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Vasoconstriction to Endogenous Endothelin-1 Is Increased in the Peripheral Circulation of Essential Hypertensive Patients

Stefano Taddei, MD

Department of Internal Medicine
University of Pisa
Pisa, Italy

Condensed Abstract Patients and Methods
Abstract Results
Introduction Discussion

Condensed Abstract

In hypertensive patients and healthy controls (n=10, each group) we tested forearm blood flow (strain-gauge venous plethysmography) changes induced by the intrabrachial infusion of TAK-044, an antagonist for endothelin ET-A/B receptors and NG-monomethyl-L-arginine, an NO-synthase inhibitor. Vasodilation to TAK-044, but not to sodium nitroprusside, was increased (p<0.01) while the vasoconstrictor response to NG-monomethyl-L-arginine, but not to norepinephrine, was decreased (p<0.01) in hypertensive patients as compared to controls. Vasodilation to TAK-044 and vasoconstriction to NG-monomethyl-L-arginine showed an inverse correlation (r= -0.56; p<0.05). In essential hypertension endothelin causes a greater degree of peripheral vasoconstriction.


To evaluate the role of endothelin-1 in control of vascular tone in essential hypertension, we tested forearm blood flow (strain-gauge venous plethysmography) changes induced by intrabrachial infusion of TAK-044 (10, 30, 100 µg/100 ml/min), an ET-A/B receptor antagonist, or sodium nitroprusside (1 and 2 µg/100 ml/min), a vasodilator acting on smooth muscle cells, in hypertensive patients and healthy controls (n=10, each group). The NO pathway was also evaluated by infusion of NG-monomethyl-L-arginine, (L-NMMA, 10, 30, 100 µg/100 ml/min), an NO-synthase inhibitor, and norepinephrine (3, 9, 30 ng/100 ml/min), as control. Finally, immunoreactive plasma endothelin-1 was measured by radioimmunoassay. In hypertensive patients, TAK-044 caused a vasodilation which was significantly (p<0.01) increased as compared to normotensive subjects. Moreover the vasoconstriction to L-NMMA was significantly (p<0.01) decreased in hypertensive patients as compared to controls. In contrast, the vascular response to sodium nitroprusside and norepinephrine and immunoreactive plasma endothelin-1 were similar in hypertensive patients and controls. In the study population, vasodilation to TAK-044 and vasoconstriction to L-NMMA showed an inverse correlation (r= -0.56; p<0.05). These results indicate that TAK-044 caused a greater degree of vasodilation in the forearm vessels of essential hypertensive patients as compared to normotensive subjects, an alteration associated with decreased tonic NO release.


Endothelin-1 (ET-1), a 21 amino acid isopeptide generated by the vascular endothelium and characterized by sustained and potent vasoconstrictor action (1), acts through specific receptors termed ETA and ETB (2). ETA receptors are represented only on smooth muscle cells and mediate contractions and growth promotion (2). In contrast, ETB receptors are located both on smooth muscle cells to evoke contractions (3) and endothelial cells to induce relaxation by production of nitric oxide (NO) (4).

In humans ET-1 could be implicated in the pathophysiology of several cardiovascular diseases including essential hypertension (5). Bosentan, a mixed ETA/B receptor antagonist, significantly lowered blood pressure values in essential hypertensive patients, suggesting a role for this peptide in the pathogenesis of hypertension (6). However a clear demonstration of a disturbance in ET-1 mediated vascular control is still lacking in essential hypertension.

In the present study we compared forearm vasodilation to TAK-044, a combined ETA/B receptor antagonist (7), in healthy controls and essential hypertensive patients in order to indirectly assess the contribution of endogenous ET-1 to vascular tone under healthy conditions and in essential hypertension. Moreover, the activity of the L-arginine-NO pathway was also evaluated.

Patients and methods

The study population included 10 normotensive controls (51.2±6.4 years; 121.4±4.3/78.2±3.2 mmHg) and 10 matched never treated essential hypertensive patients (50.6±6.8 years; 160.2±6.2/101.6±2.8 mmHg) characterized by no smoking and ethanol consumption history and similar, and within a normal range, values of plasma glucose, total and LDL cholesterol and creatinine clearance. The protocol was approved by the Ethical Committee of the University of Pisa and all patients gave written consent to the study.

Briefly, the brachial artery was cannulated for drug infusion at systemically ineffective rates, intraarterial blood pressure and heart rate monitoring. Forearm blood flow (FBF) was measured in both forearms by strain-gauge venous plethysmography. Circulation to the hand was excluded one min before FBF measurement by inflating a pediatric cuff around the wrist at suprasystolic blood pressure. Forearm volume was measured according to the water displacement method. Details concerning the method have already been published (8).

To assess ET-1 contribution to vascular tone TAK-044 (Takeda Chemical Industries Ltd, Japan), a specific inhibitor of ETA/B receptors (7,9,10), was infused into the brachial artery of normotensive subjects and essential hypertensive patients (10, 30 100 µg/100 ml forearm tissue/min; 10 minutes for each dose) (11). IC50 for TAK-044 is 3.8 nM and 130 nM for ET-A and ET-B receptors respectively in vitro, and similar in vivo (7,9). Moreover, the integrity of the NO pathway was evaluated by intrabrachial NG-monomethyl-L-arginine (L-NMMA; Clinalfa AG, Läufelfingen, Switzerland), a specific antagonist for NO-synthase (12) (10, 30 and 100 µg/100 ml forearm tissue/min; 5 minutes for each dose). As a control for non specific vascular responses, vasodilation to sodium nitroprusside (SNP, Malesci, Milan, Italy), a direct smooth muscle cell relaxant compound, (1 and 2 µg/100 ml forearm tissue/min; 5 minutes for each dose), and vasoconstriction to norepinephrine (NE, Jacopo Monaco, Venezia, Italy), a preferential a -adrenoceptor agonist, (3, 9 and 30 ng/100 ml forearm tissue/min; 5 minutes for each dose) was also evaluated. The infusion sequence was randomized and 60 minute washout was allowed between each dose-response curve. However TAK-044 was always administrated as the last infusion.

In each subject, a venous blood sample was obtained for assay of plasma immunoreactive ET concentrations (by radioimmunoassay) (13).

Data were analysed in terms of forearm vascular resistances (FVR) (calculated as the ratio between intraarterial mean pressure and FBF and expressed as standard units [SU]). Clinical characteristics of study subjects were compared by the paired Student's t test. Dose-response curves were analysed by ANOVA for repeated measures and Scheffč's test was applied for multiple comparison testing. Results were expressed as mean + SD.


Table 1 describes mean blood pressure, heart rate, experimental and contralateral FVR behaviour during each infusion.

Table 1. Behaviour of intraarterial mean blood pressure (MBP, mmHg), heart rate (HR, beats/minute),
experimental (ex) and contralateral (c) FVR (units) during infusion of TAK-044, SNP, and L-NMMA
(expressed asm g/100 ml/min), and NE (expressed as ng/100 ml/min), in normotensive subjects and
essential hypertensive patients.

Normotensive subjects

Essential hypertensive patients



FVR ex




FVR ex



90.5± 4.2

73.0± 3.9

27.1± 3.1

27.4± 3.4

109.0± 5.8

66.8± 4.1

32.1± 3.7

33.0± 3.2

TAK-044 10

90.5± 4.3

72.8± 4.4

25.2± 4.3

27.4± 3.4

108.2± 5.9

66.2± 3.1

29.2± 3.1

31.8± 3.5

TAK-044 30

90.0± 4.3

72.6± 4.2

25.1± 5.5

28.1± 3.6

108.4± 5.8

65.4± 3.4

21.3± 2.2*

32.8± 3.5

TAK-044 100

90.0± 4.6

72.5± 4.0

24.1± 5.8

27.2± 3.3

108.0± 5.9

64.6± 4.1

17.1± 2.1**

31.7± 3.6


90.4± 4.2

72.4± 4.6

29.3± 3.6

27.4± 3.4

108.9± 5.6

67.4± 3.2

35.1± 3.5

32.0± 3.3


90.5± 4.3

72.2± 4.6

13.5± 2.4**

27.4± 3.4

108.9± 5.6

67.6± 2.8

18.5± 2.4**

33.0± 3.4


90.5± 4.3

72.6± 4.4

8.9± 1.8**

27.4± 3.4

109.0± 5.6

67.4± 2.9

11.0± 1.9**

32.0± 3.5


89.8± 4.5

72.9± 3.5

27.2± 4.7

27.2± 3.3

108.1± 5.9

65.2± 4.0

31.8± 3.8

31.8± 3.4


89.8± 4.5

72.7± 3.8

33.0± 5.0

28.0± 3.5

108.2± 5.8

65.4± 3.6

34.9± 3.5

32.7± 3.5


89.9± 4.4

72.9± 3.7

39.1± 5.6*

27.4± 3.4

108.1± 5.8

65.8± 4.1

38.6± 3.6

33.7± 3.6

L-NMMA 100

90.0± 4.5

71.7± 4.2

51.9± 13.9**

27.2± 3.4

107.9± 5.8

65.8± 3.8

45.0± 4.1**

32.6± 3.5


89.9± 4.7

72.4± 4.3

29.0± 3.5

27.3± 3.3

108.1± 5.8

65.6± 4.5

36.0± 3.8

31.8± 3.5

NE 3

89.8± 4.5

72.5± 4.1

33.2± 4.4

27.3± 3.4

108.2± 5.7

65.4± 4.5

40.1± 3.9

31.8± 3.6

NE 9

89.9± 4.7

72.9± 4.0

39.9± 6.0**

27.2± 3.5

108.2± 5.6

65.0± 4.0

45.1± 4.4*

32.7± 3.3

NE 30

90.0± 4.8

72.7± 4.2

53.2± 7.6**

27.2± 3.4

108.2± 5.9

65.4± 4.0

56.9± 4.9**

31.8± 3.5

* p<0.01 or ** p<0.001 versus baseline.

In healthy subjects, TAK-044 caused a modest increase in FBF (fig 1) and decrease in FVR (-12.3±17.2%), while, in essential hypertensive patients FBF increase (fig 1) and FVR decrease (- 47.2±12.1%) was significantly (P<0.01) increased as compared to normotensive controls. L-NMMA caused a decrease in FBF (fig 1), which was significantly (p<0.01) reduced in essential hypertensive patients (FVR increase: 41.4±14%) as compared to normotensive subjects (FVR increase: 95.3±41%). Vasodilation to SNP and vasoconstriction to NE was similar in normotensive subjects and hypertensive patients. Contralateral FVR did not significantly change throughout the study (table 1).

wpe1.jpg (10912 bytes)

Figure 1. Bars show forearm vasoconstriction and vasodilation induced by intra-brachial
L-NMMA (100 µg/100 ml forearm tissue/min) and TAK-044 (100 µg/100 ml forearm
tissue/min), respectively, in normotensive subjects and essential hypertensive patients.
Results are represent as per cent FBF changes as compared to baseline. *: p<0.05 or
less, between normotensive subjects and hypertensive patients.

Considering the entire study population, vasodilation to TAK-044 was negatively (r=-0.56; p<0.05) correlated with vasoconstriction to L-NMMA (fig. 2).

wpe2.jpg (12655 bytes)

Figure 2. Scatterplot of relationship between maximum of the FBF response to
L-NMMA (x axis) and TAK-044 (y axis, bottom) in 10 normotensive subjects and
10 essential hypertensive patients. The maximum effect is calculated as per cent
increase in vasoconstriction to L-NMMA at 100 µg/100 ml forearm tissue/min
and vasodilation to TAK-044 at 100 µg/100 ml forearm tissue/min.

Plasma immunoreactive ET was similar in normotensive subjects (4.9+0.1 pg/mL) and hypertensive patients (4.3+0.1 pg/mL).


The present results show that TAK-044, a combined ETA/B receptor antagonist (7), caused a greater degree of vasodilation in the forearm vasculature of essential hypertensive patients as compared to normotensive control subjects, while the response to SNP, a direct relaxant compound, was similar in the two study subgroups. These findings indicate that in essential hypertensive patients endogenous ET-1 shows greater vasoconstrictor activity.

The possibility that this increased effect of TAK-044 in hypertensive patients might be caused merely by an increase in ET-1 production is not supported by measurement of plasma ET-1 levels. In line with previous evidence (14), plasma ET-1 concentrations were found to be similar in normotensive controls and essential hypertensive patients with normal renal function. However, since only around 20% of generated ET is secreted luminally, while the greater portion of the peptide is secreted toward the adjacent smooth muscle (15), plasma ET measurement is not a sensitive marker for tissue production of the peptide.

An alternative explanation may be related to impaired ET-1 mediated NO production in essential hypertensive patients. As previously demonstrated (9), intrabrachial TAK-044 only slightly increased FBF in normotensive subjects (around 20%), indicating that in healthy conditions endothelial ETB receptor mediated NO-dependent vasodilation almost completely counterbalances smooth muscle ETA and ETB receptor mediated vasoconstriction (16). In essential hypertension, ET-1 mediated endothelial ETB receptor stimulation leads to modest NO activation, because of the presence of impaired NO availability (8,17), thereby unmasking the vasoconstrictor effect of the peptide. In line with this possibility and in agreement with previous evidence (17), our hypertensive study population is characterized by an impairment in the NO system since the vasoconstrictor effect of L-NMMA, but not that of the unrelated vasoconstrictor NE, is decreased as compared to healthy controls. Moreover, the existence of a negative and significant correlation between the vasodilating and vasoconstricting response to TAK-044 and L-NMMA respectively, seems to indicate an association between increased vasoconstriction to endogenous ET-1 and diminished NO production. Finally, recent evidence indicates that intrabrachial infusion of BQ-788, a selective ET-B receptor antagonist, causes vasodilation and vasoconstriction in essential hypertensive patients and normotensive controls respectively (18), further supporting the existence of a differential vascular activity for ETB receptors in healthy conditions and in patients with essential hypertension.

In conclusion, in essential hypertension the vasoconstrictor activity of endogenous ET-1 is increased as compared to healthy conditions, suggesting a possible role for ET-1 in the pathogenesis of hypertension and/or its complications.



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Agostino Virdis, MD; Lorenzo Ghiadoni, MD; Isabella Sudano, MD;
Massimo Notari * MD; Antonio Salvetti, MD
Department of Internal Medicine, University of Pisa
* TAKEDA Italia Farmaceutici s.p.a. Rome, Italy

Address for correspondence:
Stefano Taddei, M.D.
Department of Internal Medicine
University of Pisa
Via Roma, 67
56100 Pisa – Italy