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Antihypertensive effects of BQ-123, a selective endothelin ETA receptor antagonist, in spontaneously hypertensive rats treated with DOCA-salt
ejp ELSEVIER
European Journal of Pharmacology 259 (1994) 339-342
Short communication
Antihypertensive effects of BQ-123, a selective endothelin ET A receptor antagonist, in spontaneously hypertensive rats treated with DOCA-salt Megumu Okada a,*, Takahiro Fukuroda a, Koji Shimamoto a, Risa Takahashi Fumihiko Ikemoto a Mitsuo Yano b Masaru Nishikibe a
a,
9
a Pharmacology and b Biochemistry, Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Tsukuba Techno-Park Oho, Okubo 3, Tsukuba Ibaraki 300-33, Japan
Received 27 April 1994; accepted 17 May 1994
Abstract
We determined the antihypertensive effects of BQ-123 (cyclO(-D-Trp-o-Asp-L-Pro-D-VaI-L-Leu-), sodium salt), a selective endothelin ETA receptor antagonist, in spontaneously hypertensive rats treated with deoxycorticosterone acetate-salt (DOCA-salt SHR). BQ-123 (1-30 mg/kg/h) decreased blood pressure in DOCA-salt SHR in a dose-dependent manner, although plasma immunoreactive endothelin-1 did not significantly increase and the maximal contractile response to endothelin-1 in the aorta significantly decreased as compared with values observed in age-matched SHR. These results suggest that endogenous endothelin-1 is involved in the maintenance of hypertension in DOCA-salt SHR, and that circulating endothelin-1 is not sufficient to reflect the physiological role of endothelin-1. Key words: Endothelin; Endothelin receptor antagonist; Hypertension; DOCA-salt SHR (deoxycorticosterone acetate-salt spontaneously hypertensive rat)
1. Introduction
Endothelin-1 (Yanagisawa et al., 1988) is thought to be a pathophysiological factor in hypertension. However, it is unknown whether this peptide plays a substantial role in the maintenance of hypertension. We recently reported that BQ-123 (cyclo(-D-Trp-D-Asp-LPro-D-Val-L-Leu-), sodium salt), a selective endothelin E T A receptor antagonist, produced a significant decrease in blood pressure in stroke-prone spontaneously hypertensive rats (SHRSP), but not in spontaneously hypertensive rats (SHR) or normotensive Wistar Kyoto rats (WKY) (Ihara et al., 1992; Nishikibe et al., 1993). We also observed that plasma immunoreactive endothelin-1 was significantly elevated in SHRSP as compared with WKY (Nishikibe et al., 1993). These results convinced us that endogenous endothelin-1 is involved at least in part in the maintenance of high blood pressure in SHRSP. Kohno et al. (1991) reported that plasma immunoreactive endothelin-1 was increased in
* Corresponding author. Tel. 81-298-77-2000, fax 81-298-77-2028. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 1 4 - 2 9 9 9 ( 9 4 ) 0 0 2 9 2 - F
hypertension due to chronic treatment with deoxycorticosterone acetate-salt in SHR (DOCA-salt SHR). Larivi~re et al. (1993a, b) demonstrated increased immunoreactive endothelin-1 content and prepro endothelin-1 gene expression in the blood vessels of DOCA-salt hypertensive rats. These observations suggest that endothelin-1 contributes to the maintenance of blood pressure in certain forms of hypertension. In the present study, we therefore examined the effects of BQ-123 on blood pressure in DOCA-salt SHR, one of the models in which increased plasma a n d / o r tissue endothelin-1 has been reported, to assess the pathophysiological role of endogenous endothelin-1. We also determined the vascular contractile response to exogenous endothelin-1 in this model.
2. Materials and methods
Male SHR (Charles River, Japan) aged 10 weeks were used. The animals were treated with D O C A (Sigma, USA) once weekly at a subcutaneous dose of 25 m g / k g and were allowed free access to 1% NaCI drinking solution for 4 weeks.
340
M. Okada et al. /European Journal of Pharmacology' 259 (1994) 339 342
2.1. Measurement of blood pressure Blood pressure was measured under conscious and unrestrained conditions. SHR treated with DOCA-salt for 4 weeks were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally). Catheters were inserted into the lower abdominal aorta through the left femoral artery for blood pressure measurement and through the left femoral vein for drug administration. Each animal was placed in an individual cage for the continuous measurement of blood pressure with a polygraph (Nihon Kohden RM-6000, Japan) after a 24-h recovery period. BQ-123 (1, 10 and 30 m g / k g / h ) or physiological saline (0.5 m l / h ) was infused intravenously throughout the experimental period. Each dose of the drug was given to each rat.
2.2. Preparation of aortic rings One ring preparation was prepared from each rat. Thoracic aortas from DOCA-salt SHR and agematched SHR were removed and dissected free of connective tissue. The arteries were cut into rings (4 mm in length) and the endothelium was removed with a wet filter. The preparations were mounted in organ baths containing 5 ml of modified Krebs solution (NaC1, 115.0; KC1, 4.7; CaCl 2, 2.5; MgC12-6H20, 1.2; K H 2 P O 4, 1.2; NaHCO3, 25.0; and glucose, 5.56 raM) and were gassed with 95% O 2 and 5% CO 2. The initial tension was 2.0 g, and the tissues were equilibrated for 90 min. They were then challenged with 1 ~M norepinephrine, and denuding of the endothelium was checked by the inability of 0.1 mM acetylcholine to relax rings precontracted with norepinephrine. The concentration-response curves for endothelin-1 were obtained by cumulative addition of endothelin-1 (1 pM-3.3 nM) to the organ bath. Contractile responses were measured using an isometric transducer (Nihon Kohden TB-651T, Japan).
2.3. Measurement of plasma immunoreactive endothelin-1 Blood samples (10 ml) from the abdominal aorta of anesthetized DOCA-salt SHR and age-matched SHR were collected in a syringe containing 10 mg ethylenediaminetetraacetic acid (EDTA) and 5000 KIU aprotinin. The blood samples were centrifuged at 2000 × g for 15 min at 4°C and the plasma was stored at -20°C until assay. The procedure of Suzuki et al. (1989) was performed to extract immunoreactive endothelin-1 from plasma. Briefly, each sample was diluted with 3 volumes of 8% acetic acid. After centrifugation, the supernatant was applied by gravity through a Sep-Pak C18 cartridge (Waters Chromatography Division, Millipore Corp., Milford, MA, USA). After the cartridge was washed with pure water (MILL1-Q, Reagent Wa-
ter System), the definitely adsorbed peptides were eluted with 50% ethanol in 0.1% trifluoroacetic acid. After the solvent was evaporated with a centrifugal evaporator (model VC-960, Taitec Co., Japan), the dry residue was dissolved in 250 t~l of buffer D (0.1 M phosphate buffer, pH 7.0 containing 10% Black Ace from Snow Bland Milk Products Co., Japan, 0.4 M NaCi and 2 mM EDTA). Sandwich enzyme immunoassay for endothelin-1 was performed using immobilized anti-endothelin-(15-21) rabbit IgG and horseradish peroxidase-labeled anti-endothelin-l-(1-21) rabbit IgG Fab (Immuno-Biological Lab. Co., Japan). This assay reacts 100% with endothelin-l-(1-21) and cross-reacts 67% with endothelin-2 and < 0.01% with endothelin-3, big endothelin-1, big endothelin-2 and big endothelin-3. The data are presented as the means + S.E. Statistical analysis of the results was performed using unpaired t-tests and values were considered to be significant at P < 0.05.
3. Results
3.1. Effects of BQ-123 on blood pressure in DOCA-salt SHR The mean blood pressure before the administration of saline and BQ-123 1, 10 and 30 m g / k g / h was 167+10, 165+14, 167+13 and 182+16 mm Hg, respectively; no significant difference was seen between these values. Intravenous infusion of BQ-123 1 and 10 m g / k g / h markedly reduced blood pressure in DOCA-salt SHR in a dose-dependent manner, but 30 m g / k g / h did not produce further reductions in blood pressure. The antihypertensive effect developed gradually and plateaued at about 2 h after infusion. The maximal change in mean blood pressure induced by BQ-123 10 and 30 m g / k g / h was about - 4 0 mm Hg (Fig. 1).
3.2. Contractile response to endothelin-1 on aortic rings The contractile response to norepinephrine (1 /zM), which elicits a maximal response in SHR, was greater in DOCA-salt SHR than in SHR (0.87 _+0.15 vs. 0.59 + 0.05 g). In contrast, the maximal vasocontraction caused by endothelin-1 decreased significantly in DOCA-salt SHR as compared with age-matched SHR. However, pD 2 values did not differ significantly between DOCA-salt SHR and age-matched SHR; they were 10.07 _+0.28 and 9.60 _+0.12, respectively (Fig. 2).
3.3. Plasma concentration of immunoreactive endothelin- 1 The mean plasma immunoreactive endothelin-1 was slightly higher in DOCA-salt SHR than in age-matched
M. Okada et al. /European Journal of Pharmacology 259 (1994) 339-342 10
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Time after infusion of BQ-123 (h) Fig. 1. Effects of intravenous infusion of BQ-123 (e: 1 m g / k g / h , n = 7; • : 10 m g / k g / h , n = 7 and • : 30 m g / k g / h , n = 6) and saline ( o : 0.5 ml/h, n = 9 ) for 4 h on mean blood pressure (MBP) in conscious DOCA-salt SHR. Each point and vertical bar represents mean and S.E., respectively.
0.8"
0.6
P
0.4
0 U.
0.2 0.0 -12
-11 -10 -9 Endothelin-1 (log M)
-8
Fig. 2. Concentration-contraction curves for endothelin-1 in SHR (e, n = 8) and DOCA-salt SHR ( o , n = 6). Responses are expressed as absolute grams of contraction. Each point and vertical bar represents mean and S.E., respectively. * P < 0.05; significantly different from the SHR group.
SHR, but the difference was not significant (5.8 + 1.4 and 3.7 + 0.6 pg/ml, respectively).
4. D i s c u s s i o n
We previously reported that BQ-123 blocked the in vivo and in vitro response to exogenous endothelin-1 (Ihara et al., 1992) and produced a significant decrease in blood pressure in SHRSP, but not in SHR and WKY (Nishikibe et al., 1993). In the present study, BQ-123 1 and 10 m g / k g / h lowered blood pressure in DOCA-salt SHR in a dose-dependent manner, but 30 m g / k g / h did not produce further reductions in blood pressure. In a preliminary experiment, we measured
341
the plasma concentration of BQ-123 in rats. The plasma concentration at an infusion rate of 1 and 10 m g / k g / h was about 0.5-5 /zM. This concentration range was able to inhibit the pressor response to endothelin-1 (1 nmol/kg i.v.) by about 40-75%. Therefore, the dose of BQ-123 used in the present study was appropriate. Furthermore, the data obtained from infusing 30 rag/ k g / h suggest that there is a limit to the physiological role of endothelin-1 in the maintenance of high blood pressure in this model. These findings indicate that endogenous endothelin-1 is one of pathogenetic factors in certain forms of hypertension in rats. In SHRSP, plasma immunoreactive endothelin-1 levels were significantly elevated compared with those of WKY (Nishikibe et al., 1993). Kohno et al. (1991) reported that plasma immunoreactive endothelin-1 was significantly higher in DOCA-salt SHR than in SHR or salt-loading SHR. In the present study, plasma immunoreactive endothelin-1 tended to be higher in DOCA-salt SHR than in SHR, although the difference was not significant. The reason for the lack of increase in plasma immunoreactive endothelin-1 in our study remains to be explained. It may be a reflection of differences between the dose of DOCA a n d / o r the length of the experimental period. We did not measure the plasma concentration of immunoreactive endothelin-1 in WKY in this experiment. However, we have demonstrated that plasma immunoreactive endothelin1 levels in WKY do not differ from those of SHR at 15 weeks of age (data not shown). It has been reported that plasma immunoreactive endothelin-1 levels in DOCA-salt hypertensive rats are not different from those in normotensive rats (Suzuki et al., 1990; Kohno et al., 1991; Nguyen et al., 1992; Larivi~re et al., 1993a). However, Larivi~re et al. (1993a, b) reported that immunoreactive endothelin-1 content and prepro endothelin-1 gene expression in the blood vessels of DOCA-salt hypertensive rats were significantly increased as compared with values in uninephrectomized rats, suggesting that endothelin-1 is involved in the maintenance of blood pressure in DOCA-salt hypertensive rats although plasma immunoreactive endothelin-I did not increase in either type of rat. In addition to these reports, the response to endothelin-1 in the thoracic aorta and mesenteric artery was significantly decreased and the binding of endothelin-1 to vascular membranes was significantly lower in DOCA-salt hypertensive rats than in uninephrectomized rats (Nguyen et al., 1992). These results led to the speculation that high levels of vascular endothelin-1 may explain the clown-regulation of vascular endothelin receptors, resulting in a depressed vasoconstrictive response to this peptide. The observation of decreased endothelin-1 binding sites without changes in binding affinity when cultured vascular smooth muscle cells were incubated with unlabeled endothelin-1 (Hirata et
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M. Okada et al. / European Journal of Pharmacology 259 (1994) 339-342
al., 1988; Roubert et al., 1990) supports this speculation. We saw a decreased response to endothelin-1, but not to norepinephrine, in the thoracic aorta of DOCA-salt SHR in comparison with age-matched SHR. We also observed that the response to endothelin-1 in the thoracic aorta of WKY did not differ from that in age-matched SHR (data not shown). This finding infers an increase in the vascular content of endothelin-1 in DOCA-salt SHR. Taken together, these observations suggest that circulating endothelin-1 may not reflect local vascular production. Indeed, endothelial cells in culture appear to release more endothelin-1 in the abluminal than in the luminal direction (Wagner et al., 1992). More recently, it was reported that phosphoramidon reduces blood pressure in DOCA-salt rats by inhibition of endothelin conversion (Vemulapalli et al., 1993). We also found in our preliminary study that BQ-123 significantly lowered blood pressure in DOCA-salt hypertensive rats (unpublished data). In conclusion, the results of the present study suggest that endogenous endothelin-1 plays, at least in part, a role in the maintenance of blood pressure in DOCA-salt SHR, and that circulating endothelin-1 is not sufficient to reflect the physiological role of endothelin-1.
References Hirata, Y., H. Yoshimi, S. Takaichi, M. Yanagisawa and T. Masaki, 1988, Binding and receptor down-regulation of novel vasoconstrictor endothelin in cultured rat vascular smooth muscle cells, FEBS Lett. 239, 13. Ihara, M., K. Noguchi, T. Saeki, T. Fukuroda, S. Tsuchida, S. Kimura, T. Fukami, K. Ishikawa, M. Nishikibe and M. Yano,
1992, Biological profile of highly potent novel endothelin antagonists selective for the ET A receptor, Life Sci. 50, 247. Kohno, M., K. Murakawa, T. Horio, K. Yokokawa, K. Yasunari, T. Fukui and T. Takeda, 1991, Plasma immunoreactive endothelin-I in experimental malignant hypertension, Hypertension 18, 93. Larivi~re, R., G. Thibault and E.L. Schiffrin, 1993a, Increased endothelin-1 content in blood vessels of deoxycorticosterone acetate-salt hypertensive but not in spontaneously hypertensive rats, Hypertension 21, 294. Larivi~re, R., R. Day and E.L. Schiffrin, 1993b, Increased expression of endothelin-I gene in blood vessels of deoxycorticosterone acetate-salt hypertensive rats, Hypertension 21,916. Nguyen, P.V., A. Parent, L.Y. Deng, J.P. Flfickiger, G. Thibault and E.L. Schiffrin, 1992, Endothelin vascular receptors and responses in deoxycorticosterone acetate-salt hypertensive rats, Hypertension 19 (Suppl. 2), II-98. Nishikibe, M., S. Tsuchida, M. Okada, T. Fukuroda, K. Shimamoto, M. Yano, K. Ishikawa and F. Ikemoto, 1993, Antihypertensive effect of a newly synthesized endothelin antagonist, BQ-123, in a genetic hypertensive model, Life Sci. 52, 717. Roubert, P., V. Gillard, P. Plas, P.E. Chabrier and P. Braquet, 1990, Down-regulation of endothelin binding sites in rat vascular smooth muscle cells, Am. J. Hypertens. 3, 310. Suzuki, N., H. Matsumoto, C. Kitada, T. Masaki and M. Fuiino, 1989, A sensitive sandwich-enzyme immunoassay for human endothelin, J. Immunol. Methods 118, 245. Suzuki, N., T. Miyauchi, Y. Tomobe, H. Matsumoto, K. Goto, T. Masaki and M. Fuiino, 1990, Plasma concentrations of endothelin-1 in spontaneously hypertensive rats and DOCA-salt hypertensive rats, Biochem. Biophys. Res. Commun. 167, 941. Vemulapalli, S., R.W. Watkins, A. Brown, J. Cook, V. Bernardino and P.J.S. Chiu, 1993, Disparate effects of phosphoramidon on blood pressure in SHR and DOCA-salt hypertensive rats, Life Sci. 53, 783. Wagner, O.F., G. Christ, J. Wojta, H. Vierhapper, S. Parzer, P.J. Nowotny, B. Schneider, W. Waldh~usl and B.R. Binder, 1992, Polar secretion of endothelin-1 by cultured endothelial cells, J. Biol. Chem. 267, 16066. Yanagisawa, M., H. Kurihara, S. Kimura, Y. Tomobe, M. Kobayashi, Y. Mitsui, Y. Yazaki, K. Goto and T. Masaki, 1988, A novel potent vasoconstrictor peptide produced by vascular endothelial cells, Nature 332, 411.
European Journal of Pharmacology 259 (1994) 339-342
Short communication
Antihypertensive effects of BQ-123, a selective endothelin ET A receptor antagonist, in spontaneously hypertensive rats treated with DOCA-salt Megumu Okada a,*, Takahiro Fukuroda a, Koji Shimamoto a, Risa Takahashi Fumihiko Ikemoto a Mitsuo Yano b Masaru Nishikibe a
a,
9
a Pharmacology and b Biochemistry, Tsukuba Research Institute, Banyu Pharmaceutical Co., Ltd., Tsukuba Techno-Park Oho, Okubo 3, Tsukuba Ibaraki 300-33, Japan
Received 27 April 1994; accepted 17 May 1994
Abstract
We determined the antihypertensive effects of BQ-123 (cyclO(-D-Trp-o-Asp-L-Pro-D-VaI-L-Leu-), sodium salt), a selective endothelin ETA receptor antagonist, in spontaneously hypertensive rats treated with deoxycorticosterone acetate-salt (DOCA-salt SHR). BQ-123 (1-30 mg/kg/h) decreased blood pressure in DOCA-salt SHR in a dose-dependent manner, although plasma immunoreactive endothelin-1 did not significantly increase and the maximal contractile response to endothelin-1 in the aorta significantly decreased as compared with values observed in age-matched SHR. These results suggest that endogenous endothelin-1 is involved in the maintenance of hypertension in DOCA-salt SHR, and that circulating endothelin-1 is not sufficient to reflect the physiological role of endothelin-1. Key words: Endothelin; Endothelin receptor antagonist; Hypertension; DOCA-salt SHR (deoxycorticosterone acetate-salt spontaneously hypertensive rat)
1. Introduction
Endothelin-1 (Yanagisawa et al., 1988) is thought to be a pathophysiological factor in hypertension. However, it is unknown whether this peptide plays a substantial role in the maintenance of hypertension. We recently reported that BQ-123 (cyclo(-D-Trp-D-Asp-LPro-D-Val-L-Leu-), sodium salt), a selective endothelin E T A receptor antagonist, produced a significant decrease in blood pressure in stroke-prone spontaneously hypertensive rats (SHRSP), but not in spontaneously hypertensive rats (SHR) or normotensive Wistar Kyoto rats (WKY) (Ihara et al., 1992; Nishikibe et al., 1993). We also observed that plasma immunoreactive endothelin-1 was significantly elevated in SHRSP as compared with WKY (Nishikibe et al., 1993). These results convinced us that endogenous endothelin-1 is involved at least in part in the maintenance of high blood pressure in SHRSP. Kohno et al. (1991) reported that plasma immunoreactive endothelin-1 was increased in
* Corresponding author. Tel. 81-298-77-2000, fax 81-298-77-2028. 0014-2999/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 0 1 4 - 2 9 9 9 ( 9 4 ) 0 0 2 9 2 - F
hypertension due to chronic treatment with deoxycorticosterone acetate-salt in SHR (DOCA-salt SHR). Larivi~re et al. (1993a, b) demonstrated increased immunoreactive endothelin-1 content and prepro endothelin-1 gene expression in the blood vessels of DOCA-salt hypertensive rats. These observations suggest that endothelin-1 contributes to the maintenance of blood pressure in certain forms of hypertension. In the present study, we therefore examined the effects of BQ-123 on blood pressure in DOCA-salt SHR, one of the models in which increased plasma a n d / o r tissue endothelin-1 has been reported, to assess the pathophysiological role of endogenous endothelin-1. We also determined the vascular contractile response to exogenous endothelin-1 in this model.
2. Materials and methods
Male SHR (Charles River, Japan) aged 10 weeks were used. The animals were treated with D O C A (Sigma, USA) once weekly at a subcutaneous dose of 25 m g / k g and were allowed free access to 1% NaCI drinking solution for 4 weeks.
340
M. Okada et al. /European Journal of Pharmacology' 259 (1994) 339 342
2.1. Measurement of blood pressure Blood pressure was measured under conscious and unrestrained conditions. SHR treated with DOCA-salt for 4 weeks were anesthetized with sodium pentobarbital (50 mg/kg, intraperitoneally). Catheters were inserted into the lower abdominal aorta through the left femoral artery for blood pressure measurement and through the left femoral vein for drug administration. Each animal was placed in an individual cage for the continuous measurement of blood pressure with a polygraph (Nihon Kohden RM-6000, Japan) after a 24-h recovery period. BQ-123 (1, 10 and 30 m g / k g / h ) or physiological saline (0.5 m l / h ) was infused intravenously throughout the experimental period. Each dose of the drug was given to each rat.
2.2. Preparation of aortic rings One ring preparation was prepared from each rat. Thoracic aortas from DOCA-salt SHR and agematched SHR were removed and dissected free of connective tissue. The arteries were cut into rings (4 mm in length) and the endothelium was removed with a wet filter. The preparations were mounted in organ baths containing 5 ml of modified Krebs solution (NaC1, 115.0; KC1, 4.7; CaCl 2, 2.5; MgC12-6H20, 1.2; K H 2 P O 4, 1.2; NaHCO3, 25.0; and glucose, 5.56 raM) and were gassed with 95% O 2 and 5% CO 2. The initial tension was 2.0 g, and the tissues were equilibrated for 90 min. They were then challenged with 1 ~M norepinephrine, and denuding of the endothelium was checked by the inability of 0.1 mM acetylcholine to relax rings precontracted with norepinephrine. The concentration-response curves for endothelin-1 were obtained by cumulative addition of endothelin-1 (1 pM-3.3 nM) to the organ bath. Contractile responses were measured using an isometric transducer (Nihon Kohden TB-651T, Japan).
2.3. Measurement of plasma immunoreactive endothelin-1 Blood samples (10 ml) from the abdominal aorta of anesthetized DOCA-salt SHR and age-matched SHR were collected in a syringe containing 10 mg ethylenediaminetetraacetic acid (EDTA) and 5000 KIU aprotinin. The blood samples were centrifuged at 2000 × g for 15 min at 4°C and the plasma was stored at -20°C until assay. The procedure of Suzuki et al. (1989) was performed to extract immunoreactive endothelin-1 from plasma. Briefly, each sample was diluted with 3 volumes of 8% acetic acid. After centrifugation, the supernatant was applied by gravity through a Sep-Pak C18 cartridge (Waters Chromatography Division, Millipore Corp., Milford, MA, USA). After the cartridge was washed with pure water (MILL1-Q, Reagent Wa-
ter System), the definitely adsorbed peptides were eluted with 50% ethanol in 0.1% trifluoroacetic acid. After the solvent was evaporated with a centrifugal evaporator (model VC-960, Taitec Co., Japan), the dry residue was dissolved in 250 t~l of buffer D (0.1 M phosphate buffer, pH 7.0 containing 10% Black Ace from Snow Bland Milk Products Co., Japan, 0.4 M NaCi and 2 mM EDTA). Sandwich enzyme immunoassay for endothelin-1 was performed using immobilized anti-endothelin-(15-21) rabbit IgG and horseradish peroxidase-labeled anti-endothelin-l-(1-21) rabbit IgG Fab (Immuno-Biological Lab. Co., Japan). This assay reacts 100% with endothelin-l-(1-21) and cross-reacts 67% with endothelin-2 and < 0.01% with endothelin-3, big endothelin-1, big endothelin-2 and big endothelin-3. The data are presented as the means + S.E. Statistical analysis of the results was performed using unpaired t-tests and values were considered to be significant at P < 0.05.
3. Results
3.1. Effects of BQ-123 on blood pressure in DOCA-salt SHR The mean blood pressure before the administration of saline and BQ-123 1, 10 and 30 m g / k g / h was 167+10, 165+14, 167+13 and 182+16 mm Hg, respectively; no significant difference was seen between these values. Intravenous infusion of BQ-123 1 and 10 m g / k g / h markedly reduced blood pressure in DOCA-salt SHR in a dose-dependent manner, but 30 m g / k g / h did not produce further reductions in blood pressure. The antihypertensive effect developed gradually and plateaued at about 2 h after infusion. The maximal change in mean blood pressure induced by BQ-123 10 and 30 m g / k g / h was about - 4 0 mm Hg (Fig. 1).
3.2. Contractile response to endothelin-1 on aortic rings The contractile response to norepinephrine (1 /zM), which elicits a maximal response in SHR, was greater in DOCA-salt SHR than in SHR (0.87 _+0.15 vs. 0.59 + 0.05 g). In contrast, the maximal vasocontraction caused by endothelin-1 decreased significantly in DOCA-salt SHR as compared with age-matched SHR. However, pD 2 values did not differ significantly between DOCA-salt SHR and age-matched SHR; they were 10.07 _+0.28 and 9.60 _+0.12, respectively (Fig. 2).
3.3. Plasma concentration of immunoreactive endothelin- 1 The mean plasma immunoreactive endothelin-1 was slightly higher in DOCA-salt SHR than in age-matched
M. Okada et al. /European Journal of Pharmacology 259 (1994) 339-342 10
-r
-10
E
E
D.
-30 ¢-
-404 e-
-50
tO
-60
"700
2
a
4
Time after infusion of BQ-123 (h) Fig. 1. Effects of intravenous infusion of BQ-123 (e: 1 m g / k g / h , n = 7; • : 10 m g / k g / h , n = 7 and • : 30 m g / k g / h , n = 6) and saline ( o : 0.5 ml/h, n = 9 ) for 4 h on mean blood pressure (MBP) in conscious DOCA-salt SHR. Each point and vertical bar represents mean and S.E., respectively.
0.8"
0.6
P
0.4
0 U.
0.2 0.0 -12
-11 -10 -9 Endothelin-1 (log M)
-8
Fig. 2. Concentration-contraction curves for endothelin-1 in SHR (e, n = 8) and DOCA-salt SHR ( o , n = 6). Responses are expressed as absolute grams of contraction. Each point and vertical bar represents mean and S.E., respectively. * P < 0.05; significantly different from the SHR group.
SHR, but the difference was not significant (5.8 + 1.4 and 3.7 + 0.6 pg/ml, respectively).
4. D i s c u s s i o n
We previously reported that BQ-123 blocked the in vivo and in vitro response to exogenous endothelin-1 (Ihara et al., 1992) and produced a significant decrease in blood pressure in SHRSP, but not in SHR and WKY (Nishikibe et al., 1993). In the present study, BQ-123 1 and 10 m g / k g / h lowered blood pressure in DOCA-salt SHR in a dose-dependent manner, but 30 m g / k g / h did not produce further reductions in blood pressure. In a preliminary experiment, we measured
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the plasma concentration of BQ-123 in rats. The plasma concentration at an infusion rate of 1 and 10 m g / k g / h was about 0.5-5 /zM. This concentration range was able to inhibit the pressor response to endothelin-1 (1 nmol/kg i.v.) by about 40-75%. Therefore, the dose of BQ-123 used in the present study was appropriate. Furthermore, the data obtained from infusing 30 rag/ k g / h suggest that there is a limit to the physiological role of endothelin-1 in the maintenance of high blood pressure in this model. These findings indicate that endogenous endothelin-1 is one of pathogenetic factors in certain forms of hypertension in rats. In SHRSP, plasma immunoreactive endothelin-1 levels were significantly elevated compared with those of WKY (Nishikibe et al., 1993). Kohno et al. (1991) reported that plasma immunoreactive endothelin-1 was significantly higher in DOCA-salt SHR than in SHR or salt-loading SHR. In the present study, plasma immunoreactive endothelin-1 tended to be higher in DOCA-salt SHR than in SHR, although the difference was not significant. The reason for the lack of increase in plasma immunoreactive endothelin-1 in our study remains to be explained. It may be a reflection of differences between the dose of DOCA a n d / o r the length of the experimental period. We did not measure the plasma concentration of immunoreactive endothelin-1 in WKY in this experiment. However, we have demonstrated that plasma immunoreactive endothelin1 levels in WKY do not differ from those of SHR at 15 weeks of age (data not shown). It has been reported that plasma immunoreactive endothelin-1 levels in DOCA-salt hypertensive rats are not different from those in normotensive rats (Suzuki et al., 1990; Kohno et al., 1991; Nguyen et al., 1992; Larivi~re et al., 1993a). However, Larivi~re et al. (1993a, b) reported that immunoreactive endothelin-1 content and prepro endothelin-1 gene expression in the blood vessels of DOCA-salt hypertensive rats were significantly increased as compared with values in uninephrectomized rats, suggesting that endothelin-1 is involved in the maintenance of blood pressure in DOCA-salt hypertensive rats although plasma immunoreactive endothelin-I did not increase in either type of rat. In addition to these reports, the response to endothelin-1 in the thoracic aorta and mesenteric artery was significantly decreased and the binding of endothelin-1 to vascular membranes was significantly lower in DOCA-salt hypertensive rats than in uninephrectomized rats (Nguyen et al., 1992). These results led to the speculation that high levels of vascular endothelin-1 may explain the clown-regulation of vascular endothelin receptors, resulting in a depressed vasoconstrictive response to this peptide. The observation of decreased endothelin-1 binding sites without changes in binding affinity when cultured vascular smooth muscle cells were incubated with unlabeled endothelin-1 (Hirata et
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al., 1988; Roubert et al., 1990) supports this speculation. We saw a decreased response to endothelin-1, but not to norepinephrine, in the thoracic aorta of DOCA-salt SHR in comparison with age-matched SHR. We also observed that the response to endothelin-1 in the thoracic aorta of WKY did not differ from that in age-matched SHR (data not shown). This finding infers an increase in the vascular content of endothelin-1 in DOCA-salt SHR. Taken together, these observations suggest that circulating endothelin-1 may not reflect local vascular production. Indeed, endothelial cells in culture appear to release more endothelin-1 in the abluminal than in the luminal direction (Wagner et al., 1992). More recently, it was reported that phosphoramidon reduces blood pressure in DOCA-salt rats by inhibition of endothelin conversion (Vemulapalli et al., 1993). We also found in our preliminary study that BQ-123 significantly lowered blood pressure in DOCA-salt hypertensive rats (unpublished data). In conclusion, the results of the present study suggest that endogenous endothelin-1 plays, at least in part, a role in the maintenance of blood pressure in DOCA-salt SHR, and that circulating endothelin-1 is not sufficient to reflect the physiological role of endothelin-1.
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