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Involvement of α-adrenoceptors in the endothelium-dependent depression of noradrenaline-induced contraction in rat aorta
European Journal of Pharmacology, 240 (1993) 195-200
195
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53226
Involvement of a-adrenoceptors in the endothelium-dependent depression of noradrenaline-induced contraction in rat aorta Kyoko Kaneko and Satoru Sunano Research Institute of Hypertension, Kinki University, Osaka-$ayama, Osaka 589, Japan
Received 20 April 1993, accepted 1 June 1993
The involvement of endothelium-derived nitric oxide (NO) in the depressant action of the endothelium on noradrenalineinduced contractions and characterization of the receptor involved in the release of NO were studied using rat aorta. The noradrenaline-induced contraction was significantly potentiated by endothelium removal and in the presence of NG-nitro-Larginine (L-NNA) or N6-monomethyl-L-arginine (L-NMMA). The contraction induced by phenylephrine was also potentiated in the presence of L-NNA. Clonidine could induce contraction only in endothelium-denuded preparations or in the presence of L-NNA. The potentiating action of L-NNA on noradrenaline-induced contractions could also be observed in the presence of yohimbine or rauwolscine, although dose-response curves were shifted to the right. The depression of noradrenaline-induced contractions observed in the presence of the endothelium was increased by repeated stimulation. The depression was prevented by L-NNA and this effect was reversed by L-arginine. These results indicate the possibility that NO can be released through stimulation of a 1- and a2-adrenoceptors on the endothelium and depresses noradrenaline-induced contractions of smooth muscle, although the contribution of the respective adrenoceptors remains to be investigated. The release of NO was increased when the stimulation was applied repeatedly. Noradrenaline-induced contraction; Endothelium; Nitric oxide (NO); a-Adrenoceptors
1. Introduction The noradrenaline-induced contraction of vascular smooth muscles is depressed in the presence of endothelium (Cocks and Angus, 1983; Egl~me et al., 1984; Carrier and White, 1985; Miller and Vanhoutte, 1985; Murakami et al., 1985; Bullock et al., 1986; Martin et al., 1986; White and Carrier, 1986; Cohen et al., 1988; Godfraind and Alosachie, 1988; Osugi et al., 1990; U r a b e et al., 1991). This indicates the inhibitory involvement of spontaneous a n d / o r stimulated release of endothelium-derived relaxing factor ( E D R F , Furchgott and Zawadzki, 1980) in noradrenaline-induced contractions. In the case of stimulated release, a 2adrenoceptors on endothelial cells are thought to be involved (Egl~me et al., 1984; Cohen et al., 1988). The endothelium-dependent depression of noradrenalineinduced contractions is m o r e marked when the contraction is evoked repeatedly (Nakaki et al., 1990; Sunano et al., 1991), which indicates that the repeated
Correspondence to: Kyoko Kaneko, Research Institute of Hypertension, Kinki University, 377-2 Ohno-higashi, Osaka-sayama, Osaka 589, Japan. Tel. 0723-66-0221 (ext. 3317), fax 0723-66-0206.
application of noradrenaline provokes an increased release of E D R F . The present experiment was performed to characterize the a - a d r e n o c e p t o r involved in the depression of noradrenaline-induced contractions and the increased depression observed after repeated stimulation rat aorta. As E D R F has been identified as nitric oxide (NO, see Ignarro, 1989; Moncada et al., 1991) and its synthesis or release can be blocked by N G-mono methyl-L-arginine ( L - N M M A ) (Palmer et al., 1988) or N G-nitro-L-arginine (L-NNA) (Mulsch and Busse, 1990; Rees et al., 1990), the influence of E D R F was studied by treating the preparation with these compounds.
2. Materials and methods Wistar-Kyoto rats, 16 to 17 weeks old, were used. The rats were killed by bleeding from the carotid artery after they had been anaesthetized with diethyl ether. The thoracic aorta was excised and adhesive fat and connective tissues were removed carefully. Ring preparations of 1 m m width were m a d e from the distal portion of the aortic arch. In about 20 preparations,
196
the endothelium was removed by rubbing the luminal surface of the aorta with a soft rubber band. These preparations were mounted in organ baths filled with a modified Tyrode's solution, using two tungsten wires of 30 ~m diameter. One wire was fixed to the organ bath and the other wire was connected to the force-displacement transducer (Shin-koh, Nagano, Japan), so that isometric tension changes could be observed. The composition of the modified Tyrode's solution was as follows (mM): NaC1, 137; KCI, 5.4; CaC12, 2.0; MgC12, 1.0; NaHCO 3, 11.9; NaHsPO4, 0.4; glucose, 5.6 and Ca(II)-EDTA, 0.026. High-K Tyrode's solution was made by replacing NaCI with equimolar KC1. The final concentration of K ÷ in the solution was 50 mM. These solutions were aerated with a mixture of 95% O 2 and 5% CO2 at 37°C. The preparations were equilibrated in the modified Tyrode's solution for 1 h and then subjected to two successive stimulations with high K solution lasting 15 min and separated by an interval of 15 min. This procedure was required to obtain a constant amplitude of the contraction in the following experiments. Dose-response curves for agonists were then made for the first application of drugs, since the
contraction was markedly decreased after the second application of the same agonist, as we reported previously (Sunano et al., 1991) and was also observed in the present experiments. The drugs used in the present experiments were noradrenaline bitartrate (Sigma, St. Louis), L-phenylephrine hydrochloride (Sigma, St. Louis), clonidine (Sigma, St. Louis), 1-(2,6-dichlorobenzylideneamino)guanidine (guanabenz) (Sigma, St. Louis), yohimbine hydrochloride (Wako, Osaka), rauwolscine hydrochloride (Research Biochemicals, MA), ONO-11113 (9,1 l-epithio- 11,12-methano-thromboxane A 2, courtesy of Ono Pharmaceutical Co., Osaka), acetylcholine chloride (Wako, Osaka), NG-monomethyl-L-arginine (L-NMMA, Sigma, St. Louis), NG-nitro-L-arginine (LNNA, Sigma, St. Louis), L-arginine hydrochloride (Sigma, St. Louis), calcium disodium ethylendiaminetetraacetate (Ca(II)-EDTA, Wako, Osaka). These drugs were dissolved in distilled water. L-NNA was dissolved in distilled water under sonication. The values obtained are expressed as means + S.E.M. and analysed by Student's t-test, considering P values less than 0.05 to be significant.
b mg
Q mg
1000
1000.
"~ 500
." ~ 10
? g
g
8
'T
. . . . . . . 8 7 6 -log [NA]
S
• . t,
0-log [ NA ]
C mg 1000
~ s00
0
? 6 5 -log l: PP~nylep~]
/.
Fig. 1. Dose-response curves for contractions induced by noradrenaline, noradrenaline in the presence of yohimbine, and phenylephrine in the absence and presence of L-NNA. L - N N A (10 - 4 M) was applied 30 min prior to the dose-response experiment. T h e slight elevation of tension at concentrations lower than 10 - 9 M is due to the elevation of basal tension induced by L-NNA. Each value represents the m e a n + S.E.M. (vertical bars). Asterisks indicate significant differences from respective values in the absence of L - N N A (control, P < 0.001). (a) Noradrenaline (NA)-induced contraction, n = 15 in the absence of L - N N A (e), n = 12 in the presence of L - N N A (o). (b) Noradrenaline (NA)-induced contraction in the presence of 10-5 M yohimbine, n = 11 in the absence of L - N N A ( ~, ), n = 10 in the presence of L - N N A ( • ). (c) Phenylephrine (Phenyleph)-induced contraction, n = 6 in the absence of L - N N A (e), n = 8 in the presence of L - N N A (©).
197 The contractile response of rat aorta with an intact endothelium to phenylephrine was also markedly potentiated in the presence of 10 -4 M L - N N A (fig. lc). The decrease in the contractions elicited by a higher concentration of phenylephrine disappeared in the presence of L-NNA, as in the case of the noradrenaline-induced contraction.
3. R e s u l t s
3.1. Effects of L-NNA on noradrenaline- and phenylephrine-induced contractions In the present study, L - N N A at a concentration of 10 -4 M blocked the relaxation of endothelium-intact preparations induced by a low concentration (lower than 10 -7 M) of acetylcholine completely and that elicited by higher concentrations (10 -5 and 10 -4 M) of acetylcholine almost completely (by 93%) (not shown). At this concentration L - N N A increased the basal tension of rat aorta with an intact endothelium slightly and potentiated the noradrenaline-induced contraction (fig. 1). The threshold concentration for tension development and the concentration at which the maximum contraction was induced were not greatly altered by L-NNA. The decrease in the contraction amplitude observed at a higher concentration of noradrenaline disappeared in the presence of L-NNA. Removal of the endothelium potentiated the noradrenalineinduced contraction and minimized the potentiating effect of L - N N A and L - N M M A (not shown). The dose-response curve for noradrenaline-induced contractions was shifted to the right in the presence of yohimbine (10 -5 M) (fig. lb). Both the threshold concentration and the EDs0 value were about 10 times higher than those in the absence of yohimbine. L - N N A also potentiated the noradrenaline-induced contraction in the presence of yohimbine. The effect of L - N N A was apparently similar to that observed in the absence of yohimbine, except for the rightward shift of the dose-response curves. Treatment with rauwolscine (10 -5 M) also did not prevent the potentiation of noradrenaline-induced contractions by L - N N A (not shown).
3.2. Effects of L-NNA on contraction and relaxation induced by a 2-adrenoceptor stimulation Clonidine could not induce contraction in preparations with an intact endothelium even at high concentrations (fig. 2a). In the presence of L - N N A (10 -4 M), however, the drug could induce contraction, although the amplitude of the contraction was lower than that induced by noradrenaline or by phenylephrine. Under these conditions clonidine could induce a contraction even when endothelium was removed. It was also observed that clonidine induced relaxation in a dose-dependent manner in the presence of prazosin (10 -5 M) and propranolol (10 -6 M) (fig. 2b). The maximum relaxation amplitude was about 50% of the precontraction amplitude induced by ONO-11113 (10 -9 M), a thromboxane A 2 analogue. The relaxing activity of clonidine was blocked by L - N N A almost completely (fig. 2b). Guanabenz also induced a similar dose-dependent relaxation under the same conditions (not shown). The relaxation produced by 10-5 M guanabenz was 44.0 + 9.0% (mean + S.E.M., n = 7) of the precontraction. Dose-dependent relaxation was also observed with noradrenaline in the presence of the same concentration of prazosin and propranolol (not shown). The maximum relaxation induced by noradrenaline was ob-
o
b
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9
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.
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5
~
i 10
~
~
~i
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Fig. 2. Clonidine-induced contraction and relaxation in the absence and presence of L-NNA. Each value indicates the mean+ S.E.M. (vertical bars). (a) Dose-response curve for clonidine-induced contraction. L-NNA (10-4 M) was applied 30 min before the start of contraction experiments, n = 4 in the absence of L-NNA (o), n = 10 in the presence of L-NNA (©), * P < 0.001. (b) Dose-response curve for clonidine-induced relaxation. The preparations were precontracted in the presence of ONO-11113 (10 _9 M). L-NNA was applied 30 min before precontraction, n = 16 in the absence of L-NNA (o), n = 7 in the presence of L-NNA (©). * P < 0.05 at 10-1° M and P < 0.001 at higher concentration.
198 I
served at 10 -4 M, and the amplitude of the relaxation was 26.2 + 5.9% (mean + S.E.M., n = 8) of the precontraction induced by ONO-11113.
6
tPhenyleph I
I Phenyleph I L-NMMA
I
3.3. Involvement of endothefium-derived NO in the deterioration of noradrenaline-induced contraction b ,
The noradrenaline-induced contraction of the aorta with intact endothelium decreased when it was elicited repeatedly (fig. 3). The contraction was elicited with 10 -5 M noradrenaline, since the deterioration was more pronounced when a high concentration (higher than 10 -6 M) of the drug was applied. The amplitude of the fifth contraction was 48.6 +__2.7% (mean + S.E.M., n -- 5) of that of the first contraction (fig. 3b). In the endothelium-denuded preparation, no deterioration of the contraction was observed after repeated application of a high concentration of the drug. The amplitude of the fifth contraction in the endotheliumdenuded preparation was 102 + 1.8% (mean + S.E.M., n = 13) of that of the first contraction. The deterioration of the noradrenaline-induced contraction of endothelium-intact preparations was prevented by 10 - 4 M L-NMMA (fig. 3a). The amplitude of the sixth contraction, which was elicited in the presence of LNMMA, was 119 _+ 3.0% (mean + S.E.M., n = 10) of the first contraction. The prevention of the deterioration by L-NMMA was counteracted by L-arginine (10 -3 M, fig. 3a) but not by D-arginine (10 -3 M). The contraction elicited in the presence of L - N M M A and L-arginine was 48 + 5.2% (mean + S.E.M., n = 13), and that in the presence of L-NMMA and D-arginine was 127 _+ 5.7% (mean + S.E.M., n = 5) of the first contraction, respectively. L-Arginine at the same concentration showed no depressant effect on noradrenaline (10 -2 M)-induced contractions. L-NMMA reversed the deterioration of noradrenaline-induced contractions 1
5
6
L-NMNIA
I
I
I
--
I
b
L-Arginine- -
I
--
tNA
I
INA
I
INA
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L-NMMA
I
20rain
Fig. 3. Effects of L - N M M A on the deterioration of noradrenalineinduced contractions as a result of repeated stimulation. 1, 5 and 6 indicate first, fifth and sixth initiation of contraction, respectively. NA: 10 -5 M noradrenaline. Noradrenaline-induced contraction was decreased markedly by repeated stimulation, as shown in (b). T h e deterioration was prevented by the presence of L - N M M A (10-4 M), as shown in (a), or reversed as shown in (b). L-Arginine (10 -3 M) restored the deterioration in the presence of L - N M M A .
Yohimbine 5
~
INA
1
INA
i
~
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INA
I
INA
~I~NMMAINA
~
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400mg
. . i
Fig. 4. Deterioration of contractions induced by phenylephrine and by noradrenaline in the presence of yohimbine. Phenyleph: 10 -5 M phenylephrine. Yohimbine: 10 -5 M yohimbine. The decreased contractions (a and b) were restored by application of L - N M M A (10 -4 M). In (b), L - N M M A was applied during (upper trace) or prior to the noradrenaline-induced contraction. Others are the same as in fig. 3.
when it was applied after the deterioration had been provoked by repeated stimulation (fig. 3b). A similar effect was observed with L-NNA (not shown). The contraction elicited by phenylephrine (10 -5 M) was also decreased in an endothelium-dependent manner when it was elicited repeatedly; the decrease was prevented by 10 -a M L-NMMA (fig. 4a) or L-NNA (not shown). In addition, yohimbine (10 -5 M, fig. 4b) and rauwolscine (10 -5 M) (not shown) did not effect the decrease in the noradrenaline-induced contraction elicited by repeated stimulation.
4. Discussion
Noradrenaline-induced contractions of rat aorta were reduced in the presence of endothelium, as has been reported in various arteries (Cocks and Angus, 1983; Egl~me et al., 1984; Carrier and White, 1985; Miller and Vanhoutte, 1985; Murakami et al., 1985; Bullock et al., 1986; Martin et al., 1986; White and Carrier, 1886; Cohen et al., 1988; Godfraind and Alosachie, 1988; Osugi et al., 1990; Urabe et al., 1991). Various mechanisms have been proposed to underlie the inhibition of the contraction by the endothelium. Of these mechanisms, the involvement of E D R F , which was reported by Furchgott and Zawadzki (1980), has been recently emphasized (Cocks and Angus, 1983; Egl~me et al., 1984; Murakami et al., 1985; Martin et al., 1986; Cohen et al., 1988; Godfraind and Alosachie, 1988; Osugi et al., 1990; Urabe et al., 1991). In the present experiments, L-NNA and L-NMMA, which block the synthesis of NO from L-arginine, were used instead of endothelium removal and its was observed that treatment with these drugs markedly potentiated
199 the contraction elicited by noradrenaline. This indicates that NO is involved in the depressant action of endothelium on the noradrenaline-induced contraction. N O is released either spontaneously (basal release; see Martin, 1988) or in response to the stimulation of the endothelial receptor by the agonist (stimulated release; see Miller et al., 1988). Since the noradrenaline-induced contraction was reduced in an endothelium-dependent manner, as will be discussed below, and because this reduction was blocked by L-NMMA or L-NNA, it is more likely that NO is released by stimulation with the drug. In the case of stimulated release, most reports are in agreement that the depression of noradrenalineinduced contractions is due to the release of E D R F through stimulation of ot2-adrenoceptors on endothelial cells (Egl~me et al., 1984; Miller and Vanhoutte, 1985; Bullock et al., 1986). This conclusion is based on the result that the contraction elicited by az-adrenoceptor agonists is depressed more strongly or almost completely by the endothelium and that az-adrenoceptor agonists can induce marked endothelium-dependent relaxation in precontracted preparations. In the present experiment with WKY aorta, clonidine failed to induce contraction in endothelium-intact preparations and induced contractions when the preparation was treated with L-NNA. This suggests that NO released by stimulation with clonidine strongly depressed the contraction elicited by the same drug. We also observed that clonidine and guanabenz brought about a relaxation of ONO-11113-precontracted preparations in a dose-dependent manner in the presence of prazosin and propranolol. In addition, noradrenaline induced a dose-dependent relaxation under the same condition. In the present experiment, we observed that the relaxation elicited by a2-adrenoceptor stimulation was blocked by treating the preparation with L-NNA rather than by removing the endothelium. These results suggest that NO is released by stimulation with noradrenaline through az-adrenoceptor activation. However, the present experiments showed that the contraction evoked by phenylephrine, and at-adrenoceptor agonist, was also markedly depressed in the presence of the endothelium and the depression was reversed by treatment with L-NNA. The dose-response curve for noradrenaline in the presence of yohimbine or rauwolscine was also potentiated by treatment with L-NNA. Although yohimbine shifted the dose-response curve for noradrenaline to the right, this may be due to non-selective blockade of al-adrenoceptors on the smooth muscle (Ruffolo et al., 1981). The results, therefore, suggest that stimulation of a~-adrenoceptors on the endothelium by noradrenaline can cause the release of NO. Although controversial results have been reported, the possibility of the involvement of
al-adrenoceptors in the release of NO by adrenoceptor agonists is consistent with reports that al-adrenoceptor stimulation increases the level of cyclic G M P in endothelium-intact aorta (Bigaud et al., 1984; Miller et al., 1985). The endothelium-dependent relaxation caused by a~-adrenoceptor stimulation in precontracted preparations, however, could not be examined because of the strong contractile response elicited by ~ - a d r e n o c e p t o r stimulation. The noradrenaline-induced contraction was reduced by repeated initiation of contraction. A similar deterioration of noradrenaline-induced contractions has been reported in the same vascular tissue (Nakaki et al., 1990; Sunano et al., 1991). The deterioration was endothelium-dependent and prevented in the presence of L-NNA or L-NMMA. The prevention by L-NNA was counteracted by the addition of L-arginine. These results indicate the involvement of NO in the deterioration. Since the deterioration was much less prominent with high-K-induced contractions (unpublished observation), it can be considered that the release of NO through a-adrenoceptor stimulation increases as a result of the repetitive application of noradrenaline. The deterioration was also observed for phenylephrineinduced contractions and noradrenaline-induced contractions in the presence of yohimbine or rauwolscine, indicating the involvement of oq-adrenoceptors on the endothelium. Thus it can be concluded that a~-adrenoceptor stimulation induced the release of NO from the endothelium and that this release increases with repeated stimulation. In summary, noradrenaline-induced contractions of rat aorta were potentiated by L-NNA or L-NMMA, indicating the inhibitory involvement of NO in the contraction induced by noradrenaline. Stimulation of both a~- and a2-adrenoceptors on the endothelium by noradrenaline can cause the release of NO and the released NO depresses the contraction elicited by noradrenaline itself. The release of NO through stimulation of a-adrenoceptors, especially al-adrenoceptors, increases with repeated stimulation. The contribution of the respective receptors to the depression, however, remains to be investigated further.
References Bigaud, M., P. Schoeffter,J.C. Stoclet and R.C. Miller, 1984, Dissociation between endothelium-mediated increases in tissue cGMP levels and modulation of aortic contractile responses, NaunynSchmied. Arch. Pharmacol. 328, 221. Bullock, G.R., S.G. Taylor and A.H. Weston, 1986, Influence of the vascular endothelium on agonist-induced contractions and relaxations in rat aorta, Br. J. Pharmacol. 89, 819. Carrier, G.O. and R.E. White, 1985, Enhancement of alpha-1 and
200 alpha-2 adrenergic agonist-induced vasocontraction by removal of endothelium in rat aorta, J. Pharmacol. Exp. Ther. 232, 682. Cocks, T.M. and J.A. Angus, 1983, Endothelium-dependent relaxation of coronary arteries by noradrenaline and serotonin, Nature 305, 627. Cohen, R.A., K.M. Zitnay, R.M. Weisbrod and B. Tesfamariam, 1987, Influence of the endothelium on tone and the response of isolated pig coronary artery to norepinephrine, J. Pharmacol. Exp. Ther. 244, 550. Egl~me, C., T. Godfraind and R.C. Miller, 1984, Enhanced responsiveness of rat isolated aorta to clonidine after removal of the endothelial cells, Br. J. Pharmacol. 81, 16. Furchgott, R.F. and J.V. Zawadzki, 1980, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288, 373. Godfraind, T. and I. Alosachie, 1988, Influence of endothelium and cyclic GMP on alpha-adrenoceptors, in: Vasodilatation: Vascular Smooth Muscle, Peptides, Autonomic Nerves, and Endothelium, ed. P.M. Vanhoutte (Raven Press, New York) p. 437. Ignarro, L.J., 1989, Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein, Circ. Res. 65, 1. Martin, W., 1988, Basal release of endothelium-derived relaxing factor, in: Relaxing and Contracting Factors, ed. P.M. Vanhoutte (The Humana Press, Clifton, NJ) p. 159. Martin, W., R.F. Furchgott, G.M. Villani and D. Jothianandan, 1986, Depression of contractile responses in rat aorta by spontaneously released endothelium-derived relaxing factor, J. Pharmacol. Exp. Ther. 237, 529. Miller, V.M. and P.M. Vanhoutte, 1985, Endothelial a2-adrenoce ptors in canine pulmonary and systemic blood vessels, Eur. J. Pharmacol. 118, 123. Miller, R.C., P. Schoeffter and J.C. Stoclet, 1985, Insensitivity of calcium-dependent endothelial stimulation in rat isolated aorta to the calcium entry blocker, flunarizine, Br. J. Pharmacol. 85, 481. Miller, R.C., V. Schini and P. Schoeffter, 1988, Modulation by the endothelium of agonist-induced contractions of vascular smooth muscle, in: Relaxing and Contraction Factors, ed. P.M. Vanhoutte (The Humana Press, Clifton, NJ) p. 241. Moncada, S., R.M.J. Palmer and E.A. Higgs, 1991, Nitric oxide:
physiology, pathophysiology, and pharmacology, Pharmacol. Rev. 43, 109. Mulsch, A. and R. Busse, 1990, N6-Nitro-L-arginine (N 5[imino(nitro-amino)-methyl]oL-ornithine) impairs endotheliumdependent dilations by inhibiting cytosolic nitric oxide synthesis from L-arginine, Naunyn-Schmied. Arch. Pharmacol. 341, 143. Murakami, K., H. Karaki and N. Urakawa, 1985, Role of endothelium in the contractions induced by norepinephrine and clonidine in rat aorta, Jpn. J. Pharmacol. 39, 357. Nakaki, T., Y. Otsuka and R. Kato, 1990, Tension-induced release of endothelium-derived relaxing factors; possible role in establishment of desensitization of norepinephrine-induced contraction in rat aorta, Jpn. J. Pharmacol. 54, 491. Osugi, S., K. Shimamura and S. Sunano, 1990, Decreased modulation by endothelium-induced contractions in aorta from strokeprone spontaneously hypertensive rats, Arch. Int. Pharmacodyn. 305, 86. Palmer, R.M.J., D.D. Rees, D.S. Ashton and S. Moncada, 1988, L-Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation, Biochem. Biophys. Res. Commun. 153, 1251. Rees, D.D., R.M.J. Palmar, R. Schulz, H.F. Hodson and S. Moncada, 1990, Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo, Br. J. Pharmacol. 101, 746. Ruffolo, R.R., J.E. Waddell, Jr. and E.L. Yaden, 1981, Postsynaptic alpha adrenergic receptor subtypes differentiated by yohimbine in tissues from the rat. Existence of alpha-2 adrenergic receptors in rat aorta, J. Pharmacol. Exp. Ther. 217, 235. Sunano, S., Y. Hosogi and K. Shimamura, 1991, Deterioration of contraction induced by repetitive application of noradrenaline and roles of endothelium in aorta from spontaneously hypertensive rats, J. Vasc. Med. Biol. 3, 251. Urabe, M., H. Kawasaki and K. Takasaki, 1991, Effect of endothelium removal on the vasoconstrictor response to neuronally released 5-hydroxytryptamine and noradrenaline in the rat isolated mesenteric and femoral arteries, Br. J. Pharmacol. 102, 85. White, R.E. and G.O. Carrier, 1986, a 1- and a2-adrenoceptor agonist-induced contraction in rat mesenteric artery upon removal of endothelium, Eur. J. Pharmacol. 122, 349.
195
© 1993 Elsevier Science Publishers B.V. All rights reserved 0014-2999/93/$06.00
EJP 53226
Involvement of a-adrenoceptors in the endothelium-dependent depression of noradrenaline-induced contraction in rat aorta Kyoko Kaneko and Satoru Sunano Research Institute of Hypertension, Kinki University, Osaka-$ayama, Osaka 589, Japan
Received 20 April 1993, accepted 1 June 1993
The involvement of endothelium-derived nitric oxide (NO) in the depressant action of the endothelium on noradrenalineinduced contractions and characterization of the receptor involved in the release of NO were studied using rat aorta. The noradrenaline-induced contraction was significantly potentiated by endothelium removal and in the presence of NG-nitro-Larginine (L-NNA) or N6-monomethyl-L-arginine (L-NMMA). The contraction induced by phenylephrine was also potentiated in the presence of L-NNA. Clonidine could induce contraction only in endothelium-denuded preparations or in the presence of L-NNA. The potentiating action of L-NNA on noradrenaline-induced contractions could also be observed in the presence of yohimbine or rauwolscine, although dose-response curves were shifted to the right. The depression of noradrenaline-induced contractions observed in the presence of the endothelium was increased by repeated stimulation. The depression was prevented by L-NNA and this effect was reversed by L-arginine. These results indicate the possibility that NO can be released through stimulation of a 1- and a2-adrenoceptors on the endothelium and depresses noradrenaline-induced contractions of smooth muscle, although the contribution of the respective adrenoceptors remains to be investigated. The release of NO was increased when the stimulation was applied repeatedly. Noradrenaline-induced contraction; Endothelium; Nitric oxide (NO); a-Adrenoceptors
1. Introduction The noradrenaline-induced contraction of vascular smooth muscles is depressed in the presence of endothelium (Cocks and Angus, 1983; Egl~me et al., 1984; Carrier and White, 1985; Miller and Vanhoutte, 1985; Murakami et al., 1985; Bullock et al., 1986; Martin et al., 1986; White and Carrier, 1986; Cohen et al., 1988; Godfraind and Alosachie, 1988; Osugi et al., 1990; U r a b e et al., 1991). This indicates the inhibitory involvement of spontaneous a n d / o r stimulated release of endothelium-derived relaxing factor ( E D R F , Furchgott and Zawadzki, 1980) in noradrenaline-induced contractions. In the case of stimulated release, a 2adrenoceptors on endothelial cells are thought to be involved (Egl~me et al., 1984; Cohen et al., 1988). The endothelium-dependent depression of noradrenalineinduced contractions is m o r e marked when the contraction is evoked repeatedly (Nakaki et al., 1990; Sunano et al., 1991), which indicates that the repeated
Correspondence to: Kyoko Kaneko, Research Institute of Hypertension, Kinki University, 377-2 Ohno-higashi, Osaka-sayama, Osaka 589, Japan. Tel. 0723-66-0221 (ext. 3317), fax 0723-66-0206.
application of noradrenaline provokes an increased release of E D R F . The present experiment was performed to characterize the a - a d r e n o c e p t o r involved in the depression of noradrenaline-induced contractions and the increased depression observed after repeated stimulation rat aorta. As E D R F has been identified as nitric oxide (NO, see Ignarro, 1989; Moncada et al., 1991) and its synthesis or release can be blocked by N G-mono methyl-L-arginine ( L - N M M A ) (Palmer et al., 1988) or N G-nitro-L-arginine (L-NNA) (Mulsch and Busse, 1990; Rees et al., 1990), the influence of E D R F was studied by treating the preparation with these compounds.
2. Materials and methods Wistar-Kyoto rats, 16 to 17 weeks old, were used. The rats were killed by bleeding from the carotid artery after they had been anaesthetized with diethyl ether. The thoracic aorta was excised and adhesive fat and connective tissues were removed carefully. Ring preparations of 1 m m width were m a d e from the distal portion of the aortic arch. In about 20 preparations,
196
the endothelium was removed by rubbing the luminal surface of the aorta with a soft rubber band. These preparations were mounted in organ baths filled with a modified Tyrode's solution, using two tungsten wires of 30 ~m diameter. One wire was fixed to the organ bath and the other wire was connected to the force-displacement transducer (Shin-koh, Nagano, Japan), so that isometric tension changes could be observed. The composition of the modified Tyrode's solution was as follows (mM): NaC1, 137; KCI, 5.4; CaC12, 2.0; MgC12, 1.0; NaHCO 3, 11.9; NaHsPO4, 0.4; glucose, 5.6 and Ca(II)-EDTA, 0.026. High-K Tyrode's solution was made by replacing NaCI with equimolar KC1. The final concentration of K ÷ in the solution was 50 mM. These solutions were aerated with a mixture of 95% O 2 and 5% CO2 at 37°C. The preparations were equilibrated in the modified Tyrode's solution for 1 h and then subjected to two successive stimulations with high K solution lasting 15 min and separated by an interval of 15 min. This procedure was required to obtain a constant amplitude of the contraction in the following experiments. Dose-response curves for agonists were then made for the first application of drugs, since the
contraction was markedly decreased after the second application of the same agonist, as we reported previously (Sunano et al., 1991) and was also observed in the present experiments. The drugs used in the present experiments were noradrenaline bitartrate (Sigma, St. Louis), L-phenylephrine hydrochloride (Sigma, St. Louis), clonidine (Sigma, St. Louis), 1-(2,6-dichlorobenzylideneamino)guanidine (guanabenz) (Sigma, St. Louis), yohimbine hydrochloride (Wako, Osaka), rauwolscine hydrochloride (Research Biochemicals, MA), ONO-11113 (9,1 l-epithio- 11,12-methano-thromboxane A 2, courtesy of Ono Pharmaceutical Co., Osaka), acetylcholine chloride (Wako, Osaka), NG-monomethyl-L-arginine (L-NMMA, Sigma, St. Louis), NG-nitro-L-arginine (LNNA, Sigma, St. Louis), L-arginine hydrochloride (Sigma, St. Louis), calcium disodium ethylendiaminetetraacetate (Ca(II)-EDTA, Wako, Osaka). These drugs were dissolved in distilled water. L-NNA was dissolved in distilled water under sonication. The values obtained are expressed as means + S.E.M. and analysed by Student's t-test, considering P values less than 0.05 to be significant.
b mg
Q mg
1000
1000.
"~ 500
." ~ 10
? g
g
8
'T
. . . . . . . 8 7 6 -log [NA]
S
• . t,
0-log [ NA ]
C mg 1000
~ s00
0
? 6 5 -log l: PP~nylep~]
/.
Fig. 1. Dose-response curves for contractions induced by noradrenaline, noradrenaline in the presence of yohimbine, and phenylephrine in the absence and presence of L-NNA. L - N N A (10 - 4 M) was applied 30 min prior to the dose-response experiment. T h e slight elevation of tension at concentrations lower than 10 - 9 M is due to the elevation of basal tension induced by L-NNA. Each value represents the m e a n + S.E.M. (vertical bars). Asterisks indicate significant differences from respective values in the absence of L - N N A (control, P < 0.001). (a) Noradrenaline (NA)-induced contraction, n = 15 in the absence of L - N N A (e), n = 12 in the presence of L - N N A (o). (b) Noradrenaline (NA)-induced contraction in the presence of 10-5 M yohimbine, n = 11 in the absence of L - N N A ( ~, ), n = 10 in the presence of L - N N A ( • ). (c) Phenylephrine (Phenyleph)-induced contraction, n = 6 in the absence of L - N N A (e), n = 8 in the presence of L - N N A (©).
197 The contractile response of rat aorta with an intact endothelium to phenylephrine was also markedly potentiated in the presence of 10 -4 M L - N N A (fig. lc). The decrease in the contractions elicited by a higher concentration of phenylephrine disappeared in the presence of L-NNA, as in the case of the noradrenaline-induced contraction.
3. R e s u l t s
3.1. Effects of L-NNA on noradrenaline- and phenylephrine-induced contractions In the present study, L - N N A at a concentration of 10 -4 M blocked the relaxation of endothelium-intact preparations induced by a low concentration (lower than 10 -7 M) of acetylcholine completely and that elicited by higher concentrations (10 -5 and 10 -4 M) of acetylcholine almost completely (by 93%) (not shown). At this concentration L - N N A increased the basal tension of rat aorta with an intact endothelium slightly and potentiated the noradrenaline-induced contraction (fig. 1). The threshold concentration for tension development and the concentration at which the maximum contraction was induced were not greatly altered by L-NNA. The decrease in the contraction amplitude observed at a higher concentration of noradrenaline disappeared in the presence of L-NNA. Removal of the endothelium potentiated the noradrenalineinduced contraction and minimized the potentiating effect of L - N N A and L - N M M A (not shown). The dose-response curve for noradrenaline-induced contractions was shifted to the right in the presence of yohimbine (10 -5 M) (fig. lb). Both the threshold concentration and the EDs0 value were about 10 times higher than those in the absence of yohimbine. L - N N A also potentiated the noradrenaline-induced contraction in the presence of yohimbine. The effect of L - N N A was apparently similar to that observed in the absence of yohimbine, except for the rightward shift of the dose-response curves. Treatment with rauwolscine (10 -5 M) also did not prevent the potentiation of noradrenaline-induced contractions by L - N N A (not shown).
3.2. Effects of L-NNA on contraction and relaxation induced by a 2-adrenoceptor stimulation Clonidine could not induce contraction in preparations with an intact endothelium even at high concentrations (fig. 2a). In the presence of L - N N A (10 -4 M), however, the drug could induce contraction, although the amplitude of the contraction was lower than that induced by noradrenaline or by phenylephrine. Under these conditions clonidine could induce a contraction even when endothelium was removed. It was also observed that clonidine induced relaxation in a dose-dependent manner in the presence of prazosin (10 -5 M) and propranolol (10 -6 M) (fig. 2b). The maximum relaxation amplitude was about 50% of the precontraction amplitude induced by ONO-11113 (10 -9 M), a thromboxane A 2 analogue. The relaxing activity of clonidine was blocked by L - N N A almost completely (fig. 2b). Guanabenz also induced a similar dose-dependent relaxation under the same conditions (not shown). The relaxation produced by 10-5 M guanabenz was 44.0 + 9.0% (mean + S.E.M., n = 7) of the precontraction. Dose-dependent relaxation was also observed with noradrenaline in the presence of the same concentration of prazosin and propranolol (not shown). The maximum relaxation induced by noradrenaline was ob-
o
b
mg 1000
1 0 °~ 0- ~
~, ~ o"
~ 217. ~
500
~ 3~' ~0,
50. .
9
.
.
.
8
7 6 -log E C I o ~ d i ~ ]
5
~
i 10
~
~
~i
- I ~ CC l ~ i ~
~i
~
~i
]
Fig. 2. Clonidine-induced contraction and relaxation in the absence and presence of L-NNA. Each value indicates the mean+ S.E.M. (vertical bars). (a) Dose-response curve for clonidine-induced contraction. L-NNA (10-4 M) was applied 30 min before the start of contraction experiments, n = 4 in the absence of L-NNA (o), n = 10 in the presence of L-NNA (©), * P < 0.001. (b) Dose-response curve for clonidine-induced relaxation. The preparations were precontracted in the presence of ONO-11113 (10 _9 M). L-NNA was applied 30 min before precontraction, n = 16 in the absence of L-NNA (o), n = 7 in the presence of L-NNA (©). * P < 0.05 at 10-1° M and P < 0.001 at higher concentration.
198 I
served at 10 -4 M, and the amplitude of the relaxation was 26.2 + 5.9% (mean + S.E.M., n = 8) of the precontraction induced by ONO-11113.
6
tPhenyleph I
I Phenyleph I L-NMMA
I
3.3. Involvement of endothefium-derived NO in the deterioration of noradrenaline-induced contraction b ,
The noradrenaline-induced contraction of the aorta with intact endothelium decreased when it was elicited repeatedly (fig. 3). The contraction was elicited with 10 -5 M noradrenaline, since the deterioration was more pronounced when a high concentration (higher than 10 -6 M) of the drug was applied. The amplitude of the fifth contraction was 48.6 +__2.7% (mean + S.E.M., n -- 5) of that of the first contraction (fig. 3b). In the endothelium-denuded preparation, no deterioration of the contraction was observed after repeated application of a high concentration of the drug. The amplitude of the fifth contraction in the endotheliumdenuded preparation was 102 + 1.8% (mean + S.E.M., n = 13) of that of the first contraction. The deterioration of the noradrenaline-induced contraction of endothelium-intact preparations was prevented by 10 - 4 M L-NMMA (fig. 3a). The amplitude of the sixth contraction, which was elicited in the presence of LNMMA, was 119 _+ 3.0% (mean + S.E.M., n = 10) of the first contraction. The prevention of the deterioration by L-NMMA was counteracted by L-arginine (10 -3 M, fig. 3a) but not by D-arginine (10 -3 M). The contraction elicited in the presence of L - N M M A and L-arginine was 48 + 5.2% (mean + S.E.M., n = 13), and that in the presence of L-NMMA and D-arginine was 127 _+ 5.7% (mean + S.E.M., n = 5) of the first contraction, respectively. L-Arginine at the same concentration showed no depressant effect on noradrenaline (10 -2 M)-induced contractions. L-NMMA reversed the deterioration of noradrenaline-induced contractions 1
5
6
L-NMNIA
I
I
I
--
I
b
L-Arginine- -
I
--
tNA
I
INA
I
INA
I
L-NMMA
I
20rain
Fig. 3. Effects of L - N M M A on the deterioration of noradrenalineinduced contractions as a result of repeated stimulation. 1, 5 and 6 indicate first, fifth and sixth initiation of contraction, respectively. NA: 10 -5 M noradrenaline. Noradrenaline-induced contraction was decreased markedly by repeated stimulation, as shown in (b). T h e deterioration was prevented by the presence of L - N M M A (10-4 M), as shown in (a), or reversed as shown in (b). L-Arginine (10 -3 M) restored the deterioration in the presence of L - N M M A .
Yohimbine 5
~
INA
1
INA
i
~
?-,.
INA
I
INA
~I~NMMAINA
~
tNA
I~NNNA
400mg
. . i
Fig. 4. Deterioration of contractions induced by phenylephrine and by noradrenaline in the presence of yohimbine. Phenyleph: 10 -5 M phenylephrine. Yohimbine: 10 -5 M yohimbine. The decreased contractions (a and b) were restored by application of L - N M M A (10 -4 M). In (b), L - N M M A was applied during (upper trace) or prior to the noradrenaline-induced contraction. Others are the same as in fig. 3.
when it was applied after the deterioration had been provoked by repeated stimulation (fig. 3b). A similar effect was observed with L-NNA (not shown). The contraction elicited by phenylephrine (10 -5 M) was also decreased in an endothelium-dependent manner when it was elicited repeatedly; the decrease was prevented by 10 -a M L-NMMA (fig. 4a) or L-NNA (not shown). In addition, yohimbine (10 -5 M, fig. 4b) and rauwolscine (10 -5 M) (not shown) did not effect the decrease in the noradrenaline-induced contraction elicited by repeated stimulation.
4. Discussion
Noradrenaline-induced contractions of rat aorta were reduced in the presence of endothelium, as has been reported in various arteries (Cocks and Angus, 1983; Egl~me et al., 1984; Carrier and White, 1985; Miller and Vanhoutte, 1985; Murakami et al., 1985; Bullock et al., 1986; Martin et al., 1986; White and Carrier, 1886; Cohen et al., 1988; Godfraind and Alosachie, 1988; Osugi et al., 1990; Urabe et al., 1991). Various mechanisms have been proposed to underlie the inhibition of the contraction by the endothelium. Of these mechanisms, the involvement of E D R F , which was reported by Furchgott and Zawadzki (1980), has been recently emphasized (Cocks and Angus, 1983; Egl~me et al., 1984; Murakami et al., 1985; Martin et al., 1986; Cohen et al., 1988; Godfraind and Alosachie, 1988; Osugi et al., 1990; Urabe et al., 1991). In the present experiments, L-NNA and L-NMMA, which block the synthesis of NO from L-arginine, were used instead of endothelium removal and its was observed that treatment with these drugs markedly potentiated
199 the contraction elicited by noradrenaline. This indicates that NO is involved in the depressant action of endothelium on the noradrenaline-induced contraction. N O is released either spontaneously (basal release; see Martin, 1988) or in response to the stimulation of the endothelial receptor by the agonist (stimulated release; see Miller et al., 1988). Since the noradrenaline-induced contraction was reduced in an endothelium-dependent manner, as will be discussed below, and because this reduction was blocked by L-NMMA or L-NNA, it is more likely that NO is released by stimulation with the drug. In the case of stimulated release, most reports are in agreement that the depression of noradrenalineinduced contractions is due to the release of E D R F through stimulation of ot2-adrenoceptors on endothelial cells (Egl~me et al., 1984; Miller and Vanhoutte, 1985; Bullock et al., 1986). This conclusion is based on the result that the contraction elicited by az-adrenoceptor agonists is depressed more strongly or almost completely by the endothelium and that az-adrenoceptor agonists can induce marked endothelium-dependent relaxation in precontracted preparations. In the present experiment with WKY aorta, clonidine failed to induce contraction in endothelium-intact preparations and induced contractions when the preparation was treated with L-NNA. This suggests that NO released by stimulation with clonidine strongly depressed the contraction elicited by the same drug. We also observed that clonidine and guanabenz brought about a relaxation of ONO-11113-precontracted preparations in a dose-dependent manner in the presence of prazosin and propranolol. In addition, noradrenaline induced a dose-dependent relaxation under the same condition. In the present experiment, we observed that the relaxation elicited by a2-adrenoceptor stimulation was blocked by treating the preparation with L-NNA rather than by removing the endothelium. These results suggest that NO is released by stimulation with noradrenaline through az-adrenoceptor activation. However, the present experiments showed that the contraction evoked by phenylephrine, and at-adrenoceptor agonist, was also markedly depressed in the presence of the endothelium and the depression was reversed by treatment with L-NNA. The dose-response curve for noradrenaline in the presence of yohimbine or rauwolscine was also potentiated by treatment with L-NNA. Although yohimbine shifted the dose-response curve for noradrenaline to the right, this may be due to non-selective blockade of al-adrenoceptors on the smooth muscle (Ruffolo et al., 1981). The results, therefore, suggest that stimulation of a~-adrenoceptors on the endothelium by noradrenaline can cause the release of NO. Although controversial results have been reported, the possibility of the involvement of
al-adrenoceptors in the release of NO by adrenoceptor agonists is consistent with reports that al-adrenoceptor stimulation increases the level of cyclic G M P in endothelium-intact aorta (Bigaud et al., 1984; Miller et al., 1985). The endothelium-dependent relaxation caused by a~-adrenoceptor stimulation in precontracted preparations, however, could not be examined because of the strong contractile response elicited by ~ - a d r e n o c e p t o r stimulation. The noradrenaline-induced contraction was reduced by repeated initiation of contraction. A similar deterioration of noradrenaline-induced contractions has been reported in the same vascular tissue (Nakaki et al., 1990; Sunano et al., 1991). The deterioration was endothelium-dependent and prevented in the presence of L-NNA or L-NMMA. The prevention by L-NNA was counteracted by the addition of L-arginine. These results indicate the involvement of NO in the deterioration. Since the deterioration was much less prominent with high-K-induced contractions (unpublished observation), it can be considered that the release of NO through a-adrenoceptor stimulation increases as a result of the repetitive application of noradrenaline. The deterioration was also observed for phenylephrineinduced contractions and noradrenaline-induced contractions in the presence of yohimbine or rauwolscine, indicating the involvement of oq-adrenoceptors on the endothelium. Thus it can be concluded that a~-adrenoceptor stimulation induced the release of NO from the endothelium and that this release increases with repeated stimulation. In summary, noradrenaline-induced contractions of rat aorta were potentiated by L-NNA or L-NMMA, indicating the inhibitory involvement of NO in the contraction induced by noradrenaline. Stimulation of both a~- and a2-adrenoceptors on the endothelium by noradrenaline can cause the release of NO and the released NO depresses the contraction elicited by noradrenaline itself. The release of NO through stimulation of a-adrenoceptors, especially al-adrenoceptors, increases with repeated stimulation. The contribution of the respective receptors to the depression, however, remains to be investigated further.
References Bigaud, M., P. Schoeffter,J.C. Stoclet and R.C. Miller, 1984, Dissociation between endothelium-mediated increases in tissue cGMP levels and modulation of aortic contractile responses, NaunynSchmied. Arch. Pharmacol. 328, 221. Bullock, G.R., S.G. Taylor and A.H. Weston, 1986, Influence of the vascular endothelium on agonist-induced contractions and relaxations in rat aorta, Br. J. Pharmacol. 89, 819. Carrier, G.O. and R.E. White, 1985, Enhancement of alpha-1 and
200 alpha-2 adrenergic agonist-induced vasocontraction by removal of endothelium in rat aorta, J. Pharmacol. Exp. Ther. 232, 682. Cocks, T.M. and J.A. Angus, 1983, Endothelium-dependent relaxation of coronary arteries by noradrenaline and serotonin, Nature 305, 627. Cohen, R.A., K.M. Zitnay, R.M. Weisbrod and B. Tesfamariam, 1987, Influence of the endothelium on tone and the response of isolated pig coronary artery to norepinephrine, J. Pharmacol. Exp. Ther. 244, 550. Egl~me, C., T. Godfraind and R.C. Miller, 1984, Enhanced responsiveness of rat isolated aorta to clonidine after removal of the endothelial cells, Br. J. Pharmacol. 81, 16. Furchgott, R.F. and J.V. Zawadzki, 1980, The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288, 373. Godfraind, T. and I. Alosachie, 1988, Influence of endothelium and cyclic GMP on alpha-adrenoceptors, in: Vasodilatation: Vascular Smooth Muscle, Peptides, Autonomic Nerves, and Endothelium, ed. P.M. Vanhoutte (Raven Press, New York) p. 437. Ignarro, L.J., 1989, Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein, Circ. Res. 65, 1. Martin, W., 1988, Basal release of endothelium-derived relaxing factor, in: Relaxing and Contracting Factors, ed. P.M. Vanhoutte (The Humana Press, Clifton, NJ) p. 159. Martin, W., R.F. Furchgott, G.M. Villani and D. Jothianandan, 1986, Depression of contractile responses in rat aorta by spontaneously released endothelium-derived relaxing factor, J. Pharmacol. Exp. Ther. 237, 529. Miller, V.M. and P.M. Vanhoutte, 1985, Endothelial a2-adrenoce ptors in canine pulmonary and systemic blood vessels, Eur. J. Pharmacol. 118, 123. Miller, R.C., P. Schoeffter and J.C. Stoclet, 1985, Insensitivity of calcium-dependent endothelial stimulation in rat isolated aorta to the calcium entry blocker, flunarizine, Br. J. Pharmacol. 85, 481. Miller, R.C., V. Schini and P. Schoeffter, 1988, Modulation by the endothelium of agonist-induced contractions of vascular smooth muscle, in: Relaxing and Contraction Factors, ed. P.M. Vanhoutte (The Humana Press, Clifton, NJ) p. 241. Moncada, S., R.M.J. Palmer and E.A. Higgs, 1991, Nitric oxide:
physiology, pathophysiology, and pharmacology, Pharmacol. Rev. 43, 109. Mulsch, A. and R. Busse, 1990, N6-Nitro-L-arginine (N 5[imino(nitro-amino)-methyl]oL-ornithine) impairs endotheliumdependent dilations by inhibiting cytosolic nitric oxide synthesis from L-arginine, Naunyn-Schmied. Arch. Pharmacol. 341, 143. Murakami, K., H. Karaki and N. Urakawa, 1985, Role of endothelium in the contractions induced by norepinephrine and clonidine in rat aorta, Jpn. J. Pharmacol. 39, 357. Nakaki, T., Y. Otsuka and R. Kato, 1990, Tension-induced release of endothelium-derived relaxing factors; possible role in establishment of desensitization of norepinephrine-induced contraction in rat aorta, Jpn. J. Pharmacol. 54, 491. Osugi, S., K. Shimamura and S. Sunano, 1990, Decreased modulation by endothelium-induced contractions in aorta from strokeprone spontaneously hypertensive rats, Arch. Int. Pharmacodyn. 305, 86. Palmer, R.M.J., D.D. Rees, D.S. Ashton and S. Moncada, 1988, L-Arginine is the physiological precursor for the formation of nitric oxide in endothelium-dependent relaxation, Biochem. Biophys. Res. Commun. 153, 1251. Rees, D.D., R.M.J. Palmar, R. Schulz, H.F. Hodson and S. Moncada, 1990, Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo, Br. J. Pharmacol. 101, 746. Ruffolo, R.R., J.E. Waddell, Jr. and E.L. Yaden, 1981, Postsynaptic alpha adrenergic receptor subtypes differentiated by yohimbine in tissues from the rat. Existence of alpha-2 adrenergic receptors in rat aorta, J. Pharmacol. Exp. Ther. 217, 235. Sunano, S., Y. Hosogi and K. Shimamura, 1991, Deterioration of contraction induced by repetitive application of noradrenaline and roles of endothelium in aorta from spontaneously hypertensive rats, J. Vasc. Med. Biol. 3, 251. Urabe, M., H. Kawasaki and K. Takasaki, 1991, Effect of endothelium removal on the vasoconstrictor response to neuronally released 5-hydroxytryptamine and noradrenaline in the rat isolated mesenteric and femoral arteries, Br. J. Pharmacol. 102, 85. White, R.E. and G.O. Carrier, 1986, a 1- and a2-adrenoceptor agonist-induced contraction in rat mesenteric artery upon removal of endothelium, Eur. J. Pharmacol. 122, 349.