Hypoxic exposure time dependently modulates endothelininduced contraction of pulmonary artery smooth muscle

Russell A. Bialecki, Carol S. Fisher, Wallace W. Murdoch, Herbert G. Barthlow, Richard B. Stow, Michael Mallamaci, and William Rumsey

Respiratory, Inflammatory, and Neurological Diseases Research Section, Zeneca Pharmaceuticals, Zeneca, Inc., Wilmington, Delaware 19850-5437

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 on beta -adrenergic receptors.

Before experimentation, vascular rings were placed under their predetermined optimal passive load (~3 g) and equilibrated in PSS for 30 min. Isometric tension developed in response to drug stimulation was measured with Grass FT03 linear tension displacement transducers and recorded on a Grass 7D polygraph.

Concentration-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.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

Additional control studies were undertaken to confirm endothelium denudation of the PA and aorta preparations used in these studies. Moderate relaxation responses were obtained with 1 µM acetylcholine in precontracted, endothelium-intact PA and aorta controls (-0.64 ± 0.09 and -0.34 ± 0.02 g, respectively; n >=  16). In comparison, relaxation responses to 1 µM acetylcholine were nearly abolished in the precontracted, endothelium-denuded PA (0.09 ± 0.01 g, n = 51) and aorta (-0.06 ± 0.10 g, n = 30) used in the present studies.

To determine whether chronic hypoxic exposure changes the response of conduit PA smooth muscle to ET, studies were designed to characterize the ET-1-induced contraction of PA obtained from rats exposed to air (control) or 14-day hypoxia. ET-1 caused concentration-dependent contraction of PA from rats exposed to air or hypoxia for 14 days (Fig. 1A). The threshold concentration of ET-1 necessary for consistent contraction of PA from rats exposed to 14 days of hypoxia was 100-fold greater than that required for PA from rats exposed to air (300 vs. 3 pM, respectively; P < 0.01, n >=  5). Contractile sensitivity to ET-1 was decreased in PA obtained from 14-day hypoxic rats compared with air controls (pD2: 9.07 ± 0.04 vs. 9.66 ± 0.05, respectively; P < 0.05, n >=  5). In addition, the magnitude of maximum contraction to 1 µM ET-1 was decreased in PA from rats exposed to 14 days of hypoxia compared with air controls (38 ± 8 vs. 92 ± 7%, respectively; P < 0.001, n >=  5).


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Fig. 1.   Concentration-response curves of endothelin-1 (ET-1) in extralobar branch pulmonary artery (PA; A) and aorta smooth muscle (B). Tissues were harvested from rats exposed to air (control) or a hypoxic environment consisting of 10% O2-90% N2 for 14 days. Responses were normalized as a percentage of the 30 mM BaCl2 reference. Points represent means ± SE; n = 6 rats. * P < 0.01 vs. hypoxic rats.

Similar studies were undertaken to evaluate the contractile response of aorta to ET-1 after exposure to air or hypoxia for 14 days (Fig. 1B). The concentration of ET-1 necessary for threshold contraction of aorta obtained from 14-day hypoxic rats was not different from that required to contract aorta from air control rats (1 vs. 0.3 nM, respectively; P = NS, n = 6). In addition, no difference was observed in the sensitivity of aorta to ET-1 from 14-day hypoxic rats compared with air controls (pD2: 8.62 ± 0.41 vs. 8.74 ± 0.21, respectively; P = NS, n = 6). Likewise, no difference was observed in the magnitude of maximum contraction to 1 µM ET-1 in aorta from 14-day hypoxic rats compared with controls (98 ± 1 vs. 95 ± 2%, respectively; P = NS, n = 6). The similarity in sensitivity and maximum contraction to ET-1 in aorta from rats exposed to hypoxia and air suggests that ET-1-induced contraction of this systemic vascular segment is not affected by 14 days of hypoxic exposure.

The effects of chronic hypoxic exposure on the sensitivity and maximum contraction of PA and aorta to other ET agonists were also determined. PA taken from rats exposed to 21 days of hypoxia demonstrated significant decreases in the sensitivity and maximum contraction to ET-1, ET-2, and ET-3 compared with responses of PA from air controls (Table 1). In addition, sarafotoxin S6c-induced contraction of PA was abolished after exposing rats to 21 days of hypoxia. Similar analyses performed with aorta from rats exposed to 21 days of hypoxia indicated that this insult was without effect on the sensitivity and maximum contraction of aorta to ET-1, ET-2, ET-3, and sarafotoxin S6b. However, a marked contraction to sarafotoxin S6c was observed in aorta from rats exposed to 21 days of hypoxia compared with air controls.

                              
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Table 1.   Comparison of sensitivity and %maximum contraction to ET-receptor agonists in PA and aorta from rats exposed to air or hypoxia for 21 days

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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Fig. 2.   Effect of hypoxic exposure time on hematocrit and mass ratio of right ventricle to left ventricle plus septum (RV/LV+S). Points represent means ± SE; n = 6. * P < 0.05 vs. day 0.

Additional studies were performed to evaluate the effects of hypoxia on plasma concentrations of ET-1 (Fig. 3). Hypoxic exposure throughout 48 days was associated with a progressive increase in immunoreactive plasma concentrations of ET-1. Immunoreactive plasma concentrations of ET-1 increased significantly at 7 days of hypoxia compared with values obtained at time 0 (15.20 ± 2.57 vs. 9.10 ± 1.89 pg/ml ET-1, respectively; P < 0.04, n > 7). The maximum plasma concentration of ET-1 observed at 48 days of hypoxia was ~3.5-fold greater than values obtained from 48-day air controls (53.21 ± 9.42 vs. 14.05 ± 3.16 pg/ml ET-1, respectively; P < 0.01, n > 3). No significant difference was observed between immunoreactive plasma concentrations of ET-1 obtained from 0-day and 48-day air control rats (P = 0.28, n > 3).


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Fig. 3.   Effect of chronic hypoxic exposure on immunoreactive plasma ET-1 (black-square) concentration. Plasma ET-1 concentration from 48-day air control rats (square ) is shown for comparison. Points represent means ± SE of duplicate determinations from >= 4 rats. * P < 0.05 vs. day 0. dagger  P < 0.05 vs. 48-day air control.

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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Fig. 4.   Effect of hypoxic exposure time on ET-1 sensitivity [negative log of molar concentration (pD2); A] and maximum contraction to 1 µM ET-1 (B) in rat extralobar branch PA smooth muscle. Maximum contraction to ET-1 is normalized to a 30 mM BaCl2 reference. Points represent means ± SE; n >=  6. * P < 0.05 vs. air controls.

Hypoxic exposure caused time-dependent changes in the magnitude of maximum contraction of PA to ET-1 (Fig. 4B). Hypoxic exposure for 1 day did not change the maximum contraction of PA to 1 µM ET-1 compared with air controls (100 ± 2 vs. 100 ± 1%, respectively; n = 8). However, maximum contraction to ET-1 decreased throughout 12 days of hypoxic exposure to a level approximating 38 ± 8% (P < 0.05, n = 6) of the BaCl2 reference response obtained from air controls. The hypoxia-induced decrease in maximum contraction of PA to ET-1 subsequently returned toward normal levels by 21 days of hypoxia and approached control values after 48 days of hypoxia (83 ± 3 and 98 ± 1% of the BaCl2 response, respectively; n = 7-10).

Additional experiments were performed to determine whether hypoxic exposure changed the magnitude of contraction to nonselective contractile agonists. Hypoxic exposure throughout 12 days caused a time-dependent decrease in the response of PA to 30 mM BaCl2 and 80 mM KCl compared with control responses measured at day 0 (Fig. 5). The maximum decreases in BaCl2 and KCl responses at 12 days of hypoxia were ~43 ± 4 and 42 ± 6% of respective control values obtained at day 0. The hypoxia-induced decrease in maximum contraction of PA to BaCl2 and KCl subsequently returned toward normal levels by 21 days of hypoxia and approximated day 0 control values after 48 days of hypoxia (BaCl2, 100 ± 10 vs. 116 ± 11%; KCl, 60 ± 8 vs. 65 ± 7% of the BaCl2 response, respectively; P = NS, n = 7-10).


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Fig. 5.   Effect of hypoxic exposure time on maximum contraction to 30 mM BaCl2 and 80 mM KCl in rat extralobar branch PA smooth muscle. Responses are normalized to the 30 mM BaCl2 reference response obtained at day 0 hypoxic exposure. Points represent means ± SE; n = 7-10. * P < 0.05 vs. day 0.

Preliminary studies indicated that hypoxic exposure for 12 days caused a 208 ± 6% increase in the index of right ventricular hypertrophy that was associated with a 0.7 ± 0.05 log unit decrease (P < 0.01, n = 6) in the sensitivity of PA to ET-1 compared with air controls. A comparison was made between the extent of right ventricular hypertrophy and the decrease in sensitivity of PA to ET-1 from rats exposed to 14 days of hypoxia (Fig. 6). The relationship between right ventricular hypertrophy and decreased sensitivity of PA to ET-1 was linear (r = 0.733; P < 0.01, n = 12) over the right ventricular hypertrophy index range of 0.311 to 0.488. A similar relationship was observed between these variables after 21 days of hypoxic exposure (r = 0.86; P < 0.01, n = 7) and 48 days of hypoxic exposure (r = 0.83; P < 0.01, n = 10).


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Fig. 6.   Relationship between mass ratio of right ventricle to left ventricle plus septum and extralobar branch PA smooth muscle sensitivity (pD2) to ET-1. Determinations were made after exposure of rats to hypoxia for 14 days. Points represent single values, with dotted line showing 95% confidence interval estimate; n = 12.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.

    ACKNOWLEDGEMENTS

We wish to thank Dr. Joseph Kelly, Jr., for expert technical assistance and Robert Caccese for editorial assistance.

    FOOTNOTES

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.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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