Introduction

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Endothelin (ET)-1 is synthesized in the lung by endothelial cells, bronchial and alveolar epithelial cells, and tissue macrophages, and plays roles in a number of important biologic processes, including smooth-muscle contraction and relaxation, the mitogenic action of smooth-muscle cells and fibroblasts, and proinflammatory actions (1, 2). ET-1 is a potent regulator of pulmonary vascular tone that acts by causing vasoconstriction or vasodilation and the proliferation of smooth-muscle cells and fibroblasts in the medial wall (1). The actions of ET-1 in the pulmonary vasculature are mediated by at least three different receptors, ET_A receptor and two ET_B receptor subtypes (1, 2, 6). ET_A receptor is found in pulmonary vascular smooth-muscle cells and mediates smooth-muscle contraction and cell proliferation (3). ET_B receptors are expressed in multiple cell types, including endothelial cells and vascular smooth-muscle cells, and cause either contraction or relaxation (1, 2). Pharmacologic studies suggest that ET_B receptor expressed in endothelium (designated ET_B1 receptor) mediates vasodilation by releasing vasodilator prostaglandins and nitric oxide and activating potassium channels, whereas ET_B receptor expressed in smooth-muscle cells (designated ET_B2 receptor) acts as a vasoconstrictor (2, 6). Because the complex actions of ET-1 depend basically on the receptor subtypes expressed in a particular vascular segment, it is important to elucidate the distribution of ET_A and ET_B receptor subtypes in the pulmonary vasculature in order to understand better the actions of ET-1 in the regulation of pulmonary circulation. The tissue distribution of ET_A and ET_B receptor subtypes in the lung has been studied by an autoradiographic technique using ¹²⁵I-endothelins combined with ET_A and ET_B receptor-specific antagonists, in situ hybridization, and immunohistochemical methods (7). These techniques, however, have limitations involving morphologic resolution or high background levels, and few studies have examined the spatial localization of receptors along the axial pathways of pulmonary vessels.

Previous studies have suggested that hypoxia variably affects basal ET-1 synthesis and the gene expression of ET-1 and ET_A and ET_B receptors. The overexpression of the messenger RNA (mRNA) for ET-1 and ET_A and ET_B receptors and the overproduction of ET-1 peptide in whole lung have been demonstrated in an experimental model of hypoxic pulmonary hypertension (12, 13). However, the role of ET-1 and its receptors in the pathogenesis of hypoxic pulmonary hypertension remains unclear because of the potential bidirectional effects of ET-1 on pulmonary vascular tone through ET_A and ET_B receptor subtypes (14). Therefore, a knowledge of the topographic changes in ET_A and ET_B receptor subtypes in the pulmonary vasculature after exposure to hypoxia is essential to understanding the pathophysiology of hypoxic pulmonary hypertension.

In the present study, we investigated the spatial localization and distribution of ET_A and ET_B receptor subtypes in normal rat pulmonary vasculature, particularly in pulmonary resistance arteries (which is the site of pulmonary blood flow regulation in normal and rapid adaptation and remodeling in disease), by immunohistochemical methods involving antibodies specific to these receptors. Furthermore, we studied the temporal changes in the mRNA transcripts and immunoreactivities of these receptors in the pulmonary vasculature after exposure to hypobaric hypoxia in order to explore the roles of these receptors in the pathogenesis of hypoxic pulmonary hypertension.

	Materials and Methods

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Animals

Adult male Sprague-Dawley rats (6 to 8 wk old, 240 to 300 g) were placed for 12 h or 3, 7, or 14 d in a specially designed hypobaric chamber. The chamber was depressurized to 380 mm Hg (oxygen concentration reduced to about 10%) and maintained on a 12-h light-dark cycle. The exposure to hypobaric hypoxia was for 24 h/d except when the chamber was opened for 10 to 15 min every 3 d for cleaning and replenishing the food and water and/or to remove rats. The rats were allowed standard laboratory chow and tap water ad libitum. Age-matched control animals were maintained in room air. The lungs were isolated from the rats after the intraperitoneal administration of 60 mg pentobarbital and intracardiac injection of 100 U heparin. Cannulas were inserted into the pulmonary artery and left atrium, and the lungs were perfused at 36 cm H₂O through the pulmonary arterial cannula with phosphate-buffered saline (PBS). The right lung was removed at the main bronchus, frozen in liquid nitrogen, and stored at 80°C for later extraction of total cellular RNA. The left lung was perfused with 4% paraformaldehyde (PFA), inflated by infusion with 4% PFA through the cannulas inserted in the trachea, and fixed in 4% PFA overnight at 4°C. After the PFA was removed by repeated instillation and withdrawal of PBS through the cannula, the lung was filled with optimal cutting temperature (OCT) compound through the tracheal cannula, embedded in OCT compound, and stored at 80°C until used for immunohistochemical studies or in situ hybridization. The development of hypoxia-induced pulmonary hypertension was followed by measuring the weight ratio of the right ventricle to the left ventricle plus the septum (RV/LV + S), as previously described (15). The number of animals in each group was four to six.

Immunohistochemistry

Frozen sections (4 µm) cut with a cryostat at 20°C were mounted on sialinized slides and incubated with 10% normal goat serum to reduce the nonspecific binding of the second antibodies. The serum was removed, and the sections were incubated with anti-ET_A or anti-ET_B receptor antibodies (IBL Inc., Gumma, Japan) at a concentration of 5 mg/ml for 12 h at 4°C. In addition, monoclonal antibodies against vascular cell adhesion molecule-1 (5 mg/ ml; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) and -smooth-muscle (-SM) actin (0.2 mg/ml; Dako, Glostrup, Denmark) were used as endothelial markers and smooth-muscle markers, respectively. The sections were further incubated with a horseradish peroxidase-labeled biotinylated goat antirabbit immunoglobulin G antibody (1/100) for 30 min. To block endogenous peroxidase activity, the sections were immersed in 0.3% hydrogen peroxide in 100% methanol and then incubated with the avidin- biotin-peroxidase complex (1/100; Vector Laboratories, Inc., Burlingame, CA) for 30 min. Subsequently, the immunoperoxidase color reaction was developed by incubation for 15 min in Tris/HCl containing 0.05% 3,3'-diaminobenzidine tetrahydrochloride and 0.01% hydrogen peroxide. The reaction was terminated by rinsing the sections in tap water. The sections were counterstained with methyl green and mounted in Marinol (Muto Pure Chemicals, Tokyo, Japan). Serial sections were stained with elastic van Gieson staining for the accurate identification of arteries and veins and to evaluate medial wall thickness. Negative controls were prepared with nonimmune serum instead of the primary antibody, or by omitting steps in the avidin-biotin- peroxidase procedure.

Anatomic segments of the pulmonary artery were defined by the classification proposed by Sasaki and colleagues (16), with a minor modification. In brief, elastic arteries and muscular arteries were distinguished by the structure of the media, and muscular arteries were subdivided into four segments according to the thickness of the smooth-muscle layers and the structures of accompanying airways: that is, thick muscular (TM; bulky media consisting of up to 10 smooth-muscle layers, equivalent to the oblique muscle segments described by Hislop and Reid [17]), ordinary muscular (OM; one or two layers of smooth muscle), partially muscular (PM; a discontinuous layer of smooth muscle), and nonmuscular (NM; pericytes instead of smooth-muscle cells) segments. The OM, PM, and NM segments corresponded roughly to the preacinar bronchus or terminal bronchioles, respiratory bronchioles, and alveolar duct, respectively, although some overlap was observed. The sections were examined by light microscopy without knowledge of the treatment groups, and the intensity of immunostaining was graded semiquantitatively from 0 to 3: Grade 0, no staining; Grade 1, focal or weak staining; Grades 2 and 3, diffuse moderate and strong staining, respectively. To assess changes in ET_A and ET_B receptor expression after exposure to hypoxia, the immunostaining grade of the pulmonary arteries was estimated in lung sections from each animal. For each rat, pulmonary arteries were grouped according to their external diameter range: over 400, 200 to 400, 120 to 200, 60 to 120, or 30 to 60 µm, corresponding roughly to elastic artery and TM, OM, PM, and NM segments of muscular artery, respectively.

Preparation of Complementary DNA (cDNA) Probes for ET_A and ET_B Receptors

Partial cDNAs for rat ET_A and ET_B receptors were synthesized by reverse transcriptase polymerase chain reaction (RT-PCR). Reverse transcription was performed using Maloney Murine Leukemia Virus (MMLV) RT (GIBCO BRL, Gaithersburg, MD), oligo(dT)_12-18 (Pharmacia Biotec, Milwaukee, WI), and 10 mg of total RNA extracted from rat lung as a template. The primers used for PCR were as follows: sense 5'-TGTTGCTGTTGTCACCAGTCC-3', antisense 5'-GAGCGCAGCTGCTGCGTGACCG-3' for ET_A receptor; and sense 5'-CTGCTGGTGCCAAACGTTTGAG-3', antisense 5'-CCATGGCTTTCTTAGGTTGTA-3' for ET_B receptor. The sequences of sense and antisense primers correspond to nucleotide (NT) residues 1153/1173 and 1330/1351 of the rat ET_A receptor cDNA, and NT residues 1203 /1224 and 1505/1525 of ET_B receptor cDNA, respectively (18, 19). The RT-PCR products, a 199-base pair (bp) rat ET_A receptor partial cDNA and a 323-bp ET_B cDNA were subcloned into TA cloning vector (Invitrogen Corp., San Diego, CA), transfected into INVaF'competent cells (Invitrogen), and amplified and purified as previously described (20). The NT sequences of the partial cDNA of the rat ET_A and ET_B receptors were confirmed by the dideoxy termination method using an ABI Model 371 autosequencer and Taq Dyedeoxy TM terminator cycle sequencing kit (Applied Biosystems, Foster City, CA). Sense and antisense riboprobes for ET_B receptor were synthesized in vitro from the cDNA clone by SP6 or T7 RNA polymerase in the presence of digoxiginin (DIG)-uridine triphosphate (UTP) (Boehringer Mannheim, Mannheim, Germany) at 37°C for 60 min after digestion with NotI or HindIII, respectively.

Northern Blot Analysis

Northern blot analysis was carried out as previously described (20). Briefly, total cellular RNA was isolated from control and experimental lungs by the guanidium thiocyanate/cesium chloride method. Ten micrograms of denatured total RNA was electrophoresed on 1% agarose formaldehyde gels and transferred to Hibond N nylon membranes (Amersham Japan, Tokyo, Japan). The RNA was cross-linked to the membranes by ultraviolet irradiation. Following prehybridization, the membranes were hybridized for 48 h at 42°C in the presence of a 199-bp, random-primed, ³²P-labeled ET_A, or a 323-bp ET_B receptor partial cDNA probe. After hybridization, the membranes were washed at a final stringency of 0.1× saline sodium citrate (SSC; 0.15 M NaCl and 0.015 M sodium citrate, pH 7.4) and 0.1% sodium dodecyl sulfate at 65°C. Autoradiography was performed at 80°C for 48 h, and the bands were quantitated by densitometry using a computer-assisted image analyzer (BAS 2000; Scanalytics, Billerica, MA). After probing with ET_A or ET_B receptor cDNAs, the membranes were stripped and reprobed with a ³²P-labeled 28S ribosomal RNA (rRNA) cDNA as a positive control in the same manner except that autoradiography was performed for 4 h. The amounts of ET_A or ET_B receptor mRNA expression are expressed as the ratio of ET_A or ET_B receptor mRNA to 28S rRNA.

In Situ Hybridization

Cryostat sections (4 µm) were permeabilized with proteinase K (2 mg/ml) for 20 min at 37°C and fixed with 4% PFA for 5 min at room temperature. The sections were immersed in 2 mg glycine in distilled water to block the aldehyde groups. After prehybridization with prehybridization mixture (10 mM Tris/HCl [pH 7.6], 600 mM NaCl, 1 mM ethylenediaminetetraacetic acid, 50% formamide, and 1× Denhardt's solution), the sections were hybridized at 44°C for 12 h with a DIG-UTP-labeled probe for ET_B receptor in the presence of 10% dextran. After the unbound riboprobe was removed under high-stringency conditions (twice in 50% formamide in 2× SSC for 30 min, twice in 2× SSC for 20 min at 50°C, and once in 0.2× SSC for 30 min at room temperature), the sections were incubated with anti-DIG alkaline phosphatase-labeled antibody for 12 h at 4°C. The location of the antibody-antigen complex was visualized as an enzyme-linked color reaction using nitroblue tetraazolium and X-phosphate (Boehringer Mannheim). Negative control experiments using a DIG-labeled sense probe for ET_B receptor showed few nonspecific stainings (data not shown).

Statistical Methods

Numerical data are means ± SEM. Statistical analyses of immunohistochemical grading and autoradiographic densitometry were done by a one-way analysis of variance using proprietary software (Statview; Abacus Concepts, Inc., Berkeley, CA). P < 0.05 was considered significant.

	Results

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Localization and Distribution of ET_A and ET_B Receptors in Normal Rat Lung

In normal rat lung, immunoreactivity to ET_A receptor was observed in the smooth-muscle layer of the bronchus and the media of the pulmonary artery and large pulmonary vein. Staining intensity was higher in the bronchus than in the pulmonary artery at the same segmental levels. The distribution of ET_A receptor along the axial pathway of the pulmonary artery was dominant in proximal segments. Some positive staining was observed in the media of elastic artery and in TM and OM segments of the muscular artery, but little staining was seen in PM or NM segments in control rats. Weak staining was also found in the media of the large pulmonary vein (Figure 1, Table 1).

View larger version (149K): [in this window] [in a new window]   View larger version (148K): [in this window] [in a new window]   Figure 1.   Representative photomicrographs of pulmonary vasculature from a control rat stained with anti-ETA (A, C, E, G, I, and K) or anti-ETB (B, D, F, H, J, and L) receptor antibodies. A and C show positive staining for ETA receptors in the media of elastic arteries and TM segments of muscular arteries, respectively. E shows weak staining for ETA receptors in the media of OM segments. G and I show little staining in the PM and NM segments, respectively. K shows weak staining for ETA receptors in the media of the pulmonary vein. B and D show positive staining for ETB receptors in the intima and weak staining in the media of elastic arteries and TM segments of muscular arteries, respectively. F and H show positive staining for ETB receptors in the intima and media of OM and PM segments of muscular arteries, respectively. J shows faint staining for ETB receptors in the endothelium of NM segments. L shows positive staining for ETB receptors in the intima of pulmonary veins. (Bar = 30 µm.)

Immunoreactive ET_B receptors were found in the smooth-muscle layer of the large bronchus and the media and intima of pulmonary vessels. Low levels of staining were also detected in the bronchial epithelium and alveolar walls, although it was difficult to define the cellular distribution in the alveolar walls. In contrast to ET_A receptor, the distribution of ET_B receptors in the media of the pulmonary artery along the axial pathway appeared to predominate in distal rather than proximal segments, whereas in the intima, ET_B receptors were more abundant in proximal segments. Some positive staining for ET_B receptors was demonstrated in the intima of elastic artery and TM segments of muscular arteries, and only faint staining was detected in OM, PM, and NM segments. Immunoreactivity for ET_B receptors was also found in the intima, but little in the media of the pulmonary vein (Figure 1, Table 1). Negative controls for ET_A or ET_B receptor immunostaining showed few nonspecific stainings (data not shown).

Effects of Hypobaric Hypoxia on ET_A and ET_B Receptor Expression

Measurements of ventricular weights showed the RV/ LV+S ratios to be 0.31 ± 0.01 in control rats, and 0.40 ± 0.02 and 0.59 ± 0.01 in rats exposed to hypoxia for 7 and 14 d, respectively (control versus 7 and 14 d, P < 0.05). Histologic findings of elastic van Gieson staining in rats exposed to hypoxia for 7 and 14 d were also consistent with previous reports of a hypoxic pulmonary hypertension model, that is, an increase in medial wall thickness of muscular arteries corresponding to the terminal and respiratory bronchioles and extension of the muscle into smaller and more peripheral arteries than in control rats (data not shown) (21). The results indicate that a significant degree of pulmonary hypertension develops after 7 and 14 d exposure to hypoxia.

Northern blot analysis was used to examine changes in the expression of ET_A or ET_B receptor mRNAs in lung tissue after exposure to hypoxia. Bands of 5.2 and 4.2 kb of ET_A receptor mRNA transcripts were expressed in normal and hypoxia-exposed rat lung (Figure 2A). Compared with control rats, the ratio of ET_A receptor mRNA to 28S rRNA was about 1.5-fold higher after 12 h exposure to hypoxia, then decreased 1.3-fold at 3 d exposure, and there was a 30 to 50% increase in the ratio after 7 and 14 d exposure (Figure 2B). A slight decrease in the ratio at 3 d exposure appeared to be slightly discrepant with immunohistochemical findings. The ET_B receptor-specific probe hybridized with a single 5-kb band in the lung (Figure 3A). Hypoxic exposure was also associated with a significant increase in the ratio of ET_B receptor mRNA to 28S rRNA in the lung. The ratio increased 4-fold after 7 d exposure (Figure 3B).

View larger version (20K): [in this window] [in a new window]   Figure 2.   Effects of exposure to hypoxia on the level of ETA receptor mRNA level in rat lungs. (A) Representative Northern blots of ETA receptor in lung tissue from rats exposed to air and hypoxia for 0.5, 3, 7, or 14 d. Two bands, 5.2 and 4.2 kb, were detected in control and hypoxia-exposed rat lung. (B) Quantitation of ETA receptor mRNA by densitometry. Data were normalized to allow for variations in RNA loading using a 28S rRNA probe. The mRNA from each animal was quantitated individually, and the means ± SEM for each group were determined. ETA receptor mRNA expression increased after exposure to hypoxia. n = 4 for each group. *P < 0.05 versus control animals.

View larger version (19K): [in this window] [in a new window]   Figure 3.   Effects of exposure to hypoxia on ETB receptor mRNA levels in rat lungs. (A) Representative northern blots of ETB receptors in lung tissue from rats exposed to air and hypoxia for 0.5, 3, 7, or 14 d. A 5-kb ETB receptor band was detected in control and hypoxia-exposed rat lung. (B) Quantitation of ETB receptor mRNA by densitometry. Data were normalized to allow for variations in RNA loading using a 28S rRNA probe. The mRNA from each animal was quantitated individually, and means ± SEM for each group were determined; ETB receptor mRNA transcripts increased after 7 and 14 d exposure to hypoxia. n = 4 for each group. *P < 0.05 versus control animals.

Following exposure of rats to hypoxia, there was a progressive increase in immunoreactivity for ET_A receptors in the media of distal segments of muscular arteries. The increase was evident by 12 h after exposure, and reached a maximum after 14 d. In control animals, little positive staining was found in the arteries of less than 120 µm in diameter (ID), which corresponds roughly to PM or NM segments of muscular arteries, whereas faint staining was observed after 12 h exposure to hypoxia. The immunoreactivity rose gradually over 14 d of exposure, and weak to moderate staining was found in newly muscularized pulmonary arteries at alveolar wall level (Figure 4). Semiquantitative evaluation showed immunoreactivity for ET_A receptor to increase by 2.5-, 5-, and 20-fold compared with controls in arteries of 120 to 200 µm, 60 to 120 µm, and 30 to 60 µm ID, respectively, after 14 d exposure to hypoxia (Figure 5). There were no significant changes in ET_A receptor immunoreactivity in elastic arteries or TM segments of muscular arteries (data not shown).

View larger version (112K): [in this window] [in a new window]   Figure 4.   Representative micrographs showing immunostaining with anti-ETA receptor and anti--SM actin antibodies of distal segments of pulmonary arteries from a control rat and hypoxia-exposed rats. A shows little staining for ETA receptors in PM segments of muscular arteries from a control rat. B and C show faint and fairly strong staining for ETA receptors in the media of PM segments from rats exposed to hypoxia for 0.5 and 14 d, respectively. D shows negative staining for ETA receptors in NM segments from a control rat. E shows faint staining for ETA receptors in pericytes of NM segments from a rat exposed to hypoxia for 0.5 d. F shows positive staining for ETA receptors in pericytes and smooth-muscle cells of NM segment extending from the proximal site in a rat exposed to hypoxia for 14 d. G and H show faint to moderate staining for -SM actin in NM segments from a control rat and a rat exposed to hypoxia for 0.5 d, respectively. I shows strong staining for -SM actin in NM segments from a 14 d-exposed rat. (Bar = 30 µm.)

View larger version (34K): [in this window] [in a new window]   Figure 5.   Time course of the effect of exposure to hypoxia on ETA receptor (A) and -SM actin (B) immunoreactivity grading in pulmonary arteries 120 to 200, 60 to 120, and 30 to 60 µm ID. ETA receptor immunoreactivity grading increased significantly after 0.5 d exposure to hypoxia and reached a maximum after 14 d for each size of vessel. The increase after 14 d exposure was greater in smaller arteries; that is, about 2.5-, 5-, and 20-fold increases compared with control rats for arteries with ID of 120 to 200, 60 to 120, and 30 to 60 µm, respectively. -SM actin immunoreactivity grading increased significantly after 7 d exposure in the arteries with 120 to 200 µm ID; it increased significantly after 3 d exposure and reached maximum after 14 d exposure in arteries with 60 to 120 and 30 to 60 µm ID. A total of 60 to 500 vessels were counted for each group (*P < 0.001).

In accordance with the elevation of ET_A receptors, immunoreactivity for -SM actin also increased in the muscular arteries corresponding to the bronchioles and alveolar ducts after exposure to hypoxia (Figure 4). Semiquantitative evaluation showed that the increase of -SM actin immunoreactivities was preceded by that of ET_A receptors; that is, a significant increase of -SM actin was found at 7 and 14 d exposure to hypoxia in arteries of 120 to 200 µm ID, and at 3, 7, and 14 d exposure in arteries of 60 to 120 and 30 to 60 µm ID (Figure 5). There were no significant changes in -SM actin immunoreactivities in larger pulmonary arteries (data not shown).

The most significant change in ET_B receptor expression associated with exposure to hypoxia was an increase in the number of ET_B receptors in the intima of distal segments of muscular arteries. The maximum increase in ET_B receptors in the intima of arteries was reached after 7 d exposure (Figure 6). Semiquantitative analysis showed immunoreactivity for ET_B receptors in the intima to increase by 1.9-, 1.8-, and 1.7-fold in arteries of 120 to 200, 60 to 120, and 30 to 60 µm ID, respectively, after 7 d exposure to hypoxia (Figure 7). Immunoreactivity for ET_B receptors in the media of muscular arteries also tended to increase after exposure to hypoxia, though the changes were not statistically significant. In agreement with these findings, in situ hybridization using ET_B receptor-specific riboprobe demonstrated the greater expression of ET_B receptor mRNA transcripts in the intima of OM, PM, and NM segments of rats exposed to hypoxia for 7 d than of control rats (Figure 8). There were no significant changes in the immunoreactivities for ET_B receptor in elastic arteries, TM segments of muscular arteries, or pulmonary veins (data not shown).

View larger version (181K): [in this window] [in a new window]   Figure 6.   Representative photomicrographs of lungs from control and hypoxia-exposed rats stained with anti-ETB receptor antibody. A shows faint staining of ETB receptor in the intima and media of OM segment of muscular arteries from a control rat. B shows strong ETB receptor immunoreactivity in the intima and medial thickening of the OM segments in a rat exposed to hypoxia for 7 d. C shows faint staining of ETB receptors in PM segment of muscular arteries from a control rat. D shows enhanced ETB receptor staining in the intima of PM segments from a rat exposed to hypoxia for 7 d. (Bar = 30 µm.)

View larger version (47K): [in this window] [in a new window]   Figure 7.   Time course of the effect of exposure to hypoxia on ETB receptor immunoreactivity grading in the endothelium of pulmonary arteries with 120 to 200, 60 to 120, and 30 to 60 µm ID. ETB receptor immunoreactivity grading increased significantly after 3, 7, and 14 d exposure to hypoxia for each vessel size. A total of 60 to 500 vessels were counted in each group (*P < 0.001).

View larger version (137K): [in this window] [in a new window]   Figure 8.   In situ hybridization of ETB receptors in the lungs of control and hypoxia-exposed rats. A and C show little staining in PM and NM segments, respectively, of muscular arteries from a control rat. B and D show positive staining for ETB receptor mRNA transcripts in the endothelium of PM and NM segments, respectively, after exposure to hypoxia for 7 d. (Bar = 30 µm.)

	Discussion

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We have demonstrated the localization and distribution of two different subtypes of ET receptor in rat lung, focusing on the pulmonary vasculature. In normal rats, ET_A receptors are localized in the media of pulmonary arteries and veins with a predominant distribution in proximal segments, although some ET_A receptors are expressed in the distal pulmonary arteries accompanying the terminal bronchioles. These data are consistent with previous pharmacologic and physiologic studies using vascular rings from main and distal pulmonary arteries that have shown attenuation of ET-1-induced contractility by selective ET_A receptor antagonists to be more significant in the large vessels than in the small vessels (22). In other words, ET-1-induced pulmonary vasoconstriction mediated by ET_A receptors may occur primarily in the proximal rather than the distal segments of the pulmonary artery. ET_B receptors are expressed in the media and intima of pulmonary vessels. In contrast to ET_A receptors, the distribution of ET_B receptors in the media of the pulmonary artery predominates in distal segments, whereas they are more abundant in the proximal segments of the endothelium. These results support previous physiologic data that suggest vasoconstrictor effects through ET_B receptors occur primarily in the distal segments of the pulmonary artery (23). Although small amounts of ET_B receptors are also expressed in the smooth-muscle layers of elastic arteries and large muscular arteries, their vasoconstrictive effects may be canceled by dilatory action mediated by ET_B receptors expressed in the endothelium.

ET-1 is known to have bidirectional actions on pulmonary vascular tone. Although there is little doubt that ET_A receptors found in the smooth-muscle layer of pulmonary vessels mediate vasoconstriction, ET_B receptor activation causes either vasoconstriction or vasodilatation in pulmonary circulation, depending on its degree of activation. Lower concentrations of ET_B receptor agonist elicit dilation, whereas higher concentrations cause constriction of the vascular smooth muscle (25). This bidirectional action may be due to the existence of two different ET_B receptor subtypes: ET_B1 receptor expressed in endothelial cells mediates vasodilatation by releasing nitric oxide and dilator prostaglandins or activating potassium channels, and ET_B2 receptor expressed in smooth-muscle cells mediates vasoconstriction by elevating intracellular Ca²⁺ concentrations (1, 2, 26). The overall response to ET-1 is believed to be determined by the balance between vasoconstriction mediated by ET_A and ET_B2 receptors, and vasodilation mediated by ET_B1 receptors. The expression of these receptors in the smooth muscle and endothelium appears to differ along the axial pathway of the pulmonary vasculature, and this heterogeneity may cause different responses to ET-1 in the proximal and distal segments of the pulmonary artery.

Double-occlusion experiments in isolated perfused rat lung suggested that vasoconstriction induced by ET-1 occurs at venous as well as arterial sites (28). In this study, some ET_A receptors, but few ET_B receptors, were found in the media of the large pulmonary vein, suggesting that ET-1-induced venoconstriction is mediated mainly by ET_A receptors, at least in the large vein. However, the participation of ET_B receptors in postcapillary vasoconstriction cannot be excluded, because we could not identify the receptor subtypes in smaller pulmonary veins in this study. ET_B receptors expressed in the endothelium of the pulmonary vein might exert dilatory effects by releasing vasodilatory mediators and facilitating ET-1 clearance, as in the pulmonary artery (29, 30).

Hypoxic exposure is associated with an increase in the number of ET_A receptors in the media of pulmonary resistance arteries corresponding to terminal and respiratory bronchioles and alveolar ducts, although the change in the proximal segments of the pulmonary artery seems minimal. The increase in ET_A receptors in resistance arteries probably plays an important role in vascular remodeling following exposure to hypoxia for the following reasons. First, ET_A receptor is known to mediate mitogenic activity to stimulate DNA synthesis and cell proliferation in smooth-muscle cells and fibroblasts (3). Second, as shown in immunohistochemical study for -SM actin, the localization of the new ET_A receptors induced by hypoxia corresponds closely to the site where smooth-muscle cell and fibroblast proliferation primarily occurs after exposure to hypoxia, namely, OM, PM, and NM segments of muscular arteries. Third, the upregulation of ET_A receptors that was evident after 12 h exposure to hypoxia preceded the increase in smooth-muscle cell proliferation and the extension of the muscle into smaller vessels (Figure 5). Finally, several studies have shown that blocking of ET_A receptors with a selective antagonist abolishes the increase in pulmonary arterial pressure and prevents the development of vascular remodeling associated with hypoxic pulmonary hypertension (31, 32). Thus, the increase in the number of ET_A receptors in resistance arteries probably facilitates the development of hypoxic pulmonary hypertension by promoting vascular remodeling.

Expression of ET_A receptor mRNA in the lung was also induced at 0.5 d exposure to hypoxia, and there was a persistent elevation at 7 and 14 d exposure, though a small discrepancy was found between mRNA expression and immunohistochemical findings at 3 d exposure. Although the reason for the discrepancy was not clear, a drop in ET_A receptor mRNA expression at Day 3 may reflect a decrease in expression in nonvascular sites.

The increases in the expression of ET_B receptor mRNA in the lung after exposure to hypoxia seem to be comparable with the results in previous studies, but the localization has not been previously reported (12, 13). In situ hybridization and immunohistochemical studies showed the most significant change in ET_B receptor expression after hypoxic exposure to be an increase in the endothelium of the distal segments of muscular arteries accompanying terminal and respiratory bronchioles and alveolar ducts. Because ET_B receptors expressed in the endothelium are thought to mediate the vasodilatory action, the increase in the number of ET_B receptors could result in an augmentation of the vasodilatory effect of ET-1 by exposure to hypoxia. This hypothesis is supported by a previous hemodynamic study that showed pulmonary vasodilatation induced by ET-1 to be more prominent in preconstricted isolated perfused lung from rats exposed to hypoxia for 21 d than in control rat lungs (33). In addition to vasodilatory activity, because ET_B receptors in the endothelium involve ET-1 clearance, an increase in the number of ET_B receptors in the endothelium may reduce ET-1 concentrations in the circulating blood and local tissues, resulting in an attenuation of ET-1-induced vasoconstriction and mitogenic action for vascular smooth-muscle cells and fibroblasts (29, 30). Furthermore, ET-1 can act as a mitogen through ET_A receptors, although ET_B receptors may mediate the inhibition of cell proliferation under certain conditions (34). Accordingly, the increase in the number of ET_B receptors in resistance arteries might attenuate the vascular remodeling induced by exposure to hypoxia. Therefore, the increase in the number of ET_B receptors in the endothelium might act as a compensatory mechanism in the development of hypoxic pulmonary hypertension through vasodilatory action and the attenuation of vascular remodeling.

We also found that the number of ET_B receptors in smooth muscle tends to increase under hypoxic conditions. It is conceivable that these receptors have vasoconstrictive action and may contribute to the pathogenesis of hypoxic pulmonary hypertension. Indeed, we and others have observed that ET_B receptors are significantly involved in ET-1-induced vasoconstriction in hypoxic pulmonary hypertensive rat lungs (35, 36). However, previous studies have demonstrated that a selective ET_A receptor antagonist, BQ123, prevents the development of hypoxic pulmonary hypertension in rats almost completely, suggesting that ET_B receptors play a minor role in this process (31, 32). Although the exact significance of ET_B receptors in smooth muscle in this pathogenesis remains uncertain, these findings suggest that vasoconstrictor action mediated by ET_B receptors may be masked by vasodilatory effects mediated by ET_B receptors that increase in number in the endothelium.

In summary, our immunohistochemical study suggests that ET-1 exerts its vasoconstrictor effect mainly through ET_A receptors in the pulmonary conduit arteries and pulmonary vein, whereas its effects are mediated by ET_B and ET_A receptors in resistance arteries in normal rats. ET_B receptor expressed in the endothelium of the entire pulmonary artery and vein are thought to contribute to the basal vasodilatory activity and clearance of endothelins. We have also demonstrated that exposure to hypoxia is associated with an increase in the number of ET_A receptors in the media of pulmonary resistance arteries, which probably plays an important role in the pathogenesis of hypoxic pulmonary hypertension. ET_B receptor is also upregulated in the endothelium of resistance artery after exposure to hypoxia, which could counteract the development of pulmonary hypertension. In this context, an ideal treatment for hypoxic pulmonary hypertension may result from a selective combined blockage of ET_A and ET_B receptors expressed in smooth-muscle cells, because blockade for ET_B receptors in the endothelium may exaggerate hypoxic pulmonary hypertension.