rat lungs demonstrates that nitric oxide gas (#{149}. NO, 70 nM) added to the perfusate containing a small amount of hemolysate. [175 /Ll of lysed red blood cells ...
I
Nitric oxide-related red cell lysate N. F. V0ELKEL,
vasoconstriction J. Y.
K. L0BEL,
WESTCOTT,
Pulmonary Hypertension Center and tDivision University of Colorado Health Sciences Center,
salt solution
(EBSS)J
triggered
profound
and
sustained vasoconstriction. Vasoconstriction was not observed when #{149} NO was added to lungs perfused with washed intact rat or human RBC or with oxyhemoglobin (Hgb 20 /LM). The presence of hemolysate in the perfusate also caused vasoconstriction in response to n-acetylcysteine (50 ELM), glutathione (10-4 M), or ascorbic acid (104 M) and potentiated greatly the vasoconstrictor response to 5 mM KCI. Not only . NO, but also nitroprusside (SNP) or L-arginine and paradoxically three #{149} NO synthesis inhibitors, including N-monomethyl Larginine, L-NAME, and nitroblue tetrazolium, which
have different mechanisms of action, each caused in the presence of hemolysate large vasoconstrictive responses. Hemolysate itself enhanced 02 consumption by slices of lung; no effects of this dose of #{149} NO on lung slice respiration were seen in the absence of hemolysate. Both Hgb and hemolysate lowered perfusate cGMP levels to the same degree suggesting that the vasoconstrictive response
was not due to unique
effects of hemolysate
on guanylyl (SOD) and
cyclase. Addition of superoxide dismutase catalase (CAT) to the hemolysate containing perfusate, or addition of a cyclooxygenase or 5-lipoxygenase inhibitor, virtually abolished the #{149} NO induced vasoconstriction. The latter data are consistent with the concept that exposure of the vasculature to hemolysate may result in the
However,
formation
of peroxynitrite.
not abolish L-arginine
the pulmonary vasoconstriction induced by or by NAC. Our data indicate that hemolysate
SOD and CAT did
has profound
effects on lung vessel tone regulation
and on
lung
mitochondrial
precise
tissue
molecular hemolysate Lobel, K.,
mechanisms likely Westcott, are
function,
responsible
yet
for
the
the
action
of
to be very complex.-Voelkel, N. F., J. Y., Burke, T. J. Nitric oxide-related
vasoconstriction in lungs perfused FASEBJ. 9, 379-386 (1995) Key Words: pulmonary vosoconstriction electron transport #{149}KCI
with
red cell lysate.
heinosylate
.
.50. © FASEB
AND T.
with
J. BIJRKE Department
of Medicine,
implicated in the endothelium-dependent control of vascular tone (6-8). . NO or a . NO-derived metabolite may account for all or part of the activity of EDRF (9), and has been implicated as a factor that alters pulmonary vessel tone during septic shock (3). Inhalation of . NO gas has been used to reduce pulmonary hypertension in adults (10) and in neonates (11). Recently, both neuroprotective and neurodestructive effects of . NO (4, 12), depending on its redox form (13), and inhibition of mitochondrial electron transport (14-16) have been reported. N-acetylcysteine (NAC), which is known to potentiate the inhibition of platelet aggregation by nitroglycerin (17)-presumably a NO-dependent effect-turns sodium nitroprusside into a neurotoxin (4) and, as we report here, causes large vasoconstriction in lungs perfused with hemolysale-containing solution. Our findings may have relevance for acute lung injury syndromes associated with hemolytic episodes such as the acute chest syndrome in sickle cell anemia (18, 19).
MATERIALS Isolated
perfused
AND
METHODS
lung preparation
rats (300 g body weight) raised on a regular diet were used for the experiments. The rats were anesthetized with an injection of sodium penlobarbital (80 mg/kg i.p.) and the lungs and heart sere isolated as described previously (20). The lungs were perfused with Earle’s balanced salt solution (EBSS) at a constant rate of 0.03 ml. g’ . min’. After a 30 mm equilibration period, pulmonary pressor responses were elicited with a bolus injection of angiotensin II (All) (Sigma, St. Louis, Mo.) (1 sg bolus) or exposure to alveolar hypoxia, which was introduced by switching from one gas bag that contained 21% oxygen to one that contained 0% oxygen (19). After testing the pressor responses to All and hypoxia, either N-acetylcysteine (NAC) (50 sM) or KCI (5 ms), trace amounts of #{149} NO (see below), agents that alter the production of nitric oxide, or inhibitors of energy metabolism including CN (10-9 NI), antimycin (5 x 10’ M), or myxothiazol (108M) were added to the perfusate. Pulmonary pressor responses were monitored in lungs perfused with EBSS alone or with EBSS plus either intact rat or human erythrocytes, or with hemolysate (175 1sl/50 ml EBSS). As a control pure hemoglobin (Hgb) was used at an identical protein concentration. Finally, catalase (50 U/mI) and Ca2, Zn2, SOD (50 U/mI) (n - 5) or the cyclooxygenase inhibitor meclofenamate (10-5 M) (n = 5), or the Sprague-Dawley
5-lipoxygenase activating protein (provided by Dr. A. Ford-Hutchinson
(FLAP), Merck
inhibitor MK 886 (l0 M) Frosst Center for Therapeutic
mitochondrial
IT IS BECOMING INCREASINGLY APPARENT THAT endotheliumderived relaxing factor (EDRF)2 (or nitric oxide (. NO)) can be physiologically beneficial or injurious (1-3). The injurious effects of . NO have been determined during noxious insults to the central nervous system (CNS) and kidney (4, 5). In the present study we provide evidence that the pulmonary circulation is also compromised by . NO under the pathophysiologic conditions associated with hemolysis. . NO has been
0892-6638/95/0009-0379/$01
in lungs perfused
of Renal Diseases and Hypertension, Denver, Colorado 80262, USA
ABSTRACT The present study in isolated rat lungs demonstrates that nitric oxide gas (#{149} NO, 70 nM) added to the perfusate containing a small amount of hemolysate [175 /Ll of lysed red blood cells (RBC) per 50 ml of Earle’s
balanced
cUi1
RFSEMCH
..
-
‘To whom correspondence and reprint requests should be addressed, at: Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Health Sciences Ctr., 4200 E. Ninth Ave., Box C-272, Denver, CO 80262, USA. 2Abbreviations: . NO, nitric oxide; NAC, N-acetyl cysteine; EDRF, Endothelium-dependent relaxing factor; CN, cyanide; EBSS, Earle’s balanced salt solution; DNP, dinitrophenol; 4-AP, 4-aminopyridine; SOD, superoxide dismutase; CAT, catalase; 00N0, peroxynitrite; Hgb, hemoglobin; CNS, central nervous system; H, hypoxia; All, angiotensin II; TXB2, thromboxane B2;
NEM,
N-ethylmaleimide.
379
RESEARCH Research,
COMMUNICATION
Quebec,
Canada) (a 5) (21), were added to the hemolysateat the onset of lung perfusion, and the effect of these on the response to trace amounts of #{149} NO gas was assessed.
containing agents
perfusate
Preparation
of hemolysate
Hemolysate was generated from human volunteer blood obtained by routine venipuncture and by centrifuging at 1000 RPM for 10 mm, decanting the buffy coat and plasma, and washing the red blood cells twice with sterile 0.9% saline solution. The washed red cells were lysed in cold distilled water (175 tl of packed cells in 100 sl of water). The cells were centrifuged and the membrane free supernatant was used for the experiments. Preparation
of
NO for addition
#{149}
to perfusate
trace amounts of #{149} NO were added to with a concentration of 800 ppm (balance ml syringe and 100 tl of the gas was injected culating perfusate volume: 50 ml; #{149} NO lung was ventilated with #{149} NO (800 ppm) Only
Measurement Enzyme
of lung perfusate
immunoassays
measure 6-keto-PGF,, the #{149} NO gas-related
the perfusate. NO from a tank nitrogen) was aspirated into a 1 into the perfusate reservoir (circoncentration: (70 nM)) or the for I mm.
eicosanoids
established
in our laboratory TXB,, and LIC4 5 mm before pulmonary pressor response.
(22) were used and at the peak
to of
Rat lung slice respiration
Vasoconstriction of All is usually
due to acute hypoxia (H) or bolus injection modest (19) and this was confirmed in the
present study (Fig. 1 and Fig. 2). Perfusion of lungs with EBSS that contained human Hgb (20 tM) resulted in a small augmentation of hypoxiaand All-induced vasoconstrictions (Fig. 2). However, the vasoconstriction elicited by 5 mM KC1 was not affected by Hgb, and NAC did not alter vascular tone (Fig. 1 and Fig. 2). In contrast, if lungs were
perfused with hemolysate-containing NAC elicited a large pressor response,
EBSS, addition of and the KCI (5 mM
-induced vasoconstrictor response was 7- to 15-fold greater than in lungs perfused with EBSS alone (Fig. 1 and Fig. 2). Addition of intact RBC (rat or human) did not produce these effects.
Effect of #{149} NO, sodium nitroprusside (SNP), L-arginine, and inhibitors of NO synthesis in hemolysate-perfused lungs Addition of #{149} NO gas, SNP, L-arginine, or tetrahydrobiopterine (not shown) caused prompt and large pressor responses in hemolysate, but not in EBSS perfused lungs
lobe of the lung was removed while perfusion was maintained. This lobe was placed in cold (4#{176}C) EBSS. Slices (0.5 mm thick) were made with One
Clark ration
oxygen studies
electrode were used to monitor oxygen consumption. All respiwere carried out at room temperature. EBSS was saturated
with room air and placed in the oximeter. The recorder was set so that 100% full-scale equaled 20.9% oxygen. Appropriate corrections for Denver’s altitude and the barometric pressure were made each day. A slice was added to the oximeter and basal 0, consumption was monitored for 5-8 mm. The initial rate of oxygen consumption was relatively fast due to the effect of warming the tissue from 4#{176}C to room temperature; the rate after two mm, however, was slower and quite constant. This latter rate was used in all experiments. In all studies, unless otherwise noted, the experimental additions (i.e., dinitrophenol (DNP), antimycin, hemolysate and/or #{149} NO) were made to the intact, perfused lungs. These respiration studies, therefore, monitored the residual effects of the in situ treatments. After each experiment, which was conducted in triplicate (i.e., on three slices from the same lung), the wet weight of the lung slice was determined and 02 consumption was reported as nmol 0,/mg wet wt/h. The basal values compare favorably with those reported by others (23).
Measurement
of perfusate
‘.J’cLJ____ H
All
to modify
H
(wHg)
All
H
All
activity
H
for multiple comassess statistical to be statistically
RESULTS
vasoconstriction
by hemolysate
Isolated lungs perfused with cell-free physiological salt solution usually have blunted vasoconstrictor responses (19).
March
1995
hemolysate
175MMhemolysate
LZ)
All of variance test to determined
ml.,
NAC
1(0
l
analysis
9
1(0
175
Data were analyzed using a two-way analysis parisons and the Student-Newman-Keuls significance. Data at the P < 0.05 level were different.
Vol.
H
4
---
380
NAC
cGMP
the hemolysate
pulmonary
lop,
s-
to characterize the activitycontained in RBC lysates in contrast to Hgb, aliquots of the hemolysate were unfrozen and either boiled in a water bath, sonicated, or dialyzed (suspended in 11 of EBSS) using a dialysis tubing that retains material with a molecular weight greater than 6000-8000. This modified hemolysate was added to perfusate to examine the question of whether these modifications would allow a NO-triggered pulmonary pressor response.
Enhanced
NAC
#{149} 1’
j
To begin
Statistical
PhysiologicalSalt Solution human hemoglobin
+ 20 tM
175 p11 hemolysats
To assess whether the presence of hemolysate or of Hgb in the perfusate affected the production of cyclic GMP, perfusate samples were taken 60 mm after onset of lung perfusion. The samples were quickly frozen and assayed for cGMP using a commercially available RIA (Amersham, Arlington Heights, Ill.). The cyclic GMP levels of lungs perfused with EBSS were compared with those perfused with EBSS + Hgb or with EBSS + hemolysate.
Attempt
PhysiologicalSalt Solution (no blood productadded)
L_d”.
Stadie Riggs microtome and if necessary were trimmed to a final shape that was between 25 and 80 mg wet wt. A Yellow Springs oximeter and a a
H
1.
Figure
___. NAC
Pulmonary
All
artery
lated rat lungs perfused
Hemolysate dialyzed
L H pressure
at constant
KCI
MW
overnight cutoff 6-8,000
tracings obtained from isoflow. Alveolar hypoxia (H), or
injection of a bolus of angiotensin II (All, I tg) routinely cause vasoconstriction (Top panel). N-acetylcysteine (NAC, 50 tM) causes no pressor response in lungs perfused with physiological salt solution (EBSS) (Top panel) or with a solution that contains hemoglobin (Hgb, 20 tM) (second panel). In contrast, lungs perfused with a solution which contains a small amount of hemolysate demonstrate a large pressor response shortly after addition of NAC (third panel), KCI (5 mM) (4th panel) or 4-aminopyridine (4-AP, 106
M)
The FASEB Journal
(5th
panel)
to the
perfusate.
VOELKEL
RESEARCH
Pap
(mmHg)
5-lipoxygenase activating protein inhibitor MK 886 (105 M) inhibited the pressor response observed after #{149} NO (70 M) addition to the perfusate. The pressure response in the cyclooxygenase blocked lungs after . NO addition (a = 5) was 16 ± 8 mmHg and in the 5-lipoxygenase blocked lungs (a = 5) was 5 ± 4 mmHg. Because both cyclooxygenase and 5-lipoxygenase blockers dramatically inhibited the . NO related contraction, we measured lung perfusate 6-ketoPGF1a (a stable prostacyclin metabolite) thromboxane B2
a: t#{149}_U II ml
NAC t50..M)
-j.
-i-.
‘
-
.5
#{149}
#{149}
S
I HO. Hmolyst
o
IP’
L_
UI
Hmoly.t
..l
COMMUNICATION
i#{176}
(TXB2), a stable metabolite of thromboxane A2 and leukotriene C4. During the NO triggered pulmonary pressor response (Ls Pap 50.6 ± 6 mmHg) (n 5) there was no significant increase in the production of any of the three measured metabolites. (Table 1). #{149}
=
D..,
C.,. IIWml.
#{149} #{149}
H
#{149} H
: L
#{149} -o-=.=.=-..------.__i u . I
____ ,j
14.O.UIyI.t.
Figure 2. Dose-dependent Pap in mmHg) in isolated either
hemolysate,
Inhibitors of mitochondrial respiration pressor response in hemolysate-perfused
#{149}
#{149} : -..
#{149}
So
ISHM)
human
Hm,.IssM.
.
Antimycin hibitors
.
pulmonary artery pressor responses ( lungs perfused with EBSS containing hemoglobin
(Hgb)
or
=
washed,
of
[5 x 10 Ml and mitochondrial
H
A.
intact
myxothiazol complex
cause large lungs (108 M), both inIII, and cyanide
NOH
RBC. Responses to bolus injection of angiotensin (A), addition of N-acetylcysteine (NAC) (B), alveolar hypoxia (C), or addition of KCI to the perfusate reservoir (D). The total perfusate volume of the noncirculating system was 50 ml. As little as 17.5 1il of packed RBC lysate added to the perfusate at the start of the perfusion promoted large vasoconstriction in response to NAC (B) or in response to KCI (D). Perfusion of the lungs with a solution containing equimolar Hgb (20 sM) or intact RBC did not mented pressor response after either NAC (B) P < 0.05, comparison is made with lungs perfused
show an augor KCI (D). with EBSS (0
sl of hemolysate)
(Fig. caused nitric large agents
__
B.
3). NO added to the preparation via the airways vasodilation (Fig. 3). Various agents known to inhibit oxide synthesis by different mechanisms each caused and sustained pressor responses. Figure 4 lists the and summarizes the magnitude of these pressure
-
.
changes.
lop,
All
H
SNP
4mb,
C.
Effect of SOD plus CAT on vasoconstriction
NO-induced a.
Addition
of SOD (50 U/ml) and CAT (50 U/ml) to the hemolysate-containing perfusate at the start of lung perfusion practically abolished the pressor response observed after addition of NO gas to the perfusate: . NO (70 nM) caused a pressor response of 26 ± 7 mmHg without SOD plus CAT (n = 4) and 3.0 ± 0.5 mmHg (n = 4) when SOD plus CAT were present (P < 0.05). However, this inhibition did not extend to attempts to inhibit the NAC (50 tM) -treated lungs. NAC caused a pressor response of 38 ± 7 mmHg (a = 4) in lungs perfused without SOD and CAT and 33 ± 5 mmHg (a = 4) in lungs perfused with SOD and CAT. Like-
wise, SOD plus CAT did not inhibit
the pressor
response
-
s-
-
All
H
f
All
H
L-Arnine (NOS.substrate)causesvasnstflctlol,
due
to addition of L-arginine. In three experiments, the change in pressure (in the presence of CAT and SOD) due to Larginine (104 M) ranged from 15 to 20 mmHg.
b#{176}min Figure
3. a) Trace
amounts
[70 nM
(see Methods)]
of
.
NO (A) or
addition
Effect
of meclofenamate
or MK 886 on
NO-induced
#{149}
vasoconstriction Addition containing
of either meclofenamate (10-5 M) to the hemolysateperfusate at the start of perfusion or of the
#{149} NO-RELATED
VASOCONSTRUCTION
IN LUNGS
(B) of Na-nitroprusside (SNP; l0 M) or of L-arginine (Larg; l0 M) (C) to the hemolysate-containing perfusate cause large pressor responses (bottom). b) #{149} NO (70 nM) added to hemolysatecontaining perfusate causes vasoconstriction which is turned into
vasodilation ppm).
by ventilating
the lungs for 1 mm with
NO gas (800
#{149}
381
RESEARCH
COMMUNICATION
60
A
mIT
50
#{149} NAC 5xlO DZnProtFkwphlX5xlO-5 #{149} L-NAME tO #{149}NMMAl0 fl SNP i05 #{149} SNO 7x108 #{149} L-azinine IO
30 20
#{149}KCNII.H
10
B
0 Figure 4. Inhibitors of NOS, as well as L-arginine, SNP and . NO and the heme oxygenases inhibitor Zn2-protoporphyrine IX (at the doses indicated) cause pulmonary pressor responses (20-45 mmHg) in lungs perfused with hemolysate-containing (175 tl/5O ml) EBSS; each bar represents n = 4 to 6.
ATIMYC
All
C M) caused
large pressor responses in hemolysateperfused lungs. (Fig. 5). Antimycin and KCN will also induce vasoconstriction in lungs perfused with salt solution; however, much higher concentrations are needed to elicit these effects (usually 10 or 10 M concentrations) (24).
(1011_109
Lung
slice
and Figure
respiration
is affected
by hemolysate
(Table
1
6)
40
130 Q
02 consumption by the slices from control lungs (perfused with EBSS) averaged 52.5 ± 2.1 nmol/mg wet wt/h (Table 2). NO alone did not alter slice respiration nor did Hgb. Hemolysate-treated lungs demonstrated an enhanced 02 consumption similar to that observed after addition of the mitochondrial inhibitor DNP (1 mM). Under these latter conditions, addition of NO to the perfusate further increased 02 consumption rate. Inspection of the 02 consumption tracing revealed a nonuniform pattern reflecting periods of 02 consumption interspersed by episodes of little or no 02 consumption (Fig. 6). Finally, antimycin (109 M), which at this dose does not alter vascular tone in EBSS perfused lungs, reduced 02 consumption by slices to 35.6 ± 2.9 nmol/mg wt/h. In the presence of hemolysate, the 02 consumption was reduced even further to levels of approximately 20% of normal. Pyruvate/malate (10 mM) addition to the slices from hemolysate-treated lungs did not stimulate respiration, but the addition of succinate (10 mM) was able to transiently and promptly stimulate oxygen consumption. These data implicate complex I as a potential site of the
20
10
#{149}
0KCN 1O9M antimycin 5.1()-9M
#{149}
hemolysate
effects.
Hemolysate
and Hgb decrease
perfusate
cGMP
T
Myxothiazol
1O8M
Figure 5. Inhibitors of mitochondrial electron transport cause large pulmonary pressor responses in lungs perfused with Earle’s solution containing hemolysate (175 sl/50 ml). Pressure tracing after addition of KCN (A) or antimycin (B) to perfusate. Mean ± SEM of pressure
responses
after
addition of either KCN, antimycin, (n = 4, for each experimental group)
myxothiazol to perfusate shown in panel C.
or
is
Effects of sonication, dialysis, boiling, and N-ethylmaleimide on hemolysate activity Boiling or sonication of the hemolysate for 10 mm or incubation with N-ethylmaleimide (NEM, 10 M) before addition to the perfusate prevented the potentiation of the 4-AP or the NO-related
and
NAC-induced
pulmonary
vasoconstric-
Perfusate levels of cGMP were assayed to compare the effects of Hgb and hemolysate (Table 3). Perfusate samples taken at 1 h after
onset
of lung
perfusion
with
EBSS
exhibited
the
highest levels of cGMP. The presence of hemolysate or Hgb reduced perfusate cGMP levels to a similar degree.
TABLE
2.
Basal
respiration in lung slices from perfused rat lungs0 EBSS
TABLE
6-Keto-PGF, Before #{149} NO After #{149} NO
TXB,
Hemoglobin Antimycin (5 x 10
LTC4
25.4
± 6.9
2.6 ± .5
21.4
± 8.3
25.6
± 8.7
4.0 ± .8
24.0
± 9.1
DNP
(1
.NO
70
Vol. 9
ml perfusate,
March 1995
mean
± 5EM (n
=
The
80.8
(2)
47.3
(2)
0Values are nmol indicate the number
FASEB Journal
± 2.1 (5) ± 1.9 (3) ± 2.9 (3)
nM
respiration responses been cut from lungs
6).
M)
52.5 48.3 35.6
mM)
measurements were
0Value s are ng/50
382
Control
1. Lung perfusate eicosanoid levels0
EBBS
Os/mg of lungs obtained
72.3
+
hemolysate
± 7.0 (5) -
13.6 ± 1.3 (3) -
97.0
wet lung tissue/h; numbers studied. For each lung
± 5.1 (4) in parentheses three replicate
and
averaged. These data represent at 10-30 mm after the slices had had been exposed to the agents in situ.
that were still present that
VOELKEL
RESEARCH COMMUNICATION However, Hemolysate + #{149}NO
Hemolysate 100
90
%
Oxygen 80
70
from
a
panel).
rate of oxygen
hemolysate However, of bursts
minimal
oxygen EBSS
treated
consumption lung
hemolysate
suggestive represents
sec-a
-30
6. The
Figure
SOD
plus
of respiration consumption containing
is .
NO
and
results
interspersed (right
20.9%
in lung slices obtained
steady
continuous
in a ragged by
periods
short
panel).
02 (room
(left tracing
100%
of
oxygen
air) at 25#{176}C.
tion. Overnight dialysis of the hemolysate destroyed its activity in respect to potentiation of pulmonary vascular responses, including the ability vasoconstriction (Fig. 1).
to elicit
a
NO
triggered
#{149}
DISCUSSION In the presence of hemolysate (but not hemoglobin), addition of NAC or KC1 to the perfusate caused large acutely reversible pressor responses (Fig. 1 and Fig. 2), as did glutathione (104 M) or ascorbic acid (104 M) (not shown). Because reducing agents have been reported to either enhance the formation of . NO or increase its half-life (25), and because the data with the reducing agents suggested to us that hemolysate and #{149}NO may have interacted to enhance vasoconstrictive responses (26), we performed studies to directly test the effects of exogenous #{149} NO in hemolysateperfused lungs. The #{149} NO donor SNP, L-arginine, (the substrate for nitric oxide synthase), or trace amounts of nitric oxide gas (see Methods) all caused large and sustained pul-
did
not
abolish
the
responses
tion. Although Archer and Hampi (30) reported that Nmonomethyl-L-arginine may act as a partial agonist for #{149} NO synthesis, nitroblue tetrazolium has not been reported
to exhibit such duality of action. Because in our studies both nitric oxide itself and various inhibitors of NOS caused large vasoconstrictions in hemolysate-perfused lungs, we speculate that hemolysate provides conditions that facilitate . NO and peroxynitrite formation as well as an imbalance between #{149} NO formation and O? removal. Under normal physiologic conditions, small amounts of . NO are produced at all times. This basal production can best be appreciated when NOS is inhibited by NOS inhibitors that cause potentiation of pulmonary vascular pressor responses. Likely in the presence of these small amounts of #{149}NO there is little if any peroxynitrite formed, because the generation of O2 is matched by endogenous detoxification systems. However, under conditions of altered redox states (4, 13, 28), there is the likelihood that
formation of O2 is increased or the elimination of O2 is reduced, or that NO is generated from ONOO (28). Provision of exogenous NO would then generate more ONOO and the associated vasoconstrictive response may not be offset by the vasodilatory effects of NO. In addition, under our experimental conditions the affinity of mitochondna! heme proteins and Fe-S clusters for NO (19) may have increased. For example, L-arginine-dependent inhibition of #{149}
#{149}
.
#{149}
macrophage
the
TABLE
#{149}
#{149}
#{149}
#{149}
pressor
response
observed
after
NO
addi-
#{149}
tion to the perfu sate. Likewise, addition of a cyclooxygenase or 5-lipoxygenase blocking agent inhibited the NO triggered vasoconstriction. Both the prostaglandin H2 synthase(cyclooxygenase) and 5-lipoxygenase-catalyzed reactions generate O2, which combines with NO to form peroxynitrite. These data taken together suggest that the NOrelated vasoconstrictor response could possibly be due to peroxynitrite (27). Perhaps SOD removed 02 and the cyclooxygenase and 5-lipoxygenase inhibitors reduced formation of O2, and thereby the effects attributable to ONOO. #{149}
#{149}
.
#{149} NO-RELATED
CAT
hum (which noncompetitively inhibits the conversion of Nw OH-L-arginine) (29) and it also caused large vasoconstric-
monary vasoconstriction in lungs perfused with hemolysatecontaining EBSS, suggesting that circulating NO itself or a NO-derived metabolite (generated in close proximity to the lung vessels) was involved in the vasoconstrictive response (Fig. 3). In contrast, when 100 t/l of NO gas (800 ppm) was injected during hypoxic vasoconstriction into the trachea of hemolysate perfused lungs, no further vasoconstriction occurred. However, during the NOinduced vasoconstriction, if the lungs were ventilated with #{149} NO (800 ppm) for 1 mm, vasodilation occurred (Fig. 3). Addition of CAT and SOD to the hemolysate-containing perfusate at the onset of lung perfusion practically abolished pulmonary
and
by addition of 50 iM NAC or L-arginine to the hemolysate-perfused lungs. Because Moro and colleagues (28) suggested (based on their data with platelets) that thiols convert peroxynitrite to #{149}NO we can only speculate that the NACand L-arginine-induced vasoconstrictions were not inhibitable by SOD or cyclooxygenase blockade because of a stoichiometry that favored the formation or stability of ONOO. For the #{149} NO- or NAC-induced vasoconstriction to occur, hemolysate was critical because no pressor response was observed when the lungs were perfused with EBSS alone or with EBSS plus pure hemoglobin. Not only . NO and NO donors, but paradoxically also the NOS inhibitors Nmonomethyl-L-arginine or L-NAME, caused large pulmonary vasoconstriction (Fig. 4). We used nitroblue tetrazocaused
VASOCONSTRUCTIONIN LUNGS
mitochondrial
complex
I and complex
II activi-
ties (15), a NO dependent inhibition of vascular smooth muscle cell mitochondrial complex I and II activities (31), and an Esc/zerichia coli endotoxin-induced (via NO) inhibition of hepatocyte mitochondrial energy metabolism (16) .
have
been
reported.
Thus,
.
NO
effects
on mitochondrial
electron transport and associated ATP generation could affect free radical production and/or transmembrane electrical potential as well as alterations in cellular calcium regulation (32), which alone or in concert could effect the profound vasoconstrictions observed in our studies. 3. Cyclic GMP perfusate
EBSS
1480 ± 215
(n = 5) Hemoglobin
(n
=
=
504
±
182
±
2806
4)
Hemolysate
(n
levels0
7)
‘Cyclic GMP perfusate levelswere measured at 45 mm after onset of lung perfusion and are expressed as fmol/ml of perfusate. 5P < 0.01, compared with EBSS.
383
RESEARCH To
COMMUNICATION
examine
whether
the
pulmonary
vasoconstriction
elicited by addition of trace amounts of NO to the hemolysate-containing perfusate may have been related to alterations of mitochondrial electron transport, respiration of slices from treated lungs was measured. Trace amounts of #{149} NO added to EBSS did not alter lung respiration whereas the uncoupler DNP increased respiration markedly (Table 2). In addition, we reasoned that metabolic inhibitors like antimycin and myxothiazol (inhibitors of complex III) and cyanide might act synergistically with the hemolysate (which we propose may alter NO availability or NO kinetics at intracellular targets). The addition of very small concentrations of antimycin, myxothiazol, or KCN (an inhibitor of the final stage of electron transport to 02) to hemolysate perfused lungs caused rapid, large vasoconstriction (Fig. 5). Slice respiration from lungs perfused with EBSS was modestly decreased by this dose of antimycin (5 x 10 M), but not by hemoglobin (Table 2). Lung slice respiration was increased by hemolysate to a similar degree as after the addition of DNP (Table 2). These data suggest that hemolysate itself may uncouple lung cell mitochondnial respiration. Addition of trace amounts of NO to the hemolysate markedly increased the lung slice mitochondnial respiration (Table 2). Succinate (10 mM) -stimulated respiration was not affected by the presence of hemolysate whereas addition of pyruvate/malate (10 mM) to slices from hemolysate-perfused lungs failed to stimulate mitochondnial respiration (data not shown), indicating that hemolysate may have altered substrate oxidation at mitochondrial complex I. Because NO has been shown to inhibit complex I and complex II activities (15), and because trace amounts of NO added to hemolysate perfused lungs increased respiration further, the combination of hemolysate and NO may have had additive or synergistic effects on complex I that lead to uncoupling of mitochondrial respiration. Alteration of mitochondrial function would be expected to have consequences for the control of the electrical membrane potential, the open/closed state of ion channels, and Ca2 kinetics in target cells such as pulmonary endothelium and/or vascular smooth muscle cells. Following this line of reasoning, we tested the effects of several concentrations (106_103 M) of a K channel blocker, 4-aminopyridine (28), in lungs perfused with or without hemolysate. In lungs perfused with hemolysate, the dose-response (vasoconstriction) relationship was dramatically shifted such that quite small K channel blocker concentrations caused pulmonary vasoconstriction. [Vasoconstniction was consistently observed after addition of 4-aminopyridine (4-AP) to the perfusate in doses as low as 10-6 M (Fig. 1 and Fig. 7)1. For comparison the dose usually required for contraction of pulmonary arteries
50-
#{149}
#{149}
#{149}
.
M (33,
34).
Although it remains unclear how hemolysate induces tissue respiratory disturbances and evokes vasoconstriction, it is clear that sonication, boiling, or overnight dialysis of the lysate or previous incubation with NEM (35) prevents both hemolysate-related potentiation of the vasoconstniction and the NO- or NAC-induced vasoconstriction. Together these data imply a role for small molecular weight substances with critical SH groups in the increased vascular reactivity. The potentiation of the KC1 and 4-AP-induced vasoconstniction by hemolysate (Fig. 1 and Fig. 7) is consistent with the concept that the vascular cells of the lungs perfused with hemolysate are depolarized with greater ease by slightly increased concentrations of perfusate (extracellular) KCI, or that the hemolysate had altered the open time probability of membrane ion channels (36, 37). In Xenopus laevis (frog oocytes) superfused with hemolysate-containing solution and #{149}
384
Vol. 9
March
1995
-
S
30
(mmHg) 20
.
#{149}
is l0
.
40.
The FASEB
S S
10.
Scnicat.d RBC
Dm.mbranes
510’6
1OJ6M
101M
1O4M
0 )( NEM
103M
(4 amino-pyrldinel Figure 7. Addition of the K-channel blocker 4-aminopyridine (4-AP) to the hemolysate containing lung perfusate causes vasoconstriction. Individual data are shown. When a large dese (103 M) of 4-AP was added to EBSS (not containing hemolysate) the pressor response was very small (0). When hemolysate was sonicated 0 or incubated with N-ethylmaleimide (NEM) (X) before addition to the perfusate, the vasoconstriction obtained with the designated 4-AP concentration was absent or very small.
clamped at +20 mV, responses 4.5 iA indicative of membrane
with maximal depolarization
currents up to were observed
U.
Mihic and A. Harris, University of Colorado Health Sciences Center, Department of Pharmacology, personal communication). Membrane depolarization or alteration of ion channel function could be a direct response to hemolysate itself or a secondary result of the bioenergetic disturbances suggested from the lung slice respiration data. Finally, because . NO-induced vasoconstriction may also
reflect
reduced
signal
transduction
due
to inhibition
of
guanylyl cyclase (38), we measured perfusate cGMP concentrations in lungs perfused with either EBSS alone or with EBSS containing either hemoglobin or hemolysate. The perfusate cGMP levels in the hemoglobin or hemolysateperfused lungs were substantially lower than those in the EBSS perfused lungs (Table 3). However, there was no significant difference in cGMP levels between hemoglobin or hemolysate perfused lungs, and only hemolysate potentiated vasoconstriction. Zn2-protoporphyrine IX, an inhibitor of both heme oxygenases and guanylyl cyclase (39), caused a large vasoconstriction in hemolysate perfused lungs (Fig. 4), suggesting that hemolysate and the heme oxygenase/guanylyl cyclase inhibitor were synergistic in regard to cGMP synthesis. We conclude that the presence of some dialyzable hemoly-
sate component of RBC has a profound effect on pulmonary vascular reactivity: either addition of NO to the perfusate-but not ventilation with NO (40)-or addition of reducing agents like NAC or of small amounts of KCI cause large pulmonary pressor responses in the presence of .
.
hemolysate.
increased
Slices from lungs
respiration,
similar
perfused
with hemolysate
to slices from
lungs
have
treated
with DNP. We speculate that the hemolysate factor (or factors) cause uncoupling of mitochondrial electron transport and that addition of . NO to the hemolysate-containing perfusate exacerbates the degree of uncoupling. These tissue respiratory events may then trigger secondary cellular events: membrane depolarization and/or increases in cellular
free Ca2 levels, leading to the observed large pulmonary vasoconstrictor responses. Direct effects of hemolysate on membrane ion channels cannot be excluded. Because cGMP Journal
VOELKEL
RESEARCH COMMUNICATION formation
was not different in lungs perfused with either pure Hgb or hemolysate, the #{149}NO-or NAC-induced vasoconstriction does not appear to be related exclusively to an impairment of lung guanylyl cyclase activity. Bolotina et al. (41) demonstrated cGMP-independent direct activation of K-channels by #{149} NO in vascular smooth muscle. One explanation for our findings could be that the activity of endothelial or smooth muscle cell K channels was altered by hemolysate. Because addition of small amounts of NO gas to the hemolysate (but not ventilation with . NO) caused vasoconstriction, we speculate that it is not . NO per se, but a NO-derived metabohite generated in the hemolysate or in the endothelial cells (42) that is responsible for the pulmonary vasoconstriction elicited by exogenous . NO. Although hemoglobin can accumulate in endothelial cells (41), and perhaps can cause endothehial cell damage, our studies with purified oxidized or reduced hemoglobin did not demonstrate synergy with #{149} NO in the generation of the disturbances in tissue respiration. Although the factor or factors that distinguish hemolysate activity from hemoglobin itself remain presently unknown, it is clear from our studies that hemolysate likely affects several variables that control pulmonary vascular tone: mitochondrial function, cyclic GMP synthesis, cell membrane potential, and the tissue redox balance. How these variables contribute, individually or syngergistically, to the dramatic alteration of lung vasculature tone regulation is presently unclear, yet these mechanisms may be relevant to hemolytic disturbances in vivo. The authors wish to acknowledge the excellent secretarial help of Ms. Rebecca Kendig. Purified human hemoglobin for the Hgb studies
was
kindly
ment,
University
in-Aid
by the
National
provided of Denver.
American
Institutes
heart of
by
Sandra
This
Eaton,
work
was
Association
Health,
Chemistry supported
and
an
ROl
Departby a Grantgrant
by the
HL43180.
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vasodilation by Circulation 88,
Received for publication Accepted .for publication
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December
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1994.
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