Involvement of the nitric oxideL-arginine and sympathetic nervous

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					                                            Life Sciences 71 (2002) 819 – 825
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Involvement of the nitric oxide/L-arginine and sympathetic nervous
   systems on the vasodepressor action of human urotensin II
                        in anesthetized rats
                                Aly M. Abdelrahman a, Catherine C.Y. Pang b,*
                        a
                         Department of Pharmacology, Faculty of Medicine, Minia University, Minia, Egypt
       b
           Department of Pharmacology and Therapeutics, Faculty of Medicine, University of British Columbia, Vancouver,
                                             British Columbia, Canada V6T 1Z3
                                        Received 30 July 2001; accepted 8 February 2002



Abstract

    This study examined if the nitric oxide (NO)/L-arginine pathway participates in and if the sympathetic nervous
system attenuates the depressor action of human urotensin II. I.V. bolus injections of human urotensin II (0.1 –30
nmol/kg) caused dose-dependent decreases in mean arterial pressure (MAP, EC50 = 2.09 F 0.8 nmol/kg; Emax =
À18 F 3 mmHg ) and increases in heart rate. The depressor response to human urotensin II (3 nmol/kg) was
attenuated by approximately 50% in rats with MAP elevated through pretreatment with NG-nitro-L-arginine
methyl ester (inhibitor of NO synthase), relative to that in rats with MAP elevated to a similar level through a
continuous infusion of noradrenaline. Autonomic blockade with i.v. injections of mecamylamine (ganglion
blocker) and propranolol (h-adrenoceptor antagonist) markedly augmented the depressor response to human
urotensin II, but almost completely attenuated the tachycardia. The results suggest that the depressor response to
human urotensin II is partially mediated via the NO/L-arginine pathway, and is suppressed by activity of the
sympathetic nervous system. Furthermore, tachycardic response to human urotensin II is primarily mediated
indirectly via baroreflex mechanisms. D 2002 Published by Elsevier Science Inc.

Keywords: Human urotensin II; Blood pressure; Heart rate; Nitric oxide; Autonomic nervous system




   *
     Corresponding author. Tel.: +1-604-822-2039; fax: +1-604-822-6012.
   E-mail address: ccypang@interchange.ubc.ca (C.C.Y. Pang).

0024-3205/02/$ - see front matter D 2002 Published by Elsevier Science Inc.
PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 1 7 4 3 - 5
820                      A.M. Abdelrahman, C.C.Y. Pang / Life Sciences 71 (2002) 819–825


Introduction

   Human urotensin II (hUT-II), a cyclic 11-amino acids peptide, has been identified as a ligand for the
GPR14 receptor, which is an orphan G-protein-coupled receptor similar to the somatostatin receptor
([1,2], see review [3]). GPR14 mRNA is present in the heart, the smooth muscle cells of the coronary
artery and aorta, as well as the endothelial cells of the coronary artery and umbilical veins [2].
Furthermore, binding sites of hUT-II have been detected in the heart and vasculature [4]. There is
evidence that hUT-II causes contraction of blood vessels in vitro. Its activity is, however, dependent on
the type of vasculature and the species from which the vessel is derived [3,5]. In the rat, hUT-II
contracted many arteries including the thoracic aorta, carotid artery, pulmonary artery and left anterior
descending coronary artery [6–8]. The hUT-II also contracted the human coronary, mammary and radial
arteries, as well as the saphenous and umbilical veins [4]. Indeed, hUT-II has been described as the most
potent human vasoconstrictor peptide yet identified, as it is more potent than endothelin, noradrenaline
and serotonin in contracting the isolated thoracic aorta [2]. Its vasoconstrictor effect in the rabbit thoracic
aorta was mediated by a phospholipase C-dependent increase in inositol phosphates [9]. In anesthetized
monkeys, hUT-II increased total peripheral resistance but caused cardiac contractile dysfunction
exemplified by an increase in left ventricular end-diastolic pressure and reductions in cardiac output
and cardiac contractility [2].
   The hUT-II, however, had no constrictor effect in the human small subcutaneous resistance arteries,
internal mammary artery, saphenous vein, and small subcutaneous vein [10], as well as the rat small
mesenteric artery [6]. Furthermore, it caused endothelium-dependent relaxation of pre-constricted left
anterior descending coronary and mesenteric arteries [6], and sustained vasodilatation in the isolated
perfused rat heart [11]. It also dilated isolated small pulmonary arteries and abdominal resistance arteries
from humans with potencies similar to that of adrenomedullin, and greater than those of sodium
nitroprusside and acetylcholine [12]. Furthermore, hUT-II decreased blood pressure and increased heart
rate in conscious rats [13]. It is, however, unclear if its tachycardic effect was direct or mediated via
autonomic reflex.
   In preliminary experiments, we have found that hUT-II causes a depressor response and tachycardia in
anesthetized rats. This study examined if the nitric oxide (NO)/L-arginine pathway contributes to and if
the sympathetic nervous system suppresses the depressor effect of hUT-II, and if human urotensin II has
a direct positive chronotropic action.


Methods


Animal preparation

   Male Sprague–Dawley rats (300–400 g) were anesthetized with thiobutabarbitone (Inactin, 100 mg/
kg i.p.). Cannulae were inserted into the femoral artery for the measurement of mean arterial pressure
(MAP) by a pressure transducer (P23DB, Gould Statham, CA), and both femoral veins for the
administration of drugs. Heart rate (HR) was derived electronically from the upstroke of the arterial
pulse pressure by a tachograph (Grass 7P4G). All cannulae were filled with heparinized normal saline
(25 i.u./ml in 0.9% NaCl).
                             A.M. Abdelrahman, C.C.Y. Pang / Life Sciences 71 (2002) 819–825                                  821

Experimental protocol

   Dose-MAP and HR response curves of hUT-II (0.1–30 nmol/kg, i.v. bolus) or equal volumes of saline
were constructed in two groups of rats (n = 5 each) at dose-intervals of 5 min.
   An additional five groups of rats (n = 6 each) were used to examine if the NO/L-arginine system
contributes to the depressor effect of hUT-II. Three groups were given hUT-II (3 nmol/kg i.v. bolus) at
10 min following pretreatment with saline (0.9% NaCl i.v. bolus), NG-nitro-L-arginine methyl ester (L-
NAME, 26 mg/kg, i.v. bolus) or noradrenaline (2 Ag/kg/min, i.v. infusion). The dose of L-NAME used
was three times the dose that caused a maximum increase in MAP in rats [14]. Noradrenaline served as a
positive control for L-NAME and the dose selected caused similar pressor response as L-NAME. An
additional two groups served as time-controls, and they received injections of an equivalent volume of
saline (instead of hUT-II) following pretreatment with L-NAME or noradrenaline. Hemodynamic




Fig. 1. Effects (mean F s.e. mean; n = 5 per group) of human urotensin II (0.1 – 30 nmol/kg) on: a) mean arterial pressure
(MAP) and b) heart rate (HR) in anesthetized rats. *Significantly different from time-control rats that received saline injections
( P < 0.05).
822                          A.M. Abdelrahman, C.C.Y. Pang / Life Sciences 71 (2002) 819–825

response to hUT-II was measured at the time of peak response, at approximately 4–5 min following i.v.
bolus injection of hUT-II and the same time points in the time-control rats.
   Another two groups of rats (n = 6 each) were used to examine if the depressor effect of hUT-II was
attenuated by activities from the sympathetic nervous system, and if hUT-II has direct positive
chronotropic action. One group was given hUT-II (3 nmol/kg, i.v. bolus) at 10 min after pretreatment
with both mecamylamine (1 mg/kg, i.v. bolus) and propranolol (0.5 mg/kg, i.v. bolus). The dose of
mecamylamine used was double that which abolished ganglionic transmission for more than 2 h [15].
The second group served as time-control, and received saline (instead of hUT-II) at 10 min after
injections of mecamylamine and propranolol. Hemodynamic response to hUT-II was also measured at
the time of peak response.

Drugs

   Inactin (thiobutabarbitone) was from Research Biochemicals International (MA, USA). Human
urotensin II (Phoenix Pharmaceuticals, CA, USA), as well as L-NAME, noradrenaline, mecamylamine
and propranolol (Sigma Chemical Co., St Louis, MO, USA) were dissolved in normal saline.

Calculation and statistical analysis

  All data are presented as mean F s.e. mean. The data were analyzed by one-way analysis of variance
(ANOVA) followed by Tukey test, with P< 0.05 selected as the criterion for statistical significance.


Results

Dose response curve of hUT-II

  Baseline MAP and HR of rats to be treated with hUT-II or saline were 92 F 8 and 88 F 6 mmHg, and
368 F 17 and 362 F 10 beats/min, respectively. I.V. injections of hUT-II caused a dose dependent decrease

Table 1
Effects (means F s.e. mean) of pretreatments with saline (0.9% NaCl), noradrenaline (2 Ag/kg/min), NG-nitro-L-arginine methyl
ester (L-NAME; 26 mg/kg), and combined mecamylamine and propanolol (Mec-Prop; 1 mg/kg and 0.5 mg/kg, respectively) on
mean arterial pressure (MAP) and heart rate (HR) in seven groups (n = 6 each) of thiobutabarbitone-anesthesized rats to be
subsequently treated with either human urotensin II (hUT-II) or saline
Groups                                MAP (mmHg)                                         HR (beats/min)
                                      Baseline                Treatment                  Baseline                  Treatment
Saline: hUT-II                        98   F   1               97   F   1                378   F   8               373   F   8
Noradrenaline: hUT-II                 90   F   3              125   F   5a               370   F   7               394   F   18
Noradrenaline: Saline                 89   F   3              120   F   5a               340   F   8               340   F   13
L-NAME: hUT-II                        92   F   4              126   F   5a               362   F   10              324   F   11
L-NAME: Saline                        85   F   4              138   F   3a               347   F   4               295   F   5a
Mec-Prop: hUT-II                      91   F   3               67   F   3a               360   F   8               334   F   14
Mec-Prop: Saline                      85   F   4               64   F   3a               345   F   10              321   F   10
   a
       Significantly different from baseline MAP and HR ( P < 0.05).
                             A.M. Abdelrahman, C.C.Y. Pang / Life Sciences 71 (2002) 819–825                                823

in MAP (EC50 = 2.09 F 0.8 nmol/kg; Emax = À18 F 3 mmHg ) and a dose-dependent increase in HR (Fig.
1A, 1B). Depressor response to hUT-II reached plateau at approximately 4–5 min following i.v. bolus
injection. MAP and HR did not change significantly in the time-control rats that received injections
of saline.

Effect of hUT-II in the absence and presence of administrations of noradrenaline, L-NAME or combined
mecamylamine and propranolol

   Baseline MAP and HR before and after the administrations of L-NAME, noradrenaline or combined
mecamylamine and propranolol are shown in Table 1. Noradrenaline increased MAP but did not alter
HR in both groups. MAP was increased in both groups given L-NAME, but HR was decreased
significantly only in one of the groups. The combined administrations of mecamylamine and propranolol
reduced MAP significantly and HR insignificantly in both groups.




Fig. 2. Effects (mean F s.e. mean; n = 6 per group) of human urotensin II (3 nmol/kg) or saline (0.9% NaCl) on: a) mean arterial
pressure (MAP) and b) heart rate (HR) in rats pretreated with saline, noradrenaline (NA; 2 Ag/kg/min), NG-nitro-L-arginine-
methyl ester (L-NAME, 26 mg/kg), or combined mecamylamine (Mec; 1 mg/kg) and propranolol (prop; 0.5 mg/kg). Closed and
open bars represent the effects of human urotensin II or saline, respectively. aSignificantly different ( P < 0.05) from human
urotensin II in the saline-pretreated group; bSignificantly different from saline in respective pretreated group. cSignificantly
different from human urotensin II in the noradrenaline-pretreated group.
824                     A.M. Abdelrahman, C.C.Y. Pang / Life Sciences 71 (2002) 819–825

   The injection of saline did not alter MAP nor HR in rats pretreated with noradrenaline, L-NAME, or
combined mecamylamine and propranolol (Fig. 2A, 2B). MAP was decreased and HR was increased to
peak values at 4–5 min after i.v. injection of hUT-II (3 nmol/kg) in rats pretreated with saline (Fig. 2A,
2B), and the effect lasted 10 min. In rats pretreated with noradrenaline, hUT-II caused a greater decrease
in MAP relative to the changes in rats pretreated with saline. In rats pretreated with L-NAME, hUT-II
caused a small and insignificant decrease in MAP, and an increase in HR which were less than that of the
corresponding changes in the rats treated with noradrenaline (Fig. 2A). In rats pretreated with
mecamylamine and propranolol, hUT-II also decreased MAP and slightly increased heart rate, the
depressor response was markedly greater, and the increase in HR was much less than those in rats
pretreated with saline (Fig. 2A, 2B).


Discussion

   The hUT-II has diverse vascular actions that ranged from vasoconstriction to vasodilatation; its
activity is dependent on the type of vessels studied and species of animals from which the vessels are
derived (see Introduction). In the present study, hUT-II caused a dose-dependent reduction in blood
pressure accompanied by tachycardia. Depressor and tachycardic effects of hUT-II in conscious rats have
been reported previously [13]. The onset of the vasodilator effect was slow (4–5 min) and the response
lasted 10 min. This is consistent with the slow dissociation of [125I]human urotensin at recombinant
GPR14 [8].
   The vasodilator effect of hUT-II was enhanced in rats with elevated MAP through a continuous
infusion of noradrenaline. In rats pretreated with L-NAME, hUT-II failed to produce a significant
decrease in blood pressure. As well, the percent decrease in MAP after L-NAME was markedly less than
that following the infusion of noradrenaline which increased MAP to the same level as that elicited by L-
NAME. On the other hand, the depressor response to hUT-II in the L-NAME-treated rats was not
significantly different than that in the saline-pretreated rats. Since L-NAME did not completely abolish
the depressor response to hUT-II, other depressor mechanisms, such as endothelium-derived hyper-
polarizing factor (EDHF), may also contribute to the depressor effect of hUT-II; however, this was not
examined in the present study. The ability of L-NAME to attenuate the depressor response to hUT-II
suggests that the depressor response is at least partially mediated via the NO/L-arginine pathway. In
support of our results, NG-nitro-L-arginine and L-NAME were shown to attenuate the vasodilator effect
of hUT-II in the isolated perfused rat heart [11] as well as the isolated left anterior descending coronary
artery, respectively [6]. Interestingly, L-NAME as well as endothelial denudation were also shown to
enhance the contractile response of hUT-II in the rat pulmonary artery, and unmask the contractile
response of hUT-II in the isolated human small pulmonary artery [7].
   In rats pretreated with mecamylamine and propranolol, hUT-II caused a markedly greater depressor
response and much less change in HR. These results show that the sympathetic nervous system
attenuates the depressor effect of hUT-II. Furthermore, the tachycardic response to hUT-II was primarily
mediated via hypotension-induced reflex alteration in autonomic activity, since the response was almost
completely inhibited by autonomic blockade.
   To summarize, hUT-II is a potent vasodilator which modestly reduces MAP and increases HR via the
activation of baroreflex mechanisms. Its vasodilator action is partially mediated via the NO/L-arginine
pathway, and attenuated by reflex sympathetic activation.
                             A.M. Abdelrahman, C.C.Y. Pang / Life Sciences 71 (2002) 819–825                                825

Acknowledgements

   The work was supported by the Heart and Stroke Foundation of B.C. and Yukon.


References

 [1] Marchese A, Heiber M, Nguyen T, Heng HH, Saldivia VR, Cheng R, Murphy PM, Tsui LC, Shi X, Gregor P. Cloning and
     chromosomal mapping of three novel genes, GPR9, GPR10, and GPR14, encoding receptors related to interleukin 8,
     neuropeptide Y, and somatostatin receptors. Genomics 1995;29(2):335 – 44.
 [2] Ames RS, Sarau HM, Chambers JK, Willette RN, Aiyar NV, Romanic AM, Louden CS, Foley JJ, Sauermelch CF,
     Coatney RW, Ao Z, Disa J, Holmes SD, Stadel JM, Martin JD, Liu WS, Glover GI, Wilson S, McNulty DE, Ellis CE,
     Elshourbagy NA, Shabon U, Trill JJ, Hay DW, Douglas SA. Human urotensin-II is a potent vasoconstrictor and agonist for
     the orphan receptor GPR14. Nature 1999;401(6750):282 – 6.
 [3] Douglas SA, Ohlstein EH. Human urotensin-II, the most potent mammalian vasoconstrictor identified to date, as a
     therapeutic target for the management of cardiovascular disease. Trends Cardiovasc Med 2000;10(6):229 – 37.
 [4] Maguire JJ, Kuc RE, Davenport AP. Orphan-receptor ligand human urotensin II: receptor localization in human tissues and
     comparison of vasoconstrictor responses with endothelin-1. Br J Pharmacol 2000;131(3):441 – 6.
 [5] Douglas SA, Ashton DJ, Sauermelch CF, Coatney RW, Ohlstein DH, Ruffolo MR, Ohlstein EH, Aiyar NV, Willette RN.
     Human urotensin-II is a potent vasoactive peptide: pharmacological characterization in the rat, mouse, dog and primate. J
     Cardiovasc Pharmacol 2000;36(5 Suppl 1):S163 – 6.
 [6] Bottrill FE, Douglas SA, Hiley CR, White R. Human urotensin-II is an endothelium-dependent vasodilator in rat small
     arteries. Br J Pharmacol 2000;130(8):1865 – 70.
 [7] MacLean MR, Alexander D, Stirrat A, Gallagher M, Douglas SA, Ohlstein EH, Morecroft I, Polland K. Contractile
     responses to human urotensin-II in rat and human pulmonary arteries: effect of endothelial factors and chronic hypoxia in
     the rat. Br J Pharmacol 2000;130(2):201 – 4.
 [8] Douglas SA, Sulpizio AC, Piercy V, Sarau HM, Ames RS, Aiyar NV, Ohlstein EH, Willette RN. Differential vaso-
     constrictor activity of human urotensin-II in vascular tissue isolated from the rat, mouse, dog, pig, marmoset and
     cynomolgus monkey. Br J Pharmacol 2000;131(7):1262 – 74.
 [9] Opgaard OS, Nothacker H, Ehlert FJ, Krause DN. Human urotensin II mediates vasoconstriction via an increase in inositol
     phosphates. Eur J Pharmacol 2000;406(2):265 – 71.
[10] Hillier C, Berry C, Petrie MC, O’Dwyer PJ, Hamilton C, Brown A, McMurray J. Effects of urotensin II in human arteries
     and veins of varying caliber. Circulation 2001;103(10):1378 – 81.
[11] Katano Y, Ishihata A, Aita T, Ogaki T, Horie T. Vasodilator effect of urotensin II, one of the most potent vasoconstricting
     factors, on rat coronary arteries. Eur J Pharmacol 2000;402(1 – 2):209 – 11.
[12] Stirrat A, Gallagher M, Douglas SA, Ohlstein EH, Berry C, Kirk A, Richardson M, MacLean MR. Potent vasodilator
     responses to human urotensin—II in human pulmonary and abdominal resistance arteries. Am J Physiol — Heart Circ
     Physiol 1992;280(2):H925 – 8.
[13] Gardiner SM, March JE, Kemp PA, Davenport AP, Bennett T. Depressor and regionally-selective vasodilator effects of
     human and rat urotensin II in conscious rats. Br J Pharmacol 2001;132(8):1625 – 9.
[14] Wang YX, Lim SL, Pang CC. Increase by NG-nitro-L-arginine methyl ester (L-NAME) of resistance to venous return in
     rats. Br J Pharmacol 1995;114:1454 – 8.
[15] Wang YX, Pang CC. Possible dependence of pressor and heart rate effects of NG-nitro-L-arginine on autonomic nerve
     activity. Br J Pharmacol 1991;103:2004 – 8.