Department of Physiology and Biophysics, Indiana University School of Medicine, Indianapolis, Indiana 46202
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ABSTRACT |
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The principal stimulus that evokes pulmonary hypertension is chronic alveolar hypoxia. Pulmonary hypertension is associated with remodeling of the vessel walls, involving hypertrophy and hyperplasia of pulmonary arterial smooth muscle (PASM) and a concomitant increase in the deposition of connective tissue, resulting in increased wall thickness. The purpose of the present study was to determine the effect of hypoxia-induced hypertension on the structure and function of PASM. Experiments were designed to determine whether hypoxia-induced pulmonary hypertension is associated with alterations in PASM: 1) reactivity to a variety of agonists, 2) contractile protein proportions and isoforms, and 3) structural properties. Young adult male rats were made hypoxic by lowering the fraction of inspired O2 (10%) for 14 days. Pulmonary arterial segments were isolated and dose-response curves to various agonists (high K+, norepinephrine, serotonin, angiotensin II, and adenosine) were generated. Gel electrophoresis was used to measure changes in the relative amounts of actin or myosin and of myosin heavy chain (MHC) isoforms. Structural changes were correlated with the pharmacological and biochemical data. Hypoxia-induced pulmonary hypertension caused a general decreased reactivity, an increase in the proportion of nonmuscle to muscle MHC isoforms in PASM, and an increase in arterial wall thickness with PASM hypertrophy or hyperplasia.
myosin heavy chain isoforms; arterial smooth muscle; decreased contractility; hypertrophy; hyperplasia
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INTRODUCTION |
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PULMONARY HYPERTENSION is a primary event leading to the development of right ventricular failure and respiratory failure. The principal stimulus that evokes pulmonary hypertension is chronic alveolar hypoxia. Local alveolar hypoxia causes an acute vasoconstriction and increased arterial resistance. When the hypoxia affects the whole lung rather than discrete regions and is chronic rather than acute, pulmonary hypertension results. Remodeling of the vessel walls, including an increase in wall thickness by hypertrophy and hyperplasia of the pulmonary arterial smooth muscle (PASM) and an increase in the deposition of connective tissue, accompanies hypoxia-induced pulmonary hypertension (3, 21, 24, 30). Whether the vascular wall remodeling precedes or occurs concomitantly with the pulmonary hypertension has not been established. Reid (24) has suggested that although vasoconstriction plays a role in some types of hypertension, ultimately, the structural changes and not smooth muscle contractility per se are responsible for the luminal reduction and maintenance of high vascular pressures.
Griffith et al. (7) reported the results of an investigation of pulmonary arterial muscle contractility and pulmonary arterial wall mechanics that support Reid's (24) suggestion. Specifically, no increase in contractility was found in PASM from rats exposed to hypoxia for 14 days. In fact, although no change was found in the tension-velocity relationship, a decrease in the active stress-developing ability was actually found in the hypertensive muscle. However, increases in passive stiffness and wall thickness were found, supporting the idea that increases in wall thickness, and connective tissue in particular, contribute to hypoxia-induced pulmonary hypertension. Despite the fact that an increase in PASM contractility is not responsible for the maintenance of hypertension, the smooth muscle may still be the cell type primarily involved in the development and indirectly in the maintenance of hypoxia-induced pulmonary hypertension. Changes in smooth muscle cell function in response to hypoxia or high transmural pressure may be responsible for the vascular wall thickening and increased connective tissue. For instance, a smooth muscle phenotypic change from a contractile to a synthetic cell type could be responsible for the majority of structural changes in the vessel wall and maintenance of the high vascular resistance. A less contractile phenotype would be accompanied by either no change or a decrease in vascular smooth muscle reactivity. Such phenotypic changes are known to occur in other models of hypertrophic or hyperplastic vascular smooth muscle (1) and could explain the source of the increased connective tissue in the walls of the hypertensive vessels.
The purpose of the present study was to determine whether chronic hypoxia-induced pulmonary hypertension alters the sensitivity and/or reactivity of the PASM to a variety of agonists, including high K+ and several physiological agonists {norepinephrine (NE), angiotensin II (ANG II), serotonin [5-hydroxytryptamine (5-HT)], and adenosine (Ado)}, and to determine whether myosin heavy chain (MHC) isoform shifts correlate with the decreased ability of PASM to develop active stress in hypoxia-induced pulmonary hypertension.
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METHODS |
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Adult (10-wk-old) Sprague-Dawley rats weighing 300-324 g were made hypoxic by lowering the fraction of inspired O2 (10%). Rats were placed in polycarbonate chambers, and N2 mixed with room air (Venturi system) was allowed to flow into the chambers at 1.5 l/min. The animals were made hypoxic by lowering the fraction of inspired O2 to 15% for 24 h and then to 10% for the remainder of the 14 days. O2 and CO2 tensions were measured with Beckman O2 (model C-2) and CO2 (model LB-1) analyzers. O2 tension was monitored continuously with a Beckman OM-15 O2 meter. PO2 was kept in the range of 75-80 Torr, and PCO2 was kept in the range of 0.3-0.5 Torr. The chambers were opened every other day for feeding and cleaning to prevent ammonia buildup. Control animals were maintained under similar conditions but were allowed to breathe room air.
After the 14-day hypoxic-exposure period, the rats were given an intraperitoneal injection of pentobarbital sodium (50 mg/kg body weight) and exsanguinated by section of the abdominal aorta. After the rats were anesthetized, blood samples for hematocrit determination were collected by insertion of a heparinized capillary tube into the area of the nasal canthus. Right ventricular pressure (PRV), an index of pulmonary arterial pressure, was measured in the rats after 2 wk of chronic hypoxia and in age-matched control animals. To measure PRV, a polyethylene catheter (inner diameter 23 µm, outer diameter 38 µm) was introduced into the right jugular vein and manipulated into the right ventricle. Pressure traces were displayed on a Hewlett-Packard patient monitor (model 78353B) that provided digital presentation of systolic, diastolic, and mean pressures. The heart and lungs were removed, and the hearts were dissected free of all atrial tissue. The ratio of the right ventricle to the left ventricle plus septum weights was determined and used as an index of the relative degree of right heart hypertrophy.
The lungs were placed in cold (4°C) Earle's balanced salt solution (EBSS; 2.4 mM CaCl2, 0.8 mM MgSO4, 5.4 mM KCl, 116.4 mM NaCl, 0.9 mM Na2HPO4, 5.5 mM D-glucose, 26.2 mM NaHCO3 and 0.03 mM phenol red sodium). The two main branches of the pulmonary artery were excised and cleaned of all visible parenchyma and connective tissue under a dissecting microcope.
Some pulmonary arterial branches were prepared for morphometrics. In these cases, the pulmonary artery and its two major branches were isolated as described above except that the dissection was performed in Ca2+-free, EGTA-containing Krebs-Henseleit buffer (to ensure that the smooth muscle was fully relaxed). Under the dissecting microscope, each branch was cut into two ring segments such that four ring segments were obtained from each rat. The arteries were held on end and kept patent with surgical steel posts while they were completely immersed in fixative (2.5% glutaraldehyde and 1.5% paraformaldehyde in 0.1 M cacodylate buffer at 4°C) for 2-4 h. Vessel segments were then postfixed in osmium tetroxide, dehydrated with alcohol, and embedded in Polybed 812. One-micrometer-thick sections were stained with toluidine blue.
Photographs were taken of each arterial segment through the ×2.5 objective of a Zeiss Ultraphot microscope. This magnification allowed visualization of the entire vessel cross section in one visual field. These whole section micrographs (final magnification ×45) were utilized to measure arterial cross-sectional area (CSA) and luminal diameter. All other light micrographs were taken through the ×10 objective, resulting in visualization of only a segment of the vessel wall at a time. These micrographs (final magnification ×208) were then arranged in a collage to recreate the entire vessel cross section and were used to measure wall thickness, medial thickness, adventitial thickness, and number of smooth muscle cell layers.
The ×45 micrographs were placed on a digitizing pad, and the inner and outer perimeters of each arterial wall were traced. With the use of planimetery software (SIGMASCAN, Jandel Scientific), two CSAs were calculated based on the inner and outer perimeters. The CSA of the vessel wall was then calculated by subtracting the inner CSA (i.e., lumen) from the outer CSA. A similar method was used to calculate the CSA of the medial layer.
The ×208 micrographs were arranged as a collage to reconstruct the images of the transverse arterial cross sections. These collages were analyzed with a drafter's T square to mark the vessel wall at points separated from each other by 90° angles. The T square was then rotated 45°, and four additional points of intersection were marked. At each of these eight points, the following measurements were made: total wall thickness, adventitial thickness, and number of smooth muscle cell layers. This process resulted in eight different independent measurements of each of these parameters for each vessel cross section. The eight measurements of each parameter were averaged for each section. Then the results, in mean values for each of the four sections of the same artery, were averaged together to give the mean wall thickness, medial thickness, adventitial thickness, and number of smooth muscle layers for each rat. The mean medial and adventitial thicknesses are expressed as percentages of the mean total wall thickness for each artery. Wall-to-lumen ratios were calculated from the wall thickness at each point divided by the mean luminal radius of the same section. Mean values from all the hypertensive arterial segments were then compared with those from control preparations.
For the reactivity studies, main pulmonary arterial rings ranging from 1.5 to 2.0 mm in diameter and from 2.5 to 3.5 mm in length were gently threaded onto horizontally oriented, fixed-position surgical steel rods (300 µm in diameter, 5 mm in length) located in the lower third of 20-ml volume glass muscle baths. Once anchored to this wire, a second wire of the same dimensions but suspended from a force transducer (Grass Instruments, Quincy, MA) that was connected to a chart recorder (Gould) was introduced into each lumen above the first wire. Each muscle bath contained 10 ml of EBSS aerated with 95% O2-5% CO2. The outer jacket of each muscle bath was connected to a circulating water bath maintained at 37°C.
In experiments in which ring segments denuded of endothelium were
required, the endothelium was mechanically removed by threading the
vessel onto a lightly sanded surgical steel rod and gently rotating the
vessel twice in each direction. The integrity of the endothelium was
tested by applying ACh (5 × 106 M) to NE-contracted
arterial rings. Relaxation in response to ACh indicated insufficient
removal of the endothelium, and data from such arterial rings were not
used. In those experiments in which tachyphylaxis was a concern, this
test of endothelial integrity was performed after washout of the last
test drug used in the experiment.
Once mounted, the arterial rings were equilibrated for 1 h at a resting tension ranging from 5.5 to 8.0 mN. The optimal resting tension for maximal active tension development (Po) was previously determined to be 7.0 ± 0.8 mN for normal rat pulmonary arterial segments (25). Because the rats used for that previous study were much older (400-500 g), this same optimal resting tension was verified in normal rat pulmonary arterial segments from rats of the same age (300-325 g) as those used for all experiments described in this study. In addition, a rather extensive preliminary study of the active versus resting tension relationship in hypoxia-induced hypertensve rat arterial rings was carried out. The hypertensive muscle has a broader resting versus active tension curve plateau region compared with the control muscle. Changing resting tension in the 7- to 16-mN range had no significant effect on active tension production in the hypertensive preparations. These findings are similar to those reported by Griffith et al. (7). Therefore, an optimal resting tension value of 7.0 mN was used as the baseline tension for all experiments unless otherwise specified. Equilibration was followed by a maximal contraction with 80 mM KCl. The peak tension developed (in mN) was normalized to the CSA of the tissue (in mN/mm2).
At the end of the pharmacological experiments, the length and width (diameter) of each vessel segment were measured. The vessel segments were then blotted, and the wet weight was obtained. Because the density of the tissue is close to one (1 mg/1 mm3), the weight of the tissue in milligrams is an estimate of the volume of tissue in cubic millimeters (i.e., density = mass/volume). The volume divided by the width of the muscle is then approximately equal to the CSA across which tension is developed (17). Po was determined for each vessel segment used in the pharmacological experiments and was defined as the maximum tension developed in response to supramaximal stimulation with 80 mM KCl. As the amount of muscle increases with thicker or longer ring segments, the absolute tension developed is also greater. Therefore, Po must be normalized to CSA for comparative purposes: Po/CSA = tension (in mN) developed in response to 80 mM KCl/[volume (in mm3)/width (in mm)].
The high-K+ solution used to obtain Po was subsequently washed out, and the muscle was allowed to relax completely. Each ring was then exposed to an agonist (5-HT, NE, ANG II, or Ado) with different randomly applied or cumulative doses (depending on the agonist). Example tension versus time tracings are shown in Fig. 1. The magnitude of each response was normalized by expressing it as a percentage of the response to 80 mM KCl (%Po).
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Each agonist solution was prepared as a high-concentration
"stock" solution, divided into 1- to 2-ml aliquots, and then
frozen at 70°C. Serial dilutions of each stock solution were
made on the day of the experiment, providing a series of agonist
solutions that covered a range of several orders of magnitude (i.e.,
10
10 to
10
3 M). For agonists that
showed no tachyphylaxis (NE and Ado), it was possible to give additive
doses to a given vessel without washing between doses. Serial-dilution
solutions were added to the smooth muscle baths in small aliquots such
that after each dose was added, the final concentration of the agonist
was 10 times higher than the previous dose. For the other agonists
(5-HT and ANG II), a single dose-response protocol (i.e., washing
between doses) was utilized to ensure that tachyphylaxis of the muscle did not cause error in the dose-response curves generated.
NE stock solution was made by dissolving 42.25 g of NE HCl (mol wt
205.6; Arterenol, Sigma) in 50 ml of sterile 0.9% NaCl. One hour after washout of the
high-K+ solution, when tension
reached a steady baseline, aliquots of this solution were added to the
tissue bath in the following order of cumulative final doses:
1010,
10
9,
10
8,
10
7,
10
6,
10
5, and 5 × 10
5 M.
ANG II stock solution was made by dissolving 5 mg of ANG II (human
synthetic form acetate salt, mol wt 1,046.3; Sigma) in 5 ml of sterile
H2O. One hour after washout of the
high-K+ solution, when tension
reached a steady baseline, a single dose of ANG II was added to each
bath. Because ANG II was one of the agonists that resulted in
tachyphylaxis, a cumulative dose-response protocol was not used. For
the single dose-response experiments, aliquots of ANG II were added to
the tissue bath to provide one of the following final concentrations of
ANG II for each arterial ring:
106,
10
7, 2 × 10
8,
10
9,
10
10, and
10
11 M.
5-HT stock solution was made by dissolving 0.1937 g of 5-HT (creatinine
sulfate complex, mol wt 387.4; Sigma) in 3 ml of sterile H2O + 1 ml of EBSS. Then 0.100 ml
of 5 N NaOH was added to bring the solution to a pH close to 7.4 (i.e.,
within the pH range of the phenol red sodium indicator that is a
component of EBSS). One hour after washout of the
high-K+ solution, when tension
reached a steady baseline, aliquots of these solutions were added to
the tissue bath to create one of the following final concentrations of
5-HT in the tissue bath: 107,
10
6,
10
5, 2 × 10
5,
10
4, or
10
3 M.
Ado stock solution was made by dissolving 116.3 mg of Ado (hemisulfate
form, mol wt 316.3; Sigma) in 10 ml of sterile
H2O. One hour after washout of the
high-K+ solution, when tension
reached a steady baseline, vessels were precontracted with
105 M NE. As soon as the
response to NE reached a plateau, aliquots of the Ado solution were
added to the tissue bath in a sequential manner to reach cumulative
concentrations of Ado as follows:
10
8,
10
7,
10
6,
10
4, and
10
3 M.
KCl stock solution was made by dissolving 18.64 g of KCl (mol wt 58.44; Sigma) in 100 ml of sterile H2O and was stored at 4°C until used. For the initial maximal contraction in each pharmacological experiment, 320 µl of the KCl solution were added to each bath to give a final concentration of 80 mM KCl, previously shown to be more than sufficient to ensure a maximum isometric contraction (25).
To determine whether hypoxia-induced pulmonary hypertension or removal of the endothelium resulted in changes in PASM reactivity or sensitivity to various physiological agents, dose-response curves of the vasoconstrictors NE, ANG II, and 5-HT and to the vasodilator Ado were generated and compared.
In other experiments, the main pulmonary arterial rings were prepared
for MHC isoform and actin quantification. The main pulmonary arteries
were dissected as described for the isometric tension studies. Due to
the small absolute amount of muscle in rat pulmonary arteries, the two
main branches and the trunk were used, and the muscle tissue of
pulmonary arteries from four to five rats was combined for each sample.
The arterial tissue was frozen in liquid N2, pulverized, acetone dried,
desiccated under a low vacuum, and then stored at 70°C until
used. For each experiment, 0.6 mg of each sample was dissolved in 100 µl of SDS-gel dissociation medium. The mixture was heated at
100°C for 30 min and centrifuged, and the supernatant was then
applied to 5% acrylamide-0.75%
N,N'-methylene-bis-acrylamide (BIS; 1.50% BIS
for actin) gels with the buffer system of Porzio and Pearson (23). The
samples were subjected to electrophoresis along with
heavy-molecular-weight and BSA standards at 300 V and constant voltage
at 10°C for ~2-3 h for separation of actin and myosin and
for 5 h for MHC isoform separation. After electrophoresis, the gels
were stained with Coomassie blue, and myosin content was determined by
quantitative densitometric scanning.
Student's t-test was used when comparing any two mean values for the hypertensive and control data. All results are expressed as mean values ± SE, and P < 0.05 is indicative of mean values that are significantly different from one another. Differences between any two cumulative dose-response curves were demonstrated with a multiple (repeated)-measures ANOVA. For dose-response curves obtained from individual (rather than cumulative) dose-response experiments, a two-way ANOVA for independent measures was utilized. To compare any two mean values at a given dose, Student's t-test was used. Myosin isoform ratios were compared with one-way ANOVA followed by the Newman-Keuls test.
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RESULTS |
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The mean hematocrit for hypoxic rats (57.0 ± 1.0%;
n = 9) is significantly
greater than that of age-matched control rats (42.0 ± 1.5%;
n = 13;
P < 105). The right
ventricle-to-left ventricle plus septum weight ratio for hypoxic rats
(n = 24) is also significantly greater
than that for control rats (0.40 ± 0.01 and 0.25 ± 0.01, respectively; n = 18;
P < 10
6). The mean hypoxic
rat (n = 4)
PRV of 16.0 ± 0.7 mmHg is
significantly higher than the mean control rat
(n = 6)
PRV of 9.5 ± 0.3 mmHg (P < 0.00005).
Both hypertensive (n = 4) and control (n = 4) pulmonary arteries demonstrated a loose outer adventitial connective tissue layer and inner organized smooth muscle layers. But only the hypertensive arteries had a poorly organized layer of cells between the inner organized smooth muscle layers and the outer connective tissue layer. Enlarged views of histological sections (×208) of arteries are shown in Fig. 2. The increase in the number of smooth muscle cell layers and the presence of an amorphous layer with either nonmuscle or migrated smooth muscle cells in the hypertensive arteries are evident in these micrographs. Figure 2B shows palisading nuclei within the inner, more organized smooth muscle cell layers, suggesting that the smooth muscle cells were cut in cross section and lie parallel to the longitudinal axis of the vessels. The amorphous layer does not show such palisading nuclei, and the direction in which these cells are oriented could not be determined. None of the sections demonstrates any clear changes in the intima associated with hypoxia-induced hypertension. There was no evidence of intimal proliferation, and no atherosclerotic changes were seen with light microscopy.
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The mean CSA for control and hypertensive pulmonary arteries and other
morphometric values determined from light micrographs are shown in
Table 1. The mean CSA of hypertensive
pulmonary arteries measured from light micrographs is approximately
double the mean CSA of control pulmonary arteries. The mean
hypertensive pulmonary arterial wall thickness and adventitial and
medial thicknesses are all significantly greater than the mean control
thicknesses. Even when the medial thickness of the hypertensive
pulmonary arteries was measured without including the outer amorphous
muscle layer (media outer layer), the mean is significantly
greater than that of the control arteries. The mean number of smooth
muscle cell layers is increased, whereas the thickness of the cell
layers, excluding the outer amorphous layer, is unchanged in the
hypertensive pulmonary arteries relative to the control arteries. The
mean luminal radius is also not different between the hypertensive and
control arteries. Although there is a significant increase in the mean
adventitial thickness in the hypertensive pulmonary arteries, the
proportion (in percent) of the wall that is composed of adventitia in
the hypertensive arteries is not significantly different from that in
the control arteries. Similarly, the proportion of the medial thickness
relative to the wall thickness is unchanged in the hypertensive
arteries relative to that in the control arteries. Although the radius
of the lumen is not different, the wall thickness is increased in the
hypertensive pulmonary arteries as reported above, and, therefore, the
wall-to-lumen ratio in the hypertensive arteries is significantly
greater than that in the control arteries.
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Control vessels with and without endothelium responded to doses of NE 10
6 M with sustained
contractions for >1 h. Hypertensive vessels (n = 49) required a dose of
10
5 M NE to evoke a
sustained response. The NE dose-response curves for pulmonary arterial
preparations with and without endothelium are shown in Fig.
3A. There
is a general upward shift in the dose-response curve for pulmonary
arteries without endothelium (F = 23.987; P < 0.001). The NE
dose-response curves for hypertensive and control pulmonary arteries
obtained from single-dose experiments are shown in Fig.
3B. There is a general downward shift
in the hypertensive dose-response curve relative to the control curve
(F = 116.878; P < 0.001). The mean maximum
response of the hypertensive arteries (95.9 ± 8.4%Po;
n = 9) is significantly lower than the
mean control response to the same dose (133 ± 0.2%Po;
n = 6;
P < 0.05). The NE dose-response
curves for control pulmonary arteries (with endothelium) exposed to the
same range of NE doses as those used above but under acute normoxic and
hypoxic conditions are shown in Fig. 3C. The
PO2 measured in the hypoxic tissue
baths was 28.3 ± 1.2 Torr
(n = 4 arteries). Acute in vitro
hypoxia resulted in a general downward shift in the NE dose-response
curve relative to the control curve (F = 140.839; P < 0.001).
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The ANG II dose-response curves for pulmonary arteries with and without
endothelium are shown in Fig.
4A. There is a general upward shift in the dose-response curve for denuded vessels compared with control arteries (F = 23.987;
P < 0.001). The mean maximum response of the arteries without endothelium (70.2 ± 6.8%Po;
n = 10) occurred at
108 M ANG II and is
significantly greater than that of control arteries with endothelium
(29.6 ± 8.1%Po;
n = 6;
P < 0.05) at the same dose. The ANG
II dose-response curves for hypertensive and control pulmonary arteries
(both with endothelium) are shown in Fig.
4B. There is a general downward shift
in the hypertensive dose-response curve relative to the control curve,
as in the case of NE (F = 48.024;
P < 0.001). The mean maximum
response of the hypertensive arteries to ANG II (17.3 ± 3.0%Po;
n = 8) is significantly different from
that of the control arteries at the same dose (36.2 ± 9.3%Po; n = 5;
P < 0.05).
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The mean dose-response curves for 5-HT in pulmonary arteries with and without endothelium are shown in Fig. 5A. There is a general upward shift in the dose-response curve for vessels without endothelium (F = 47.34; P < 0.001). The mean maximum response to 5-HT is 95.6 ± 2.4%Po in the segments without endothelium (n = 3), which is significantly greater than the mean maximum response for control segments (46.3 ± 5.8%Po; n = 6; P < 0.05). The 5-HT dose-response curves for hypertensive and control pulmonary arteries (both with endothelium) are shown in Fig. 5B. There is no significant difference between the hypertensive and control dose-response curves for 5-HT (F = 3.33; P > 0.05). The mean maximum response of the hypertensive arteries to 5-HT (61.4 ± 8.9%Po; n = 3) is not significantly different from the mean control response at the same dose (53.0 ± 9.4%Po; n = 6; P > 0.05).
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No response to Ado could be elicited in vessels unless they were
actively precontracted. Arterial segments
(n = 4) at resting tension
did not respond to any dose of Ado. NE was chosen as the agonist for
precontracting the vessels because it provided a reproducible response
to a given dose and a relatively sustained plateau (i.e., >1 h for
doses > 106 M;
n = 21). Doses of
10
8 to
10
3 M Ado did not elicit
any response in the resting vessels but resulted in either contraction
or relaxation of NE-precontracted pulmonary arteries depending on the
Ado concentration as previously reported (26). At low doses of Ado, the
response varied from slight relaxation to slight contraction. At higher
doses of Ado, only relaxation occurred. The response to a given dose of
Ado was usually maintained regardless of whether it resulted in an increase or decrease in tension. Only control vessels displayed contraction in response to low doses of Ado.
The effect of endothelium removal on the response of pulmonary arterial
muscle to Ado has been previously reported (26). Briefly, there was a
slight contractile response at
106 M Ado in vessels with
intact endothelium (4.1 ± 2.2%Po;
n = 10; P < 0.05). No contractile response
to Ado was observed for rat pulmonary arteries without endothelium.
Vessels denuded of endothelium responded with relaxation over the range
of 10
7 to
10
3 M Ado. Higher doses of
Ado (
10
4 M) resulted in
relaxation of the same magnitude in both vessels with intact
endothelium and vessels without endothelium. The mean Ado dose-response
curves for hypertensive and control pulmonary arteries are shown in
Fig. 6. Unlike control arteries, no
contractile response to Ado was observed for hypertensive rat pulmonary
arteries. The hypertensive pulmonary arteries responded to Ado with
relaxation over the dose range of
10
8 to
10
3 M. At the highest dose
of Ado (10
3 M), the
hypertensive arteries did not demonstrate as much relaxation (
42.2 ± 4.9%; n = 5) as
the control arteries.
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The KCl dose-response curves from hypertensive and control pulmonary arteries are shown in Fig. 7. Arterial ring segments were exposed to KCl doses in the range of 20-120 mM, and the active responses are expressed as a percentage of the maximal response to 80 mM KCl. This method of normalization of the responses allows for comparison of the mean dose-response curves to ensure that when the rats were made hypertensive, the sensitivity and relative responsiveness to KCl were not altered. The curves are almost superimposable, and there is no significant difference in the response at any dose of KCl (P > 0.05). Tension-generating ability was determined by comparing responses normalized to the amount of smooth muscle (i.e., CSA) as shown in Fig. 8. The hypertensive pulmonary arterial muscle tension-generating ability is reduced compared with control preparations when normalized for the proportionate amount of muscle in the tissue CSA (P < 0.05).
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Analyses of densitometric scans of the 1.5% BIS-SDS-polyacrylamide gels showed that there was no difference in the amount of actin relative to the amount of total protein (i.e., peak areas/mg dry weight of total protein) in hypertensive compared with control myofibrillar protein (1,100 ± 120 and 988 ± 110 mm2/mg protein, respectively). Similarly, there is no difference in the actin-to-myosin ratios of hypertensive PASM (2.19 ± 0.12) and the control PASM (1.90 ± 0.14). Scans of the 0.75% BIS-SDS-polyacrylamide gels revealed four distinct myosin isoforms for both control and hypertensive pulmonary arterial muscles (20). The four MHCs have molecular masses of ~204, 200, 196-198, and 190 kDa (MHC204, MHC200, MHC196, and MHC190, respectively). Other investigators have identified four isoforms in other smooth muscles (5) and the latter two isoforms in nonmuscle cells (16). The relative amounts of these various isoforms in hypertensive and control pulmonary arterial muscles are compared in Table 2. The hypertensive and control ratios of MHC200 to MHC204 are not significantly different (P > 0.05). However, the MHC190+196/MHC200+204 ratio is higher in the hypertensive than in the control pulmonary arterial muscle (P < 0.05).
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DISCUSSION |
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Results of this study show that after 14 days of hypoxia there is an increase in the adventitial and medial thicknesses but no change in the percent contribution of the media to the wall thickness in rat pulmonary arterial walls. Similar results have been previously reported (7). Reid (24) found approximately a doubling in thickness of the arterial smooth muscle in all the muscular arteries as well as an extension of muscle into previously nonmuscular peripheral arteries. This doubling of muscle means a greater absolute amount of muscle. An increase in the mean number of layers of smooth muscle from 3.1 in control arteries to 5.2 in hypertensive pulmonary arteries, with no increase in the thickness of each muscle layer, as shown in this study, suggests smooth muscle hyperplasia has occurred at this stage (14 days of hypoxia). The luminal radius is not altered in the hypertensive arteries, but the adventitial layer is almost double that observed for control arteries. In addition, there is an increase in the number of cells in the adventitial layer close to the media. These cells are not organized into a layer with uniform thickness like the medial layers nor are the elastic fibers organized into a continuous concentric band, suggesting that either hyperplasia of smooth muscle is not occurring on the luminal side of the vessel but rather on the adventitial side, at least in the large hilar arteries utilized in this study, or nonmuscle cells such as fibroblasts are proliferating in the adventitia of hypertensive arteries.
A response to a given agonist depends on the number of receptors, affinity of the receptors, intracellular coupling mechanisms, and, ultimately, the number of tension-generating sites (i.e., actin and myosin interactions) in vascular smooth muscle. Normalizing the Po produced to the tissue CSA provides an index of the number of tension-generating sites per unit of wall CSA. However, this method of normalization does not take into consideration changes in vessel wall composition. A method of normalization that allows for comparisons of changes in reactivity to the specific agonist regardless of changes in wall structure is to express the response as a proportion of a maximal contraction for the preparation in response to membrane depolarization (%Po). Normalization of agonist responses to the magnitude of the maximal KCl response allows for determination of whether differences in the active responses to various agonists are specific to a particular agonist or are due to a general alteration in contractility.
The pulmonary arterial segments from both control and hypertensive rats
responded to the same range of NE doses (i.e.,
1010 to
10
4 M), with a peak
response at 10
5 to
10
4 M, in agreement with
that reported by other investigators (13). The general upward shift in
the NE dose-response curve for endothelium-denuded vessels is likely
simply due to a decrease in endothelium-derived relaxing factor because
there is increased reactivity of the PASM to all contractile agonists
(ANG II, NE, and 5-HT) investigated when the endothelium is rendered
nonfunctional. The fact that sustained tension in the hypertensive
vessels required a higher dose
(
10
5 M) than in control
vessels suggests that there may be a change in receptor function or a
coupling when the smooth muscle is made chronically hypoxic.
-Adrenergic receptors could play a greater role in hypertensive
compared with control rats, and, therefore, a higher dose of NE would
be required to overcome the relaxation effects of the
-receptors.
This idea is supported by the fact that, after 5 days to 2 wk of
hypoxia, pulmonary arteries show less vasoconstriction in response to
NE (12, 19, 22) and that the vasoconstriction is augmented with
-receptor blockade (12, 22). A decrease in ability to develop
tension relative to the CSA could be due to the loss of contractile
function in some of the smooth muscle cells and/or a
disproportionate increase in connective tissue. However, the NE
responses were normalized to Po,
and, therefore, the decrease in the absolute ability of the smooth
muscle to develop tension cannot account for the decrease in NE
reactivity observed. Therefore, a change in NE-receptor function, such
as receptor downregulation, must have occurred with the development of
pulmonary hypertension. A decreased vasoconstriction (decreased
vascular resistance) in response to NE has also been reported in
isolated lungs from chronically hypoxic rats (22).
A decrease in reactivity to NE was also observed in isolated pulmonary
arteries from control rats that were exposed to acute hypoxia (i.e., in
vitro hypoxia). This is possibly due to a generalized decrease in
reactivity in response to all agonists during acute hypoxia because
Lloyd (11) showed that hypoxic media decreased the response to
electrical stimulation, 5-HT, NE, ANG,
K+, and ACh without altering
resting tension. Harabin et al. (8) found a decrease in reactivity to
PGF2 and ANG II in isolated perfused pig lungs exposed to an inspired
PO2 < 30 Torr. But these
investigators found no change in reactivity to KCl and therefore
concluded that it was unlikely that the supply of ATP available for
contraction was limiting. A decrease in
PO2 results in membrane
depolarization and an increased voltage-dependent permeability to
Ca2+ (6). It is possible that,
because the membrane is already partly depolarized by hypoxia, the
amount of further depolarization due to other stimuli is limited, and
hence the magnitude of the response to stimuli other than hypoxia is
reduced. Another hypothesis for the decreased reactivity to NE is that
hypoxia may result in the release of mediators that bind
nonspecifically to and result in "heterologous" desensitization
of the adrenergic receptors (10). Or such mediators might cause
downregulation of the adrenergic receptors. Last, endothelium-derived
factors such as endothelium-derived relaxing factor (NO) or
endothelium-derived hyperpolarizing factor, reactive oxygen species, or
NO from smooth muscle might be released in greater quantity after
chronic hypoxia or a return to normoxia, thus compromising force
development in response to high K+
or contractile agonists. The mechanisms for the decrease in
responsiveness to NE during acute hypoxia and chronic hypoxia may or
may not be the same. Important to note is that the literature is not in complete agreement about the effect of chronic hypoxia-induced pulmonary hypertension on NE reactivity, but this is likely due to
differences in methodology. For example, McMurtry et al. (14) found an
increase in response to NE in isolated perfused lungs from rats with
chronic hypoxia-induced pulmonary hypertension. However, their data
were not normalized and reflected the responses of the entire pulmonary
circulation rather than those of isolated arterial muscle.
From the pilot studies with ANG II, it appeared that the peak response
occurred at an ANG II dose of
107 M, and higher doses
actually resulted in lower responses. Therefore, the range of doses was
set at 10
10 to
10
6 M to ensure that a
supramaximal dose was included for the purpose of demonstrating a
maximum peak. However, the great variability in the magnitude for the
responses of ANG II resulted in mean curves that do not have a clear
maximum over the range of doses tested. When several higher doses were
tested, the response was always submaximal. Similar dose ranges have
been used by other investigators (13). It is worth noting that McMurtry
et al. (13) did not use a supramaximal dose for ANG II, whereas Chand and Altura (2) did show a supramaximal response to ANG II at 10
7 M.
The ANG II dose-response curve of the hypertensive arteries is significantly shifted downward compared with the control curve, demonstrating a decrease in reactivity to ANG II in the arteries from rats exposed to chronic hypoxia. McMurtry et al. (13) also showed a decreased response to ANG II in both isolated perfused lungs and isolated main pulmonary arterial segments after exposure to chronic hypoxia. The possible explanations for the decrease in reactivity may be similar to those for NE, including heterologous (i.e., nonspecific) desensitization or receptor downregulation.
The 5-HT dose-response curve for the hypertensive PASM was not significantly different from that for the control PASM. 5-HT was the only agonist that did not lose its potency in eliciting a contraction as a result of chronic hypoxia. Downregulation of receptors with hypoxia may be receptor specific, or perhaps only certain intracellular coupling mechanisms are affected by chronic hypoxia.
Previously, Roepke et al. (26) reported that the predominant effect of Ado in the pulmonary circulation is vasodilation. Results of the present study show that hypertensive pulmonary arterial rings also relax in response to Ado. However, the relaxation induced by high doses of Ado was not as great in the hypertensive rings as in either endothelium-denuded or -intact control PASM rings. The decreased ability to relax in response to Ado may be contributing to the pulmonary hypertension.
Hypertensive rat pulmonary arteries show no difference in absolute maximum tension development (i.e., response to high K+) from control arteries. This is surprising at first glance because pulmonary hypertension is associated with hypertrophy of the smooth muscle. McMurtry et al. (13) demonstrated that the pressor response to KCl is diminished in both isolated perfused lungs and isolated main pulmonary arterial segments from rats with chronic hypoxia-induced pulmonary hypertension. Because morphometric measurements in this study showed that there is an increase in CSA of the medial layer, it is best to normalize the KCl responses to the CSA. The finding that the CSA is doubled in the hypertensive vessels is supported by the fact that other investigators (7, 24) have shown a similar doubling of CSA. Results of this study show that the mean Po normalized in this manner (i.e., Po/CSA) is decreased in the hypertensive PASM. A decreased Po/media CSA may be explained by an increase in the proportion of noncontractile to contractile smooth muscle (i.e., a phenotypic change) and/or a disproportionate increase in connective tissue in the hypertensive arteries.
Results of this study show the presence of four protein bands on SDS gels that closely correspond in molecular mass to those of myosin. The two densest bands (204 and 200 kDa) are the two forms of MHC (MHC200 and MHC204, respectively) consistently found in a variety of mammalian smooth muscles (17, 18, 27, 29). Although these two MHCs are present in most smooth muscle tissues, the ratio of the amount of MHC204 to MHC200 varies depending on the specific smooth muscle tissue type from which myosin is isolated (16, 29) and the animal's age (4, 15). Two other bands appear on the gels at the 190- and ~196- to 198-kDa positions. Degradation experiments have ruled out the possibility that the 190-kDa protein is a degradation product of MHC204 and MHC200. Eddinger and Murphy (4) reported an MHC of 190 kDa that they isolated "most readily" from uterine tissue of the guinea pig and mouse, and it has also been isolated from aortic tissue from both of these species. These investigators claim that it has not been isolated from the rat, rabbit, or pig, although the rat pulmonary artery was not investigated. Another protein band that has a mobility intermediate between MHC200 and MHC190, at ~196-198 kDa, has been reported (9, 27, 28). The 190- and 196- to 198-kDa MHCs have been described as nonmuscle MHCs and have been labeled nonmuscle myosin. Although in this study no change in the MHC200/MHC204 ratio was found in the hypoxia-induced pulmonary hypertensive pulmonary arterial smooth muscle, an increase in the ratio of MHC190+196 to MHC200+204 in the hypertensive PASM did occur. Two clearly defined smooth muscle phenotypes have been reported: the contractile and the synthetic phenotypes (1). There is a relatively greater amount of nonmuscle myosin (i.e., MHC190 and MHC196) in tissues from young animals, and the ratio of nonmuscle to muscle (MHC204 and MHC200, respectively) myosin decreases with age (4, 15). An abundance of nonmuscle myosin may represent a less differentiated state of smooth muscle cells in the case of development or that more of the muscle is in the synthetic phenotype. On the other hand, expression of nonmuscle myosin could just as easily be due to an increase in the number of nonmuscle cells, a possibility that cannot be contradicted without definitive staining in situ. However, a definitive immunohistochemical stain distinguishing synthetic smooth muscle from nonmuscle cells such as fibroblasts has not yet been identified.
In conclusion, the main findings of this investigation may be summarized as decreased reactivity, decreased tension production per unit of CSA of muscle despite hyperplasia, and an increase in the proportion of nonmuscle myosin to muscle myosin isoforms in the pulmonary arterial wall with pulmonary hypertension. The cause-and-effect relationship between these parameters remains to be established.
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ACKNOWLEDGEMENTS |
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We thank Marlene Brown for expert typing of this paper and Peggy Harger-Allen and Sally Debono for excellent technical assistance in production of the micrographs utilized in this study.
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FOOTNOTES |
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The work presented in this manuscript was funded by National Heart, Lung, and Blood Institute Grant HL-40894.
J. E. Roepke was the recipient of an American Heart Association-Indiana Affiliate Fellowship. N. H. Oberlies was the recipient of an Indiana University School of Medicine Undergraduate Biomedical Research Program Scholarship.
Address for reprint requests: C. S. Packer, Dept. of Physiology and Biophysics, Indiana Univ. School of Medicine, 635 Barnhill Dr., Indianapolis, IN 46202-5120.
Received 28 April 1995; accepted in final form 2 February 1998.
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