central role for K+ channels in
perinatal pulmonary vasodilation (11, 31). Furthermore, inhibition of
K Ca channels in cultured fetal
PASMCs causes membrane depolarization and an increase in cytosolic
Ca2+ concentration (12). A
patch-clamp study (11) showed that an increase in
PO2 in freshly isolated fetal PASMCs
increases K+ efflux through
K Ca channels and causes
membrane hyperpolarization. In contrast, in adult PASMCs, the
O2-sensitive
K+ channel appears to be a
4-aminopyridine (4-AP)-sensitive voltage-dependent K+
(K v) channel (6, 35).
Although these observations support the importance of PASMC
K+ channels in the regulation of
perinatal pulmonary vascular tone, they suggest that it is not the same
K+ channel that responds to
O2 in the fetus and adult.
Maturation-related differences in the capacity of the pulmonary
vasculature to respond to changes in
PO2 have been previously described
(2, 20, 36). For example, a 4- to 6-mmHg increase in
PO2 in the late-gestation ovine fetus causes a three- to fourfold increase in pulmonary blood flow (4), whereas in the early-gestation animal, there is no increase in pulmonary blood flow (23). Pulmonary vasodilation caused by endothelium-dependent nitric oxide (NO), a vasoactive mediator that is
essential for the normal transition of the pulmonary vasculature (1,
20), also increases with maturation (2). NO causes pulmonary
vasodilation, at least in part, through activation of K Ca channels (5). If PASMC
K+ channels play a central role in
the regulation of perinatal pulmonary vascular tone, then
maturation-related changes in PASMC
K+-channel activity might also be expected.
To investigate changes in functional expression of
K+ channels between the fetal and
adult pulmonary circulations, whole cell studies with conventional and
amphotericin B-perforated patch-clamp techniques were done in freshly
dispersed PASMCs from late-gestation fetal and adult sheep. To study
basal physiological K+-channel
activity and examine the effects of acute hypoxia on that activity, an
environment with a similar PO2 to
that seen in the SMCs under study was established. For the fetal
PASMCs, the PO2 is normally low
(~20 mmHg), whereas in the adult, resistance PASMCs have a
PO2 of ~140 mmHg. Thus patch-clamp studies were initially done with cells perfused with hypoxia for the
fetus and normoxia for the adult. Because fetal
K+-channel activity is known to
increase on switching from hypoxia to normoxia (11), studies were also
done with normoxic fetal PASMCs to determine whether normoxic currents
were sensitive to acute hypoxia.
Whole Cell Patch Clamp
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
36 ± 3 mV and could be depolarized by
charybdotoxin (100 nM) or tetraethylammonium chloride (5 mM; both
blockers of Ca2+-dependent
K+ channels) but not by
4-aminopyridine (4-AP; 1 mM; blocker of voltage-gated
K+ channels) or glibenclamide (10 µM; blocker of ATP-dependent K+
channels). In hypoxia, chelation of intracellular
Ca2+ by 5 mM
1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic
acid further reduced the amplitude of the whole cell
K+ current and prevented
spontaneously transient outward current activity. Under these
conditions, the remaining current was partially inhibited by 1 mM 4-AP.
K+ currents of fetal PASMCs
maintained in normoxia were not significantly reduced by acute hypoxia.
In normoxic adult PASMCs, whole cell K+ currents were large and RMP was
49 ± 3 mV. These 4-AP-sensitive K+ currents were partially
inhibited by exposure to acute hypoxia. We conclude that the
K+ channel regulating RMP in the
ovine pulmonary circulation changes after birth from a
Ca2+-dependent
K+ channel to a voltage-dependent
K+ channel. The
maturational-dependent differences in the mechanism of the response to
acute hypoxia may be due to this difference in
K+ channels.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
Whole cell recordings. Gentle
trituration produced a suspension of single cells that were pipetted
into a perfusion chamber on the stage of an inverted microscope for
conventional whole cell (17) and perforated patch-clamp (27) studies.
After a brief period to allow partial adherence to the bottom of the
recording chamber, cells were perfused with either a hypoxic (fetal
PASMCs) or normoxic (fetal and adult PASMCs) solution composed of (in mM) 115 NaCl, 25 NaHCO3, 4.2 KCl,
1.2 KH2PO4,
1.5 CaCl2, and 10 HEPES (pH 7.4 with NaOH). Hypoxic solutions (PO2
~35 mmHg) were bubbled with 3.5%
CO2-balance
N2, whereas normoxic solutions
(PO2 ~160 mmHg) were bubbled with
20% O2-3.5% CO2-balance
N2.
O2 levels were measured with a
Ciba Corning blood gas analyzer from samples taken from the bath during
perfusion. Electrodes (resistance 2-3 M) were fire polished and
filled with a solution composed of (in mM) 140 KCl, 1.0 MgCl2, 10 HEPES and 1.0 EGTA or 5 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA; see below) (pH 7.2 with KOH). For perforated patch-clamp studies, amphotericin B (120 µg/ml) was included in the pipette solution. Capacitance was corrected for, and perforation was monitored by changes in membrane potential [observed in current
(I) = 0 mode on the patch-clamp
amplifier] and changes in series resistance. Recordings were not
made until series resistance was <15 M
to minimize voltage errors.
The small currents recorded in the fetal cells usually made series
resistance compensation unnecessary. In the adult cells, series
resistance was further minimized by electronic compensation.
Outward currents were recorded from fetal and adult cells with the
perforated patch-clamp technique. For these studies, cells were voltage
clamped at a holding potential of 70 mV, and currents were
evoked by +10- or +20-mV steps to more positive potentials, with test
pulses of 100- or 200-ms duration.
A previous study (8) on spontaneously transient outward current (STOC)
activity show that the activity can be inhibited by high (1-10 mM)
intracellular concentrations of the
Ca2+ chelator EGTA. To test the
Ca2+ sensitivity of outward
currents recorded from hypoxic fetal cells, conventional whole cell
recordings were made with 5 mM BAPTA (a Ca2+ chelator with a faster
Ca2+-binding time than EGTA) (32)
included in the pipette solution. For some of these studies, cells were
also voltage clamped at a holding potential of 10 mV to
inactivate K v channels and
then depolarized to more positive potentials in +10-mV steps. For all patch-clamp studies, currents were filtered at 1 kHz and sampled at 2.5 kHz.
Membrane potentials were recorded with the perforated patch-clamp technique in current-clamp mode at the resting membrane potential (RMP; measured as zero potential) of each cell. Membrane potential stability was always determined for at least 1 min before any recording. Data were recorded and analyzed with pClamp 6.02 software (Axon Instruments, Foster City, CA). Drugs dissolved in the extracellular perfusate were applied to the cells via gravity perfusion at a rate of 2 ml/min. All experiments were performed at 22°C.
Drugs Used
4-AP (inhibitor of K v channels), tetraethylammonium chloride (TEA; inhibitor of K Ca channels), and glibenclamide (inhibitor of ATP-dependent K+ channels) were obtained from Sigma (St. Louis, MO), whereas charybdotoxin (CTX; inhibitor of K Ca channels) was obtained from RBI (Natick, MA). Drug solutions were adjusted to pH 7.4. All drugs were dissolved in normal saline except glibenclamide, which was dissolved in ethanol and then diluted in normal saline. The diluted ethanol vehicle was tested and found to have no effect on K+-channel activity (data not shown).Statistical Analysis
Intergroup differences were assessed by a factorial analysis of variance with post hoc analysis with Fisher's least significant difference test. Data are expressed as means ± SE. Membrane potential data were compared with Student's unpaired t-test. P values < 0.05 were considered significant. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
K+-Channel Activity in Fetal and Adult PASMCs
Basal whole cell K+-current (IK) values were recorded from fetal PASMCs in hypoxia and normoxia and from adult PASMCs in normoxia. IK recorded in fetal PASMCs in hypoxia was small (473 ± 151 pA at +50 mV; n = 11; Fig. 1A) and had the characteristics of STOCs (Fig. 1B) as previously described (14). STOC activity could be recorded with both conventional whole cell and perforated patch-clamp techniques but gradually declined over time under the conventional configuration (data not shown). Pharmacological studies on STOC activity were therefore done with the perforated patch clamp unless otherwise stated. When the cells were held at a steady-state potential of
|
|
Because CTX has been shown to block at least two
K v channels (16), the
Ca2+ sensitivity of the
IK activity in
hypoxic fetal PASMCs was also tested. With the use of the conventional
whole cell patch clamp, cells were dialyzed with the rapid
Ca2+ chelator BAPTA (5 mM). Under
these conditions, whole cell
IK was further
reduced (202 ± 44 pA at +50 mV; n = 4 cells; Fig. 1A), and there was
no evidence of STOC activity. The remaining outward current (~40% of
IK recorded from
hypoxic fetal PASMCs with the perforated patch clamp) could be
inhibited by 1 mM 4-AP (81.4 ± 2.8% at +50 mV;
n = 3; Fig.
3A).
Therefore, a small component of hypoxic fetal
IK was due to
activation of K v channels.
Even with BAPTA in the pipette,
K Ca-channel activity could be
recorded by holding cells at a potential of 10 mV to inactivate
K v-channel activity. This
channel activity was inhibited by 100 nM CTX (Fig. 3B). Because Cornfield et al.
(11) have previously shown, using the perforated
patch-clamp technique, that hypoxic fetal
IK levels are
increased by normoxia, BAPTA-dialyzed cells were exposed to 4 min of
normoxia, and the effects on
K Ca channels were recorded at
potentials positive to
10 mV. Under these conditions,
K Ca-channel activity was
increased in normoxia (Fig. 3B).
|
IK recorded from
normoxic fetal PASMCs was larger (876 ± 261 pA at +50 mV;
n = 5: Fig.
1A) than
IK recorded from
hypoxic PASMCs and without STOC activity but was still inhibited by TEA
(data not shown).
IK values
recorded in normoxia from adult PASMCs were larger than those recorded
from either fetal preparation (1,389 ± 214 pA at +50 mV;
n = 11 cells; Fig.
1A), with no significant difference in cell capacitance (15.9 ± 1.1 pF,
n = 11; 17.8 ± 0.9 pF,
n = 5; and 17.7 ± 1.5 pF,
n = 11 for hypoxic fetal
cells, normoxic fetal cells, and normoxic adult cells, respectively). Outward currents in the adult PASMCs had no STOC activity and were
characteristic of delayed rectifier activity (Fig.
1B). At positive
membrane potentials, currents could be inhibited by both 1 mM 4-AP
(61.1 ± 7.4% at +50 mV; n = 6 cells) and 5 mM TEA (33.3 ± 2.7% at +50 mV;
n = 4 cells), suggesting that, as in
the fetal cells, both K v and
K Ca channels were present but
that K v channels were
predominant. TEA had little effect on
IK recorded at
negative membrane potentials, in contrast to 4-AP that completely
inhibited currents evoked at these potentials (Fig.
4). Figure
4B shows the percentage of
IK inhibited by 5 mM TEA at potentials of 10 and +50 mV in both fetal and adult
PASMCs and clearly demonstrates the decrease in contribution of
K Ca channels to
IK during
maturation.
|
RMP in Fetal and Adult PASMCs
Average RMP recorded from fetal cells in hypoxia was
|
Effect of Hypoxia on IK in Fetal and Adult PASMCs
Exposure of normoxic fetal PASMCs to 3 min of hypoxia had little effect on IK (15.1 ± 9% inhibition at +10 mV; n = 4; not significant; Fig. 6). In contrast, IK recorded from adult PASMCs was partially inhibited by acute exposure to hypoxia (33.5 ± 6% inhibition at +10 mV; P < 0.05; n = 5; Fig. 6).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The mechanism by which hypoxia contributes to the low-flow, high-resistance fetal pulmonary vasculature remains uncertain as does the full mechanism for the dilation of the pulmonary vasculature that occurs at birth. Although the mechanical effects of ventilation and endothelium-dependent dilator factors such as NO play an important role (20), there is also evidence that PASMCs are involved (11, 12). In the adult pulmonary vasculature, hypoxia has been shown to inhibit K v channels in PASMCs to initiate the constriction involved in matching ventilation and perfusion (6, 35). A previous study (11) with fetal PASMCs suggested that the K+ channel controlling RMP and activated by O2 is Ca2+ sensitive. To fully determine the roles of the K v and K Ca channels in both fetal and adult PASMCs, electrophysiological studies of their K+-channel activity were done in both hypoxia and normoxia.
Our data suggest that there are maturational changes in K+-channel expression between the fetus and adult. The K+ channel controlling RMP in the hypoxic environment of the developing fetus appears to be a K Ca channel because membrane potentials recorded from fetal PASMCs were only depolarized by CTX and TEA (K Ca-channel blockers) (Fig. 5). Outward currents in hypoxic fetal PASMCs are characteristic of STOCs. STOCs are likely to be due to the activation of K Ca channels by quantal releases of Ca2+ from the sarcoplasmic reticulum (24). Their activity gradually declined under conditions of cell dialysis with the conventional whole cell patch clamp and could be prevented by chelation of intracellular Ca2+ with high concentrations of BAPTA. The presence of STOCs may reflect the high-tone environment of the fetal pulmonary vasculature because they are also found in other high-resistance beds such as the adult cerebral circulation (24). The physiological significance for STOCs in the developing fetal pulmonary circulation is unknown but may provide a moderating influence that prevents fetal pulmonary vascular resistance from rising too high. The patch-clamp evidence that K Ca-channel activity controls RMP and hence helps to determine pulmonary vascular tone in the fetus is consistent with previous data (12) showing that blockade of K Ca channels in single fetal PASMCs increases cytosolic Ca2+ concentration. It should be noted, however, that in the intact, acutely instrumented fetus, K Ca-channel blockade has not been conclusively shown to affect basal pulmonary vascular tone. This apparent contradiction may be due to the presence of vasoactive mediators such as NO (1) or prostacyclin (18) in the intact pulmonary circulation. These vasodilator agents may antagonize the constrictor effect of K Ca-channel blockade in the whole animal. Indeed, initial patch-clamp studies support this interpretation by showing that NO can overcome the blocking effect of CTX on single K Ca channels (Reeve, unpublished observations).
Although the K Ca channel
appears to control RMP, there is some
K v-channel activity in hypoxic
fetal PASMCs as indicated by partial inhibition of
IK by 4-AP (Fig.
3A). In the normoxic adult PASMCs,
both K v and
K Ca channels contribute to
IK, but here
K v-channel activity is
predominant and controls RMP (Figs. 4 and 5). Because hypoxia has been
reported to inhibit one or more
K v channels to initiate
pulmonary vasoconstriction, the maturation-related increase in the
capacity of the pulmonary vasculature to respond to hypoxia may be due
to increasing K v-channel
activity with age. Exposure to chronic hypoxia can downregulate some
K v channels in rat PASMCs (29,
33) and upregulate K Ca-channel activity in human PASMCs (25). Chronic hypoxia is also known to
depolarize the RMP of cells to similar values as those recorded from
hypoxic fetal PASMCs (29). Because
PO2 in the fetus is normally low, it
is possible that prolonged exposure to hypoxia downregulates
K v-channel activity, resulting
in a depolarized membrane potential and hence dominant
K Ca-channel activity. It has
also been shown in cultured cell lines that prolonged depolarization
can downregulate K v-channel
expression, providing an additional pathway by which
K v-channel activity may be
modulated in the constricted fetal pulmonary circulation (19).
Furthermore, in systemic hypertension, where basal tone is increased as
the blood vessels constrict, there is a compensatory increase in the expression of K Ca channels
(21). Importantly, although
K v-channel activity was
observed in hypoxic fetal PASMCs, it was only recorded when cells were
depolarized from a holding potential of 70 mV. At a more
depolarized holding potential closer to the RMP of these cells,
K v-channel activity was
inhibited and only K Ca-channel activity was recorded (Fig. 3).
Because normoxia hyperpolarizes fetal PASMC membrane potential to values similar to those recorded from normoxic adult PASMCs, it could be considered that normoxia simply brings the fetal pulmonary circulation to membrane potentials where K v channels are active. In this case, hypoxia might be expected to inhibit IK recorded from normoxic fetal PASMCs. However, our data do not support this, with little effect of hypoxia observed on IK recorded from fetal PASMCs that had been normoxic for 2-6 h. In contrast, IK values recorded from normoxic adult PASMCs were partially inhibited by acute hypoxia (Fig. 6). On reflection, these data might not be considered surprising. In the whole lung, chronic exposure to hypoxia prevents subsequent pulmonary vasoconstriction to acute hypoxic challenges. Animals must be returned to normoxia for at least 24 h before this acute hypoxic vasoconstriction returns (D. P. Nelson and E. K. Weir, unpublished observations), suggesting that there is some delay in the recovery of the mechanism by which acute hypoxia is sensed. The short exposure of the fetal PASMCs to normoxia before their exposure to acute hypoxia may not have allowed for this conversion to occur. The increase in the proportion of IK sensitive to 4-AP in the adult suggests an increase in K v-channel activity that may account for the larger whole cell currents even between normoxic fetal and normoxic adult cells. Although acute normoxia activates K Ca channels to initiate membrane hyperpolarization, long-term normoxia may allow the upregulation of K v channels and development of the mechanism by which acute hypoxia is sensed.
In summary, these studies show that there are maturational changes in K+-channel activity in the pulmonary circulation and that the change in the channel controlling the RMP between the hypoxic fetal and normoxic adult circulations may parallel the different O2-sensing mechanisms of each bed.
![]() |
ACKNOWLEDGEMENTS |
---|
H. L. Reeve was supported by National Heart, Lung, and Blood Institute Grant R29-HL-59182-01 and is the 1997 Giles F. Filley Awardee. E. K. Weir and S. L. Archer were supported by Department of Veterans Affairs Merit Review funding. S. L. Archer was also supported by an American Heart Association Grant-in-Aid (National) Award. D. N. Cornfield was supported by American Heart Association Clinician-Scientist Award 93004240 and a University of Minnesota Children's Scholar Foundation Award.
![]() |
FOOTNOTES |
---|
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. §1734 solely to indicate this fact.
Address for reprint requests: H. L. Reeve, Research 151, VA Medical Center, 1 Veteran's Dr., Minneapolis, MN 55417.
Received 2 June 1998; accepted in final form 20 August 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Abman, S. H.,
B. A. Chatfield,
S. L. Hall,
and
I. F. McMurtry.
Role of endothelium-derived relaxing factor during transition of pulmonary circulation at birth.
Am. J. Physiol.
259 (Heart Circ. Physiol. 28):
H1921-H1927,
1990
2.
Abman, S. H.,
B. A. Chatfield,
D. M. Rodman,
S. L. Hall,
and
I. F. McMurtry.
Maturational changes in endothelium-derived relaxing factor of ovine pulmonary arteries in vitro.
Am. J. Physiol.
260 (Lung Cell. Mol. Physiol. 4):
L280-L285,
1991
3.
Abman, S. H.,
P. F. Shanley,
and
F. J. Accurso.
Failure of postnatal adaptation of the pulmonary circulation after chronic intrauterine pulmonary hypertension in fetal lambs.
J. Clin. Invest.
83:
1849-1858,
1989[Medline].
4.
Accurso, F.,
B. Albert,
R. Wilkening,
R. Peterson,
and
G. Meschia.
Time-dependent response of fetal pulmonary blood flow to an increase in fetal oxygen tension.
Respir. Physiol.
63:
43-52,
1986[Medline].
5.
Archer, S. L.,
J. Huang,
V. Hampl,
D. P. Nelson,
P. J. Shultz,
and
E. K. Weir.
Nitric oxide and cGMP cause vasorelaxation by activation of a charybdotoxin-sensitive K channel by cGMP-dependent protein kinase.
Proc. Natl. Acad. Sci. USA
91:
7583-7587,
1994[Abstract].
6.
Archer, S. L.,
J. M. C. Huang,
H. L. Reeve,
V. Hampl,
S. Tolarova,
E. Michelakis,
and
E. K. Weir.
The differential distribution of electrophysiologically distinct myocytes in conduit and resistance arteries determines their response to nitric oxide and hypoxia.
Circ. Res.
78:
431-442,
1996
7.
Belik, J.,
A. J. Halayko,
K. Rao,
and
N. L. Stephens.
Fetal ductus arteriosus ligation. Pulmonary vascular smooth muscle biochemical and mechanical changes.
Circ. Res.
72:
588-596,
1993[Abstract].
8.
Benham, C. D.,
and
T. B. Bolton.
Spontaneous transient outward currents in single visceral and vascular smooth muscle cells of the rabbit.
J. Physiol. (Lond.)
381:
385-406,
1986[Abstract].
9.
Cassin, S.,
G. S. Dawes,
J. C. Mott,
B. B. Ross,
and
L. B. Strang.
The vascular resistance of the fetal and newly ventilated lung of the lamb.
J. Physiol. (Lond.)
171:
61-79,
1964.
10.
Cornfield, D.,
B. Chatfield,
J. McQueston,
I. McMurtry,
and
S. Abman.
Effects of birth-related stimuli on L-arginine-dependent pulmonary vasodilation in ovine fetus.
Am. J. Physiol.
262 (Heart Circ. Physiol. 31):
H1474-H1481,
1992
11.
Cornfield, D. N.,
H. L. Reeve,
S. Tolarova,
E. K. Weir,
and
S. L. Archer.
Oxygen causes fetal pulmonary vasodilation through activation of a calcium-dependent potassium channel.
Proc. Natl. Acad. Sci. USA
93:
8089-8094,
1996
12.
Cornfield, D. N.,
T. Stevens,
I. F. McMurtry,
S. H. Abman,
and
D. M. Rodman.
Acute hypoxia increases cytosolic calcium in fetal pulmonary artery smooth muscle cells.
Am. J. Physiol.
265 (Lung Cell. Mol. Physiol. 9):
L53-L56,
1993
13.
Dawes, G. S.,
J. C. Mott,
J. G. Widdicombe,
and
D. G. Wyatt.
Changes in the lungs of the newborn lamb.
J. Physiol. (Lond.)
121:
141-162,
1953.
14.
Emmanouilides, G.,
A. Moss,
E. Duffie,
and
F. Adams.
Pulmonary arterial pressure changes in human infants from birth to 3 days of age.
J. Pediatr.
65:
327-333,
1964.
15.
Fineman, J. R.,
J. Wong,
F. C. Morin,
L. M. Wild,
and
S. J. Soifer.
Chronic nitric oxide inhibition in utero produces persistent pulmonary hypertension in newborn lambs.
J. Clin. Invest.
93:
2675-2683,
1994[Medline].
16.
Grissmer, S.,
A. N. Nguyen,
J. Aiyar,
D. C. Hanson,
R. J. Mather,
G. A. Gutman,
M. J. Karmilowicz,
D. D. Auperin,
and
K. G. Chandy.
Pharmacological characterization of five cloned voltage-gated K+ channels, types Kv1.1, 1.2, 1.3, 1.5 and 3.1, stably expressed in mammalian cell lines.
Mol. Pharmacol.
45:
1227-1234,
1994[Abstract].
17.
Hamill, O. P.,
A. Marty,
E. Neher,
B. Sakmann,
and
F. J. Sigworth.
Improved patch-clamp techniques for high resolution current recoding from cells and cell-free membrane patches.
Pflügers Arch.
391:
85-100,
1981[Medline].
18.
Leffler, C. W.,
T. L. Tyler,
and
S. Cassin.
Effect of indomethacin on pulmonary vascular response to ventilation of fetal goats.
Am. J. Physiol.
235 (Heart Circ. Physiol. 4):
H346-H351,
1978.
19.
Levitan, E. S.,
R. Gealy,
J. S. Trimmer,
and
K. Takimoto.
Membrane depolarization inhibits Kv1.5 voltage-gated K+ channel gene transcription and protein expression in pituitary cells.
J. Biol. Chem.
270:
6036-6041,
1995
20.
Liu, S. F.,
A. A. Hislop,
S. G. Haworth,
and
P. J. Barnes.
Developmental changes in endothelium-dependent pulmonary vasodilation in pigs.
Br. J. Pharmacol.
106:
324-330,
1992[Abstract].
21.
Liu, Y.,
A. G. Hudetz,
H.-G. Knaus,
and
N. J. Rusch.
Increased expression of Ca2+-sensitive K+ channels in the cerebral microcirculation of genetically hypertensive rats. Evidence for their protection against cerebral vasospasm.
Circ. Res.
82:
729-737,
1998
22.
McQueston, J. A.,
J. P. Kinsella,
D. D. Ivy,
I. F. McMurtry,
and
S. H. Abman.
Chronic pulmonary hypertension in utero impairs endothelium-dependent vasodilation.
Am. J. Physiol.
268 (Heart Circ. Physiol. 37):
H288-H294,
1995
23.
Morin, F.,
E. Eagan,
W. Ferguson,
and
C. E. G. Lundgren.
Development of pulmonary vascular response to oxygen.
Am. J. Physiol.
254 (Heart Circ. Physiol. 23):
H542-H546,
1988
24.
Nelson, M. T.,
H. Cheng,
M. Rubart,
L. F. Santana,
A. D. Bonev,
H. J. Knot,
and
W. J. Lederer.
Relaxation of arterial muscle by calcium sparks.
Science
270:
633-637,
1995[Abstract].
25.
Peng, W.,
J. R. Hoidal,
S. V. Karwande,
and
I. S. Farrukh.
Effect of chronic hypoxia on K+ channels: regulation in human pulmonary vascular smooth muscle cells.
Am. J. Physiol.
272 (Cell Physiol. 41):
C1271-C1278,
1997
26.
Post, J. M.,
J. R. Hume,
S. L. Archer,
and
E. K. Weir.
Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction.
Am. J. Physiol.
262 (Cell Physiol. 31):
C882-C890,
1992
27.
Rae, J.,
K. Cooper,
G. Gates,
and
M. Watsky.
Low access resistance perforated patch recordings using amphotericin B.
J. Neurosci. Methods
37:
15-26,
1991[Medline].
28.
Rudolph, A.
Distribution and regulation of blood flow in the fetal and neonatal lamb.
Circ. Res.
57:
811-821,
1985[Medline].
29.
Smirnov, S. V.,
T. P. Robertson,
J. P. T. Ward,
and
P. I. Aaronson.
Chronic hypoxia is associated with reduced delayed rectifier K+ current in rat pulmonary artery muscle cells.
Am. J. Physiol.
266 (Heart Circ. Physiol. 35):
H365-H370,
1994
30.
Soifer, S. J.,
R. D. Loitz,
C. Roman,
and
M. A. Heymann.
Leukotriene end organ antagonists increase pulmonary blood flow in fetal lambs.
Am. J. Physiol.
249 (Heart Circ. Physiol. 18):
H570-H576,
1985[Medline].
31.
Tristani-Firouzi, M.,
E. B. Martin,
S. Tolarova,
E. K. Weir,
S. L. Archer,
and
D. N. Cornfield.
Ventilation-induced pulmonary vasodilation at birth is modulated by potassium channel activity.
Am. J. Physiol.
271 (Heart Circ. Physiol. 40):
H2353-H2359,
1996
32.
Tsien, R. Y.
New calcium indicators and buffers with high selectivity against magnesium and protons: design, synthesis and properties of prototype structures.
Biochemistry
19:
2396-2404,
1980[Medline].
33.
Wang, J.,
M. Juhaszova,
L. J. Rubin,
and
X.-Y. Yuan.
Hypoxia inhibits gene expression of voltage-gated K+ channel -subunits in pulmonary artery smooth muscle cells.
J. Clin. Invest.
100:
2347-2353,
1997
34.
Wild, L. M.,
P. A. Nickerson,
and
F. C. Morin.
Ligating the ductus arteriosus before birth remodels the pulmonary vasculature of the lamb.
Pediatr. Res.
25:
251-257,
1989[Abstract].
35.
Yuan, X.-J.,
W. F. Goldman,
M. L. Tod,
J. Rubin,
and
M. P. Blaustein.
Hypoxia reduces potassium currents in cultured rat pulmonary but not mesenteric arterial myocytes.
Am. J. Physiol.
264 (Lung Cell. Mol. Physiol. 8):
L116-L123,
1993
36.
Zellers, T.,
and
P. Vanhoutte.
Endothelium-dependent relaxations of piglet pulmonary arteries augment with maturation.
Pediatr. Res.
30:
176-180,
1991[Abstract].