Platelet-activating factor modulates pulmonary
vasomotor tone in the perinatal lamb
BASIL O. IBE, SUE HIBLER, AND J. USHA RAJ
Department of Pediatrics, University of California, Los Angeles, School of Medicine,
Harbor-UCLA Medical Center, Torrance, California 90502
lung; platelet-activating factor antagonist; hypoxia; pulmonary circulation; pulmonary vascular resistance
changes in vascular reactivity or vasomotor tone (17),
and the stimulation of endothelial cells to release nitric
oxide (25, 27), among other effects. In a previous study
(1), direct infusion of PAF into the left pulmonary
artery of chronically catheterized preterm fetal lambs
resulted in pulmonary vasodilation without changes in
pulmonary arterial pressure. This suggests that, in the
pulmonary circulation, exogenously administered PAF
may act as a vasodilator or vasoconstrictor, depending
on the level of baseline tone; if baseline tone is high,
exogenous PAF acts as a vasodilator and, if baseline
tone is low, exogenous PAF acts as a constrictor. Physiological effects of endogenous PAF will depend on its
net production and the distribution and/or the numbers
of PAF receptors in the vessels, which may be dependent on the age of the animal and on the species. In this
study, we have used the specific PAF-receptor antagonist WEB-2170 (18) to study the role of endogenous
PAF in maintenance of basal vasomotor tone in both
the pulmonary and systemic circulations during the
perinatal period. Our hypothesis was that a drop in
PAF level after birth may be one method by which a fall
in pulmonary vascular resistance postnatally is facilitated.
METHODS
Animal Preparation
PLATELET-ACTIVATING FACTOR (PAF) is a potent phospholipid mediator with a wide spectrum of biological
properties (31). It is synthesized by many types of cells,
including platelets and lung epithelial and endothelial
cells. After a stimulus, 1–0-alkylphospholipids released
from membrane are acted on by phospholipase A2
(PLA2 ) to form lyso-PAF (2, 31). The lyso-PAF so formed
may be acted on by acetyltransferase to form PAF. PAF
synthesized in vivo may produce any of the physiological effects of PAF, or it may be catabolized by acetylhydrolase, the PAF catabolic enzyme, to form lyso-PAF.
The lung is a major target of the biological activities
of PAF (11). Infusion of PAF into the pulmonary circulation increases total pulmonary vascular resistance (16).
In spontaneously breathing sheep, administration of
PAF induces pulmonary vasoconstriction and increases
transvascular fluid filtration, which can be reversed if
PAF treatment is discontinued (6, 10). Other reports
have demonstrated that PAF plays a significant role in
fetal lung development and maturation (4, 19). This
suggests that, apart from being an inflammatory mediator, PAF does have some beneficial physiological functions. The interaction of PAF with its receptor involves
a series of complex biochemical events that may lead to
an increase in intracelluar calcium concentration (34),
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Near-term pregnant ewes of 139–146 days gestation, term
being 150 days, were premedicated with ketamine hydrochloride (1 g im) and atropine sulfate (1.6 mg im). An endotracheal tube was tied into the trachea, and catheters were
placed in the external jugular vein and femoral artery. The
ewe was mechanically ventilated with oxygen-enriched air by
using a piston-type ventilator (Harvard Instruments). Tidal
volume was adjusted to maintain arterial PCO2 between 30
and 35 Torr. Arterial PO2 was kept above 80 Torr. Anesthesia
was maintained with a continuous infusion of ketamine
hydrochloride (500–750 mg/h). Adequacy of anesthesia in the
ewe was judged by the presence of a stable heart rate and
blood pressure that were unchanged by painful stimuli. The
ewe received intravenous fluids (Ringer lactate and 10%
dextrose) continuously at a rate of ,500 ml/h. After induction
of adequate anesthesia, a hysterotomy was performed.
Fetal Lambs
A total of eight fetal lambs were studied. After delivery of
the fetal head and neck through the uterine incision, a glove
filled with warm saline was placed over the animal’s head to
prevent air breathing. Sedation was provided with intramuscular ketamine (25 mg/kg) and acepromazine (0.5 mg/kg).
Subcutaneous lidocaine (2%) was used for local anesthesia. A
fetal skin incision was made on the right side of the neck, and
a catheter was placed in the carotid artery. A balloon-tipped
thermodilution catheter (5-F), floated into the main pulmo-
8750-7587/98 $5.00 Copyright r 1998 the American Physiological Society
1079
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Ibe, Basil O., Sue Hibler, and J. Usha Raj. Plateletactivating factor modulates pulmonary vasomotor tone in the
perinatal lamb. J. Appl. Physiol. 85(3): 1079–1085, 1998.—
Eight near-term fetal lambs were studied acutely in utero to
determine role of platelet-activating factor (PAF) in the
regulation of vasomotor tone in systemic and pulmonary
circulations in the immediate perinatal period. Four fetal
lambs were studied predelivery and 2 h postdelivery to
determine circulating PAF levels. Aortic and pulmonary
arterial pressures and cardiac output were measured continuously, and systemic and pulmonary vascular resistances were
calculated. Left pulmonary arterial blood flow was also
measured in four fetal lambs. After delivery and oxygenation,
circulating PAF levels fell significantly. When WEB-2170, a
specific PAF-receptor antagonist, was infused to block effect
of endogenous PAF in the eight near-term fetal lambs,
systemic vascular resistance fell 30% but pulmonary vascular
resistance fell dramatically by 68%. Specificity of WEB-2170
was tested in juvenile lambs and was found to be very specific
in lowering vasomotor tone only when tone was elevated by
action of PAF. Our data show that endogenous PAF levels in
the fetus contribute to maintain a high basal systemic and
pulmonary vasomotor tone and that a normal fall in circulating PAF levels after birth and oxygenation may facilitate fall
in pulmonary vascular resistance at birth.
1080
PAF MODULATION OF PERINATAL PULMONARY CIRCULATION
Juvenile Lambs
Juvenile lambs were used to probe the specificity of WEB2170 as a PAF-receptor antagonist. Lambs were premedicated with ketamine hydrochloride (25 mg/kg im) and atropine sulfate (40 µg/kg im). Subcutaneous lidocaine (2%) was
used for local anesthesia. A catheter was inserted into the
carotid artery, and a 7-F balloon-tipped thermodilution catheter (Baxter Health Care) was floated into the pulmonary
artery via the internal jugular vein. An additional catheter
was placed in the right atrium via the saphenous vein. The
lambs were allowed at least 24 h to recover from operative
stress before study protocols were begun. During the studies,
lambs were awake, unanesthetized, breathing spontaneously,
and resting quietly in slings. Body temperature was maintained at 39–40°C with an overhead warmer, if necessary. An
interval of at least 24 h was allowed between studies in each
lamb. Between studies, catheters were flushed daily, filled
with heparin, and capped. Oxacillin (10 mg/kg im) and
gentamicin (2 mg/kg im) were administered twice daily.
International, Harbor City, CA) with flow controlled by
gravity. Columns were prewashed with 2 ml methanol followed by 5 ml of 10% acetic acid. Plasma samples that were
spiked with 10,000 counts/min of [3H]PAF to monitor the
recovery of PAF from the plasma were diluted with an equal
volume of 20% acetic acid and loaded on the column. The
column was then washed sequentially with 2 ml of 10% acetic
acid and 3 3 2 ml of ethyl acetate. PAF was eluted from the
column with 6 ml of methanol in a polypropylene plastic tube.
Further cleanup of the extracted PAF was done by adding 0.1
g of DEAE-cellulose to each sample and then extracting the
PAF with a mixture of chloroform-methanol-water (1:1:0.9).
Recovery of radioactivity from the spiked samples was between 92 and 95%. Sample extracts were then dried under a
stream of nitrogen and taken up in 1 ml of the RIA buffer. The
extraction procedure was tested for specificity in alkylphospholipid extraction. For this test, samples dried with a stream of
nitrogen were taken up in 200 µl of chloroform and purified by
TLC on glass-silica gel plates (J. T. Baker, Phillipsburg, NJ)
by using chloroform-methanol-water (65:35:5) as the developing solvent. Purified PAF was tested for retention of biological
activity by the serotonin-release method before measurement
by 125I RIA.
PAF RIA. PAF was measured by RIA with an equimolar
solution of the hexadecyl and octadecyl analogs as standard.
Purified PAF was redissolved in the RIA buffer provided with
the assay kit (DuPont-New England Nuclear), and the assay
was performed as described by the vendor. The assay was
tested for linearity and for intra-assay and interassay variabilities. The variabilities were ,10%. The cross-reactivity of
the antibody with arachidonyl-PAF, carbamyl-PAF, lyso-PAF,
phosphatidylcholine, and WEB-2170 were ,0.5%. The amount
of metabolite measured was corrected for recovery and expressed as nanograms per milliliter of plasma.
Drug Preparation
WEB-2170 (gift of Boehringer Ingelheim) was dissolved in
normal saline, and pH was adjusted to 5.6 with NaOH. PAF
(Biomol, Plymouth Meeting, PA) was dissolved in ethanol,
and then aliquots were mixed with 0.1% bovine serum
albumin in distilled water. Angiotensin II (Sigma Chemical,
St. Louis, MO) was diluted with normal saline.
Physiological Measurements
Pressures in the aorta and pulmonary artery were measured continuously with Statham P23 transducers and recorded on a polygraph recorder (model R511A, Sensormedics).
Heart rate was determined from the phasic pressure tracing
and recorded at 15-min intervals. Cardiac output was determined by the thermodilution method (model SP1453, Gould)
at ,20-min intervals, in triplicate, and the average value was
recorded. Left pulmonary arterial blood flow was measured
continuously by using a Transonic flowmeter (model T108)
connected to a size 4S perivascular flow probe in four fetal
lambs. Arterial pH, gas tensions (model BMS3MIC2, Radiometer, Copenhagen, Denmark), hematocrit (Readacrit centrifuge), and glucose concentration (Dextrostix) were monitored
every 30 min.
Experimental Protocols
Measurement of PAF
Extraction of PAF. Plasma samples were prepared from
blood drawn from fetal lambs before and after delivery.
Plasma samples were extracted for PAF measurement, as
recommended by DuPont-New England Nuclear (Boston,
MA) on BOND-ELUT C18 extraction columns (Analytichem
Physiological role of endogenous PAF in fetal lambs, before
and after birth. Eight fetal lambs, 142–143 days gestation,
were studied in utero. After insertion of catheters and placement of flow probes (4 of 8 lambs), baseline hemodynamic
variables were monitored for 10–15 min. Arterial pH and
blood-gas tensions were measured. If arterial pH, PO2, and
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nary artery via the internal jugular vein, was used to
determine cardiac output and monitor core temperature.
After a left thoracotomy to expose the heart and great vessels,
an ultrasonic flow probe (4 mm) was placed around the left
pulmonary artery in four of eight lambs. During the studies,
the fetal body remained partially in the uterus and the fetus
was covered with warm moist towels.
Five of the fetal lambs were studied after delivery. After
delivery of the fetal head and neck through the uterine
incision, an endotracheal tube was tied into the trachea. Fetal
lung fluid was aspirated via the endotracheal tube (usually 10
ml/kg body wt) and discarded before delivery. Once delivered,
the lamb was weighed and dried, and mechanical ventilation
(Healthdyne Infant Ventilator) was initiated immediately.
Sedation was provided with ketamine (25 mg/kg) and
acepromazine (0.5 mg/kg). Subcutaneous lidocaine (2%) was
used for local anesthesia. The umbilical artery was cannulated with a 5-F polyvinyl catheter that was advanced into
the descending aorta. After a neck incision, a balloon-tipped
thermodilution catheter (5-F) was placed in the internal
jugular vein and floated into the pulmonary artery. An
additional catheter was placed in the right atrium via the
saphenous vein. During mechanical ventilation, inspired
oxygen concentration was adjusted to maintain arterial PO2
.90 Torr. Respiratory rate was kept between 20 and 30
breaths/min, and the peak inspiratory pressure was adjusted
to keep arterial PCO2 between 30 and 40 Torr. End-expiratory
pressure was kept constant at 2 cmH2O. Body temperature,
monitored by a rectal probe, was maintained between 39 and
40°C with a heating pad and a heat lamp, if necessary. An
infusion of 10% dextrose solution (4–5 ml · kg21 · h21 ) was
given to maintain Dextrostix .45 mg/100 ml, and any blood
withdrawn was replaced with autologous whole blood (obtained from the placenta). We waited for a period of 3–6 h,
before initiation of the experimental protocol, to allow adequate time for the lamb to undergo successful extrauterine
cardiopulmonary transition. During this time, vascular pressures were monitored continuously and arterial blood pH and
gas tensions were measured every 30 min.
PAF MODULATION OF PERINATAL PULMONARY CIRCULATION
Torr, respectively, at baseline and remained unchanged
after infusion of WEB-2170. Hematocrit (Hct) was 42 6
1%. In the five lambs that received WEB-2170 in
incremental doses, the mean dose at which we first saw
a hemodynamic effect was 10 6 1 mg/kg. The maximum
effect occurred at a mean dose of 32 6 5 mg/kg, with
additional doses of up to 47 6 6 mg/kg (200 mg total
dose) having no further effect. The mean arterial pH,
PCO2, and PO2 after infusion of WEB-2170 were 7.34 6
0.02, 27 6 2 Torr, and 39 6 1 Torr, respectively, and they
were not different from the values before the infusion of
WEB-2170.
Figure 1 summarizes mean values for aortic and
pulmonary arterial pressures in fetal lambs at baseline
and maximum response after infusion of WEB-2170 in
a dose of 48.5 mg/kg (200-mg total dose). Aortic pressure fell 17 6 3% from 61 6 4 to 51 6 3 mmHg, and
pulmonary arterial pressure fell 20 6 3% from 60 6 5 to
48 6 4 mmHg. The nadir of the fall in aortic and
pulmonary arterial pressures occurred at ,1 min after
infusion of WEB-2170, and pressures remained at this
lower level. Baseline heart rate and cardiac output
were 141 6 10 beats/min and 267 6 22 ml · kg21 · min21,
respectively. Cardiac output increased significantly to
340 6 31 ml · kg21 · min21 after WEB-2170. Heart rate
was unaffected and was 148 6 10 beats/min. The Hct
remained stable at 42 6 1%. Calculated systemic
vascular resistance decreased significantly by 33 6 4%
from 0.241 6 0.029 to 0.161 6 0.022 mmHg · min ·
ml21 · kg.
In the four fetal lambs in which left pulmonary
arterial blood flow was measured (Fig. 2), pulmonary
arterial pressure fell 15 6 2% from 53 6 3 to 45 6 2
mmHg, whereas left pulmonary artery flow increased
157 6 13% from 23 6 8 to 60 6 20 ml/min after infusion
of WEB-2170 (49 6 4 mg/kg; 200-mg total dose) into the
pulmonary artery, showing an almost 68% decrease in
calculated pulmonary vascular resistance.
In the five fetal lambs that were delivered and
ventilated, mean arterial pH after birth was 7.39 6
0.04 and PO2 and PCO2 were 133 6 13 and 24 6 3 Torr,
respectively. Cardiac output and heart rate were 215
ml·kg21 ·min21 and 138 beats/min, respectively. Figure 3
Data Analysis
All data are means 6 SE. The data were analyzed statistically by using a two-tailed Student’s t-test. For example, a
t-test was used to discover the differences in pulmonary
arterial pressures during pre- and post-WEB-2170 treatment
of lamb lungs. For multiple comparisons, a one-way ANOVA
was performed followed by Tukey’s post hoc test. A P , 0.05
was considered significant.
RESULTS
Physiological Role of Endogenous PAF in Fetal Lambs,
Before and After Birth
Fetal lambs. The mean arterial pH was 7.35 6 0.02
and CO2 and O2 gas tensions were 28 6 1 and 40 6 1
Fig. 1. Changes in pulmonary arterial (PA) and aortic (Aorta)
pressures after infusion of WEB-2170 in fetal lambs. Values are
means 6 SE for 5 lambs. Pulmonary artery and aortic pressures were
measured before (Pre-WEB) and after (Post-WEB) administration of
WEB-2170. * Different from Pre-WEB, P , 005.
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PCO2 were within the physiological range (pH 7.32–7.42, PO2
25–32 Torr, PCO2 38–45 Torr) and mean arterial pressures
remained stable during this time period, the fetus was
studied. In four lambs, the PAF receptor-antagonist, WEB2170, was infused into the pulmonary artery in incremental
doses ranging from 20 to 200 mg. An effect was first seen at a
total dose of 40 mg. Doses up to 160 mg continued to have an
effect. From 160 to 200 mg (47 6 6 mg/kg) no further effect
was seen. On the basis of these results, the remaining four
lambs received a 200-mg (55 6 2 mg/kg) bolus of WEB-2170
into the pulmonary artery. In all lambs hemodynamic measurements were continued for 15–20 min. In two animals,
PAF (400 ng) was infused into the pulmonary artery at 10–15
min after WEB-2170. Effectiveness of WEB-2170 was tested
by intravenous infusion of PAF (400 ng) in two animals.
Postdelivery study of fetal lambs. Five lambs were delivered and mechanically ventilated. After a postbirth stabilization period of 4 6 1 h, baseline (pre-WEB-2170) measurements of aortic and pulmonary arterial pressures and cardiac
output were obtained. PAF levels were measured before and
after birth.
Specificity of the PAF-receptor antagonist WEB-2170. Specificity of the WEB-2170 compound was tested in juvenile
lambs. Six lambs (age 6 6 1 wk, body weight 11 6 2 kg) were
studied. After a stable baseline period of at least 45 min,
during which time pressures did not vary by .10% of mean
values, the experimental protocols were begun.
Group I (n 5 4). Specificity of WEB-2170 was tested in this
group of lambs. A PAF infusion was begun at 1 µg · kg21 · h21
and increased as needed to obtain a minimum 30-min period
of stable pulmonary arterial pressure approximately double
that of baseline. The infusion rate required varied from 1 to
10 µg · kg21 · h21 (mean 4 µg · kg21 · h21 ).
Group II (n 5 4). Pulmonary vascular resistance was
elevated in this group of lambs with hypoxia. Pulmonary
arterial pressure was elevated to approximately double that
of baseline by allowing the lambs to breathe a hypoxic gas
mixture (air and nitrogen) that was blown into a plastic bag
loosely fitted over the lamb’s head. Frequent arterial blood
gases were monitored to maintain PO2 ,50 Torr.
Group III (n 5 5). Both pulmonary and systemic vascular
resistances were elevated in this group of lambs with angiotensin II infusion. Angiotensin II was infused at a dose of
15–50 µg · kg21 · min21 to elevate aortic pressure at least 50%
and pulmonary arterial pressure by at least 30%.
In all lambs, after a steady-state period of ,30 min of
elevated aortic and pulmonary arterial pressures, WEB-2170
was infused into the aorta in incremental doses ranging from
5 to 50 mg/kg. Hemodynamic measurements were continued
for an additional 10–15 min.
1081
1082
PAF MODULATION OF PERINATAL PULMONARY CIRCULATION
Fig. 2. Changes in pulmonary arterial pressure and flow rate after
infusion of WEB-2170 in fetal lambs. Pressure and flow rate were
measured before and after administration of WEB-2170. Values are
means 6 SE for 4 lambs. * Different from Pre-WEB, P , 0.05.
Specificity of WEB-2170
Group I juvenile lambs. In group I juvenile lambs,
arterial pH averaged 7.45 6 0.02 and PO2 and PCO2
were 88 6 12 and 33 6 3 Torr, respectively, at baseline
Fig. 3. Changes in pulmonary arterial and aortic pressures after
infusion of WEB-2170 in newborn lambs. Values are means 6 SE for
5 lambs. Pulmonary arterial and aortic pressures were measured
before and after administration of WEB-2170. * Different from PreWEB, P , 0.05.
and did not change significantly throughout the study.
The Hct was 32 6 2%.
Table 1 shows a summary of the mean values for
aortic pressure, pulmonary arterial pressure, heart
rate, cardiac output, systemic vascular resistance, and
pulmonary vascular resistance for baseline, steadystate PAF, and PAF 1 WEB-2170. Baseline aortic
pressure averaged 81 6 5 mmHg and did not change
significantly with PAF infusion or after treatment with
WEB-2170. Pulmonary arterial pressure increased significantly with PAF infusion and then fell close to
baseline value after infusion of 5 mg/kg WEB-2170.
Additional WEB-2170 (50 mg/kg) had no further effect.
Baseline heart rate and cardiac output were 122 6 7
beats/min and 265 6 20 ml · kg21 · min21, respectively.
There was no significant change in heart rate and
cardiac output after the infusion of PAF. However, after
infusion of WEB-2170 (5 mg/kg), heart rate and cardiac
output increased significantly. Further increases in
heartbeat and flow rate were observed with the higher
dose of WEB-2170 (50 mg/kg) administered. Heart rate
and cardiac output increased with infusion of WEB2170, especially at the higher dose. Calculated systemic
vascular resistance increased by 10% with PAF infusion, whereas calculated pulmonary vascular resistance was nearly doubled. After treatment with WEB2170 (5 mg/kg) systemic and pulmonary vascular
resistances fell to baseline levels. Additional WEB2170 infusion (50 mg/kg) had no further effect.
Group II juvenile lambs. In group II juvenile lambs,
baseline arterial blood pH was 7.45 6 0.03 and PO2 and
PCO2 were 36 6 3 and 77 6 2 Torr, respectively. During
steady-state hypoxia, arterial blood pH was 7.54 6
0.02, PO2 and PCO2 were 37 6 3 and 25 6 2 Torr,
respectively, and remained unchanged after infusion of
WEB-2170. The Hct was 33 6 1% during the study.
Table 2 summarizes the mean values for aortic pressure, pulmonary arterial pressure, heart rate, cardiac
output, systemic and pulmonary vascular resistances
for baseline, steady-state hypoxia, and hypoxia 1 WEB2170. Baseline aortic and pulmonary arterial pressures
were similar to the baseline values for group I lambs
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shows changes in pulmonary arterial and aortic pressures after infusion of WEB-2170 in these five newborn
lambs. Aortic pressure fell by 31 6 3% from 58 6 4 to
40 6 2 mmHg. The pulmonary arterial pressure fell by
18 6 3% from 40 6 4 to 33 6 2 mmHg. Calculated
systemic and pulmonary vascular resistances (Fig. 4)
were 0.284 6 0.041 and 0.189 6 0.019 mmHg · min ·
ml21 · kg before infusion of WEB-2170 and changed by
48% to 0.163 6 0.19 mmHg · min · ml21 · kg for systemic
resistance and by 30% to 0.132 6 0.020 mmHg · min ·
ml21 · kg for pulmonary vascular resistance. The postWEB-2170 arterial blood-gas values remained stable at
pH 7.32 6 0.04, PCO2 29 6 4 Torr, and PO2 131 6 18 Torr.
Circulating levels of PAF in fetal lambs, before and
after birth. The concentration of PAF in plasma of fetal
lambs before delivery was 6.43 6 3.48 ng/ml. The
concentration of PAF in plasma of the lambs 90 min
after delivery was 1.31 6 1.03 ng/ml. The concentration
of PAF was significantly higher in plasma of fetal lambs
than in plasma of the immediate-newborn lambs.
Fig. 4. Changes in calculated systemic and pulmonary vascular
resistances after infusion of WEB-2170 in newborn lambs. Data are
means 6 SE for 5 lambs. Changes in vascular resistances were
calculated from measurements obtained before and after administration of WEB-2170. * Different from Pre-WEB, P , 0.05.
1083
PAF MODULATION OF PERINATAL PULMONARY CIRCULATION
Table 1. Effect of WEB-2170 on systemic and pulmonary circulations in lambs during PAF infusion
Vascular Resistance,
mmHg · min · ml21 · kg
Pressure, mmHg
Steady-State Period
HR,
beats/min
Aortic
Pulmonary
CO,
ml · kg21 · min21
Systemic
Pulmonary
Baseline
PAF infusion
PAF 1 WEB-2170 (5 mg/kg)
PAF 1 WEB-2170 (50 mg/kg)
122 6 7
146 6 20
165 6 10†
183 6 11†
81 6 5
86 6 6
84 6 4
82 6 3
13 6 1
27 6 4*
16 6 1
16 6 1
265 6 20
247 6 4
291 6 24
314 6 45‡
0.309 6 0.022
0.353 6 0.037*
0.295 6 0.027
0.280 6 0.038
0.050 6 0.002
0.114 6 0.019*
0.057 6 0.005
0.054 6 0.006
Values are means 6 SE for four 5- to 7-wk-old lambs. PAF, platelet-activating factor; HR, heart rate; CO, cardiac output. * Different from
baseline and PAF 1 WEB (5 and 50 mg/kg), P , 0.05. † Different from baseline, P , 0.05. ‡ Different from baseline and PAF infusion, P , 0.05.
DISCUSSION
PAF is an important phospholipid mediator synthesized in response to endotoxemia (5) and other endogenous and exogenous stimuli. PAF also causes decreased lung compliance and increased airway
resistance (5, 16). Among its other wide-ranging physiological as well as pathological effects in vivo, PAF is a
potent vasoactive mediator in the pulmonary and systemic circulations (7, 9). It has been shown that exogenous PAF induces pulmonary and systemic vasoconstriction, when baseline vasomotor tone is low, which
can be abolished by administration of a specific PAFreceptor antagonist (26, 35). However, the role of
endogenous PAF in modulation of the pulmonary and
systemic circulations in the normal fetus and newborn
is not well understood and therefore needs further
examination.
In this report, we have investigated the modulation
of perinatal ovine pulmonary and systemic vasomotor
tone by PAF. To study the role of endogenous PAF in the
fetus and newborn, we used WEB-2170, a potent and
highly specific PAF-receptor antagonist (14, 18, 22) to
unmask the physiological role of PAF. Inhibition of
endogenous PAF activity in the fetal lamb resulted in a
30% decrease in systemic vascular resistance and a
much greater reduction (68%) in pulmonary vascular
resistance. This shows that endogenous PAF acts both
as a pulmonary and systemic vasoconstrictor, with a
much greater role as a pulmonary vasoconstrictor in
the fetus. Inhibition of this potent vasoconstrictor at its
receptor resulted in a decrease in vasomotor tone.
Because the infusion of WEB-2170 did not result in
changes in arterial pH, PCO2, and PO2, we can conclude
that the decrease in systemic and pulmonary vascular
tone was not due to changes in blood pH and blood-gas
tensions. The dose of WEB-2170 used in our infusion
study is five to six times higher than the dose of
inhibitors (e.g., indomethacin) usually used to inhibit
Table 2. Effect of WEB-2170 on systemic and pulmonary circulations in lambs during hypoxia
Vascular Resistance,
mmHg · min · ml21 · kg
Pressure, mmHg
Steady-State Period
HR,
beats/min
Aortic
Pulmonary
CO,
ml · kg21 · min21
Systemic
Pulmonary
Baseline
Hypoxia
Hypoxia 1 WEB-2170 (50 mg/kg)
120 6 15
128 6 15
163 6 10†
81 6 5
94 6 6
90 6 6
13 6 1
31 6 1*
31 6 1*
208 6 19
250 6 13
247 6 27
0.419 6 0.052
0.393 6 0.015
0.365 6 0.038
0.065 6 0.012
0.119 6 0.007*
0.124 6 0.014*
Values are means 6 SE for four 5- to 7-wk-old lambs. * Different from baseline, P , 0.05. † Different from baseline and hypoxia, P , 0.05.
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(81 6 5 and 13 6 1 mmHg, respectively). With hypoxia,
aortic pressure remained unchanged and pulmonary
artery pressure was more than doubled. WEB-2170 (50
mg/kg) had no effect on either aortic or pulmonary
arterial pressures during hypoxia. Heart rate and
cardiac output remained unchanged from baseline values; however, after treatment with WEB-2170, heart
rate increased significantly to 163 6 10 beats/min.
Calculated systemic and pulmonary vascular resistances were 0.419 6 0.052 and 0.065 6 0.012
mmHg · min · ml21 · kg at baseline. Systemic vascular
resistance was unaffected by hypoxia or WEB-2170;
however, pulmonary vascular resistance was doubled
with hypoxia and remained elevated after treatment
with WEB-2170.
Group III lambs. In group III juvenile lambs, mean
arterial pH was 7.47 6 0.03 and PO2 and PCO2 were
80 6 3 and 36 6 1 Torr, respectively, at baseline and
remained unchanged throughout the study period. The
Hct was 29 6 3%. Table 3 summarizes the mean values
for aortic pressure, pulmonary arterial pressure, heart
rate, cardiac output, systemic pulmonary vascular resistances for baseline, steady-state angiotensin II, and
after WEB-2170 (50 mg/kg). During angiotensin II
infusion (25–50 µg · kg21 · min21 ), mean aortic and pulmonary arterial pressures increased 58 and 54%, respectively, over baseline values. Infusion of WEB-2170
during angiotensin II infusion had no effect on aortic or
pulmonary arterial pressures. Mean cardiac output
during the study period was 243 ml · kg21 · min21. Heart
rate increased slightly from 110613 to 138 6 18
beats/min, but it did not change significantly with
angiotensin II infusion. Calculated systemic and pulmonary vascular resistance increased by 59 and 57%,
respectively, with angiotensin II infusion. Infusion of
WEB-2170 (50 mg/kg) during angiotensin II infusion
had no effect on aortic pressure, pulmonary artery
pressure, calculated systemic vascular resistance and
calculated pulmonary vascular resistance.
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PAF MODULATION OF PERINATAL PULMONARY CIRCULATION
Table 3. Effect of WEB-2170 on systemic and pulmonary circulations in lambs during angiotensin II infusion
Vascular Resistance,
mmHg · min · ml21 · kg
Pressure, mmHg
Steady-State Period
HR,
beats/min
Aortic
Pulmonary
CO,
ml · kg21 · min21
Systemic
Pulmonary
Baseline
Angiotensin II infusion
Angiotensin II 1 WEB-2170 (50 mg/kg)
110 6 13
138 6 18
154 6 13*
74 6 2
117 6 13
111 6 4
13 6 1
20 6 2*
19 6 2*
243 6 27
243 6 27
243 6 27
0.320 6 0.057
0.510 6 0.107*
0.478 6 0.078*
0.056 6 0.008
0.088 6 0.014*
0.083 6 0.012*
Values are means 6 SE for five 5- to 7-wk-old lambs. * Different from baseline, P , 0.05.
(1). The high circulating plasma level of PAF may be
indicative of a high tissue production in the lung of the
fetus, which may serve to maintain high pulmonary
vascular resistance in utero. A successful transition to
the immediate neonatal period is facilitated by a decrease in the endogenous production of this potent
vasoconstrictor in the system.
Pathological conditions such as persistent pulmonary hypertension of the newborn (PPHN) are associated with high circulating levels of PAF (7), which fall
to the normal range with an improvement in the
clinical condition. The concentration of PAF in plasma
of PPHN patients was ,10 times more than that in
normal controls. However, concentration of PAF in
plasma of non-PPHN controls was 1.6 6 0.07 ng/ml
plasma (7) and is similar to the values (1.31 6 1.03
ng/ml plasma) we obtained in the immediate newborn
ovine plasma. Our findings regarding a high circulating
level of PAF in the fetus with a significant fall at birth
suggest that PAF plays a crucial role in maintaining a
high pulmonary vascular resistance in utero.
Pulmonary vascular resistance in utero is high, and
pulmonary blood flow is 8–10% of total combined
ventricular cardiac output (28, 30). At birth, with the
onset of ventilation, pulmonary vascular resistance
falls dramatically and pulmonary blood flow increases
such that the entire cardiac output passes through the
pulmonary circulation. The exact mechanisms controlling these changes are not well understood. Certainly,
the change in oxygen tension to which the lung vessels
are exposed after birth most likely does play some role.
However, changes in production and release of some
endogenous vasoactive mediators in the lung may be
equally important. For instance, eicosanoids, the metabolites of arachidonic acid, have been shown to play
significant roles in the control of pulmonary vasomotion in the fetus and newborn (28). There is an increase
in the production of prostacylin by the lung at the onset
of breathing (21), and an increase in nitric oxide, a
potent vasodilator, has also been shown to play a
significant role in postnatal changes in the pulmonary
circulation (12, 24).
Regulation of the fetal and immediate postnatal
pulmonary circulation portends a preference for factors
producing active vasoconstriction and those producing
vasodilation, respectively. In the fetus, it seems that
the balance is tipped toward factors producing vasoconstriction. PAF is a potent vasoactive mediator in the
pulmonary circulation (5, 8, 16, 23, 32). Our data
suggest that PAF is also one of the active vasoconstrictors operating in the fetal pulmonary circulation. Fall
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other endogenous vasoactive mediators such as prostacyclin or thromboxane A2. WEB-2170 is a highly lipophilic compound that may be easily absorbed by lipid
membranes, thereby making it less available for binding to the PAF receptor. It is possible that the high
concentration of WEB-2170 needed in our study resulted in an optimal amount of the drug being available
to bind to the PAF receptor. This explanation is plausible because infusion of more antagonist after the
maximum effect had been attained did not produce any
further decrease in systemic or pulmonary vascular
resistances. This demonstrates that the observed effect
was due to inhibition of PAF binding to its receptor in
the lung rather than due to a nonspecific interaction of
WEB-2170 at the PAF receptor.
In a previous report, infusion of exogenous PAF into
preterm lambs led to vasodilation of the fetal pulmonary circulation (1). This apparent discrepancy is explainable by the differences in experimental conditions.
When we block the effect of endogenous PAF, we are
truly unmasking its physiological role. However, infusion of exogenous PAF will merely demonstrate the
effect of exogenous PAF on a pulmonary vascular bed
that is constricted. There were also differences in
gestational age, 122- to 130-day-gestation fetuses employed in the study infusing exogenous PAF (1) as
opposed to 139- to 146-day-gestation fetuses used in
our study. Furthermore, it is possible that exogenous
PAF, by acting directly on the PAF receptor or indirectly
by stimulating release of other vasoactive mediators,
such as eicosanoids (3, 13, 20, 33) and/or nitric oxide
(27), may result in different hemodynamic effects in the
pulmonary circulation of the fetus and newborn. For
instance, in the ferret, PAF induces relaxation of pulmonary arteries but contraction of pulmonary veins (15).
We found that circulating levels of endogenous PAF are
high in the fetal lamb, whether preterm or near term,
and that this level drops significantly after birth. For
instance, when we measured plasma PAF concentration in very-preterm lambs (122–123 days gestation;
n 5 5) that were delivered and mechanically ventilated
for ,2 h, the pre- and postdelivery plasma PAF levels
were 2.09 6 1.04 (SE) and 0.32 6 0.41 ng/ml plasma,
respectively. The pre- and postdelivery plasma PAF
concentrations in near-term fetal lambs that we studied and have reported here are over three times higher
than the plasma PAF concentrations in very-preterm
fetal lambs. The higher plasma PAF concentration in
near-term fetal lambs compared with that in verypreterm fetal lambs may be one other reason for the
difference in our findings from those of Accurso et al.
PAF MODULATION OF PERINATAL PULMONARY CIRCULATION
in pulmonary vascular resistance at birth could result
from decreased synthesis of vasoconstrictors and/or
increased synthesis of other vasodilators, such as prostacyclin (21) and endogenous nitric oxide (12), or from
enhanced removal by catabolism of the vasoconstrictor
PAF (29). We have previously reported that oxygenation results in an increase in catabolism of PAF, which
may be one additional mechanism by which there is
decrease in amount of vasoconstrictors at birth.
We thank J. Morris for help with the experiments and M. Unutoa
for secretarial assistance.
This work was supported in part by National Heart, Lung, and
Blood Institute Grants HL-38438 and HL-47804.
Address for reprint requests: B. O. Ibe, Dept. of Pediatrics,
Harbor-UCLA Medical Center, RB-1, 1124 W. Carson St., Torrance,
CA 90502.
Received 24 July 1997; accepted in final form 4 May 1998.
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