BLOOD PRESSURE
1992; 1: 45-49
Contractions to Endothelin in Normotensive and Spontaneously Hypertensive Rats: Role of Endothelium and Prostaglandins WOLFGANG AUCH-SCHWELK and PAUL M. VANHOUTTE
’
From Baylor College of Medicine. Houston, Texas. U.S.A. and German Heart Institute, Berlin, Germany
Blood Press Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
Auch-Schwek W, Vanhoutte PM. Contractions to endothelin in normotensiue and spontaneously hypertensitie rats: Role of endothelium and prostaglandins. Blood Pressure 1992; 1: 45-49. Contractions to endothelin-1 in aortas of the spontaneously hypertensive rats (SHR) were compared with those of normotensive controls (WKY); rings with and without endothelium were studied in organ chambers. Contractions to endothelin were smaller in aortas of SHR compared to WKY, whether the endothelium was present or not. The presence of a functional endothelium reduced contractions to the peptide in both strains. Endothelium-dependent relaxations to acetylcholine and endothelium-independent relaxations to nitric oxide were observed in rings from both strains during contraction with endothelin. Indomethacin reduced the contractions to endothelin in the aorta from SHR with endothelium, but not in those without endothelium; it did not significantly affect endothelin-induced contractions in rings of W K Y with or without endothelium. These experiments demonstrate that contractions of the vascular smooth muscle to endothelin are reduced in the aorta of the SHR. The basal and stimulated release of endothelium-derived relaxing factor inhibits contractions to endothelin in the aorta from both strains. The inhibitor of cyclooxygenase indomethacin does not prevent the response of the vascular smooth muscle to endothelin; however, endothelin may stimulate the release of an indomethacin-sensitive endothelium-derived contracting factor in the SHR aorta. Ke-v w0rd.s: endothelin, endothelium-derived relaxing factor, endothelium-derived contracting factor, cyclooxygenase. rat aorta, spontaneously Iiypertensitie rat.
INTRODUCTION Endothelium-dependent contractions to acetylcholine have been observed in the aorta of the spontaneously hypertensive rat (SHR), but not in that of normotensive controls (WKY) [I]. They are prevented by cyclooxygenase inhibitors [l]. Endothelin-1 is a potent vasoconstrictor peptide isolated from cultured endothelial cells [2]. The present experiments were designed to determine whether or not the endothelium demonstrates the responsiveness to endothelin- 1 differently in the aorta of the SHR and the WKY. The findings suggest that in the SHR, but not in the WKY, the peptide causes the release of an indomethacin-sensitive endothelium-derived contracting factor. MATERIALS AND METHODS Experiments were performed on isolated aortas of male spontaneously hypertensive rats (30-34 weeks old) and age- and weight-matched normotensive Wistar-Kyoto rats (Harlan, Indianapolis, IN; body weight 378 f25, n = 7, and 353 f40 g, n = 7, respectively). Systolic blood pressure was determined by an indirect tail cuff method in the unanesthetized animals prior to the experiments (208f9 and 125+3 mmHg, for the SHR and WKY, respectively, p < 0.05). The aorta was excised under anesthesia (sodium pentobarbital 50 mg/kg, i.p.). The thoracic part of the aorta was cut into 4 rings of 6 mm length. In some of the rings the endothelium was
removed by gently rubbing the intimal surface with the tip of a small forceps [l]. The rings were suspended in organ chambers between a clip and a force transducer (UTC-2, Gould Inc., Cleveland, OH) by two stainless steel wires inserted into the lumen of the vessels. Changes in isometric force were measured. The organ chambers were filled with 25 ml modified Krebs-Ringer bicarbonate solution [of the following composition (mM): NaCl, 118; KCl, 4.7; CaC12, 2.5; MgS04, 1.2; KH2P04,1.2; NaHCO3,25.0; EDTA calcium disodium 0.026; glucose, 11.1 (control solution)] kept at 37°C and aerated with a 95% 0 2 and 5% C02 gas mixture. The preparations were set individually at the optimal point of their length-tension relationship as determined by repeated exposure to norepinephrine (3 x lo p7 M, Table I). The presence of the endothelium was confirmed by the occurrence of relaxations to acetylcholine (1 OP7 M) in rings contracted with norepinephrine (3 x lop7M (1)). At the beginning of the experiment all rings were exposed to 60 mM KCl (isotonic solution). Then all rings were incubated for 45 min with phentolamine ( lop6M) and propanolol(5 x M) to exclude indirect effects of endothelin through the release of norepinephrine from perivascular nerve endings. In some rings indomethacin ( l o P 5M) was present during the incubation period and throughout the experiment to prevent the synthesis of vasoactive prostanoids. These drugs did not affect basal tension. Endothelin-1 (lo-” to l o p 7M) was added in a cumulative manner.
46
W. Auch-Schwelk and P . M . Vunhoutte
ENDOTHELIN AND HYPERTENSION
Table I. Experimental conditions (Means fSEM)
RAT AORTA WITHOUT ENDOTHELIUM
With endothelium Basal tension in g 5.8 f0.5 SHR 5.1 f0.6 WKY Contraction to K C L (60 m M ) , in g SHR 3.88 f0.15 4.26k0.29 WKY n (14)
Without endothelium
=
5.9 f 0.5 5.8 k 0.4
8
3.34k0.23 3.15 10.36 (14)
W
ia
I 160
0
WKY
0
SHR
No7
60
When the response of the rings was stable, acetylcholine and nitric oxide were added to rings with and without endothelium, respectively.
I
-10
-11
-8
-0
-I
Blood Press Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
ENDOTHELIN (log M)
Drugs
The following drugs were used: acetylcholine hydrochloride, porcine endothelin- 1 (Peptides International, Louisville, Kentucky), indomethacin, (1)-norepinephrine, phentolamine hydrochloride, propranolol hydrochloride. Unless otherwise specified all drugs were obtained from Sigma (St. Louis, MO); they were prepared in distilled water except for indomethacin, which was dissolved by sonication in NaIC03 ( lop5M). Preparation of nitric oxide: A gas bulb fitted with a rubber injection septum was filled with nitric oxide from a cylinder (Union Carbide, Chicago, IL). 100 pl were removed with a syringe and injected into another gas bulb filled with 100 ml of distilled water, which had been previously gassed with helium for approximately 3 h, giving a stock solution of nitric oxide of 4 x M ~31. Calculations Contractions were expressed as absolute values (g) or as percent of contractions to KCl (60 mM), which were comparable in tissues from SHR and WKY (Table I). EDSOvalues were calculated as the negative logarithm of the concentration (determined in the individual rings), which caused 50% of the maximal contraction to endothelin. Relaxations to acetylcholine or nitric oxide were expressed as percent of the initial contraction to endothelin ( M). Student's t-test for paired observations was used for statistical comparison of rings from the same rat; the t-test for unpaired observations was used for comparisons between the two strains. Mean values from more than two groups were compared by one-way analysis of variance.
RESULTS Endothelin (lo-" to
M) caused concentration-
Fig. I . Contractions to endothelin (lo-" to M) in the aorta without endothelium from WKY and SHR. Contractions are expressed as percent of the maximal contraction to KCI (60 mM) and shown as meanskSEM. *indicates significant difference between arteries from WKY and SHR ( p < 0.05).
dependent contractions in all preparations. The maximal contraction was significantly smaller in rings with and without endothelium from the SHR compared to the WKY; the EDSOfor endothelin was not significantly different in the aorta of the two strains (Figs 1 and 2, Table 11). Removal of the endothelium intensified contractions to endothelin aortas from both strains. The ED50 to the peptide was lower after removal of the endothelium in rings from WKY, but not from SHR (Table 11).
ENDOTHELIN AND PROSTAGLANDINS RAT AORTA WITH ENDOTHELIUM
loo
1
...._. WKY ~
CONTROL 0
a
0 z
20 401
*
0-I
5HR
0
INWMETHACIN
cd I
-11
-10
-0
-0
-7
ENDOTHELIN (log M)
Fig. 2. Effect of indomethacin M) on contractions to M) in the aorta from WKY and endothelin (lo-" to SHR with endothelium. Contractions are expressed as percent of the maximal contraction to KCI (60 mM) and shown as meansf SEM. *indicates significant difference from control in SHR rings ( p < 0.05).
Endothelin and SHR aorta
47
Table 11. EDso values and maximal response (in % KCI) to endothelin in the thoracic aorta of WKY and SHR rats ~~
Maximal contraction
EDso WKY
SH R
WKY
SHR
without endothelium
8.17k0.07 8.46 k0.1Od
8.36k0.08 8.46k0.13
93.3f6.3 143.1 f 14.8"
53.6 f 1 3.7b 95.8 f 1 1.2ab
Indomethacin with endothelium without endothelium
8. I4 f0.09 8.36 f0.07d
8.02 f0.07' 8.18 f0.09
82.2 f4.4 122.7f9. l a
33.8 f9.1bc 86.1 f 1 5.7ab
Blood Press Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
Control with endothelium
EDSO values represent the negative logarithm of the concentration determined in each individual ring, which gives 50% of the maximal contraction to endothelin. Data are shown as meansf SEM. "significant different from rings with endothelium. bsignificantdifferent from WKY. 'significant different from untreated rings.
Acetylcholine (3 x lop5 M) caused comparable relaxations in rings with endothelium from both strains contracted with endothelin (lo-' M; WKY 89.4f2.6%, SHR 101.3f6.2%) Nitric oxide (3 x lop6 M) caused relaxations in rings without endothelium of aortas from both strains. The relaxations were biphasic: a rapid transient relaxation was followed by a smaller stable reduction in tension; only the maximal peak relaxation was compared. The relaxations were significantly larger in the SHR than in the WKY (WKY 89. I f5.6, SHR 108.2 f 10.6, p < 0.05). Indomethacin ( M) reduced contractions to endothelin in rings of SHR with endothelium, but not those in rings without endothelium; it did not affect contractions in rings of WKY, whether the endothelium was present or not (Fig. 2, Table 11).
DISCUSSION The present study confirms that endothelin is a vasoconstrictor in the isolated aorta of the SHR. The data obtained in the aorta from WKY rats without endothelium are in accordance with previous determinations of the biological activity of porcine endothelin in the aorta of that species [4]. Contractions to endothelin are reduced in the aorta of SHR rats compared to that of normotensive controls. This reduced responsiveness in the SHR aorta is at variance with the augmented responses reported by other investigators in the renal and mesenteric arteries of the SHR [5, 6, 71, but in agreement with the results reported by different laboratories comparing the responsiveness of the aorta of WKY and SHR to
endothelin [7-10]. The cause for the reduction in responsiveness to endothelin observed in the aorta is unknown. It cannot be explained by the altered function of the endothelium in hypertension [ 1 1,12,1J, since the reduction can be observed both in rings with and without endothelium. An altered production of vasoactive prostanoids [13] is unlikely to be the cause for reduction of contractions, since the inhibitor of cyclooxygenase, indomethacin, augmented rather than reduced the difference between the two strains. Since the response to high potassium was comparable in the SHR and WKY aortas, a major defect in the contractile process cannot be held responsible. Thus the smaller contraction in the aorta of the SHR is most likely due to a reduced responsiveness to endothelin in the vascular smooth muscle of the SHR. Theoretically, a chronically augmented exposure to endothelin might lead to a desensitization of receptors; however, measurement of plasma endothelin- 1 levels revealed similar levels in genetically hypertensive rats compared to normotensive controls [14, 151. The presence of endothelium inhibits contractions to most agonists in the rat aorta [16, 171. In the present study the inhibitory effect of the endothelium on contractions to endothelin could be demonstrated in aortas from both SHR and WKY. Since this inhibition can be observed after treatment with indomethacin, prostacyclin is not likely to be involved. It can be attributed to the basal release of endothelium-derived relaxing factor(s). In support of this interpretation, acetylcholine causes endothelium-dependent relaxations in rings from both strains. Nitric oxide, which is probably a major relaxing factor released by the endothelium [18, 19, 31, also inhibits contractions to
Blood Press Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
48
W . Auch-Schwelk and P . M . Vanhoutte
endothelin in both strains. The marginally increased response to nitric oxide in rings from SHR is explained best by the smaller contraction in response to the higher concentrations of endothelin used t o contract the preparations. The release by endothelin of contracting prostanoids from the smooth muscle of the rat aorta has been suggested by experiments using thromboxane A*/ prostaglandin HZreceptor antagonists [20].The present data do not provide any evidence for the release of vasoactive prostaglandins in response to endothelin- 1 in preparations without endothelium. However, the present experiments suggest that products of cyclooxygenase are involved in the modulatory influence of the endothelium in response to endothelin in the S H R , but not in the WKY. This interpretation is prompted by the observation that indomethacin (in a concentration known to abolish the production of prostanoids in the SHR-aorta [21]) reduces contractions in rings with but not without endothelium in the hypertensive strain. One explanation could be that exogenous endothelin stimulates the release of a contracting product of cyclooxygenase (e.g. thromboxane AZ [ 131) from the endothelium, which masks the inhibitory effect of nonprostanoid endothelium-derived relaxing factor. The response to endothelin in the S H R aorta is modified by the endothelium in a way similar t o the response to acetylcholine a n d serotonin. These substances cause larger contractions in the aorta of SHR compared to W K Y , presumably because they release endothelium-derived contracting factor which can be inhibited by indomethacin [22-261. Since endothelin is a peptide it is unlikely that its synthesis or release is regulated by cyclooxygenase. The direct effects of endothelin on the vascular smooth muscle are not prevented by indomethacin as is the case for oxygen-derived free radicals, which can be regarded as endothelium-derived contracting factor [27, 28, 23, 241. Taken together with the reduced responsiveness to the vasoconstrictor effect of the peptide, this does not support the concept that endothelin is the mediator of endothelium-dependent contractions in the S H R aorta.
ACKNOWLEDGEMENTS The authors thank Mr. Greg Brandt for technical assistance in the blood pressure measurements, Mrs. Helen Hendrickson and Mr. Robert Lorenz for preparing the figures and Mrs. Kathleen Kros for preparing the manuscript. This work was supported in part by the National Institute of Health, HL 35614. Dr. Auch-Schwelk was supported by the Deutsche Forschungsgemeinschaft (Au 76/1- I).
REFERENCES 1 . Liischer TF, Vanhoutte PM. Endothelium-dependent contractions to acetylcholine in the aorta of the spontaneously hypertensive rat. Hypertension 1986; 8: 344-8. 2, Yanagisawa M, Kurihara H, Kimura S, et al. Endothelin: A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 332: 41 1-5. 3. Palmer RMJ, Ferridge AG, Moncada S . Nitric oxide release accounts for the biological activity of endothehum-derived relaxing factor. Nature (Lond) 1987; 327: 524-5. 4. Yanagisawa M, Inoue A, Ishikawa T, et al. Primary structure, synthesis, and biological activity of rat endothelin, an endothelium-derived vasoconstrictor peptide. Proc Natl Acad Sci (USA) 1988; 85: 6964-7. 5. Tomobe Y, Miyauchi T, Saito A, et al. Effects of endothelin on the renal artery from spontaneously hypertensive and Wistar Kyoto rats. Eur J Pharmacol 1988; 152: 373-4. 6. Miyauchi T, Ishikawa T, Tomobe Y, et al. Characteristics of pressor response to endothelin in spontaneously hypertensive and Wistar-Kyoto rats. Hypertension 1989; 14: 427-34. 7. Criscione L, Nellis P, Riniker B, et al. Reactivity and sensitivity of mesenteric vascular beds and aortic rings of spontaneously hypertensive rats to endothelin: effects of calcium entry bIockers. Br J Pharmacol 1990; I00 31-6. 8. Wright CE, Fozard JR. Differences in regional vascular sensitivity to endothelin- 1 between spontaneously hypertensive and normotensive Wistar Kyoto rats. Br J Pharmacol 1990; 100: 107-13. 9. Clozel M. Endothelin sensitivity and receptor binding in the aorta of spontaneously hypertensive rats. J Hypertens 1989; 7: 913-7. 10. Wu CC, Bohr DF. Role of the endothelium in the response to endothelin in hypertension. Hypertension 1991; 16: 677-81. 1 1 . Winquist RJ, Bunting PB, Baskin EP, Wallace AA. Decreased endothelium-dependent relaxation in New Zealand genetic hypertensive rats. J Hypertens 1984; 2: 541-6. 12. Konishi M, Su C. Role of endothelium in dilator responses of spontaneously hypertensive rat arteries. Hypertension 1983; 5: 881-6. 13. De Nucci G, Thomas R, D’Orleans-Juste, P, et al. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Natl Acad Sci USA 1988; 85: 9797-9800. 14. Suzuki N, Miyauchi T, Tomobe Y, et al. Plasma concentrations of endothelin-1 in spontaneously hypertensive rats and DOCA-salt hypertensive rats. Biochem Biophys Res Commun 1990; 167: 941-7. 15. Kohno M, Murakawa K, Horio T, et al. Plasma immunoreactive endotheiin- 1 in experimental malignant hypertension. Hypertension 1991; 18: 93-100. 16. Martin W, Furchgott RF, Villani GM, Jothianandan D. Depression of contractile responses in rat aorta by spontaneously released endothelium-derived relaxing factor. J Pharmacol Exp Ther 1985; 232: 708-16. 17. Bullock GR, Taylor SG, Weston AH. Influence of the vascular endothelium on agonist-induced contractions and relaxations in rat aorta. Br J Pharmacol 1986; 89: 8 19-30.
Blood Press Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
Endothelin and SHR aorta
18. Furchgott RF. Studies on relaxation of rabbit aorta by sodium nitrite: The basis for the proposal that the acidactivatable inhibitory factor from bovine retractor penis is inorganic and the endothelium derived relaxing factor is nitric oxide. In: Vanhouette PM, ed. Vasodilatation: Vascular smooth muscle, peptides, autonomic nerves, and endothelium. New York: Raven Press, 1988: 401-14. 19 Ignarro LJ. Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein. Circ Res 1988; 65: 1-21. 20. Reynolds EE, Mok LL. Role of thromboxane A2/ prostaglandin H2 receptor in the vasoconstrictor response of rat aorta to endothelin. J Pharmacol Exp Ther 1990; 252: 915-21. 21. Luscher TF, Romero JC, Vanhoutte PM. Bioassay of endothelium-derived vasoactive substances in the aorta of normotensive and spontaneously hypertensive rats. J Hypertens 1986; 4 (Suppl 6): S81-S3. 22. Liischer TF, Vanhoutte PM. Endothelium-dependent responses to platelets and serotonin in spontaneously hypertensive rats. Hypertension 1986; 8 (Suppl. 11): 5560. 23. Auch-Schwelk W, Katusic ZS, Vanhoutte PM. Contractions to oxygen-derived free radicals are augmented in the SHR aorta. Hypertension 1989; 13: 859-64.
49
24. Auch-Schwelk W, Katusic ZS, Vanhoutte PM. Thromboxane A2 receptor antagonists inhibit endotheliumdependent contractions. Hypertension 1990; 15: 699-703. 25. Auch-Schwelk W, Katusic Z , Vanhoutte PM. Nitric oxide inactivates endothelium-derived contracting factor in the rat aorta. Hypertension 1992; 19: 442-5. 26. Auch-Schwelk W, Vanhoutte PM. Endothelium-derived contracting factor released by serotonin in the aorta of the SHR. Am J Hypertens 1991; 4: 769-72. 27. Vanhoutte PM, Katusic ZS. Endothelium-derived contracting factor: endothelin and/or superoxide anion? TIPS 1988; 9: 229-30. 28. Katusic ZS, Vanhoutte PM. Superoxide anion is an endothelium-derived contracting factor. Am J Physiol 1989; 257: H33-H373. Submitted February 2, 1992; accepted February 25, 1992 Address for correspondence: Paul M. Vanhoutte Baylor College of Medicine Houston, TX 77030 U.S.A.
1992; 1: 45-49
Contractions to Endothelin in Normotensive and Spontaneously Hypertensive Rats: Role of Endothelium and Prostaglandins WOLFGANG AUCH-SCHWELK and PAUL M. VANHOUTTE
’
From Baylor College of Medicine. Houston, Texas. U.S.A. and German Heart Institute, Berlin, Germany
Blood Press Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
Auch-Schwek W, Vanhoutte PM. Contractions to endothelin in normotensiue and spontaneously hypertensitie rats: Role of endothelium and prostaglandins. Blood Pressure 1992; 1: 45-49. Contractions to endothelin-1 in aortas of the spontaneously hypertensive rats (SHR) were compared with those of normotensive controls (WKY); rings with and without endothelium were studied in organ chambers. Contractions to endothelin were smaller in aortas of SHR compared to WKY, whether the endothelium was present or not. The presence of a functional endothelium reduced contractions to the peptide in both strains. Endothelium-dependent relaxations to acetylcholine and endothelium-independent relaxations to nitric oxide were observed in rings from both strains during contraction with endothelin. Indomethacin reduced the contractions to endothelin in the aorta from SHR with endothelium, but not in those without endothelium; it did not significantly affect endothelin-induced contractions in rings of W K Y with or without endothelium. These experiments demonstrate that contractions of the vascular smooth muscle to endothelin are reduced in the aorta of the SHR. The basal and stimulated release of endothelium-derived relaxing factor inhibits contractions to endothelin in the aorta from both strains. The inhibitor of cyclooxygenase indomethacin does not prevent the response of the vascular smooth muscle to endothelin; however, endothelin may stimulate the release of an indomethacin-sensitive endothelium-derived contracting factor in the SHR aorta. Ke-v w0rd.s: endothelin, endothelium-derived relaxing factor, endothelium-derived contracting factor, cyclooxygenase. rat aorta, spontaneously Iiypertensitie rat.
INTRODUCTION Endothelium-dependent contractions to acetylcholine have been observed in the aorta of the spontaneously hypertensive rat (SHR), but not in that of normotensive controls (WKY) [I]. They are prevented by cyclooxygenase inhibitors [l]. Endothelin-1 is a potent vasoconstrictor peptide isolated from cultured endothelial cells [2]. The present experiments were designed to determine whether or not the endothelium demonstrates the responsiveness to endothelin- 1 differently in the aorta of the SHR and the WKY. The findings suggest that in the SHR, but not in the WKY, the peptide causes the release of an indomethacin-sensitive endothelium-derived contracting factor. MATERIALS AND METHODS Experiments were performed on isolated aortas of male spontaneously hypertensive rats (30-34 weeks old) and age- and weight-matched normotensive Wistar-Kyoto rats (Harlan, Indianapolis, IN; body weight 378 f25, n = 7, and 353 f40 g, n = 7, respectively). Systolic blood pressure was determined by an indirect tail cuff method in the unanesthetized animals prior to the experiments (208f9 and 125+3 mmHg, for the SHR and WKY, respectively, p < 0.05). The aorta was excised under anesthesia (sodium pentobarbital 50 mg/kg, i.p.). The thoracic part of the aorta was cut into 4 rings of 6 mm length. In some of the rings the endothelium was
removed by gently rubbing the intimal surface with the tip of a small forceps [l]. The rings were suspended in organ chambers between a clip and a force transducer (UTC-2, Gould Inc., Cleveland, OH) by two stainless steel wires inserted into the lumen of the vessels. Changes in isometric force were measured. The organ chambers were filled with 25 ml modified Krebs-Ringer bicarbonate solution [of the following composition (mM): NaCl, 118; KCl, 4.7; CaC12, 2.5; MgS04, 1.2; KH2P04,1.2; NaHCO3,25.0; EDTA calcium disodium 0.026; glucose, 11.1 (control solution)] kept at 37°C and aerated with a 95% 0 2 and 5% C02 gas mixture. The preparations were set individually at the optimal point of their length-tension relationship as determined by repeated exposure to norepinephrine (3 x lo p7 M, Table I). The presence of the endothelium was confirmed by the occurrence of relaxations to acetylcholine (1 OP7 M) in rings contracted with norepinephrine (3 x lop7M (1)). At the beginning of the experiment all rings were exposed to 60 mM KCl (isotonic solution). Then all rings were incubated for 45 min with phentolamine ( lop6M) and propanolol(5 x M) to exclude indirect effects of endothelin through the release of norepinephrine from perivascular nerve endings. In some rings indomethacin ( l o P 5M) was present during the incubation period and throughout the experiment to prevent the synthesis of vasoactive prostanoids. These drugs did not affect basal tension. Endothelin-1 (lo-” to l o p 7M) was added in a cumulative manner.
46
W. Auch-Schwelk and P . M . Vunhoutte
ENDOTHELIN AND HYPERTENSION
Table I. Experimental conditions (Means fSEM)
RAT AORTA WITHOUT ENDOTHELIUM
With endothelium Basal tension in g 5.8 f0.5 SHR 5.1 f0.6 WKY Contraction to K C L (60 m M ) , in g SHR 3.88 f0.15 4.26k0.29 WKY n (14)
Without endothelium
=
5.9 f 0.5 5.8 k 0.4
8
3.34k0.23 3.15 10.36 (14)
W
ia
I 160
0
WKY
0
SHR
No7
60
When the response of the rings was stable, acetylcholine and nitric oxide were added to rings with and without endothelium, respectively.
I
-10
-11
-8
-0
-I
Blood Press Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
ENDOTHELIN (log M)
Drugs
The following drugs were used: acetylcholine hydrochloride, porcine endothelin- 1 (Peptides International, Louisville, Kentucky), indomethacin, (1)-norepinephrine, phentolamine hydrochloride, propranolol hydrochloride. Unless otherwise specified all drugs were obtained from Sigma (St. Louis, MO); they were prepared in distilled water except for indomethacin, which was dissolved by sonication in NaIC03 ( lop5M). Preparation of nitric oxide: A gas bulb fitted with a rubber injection septum was filled with nitric oxide from a cylinder (Union Carbide, Chicago, IL). 100 pl were removed with a syringe and injected into another gas bulb filled with 100 ml of distilled water, which had been previously gassed with helium for approximately 3 h, giving a stock solution of nitric oxide of 4 x M ~31. Calculations Contractions were expressed as absolute values (g) or as percent of contractions to KCl (60 mM), which were comparable in tissues from SHR and WKY (Table I). EDSOvalues were calculated as the negative logarithm of the concentration (determined in the individual rings), which caused 50% of the maximal contraction to endothelin. Relaxations to acetylcholine or nitric oxide were expressed as percent of the initial contraction to endothelin ( M). Student's t-test for paired observations was used for statistical comparison of rings from the same rat; the t-test for unpaired observations was used for comparisons between the two strains. Mean values from more than two groups were compared by one-way analysis of variance.
RESULTS Endothelin (lo-" to
M) caused concentration-
Fig. I . Contractions to endothelin (lo-" to M) in the aorta without endothelium from WKY and SHR. Contractions are expressed as percent of the maximal contraction to KCI (60 mM) and shown as meanskSEM. *indicates significant difference between arteries from WKY and SHR ( p < 0.05).
dependent contractions in all preparations. The maximal contraction was significantly smaller in rings with and without endothelium from the SHR compared to the WKY; the EDSOfor endothelin was not significantly different in the aorta of the two strains (Figs 1 and 2, Table 11). Removal of the endothelium intensified contractions to endothelin aortas from both strains. The ED50 to the peptide was lower after removal of the endothelium in rings from WKY, but not from SHR (Table 11).
ENDOTHELIN AND PROSTAGLANDINS RAT AORTA WITH ENDOTHELIUM
loo
1
...._. WKY ~
CONTROL 0
a
0 z
20 401
*
0-I
5HR
0
INWMETHACIN
cd I
-11
-10
-0
-0
-7
ENDOTHELIN (log M)
Fig. 2. Effect of indomethacin M) on contractions to M) in the aorta from WKY and endothelin (lo-" to SHR with endothelium. Contractions are expressed as percent of the maximal contraction to KCI (60 mM) and shown as meansf SEM. *indicates significant difference from control in SHR rings ( p < 0.05).
Endothelin and SHR aorta
47
Table 11. EDso values and maximal response (in % KCI) to endothelin in the thoracic aorta of WKY and SHR rats ~~
Maximal contraction
EDso WKY
SH R
WKY
SHR
without endothelium
8.17k0.07 8.46 k0.1Od
8.36k0.08 8.46k0.13
93.3f6.3 143.1 f 14.8"
53.6 f 1 3.7b 95.8 f 1 1.2ab
Indomethacin with endothelium without endothelium
8. I4 f0.09 8.36 f0.07d
8.02 f0.07' 8.18 f0.09
82.2 f4.4 122.7f9. l a
33.8 f9.1bc 86.1 f 1 5.7ab
Blood Press Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
Control with endothelium
EDSO values represent the negative logarithm of the concentration determined in each individual ring, which gives 50% of the maximal contraction to endothelin. Data are shown as meansf SEM. "significant different from rings with endothelium. bsignificantdifferent from WKY. 'significant different from untreated rings.
Acetylcholine (3 x lop5 M) caused comparable relaxations in rings with endothelium from both strains contracted with endothelin (lo-' M; WKY 89.4f2.6%, SHR 101.3f6.2%) Nitric oxide (3 x lop6 M) caused relaxations in rings without endothelium of aortas from both strains. The relaxations were biphasic: a rapid transient relaxation was followed by a smaller stable reduction in tension; only the maximal peak relaxation was compared. The relaxations were significantly larger in the SHR than in the WKY (WKY 89. I f5.6, SHR 108.2 f 10.6, p < 0.05). Indomethacin ( M) reduced contractions to endothelin in rings of SHR with endothelium, but not those in rings without endothelium; it did not affect contractions in rings of WKY, whether the endothelium was present or not (Fig. 2, Table 11).
DISCUSSION The present study confirms that endothelin is a vasoconstrictor in the isolated aorta of the SHR. The data obtained in the aorta from WKY rats without endothelium are in accordance with previous determinations of the biological activity of porcine endothelin in the aorta of that species [4]. Contractions to endothelin are reduced in the aorta of SHR rats compared to that of normotensive controls. This reduced responsiveness in the SHR aorta is at variance with the augmented responses reported by other investigators in the renal and mesenteric arteries of the SHR [5, 6, 71, but in agreement with the results reported by different laboratories comparing the responsiveness of the aorta of WKY and SHR to
endothelin [7-10]. The cause for the reduction in responsiveness to endothelin observed in the aorta is unknown. It cannot be explained by the altered function of the endothelium in hypertension [ 1 1,12,1J, since the reduction can be observed both in rings with and without endothelium. An altered production of vasoactive prostanoids [13] is unlikely to be the cause for reduction of contractions, since the inhibitor of cyclooxygenase, indomethacin, augmented rather than reduced the difference between the two strains. Since the response to high potassium was comparable in the SHR and WKY aortas, a major defect in the contractile process cannot be held responsible. Thus the smaller contraction in the aorta of the SHR is most likely due to a reduced responsiveness to endothelin in the vascular smooth muscle of the SHR. Theoretically, a chronically augmented exposure to endothelin might lead to a desensitization of receptors; however, measurement of plasma endothelin- 1 levels revealed similar levels in genetically hypertensive rats compared to normotensive controls [14, 151. The presence of endothelium inhibits contractions to most agonists in the rat aorta [16, 171. In the present study the inhibitory effect of the endothelium on contractions to endothelin could be demonstrated in aortas from both SHR and WKY. Since this inhibition can be observed after treatment with indomethacin, prostacyclin is not likely to be involved. It can be attributed to the basal release of endothelium-derived relaxing factor(s). In support of this interpretation, acetylcholine causes endothelium-dependent relaxations in rings from both strains. Nitric oxide, which is probably a major relaxing factor released by the endothelium [18, 19, 31, also inhibits contractions to
Blood Press Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
48
W . Auch-Schwelk and P . M . Vanhoutte
endothelin in both strains. The marginally increased response to nitric oxide in rings from SHR is explained best by the smaller contraction in response to the higher concentrations of endothelin used t o contract the preparations. The release by endothelin of contracting prostanoids from the smooth muscle of the rat aorta has been suggested by experiments using thromboxane A*/ prostaglandin HZreceptor antagonists [20].The present data do not provide any evidence for the release of vasoactive prostaglandins in response to endothelin- 1 in preparations without endothelium. However, the present experiments suggest that products of cyclooxygenase are involved in the modulatory influence of the endothelium in response to endothelin in the S H R , but not in the WKY. This interpretation is prompted by the observation that indomethacin (in a concentration known to abolish the production of prostanoids in the SHR-aorta [21]) reduces contractions in rings with but not without endothelium in the hypertensive strain. One explanation could be that exogenous endothelin stimulates the release of a contracting product of cyclooxygenase (e.g. thromboxane AZ [ 131) from the endothelium, which masks the inhibitory effect of nonprostanoid endothelium-derived relaxing factor. The response to endothelin in the S H R aorta is modified by the endothelium in a way similar t o the response to acetylcholine a n d serotonin. These substances cause larger contractions in the aorta of SHR compared to W K Y , presumably because they release endothelium-derived contracting factor which can be inhibited by indomethacin [22-261. Since endothelin is a peptide it is unlikely that its synthesis or release is regulated by cyclooxygenase. The direct effects of endothelin on the vascular smooth muscle are not prevented by indomethacin as is the case for oxygen-derived free radicals, which can be regarded as endothelium-derived contracting factor [27, 28, 23, 241. Taken together with the reduced responsiveness to the vasoconstrictor effect of the peptide, this does not support the concept that endothelin is the mediator of endothelium-dependent contractions in the S H R aorta.
ACKNOWLEDGEMENTS The authors thank Mr. Greg Brandt for technical assistance in the blood pressure measurements, Mrs. Helen Hendrickson and Mr. Robert Lorenz for preparing the figures and Mrs. Kathleen Kros for preparing the manuscript. This work was supported in part by the National Institute of Health, HL 35614. Dr. Auch-Schwelk was supported by the Deutsche Forschungsgemeinschaft (Au 76/1- I).
REFERENCES 1 . Liischer TF, Vanhoutte PM. Endothelium-dependent contractions to acetylcholine in the aorta of the spontaneously hypertensive rat. Hypertension 1986; 8: 344-8. 2, Yanagisawa M, Kurihara H, Kimura S, et al. Endothelin: A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature 1988; 332: 41 1-5. 3. Palmer RMJ, Ferridge AG, Moncada S . Nitric oxide release accounts for the biological activity of endothehum-derived relaxing factor. Nature (Lond) 1987; 327: 524-5. 4. Yanagisawa M, Inoue A, Ishikawa T, et al. Primary structure, synthesis, and biological activity of rat endothelin, an endothelium-derived vasoconstrictor peptide. Proc Natl Acad Sci (USA) 1988; 85: 6964-7. 5. Tomobe Y, Miyauchi T, Saito A, et al. Effects of endothelin on the renal artery from spontaneously hypertensive and Wistar Kyoto rats. Eur J Pharmacol 1988; 152: 373-4. 6. Miyauchi T, Ishikawa T, Tomobe Y, et al. Characteristics of pressor response to endothelin in spontaneously hypertensive and Wistar-Kyoto rats. Hypertension 1989; 14: 427-34. 7. Criscione L, Nellis P, Riniker B, et al. Reactivity and sensitivity of mesenteric vascular beds and aortic rings of spontaneously hypertensive rats to endothelin: effects of calcium entry bIockers. Br J Pharmacol 1990; I00 31-6. 8. Wright CE, Fozard JR. Differences in regional vascular sensitivity to endothelin- 1 between spontaneously hypertensive and normotensive Wistar Kyoto rats. Br J Pharmacol 1990; 100: 107-13. 9. Clozel M. Endothelin sensitivity and receptor binding in the aorta of spontaneously hypertensive rats. J Hypertens 1989; 7: 913-7. 10. Wu CC, Bohr DF. Role of the endothelium in the response to endothelin in hypertension. Hypertension 1991; 16: 677-81. 1 1 . Winquist RJ, Bunting PB, Baskin EP, Wallace AA. Decreased endothelium-dependent relaxation in New Zealand genetic hypertensive rats. J Hypertens 1984; 2: 541-6. 12. Konishi M, Su C. Role of endothelium in dilator responses of spontaneously hypertensive rat arteries. Hypertension 1983; 5: 881-6. 13. De Nucci G, Thomas R, D’Orleans-Juste, P, et al. Pressor effects of circulating endothelin are limited by its removal in the pulmonary circulation and by the release of prostacyclin and endothelium-derived relaxing factor. Proc Natl Acad Sci USA 1988; 85: 9797-9800. 14. Suzuki N, Miyauchi T, Tomobe Y, et al. Plasma concentrations of endothelin-1 in spontaneously hypertensive rats and DOCA-salt hypertensive rats. Biochem Biophys Res Commun 1990; 167: 941-7. 15. Kohno M, Murakawa K, Horio T, et al. Plasma immunoreactive endotheiin- 1 in experimental malignant hypertension. Hypertension 1991; 18: 93-100. 16. Martin W, Furchgott RF, Villani GM, Jothianandan D. Depression of contractile responses in rat aorta by spontaneously released endothelium-derived relaxing factor. J Pharmacol Exp Ther 1985; 232: 708-16. 17. Bullock GR, Taylor SG, Weston AH. Influence of the vascular endothelium on agonist-induced contractions and relaxations in rat aorta. Br J Pharmacol 1986; 89: 8 19-30.
Blood Press Downloaded from informahealthcare.com by Queen's University on 01/05/15 For personal use only.
Endothelin and SHR aorta
18. Furchgott RF. Studies on relaxation of rabbit aorta by sodium nitrite: The basis for the proposal that the acidactivatable inhibitory factor from bovine retractor penis is inorganic and the endothelium derived relaxing factor is nitric oxide. In: Vanhouette PM, ed. Vasodilatation: Vascular smooth muscle, peptides, autonomic nerves, and endothelium. New York: Raven Press, 1988: 401-14. 19 Ignarro LJ. Biological actions and properties of endothelium-derived nitric oxide formed and released from artery and vein. Circ Res 1988; 65: 1-21. 20. Reynolds EE, Mok LL. Role of thromboxane A2/ prostaglandin H2 receptor in the vasoconstrictor response of rat aorta to endothelin. J Pharmacol Exp Ther 1990; 252: 915-21. 21. Luscher TF, Romero JC, Vanhoutte PM. Bioassay of endothelium-derived vasoactive substances in the aorta of normotensive and spontaneously hypertensive rats. J Hypertens 1986; 4 (Suppl 6): S81-S3. 22. Liischer TF, Vanhoutte PM. Endothelium-dependent responses to platelets and serotonin in spontaneously hypertensive rats. Hypertension 1986; 8 (Suppl. 11): 5560. 23. Auch-Schwelk W, Katusic ZS, Vanhoutte PM. Contractions to oxygen-derived free radicals are augmented in the SHR aorta. Hypertension 1989; 13: 859-64.
49
24. Auch-Schwelk W, Katusic ZS, Vanhoutte PM. Thromboxane A2 receptor antagonists inhibit endotheliumdependent contractions. Hypertension 1990; 15: 699-703. 25. Auch-Schwelk W, Katusic Z , Vanhoutte PM. Nitric oxide inactivates endothelium-derived contracting factor in the rat aorta. Hypertension 1992; 19: 442-5. 26. Auch-Schwelk W, Vanhoutte PM. Endothelium-derived contracting factor released by serotonin in the aorta of the SHR. Am J Hypertens 1991; 4: 769-72. 27. Vanhoutte PM, Katusic ZS. Endothelium-derived contracting factor: endothelin and/or superoxide anion? TIPS 1988; 9: 229-30. 28. Katusic ZS, Vanhoutte PM. Superoxide anion is an endothelium-derived contracting factor. Am J Physiol 1989; 257: H33-H373. Submitted February 2, 1992; accepted February 25, 1992 Address for correspondence: Paul M. Vanhoutte Baylor College of Medicine Houston, TX 77030 U.S.A.
