Division of Pulmonary and Critical Care Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21224
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ABSTRACT |
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In isolated porcine pulmonary arterioles with endothelium, intraluminal diameter measured at a transmural pressure of 20 mmHg decreased spontaneously from 233 ± 11 to 171 ± 12 µm in 135 min. This intrinsic constriction was not prevented by indomethacin, tetraethylammonium, or superoxide dismutase. Indomethacin plus NG-nitro-L-arginine methyl ester caused initial constriction and BQ-123 or BQ-123 plus BQ-788 caused initial dilation, but these treatments did not prevent subsequent progressive constriction. In pulmonary arterioles with endothelium exposed to calcium-free conditions and pulmonary arterioles without endothelium, the intraluminal diameter measured at a transmural pressure of 20 mmHg was constant at 239 ± 16 and 174 ± 7 µm, respectively. Thus the spontaneous development of tone in isolated pulmonary arterioles required extracellular calcium and resulted from 1) time-independent smooth muscle contraction caused by mechanisms intrinsic to smooth muscle and 2) time-dependent contraction caused by decreasing activity of endothelium-derived relaxing factors other than nitric oxide, vasodilator prostaglandins, and hyperpolarizing factors acting on calcium-dependent potassium channels or increasing activity of endothelium-derived contracting factors other than endothelin-1, vasoconstrictor prostaglandins, and superoxide anions. Further investigation is indicated to identify these unknown mechanisms and determine their role in pulmonary vasoreactivity.
endothelium-derived relaxing factor; endothelium-derived contracting factor; nitric oxide; prostaglandins; endothelin-1; superoxide anions; extracellular calcium concentration; pig
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INTRODUCTION |
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VASOMOTOR RESPONSES having the greatest impact on pulmonary vascular resistance and ventilation-perfusion matching are thought to occur in small, distal "resistance" pulmonary arteries rather than in large, proximal "conduit" arteries; therefore, isolated small pulmonary arteries are being used with increasing frequency to study the mechanisms of pulmonary vasoreactivity, and the characteristics of these vessels are becoming better appreciated (14, 22, 23, 27, 28). Recent studies (24, 25, 27) in isolated cat and pig pulmonary arteries 200-600 µm in diameter revealed that these resistance vessels developed intrinsic tone, a phenomenon that is not typical of larger conduit pulmonary arteries. Because baseline tone and the manner in which it is generated can exert profound effects on vasomotor responses (1, 3, 20, 33), the mechanisms by which isolated small pulmonary arteries develop intrinsic tone could be important determinants of vasoreactivity in this tissue.
In general, development of intrinsic tone could involve mechanisms intrinsic to vascular smooth muscle, endothelium, or both. For example, tone could develop because a progressive decrease in the activity of endothelium-derived relaxing factors (EDRFs) unmasked a contraction generated by mechanisms intrinsic to smooth muscle. Alternatively, a progressive increase in the activity of endothelium-derived contracting factors (EDCFs) could cause otherwise quiescent smooth muscle to contract. In isolated systemic arteries where intrinsic tone develops routinely and has long been used as a criterion of preparation acceptability (19, 21, 34), only a few studies have been reported, and these yielded inconsistent results. In coronary arteries, Rubanyi (32) found that development of intrinsic tone depended on the endothelium and elevation of transmural pressure (Ptm), whereas Cohen et al. (6) found that intrinsic tone was attenuated by the endothelium. In cerebral arterioles, intrinsic tone was decreased by reductions in Ptm and increased by inhibitors of nitric oxide (NO) synthase (NOS) (19, 34). To our knowledge, the only experiments investigating the mechanisms of intrinsic tone in small pulmonary arteries were those of Madden et al. (27), who demonstrated a requirement for extracellular calcium.
We performed the present study to determine the magnitude and
mechanisms of intrinsic tone in porcine pulmonary arteries < 300 µm
in diameter. Our results demonstrate that intrinsic constriction in
these vessels was time dependent and quantitatively significant, achieving ~35% of maximum possible vasoconstriction at 135 min under
control conditions and ~70% of maximum vasoconstriction when
modulation by NO and vasodilator prostaglandins was blocked. In
addition, our results confirm the dependence of this response on
extracellular calcium and suggest that the initial spontaneous tone was
generated by mechanisms intrinsic to smooth muscle and modulated by
endothelium-derived NO and endothelin (ET)-1, whereas subsequent
progressive tone development was due to altered activity of
endothelium-derived vasoactive factors other than NO, prostaglandins, hyperpolarizing factors acting on calcium-dependent potassium channels,
ET-1, or superoxide anions (O2·). Further investigation is warranted to identify these unknown mechanisms and determine their role in pulmonary vasoreactivity.
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METHODS |
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Preparation of isolated pulmonary arterioles. All protocols and procedures were approved by the Animal Care and Use Committee in accordance with National Institutes of Health guidelines. Male pigs (20-25 kg) were anesthetized with ketamine (30 mg/kg im) followed by pentobarbital sodium (12.5 mg/kg iv). After exsanguination via the femoral arteries, the lungs were rapidly removed and placed in ice-cold Krebs-Ringer bicarbonate solution containing (in mM) 118.3 NaCl, 4.7 KCl, 1.2 MgSO4, 1.2 KH2PO4, 2.5 CaCl2, 25.0 NaHCO3, and 11.1 glucose. Under a dissecting microscope, pulmonary arterioles ~100-150 µm in diameter and 1 mm long were isolated from seventh-generation branches of intrapulmonary arteries. The vessels were placed in a microvascular chamber, cannulated at one end with a glass micropipette, and secured with 12-0 nylon monofilament suture. Krebs-Ringer bicarbonate solution was infused slowly through the pipette until the arteriole was completely filled. The other end of the vessel was then cannulated with a second micropipette prefilled with Krebs-Ringer bicarbonate solution. Both cannulas were connected to a reservoir that could be raised or lowered to control Ptm. Ptm was measured with a pressure transducer positioned at the level of the vessel lumen. In the microvascular chamber, the vessels were superfused with Krebs-Ringer bicarbonate solution (total volume 100 ml) gassed with 16% O2-5% CO2-balance N2 (pH 7.35-7.45), maintained at 37°C, and recirculated except as noted in Experimental protocols. The chamber was placed on the stage of an inverted microscope (Nikon TMS-F) connected to a video camera (Panasonic CCTV camera). The vascular image was projected onto a video monitor, and the intraluminal diameter (ID) was continuously measured with a video dimension analyzer (Living Systems Instrumentation, Burlington, VT). The ID and Ptm were recorded with a recorder (Gould, Cleveland, OH). In some vessels, endothelial cells were disrupted by gently rubbing the intraluminal surface with a steel wire (70 µm in diameter). These vessels were then perfused with 2 ml of air bubbles followed by 2 ml of Krebs-Ringer bicarbonate solution (perfusion pressure < 5 mmHg) before being mounted in the microvascular chamber.
Experimental protocols. Initially,
isolated pulmonary arterioles were allowed to equilibrate in the
microvascular chamber for 20 min at a
Ptm of 5 mmHg.
Ptm was then increased to 20 mmHg in 5-mmHg steps at 5- to 7-min intervals and thereafter held constant. Measurements of the ID at Ptm of
20 mmHg (ID20) began 5 min after the Ptm was increased to 20 mmHg
(time 0) and continued until the end
of the experiment. This Ptm was
chosen because it approximated the normal mean pulmonary arterial
pressure in the pig (37). To assess viability, the vessels were exposed
to KCl (60 mM) at 20 min and the thromboxane
A2 agonist U-46619
(108 M) followed by ACh
(10
6 M) at 50 min. In each
case, the agonists were washed out of the microvascular chamber by 20 min of nonrecirculating superfusion with fresh Krebs-Ringer bicarbonate
solution after the responses had stabilized (5-10 min). Thus,
during the first 75 min of the experiment, the superfusate was
recirculated for 35 min and not recirculated for 40 min. In most
arterioles, KCl was administered again at the end of the experiment. In
some untreated control arterioles with (Endo+;
n = 6) and without
(Endo
; n = 6) endothelium, KCl
and U-46619-ACh were administered only at the end of the experiment. Control Endo+ (n = 19) and Endo
(n = 8) arterioles, which
received no other treatment, were considered acceptable if
1) the contractile responses
(decreased ID20) to KCl and
U-46619 were
30% of the immediately preceding control
ID20 and
2) the dilator responses (increased
ID20) to ACh were
50 (Endo+
vessels) or
0% (Endo
vessels) of the U-46619 response.
Experimental Endo+ arterioles were continuously exposed to one of the
following treatments from the time of mounting in the microvascular
chamber: 1)
Ca2+-free Krebs-Ringer bicarbonate
solution containing EGTA
(103 M;
n = 6);
2) indomethacin
(10
5 M;
n = 6);
3) indomethacin
(10
5 M) plus
NG-nitro-L-arginine
methyl ester (L-NAME; 3 × 10
5 M;
n = 6);
4) indomethacin
(10
5 M) plus
L-NAME (3 × 10
5 M) + L-arginine
(10
4 M;
n = 6);
5)
L-arginine
(10
4 M;
n = 5);
6) tetraethylammonium chloride (TEA;
5 × 10
3 M;
n = 4);
7) charybdotoxin (CTX;
10
7 M;
n = 4);
8) BQ-123 (3 × 10
6 M;
n = 6);
9) BQ-788 (3 × 10
6 M;
n = 6);
10) BQ-123 (3 × 10
6 M) plus BQ-788 (3 × 10
6 M;
n = 6); and
11) superoxide dismutase (SOD; 150 U/ml; n = 5), where
n is the number of animals. We also
determined the effects of L-NAME
(3 × 10
5 M)
administered 75 min into the experiment to Endo+
(n = 9) and Endo
(n = 5) arterioles pretreated with
indomethacin (10
5 M).
Acceptance criteria on viability testing with KCl and U-46619-ACh for
the experimental arterioles were the same as for the control arterioles
except that 1) the responses to KCl
and U-46619-ACh were not required in Endo+ arterioles exposed to
calcium-free conditions and 2) the
dilator responses to ACh in Endo+ arterioles exposed to indomethacin
plus L-NAME had to be
0% of
the U-46619 response.
In additional experiments, we measured the contractile responses to
progressively increasing superfusate concentrations of ET-1
(1011 to
10
8 M) in untreated control
Endo+ arterioles (n = 6) and Endo+
arterioles treated with BQ-123 (3 × 10
6 M;
n = 4), BQ-788 (3 × 10
6 M;
n = 5), or both BQ-123 and BQ-788
(n = 9). After each administration of
ET-1, sufficient time was allowed for the vessels to achieve a stable
ID20. These experiments were
performed either at the end of the time-course experiments described
above or in separate studies.
Drugs. ACh, indomethacin,
L-NAME,
L-arginine, TEA, BQ-788, and
bovine copper-zinc SOD were purchased from Sigma (St. Louis, MO); CTX
was from from Peptides International (Louisville, KY); BQ-123 and ET-1
were from American Peptide (Sunnyvale, CA); and U-46619 was from Cayman
Chemical (Ann Arbor, MI). Stock solutions of ACh,
L-NAME,
L-arginine, TEA, CTX, SOD, and
ET-1 were prepared each day in deionized water and stored at 4°C
until used. Indomethacin was dissolved each day in an aqueous solution
of NaHCO3 (2.35 × 102 M). BQ-123 and BQ-788
were dissolved in absolute ethanol, stored at
60°C, and, on
the day of the experiment, diluted as necessary with deionized water.
Concentrations are expressed as final molar concentrations in the superfusate.
Data analysis. Student's t-test or one- or two-factor analyses of variance were used for statistical analysis as appropriate. When significant F-ratios were obtained with analysis of variance, Dunnett's test was used to compare individual means with a control value, and least significant differences were used to compare means at a specific time or level of treatment. P values < 0.05 were assumed to indicate significance. Values are means ± SE.
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RESULTS |
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Table 1 shows the average
responses to KCl and U-46619-ACh administered at the beginning of the
experiment to test vessel viability. KCl (60 mM) elicited vigorous
contractile responses in Endo+ and Endo arterioles but not in
Endo+ arterioles exposed to calcium-free conditions. During exposure to
U-46619, ACh (10
6 M) caused
vasodilation in all groups except the Endo
arterioles and Endo+
arterioles exposed to L-NAME or
calcium-free conditions. When viability testing was delayed until the
end of the experiment, KCl and U-46619 caused contraction in Endo+ and
Endo
arterioles, but ACh reversed the U-46619-induced
contraction only in Endo+ arterioles (data not shown).
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As shown in Fig. 1,
ID20 in Endo+ arterioles decreased
progressively and spontaneously from 233 ± 11 µm at
time 0 to 171 ± 12 µm at the end
of the experiment (135 min). This intrinsic contraction did not occur
in arterioles exposed to calcium-free conditions in which
ID20 remained at its baseline
value of 238 ± 10 µm. If this value represented the
maximum (fully relaxed) ID20 in
these vessels and the minimum (fully constricted)
ID20 value was ~35 µm (Table
1), intrinsic vasoconstriction achieved ~35% of maximum possible
vasoconstriction at 135 min.
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Although ID20 at
time 0 was the same in untreated Endo+
arterioles and Endo+ arterioles exposed to calcium-free conditions, it
was markedly diminished in Endo vessels (169 ± 21 µm).
This contraction of Endo
arterioles, which began immediately
after endothelial denudation under the dissecting microscope, remained constant throughout the experiment. As a result,
ID20 was the same in Endo
and Endo+ vessels at 135 min. Similar results were obtained in Endo+
and Endo
arterioles in which viability testing with KCl and
U-46619-ACh was delayed until the end of the experiment (Fig.
2).
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Indomethacin (105 M), an
inhibitor of cyclooxygenase (9, 38), did not alter the constrictor
responses to KCl or U-46619, the dilator responses to ACh, or the
development of intrinsic tone (Table 1, Fig.
3). Treatment with both indomethacin and L-NAME (3 × 10
5 M), an inhibitor of NOS
(2, 11), abolished the vasodilator response to ACh (Table 1) and
decreased ID20 at
time 0 but did not prevent further
intrinsic vasoconstriction (Fig. 3). In these vessels,
ID20 decreased from 194 ± 17 µm at 0 min to 99 ± 21 µm at 135 min. When
10
4 M
L-arginine, a substrate for NOS
(11), was included as pretreatment with indomethacin and
L-NAME, vasodilator responses to
ACh were restored (Table 1) and intrinsic constriction was markedly
attenuated (Fig.
4A).
In Endo+ arterioles treated with
L-arginine alone, ACh caused
vasodilation (Table 1) and development of intrinsic tone was abolished
(Fig. 4B).
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TEA, a nonspecific potassium-channel antagonist (8, 17), did
not alter intrinsic vasoconstriction (Fig.
5A).
CTX, a specific inhibitor of calcium-dependent potassium channels (8,
17), did not alter ID20 at 0 min,
but it enhanced intrinsic tone at 0-20 min and inhibited intrinsic
tone at 70-135 min (Fig. 5A). Neither agent altered the vasoconstrictor response to KCl at the beginning of the experiment (Table 1); however, CTX had abolished this
response by the end of the experiment (Fig.
5B).
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Figure 6A
directly compares Endo+ arterioles pretreated with indomethacin plus
L-NAME with Endo
arterioles. Development of intrinsic tone was greater in Endo+
arterioles treated with indomethacin plus
L-NAME. Figure
6B shows the effects of delayed
administration of L-NAME (75 min) in Endo+ and Endo
arterioles pretreated with indomethacin.
L-NAME caused contraction in
Endo+ arterioles but had no effect in Endo
arterioles.
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As shown in Fig. 7, ET-1
(1011 to
10
8 M) caused a
concentration-dependent contraction in Endo+ arterioles, which was
blocked by the ETA-receptor
antagonist BQ-123 (3 × 10
6 M) but not by the
ETB-receptor antagonist BQ-788 (3 × 10
6 M). In
arterioles treated with both BQ-123 and BQ-788, high concentrations of
ET-1 caused a slight vasoconstriction, but this contractile response
was still markedly reduced compared with that in control Endo+
arterioles.
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Figure 8 shows the effects of ET-1-receptor
antagonists on the time course of
ID20. BQ-123 (3 × 106 M) increased
ID20 at 0 min (262 ± 23 µm);
however, it did not alter subsequent intrinsic vasoconstriction
(ID20 = 225 ± 28 µm at 135 min). BQ-788 (3 × 10
6
M) did not alter ID20 at 0 min
(256 ± 15 µm) but increased subsequent intrinsic vasoconstriction
(ID20 = 134 ± 21 µm at 135 min). Treatment with BQ-123 and BQ-788 together
increased both ID20 at
time 0 (284 ± 15 µm) and
subsequent constriction (ID20 = 178 ± 14 µm at 135 min).
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SOD (150 U/ml) did not affect the time course of
ID20 (Fig.
9).
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DISCUSSION |
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Control Endo+ pulmonary arterioles constricted in response to KCl and
U-46619 and dilated in response to ACh after precontraction with
U-46619 (Table 1). Arterioles subjected to endothelial denudation maintained vigorous constrictor responses to KCl and U-46619 but did
not exhibit dilator responses to ACh. These results indicate that
contractile function and receptor-linked signal transduction were
intact in both Endo+ and Endo arterioles, whereas endothelial function was intact in only Endo+ arterioles.
As shown in Fig. 1, Endo+ arterioles exhibited progressive constriction, with ID20 decreasing from 233 ± 11.0 µm at 0 min to 170 ± 12.0 µm at 135 min. Most of this constriction occurred during the first 75 min of the experiment. Because superfusate was not recirculated for 40 of these 75 min, the decrease in ID20 is unlikely to have been caused by a time-dependent change in superfusate composition, such as increasing osmolality due to evaporation. Rather, it must have resulted from factors or mechanisms intrinsic to the vessel.
The time course of ID20 in Endo+
arterioles differed markedly from that in Endo arterioles, which
were already constricted at 0 min
(ID20 = 169 ± 21.4 µm) and
did not exhibit a significant change in
ID20 thereafter. Constriction of
Endo
arterioles at time 0 is
more likely to have resulted from endothelium removal than vascular
smooth muscle damage due to the denudation procedure because
ID20 decreased rapidly in response
to KCl and U-46619 and returned promptly to baseline on washout of
these agonists, indicating normal smooth muscle function. Furthermore,
the differences between Endo+ and Endo
arterioles were present
when viability testing was delayed until the end of the experiment
(Fig. 2), indicating that they were not the result of transient
exposures to KCl, U-46619, or ACh. We conclude that the progressive
development of tone seen in control Endo+ pulmonary arterioles was the
product of two influences: 1) smooth
muscle contraction already established at time
0 by mechanisms intrinsic to smooth muscle and
2) endothelial antagonism of smooth
muscle contraction, which was present at time
0 and progressively diminished thereafter.
In Endo+ arterioles exposed to calcium-free conditions, vasoconstrictor responses to KCl were absent (Table 1). This finding is consistent with a previous work (5) that indicated that KCl causes smooth muscle contraction primarily via membrane depolarization and calcium influx through voltage-dependent calcium channels. In contrast, vasoconstriction induced by U-46619, although diminished, was still present in these arterioles (Table 1). This finding is consistent with previous results (7) that suggested that vasoconstrictor responses to U-46619 utilize calcium released from intracellular stores. As shown in Fig. 1, Endo+ arterioles exposed to calcium-free conditions remained dilated throughout the experiment (ID20 = 240 ± 16.1 µm), confirming previous studies (13, 15, 27) in both pulmonary and systemic arteries. These results indicate that the smooth muscle or endothelial mechanisms responsible for intrinsic constriction in Endo+ pulmonary arterioles required extracellular calcium.
Studies of the mechanisms responsible for intrinsic tone in systemic
arteries have not been conclusive. Some (6, 30) suggested that
constriction was caused by mechanisms intrinsic to smooth muscle,
whereas others (19, 31) concluded that endothelial mechanisms played a
major role. Because short periods of decreased Ptm reduced intrinsic tone in
cerebral arteries (34), the mechanisms responsible for intrinsic
constriction could be similar to those mediating myogenic responses
(29). For example, depolarization and calcium influx in smooth muscle
due to stretch-induced activation of ion channels (12, 29) could
explain the constriction we observed at time
0 in Endo arterioles. Alternatively, stretch could act on endothelium, altering the release of EDRFs such as NO and
vasodilator prostaglandins or EDCFs such as ET, vasoconstrictor prostaglandins, and superoxide anions (12, 18, 29, 32). To evaluate the
roles of EDRFs and EDCFs, we determined the effects of
1) indomethacin, an inhibitor of
cyclooxygenase, which catalyzes production of prostaglandins from
arachidonic acid; 2)
L-NAME, an inhibitor of NOS,
which catalyzes production of NO from
L-arginine; 3) TEA and CTX, antagonists of
calcium-dependent potassium channels, which may mediate the
vasodilating effects of endothelium-derived hyperpolarizing factors
(EDHFs); 4) BQ-123 and BQ-788,
antagonists of ETA and
ETB receptors, respectively; and
5) SOD, which catabolizes superoxide anions.
As shown in Fig. 3, indomethacin did not alter development of intrinsic
tone in pulmonary arterioles. This cyclooxygenase inhibitor has been
shown to block prostaglandin production and contractile responses to
arachidonic acid in systemic vascular tissue at concentrations less
than or equal to that used in the present study
(105 M) (9, 35, 38).
Furthermore, indomethacin did not alter either the contractile
responses to KCl and U-46619 or the dilator responses to ACh (Table 1),
indicating the absence of nonspecific effects. We conclude that
prostaglandins did not mediate intrinsic constriction in isolated
pulmonary arterioles.
To evaluate the role of the EDRF NO, we determined the effects of L-NAME, an inhibitor of NOS (2, 11). As shown in Table 1, ACh caused a marked dilation in pulmonary arterioles treated with indomethacin but not in arterioles treated with indomethacin plus L-NAME. ACh-induced vasodilation was restored when L-arginine was added to indomethacin plus L-NAME. Vasoconstrictor responses to KCl and U-46619 were present in all three groups. These results indicate that L-NAME inhibited NO synthesis and did not impair contractile function.
The effects of L-NAME on the time course of ID20 are shown in Fig. 3. At 0 min, Endo+ arterioles treated with L-NAME plus indomethacin were markedly constricted compared with both untreated and indomethacin-treated Endo+ arterioles; however, L-NAME did not prevent the subsequent progressive decline in ID20. These results indicate that although NO modulated vasomotor tone at time 0, a decrease in NO-mediated relaxation did not cause the progressive vasoconstriction that occurred from 0 to 135 min.
The addition of L-arginine
(104 M) to indomethacin
plus L-NAME (Fig.
4A) or treatment with
L-arginine alone (Fig.
4B) abolished intrinsic tone. These
results suggest that NO production in these vessels was limited by
L-arginine concentration and
that enhancement of NO production could overcome the mechanisms
responsible for intrinsic constriction.
EDHF is an unknown vasodilator that may act by opening calcium-dependent potassium channels in smooth muscle (8); however, 5 mM TEA, which blocks these channels and the effects of EDHF (8, 17), did not alter intrinsic vasoconstriction in pulmonary arterioles (Fig. 5A). CTX, a more specific antagonist of calcium-dependent potassium channels (8, 17), did inhibit intrinsic constriction (Fig. 5A), but unlike TEA, it also abolished the vasoconstrictor responses to KCl at the end of the experiment (Fig. 5B). These results suggest that the effects of CTX were due to nonspecific toxicity. Thus our data provide no evidence that intrinsic vasoconstriction was caused by decreasing activity of an EDHF acting on calcium-dependent potassium channels.
As illustrated in Fig. 6A, intrinsic
constriction was more severe in Endo+ arterioles treated with
L-NAME plus indomethacin than in
untreated Endo arterioles. This difference could be due to
smooth muscle vasodilator mechanisms in Endo
arterioles or unopposed EDCF activity in Endo+ arterioles treated with
L-NAME plus
indomethacin. To assess the role of smooth muscle type II NOS, which
may have been induced by cytokines or other factors (36, 39), we
administered L-NAME at 75 min to
Endo
and Endo+ arterioles pretreated with indomethacin (Fig.
6B).
L-NAME had no effect
in Endo
arterioles but caused a marked constriction in Endo+
arterioles. These results indicate that NOS was absent or inactive in
smooth muscle.
It seems more likely that the differences shown in Fig. 6A were due to increasing EDCF activity. The magnitude of tone generated by these contractile factors was impressive. Assuming again that arterioles exposed to calcium-free conditions were maximally relaxed (Fig. 1) and that fully constricted arterioles had an ID20 of ~35 µm (Table 1), intrinsic vasoconstriction in the absence of modulation by NO and vasodilator prostaglandins (Fig. 6A) achieved ~70% of the maximum potential constriction at 135 min. The EDCFs involved could be ET or superoxide anions (18, 26) but not vasoconstrictor prostaglandins. To evaluate these possibilities, we studied arterioles exposed to ET antagonists or SOD.
The ETA-receptor antagonist BQ-123
abolished the constrictor responses to ET-1, whereas the
ETB-receptor antagonist BQ-788 (3 × 106 M) had no
effect (Fig. 7). When these antagonists were administered together,
contraction to ET-1 was still inhibited, but the vessels were more
contracted than after BQ-123 alone. These results indicate that
ET-1-induced constriction of porcine pulmonary arterioles was mediated
by ETA rather than
ETB receptors. In contrast,
previous results (16) in rat intrapulmonary arteries suggested that
ET-1-induced vasoconstriction was mediated by both
ETA and
ETB receptors. This difference may
be due to species-dependent differences in receptor density (10).
Indeed, in the presence of BQ-123, ET-1 caused vasodilation in pig
pulmonary arterioles (Fig. 7), suggesting that activation of
ETB receptors mediated
vasodilation, perhaps by increasing release of NO from the endothelium
as previously suggested (16).
BQ-123 alone or in combination with BQ-788 increased ID20 at 0 min, suggesting that ETA receptor-mediated constriction contributed to the initial vasomotor tone; however, neither BQ-123 nor BQ-788 prevented subsequent progressive constriction (Fig. 8), indicating that this response was not due to activation of ETA or ETB receptors. Indeed, BQ-788 accelerated the decline in ID20, suggesting that ETB receptors exerted a vasodilatory influence that modulated progressive constriction.
At concentrations of 100-200 U/ml, SOD eliminated or markedly reduced release of superoxide anions from vascular tissues (4, 40). In preliminary studies of isolated porcine coronary arterioles exposed to anoxia-reoxygenation, we found that SOD (150 U/ml) caused a 70% decrease in superoxide anion release as assessed by lucigenin-enhanced chemilumenescence (data not shown). However, this concentration of SOD had no effect on the time course of ID20 in pulmonary arterioles (Fig. 9), indicating that intrinsic tone was not due to superoxide anions. These results do not eliminate the possibility that intrinsic constriction was mediated by H2O2, the product of SOD-induced catabolism of superoxide anions; however, in this case, SOD would be expected to enhance intrinsic constriction, which did not occur.
The development of intrinsic tone in pig pulmonary arterioles was
similar to that described by Madden et al. (27) in cannulated cat
pulmonary arterioles 200-600 µm in diameter. The ID measured at
a Ptm of 10 mmHg decreased
spontaneously by 23.9 ± 4.4% over 60-90 min. In contrast,
Leach et al. (23) were unable to demonstrate intrinsic tone in rat
pulmonary arterioles of similar size. In this study, resting isometric
tension, adjusted to be equivalent to a
Ptm of 30 mmHg, was not altered by
exposure to calcium-free conditions or several vasodilator agents. This
disparity may be due to differences in species, preparation,
experimental conditions, or other factors. For example, it is likely
that rat arterioles 200 µm in diameter were obtained from more
proximal loci in the pulmonary vasculature than in pig or cat
arterioles of the same diameter. Endothelium-dependent vasodilation was
reduced or absent in rat pulmonary arterioles (23), whereas Endo+
arterioles that did not dilate at least 50% in response to
106 M ACh were excluded in
our study. The higher resting Ptm
and the isometric conditions used in the rat arterioles may have
activated mechanisms not operative at lower constant levels of
Ptm. Such considerations also lead
to important questions about the role of intrinsic pulmonary arteriolar
tone in the intact animal. Further investigation will be necessary to
obtain answers.
In summary, our results demonstrate that development of intrinsic tone in isolated pulmonary arterioles was dependent on time and extracellular calcium concentration. Initially, tone was generated by mechanisms intrinsic to vascular smooth muscle and modulated by endothelium-derived NO and ET-1 but not by prostaglandins; however, subsequent progressive constriction could not be explained by these mechanisms. Rather, our results indicate that progressive development of intrinsic tone was caused by 1) decreased activity of an EDRF other than NO, vasodilator prostaglandins, and EDHFs acting via calcium-dependent potassium channels or 2) increased activity of an EDCF other than vasoconstrictor prostaglandins, ET-1, or superoxide anions. Additional research is indicated to determine the identity of the unknown mechanisms responsible for intrinsic tone, their role in pulmonary vasoreactivity, and whether they are unique to pulmonary vessels.
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ACKNOWLEDGEMENTS |
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This study was supported by National Heart, Lung, and Blood Institute Grant HL-51912 and a research grant from the American Lung Association (to Q. Liu).
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FOOTNOTES |
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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: Q. Liu, Div. of Pulmonary and Critical Care Medicine, The Johns Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (E-mail: qliu{at}welchlink.welch.jhu.edu).
Received 19 November 1998; accepted in final form 8 February 1999.
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REFERENCES |
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1.
Bennie, R. E.,
C. S. Packer,
D. R. Powell,
N. Jin,
and
R. A. Rhoades.
Biphasic contractile response of pulmonary artery to hypoxia.
Am. J. Physiol.
261 (Lung Cell. Mol. Physiol. 5):
L156-L163,
1991
2.
Bishop-Bailey, D.,
S. W. Larkin,
T. D. Warner,
G. Chen,
and
J. A. Mitchell.
Characterization of the induction of nitric oxide synthase and cyclo-oxygenase in rat aorta in organ culture.
Br. J. Pharmacol.
121:
125-133,
1997[Abstract].
3.
Bonnet, P.,
J. A. Argibay,
and
D. Garnier.
Contractile responses to hypoxia of isolated rings from the left branch of rabbit pulmonary artery.
Fundam. Clin. Pharmacol.
3:
115-126,
1989[Medline].
4.
Brandes, R. P.,
M. Barton,
K. M. Philippens,
G. Schweitzer,
and
A. Mugge.
Endothelial-derived superoxide anions in pig coronary arteries: evidence from lucigenin chemiluminescence and histochemical techniques.
J. Physiol. (Lond.)
500:
331-342,
1997[Abstract].
5.
Chen, X. L.,
and
C. M. Rembold.
Phenylephrine contracts rat tail artery by one electromechanical and three pharmacomechanical mechanisms.
Am. J. Physiol.
268 (Heart Circ. Physiol. 37):
H74-H81,
1995
6.
Cohen, R. A.,
K. M. Zitnay,
R. M. Weisbrod,
and
B. Tesfamariam.
Influence of the endothelium on tone and the response of isolated pig coronary artery to norepinephrine.
J. Pharmacol. Exp. Ther.
244:
550-555,
1988[Abstract].
7.
Drummond, R. M.,
and
R. M. Wadsworth.
Contraction of the sheep middle cerebral, pulmonary and coronary arteries initiated by release of intracellular calcium.
J. Auton. Pharmacol.
14:
109-121,
1994[Medline].
8.
Feletou, M.,
and
P. M. Vanhoutte.
Endothelium-derived hyperpolarizing factor.
Clin. Exp. Pharmacol. Physiol.
23:
1082-1090,
1996[Medline].
9.
Fiscus, R. R.,
and
D. C. Dyer.
Effects of indomethacin on contractility of isolated human umbilical artery.
Pharmacology
24:
328-336,
1982[Medline].
10.
Fukuroda, T.,
M. Kobayashi,
S. Ozaki,
M. Yano,
T. Miyauchi,
M. Onizuka,
Y. Sugishita,
K. Goto,
and
M. Nishikibe.
Endothelin receptor subtypes in human versus rabbit pulmonary arteries.
J. Appl. Physiol.
76:
1976-1982,
1994
11.
Guo, J. P.,
T. Murohara,
M. Buerke,
R. Scalia,
and
A. M. Lefer.
Direct measurement of nitric oxide release from vascular endothelial cells.
J. Appl. Physiol.
81:
774-779,
1996
12.
Harder, D. R.
Pressure-induced myogenic activation of cat cerebral arteries is dependent on intact endothelium.
Circ. Res.
60:
102-107,
1987[Abstract].
13.
Harder, D. R.,
R. Gilbert,
and
J. H. Lombard.
Vascular muscle cell depolarization and activation in renal arteries on elevation of transmural pressure.
Am. J. Physiol.
253 (Renal Fluid Electrolyte Physiol. 22):
F778-F781,
1987
14.
Harder, D. R.,
J. A. Madden,
and
C. Dawson.
Hypoxic induction of Ca2+-dependent action potentials in small pulmonary arteries of the cat.
J. Appl. Physiol.
59:
1389-1393,
1985
15.
Hayashida, N.,
K. Okui,
and
Y. Fukuda.
Modulation of vascular dynamics by spontaneous contraction of smooth muscle in the isolated carotid artery of the rat.
Jpn. J. Physiol.
37:
183-196,
1987[Medline].
16.
Higashi, T.,
T. Ishizaki,
K. Shigemori,
T. Nakai,
S. Miyabo,
T. Inui,
and
T. Yamamura.
Pharmacological heterogeneity of constrictions mediated by endothelin receptors in rat pulmonary arteries.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L287-L293,
1997
17.
Hille, B.
Structure and function.
In: Ionic Channels of Excitable Membranes (2nd ed.). Sunderland, MA: Sinauer Associates, 1992, chapt. 16, p. 423-444.
18.
Katusic, Z. S.,
and
P. M. Vanhoutte.
Superoxide anion and endothelial regulation of arterial tone.
Semin. Perinatol.
15:
30-33,
1991[Medline].
19.
Kimura, M.,
H. H. Dietrich,
and
R. G. Dacey, Jr.
Nitric oxide regulates cerebral arteriolar tone in rats.
Stroke
25:
2227-2234,
1994[Abstract].
20.
Kovitz, K. L.,
T. D. Aleskowitch,
J. T. Sylvester,
and
N. A. Flavahan.
Endothelium-derived contracting and relaxing factors contribute to hypoxic responses of pulmonary arteries.
Am. J. Physiol.
265 (Heart Circ. Physiol. 34):
H1139-H1148,
1993
21.
Kuo, L.,
W. M. Chilian,
and
M. J. Davis.
Coronary arteriolar myogenic response is independent of endothelium.
Circ. Res.
66:
860-866,
1990[Abstract].
22.
Leach, R. M.,
T. P. Robertson,
C. H. Twort,
and
J. P. Ward.
Hypoxic vasoconstriction in rat pulmonary and mesenteric arteries.
Am. J. Physiol.
266 (Lung Cell. Mol. Physiol. 10):
L223-L231,
1994
23.
Leach, R. M.,
C. H. Twort,
I. R. Cameron,
and
J. P. Ward.
A comparison of the pharmacological and mechanical properties in vitro of large and small pulmonary arteries of the rat.
Clin. Sci. (Colch.)
82:
55-62,
1992[Medline].
24.
Liu, Q.,
L. A. Shimoda,
J. S. K. Sham,
and
J. T. Sylvester.
Spontaneous tone of isolated pulmonary arterioles (Abstract).
Circulation
96:
I-246,
1997.
25.
Liu, Q.,
C. M. Wiener,
and
N. A. Flavahan.
Superoxide and endothelium-dependent constriction to flow in porcine small pulmonary arteries.
Br. J. Pharmacol.
124:
331-336,
1998[Abstract].
26.
Luscher, T. F.,
C. M. Boulanger,
Y. Dohi,
and
Z. H. Yang.
Endothelium-derived contracting factors.
Hypertension
19:
117-130,
1992[Abstract].
27.
Madden, J. A.,
A. al-Tinawi,
E. Birks,
P. A. Keller,
and
C. A. Dawson.
Intrinsic tone and distensibility of in vitro and in situ cat pulmonary arteries.
Lung
174:
291-301,
1996[Medline].
28.
Madden, J. A.,
C. A. Dawson,
and
D. R. Harder.
Hypoxia-induced activation in small isolated pulmonary arteries from the cat.
J. Appl. Physiol.
59:
113-118,
1985
29.
Meininger, G. A.,
and
M. J. Davis.
Cellular mechanisms involved in the vascular myogenic response.
Am. J. Physiol.
263 (Heart Circ. Physiol. 32):
H647-H659,
1992
30.
Nyborg, N. C.,
and
P. J. Nielsen.
The level of spontaneous myogenic tone in isolated human posterior ciliary arteries decreases with age.
Exp. Eye Res.
51:
711-715,
1990[Medline].
31.
Rinaldi, G.,
and
D. Bohr.
Endothelium-mediated spontaneous response in aortic rings of deoxycorticosterone acetate-hypertensive rats.
Hypertension
13:
256-261,
1989[Abstract].
32.
Rubanyi, G. M.
Endothelium-dependent pressure-induced contraction of isolated canine carotid arteries.
Am. J. Physiol.
255 (Heart Circ. Physiol. 24):
H783-H788,
1988
33.
Schachinger, V.,
and
A. M. Zeiher.
Quantitative assessment of coronary vasoreactivity in humans in vivo. Importance of baseline vasomotor tone in atherosclerosis.
Circulation
92:
2087-2094,
1995
34.
Takayasu, M.,
and
R. G. Dacey, Jr.
Spontaneous tone of cerebral parenchymal arterioles: a role in cerebral hyperemic phenomena.
J. Neurosurg.
71:
711-717,
1989[Medline].
35.
Toda, N.
Mechanisms of contracting action of oxyhemoglobin in isolated monkey and dog cerebral arteries.
Am. J. Physiol.
258 (Heart Circ. Physiol. 27):
H57-H63,
1990
36.
Villamor, E.,
F. Perez-Vizcaino,
T. Ruiz,
J. C. Leza,
M. Moro,
and
J. Tamargo.
Group B streptococcus and E. coli LPS-induced NO-dependent hyporesponsiveness to noradrenaline in isolated intrapulmonary arteries of neonatal piglets.
Br. J. Pharmacol.
115:
261-266,
1995[Abstract].
37.
Will, D. H.,
C. H. Frith,
I. F. McMurtry,
and
D. J. MacCarter.
Effects of hypertension and hypoxemia on arterial metabolism and structure.
Adv. Exp. Med. Biol.
22:
185-203,
1972[Medline].
38.
Wohlfeil, E. R.,
and
W. B. Campbell.
25-Hydroxycholesterol enhances eicosanoid production in cultured bovine coronary artery endothelial cells by increasing prostaglandin G/H synthase-2.
Biochim. Biophys. Acta
1345:
109-120,
1997[Medline].
39.
Wong, H. R.,
J. D. Finder,
K. Wasserloos,
C. J. Lowenstein,
D. A. Geller,
T. R. Billiar,
B. R. Pitt,
and
P. Davies.
Transcriptional regulation of iNOS by IL-1 in cultured rat pulmonary artery smooth muscle cells.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L166-L171,
1996
40.
Zweier, J. L.,
R. Broderick,
P. Kuppusamy,
S. Thompson-Gorman,
and
G. A. Lutty.
Determination of the mechanism of free radical generation in human aortic endothelial cells exposed to anoxia and reoxygenation.
J. Biol. Chem.
269:
24156-24162,
1994