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(Circulation. 1995;92:357-363.)
© 1995 American Heart Association, Inc.

Articles

Endothelin ET_A and ET_B Receptors Cause Vasoconstriction of Human Resistance and Capacitance Vessels In Vivo

William G. Haynes, BSc, MD, MRCP; Fiona E. Strachan, BN, RGN; David J. Webb, MD, FRCP, FFPM

From the University of Edinburgh (UK), Department of Medicine, Western General Hospital.

Correspondence to Dr David J. Webb, University Department of Medicine, Western General Hospital, Edinburgh EH4 2XU, UK. E-mail d.j.webb@ed.ac.uk.

	Abstract

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Background The role of endothelin ET_B receptors in mediating vasoconstriction in humans is unclear. As yet, there have been no in vivo studies in resistance vessels, and in vitro data have been contradictory. We therefore investigated the function of ET_B receptors in vivo in human forearm resistance and hand capacitance vessels using endothelin-1 as a nonselective agonist at ET_A and ET_B receptors and endothelin-3 and sarafotoxin S6c as selective agonists at the ET_B receptor.

Methods and Results A series of single-blind studies were performed, each in six healthy men. Brachial artery infusion of endothelin-1 and endothelin-3 caused slow-onset dose-dependent forearm vasoconstriction. Although endothelin-3 caused significantly less forearm vasoconstriction than endothelin-1 at low doses, vasoconstriction was similar to the two isopeptides at the highest dose (60 pmol/min). Endothelin-3 caused transient forearm vasodilatation at this dose, whereas endothelin-1 showed only a nonsignificant trend toward causing early vasodilatation. Intra-arterial sarafotoxin S6c caused a progressive reduction in forearm blood flow, although less than that to endothelin-1 (P=.04). Dorsal hand vein infusion of sarafotoxin S6c caused local venoconstriction that was also less than that to endothelin-1 (P=.002).

Conclusions Selective ET_B receptor agonists cause constriction of forearm resistance and hand capacitance vessels in vivo in humans, suggesting that both ET_A and ET_B receptors mediate vasoconstriction. Hence, antagonists at both ET_A and ET_B receptors, or inhibitors of the generation of endothelin-1, may be necessary to completely prevent vasoconstriction to endogenously generated endothelin-1.

Key Words: endothelin • vasoconstriction • vessels

	Introduction

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The endothelins are a family of 21-amino-acid peptides with potent and characteristically sustained vasoconstrictor and vasopressor actions.¹ Endothelin-1 is the predominant isopeptide generated by the vascular endothelium.² Endothelin-2 and endothelin-3 are more difficult to detect in humans and are probably less important in their cardiovascular effects.

Two specific receptors for the endothelins have been isolated by in vitro expression of cloned human cDNA.^{3 4} The ET_A receptor has a high affinity for endothelin-1, with a K_i of 0.6 nmol/L for endothelin-1 compared with 140 nmol/L for endothelin-3.⁵ ET_A receptor mRNA was initially reported to be highly expressed in human aorta but not cultured human endothelial cells, suggesting selective vascular expression of this receptor in smooth muscle cells.³ The ET_B receptor has equal affinity for all three endothelins, with K_i values for endothelin-1 and endothelin-3 of 0.12 and 0.06 nmol/L, respectively.⁵ The ET_B receptor has been reported to be highly expressed in cultured endothelial cells⁴ but not vascular smooth muscle cells.⁶

On the basis of the greater vasoconstrictor potency of endothelin-1 than endothelin-3 and the apparently exclusive expression of ET_A receptors in vascular smooth muscle, vasoconstriction to endothelin-1 was initially thought to be mediated solely by vascular smooth muscle cell ET_A receptors. Vascular ET_B receptors located on endothelial cells were thought only to mediate generation of endothelium-derived dilator substances. More recent evidence suggests that ET_B receptor mRNA is expressed in human vascular smooth muscle obtained from the aorta, pulmonary artery, and coronary artery,⁷ consistent with a potential vasoconstrictor role for this receptor. Indeed, in animals, there is functional evidence for ET_B receptor–mediated vasoconstriction in vitro, particularly in venous tissue.^{8 9 10 11 12 13} In addition, selective ET_B receptor agonists have pressor effects in animals in vivo.^{12 14 15 16} However, the contribution of ET_B receptors to vasoconstriction is variable and appears to depend markedly on species, vessel type, and vessel size.¹⁷ Furthermore, the functional significance of such vascular smooth muscle ET_B receptors in humans is unclear, with in vitro studies reporting that ET_B receptors make either a minimal^{11 17 18 19 20 21 22 23 24} or, at most, a moderate contribution^{25 26 27} to vasoconstriction, depending on the types of vessel studied.

The relevance of this issue is emphasized by the recent development of both selective and nonselective antagonists at ET_A and ET_B receptors. For example, selective ET_A receptor antagonists will block vasoconstriction mediated by ET_A receptors but may not block all constriction to endothelin-1 if there are also vasoconstrictor ET_B receptors. However, if the putative constrictor ET_B receptor is relatively unimportant in humans, then blocking both ET_A and ET_B receptors may cause less vasodilatation than blocking the ET_A receptor alone, because such receptor antagonists will also block the endothelial dilator ET_B receptor.

In view of the inconsistent results with and the potential disadvantages of in vitro studies, we investigated the function of endothelin ET_A and ET_B receptors in blood vessels in vivo in humans. We used endothelin-1 as a nonselective agonist at ET_A and ET_B receptors and endothelin-3 and sarafotoxin S6c as selective ET_B receptor agonists; these peptides have about 2000- and 300 000-fold selectivity, respectively, for the ET_B over the ET_A receptor.^{5 28} Using locally active doses of these agents, we assessed responses both of resistance vessels, using brachial artery administration,²⁹ and of capacitance vessels, using dorsal hand vein administration.^{30 31 32} We used local doses of peptides so that interpretation of the results would not be confounded by direct effects of systemic administration on kidney, heart, or brain or by reflex effects consequent to changes in blood pressure.

	Methods

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Subjects
Twenty-four healthy male subjects between 22 and 38 years of age participated in these studies, which were conducted with the approval of the Lothian Medicine and Clinical Oncology Ethics of Medical Research Subcommittee and with the written informed consent of each subject. No subject received vasoactive or nonsteroidal anti-inflammatory drugs in the week before each phase of the study, and all abstained from alcohol for 24 hours and from food, caffeine-containing drinks, and cigarettes for at least 3 hours before any measurements were made. All studies were performed in a quiet room maintained at a constant temperature of between 22°C and 25°C.

Drugs
Pharmaceutical-grade endothelin-1 (Clinalfa, NovaBiochem), endothelin-3 (Clinalfa), and sarafotoxin S6c (Sigma Chemical Co Ltd) were administered. A single dose of each peptide was used in individual studies because the slow onset and long-lasting action of the endothelin isopeptides preclude the use of repeated doses in a single study to examine conventional dose-response relations.³³ The peptides were dissolved in physiological saline (0.9%; Baxter Healthcare Ltd).

Intra-arterial Administration
The left brachial artery was cannulated under local anesthesia (1% lignocaine; Astra Pharmaceuticals) with a 27–standard wire gauge steel needle (Coopers Needle Works) attached to a 16-gauge epidural catheter (Portex Ltd). Patency was maintained by infusion of 0.9% physiological saline via a Welmed P1000 syringe pump (Welmed Clinical Care Systems). The total rate of intra-arterial infusion was maintained constant throughout all intra-arterial studies at 1 mL/min.

Intravenous Administration
A vein on the dorsum of the left hand was cannulated in the direction of flow with a 23-gauge butterfly needle (Abbott) attached to a 16-gauge epidural catheter, without use of local anesthesia. The same vein was used in each subject for each individual study. Patency was maintained by infusion of 0.9% physiological saline via a Welmed P1000 syringe pump. The total rate of intravenous infusion was maintained constant throughout all studies at 0.25 mL/min.

Measurements
Forearm Blood Flow
Blood flow was measured in the infused and noninfused forearms by venous occlusion plethysmography³⁴ using indium/gallium-in-Silastic strain gauges²⁹ that were securely applied to the widest part of each forearm. The hands were excluded from the circulation during each measurement period by inflation of a wrist cuff to 220 mm Hg. Upper-arm cuffs were intermittently inflated to 40 mm Hg for 10 in every 15 seconds to temporarily prevent venous outflow from the forearm and thus obtain plethysmographic recordings. Recordings of forearm blood flow were made repeatedly over 3-minute periods unless otherwise stated. Voltage output from a dual-channel Vasculab SPG 16 strain-gauge plethysmograph (Medasonics Inc) was transferred to a Macintosh personal computer (Classic II, Apple Computer Inc) with a MacLab analog-to-digital converter and CHART software (v. 3.2.8; both from AD Instruments). Calibration was achieved by use of the internal standard of the Vasculab plethysmography unit.

Dorsal Hand Vein Diameter
The left hand was supported above the level of the heart by means of an arm rest. The ID of the dorsal hand vein, distended by inflation of an upper arm cuff to 30 mm Hg, was measured by the technique of Aellig.³⁰ In brief, a magnetized lightweight rod rested on the summit of the infused vein 1 cm downstream from the tip of the infusion cannula. This rod passed through the core of a linear variable differential transformer (LVDT; model 025 MHR, Lucas Schaevitz Inc) supported above the hand by a small tripod, the legs of which rested on areas of the dorsum of the hand free of veins. If venoconstriction occurred while this cuff was inflated or if the cuff was deflated with consequent emptying of the vein, there was a downward displacement of the lightweight rod that caused a linear change in the voltage generated by the LVDT. The voltage output from the LVDT was transferred to a Macintosh personal computer by use of a MacLab analog-to-digital converter and CHART software. Standard displacements were used to calibrate the LVDT to determine the ID of the vein.

Blood Pressure
A well-validated semiautomated noninvasive oscillometric sphygmomanometer (Takeda UA 751, Takeda Medical Inc) was used to make duplicate measurements of blood pressure in the noninfused arm.³⁵

Study Design
Four single-blind studies were performed, with the experimental subjects but not the investigators blinded to the peptide and dose administered in each study.

Forearm Resistance Bed Protocols
Subjects rested recumbent throughout each study. Strain gauges and arm cuffs were applied, and the left brachial artery cannula was sited. Saline was infused for 30 minutes, during which two measurements of forearm blood flow were made (at -20 and -10 minutes). Blood pressure was measured immediately after each forearm blood flow measurement, thereby avoiding any effect on forearm blood flow measurements of the venous congestion caused by this procedure.³⁶ Three protocols were then followed, each in separate groups of subjects, as follows.

Protocol 1: Low-dose intra-arterial endothelin-1 and endothelin-3. On four separate occasions, in random order, six subjects received brachial artery infusion of endothelin-1 and endothelin-3 at 1 and 5 pmol/min, each for 60 minutes. The choice of doses was based on previous work showing, in vivo, that 5 pmol/min of endothelin-1 causes slow-onset vasoconstriction in human forearm resistance vessels, reducing blood flow by 40%.^{29 33} Forearm blood flow was recorded from 3 minutes before to 5 minutes after the endothelin infusion was begun. Thereafter, measurements were made at 5-minute intervals for 60 minutes. Blood pressure was measured 60 minutes after the infusion was begun.

Protocol 2: High-dose intra-arterial endothelin-1 and endothelin-3. On two separate occasions, in random, balanced order, six subjects received endothelin-1 and endothelin-3 via the brachial artery at 60 pmol/min for 5 minutes, followed by physiological saline for 55 minutes. Because no significant vasodilatation had been observed in previous studies using intra-arterial endothelin-1 at 5 pmol/min,^{29 33} we chose a dose of 60 pmol/min with the intention of stimulating sufficient endothelial generation of dilator substances to cause vasodilatation before the development of vasoconstriction. Forearm blood flow was recorded from 3 minutes before to 10 minutes after the endothelin infusion was begun. Thereafter, measurements were made at 5-minute intervals for 60 minutes. Blood pressure was measured 10 and 60 minutes after the infusion was begun.

Protocol 3: Intra-arterial endothelin-1 and sarafotoxin S6c. On two separate occasions, in random, balanced order, six subjects received endothelin-1 and sarafotoxin S6c via the brachial artery at 5 pmol/min for 60 minutes. Forearm blood flow was recorded from 3 minutes before to 5 minutes after peptide infusion was begun. Thereafter, measurements were made at 5-minute intervals for 60 minutes. Blood pressure was measured at 60 minutes, just before the infusion was terminated.

Hand Vein Studies
Protocol 4: Intravenous endothelin-1 and sarafotoxin S6c. Six subjects were studied on two separate occasions, in random, balanced order. Subjects rested semirecumbent throughout. The dorsal hand vein cannula and the LVDT were sited. Saline was infused for 30 minutes, during which vein diameter was measured every 5 minutes. Endothelin-1 and sarafotoxin S6c were infused at 5 pmol/min for 60 minutes, with measurements of vein diameter every 5 minutes. The choice of this dose was based on previous work that showed, in vivo, that endothelin-1 at 5 pmol/min causes slow-onset venoconstriction of 60% in human skin capacitance vessels.^{29 31} Blood pressure was measured twice before the dose was given and at 60 minutes, just before the infusion was terminated.

Data Analysis and Statistics
Plethysmographic data listings were extracted from the CHART data files, and forearm blood flows were calculated for individual venous occlusion cuff inflations by use of a template spreadsheet (EXCEL 4.0; Microsoft Ltd). Because wrist cuff inflation results in a transient forearm vasoconstriction,³⁷ recordings made in the first 60 seconds after wrist cuff inflation were not used for analysis. Usually, the last five flow recordings in each 3-minute measurement period were calculated and averaged for the infused and noninfused arms. However, to detect early transient changes in blood flow, every recording made immediately before and after the start of peptide infusion was analyzed. Basal blood flow was taken as the average of all flow recordings made in the 2 minutes before infusion of peptides was begun. The intersubject, intrasubject (interstudy), and intrasubject (intrastudy) coefficients of variation for basal forearm blood flow measurements in our laboratory are 51%, 33%, and 14%, respectively. The intersubject, intrasubject (interstudy), and intrasubject (intrastudy) coefficients of variation for the basal ratio of blood flow between infused and noninfused arms in our laboratory are 22%, 15%, and 8%, respectively. Therefore, to reduce the variability of blood flow data, the ratio of flows in the two arms was calculated for each time point, in effect using the noninfused arm as a contemporaneous control for the infused arm.³⁸ Forearm blood flow results are shown as a percentage change from basal in the ratio of blood flow between infused and noninfused arms.²⁹

Basal vein diameter was calculated as the mean of the last three measurements before the start of the peptide infusion, expressed in millimeters. The intersubject, intrasubject (interstudy), and intrasubject (intrastudy) coefficients of variation for basal hand vein diameter measurements in our laboratory are 43%, 26%, and 5%, respectively. Given the high intersubject and interstudy variability in hand vein diameter, responses after infusion of peptides are expressed as percentage change in vein diameter from basal.³² Duplicate blood pressure measurements were averaged at each time point. Basal blood pressure was taken as the average of the second set of measurements made before infusion of peptides.

To obtain an estimate of the contribution of ET_B receptors to vasoconstriction, the ratio of constriction to the ET_B agonist compared with constriction to endothelin-1 was calculated for each subject at the 60-minute time point. Because these data had a skewed distribution, ratios were logarithmically transformed for statistical analysis. Data are shown as mean values, with 95% confidence intervals (CI) shown in the text and SEM in the figures. Data were examined by a repeated-measures ANOVA with statistical testing of overall significance by Scheffé's F test (ANOVA) using STATVIEW 512⁺ software (Brainpower Inc) for the Apple Macintosh personal computer.

	Results

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Basal blood pressure, heart rate, forearm blood flow, and vein diameter were similar on the different study days, and there was no significant difference in basal forearm blood flow between the infused and noninfused arms (Tables 1 and 2). Blood pressure, heart rate, and blood flow in the noninfused arm did not change significantly after infusion of any study agent, confirming that drug effects were confined to the infused arm (Tables 1 and 2).

View this table: [in this window] [in a new window]   Table 1. Mean Arterial Pressure, Heart Rate, and Forearm Blood Flows Before and After Brachial Artery Administration of Peptides in the Two Study Protocols (1 and 2) Comparing Endothelin-1 and Endothelin-3

View this table: [in this window] [in a new window]   Table 2. Mean Arterial Pressure, Heart Rate, Forearm Blood Flow, and Hand Vein Size Before and After Administration of Peptides in the Two Study Protocols (3 and 4) Comparing Endothelin-1 and Sarafotoxin S6c

Protocol 1: Low-Dose Intra-arterial Endothelin-1 and Endothelin-3
Endothelin-1 at 1 pmol/min caused a modest but significant forearm vasoconstriction, with an 11% reduction in forearm blood flow at 60 minutes (CI, -22% to -1%; ANOVA, P=.02; Fig 1). Endothelin-3 at 1 pmol/min tended to decrease forearm blood flow, with a 5% reduction in blood flow at 60 minutes, but this was not significant (CI, -14% to +3%; ANOVA, P=.163; Fig 1). There was no significant difference between the responses to endothelin-1 and endothelin-3 at 1 pmol/min (ANOVA, P=.454). There was no significant vasodilatation early in the course of infusion of either peptide. The average ratio of forearm vasoconstriction to endothelin-3 and endothelin-1 was 0.16, although this estimate had wide CIs (CI, 0.03 to 0.98).

View larger version (29K): [in this window] [in a new window]   Figure 1. Graph. Six subjects received brachial artery infusion of endothelin-3 (, 1 pmol/min; , 5 pmol/min) and on a separate occasion, endothelin-1 (, 1 pmol/min; , 5 pmol/min), each for 60 minutes. Shaded bar indicates period of infusion of endothelin isopeptides. Endothelin-1 caused significant forearm vasoconstriction at both doses, whereas the effect of endothelin-3 was significant only at 5 pmol/min. For clarity, error bars have been removed from that part of the figure showing results for the first 5 minutes of peptide infusion. ia indicates intra-arterial.

Endothelin-1 at 5 pmol/min caused substantial forearm vasoconstriction, with a 40% reduction in forearm blood flow at 60 minutes (CI, -52% to -28%; ANOVA, P=.0002; Fig 1). The same dose of endothelin-3 also significantly reduced forearm blood flow, with a 25% reduction in blood flow at 60 minutes (CI, -36% to -13%; ANOVA, P=.001; Fig 1). There was significantly greater vasoconstriction after endothelin-1 than endothelin-3 (ANOVA, P=.04). There was no significant vasodilatation early in the course of infusion of either peptide. The average ratio of forearm vasoconstriction to endothelin-3 and endothelin-1 was 0.58 (CI, 0.39 to 0.87).

Protocol 2: High-Dose Intra-arterial Endothelin-1 and Endothelin-3
Endothelin-1, at 60 pmol/min for 5 minutes, caused a trend to transient nonsignificant forearm vasodilatation in the first 2 minutes of infusion, with a maximum increase of 16% (CI, -7% to +23%; Fig 2) at 2 minutes. Thereafter, vasoconstriction occurred, with the maximum decrease in blood flow occurring at 10 minutes (-28%; CI, -48% to -9%), although flow was still reduced after 60 minutes (-17%; CI, -30% to -4%). Endothelin-3 caused significant early forearm vasodilatation, with a maximum increase in flow of 24% at 3 minutes (CI, +4% to +43%; Fig 2). Forearm vasoconstriction occurred after 10 minutes, with a maximum reduction in blood flow of 24% at 60 minutes (CI, -43% to -5%). There was a significant difference between the overall responses to endothelin-1 and endothelin-3 over the 60 minutes after bolus administration of isopeptide (ANOVA, P=.04). However, maximum vasoconstriction to the isopeptides was similar (Fig 2). The average ratio of forearm vasoconstriction to endothelin-3 and endothelin-1 was 0.82, although this estimate had wide CIs (CI, 0.13 to 5.07).

View larger version (23K): [in this window] [in a new window]   Figure 2. Graph. Six subjects received brachial artery infusion of endothelin-1 (, 60 pmol/min) and on a separate occasion, endothelin-3 (, 60 pmol/min), each for 5 minutes. Shaded area indicates period of infusion of endothelin isopeptides. Forearm vasodilatation occurred initially with endothelin-3 but not with endothelin-1. Both isopeptides then caused vasoconstriction of similar degree. ia indicates intra-arterial.

Protocol 3: Intra-arterial Endothelin-1 and Sarafotoxin S6c
Endothelin-1 at 5 pmol/min did not cause early vasodilatation but did produce slow-onset forearm vasoconstriction, with a maximum reduction in forearm blood flow of 48% at 60 minutes (CI, -60% to -37%; ANOVA, P=.0001; Fig 3). There was no significant vasodilatation to sarafotoxin S6c early in the course of the infusion, although there may have been a trend for this to occur (Fig 3). Like endothelin-1, sarafotoxin S6c caused slow-onset forearm vasoconstriction (ANOVA versus basal, P=.002; Fig 3). However, the maximum change in blood flow with sarafotoxin S6c at 60 minutes (-25%; CI, -37% to -13%) was significantly less than that to endothelin-1 (ANOVA, P=.04). The average ratio of forearm vasoconstriction to sarafotoxin S6c and endothelin-1 was 0.48 (CI, 0.30 to 0.75).

View larger version (23K): [in this window] [in a new window]   Figure 3. Graph. Six subjects received brachial artery infusion of endothelin-1 (, 5 pmol/min) and on a separate occasion, sarafotoxin S6c (, 5 pmol/min), each for 60 minutes. Shaded bar indicates period of infusion of peptides. Both peptides caused significant forearm vasoconstriction, although the effect of sarafotoxin S6c was less than that of endothelin-1. ia indicates intra-arterial.

Protocol 4: Intravenous Endothelin-1 and Sarafotoxin S6c
Endothelin-1 caused a slow-onset and marked decrease in hand vein diameter, with a maximal reduction at 60 minutes (-68%; CI, -84% to -52%; ANOVA, P=.001; Fig 4). Sarafotoxin S6c also caused venoconstriction, although the maximum decrease in hand vein size at 60 minutes (-19%; CI, -29% to -9%; ANOVA versus basal, P=.003; Fig 4) was significantly less than that to endothelin-1 (ANOVA, P=.002). The average ratio of venoconstriction to sarafotoxin S6c and endothelin-1 was 0.25 (CI, 0.14 to 0.44).

View larger version (21K): [in this window] [in a new window]   Figure 4. Graph. Six subjects received dorsal hand vein infusion of endothelin-1 (, 5 pmol/min) and on a separate occasion, sarafotoxin S6c (, 5 pmol/min), each for 60 minutes. Shaded bar indicates period of infusion of peptides. Both peptides caused significant venoconstriction, although the effect of sarafotoxin S6c was less than that of endothelin-1. iv indicates intravenous.

	Discussion

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These studies show that selective agonists at endothelin ET_B receptors constrict forearm resistance and hand capacitance vessels in vivo in humans. In addition, high doses of endothelin-3, and perhaps of endothelin-1, cause transient forearm vasodilatation. These results suggest an important role for ET_B receptors in mediating the vascular effects of endothelin-1. It is possible that different findings might have been obtained if other vascular beds had been studied. However, responses in human forearm resistance vessels and dorsal hand veins are thought to be broadly representative of responses in other resistance and capacitance beds.³⁹ Given that resting forearm blood flow is 50 mL/min,³⁹ doses of 1, 5, and 60 pmol/min of peptide should achieve local concentrations of 0.02, 0.1, and 1 nmol/L, respectively. Endothelin-1 would be expected to act equally on both ET_A and ET_B receptors at these concentrations, while endothelin-3 would be expected to be relatively selective for the ET_B receptor, because this isopeptide has a K_i at ET_A receptors of about 140 nmol/L.⁵ Sarafotoxin S6c at 5 pmol/min should have been highly selective for the ET_B receptor, because the calculated concentration in forearm blood (0.1 nmol/L) is at least 70 000-fold lower than its K_i at ET_A receptors (>7300 nmol/L).⁵

Administration of endothelin-3 at 60 pmol/min caused significant early forearm vasodilatation, and there was also a tendency for similar transient vasodilatation to occur with endothelin-1, although this was not statistically significant. Vasodilatation is likely to have been due to activation of ET_B receptors on endothelial cells, causing generation of endothelium-derived dilator substances.³¹ The apparent absence of significant vasodilatation to high-dose endothelin-1 may have been due to additional early vasoconstriction mediated by ET_A receptors masking dilatation, although it should be noted that the CIs at these time points were sufficiently wide for an 20% vasodilatation to endothelin-1 to have been missed by chance. Lower doses of endothelin-1 and sarafotoxin S6c failed to cause early vasodilatation. The lack of vasodilatation to endothelin-1 contrasts with previous findings,⁴⁰ possibly because of differences in doses used and experimental design. In view of the relatively high doses required to cause vasodilatation, it is likely that vasodilatation to the endothelins represents a pharmacological rather than a physiological phenomenon. Because human dorsal hand veins have no basal tone, it is not possible to demonstrate whether endothelin-1 or sarafotoxin S6c causes venodilatation without preconstriction of the vein. Previous work has shown no venodilatation to endothelin-1 or endothelin-3 in preconstricted dorsal hand veins.⁴¹ Nonetheless, inhibition of prostaglandin but not nitric oxide generation potentiates venoconstriction to endothelin-1 in vivo in humans.³¹ Thus, the venous endothelium may generate vasodilator substances in response to endothelin, but the vasodilator effects of such substances appear to be masked by the simultaneous direct venoconstriction caused by the peptide and serve only to modulate venoconstriction.

Given that both endothelin-3 and sarafotoxin S6c caused vasoconstriction, our results suggest the presence of vasoconstrictor ET_B receptors. However, constriction to the ET_B agonists was almost always less than that to the nonselective ET_A and ET_B agonist endothelin-1, implying that both ET_A and ET_B receptors contribute to vasoconstriction. The 95% CIs of the ratio of forearm vasoconstriction to sarafotoxin S6c and endothelin-1 are consistent with ET_B receptors contributing substantially to constriction, accounting for between 30% and 75% of the response to endothelin-1. Although the magnitude of the ET_B contribution in vitro appears to differ between vessels,¹⁷ the similarity of responses in forearm resistance vessels and cutaneous capacitance vessels of the hand suggests that ET_B receptors may be of widespread functional importance in human blood vessels.

Our finding of ET_B receptor–mediated vasoconstriction of resistance vessels contrasts with some in vitro studies that suggest little contribution of ET_B receptors to constriction of human arteries.^{11 17 18 19 20 21 22 23 24} This difference may reflect the fact that we examined responses in an intact resistance bed, because ET_B receptor–mediated vasoconstriction appears to play a relatively greater role in smaller vessels, particularly those responsible for determining resistance.^{17 42} All of the in vitro studies in which human vessels exhibited little or no ET_B–mediated arterial vasoconstriction examined vessels >400 µm in diameter.^{11 17 18 19 20 21 22 23 24} In addition to the influence of vessel size on the contribution of ET_B receptors, there may be regional differences. Local injection of the ET_A antagonist PD147953 has been shown to completely prevent vasoconstriction of human skin vessels caused by intradermal injection of endothelin-1, suggesting that vasoconstriction is mediated mainly by ET_A receptors in this microvascular bed.⁴³ The effects of sarafotoxin S6c, compared with those of endothelin-1, were less in hand veins than in forearm resistance vessels, despite in vitro evidence from animal vessels that responses to ET_B agonists are greater in veins than arteries.^{8 9 12 13} This may reflect a true species difference, because endothelin-1 is about eightfold more potent as a venoconstrictor than endothelin-3 in human hand veins,⁴¹ which also suggests that ET_A receptors predominate in these vessels.

Although vasoconstriction to the ET_B agonists endothelin-3 and sarafotoxin S6c is most likely to be caused by stimulation of vascular smooth muscle ET_B receptors, there are alternative explanations. First, ET_B receptors may be confined to endothelial cells but cause late-onset vasoconstriction through stimulation of the generation of endothelium-derived vasoconstrictor agents. These substances might include constrictor prostanoids or even endothelin-1, because endothelin-3 is known to stimulate production of endothelin-1 in vitro.⁴⁴ Second, some of the effects of endothelin-3 could have been mediated by a putative ET_C (endothelin-3–selective) receptor situated on endothelial cells. However, although there is evidence from binding⁴⁴ and functional⁴⁵ studies to support the existence of an endothelin-3–selective receptor in the vasculature, and a potential candidate has been identified in Xenopus laevis melanophores,⁴⁶ this receptor has not been identified in humans. Any contribution from the putative ET_C receptor will depend on its isolation and pharmacological characterization. Third, there may be receptor-mediated clearance of endogenously generated endothelin-1 by ET_B receptors, as has been shown in animals.⁴⁷ If this were the case, ET_B agonists might prevent local clearance of endothelin-1, which would then act on ET_A receptors to cause vasoconstriction. However, this possibility appears highly unlikely, given that ET_A antagonists do not influence vasoconstriction to sarafotoxin S6c in vitro.^{8 10 13 26 48} In future, studies with selective ET_B receptor antagonists should clarify this issue, because such agents would be expected to potentiate responses to endothelin-1 if ET_B receptor–mediated clearance of endothelin-1 does occur.

Thus, the most likely explanation for our results is that there are functionally active ET_A and ET_B receptors on vascular smooth muscle cells causing vasoconstriction, to both of which endothelin-1 would have access. These findings have implications for the future development of antiendothelin therapies, because they suggest that full inhibition of vasoconstriction to endogenously generated endothelin-1 may be obtained only by use of either combined ET_A/B endothelin receptor antagonists⁴⁹ or inhibitors of endothelin generation.²⁹

	Acknowledgments

 
This work was supported by a grant from the Biomedical Research Committee of the Scottish Home and Health Department. We wish to thank E. Stanley and Dr N. Lannigan of the Pharmacy Department at the Western General Hospital for preparing ampoules of endothelin-1, endothelin-3, and sarafotoxin S6c. We thank Dr Gillian Gray for her helpful discussions regarding the manuscript.

Received November 17, 1994; revision received January 23, 1995; accepted January 28, 1995.

	References

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				F. E. Strachan, J. C. Spratt, I. B. Wilkinson, N. R. Johnston, G. A. Gray, and D. J. Webb Systemic Blockade of the Endothelin-B Receptor Increases Peripheral Vascular Resistance in Healthy Men Hypertension, January 1, 1999; 33(1): 581 - 585. [Abstract] [Full Text] [PDF]

				C. Y. Pang, J. Zhang, H. Xu, J. E. Lipa, C. R. Forrest, and P. C. Neligan Role and mechanism of endothelin-B receptors in mediating ET-1-induced vasoconstriction in pig skin Am J Physiol Regulatory Integrative Comp Physiol, October 1, 1998; 275(4): R1066 - R1074. [Abstract] [Full Text] [PDF]

				T. J Rabelink, E. S.G Stroes, K.P. Bouter, and P. Morrison Endothelin blockers and renal protection: a new strategy to prevent end-organ damage in cardiovascular disease? Cardiovasc Res, September 1, 1998; 39(3): 543 - 549. [Full Text] [PDF]

				R. Choussat, L. Hittinger, F. Barbe, G. Maistre, A. Carayon, B. Crozatier, and J. Su Acute effects of an endothelin-1 receptor antagonist bosentan at different stages of heart failure in conscious dogs Cardiovasc Res, September 1, 1998; 39(3): 580 - 588. [Abstract] [Full Text] [PDF]

				M. Iglarz, K. Matrougui, B. I. Levy, and D. Henrion Chronic blockade of endothelin ETA receptors improves flow dependent dilation in resistance arteries of hypertensive rats Cardiovasc Res, September 1, 1998; 39(3): 657 - 664. [Abstract] [Full Text] [PDF]

				J. P Noon, B. R Walker, M. F Hand, and D. J Webb Impairment of forearm vasodilatation to acetylcholine in hypercholesterolemia is reversed by aspirin Cardiovasc Res, May 1, 1998; 38(2): 480 - 484. [Abstract] [Full Text] [PDF]

				M. C. Verhaar, F. E. Strachan, D. E. Newby, N. L. Cruden, H. A. Koomans, T. J. Rabelink, and D. J. Webb Endothelin-A Receptor Antagonist–Mediated Vasodilatation Is Attenuated by Inhibition of Nitric Oxide Synthesis and by Endothelin-B Receptor Blockade Circulation, March 3, 1998; 97(8): 752 - 756. [Abstract] [Full Text] [PDF]

				T. E. Rasmussen, M. Jougasaki, T. Supaporn, J. W. Hallett Jr., D. P. Brooks, and J. C. Burnett Jr. Cardiovascular actions of ET-B activation in vivo and modulation by receptor antagonism Am J Physiol Regulatory Integrative Comp Physiol, January 1, 1998; 274(1): R131 - R138. [Abstract] [Full Text] [PDF]

				M. Adner, E. Uddman, L. O. Cardell, and L. Edvinsson Regional variation in appearance of vascular contractile endothelin-B receptors following organ culture Cardiovasc Res, January 1, 1998; 37(1): 254 - 262. [Abstract] [Full Text] [PDF]

				D. Hasdai, V. Mathew, R. S. Schwartz, L. A. Smith, D. R. Holmes Jr, Z. S. Katusic, and A. Lerman Enhanced Endothelin-B-Receptor–Mediated Vasoconstriction of Small Porcine Coronary Arteries in Diet-Induced Hypercholesterolemia Arterioscler. Thromb. Vasc. Biol., November 1, 1997; 17(11): 2737 - 2743. [Abstract] [Full Text]

				M. P. Love, W. G. Haynes, G. A. Gray, D. J. Webb, and J. J.V. McMurray Vasodilator Effects of Endothelin-Converting Enzyme Inhibition and Endothelin ETA Receptor Blockade in Chronic Heart Failure Patients Treated With ACE Inhibitors Circulation, November 1, 1996; 94(9): 2131 - 2137. [Abstract] [Full Text]

				S. N. A. Hussain Regulation of ventilatory muscle blood flow J Appl Physiol, October 1, 1996; 81(4): 1455 - 1468. [Abstract] [Full Text] [PDF]

				W. G. Haynes, C. J. Ferro, K. P. J. O'Kane, D. Somerville, C. C. Lomax, and D. J. Webb Systemic Endothelin Receptor Blockade Decreases Peripheral Vascular Resistance and Blood Pressure in Humans Circulation, May 15, 1996; 93(10): 1860 - 1870. [Abstract] [Full Text]

				J. P.J. Halcox, K. R.A. Nour, G. Zalos, and A. A. Quyyumi Coronary Vasodilation and Improvement in Endothelial Dysfunction With Endothelin ETA Receptor Blockade Circ. Res., November 23, 2001; 89(11): 969 - 976. [Abstract] [Full Text] [PDF]

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