Role of wall tension in hypoxic responses of isolated rat
pulmonary arteries
Masami
Ozaki1,
Carol
Marshall2,
Yoshikiyo
Amaki1, and
Bryan E.
Marshall2
2 Department of Anesthesia,
University of Pennsylvania, Philadelphia, Pennsylvania 19104; and
1 Department of Anesthesia, Jikei
University School of Medicine, Tokyo 105-8461, Japan
 |
ABSTRACT |
The changes in
force developed during 40-min exposures to hypoxia (37 ± 1 mmHg)
were recorded in large (0.84 ± 0.02-mm-diameter) and small (0.39 ± 0.01-mm-diameter) intrapulmonary arteries during combinations of
mechanical wall stretch tensions (passive + active myogenic
components), equivalent to transmural vascular pressures of 5, 15, 30, 50, and 100 mmHg, and active (vasoconstriction) tensions, stimulated by
PGF2
in doses of 0, 25, 50, and
75% effective concentrations. Constriction was observed in all
arteries during the first minute; however, at any active tension, the
pattern of the subsequent response was a function of the stretch
tension. At 5, 15, and 30 mmHg, the constriction decreased slightly at 5 min and then increased again to remain constrictor throughout. At 50 and 100 mmHg, the initial constriction was followed by persistent dilation. Hypoxic constrictor responses, most resembling those observed
in lungs in vivo and in vitro, were observed when the mechanical
stretch wall tension was equivalent to 15 or 30 mmHg and the dose of
PGF2
was 25 or 50% effective
concentration. These observations reconcile many apparently
contradictory results reported previously.
hypoxic pulmonary vasoconstriction; wall stress; prostaglandin
F2
; mechanical stretch; myogenic tone
 |
INTRODUCTION |
HYPOXIC PULMONARY VASOCONSTRICTION (HPV) has been
established as an important regulatory mechanism both for reducing
arterial hypoxemia in the presence of abnormal ventilation-perfusion
ratios and as a cause of pulmonary hypertension in many chronic and
acute cardiopulmonary disease states (3, 19). When a local region of
the lung becomes hypoxic in vivo, the HPV response is characterized by
a rapid and persistent vasoconstriction of the pulmonary arteries (PAs)
that remains essentially constant for at least 4 h (5). When isolated
rat lungs are ventilated and perfused in vitro and exposed to hypoxia,
a similarly rapid constrictor response is observed, with a maximum
constriction at 2-5 min and a small 10-20% decline over the
next 10 min, and thereafter constriction is sustained for at least 40 min (16, 24). Although there is little disagreement about the form of
these responses, those reported for isolated rat PAs are quite
disparate. The only consistent observation has been an initial rapid
constriction, but thereafter, investigators (1, 10, 12, 31, 32)
reported a variable vasodilation that often more than abolished the
initial constriction and may or may not be succeeded by a slow
constriction developing over many minutes.
Isolated PAs are convenient for the study of the physiological
properties, pharmacological influences, and nature of the mechanism of
HPV. However, the form of these responses to hypoxia in isolated rat
PAs is so variable and differs so much from the whole lung responses
that the applicability of the results to HPV in general remains
uncertain. In considering the experimental designs of many
investigators, differences in three principal variables became apparent. The first was the size of the PA, the second was the choice
and dose of the vasoconstrictor used to generate an active tension
before eliciting HPV, and the third was the passive or mechanical
stretch tension maintained throughout the study. Although there have
been reports on the influence of arterial size (12, 13) and
vasoconstrictor choice (14), the role of active and passive tensions on
the responses to hypoxia has not been investigated systematically.
Transient initial hypoxic constrictor responses have been observed in
all rat PAs including the main extrapulmonary and large and small
intrapulmonary arteries, and this has led some to the belief that size
is not important. However, differences in the form of the response
observed in small and large arteries (13) and the fact that HPV in vivo
results from constriction of small PAs of <600 µm in diameter (6)
undermine this conclusion.
The choice of mechanical stretch tension has generally been derived
directly from the techniques used for systemic arteries. Some
investigators observed the responses to repeated doses of KCl as the
arterial ring (or strip) was increasingly stretched. The smallest
passive tension that elicited a maximum response to KCl is then used
for the study of HPV. However, the passive tensions used have varied
widely (0.5-3 g, even in the same sized arteries), and the
assumption that this tension is entirely passive with no active
myogenic component and is also optimum for HPV has not been formally tested.
Finally, in lungs in vitro, it has been shown that HPV responses are
more readily and consistently observed when the PAs are preconstricted.
This practice was therefore introduced to the study of HPV in isolated
PAs, but despite studies demonstrating that hypoxic responses are
influenced by the choice of vasoconstrictor (14, 25), the
dose and choice of specific vasoconstrictor in most publications are
probably not regarded as a critical variable. For systemic arteries,
Mulvany and Halpern (20) drew attention to the importance of
standardizing wall tension, and these principles were applied to PAs by
Leach and colleagues (12-14) in a series of elegant studies that
form the basis for the present work. Those studies established not only
that the form of the responses could be altered by changing stretch
tension but also demonstrated that out of the many vasoconstrictor
agents that have been used, only prostaglandin
F2
(PGF2
) had comparable
dose-response curves in both small and large PAs, presumably because
the receptor density, affinity, and endothelium-dependent actions of
PGF2
were less variable with
artery size than those of other agents (14).
The present work has systematically examined the influence of stretch
(imposed by stretching the artery mechanically) and active
(imposed by PGF2
) wall
tensions on large (
1-mm-diameter) and small (
400-µm-diameter)
intrapulmonary arteries exposed to 40 min of hypoxia. The results
demonstrate that, independent of size, there is a systematic and
dramatic change in the form of the hypoxic response that is determined
primarily by the stretch tension, whereas the active tension has a more
subtle influence.
 |
MATERIALS AND METHODS |
PA isolation. The protocols for the
preparation and care of the rats used in these studies were approved by
the University of Pennsylvania Animal Care and Use Committee. Male
Wistar rats were anesthetized with 10 mg of Ketalar (ketamine
hydrochloride) intramuscularly and 50 mg/kg of pentobarbital sodium
intraperitoneally. The chest was opened in the midline, and heparin
sulfate (200 units) was injected into the right ventricle to
anticoagulate the blood. The rats were exsanguinated, and the heart and
lungs were removed en bloc and immersed in ice-cold Hanks' balanced salt solution containing (in mM) 1.3 CaCl2, 5.0 KCl, 0.5 MgCl2 · 6H2O,
0.3 KH2PO4,
0.4 MgSO4 · 7H2O,
138 NaCl, 4.0 NaHCO3, 0.3 Na2HPO4, 5.6 D-glucose, and 0.03 phenol red.
With the aid of a dissecting microscope the first (large)- and second
(small)-generation PAs were immediately isolated. The arteries were cut
into ring segments ~1 mm in length and threaded with two wires
(25-µm-diameter tungsten steel). At the end of each study, the wet
weight of the arterial segment was recorded.
Experimental circuit. The perfusate
circuit and small-vessel myograph are similar to those described by
others (14, 20). The arteries were mounted on a small-vessel myograph,
bathed in circulating Earle's balanced salt solution containing (in
mM) 1.8 CaCl2, 5.3 KCl, 0.8 MgSO4 (anhydrous), 117 NaCl, 26.0 NaHCO3, 1.0 NaH2PO4 · H2O,
5.6 D-glucose, and 0.03 phenol
red. The perfusate was circulated through one of two water-jacketed
reservoirs at 19 ml/min by a Harvard Apparatus model 1203 peristaltic
pump. In the reservoirs, the Earle's balanced salt solution was gassed by either the normoxic (21% oxygen-5% carbon dioxide-balance
nitrogen) or hypoxic (0% oxygen-5% carbon dioxide-balance nitrogen)
gas mixture, and the temperature of the water jacket was regulated by a
Haake model FE2 thermostat/pump to maintain the perfusate temperature
at 37°C.
The arterial segment was suspended by the two wires in a small-vessel
myograph based on that described by Mulvany and Halpern (20). Each wire
was attached horizontally to stainless steel "jaws," with one end
fixed to a calibrated force transducer (Kulite BG-10GM, Kulite
Semiconductor Products, Ridgefield, NJ) and the other to a calibrated micrometer.
Stretch tension determination. For
every arterial segment, the circumference-wall tension relationship was
initially determined so that stretch tensions could be accurately
selected. At the start of each study, the micrometer was adjusted so
that the two wires overlapped as viewed from the monocular scope
situated perpendicularly above. After a 30-min equilibration period,
the length and diameter of the mounted strip were measured with a
monocular scope grid when no tension was imposed. The micrometer was
then adjusted to stretch the PA circumferentially until the recorder
detected force development. The micrometer was thereafter adjusted to
stretch the PA in a stepwise manner while the distance between the
wires (f; in µm) and the
force developed were recorded until the wall tension was ~2 mN/mm.
The circumference of the PA
(Cin; in µm) was calculated from
|
(1)
|
where
d is the diameter of the wire (25 µm). The relationship between the circumference and the wall tension
was fit with a simple exponential function (13, 19) of the form
|
(2)
|
where
T (in mN/mm) is the wall tension calculated by dividing the recorded
force (in mN) by twice the measured length of the PA segment (in mm),
a is the intercept, and
b is the slope.
With the assumption that the arterial wall thickness was much less than
the diameter and that the form of the wall curvature was not critical,
the Laplace equation relates wall tension, circumference, and the
effective transmural pressure (P; in mmHg; 1 mN
7.5 mmHg)
|
(3)
|
For
example, for an effective transmural pressure of 30 mmHg, T = 0.64Cin. The wall
tension from Eq. 3 was calculated for each Cin recorded
for an artery, and the line obtained was superimposed on the curve
generated from Eq. 2 for that artery.
The line intersected the curve at the point where the observed wall
tension corresponded to an effective intravascular pressure of 30 mmHg.
This procedure was repeated for different arteries for effective
transmural pressures of 5, 15, 30, 50, and 100 mmHg so that for each
specific artery, the individual circumference, and therefore stretch
wall tension corresponding to a particular effective transmural
pressure, could be selected for subsequent studies.
The following two preliminary studies were performed: the first to
establish the stretch wall tension dependence of the response to KCl
and the second to determine the dose-response relationship for the
active wall tension generated by
PGF2
.
Stretch tension with 75 mM KCl
challenge. Large and small PAs were prepared as in
Stretch tension
determination to establish the
circumference-wall tension relationship, and then, after a 30-min
resting period, the perfusate was replaced with one containing 75 mM
KCl (NaCl was reduced by 75 mM to preserve osmolality, and the
temperature and normoxic gas tensions were unchanged). The contractile
response was observed for 2 min; then the KCl was washed out, and the
PAs returned to baseline tension. This maneuver was repeated as the
circumference of the PA was increased in a stepwise manner until the
active tension developed in response to KCl did not increase further.
The active wall tension was calculated by subtracting the stretch wall
tension from the total wall tension recorded at each
Cin.
PGF2
dose-response
curves.
PGF2
was selected as the source
of the imposed active wall tension for this work because, in contrast
to many other vasoconstrictors, the dose-response curves for small and
large rat PAs were reported to be similar (14). Small and large
arteries were prepared as described in Stretch tension
determination, after which a stretch tension
corresponding to an effective transmural pressure of either 5, 15, or
30 mmHg was imposed. The active wall tension change was recorded with
the addition of PGF2
to achieve
1, 5, 10, 50, and 100 µM concentrations. The dose-response curves
were fit with an equation of the form
|
(4)
|
where
Robs and
Rmax are the observed and maximum
responses respectively; D is the
PGF2
concentration (in mol/l),
pD is the negative logarithm of
EC50, and
S is the slope at the
EC50.
Responses to hypoxia with variable stretch and active
tensions. In each large and small PA, the
circumference-wall tension relationship was determined under normoxic
conditions as described in Stretch tension
determination, and a stretch tension
corresponding to an effective transmural pressure, selected in random
order, of either 5, 15, 30, 50, or 100 mmHg was maintained as the
baseline stretch tension throughout the rest of the experiment. A
single 2-min response to 75 mM KCl was determined by replacing the
perfusate as described in the first preliminary study, after which the
KCl was washed out and baseline conditions were restored. To establish a stable hypoxic response, the artery was then challenged with two
6-min exposures to hypoxia, separated by 6 min of normoxia. This was
achieved by rapidly exchanging the perfusate in the bath at the same
time that the hypoxic or normoxic reservoir was selected. The
subsequent experimental observations consisted of recording the wall
tension changes during exposure to 40 min of hypoxia in the presence of
either 0, 25, 50, or 75% cumulative effective concentrations
(EC25,
EC50, and
EC75, respectively) of
PGF2
. Each 40-min hypoxic
exposure was followed by 10 min of normoxia during which the next dose
of PGF2
was administered. After the last hypoxic response, the
PGF2
was washed out until the
baseline tension was reestablished and a final challenge with 75 mM KCl
was recorded.
Myogenic tone with mechanical stretch.
Hypoxic dilation was observed in the preceding studies when mechanical
stretch was equivalent to 50 or 100 mmHg, but the
PGF2
concentration was zero.
The simplest explanation for this is that increased passive tension by
mechanical stretch was associated with the development of myogenic tone
that was abolished by hypoxia. The following two additional studies
investigated this hypothesis.
In the first study, the conditions were normoxic throughout. Large and
small PAs were prepared as before, and after the length-tension relationship was determined, they were subjected to stretch wall tension corresponding to either 30 or 50 mmHg. Without further manipulations or additions, the wall tension was recorded for 40 min,
after which the response to
PGF2
(10
6 M) was recorded. The
normoxic perfusate solution was then replaced with a normoxic
Ca2+-free relaxing solution
[composition in mM: 4.7 KCl, 1.17 MgSO4 (anhydrous), 119 NaCl, 26 NaHCO3, 1.18 KH2PO4,
1.0 D-glucose, 0.026 EDTA, 1 EGTA, and 0.03 phenol red], and the artery was stimulated with
PGF2
(10
6 M) to deplete internal
Ca2+ stores. This latter procedure
was repeated, and the final perfusate was replaced with normoxic
relaxing solution to which papaverine (10
5 M) was added. The wall
tension was again recorded for 40 min, after which the absence of a
response to PGF2
was confirmed by the addition of PGF2
(10
6 M). The changes in
wall tensions were compared in the absence and presence of a relaxing solution.
For the second study, large and small arteries were prepared as above
and subjected to a mechanical stretch wall tension equivalent to 50 mmHg only. After the responses to KCl (75 mM) and to three 6-min
exposures to hypoxia were recorded, the arteries were allowed to
equilibrate at normoxia for an additional 5 min. The perfusate was
replaced with Ca2+-free relaxing
solution and stimulated with
PGF2
(10
6 M) twice. The
perfusate was then replaced with
Ca2+-free relaxing solution
containing papaverine (10
5
M). After a further 15 min of equilibration, hypoxia was established, and the wall tension was recorded for 40 min. Finally, the absence of
response to PGF2
was confirmed.
The changes in wall tension when the perfusate was made hypoxic were
compared with the normoxic baseline.
Drugs and solutions. Heparin was
obtained from Elkins-Sinn (Cherry Hill, NJ); ketamine hydrochloride was
from Fort Dodge Laboratories; pentobarbital sodium was Abbott
Laboratories (North Chicago, IL); PGF2
(9,11-dideoxy-9a,11a-epoxymethanoprostaglandin
F2
) and papaverine were from
Sigma (St. Louis, MO); Earle's balanced salt solution and Hanks'
balanced salt solution were from GIBCO BRL, Life Technologies (Grand
Island, NY).
Statistics. The values reported are
expressed as means ± SE. Areas under the hypoxic response
recordings and the changes in wall tensions at 1, 5, and 40 min were
analyzed by two-way repeated-measures ANOVA, with the significance of
the difference between means tested by
t-test with the Bonferroni correction.
Paired t-tests were used as described
in the text. A P value of <0.05 was
considered significant.
 |
RESULTS |
Mechanical stretch wall tension with 75 mM KCl
challenge. In the first preliminary study, for both
large and small arteries, the response to stimulation with 75 mM KCl
revealed an increase in developed active tension with each stepwise
increment of the mechanical stretch wall tension until a maximum
response was achieved, and further increments of stretch tension were
accompanied by a decreased response. For large and small arteries, the
maximum active responses of 1.67 ± 0.13 and 1.74 ± 0.41 mN/mm,
respectively, were observed at stretch tensions of 470 ± 50 and 356 ± 83 mg, respectively. To allow for comparisons, these data were
normalized by expressing the responses of each artery as a percentage
of the maximum active tension developed [active tension
(%Max)] and the circumferences as the ratio
(Cin/Cmax)
of the Cin to the
circumference at the maximum active tension
(Cmax). These
normalized data are shown in Fig. 1. The
maximum stretch force used was 1,579 ± 54 and 1,131 ± 156 mg
for the large and small arteries, respectively.

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Fig. 1.
Influence of mechanical stretch wall tension on response to 75 mM KCl.
Results of this preliminary study have been normalized to allow
comparison of large and small pulmonary arteries (PAs). Active tension
is observed active tension developed and expressed as percent maximum
active tension developed (%Max).
Cin/Cmax,
ratio of circumference observed at a particular stretch tension to
circumference when KCl response was maximal. Maximal active tensions
were developed at
Cin/Cmax = 1, when stretch forces were 470 ± 50 and 356 ± 83 mg for large and small PAs, respectively. Active tension development
was reduced at greater or lesser stretch forces.
|
|
PGF2
dose-response
curves.
In the second preliminary study, small and large arteries were prepared
with imposed stretch tensions equivalent to transmural pressures of 5, 15, or 30 mmHg and n = 9 arteries for
each of these six groups. Each artery was exposed to 1, 5, 10, 50, and 100 µmol/l of PGF2
, and the
slope and negative logarithm of the
EC50 coefficient (pD) for the
sigmoid dose-response relationship were derived. There were no
significant differences between the dose-response curve coefficients
for the three stretch tensions in either the large or small arteries
(data not shown), and, therefore, the results were combined in Table
1. For the subsequent studies, at all
stretch tensions (including 50 and 100 mmHg), the following concentrations of PGF2
were
derived from these equations. For large arteries, concentrations of
PGF2
of 0.50, 1.65, and 8.54 µmol/l were used to achieve active tensions corresponding to
EC25,
EC50, and
EC75, respectively, and for the
small arteries these concentrations were 0.27, 1.01, and 8.06 µmol/l,
respectively.
Responses to hypoxia with variable stretch and active
tensions. Each large or small artery was studied at
only one of the five different stretch tensions and with
n = 12 arteries/group; the reported
data are therefore from a total of 120 arteries. At rest, the diameter
of the large arteries was 0.84 ± 0.02 mm and of the small arteries
was 0.39 ± 0.01 mm. For comparison with previous reports, the total
stretch force (in mg) measured at the transducer for each of the groups
is shown in Fig. 2. The perfusate gas
composition during normoxia was PO2
of 133 ± 1 mmHg, PCO2 of 38 ± 2 mmHg, and pH of 7.39 ± 0.02 and during hypoxia was
PO2 of 37 ± 1 mmHg,
PCO2 of 37 ± 1 mmHg, and pH of
7.39 ± 0.01.

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Fig. 2.
Relationship between mechanical stretch wall force and equivalent
transmural vascular pressure of large and small PAs. P5, P10, P30, P50,
and P100, transmural pressure of 5, 15, 30, 50, and 100 mmHg,
respectively. Values are means ± SE. It is noted that forces < 500 mg for large and 300 mg for small PAs correspond to transmural
pressures in physiological range, whereas forces > 700 mg for large
and 500 mg for small PAs correspond to pulmonary hypertension.
|
|
From the continuous recordings of transducer force during the 40-min
hypoxic exposure, the changes in force were calculated at the end of
the first min and at 5-min intervals for 40 min. These data, expressed
as the changes in (delta) wall tension (in mN/mm), are the changes from
baseline when the baseline varied with the specific combined stretch
and active wall tensions. The means (±SE) for the delta wall
tensions are summarized in Fig. 3 for the
large arteries and Fig. 4 for the small
arteries. Note that from the wet weight of the artery at the end of the
experiment, the volume and, therefore, the wall thickness were
calculated. From this value, wall stress (in
mN/mm2) was derived; however,
this additional normalization did not reduce variability, and these
data are not presented. The mean KCl response for all arteries at the
end of the experiment (0.82 ± 0.5 mN/mm) was slightly and
significantly greater than that at the beginning (0.73 ± 0.03 mN/mm), and, therefore, the basic reactivity of the arterial
contractile apparatus was preserved throughout the 4-h experimental
period.

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Fig. 3.
Responses of large PAs to 40 min of hypoxia. Each point is mean ± SE of change in (delta) wall tension developed to 5 different stretch
tensions during course of exposure; n = 12 arteries. A: no added
PGF2 .
B: 25% effective concentration
(EC25) of
PGF2 .
C: 50% effective concentration
(EC50) of
PGF2 .
D: 75% effective concentration
(EC75) of
PGF2 . Phase 1 constriction is
observed at 1 min, but subsequent response varies with imposed stretch
and active wall tensions. See text for further discussion.
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Fig. 4.
Responses of small PAs to 40 min of hypoxia. Each point is mean ± SE of delta wall tension developed to 5 different stretch tensions
during course of exposure; n = 12 arteries. A: no added
PGF2 .
B:
EC25 of
PGF2 .
C:
EC50 of
PGF2 .
D:
EC75 of
PGF2 . Phase 1 constriction is
observed at 1 min, but subsequent response varies with imposed
mechanical stretch and active wall tensions. See text for further
discussion.
|
|
Overall, a clear general pattern was revealed. All the response curves
have an early or phase 1 constriction during the first minute
irrespective of the stretch tension or concentration of PGF2
. This was followed by
variable phase 2 dilation at 5 min, which was greater with increased
stretch wall tension and somewhat enhanced by increasing
PGF2
. After 5 min, there was a
marked contrast between the phase 3 constrictor responses evident when
the stretch tensions were equivalent to 30 mmHg or less and the
sustained dilator response observed for wall tensions was equivalent to
50 mmHg or greater. Thus, for equivalent transmural pressures of 30 mmHg or less, the responses were characterized as triphasic and
predominantly constrictor, whereas for pressures of 50 mmHg or greater,
the responses were biphasic, with a prolonged phase 2 dilation. These
responses were influenced by
PGF2
such that for both large
and small arteries the greatest phase 3 constriction and the greatest
phase 2 dilation occurred when the
EC25 of
PGF2
was used.
More detailed analysis of these responses revealed that whereas for
large and small arteries, the responses at 1 min were constrictor when
the stretch tension was 30 mmHg or less, at greater stretch tensions,
the response was often not significantly different from zero,
particularly for large arteries. Furthermore, the small arteries reveal
that the phase 1 responses were significantly more positive with all
concentrations of PGF2
than
when it was zero. By 5 min for both large and small arteries, most of the responses were significantly less than those at 1 min when the
concentration of PGF2
was
EC50 or
EC75. By 40 min, at the end of the
hypoxic exposure, the responses for both large and small arteries were
significantly dilator for stretch tensions equivalent to 50 or 100 mmHg
compared with the constrictor responses with stretch tensions
equivalent to 5, 15, or 30 mmHg.
To quantitate the constrictor or dilator form of the responses, the
areas under the delta wall tension curves were calculated and are shown
for large and small arteries in Table 2.
For both large and small arteries and at all
PGF2
concentrations, the
dilator responses observed for equivalent transmural pressures of 50 or
100 mmHg were significantly different from the constrictor responses
observed for those arteries exposed to equivalent transmural pressures
of 30 mmHg or less. Also in both large and small arteries, the greatest
constrictor response was observed with the combination of stretch
tension equivalent to 30 mmHg and active tension due to
EC25 of
PGF2
, and the greatest dilator
responses were observed with the combination of stretch tension
equivalent to 50 mmHg and active tension due to
EC25 of
PGF2
.
Because there are no striking differences between large and small
arteries in this study, the results from all arteries have been
combined for each combination of active and stretch tensions, and the
results are shown in a contour plot (Fig.
5). Statistical analysis of these data
confirm the significance of the stretch tension as the major
determinant of the predominantly dilator response when the equivalent
transmural pressure is 50 mmHg or greater and also reveals that
EC25 of
PGF2
supports greater constrictor responses at stretch wall tensions equivalent to 30 mmHg
and greater dilator responses at a stretch wall tension equivalent to
50 mmHg.

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Fig. 5.
Contour plot summarizing influence of mechanical stretch and active
tensions on overall response to hypoxia of all arteries tested. Stretch
wall tension is expressed as equivalent transmural pressure of imposed
mechanical stretch tension. Active wall tension is
ECx of
PGF2 used to exert active
preconstriction. Contours indicate area (in
mN · min · mm 1)
under curves calculated as in Table 2 but combined for large and small
PAs. There is a sharp transition from constrictor (Cons) to dilator
(Dilation) response as mechanical stretch tension equivalent is changed
from 30 to 50 mmHg. The more subtle influence of preconstrictor
PGF2 is also revealed by peak
and trough corresponding to constrictor and dilator responses at
EC25 to
EC50 of
PGF2 .
|
|
Myogenic tone with mechanical stretch.
The results from both studies in large and small arteries are
summarized in Fig. 6. During normoxia, when
the stretch tension was equivalent to 30 mmHg, there were no
significant differences in the wall tension throughout the 40 min
whether the normal perfusate or the relaxing solution was present. This
result indicates that stretch corresponding to a wall tension of 30 mmHg or less is not associated with the development of myogenic tone
and that the wall tension is entirely passive. In contrast, when the
stretch wall tension was equivalent to 50 mmHg, the addition of the
relaxing solution was associated with significant decreases in wall
tension throughout the 40 min of normoxia in both large and small
arteries. The addition of hypoxia to the relaxing solution did not
further alter the wall tension. The reductions in wall tension observed
with the relaxing solution at increased stretch in this study
correspond closely to the dilations that were observed during hypoxia
at increased mechanical stretch in the main study. These results
suggest that active myogenic tone develops in arteries subjected to
wall tensions equivalent to 50 mmHg or more and that both this myogenic
tone and phase 3 hypoxic constriction are abolished when hypoxia is imposed.

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Fig. 6.
Summary of results of studies of myogenic tone with varying stretch
wall tension. A and
B: small arteries
(n = 6) with stretch tensions of 30 and 50 mmHg, respectively. C and
D: large arteries
(n = 6) with stretch
tensions of 30 and 50 mmHg, respectively. Horizontal dashed line,
starting baseline. Values are means ± SE. For both small and large
PAs, when stretch tension was 30 mmHg
(A and
C), removal ( ) of
Ca2+ and addition (+) of
papaverine (Pap) did not significantly alter wall tension, and,
therefore, imposed tension was entirely passive and myogenic tone was
absent. When mechanical stretch tension was increased to 50 mmHg
(B and
D), addition of relaxing solution
significantly reduced wall tension, and this reduction was the same
under normoxic and hypoxic (Hypox) conditions. Therefore, stretch wall
tensions of 50 mmHg or more are associated with generation of myogenic
tone, and final wall tension has a passive and a small active
component.
|
|
 |
DISCUSSION |
The present studies have shown that variations in mechanical stretch
and active wall tensions applied to isolated large and small rat PAs
determine the overall form of the response to 40 min of hypoxia.
Stretch wall tension was identified as the principal variable; when the
applied tension was equivalent to a transmural pressure of 30 mmHg or
less, the response was predominantly constrictor, and when the
equivalent transmural pressure was 50 mmHg or greater, the response was
predominantly dilator. Active preconstriction by
PGF2
had a more subtle
influence such that constrictor or dilator responses were enhanced when
PGF2
was present in small
(EC25) or moderate
(EC50) concentrations. For both
the large and small PAs, the most active and sustained constrictor responses were obtained when the stretch applied was equivalent to 15 or 30 mmHg combined with preconstriction with
EC25 or
EC50 of
PGF2
. These observations
therefore confirm the hypothesis that HPV is a property of all
pulmonary arteries irrespective of size, a conclusion consistent with
the observations that hypoxic constriction is observed in vascular
smooth muscle cells isolated from large or small PAs (15, 21).
Although the general constrictor or dilator nature of the overall
hypoxic response, represented by the areas under the curves (Table 2,
Fig. 5), is the most obvious result, the details of the responses
throughout the 40 min of hypoxia (Figs. 3 and 4) reveal that the
initial, transient (first 1 min of hypoxia), phase 1 constriction is
present in all preparations and is generally followed by a phase 2 dilation (at 5 min of hypoxia). The principal differences between the
responses are therefore due to the phase 3 constrictor response. The
dilator phase 2 at lower stretch tensions is seen only as a dip in an
otherwise continuous constrictor response. But at the higher stretch
tensions, the phase 3 constriction is totally abolished so that the
dilator response more than reverses the entire initial constriction.
These data therefore confirm the suggestions of others (1, 12) that two
constrictor phases separated by a dilator phase can be observed in the
hypoxic response of isolated rat PAs. But different investigators have
reported inconsistencies in these phases.
The apparent contradictions are of two sorts. The first is that many
early investigators (8, 18, 25, 32) tested the hypoxic responses in rat
PAs with only 5- to 10-min exposures to hypoxia, probably because the
response was poorly sustained. Those studies have therefore
investigated only the phase 1 constrictor responses, and although these
responses are less sensitive to the stretch forces applied to the
arteries and the pharmacological properties reported may play a role in
the initial response to HPV, there is little evidence that the results
are useful for the interpretation of the prolonged HPV responses of
whole lungs. For example, several studies (1, 8, 10, 20, 22, 23, 25,
31) have demonstrated that removal of the endothelium or inhibition of
nitric oxide synthase abolishes or attenuates the phase 1 constriction
in isolated arteries and has little or no effect on phase 3 constriction. But nitric oxide synthase inhibition enhances the HPV
responses of intact lungs in vivo (7, 27) or in vitro (9) without
changing the character of the response, and therefore the phase 3 constrictor responses are probably more representative of the
physiologically (and clinically) relevant HPV response. This outcome
remains controversial because others (11, 30) have concluded that phase
3 is endothelium dependent in isolated PAs, which Ward and Robertson
(30) attribute to an endothelium-derived contracting factor. Although
endothelial products are clearly important modulators of the final
response to hypoxia, there is uncertainty about whether any of the
phases are specifically endothelium dependent (17).
The second type of apparent contradiction concerns the phase 3 response
that different authors (1, 10, 12) have reported to be absent (Fig.
7A),
slow and delayed (Fig. 7B), or
partially merged (Fig. 7C) with the
phase 1 response. Analysis of these and other reports reveals that the
weak, delayed, or absent phase 3 responses were observed when stretch
tensions ranged from 0.7 to 3.0 g, whereas the strongest responses,
those reported by Leach et al. (14), were observed at the lowest
stretch tension employed by these authors, which was equivalent to a
transmural pressure of 30 mmHg. The data from the present studies
(Fig. 2) demonstrate that forces of <500 mg for large arteries
and 300 mg for small arteries correspond to transmural pressures in the
physiological range, whereas forces > 700 mg for large arteries and
500 mg for small arteries correspond to pulmonary hypertension. Thus we
hypothesize that the characteristics of each of the published reports
can now be understood primarily in terms of the stretch tensions
applied in preparing the arteries. For most reported studies, the
stretch wall tensions were equivalent to severe pulmonary hypertension, often >100 mmHg, and these are conditions known to inhibit HPV in
lungs (2, 4).

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Fig. 7.
Reported hypoxic responses of isolated PAs record a consistent phase 1 constrictor response but a variable phase 3 response relative to
baseline (horizontal dotted line). A:
phase 1 is succeeded by full phase 2 dilation, and phase 3 response is
absent (10). B: phase 1 is succeeded
by phase 2 dilation, but a phase 3 constriction develops late (1).
C: response is constrictor throughout,
with phase 1 and 3 constrictor responses overlapping and phase 2 dilation appearing as a notch between them (12).
|
|
A remaining source of variability is associated with the practice of
preconstriction before testing with hypoxia; a wide range of doses and
different agents have been employed, and it is clear that not all
agents are equivalent in arteries of different sizes. Our choice of
PGF2
was based on the studies
of Leach et al. (14), who reported that, in Cummin Sprague Europe rats,
the dose-response curves for small and large arteries were more similar with PGF2
than with
norepinephrine or serotonin. However, we observed in Wistar rats that
although the maximum response to KCl was similar in both arteries, the
maximum response to PGF2
in the
small arteries was about one-half of that in the large arteries. It
should also be noted that the increasing doses of PGF2
were not randomized in the
present studies, and although the responses to incremental doses were
in the ranges expected from the dose-response curves developed in the
second preliminary study, nevertheless changes in responsivity to
PGF2
cannot be ruled out. The
present observations with PGF2
were consistent for both large and small arteries, and we suggest not
only that constrictor and dilator responses to hypoxia were more active in the presence of small or moderate concentrations of a
vasoconstrictor agent but also, under the right conditions of stretch
tension, that a sustained constrictor response to hypoxia was observed with no added constrictor agent.
The phase 2 dilation may be interpreted in two ways. If phase 1 and 3 constrictions are the only active responses, then phase 2 will vary as
the relative speed of onset and persistence of these constrictor
responses alter. Conversely, if phases 1-3 are all active
responses, then the strength of the dilation is an additional
independent variable. Either view can satisfy the present observations,
but recent investigations support active mechanisms for all three
phases. Under this hypothesis, phase 1 constriction coincides with
increased intracellular Ca2+
attributed to partial depolarization, permitting
Ca2+ entry (28) and
Ca2+-induced
Ca2+-release from sarcoplasmic
reticulum stores (26, 29). Phase 2 dilation corresponds to a reuptake
of sarcoplasmic Ca2+ through the
activity of sarcoplasmic reticulum pumps (29). Phase 3 is least
understood but seems most consistent with increased force
sensitization where contraction increases while
intracellular Ca2+ remains
constant (26, 33). Although it remains for future studies to clarify
the precise nature of the mechanisms responsible for the
phases, this synthesis provides a useful foundation. On this basis, the
hypoxic dilation associated with excessive mechanical stretch wall
tensions is attributable to a loss of force sensitization.
HPV in intact lungs, both in vivo and in vitro, is characterized by a
sustained constrictor response, and it appears desirable to select
conditions for the study of HPV in isolated PAs that are consistent
with a similar form of response. The mechanical stretch forces imposed
on the arterial segments are composed of both passive and myogenic
active components and should therefore not exceed the equivalent of an
intravascular pressure of 30 mmHg. For large arteries in the present
work, that coincided with forces less than or equal to the lowest wall
tension consistent with a maximal response to 75 mM KCl (475 mg), but
in small arteries, the optimum force for the hypoxic response (250 mg)
was significantly less than that observed for the maximum KCl (356 mg).
To ensure that consistently constrictor responses to hypoxia are
present, the safest course may be to select a stretch force that is
substantially lower, perhaps 50-70%, of that at which the maximal
response to KCl is observed.
In summary, the present work has systematically examined the influence
of active and stretch (passive + myogenic) wall tensions on the
responses to hypoxia of isolated large and small rat PAs. The studies
demonstrated that although the transient phase 1 constrictor response
was relatively insensitive to wall tension, the phase 3 constrictor
response was so critically determined by it that a maximal constrictor
response was observed at a force equivalent to a transmural pressure of
30 mmHg or less, and the constrictor response was replaced by a dilator
response when the equivalent stretch transmural pressure was 50 mmHg or
greater. The responses were most active when the arteries were
preconstricted by EC25 or
EC50 of
PGF2
. The present observations
not only define the conditions required for isolated PAs to
reproduce more closely the HPV response observed in intact lungs
but also serve to reconcile many previously apparently contradictory results.
 |
ACKNOWLEDGEMENTS |
We acknowledge the technical assistance provided by Q. C. Meng in
the conduct of these studies.
 |
FOOTNOTES |
This work was supported by National Institute of General Medical
Sciences Grant GM-29628.
Address for reprint requests: B. E. Marshall, Center for Research in
Anesthesia, 781 Dulles, Hospital of the Univ. of Pennsylvania,
Philadelphia, PA 19104.
Received 15 December 1997; accepted in final form 9 September
1998.
 |
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