687
Monohydroxyeicosatetraenoic Acids (5-HETE
and 15-HETE) Induce Pulmonary
Vasoconstriction and Edema
Kenneth E. Burhop, William M. Selig, and Asrar B. Malik
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5-, 15-, and 12-HETE (monohydroxyeicosatetraenoic acids) are products of the lipoxygenation of
arachidonic acid. We investigated their role as possible mediators of pulmonary vasoactivity and
pulmonary edema. Pulmonary artery pressure (?„), capillary pressure (P ap ), the change in lung wet
weight (Awt) from baseline, and capillary filtration coefficient (K,) (as a measure of vascular
permeability) were determined following an intravenous injection of each mono-HETE in lungs
perfused at constant flow with either a phosphate-buffered Ringer's-albumin solution (PBR) or diluted
blood. Injection of 2 (xg of each compound into the pulmonary artery of lungs perfused with either
PBR or diluted blood did not produce any effect. However, in PBR-perfused lungs, 4 (ig 15-HETE
induced increases in Pp., P^., and lung wet weight (p<0.05), which were greater than the increases
observed after 4 jig 5-HETE. Kr increased following both 5- and 15-HETE. The pulmonary vasoconstrictor and edemagenic responses were attenuated by increasing perfusate albumin concentration from 0.5 to 1.5 g%. In contrast, 12-HETE (4 |i.g) had no effect on these parameters. In
blood-perfused lungs, the pulmonary vascular responses to all HETE compounds (4 |xg) were
attenuated. In both RingerValbumin-perfused and blood-perfused lungs, the relative magnitude of
the hemodynamic and fluid filtration responses to each mono-HETE were as follows: 15-HETE >
5-HETE > 12-HETE. In conclusion, the pulmonary vasoconstrictor and edemagenic effects of 5- and
15-HETE occur independently of blood-formed elements. 15-HETE causes greater pulmonary
vasoconstriction and edema than 5-HETE. Both 5- and 15-HETE induce pulmonary edema, probably
as a result of increased lung vascular permeability. The results indicate that 5- and 15-HETE are potent
pulmonary inflammatory mediators. (Circulation Research 1988;62:687-698)
T
he monohydroxyeicosatetraenoic acids (HETEs)
are a group of lipoxygenase products of arachidonic acid metabolism'"5 that include 5-hydroxy-5,8,10,14-eicosatetraenoic acid (5-HETE), 15hydroxy-5,8,ll,13-eicosatetraenoic acid (15-HETE),
and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12HETE) (Figure 1). HETEs may have a role in the
initiation or potentiation of the inflammatory process6"8
and in the stimulation' or inhibition10"13 of synthesis of
other lipoxygenase pathways. HETEs are produced by
a variety of cell types including neutrophils,'-3-41415
platelets,2-16 macrophages,17"19 mast cells,9-20 and eosinophils,21 all of which may be involved in the inflammatory response. HETEs are generated in sufficient
quantities to be detected in vivo in animal models of
diffuse lung injury, such as following infusion of
endotoxin22"24 or thrombin,23 and attain maximum
detectable concentrations coincident with increased
lung vascular permeability.24-2515-HETE has also been
detected in human airways2* and was shown to have a
potent agonist effect on canine tracheal mucus
secretion.27
From the Department of Physiology, Albany Medical College of
Union University, Albany, New York.
Supported by Program Project HL 32418. K.E.B. and W.M.S.
were Postdoctoral Fellows in Pulmonary Research supported by
National Institutes of Health, USPHS grant HL 07529.
Address for correspondence: Dr. A.B. Malik, Department of
Physiology, Albany Medical College, 47 New Scotland Avenue,
Albany, NY 12208.
Received October 27, 1986; accepted October 8, 1987.
The role of mono-HETEs in mediating changes in
pulmonary hemodynamics and vascular permeability is
not clear. The purpose of the present study was: 1) to
compare the pulmonary microvascular responses to
injection of 5-, 15-, and 12-HETE in the isolated
Ringer's-albumin-perfused guinea pig lung; 2) to
compare the responses of the mono-HETEs in bloodperfused lungs to examine the role of blood-formed
elements in the response; and 3) to evaluate the role of
mono-HETEs as possible mediators of pulmonary
edema.
Materials and Methods
Isolated, Perfused Guinea Pig Lung
Guinea pigs (400-500 g) were anesthetized with
sodium pentobarbital (50 mg/kg i.v.; Abbott, Chicago,
Illinois) and tracheotomized. Following a thoracotomy, intracardiac heparin (700 U/kg, Invenex, Chagrin
Falls, Ohio) was administered, and the animals were
exsanguinated. The lungs and heart were removed
together and suspended from one end of a counterweighted beam balance to monitor lung weight. The
pulmonary artery and left atrium were cannulated.
Perfusion was begun at a low flow rate within 5 minutes
of pneumothorax using a peristaltic roller pump (model
1215, Harvard Apparatus, Millis, Massachusetts).
Airway pressure was maintained at 1 cm H2O with 95%
O2-5% CO2, and venous outflow pressure was set at 3
cm H2O so all lungs remained in the Zone III condition
to minimize changes in vascular surface area. Lungs
were covered with Saran wrap to prevent evaporative
688
Circulation Research
Vol 62, No 4, April 1988
Phosphollpld
Prostaglandlns
15-HPETE
12HPETE
COOH
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LTB4
12 HETB
FIGURE 1. Pathways and products of the arachidonic acid cascade. Included are structures of 5-, 15-, and 12-hydroxyeicosatetraenoic
acids (HETEs).
fluid loss. Recirculation of perfusate (reservoir volume
250 ml) at 28 ml/min was begun after lungs had been
washed with approximately 500 ml perfusate to clear
the pulmonary circulation of blood.
Details of the perfusion system used in the present
study have been previously described.28 Continuous
pulmonary wet weight recordings were made on a
three-channel Gould recorder (model 2200S, Cleveland, Ohio) calibrated so that a 0.5-g weight change
resulted in a 5-cm recorder pen deflection. Pulmonary
arterial and left atrial pressures were continuously
monitored with transducers (Statham P50 and Gould
P23ID) connected to catheters (PE90) placed in the
pulmonary artery and left atrium. All monitored
variables were stable, and lungs without intervention
remained isogravimetric for up to 2 hours. All studies
were carried out for a 70-minute period.
The perfusate used consisted of either a phosphatebuffered Ringer's solution (PBR) containing 0.5%
bovine serum albumin (Fraction V, Sigma Chemical,
St. Louis, Missouri), PBR containing 1.5% bovine
serum albumin (Fraction V, Sigma), or blood diluted
with PBR. Blood was obtained from heparinized (700
U/kg) donor guinea pigs by direct cardiac puncture.
The heparinized blood was diluted (1:3 vohvol) with
PBR to a final hematocrit of 13 ± 2% and a final protein
concentration of 1.5±0.2%. In a separate group of
experiments, the effects of varying protein concentrations in the blood on the pulmonary microvascular
response to HETEs were examined because protein
binding of HETEs14 may determine the magnitude of
the response. For these studies, heparinized blood was
obtained from donor guinea pigs in the manner
described above. The whole blood was centrifuged at
l,500g at 4° C for 10 minutes, varying amounts of the
plasma were drawn off, and the blood cells were
reconstituted back to the original volume with PBR
containing 0.5% bovine serum albumin. This "protein
poor" blood cell suspension was then diluted with PBR
(1:3 vol:vol) to a final hematrocrit of 15 ± 1%, and the
protein concentration of this blood perfusate was
determined.29 The Ringer's solution in all cases contained the following constituents (mM): NaCl 137,
CaCl2 1.8, MgCl2 1.05, KC1 2.68, NaHCO3 0.06,
NaH2PO4 0.130, Na2HPO4 0.869, and dextrose 5.55.
The warmed (38° C) perfusates were continuously
gassed in the outflow reservoir with 95% O2-5% CO 2 ,
and pH, Pco 2 , and Po2 were periodically monitored
with a Radiometer ABL2 blood gas analyzer (Copenhagen, Denmark).
Pulmonary Capillary Pressure and Segmental
Vascular Resistance
Pulmonary capillary pressure (P^) was estimated
using the double-occlusion technique of Linehan et al.30
The double-occlusion technique consisted of a brief
(2-3-second) simultaneous occlusion of both arterial
inflow and venous outflow during which the arterial
pressure (P,J decreased and venous pressure (Pv)
increased to an equilibrium pressure that approximated
Pap-30"3' P ^ was used to partition resistance into
upstream, or arterial (RJ, and downstream, or venous
), components from the following equations where
equals flow:
8
Burhop el al
P
R =
and
f
689
lungs were dried to a constant weight at 70° C. K, was
expressed in units of ml/(min-cm H 2 Og dry lung wt).
— P
r
c«p
0
P™ -
Pulmonary Inflammatory Effects of Mono-HETEs
p.
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Capillary Filtration Coefficient
The capillary filtration coefficient (Kf) was measured
at specific intervals in all experimental groups under
Zone III conditions. Following an isogravimetric period, the outflow pressure was rapidly elevated by 3 cm
H2O for 5 minutes. P ^ was measured immediately
before the increase in Pv and at the end of the 5-minute
elevation of Pv to obtain the change. The resulting
increase in lung weight corresponds to a twocompartment model: a rapid component attributed to
vascular filling and a slower component representing
an increase in the interstitial volume attributed to transvascular fluid filtration.32 Kf was determined according
to the method of Drake et al.32 The rate of lung weight
gain was calculated for each minute following the
rise in Pv and was expressed as a semilogarithmic function over time. The slow component of weight gain
(corresponding to the increase in interstitial volume) was
extrapolated to time 0 to obtain an estimate of the fluid
filtration rate, which was divided by the change in P^
to obtain Kr. At the end of each experiment, nonpulmonary tissue was dissected from the lungs, and the
Experimental Protocols
A bolus injection of either 2 or 4 ^g of each monoHETE (5-, 15-, and 12-HETE) was made directly into
the pulmonary artery of either PBR- or blood-perfused
lungs following the baseline measurements. All parameters were then monitored for up to 70 minutes postHETE injection. In control studies, hemodynamic or
fluid filtration parameters did not change significantly
for up to 2 hours following injection of saline in control
lungs (« = 3). 15-HETE (obtained from The Upjohn
Company, Kalamazoo, Michigan) was dissolved in
hexane:ethanol (3:1) to a final concentration of 1.0
mg/ml, 5-HETE (U-68687, Upjohn) was dissolved in
25% EtoAc/hexane to a final concentration of 5.0
mg/ml, and 12-HETE (Biomol, Philadelphia, Pennsylvania) was dissolved in ethanol to a final concentration
of 80.0 jig/ml. All the mono-HETEs used in these
studies were routinely examined by reverse phase
high-pressure liquid chromatography (HPLC) against
appropriate standards prior to and after each series of
experiments to ensure purity of the compounds.
Statistical Analysis
Data are expressed as mean±SEM. Statistical
analysis in the perfusion experiments was performed
using repeated measures analysis of variance33 fol lowed
by multiple comparisons testing using the Bonferroni
t test.33 Statistical significance was accepted at/? < 0.05.
20 r
15
Pobnoury
Artery Preuure 10
(cm H,0)
> 5-HETE
o—O15-HETE
D-O12-HETE
Putmowy
Cipfflvy
Preuure
(cm H,0)
FIGURE 2. Effect of 5-, 15-, and 12-hydroxyeicosatetraenoic acids (HETEs) (4 \ig) on pulmonary arterial
and capillary pressure in isolated guinea pig lungs
perfused with a phosphate-buffered Ringer's solution
containing 0.5% albumin (n=6 per group). Values are
mean±SEM. *Different (p<0.05) from baseline.
690
Circulation Research
Vol 62, No 4, April 1988
O.Z5 r
I.W
Mntoury
ArtnW
mmnci
(cm
0.1S
CIS
FIGURE 3. Changes in pulmonary arterial and pulmonary
venous resistances observed following injection of 5-, 15-,
and 12-hydroxyeicosatetraenoic acids (HETEs) (4 \ig) in
isolated guinea pig lungs perfused with a phosphate-buffered
Ringer's solution containing 0.5 g% albumin (n = 6 per
group). Values are mean ± SEM. 'Different (p < 0.05) from
baseline.
•~»5HFTE
o-oU-HETE
0
0.25
0.M
1.15
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(eaH,0-«n-iBta)0.10
0.05
•j
IB
U
U
50
Results
15-HETE
As seen in Figure 2, the injection of 4 \ig 15-HETE
into isolated guinea pig lungs perfused with PBR
containing 0.5% albumin increased (p<0.05) P,,, (top)
and P ap (bottom) within 20 minutes postinjection. P^
and P continued to rise and reached maximum values
(mean ± SEM) of 15.2 ±2.3 and 9.4 ±0.9 cm H2O,
respectively, at the end of the 70-minute experimental
period. 15-HETE produced increases (/?<0.05) in both
R, (Figure 3; top) and Rv (Figure 3; bottom). The
increases in R, and Rv were similar in magnitude and
paralleled the rises in P^ and P . Lung wet weight
gradually increased over time following injection of
15-HETE (Figure 4) and reached a maximum value
(mean ±SEM) of 2.3 ±0.8 g over baseline (control
guinea pig lung wet weight at baseline, 3.0±0.1 g) at
TO
the end of the 70-minute experimental period. The final
lung wet/dry weight ratio (mean ± SEM) was 10.1
±1.0. As seen in Figure 5, Kf increased (p<0.05)
approximately threefold over baseline at 70 minutes
postinjection of 15-HETE. The increase in Kf closely
paralleled the rise in lung wet weight. In two of the
experiments (data not included in Figure 5), the lung
wet weight increased at such a rapid rate by 70 minutes
postinjection of 15-HETE that Kf determinations were
not possible.
Injection of the same volume of the 15-HETE vehicle
(i.e., 4 (JLI hexane:ethanol diluted 3:1) into control lungs
(n = 6) produced relatively small changes (compared
with injection of 15-HETE) in all parameters measured
(Table 1). The final lung wet/dry weight ratio (mean
± SEM) was 7.8 ± 0.5. The magnitude of the changes
in the vehicle control group were similar to the saline
control group, and the values at 70 minutes postvehi-
5-HETE
O-O13HTTE
O-CI12-HETE
FIGURE 4. The change in lung wet weight from baseline
in isolated guinea pig lungs perfused with a phoshatebuffered Ringer's solution containing 0.5 g% albumin
(n = 6 per group) following injection of 5-, 15-, and
12-hydroxyeicosatetraenoic acids (HETEs) (4 \ig). Values are mean ± SEM. 'Different (p < 0.05) from baseline.
Change in
Lung Wet
Weight From
Baseline (g)
0.5 -
Burhop et al
Pulmonary Inflammatory Effects of Mono-HETEs
691
ROOSTS
16
14
5HETE
O—o 15-HETE
D—a 12-HETE
12
FIGURE 5. Upper panel: Effects of 5-,
15-, and 12-hydroxyeicosatetraenoic acids
(HETEs) (4 \ig) on capillary filtration
coefficient (Kf) in isolated guinea pig lungs
perfused with phosphate-buffered Ringer's
solution containing 0.5 g% albumin (n = 4
per group). Values are mean ± SEM.
'Different fp < 0.05) from baseline. Lower
panel: Effects of 5-, 15-, and 12-HETE (4
\ig) on Kf in isolated guinea pig lungs
perfused with guinea pig blood diluted 3:1
(volfvol) with phosphate-buffered Ringer's
solution (n = 4 per group). Protein concentration of theperfusate is 1.5g%. Values are
mean ±SEM. *Different (p<0.05) from
baseline.
10
l 6
o
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BLOOD
10
20
30
50
70
ThM(mta)
cle were less than the 15-HETE challenged group
(p<0.05).
In lungs perfused with diluted blood (final protein
concentration 1.5 g%), the changes in hemodynamics
and fluid filtration induced by injection of 4 \ig
15-HETE (Figures 6, 7, and 8) were attenuated
compared with the responses in lungs perfused with
PBR (0.5% albumin). Following injection of 15HETE, P,,, increased from a baseline value of 9.4 ± 0.5
to 11.1 ±0.4 cm H2O within 3 minutes and remained
elevated (p < 0.05) (2-3 cm H2O greater than baseline)
for the duration of the study. P^ also increased from
a baseline of 6.0 ± 0.2 to a maximum value of 8.7 ± 0.6
cm H2O at the end of the study (p<0.05). There was
no change in R, following the 15-HETE injection,
while there was an immediate increase (p<0.05) in R,
(Figure 7). In blood-perfused lungs, injection of
15-HETE caused a small increase in lung wet weight
of 0.43 ±0.21 g by the end of the 70-minute experimental period (Figure 8), but there was no change in
Kf from baseline (Figure 5). The vehicle alone did not
produce any significant change in the measured
parameters.
The pulmonary hemodynamic and fluid filtration
responses to injection of 4 (xg 15-HETE into lungs
perfused with PBR containing 1.5 g% albumin (i.e.,
protein concentration the same as the concentration
of the diluted blood perfusate and three times the
TABLE 1. Response to Injection of 4 /il 15-HETE Vehicle (Hexane/ETOH; 3 = 1) Into Rlnger's-Albumin (0.5%)-Perfused
Isolated Lungs
Time postinjection (minutes)
70
50
1
30
3
10
20
Baseline
5
8.9±0.8*
8.5±0.8*
7.7 + 0.5
7.7±0.4
7.8 + 0.5*
7.4±0.5
7.3+0.7
7.7 + 0.6
5.8±0.4*
6.2±0.5*
—
5.2±0.3
5.3±0.3
5.2±0.3
5.0±0.3
5.2±0.4
4.6
+
0.3
c^
0.10±0.02
—
0.08 + 0.02 0.09±0.01 0.08 ±0.02 0.09±0.02 0.09 ±0.02 0.10±0.02
0.08 ±0.02
R,
0.14±0.02*
—
0.09 + 0.02
0.10±0.02 0.10 + 0.02 0.10±0.02 0.10±0.01 0.10±0.01 0.12±0.02
Rv
0
0
0.02 ±0.02 0.07 ±0.04 0.09 + 0.05 0.19 ±0.09*
0
0
0
AWt
5.05 ±0.42
4.73±0.94 3.78±0.77 4.41 ±0.55 4.82±0.37
3.74±0.33
—
—
—
Kf
Pp,, pulmonary arterial pressure (cm H2O); P^p, pulmonary capillary pressure (cm H2O); R,, pulmonary arterial resistance (cm H20/(ml/
min)); RY, pulmonary venous resistance (cm H2O/(iru7min)); AWt, change in lung wet weight from baseline (g); Kf, capillary filtration
coefficient (ml/(min-cm H2O-g lung dry wt)x 10~^); HETE, monohydroxyeicosatetraenoic acid. Values are mean± SEM; n = 3.
•Different (p<0.05) from baseline.
Pp.
P
6.9±0.4
692
Circulation Research
Vol 62, No 4, April 1988
IS
10
FIGURE 6. Effects of 5-, 15-, and 12-hydroxyeicosatetraenoic acids (HETEs) (4 \tg) on pulmonary arterial and
capillary pressure in isolated guinea pig lungs perfused
with diluted blood fn = 5 per group). Protein concentration of perfusate is 1.5 g%. Values are mean±SEM.
'Different (p < 0.05) from baseline.
•-•5HETE
o-oiSHETE
D-D1I-HETE
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io
Cantor
S 10
50
20
70
TWE(o
PBR-0.5% albumin discussed above) are summarized in Table 2. The relative magnitude of changes
in each hemodynamic parameter was similar to that
seen in blood-perfused lungs. The change in lung
wet weight from baseline (0.35 ±0.049 g) at the
end of the experiment was also similar to those
observed in blood-perfused lungs given 15-HETE
(0.43 ±0.219 g), and there also was no change
inK f .
In those lungs in which the blood perfusate contained
varying concentrations of protein (the perfusate protein
concentration ranged from 0.5 to 0.8 g/100 ml), the
0.M r
0.20
0.15
ArtoM
0.10
0.05
•-•S-HETE
O-015-HETE
0
0.25
D-O12-HETE
0.20
0.15
Vtnon
0.10
OilS
5 10
20
30
TME(Bta)
50
70
FIGURE 7. Changes in pulmonary arterial and pulmonary venous resistances observed following injection of
5-, 15-, and 12-hydroxyeicosatetraenoic acids (HETEs)
(4 \Lg) in isolated guinea pig lungs perfused with diluted
blood (n= 5 per group). Protein concentration of the
perfusate is 1.5 g%. Values are mean ± SEM. *Different
(p < 0.05) from baseline.
Burhop et al
Pulmonary Inflammatory Effects of Mono-HETEs
693
3.0 p
2.5 5-HETE
o—o 15-HETE
a-a 12-HETE
2.0
Chflojt In
LtmgWtt
WttgMFrem 1.0
i (I)
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injection of 4 ^g 15-HETE produced increases in
pressures and resistances similar to those in lungs
perfused with diluted blood containing 1.5 g% protein
(Table 3). However, the increases in lung wet weight
and Kf produced by 15-HETE challenge were greater
at the lower protein concentration (Table 3). At a
protein concentration of 0.8 g/100 ml, the increases in
weight and Kf were abolished to the same degree as in
the lungs perfused with diluted blood (1.5 g% protein
concentration), while the responses were evident at a
protein concentration of 0.5 g% (Table 3).
Injection of 2 ng 15-HETE (« = 4) did not produce
significant changes in the measured parameters with
any of the different perfusates used (i.e., PBR or
diluted blood).
5-HETE
Injection of 4 ^g 5-HETE into lungs perfused with
PBR containing 0.5% albumin caused small but
significant increases in P^ and P^ by the end of the
70-minute experimental period (Figure 2). P^ increased
from a baseline of 7.1 ±0.3 to 9.0±0.6 cm H2O
(mean ± SEM) at 70 minutes postinjection, while P,,,,
increased from a baseline of 5.2±0.3 to 7.1 ±0.4 cm
H2O at 70 minutes postinjection. 5-HETE did not cause
any change in R, but did increase Rv from a baseline
of 0.11 ±0.01 to 0.18 ±0.02 cm H20/(ml/min) by the
end of the study (Figure 3). As seen in Figure 4, the
lung wet weight increased within 50 minutes following
FIGURE 8. Change in lung wet weight from baseline in
isolated guinea pig lungs perfused with diluted blood
(n = 5 per group) following injection of 5-, 15-, and
12-hydroxyeicosatetraenoic acids (HETEs) (4 \xg). Protein concentration of perfusate is 1.5 g%. Values are
mean + SEM. *Different fp < 0.05) from baseline.
injection of 5-HETE and reached a maximum value of
1.30 ± 0.43 g over baseline (baseline lung wet weight,
3.0 ±0.1 g) by 70 minutes postinjection. The lung
wet/dry weight ratio (mean ± SEM) was 9.6 ± 0.5. As
seen in Figure 5, Kf increased (p<0.05) within 10
minutes postinjection of 5-HETE and reached its
maximum value (3.5 times baseline) at 70 minutes
postinjection. Figure 9 shows a tracing from a representative experiment illustrating a K, determination for
control (baseline) and a Kf measurement made at 50
minutes postchallenge with 4 p,g 5-HETE.
Injection of an equal volume of the vehicle in which
the 5-HETE was dissolved did not produce significant changes in any measured parameter: Pp, was
7.0 ±0.7 cm H2O at baseline, and the value at 70
minutes after the vehicle was 7.9 ±0.3 cm H2O; the
lung weight increased only 0.10 ± 0.07 g; and baseline
Pop w a s 4.7 ±0.4 cm H2O compared with the 70
minutes postvehicle value of 5.7±0.8 cm H2O.
In lungs perfused with diluted blood (protein concentration 1.5 g%), 5-HETE produced small but
significant (p<0.05) increases in P,,, from a baseline
of 10.6 ±0.3 to a 70-minute value of 12.8 ±0.5 cm
H2O and in P^ from a baseline value of 6.9 ± 0.3 to a
70-minute value of 8.7 ± 0.5 cm H2O (Figure 6). There
was no change in R, following injection of 5-HETE,
but there was a small (p<0.05) increase in Rv (Figure
7) within 50 minutes postinjection of 5-HETE. As seen
in Figure 8, lungs perfused with diluted blood remained
TABLE 2. Effects of 15-HETE (4 ^g) In Lungs Perfused With Ringer's-Albumin (1.5 g%) Solution
Time postinjection (minutes)
Baseline
1
10
20
30
5
70
50
6.5±0.7
7.0 + 0.7
7.1+0.7
7.1+0.6
7.0 + 0.6
7.2 + 0.7
7.1+0.5
8.2 + 0.5*
7.6 + 0.5
Pp.
—
4.4±0.3
4.6 + 0.2
4.9 + 0.3
4.6 + 0.2
5.7 + 0.4*
4.8 + 0.3
4.9 + 0.3
5.1+0.2
p«p
—
0.07±0.01
0.08
+
0.02
0.08
+
0.01
0.09
+ 0.02
0.08
+
0.01
0.09
+
0.01
0.09
+
0.02
0.09
+
0.01
K
—
0.08 ±0.01
Rv
0.09 + 0.01 0.10 + 0.02 0.10 + 0.01 0.09 + 0.01 0.09 + 0.01
0.11+0.01* 0.13 + 0.01*
0
AWt
0
0.10 + 0.02 0.19 + 0.04* 0.25 + 0.03* 0.35+0.04*
0
0
0
4.39 + 0.66
5.14+0.65 4.20 + 0.89 3.00 + 0.23
—
5.55 + 0.90
4.48+0.52
—
—
K,
Pp., pulmonary arterial pressure (cm H2O); P,^, pulmonary capillary pressure (cm H2O); R,, pulmonary arterial resistance (cm H2O/(rru7
min)); Rv, pulmonary venous resistance (cm H2O/(rru7min)); AWt, change in lung wet weight from baseline (g); Kf, capillary filtration
coefficient (ml/(min-cm H2O-g lung dry wt)x 10"*); HETE, monohydroxyeicosatetraenoic acid. Values are mean + SEM; n = 3.
•Different (p<0.05) from baseline.
3
694
Circulation Research
Vol 62, No 4, April 1988
TABLE 3. Effects of 15-HETE (4 ^g) in Lungs Perfused With Diluted Blood With Varying Concentrations
of Proteins
Experiment
Baseline
Protein
70 minutes
postinjection
Baseline
70 minutes
postinjection
Baseline
70 minutes
postinjection
15.4
10.8
0.6
10.3
6.7
18.0
13.7
0.8
11.3
6.7
13.7
10.1
0.5
12.0
6.9
0.18
0.15
0
2.47
R.
Rv
AWt
0.15
0.38
1.22
11.96
0.12
0.13
0
2.86
0.16
0.28
1.84
9.08
0.13
0.25
0.19
3.56
0.16
0.12
0
1.92
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
Kf
Whole guinea pig blood was centrifuged, varying amounts of plasma were drawn off, and cells were diluted to a
prescribed volume with a phosphate-buffered Ringer's solution containing 0.5% bovine serum albumin.
Protein, protein concentration of isolated lung perfusate (g/100 ml); Pp., pulmonary arterial pressure (cm H2O); P^p,
pulmonary capillary pressure (cm H2O); R,, pulmonary arterial resistance (cm H20/(ml/min)); RV, pulmonary venous
resistance (cm H2O/(ml/min)); AWt, change in lung wet weight from baseline (g); Kf, capillary filtration coefficient
(ml/(min-cm H 2 Og lung dry wt)x 10~2); HETE, monohydroxyeicosatetraenoic acid.
All values represent individual data points collected either during baseline or at 70 minutes postinjection of a
bolus of 15-HETE in each individual experiment.
isogravimetric for the entire 70-minute experimental
period following injection of 4 p,g 5-HETE, and there
also was no change in Kf (Figure 8).
The pulmonary hemodynamic and fluid filtration
responses to injection of 4 jxg 5-HETE into lungs
perfused with PBR containing 1.5% albumin are
summarized in Table 4. The relative magnitude of
change over time in each hemodynamic parameter was
®
Awt during Ki
mMiurmtnt
10
lOr
•H—Pwp
* * * •
cmHjO
P.
cmHiO
•JV
10
Peap"
Pp.
cm HjO
h
cmHjO
0
o1-
u
MJnuin
HlnutH
FIGURE 9. Typical responses of an isolated guinea pig lung (perfused with phosphate-buffered Ringer's solution containing 0.5 g%
albumin) at baseline (Panel A) and at 50 minutes postinjection of 4 iig 5-hydroxyeicosatetraenoic acid (HETE) (Panel B). Data for
change in weight (Awt, g) produced during capillary filtration coefficient (Kf) determination, mean pulmonary arterial pressure (P^,
cm H2O), mean pulmonary venous pressure (P,, cm H2O), and capillary pressure (P^, cm H2O) as estimated by double-occlusion
technique are shown. Paper speed of recorder was transiently increased during P^ measurements to aid in analysis. Evident is increase
in Kf (increased slope of weight gain) and, thus, change in vascular permeability to water produced by 5-HETE.
Burhop et al
Pulmonary Inflammatory Effects of Mono-HETEs
695
TABLE 4. Effects of 5-HETE (4 /xg) in Lungs Perfused With Rlnger's-Albumln (1.5 g%) Solution
Time postinjection (minutes)
50
1
20
70
10
Baseline
3
5
30
7.6±0.7
7.6±0.6
8.2±0 .6
8.0±0.5
7.8 + 0.6
7.6±0.7
7.1 ±0.9
7.8±0.1
7.8±0.8
Pp.
P
—
5.2±0.2
6.0±0 .7*
4.8±0.1
5.2±0.3
5.2 + 0.1
5.1 ±0.1
6.2±0.8*
5.2 + 0.1
^
—
0.08 ±0.03
0.09±0.03 0.09±0.03 0.08 + 0.02 0.09±0.03 0.09±0.01 0.08 ±0 .01
0.06±0.01
K
—
0.11 ±0.03*
0.06±0.01
0.08±0.01 0.08 ±0.01 0.08 ±0.01 0.06±0.01 0.08 ±0.01 0.10±0 .02*
R,
AWt
0
0
0.12±0.08
0
0
0
0
0
0
3.57
+
0.56
3.16±0.87
3.34±0.32
2.65±0.15
4.07
±0
3.34±0.10
.68
—
—
—
P_, pulmonary arterial pressure (cm H2O); P,^, pulmonary capillary pressure (cm H2O); R,, pulmonary arterial resistance (cm H2O/(rruV
min)); Rv, pulmonary venous resistance (cm H2O/(ml/min)); AWt, change in lung wet weight from baseline (g); Kf, capillary filtration
coefficient (ml/(min-cm H2O-g lung dry wt)x 10"*); HETC, monohydroxyeicosatetraenoic acid. Values are mean + SEM; n = 3.
•Different (p<0.05) from baseline.
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very similar to that seen in the blood-perfused lungs
containing 1.5 g% protein. There was also no significant change from baseline in either Kf or lung wet
weight at the end of the experiment.
In those lungs in which the perfusate contained
diluted blood with concentrations of protein varying
from 0.5 to 0.7 g%, the injection of 4 ng 5-HETE
produced increases in pulmonary hemodynamics that
were similar to those in lungs perfused with blood
containing 1.5 g% protein (Table 5). As seen in Table
5, in the range of concentrations of protein examined,
the increases in lung wet weight produced by injection
of 5-HETE were not affected by the concentration of
protein present.
Injection of 2 \x.g 5-HETE (n = 3) in either bloodperfused or PBR-albumin-perfused lungs did not cause
any significant change in any measured parameter.
12-HETE
As seen in Figures 2, 3, and 4, the injection of 4 \ig
12-HETE into lungs perfused with PBR-albumin did
not cause any significant change in any measured
parameter. 12-HETE also did not alter the lung wet/dry
weight ratio and Kf (Figures 2, 3, 4, and 5). Injection
of 12-HETE (4 u,g) into lungs perfused with diluted
blood also did not produce any change in any measured
parameter (Figures 5, 6, 7, and 8).
Discussion
In isolated guinea pig lungs perfused with PBR
containing 0.5% albumin, injections of either 5-HETE
or 15-HETE (4 jig) produced pulmonary vasoconstriction and edema. The edema may be the result of the rise
in the vascular permeability in these lungs as evidenced
by the increase in Kf, a measure of the lung vascular
permeability to water. These changes were dosedependent because a 2-^,g injection of 5- or 15-HETE
did not have an effect. The increased pulmonary
vascular permeability and lung water content observed
with 5- and 15-HETE is in contrast to the effects of other
lipoxygenase metabolites such as leukotrienes C4 and
D4 (LTQ and LTD4), which do not result in an increase
in vascular permeability and edema.34 Also, in contrast
to 5- and 15-HETE, 12-HETE did not produce changes
in pulmonary hemodynamics or lung fluid filtration.
Based on magnitude of increases in R v , the monoHETEs may be ranked in order of potency as follows:
15-HETE > 5-HETE > 12-HETE.
TABLE 5. Effects of 5-HETE (4 Mg) in Lungs Perfused With Diluted Blood With Varying Concentrations
of Protein
Experiment
3
1
:2
70 minutes
70 minutes
70 minutes
Baseline
Baseline
postinjection
postinjection
Baseline
postinjection
Protein
0.7
0.5
0.6
—
—
—
12.2
10.0
14.7
12.5
8.3
11.0
PP.
8.2
6.6
10.3
9.8
5.2
8.1
P-P
0.12
0.16
0.14
0.11
0.10
0.10
R.
0.14
0.26
0.18
0.24
0.08
0.20
Rv
0.41
AWt
0
0
0
0.22
0.76
3.46
3.00
3.62
4.28
4.13
2.27
Kf
Whole guinea pig blood was centrifuged, varying amounts of plasma were drawn off, and cells were diluted to a
prescribed volume with a phosphate-buffered Ringer's solution containing 0.5% bovine serum albumin.
Protein, protein concentration of isolated lung perfusate (g/100 ml); Pp., pulmonary artery pressure (cm H2O); P,^,
pulmonary capillary pressure (cm H2O); R,, pulmonary arterial resistance (cm H20/(ml/min)); Rv, pulmonary venous
resistance (cm H2O/(ml/min)); AWt, change in lung wet weight from baseline (g); Kf, capillary filtration coefficient
(ml/(min-cm H ^ - g lung dry wt)x 10"2); HETE, monohydroxyeicosatetraenoic acid.
All values represent individual data points collected either during baseline or at 70 minutes postinjection of a 4-jtg
bolus of 5-HETE in each individual experiment.
696
Circulation Research
Vol 62, No 4, April 1988
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The greater increases in P,. and P ^ with 15-HETE
than with 5-HETE corresponded to greater increases in
both R, and Rv. The constrictor effects of 5-HETE were
confined to the pulmonary venous segment of the
vasculature. The increases in vascular resistance observed with 5- and 15-HETE were independent of
changes in the circulating concentrations of either
thromboxane or prostacyclin over time (unpublished
observation), which is in contrast to pulmonary vasoconstriction induced by LTQ and LTD4, whose pulmonary vasoactive effects are mediated in some species
by the release of thromboxane A2.35
15-HETE produced a greater increase in lung wet
weight than 5-HETE. The greater rise in P ^ induced
by 15-HETE may partially explain the greater transvascular fluid filtration (as assessed by lung wet weight
gain over time); however, both 15- and 5-HETE caused
significant increases in K f . The greater increase in lung
wet weight over time produced by 15-HETE may be the
result of both increased pulmonary vessel wall permeability to water and capillary hydrostatic pressure,
while 5-HETE appears to cause pulmonary edema
primarily through an increase in vascular permeability.
Both compounds produced significant increases in
wet/dry lung weight compared with lungs challenged
with either 12-HETE or vehicle controls.
The mechanism by which 5- and 15-HETE increase
pulmonary vascular permeability is not apparent from
these studies. It is known that 12-HETE (the major
platelet lipoxygenase metabolite), 5-HETE (the major
neutrophil lipoxygenase monohydroxy fatty acid metabolite), and 15-HETE (a hydroxylated fatty acid
product produced primarily by neutrophils) are rapidly
incorporated into the phospholipids and trigrycerides
of human neutrophils,14-3* mouse peritoneal macrophages,17 mouse macrophage-like tumor cells,18 cultured bovine aortic endothelial and smooth muscle
cells,37 and human umbilical vein endothelial cells.38
The time required for maximal uptake of mono-HETEs
by cells is between 15 and 30 minutes following
exposure.17'36'37 The increases in pulmonary vascular
pressures and lung wet weight produced by either 5- or
15-HETE in the present study also reached significant
levels between 15 and 30 minutes following injection.
The incorporation and substitution of these monohydroxy fatty acid products of leukocytes and platelets
could alter the functional properties and characteristics
of cell membranes,1417-37 such as membrane fluidity,25
as well as induce protein kinase C activation,39 which
may explain the increase in vascular permeability to
water. Another result of incorporation of these monoHETEs into cellular membranes may be their removal
from the circulation17-36"38 and their metabolism to other
polar products.17-23
The results of the experiments using blood as the
lung perfusate demonstrated that blood-formed elements do not amplify the pulmonary vasoactive and
microvascular fluid filtration responses to injection of
5-, 15-, and 12-HETE. When the lungs were perfused
with blood containing 1.5 g% protein rather than with
the Ringer's solution containing 0.5 g% protein, the
pulmonary hemodynamic changes produced by the
mono-HETEs were markedly attenuated and the increases in lung wet weight and Kf were abolished.
These experiments suggest that the reduction of the
pulmonary vascular response to the mono-HETEs in
blood-perfused lungs may be related to protein binding
of the HETEs.
Two separate experiments were performed to examine if the pulmonary vascular response to the HETEs
was reduced by the perfusate protein concentration
(i.e., albumin binding) or metabolism of HETEs by
blood-formed elements. The first experiment used PBR
containing 1.5 g% (versus 0.5 g%) albumin. This
protein concentration approximated the mean protein
concentration of the blood perfusate. Injection of the
same dosage of each HETE (4 \ig) into lungs perfused
with this higher concentration of albumin produced
responses similar to those observed in blood-perfused
lungs. Pulmonary vascular resistance as well as the lung
wet weight and K, values were attenuated to the same
degree as with the blood perfusate. Administration of
12 jxg 5- and 15-HETE at the end of two experiments
(a threefold increase in dosage from 4 to 12 u.g
correlating with the similar threefold increase in
albumin concentration from 0.5 to 1.5 g%) produced
a response similar to that seen in lungs perfused with
the lower 0.5 g% albumin concentration (i.e., lung wet
weight and vascular pressures increased) (unpublished
observations). The second experiment involved reducing the protein concentration of the blood perfusate. We
accomplished this by centrifuging the whole blood,
removing the plasma, and adding varying concentrations of albumin plus PBR to the blood cells. The
increases in lung wet weight and Kf produced by
15-HETE were attenuated as the protein concentration
increased (threshold concentration was approximately
0.7 g%). At a protein concentration of 0.5 g% in the
blood perfusate, the increases in lung weight and Kf
with 15-HETE were similar to those seen in the lungs
perfused with PBR containing 0.5% albumin. On the
other hand, injection of 4 (xg 5-HETE into lungs
perfused with blood (protein concentrations ranging
from 0.5 to 0.7 g%) resulted in responses similar to
lungs perfused with blood having a higher protein
concentration. In both blood-perfused experiments, the
increases in lung wet weight were less than those seen
in lungs perfused with the cell-free PBR solution
containing 0.5% albumin, and there was no significant
change in Kf from baseline. Therefore, the magnitude
of the pulmonary vascular permeability response to
15-HETE is 1 imited primarily by the amount of protein
present in the perfusate, and the response to 5-HETE
is limited by both the perfusate protein concentration
(i.e., albumin binding) and by the removal of 5-HETE
by blood-formed elements.
12-HETE did not have any effects on hemodynamics
or fluid balance in lungs perfused with either PBR
containing albumin or blood. This lower level of
potency of 12-HETE compared with other monoHETEs has also been reported for other properties of
12-HETE such as 12-HETE's chemotactic ability for
Burhop et al
Downloaded from http://circres.ahajournals.org/ by guest on June 14, 2017
neutrophils'5-40 and eosinophils.21 In contrast to both 5and 15-HETE, which are both hydroxylated fatty acids
produced primarily by porymorphonuclear leukocytes
(PMNs), 12-HETE is produced primarily by platelets
and is the major lipoxygenase metabolite of platelets.
This finding is consistent with observations that lung
vascular injury and subsequent edema formation in a
variety of models involves PMN-endothelial interactions, and not platelets.35
In conclusion, the present study indicates that both
5- and 15-HETE are pulmonary vasoconstrictors (15HETE > 5 -HETE) and that both mono-HETEs increase
lung vascular permeability. The pulmonary vascular
effects of 5- and 15-HETE are not amplified by the
presence of blood-formed elements, but rather, the
effects are attenuated in blood-perfused lungs. The
attenuation following 15-HETE appears to be the result
of albumin binding, while the attenuation of the
response to 5-HETE challenge in blood may be due to
both albumin binding and metabolism of 5-HETE by
blood-formed elements. The increased pulmonary
vascular permeability and lung water content observed
with 5- and 15-HETE are marked, and these changes
contrast with other lipoxygenase metabolites such as
LTC4 and LTD4, which do not increase vascular
permeability and lung water content/ 1 12-HETE, in
contrast to 5- and 15-HETEs, does not directly increase
pulmonary vascular resistance or lung fluid filtration.
The mono-HETEs are generated in sufficient quantities
to be detected in vivo in animal models of acute lung
injury22"25 and attain maximum detectable concentrations coincident24'25 with increased vascular permeability. These lipoxygenation products are locally generated by inflammatory cells such as neutrophils, and
their microvascular effects may be modulated by the
local concentrations of albumin and blood-formed
elements in the microvessel. The present study indicates that both 5-HETE and 15-HETE are pulmonary
inflammatory mediators and may be involved in the
neutrophil-dependent pulmonary vascular injury and
edema.
Acknowledgments
The authors thank Dr. Herbert Johnson at The
Upjohn Company for providing the 5- and 15-HETEs
used in these experiments and Dr. William Jubiz at the
Albany Veterans Administration Hospital for HPLC
analysis of the HETEs.
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KEY WORDS • monohydroxyeicosatetraenoic acids • isolated
guinea pig lungs • lipoxygenase metabolites • pulmonary
vasoconstriction • pulmonary hemodynamics • capillary
filtration coefficient • albumin-Ringer's perfusate • blood
perfusate • hematocrit
Monohydroxyeicosatetraenoic acids (5-HETE and 15-HETE) induce pulmonary
vasoconstriction and edema.
K E Burhop, W M Selig and A B Malik
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Circ Res. 1988;62:687-698
doi: 10.1161/01.RES.62.4.687
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