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1 Division of Clinical
Pathophysiology and 2 Laboratory
of Molecular Biology, The present
study was aimed at examining the role of nitric oxide (NO) in the
hypoxic contraction of isolated small pulmonary arteries (SPA) in the
rat. Animals were treated with either saline (sham experiments) or
Escherichia coli lipolysaccharide
[LPS, to obtain expression of the inducible NO synthase (iNOS) in
the lung] and killed 4 h later. SPA (300- to 600-µm outer
diameter) were mounted as rings in organ chambers for the recording of
isometric tension, precontracted with PGF2
pulmonary circulation; hypoxia; vascular endothelium; nitric oxide; endotoxin; rat
THE IN VITRO CONTRACTILE response of pulmonary arteries
isolated from rat lungs has been used as a model of hypoxic pulmonary vasoconstriction (HPV) (23). This in vitro form of HPV is biphasic, consisting of a transient (phase 1, lasting 5-10 min) followed by
a sustained (phase 2) contraction (9, 13, 14, 33). Most authors report
an important influence of the endothelium on these responses,
especially phase 2. The mechanisms of such endothelium dependence are
not well understood. Several studies of isolated pulmonary arteries
found that the endothelium-dependent component of phase 1 was
suppressed by in vitro treatment with inhibitors of nitric oxide (NO)
synthesis from L-arginine
(L-arginine-NO pathway) (5, 17,
22, 33). Much less information is available on the implication of this
pathway in phase 2 contraction. One group reported its suppression by
NG-nitro-L-arginine methyl ester
(L-NAME), an inhibitor of NO
synthase (NOS) or by oxyhemoglobin, a scavenger of NO, in large
(extrahilar) pulmonary arteries in the rat (33). This effect of
L-NAME was not observed by others (14). Thus the role of
the L-arginine-NO pathway in phase 2 contraction
remains uncertain.
Endotoxin [lipopolysaccharide (LPS)] is present in the
outer wall of gram-negative bacteria. Exposure to LPS, either in vivo or in vitro, results in abnormalities of vascular reactivity (6, 18,
25, 32) related in part to expression of the inducible isoform of NOS
(iNOS) in the endothelium and vascular smooth muscle (6, 27, 34), which
leads to massive production of NO over extended time. The effects of
these events on the hypoxic contractile behavior of isolated vessels
have received little attention (34).
The present study had the following objectives:
1) to examine the role of NO and the
L-arginine-NO pathway in the
endothelium-dependent HPV of small intrapulmonary arteries (SPA)
isolated in the rat; 2) to determine
how this role would be modified in vessels expressing iNOS; and
3) to define more precisely the
level of hypoxia required to elicit HPV in these conditions.
The study was approved by the State Committee controlling Animal Experimentation.
In Vivo Part of Experiment
ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
, and exposed
to either severe (bath PO2 8 ± 3 mmHg) or milder (21 ± 3 mmHg) hypoxia. In SPA from sham-treated
rats, contractions elicited by severe hypoxia were completely
suppressed by either endothelium removal or preincubation with an NOS
inhibitor [NG-nitro-L-arginine
methyl ester (L-NAME),
10
3 M]. In SPA from
LPS-treated rats, contractions elicited by severe hypoxia occurred
irrespective of the presence or absence of endothelium and were largely
suppressed by L-NAME. The milder
hypoxia elicited no increase in vascular tone. These results indicate
an essential role of NO in the hypoxic contractions of precontracted
rat SPA. The endothelium independence of HPV in arteries from
LPS-treated animals appears related to the extraendothelial expression
of iNOS. The severe degree of hypoxia required to elicit any
contraction is consistent with a mechanism of reduced NO production
caused by a limited availability of
O2 as a substrate for NOS.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
1 · h1)
to maintain an appropriate level of anesthesia, as judged by the
interdigital reflex. Four hours after the bolus of LPS or saline was
started, anesthesia was deepened with pentobarbital (20 mg/kg iv), the
thorax was rapidly opened, and the heart and lungs were removed and
immediately placed in ice-cold Earle's balanced salt solution (EBSS;
see below for composition). Throughout the in vivo part of the
experiment, the animal received 50%
O2 through a tracheal cannula
inserted to facilitate spontaneous breathing. Esophageal temperature
was monitored and kept at 37-38°C.
In Vitro Contractility Studies
The left lung was dissected in EBSS at 4°C with a dissecting microscope and microsurgical instruments. The intrapulmonary bronchial tree was opened and then gently lifted from the underlying arteries. The first-order artery was cut longitudinally to allow threading of a thin wire (80 µm) into the lumen of second-order branches for in situ endothelium removal when required. The third and fourth branches (counting from the hilus) were systematically used. Rings (1- to 1.5-mm long, outer diameter of 300-600 µm) were cut from the most proximal part of these branches. This precise location was chosen for its reproducibility and because manipulation of the rings was easier and quicker compared with more distal sampling. The rings were transferred into organ chambers heated at 37°C and mounted on two vertical stainless steel needles (diameter 80 µm). One of the needles was directly attached to the chamber floor and the other to a strain gauge (UL20-GR, Minebea) for the measurement of isometric force.The 2-ml chambers were continuously perfused with EBSS containing
L-arginine
(104 M) and gentamicin (2.5 mg/ml). EBSS was gassed with the required concentration of
O2 plus 6%
CO2 in
N2, to obtain a pH between 7.36 and 7.40. Gassed EBSS was supplied from a pair of heated 50-ml
reservoirs, one bubbled with 21%
O2 and the other with an hypoxic
mixture (either 2.5% or 0%). The liquid was transferred in and out of
the chambers at a constant rate of 4 ml/min by means of two peristaltic
pumps (IPN4, Ismatec, Zürich, Switzerland). Outflow occurred
through an opening located above the chamber floor at a maximal rate
set higher than the inflow rate so that the volume of fluid within the
chamber remained constant (2 ml). Depending on the protocol stage (see
below), the outflow was either discarded (open-circuit configuration)
or recycled to the reservoirs (closed circuit). The chamber lid, made
of Plexiglas, had an opening for the transducer arm. To minimize
contamination by room air, we continuously flushed the gas phase within
the chamber with 60 ml/min of a mixture identical in composition to
that used for the gassing of EBSS. All inflow tubings were stainless
steel except the plastic tubes inside the peristaltic pump. At any
time, the chamber PO2 could be
measured by directing part of the outflow (1.6 ml/min) through
stainless steel tubing to an oxygen electrode (OX1100, Schott
Geräte, Hofheim, Germany) placed in-line within the circuit, 5 cm
downstream from the chamber. Immediately after each
PO2 reading, calibration was checked
by driving fluid previously gassed for at least 30 min with either 0%
or 21% O2 through a path made
entirely of stainless steel tubing, into the electrode at the same flow
rate used for measurement. Chamber hypoxia was achieved in the
closed-circuit configuration by rapidly switching perfusion from the
normoxic to the hypoxic reservoir. Chamber
PO2 stabilized in 10 min, with 90% of the change occurring in the first 3 min.
In many similar studies, the chamber PO2 has been measured with a blood gas analyzer (BGA) rather than an in-line electrode (1, 14, 34). We preferred the latter method because, considering the limited solubility of O2 in water, samples of hypoxic electrolyte solutions could easily be contaminated by the atmospheric PO2 when introduced into a BGA. Indeed, we compared the values of bath PO2 simultaneously obtained from the in-line electrode and from a BGA (AVL 945) installed in the same room, thus allowing transfer of samples within a few seconds using a glass capillary tube. The PO2 measured with both methods (expressed in mmHg as mean ± SE of 3 determinations) agreed closely in normoxic (EBSS gassed with 21% O2: BGA 142 ± 0.6, in-line electrode 140 ± 0.3) but not in hypoxic (2.5% O2: BGA 48 ± 0.8, in-line electrode 22 ± 0.0; 0% O2: BGA 35 ± 0.8, in-line electrode 7 ± 0.6) conditions. Even when measured with the in-line electrode, the chamber PO2 in hypoxia was somewhat higher than predicted on the basis of O2 content in the gas used for equilibration, likely reflecting contamination by atmospheric O2 downstream from the reservoirs.
After being mounted into the organ chamber, the arteries were left to
rest for 30 min and then stretched to a passive tension equivalent to a
transmural pressure of 15 mmHg for vessels of that size, according to a
previously described procedure (13). The transmural pressure was chosen
to approximate the normal value of pulmonary artery pressure in vivo.
Preliminary experiments also showed that this degree of preload was
optimal, i.e., it led to maximal force development after exposure to 80 mM KCl. Thirty minutes after stretching, the following sequence of
steps was initiated. 1) The maximal
tension developed during a 10-min exposure to 80 mM KCl
(Tmax) was
determined. 2) A partial cumulative concentration-response curve to PGF2 was constructed
until the tone exceeded 20% of
Tmax; ACh
(105 M) was then added to
the bath and the induced relaxation was taken as an index of
endothelial integrity. 3) In
experiments requiring the inhibition of NOS, the liquid in the whole
circuit was replaced by a solution of the same composition with the
addition of L-NAME
(103 M, pH adjusted to
7.36-7.40). 4) Whether or not
L-NAME had been added at
step 3, a second partial
concentration-response curve was obtained, from which the
PGF2 concentration able to induce an active tension
equal to 20% of
Tmax
(EC20) was determined. 5) These arteries required a certain
amount of preinduced tone to respond to hypoxia (13, 14); therefore, a
concentration of PGF2 equal to the
EC20 was added to the liquid in
the circuit and then slightly adjusted if necessary until a stable precontraction between 20 and 35% of
Tmax was
obtained. 6) The vessels were
exposed to hypoxia for 35 min and then reoxygenated. At the end of each
experiment, the arteries were systematically fixed in 4% Formalin.
Subsequent standard histological examination was performed in some
intact and endothelium-denuded vessels.
All steps before step 6 were carried
out in normoxia. The partial cumulative dose-response curves
(steps 2 and
4) were obtained by stopping flow
through the circuit and then sequentially adding increasing amounts of
PGF2 dissolved in EBSS directly into the 2-ml chamber
through a small hole in the lid to achieve the following
concentrations: 106, 3 × 106,
105, 3 × 105, and
104 M. Thorough mixing was
ensured by continuous stirring for 3 min before tension was read. In
step 2, the procedure was stopped as
soon as tension exceeded 20% of
Tmax. In
step 4, only the three lowest
concentrations of PGF2 were systematically tested; the
higher ones were given only if required to obtain a tension >20% of
Tmax. We chose
not to complete the dose-response curves because tension frequently did
not plateau at 104 M
PGF2 and because the use of higher concentrations would have been extremely expensive.
In preliminary experiments, we determined that the pattern of hypoxic
contraction was the same with either KCl or PGF2 as the
precontracting agent (data not shown). PGF2 was chosen because of the stable tension achieved with this agent in
step 5.
Two rings per rat were studied, one with and the other without L-NAME. Both rings from the same animal were either endothelium intact or endothelium denuded.
Two series of experiments were successively performed with identical protocols, except for step 6. In series 1, two levels of hypoxia (2.5% and 0% O2) were applied for 35 min each, with 15 min of reoxygenation in between. The milder hypoxia was applied first. In series 2, a single 35-min severe hypoxia (0% O2) was applied. Series 2 was carried out to exclude the possible influence of ischemia-reperfusion events on the contractile response to 0% O2.
iNOS Activity
Rats were treated and killed as described in In Vivo Part of Experiment. All second-order arteries from the left lung were dissected, immediately frozen in liquid nitrogen, and stored at
80°C. Subsequently, iNOS
activity was determined in tissue homogenates from the
calcium-independent conversion of
L-[3H]arginine
to
L-[3H]citrulline
using a previously described micromethod (24). Only endothelium-intact
arteries were studied.
Solutions and Chemicals
All reagents were from Sigma (Buchs, Switzerland) with the exception of PGF2 (Cayman Chemical, Ann Arbor, MI).
PGF2 (103 M
in EBSS) and ACh (5.5 × 103 M in saline) stock
solutions were prepared, aliquoted, and stored at 20°C. The
working solutions were prepared each day from either dry powder or
frozen aliquots, as appropriate. EBSS had the following composition (in
mM): 116.3 NaCl, 5.3 KCl, 26.1 NaHCO3, 1.8 CaCl2, 1.0 NaH2PO4,
0.8 MgSO4, 5.5 D-glucose, and 0.03 phenol red
(sodium salt; as pH indicator). The 80 mM KCl solution used for the
determination of
Tmax was
identical to EBSS except for the isosmolar substitution of KCl for
NaCl. L-Arginine
(104 M) and gentamicin (2.5 mg/ml) were systematically added to both EBSS and 80 mM KCl.
Experimental Design and Data Analysis
All designs were full factorial with randomization. As explained, different arteries from the same animal were tested in the presence or absence of L-NAME, and endothelium-intact and endothelium-denuded vessels always came from different animals. Results were analyzed accordingly, using the appropriate analysis of variance model. When the F-value for an effect was globally significant, planned pairwise comparisons were made with modified t-tests (i.e., Fisher's protected least significant difference). The
-level of all
statistical tests was set at 0.05. Computations were performed with the
JMP software (SAS Institute, Cary, NC). All data are given as means ± SE unless stated otherwise. Sample size is systematically indicated.
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In Vitro Contractility of SPA
Table 1 shows mean Tmax and responses to ACh obtained in the two series of experiments. Tmax was lower in endothelium-denuded compared with endothelium-intact vessels, although this effect only reached statistical significance in one subgroup of series 1. Standard histological examination of endothelium-denuded rings (not shown) revealed no visible damage to the smooth muscle layer. We did observe a small number of breaks in the lamina elastica interna, which nevertheless remained clearly visible and sharply delineated around most of the intimal circumference. Mean Tmax was generally somewhat lower in the presence than in the absence of LPS treatment.
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In endothelium-intact vessels from sham-treated rats,
105 M ACh caused an
immediate and intense relaxation. This response was blunted, although
still clearly obtained, in vessels from LPS animals. As expected,
endothelium removal essentially abolished any vasomotor effect of ACh.
Figure 1 shows the influence of the various
conditions, in normoxia, on sensitivity of the rings to the
vasoconstrictor effect of PGF2. Pooled data are
presented for brevity, because behavior in the two series of
experiments was homogeneous. In the absence of prior treatment with
LPS, EC20 was drastically reduced
(almost 10-fold) by endothelium removal. In endothelium-intact rings, a
reduction of the same magnitude was achieved with the NOS inhibitor L-NAME. By contrast,
L-NAME did not change the
EC20 of endothelium-denuded rings.
Treatment of the animals with LPS was associated with a large increase
in EC20 in both the presence and
the absence of endothelium. This effect of LPS was largely reversed by
L-NAME, although not completely
in endothelium-intact rings.
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As usual in this preparation (14, 29, 35), hypoxic contractions could
not be obtained without the prior induction of submaximal active
tension with a vasoconstrictor agonist, and PGF2 was
used for that purpose (precontraction, step
5 of the protocol). By design, the mean level of
precontraction was relatively uniform between experimental groups
(lowest and highest values, expressed as % of
Tmax: 22 ± 1 and 30 ± 2). Thus the concentrations of PGF2 used
for precontraction (i.e., present in the bath during hypoxia) varied
between conditions, according to the pattern predicted by Fig. 1.
Precontraction tone was absolutely stable during the 10 min preceding
hypoxia (not shown).
The effects of two levels of hypoxia on vessel tone in the various conditions are displayed in Figs. 2 and 3. Major changes in tone only occurred on equilibration of the organ bath with 0% O2 [PO2: 8 ± 3 mmHg (mean ± SD)] but not with 2.5% O2 (21 ± 3 mmHg).
Severe hypoxia (0% O2).
The effects of 0% O2 were
essentially the same in the two series of experiments.
Only results from
series 2 are presented here (Fig. 2).
In the absence of treatment with LPS, severe hypoxia induced a typical
pattern consisting of a transient contraction (phase 1) followed by
transient partial relaxation and then by a sustained progressive
contraction starting 15-20 min after the onset of hypoxia (phase
2). Phase 2 contraction was rapidly reversed on reoxygenation (not
shown). This time course was profoundly altered by endothelium removal,
exposure to L-NAME, or both,
with no difference noted in the effects of these three interventions: phase 1 and phase 2 contractions were abolished and replaced by progressive relaxation. There was no statistically significant effect
of treatment with LPS on the hypoxic contraction induced by 0%
O2 in endothelium-intact rings not
exposed to L-NAME. However, in
sharp contrast with findings in the absence of LPS, the hypoxic contraction was not abolished or even significantly modified by endothelium removal. The hypoxic contraction of vessels from
endotoxemic animals was markedly suppressed by
L-NAME. This suppression was total in endothelium-denuded rings, whereas endothelium-intact rings
exposed to L-NAME retained at
all time points a hypoxic tone slightly higher in the presence compared
with the absence of treatment with LPS.
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Milder hypoxia (2.5% O2).
As stated, there was essentially no contraction with this level of
hypoxia (Fig. 3). There were, however, some
statistically significant differences between conditions, all of which
were in the same direction observed with severe hypoxia, although of much smaller amplitude.
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iNOS Activity
This activity, as measured from the calcium-independent conversion of labeled L-arginine into L-citrulline in homogenates of endothelium-intact SPA, was massively enhanced by in vivo treatment with LPS (Fig. 4).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The principal finding of this study was the complete suppression by NOS inhibition of both phase 1 and phase 2 hypoxic contraction in precontracted SPA in the rat. Furthermore, both phases required an intact endothelium in the absence but not in the presence of prior in vivo treatment with LPS. Finally, severe hypoxia (i.e., a bath PO2 well below 20 mmHg) was required to elicit these contractions.
Contractile Behavior in Normoxia
Sham experiments. Under all conditions, Tmax was lower than usually reported in pulmonary arteries of this size (13, 14, 29, 35; Table 1), raising the concern of possible tissue damage inflicted in the course of our study. In endothelium-intact vessels, the vigorous relaxation elicited by ACh argues against this possibility. Furthermore, additional experiments (not shown) indicated that rings sampled more distally than in the principal experiments had a substantially higher Tmax. As mentioned (see METHODS), our standard procedure was to excise rings from second-order branches in close proximity to the junction with the first-order artery. In this location, smooth muscle cells might either be less abundant or have a noncircular orientation in comparison with a more distal site.
Some damage to smooth muscle may have been present in endothelium-denuded arteries, which had a lower Tmax than their endothelium-intact counterparts, especially in the experiments of series 1 (Table 1); in series 2, this effect of endothelium removal was smaller and not statistically significant. Rings in which the endothelium had been rubbed had only very limited histological evidence of damage and did disclose the expected increase in sensitivity to a vasoconstrictor agonist (i.e., a lower EC20 for PGF2; Fig. 1).
In endothelium-denuded arteries,
L-NAME did not modify the
EC20 for PGF2. In
endothelium-intact vessels, in contrast, inhibition of NOS increased
the sensitivity to PGF2 severalfold, indeed to exactly
the same extent as did endothelium removal (Fig. 1). Taken together,
these observations indicate that, in agreement with the scant data
available in rat SPA (14, 29), the
L-arginine-NO pathway plays an
essential role in the modulation of vascular tone by the endothelium
under the conditions of the present study.
Effects of treatment with LPS.
In many species, in vivo treatment with LPS has a depressor effect on
vascular contractility. In rat isolated aortas (3, 6, 10, 31), other
systemic vessels (25), or large extrahilar pulmonary arteries (6, 34),
this defect has been related to hyperactivation of the
L-arginine-NO pathway caused by
the stimulated expression of iNOS in the vascular wall. Our results provide two arguments for the same scenario in SPA. Four hours after
LPS treatment, iNOS was massively expressed in these vessels (Fig. 4).
Furthermore, the marked decrease in sensitivity to the vasoconstrictor
action of PGF2 induced by LPS treatment in both
endothelium-intact and endothelium-denuded rings was essentially reversed by L-NAME (Fig. 1). The
hypocontractility of rings from LPS-treated rats may have been
augmented by the presence of 100 µM
L-arginine in the bath, because
production of NO in tissues expressing iNOS is dependent on an
exogenous supply of this substrate (3, 26).
in
endothelium-denuded rings from LPS- versus sham-treated rats (Fig. 1).
Concomitant enzyme induction in the endothelium could also have
occurred (11).
The reversal by L-NAME of
LPS-induced hypocontractility was complete in endothelium-denuded
vessels. With the endothelium intact,
L-NAME drastically reduced but
did not completely abolish the difference in sensitivity to
PGF2 between rings from LPS- and sham-treated rats.
Assuming that LPS stimulated the expression of iNOS in the endothelium,
this residual difference could be related to the lower potency of
L-NAME as an inhibitor of iNOS, as opposed to constitutive endothelial NOS (ecNOS) (28).
Alternatively, LPS could have stimulated the endothelial production of
a vasodilator unrelated to NO. Whatever the explanation, the magnitude
of LPS effects not inhibited by
L-NAME was relatively small. The
blunting by LPS of
Tmax and of
endothelium-dependent vasodilation to ACh is in accordance with a large
body of experimental work in different vessels and different species
(18, 25, 31, 32).
Hypoxic Contractile Responses
Level of hypoxia required to elicit contraction in vitro. A first feature of all hypoxic contractions observed in the present study was the very low PO2 required to elicit these responses. Indeed, the vigorous contractions observed with severe hypoxia (bath PO2 8 ± 3 mmHg; Fig. 2) were essentially absent with a slightly higher PO2 (21 ± 3 mmHg; Fig. 3). The ability of severe hypoxia to elicit contraction of isolated pulmonary arteries is consistent with a substantial amount of experimental work in the rat (13, 17, 23, 29) and other species (8, 12). On the other hand, the lack of response to a slightly milder hypoxia may seem to contradict several reports of contractions obtained in isolated rat pulmonary arteries submitted to a bath PO2 of 23 (34), 33 (1, 14), 44 (23), or even 57 (33) mmHg.
There are two nonmutually exclusive explanations for this apparent discrepancy. First, the bath PO2 may have been overestimated in some studies (1, 14, 34) because of the use of a BGA rather than an in-line PO2 electrode, as explained in METHODS. This potential error did not occur in the study by Rodman and associates (23). However, as these authors note, extrapolation from bath to cellular PO2 may be difficult because of the problems of unstirred layers and diffusion barriers. Such problems may have been minimized in the present work because of the continuous perfusion of organ chambers during hypoxia and the small size of the arteries examined. Viewed against this background, our data strongly support the idea that a severe level of hypoxia, indeed, close to anoxia, is required to trigger a further increase in the tone of precontracted rat SPA.Sham experiments. In our experiments, the contraction elicited by severe hypoxia in intact precontracted vessels followed the typical biphasic pattern reported by other studies of either large (1, 14) or small (13, 14, 29) pulmonary arteries in the rat (Fig. 2). Both the early transient phase 1 and the later sustained phase 2 were completely suppressed by removal of the endothelium. In the case of phase 2, this is consistent with most observations made by others (13, 14, 33, 35). On the other hand, phase 1 was either partially (13, 14) or totally (35) endothelium independent in studies of small arteries, including one from our own laboratory (13), at some variance with our present findings. The reasons for this discrepancy are not clear. It could be caused by the possible damage inflicted on smooth muscle during endothelium removal, as discussed in Contractile Behavior in Normoxia. The suppression of phase 1 by L-NAME in endothelium-intact vessels (discussed below) argues against this possibility. Alternatively, unrecognized variables could be involved, such as the exact kinetics of PO2 change or the genetic background of the animal.
Several studies in large rat pulmonary arteries found that NOS inhibitors or scavengers of NO abolish (5, 22, 33) or diminish (17) phase 1 and suppress phase 2 (33). Analog information in rat SPA is less abundant. Teng and Barer (29) reported a considerable blunting of phase 1 contraction when such arteries were preincubated with L-NAME (100 µM) but provided no information on phase 2. The present experiments support and complement this study: not only phase 1 but also phase 2 was totally abolished by L-NAME (1 mM) (Fig. 2). This cumulated evidence obtained with pharmacological probes implies that the L-arginine-NO pathway plays a fundamental role at all phases of hypoxic contractions in both large and small pulmonary arteries from nonendotoxemic rats. At variance with this conclusion, Leach and co-workers (14) found that neither phase 1 nor phase 2 was affected by the NOS inhibitor NG-nitro-L-arginine (L-NNA), whether in large or small rat pulmonary arteries. These authors used a relatively low concentration of L-NNA (10 µM), which efficiently blocked relaxation to ACh but failed to significantly augment the contractile response to a vasoconstrictor agonist, in contradistinction to effects noted by others (3, 6, 26, 29, 33, 34) and ourselves (Fig. 1) with higher concentrations (100 µM to 1 mM) of various L-arginine analogs including L-NNA (5, 31). Dose-response studies in rat aortas indicate that higher concentrations of NOS inhibitors may be required for the augmentation of agonist-induced contraction, as opposed to the inhibition of endothelium-dependent relaxation (19), raising the possibility that basal and agonist-stimulated endothelial production of NO may be differentially affected by NOS inhibitors (20). In short, results by Leach and co-workers (Fig. 9 in Ref. 14) would be reconciled with ours (Fig. 2) if it is assumed that in their study NOS inhibition was insufficient to suppress the basal release of NO.Effects of treatment with LPS.
The effects of in vivo endotoxemia on the hypoxic contractile behavior
of isolated pulmonary vessels have been explored for the first time in
the present study. We found that precontracted SPA from LPS-treated
rats retained the ability to increase tone on severe hypoxia (Fig. 2).
In contrast with observations in arteries from sham-treated rats, this
hypoxic contraction was not altered by endothelium removal. There was a
remarkable parallel between the effects of NOS inhibition on the
sensitivity to PGF2 in normoxia (Fig. 1) and on the time
course of vascular tone during severe hypoxia (Fig. 2): whenever
L-NAME decreased the
EC20 (i.e., in all conditions
except endothelium-denuded arteries of sham-treated rats), it also
suppressed or largely diminished the subsequent contraction in response
to severe hypoxia. These data imply a major role of the
L-arginine-NO pathway in all
hypoxic contractions observed in the present study. The endothelium
independence of hypoxic contractions in arteries from LPS-treated rats
simply reflects the activation of this pathway at an extraendothelial site, presumably because of the expression of iNOS, as discussed in
Contractile Behavior in Normoxia. This
result is consistent with the work of Zelenkov and co-workers (34), who
found in the rat that endothelium-denuded large pulmonary arteries did not contract on hypoxia unless incubated with LPS in vitro. We extended
these findings in two ways: we demonstrated such effects in more
peripheral pulmonary arteries differently exposed to LPS (i.e., in
vivo), and we showed that, at least in our conditions, the
PO2 required to trigger hypoxic
contraction was not modified by treatment with LPS (Figs. 2 and 3).
Further Comments
The nature of the link between the L-arginine-NO pathway and the hypoxic contractions observed in precontracted pulmonary arteries is uncertain. In theory, hypoxia could either inhibit NOS or act distally to the generation of NO. Recently, the effects of O2 concentration on NOS activity have been investigated, using purified bovine aortic ecNOS and iNOS from cultured RAW 264.7 macrophages; for these two isoforms of NOS, the Michaelis constant values for O2 were 7.7 and 6.3 µM, respectively, equivalent under normal atmospheric pressure to PO2 of 6 and 5 mmHg (21). If rat isoforms of NOS behave similarly, severe hypoxia under the conditions of our experiments (i.e., a bath PO2 of 8 mmHg, necessarily associated with a lower intracellular PO2 because of diffusion barriers) would obviously inhibit NO production by the mere limitation of the O2 substrate, whereas this effect would be less likely with milder hypoxia (bath PO2 21 mmHg). Thus, although inhibition of NOS by severe hypoxia cannot be ascertained from our data, it would be consistent with the differential effects of the two levels of hypoxia tested in the present study. Further studies would be required to dissect out the interactions of hypoxia with the L-arginine-NO pathway in vascular tissue.In conclusion, our data highlight several important differences between hypoxic contractions exhibited in vitro by precontracted SPA in the rat and the usual characteristics of HPV in the intact lung. A much lower PO2 is required to elicit vasoconstriction in vitro than in perfused rat lungs (16) or in vivo (2). Furthermore, although pharmacological NOS inhibitors abolish or reduce HPV in isolated rat arteries, they enhance it in the intact lung (7, 15). Finally, in contrast with several observations made in the intact pulmonary circulation (4, 30), in vivo endotoxemia does not suppress hypoxic contraction in isolated endothelium-intact arteries. These differences must be borne in mind when using isolated pulmonary arteries in the rat as a tool to investigate the mechanisms of HPV.
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ACKNOWLEDGEMENTS |
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The authors gratefully acknowledge the help of Urs Rüegg, of the School of Pharmacy at the University of Lausanne, who provided the small vessel myograph used in these studies. The authors thank Bernard Waeber for helpful discussions and for critical review of the manuscript. Camille Anglada, Martine Vaglio, Antoinette Ney, and Christian Durussel provided outstanding technical assistance, and Françoise Bilat provided excellent secretarial help.
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FOOTNOTES |
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This work was funded by a grant from the Swiss National Science Foundation (31-40886.94).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: F. Feihl, Div. of Clinical Pathophysiology and Medical Teaching, BH19-313, Lausanne Univ. Hosp., 1011 Lausanne, Switzerland (E-mail: Francois.Feihl{at}chuv.hospvd.ch).
Received 20 July 1998; accepted in final form 28 December 1998.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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