Mediators of alkalosis-induced relaxation in pulmonary
arteries from normoxic and chronically hypoxic piglets
John B.
Gordon,
Ted R.
Halla,
Candice D.
Fike, and
Jane A.
Madden
Departments of Pediatrics and Neurology, Medical College of
Wisconsin and Zablocki Veterans Affairs Medical Center, Milwaukee,
Wisconsin 53226
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ABSTRACT |
Alkalosis-induced relaxation was measured in
precontracted arterial rings from 1-wk-old piglets exposed to normoxia
or to 3 days of chronic hypoxia. In normoxic piglet arteries,
alkalosis-induced relaxation was blunted in arteries without functional
endothelium and in arteries treated with nitric oxide synthase or
guanylate cyclase inhibitors but not in arteries treated with
cyclooxygenase inhibitors or Ca2+-
and ATP-dependent K+-channel
inhibitors. Inhibition of voltage-dependent
K+ channels with
10
3 M 4-aminopyridine also
failed to block alkalosis-induced relaxation. 4-Aminopyridine at
10
2 M did
block the response, but this may have been due to sustained vascular
smooth muscle depolarization. Arteries from hypoxic piglets exhibited
greater contraction to the thromboxane mimetic U-46619, decreased
endothelium-dependent relaxation, and blunted alkalosis-induced relaxation. The residual relaxation was eliminated by nitric oxide synthase but not by cyclooxygenase or voltage-dependent
K+-channel inhibition.
Alkalosis-induced relaxation of newborn piglet pulmonary arteries
appears to be mediated by the nitric oxide-cGMP pathway and is
attenuated after 3 days of hypoxia, likely because of decreased nitric
oxide activity.
pulmonary hypertension; newborn; endothelium-derived relaxing
factors; potassium channels
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INTRODUCTION |
HYPOCAPNIC ALKALOSIS is widely used in treating severe
neonatal and pediatric pulmonary hypertension (3, 8, 26). This practice
is based on studies showing that alkalosis causes acute pulmonary
vasodilation (9, 12, 17, 18, 34-36, 41). However, the mediator(s)
of alkalosis-induced pulmonary vasodilation remains uncertain (1, 12,
18, 25, 41, 42). Furthermore, responses to alkalosis have generally
been measured in the lungs or vessels from normal animals after
vasomotor tone was acutely raised (9, 12, 17, 18, 34-36, 41).
Neither the pulmonary vascular responses to alkalosis nor the mediators
involved have been studied in a model of preexistent pulmonary
hypertension, the condition for which alkalosis therapy is often used.
In some studies, alkalosis-induced pulmonary vasodilation appeared to
be mediated by endothelium-derived modulators such as prostacyclin (18,
41) and nitric oxide (2). However, others (12, 25, 42) found that
neither of these modulators played a role. More recently, it has been
suggested that activation of K+
channels, particularly voltage-dependent
K+
(KV) channels, may contribute to
alkalosis-induced pulmonary vasodilation (1, 39). Because both
endothelium-derived modulator activity and
K+-channel activation are altered
by pulmonary hypertension (11, 21, 28, 33, 37), we hypothesized that
the magnitude and/or mediator(s) of alkalosis-induced pulmonary
vasodilation would differ between normal and pulmonary hypertensive
animals. To test this hypothesis, responses to alkalosis were measured
under control conditions and after inhibition of endothelium-derived
modulators or K+-channel
activation in pulmonary arteries from normoxic (Norm) and chronically
hypoxic (Hyp) 1-wk-old piglets.
 |
METHODS |
Preparation. This study was approved
by the Institutional Animal Care and Use Committees of the Medical
College of Wisconsin and the Zablocki Veterans Affairs Medical Center
(Milwaukee, WI). Responses to alkalosis were measured in 186 rings from
35 Norm piglets and 55 rings from 7 Hyp piglets. The Hyp piglets were maintained in 12% O2 for 3 days
before study, and all piglets were euthanized at 1 wk of age. Fike and
Kaplowitz (10, 11) previously showed that this relatively
brief chronic Hyp exposure leads to reactive pulmonary hypertension
(10, 11). At the time of study, the piglets were anesthetized with
ketamine (40 mg/kg im), acepromazine (1.5 mg/kg im), and pentobarbital
sodium (25 mg/kg ip). Heparin (5,000 units iv) was administered, and the piglets were euthanized by exsanguination. The lungs and heart were
removed en bloc, and the lungs were then immersed in cold modified
Krebs solution (MKS) and refrigerated at 4°C.
At the time of study, fourth- to fifth-generation pulmonary arterial
segments (
0.5-mm diameter) were identified and gently dissected from
the lungs. The segments were cut into 2-mm-long rings, and small
stainless steel hooks were inserted through the lumen. The upper hook
was suspended from an isometric force transducer (model 52-9529, Harvard Apparatus, South Natick, MA), and the lower hook was attached
to the base of a 10-ml organ chamber (Harvard Apparatus). The organ
baths were filled with MKS maintained at 37.5°C and initially
bubbled with 6% CO2 and 21%
O2 to achieve a pH of 7.40. The
transducer was mounted on a micromanipulator (model 55020, Stoelting,
Chicago, IL), allowing adjustment of resting tension as needed. Vessel
tension was constantly measured and recorded on flatbed recorders
(model L6514-4, Linseis, Princeton Junction, NJ).
Protocol. In preliminary KCl
length-tension experiments (data not shown), we found that the optimal
resting tension for 0.5-mm-diameter piglet pulmonary arteries was 1 g.
Therefore, the resting tension was set at 1 g in all subsequent
experiments. The arteries were allowed to equilibrate at the resting
tension for 60-90 min, then responses to three successive
challenges with 40 mM KCl were measured to assess vascular smooth
muscle integrity. The arteries were washed with fresh MKS and allowed
to stabilize at the resting tension for 20 min between each KCl
challenge. After the final wash, the arteries were submaximally
contracted with the thromboxane mimetic U-46619. In arteries from Norm
piglets, a mean concentration of 8.7 × 10
8 ± 1.1 × 10
8 M was used for this
initial challenge with U-46619 (termed U-46619 challenge 1) because
preliminary experiments had shown that 80% of maximal contraction
occurred at a concentration of U-46619 between 3 × 10
8 and
10
7 M. However, arteries
from Hyp piglets were more responsive to U-46619
challenge 1, so a significantly lower
mean concentration of U-46619 (2.5 × 10
8 ± 0.1 × 10
8 M;
P < 0.001) was used. Arteries that
failed to exhibit an increase in tension of at least 200 mg/mg vessel
wt in response to the third challenge with 40 mM KCl or to U-46619
challenge 1 were excluded from further study.
After a stable response to U-46619 challenge
1 was achieved (
20 min), relaxation in response to
bradykinin (BK; 10
9 M) or
acetylcholine (ACh; 10
7 M)
was measured to test for endothelial function. Arteries from Norm
piglets were assigned to control (Con) or inhibitor-treated groups
described in Study groups
if BK caused a >40% decrease and/or ACh caused a >25%
decrease in the U-46619 challenge 1 response. If BK or ACh caused a <15% decrease in the U-46619
challenge 1 response, the arteries
were deemed to have nonfunctional endothelium and were assigned to the
endothelium-negative (Endo
) group. In arteries from Hyp piglets,
relaxation to ACh was abolished (see Study
groups) and responsiveness to BK was blunted.
Therefore, the arteries from Hyp piglets were deemed to have functional
endothelium if BK caused a >30% decrease in the U-46619
challenge 1 response.
After the arteries were tested for endothelial function, the arteries
were washed with fresh MKS and assigned to only one study group (Tables
1 and 2) for
the remainder of the experiment. The arteries were allowed to stabilize
for a further 15-30 min after addition of the inhibitor, and the
resting tension was measured. Submaximal contraction was then again
induced with U-46619 (termed U-46619 challenge
2). The mean concentration of U-46619
challenge 2 was also significantly
higher in arteries from Norm piglets compared with those from Hyp
piglets (1.1 × 10
7 ± 2.0 × 10
8 and
3.9 × 10
8 ± 0.2 × 10
8 M,
respectively; P = 0.02). After a
stable U-46619 challenge 2-induced
increase in tension occurred (
20 min), the bath was made alkalotic
by changing the gas mixture to 21%
O2-2%
CO2-balance N2. The pH stabilized at
7.65-7.70 within 5 min, and a stable tension was measured after 20 min. The gas mixture was then returned to 21%
O2 and 6%
CO2, resulting in a rapid return
to normal pH (7.40 ± 0.002), and the resultant tension was again
measured. At the end of each experiment, arterial rings were blotted
dry and weighed. All tension measurements are expressed as milligrams per milligram of vessel weight.
Study groups. The arteries from Norm
piglets were assigned to 12 study groups as shown in Table 1. The
arteries from Hyp piglets were assigned to the five study groups
indicated in Table 2. The concentrations of inhibitors and
other criteria used to assign rings to the different groups in this
study were based on reports in the literature and our preliminary
experiments. In Con arteries from Norm piglets (Norm Con), BK caused a
73.3 ± 4.3% and ACh caused a 56.4 ± 7.0% decrease
in the U-46619 challenge 1 response,
indicating intact endothelial function. In contrast, arterial rings
without functional endothelium (Norm Endo
) exhibited decreases
of 11.3 ± 2.3 and 11.1 ± 1.6% in response to BK and ACh,
respectively. The addition of
10
4 M
N
-nitro-L-arginine
(L-NNA) to the bath
of Norm L-NNA arteries also reduced BK-induced relaxation to 10.7 ± 2.5%, indicating effective inhibition of endothelium-dependent relaxation as previously reported in a study (13) on newborn pulmonary vessels. Arteries treated with
either 10
6
(n = 8) or
10
5 M
(n = 2)
[1H-(1,2,4)oxadiazolo(4,3,-a)quinoxaline-1-one]
(ODQ) exhibited similar blunting of the responses to BK (mean
13.3 ± 5.4%). In addition, relaxation in response to
10
4 M sodium nitroprusside
was significantly reduced (39.7 ± 9.8% in Con arteries vs. 1.0 ± 1.6% in those treated with
10
6 M ODQ). Thus, as in
previous studies (6, 14), both
10
6 and
10
5 M ODQ blocked the
nitric oxide-cGMP pathway. Therefore, arteries treated with
10
6 and
10
5 M ODQ were combined
into a single Norm ODQ group.
Indomethacin (Indo) at 10
5
M or meclofenamate (Mec) at
10
5 M has been used to
inhibit cyclooxygenase (Cyclo) activity in previous studies (22, 27,
38), and we found that 10
5
M Mec significantly attenuated arachidonic acid-induced relaxation in
preliminary experiments (data not shown). Responses to both BK and
alkalosis were similar in Norm Mec (9 arteries from 5 piglets) and Norm
Indo (8 arteries from 5 piglets) groups. For example, alkalosis-induced
relaxation was 40.9 ± 9.8 and 44.4 ± 11.5%, respectively.
Therefore, data from both groups were combined into a single
Cyclo-inhibited group (Norm Cyclo). Only
10
5 M Mec was used to
inhibit Cyclo activity in arteries from Hyp piglets (Hyp Cyclo).
Preliminary experiments also showed that both
10
6 and
10
5 M glibenclamide (Glib)
blocked relaxation due to the ATP-dependent K+
(KATP)-channel opener cromakalim
(data not shown). Because previous studies (7, 32) used this
concentration range of Glib, Norm Glib arteries were treated with
5 × 10
6 M
Glib to evaluate the contribution of
KATP channels to alkalosis-induced relaxation in this study. Previous studies (5, 20) have shown that
iberiotoxin (IbTX) in concentrations ranging from
10
9 to
10
7 M inhibited
Ca2+-dependent
K+
(KCa)-channel activation.
We measured responses to alkalosis in arteries after adding
10
9
(n = 2),
10
8
(n = 5), 3 × 10
8
(n = 3), and
10
7
(n = 4) M IbTX to the bath. Alkalosis
caused a 40.6 ± 16.5% relaxation in arteries treated with
10
9 M IbTX vs. 37.4 ± 8.8, 36.4 ± 12, and 35.7 ± 12.8%, respectively, in those
treated with the three higher concentrations. Although there were no
significant differences between the four concentrations used, only
arteries treated with
>10
9 M IbTX, to minimize
the possibility that insufficient inhibitor was present to block the
KCa channels, were included
when the effects of alkalosis on the Norm IbTX group were studied.
KV-channel inhibition has been
described with a range of
10
3 to
10
2 M 4-aminopyridine
(4-AP) in studies of pulmonary arterial smooth muscle cells and
pulmonary vessels (29, 37, 43, 44). However, high concentrations of
4-AP may result in depolarization due to non-KV-dependent mechanisms (19).
Therefore, we measured the responses to alkalosis in arteries from both
Norm and Hyp piglets after the addition of either
10
3 or
10
2 M 4-AP to the bath. In
addition, the effects of depolarization on alkalosis-induced relaxation
were also assessed in arteries from Norm piglets treated with the
Na+-K+-ATPase
inhibitor ouabain (Ouab;
10
3 M) and arteries
precontracted with 40 mM KCl rather than with U-46619.
Preparation of drugs and solutions.
The MKS consisted of (in mM) 120 NaCl, 4.7 KCl, 1.7 NaH2PO4,
0.72 MgSO4 · 7H2O,
2.5 CaCl2 · 2H2O,
20 NaHCO3, and 10 glucose. KCl
challenges were achieved by substituting MKS with 80 mM NaCl and 40 mM
KCl, thus maintaining isosmolar conditions. This solution was also used
in those arteries in which responses to alkalosis were measured after
precontraction with 40 mM KCl rather than with U-46619. ACh, BK, IbTX,
sodium nitroprusside, Ouab (all from Sigma, St. Louis, MO), and Mec
(BIOMOL Research Laboratories, Plymouth Meeting, PA) were prepared in normal saline. The stock solutions of Indo (Sigma) and U-46619 (BIOMOL)
were prepared in ethanol and then added to normal saline. Glib, ODQ,
and cromakalim (all from Sigma) were prepared in dimethyl sulfoxide.
L-NNA (Sigma) was dissolved in
normal saline with 12 N HCl. 4-AP (Sigma) was also dissolved in saline,
and HCl was added to titrate the pH back to 7.40. Neither addition of
the different drugs nor their vehicles in the volumes (5-100 µl)
used in this study altered the pH of the MKS. All drug concentrations are expressed as the final molar concentrations in the organ bath.
Data analysis. All data are expressed
as means ± SE. The number of piglets and arteries studied under
each condition are indicated in Figs. 1-9 and Tables 1-3.
Absolute tension and changes in tension in response to the various
interventions are expressed in milligrams per milligram of vessel
weight. The percentage of alkalosis-induced relaxation
[calculated as (decrease in tension during alkalosis divided by
increase in tension in response to U-46619 challenge 2) × 100] was also compared among groups
to facilitate comparisons among groups with differing absolute tensions
during U-46619 challenge 2. All groups
were compared by one- or two-way ANOVA and Tukey's or the least
significant difference test as appropriate. Differences were considered
significant at P < 0.05.
 |
RESULTS |
Alkalosis-induced relaxation in arteries from Norm
piglets. There were no differences in piglet or artery
weight, artery diameter, initial resting tension, tension after the
third challenge with 40 mM KCl, or tension after U-46619
challenge 1 to the bath. Table 3 shows the mean preparation
characteristics for all Norm and Hyp piglets. Figure
1 illustrates the effects of
endothelium-derived modulators on the absolute tension measurements
made under the four study conditions. The tension measured under
resting conditions and after the U-46619 challenge
2 to the bath was significantly higher in Norm
Endo
arteries compared with Norm Con arteries (Fig. 1). U-46619
challenge 2 also caused a greater
increase in tension in arteries treated with a nitric oxide synthase
inhibitor (Norm L-NNA) or a
guanylate cyclase inhibitor (Norm ODQ) but not in arteries treated with
Cyclo inhibitors (Norm Cyclo; Fig. 1). In Norm Con and Norm Cyclo
arteries, tension decreased significantly in response to alkalosis,
then returned to the previous U-46619 challenge
2 level when normocarbia was restored. In contrast, tension did not change in response to alkalosis in Norm Endo
, Norm L-NNA, or Norm ODQ arteries
(Fig. 1). To facilitate comparison of the effects of alkalosis among
groups with differing absolute resting and U-46619
challenge 2-induced tensions, the
percent decrease in the U-46619 challenge
2 response during alkalosis was also measured and is
illustrated as percent alkalosis-induced relaxation (Fig.
2). The percent alkalosis-induced
relaxation was similar in Norm Cyclo and Norm Con arteries but was
significantly blunted in Norm Endo
, Norm
L-NNA, and Norm ODQ arteries.

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Fig. 1.
In normoxic (Norm) piglets, tension measured under resting conditions
was significantly higher ( ) in endothelium-negative
(Endo ) and nitric oxide synthase-inhibited [with
N -monomethyl-L-arginine
(L-NNA)] arteries compared
with control (Con) arteries. Tension measured after 2nd submaximal
contraction induced by U-46619 (U-46619 challenge
2) was significantly higher (**) in Norm Endo ,
Norm L-NNA, and Norm guanylate
cyclase-inhibited (with ODQ) arteries compared with Norm Con and Norm
cyclooxygenase (Cyclo)-inhibited arteries. Alkalosis caused a
significant decrease in tension in Norm Con and Norm Cyclo arteries (*)
but not in Norm Endo , Norm
L-NNA, or Norm ODQ arteries.
Nos. in parentheses, no. of arteries, piglets.
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Fig. 2.
In Norm piglets, alkalosis-induced relaxation, calculated as percent
decrease in U-46619-induced tension during alkalosis, was significantly
blunted in Norm Endo , Norm
L-NNA, and Norm ODQ arteries
( ). Nos. in parentheses, no. of arteries, piglets.
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The effects of K+-channel
inhibition on absolute tension are shown in Fig.
3. U-46619 challenge
2 caused a significant increase in tension in all
groups. Tension measurements in
KATP channel-inhibited (Norm Glib)
and KCa channel-inhibited
(Norm IbTX) arteries did not differ from Norm Con arteries (Fig.
3A). In contrast, comparison of
tension measurements made in KV
channel-inhibited (Norm 4-AP 10
3 M and Norm 4-AP
10
2 M) and Norm Con
arteries approached significance (ANOVA interaction term,
P = 0.07; Fig.
3B). However, only arteries treated
with 10
2 M 4-AP had a
blunted response to alkalosis when the percent alkalosis-induced relaxation was compared in all groups (Fig.
4).

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Fig. 3.
A: in Norm piglets, tension measured
under resting conditions and after U-46619 challenge
2 did not differ between ATP-dependent
K+ or
Ca2+-dependent
K+ channel-inhibited [with
glibenclamide (Glib) or iberiotoxin (IbTX), respectively] and
Norm Con arteries. Alkalosis caused a significant decrease in tension
in all groups (*). B: there was no
difference in tension measured under resting conditions or after
U-46619 challenge 2 among Norm Con and
voltage-dependent K+
channel-inhibited [with
10 3 and
10 2 M 4-aminopyridine
(4-AP)] arteries. Alkalosis-induced relaxation was significant in
Norm Con and Norm 4-AP 10 3
M arteries. Nos. in parentheses, no. of arteries, piglets.
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Fig. 4.
In Norm piglets, alkalosis-induced relaxation, calculated as percent
decrease in U-46619-induced tension during alkalosis, was significantly
blunted only in 4-AP 10 2
arteries ( ). Nos. in parentheses, no. of arteries, piglets.
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Figure 5 illustrates the effects of
alkalosis in Norm 4-AP 10
2
M arteries, arteries treated with the
Na+-K+-ATPase
inhibitor Ouab (Norm Ouab), and those precontracted with 40 mM KCl
(Norm KCl) rather than with U-46619 challenge
2. Absolute tension was significantly higher under all
experimental conditions in Norm 4-AP
10
2 M arteries compared
with Norm Ouab or Norm KCl arteries (Fig. 5A). However, alkalosis failed to
reduce tension in any of these groups, and the percent
alkalosis-induced relaxation was significantly blunted in all
groups compared with the Norm Con group (Fig.
5B).

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Fig. 5.
A: in Norm piglets, tension was
significantly higher in Norm 4-AP
10 2 M compared with Norm
ouabain (Ouab)-inhibited or Norm KCl-contracted arteries, but alkalosis
had no effect on absolute tension in any group.
B: percent relaxation was
significantly decreased in all groups compared with Norm Con group
( ). Nos. in parentheses, no. of arteries, piglets.
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Alkalosis-induced relaxation in arteries from Hyp
piglets. There were no differences in piglet or artery
weight, artery diameter, initial resting tension, tension after 40 mM
KCl, or tension in response to U-46619 challenge
1 among the 55 arteries from the 7 Hyp piglets. Table 3
shows the mean preparation characteristics for all Norm and Hyp
piglets. Although the Hyp piglets weighed significantly less than the
Norm piglets, both the weight and diameter of the arteries were the
same as those from Norm piglets (Table 3). The increase in tension in
response to both the final challenge with 40 mM KCl and the U-46619
challenge 1 was significantly higher
in arteries from Hyp compared with Norm piglets (Fig.
6A) even
though a significantly higher concentration of U-46619 was used in the
Norm piglets (8.7 × 10
8 ± 1.1 × 10
8 and 2.5 × 10
8 ± 0.1 × 10
8 M, respectively;
P < 0.001). Moreover, BK and ACh
caused significantly less relaxation in arteries from Hyp than from
Norm piglets (Fig. 6B).

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Fig. 6.
A: change in tension in response to
3rd challenge with 40 mM KCl or 1st submaximal contraction induced by
U-46619 (U-46619 challenge 1; #1) was
significantly higher in Hyp Con compared with Norm Con arteries (*).
Nos. in parentheses, no. of arteries, piglets.
B: percent relaxation in
response to bradykinin (BK) and ACh was significantly less in Hyp Con
compared with Norm Con arteries ( ).
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Resting tension measured before U-46619 challenge
2 was similar in Norm Con compared with Hyp Con
arteries (Fig.
7A).
However, tension measured during U-46619 challenge
2 was significantly higher in Hyp Con arteries (Fig.
7A) even though the concentration of
U-46619 challenge 2 was significantly
higher in Norm Con than in Hyp Con arteries (1.1 × 10
7 ± 2.0 × 10
8 and 3.9 × 10
8 ± 0.2 × 10
8 M U-46619,
respectively). Alkalosis caused a significant decrease in tension in
both groups (Fig. 7A). However, the
percent alkalosis-induced relaxation was significantly blunted in Norm
Con compared with Hyp Con arteries (Fig.
7B).

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Fig. 7.
A: tension measured under resting
conditions did not differ between Norm Con and Hyp Con arteries, but
tension was significantly greater after U-46619
challenge 2 to Hyp Con arteries (**).
Alkalosis caused a significant decrease in tension in both groups (*).
B: however, percent relaxation in
response to alkalosis was significantly lower in Hyp Con compared with
Norm Con arteries ( ). Nos. in parentheses, no. of arteries,
piglets.
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In contrast to arteries from Norm piglets (Fig.
1B), neither resting tension nor
tension measured after U-46619 challenge 2 differed between Hyp Con and Hyp
L-NNA arteries from Hyp piglets (Fig.
8A).
However, as in Norm piglets, alkalosis had no effect on tension in Hyp
L-NNA arteries and the effects
of alkalosis were similar in Hyp Con and Hyp Cyclo arteries (Figs.
8A and 9). Moreover, alkalosis-induced relaxation was significantly inhibited in
Hyp 4-AP 10
2 M arteries
(Figs. 8B and 9). However, the effects
of 10
3 M 4-AP did approach
significance in the Hyp 4-AP
10
3 M arteries
(Figs. 8B and 9).

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Fig. 8.
A: in Hyp piglets, resting tension and
tension after U-46619 challenge 2 did
not differ between Hyp Con, Hyp
L-NNA, and Hyp Cyclo arteries.
However, alkalosis reduced tension in Hyp Con and Hyp Cyclo arteries
(*) but not in Hyp L-NNA
arteries. Mec, meclofenamate. B:
tension was higher in Hyp 4-AP
10 2 M arteries (**), and
alkalosis had no effect in that group. Differences between Hyp 4-AP
10 3 M and Hyp Con arteries
approached but did not reach significance
(* P = 0.07). Nos. in parentheses,
no. of arteries, piglets.
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Fig. 9.
In Hyp piglets, alkalosis-induced relaxation was significantly
attenuated in Hyp L-NNA and Hyp
4-AP 10 2 M arteries
( ). Nos. in parentheses, no. of arteries, piglets.
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DISCUSSION |
This study identifies the relative contribution of endothelium-derived
modulators and K+-channel
activation to alkalosis-induced relaxation in isolated, 0.5-mm-diameter
pulmonary arteries from Norm piglets. In addition, the magnitude and
mediators of alkalosis-induced vasodilation were determined for the
first time in an animal model of preexistent pulmonary hypertension.
Endothelium-derived nitric oxide appeared to be the predominant
mediator of alkalosis-induced relaxation in arteries from both Norm
piglets and piglets with pulmonary hypertension due to 3 days of
chronic hypoxia. However, alkalosis-induced relaxation was blunted in
arteries from chronically Hyp piglets. This was likely due to decreased
endothelium-derived nitric oxide activity because endothelium-derived
relaxation to ACh and BK was also diminished in the arteries from
chronically Hyp piglets. Although alkalosis-induced relaxation was
inhibited by 10
2 M 4-AP in
arteries from both Norm and Hyp piglets, this was likely due to a
nonspecific effect of 4-AP rather than to
KV-channel inhibition.
Mediators of alkalosis-induced relaxation in arteries
from Norm piglets. Our finding that alkalosis-induced
relaxation was blunted in arteries without functional endothelium
suggests that the response is endothelium dependent (Figs. 1 and 2).
Moreover, the significant effects of nitric oxide synthase and
guanylate cyclase inhibition, but lack of effect of Cyclo inhibition,
suggest that the nitric oxide-cGMP pathway mediates alkalosis-induced vasodilation in pulmonary arteries from Norm piglets. How alkalosis enhances nitric oxide activity is not known, but because alkalosis increases cytosolic Ca2+ in
several cell types (15, 40), it may also increase endothelial cytosolic
Ca2+, a potent stimulus for
endothelial nitric oxide synthesis (23). Increased cytosolic
Ca2+ can also stimulate
endothelial prostacyclin synthesis. However, Cyclo inhibition had no
effect on alkalosis-induced relaxation in this study, suggesting that
prostacyclin played little role in alkalosis-induced relaxation in
pulmonary arteries from Norm piglets.
Our data are consistent with those obtained in bovine aortic
endothelial cells showing that alkalosis caused increased release of a
nonprostanoid endothelium-derived relaxing factor (24) and with
preliminary data presented by Aschner and Smith (2) suggesting that nitric oxide mediated alkalosis-induced relaxation of
piglet pulmonary arteries. In addition, Indo failed to block alkalosis-induced vasodilation in previous studies (12, 25) of intact
lambs. In contrast, however, nitric oxide did not seem to be involved
in alkalosis-induced vasodilation of adult rabbit lungs (42) or intact
lambs (12) and prostacyclin did appear to mediate the vasodilator
response to alkalosis in mature rats (41) and intact piglets (18). Thus
the contribution of different endothelium-derived modulators to
alkalosis-induced vasodilation may vary with species and/or
age. This conclusion is supported by studies showing that basal nitric
oxide and prostacyclin activities differed between species (4) and that
both nitric oxide and dilator prostaglandin activities changed with age
(16, 30).
Several lines of evidence led us to investigate the contribution of
K+ channels to alkalosis-induced
vascular relaxation in piglet pulmonary arteries. In recent patch-clamp
studies of canine (1) and rat (39) pulmonary arterial smooth muscle
cells, alkalosis promoted KV-channel activation, and in
newborn piglets, KATP channels
appeared to contribute to basal pulmonary vasomotor tone (32). In
addition, alkalosis increases vascular smooth muscle cytosolic
Ca2+ concentration (40), a potent
stimulus for KCa-channel
activation. However, neither IbTX at concentrations sufficient to
inhibit KCa-channel
activation (5, 20) nor Glib at concentrations sufficient to inhibit
KATP-channel activation (7, 32)
altered the response to alkalosis (Figs.
3A and 4). 4-AP at
10
3 M, a concentration
thought to inhibit KV channels
(29, 37), also failed to significantly alter the response to alkalosis
(Figs. 3B and 4). In contrast,
10
2 M 4-AP did
significantly blunt the response to alkalosis. Even though this may
indicate that KV-channel
activation contributed to alkalosis-induced relaxation,
10
2 M 4-AP has several
nonspecific effects, including inhibition of
Ca2+-ATPase and
Na+-K+-ATPase
(19). Our observation that alkalosis failed to induce relaxation in
arteries treated with Ouab or precontracted with KCl (Fig. 5) provides
indirect support for the hypothesis that 10
2 M 4-AP blunted
alkalosis-induced relaxation by causing sustained vascular smooth
muscle depolarization rather than
KV-channel inhibition. Further
studies on isolated vessels and intact lungs from piglets and other
species are needed to determine whether the
KV-channel activation described in
patch-clamp studies (1, 39) plays a physiologically significant role in
alkalosis-induced vasodilation.
Effects of chronic hypoxia on piglet pulmonary
arteries. The finding that pulmonary arteries from
piglets exposed to 3 days of 12%
O2 exhibited greater reactivity to
both KCl and U-46619 challenge 1 than
did arteries of a similar size and generation from Norm piglets (Fig.
6A) is consistent with previous
observations by Fike and Kaplowitz (10) that Hyp pulmonary vascular
reactivity was enhanced after 3 days of chronic hypoxia. The enhanced
reactivity to KCl and U-46619 together with our finding that the
responses to the endothelium-dependent dilators BK and ACh were blunted and abolished, respectively, in arteries from Hyp piglets (Fig. 6B) suggests that nitric oxide
activity was decreased in pulmonary arteries from piglets exposed to 3 days of chronic hypoxia.
Magnitude and mediators of alkalosis-induced
relaxation after chronic hypoxia. Alkalosis-induced
relaxation was significantly blunted in arteries from Hyp piglets
studied under Con conditions (Fig. 7). This was also likely a
consequence of decreased nitric oxide activity. However, nitric oxide
synthase inhibition further reduced alkalosis-induced relaxation from
25 to <1% (Figs. 8A and 9),
suggesting that the vasodilator response was mediated by residual
nitric oxide activity. As with the arteries from Norm piglets,
10
2 M 4-AP also attenuated
the response to alkalosis in arteries from Hyp piglets (Figs.
8B and 9). However, unlike the
arteries from Norm piglets, the effects of
10
3 M 4-AP approached
significance (Figs. 8B and 9), raising
the possibility that KV channels
might play a role in the arteries from Hyp piglets.
Whether alkalosis-induced vasodilation would be further reduced or
abolished in a model of more severe pulmonary hypertension remains to
be determined. A previous study by Fike and Kaplowitz (11)
demonstrating a greater decrease in nitric oxide synthase after 10 days
of chronic hypoxia suggested that nitric oxide would contribute less to
alkalosis-induced relaxation in piglet pulmonary arteries as pulmonary
hypertension due to chronic hypoxia worsened. However, other
vasodilator modulators such as epoxygenase metabolites of arachidonic
acid (31) may contribute to alkalosis-induced vasodilation in more
severe pulmonary hypertension.
Summary. Our data suggest that
hypocarbic alkalosis-induced vascular relaxation is mediated
predominantly by nitric oxide in isolated pulmonary arteries from
newborn piglets and that this response is attenuated after 3 days of
chronic hypoxia. Although this calls into question the efficacy of
alkalosis therapy in the treatment of neonatal and pediatric pulmonary
hypertension, several issues need yet to be addressed. For example, the
contribution of both endothelium-derived modulators and
K+-channel activation to
alkalosis-induced vasodilation must be examined in isolated lungs
and/or intact animals to evaluate their roles in a more
physiological preparation. In addition, interspecies variability in the
consequences of more prolonged hypoxia and other hypertensive stimuli
on alkalosis-induced vasodilation must be evaluated. Finally,
understanding how alkalosis leads to increased synthesis of nitric
oxide or other modulators of vasomotor tone may lead to a more
effective treatment strategy for infants and children with severe
pulmonary hypertension.
 |
ACKNOWLEDGEMENTS |
This study was supported by grants from the Children's Hospital of
Wisconsin Foundation (Milwaukee) and the Medical College of Wisconsin (Milwaukee).
 |
FOOTNOTES |
C. D. Fike was funded by a March of Dimes Research Grant. J. A. Madden
was supported by funds from Veterans Affairs Medical Research.
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: J. B. Gordon, Children's Hospital of
Wisconsin, Critical Care Section, MS 681, PO Box 1997, Milwaukee, WI
53201.
Received 15 June 1998; accepted in final form 12 October 1998.
 |
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