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1 Department of Cardiovascular Pharmacology, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania 19406-0939; 2 Division of Neuroscience and Biomedical Systems, Institute of Biomedical and Life Sciences, Glasgow University, Glasgow G12 8QQ; and 3 Departments of Cardiology, 4 Cardiothoracic Surgery, and 5 Surgery, Glasgow Western Infirmary, Glasgow G11 6NT, United Kingdom
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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The peptide human
urotensin-II (hUT-II) and its receptor have recently been cloned. The
vascular function of this peptide in humans, however, has yet to be
determined. Vasoconstrictor and vasodilator responses to hUT-II were
investigated in human small muscular pulmonary arteries [~170 µm
internal diameter (ID)] and human abdominal resistance arteries
(~200 µm ID). Vasodilator responses were investigated in
endothelin-1 (3 nM) precontracted vessels and, in the small pulmonary
vessels, compared with the known vasodilators adrenomedullin, sodium
nitroprusside, and acetylcholine. In human small pulmonary
arteries, hUT-II did not induce vasoconstriction but was a potent
vasodilator [
log M concentration causing 50% of the maximum
vasodilator effect (pIC50) 10.4 ± 0.5; percentage of
reduction in tone (Emax) 81 ± 8% (vs.
23 ± 11% in time controls), n = 5]. The order
of potency for vasodilation was human urotensin-II = adrenomedullin (pIC50 10.1 ± 0.4, n = 6) > sodium nitroprusside (pIC50 7.4 ± 0.2, n = 6) = acetylcholine (pIC50 6.8 ± 0.3, n = 6). In human abdominal arteries, hUT-II did
not induce vasoconstriction but was a potent vasodilator
[pIC50 10.3 ± 0.7; Emax
96 ± 8% (vs. 43 ± 16% in time controls),
n = 4]. This is the first report that hUT-II is a
potent vasodilator but not a vasoconstrictor of human small pulmonary
arteries and systemic resistance arteries.
vasoconstriction; adrenomedullin; sodium nitroprusside; acetylcholine; vasodilators
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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HUMAN UROTENSIN-II (hU-II) is a recently cloned human cyclic peptide and present in human cardiac tissue and arteries. Rat, mouse, and porcine isoforms of U-II have also recently been cloned (2, 10). A receptor for hU-II has very recently been described in humans, and hU-II mediates vasoconstriction in many arteries from nonhuman primates, including coronary, large pulmonary, and carotid arteries (1). It is a magnitude more potent than endothelin-1 (ET-1) and, hence, the most potent mammalian vasoconstrictor identified so far. Ames et al. (1) also noted that low doses of hU-II decrease total peripheral resistance in the anesthetised monkey, and fish U-II has been shown to vasodilate isolated rat aortas (3).
We recently reported that hU-II is a potent vasoconstrictor in rat main
pulmonary arteries. It did not however, constrict human small muscular
pulmonary arteries (6). However, when these vessels were
pretreated with the nitric oxide synthase inhibitor N
-nitro-L-arginine methyl ester
(L-NAME), 3 of 10 vessels did contract to hU-II. Here, we
investigated the possibility that hU-II induces vasodilation, which
would obscure any vasoconstrictor response. We have examined the
ability of hU-II to relax preconstricted human small muscular pulmonary
arteries and compared responses with the following known vasodilators:
adrenomedullin (ADM), sodium nitroprusside (SNP), and acetylcholine
(ACh). We subsequently examined vasodilator responses to hU-II in human
abdominal resistance arteries.
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Protocols were approved by the West Ethics Committee, and patients gave written informed consent.
Macroscopically normal small muscular pulmonary arteries [~170 µm internal diameter (ID)] were dissected from lung tissue removed during bronchial carcinoma removal (male patients, age range 35-75). Human abdominal resistance arteries (~200 µm ID) were dissected from abdominal adipose tissue biopsies obtained from otherwise healthy, male patients (age range 35-75) undergoing hernia repair. All vessels were isolated and mounted as ring preparations in isometric wire myographs. Tension was applied to vessels to give a transmural pressure equivalent to ~12-16 mmHg (small pulmonary arteries) or 90 mmHg (systemic arteries) to simulate in vivo pressures.
All vessels were bathed in Krebs-buffer solution at 37°C with a constant supply of 16% O2-5% CO2-balance N2 to mimic in vivo PO2 values (bath PO2 ~ 120 mmHg). All vessels have walls <1.5 µm thick, and, hence, tissue diffusional problems are not encountered with active bubbling.
Experimental protocols. After 45 min of equilibration, the response to 50 mM KCl was determined. Vessels were constricted with 0.1 µM 5-hydroxytryptamine (5-HT), and 1 µM ACh was added to test for endothelial integrity. Protocols were subsequently applied to study either vasoconstrictor or vasodilator activity.
Vasoconstrictor responses. Cumulative concentration-response curves (CCRCs) were constructed to hU-II (1 pM-0.1 µM). Concentrations were administered every 3 min (with 11 concentrations giving a total time course of ~33 min).
Vasodilator responses. Vessels were preconstricted with 3 nM ET-1. CCRCs were constructed to hU-II (1 pM-0.1 µM). In the small pulmonary arteries, responses were also compared with those to ACh (1 nM-10 µM), SNP (10 nM-10 µM), and ADM (1 pM-0.1 µM). Stable responses were allowed to develop. Maximum responses to ADM and hU-II were stable after 5-6 min, and, hence, concentrations were administered every 6-7 min (with 11 concentrations giving a total time course of ~70-80 min). Maximum responses to ACh and SNP were achieved after 3-4 min, and concentrations were administered every 4-5 min (with 9 concentrations, giving a total time course of ~36-45 min). From each lung or abdominal vessel, adjacent vessels were set up and preconstricted with ET-1, but no vasodilator was subsequently added; these served as time controls.
Statistical analysis of data.
All responses to hU-II were calculated as both a percentage of the
initial response to 3 nM ET-1 to calculate the percentage of reduction
in tone induced (Emax) and as a percentage of
the maximum response achieved. The log M concentration causing 50% of the maximum vasodilator effect in each vessel (pIC50)
values were subsequently calculated from each individual experiment by British Broadcasting Corporation microcomputer graphical interpolation. Initial responses to ACh were assessed as a percentage of the preconstriction to 5-HT. Statistical comparisons of unpaired data sets
were carried out by one-way analysis of variance (ANOVA) followed by
Tukey's multicomparisons test, with P < 0.05 considered significant. Comparisons were also made with time control
data obtained over the same periods of time as the experimental
procedures were conducted.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Human small muscular pulmonary arteries (n = 5). The vasoconstrictor response to 50 mM KCl was 1.08 ± 0.22 mN. The vasoconstrictor response to 0.1 µM 5-HT was 65 ± 9% of the response to 50 mM KCl. ACh (1 µM) caused a 61 ± 10% reduction in 5-HT-induced tone. The vasoconstriction to 3 nM ET-1 was 171 ± 37% of the response to 50 mM KCl. The internal diameter (ID) for the hU-II-treated vessels was 163 ± 15 µm, and the ID for the time controls was 184 ± 15 µm.
hU-II did not induce vasoconstriction in any vessel (n = 10). It did, however, induce a vasodilation (Fig. 1). The pIC50 value for the response to hU-II was 10.4 ± 0.5. Figure 1 and Table 1 compare the responses to hU-II with other known vasodilators. hU-II was equipotent with ADM, and it can be seen that both are extremely potent vasodilators of small pulmonary arteries. The order of potency was hU-II = ADM > SNP = ACh. For ADM, SNP, and ACh, all responses were significantly different from time controls, and the maximum fall off in tone with time was not significantly different among groups: ADM 20 ± 6%, SNP 24 ± 7%, and ACh 21 ± 6%. There were no significant differences in the maximum vasodilation achieved at the concentration ranges used. It should be noted that higher concentrations were used in limited experiments for all vasodilators to ensure that a maximum response had been achieved.
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Human abdominal resistance vessels (n = 4). The vasoconstrictor response to 50 mM KCl was 3.15 ± 1.22 mN. The vasoconstrictor response to 0.1 µM 5-HT was 67 ± 18% of the response to 50 mM KCl. ACh (1 µM) caused a 56 ± 9% reduction in 5-HT-induced tone. The vasoconstriction to 3 nM ET-1 was 146 ± 26% of the response to 50 mM KCl. The ID for the hU-II-treated vessels was 191 ± 23 µm, and the ID for the time controls was 194 ± 27 µm.
hU-II did not induce a vasoconstriction in any vessels tested. In light of the results in the small pulmonary arteries, we therefore investigated vasodilator responses. hU-II induced a concentration-dependent vasodilation. The pIC50 value for the response to hU-II was 10.3 ± 0.7. The maximum vasodilation achieved was 94 ± 6% and not significantly different from that to hU-II in the small pulmonary arteries (Fig. 2 and Table 1).
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Time controls. Figure 2 illustrates the fall off in vascular tone during the time course of each experiment. This was significantly different from the hU-II response data at every time point of hU-II administration [for both small pulmonary and abdominal resistance arteries, P < 0.05 (response to 1 pM hU-II vs. time control) to P < 0.001 (response to 0.1 µM hU-II vs. time control)].
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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This is the first report that hU-II is a potent vasodilator of
human small pulmonary and systemic resistance vessels. Less profound
vasodilator responses to fish U-II in the rat aorta have been reported
(3). hU-II has been shown to be a potent vasoconstrictor in other species (1). Indeed, we have previously shown
that hU-II is a potent vasoconstrictor in the rat main pulmonary
artery, being some four times more potent than ET-1 (6).
hU-II did not, however, constrict smaller pulmonary arteries of the rat nor did it contract human small pulmonary arteries under normal conditions, as verified here. ET-1 is a potent vasoconstrictor of human
pulmonary resistance arteries having a log M concentration giving
50% of maximum response (pEC50) value of
8.1-8.3 (7, 8). The current results suggest,
therefore, that hU-II and ET-1, at least in the human circulation, may
play differential roles in the regulation of vascular tone and resistance.
The results presented here suggest that any vasoconstrictor effect of hU-II in the small pulmonary and abdominal arteries may be masked by the potent vasodilator effects of hU-II. The "anatomically diverse" contractile profile of hU-II is consistent with previous observations made in the rat. In the rat, the vasoconstrictor activity of hU-II is limited to the thoracic aorta, and hU-II has no effect on the rat abdominal aorta or femoral and renal arteries (1).
hU-II has been shown to constrict all nonhuman primate arteries tested to date, although the vessels studied by Ames et al. in 1999 (1) were all large conduit arteries. The current study may suggest, however, that there is species variation in responses to hU-II and that vasodilator responses may predominate in certain human resistance arteries.
The pEC50 for the contractile response to hU-II ranges from 8.55 (in the rat pulmonary artery; see Ref. 6) to 9-9.4 (in nonhuman primate arteries; see Ref. 1). As the pIC50 for the vasodilator effect observed here is 10.2-10.4, this may suggest that more than one receptor subtype exists for hU-II, but this requires molecular confirmation. Parallels can be drawn with the pharmacology of ET-1, which is both a potent vasoconstrictor and a vasodilator, activating two receptor subtypes (4).
We have previously published (5a) preliminary studies indicating that ADM is an extremely potent vasodilator in the human small pulmonary artery compared with resistance vessels obtained from buttock biopsies where the pIC50 is only ~7.7. Plasma ADM levels range from ~5 pM in normal subjects to 10-14 pM in patients with cardiovascular and pulmonary arterial disease (5, 9, 11, 12). The results presented here indicate that these concentration will have a direct effect on pulmonary resistance arteries to produce vasodilation. ADM is likely, therefore, to regulate pulmonary vascular tone, and increased levels in cardiopulmonary disease would provide a compensatory vasodilator mechanism. hU-II (3 nmol/kg iv) has been shown to induce significant increases in total peripheral resistance in the monkey (1).
We previously considered that ADM was probably the most potent small pulmonary artery vasodilator (5a), but here we show that hU-II is equipotent to ADM and also equipotent in the human abdominal resistance artery. Both are magnitudes more potent than SNP and ACh, which act through endothelium-independent and -dependent mechanisms, respectively. The mechanism of hU-II and ADM-induced vasodilation in human resistance vessels is still unclear and under investigation.
In conclusion, therefore, this is the first report to suggest that hU-II may be a potent vasodilator of human small pulmonary arteries and systemic resistance arteries being devoid of contractile activity.
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ACKNOWLEDGEMENTS |
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This work was funded by the Wellcome Trust, United Kingdom.
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FOOTNOTES |
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Address for reprint requests and other correspondence: M. R. MacLean, Div. of Neuroscience and Biomedical Systems, Institute of Biomedical and Life Sciences, West Medical Bldg., Glasgow Univ., Glasgow G12 8QQ, UK (E-mail: M.MacLean{at}bio.gla.ac.uk).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 September 2000; accepted in final form 18 October 2000.
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REFERENCES |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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