Br. J. Pharmacol.
Br.
J.
Pharmacol.
(1990), ", 107-112 (1990),
99,
107-112
,'-.
©
MacmiHan Press Ltd, 1990
Macmillan
Press
Ltd,
1990
Regional haemodynamic effects of endothelin- 1 and endothelin-3 in conscious Long Evans and Brattleboro rats 1S.M. Gardiner, A.M. Compton & T. Bennett Department of Physiology & Pharmacology, Medical School, Queen's Medical Centre, Nottingham NG7 2UH
1 The regional haemodynamic effects of bolus doses (4 and 40 pmol) and infusions (12 and 120 pmol h1) of endothelin-1 and endothelin-3 were assessed in conscious, Long Evans and Brattleboro (i.e. vasopressin-deficient) rats, chronically-instrumented with pulsed Doppler flow probes. 2 In both strains of rat the lower bolus dose of endothelin-1 caused only a slight pressor effect, but there were marked renal and mesenteric vasoconstrictions and hindquarters vasodilatation. 3 The lower bolus dose of endothelin-3 did not affect blood pressure significantly, although the changes in regional haemodynamics were qualitatively similar to those seen following endothelin-1 in Long Evans and Brattleboro rats. 4 The higher dose of endothelin-1 caused an initial hypotension accompanied by substantial hindquarters vasodilatations in Long Evans and Brattleboro rats. Subsequently, in both strains, there was a rise in blood pressure accompanied by renal, mesenteric and hindquarters vasoconstrictions. 5 The higher bolus dose of endothelin-3 caused initial hypotension and hindquarters vasodilatation similar to those seen with endothelin-1. However, the subsequent pressor effect was less with endothelin-3, as was the renal vasoconstriction, and it did not cause any increase in hindquarters vascular resistance. 6 Infusion of endothelin-l at the lower rate (12 pmol h- 1) caused renal and mesenteric vasoconstrictions in both strains of rat, whereas endothelin-3 at this rate caused only mesenteric vasoconstriction. 7 Infusion of endothelin-1 at the higher rate (120 pmolh 1) caused progressive hypertension and vasoconstrictions in all three vascular beds studied; these were similar in both strains of rat. Endothelin-3 had a smaller pressor effect and a lesser constrictor action on the renal and mesenteric vascular beds; it did not constrict the hindquarters vascular bed. 8 These results show, that in conscious Long Evans and Brattleboro rats, the initial depressor effects of the higher bolus doses of endothelin-1 and -3 were similar, and, hence, not influenced by the absence of endogenous vasopressin. Endothelin-1 and -3 appear equipotent in their initial hyperaemic vasodilator effects in the hindquarters vasculature in both strains, making it unlikely that this effect is dependent on the release of atrial natriuretic peptide (ANP), since ANP does not cause significant increases in hindquarters blood flow in Brattleboro rats. The greater delayed pressor effect of endothelin-1 is associated with its more marked vasoconstrictor effects on renal and mesenteric vascular beds and is accentuated, relative to endothelin-3, by the lack of a constrictor effect of endothelin-3 in the hindquarters vasculature.
Introduction Following the original description of the potent pressor effects of porcine endothelin by Yanagisawa et al. (1988a), much interest has been shown in the cardiovascular actions of this peptide, the original form of which is now known as endothelin-1 (Inoue et al., 1989). Studies of the in vivo haemodynamic effects of endothelin-1 in conscious Wistar rats have shown that it can cause initial vasodilatations in the hindquarters and carotid vascular beds and also particularly marked renal and mesenteric vasoconstrictions (Gardiner et al., 1989a). Similar effects have been seen in anaesthetized, ganglion-blocked hypertensive rats (Wright & Fozard, 1988). Recently, some in vivo cardiovascular effects of the homologous peptide originally designated as rat endothelin (now known as endothelin-3) have been described (Yanagisawa et al., 1988b; Inoue et al., 1989). Like endothelin-1, endothelin-3 causes initial hypotension followed by a pressor effect in conscious rats, although the in vitro vasoconstrictor actions of endothelin-3 are less than those of endothelin-1 (Yanagisawa et al., 1988b); indeed, endothelin-3 has been found to be a vasodilator in the preconstricted mesenteric vasculature of the rat in vitro (Warner et al., 1989a). The latter observation is consistent with the finding that the hypotensive response to endothelin-3 was greater than that to endothelin-1, although these measurements were made in urethane-anaesthetized rats treated with atropine, propranolol and bunazocine (Inoue et '
Author for correspondence.
al., 1989). In rats anaesthetized with fentanyl, fluanisone and midazolam, endothelin-1 and endothelin-3 have been found to exert similar initial hypotensive effects, although the former peptide had about three times the pressor activity of the latter (Spokes et al., 1989). In anaesthetized cats, also, endothelin-1 is a more potent pressor agent than endothelin-3 (Minkes et al., 1989), but it is notable that the sensitivity of this model to endothelin-1 is much less than that of conscious rats (Gardiner et al., 1989a). Furthermore, anaesthetized cats show no pressor responses to endothelin-3 (Minkes & Kadowitz, 1989), unlike conscious (Yanagisawa et al., 1988b) and anaesthetized (Inoue et al., 1989) rats. To date there have been no systematic studies of the detailed regional haemodynamic effects of endothelin-1 and endothelin-3 in the same conscious animals. The present work provides these data in normal, Long Evans rats and in Brattleboro (i.e. vasopressin-deficient) rats. The latter animals were studied since it was feasible that release of vasopressin in response to endothelin-induced hypotension might contribute to the regional haemodynamic changes in Long Evans rats. In addition, any differences in sensitivity to the vasoconstrictor actions of endothelins between Long Evans and Brattleboro rats could be of relevance to our understanding of the role of endogenous endothelin in cardiovascular regulation. Furthermore, since it has been suggested that release of atrial natriuretic peptide might be responsible for the hypotensive and vasodilator effects of endothelin-1 (Winquist et al., 1989), comparison of the responses of Long Evans and Brattleboro rats to this peptide should provide results germane to this hypoth-
108
S.M. GARDNER et al.
esis because atrial natriuretic peptide increases hindquarters blood flow in Long Evans, but not in Brattleboro rats (Gardiner et al., 1988). Some of these results have been presented to the British Pharmacological Soc~ety (Gardiner et al., 1989b).
Methods All experiments were carried out on male, Long Evans and homozygous Brattleboro rats (350-450g). Under anaesthesia (sodium methohexitone (Brietal, Lilly), 60 mg kg-1 i.p., supplemented as required), miniaturized pulsed Doppler probes (Haywood et al., 1981) were sutured around the left renal and superior mesenteric arteries and around the distal abdominal aorta (below the level of the ileocaecal artery). The probe wires were passed through a small incision in the left flank and fed subcutaneously to emerge at the back of the neck where they were sutured in place. Animals were given an intramuscular injection of ampicillin (Penbritin 7mg kg- 1) and allowed to recover for at least 7 days with free access to food and water. Then, animals with acceptable signals (signal:noise > 20:1) from all 3 probes were briefly anaesthetized (sodium methohexitone 40mgkg-1) and had intravenous (right jugular vein) and intraarterial (distal abdominal aorta via ventral caudal artery) catheters implanted. The catheters, filled with heparinized saline (30uml-1), were fed subcutaneously to emerge at the same point as the probe wires. The latter were soldered into a microconnector (Microtech Inc., Boothwyn, PA, U.S.A.) that was clamped into a harness worn by the rat. A flexible spring attached to the harness carried the catheters within it and the connecting lead to the Doppler flowmeter (Crystal Biotech VFI sytem) was taped to the spring to support it; the spring was attached to a counter-balanced, universally-jointed, lever system. At least 24 h later, continuous recordings were started (on a Gould ES1000 system) of phasic and mean blood pressures, of instantaneous heart rate and of phasic and mean Doppler shift signals from the renal, mesenteric and hindquarters probes. Under the conditions of our experiments, percentage changes in the Doppler shift signals are a good index of volume flow changes (Haywood et al., 1981; Gardiner et al., 1988) and together with mean blood pressure (BP) can be used to calculate percentage changes in vascular resistances. Experiments were run on two consecutive days. Long Evans (n = 8) and Brattleboro (n = 8) rats were randomised to receive either endothelin-1 or endothelin-3 on the first day. Each peptide was given as a bolus injection at a low dose (4 pmol in 0.1 ml) followed, at least 60 min later, by a 60 min infusion (12 pmol h-1 in 0.3 ml). At least 1 h after the end of
the infusion, a bolus injection (40 pmol in 0.1 ml) was given; the higher dose infusion (120pmolh-' in 0.3ml) was not started until at least 90 min later. In order to assess the effects of bolus injections, measurements were made 0.5, 1, 5 and o min after injection; with the higher dose, measurements were made also at 20 min. When peptides were given by infusion, measurements were made at 5, 10, 20, 30, 40, 50 and 60 min during infusion and at the same time points after the infusion had been stopped. Bolus injections or infusions of the vehicle had no systematic effects.
Peptides Both peptides were obtained from the Peptide Institute Inc. (European Distributor: Scientific Marketing Associates, London) and were reconstituted in sterile isotonic saline containing 1% bovine serum albumin.
Data analysis Data were subjected to non-parametric, two-way analysis of variance (Friedman's test) to detect changes relative to baseline. Comparisons between groups were made by use of Wilcoxon's rank sum test or the Mann-Whitney U test, as appropriate. Comparisons between Doppler shift changes with endothelin-1 and endothelin-3 were not analysed statistically since mean blood pressure changes were often different with the two peptides, and hence the resistance changes were the variables of importance.
Results
Regional haemodynamic effects of endothelin-J and endothelin-3 in Long Evans rats The lower bolus dose (4pmol) of endothelin-1 had no significant effects on heart rate although there was a small transient increase in mean BP (Table 1). However, there were significant reductions in renal and mesenteric blood flows accompanying an initial hindquarters hyperaemia (Table 1), indicating the occurrence of renal and mesenteric vasoconstrictions and hindquarters vasodilatation (Table 1). The dissociation between systemic BP and regional haemodynamic changes was even more apparent following the lower dose of endothelin-3, since there was no change in mean BP although there were significant regional haemodynamic effects (Table 1). The renal vasoconstrictor effect of endothelin-1 was greater than that of endothelin-3 (Table 1).
Table 1 Cardiovascular changes following i.v. bolus injections (4pmol) of endothelin-l (ET-1) or endothelin-3 (ET-3) in conscious, Long Evans rats Time after injection (min) 1.0
5.0
10.0
Peptide Et-3
Et-l Heart rate (beats-min -1) Mean BP (mmHg) Doppler shift (%) Renal Mesenteric Hindquarters Vascular resistance (%) Renal Mesenteric Hindquarters
Et-l
Et-]
Et-3
Et-]
-4+7 0+ 1
12+ 11 0+2
-9+8
-10 + 3* -5 + 2 -2 + 3
-5 + 1* 1+3 -3 + 6
-5 + 3 0+ 1 -6 +4
-2 + 2 0+4 0+7
13 + 4* 6+2 3+4
5 + 2* 0+4 6+ 8
7+4 1+3 8+6
2+2 2+4 5 8
21 + 12 3+4
19 + 11 5 5
-9+ 14 6+2*
4+8 3+2
-7 + 3* -31 + 2* 32 + 7*
-3 + 2 -20 ± 5* 27 + 7*
-15 + 3* -14 + 2* 0+5
-5 + 2 -11 + 3 8+ 5
12 + 4* 50 9* -20 +5*
9+7 36 + 14* -17 + 4*
26 + 4* 24 + 5* 8+5
Values are given as mean ± s.e.mean (n = 8). * P < 0.05 versus baseline (Friedman's test). t
Et-3
P < 0.05 endothelin-1 versus endothelin-3 (Wilcoxon's test).
8 + 3*t 16 + 4* -3 +6
0+3
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The higher bolus dose (40 pmol) of endothelin-1 caused an initial tachycardia, hypotension and falls in renal and mesenteric blood flows, together with hindquarters hyperaemia (Table 2). However, only the initial change in hindquarters vascular resistance was significant (Table 2). Thereafter, mean BP increased and there was a bradycardia, in association with both reductions in renal and mesenteric blood flows (Table 2) and increases in resistance in all three vascular beds (Table 2). Endothelin-3 caused initial hypotension and tachycardia similar to those seen with endothelin-1 (Table 2), although the former peptide caused early renal, as well as hindquarters, vasodilatations (Table 2). Subsequently the pressor and renal and mesenteric vasoconstrictor effects of endothelin-3 were less than those of endothelin-1, and there was no increase in hindquarters vascular resistance following endothelin-3 administration (Table 2). Infusion, at the lower rate (12pmolh-1), of endothelin-1 had a slight pressor effect (+7 + 2 mmHg, 20 min into infusion), accompanied by increases in renal and mesenteric vascular resistances (maximum change + 16 + 3% and + 13 + 4%, respectively). Infusion of the same dose of endothelin-3 did not affect mean BP and increased only mesenteric vascular resistance (maximum change + 14 + 3%). Endothelin-1, infused at a rate of 120 pmol h1, caused bradycardia and a progressive increase in mean BP (Table 3) associated with marked reductions in renal and mesenteric blood flows, but only a slight reduction in hindquarters flow (Table 3). These changes were associated with vasoconstriction in all 3 vascular beds (Table 3). After the infusion was stopped the vasoconstrictions reversed more rapidly than the pressor effect (Table 3). Infusion of endothelin-3 had a modest pressor effect that was not associated with a significant bradycardia (Table 3). There were renal and mesenteric vasoconstrictions, but no change in hindquarters vascular resistance (Table 3). All changes reversed within IOmin of the end of the infusion (Table 3). The pressor and vasoconstrictor effects of endothelin-l infusion were greater than those of endothelin-3 infusion (Table 3).
Regional haemodynamic effects of endothelin-1 and endothelin-3 in Brattleboro rats The qualitative responses to the lower bolus doses (4pmol) of endothelin-l and endothelin-3 in Brattleboro rats (Table 4) were similar to those in Long Evans rats (Table 1), although
the mesenteric vasoconstrictor effect of endothelin-1 was less prolonged in Long Evans (Table 1) than in Brattleboro rats (Table 4). Furthermore, the renal vasoconstrictor effects of endothelin-l and endothelin-3 were similar in the latter strain
(Table 4), whereas endothelin-3 had a less marked effect than endothelin-1 in Long Evans rats (Table 1). The higher bolus doses (40pmol) of endothelin-l and endothelin-3 caused initial hypotension and tachycardia (Table 5), accompanied by reductions in renal (endothelin-1 only) and mesenteric flows and an increase in hindquarters flow (Table 5); for both peptides, the change in hindquarters vascular resistance was significant but the initial increase in mesenteric vascular resistance was significant for endothelin-3 only (Table 5). Thereafter, there were bradycardias and increases in mean BP. The pressor effect of endothelin-1 was greater than that of endothelin-3 (Table 5), as were the renal and hindquarters vasoconstrictor effects (Table 5), although the increases in mesenteric vascular resistance were similar with both peptides (Table 5), unlike the situation in Long Evans rats (Table 2). Infusion of endothelin-1 at the lower rate (12 pmol h') had no significant effects on heart rate or mean BP but caused significant renal and mesenteric vasoconstrictions (+ 15 + 5% and + 17 ± 5%, respectively; maximum changes). Infusion of endothelin-3 caused mesenteric vasoconstriction only (maximum change + 23 + 3%). Endothelin-1 when infused at a rate of 120 pmol h-, caused progressive bradycardia and hypertension accompanied by reductions in renal and mesenteric blood flows (Table 6). There were vasoconstrictions in the renal mesenteric and hindquarters vascular beds (Table 6). All cardiovascular effects of endothelin-1 had reversed by 40min after the infusion (Table 6). This picture was not different from that seen in Long Evans rats (Table 3). Infusion of endothelin-3 had significantly smaller pressor and vasoconstrictor effects than endothelin-1 (Table 6), and the pattern of differences in regional haemodynamics was similar to that seen in Long Evans rats (Table 3).
Discussion The present work shows that bolus administration of a high dose of endothelin-1 or endothelin-3 causes similar falls in mean BP and similar hindquarters vasodilatations, and the effects are not different in Long Evans and Brattleboro rats, indicating that the haemodynamic profile is not overtly influenced by cardiovascular actions of endogenous vasopressin in the former strain. These findings do not support the proposition that endothelin-3 is a more potent hypotensive (Inoue et al., 1989) or vasodilator (Warner et al., 1989a) agent than endothelin-1, at least under the conditions of our experiments. The only vascular bed in which endothelin-3 administration was followed by a fall in vascular resistance not seen with
Table 4 Cardiovascular changes following i.v. bolus injection (4pmol) of endothelin-l (Et-1) or endothelin-3 (Et-3) in conscious, Brattleboro rats Time after injection (min) 1.0 5.0
0.5
10.0
Peptide Heart rate (beats min -1) Mean BP (mmHg) Doppler shift (%) Renal Mesenteric
Et-l
Et-3
6+9
6+9 2+4
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Et-3
Et-l
Et-3
Et-]
Et-3
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-14 + 7 5+2
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-9+8
0 +1
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1+4 -2+2
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1 +15
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-13 ± 3* -20 + 4* 21 + 10*
-9 + 2* 10 + 4 7 +5
-9 + 2* -12 ± 2* -3 + 5
-6 + 3 2+5 2 +5
-2 + 3 -12 + 3* 6+ 7
-5 ±4 6i6 3i5
Vascular resistance (%) Renal Mesenteric Hindquarters
2+4 21 + 7* -16 + 5*
10 + 8 33 + 7* -16 + 5*
21 + 5* 33 + 9* -10 + 5
16 + 6* 17 + 5* 0+ 7
10 + 4* 14 + 3* 4+6
7+4 -1 + 5 0+ 7
1 +3 12 + 4* -5+5
5 +4 -6+4 -4+ 5
Values are mean + s.e.mean (n = 8). * P < 0.05 versus baseline (Friedman's test).
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112
S.M. GARDNER et al.
endothelin-1 was the kidney, and only in Long Evans rats (Table 2); in that situation renal blood flow was not increased, so it is feasible the vasodilatation was an autoregulatory phenomenon. It is clear from the present work with the lower bolus dose that the regional haemodynamic effects of endothelin-1 or -3 can be substantial when there is little change in mean BP. Furthermore, the hindquarters vasodilator effects of these peptides are not necessarily associated with hypotension, and are not seen when the peptides are infused. While it is feasible that bolus doses of endothelin-1 or endothelin-3 cause hindquarters vasodilatation by releasing eicosanoids or endothelium-derived relaxing factor (De Nucci et al., 1989; Warner et al., 1989b), we have been unable to block this effect with either N0-monomethy-L-arginine, an inhibitor of nitric oxide synthesis by endothelial cells (Gardiner et al., 1989c), or with indomethacin (Gardiner, S.M., Compton, A.M. & Bennett, T., unpublished observations). Indeed, recent findings indicate that indomethacin reduces the contractile effects of endothelin-I on rat aorta (Eglen et al., 1989). In addition, it seems unlikely that the hindquarters hyperaemic responses to bolus doses of endothelin-1 are dependent upon release of atrial natriuretic peptide (Winquist et al., 1989), since bolus doses of this peptide do not cause significant increases in hindquarters blood flow in Brattleboro rats (Gardiner et al., 1988). The results with the higher rate of infusion of endothelin-1
demonstrate that this peptide has marked vasoconstrictor effects in mesenteric, renal and hindquarters vascular beds, the ranking of effect descending in that order. Overall, there was a striking consistency of effect of endothelin-1 in Long Evans and Brattleboro rats, as there was for endothelin-3. In both strains the latter peptide had significantly less pressor and vasoconstrictor action than endothelin-l. While it is possible that endothelin-3 is a more potent activator of dilator mechanisms that oppose its vasoconstrictor action, the similarity of effect of the two peptides in causing initial hindquarters vasodilatation raises the possibility that endothelin-3 is less effective at activating vasoconstrictor mechanisms than endothelin-1 (Warner et al., 1989b). Interestingly, the relative lack of vasoconstrictor effects of endothelin-3 was most marked in the hindquarters, since under no circumstances did endothelin-3 cause a significant increase in resistance in this vascular bed, whereas the higher bolus dose and the higher rate of infusion of endothelin-1 did. Although endothelin-1 had strong vasoconstrictor effects, even following a 60 min infusion of a high dose of this peptide all the haemodynamic effects had disappeared within 40 min. Hence, the relative irreversibility of the effects of this peptide in vitro, and in anaesthetized animals (Yanagisawa et al., 1988a) does not reflect the situation in the intact, conscious rat.
References DE NUCCI, G., THOMAS, R., D'ORLEANS-JUSTE, P., ANTUNES, E.,
WALDER, C., WARNER, T.D. & VANE, J.R. (1988). Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endotheliumderived relaxing factor. Proc. Natl. Acad. Sci. U.S.A., 85, 97979800. EGLEN, R.M., MICHEL, A.D., SHARIF, N.A., SWANK, S.R. & WHITING, R.L. (1989). The pharmacological properties of the peptide, endothelin. Br. J. Pharmacol., 97, 1297-1307. GARDINER, S.M., COMPTON, A.M. & BENNETT, T. (1988). Regional haemodynamic effects of depressor neuropeptides in conscious, unrestrained, Long Evans and Brattleboro rats. Br. J. Pharmacol., 95, 197-208. GARDINER, S.M., COMPTON, A.M. & BENNETT, T. (1989a). Regional haemodynamic effects of endothelin-1 in conscious, unrestrained, Wistar rats. J. Cardiovasc. Pharmacol., 13, suppl. 5, S202-S204. GARDINER, S.M., COMPTON, A.M. & BENNETT, T. (1989b). Haemodynamic effects of endothelin-1 and endothelin-3 in conscious Long Evans rats. Br. J. Pharmacol., 98, 624P. GARDINER, S.M., COMPTON, A.M., BENNETT, T., PALMER, R.M.J. &
MONCADA, S. (1989c). The effect of NG-monomethyl-L-arginine (L-NMMA) on the haemodynamic actions of endothelin-1 in conscious Long Evans rats. Br. J. Pharmacol., 98, 623P. HAYWOOD, J.R., SHAFFER, R.A., FASTENOW, C., FINK, G.D. &
BRODY, M.J. (1981). Regional blood flow measurement with pulsed Doppler flowmeter in conscious rat. Am. J. Physiol., 241, H273H278. INOUE, A., YANAGISAWA, M., KIMURA, S., KASUYA, Y., MIYAUCHI,
T., GOTO, K. & MASAKI, T. (1989). The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc. Nat!. Acad. Sci. U.S.A., 86, 2863-2867. MINKES, R.K., COY, D.H., MURPHY, W.A., McNAMARA, D.B. & KADO-
WITZ, P.J. (1989). Effects of porcine and rat endothelin and an
analog on blood pressure in the anaesthetized cat. Eur. J. Pharmacol., 164, 571-575. MINKES, R.K. & KADOWITZ, P.J. (1989). Differential effects of rat endothelin on regional blood flow in the cat. Eur. J. Pharmacol., 165, 161-164. SPOKES, R.A., GHATEI, M.A. & BLOOM, S.R. (1989). Studies with endothelin-3 and endothelin-1 on rat blood pressure and isolated tissues: evidence for multiple endothelin receptor subtypes. J. Cardiovasc. Pharmacol., 13, suppl. 5, S191-S192. WARNER, T.D., DE NUCCI, G. & VANE, J.R. (1989a). Rat endothelin is a vasodilator in the isolated perfused mesentery of the rat. Eur. J. Pharmacol., 159, 325-326. WARNER, T.D., MITCHELL, J.A., DE NUCCI, G. & VANE, J.R (1989b). Endothelin-1 and endothelin-3 release EDRF from isolated perfused arterial vessels of the rat and rabbit. J. Cardiovasc. Pharmacol., 13, suppl. 5, S85-S88. WINQUIST, R.J., SCOTT, A.L. & VLASUK, G.P. (1989). Enhanced release of atrial natriuretic factor by endothelin in atria from hypertensive rats. Hypertension, 14, 111-114. WRIGHT, C.E. & FOZARD, J.R. (1988). Regional vasodilation is a prominent feature of the haemodynamic response to endothelin in anesthetized spontaneously hypertensive rats. Eur. J. Pharmacol., 155, 201-203. YANAGISAWA, M., INOUE, A., ISHIKAWA, T., KASUYA, Y, KIMURA, S., KUMAGAYE, S-I., NAKAJIMA, K., WATANABE, TX., SAKIBARA, S.,
GOTO, K. & MASAKI, T. (1988b). Primary structure, synthesis, and biological activity of rat endothelin, and endothelium-derived vasoconstrictor peptide. Proc. Nati. Acad. Sci. U.S.A., 85, 69646967. YANAGISAWA, M., KURIHARA, H., KIMURA, S., TOMOBE, Y., KOBAYASHI, M., MITSUI, Y., YAZAKI, Y., GOTO, K. & MASAKI, T. (1988a).
A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature, 332, 411-415.
(Received August 8, 1989 Accepted September 13, 1989)
Br.
J.
Pharmacol.
(1990), ", 107-112 (1990),
99,
107-112
,'-.
©
MacmiHan Press Ltd, 1990
Macmillan
Press
Ltd,
1990
Regional haemodynamic effects of endothelin- 1 and endothelin-3 in conscious Long Evans and Brattleboro rats 1S.M. Gardiner, A.M. Compton & T. Bennett Department of Physiology & Pharmacology, Medical School, Queen's Medical Centre, Nottingham NG7 2UH
1 The regional haemodynamic effects of bolus doses (4 and 40 pmol) and infusions (12 and 120 pmol h1) of endothelin-1 and endothelin-3 were assessed in conscious, Long Evans and Brattleboro (i.e. vasopressin-deficient) rats, chronically-instrumented with pulsed Doppler flow probes. 2 In both strains of rat the lower bolus dose of endothelin-1 caused only a slight pressor effect, but there were marked renal and mesenteric vasoconstrictions and hindquarters vasodilatation. 3 The lower bolus dose of endothelin-3 did not affect blood pressure significantly, although the changes in regional haemodynamics were qualitatively similar to those seen following endothelin-1 in Long Evans and Brattleboro rats. 4 The higher dose of endothelin-1 caused an initial hypotension accompanied by substantial hindquarters vasodilatations in Long Evans and Brattleboro rats. Subsequently, in both strains, there was a rise in blood pressure accompanied by renal, mesenteric and hindquarters vasoconstrictions. 5 The higher bolus dose of endothelin-3 caused initial hypotension and hindquarters vasodilatation similar to those seen with endothelin-1. However, the subsequent pressor effect was less with endothelin-3, as was the renal vasoconstriction, and it did not cause any increase in hindquarters vascular resistance. 6 Infusion of endothelin-l at the lower rate (12 pmol h- 1) caused renal and mesenteric vasoconstrictions in both strains of rat, whereas endothelin-3 at this rate caused only mesenteric vasoconstriction. 7 Infusion of endothelin-1 at the higher rate (120 pmolh 1) caused progressive hypertension and vasoconstrictions in all three vascular beds studied; these were similar in both strains of rat. Endothelin-3 had a smaller pressor effect and a lesser constrictor action on the renal and mesenteric vascular beds; it did not constrict the hindquarters vascular bed. 8 These results show, that in conscious Long Evans and Brattleboro rats, the initial depressor effects of the higher bolus doses of endothelin-1 and -3 were similar, and, hence, not influenced by the absence of endogenous vasopressin. Endothelin-1 and -3 appear equipotent in their initial hyperaemic vasodilator effects in the hindquarters vasculature in both strains, making it unlikely that this effect is dependent on the release of atrial natriuretic peptide (ANP), since ANP does not cause significant increases in hindquarters blood flow in Brattleboro rats. The greater delayed pressor effect of endothelin-1 is associated with its more marked vasoconstrictor effects on renal and mesenteric vascular beds and is accentuated, relative to endothelin-3, by the lack of a constrictor effect of endothelin-3 in the hindquarters vasculature.
Introduction Following the original description of the potent pressor effects of porcine endothelin by Yanagisawa et al. (1988a), much interest has been shown in the cardiovascular actions of this peptide, the original form of which is now known as endothelin-1 (Inoue et al., 1989). Studies of the in vivo haemodynamic effects of endothelin-1 in conscious Wistar rats have shown that it can cause initial vasodilatations in the hindquarters and carotid vascular beds and also particularly marked renal and mesenteric vasoconstrictions (Gardiner et al., 1989a). Similar effects have been seen in anaesthetized, ganglion-blocked hypertensive rats (Wright & Fozard, 1988). Recently, some in vivo cardiovascular effects of the homologous peptide originally designated as rat endothelin (now known as endothelin-3) have been described (Yanagisawa et al., 1988b; Inoue et al., 1989). Like endothelin-1, endothelin-3 causes initial hypotension followed by a pressor effect in conscious rats, although the in vitro vasoconstrictor actions of endothelin-3 are less than those of endothelin-1 (Yanagisawa et al., 1988b); indeed, endothelin-3 has been found to be a vasodilator in the preconstricted mesenteric vasculature of the rat in vitro (Warner et al., 1989a). The latter observation is consistent with the finding that the hypotensive response to endothelin-3 was greater than that to endothelin-1, although these measurements were made in urethane-anaesthetized rats treated with atropine, propranolol and bunazocine (Inoue et '
Author for correspondence.
al., 1989). In rats anaesthetized with fentanyl, fluanisone and midazolam, endothelin-1 and endothelin-3 have been found to exert similar initial hypotensive effects, although the former peptide had about three times the pressor activity of the latter (Spokes et al., 1989). In anaesthetized cats, also, endothelin-1 is a more potent pressor agent than endothelin-3 (Minkes et al., 1989), but it is notable that the sensitivity of this model to endothelin-1 is much less than that of conscious rats (Gardiner et al., 1989a). Furthermore, anaesthetized cats show no pressor responses to endothelin-3 (Minkes & Kadowitz, 1989), unlike conscious (Yanagisawa et al., 1988b) and anaesthetized (Inoue et al., 1989) rats. To date there have been no systematic studies of the detailed regional haemodynamic effects of endothelin-1 and endothelin-3 in the same conscious animals. The present work provides these data in normal, Long Evans rats and in Brattleboro (i.e. vasopressin-deficient) rats. The latter animals were studied since it was feasible that release of vasopressin in response to endothelin-induced hypotension might contribute to the regional haemodynamic changes in Long Evans rats. In addition, any differences in sensitivity to the vasoconstrictor actions of endothelins between Long Evans and Brattleboro rats could be of relevance to our understanding of the role of endogenous endothelin in cardiovascular regulation. Furthermore, since it has been suggested that release of atrial natriuretic peptide might be responsible for the hypotensive and vasodilator effects of endothelin-1 (Winquist et al., 1989), comparison of the responses of Long Evans and Brattleboro rats to this peptide should provide results germane to this hypoth-
108
S.M. GARDNER et al.
esis because atrial natriuretic peptide increases hindquarters blood flow in Long Evans, but not in Brattleboro rats (Gardiner et al., 1988). Some of these results have been presented to the British Pharmacological Soc~ety (Gardiner et al., 1989b).
Methods All experiments were carried out on male, Long Evans and homozygous Brattleboro rats (350-450g). Under anaesthesia (sodium methohexitone (Brietal, Lilly), 60 mg kg-1 i.p., supplemented as required), miniaturized pulsed Doppler probes (Haywood et al., 1981) were sutured around the left renal and superior mesenteric arteries and around the distal abdominal aorta (below the level of the ileocaecal artery). The probe wires were passed through a small incision in the left flank and fed subcutaneously to emerge at the back of the neck where they were sutured in place. Animals were given an intramuscular injection of ampicillin (Penbritin 7mg kg- 1) and allowed to recover for at least 7 days with free access to food and water. Then, animals with acceptable signals (signal:noise > 20:1) from all 3 probes were briefly anaesthetized (sodium methohexitone 40mgkg-1) and had intravenous (right jugular vein) and intraarterial (distal abdominal aorta via ventral caudal artery) catheters implanted. The catheters, filled with heparinized saline (30uml-1), were fed subcutaneously to emerge at the same point as the probe wires. The latter were soldered into a microconnector (Microtech Inc., Boothwyn, PA, U.S.A.) that was clamped into a harness worn by the rat. A flexible spring attached to the harness carried the catheters within it and the connecting lead to the Doppler flowmeter (Crystal Biotech VFI sytem) was taped to the spring to support it; the spring was attached to a counter-balanced, universally-jointed, lever system. At least 24 h later, continuous recordings were started (on a Gould ES1000 system) of phasic and mean blood pressures, of instantaneous heart rate and of phasic and mean Doppler shift signals from the renal, mesenteric and hindquarters probes. Under the conditions of our experiments, percentage changes in the Doppler shift signals are a good index of volume flow changes (Haywood et al., 1981; Gardiner et al., 1988) and together with mean blood pressure (BP) can be used to calculate percentage changes in vascular resistances. Experiments were run on two consecutive days. Long Evans (n = 8) and Brattleboro (n = 8) rats were randomised to receive either endothelin-1 or endothelin-3 on the first day. Each peptide was given as a bolus injection at a low dose (4 pmol in 0.1 ml) followed, at least 60 min later, by a 60 min infusion (12 pmol h-1 in 0.3 ml). At least 1 h after the end of
the infusion, a bolus injection (40 pmol in 0.1 ml) was given; the higher dose infusion (120pmolh-' in 0.3ml) was not started until at least 90 min later. In order to assess the effects of bolus injections, measurements were made 0.5, 1, 5 and o min after injection; with the higher dose, measurements were made also at 20 min. When peptides were given by infusion, measurements were made at 5, 10, 20, 30, 40, 50 and 60 min during infusion and at the same time points after the infusion had been stopped. Bolus injections or infusions of the vehicle had no systematic effects.
Peptides Both peptides were obtained from the Peptide Institute Inc. (European Distributor: Scientific Marketing Associates, London) and were reconstituted in sterile isotonic saline containing 1% bovine serum albumin.
Data analysis Data were subjected to non-parametric, two-way analysis of variance (Friedman's test) to detect changes relative to baseline. Comparisons between groups were made by use of Wilcoxon's rank sum test or the Mann-Whitney U test, as appropriate. Comparisons between Doppler shift changes with endothelin-1 and endothelin-3 were not analysed statistically since mean blood pressure changes were often different with the two peptides, and hence the resistance changes were the variables of importance.
Results
Regional haemodynamic effects of endothelin-J and endothelin-3 in Long Evans rats The lower bolus dose (4pmol) of endothelin-1 had no significant effects on heart rate although there was a small transient increase in mean BP (Table 1). However, there were significant reductions in renal and mesenteric blood flows accompanying an initial hindquarters hyperaemia (Table 1), indicating the occurrence of renal and mesenteric vasoconstrictions and hindquarters vasodilatation (Table 1). The dissociation between systemic BP and regional haemodynamic changes was even more apparent following the lower dose of endothelin-3, since there was no change in mean BP although there were significant regional haemodynamic effects (Table 1). The renal vasoconstrictor effect of endothelin-1 was greater than that of endothelin-3 (Table 1).
Table 1 Cardiovascular changes following i.v. bolus injections (4pmol) of endothelin-l (ET-1) or endothelin-3 (ET-3) in conscious, Long Evans rats Time after injection (min) 1.0
5.0
10.0
Peptide Et-3
Et-l Heart rate (beats-min -1) Mean BP (mmHg) Doppler shift (%) Renal Mesenteric Hindquarters Vascular resistance (%) Renal Mesenteric Hindquarters
Et-l
Et-]
Et-3
Et-]
-4+7 0+ 1
12+ 11 0+2
-9+8
-10 + 3* -5 + 2 -2 + 3
-5 + 1* 1+3 -3 + 6
-5 + 3 0+ 1 -6 +4
-2 + 2 0+4 0+7
13 + 4* 6+2 3+4
5 + 2* 0+4 6+ 8
7+4 1+3 8+6
2+2 2+4 5 8
21 + 12 3+4
19 + 11 5 5
-9+ 14 6+2*
4+8 3+2
-7 + 3* -31 + 2* 32 + 7*
-3 + 2 -20 ± 5* 27 + 7*
-15 + 3* -14 + 2* 0+5
-5 + 2 -11 + 3 8+ 5
12 + 4* 50 9* -20 +5*
9+7 36 + 14* -17 + 4*
26 + 4* 24 + 5* 8+5
Values are given as mean ± s.e.mean (n = 8). * P < 0.05 versus baseline (Friedman's test). t
Et-3
P < 0.05 endothelin-1 versus endothelin-3 (Wilcoxon's test).
8 + 3*t 16 + 4* -3 +6
0+3
Et-3 3+ 15 1 +2
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The higher bolus dose (40 pmol) of endothelin-1 caused an initial tachycardia, hypotension and falls in renal and mesenteric blood flows, together with hindquarters hyperaemia (Table 2). However, only the initial change in hindquarters vascular resistance was significant (Table 2). Thereafter, mean BP increased and there was a bradycardia, in association with both reductions in renal and mesenteric blood flows (Table 2) and increases in resistance in all three vascular beds (Table 2). Endothelin-3 caused initial hypotension and tachycardia similar to those seen with endothelin-1 (Table 2), although the former peptide caused early renal, as well as hindquarters, vasodilatations (Table 2). Subsequently the pressor and renal and mesenteric vasoconstrictor effects of endothelin-3 were less than those of endothelin-1, and there was no increase in hindquarters vascular resistance following endothelin-3 administration (Table 2). Infusion, at the lower rate (12pmolh-1), of endothelin-1 had a slight pressor effect (+7 + 2 mmHg, 20 min into infusion), accompanied by increases in renal and mesenteric vascular resistances (maximum change + 16 + 3% and + 13 + 4%, respectively). Infusion of the same dose of endothelin-3 did not affect mean BP and increased only mesenteric vascular resistance (maximum change + 14 + 3%). Endothelin-1, infused at a rate of 120 pmol h1, caused bradycardia and a progressive increase in mean BP (Table 3) associated with marked reductions in renal and mesenteric blood flows, but only a slight reduction in hindquarters flow (Table 3). These changes were associated with vasoconstriction in all 3 vascular beds (Table 3). After the infusion was stopped the vasoconstrictions reversed more rapidly than the pressor effect (Table 3). Infusion of endothelin-3 had a modest pressor effect that was not associated with a significant bradycardia (Table 3). There were renal and mesenteric vasoconstrictions, but no change in hindquarters vascular resistance (Table 3). All changes reversed within IOmin of the end of the infusion (Table 3). The pressor and vasoconstrictor effects of endothelin-l infusion were greater than those of endothelin-3 infusion (Table 3).
Regional haemodynamic effects of endothelin-1 and endothelin-3 in Brattleboro rats The qualitative responses to the lower bolus doses (4pmol) of endothelin-l and endothelin-3 in Brattleboro rats (Table 4) were similar to those in Long Evans rats (Table 1), although
the mesenteric vasoconstrictor effect of endothelin-1 was less prolonged in Long Evans (Table 1) than in Brattleboro rats (Table 4). Furthermore, the renal vasoconstrictor effects of endothelin-l and endothelin-3 were similar in the latter strain
(Table 4), whereas endothelin-3 had a less marked effect than endothelin-1 in Long Evans rats (Table 1). The higher bolus doses (40pmol) of endothelin-l and endothelin-3 caused initial hypotension and tachycardia (Table 5), accompanied by reductions in renal (endothelin-1 only) and mesenteric flows and an increase in hindquarters flow (Table 5); for both peptides, the change in hindquarters vascular resistance was significant but the initial increase in mesenteric vascular resistance was significant for endothelin-3 only (Table 5). Thereafter, there were bradycardias and increases in mean BP. The pressor effect of endothelin-1 was greater than that of endothelin-3 (Table 5), as were the renal and hindquarters vasoconstrictor effects (Table 5), although the increases in mesenteric vascular resistance were similar with both peptides (Table 5), unlike the situation in Long Evans rats (Table 2). Infusion of endothelin-1 at the lower rate (12 pmol h') had no significant effects on heart rate or mean BP but caused significant renal and mesenteric vasoconstrictions (+ 15 + 5% and + 17 ± 5%, respectively; maximum changes). Infusion of endothelin-3 caused mesenteric vasoconstriction only (maximum change + 23 + 3%). Endothelin-1 when infused at a rate of 120 pmol h-, caused progressive bradycardia and hypertension accompanied by reductions in renal and mesenteric blood flows (Table 6). There were vasoconstrictions in the renal mesenteric and hindquarters vascular beds (Table 6). All cardiovascular effects of endothelin-1 had reversed by 40min after the infusion (Table 6). This picture was not different from that seen in Long Evans rats (Table 3). Infusion of endothelin-3 had significantly smaller pressor and vasoconstrictor effects than endothelin-1 (Table 6), and the pattern of differences in regional haemodynamics was similar to that seen in Long Evans rats (Table 3).
Discussion The present work shows that bolus administration of a high dose of endothelin-1 or endothelin-3 causes similar falls in mean BP and similar hindquarters vasodilatations, and the effects are not different in Long Evans and Brattleboro rats, indicating that the haemodynamic profile is not overtly influenced by cardiovascular actions of endogenous vasopressin in the former strain. These findings do not support the proposition that endothelin-3 is a more potent hypotensive (Inoue et al., 1989) or vasodilator (Warner et al., 1989a) agent than endothelin-1, at least under the conditions of our experiments. The only vascular bed in which endothelin-3 administration was followed by a fall in vascular resistance not seen with
Table 4 Cardiovascular changes following i.v. bolus injection (4pmol) of endothelin-l (Et-1) or endothelin-3 (Et-3) in conscious, Brattleboro rats Time after injection (min) 1.0 5.0
0.5
10.0
Peptide Heart rate (beats min -1) Mean BP (mmHg) Doppler shift (%) Renal Mesenteric
Et-l
Et-3
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-9 + 2* 10 + 4 7 +5
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-5 ±4 6i6 3i5
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10 + 8 33 + 7* -16 + 5*
21 + 5* 33 + 9* -10 + 5
16 + 6* 17 + 5* 0+ 7
10 + 4* 14 + 3* 4+6
7+4 -1 + 5 0+ 7
1 +3 12 + 4* -5+5
5 +4 -6+4 -4+ 5
Values are mean + s.e.mean (n = 8). * P < 0.05 versus baseline (Friedman's test).
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S.M. GARDNER et al.
endothelin-1 was the kidney, and only in Long Evans rats (Table 2); in that situation renal blood flow was not increased, so it is feasible the vasodilatation was an autoregulatory phenomenon. It is clear from the present work with the lower bolus dose that the regional haemodynamic effects of endothelin-1 or -3 can be substantial when there is little change in mean BP. Furthermore, the hindquarters vasodilator effects of these peptides are not necessarily associated with hypotension, and are not seen when the peptides are infused. While it is feasible that bolus doses of endothelin-1 or endothelin-3 cause hindquarters vasodilatation by releasing eicosanoids or endothelium-derived relaxing factor (De Nucci et al., 1989; Warner et al., 1989b), we have been unable to block this effect with either N0-monomethy-L-arginine, an inhibitor of nitric oxide synthesis by endothelial cells (Gardiner et al., 1989c), or with indomethacin (Gardiner, S.M., Compton, A.M. & Bennett, T., unpublished observations). Indeed, recent findings indicate that indomethacin reduces the contractile effects of endothelin-I on rat aorta (Eglen et al., 1989). In addition, it seems unlikely that the hindquarters hyperaemic responses to bolus doses of endothelin-1 are dependent upon release of atrial natriuretic peptide (Winquist et al., 1989), since bolus doses of this peptide do not cause significant increases in hindquarters blood flow in Brattleboro rats (Gardiner et al., 1988). The results with the higher rate of infusion of endothelin-1
demonstrate that this peptide has marked vasoconstrictor effects in mesenteric, renal and hindquarters vascular beds, the ranking of effect descending in that order. Overall, there was a striking consistency of effect of endothelin-1 in Long Evans and Brattleboro rats, as there was for endothelin-3. In both strains the latter peptide had significantly less pressor and vasoconstrictor action than endothelin-l. While it is possible that endothelin-3 is a more potent activator of dilator mechanisms that oppose its vasoconstrictor action, the similarity of effect of the two peptides in causing initial hindquarters vasodilatation raises the possibility that endothelin-3 is less effective at activating vasoconstrictor mechanisms than endothelin-1 (Warner et al., 1989b). Interestingly, the relative lack of vasoconstrictor effects of endothelin-3 was most marked in the hindquarters, since under no circumstances did endothelin-3 cause a significant increase in resistance in this vascular bed, whereas the higher bolus dose and the higher rate of infusion of endothelin-1 did. Although endothelin-1 had strong vasoconstrictor effects, even following a 60 min infusion of a high dose of this peptide all the haemodynamic effects had disappeared within 40 min. Hence, the relative irreversibility of the effects of this peptide in vitro, and in anaesthetized animals (Yanagisawa et al., 1988a) does not reflect the situation in the intact, conscious rat.
References DE NUCCI, G., THOMAS, R., D'ORLEANS-JUSTE, P., ANTUNES, E.,
WALDER, C., WARNER, T.D. & VANE, J.R. (1988). Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endotheliumderived relaxing factor. Proc. Natl. Acad. Sci. U.S.A., 85, 97979800. EGLEN, R.M., MICHEL, A.D., SHARIF, N.A., SWANK, S.R. & WHITING, R.L. (1989). The pharmacological properties of the peptide, endothelin. Br. J. Pharmacol., 97, 1297-1307. GARDINER, S.M., COMPTON, A.M. & BENNETT, T. (1988). Regional haemodynamic effects of depressor neuropeptides in conscious, unrestrained, Long Evans and Brattleboro rats. Br. J. Pharmacol., 95, 197-208. GARDINER, S.M., COMPTON, A.M. & BENNETT, T. (1989a). Regional haemodynamic effects of endothelin-1 in conscious, unrestrained, Wistar rats. J. Cardiovasc. Pharmacol., 13, suppl. 5, S202-S204. GARDINER, S.M., COMPTON, A.M. & BENNETT, T. (1989b). Haemodynamic effects of endothelin-1 and endothelin-3 in conscious Long Evans rats. Br. J. Pharmacol., 98, 624P. GARDINER, S.M., COMPTON, A.M., BENNETT, T., PALMER, R.M.J. &
MONCADA, S. (1989c). The effect of NG-monomethyl-L-arginine (L-NMMA) on the haemodynamic actions of endothelin-1 in conscious Long Evans rats. Br. J. Pharmacol., 98, 623P. HAYWOOD, J.R., SHAFFER, R.A., FASTENOW, C., FINK, G.D. &
BRODY, M.J. (1981). Regional blood flow measurement with pulsed Doppler flowmeter in conscious rat. Am. J. Physiol., 241, H273H278. INOUE, A., YANAGISAWA, M., KIMURA, S., KASUYA, Y., MIYAUCHI,
T., GOTO, K. & MASAKI, T. (1989). The human endothelin family: three structurally and pharmacologically distinct isopeptides predicted by three separate genes. Proc. Nat!. Acad. Sci. U.S.A., 86, 2863-2867. MINKES, R.K., COY, D.H., MURPHY, W.A., McNAMARA, D.B. & KADO-
WITZ, P.J. (1989). Effects of porcine and rat endothelin and an
analog on blood pressure in the anaesthetized cat. Eur. J. Pharmacol., 164, 571-575. MINKES, R.K. & KADOWITZ, P.J. (1989). Differential effects of rat endothelin on regional blood flow in the cat. Eur. J. Pharmacol., 165, 161-164. SPOKES, R.A., GHATEI, M.A. & BLOOM, S.R. (1989). Studies with endothelin-3 and endothelin-1 on rat blood pressure and isolated tissues: evidence for multiple endothelin receptor subtypes. J. Cardiovasc. Pharmacol., 13, suppl. 5, S191-S192. WARNER, T.D., DE NUCCI, G. & VANE, J.R. (1989a). Rat endothelin is a vasodilator in the isolated perfused mesentery of the rat. Eur. J. Pharmacol., 159, 325-326. WARNER, T.D., MITCHELL, J.A., DE NUCCI, G. & VANE, J.R (1989b). Endothelin-1 and endothelin-3 release EDRF from isolated perfused arterial vessels of the rat and rabbit. J. Cardiovasc. Pharmacol., 13, suppl. 5, S85-S88. WINQUIST, R.J., SCOTT, A.L. & VLASUK, G.P. (1989). Enhanced release of atrial natriuretic factor by endothelin in atria from hypertensive rats. Hypertension, 14, 111-114. WRIGHT, C.E. & FOZARD, J.R. (1988). Regional vasodilation is a prominent feature of the haemodynamic response to endothelin in anesthetized spontaneously hypertensive rats. Eur. J. Pharmacol., 155, 201-203. YANAGISAWA, M., INOUE, A., ISHIKAWA, T., KASUYA, Y, KIMURA, S., KUMAGAYE, S-I., NAKAJIMA, K., WATANABE, TX., SAKIBARA, S.,
GOTO, K. & MASAKI, T. (1988b). Primary structure, synthesis, and biological activity of rat endothelin, and endothelium-derived vasoconstrictor peptide. Proc. Nati. Acad. Sci. U.S.A., 85, 69646967. YANAGISAWA, M., KURIHARA, H., KIMURA, S., TOMOBE, Y., KOBAYASHI, M., MITSUI, Y., YAZAKI, Y., GOTO, K. & MASAKI, T. (1988a).
A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature, 332, 411-415.
(Received August 8, 1989 Accepted September 13, 1989)
