European Journal of Pharmacology, 229 (19921 137 142
137
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52806
Effect of endothelin-3 on cytosolic calcium level in vascular endothelium and on smooth muscle contraction Sri Agus Sudjarwo, Masatoshi Hori and Hideaki Karaki
Department of Veterinao' Pharmacology, FaculO,of Agriculture, The Uni~'ersityof Tokyo, Bunkyo-ku, Tokyo 113, Japan Received 7 September 1992, accepted 22 September 1992
In isolated rat aorta, endothelin (ET)-3 increased cytosolic Ca 2+ ([Ca2+] i) in the endothelium at a concentration (100 nM) which had little effect on muscle resting tone. In the absence of external Ca 2 ~, ET-3 still transiently increased endothelial [Ca2+]i. Verapamil (10 p.M) did not change the effects of ET-3. In aortas stimulated with 100 nM norepinephrine, 100 nM ET-3 relaxed the muscle with an increase in endothelial [Ca2+]i. An inhibitor of nitric oxide synthase, 100 p.M N~;-monomethyl-Larginine, inhibited the relaxant effect of ET-3 but not the increase in endothelial [Ca z+ ]i. In the absence of the endothelium or in the presence of an antagonist of ET B receptors, 3 # M IRL 1038, the ET-3-induced increase in endothelial [Ca2+]i and relaxation of norepinephrine-induced contraction were inhibited. Under these conditions, ET-3 increased smooth muscle [Ca 2 +]i and induced contraction, both of which were inhibited by an inhibitor of ET A receptors, 3/xM BQ123. These results suggest that ET-3 acts on ET u receptors in the vascular endothclium to increase [Ca 2 ~]i by releasing Ca 2 ~ from storage sites and by opening non-L type Ca 2+ channels, activates nitric oxide synthase, releases nitric oxide and relaxes vascular smooth muscle. Although ET-3 also activates ET A receptors in smooth muscle to induce contraction, this effect is overcome by the relaxant effect mediated by ET B receptors.
Endothelin-3; ETr3 receptors: Endothelium-dependent relaxation; Ca 2+ levels (cytosolic); Vascular (rat aorta); (Relaxation)
1. Introduction T h e newly f o u n d vasoactive p e p t i d e s , e n d o t h e l i n s , have t h r e e isoforms, e n d o t h e l i n ( E T ) - I , ET-2 and ET-3 ( Y a n a g i s a w a et al., 1988; I n o u e et al., 1989). T h e s e e n d o t h e l i n s act on E T A r e c e p t o r s , which are relatively selectively a c t i v a t e d by ET-1 a n d E T - 2 ( A r a i et al., 1990), a n d on E T B r e c e p t o r s , which a r e a c t i v a t e d nonselectively by t h e t h r e e e n d o t h e l i n isoforms ( S a k u r a i et al., 1990). E T A r e c e p t o r s are f o u n d in vascular s m o o t h m u s c l e w h e r e a s E T u r e c e p t o r s a r e f o u n d in nerves a n d t h e v a s c u l a r e n d o t h e l i u m ( F u j i t a n i et al., 1992; S a k u r a i et al., 1992). It has b e e n shown that e n d o t h e l i n s transiently relax v a s c u l a r s m o o t h muscle, possibly by releasing e n d o t h e l i u m - d e r i v e d relaxing f a c t o r ( E D R F ) ( D e Nucci et al., 1988; S a k a t a et al., 1989). E D R F is r e l e a s e d from the v a s c u l a r e n d o t h e l i u m a n d relaxes s m o o t h muscle by i n c r e a s i n g the cyclic
Correspondence to: H. Karaki, Department of Veterinary Pharmacology, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan.
G M P c o n t e n t ( F u r c h g o t t , 1984). A t least some of the effects of E D R F are d u e to nitric oxide synthesized from L - a r g i n i n e by a Ca 2+- a n d c a l m o d u l i n - d e p e n d e n t enzyme, nitric oxide synthase (for review see M o n c a d a et al., 1991). In o r d e r to u n d e r s t a n d the effects of e n d o t h e l i n s on b l o o d vessels, it is i m p o r t a n t to m e a s u r e the functions of e n d o t h e l i u m a n d s m o o t h muscle simultaneously. Using i s o l a t e d rat a o r t a strips, Sato et al. (1990) rep o r t e d a m e t h o d to m e a s u r e cytosolic Ca 2+ levels ([ Ca2+ ]i) in e n d o t h e l i a l cells a n d muscle tension simult a n e o u s l y a n d f o u n d that t h e r e was a positive c o r r e l a tion b e t w e e n the i n c r e a s e in e n d o t h e l i a l [Ca2+] i and the e n d o t h e l i u m - d e p e n d e n t r e l a x a t i o n due to c a r b a chol and histamine. In the p r e s e n t e x p e r i m e n t s , we e x a m i n e d the effects of ET-3, an a n t a g o n i s t of E T B r e c e p t o r s , I R L 1038 ( U r a d e et al., 1992), and an antagonist of E T A r e c e p t o r s , BQ123 ( I h a r a et al., 1992), on e n d o t h e l i a l [Ca2+]~ a n d muscle t o n e in isolated rat a o r t a strips. O u r results suggest that ET-3 acts on ET~ r e c e p t o r s , i n c r e a s e s e n d o t h e l i a l [Ca 2 +]i, activates nitric oxide synthase, r e l e a s e s nitric oxide and relaxes vascular s m o o t h muscle.
138 2. Materials and methods
2.1. Preparations, solutions and measurement of muscle tension The thoracic aorta was isolated from male Wistar rats (250-300 g), cut into spiral strips (1-2 mm in width and 5 - 7 m m in length) and placed in normal physiological salt solution which contained (raM): NaCI 136.9, KCI 5.4, CaCI 2 1.5, MgCI 2 1.0, NaHCO~ 23.8, ethylenediamine-tetraacetic acid 0.01 and glucose 5.5. In some experiments, the endothelium was removed by gently rubbing the intimal surface with a finger moistened with the above solution. A high K ÷ solution was made by substituting 69.6 mM NaC1 with equimolar KC1. Ca2+-free solution was made by removing CaCl~ and adding 0.5 mM E G T A . Thesc solutions were saturated with a mixture of 95% O 2 and 5% CO 2 at 37°C and pH 7.4. Muscle tension was recorded isometrically with a force-displacement transducer. Each muscle strip was attached to a holder under a resting tension of 1 g and equilibrated for 60-90 min in a 10-ml muscle bath until the contractile response to the high K + solution had become stable. The functional integrity of the vascular endothelium was assessed by measuring whether 1 /xM carbachol induced almost complete relaxation ( > 90%) in aortas stimulated with 100 nM norepinephrine (Karaki and Nagase, 1987).
1989) and also because we measured [Ca2+]i simultaneously in different tissues (smooth muscle and endothelium) with different fura-2 contents. The ratio of F340 to F380 (R340/380) was used as an indicator of [Ca2+]i , and a quantitative comparison of [Ca2+]i was made by taking resting and high K+-stimulated [Ca -~~]i as 0 and 100%, respectively, unless stated otherwise. 2.3. Chemicals ET-3 was purchased from the Peptidc Institute (Osaka, Japan). Other chemicals used were an E T B antagonist, I R L 1038 ([Cysll-Cys 15] ET-I (11-21)), an ET a antagonist, BQ123 (cyclo[-Asp-Pro-Val-Leu-Trp-]) (donated by Ciba-Geigy Japan, Takarazuka, Japan), verapamil hydrochloride, NG-monomethyl-L-argininc (L-NMMA; Sigma Chemicals, St. Louis, MO, U.S.A.) and norepinephrinc bitartrate (Wako Pure Chemicals, Tokyo, Japan). Preliminary experiments confirmed that these chemicals did not interfere with fura-2 fluorescence. 2.4. Statistics The results of the experiments are expressed as means _+ S.E.M. Unpaired Student's t-test was used for statistical analysis of the results.
3. Results 2.2. Simultaneous measurement of muscle tension and
cytosolic Ca: + let'el
3.1. £ffects q[ ET-3 on resting muscle
Muscle strips were incubated with 5 /xM of the acetoxymcthyl ester of fura-2 ( f u r a - 2 / A M ) for 3 - 5 h at room temperature. The noncytotoxic detergent, cremophor EL (0.02%), was also added to increase the solubility of f u r a - 2 / A M . Experiments were performed with a fluorimeter (CAF-100, Japan Spectroscopic, Tokyo, Japan) as described previously (Sat() et al., 1990). The muscle strip was held horizontally in a temperature-controlled organ bath. One end of the muscle strip was connected to a force-displacement transducer to monitor mechanical activity. The endothelial surface of the strip was excited alternately by light at 340 nm and 380 nm (48 He) and the intensity of fluorescence at 500 nm (F340 and F380) was measured at the endothelial surface. Ca ~+ signals thus obtained derive from both the endothelium and smooth muscle cells. In some experiments, the endothelium was removed to measure [Ca2+]i in smooth muscle cells. By comparing the total and the smooth muscle [Ca2+]i, it is possible to estimate changes in endothelial [Ca2+]~ (Sato et al., 199(I). Absolute [Ca2+]i was not calculated because the dissociation constant of fura-2 for Ca 2+ might bc diffcrcnt from that obtained in vitro (Karaki,
As shown in fig. 1A, 100 nM ET-3 increased [Ca2+]i to 103.2 _+ 23.5% (n = 5) without changing muscle tone. Carbachol (1 /xM) also increased [Ca2+]i to 199.0 _+ 45.8% (n = 6) without changing muscle tension. In contrast, high K + increased both [Ca2+]i and muscle tension (to 100%, respectively). An antagonist of ET B receptors, 3 # M I R L 1038, did not change the effects of high K + and carbachol (n - 4 each). In the presence of 3 /xM 1RL 1038, however, 1(10 nM ET-3 increased both [Ca2+]~ (48.9_+ 8.4%, n = 4) and muscle tension (46.8_+ 4.6%, n = 4), and these changes were completely inhibited by an antagonist of ET A receptors, 3 # M BQI23 (n = 4 each). The same concentration of BQ123 did not modify the effects of high K + and carbachol (n = 4 each). In endothelium-denuded muscle (fig. 1B), high K" also increased [CaZ+] i and muscle tension (to 100%, respectively). Carbachol (1 /xM) was ineffective (n = 5) whereas 100 nM ET-3 increased [Ca~+]i to 57.9_+ 16.5% (n = 4) and muscle tension to 70.8 ± 11.4~: (n = 4). The effects of ET-3 were completely inhibited by 3 /xM BQ123 whereas 3 /xM I R L 1038 was ineffective (n = 3 each).
139 A. End
(+) - Ioo
A. End (+)
(%)
B. End (-)
R340/3B0
0 -loo(%)
Tension
0.5 g KCI
ET-3
KCI
R340/380
~
~ o
CCh
/
5 min B. End
(-)
Tension ~
t
R340/380
Tens,on/ l ~ ~ ET-3
KCI
0.5g
NE ET-3 CCh
/
KCI
0
CCh
Fig. 1. Effects of ET-3 (100 nM) and carbachol (CCh, 1 /xM) on cytosolic Ca2+ level (upper trace, expressed as R340/380) and muscle tension (lower trace) in rat aortas with and without endothelium (A: End(+) and B: End(-), respectively). 100% represents eytosolic Ca2+ stimulated by high K +. The trace shows typical results from one of four experiments. Figure 2 A shows the effects of ET-3 and carbachol on endothelial [Ca2+]i in the absence of external Ca 2+. Addition of 100 n M ET-3 transiently increased [Ca2+]~ to 64.8 _+ 6.2% (n = 4). Sequential addition of 1 /xM carbachol again transiently increased [Ca2+] i to 74.4 _+ 20.4% (n = 4). W h e n applied first, carbachol transiently increased [Ca2+] i to 80.3 _+ 12.8% (n = 4), which was not different from the value m e a s u r e d in the strips p r e t r e a t e d with 100 nM ET-3. T h e increase in [Ca2+]~ was not followed by a c h a n g e in muscle tone. As shown in fig. 2B, neither 100 n M ET-3 nor 1 /xM carbachol c h a n g e d [Ca2+]i in the absence of the e n d o t h e l i u m (n = 4).
NE
ET-3 CCh
5 min
Fig. 3. Effects of ET-3 (100 nM) and carbachol (CCh, 1 /xM) on cytosolic Ca 2+ level (upper trace, expressed as R340/380) and muscle tension (lower trace) in rat aortas with and without endothelium (A: End( + ) and B; End(- ), respectively) in the presence of 100 nM norepinephrine (NE). 100% represents cytosolic Ca2+ stimulated by high K +. The trace shows typical results from one of four to five experiments.
iment, [Ca2+]i and muscle tension m e a s u r e d 10 min after the addition of n o r e p i n e p h r i n e were taken as 100%. Addition of 100 nM ET-3 transiently relaxed the sustained contraction induced by n o r e p i n e p h r i n e to 61.6 _+ 8.5% (n = 5). This change followed a transient increase in [Ca2+]i to 159.4 _+ 26.9% (n = 5). Sequential addition of 1 /xM carbachol almost completely relaxed the muscle to 10.4 _+ 5.9% (n = 5) and increased [Ca2+]i to 253.2_+ 36.7% (n = 5). In e n d o t h e l i u m - d e n u d e d muscle (fig. 3B), 100 nM ET-3 and 1 /xM carbachol were ineffective although n o r e p i n e p h r i n e increased both [Ca2+] i and muscle tension (n = 4).
3.3• Effects of verapamil and L-NMMA 3.2. Effects of ET-3 on norepinephrine-stimulated muscle As shown in fig. 3A, 100 nM n o r e p i n e p h r i n e increased both [Ca2+]i and muscle tension. In this experA. End (+)
B. End (-) - I oo (%)
R340/380
~ 0
• Tension
~
~f
0 Ca
0.5 g . .
KCI
ET-3 CCh
KCI
ET-3 CCh
5 min
Fig. 2. Effectsof ET-3(100nM)and carbachol(CCh, 1 /zM)on cytosolic Ca2+ level(upper trace, expressed as R340/380)and muscle tension (lower trace) in rat aortas with and without endothelium (A: End(+) and B: End(-), respectively) in the absence of external Ca 2+ (0 Ca; with 0.5 mM EGTA). 100% represents cytosolic Ca 2+ stimulated by high K +. The trace shows typical results from one of four to six experiments.
Figure 4 shows the effects of 10 /xM verapamil and 100 /xM L - N M M A on the ET-3-induced changes in [Ca2+]~ and muscle tension in aortas stimulated with 100 nM norepinephrine. Varapamil c h a n g e d neither the relaxation nor the [Ca2+]i stimulated by ET-3. T h e same concentration of verapamil almost completely inhibited the increase in [Ca2+]i elicited by high K + and n o r e p i n e p h r i n e in the absence of the endothelium, as reported previously (Sato et al., 1988; Ozaki et al., 1990; Karaki et al., 1991). However, 10 /xM verapamil did not inhibit the norepinephrine-stimulated [Ca2+]i in the presence of the endothelium (n = 4). Figure 4 also show that L - N M M A inhibited the ET-3-induced relaxation although it did not change the ET-3-stimulated [Ca2+] i. Verapamil and L - N M M A had similar effects on the carbachol-induced changes. Figure 5 shows the concentration-response curves for the relaxant effect of ET-3 in 100 nM norepinephrine-stimulated aortas. ET-3 induced half-maxim u m relaxation at 13.8 nM, maximum relaxation being obtained with 100 nM ET-3 (68.6 _+ 4.8%, n = 4). A
140 400 120
ET-3 caused concentration-dependent contraction of smooth muscle. IRL 1{)38 (3 /,M) and 100 # M LNMMA almost completely inhibited the relaxation induced by 1-100 nM ET-3. However, neither 1() /xM verapami[ (fig. 5) nor 3 # M BQI23 ( n - 4 , data not shown) changed the relaxant effect of ET-3.
4. Discussion
lI=
0
0
Control
+ Ver
L
+ Control L-NMMA J [
+ Ver
ET-3
+ L-NMMA I
CCh
Fig. 4. Effects of 100 nM ET-3 and I /aM carbachol on cylosolic ('a 3~ level ([('
137
© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00
EJP 52806
Effect of endothelin-3 on cytosolic calcium level in vascular endothelium and on smooth muscle contraction Sri Agus Sudjarwo, Masatoshi Hori and Hideaki Karaki
Department of Veterinao' Pharmacology, FaculO,of Agriculture, The Uni~'ersityof Tokyo, Bunkyo-ku, Tokyo 113, Japan Received 7 September 1992, accepted 22 September 1992
In isolated rat aorta, endothelin (ET)-3 increased cytosolic Ca 2+ ([Ca2+] i) in the endothelium at a concentration (100 nM) which had little effect on muscle resting tone. In the absence of external Ca 2 ~, ET-3 still transiently increased endothelial [Ca2+]i. Verapamil (10 p.M) did not change the effects of ET-3. In aortas stimulated with 100 nM norepinephrine, 100 nM ET-3 relaxed the muscle with an increase in endothelial [Ca2+]i. An inhibitor of nitric oxide synthase, 100 p.M N~;-monomethyl-Larginine, inhibited the relaxant effect of ET-3 but not the increase in endothelial [Ca z+ ]i. In the absence of the endothelium or in the presence of an antagonist of ET B receptors, 3 # M IRL 1038, the ET-3-induced increase in endothelial [Ca2+]i and relaxation of norepinephrine-induced contraction were inhibited. Under these conditions, ET-3 increased smooth muscle [Ca 2 +]i and induced contraction, both of which were inhibited by an inhibitor of ET A receptors, 3/xM BQ123. These results suggest that ET-3 acts on ET u receptors in the vascular endothclium to increase [Ca 2 ~]i by releasing Ca 2 ~ from storage sites and by opening non-L type Ca 2+ channels, activates nitric oxide synthase, releases nitric oxide and relaxes vascular smooth muscle. Although ET-3 also activates ET A receptors in smooth muscle to induce contraction, this effect is overcome by the relaxant effect mediated by ET B receptors.
Endothelin-3; ETr3 receptors: Endothelium-dependent relaxation; Ca 2+ levels (cytosolic); Vascular (rat aorta); (Relaxation)
1. Introduction T h e newly f o u n d vasoactive p e p t i d e s , e n d o t h e l i n s , have t h r e e isoforms, e n d o t h e l i n ( E T ) - I , ET-2 and ET-3 ( Y a n a g i s a w a et al., 1988; I n o u e et al., 1989). T h e s e e n d o t h e l i n s act on E T A r e c e p t o r s , which are relatively selectively a c t i v a t e d by ET-1 a n d E T - 2 ( A r a i et al., 1990), a n d on E T B r e c e p t o r s , which a r e a c t i v a t e d nonselectively by t h e t h r e e e n d o t h e l i n isoforms ( S a k u r a i et al., 1990). E T A r e c e p t o r s are f o u n d in vascular s m o o t h m u s c l e w h e r e a s E T u r e c e p t o r s a r e f o u n d in nerves a n d t h e v a s c u l a r e n d o t h e l i u m ( F u j i t a n i et al., 1992; S a k u r a i et al., 1992). It has b e e n shown that e n d o t h e l i n s transiently relax v a s c u l a r s m o o t h muscle, possibly by releasing e n d o t h e l i u m - d e r i v e d relaxing f a c t o r ( E D R F ) ( D e Nucci et al., 1988; S a k a t a et al., 1989). E D R F is r e l e a s e d from the v a s c u l a r e n d o t h e l i u m a n d relaxes s m o o t h muscle by i n c r e a s i n g the cyclic
Correspondence to: H. Karaki, Department of Veterinary Pharmacology, Faculty of Agriculture, The University of Tokyo, Bunkyo-ku, Tokyo 113, Japan.
G M P c o n t e n t ( F u r c h g o t t , 1984). A t least some of the effects of E D R F are d u e to nitric oxide synthesized from L - a r g i n i n e by a Ca 2+- a n d c a l m o d u l i n - d e p e n d e n t enzyme, nitric oxide synthase (for review see M o n c a d a et al., 1991). In o r d e r to u n d e r s t a n d the effects of e n d o t h e l i n s on b l o o d vessels, it is i m p o r t a n t to m e a s u r e the functions of e n d o t h e l i u m a n d s m o o t h muscle simultaneously. Using i s o l a t e d rat a o r t a strips, Sato et al. (1990) rep o r t e d a m e t h o d to m e a s u r e cytosolic Ca 2+ levels ([ Ca2+ ]i) in e n d o t h e l i a l cells a n d muscle tension simult a n e o u s l y a n d f o u n d that t h e r e was a positive c o r r e l a tion b e t w e e n the i n c r e a s e in e n d o t h e l i a l [Ca2+] i and the e n d o t h e l i u m - d e p e n d e n t r e l a x a t i o n due to c a r b a chol and histamine. In the p r e s e n t e x p e r i m e n t s , we e x a m i n e d the effects of ET-3, an a n t a g o n i s t of E T B r e c e p t o r s , I R L 1038 ( U r a d e et al., 1992), and an antagonist of E T A r e c e p t o r s , BQ123 ( I h a r a et al., 1992), on e n d o t h e l i a l [Ca2+]~ a n d muscle t o n e in isolated rat a o r t a strips. O u r results suggest that ET-3 acts on ET~ r e c e p t o r s , i n c r e a s e s e n d o t h e l i a l [Ca 2 +]i, activates nitric oxide synthase, r e l e a s e s nitric oxide and relaxes vascular s m o o t h muscle.
138 2. Materials and methods
2.1. Preparations, solutions and measurement of muscle tension The thoracic aorta was isolated from male Wistar rats (250-300 g), cut into spiral strips (1-2 mm in width and 5 - 7 m m in length) and placed in normal physiological salt solution which contained (raM): NaCI 136.9, KCI 5.4, CaCI 2 1.5, MgCI 2 1.0, NaHCO~ 23.8, ethylenediamine-tetraacetic acid 0.01 and glucose 5.5. In some experiments, the endothelium was removed by gently rubbing the intimal surface with a finger moistened with the above solution. A high K ÷ solution was made by substituting 69.6 mM NaC1 with equimolar KC1. Ca2+-free solution was made by removing CaCl~ and adding 0.5 mM E G T A . Thesc solutions were saturated with a mixture of 95% O 2 and 5% CO 2 at 37°C and pH 7.4. Muscle tension was recorded isometrically with a force-displacement transducer. Each muscle strip was attached to a holder under a resting tension of 1 g and equilibrated for 60-90 min in a 10-ml muscle bath until the contractile response to the high K + solution had become stable. The functional integrity of the vascular endothelium was assessed by measuring whether 1 /xM carbachol induced almost complete relaxation ( > 90%) in aortas stimulated with 100 nM norepinephrine (Karaki and Nagase, 1987).
1989) and also because we measured [Ca2+]i simultaneously in different tissues (smooth muscle and endothelium) with different fura-2 contents. The ratio of F340 to F380 (R340/380) was used as an indicator of [Ca2+]i , and a quantitative comparison of [Ca2+]i was made by taking resting and high K+-stimulated [Ca -~~]i as 0 and 100%, respectively, unless stated otherwise. 2.3. Chemicals ET-3 was purchased from the Peptidc Institute (Osaka, Japan). Other chemicals used were an E T B antagonist, I R L 1038 ([Cysll-Cys 15] ET-I (11-21)), an ET a antagonist, BQ123 (cyclo[-Asp-Pro-Val-Leu-Trp-]) (donated by Ciba-Geigy Japan, Takarazuka, Japan), verapamil hydrochloride, NG-monomethyl-L-argininc (L-NMMA; Sigma Chemicals, St. Louis, MO, U.S.A.) and norepinephrinc bitartrate (Wako Pure Chemicals, Tokyo, Japan). Preliminary experiments confirmed that these chemicals did not interfere with fura-2 fluorescence. 2.4. Statistics The results of the experiments are expressed as means _+ S.E.M. Unpaired Student's t-test was used for statistical analysis of the results.
3. Results 2.2. Simultaneous measurement of muscle tension and
cytosolic Ca: + let'el
3.1. £ffects q[ ET-3 on resting muscle
Muscle strips were incubated with 5 /xM of the acetoxymcthyl ester of fura-2 ( f u r a - 2 / A M ) for 3 - 5 h at room temperature. The noncytotoxic detergent, cremophor EL (0.02%), was also added to increase the solubility of f u r a - 2 / A M . Experiments were performed with a fluorimeter (CAF-100, Japan Spectroscopic, Tokyo, Japan) as described previously (Sat() et al., 1990). The muscle strip was held horizontally in a temperature-controlled organ bath. One end of the muscle strip was connected to a force-displacement transducer to monitor mechanical activity. The endothelial surface of the strip was excited alternately by light at 340 nm and 380 nm (48 He) and the intensity of fluorescence at 500 nm (F340 and F380) was measured at the endothelial surface. Ca ~+ signals thus obtained derive from both the endothelium and smooth muscle cells. In some experiments, the endothelium was removed to measure [Ca2+]i in smooth muscle cells. By comparing the total and the smooth muscle [Ca2+]i, it is possible to estimate changes in endothelial [Ca2+]~ (Sato et al., 199(I). Absolute [Ca2+]i was not calculated because the dissociation constant of fura-2 for Ca 2+ might bc diffcrcnt from that obtained in vitro (Karaki,
As shown in fig. 1A, 100 nM ET-3 increased [Ca2+]i to 103.2 _+ 23.5% (n = 5) without changing muscle tone. Carbachol (1 /xM) also increased [Ca2+]i to 199.0 _+ 45.8% (n = 6) without changing muscle tension. In contrast, high K + increased both [Ca2+]i and muscle tension (to 100%, respectively). An antagonist of ET B receptors, 3 # M I R L 1038, did not change the effects of high K + and carbachol (n - 4 each). In the presence of 3 /xM 1RL 1038, however, 1(10 nM ET-3 increased both [Ca2+]~ (48.9_+ 8.4%, n = 4) and muscle tension (46.8_+ 4.6%, n = 4), and these changes were completely inhibited by an antagonist of ET A receptors, 3 # M BQI23 (n = 4 each). The same concentration of BQ123 did not modify the effects of high K + and carbachol (n = 4 each). In endothelium-denuded muscle (fig. 1B), high K" also increased [CaZ+] i and muscle tension (to 100%, respectively). Carbachol (1 /xM) was ineffective (n = 5) whereas 100 nM ET-3 increased [Ca~+]i to 57.9_+ 16.5% (n = 4) and muscle tension to 70.8 ± 11.4~: (n = 4). The effects of ET-3 were completely inhibited by 3 /xM BQ123 whereas 3 /xM I R L 1038 was ineffective (n = 3 each).
139 A. End
(+) - Ioo
A. End (+)
(%)
B. End (-)
R340/3B0
0 -loo(%)
Tension
0.5 g KCI
ET-3
KCI
R340/380
~
~ o
CCh
/
5 min B. End
(-)
Tension ~
t
R340/380
Tens,on/ l ~ ~ ET-3
KCI
0.5g
NE ET-3 CCh
/
KCI
0
CCh
Fig. 1. Effects of ET-3 (100 nM) and carbachol (CCh, 1 /xM) on cytosolic Ca2+ level (upper trace, expressed as R340/380) and muscle tension (lower trace) in rat aortas with and without endothelium (A: End(+) and B: End(-), respectively). 100% represents eytosolic Ca2+ stimulated by high K +. The trace shows typical results from one of four experiments. Figure 2 A shows the effects of ET-3 and carbachol on endothelial [Ca2+]i in the absence of external Ca 2+. Addition of 100 n M ET-3 transiently increased [Ca2+]~ to 64.8 _+ 6.2% (n = 4). Sequential addition of 1 /xM carbachol again transiently increased [Ca2+] i to 74.4 _+ 20.4% (n = 4). W h e n applied first, carbachol transiently increased [Ca2+] i to 80.3 _+ 12.8% (n = 4), which was not different from the value m e a s u r e d in the strips p r e t r e a t e d with 100 nM ET-3. T h e increase in [Ca2+]~ was not followed by a c h a n g e in muscle tone. As shown in fig. 2B, neither 100 n M ET-3 nor 1 /xM carbachol c h a n g e d [Ca2+]i in the absence of the e n d o t h e l i u m (n = 4).
NE
ET-3 CCh
5 min
Fig. 3. Effects of ET-3 (100 nM) and carbachol (CCh, 1 /xM) on cytosolic Ca 2+ level (upper trace, expressed as R340/380) and muscle tension (lower trace) in rat aortas with and without endothelium (A: End( + ) and B; End(- ), respectively) in the presence of 100 nM norepinephrine (NE). 100% represents cytosolic Ca2+ stimulated by high K +. The trace shows typical results from one of four to five experiments.
iment, [Ca2+]i and muscle tension m e a s u r e d 10 min after the addition of n o r e p i n e p h r i n e were taken as 100%. Addition of 100 nM ET-3 transiently relaxed the sustained contraction induced by n o r e p i n e p h r i n e to 61.6 _+ 8.5% (n = 5). This change followed a transient increase in [Ca2+]i to 159.4 _+ 26.9% (n = 5). Sequential addition of 1 /xM carbachol almost completely relaxed the muscle to 10.4 _+ 5.9% (n = 5) and increased [Ca2+]i to 253.2_+ 36.7% (n = 5). In e n d o t h e l i u m - d e n u d e d muscle (fig. 3B), 100 nM ET-3 and 1 /xM carbachol were ineffective although n o r e p i n e p h r i n e increased both [Ca2+] i and muscle tension (n = 4).
3.3• Effects of verapamil and L-NMMA 3.2. Effects of ET-3 on norepinephrine-stimulated muscle As shown in fig. 3A, 100 nM n o r e p i n e p h r i n e increased both [Ca2+]i and muscle tension. In this experA. End (+)
B. End (-) - I oo (%)
R340/380
~ 0
• Tension
~
~f
0 Ca
0.5 g . .
KCI
ET-3 CCh
KCI
ET-3 CCh
5 min
Fig. 2. Effectsof ET-3(100nM)and carbachol(CCh, 1 /zM)on cytosolic Ca2+ level(upper trace, expressed as R340/380)and muscle tension (lower trace) in rat aortas with and without endothelium (A: End(+) and B: End(-), respectively) in the absence of external Ca 2+ (0 Ca; with 0.5 mM EGTA). 100% represents cytosolic Ca 2+ stimulated by high K +. The trace shows typical results from one of four to six experiments.
Figure 4 shows the effects of 10 /xM verapamil and 100 /xM L - N M M A on the ET-3-induced changes in [Ca2+]~ and muscle tension in aortas stimulated with 100 nM norepinephrine. Varapamil c h a n g e d neither the relaxation nor the [Ca2+]i stimulated by ET-3. T h e same concentration of verapamil almost completely inhibited the increase in [Ca2+]i elicited by high K + and n o r e p i n e p h r i n e in the absence of the endothelium, as reported previously (Sato et al., 1988; Ozaki et al., 1990; Karaki et al., 1991). However, 10 /xM verapamil did not inhibit the norepinephrine-stimulated [Ca2+]i in the presence of the endothelium (n = 4). Figure 4 also show that L - N M M A inhibited the ET-3-induced relaxation although it did not change the ET-3-stimulated [Ca2+] i. Verapamil and L - N M M A had similar effects on the carbachol-induced changes. Figure 5 shows the concentration-response curves for the relaxant effect of ET-3 in 100 nM norepinephrine-stimulated aortas. ET-3 induced half-maxim u m relaxation at 13.8 nM, maximum relaxation being obtained with 100 nM ET-3 (68.6 _+ 4.8%, n = 4). A
140 400 120
ET-3 caused concentration-dependent contraction of smooth muscle. IRL 1{)38 (3 /,M) and 100 # M LNMMA almost completely inhibited the relaxation induced by 1-100 nM ET-3. However, neither 1() /xM verapami[ (fig. 5) nor 3 # M BQI23 ( n - 4 , data not shown) changed the relaxant effect of ET-3.
4. Discussion
lI=
0
0
Control
+ Ver
L
+ Control L-NMMA J [
+ Ver
ET-3
+ L-NMMA I
CCh
Fig. 4. Effects of 100 nM ET-3 and I /aM carbachol on cylosolic ('a 3~ level ([('
