Br. J. Pharmacol. (1990), 100, 107-113
C) Macmillan Press Ltd, 1990
Differences in regional vascular sensitivity to endothelin- 1 between spontaneously hypertensive and normotensive Wistar-Kyoto rats 'Christine E. Wright & 2John R. Fozard Preclinical Research, Sandoz Pharma A.G., CH 4002 Basel, Switzerland 1 The systemic and regional haemodynamic effects of porcine endothelin-1 (endothelin) have been measured in anaesthetized spontaneously hypertensive (SH) rats rendered areflexic by ganglion blockade; comparisons were made with age-matched Wistar-Kyoto (WKY) control animals. 2 In both SH and WKY rats endothelin (0.1-1 nmol kg-1 i.v.) elicited an initial, short-lived (
L.I
IF *\;
0
E 50
-0 V 0
//
/i
u
a
L
0
-100L
:--
9 8 -log [Et] (M)
10
01
5
10 20 40 60
Time (min)
Et 1
10204060
5
01
Time (min)
Et 1
Figure 4 Effects of endothelin 1 nmol kg'- i.v. bolus (Et 1) on circulatory variables in WKY (a, n = 16) and SH (b, n = 16) rats. For key to variables see legend to Figure 2.
strain. With 0.3 nmol kg- of the peptide, renal vascular conductance fell within 30s by 19.5 + 6.7 and 15.0 + 9.0% in SH and WKY rats, respectively. The highest dose of endothelin, 1 nmol kg1- , resulted in initial falls in renal vascular conductance of 82.3 + 4.6 and 75.9 + 4.7% in SH and WKY rats, respectively. This renal vasoconstriction was maintained for more than 20 min in both rat strains, with conductance values gradually returning to resting baseline over 60-80min (Figure 4). Mesenteric vascular conductance fell significantly with endothelin 0.1-1 nmol kg-' in both SH and WKY rats. Mean values in SH and WKY rats respectively were: 0.1 nmol kg 1, -16.9 + 9.5 and -35.8 + 5.8; 0.3 nmol kg- , - 24.1 + 10.0 and -41.5 + 7.4; and lnmolkg-1, -26.1 + 16.7 and -65.9 + 6.8%. The vasoconstriction at the latter dose was significantly greater in the WKY rats. Similar to the renal vascular response, the mesenteric vasoconstriction was of long
a 300 r
b A
a) 0 C
co
0
2200
V
A/
)i
0
_0))
,100
0
20
Co
u
K
-V
v
0.01 a)
0.1
03
1
1
3
10
d
c
Pre 8 7 6 5 -log [ACh] (M)
10
9
8
-log [Et] (M)
Pre 8 7 6 5
-log [ACh] (M)
Figure 6 Contractile effects of endothelin (Et) on aortae taken from SH rats and WKY control animals; effects of endothelial removal. (a) Sample tracing (data from SH rat) to illustrate the experimental design. A cumulative dose-response curve to endothelin was established and the results expressed in terms of a supramaximal response to a prior injection of phenylephrine (PE). Twenty minutes after addition of the highest dose of endothelin (3 x 10-8M), a cumulative doseresponse curve to acetylcholine (ACh) or the vehicle for ACh (saline; not illustrated) was established. (b and c) Mean data from a series of such experiments comparing tissues from WKY (b) and SH (c) rats. (U) Intact aortae; (EJ) aortae denuded of endothelium; (0) and (0) indicate effects of the vehicle for acetylcholine (saline). n = 10 for endothelin concentration-response curves; 6 for acetylcholine concentration-response curves and 4 for effects of saline. The mean responses (mg; in each case, n = 10) to phenylephrine, 10- m, were: WKY, intact aorta 2826 + 175; WKY, aorta denuded of endothelium 2924 + 163; SHR, intact aorta 2084 + 147; SHR, aorta denuded of endothelium 2333 + 169.
duration and returned to pre-endothelin values only after approximately 60 min (Figure 4).
Effects of acetylcholine and sodium nitroprusside Bolus of acetylcholine (0.01-1 gg kg 1, injections endothelium-dependent vasodilator) or nitroprusside (0.31Ogg kg-1, endothelium-independent vasodilator) revealed no marked differences between SH and WKY rats with respect to the vasodilatation in the hindquarter bed (Figure 5). Similarly, in the carotid vascular bed there was no significant difference between rat strains in the acetylcholine-induced dilatation. However, with nitroprusside, the highest dose elicited a greater increase in carotid vascular conductance in SH than in WKY rats. The effects of both dilator agents on blood pressure were similar showing no difference between rat groups. Acetylcholine lgkg-t caused changes of -33.4 + 2.9 and -26.0 + 3.6 mmHg, and nitroprusside lOpgkg- elicited falls of -34.6 + 2.8 and -30.7 + 2.1 mmHg in SH and WKY rats respectively.
0
Effects of angiotensin II
ICo c;
V
-0 0
0~
CT
Kr
0.01
0. l
0.3
l
3
lo0
Acetylcholine Nitroprusside (jig kg-') (vhg kg-1) Figure 5 Effects of acetylcholine (a,c) and nitroprusside (b,d) i.v. bolus injections on carotid (a,b) and hindquarter (Hq) (c,d) vascular conductances in SH (A, n 8) and WKY rats (0, n = 8). Error bars are average s.e.mean (see Methods). =
Angiotensin 11 (0.01-0.1lugkg-') administration resulted in greater increases in blood pressure in SH compared with WKY rats. Average increases in SH and WKY rats respectively were: 0.01 jIgkg-, 21 + 2 and 13 + 1; 0.03pgkg-1, 34 + 3 and 20 + 2; and 0. Igkg-1, 53 + 4 and 36 + 3 mmHg. However, there were no significant differences between rat strains in the decreases in renal and mesenteric vascular conductances. For instance, decreases in renal vascular conductance with angiotensin II injections ranged from -20 + 4 with the lowest dose to -79 + 7% with the highest dose in SH corresponding with values in WKY rats of -27 + 5 and -73 + 8%.
REGIONAL VASCULAR SENSITIVITY TO ENDOTHELIN
Effects of indomethacin In a number of additional SH rats, prepared identically to the main group, indomethacin 5 mg kg1- was administered either i.p. 60 min before endothelin injection (n= 5), or as an i.v. bolus 10min before endothelin nmol kg' (n = 1). Neither route of indomethacin administration appeared to have any effect on the biphasic response to endothelin (1 nmol kg 1) in these animals (Figure 1).
Sensitivity of aortae with and without endothelium to endothelin: comparison between SH rats and WK Y controls Endothelin, 10- 103 10-8 M, induced slowly developing and sustained contraction of aortic rings with or without intact endothelium from both SH rats and WKY control animals (Figure 6). Removal of the endothelium from rings taken from WKY rats enhanced to a small extent, but significantly (P < 0.05), the sensitivity to endothelin, as indicated by pEC50 (M) values of 8.47 + 0.05 and 8.71 + 0.10 (in both cases n = 10) for the rings with and without endothelium, respectively (Figure 6b). Rings from SH rats with intact endothelium were also significantly (P < 0.005) less responsive to endothelin than denuded tissues, as indicated by pEC50 (M) values of 8.42 + 0.07 and 8.82 + 0.09 respectively (in both cases n = 10; Figure 6c). Acetylcholine did not relax tissues contracted with endothelin in the absence of endothelium; in intact tissues, acetylcholine relaxed tissues from both SH rats and WKY controls (Figure 6b and c). The pIC50 concentrations (M; calculated taking the response to 10- 5M acetylcholine as maximum) were not significantly different (7.16 + 0.08 and 7.03 + 0.17 respectively; in each case, n = 6) although the maximum inhibition was somewhat greater in the tissues from SH than those from WKY rats (Figure 6b and c). x
Discussion The present results emphasize the complexity of the cardiovascular effects of endothelin which is increasingly being documented in a number of species (see Introduction). However, unlike the majority of studies published to date, the present results were obtained in animals rendered areflexic by administration of the ganglion blocking agent, mecamylamine. This allows interpretation of the effects of endothelin uncomplicated by intact autonomic cardiovascular reflexes. This is particularly important when comparing cardiovascular responses of SH rats with other rat strains because of the alterations in reflex buffering capacity well known to occur in hypertension (see, e.g. Wright et al., 1987). Under our experimental conditions, the effects of endothelin were qualitatively similar in SH and WKY rats. The initial short-lived falls in blood pressure were associated with similarly short-lived vasodilator responses in both carotid and hindquarter vascular beds and it seems likely that the latter are the basis of the former. The poor correlation between blood pressure fall and the conductance changes in the carotid and hindquarters vascular beds almost certainly reflects, to a large extent, the complexity of action of endothelin and, in particular, the differential contribution of the vasoconstrictor component at the different dose levels. However, since information on conductance changes is available for only four vascular beds, precise explanation of the poor correlation is not possible. The lack of any associated tachycardia (clearly seen in SH rats with normal reflexes-Winquist et al., 1989a) testifies to the adequacy of ganglion blockade and rules out the possibility that the vasodilatation results from modification of on-going autonomic tone and, in particular, the prejunctional inhibition of sympathetic neurotransmitter release (Wiklund et al., 1988).
111
The sustained rise in blood pressure which superseded the initial fall was associated with vasoconstriction, particularly in the renal and mesenteric vasculature but also, once the vasodilatation had waned, in the carotid and hindquarters beds. The intense, long-lasting renal vasoconstriction at the higher doses is particularly noteworthy. The same phenomenon has been observed in several species (Cocks et al., 1989; Minkes & Kadowitz, 1989) and in vitro (Firth et al., 1988; Cairns et al., 1988) and is associated with a marked decline in renal function (Lopez-Farr6 et al., 1989). On this basis endothelin has been implicated as a mediator in the pathogenesis of acute renal failure (Firth et al., 1988). Despite qualitative similarities, the effects of endothelin differed quantitatively in SH compared to WKY rats. In particular, both the initial falls in MAP and the associated vasodilator responses of the hindquarter and carotid vascular beds were greater in SH than WKY rats. Although the differences may in part reflect differences in baseline values between the two strains (MAP was significantly higher, hindquarter conductance was significantly lower in SH compared to WKY rats-Table 1), this seems unlikely to be the sole explanation for our observations. Thus, the greatest differences were seen in the carotid vascular bed where baseline values did not differ significantly between the two strains. Moreover, neither the vasodilator responses to an endothelium-dependent vasodilator, acetylcholine, nor those to the directly acting agent, sodium nitroprusside, differed markedly in SH compared to WKY rats (Figure 5). Thus the vasodilator component of the cardiovascular response to endothelin appears to be increased selectively in the spontaneously hypertensive state. The mechanism of the vasodilator response to endothelin in vivo has not been established, but it seems unlikely to reflect a direct action on vascular smooth muscle. Thus, no direct relaxation of arterial segments from a variety of species has ever been demonstrated (Cocks et al., 1989; Eglen et al., 1989; Saito et al., 1989). On the other hand, endothelin has been shown to dilate the rat perfused mesenteric vascular bed when tone was induced by methoxamine (Warner et al., 1989a,b; Randall et al., 1989) and to release the vasodilator eicosanoid, prostacyclin, from rat perfused lungs in vitro (de Nucci et al., 1988). In the perfused mesenteric vascular bed, vasodilatation could be inhibited by removal of the endothelium, methylene blue or haemoglobin indicating indirect effects of endothelin mediated by the release of endothelium-derived releasing factor(s), such as EDRF (Warner et al., 1989a,b; Randall et al., 1989). However, other experiments have failed to confirm the vasodilator effects of endothelin in the rat isolated perfused mesenteric arterial bed (Eglen et al., 1989; Tabuchi et al., 1989), an observation which would be consistent with the present data showing endothelin to induce exclusively vasoconstrictor responses in the mesenteric vascular bed in vivo (Figures 1-4). Moreover, Gardiner et al. (1989b) were not able to block endothelin-induced vasodilatation in conscious normotensive rats by administration of NG-monomethyl-L-arginine (L-NMMA) at a dose adequate to increase peripheral vascular resistance by suppressing the generation of a major component of EDRF, endothelium-derived nitric oxide (EDNO) (Gardiner et al., 1989c; see also Whittle et al., 1989). Finally, the present data showing the cardiovascular response to endothelin in the SH rat to be unaffected by pretreatment with indomethacin (Figure 1) render it unlikely that prostacyclin is playing a major role in the vasodilator response in vivo, at least under the conditions of the present experiment. Despite this, but in confirmation of the data of Eglen et al. (1989) and Godfraind et al. (1989), the endothelium does modulate the vasoconstrictor response to endothelin in rat isolated aorta (Figure 6). Intriguingly, in our experiments a somewhat greater increase in vasoconstrictor sensitivity due to endothelium removal was seen in tissues from SH rats (Figure 6), which could conceptually be used to support a greater role for endothelium-mediated vasodilator responses as the basis of the differential effects seen in SH and WKY rats in vivo. However, the in vitro effect most likely reflects not
112
C.E. WRIGHT & J.R. FOZARD
the release by endothelin of EDRF but physiological antagonism of endothelin by a basal EDRF-mediated vasodilator tone, since similar effects are manifested against a variety of other vasoconstrictor agents (Martin et al., 1986; Alosachie & Godfraind, 1988). In seeking alternative explanations for the mechanism of the vasodilator action of endothelin, one obvious possibility is the release of an EDRF other than EDNO (see e.g. Taylor & Weston, 1989). A further possibility is the release of atrial natriuretic peptide (ANP). Such a release has been demon-
strated both from cultured neonatal rat atrial cardiocytes (Fukuda et al, 1988) and adult rat atria in vitro, where release was greater in tissues taken from SH rats than WKY controls (Winquist et al., 1989b). However, unlike endothelin, the fall in blood pressure induced by the acute administration of ANP in the rat is not generally associated with a decrease in systemic vascular resistance (Lappe et al., 1985; Waeber et al., 1986; Gardiner et al., 1990). Clearly the precise mechanism of the vasodilator action of endothelin awaits further elucidation.
References ALOSACHIE, I. & GODFRAIND, T. (1988). The modulatory role of vascular endothelium in the interaction of agonists and antagonists with a-adrenoceptors in the rat aorta. Br. J. Pharmacol., 95, 619-629.
LIPPTON, H., GOFF, J. & HYMAN, A. (1988). Effects of endothelin in the systemic and renal vascular beds in vivo. Eur. J. Pharmacol., 155, 197-199.
CAIRNS, S.L., ROGERSON, M., FAIRBANKS, L.D., WESTWICK, J. &
(1989). Effect of endothelin on renal function in rats. Eur. J. Pharmacol., 163, 187-189.
NEILD, G.H. (1988). Endothelin and cyclosporin nephrotoxicity. Lancet, i, 1496-1497. COCKS, T.M., BROUGHTON, A., DIB, M., SUDHIR, K. & ANGUS, J.A.
(1989). Endothelin is blood vessel selective: studies on a variety of human and dog vessels in vitro and on regional blood flow in the conscious rabbit. Clin. Exp. Pharmacol. Physiol., 16, 243-246.
LOPEZ-FARRE, A., MONTANES, I., MILLAS & LOPEZ-NOVOA, J.M. MARTIN, W., FURCHGOTT, R.F., WILLANI, G.M. & JOTHIANDANAN,
D. (1989). Depression of contractile responses by spontaneously released endothelium-derived relaxing factor. J. Pharmacol. Exp. Ther., 237, 538-599. MINKES, R.K., COY, D.H., MURPHY, W.A., McNAMARA, D.B. & KADO-
R.L. (1989). The pharmacological properties of the peptide endothelin. Br. J. Pharmacol., 97, 1297-1307.
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. RANDALL, M.D., DOUGLAS, S.A. & HILEY, C.R. (1989). Vascular activities of endothelin-1 and some alanyl substituted analogues in resistance beds of the rat. Br. J. Pharmacol., 98, 685-699.
FIRTH, J.D., RATCLIFFE, P.J., RAINE, A.E.G. & LEDINGHAM, J.G.G.
SAITO, A., SHIBA, R., KIMURA, S., YANAGISAWA, M., GOTO, K. &
(1988). Endothelin: an important factor in acute renal failure? Lancet, i, 179-1181.
MASAKI, T. (1989). Vasoconstrictor response of large cerebral arteries of cats to endothelin, and endothelium-derived vasoactive peptide. Eur. J. Pharmacol., 162, 353-358.
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,
FUKUDA, Y., HIRATA, Y., YOSHIMA, H., KOJIMA, T., KOBAYASHI, Y.,
YANAGISAWA, M. & MASAKI, T. (1988). Endothelin is a potent secretogogue for atrial natriuretic peptide in cultured rat atrial myocytes. Biochem. Biophys. Res. Commun., 155, 167-172. GARDINER, S.M., COMPTON, A.M. & BENNETT, T. (1989a). Regional haemodynamic effects of endothelin-1 in conscious, unrestrained, Wistar rats. J. Cardiovasc. Pharmacol., 13, S202-S204. GARDINER, S.M., COMPTON, A.M., BENNETT, T., PALMER, R.M.J. &
MONCADA, S. (1989b). The effects of NG-monomethyl-L-arginine (L-NMMA) on the haemodynamic actions of endothelin-1 in conscious Long-Evans rats. Br. J. Pharmacol., 98, 626P. GARDINER, S.M., COMPTON, A.M., BENNETT, T., PALMER, R.M.J. &
MONCADA, S. (1989c). Haemodynamic effects of N0-monomethylL-arginine (L-NMMA) in conscious Long-Evans rats. Br. J. Pharmacol., 98, 623P. GARDINER, S.M., COMPTON, A.M. & BENNETT, T. (1990). Regional haemodynamic effects of endothelin-1 and endothelin-3 in conscious Long Evans and Brattleboro rats. Br. J. Pharmacol., 99, 107-112. GIVEN, M.B., LOWE, R.F., LIPPTON, H., HYMAN, A.L., SANDER, G.E. & GILES, T.D. (1989). (Hemodynamic actions of endothelin in con-
scious and anesthetized dogs. Peptides, 10, 41-44. GODFRAIND, T., MENNING, D., MOREL, N. & WIBO, M. (1989). Effect of endothelin-1 on calcium channel gating by agonists in vascular smooth muscle. J. Cardiovasc. Pharmacol., 13, SI 12-S1 17. HAN, S.P., TRAPANI, A.J., FOK, K.F., WESTFALL, T.C. & KNUEPFER,
M.M. (1989). Effects of endothelin on regional hemodynamics in conscious rats. Eur. J. Pharmacol., 159, 303-305. 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. KITAYOSHI, T., WATANABE, T. & SHIMAMOTO, N. (1989). Cardiovascular effects of endothelin in dogs: positive inotropic action in vivo. Eur. J. Pharmacol., 166, 519-522. LAPPE, R.W., SMITS, J.F.M., TODT, J.A., DEBETS, J.J.M. & WENDT, R.L. (1985). Failure of atriopeptin II to cause arterial vasodilation in
the conscious rat. Circ. Res., 56, 606-612.
I., SUMMER, W.R. & HYMAN, A.L. (1989). Endothelin-a new family of endothelium-derived peptides with widespread biological properties. Life Sci., 45, 1499-1513.
LE MONNIER DE COUVILLE, A.-C., LIPPTON, H.L., CAVERO,
TABUCHI, Y., NAKAMARU, M., RAKUGI, H., NAGANO, M. &
OGIHARA, T. (1989). Endothelin enhances adrenergic vasoconstriction in perfused rat mesenteric arteries. Biochem. Biophys. Res. Commun., 159,1304-1308. TAYLOR, S.G. & WESTON, A.H. (1989). Endothelium-derived hyperpolarizing factor: a new endogenous inhibitor from the vascular endothelium. Trends Pharmacol. Sci., 9, 272-274. TOMOBE, Y., MIYAUCHI, T., SAITO, A., YANAGISAWA, M., KIMURA,
S., GOTO, K. & MASAKI, T. (1988). Effects of endothelin on the renal artery from spontaneously hypertensive and Wistar Kyoto rats. Eur. J. Pharmacol., 152, 373-374. TSUCHIYA, K., NARUSE, M., SANAKA, T., NARUSE, K., NITTA, K.,
DEMURA, H. & SUGINO, M. (1989). Effects of endothelin on regional blood flow in dogs. Eur. J. Pharmacol., 166, 541-543. WAEBER, B., MATSUEDA, G.R., AUBERT, J.-F., NUSSBAUMER, J. &
BRUNNER, H.R. (1986). The haemodynamic response of conscious normotensive rats to atriopeptin III: lack of a role of the parasympathetic nervous system. Eur. J. Pharmacol., 125, 177-184. WARNER, T.D., MITCHELL, J.A., DE NUCCI, G. & VANE, J.R. (1989a). Endothelin-1 and endothelin-3 release EDRF from isolated perfused arterial vessels of the rat and rabbit. J. Cardiovasc. Pharmacol., 13, S85-S88. WARNER, T.D., DE NUCCI, G. & VANE, J.R. (1989b). Rat endothelin is a vasodilator in the isolated perfused mesentery of the rat. Eur. J. Pharmacol., 159, 325-326. WHITTLE, B.J.R., LOPEZ-BELMONTE, J. & REES, D.D. (1989). Modulation of the vasodepressor actions of acetylcholine, substance P and endothelin in the rat by a specific inhibitor of nitric oxide formation. Br. J. Pharmacol., 98, 646-652. WIKLUND, N.P., OHLEN, A. & CEDERQUIST, B. (1988). Inhibition of adrenergic neuroeffector transmission by endothelin in the guineapig femoral artery. Acta Physiol. Scand., 134, 311-312. WINQUIST, R.J., BUNTING, P.B., GARSKY, V.M., LUMMA, P.K. & SCHOFIELD, T.L. (1989a). Prominent depressor response to endothelin in spontaneously hypertensive rats. Eur. J. Pharmacol., 163, 199-203. WINQUIST, R.J., SCOTT, A.L. & VLASUK, G.P. (1989b). Enhanced release of atrial natriuretic factor by endothelin in atria from hypertensive rats. Hypertension, 14, 111-114. WRIGHT, C.E., ANGUS, J.A. & KORNER, P. (1987). Vascular amplifier properties in renovascular hypertension in conscious rabbits: and
REGIONAL VASCULAR SENSITIVITY TO ENDOTHELIN hindquarter responses to constrictor and dilator stimuli. Hypertension, 9, 122-131. WRIGHT, C.E. & FOZARD, J.R. (1988). Regional vasodilation is a prominent feature of the haemodynamic response to endothelin in anaesthetized, spontaneously hypertensive rats. Eur. J. Pharmacol., 155, 201-203. WRIGHT, C.E. & FOZARD, J.R. (1989). Regional vascular responses to endothelin in spontaneously hypertensive rats: comparison with Wistar-Kyoto controls. Br. J. Pharmacol., 97, 394P.
113
YANAGISAWA, M., KURIHARA, H., KIMURA, S., TOMOBE, Y., KOBAYASHI, M., MITSUI, Y., YAZAKI, Y., GOTO, K. & MASAKI, T. (1988).
A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature, 332, 411-415. YANAGISAWA, M. & MASAKI, T. (1989). Endothelin, a novel endothelium-derived peptide. Pharmacological activities, regulation and possible roles in cardiovascular control. Biochem. Pharmacol., 38, 1877-1883.
(Received September 21, 1989 Revised December 7,1989 Accepted December 18, 1989)
C) Macmillan Press Ltd, 1990
Differences in regional vascular sensitivity to endothelin- 1 between spontaneously hypertensive and normotensive Wistar-Kyoto rats 'Christine E. Wright & 2John R. Fozard Preclinical Research, Sandoz Pharma A.G., CH 4002 Basel, Switzerland 1 The systemic and regional haemodynamic effects of porcine endothelin-1 (endothelin) have been measured in anaesthetized spontaneously hypertensive (SH) rats rendered areflexic by ganglion blockade; comparisons were made with age-matched Wistar-Kyoto (WKY) control animals. 2 In both SH and WKY rats endothelin (0.1-1 nmol kg-1 i.v.) elicited an initial, short-lived (
L.I
IF *\;
0
E 50
-0 V 0
//
/i
u
a
L
0
-100L
:--
9 8 -log [Et] (M)
10
01
5
10 20 40 60
Time (min)
Et 1
10204060
5
01
Time (min)
Et 1
Figure 4 Effects of endothelin 1 nmol kg'- i.v. bolus (Et 1) on circulatory variables in WKY (a, n = 16) and SH (b, n = 16) rats. For key to variables see legend to Figure 2.
strain. With 0.3 nmol kg- of the peptide, renal vascular conductance fell within 30s by 19.5 + 6.7 and 15.0 + 9.0% in SH and WKY rats, respectively. The highest dose of endothelin, 1 nmol kg1- , resulted in initial falls in renal vascular conductance of 82.3 + 4.6 and 75.9 + 4.7% in SH and WKY rats, respectively. This renal vasoconstriction was maintained for more than 20 min in both rat strains, with conductance values gradually returning to resting baseline over 60-80min (Figure 4). Mesenteric vascular conductance fell significantly with endothelin 0.1-1 nmol kg-' in both SH and WKY rats. Mean values in SH and WKY rats respectively were: 0.1 nmol kg 1, -16.9 + 9.5 and -35.8 + 5.8; 0.3 nmol kg- , - 24.1 + 10.0 and -41.5 + 7.4; and lnmolkg-1, -26.1 + 16.7 and -65.9 + 6.8%. The vasoconstriction at the latter dose was significantly greater in the WKY rats. Similar to the renal vascular response, the mesenteric vasoconstriction was of long
a 300 r
b A
a) 0 C
co
0
2200
V
A/
)i
0
_0))
,100
0
20
Co
u
K
-V
v
0.01 a)
0.1
03
1
1
3
10
d
c
Pre 8 7 6 5 -log [ACh] (M)
10
9
8
-log [Et] (M)
Pre 8 7 6 5
-log [ACh] (M)
Figure 6 Contractile effects of endothelin (Et) on aortae taken from SH rats and WKY control animals; effects of endothelial removal. (a) Sample tracing (data from SH rat) to illustrate the experimental design. A cumulative dose-response curve to endothelin was established and the results expressed in terms of a supramaximal response to a prior injection of phenylephrine (PE). Twenty minutes after addition of the highest dose of endothelin (3 x 10-8M), a cumulative doseresponse curve to acetylcholine (ACh) or the vehicle for ACh (saline; not illustrated) was established. (b and c) Mean data from a series of such experiments comparing tissues from WKY (b) and SH (c) rats. (U) Intact aortae; (EJ) aortae denuded of endothelium; (0) and (0) indicate effects of the vehicle for acetylcholine (saline). n = 10 for endothelin concentration-response curves; 6 for acetylcholine concentration-response curves and 4 for effects of saline. The mean responses (mg; in each case, n = 10) to phenylephrine, 10- m, were: WKY, intact aorta 2826 + 175; WKY, aorta denuded of endothelium 2924 + 163; SHR, intact aorta 2084 + 147; SHR, aorta denuded of endothelium 2333 + 169.
duration and returned to pre-endothelin values only after approximately 60 min (Figure 4).
Effects of acetylcholine and sodium nitroprusside Bolus of acetylcholine (0.01-1 gg kg 1, injections endothelium-dependent vasodilator) or nitroprusside (0.31Ogg kg-1, endothelium-independent vasodilator) revealed no marked differences between SH and WKY rats with respect to the vasodilatation in the hindquarter bed (Figure 5). Similarly, in the carotid vascular bed there was no significant difference between rat strains in the acetylcholine-induced dilatation. However, with nitroprusside, the highest dose elicited a greater increase in carotid vascular conductance in SH than in WKY rats. The effects of both dilator agents on blood pressure were similar showing no difference between rat groups. Acetylcholine lgkg-t caused changes of -33.4 + 2.9 and -26.0 + 3.6 mmHg, and nitroprusside lOpgkg- elicited falls of -34.6 + 2.8 and -30.7 + 2.1 mmHg in SH and WKY rats respectively.
0
Effects of angiotensin II
ICo c;
V
-0 0
0~
CT
Kr
0.01
0. l
0.3
l
3
lo0
Acetylcholine Nitroprusside (jig kg-') (vhg kg-1) Figure 5 Effects of acetylcholine (a,c) and nitroprusside (b,d) i.v. bolus injections on carotid (a,b) and hindquarter (Hq) (c,d) vascular conductances in SH (A, n 8) and WKY rats (0, n = 8). Error bars are average s.e.mean (see Methods). =
Angiotensin 11 (0.01-0.1lugkg-') administration resulted in greater increases in blood pressure in SH compared with WKY rats. Average increases in SH and WKY rats respectively were: 0.01 jIgkg-, 21 + 2 and 13 + 1; 0.03pgkg-1, 34 + 3 and 20 + 2; and 0. Igkg-1, 53 + 4 and 36 + 3 mmHg. However, there were no significant differences between rat strains in the decreases in renal and mesenteric vascular conductances. For instance, decreases in renal vascular conductance with angiotensin II injections ranged from -20 + 4 with the lowest dose to -79 + 7% with the highest dose in SH corresponding with values in WKY rats of -27 + 5 and -73 + 8%.
REGIONAL VASCULAR SENSITIVITY TO ENDOTHELIN
Effects of indomethacin In a number of additional SH rats, prepared identically to the main group, indomethacin 5 mg kg1- was administered either i.p. 60 min before endothelin injection (n= 5), or as an i.v. bolus 10min before endothelin nmol kg' (n = 1). Neither route of indomethacin administration appeared to have any effect on the biphasic response to endothelin (1 nmol kg 1) in these animals (Figure 1).
Sensitivity of aortae with and without endothelium to endothelin: comparison between SH rats and WK Y controls Endothelin, 10- 103 10-8 M, induced slowly developing and sustained contraction of aortic rings with or without intact endothelium from both SH rats and WKY control animals (Figure 6). Removal of the endothelium from rings taken from WKY rats enhanced to a small extent, but significantly (P < 0.05), the sensitivity to endothelin, as indicated by pEC50 (M) values of 8.47 + 0.05 and 8.71 + 0.10 (in both cases n = 10) for the rings with and without endothelium, respectively (Figure 6b). Rings from SH rats with intact endothelium were also significantly (P < 0.005) less responsive to endothelin than denuded tissues, as indicated by pEC50 (M) values of 8.42 + 0.07 and 8.82 + 0.09 respectively (in both cases n = 10; Figure 6c). Acetylcholine did not relax tissues contracted with endothelin in the absence of endothelium; in intact tissues, acetylcholine relaxed tissues from both SH rats and WKY controls (Figure 6b and c). The pIC50 concentrations (M; calculated taking the response to 10- 5M acetylcholine as maximum) were not significantly different (7.16 + 0.08 and 7.03 + 0.17 respectively; in each case, n = 6) although the maximum inhibition was somewhat greater in the tissues from SH than those from WKY rats (Figure 6b and c). x
Discussion The present results emphasize the complexity of the cardiovascular effects of endothelin which is increasingly being documented in a number of species (see Introduction). However, unlike the majority of studies published to date, the present results were obtained in animals rendered areflexic by administration of the ganglion blocking agent, mecamylamine. This allows interpretation of the effects of endothelin uncomplicated by intact autonomic cardiovascular reflexes. This is particularly important when comparing cardiovascular responses of SH rats with other rat strains because of the alterations in reflex buffering capacity well known to occur in hypertension (see, e.g. Wright et al., 1987). Under our experimental conditions, the effects of endothelin were qualitatively similar in SH and WKY rats. The initial short-lived falls in blood pressure were associated with similarly short-lived vasodilator responses in both carotid and hindquarter vascular beds and it seems likely that the latter are the basis of the former. The poor correlation between blood pressure fall and the conductance changes in the carotid and hindquarters vascular beds almost certainly reflects, to a large extent, the complexity of action of endothelin and, in particular, the differential contribution of the vasoconstrictor component at the different dose levels. However, since information on conductance changes is available for only four vascular beds, precise explanation of the poor correlation is not possible. The lack of any associated tachycardia (clearly seen in SH rats with normal reflexes-Winquist et al., 1989a) testifies to the adequacy of ganglion blockade and rules out the possibility that the vasodilatation results from modification of on-going autonomic tone and, in particular, the prejunctional inhibition of sympathetic neurotransmitter release (Wiklund et al., 1988).
111
The sustained rise in blood pressure which superseded the initial fall was associated with vasoconstriction, particularly in the renal and mesenteric vasculature but also, once the vasodilatation had waned, in the carotid and hindquarters beds. The intense, long-lasting renal vasoconstriction at the higher doses is particularly noteworthy. The same phenomenon has been observed in several species (Cocks et al., 1989; Minkes & Kadowitz, 1989) and in vitro (Firth et al., 1988; Cairns et al., 1988) and is associated with a marked decline in renal function (Lopez-Farr6 et al., 1989). On this basis endothelin has been implicated as a mediator in the pathogenesis of acute renal failure (Firth et al., 1988). Despite qualitative similarities, the effects of endothelin differed quantitatively in SH compared to WKY rats. In particular, both the initial falls in MAP and the associated vasodilator responses of the hindquarter and carotid vascular beds were greater in SH than WKY rats. Although the differences may in part reflect differences in baseline values between the two strains (MAP was significantly higher, hindquarter conductance was significantly lower in SH compared to WKY rats-Table 1), this seems unlikely to be the sole explanation for our observations. Thus, the greatest differences were seen in the carotid vascular bed where baseline values did not differ significantly between the two strains. Moreover, neither the vasodilator responses to an endothelium-dependent vasodilator, acetylcholine, nor those to the directly acting agent, sodium nitroprusside, differed markedly in SH compared to WKY rats (Figure 5). Thus the vasodilator component of the cardiovascular response to endothelin appears to be increased selectively in the spontaneously hypertensive state. The mechanism of the vasodilator response to endothelin in vivo has not been established, but it seems unlikely to reflect a direct action on vascular smooth muscle. Thus, no direct relaxation of arterial segments from a variety of species has ever been demonstrated (Cocks et al., 1989; Eglen et al., 1989; Saito et al., 1989). On the other hand, endothelin has been shown to dilate the rat perfused mesenteric vascular bed when tone was induced by methoxamine (Warner et al., 1989a,b; Randall et al., 1989) and to release the vasodilator eicosanoid, prostacyclin, from rat perfused lungs in vitro (de Nucci et al., 1988). In the perfused mesenteric vascular bed, vasodilatation could be inhibited by removal of the endothelium, methylene blue or haemoglobin indicating indirect effects of endothelin mediated by the release of endothelium-derived releasing factor(s), such as EDRF (Warner et al., 1989a,b; Randall et al., 1989). However, other experiments have failed to confirm the vasodilator effects of endothelin in the rat isolated perfused mesenteric arterial bed (Eglen et al., 1989; Tabuchi et al., 1989), an observation which would be consistent with the present data showing endothelin to induce exclusively vasoconstrictor responses in the mesenteric vascular bed in vivo (Figures 1-4). Moreover, Gardiner et al. (1989b) were not able to block endothelin-induced vasodilatation in conscious normotensive rats by administration of NG-monomethyl-L-arginine (L-NMMA) at a dose adequate to increase peripheral vascular resistance by suppressing the generation of a major component of EDRF, endothelium-derived nitric oxide (EDNO) (Gardiner et al., 1989c; see also Whittle et al., 1989). Finally, the present data showing the cardiovascular response to endothelin in the SH rat to be unaffected by pretreatment with indomethacin (Figure 1) render it unlikely that prostacyclin is playing a major role in the vasodilator response in vivo, at least under the conditions of the present experiment. Despite this, but in confirmation of the data of Eglen et al. (1989) and Godfraind et al. (1989), the endothelium does modulate the vasoconstrictor response to endothelin in rat isolated aorta (Figure 6). Intriguingly, in our experiments a somewhat greater increase in vasoconstrictor sensitivity due to endothelium removal was seen in tissues from SH rats (Figure 6), which could conceptually be used to support a greater role for endothelium-mediated vasodilator responses as the basis of the differential effects seen in SH and WKY rats in vivo. However, the in vitro effect most likely reflects not
112
C.E. WRIGHT & J.R. FOZARD
the release by endothelin of EDRF but physiological antagonism of endothelin by a basal EDRF-mediated vasodilator tone, since similar effects are manifested against a variety of other vasoconstrictor agents (Martin et al., 1986; Alosachie & Godfraind, 1988). In seeking alternative explanations for the mechanism of the vasodilator action of endothelin, one obvious possibility is the release of an EDRF other than EDNO (see e.g. Taylor & Weston, 1989). A further possibility is the release of atrial natriuretic peptide (ANP). Such a release has been demon-
strated both from cultured neonatal rat atrial cardiocytes (Fukuda et al, 1988) and adult rat atria in vitro, where release was greater in tissues taken from SH rats than WKY controls (Winquist et al., 1989b). However, unlike endothelin, the fall in blood pressure induced by the acute administration of ANP in the rat is not generally associated with a decrease in systemic vascular resistance (Lappe et al., 1985; Waeber et al., 1986; Gardiner et al., 1990). Clearly the precise mechanism of the vasodilator action of endothelin awaits further elucidation.
References ALOSACHIE, I. & GODFRAIND, T. (1988). The modulatory role of vascular endothelium in the interaction of agonists and antagonists with a-adrenoceptors in the rat aorta. Br. J. Pharmacol., 95, 619-629.
LIPPTON, H., GOFF, J. & HYMAN, A. (1988). Effects of endothelin in the systemic and renal vascular beds in vivo. Eur. J. Pharmacol., 155, 197-199.
CAIRNS, S.L., ROGERSON, M., FAIRBANKS, L.D., WESTWICK, J. &
(1989). Effect of endothelin on renal function in rats. Eur. J. Pharmacol., 163, 187-189.
NEILD, G.H. (1988). Endothelin and cyclosporin nephrotoxicity. Lancet, i, 1496-1497. COCKS, T.M., BROUGHTON, A., DIB, M., SUDHIR, K. & ANGUS, J.A.
(1989). Endothelin is blood vessel selective: studies on a variety of human and dog vessels in vitro and on regional blood flow in the conscious rabbit. Clin. Exp. Pharmacol. Physiol., 16, 243-246.
LOPEZ-FARRE, A., MONTANES, I., MILLAS & LOPEZ-NOVOA, J.M. MARTIN, W., FURCHGOTT, R.F., WILLANI, G.M. & JOTHIANDANAN,
D. (1989). Depression of contractile responses by spontaneously released endothelium-derived relaxing factor. J. Pharmacol. Exp. Ther., 237, 538-599. MINKES, R.K., COY, D.H., MURPHY, W.A., McNAMARA, D.B. & KADO-
R.L. (1989). The pharmacological properties of the peptide endothelin. Br. J. Pharmacol., 97, 1297-1307.
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. RANDALL, M.D., DOUGLAS, S.A. & HILEY, C.R. (1989). Vascular activities of endothelin-1 and some alanyl substituted analogues in resistance beds of the rat. Br. J. Pharmacol., 98, 685-699.
FIRTH, J.D., RATCLIFFE, P.J., RAINE, A.E.G. & LEDINGHAM, J.G.G.
SAITO, A., SHIBA, R., KIMURA, S., YANAGISAWA, M., GOTO, K. &
(1988). Endothelin: an important factor in acute renal failure? Lancet, i, 179-1181.
MASAKI, T. (1989). Vasoconstrictor response of large cerebral arteries of cats to endothelin, and endothelium-derived vasoactive peptide. Eur. J. Pharmacol., 162, 353-358.
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,
FUKUDA, Y., HIRATA, Y., YOSHIMA, H., KOJIMA, T., KOBAYASHI, Y.,
YANAGISAWA, M. & MASAKI, T. (1988). Endothelin is a potent secretogogue for atrial natriuretic peptide in cultured rat atrial myocytes. Biochem. Biophys. Res. Commun., 155, 167-172. GARDINER, S.M., COMPTON, A.M. & BENNETT, T. (1989a). Regional haemodynamic effects of endothelin-1 in conscious, unrestrained, Wistar rats. J. Cardiovasc. Pharmacol., 13, S202-S204. GARDINER, S.M., COMPTON, A.M., BENNETT, T., PALMER, R.M.J. &
MONCADA, S. (1989b). The effects of NG-monomethyl-L-arginine (L-NMMA) on the haemodynamic actions of endothelin-1 in conscious Long-Evans rats. Br. J. Pharmacol., 98, 626P. GARDINER, S.M., COMPTON, A.M., BENNETT, T., PALMER, R.M.J. &
MONCADA, S. (1989c). Haemodynamic effects of N0-monomethylL-arginine (L-NMMA) in conscious Long-Evans rats. Br. J. Pharmacol., 98, 623P. GARDINER, S.M., COMPTON, A.M. & BENNETT, T. (1990). Regional haemodynamic effects of endothelin-1 and endothelin-3 in conscious Long Evans and Brattleboro rats. Br. J. Pharmacol., 99, 107-112. GIVEN, M.B., LOWE, R.F., LIPPTON, H., HYMAN, A.L., SANDER, G.E. & GILES, T.D. (1989). (Hemodynamic actions of endothelin in con-
scious and anesthetized dogs. Peptides, 10, 41-44. GODFRAIND, T., MENNING, D., MOREL, N. & WIBO, M. (1989). Effect of endothelin-1 on calcium channel gating by agonists in vascular smooth muscle. J. Cardiovasc. Pharmacol., 13, SI 12-S1 17. HAN, S.P., TRAPANI, A.J., FOK, K.F., WESTFALL, T.C. & KNUEPFER,
M.M. (1989). Effects of endothelin on regional hemodynamics in conscious rats. Eur. J. Pharmacol., 159, 303-305. 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. KITAYOSHI, T., WATANABE, T. & SHIMAMOTO, N. (1989). Cardiovascular effects of endothelin in dogs: positive inotropic action in vivo. Eur. J. Pharmacol., 166, 519-522. LAPPE, R.W., SMITS, J.F.M., TODT, J.A., DEBETS, J.J.M. & WENDT, R.L. (1985). Failure of atriopeptin II to cause arterial vasodilation in
the conscious rat. Circ. Res., 56, 606-612.
I., SUMMER, W.R. & HYMAN, A.L. (1989). Endothelin-a new family of endothelium-derived peptides with widespread biological properties. Life Sci., 45, 1499-1513.
LE MONNIER DE COUVILLE, A.-C., LIPPTON, H.L., CAVERO,
TABUCHI, Y., NAKAMARU, M., RAKUGI, H., NAGANO, M. &
OGIHARA, T. (1989). Endothelin enhances adrenergic vasoconstriction in perfused rat mesenteric arteries. Biochem. Biophys. Res. Commun., 159,1304-1308. TAYLOR, S.G. & WESTON, A.H. (1989). Endothelium-derived hyperpolarizing factor: a new endogenous inhibitor from the vascular endothelium. Trends Pharmacol. Sci., 9, 272-274. TOMOBE, Y., MIYAUCHI, T., SAITO, A., YANAGISAWA, M., KIMURA,
S., GOTO, K. & MASAKI, T. (1988). Effects of endothelin on the renal artery from spontaneously hypertensive and Wistar Kyoto rats. Eur. J. Pharmacol., 152, 373-374. TSUCHIYA, K., NARUSE, M., SANAKA, T., NARUSE, K., NITTA, K.,
DEMURA, H. & SUGINO, M. (1989). Effects of endothelin on regional blood flow in dogs. Eur. J. Pharmacol., 166, 541-543. WAEBER, B., MATSUEDA, G.R., AUBERT, J.-F., NUSSBAUMER, J. &
BRUNNER, H.R. (1986). The haemodynamic response of conscious normotensive rats to atriopeptin III: lack of a role of the parasympathetic nervous system. Eur. J. Pharmacol., 125, 177-184. WARNER, T.D., MITCHELL, J.A., DE NUCCI, G. & VANE, J.R. (1989a). Endothelin-1 and endothelin-3 release EDRF from isolated perfused arterial vessels of the rat and rabbit. J. Cardiovasc. Pharmacol., 13, S85-S88. WARNER, T.D., DE NUCCI, G. & VANE, J.R. (1989b). Rat endothelin is a vasodilator in the isolated perfused mesentery of the rat. Eur. J. Pharmacol., 159, 325-326. WHITTLE, B.J.R., LOPEZ-BELMONTE, J. & REES, D.D. (1989). Modulation of the vasodepressor actions of acetylcholine, substance P and endothelin in the rat by a specific inhibitor of nitric oxide formation. Br. J. Pharmacol., 98, 646-652. WIKLUND, N.P., OHLEN, A. & CEDERQUIST, B. (1988). Inhibition of adrenergic neuroeffector transmission by endothelin in the guineapig femoral artery. Acta Physiol. Scand., 134, 311-312. WINQUIST, R.J., BUNTING, P.B., GARSKY, V.M., LUMMA, P.K. & SCHOFIELD, T.L. (1989a). Prominent depressor response to endothelin in spontaneously hypertensive rats. Eur. J. Pharmacol., 163, 199-203. WINQUIST, R.J., SCOTT, A.L. & VLASUK, G.P. (1989b). Enhanced release of atrial natriuretic factor by endothelin in atria from hypertensive rats. Hypertension, 14, 111-114. WRIGHT, C.E., ANGUS, J.A. & KORNER, P. (1987). Vascular amplifier properties in renovascular hypertension in conscious rabbits: and
REGIONAL VASCULAR SENSITIVITY TO ENDOTHELIN hindquarter responses to constrictor and dilator stimuli. Hypertension, 9, 122-131. WRIGHT, C.E. & FOZARD, J.R. (1988). Regional vasodilation is a prominent feature of the haemodynamic response to endothelin in anaesthetized, spontaneously hypertensive rats. Eur. J. Pharmacol., 155, 201-203. WRIGHT, C.E. & FOZARD, J.R. (1989). Regional vascular responses to endothelin in spontaneously hypertensive rats: comparison with Wistar-Kyoto controls. Br. J. Pharmacol., 97, 394P.
113
YANAGISAWA, M., KURIHARA, H., KIMURA, S., TOMOBE, Y., KOBAYASHI, M., MITSUI, Y., YAZAKI, Y., GOTO, K. & MASAKI, T. (1988).
A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature, 332, 411-415. YANAGISAWA, M. & MASAKI, T. (1989). Endothelin, a novel endothelium-derived peptide. Pharmacological activities, regulation and possible roles in cardiovascular control. Biochem. Pharmacol., 38, 1877-1883.
(Received September 21, 1989 Revised December 7,1989 Accepted December 18, 1989)
