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Cardiovascular Research 1997 35(2):360-367; doi:10.1016/S0008-6363(97)00103-X
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Copyright © 1997, European Society of Cardiology

Down-regulation of endothelin B receptors in autogenous saphenous veins grafted into the arterial circulation

Daihiko Eguchia, Junji Nishimuraa, Sei Kobayashia, Kimihiro Komorib, Keizo Sugimachib and Hideo Kanaidea,*

aDivision of Molecular Cardiology, Research Institute of Angiocardiology, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-82, Japan
bDepartment of Surgery II, Faculty of Medicine, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-82, Japan

* Corresponding author. Tel.: +81 (92) 642-5549; fax: +81 (92) 642-5552.

Received 28 November 1996; accepted 8 April 1997


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 
Objectives: It has been postulated that endothelin (ET) might be involved in the development of atherosclerotic vascular lesions. The present study was done to characterize changes in the contractility and ET receptor subtypes in the autogenous saphenous vein graft (VG). Methods: The rabbit saphenous vein (SV) was grafted into the ipsilateral femoral artery (FA), and at 4 weeks after the operation, VG was harvested. In the medial layer samples of SV, VG and FA, the cytosolic Ca2+ concentration ([Ca2+]i) and force were monitored using front-surface fluorometry of fura-PE3, and mRNA expression of ET receptors was evaluated using the reverse transcription polymerase chain reaction. Results: ET-1 (10–7 M) developed force in SV, VG and FA, to the same extent. Sarafotoxin (S6c; 10–7 M) developed force in the SV to the same extent as ET-1. However, S6c did not develop force in FA, and slight force developed in VG. Contractions induced by ET-1 were associated with increases in [Ca2+]i. FA expressed ETA receptor mRNA predominantly, and SV expressed both ETA and ETB receptors mRNAs. In VG, the expression of ETB receptor mRNA was markedly reduced, but expression of ETA receptor mRNA remained unchanged. Conclusions: Functioning ETB receptors and their mRNA are down-regulated when veins are grafted into the arterial circulation. All these changes in gene expression and function are part of adaptive responses known as ‘arterialization’.

KEYWORDS Endothelin; Autogenous vein graft; Rabbit, saphenous vein; ETA/ETB receptor; mRNA; Vascular smooth muscle; Calcium, cytosolic concentration


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 References
 
Endothelin-1 (ET-1) is a vasoconstrictor peptide isolated from the supernatant of cultured porcine endothelial cells [1]. Functional activities of ET are mediated via binding to specific cell-surface receptors. There are at least two subtypes of vascular ET receptors— ETA and ETB [2]. It has been considered that stimulation of ETA receptors on vascular smooth muscle cells leads to vasoconstriction, whereas activation of ETB receptors on endothelial cells elicits vasodilation [3]. However, specific ligands for ETA and ETB receptors revealed the presence of ETB receptors on smooth muscle cells which mediate contraction in different blood vessels [4–6]. The ETB-receptor-mediated vasoconstriction may play an important role in the control of vascular tone on the low pressure side of the circulation such as is the case in venous and pulmonary circulations [7].

In addition to its role as a vasoconstrictor, ET-1 was reported to have a proliferating action on vascular smooth muscle cells [8, 9], and it has been postulated that ET-1 might be involved in the development of atherosclerotic vascular lesions. There is some evidence suggesting a role for ET-1 as a marker and/or a participant in human cardiovascular disorders [10, 11]. Although more study is needed on the receptor subtype responsible for proliferating activity, changes in ET receptor expression in vivo have been noted in pathological states such as balloon angioplasty in the rat [12], early atherosclerosis in cholesterol-fed hamsters [13], and arteriovenous fistula in the dog [14, 15].

Autogenous vein grafting into the arterial circulation is one of the pathological states in which smooth muscle cell proliferation and intimal hyperplasia occur. Although intimal and medial hyperplasia can be causative factors of graft failure [16], these structural alterations may afford vein grafts (VGs) dynamic intensity to withstand high arterial pressure and, hence, would be a compensatory adaptive response. In addition to these morphological changes, alterations in vasomotor function of VGs have been described [17–20]. However, whether or not smooth muscle contractility in response to ET-1 and expression of ET receptor subtypes change in veins transplanted into arterial circulation has not been given attention in the literature.

The purpose of this study was to determine alterations in ET receptors on venous smooth muscle within the arterial circulation. To characterize the receptors pharmacologically, we used endothelin-1 (an ETA/ETB non-selective agonist) and Sarafotoxin (S6c) (an ETB selective agonist). S6c is an extremely selective ligand of ETB receptor, and binds it with an affinity several hundred-fold that of the ETA receptor. We also detected ET receptor mRNA on native and grafted venous smooth muscle, using the reverse transcription polymerase chain reaction (RT-PCR). We also examined the partial sequences of ETA and ETB receptor mRNA in the rabbit. To our knowledge, this is the first report on the ET receptor subtype in autogenous VG and the simultaneous measurement of the [Ca2+]i and tension in VG.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 References
 
2.1 Surgical procedures
The investigation conformed with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 86-23, revised 1985). Male New Zealand White rabbits, weighing approximately 2.5 kg were anesthetized with an intramuscular injection of xyladine (10 mg/kg) and ketamine hydrochloride (40 mg/kg). Anesthesia was maintained with an additional intramuscular injection of ketamine hydrochloride (15 mg/kg) every 30 min. A longitudinal lateral incision was made in the right thigh and the lateral saphenous vein (SV) was exposed. Approximately 2 cm segment of the SV was taken for the autogenous VG. The vein was emptied of blood by gravity flow through the proximal end. Harvesting of the SV for graft was completed with meticulous care to avoid injury to the vein wall. The harvested SV was preserved in heparinized saline (5 U/ml) for 20 min before grafting, at room temperature. The lateral thigh incision was then closed with running 2-0 nylon and a longitudinal medial incision was made in the upper thigh to expose the femoral artery (FA). FA was clamped proximally and distally with single clamps. The segment, 1 cm in length, distal to the orifice of the lateral circumflex femoral artery, was resected and replaced with the harvested autogenous SV. The SV for graft was anastomosed in a reversed end-to-end fashion with interrupted 10-0 monofilament sutures, under microscopic vision. The time from vein excision to establishment of blood flow after completion of the anastomosis did not exceed 60 min. All surgical procedures were performed in an aseptic manner, and the anticoagulant was not administered because it affects the patency rate and the degree of the intimal thickening of the VG [21]. The incision was then closed with interrupted 4-0 nylon in the subcutaneous layer and running 2-0 nylon in the cutaneous layer. After recovery from anesthesia, none of the animals exhibited impairment of mobility of the hindlimbs and infection was nil.

2.2 Tissue preparation
At 4 weeks after implantation of the autogenous VG, the rabbits were anesthetized in the same manner and the VG were harvested. FA and SV from the left limb were also harvested, as control tissues. The excised vessels were placed in physiological salt solution (PSS). The adventitia and fat were removed, under a binocular microscope. The endothelial cells were then removed by gently rubbing the intraluminal surface with a cotton swab wetted with PSS solution. Medial preparations of SV, VG and FA were opened longitudinally and then cut into approximately 1.5x3 mm circular strips.

2.3 Measurement of force and [Ca2+]i
The medial strips of SV, FA and VG were loaded with [Ca2+]i indicator dye, fura-PE3 in the form of acetoxymethyl ester (fura-PE3/AM) as we reported previously [22]. Then the loaded medial strips were mounted vertically in a quartz organ bath, and isometric force development was measured by a force-transducer (strain gauge TB-612T, Nihon Kohden, Japan). During the 1 h equilibration period, the strips were stimulated with 118 mM K+ every 15 min, and the resting load was increased in a stepwise manner to obtain the maximal force development. The resting load for the measurements thus obtained was approximately 250 mg for SV and VG and 350 mg for FA. After obtaining the resting load, the steady responsiveness of each strip to 118 mM K was obtained before addition of each agonist (ET-1, S6c) to the organ bath. Changes in [Ca2+]i were simultaneously monitored as fluorescence ratio during measurement of the force, using front-surface fluorometry as we reported previously [22]. The force development and the fluorescence ratio were expressed as a percentage, while assigning the values at rest in PSS (5.9 mM K+) and 118 mM K+ PSS to be 0% and 100%, respectively. Data on fluorescence ratio and force were stored in a Macintosh computer, using a data acquisition system (MacLab).

2.4 Measurement of ETA and ETB receptor mRNA by RT-PCR
Total RNA was isolated from the smooth muscle cells from FA, SV and VG, according to Chomczynski and Sacchi [23]. Care was taken not to include either the endothelium or adventitia during the tissue trimming. Contaminating genomic DNA, if any, was digested by RNase-free DNase. First-strand cDNA was synthesized using as template total RNA. The total RNA (1 µg) was incubated at 37°C for 60 min with a mixture (total volume = 20 µl) of 200 units of M-MLV reverse transcriptase, 1x RT buffer, 10 mM DTT, 0.5 mM of each dNTP (dATP, dCTP, dTTP, dGTP), 20 units of RNase inhibitor and 50 nM of antisense primers (Table 1, RT primer). For PCR amplification, an aliquot (1 µl) of the RT product was mixed with 0.5 unit of Taq DNA polymerase, 500 nM each of sense and antisense primers (Table 1, PCR primers) in a buffer containing 20 mM Tris HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2, 100 µg/ml BSA and 200 µM each of dNTPs in a 11 µl volume. The thermal cycle profile used in this study was (1) denaturing for 30 s at 94°C, (2) annealing primers for 90 s at 55°C and (3) extending the primers for 30 s at 72°C. PCR amplification was performed for 35 cycles. Since sequences of the rabbit ETA and ETB receptor mRNA have not been documented, sequences of sense and antisense primers were designed based on those of the rat and the pig, from conserved regions among the bovine [24], rat [25]and human [26]ETA and ETB receptor sequences (Table 1). The expected size of the PCR products for ETA receptor and ETB receptor were 188 and 304 bp, respectively. A portion (10 µl) of the PCR mixture was electrophoresed in a 3% agarose gel in TAE buffer. The gel was stained with ethidium bromide and photographed. Direct sequencing of the PCR products for ETA and ETB receptors of the rabbit was done by Sawady Technology (Tokyo, Japan) using an Autosequencer (373S, ABI, Foster City, CA).


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  		Table 1 Oligonucleotide primers for RT-PCR

 
2.5 Drugs and solutions
PSS consisted of the following composition (in mM): NaCl 123, KCl 4.7, NaHCO3 15.5, KH2PO4 l.2, MgCl2 l.2, CaCl2 1.25, and D-glucose 11.5. High K+ PSS was identical to normal PSS, except for an equimolar substitution of KCl for NaCl. All solutions were gassed with a mixture of 95% O2 and 5% CO2 (pH adjusted to 7.4 at 37°C). ET-1 and S6c were obtained from the Peptide Institute Co. Ltd. (Osaka, Japan). Ketamine hydrochloride was obtained from Sankyo Co. (Tokyo, Japan). Fura-PE3/AM was from the Texas Fluorescence Laboratory (Austin, TX, USA). Xyladine hydrochloride was from SIGMA (St. Louis, MO, USA). M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase, 5x RT buffer, and 0.1 M DTT (dithiothreitol) were from BRL (Gaithersburg, MD, USA). NusieveTM 3:1 agarose and dNTPs (dATP, dCTP, dTTP, dGTP) were from TaKaRa (Kyoto, Japan). RNase inhibitor was purchased from Toyobo (Osaka, Japan). Taq DNA polymerase was from Pharmacia Biotech (Uppsala, Sweden). RQ1 RNase-free DNase was from Promega (Madison, WI, USA). All other chemicals were of the highest grade commercially available. The oligonucleotides for primers were synthesized by Sawady Technology (Tokyo, Japan).

2.6 Data analysis
All data for the simultaneous measurements of [Ca2+]i and force were collected using a computerized data acquisition system (MacLab, Analog Digital instruments, Castle Hill, Australia: Macintosh, Apple Computer, Cupertino, CA, USA). Data on representative traces shown in this report were directly printed out from the computer to a laser printer (LaserWriter II NTX-J, Apple Computer). The measured values were expressed as the means±s.e. (n = number of the experiments). For each experiment, a vascular strip from a different animal (n = 4–6) was used. For comparison, an unpaired Student t-test was used and P-values of less than 0.05 were considered to be significant.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 References
 
3.1 Effect of ET-1 on the [Ca2+]i and force in VG strips
Fig. 1 shows a representative recording of changes in the fluorescence ratio (F340/F380) and force development induced by 10–7 M ET-1 in PSS in VG. When the VG strip was exposed to 10–7 M ET-1, [Ca2+]i rose rapidly to reach a steady state. The peak levels of the [Ca2+]i elevations induced by 10–7 M ET-1 were 94±6.0% (n = 4) of those observed during 118 mM K+ depolarization. After the application of 10–7 M ET-1, the force also developed rapidly and reached a maximum within 5 min. The peak level of force development was 122±7.6% (n = 4).


Figure 1
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  		Fig. 1 Representative recordings of the effects of ET-1 on [Ca2+]i and force of VG in normal PSS. Representative recordings of changes in the fluorescence ratio ([Ca2+]i) (upper trace) and force (lower trace) induced by 118 mM K+-depolarization and 10–7 M ET-1.

 
3.2 ET-1- and S6c-induced contractions in the FA, SV and VG
Fig. 2 shows representative recordings of the contraction induced by ET-1 and S6c in FA (panel A), SV (panel B) and VG (panel C). After recording the 100% response levels by depolarization with 118 mM K+ PSS, 10–7 M S6c and then ET-1 were applied in normal PSS. Absolute levels of force developed by 118 mM K+ depolarization in FA, SV and VG were 1.83±0.05, 0.69±0.04 and 0.92±0.01 g, respectively (P<0.01). In SV strips, the 118 mM K+-induced contraction was biphasic, initially transient and sustained. The overshoot seen in SV became smaller in VG strips. The applications of 10–7 M S6c to SV and VG induced transient contractions, while 10–7 M S6c induced no contraction in FA. ET-1 induced contractions in all three vessels, to a similar extent. The maximal force developments induced by S6c and S6c plus ET-1 were 0 and 136±6.5% in FA, 131±5.7 and 143±9.2% in SV, and 36±6.5 and 132±6.5% in VG, respectively (n = 6). The force development induced by S6c in VG was significantly smaller than that in SV (P<0.001) and significantly greater than that in FA (P<0.001), while there was no difference in the force development induced by ET-1 among these three vessels (Fig. 3).


Figure 2
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  		Fig. 2 Representative recordings of the effects of 10–7 M S6c and ET-1 on the force of FA (A), SV (B) and VG (C). After recording the 100% response levels by depolarization with 118 mM K+ PSS, 10–7 M S6c and ET-1 were applied, in that order.

 

Figure 3
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  		Fig. 3 Summary of forces development induced by S6c and ET-1 in FA, SV and VG, as determined in Fig. 2. Values are the means±s.e. of number of experiments (n = 6), expressed in percentage assigning the contraction induced by 118 mM K+ at 100%. The force development induced by S6c in VG was markedly reduced compared with that in SV (P<0.001).

 
3.3 Detection of ETA and ETB receptor mRNA by RT-PCR
Fig. 4 shows evidence for detection of ETA, ETB receptors and β-actin mRNAs by RT-PCR, using total RNA prepared from smooth muscle of FA, SV and VG, and the primers for the ETA, ETB receptors and β-actin. ETA receptor bands of the expected size (188 bp) could be detected by RT-PCR, in all these blood vessels, but only when cDNA was added (Fig. 4A). However, as shown in Fig. 4B, the ETB receptor bands of the expected size (304 bp) could be detected in SV and VG, and these bands were not detected in FA. FA expressed ETA receptor mRNA predominantly, and SV expressed both ETA and ETB receptor mRNAs. In VG, the expression of ETB receptor mRNA was almost invisible, although the ETA receptor mRNA was detected to the same extent as in FA and SV. RT-PCR for β-actin, as an internal control, was also done to estimate the relative abundance of ETA and ETB receptors mRNA (Fig. 4C). As seen in Fig. 4C, the expression of β-actin mRNA did not differ among FA, SV and VG, compared to that of ETA, ETB receptor mRNA. In this experiment, we used 3 preparations from 3 different rabbits. PCR amplifications for ETA, ETB receptor and β-actin were seen for 35, 35 and 25 cycles, respectively. Possible amplifications of the genomic ETA and ETB sequence could be excluded since a band of the expected size was detected only when reverse transcriptase was added (data not shown). To assess the specificity of the RT-PCR for ETA and ETB receptor mRNA, sequences of the PCR products were determined directly. The nucleotide and amino acid sequences for this PCR product are shown in Fig. 5. Similarities of these sequences to the corresponding region of the bovine, human and rat ETA/ETB receptor cDNA sequences were 90.28/91.67, 93.75/93.56 and 88.19/86.74%, respectively. Amino acid sequences also showed 97.87/96.55, 100/100 and 100/98.85% similarity to those of bovine, human and rat ETA/ETB receptors, respectively. Thus, the PCR product obtained by ETA/ETB receptor primers in the present study was considered to be derived from rabbit ETA/ETB receptor cDNA.


Figure 4
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  		Fig. 4 The expression of ETA (A), ETB (B) and β-actin (C) mRNA in FA, SV and VG determined by RT-PCR. mRNA from each vessel was obtained from a single animal. FA expressed ETA receptor mRNA exclusively; SV expressed both ETA and ETB receptor mRNAs. In VG, the expression of ETB receptor mRNA was markedly reduced, whereas ETA receptor mRNA was expressed to an extent similar to that in FA. Lanes 4 and 8 represent DNA size marker (M; ØX174/Hinc II digest). Size of each band is given on the right of each photograph.

 

Figure 5
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  		Fig. 5 Partial nucleotide and amino acid sequences of the rabbit ETA (A) and ETB (B) receptors.

 

   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
References
 
We examined alterations in ET receptor expression in autogenous VG, both functionally and using molecular techniques. For comparison, ET receptor expression in SV and FA was also evaluated. The major findings are: (1) S6c induced a small contraction in VG compared with that in SV, while S6c induced no contraction in FA; (2) SV expressed both ETA and ETB receptors, while FA and VG expressed predominantly ETA receptors. The latter result means that when SV has been grafted into the arterial circulation, down-regulation of functioning ETB receptors occurs, whereas the expression of ETA receptors remains unchanged. This change in gene expression and function is one of the adaptive responses of veins implanted into the arterial circulation, namely ‘arterialization’ [27].

The autogenous saphenous VG is currently one suitable conduit for small-caliber arterial reconstruction for arterial occlusive disease, including ischemic heart disease. In VG, structural changes in the vascular wall occur along with the time course after implantation. We previously reported that intimal thickening was clearly distinct 1 week after grafting, developed progressively in 2 to 3 weeks, and then reached a steady state of the progression at 4 weeks after grafting [28, 29]. In addition, it has been reported that the graft surface, including the site of anastomosis, was almost completely covered with endothelial cells, and appeared morphologically normal [30]. We thus considered that the period of 4 weeks after implantation is sufficient to recover from tissue damage sustained at surgery. Therefore, we concluded that the best time for harvesting the SV graft is 4 weeks after implantation. Other investigators demonstrated that autogenous VGs also undergo concomitant functional or biochemical adaptation to the arterial environment [17–20]. These studies indicated that VGs have a decreased sensitivity to norepinephrine and histamine [19], and that they develop a constrictive response to 5-HT [17, 31]. Furthermore, endothelium-dependent relaxation in response to acetylcholine, ADP and histamine is impaired in VG [18, 20, 32]. Our study provides the first evidence that venous smooth muscle cells implanted into the arterial circulation undergo alterations in ET receptor expression, similar to that in arteries. In the present study, we removed the endothelium from the vessel samples in order to determine alterations in ET receptors solely on venous smooth muscle cells.

As shown in Fig. 1, ET-1 produced a greater force development than expected from the contraction induced by high K+ depolarization in VG. As we reported that ET-1 also increases Ca2+ sensitivity of the contractile apparatus in coronary arterial strips [33], the present results can be explained by such mechanisms. However, we cannot evaluate the Ca2+ sensitivity in the VG critically because we measured only one point (10–7 M of ET-1) on the dose–response curve. The present report is apparently the first of simultaneous measurements of [Ca2+]i and tension of VG.

Regarding the distribution of ET receptors in vascular smooth muscle, it has been postulated that ETA receptors predominate on the arterial side, whereas ETA and ETB receptors co-exist on the venous side [34]. The SV has been found to carry heterogeneous populations of ET receptors. Webb et al. reported that the ET receptor population in rabbit SV is composed of approximately 70% ETA receptors and 30% ETB receptors [35]. Our present results support this notion. Whether the significant expression of ETB receptors on the venous smooth muscle is due to low pressure, low oxygen tension or other factors is not well understood. We also examined the expression of ET receptors in the pulmonary artery and vein, and elucidated that ETA receptors predominate in the pulmonary artery, while both ETA and ETB receptors coexist in the pulmonary vein, which is perfused with high oxygen tension blood, under non-pulsatile flow with low pressure (Sakihara et al., submitted). Furthermore, Adner et al. demonstrated that, when the vessel segment was exposed to no pulsating blood flow and no nervous or humoral stimulation in the organ culture bath, the contractile responses through ETB receptors and expression of ETB receptor mRNA were increased [36]. They suggested that the up-regulation of ETB receptors in cultured vessel segments could be due to the loss of pressure in the vascular wall. Thus, it is tempting to speculate that the major determinant of the expression of ETB receptors in venous smooth muscle may be the low shear stress and low blood pressure. Modulation of ET receptors in vascular smooth muscle cells by chronic changes in local physical or chemical environments was reported. Miller and Michener reported that elevation in blood flow and oxygen tension decreased ET receptors in the rabbit femoral vein distal to the arterio-venous fistula [14], whereas Barber et al. found that these conditions increased ET receptors, especially the ETB receptor, in femoral arteries [15]. The latter report is inconsistent with our speculation that the increased pressure in the vascular wall causes down-regulation of ETB receptor. This discrepancy seemed to be due to the differences in the animals and vessels used.

In vascular remodeling, including balloon angioplasty or vein grafting, as in the case of the morphological changes, alterations in ET receptors may change temporarily. Smooth muscle cells in the early stage of vascular remodeling are the synthetic phenotype, and those in the late stage are the contractile phenotype. In an in-vitro study, intact rat aortic smooth muscle cells, which represent the contractile phenotype, predominantly expressed ETA receptor, whereas cultured rat aortic smooth muscle cells, which represent the synthetic phenotype, preferentially expressed ETB receptor [37]. Wang et al. demonstrated using rat balloon-injured carotid arteries that the levels of ETA and ETB receptor mRNA were markedly elevated in early postoperative days, and then decreased to slightly elevated levels by 14 days [12]. Although the expression of ET receptor on smooth muscle cells after balloon angioplasty was unclear because of the co-existence of endothelium in this study, it is possible to speculate that up-regulation of ETA and/or ETB receptors on venous smooth muscle cells immediately after vein grafting does occur. Observations of the time course of alterations in ET receptors after vein grafting are needed to clarify this.

In summary, when veins are transplanted into the arterial circulation, ETB receptors on venous smooth muscle cells are down-regulated as a result of ‘arterialization’. Although the mechanisms and the meaning of this event remains to be elucidated, the present results may pave the way to a better understanding of the physiology of VG.

Time for primary review 39 days.


   Acknowledgements
 
We thank M. Ohara for helpful comments on the manuscript. This study was supported in part by Grants-in-Aid for Developmental Scientific Research (No. 06557045), for General Scientific Research (Nos. 07407022, 07833008) and for Creative Basic Research Studies of Intracellular Signaling Network from the Ministry of Education, Science, Sports and Culture, Japan, by Grants from the Japan Research Foundation of Clinical Pharmacology, and by the Kimura Memorial Heart Foundation Research Grant. We also thank Ms. K. Kajishima for her secretarial services.


   References
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 References
 

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N. R. Tykocki, C. E. Gariepy, and S. W. Watts
Endothelin ETB Receptors in Arteries and Veins: Multiple Actions in the Vein
J. Pharmacol. Exp. Ther., June 1, 2009; 329(3): 875 - 881.
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