Departments of 1 Pediatrics (Critical Care), 2 Physiology, and 3 Neurology, Medical College of Wisconsin and Zablocki Veterans Administration Medical Center, Milwaukee, Wisconsin 53226
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ABSTRACT |
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We previously found that
nitric oxide synthase (NOS) inhibition fully blocked alkalosis-induced
relaxation of piglet pulmonary artery and vein rings. In contrast, NOS
inhibition alone had no effect on alkalosis-induced pulmonary
vasodilation in isolated piglet lungs. This study sought to identify
factors contributing to the discordance between isolated and in situ
pulmonary vessels. The roles of pressor stimulus (hypoxia vs. the
thromboxane mimetic U-46619), perfusate composition (blood vs.
physiological salt solution), and flow were assessed. Effects of NOS
inhibition on alkalosis-induced dilation were also directly compared in
150-350-µm-diameter cannulated arteries and
150-900-µm-diameter, angiographically visualized, in situ
arteries. Finally, effects of NOS inhibition on alkalosis-induced
vasodilation were measured in intact piglets. NOS inhibition with
N-nitro-L-arginine fully
abolished alkalosis-induced vasodilation in all cannulated
arteries but failed to alter alkalosis-induced vasodilation in intact
lungs. The results indicate that investigation of other factors, such
as perivascular tissue (e.g., adventitia and parenchyma) and remote
signaling pathways, will need to be carried out to reconcile this
discordance between isolated and in situ arteries.
newborn piglet; pressor stimulus; flow; perfusate; cannulated arteries; isolated lungs; angiography
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INTRODUCTION |
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THE RELATIVE SIMPLICITY of isolated pulmonary vessel preparations compared with isolated lungs or intact animals has facilitated delineation of some aspects of pulmonary vasomotor control (10, 16, 18, 19, 21). However, determining which mechanism or mechanisms identified in the more reductionist preparations are relevant to the whole organ or organism can be difficult (5). Pulmonary vascular responses to several physiologically important stimuli, including flow (3, 33), pressure (24), and hypoxia (19, 27, 28, 36, 41, 42), appear to differ between isolated vessel and intact lung preparations. In addition, we found that the mediators of alkalosis-induced pulmonary vasodilation differ between isolated and in situ vessels. In preconstricted pulmonary artery (10) and vein (16) rings, alkalosis-induced relaxation could be accounted for completely by the nitric oxide-cGMP (NO-cGMP) pathway. In contrast, in hypoxic piglet lungs, nitric oxide synthase (NOS) inhibition alone had no effect on alkalosis-induced pulmonary vasodilation (38). Instead, combined inhibition of NOS, cyclooxygenase, and calcium-dependent potassium (KCa) channels was required to block vasodilation. These data suggest a need for an isolated vessel preparation that more closely mimics in situ vessels to better delineate mechanisms underlying pulmonary vasomotor responses that are relevant to the whole organ.
As a step toward developing such a preparation, we sought to identify
factors contributing to the discordant responses of intact lungs and
current isolated vessel preparations. We took advantage of the
differing effects of NOS inhibition on alkalosis-induced vasodilation
seen in our previous studies of isolated vascular rings (10,
16) and lungs (38) to test the hypothesis that differences in pressor stimulus (the thromboxane mimetic U-46619 in
isolated vessels vs. hypoxia in lungs), perfusate composition [physiological salt solution (PSS) in isolated vessels vs. blood in
lungs], or flow (absent in isolated vessels vs. present in lungs) lead to the discordance between preparations. Because tension measurements made in the relatively large (500-µm-diameter) vascular rings used in our previous studies (10, 16) may not
predict the diameter changes of smaller resistance arteries, the effect of NOS inhibition on alkalosis-induced vasodilation was also measured in small (150- to 350-µm external diameter), cannulated pulmonary arteries in this study. In addition, since even these small cannulated arteries are not necessarily those responsible for resistance changes
measured in the intact lung, we also measured the effects of NOS
inhibition on alkalosis-induced vasodilation of in situ pulmonary
arteries in the same diameter range with X-ray angiography. Finally,
the effect of NOS inhibition on alkalosis-induced vasodilation in
intact piglets was measured to ascertain that in vivo responses to
alkalosis and N-nitro-L-arginine
(LNA) were similar to those described in isolated lungs
(38).
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METHODS |
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The study was approved by the Institutional Animal Care and Use Committees of the Medical College of Wisconsin and the Zablocki Veterans Administration Medical Center in Milwaukee. A total of 46 piglets was used in the following protocols: intact animals (n = 4), isolated lungs vasoconstricted with U-46619 (n = 4), isolated lungs perfused with a blood-free solution (n = 8), isolated lungs studied at different flow rates (n = 8), isolated lungs studied with angiography to identify in situ artery responses (n = 28 arteries from 6 piglets), cannulated arteries pressurized with blood or blood-free solutions (n = 17 arteries from 12 piglets), and cannulated arteries perfused with PSS to study the effects of flow (n = 4 arteries from 4 piglets). All piglets were initially anesthetized with 40 mg/kg im ketamine, 1.5 mg/kg im acepromazine, and 25 mg/kg ip pentobarbital as previously described (10, 12, 38). Piglets used for isolated lung or vessel studies were then heparinized (5000 units iv) and exsanguinated through a carotid artery catheter. Piglets used for intact animal studies were given an additional dose of 5 mg/kg iv pentobarbital and then 2-4 mg/kg iv every hour for the duration of the study.
Intact piglet protocol.
In our previous studies, NOS inhibition alone blocked alkalosis-induced
relaxation in isolated vessel rings (10, 16) but had no
effect on alkalosis-induced vasodilation in whole lungs (38). This protocol was designed to test the hypothesis
that responses of intact animals to NOS inhibition were similar to those of isolated lungs. After anesthesia, a tracheostomy tube was
placed, and a carotid artery catheter and a flow-directed pulmonary
artery catheter (model 132F5, Baxter Healthcare) were inserted. Piglets
were ventilated (model 713, Harvard Apparatus) with a tidal volume of
20 ml/kg (peaked inspiratory pressure 14-18 cmH2O),
rate of 20 breaths/min, and initial gas mixture of 28% O2
with 3-4% CO2, to achieve a PaO2
140 Torr, PaCO2
40 Torr, and pH
7.40 (termed
normocapnic normoxia in this study). In this protocol and all isolated
lung studies, the inhaled and/or exhaled gas mixtures were monitored
(model CD3A CO2 monitor and S3A O2 monitor, AEI
Technologies) and blood gases measured at each study condition (model
278 blood gas analyzer, Ciba-Corning). After 40-60 min, stable
control normocapnic normoxic hemodynamics were measured as described
below. The inspired O2 was then reduced to 12% achieving a
PaO2
40 Torr without altering the
PaCO2 and pH. After 15 min, stable normocapnic hypoxic
hemodynamics were measured. The inspired CO2 was then
reduced to 0%, resulting in a decrease in PaCO2 to
~22 Torr with a concomitant increase in pH to ~7.60. Neither tidal
volume nor ventilator rate was altered. After 15 min, stable alkalotic
hypoxic hemodynamics were measured. The gas mixture was then returned
to the normocapnic normoxic mixture, and 30-100 mg/kg of LNA were
infused intravenously over a 10- to 20-min period to block NOS
activity. After a further 20-min stabilization period, the sequence of
normocapnic normoxia, normocapnic hypoxia, and alkalotic hypoxia was
repeated, and hemodynamic measurements were made. Responses
to the endothelium-dependent vasodilators acetylcholine (ACh,
10
6 M) or bradykinin (BK 10
8 M) were
measured before and after LNA to confirm NOS inhibition. After
completion of each experiment, 100 mg/kg of pentobarbital were
administered intravenously, and the piglets were euthanized with
supersaturated KCl.
Isolated lung protocols. The isolated lung preparation has been described before (1, 27, 28, 38). Briefly, a tracheostomy tube was placed and the chest opened by a midline sternotomy. The ductus arteriosus was ligated, and the pulmonary artery and left atrium were cannulated. The lungs were then removed from the chest, and the cannulas were attached to a perfusion system. Perfusate exited the left atrium to a reservoir from where it was pumped (Masterflex model 7523-20) through a heat exchanger (Sci Med Pediatric, Sci Med Life Systems) and bubble trap and then into the pulmonary artery. Lungs were ventilated at a tidal volume of ~15 ml/kg (peak inspiratory pressure of 11-16 cmH2O) at a rate of 20 breaths/min. The gas mixtures were 21% O2 for normoxia and 6% O2 for hypoxia with 6-6.5% CO2 for normocapnia (pH ~7.40) or 3% CO2 for alkalosis (pH ~7.60).
Vasoconstriction with U-46619 in lungs.
In our previous studies, NOS inhibition fully blocked alkalosis-induced
relaxation of vessel rings that were preconstricted with the
thromboxane mimetic U-46619 (10, 16) but had no effect on
alkalosis-induced vasodilation in isolated lungs that were preconstricted with hypoxia (38). To test the hypothesis
that differences in pressor stimulus contributed to this discordance, the effect of NOS inhibition on alkalosis-induced vasodilation was
measured in lungs preconstricted with U-46619. Lungs were perfused with
a 50:50 mixture of autologous blood and Ringer's lactate containing
3% dextran 70 (hematocrit ~15%). Perfusate flow was gradually
increased to 100 ml · kg1 · min
1
over the first 40 min, and when the Ppa had reached a
steady value for >5 min, the stable normoxic normocapnic
Ppa was measured. U-46619 was then infused at 0.01 µg · kg
1 · min
1
to induce vasoconstriction during normoxic normocapnia. After 15 min,
the Ppa had reached a stable plateau, and we switched the
gas mixture to normoxic hypocapnia while maintaining the U-46619 infusion. After 15 min of alkalosis, Ppa reached a steady
nadir, and the stable alkalotic Ppa was measured. The
U-46619 infusion was then stopped, and the lungs returned to normoxic
normocapnia. LNA (10
3 M) was added to the reservoir to
block NOS activity as previously described (11, 38), and
after 15 min, the stable normoxic normocapnic Ppa was
measured again. The U-46619 infusion was then resumed, and a stable
increase in normoxic normocapnic Ppa was measured after 15 min. Finally, hypocapnia was resumed, and the alkalotic Ppa
was measured during U-46619 infusion. As with the intact piglet
protocol, responses to ACh were measured before and after adding LNA to
confirm NOS inhibition in several experiments.
Blood-free perfusate in lungs.
In our previous studies, NOS inhibition fully blocked alkalosis-induced
relaxation of rings that were bathed in PSS (10, 16) but
had no effect on alkalosis-induced vasodilation in lungs that were
perfused with the autologous blood Ringer's lactate mixture
(38). To test the hypothesis that differences in perfusate contributed to the discordant responses, this protocol measured the
effect of NOS inhibition on alkalosis-induced vasodilation in lungs
perfused blood-free perfusate. Lungs were prepared as described above
but were perfused with a blood-free perfusate consisting of Ringer's
lactate with 3% dextran 70. At the beginning of each experiment, flow
was initiated and perfusate discarded as it exited the left atrium
until it was clear and the hematocrit was <0.1%. Recirculation
of the perfusate was then initiated and slowly increased to 100 ml · kg1 · min
1
over 30-40 min. Lungs were divided into control
(n = 4) and LNA (10
3 M, n = 4) groups. In both groups, stable normocapnic normoxic Ppa was measured after ~60 min, the lungs were made
hypoxic, and the normocapnic hypoxic Ppa was measured after
15 min. Finally, the lungs were made alkalotic, and the alkalotic
hypoxic Ppa was measured after 15 min. Responses to ACh
were measured to confirm NOS inhibition in LNA-treated lungs.
High and low flow in lungs.
In our previous studies, lungs were perfused at a flow rate of 100 ml · kg body
wt1 · min
1
(38). In contrast, vessel rings were bathed in PSS and
exposed to no flow. To test the hypothesis that differences in flow may alter the contribution of NO to the response to alkalosis, this protocol measured the effects of alkalosis in control
(n = 5) and LNA (10
3 M, n = 3) lungs at high and low flow rates. After 40 min of perfusion at 150 ml · kg
1 · min
1,
the gas mixture was switched to normocapnic hypoxia. The flow was set
at either 150 ml · kg
1 · min
1
or 50 ml · kg
1 · min
1
while Ppa was maintained similar at either flow rate by
raising or lowering the reservoir, thus achieving a higher or lower
left atrial pressure (Pla). The effects of alkalosis were
then measured by reducing the CO2 to 3%. Lungs were then
returned to normocapnic hypoxia, and the other flow rate was initiated.
The normocapnic and alkalotic Ppa during hypoxia were then
measured again. Because flow rate and Pla differed in this
protocol, data are expressed as PVRI, which was calculated as
(Ppa
Pla)/flow rate indexed to body weight.
Angiography of in situ arteries in isolated lungs.
We previously measured the effects of alkalosis with and without LNA on
resistance changes in whole lungs (38) and on tension changes in single, 500-µm-diameter vascular rings (10,
16). To provide a more direct comparison between isolated and in
situ arteries, X-ray angiography was used to determine whether NOS inhibition alone blocks alkalosis-induced vasodilation of in situ 150-900-µm diameter arteries in the intact lung. Lungs from six piglets were isolated as described above and perfused with autologous blood with Ringer's lactate and 3% detran 70 mixture. In four of the
lungs (2 control, 2 LNA), only the left lobe was perfused (50 ml · kg1 · min
1),
whereas in the other two (2 LNA), the whole lung was perfused at 100 ml · kg
1 · min
1.
After perfusion was initiated, lungs were divided into control (n = 2 lungs, 10 arteries) and LNA (10
3
M, n = 4 lungs, 18 arteries). After 40-60 min of
ventilation with normocapnic normoxia, Ppa reached a steady
pressure, and the stable Ppa was measured. Angiographic
images were collected as previously described (1).
Briefly, the ventilator was stopped in end expiration, and a bolus of
2-5 ml of nonionic, radiopaque contrast medium, 61% iopamidol
(Isovue-300; Bracco Diagnostics, Princeton, NJ), was introduced into
the pulmonary artery without altering flow. Digital images (8 bit,
512 × 512) were captured at 30 frames/s with a Silicon Mounted
Digital 1M-15 CCD camera and Imaging Technologies IM-PCI frame grabber.
U-46619 was then infused at 0.003-0.005
µg · kg
1 · min
1
in control lungs and 0.0015-0.003
µg · kg
1 · min
1
in LNA lungs to cause a small increase in perfusion pressure (2-4
mmHg) and a decrease in artery diameters. After 10 min, a stable
Ppa plateau occurred, and angiographic images were again collected. Alkalosis was then induced with 3% CO2, and
after 10-15 min, a stable nadir in Ppa occurred, and
another bolus was administered. Finally, lungs were returned to
normocapnia, and after a stable increase in Ppa was seen, a
final bolus was administered. The change in Ppa in response
to ACh was then measured to confirm NOS inhibition in the LNA-treated lungs.
Isolated vessel protocols.
After administering heparin and exsanguinating the piglets, we opened
the chests and removed the lungs (10, 16). The lobar pulmonary arteries were identified and traced until small,
150-350-µm external diameter branches (4th-5th generation) were
seen. These were carefully dissected, and 2-cm-long segments were
attached at each end to glass cannulas. The side branches were then
ligated, and the intravascular pressure was raised to 10 mmHg by
raising the inflow reservoir as previously described (24).
Arteries were initially pressurized and bathed in a PSS solution. The
bath PSS was initially bubbled with a 6% CO2 and 21%
O2 solution, and vessel diameter was measured with a color
video camera (Panasonic Digital 5000) mounted on a stereo microscope
(Olympus SZ-STB1) that projected the artery on a video monitor (Sony
PVM-1390). The external diameter was measured using a video scaler
(FORA-IV 550). Increases or decreases in diameter reflect vasodilation or vasoconstriction, respectively. In all studies, vascular smooth muscle function was first established by documenting vasoconstriction to three challenges with 40 mM KCl, and endothelial function was then
documented by demonstrating relaxation to 107 M ACh.
Vessels failing to demonstrate both vascular smooth muscle and
endothelial integrity were excluded from further study.
Cannulated arteries pressurized with PSS or blood. In our previous study, NOS inhibition blocked alkalosis-induced relaxation of 500-µm-diameter artery and vein rings bathed in PSS (10, 16) but not in whole lungs perfused with blood (38). This protocol tests the hypotheses that the size of the vessels, the eccentric tension applied by wires stretching rings, and/or the use of PSS instead of blood may have contributed to the discordant effects of NOS inhibition by measuring the effects of NOS inhibition in small, cannulated, 150-350-µm external diameter arteries pressurized with PSS or blood.
Arteries were divided into two groups: those pressurized with PSS (n = 7 arteries from 5 piglets) and those pressurized with blood (n = 10 arteries from 7 piglets). In all arteries, baseline normocapnic, normoxic diameters were measured, U-46619 was added to the bath to achieve a concentration of 10Arteries with flow.
The final protocol further tests the hypothesis that flow contributes
to the discordant responses of arteries and lungs seen in our previous
studies. The responses to alkalosis were compared with and without
104 M LNA in cannulated arteries perfused at a constant
flow rate with PSS as previously described by Madden and Christman
(23). Arteries (n = 4 from 4 piglets) were
cannulated as described above. After verifying smooth muscle and
endothelial function with KCl and ACh, flow was initiated at
0.2-0.5 ml/min using a microprocessor-controlled roller pump
(Masterflex model 7524-10) while maintaining an intraluminal pressure
of 10 mmHg. After measuring the baseline normoxic, normocapnic diameter, we added U-46619 to the bath, and diameter was again measured. The gas mixture was then made alkalotic, and the diameter was
measured again. The vessels were then washed, and 10
4 M
LNA was added to the bath. The normocapnic, normoxic baseline diameter,
U-46619 diameter, and alkalotic diameter were then measured again. NOS
inhibition was documented by demonstrating blockade of ACh-induced
dilation after the addition of LNA.
Chemicals and solutions. Lungs were perfused with autologous blood mixed with 3% dextran 70 (Sigma) in Ringer's lactate or with the 3% dextran 70 and Ringer's lactate alone. The angiographic studies were performed with 61% iopamidol (Isovue-300). Arteries were bathed in PSS composed of (in mM) 120 NaCl, 4.7 KCl, 1.7 NaH2PO4, 0.72 MgSO4 7 H2O, 2.5 CaCl2 2 H2O, 20 NaHCO3, and 10 glucose. KCl challenges were achieved by modifying the NaCl and KCl in the PSS (80 mM NaCl and 40 mM KCl), thus maintaining isoosmolar conditions. Arteries were pressurized with either PSS or with autologous blood. ACh (Sigma Chemical) was prepared in normal saline, and LNA (Sigma Chemical) was dissolved in normal saline, to which a few drops of 12 N HCl were added, and then the pH was titrated back to ~7.4 with 1 N NaHCO3. U-46619 (Biomol Research Laboratories) was initially prepared in ethanol and then diluted further with normal saline. All drug concentrations are expressed as final molar concentrations in the vessel chamber or perfusion system.
Statistical analysis.
All data are expressed as means ± SE. Comparisons within single
groups were made by repeated measures ANOVA, and when P < 0.05, the least significant difference (LSD) test was used for post
hoc identification of differences between specific conditions. For
comparisons among groups, either simple ANOVA or a two-way repeated
measures ANOVA was used as appropriate. Again, the LSD was used for
post hoc testing when the ANOVA was significant. In addition to raw
data comparisons, the percent alkalosis-induced decrease in the pressor
response was calculated for the isolated arteries as (diameter during
normocapnic U-46619 diameter during alkalosis U-46619)
(diameter during normocapnic U-46619
diameter during
normocapnic baseline conditions) × 100%. All data analyses were
performed using SigmaStat statistical software version 2.03 (SPSS,
Chicago, IL).
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RESULTS |
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In all protocols, blood gases measured during normocapnia,
hypocapnia, normoxia, or hypoxia were similar during their respective control and LNA-treated conditions (Table
1). The concentrations of LNA used have
previously been shown to block NOS activity (10, 38), but to demonstrate NOS inhibition in this study, responses to ACh or BK were measured in the 4 intact piglets, in 16 of the isolated lung experiments, and in 17 of the cannulated artery studies.
LNA significantly reduced the endothelium-dependent responses in both
the intact lungs and isolated vessels (Table
2).
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Intact piglets.
Under control conditions (i.e., before LNA infusion), hypoxia increased
Ppa and PVRI, and alkalosis attenuated the hypoxic response
(Fig. 1). Infusion of LNA caused a
decrease in CI (Fig. 1) and an increase in Ppa and
PVRI compared with control conditions (Fig. 1). However, the responses
to hypoxia and alkalosis were similar to those occurring under control
conditions (Fig. 1). Thus as in hypoxic isolated lungs
(38), NOS inhibition alone failed to block
alkalosis-induced vasodilation in hypoxic intact piglets.
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Effects of pressor stimulus in lungs.
Because flow was normalized to body weight and maintained constant
throughout the experiments, changes in Ppa reflect changes in PVRI. Under control conditions, U-46619 increased normocapnic Ppa and alkalosis attenuated the pressor response (Fig.
2). The results were essentially the same
after adding LNA, although all Ppa was higher than its
respective control values (Fig. 2). Thus as in the hypoxic intact
piglets (Fig. 1) and our previous study of hypoxic piglet lungs
(38), NOS inhibition alone failed to block
alkalosis-induced vasodilation in U-46619-treated lungs.
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Effects of blood-free perfusate in lungs.
In this protocol, isolated lungs were studied under either control or
LNA conditions. Again, flow was constant, so changes in Ppa
reflect changes in PVRI. Normoxic Ppa was similar in both groups (Fig. 3). Hypoxia increased
Ppa more in the LNA compared with control lungs, but
alkalosis attenuated the hypoxic responses similarly in both groups
(Fig. 3). Thus NOS inhibition alone did not block alkalosis-induced
vasodilation in lungs perfused with a blood-free perfusate.
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Responses of in situ arteries.
In this protocol, both Ppa and in situ artery diameters
were measured during constant flow in control and LNA-treated lungs. All Ppa were significantly higher in LNA than in control
lungs (Fig. 4). In both groups, U-46619
caused a small, but significant, increase in Ppa that was
abolished during alkalosis and restored when the lungs were returned to
normocapnia (Fig. 4). Baseline normocapnic artery diameters were
greater in control than LNA arteries, indicating relative
vasoconstriction of the LNA arteries (Fig. 4). In both groups, U-46619
significantly decreased artery diameters, indicating vasoconstriction.
This was attenuated by alkalosis and restored when the lungs were
returned to normocapnia (Fig. 4). To assess whether the effects of LNA
on alkalosis-induced vasodilation differed between larger and smaller
arteries, they were divided into >350- or <350-µm-diameter groups.
Alkalosis attenuated the vasoconstrictor response to U-46619 in both
size ranges of LNA-treated arteries (Fig. 4). Thus NOS inhibition alone did not block the decrease in resistance of whole lungs or the increase
in diameter of large and small in situ arteries during alkalosis.
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Responses of cannulated arteries pressurized with PSS or blood.
Responses to U-46619 and alkalosis were initially measured under
control conditions in all arteries to confirm alkalosis-induced vasodilation (data not shown). They were then randomly assigned to
control or LNA groups, and responses to U-46619 and alkalosis were
again measured. The fact that baseline diameter was greater in PSS LNA
arteries than PSS control arteries (Fig.
5A), but baseline diameter was
greater in blood control than blood LNA arteries (Fig. 5C),
was a matter of chance since there was either a decrease or no change
in diameter in all arteries treated with LNA. In PSS-pressurized (Fig.
5A) and blood-pressurized (Fig. 5C) control arteries, U-46619 decreased vessel diameter, indicating
vasoconstriction. This response was attenuated by alkalosis. In
contrast, PSS- and blood-pressurized arteries treated with LNA
constricted to U-46619 but did not dilate during alkalosis. The % decrease in the U-46619 response in cannulated arteries was
significantly reduced by LNA, regardless of whether they were
pressurized with PSS or blood (Fig. 5, B and
D). Thus in contrast to the in situ arteries (Fig. 4), LNA
completely blocked the vasodilator response to alkalosis in the
cannulated arteries.
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Effects of flow in isolated lungs.
Alkalosis attenuated the normocapnic, hypoxic PVRI measured at flow
rates of either 50 or 150 ml · kg1 · min
1
to a similar extent in both the control and LNA lungs (Fig.
6A). Thus NOS inhibition alone
did not block alkalosis-induced attenuation of hypoxic PVRI at high or
low flow rates in whole lungs.
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Effects of flow in cannulated arteries. In cannulated, perfused arteries (Fig. 6B), neither the pressor responses to U-46619 nor the dilator responses to alkalosis were as great as in pressurized arteries (Fig. 5, A and C). However, as in the PSS-pressurized arteries, alkalosis decreased the U-46619 response under control conditions, but not after adding LNA (Fig. 6C). Thus as in the arteries studied without flow (Fig. 5), NOS inhibition appeared to block alkalosis-induced vasodilation of cannulated arteries studied with flow.
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DISCUSSION |
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We previously reported that alkalosis-induced relaxation in piglet pulmonary artery and vein rings was mediated solely by the NO-cGMP pathway (10, 16). Neither cyclooxygenase products nor K+ channel opening contributed to the response (10, 16). These studies and others showing that alkalosis 1) enhanced Ca2+ entry into cultured endothelial cells (39), 2) increased in vitro NOS activity at a pH of 7.50 (8), and 3) enhanced NO synthesis in cultured human pulmonary artery endothelial cells (26) are consistent with the hypothesis that alkalosis-induced vasodilation is mediated by NO. However, in a subsequent study of isolated newborn piglet lungs, we found that NOS inhibition alone failed to alter alkalosis-induced vasodilation (38). Instead, combined inhibition of NOS, cyclooxygenase, and KCa channels was required to block the response in intact lungs (38). The primary goal of the current study was to identify factor(s) contributing to the apparent discordance in mediators of alkalosis-induced vasodilation between isolated and in situ pulmonary vessels.
To this end, we first sought to exclude any effect of pressor stimulus on the discordance. Alkalosis-induced pulmonary vasodilation can be appreciated only when vessels have tone. Therefore, responses have generally been measured after inducing acute vasoconstriction (9, 12, 27, 31, 40). In our previous studies, hypoxia was used to induce acute vasoconstriction in lungs (38), but the thromboxane mimetic U-46619 was used to preconstrict arterial and venous rings (10, 16). Because acute hypoxia has been shown to reduce NOS activity (20) and a decrease in basal NOS synthesis might minimize the effects of NOS activation in response to alkalosis, we hypothesized that the differences in pressor stimuli might contribute to the discordance between preparations. However, in the current study, NOS inhibition failed to inhibit alkalosis-induced vasodilation in lungs preconstricted with either U-46619 (Figs. 2 and 4) or hypoxia (Figs. 1, 3, and 6A). Thus it does not appear that differences in pressor stimulus contributed to the discordance in mediators of alkalosis-induced vasodilation between lungs and vessels.
A second candidate factor that we investigated was the type of perfusate. In our previous study of isolated lungs (38) as well as intact piglets (Fig. 1) and several isolated lung protocols in the current study (Figs. 3, 4, and 6A), lungs were perfused with blood. In contrast, in most isolated vessel studies, including our own, vessels are bathed in PSS (10, 16, 19, 24, 33, 36, 41, 42). Because NO is rapidly inactivated in the presence of red blood cells, we hypothesized that the effects of increased NO synthesis might be more pronounced in preparations bathed or perfused with PSS, thus accounting for the discordance between vessels and lungs. However, despite reducing the hematocrit <1%, NOS inhibition alone failed to block alkalosis-induced vasodilation in blood-free isolated lungs (Fig. 3). Moreover, LNA inhibited alkalosis-induced vasodilation to a similar degree in cannulated arteries pressurized with PSS (Fig. 5, A and B) or blood (Fig. 5, C and D). Thus the presence or absence of blood did not appear to contribute to the discordance in mediators of alkalosis-induced vasodilation between preparations. This observation is consistent with studies of hypoxic pulmonary vasoconstriction in isolated piglet lungs showing that the response was unaffected by hematocrit (15). However, it should be noted that the influence of blood on pulmonary vascular responses may vary with species, because the same investigators found that the vigor of hypoxic pulmonary vasoconstriction did vary with hematocrit in cats and rats (15). Moreover, red blood cells seemed necessary for NO-mediated responses in isolated rabbit lungs (35).
Another candidate factor considered in this study was vessel size and type of isolated vessel preparation. In our previous studies, we examined the mediators of alkalosis-induced relaxation in 500-µm-diameter arterial and venous rings (10, 16). However, isolated vessel ring preparations have been criticized on the basis of: 1) their relatively large size compared with small resistance arteries in the lung, 2) the eccentric resting tension applied to the arteries by wires compared with the circumferential tension applied by intravascular pressure in vivo, and 3) the fact that the PSS bathing the luminal and abluminal sides of rings are the same. In an effort to address some of these criticisms, we measured the effects of NOS inhibition on alkalosis-induced dilation in 150- to 350-µm external diameter, cannulated arteries pressurized with PSS or blood. However, as in the isolated vascular rings (10, 16), LNA completely blocked responses to alkalosis in all of the cannulated artery preparations (Figs. 5, A-D, and 6B). Thus the discordance between isolated vessels and intact lungs does not appear to be related to the type of isolated vessel preparation.
One problem with comparing responses of isolated vessels and whole lungs is that changes in diameter or tension in individual vessels of a particular size range may not reflect the composite effects responsible for resistance changes across the whole lung. Therefore, in this study we measured the effects of alkalosis with and without NOS inhibition on in situ arteries within the intact lung. A lower U-46619 infusion rate was used in this experiment because the venous constriction that occurs with higher doses causes upstream distension of arteries, which complicates interpretation of vessel diameter changes. In contrast to the cannulated arteries (Figs. 5, A-D, and 6B), NOS inhibition had no effect on the vasodilatory effects of alkalosis on either 150-350 or 350- to 900-µm-diameter in situ arteries (Fig. 4). Thus direct comparison of similarly sized isolated and in situ arteries continued to demonstrate markedly different mediators of alkalosis-induced vasodilation.
A final candidate factor considered in this study was the influence of flow on the mediators of alkalosis-induced vasodilation. Because flow increases NO synthesis, we hypothesized that NOS activation associated with alkalosis might not be apparent under conditions of flow. To test this hypothesis, we measured the effects of NOS inhibition on alkalosis-induced vasodilation at high and low flow in isolated lungs (Fig. 6A). As in previous studies by others (14), PVRI was higher at low flow, and NOS inhibition accentuated hypoxic pulmonary vasoconstriction more at high than low flow, indicating that NO synthesis was greater at high flow (Fig. 6A). However, LNA did not attenuate alkalosis-induced vasodilation at either flow rate. To further evaluate the effects of flow, we also measured responses to alkalosis in a perfused cannulated arteries. With or without flow, LNA blocked the response to alkalosis in the cannulated arteries (Fig. 6B). Thus flow does not appear to be a significant contributor to the discordance in mediators of alkalosis-induced vasodilation between isolated and in situ vessels.
In summary, LNA (30-100 mg/kg in intact piglets or
103 M in isolated lungs) failed to attenuate the
alkalosis-induced decrease in PVRI in intact piglets or isolated lungs
studied under a variety of conditions (Figs. 1-4, and 6A).
Nor did it alter alkalosis-induced vasodilation of in situ arteries
(Fig. 4). In contrast, 10
4 M LNA completely blocked
alkalosis-induced vasodilation in all cannulated artery protocols
(Figs. 5, A-D, and 6B). The mediators of
endothelium-dependent vasodilation also appeared to differ between
isolated and in situ vessels in this study. In arteries, endothelium-dependent vasodilation was completely abolished by LNA
(Table 2). In contrast, although vasodilation was significantly reduced
by LNA in the isolated lungs and intact piglets, ACh and BK still
elicited an ~50% decrease in Ppa or PVRI (Table 2). This
residual vasodilator response could indicate that LNA failed to fully
block NOS activity in the lungs. However, this seems unlikely since LNA
1) significantly increased normoxic PVRI or Ppa
(Figs. 1-4 and 6A), 2) significantly
increased responses to hypoxia and U-46619 (Figs. 1-3 and
6A), and 3) abolished the flow-dependent decrease
in hypoxic PVRI (Fig. 6A). Moreover, the concentration of
LNA used in the current study has been shown to block NOS activity (7, 25, 29) without completely eliminating
endothelium-dependent vasodilation, which, at least in some cases, is
apparently mediated by prostacyclin or hyperpolarization (6,
17). Other physiologically important stimuli also result in
discordant responses between in situ and isolated vessels. For example,
increased flow typically causes acute vasodilation in intact lungs due
to shear-induced increases in NO and PG synthesis (2, 3,
37), but it causes constriction of cannulated pulmonary vessels
(33). Similarly, increased pressure caused dilation
of cat lungs but constriction of cannulated arteries (24).
Finally, hypoxia (30-50 Torr O2) alone typically
causes a brisk and sustained increase in PVRI in the whole lung
(27, 28) but requires preconstriction with another agonist
and is often biphasic or unsustained in isolated vessels (19, 36,
41, 42).
The current study excluded several factors hypothesized to contribute to the discordant responses of isolated vessels and intact lungs. Neither pressor stimulus, type of perfusate, size, or vessel (in a range of 150- to 900-µm diameter) type of isolated vessel preparation (ring vs. cannulated) nor flow contributed to the differing effects of NOS inhibition on alkalosis-induced vasodilation in vessels and whole lungs. Nor could the anesthetics we used be implicated in the discordance because the same agents were used in all preparations. Several other factors may play a role. For example, perivascular tissue may be disrupted in isolated vessels, and the intact adventitia (13) and/or pulmonary parenchyma (22) may contribute to responses of in situ arteries. Distant signaling along the vascular tree may also contribute to the responses of in situ vessels (30, 32, 34). In addition, rhythmic distension of lungs could result in different mediators than occur in vessels simply bathed in PSS. Further investigation of these and other factors potentially contributing to the discordant responses of current isolated vessel and intact lung preparations is needed to develop an isolated vessel preparation with which to identify physiologically relevant mechanisms underlying pulmonary vasomotor responses.
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ACKNOWLEDGEMENTS |
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This work was supported by an American Heart Association (Northland Affiliate) Grant-in-Aid awarded to J. B. Gordon and by the Department of Pediatrics of the Medical College of Wisconsin. M. A. VanderHeyden and E. P. Cortez were supported by the Elaine Kohler Fund and the Steigleder Endowment Fund. C. A. Dawson was supported by National Heart, Lung, and Blood Institute Grant HL-19298, the W. M. Keck Foundation, and the Department of Veterans Affairs.
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. B. Gordon, Children's Hospital of Wisconsin, Critical Care Section, MS 681, 9000 W. Wisconsin Ave., Milwaukee, WI 53226 (E-mail: jgordon{at}mcw.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
First published January 24, 2003;10.1152/ajplung.00402.2002
Received 25 November 2002; accepted in final form 15 January 2003.
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