Respiratory, Inflammatory, and Neurological Diseases Research Section, Zeneca Pharmaceuticals, Zeneca, Inc., Wilmington, Delaware 19850-5437
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ABSTRACT |
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Endothelins (ETs) have been implicated in the pathogenesis of hypoxia-induced pulmonary hypertension. We determined whether hypoxic exposure of rats (10% O2-90% N2, 1 atm, 1-48 days) altered contraction to ET in isolated segments of endothelium-denuded extralobar branch pulmonary artery (PA) and aorta. Hypoxic exposure increased hematocrit, right ventricular hypertrophy, and ET-1 plasma concentration. Hypoxia also caused a sustained decrease in PA but not in aorta sensitivity to ET-1. In comparison, hypoxic exposure throughout 12 days decreased time dependently the maximum contraction of PA to ET-1, BaCl2, and KCl. The hypoxia-induced decrease in maximum contraction of PA to ET-1 returned toward normal levels by 21 days and approximated control levels by 48 days. After 14 days of hypoxia, right ventricular hypertrophy correlated with decreased sensitivity of PA to ET-1. After 21 days of hypoxia, PA sensitivity to ET-2 and ET-3 was decreased, and sarafotoxin S6c-induced contraction was abolished. In conclusion, hypoxic exposure time dependently modulates the responsiveness of PA smooth muscle to ETs, BaCl2, and KCl. The hypoxia-induced changes in tissue responsiveness to ET-1 may be associated with increased plasma concentrations of this peptide.
aorta; hypoxia; sarafotoxin S6b; sarafotoxin S6c; pulmonary hypertension
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INTRODUCTION |
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ENDOTHELINS (ETs) represent a family of potent vasoconstrictor peptides with comitogenic properties that were isolated originally from the conditioned medium of vascular endothelial cells (23). ETs exist in three isoforms: ET-1, ET-2, and ET-3. These peptides share structural homology with the snake venom toxins sarafotoxin S6b and S6c (8). Two major ET-receptor subtypes have been described: ETA (selective for ET-1) and ETB (possessing equal affinity for all 3 ET isoforms). ETs cause sustained, concentration-dependent contraction that is mediated predominantly via the ETA-receptor subtype in normal rat and human conduit pulmonary artery (PA) smooth muscle preparations (1, 4, 5).
Little information is available about the effects of chronic hypoxia on the ET-induced response of isolated rat PA smooth muscle. Studies of the effects of acute hypoxic exposure in vitro indicate that vasoconstrictor sensitivity to ET-1 is unchanged in isolated conduit PA of the rat (21). However, extralobar PA taken from rats exposed to 14 days of hypoxia demonstrates increased sensitivity to ET-1 (12). Thus far, the progressive effects of hypoxic exposure on the responsiveness of PA smooth muscle to ET have not been characterized.
The objective of these studies was to evaluate the vasoconstrictor effects of ET in extralobar PA and aorta smooth muscle in vitro after exposing rats to chronic hypoxia. Our preliminary studies demonstrated that 14 days of hypoxia increased both right ventricular hypertrophy and hematocrit, whereas PA sensitivity and the maximum response to ET-1 were decreased. The time course of hypoxia-induced changes in PA responsiveness to ET-1 was then determined. Additional studies evaluated the effects of hypoxia on plasma concentrations of immunoreactive ET-1 as well as the contraction of PA and aorta to other ET-receptor agonists and the nonselective vasoconstrictors BaCl2 and KCl.
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MATERIALS AND METHODS |
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Chronic hypoxic exposure. Normal male Sprague-Dawley rats (300-350 g) were exposed to 10% O2-90% N2 (22°C, 1 atm) for 0, 1, 7, 12, 14, 21, 28, and 48 days in a ventilated chamber (135-liter volume). The chamber was flushed continuously at a minimum rate of 3.8 l/min of the hypoxic gas mixture to prevent accumulation of CO2, NH3, and water vapor. The exposure was 24 h/day except when the chamber was opened for 10-15 min once or twice a day to remove rats and/or to clean the chamber and replenish food and water. Normal age-matched control animals were exposed to room air for equal periods. All rats were exposed to a 12:12-h light-dark cycle and were provided rat chow and water ad libitum.
Isometric tension studies. Rats were killed by decapitation and exsanguination. Segments of extralobar right and left branch PA and aorta were removed from each animal and placed in oxygenated (95% O2-5% CO2) physiological salt solution (PSS) composed of (in mM) 119 NaCl, 4.7 KCl, 1.6 CaCl2, 1.2 KH2PO4, 1.2 MgSO4, 22.6 NaHCO3, 5 dextrose, and 0.03 EDTA, maintained at pH 7.4. The tissues were stripped of adherent fat and connective tissue. Ring segments (2-mm long, 2- to 3-mm ID) of each vascular tissue were cut and denuded of endothelium by gentle abrasion of the intimal surface.
To monitor smooth muscle function, ring segments of each vascular tissue were prepared for isometric tension studies as previously described (2). Briefly, each vascular ring was mounted on two stainless steel pins (130-µm radius) and submerged in a 5-ml organ chamber containing PSS. The water-jacketed chambers were maintained at 37°C, and the PSS was oxygenated continuously with 95% O2-5% CO2. ET-induced stimulation of intramural adrenergic neurons has been shown to increase neuronal intracellular Ca2+ concentration and contract neighboring smooth muscle via a mechanism that may involve the release of diffusible neurotransmitters including norepinephrine (17). Therefore, the buffer was supplemented with 1 µM propranolol to reduce the influence of norepinephrine onConcentration-response curves.
After the equilibration period and before construction of agonist
concentration-response curves, tissues were exposed to 80 mM KCl to
assess viability. During the plateau phase of this contraction, denudation of vascular endothelium was confirmed by the lack of relaxation to 1 µM acetylcholine. The tissues were then washed until
the tension returned to baseline levels. The tissues were equilibrated
an additional 60 min until the start of the experiment. Agonist
concentration-response curves were constructed by their cumulative
addition to the organ bath in threefold increments. Each drug
concentration was left in contact with the tissue until the response
reached a stable level before a force measurement was made. At the end
of the experiment, tissues were exposed to 30 mM
BaCl2 as a reference because
preliminary studies demonstrated that this agonist caused greater
contraction of PA and aorta than that attained with 80 mM KCl. Changes
in isometric tension development were calculated and analyzed with
Bioreport software (Modular Instruments, Malvern, PA). Contractile
responses were computed from the change in wall stress
(g/cm2) developed with the
agonist as previously described (2) and expressed as a percentage of
the maximum response to the agonist itself or to that obtained with the
30 mM BaCl2 reference. A
single-agonist concentration-response curve was constructed for each
tissue. Paired segments of rat PA and aorta were studied for
comparison. Right and left branch PAs (RPA and LPA, respectively) were
used for these and other studies because preliminary data showed no difference in their response to ET-1 [log
EC50: LPA, 9.51 ± 0.04; RPA,
9.48 ± 0.07; %maximum response to 1 µM ET-1: LPA, 94 ± 2; RPA, 91 ± 3; P = not significant
(NS), n = 14 rats].
Indexes of hypoxia-induced pulmonary hypertension. The severity of hypoxia-induced pulmonary hypertension in the rats used for the various studies was determined partly by measuring hematocrit. For this, blood was collected at the time of death into standard microhematocrit capillary tubes and analyzed in a HemataStat-II microcentrifuge (Separation Technology, Altamonte Springs, FL). In addition, ventricular mass was determined after the hearts were removed and dissected to isolate the free wall of the right ventricle from the left ventricle plus septum. The ventricles were blotted dry and tissue wet weights were determined. The mass ratio of right ventricle over left ventricle plus septum was used as an index of right ventricular hypertrophy.
Radioimmunoassay of plasma ET-1.
Rats were anesthetized with 60%
CO2-40%
O2 for 4 min, and blood (~2 ml)
was collected from the orbital sinus into iced tubes containing 7.5 mg
EDTA and aprotinin (500 KIU/ml) at 0, 7, 14, 21, 28, and 48 days. The
plasma was stored at 70°C. Plasma immunoreactive ET-1
concentration was measured by RIA with a commercially available kit
(Phoenix Pharmaceuticals, Mountain View, CA). Plasma for ET-1 extraction was acidified with 0.1% trifluoroacetic acid (TFA; high-performance liquid chromatography grade), centrifuged at 1,800 g for 30 min at 6°C, and applied
to Sep-Pak C18 columns (Waters
Associates, Milford, MA) that had been activated by washing with 2 ml
of methanol followed by 2 ml of 0.1% TFA. The columns containing
sample were then washed twice with 2 ml of 0.1% TFA. ET-1 was eluted
from the column with 2 × 2 ml of 75% acetonitrile in 0.1% TFA.
The eluent was collected in polypropylene tubes and evaporated to
dryness under N2. Samples were
then reconstituted in RIA buffer and analyzed with rabbit anti-ET-1
antiserum. The anti-ET-1 antiserum was 7% cross-reactive with ET-2 and
ET-3, <17% cross-reactive with human and porcine big ET-1, and 3%
cross-reactive with mouse vasoactive intestinal contractor. There was
no cross-reactivity with human Big ET 22-38, human
atrial natriuretic peptide, porcine brain natriuretic peptide, and
sarafotoxin S6b. Recovery from the Sep-Pak columns averaged 82%, and
the sensitivity of the assay for ET-1 was 1.5-2 pg/tube.
Analysis of data. Statistical significance was determined by analysis of variance or the Student's t-test for paired data when appropriate. P values < 0.05 were considered significant. The EC50 value (the concentration of agonist that produced one-half of the maximal contractile response) was used as an index of tissue sensitivity. The EC50 value for each agonist was determined from log-logit plots of individual concentration-response curves and averaged geometrically for each agonist within each group. For statistical analyses, EC50 values were analyzed and reported herein as the negative log of the molar concentration (pD2). Data are presented as means ± SE. In all cases, n equals the number of individual animals studied.
Reagents. The agonists, ET-1, ET-2, ET-3, sarafotoxin S6b, and sarafotoxin S6c, were obtained from Bachem (Torrance, CA). Lyophilized agonists were dissolved (100 µM) in deionized distilled H2O (dH2O) with 0.1% BSA (fraction V) and stored at 4°C for <10 days as stock aliquots from which daily solutions were prepared. Propranolol, EGTA, acetylcholine, and BSA were purchased from Sigma (St. Louis, MO). Drugs were dissolved in dH2O, kept on ice, and added to the muscle baths in 35-µl volumes or less to give the final concentrations reported and maximal bath dilutions of 1:7,000.
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RESULTS |
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ET-1-mediated contraction.
Endothelium-denuded PA rings from hypoxic rats studied under some
experimental conditions may have spontaneous contractile tone. Control
experiments were performed to ensure that spontaneously developed tone
would not compromise the comparison of responses between PA from normal
and hypoxic rats. For this, PA segments from 14-day hypoxic rats and
normal air controls were equilibrated in PSS. The buffer was then
replaced with Ca2+-free PSS
containing 2 mM EGTA. No significant relaxation response was observed
in PA from either 14-day hypoxic or air control rats [hypoxia
vs. control, change from baseline (g), 0.10 ± 0.04 vs.
0.15 ± 0.09; P = NS,
n = 6]. Thus no
difference was apparent in the resting tensions of these preparations.
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Effect of hypoxia on indexes of pulmonary hypertension. To directly correlate the effects of hypoxia with altered vasoconstrictor responses, hematocrit and right ventricular hypertrophy were evaluated as indexes of pulmonary hypertension with the same animals from which arterial rings were obtained for tension studies. We observed an increase (P < 0.05) in hematocrit with 7-day hypoxic exposure (Fig. 2). Longer hypoxic exposure times progressively increased hematocrit throughout 48 days, with a maximum value that was twofold greater than that obtained from rats at day 0 (92 ± 2 vs. 47 ± 2%, respectively; P < 0.05, n = 6). A similar trend was observed with increasing right ventricular hypertrophy throughout 48-day hypoxic exposure (Fig. 2), with a maximum 2.25-fold increase in the mass ratio of right ventricle over left ventricle plus septum compared with control samples measured at time 0 (0.54 ± 0.04 vs. 0.24 ± 0.01, respectively; P < 0.05, n = 6).
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Effect of hypoxic exposure time on PA responsiveness to ET-1. Studies were undertaken to compare the sensitivity and magnitude of maximum contraction of PA to ET-1 after various times of hypoxic exposure (Fig. 4A). Hypoxic exposure for 1 day caused an initial increase (P < 0.05) in PA sensitivity to ET-1 compared with air controls. However, continued exposure to hypoxia over 12 days caused a progressive decrease (P < 0.05) in the sensitivity of PA to ET-1 that was sustained throughout 48 days. In contrast, the sensitivity of aorta to ET-1 was not altered throughout 48 days of hypoxia (exposure time, aorta pD2 value: 0 days, 9.41 ± 0.16; 7 days, 9.31 ± 0.18; 14 days, 9.41 ± 0.15; 21 days, 9.13 ± 0.19; 48 days, 8.89 ± 0.32; n > 9, P = NS).
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DISCUSSION |
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The principal finding of this study was that hypoxic exposure of rats caused time-dependent variations in PA sensitivity and maximum contraction to ET-1. Changes in the magnitude of maximum contraction of PA to BaCl2 and KCl paralleled those observed with ET-1. These functional modifications were associated with progressive increases in hematocrit, right ventricular hypertrophy, and immunoreactive plasma concentrations of ET-1 throughout 48-day hypoxic exposure, consistent with the development of pulmonary hypertension. The increased right ventricular hypertrophy that occurred after hypoxic exposure for 14 days or more correlated with the reduced sensitivity of PA to ET-1. Additional studies indicated decreased sensitivity of PA to ET-2 and ET-3 as well as an abrogated contractile response to sarafotoxin S6c after 21-day hypoxia. Comparative studies indicated tissue-specific variations in the smooth muscle response to ET after chronic hypoxic exposure. Unlike PA, the sensitivity and maximum contraction of aorta to ET-1 did not change with chronic hypoxic exposure.
To our knowledge, this is the first study to report the progressive effects of hypoxia on the responsiveness of endothelium-denuded PA to ET-1. Other investigators have used similar preparations with an intact endothelium to show that 1-h hypoxic exposure in vitro does not alter ET-1 sensitivity (21). In addition, increased sensitivity to ET-1 has been reported with endothelium-intact conduit PA taken from rats exposed to 14-day hypoxia (12). The reason for these disparities compared with our findings is not clear but may be related to the use of different experimental conditions, including the O2 tension applied in vitro, as well as rat species and age. At present, we cannot exclude the additional possibility that these other studies were complicated by concomitant hypoxia-induced changes in endothelial cell metabolism that affected the functional responses of PA in vitro.
Chronic hypoxia resulted in progressive increases in both right ventricular hypertrophy and hematocrit. The elevated hematocrit was likely associated with an increase in blood viscosity. In the rat, increased hematocrit may account for as much as 50% of the rise in PA pressure associated with chronic hypoxia (3). However, the relative contribution of the hematocrit rise to the right ventricular hypertrophy and elevation in immunoreactive plasma concentration of ET-1 is not certain. Serial determinations of hematocrit and PA sensitivity to ET-1 showed that the majority of the sensitivity change occurred during the first 2 days of hypoxia at a time when the hematocrit increased from ~47 to 54%. Progressive increases in hematocrit were observed during the next 46 days, with relatively little additional effect on PA sensitivity to ET-1. Thus the rise in hematocrit did not parallel the change in PA sensitivity to ET-1, suggesting that changes in arterial sensitivity were caused by factors other than, or in addition to, a rise in hematocrit. Interestingly, right ventricular hypertrophy increased markedly during the first 12 days of hypoxic exposure, with an additional modest increase throughout the remaining experimental time course. Although these increases in right ventricular hypertrophy did not correlate with the time course of hypoxia-induced changes in maximum contraction of PA to ET-1, paired analyses indicated that the extent of right ventricular hypertrophy that occurred after hypoxic exposure for 14 days or more correlated with the decreased sensitivity of PA to ET-1. In addition, right ventricular hypertrophy did correlate positively with the time course of increases in immunoreactive plasma concentrations of ET-1.
Functional studies have previously demonstrated that ET-induced contraction is mediated via both ETA- and ETB-receptor subtypes in normal rat conduit PA smooth muscle and that the ETA receptor is dominant (1). ETB receptors have been described in pulmonary resistance vessels of rat and human (11, 14). The present studies confirm and extend our earlier findings. Both ET-1 and sarafotoxin S6c, agonists relatively selective for ETA and ETB receptors, respectively, caused contraction of PA from 21-day air controls. Pulmonary arterial smooth muscle from 21-day hypoxic rats demonstrates an approximate one-half log unit decrease in sensitivity to ET-1, ET-2, and ET-3 compared with air controls. Although the sensitivity of PA decreased, the rank order of sensitivity, i.e., ET-1 > ET-2 >> ET-3, remained consistent with the classic definition for the ETA receptor. Interestingly, 21-day hypoxia abolished the PA response to sarafotoxin S6c, suggesting that chronic hypoxic exposure has a differential effect on ETA receptor- and ETB receptor-mediated contraction in this preparation.
The selective changes that we observed in ET pharmacology were contrasted by those of a nonselective nature. Results showed time-dependent changes in the maximum contraction of PA to the nonselective vasoconstrictors BaCl2 and KCl. Thus the hypoxia-induced decrease in PA responsiveness to ET-1 is not specific. This observation is consistent with a report showing enhanced K+ and Ca2+ channel-dependent relaxation of PA after exposing rats to hypoxia for 3 days (16), an effect that may reduce ET-1-, BaCl2-, and KCl-induced contraction. However, we caution that the present results should not be generalized to other vasoconstrictors, given a report of acute hypoxia-induced decreases in both the potency and maximum contractile effects of phenylephrine but increased maximum contraction to norepinephrine in isolated rabbit PA (13).
Of additional interest in our studies was the observation that 21-day hypoxia caused significant sarafotoxin S6c-induced contraction of aorta but did not change the sensitivity to other ET agonists, suggesting an enhanced ETB receptor-mediated response after chronic hypoxia. These findings support the notion of site-selective differences in the effect of hypoxia on the response to ET. Additional studies are necessary to elucidate the mechanism of hypoxia-induced decreases in ETB receptor-mediated contraction of PA smooth muscle and the coordinate increase in ETB receptor-mediated contraction of aorta.
In general, the molecular mechanism(s) of agonism is varied and, for many receptor systems, is not completely understood. Conceptually, it has been convenient to categorize agonist properties based on their ability to associate with the receptor and change tissue function (7, 18). Measures of sensitivity and maximum contraction to agonists have been commonly used to provide indirect estimates of agonist affinity and efficacy, respectively. Changes in tissue sensitivity to an agonist may be due to many factors, including alterations in receptor number, receptor affinity, or metabolism of the agonist. In comparison, changes in the maximum response to an agonist may be attributable to these factors as well as to differences in receptor coupling to cellular second messengers. In the present studies, the time course of hypoxia-induced change in PA sensitivity to ET-1 differed from the time course of change in maximum contraction. We observed that 1-day hypoxia increased the sensitivity of isolated PA to ET-1, whereas hypoxic exposure for 2-48 days caused a sustained decrease in the sensitivity of PA to this agonist. In comparison, maximum contraction of PA to ET-1 did not increase at 1-day hypoxia but rather decreased throughout 12-day hypoxia to its nadir and approached values approximating air controls by 21 days. Differences in the time course of hypoxia-induced changes in PA sensitivity and maximum contraction to ET-1 may suggest a dissociation between the regulatory mechanisms that control these parameters.
The mechanism underlying the hypoxia-induced decrease in PA sensitivity to ET is not clear. The present results show significant increases in immunoreactive plasma concentrations of ET-1 over the same 7- to 48-day period in which PA sensitivity to ET-1 was decreased. It is unlikely that plasma concentrations of the peptide per se are responsible for these effects. Secretion of ET-1 has been reported to occur predominantly via an abluminal route; thus plasma concentrations of ET-1 likely reflect an underestimation of local tissue concentrations of the peptide, i.e., at the level of the smooth muscle cell (19). In addition, a recent report has demonstrated that 28-day hypoxic exposure of rats increases ET-1 mRNA of PA and ET-1 content of whole lung, whereas ET-1 mRNA was unchanged in thoracic aorta (10). This study and the results of the present one support the concept that hypoxia induces tissue-specific differences in ET-1 content and functional responsiveness.
Additional support for tissue-specific differences in the responsiveness to ET-1 is provided by previous work. ET-1 binds to its receptors with subnanomolar affinity in a pseudoirreversible manner (15, 20, 22). Consequently, this would reduce available receptors for subsequent ligand-receptor interactions. Preincubation of cultured vascular smooth muscle with ET-1 for 1 day decreased the number of ET-1-specific binding sites (6). Moreover, preincubation of isolated vascular preparations with 0.01 µM sarafotoxin S6c abolished ETB receptor-mediated contraction in vitro (9). In our studies, binding of locally elevated endogenous concentrations of ET-1 to ETA and ETB receptors in the hypoxic rat PA may have reduced the sensitivity of this tissue to exogenously applied ET-1 in vitro. Consistent with the above explanation, the observed lack of desensitization of hypoxic aorta may be attributable to lower endogenous concentrations of ET-1 within this tissue.
In summary, hypoxic exposure of rats caused time-dependent variations in the sensitivity and maximum contraction of PA smooth muscle but not of aorta to ET-1. These changes were associated with progressive increases in hematocrit, right ventricular hypertrophy, and immunoreactive plasma concentrations of ET-1 throughout 48-day hypoxic exposure, consistent with the development of pulmonary hypertension. Increased right ventricular hypertrophy that occurred at or beyond 14-day hypoxia correlated positively with decreased sensitivity of PA to ET-1. Prolonged hypoxic exposure also attenuated the response to other ET-receptor agonists as well as to BaCl2 and KCl, indicating nonspecific alterations of PA smooth muscle responsiveness to vasoconstrictors after hypoxic exposure. In addition, the decreased responsiveness of PA to ETs that we observed after hypoxic exposure was unique. ET-1 sensitivity of aorta from rats exposed to hypoxia for 21 days was not different from air controls. Interestingly, and unlike PA, aorta from 21-day hypoxic rats showed increased sensitivity and maximum contraction to sarafotoxin S6c. These findings indicate that chronic hypoxic exposure in vivo results in site-selective differences in ET-receptor pharmacology in vitro, including changes in tissue responsiveness to ET-1.
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ACKNOWLEDGEMENTS |
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We wish to thank Dr. Joseph Kelly, Jr., for expert technical assistance and Robert Caccese for editorial assistance.
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FOOTNOTES |
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Address for reprint requests: R. A. Bialecki, Respiratory, Inflammatory, and Neurological Research Section, BMRL-233, Zeneca Pharmaceuticals, 1800 Concord Pike, PO Box 15437, Wilmington, DE 19850-5437.
Received 20 March 1997; accepted in final form 13 January 1998.
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