K+-channel blockade inhibits
shear stress-induced pulmonary vasodilation in the ovine fetus
Laurent
Storme1,
Robyn L.
Rairigh2,
Thomas A.
Parker2,
David N.
Cornfield3,
John P.
Kinsella2, and
Steven H.
Abman2
1 Service de Medecine
Neonatale, Hôpital Jeanne de Flandre, CHRU de
Lille, 59110 Lille, France;
2 Pediatric Heart Lung Center,
Department of Pediatrics, University of Colorado School of
Medicine, Denver, Colorado 80218; and
3 Department of Pediatrics,
University of Minnesota School of Medicine, Minneapolis, Minnesota
55455
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ABSTRACT |
To determine whether
K+-channel activation mediates
shear stress-induced pulmonary vasodilation in the fetus, we studied
the hemodynamic effects of
K+-channel blockers on basal
pulmonary vascular resistance and on the pulmonary vascular response to
partial compression of the ductus arteriosus (DA) in chronically
prepared late-gestation fetal lambs (128-132 days gestation).
Study drugs included tetraethylammonium (TEA;
Ca2+-dependent
K+-channel blocker), glibenclamide
(Glib; ATP-dependent K+-channel
blocker), charybdotoxin (CTX; preferential
high-conductance Ca2+-dependent
K+-channel blocker), apamin (Apa;
low-conductance Ca2+-dependent
K+-channel blocker), and
4-aminopyridine (4-AP; voltage-dependent K+-channel blocker). Catheters
were inserted in the left pulmonary artery (LPA) for selective drug
infusion and in the main pulmonary artery, aorta, and left atrium to
measure pressure. An inflatable vascular occluder was placed around the
DA. LPA flow was measured with an ultrasonic flow transducer. Animals
were treated with saline, high- or low-dose TEA, Glib, Apa, CTX, CTX
plus Apa, or 4-AP injected into the LPA. DA compression caused a
time-related decrease in pulmonary vascular resistance in the control,
Glib, Apa, CTX, CTX plus Apa, and low-dose TEA groups but not in the high-dose TEA and 4-AP groups. These data suggest that pharmacological blockade of Ca2+- and
voltage-dependent K+-channel
activity but not of low-conductance
Ca2+- and ATP-dependent
K+-channel activity attenuates
shear stress-induced fetal pulmonary vasodilation.
nitric oxide; potassium channels; pulmonary circulation; blood
flow; lambs; endothelium-derived relaxing factor; pulmonary
hypertension
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INTRODUCTION |
THE FETAL PULMONARY CIRCULATION is characterized by
high resistance and low blood flow. At birth, pulmonary blood flow
increases dramatically 8- to 10-fold, and pulmonary vascular resistance (PVR) drops immediately (36). Three main factors contribute to the
increase in pulmonary blood flow at birth: ventilation of the lung
(39), increased O2 (7), and
increased shear stress (1). Vasoactive mediators released from the
endothelium, especially nitric oxide (NO), play a major role during the
transition at birth (3, 11). Inhibition of NO synthesis
can attenuate the postnatal adaptation of the pulmonary circulation,
including the vasodilator effect of increased shear stress at birth (3,
20). Although NO stimulates vascular smooth muscle cell guanylate
cyclase (6), the mechanism whereby increased guanosine
3',5'-cyclic monophosphate (cGMP) regulates pulmonary
vascular tone remains incompletely understood.
Evidence suggests a primary role for ion channel activity, including
K+ channels, in the regulation of
pulmonary vascular tone and in NO-mediated vasodilation (5, 26).
Activation of K+ channels
increases K+ efflux, causing
smooth muscle cell membrane hyperpolarization and inactivation of
voltage-operated Ca2+ channels.
Closure of voltage-operated Ca2+
channels decreases smooth muscle cell cytosolic
Ca2+ concentrations and causes
vasodilation (29). Various types of
K+ channels, including ATP,
Ca2+, and voltage dependent, are
present in smooth muscle cells and play critical physiological roles in
vascular regulation (30). K+-channel activity can be
modulated by several physiological stimuli such as
O2 tension (22), NO (5, 26), or
endothelium-derived hyperpolarization factor (EDHF) (10, 27, 28).
Furthermore, recent studies (16, 40) have demonstrated that, in the
perinatal lung, O2- and
ventilation-induced pulmonary vasodilation are partly dependent on the
activation of K+ channels. Thus
evidence exists that K+ channels
may be involved in the regulation of vascular tone in the pulmonary
circulation during fetal life. However, the effects of
K+-channel activity on shear
stress-induced vasodilation in the perinatal lung have not been studied.
Therefore, we hypothesized that stimulation of
K+-channel activity can modulate
the effects of shear stress on the pulmonary circulation in utero. To
test this hypothesis, we studied the effects of
K+-channel antagonists on basal
fetal pulmonary vascular tone and on the hemodynamic response to
partial acute compression of the ductus arteriosus (DA) in chronically
prepared late-gestation fetal lambs. Partial compression of the DA
increases mean pulmonary arterial pressure (PAP) and pulmonary artery
blood flow (1). PVR progressively falls during the first
30-60 min of partial DA compression due to a mechanical increase
in blood flow and an increase in sheer stress. A past study (17) showed
that a flow- or shear stress-induced pulmonary vasodilation response can be blocked by a nonselective NO synthase antagonist in this model.
To study the potential role of
K+-channel activity in this
response, we tested the effects of diverse K+-channel blockers on fetal PVR
under basal conditions and on stimulation during DA compression.
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MODELS AND METHODS |
Animal Preparation
All animal procedures and protocols used in this study were previously
reviewed and approved by the Animal Care and Use Committee at the
University of Colorado Health Sciences Center. Twenty-two mixed breed
(Columbia-Rambouillet) pregnant ewes between 124 and 128 days gestation
(term, 147 days) were fasted for 48 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 surgery. Under sterile conditions, the fetal lamb's
left forelimb was delivered through a uterine incision. A skin incision
was made under the left forelimb after local infiltration with
lidocaine (2 ml, 1% solution). Polyvinyl catheters (20 gauge) were
advanced into the ascending aorta and the superior vena cava after
insertion in the axillary artery and vein, respectively. A left
thoracotomy exposed the heart and great vessels. Catheters were
inserted into the left pulmonary artery (LPA; 22 gauge), main pulmonary artery (20 gauge), and left
atrium (20 gauge) by direct puncture through purse-string sutures and
secured as described previously (2). An inflatable vascular occluder
(outer circumference 10 mm; In Vivo Metric, Healdsburg,
CA) was placed loosely around the DA after gentle dissection of
adherent connective tissue with cotton-tipped swabs. An ultrasonic flow
transducer (size 6; Transonic Systems, Ithaca, NY) was placed around
the LPA to measure blood flow. The uteroplacental circulation was kept intact, and the fetus was gently replaced in the
uterus. An additional catheter was placed in the amniotic cavity to
measure pressure. Ampicillin (500 mg) was added to the amniotic cavity
before closure of the hysterotomy. The flow transducer, catheters, and
occluder were exteriorized through a subcutaneous tunnel to an external
flank pouch. The ewes recovered rapidly from surgery, generally
standing in their pens within 6 h. Food and water were provided ad
libitum. Catheters were maintained by daily infusions of 2 ml of
heparinized saline (20 U/ml). Catheter positions were verified at
autopsy. Studies were performed after a minimum recovery time of 48 h.
Physiological Measurements
The flow transducer cable was connected to an internally calibrated
flowmeter (T201, Transonic Systems, Ithaca, NY) for continuous measurement of LPA blood flow. The output filter of the flowmeter was
set at 100 Hz. The absolute value of flow was determined from the mean
of phasic blood flow signals (at least 30 cardiac cycles), with zero
blood flow defined as the measured flow value immediately before the
beginning of systole (4). Main pulmonary arterial, aortic, left atrial,
and amniotic catheters were connected to a blood pressure transducer
(TSD104, BIOPAC Systems, Santa Barbara, CA). The
pressure and flow signals were continuously recorded and processed on a
computer (PowerMac 7100/80 AV) with an
analog-to-digital converter system (BIOPAC Systems). Data were sampled
at a rate of 200 samples/s. Pressures were referenced to the amniotic
cavity pressure. Calibration of the pressure transducers was performed with a mercury column manometer. Heart rate was determined from the
phasic pulmonary blood flow signal. PVR in the left lung was calculated
as the difference between mean pulmonary arterial and left atrial (LAP)
pressures divided by mean left pulmonary blood flow. Blood samples from
the main pulmonary artery catheter were used for blood gas analysis and
O2 saturation measurements (OSM 3 hemoximeter and ABL 520, Radiometer, Copenhagen, Denmark).
Experimental Design
Three different experimental protocols were included in this study:
1) the effects of K+-channel antagonists on
basal pulmonary tone, 2) the effects
of K+-channel antagonists on the
hemodynamic response to partial DA compression, and
3) the effects of a nonspecific
vasoconstrictor, the thromboxane analog U-46619, on basal pulmonary
tone and on the hemodynamic response to partial DA compression. The
duration of each experiment was 90 min. Physiological variables,
including PAP, LAP, aortic pressure (AoP), amniotic pressure, and LPA
blood flow were recorded at 10-min intervals. Main
pulmonary arterial blood gas tensions and pH were monitored at 0, 60, and 90 min. In the control group, saline was infused throughout the
study period. In treated animals, saline was first infused from 0 to 20 min and from 50 to 90 min of the study period; study drugs were infused
between 20 and 50 min. All study drugs, including saline, were infused
into the LPA at a rate of 0.1 ml/min. Five different
K+-channel blockers were used:
tetraethylammonium [TEA; low dose (0.2 mg/min), preferential
Ca2+-sensitive
K+-channel blocker, and high-dose
(1 mg/min), which may inhibit Ca2+-sensitive and
voltage-sensitive K+ channels at
this concentration (33)], glibenclamide (Glib; 1 mg/min; an
ATP-sensitive K+-channel blocker),
charybdotoxin (CTX; 1 µg/min; a specific high-conductance Ca2+-sensitive
K+-channel blocker), apamin (Apa;
10 µg/min; a specific low-conductance Ca2+-sensitive
K+-channel blocker),
4-aminopyridine (4-AP; 0.1 mg/min; a voltage-sensitive K+-channel blocker), and the
combination of CTX and Apa. Drugs were studied in random order. The
selection of these doses was based on extensive preliminary studies,
including the demonstration that Glib blocks
cromakalim-induced fetal vasodilation (18), and the
effects of these agents on O2- and
ventilation-induced vasodilation (40). The dose of 4-AP was based on
the observation that higher doses (0.6 mg/min) caused severe acidosis
and arrythmias. The interval between each study drug infusion was at
least 24 h.
Protocol 1: Hemodynamic effects of
K+-channel
blockers on basal fetal PVR.
To investigate the role of
K+-channel activity on basal
pulmonary tone, we compared the effects of 30-min infusions of
different K+-channel blockers on
PAP, AoP, flow, and PVR values to the 30-min baseline values.
Protocol 2: Pulmonary hemodynamic effects of
K+-channel
blockade during DA compression.
To investigate the role of
K+-channel activity on the
hemodynamic response to partial compression of DA, we partially
inflated the DA occluder 10 min after the beginning of the infusion of drugs, and DA occlusion was continued for 30 min. The degree of inflation was set to increase mean PAP by 15 mmHg from its baseline value. Mean PAP was kept constant throughout the compression period by
small adjustments of the occluder. After 30 min of DA compression, the
occluder was deflated.
Protocol 3: Hemodynamic effect of nonspecific pharmacological
elevation of basal PVR during DA compression.
To ensure that nonspecific vasoconstriction does not alter the
hemodynamic response to partial compression of DA, the effects of the
thromboxane analog U-46619 were studied under basal conditions (protocol 1) and during DA
compression (protocol 2). The
dose of U-46619 (0.5 µg/min) selected for study was based on
preliminary studies, which showed a 20-30% rise in PVR, to mimic
the increase in basal PVR after high-dose TEA and 4-AP (see
RESULTS).
Drug Preparation
TEA, 4-AP, CTX, Apa, and U-46619 (all from Sigma, St. Louis, MO) were
dissolved in normal saline. The drug concentrations were high-dose TEA,
10 mg/ml; low-dose TEA, 2 mg/ml; 4-AP, 1 mg/ml; CTX, 10 µg/ml; Apa,
100 µg/ml; and U-46619, 5 µg/ml. Glib (Upjohn, Kalamazoo, MI) was
dissolved in DMSO and diluted in a physiological salt solution to
achieve a final concentration of 1 mg/ml. The pH of this solution was
7.4, and the final DMSO concentration was 1%.
Data Analysis
The results are presented as means ± SE. The data were analyzed
with repeated-measures and factorial analyses of variance. Intergroup
differences were analyzed with Fisher's least significant test (Stat
View 4.1 for Macintosh, Abacus Concepts, Berkeley, CA). A
P < 0.05 was considered significant.
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RESULTS |
Protocol 1: Hemodynamic Effects of
K+-Channel
Blockers on Basal Fetal PVR
Intrapulmonary infusion of
Ca2+-dependent
K+-channel blockers, including
low-dose TEA (n = 5 animals), CTX
(n = 4 animals), and CTX plus Apa
(n = 4 animals), did not change mean
basal PAP, LPA blood flow, or PVR (Fig. 1)
or arterial blood gas, mean AoP, or heart rate (Table
1). High-dose TEA
(n = 6 animals) and 4-AP (n = 9 animals) decreased LPA blood
flow and increased PVR by 15 and 20%, respectively
(P < 0.05; Fig.
2). High-dose TEA and 4-AP did not increase
PAP. Arterial O2 saturation and
arterial PO2
(PaO2) decreased
during 4-AP infusion (P < 0.05; Table 1).

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Fig. 1.
Effects of Ca2+-dependent
K+-channel antagonists on basal
pulmonary vascular tone. Low-dose tetraethylammonium (TEA; 0.2 mg/min),
charybdotoxin (CTX), and CTX+apamin (Apa) infusions into left pulmonary
artery (LPA) did not change mean pulmonary arterial pressure (PAP),
left pulmonary blood flow, and pulmonary vascular resistance (PVR).
Data are means ± SE.
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Table 1.
Blood gas and hemodynamic measurements during baseline and after 30 min
of K+-channel antagonist infusion in control and
treated animals
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Fig. 2.
Effects of voltage-dependent or nonspecific
K+-channel antagonists on basal
pulmonary vascular tone. High-dose TEA (1 mg/min) or
4-aminopyridine (4-AP; 0.1 mg/min) infusion into LPA
induced increased PVR. Data are means ± SE.
* P < 0.05 for treatment vs.
baseline period.
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Protocol 2: Pulmonary Hemodynamic Effects of
K+-Channel Blockade During DA Compression
In control studies (n = 12 animals),
partial compression of the DA rapidly increased mean PAP by 33 ± 3% (from 45 ± 1 to 60 ± 1 mmHg). LPA blood flow progressively
increased from 90 ± 6 to 195 ± 15 ml/min at 30 min
(P < 0.05). PVR in the left lung decreased during the DA compression period by 39 ± 5% (from 0.51 ± 0.03 to 0.30 ± 0.02 mmHg · ml
1 · min;
P < 0.05; Fig.
3). During DA compression, the heart rate increased from 154 ± 10 to 174 ± 6 beats/min
(P < 0.05), and mean AoP did not change (Table 2). Mean LAP
before compression was 2 ± 1 mmHg and did not change during DA
compression. PaO2 fell slightly from
18.7 ± 0.8 (baseline) to 17.2 ± 0.06 Torr
after 30 min of DA compression
(P < 0.05). Arterial pH also
decreased slightly from 7.38 ± 0.01 to 7.36 ± 0.01 units after
30 min (P < 0.05; Table 2).

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Fig. 3.
Effects of voltage-dependent or nonspecific
K+-channel antagonists on response
during partial compression of ductus arteriosus (DA). Data are means ± SE. Despite similar increases in PAP, flow was lower and PVR was
higher in groups treated with 4-AP and high-dose TEA (1 mg/min) than in
control group during DA compression. Furthermore, PVR did not change
during DA compression compared with baseline period in treated groups
(ANOVA for repeated measures).
* P < 0.05 for treated vs.
control group.
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Table 2.
Blood gas and hemodynamic measurements during baseline and after 30 min
of DA compression in control and treated animals
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Despite an identical increase in mean PAP during DA compression, the
rise in LPA blood flow was attenuated and the decrease in PVR was
abolished during infusions of high-dose TEA
(n = 6 animals) or 4-AP
(n = 8 animals; to inhibit
voltage-dependent K+-channels;
Fig. 3). The rise in pulmonary blood flow during ductal compression was also attenuated by CTX
(n = 4 animals), low-dose TEA
(n = 5 animals; to inhibit
Ca2+-activated
K+-channels; Fig.
4), and CTX plus Apa
(n = 4 animals; to inhibit low- and
high-conductance Ca2+-activated
K+ channels; Fig.
5). Although PVR decreased during DA
compression in these groups (Figs. 4 and 5), it was higher than control
values at 30 min (P < 0.05). PVR did
not change with high-dose TEA and 4-AP treatment during DA compression
compared with baseline (Fig. 3). Neither Apa (Fig. 5) nor Glib (Fig.
6) altered pulmonary blood flow or PVR.
Mean AoP, LAP, arterial blood gas tensions, and pH did not change in
low-dose TEA, CTX, Apa, and CTX plus Apa groups (Table 2). pH and
PaO2 decreased at the end of the
ductus compression in high-dose TEA and 4-AP groups,
respectively (Table 2).

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Fig. 4.
Effects of Ca2+-dependent
K+-channel antagonists on response
to partial compression of DA. Data are means ± SE. Despite a
similar increase in PAP, low-dose TEA (0.2 mg/min) or CTX infusion into
LPA significantly attenuated rise in flow and decrease in PVR induced
by DA compression compared with control group
(n = 12 animals).
* P < 0.05 for treated vs.
control group.
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Fig. 5.
Effects of low-conductance or combination of low- and high-conductance
Ca2+-dependent
K+-channel antagonists on response
to partial DA compression. Data are means ± SE. At 30 min of DA
compression, flow was lower, and PVR was higher in group infused with
Apa + CTX than in control group. Apa infusion alone did not alter
vasodilator response to DA compression.
* P < 0.05 for treated vs.
control group.
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Fig. 6.
Effects of ATP-dependent
K+-channel antagonism on response
to partial compression of DA. Data are means ± SE; n,
no. of animals. Glibenclamide did not change hemodynamic effects of DA
compression.
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Protocol 3: Hemodynamic Effect of Nonspecific Pharmacological
Elevation of Basal PVR During DA Compression
Infusion of the thromboxane analog U-46619 (0.5 µg/min;
n = 4 animals) increased PVR by 30%
(Fig. 7). However, DA compression increased
LPA blood flow and decreased PVR after U-46619 treatment (P < 0.05). There was no difference
in LPA blood flow and PVR among treated and control groups (Fig. 7).

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Fig. 7.
Effects of nonspecific vasoconstriction on response to partial
compression of DA. Data are means ± SE; n, no. of
animals. U-46619 infusion into LPA decreased flow and increased PVR but
did not change PAP. Despite elevation of basal PVR, U-46619 infusion
did not alter vasodilator response during DA compression.
* P < 0.05 for treated vs.
control group.
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DISCUSSION |
In this study, we hypothesized that activation of
K+ channels modulates basal PVR
and the effects of shear stress on pulmonary vascular tone during fetal
life. To test this hypothesis, we studied the effects of
K+-channel blockade on basal
pulmonary vascular tone and the vasodilator response due to compression
of the DA in the ovine fetus. Although Ca2+-dependent
K+-channel antagonists (low-dose
TEA and CTX) did not change basal fetal pulmonary blood flow or PVR,
these drugs attenuated pulmonary vasodilation during DA compression.
Furthermore, treatment with 4-AP, a voltage-dependent
K+-channel antagonist, and
high-dose TEA, a Ca2+- and
voltage-dependent K+-channel
blocker, increased basal pulmonary vascular tone and blocked the
dilator response during DA compression. To ensure that the inhibition
of DA compression-induced vasodilation was not simply due to increased
basal PVR, the response to DA compression was measured during treatment
with a nonspecific vasoconstrictor, U-46619. We found that the increase
in basal pulmonary vascular tone during infusion of U-46619 did not
alter the vasodilator response to DA compression. Pulmonary
vasodilation during DA compression was not attenuated by ATP-dependent
and low-conductance Ca2+-dependent
K+-channel blockers. Overall,
these results support the hypothesis that activation of both
Ca2+- and voltage-dependent
K+ channels modulates the effects
of shear stress on pulmonary circulation during the fetal life.
This study provides new information regarding mechanisms underlying
shear stress-induced vasodilation in the perinatal lung. Flow-mediated
vasodilation may play a major role in postnatal adaptation of the
pulmonary circulation (17). At birth, the pulmonary circulation
undergoes an immediate rise in blood flow induced by ventilation that
leads to capillary dilation and distension, along with increased
arterial and alveolar O2 tensions
(7, 39). The shear stress exerted along the endothelial surface by
acute elevation of blood flow may potentially contribute to enhance and
sustain the pulmonary vasodilation at birth (17). Pulmonary
vasodilation in response to ventilation and increased O2 abruptly increases lung blood
flow, which increases shear stress and stimulates flow-dependent
dilation in resistance vessels. The propagation of vasodilation to
resistance vessels may potentially be an important step to cause
further elevation of pulmonary blood flow during the early postnatal
period (34). Shear stress stimulates vasoactive mediator release from
the endothelium, especially NO, prostacyclin, and EDHF release (2, 15,
17, 21, 35). Cornfield et al. (17) previously showed that
NO synthase inhibition blocks fetal pulmonary artery vasodilation
during DA compression, suggesting an important role for NO release in
this model. As suggested by this current study, activation of
non-ATP-dependent K+ channels
modulates shear stress-induced vasodilation in the perinatal lung.
Furthermore, both Ca2+- and
voltage-dependent K+ channels may
contribute to basal fetal pulmonary vasular tone because
K+-channel blockade with high-dose
TEA and 4-AP increased PVR under resting conditions.
Previous studies (16, 40) have demonstrated that
O2 and ventilation cause pulmonary
vasodilation through activation of non-ATP-sensitive
K+ channels at birth. TEA infusion
attenuated the increase in LPA blood flow in response to mechanical
ventilation and increased O2,
whereas Glib, an ATP-sensitive
K+-channel antagonist, had no
effect. Iberiotoxin, a
Ca2+-dependent
K+-channel antagonist, decreased
O2-induced fetal pulmonary
vasodilation. In this study (40), TEA was not found to increase basal
fetal pulmonary vascular tone. However, the infusion of TEA was
performed 1 h after surgery in the previous study. In the current
study, a recovery period of at least 48 h after surgery was provided to
ensure hemodynamic stabilization before study. This difference between
acute and chronically instrumented fetal lambs may explain the
divergent findings.
Other investigators have previously reported that flow-induced
vasodilation involves activation of
K+ channels in vitro and may
involve NO production. Cooke et al. (14) showed that NO release due to
shear stress is dependent on activation of
Ca2+-sensitive
K+ channels of the endothelial
cell of rabbit iliac arteries, as demonstrated by inhibition of
flow-mediated vasodilation by TEA, CTX, and iberiotoxin. In contrast,
Apa, an antagonist of the low-conductance Ca2+-dependent
K+ channel, and Glib had no
effect. Ohno et al. (31) demonstrated that activation of TEA-sensitive
K+ channels stimulated NO release
from bovine aortic endothelial cells in response to increased flow.
Hutcheson and Griffith (25) found that pulse frequency-related NO
release was attenuated by Apa, TEA, and CTX but not by Glib. NO release
stimulated by increased viscosity was reduced by TEA, CTX, and Glib but
not by Apa. Shear stress has also been shown to activate inward
rectifier K+
channels (41). Thus various K+
channels may be involved in the response to shear stress. Heterogeneity in the distribution of ion channels in endothelial or smooth muscle cells from different sources of vessels or species-related differences may partially explain discrepancies between studies (23). Our in vivo
studies cannot distinguish between endothelial and
smooth muscle cell roles in this response.
These data suggest that high-conductance
Ca2+- and voltage-dependent
K+ channels can modulate
flow-induced vasodilation in the fetal lung. Our laboratory previously
reported that inhibition of NO (17) and prostaglandin (1) release also
blunts vasodilation in this model. Recent studies (5,
8-10, 12, 19, 25, 26, 42) have demonstrated links between vasodilation
caused by NO, prostacyclin, and EDHF and activation of
K+ channels in vascular smooth
muscle cells. In pulmonary arterial smooth muscle cells, NO-induced
vasodilation involves activation of a cGMP-sensitive protein kinase
that increases the opening probability of
K+ channels, with subsequent
decreases in smooth muscle cell
Ca2+ content (5, 8, 26, 42). NO
may also have non-cGMP mechanisms of vasodilation caused by direct
hyperpolarization of smooth muscle cells that causes closure of
voltage-dependent Ca2+ channels
and vasodilation (38). CTX can inhibit relaxation of rabbit aorta to NO
after blockade of soluble guanylate cyclase with methylene blue (9).
EDHF and prostacyclin can also hyperpolarize smooth muscle cells
through activation of K+ channels
(10, 12, 19). Thus blood flow can induce vasodilation through
activation of K+ channels in both endothelial and smooth
muscle cells. However, our data cannot determine whether
shear stress activates K+ channels, leading to enhanced
release of some modulator (e.g., NO), causing vasodilation (32), or
whether the relaxation response to shear stress was directly mediated
by pulmonary arterial smooth muscle cell
K+-channel activation.
This study has several limitations. First, the specificity of
pharmacological blockade is based on published studies, but the
specific effects of the K+-channel
blockers during in vivo studies are incompletely identified. A
concentration of TEA between 0.2 and 3 mM selectively inhibits Ca2+-dependent
K+ channels in vitro (30), and
higher concentrations can inhibit both ATP- and voltage-dependent
K+ channels. High-dose TEA
infusion has been considered as either a preferential
Ca2+-dependent (16) or a
nonspecific K+-channel antagonist
(40). Low-dose TEA infusion likely results in preferential
Ca2+-dependent
K+-channel blockade, whereas TEA
loses its specificity at high doses. In our study, the responses to
low-dose TEA and CTX were similar. CTX is a potent antagonist of
high-conductance Ca2+-dependent
K+ channels. Although CTX inhibits
voltage-dependent K+ channels in
lymphocytes, CTX acts exclusively on
Ca2+-dependent
K+ channels in arterial smooth
muscle (30). Furthermore, 4-AP is known as a selective inhibitor of
voltage-dependent K+ channels but
may also inhibit ATP-dependent K+
channels (30). Because Glib, a highly selective ATP-dependent K+-channel antagonist, did not
alter the response to DA compression, we speculate that the effects of
4-AP may be related to voltage-dependent K+-channel blockade. The similar
responses of high-dose TEA and 4-AP on basal PVR and vasodilation
during DA compression support the hypothesis that high-dose TEA can
inhibit both Ca2+- and
voltage-dependent K+ channels. Our
ability to test a higher dose of TEA was limited by adverse effects.
Apa infusion had no effect on the response to DA compression. The dose
of Apa used in this study (300 or 100-150 µg/kg)
could be insufficient; however, Apa has previously been shown to
attenuate the effects of
-adrenoreceptor agonists in guinea pigs at
a dose of 40 µg/kg (13). Glib, at a dose of 1 mg/min, inhibits fetal
pulmonary vasodilation to lemakalim, a specific ATP-dependent
K+-channel agonist, suggesting
that the dose of Glib in this study was adequate (18).
In summary, voltage-dependent
K+-channel antagonism increased
basal pulmonary vascular tone in the ovine fetus. Both voltage- and
Ca2+-dependent
K+-channel antagonists blunted the
vasodilator response during partial ductus compression. Low-conductance
Ca2+- and ATP-dependent
K+ channels had no effect. These
findings suggest that 1)
voltage-dependent K+ channels are
involved in the modulation of basal pulmonary vascular tone and
2) shear stress-induced vasodilation
is mediated by activation of both voltage- and
Ca2+-dependent
K+ channels.
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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: S. H. Abman, Pediatric Heart Lung
Center, Dept. of Pediatrics, 1056 East 19th Ave., Univ. of Colorado
School of Medicine, Denver, CO 80218-1088.
Received 5 June 1998; accepted in final form 16 October 1998.
 |
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