European Journal of Pharmacology, 179 (1990) 207-210
207
Elsevier EJP 20593
Short communication
Phorbol ester-induced release of endothelium-derived relaxing factor Kiyoshi S a k a t a a n d Hideaki K a r a k i Department of Veterinary Pharmacology, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received 26 January 1990, accepted 13 February 1990
The effects of a phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), on contraction of isolated vascular smooth muscle of rat aorta were examined. DPB (100 nM-1 ~tM) induced a concentration-dependent contraction in resting aorta. DPB (3-100 nM) also induced a concentration-dependent relaxation of the norepinephrine-induced contraction. The contractile effect of DPB was potentiated whereas the relaxant effect was inhibited by endothelium removal or by methylene blue, suggesting that lower concentrations of DPB release endothelium-derived relaxing factor to inhibit contraction in rat aorta. Smooth muscle (vascular); Aorta (rat); Phorboi ester; EDRF (endothelium-derived relaxing factor); Protein kinase C
1. Introduction
2. Materials and methods
Phorbol esters, the activators of protein kinase C (Castagna et ai., 1982), have been widely used to examine the role of this protein kinase in biological systems. It has been shown that phorbol esters induce contraction in isolated blood vessels (Rasmussen et al., 1984). Furthermore, phorbol esters inhibit the vasodilatation caused by endothelium-derived relaxing factor (EDRF) (Weinheimer et al., 1986; Lewis and Henderson, 1987; De Nucci et al., 1988). In the present experiments with isolated rat aorta we found that phorbol ester has dual effects, a vasodilator effect at lower concentrations which is dependent on endothelium of the blood vessel and a vasoconstrictor effect at higher concentrations which is attenuated in the presence of endothelium,
Male Wistar rats, weighing about 250 g, were stunned and bled. The thoracic aorta was dissected out and strips, width 2-3 mm and length 5-8 mm, were prepared. In some experiments, the endothelium was removed by gently rubbing the intimal surface with a finger moistened with physiological salt solution (Furchgott and Zawadzki, 1980). Each muscle strip was attached to a holder under a resting tension of 0.5 g was equilibrated for 60 min in the bathing solution. Contractile tension was recorded isometrically. It was verified for each preparation that 1/~M carbachol induced almost complete (more than 80%) relaxation of the 100 nM norepinephrine-induced contraction in order to check the functional integrity of the endothelium. In the muscle strips from which the endothelium had been rubbed off, the 1 ptM
Correspondence to: H. Karaki, Department of Veterinary Pharmacology, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan.
carbachol-induced relaxation was less than 10% of the norepinephrine-induced contraction. The concentration of phorbol ester required to induce a half-maximum effect (ECs0) was determined by cumulative addition of phorbol ester. The results were expressed as means 5= S.E.M. At least four
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
2O8 muscle strips were used to calculate a m e a n value. Statistical significance was e x a m i n e d using Stud e n t ' s t-test a n d a P value less than 0.01 was taken as showing a significant difference. T h e physiological salt solution ( p H 7.4, 3 7 ° C ) c o n t a i n e d ( m M ) 136.9 NaCI, 5.4 KC1, 1.5 CaC12, 1.0 MgCI 2, 23.8 N a H C O 3 a n d 5.5 glucose. H i g h - K solution was m a d e b y substituting the a p p r o p r i a t e a m o u n t of NaCI with KC1. These solutions were s a t u r a t e d with a 95% 0 2 a n d 5% C O 2 mixture. T h e drugs used were 1 - n o r e p i n e p h r i n e bitartrate, m e t h y l e n e blue, s o d i u m n i t r o p r u s s i d e ( W a k o Pure C h e m icals, Osaka), c a r b a m y l c h o l i n e chloride (carbachol, T o k y o Kasei, Tokyo), i n d o m e t h a c i n (Sigma, St. Louis, M O ) , 1 2 - d e o x y p h o r b o l 1 3 - i s o b u t y r a t e (DPB)., p h o r b o l 12,13-dibutyrate (PDBu), a n d p h o r b o l 12, 1 3 - d i d e c a n o a t e ( P D D ) ( F u n a k o s h i , Tokyo).
3. Results A s shown in fig. l a , D P B i n d u c e d a c o n c e n t r a t i o n - d e p e n d e n t increase in muscle tone with an ECs0 of 183.2 nM. E n d o t h e l i u m removal or 1 # M m e t h y l e n e blue (5-min p r e i n c u b a t i o n ) shifted the c o n c e n t r a t i o n - r e s p o n s e curve for D P B to the left w i t h o u t c h a n g i n g the m a x i m u m contractile tension. The ECso value in the absence of e n d o thelium was 54.6 n M a n d that in the presence of m e t h y l e n e blue was 55.0 nM. As shown in fig 2a, 100 n M D P B relaxed the s u s t a i n e d c o n t r a c t i o n i n d u c e d b y 100 n M n o r e p i n e p h r i n e . T h e i n h i b i t o r y effect increased as the D P B c o n c e n t r a t i o n was increased from 3 to 100 n M (fig. l b ) . A further increase in the D P B c o n c e n t r a t i o n to 1 /xM decreased the i n h i b i t o r y effect. T h e n o r e p i n e p h r i n e - i n d u c e d c o n t r a c t i o n was p o t e n t i a t e d in the absence of e n d o t h e l i u m (fig. 2b) or in the presence of 1 /zM m e t h y l e n e blue (fig. 2c) as r e p o r t e d previously ( N a g a s e et al., 1987). T h e a d d i t i o n of 100 n M D P B d i d not relax, b u t instead p o t e n t i a t e d to n o r e p i n e p h r i n e - i n d u c e d c o n t r a c t i o n in these muscles (fig. 2b a n d c). T h e effect on n i t r o p r u s s i d e was not altered b y e n d o thelium removal or b y a brief p r e i n c u b a t i o n ( < 10 min) with m e t h y l e n e blue (fig. 2b a n d c). H o w ever, p r o l o n g e d i n c u b a t i o n with m e t h y l e n e blue
~ot~.~÷) .wth~.~b~ ~.--~~
200 ~ "~ lo0
o 10o b ~ / / ~ , a o t ~ , ~ + ) .~ 50 .~
9
8
r
6
Fig. 1. Contractile and relaxant effects of DPB in rat aorta. (a) Contractile effect. (@) Aorta with endothelium; (©) aorta without endothelium; (11) aorta with endothelium which was pretreated with 1 /tM methylene blue for 5 min. Carbachol (1 ~M) relaxed the 100 nM norepinephrine-induced contraction by 90.6+ 2.6% (n = 8) in the presence of endothelium and by 5.0+4.4% (n = 4) in the absence of endothelium, as shown in fig. 2a and b. DPB was added cumulatively when the muscle tension reached a steady level (usually within 20 min). The ECso value for DPB was 183.2 nM (95% confidence limit: 181.6-184.8nM, n =4) in the presence of endothelium, 54.6 nM (95% confidence limit: 54.3-54.8 nM, n = 4) in the absence of endothelium, and 55.0 nM (95% confidence limit: 54.7-55.2 nM, n = 4) in the presence of methylene blue. The muscle tension induced by the 65.4 mM K+-induced contraction was taken as 100%. (b) Relaxant effect of DPB in the norepinephrine-stimulated aorta with endothelium. DPB was added during the 100 nM norepinephrine-induced sustained contraction. A concentration-response curve was obtained from a single application of DPB, as shown in fig. 2a. The ECs0 value for the vasodilator effect of DPB was 25.6 nM (95% confidence limit: 14.5-48.0nM, n =4).
( > 60 min) inhibited the effect of n i t r o p r u s s i d e ( d a t a not shown). In contrast, 1 0 / ~ M i n d o m e t h a cin did not change the v a s o d i l a t o r effect of D P B (n = 4, d a t a not shown). In a o r t a with e n d o t h e l i u m , 10, 30 a n d 100 n M P D B u relaxed the 100 n M n o r e p i n e p h r i n e - i n d u c e d c o n t r a c t i o n b y 12.9 + 6.3, 32.7 + 10.9 a n d 8.4 + 3.3%, respectively (n = 4 each). A higher c o n c e n t r a t i o n of P D B u (1 ~tM) i n d u c e d a c o n t r a c -
209
a. Endothelium(+)
i
i n d u c e d c o n t r a c t i o n b y 1 /~M c a r b a c h o l o r 1 /tM s o d i u m n i t r o p r u s s i d e was i n h i b i t e d in the presence
i
!~";~:k....~_~ Nitr°prusside ]
/f"~'~k
J. . . . .
2rain
J
. . . . . . . . . .
.
.
Norepinephrlne
of 1 /tM D P B (n = 2 each, d a t a not shown). 03°
. . . .
.
4. Discussion
Norepinephrine
Carbachol
DPB
Nitroprusstde
b. Endotbelium(-) . "\
'
I ,
I
-J/
-~ . . . . . . , Norepinephrine
_ _ ~ --~"~------~ _
,. . . . . . . . . . Norepinephrlne
Carbachol
0.3g ,
DPB
Nitroprusside
, c. Endotheliumt+)
i~! l _.:. . ~. . . . . .
Norepinephrine
L.__l
Carbachol
~
f
I
i
'k
~ _~_i . . . . . . . . . . . . . Methylene blue L Noreplnephrine ,
0.3g _----=____ , ,
DPB
Fig. 2. Effects of 1 /IM carbachol (CCh), 100 nM DPB and 1 #M sodium nitroprusside on the contraction induced by 100 nM norepinephrine (NE) in aorta with (a) and without endothelium (b). In the presence of endothelium, carbachol, DPB and nitroprusside relaxed the norepinephrine-induced contraction by 92.5 4-2.5% (n = 4), 79.8 4-5.9% (n = 4) and 100% (n = 4), respectively. In the absence of endothelium, neither carbachol nor DPB induced relaxation although nitroprusside completely inhibited the contraction. In (c), I p.M methylene blue was added 2 rain before the addition of norepinephrine to the aorta with endothelium. DPB did not relax the muscle in the presence of methylene blue but nitroprusside inhibited the contraction.
tion in a o r t a with or without e n d o t h e l i u m . In contrast, P D D (10 nM-1 # M ) d i d not relax the n o r e p i n e p h r i n e - i n d u c e d c o n t r a c t i o n nor d i d it ind u c e a c o n t r a c t i o n in a o r t a with or without e n d o thelium (n = 4 each, d a t a not shown), T h e relaxation of the 100 n M n o r e p i n e p h r i n e -
In the isolated rat aorta, 100 nM-1 /xM D P B showed a c o n t r a c t i l e effect which was p o t e n t i a t e d b y endothelium removal. This result led us to e x a m i n e t h e effect of D P B on e n d o t h e l i u m . W h e n a d d e d d u r i n g the s u s t a i n e d c o n t r a c t i o n i n d u c e d b y n o r e p i n e p h r i n e , 3-100 n M D P B i n d u c e d conc e n t r a t i o n - d e p e n d e n t relaxation. W h e n the e n d o thelium was removed, the v a s o d i l a t o r effect was abolished, suggesting that D P B acts on the e n d o thelium. It has been shown that e n d o t h e l i u m releases p r o s t a g l a n d i n s (Toda, 1984). In the p r e s e n t experiments, however, the D P B - i n d u c e d relaxation was not affected b y i n d o m e t h a c i n , suggesting that release of the inhibitory prostaglandins was not involved. Furchgott and Zawadzki (1980) rep o r t e d that m u s c a r i n i c agonists act on the vascular e n d o t h e l i u m a n d release E D R F . A f t e r a brief i n c u b a t i o n ( < 10 min) with m e t h y l e n e blue, a selective i n h i b i t o r of E D R F - m e d i a t e d r e l a x a t i o n ( M a r t i n et al., 1985), the effects of D P B a n d c a r b a c h o l were inhibited a l t h o u g h the effect of n i t r o p r u s s i d e was i n h i b i t e d o n l y after a p r o l o n g e d i n c u b a t i o n ( > 60 rain). M e t h y l e n e blue also augm e n t e d the c o n t r a c t i o n s i n d u c e d by DPB. These results suggest that D P B releases E D R F . A n o t h e r p h o r b o l ester, PDBu, also i n d u c e d rel a x a t i o n at 10-100 n M a n d c o n t r a c t i o n at 1 / x M in a o r t a with e n d o t h e l i u m . In contrast, a p h o r b o l ester which does not activate p r o t e i n kinase C, P D D ( C a s t a g n a et al., 1982), was ineffective. These results suggest that the effects of D P B a n d P D B u are a t t r i b u t a b l e to the a c t i v a t i o n of p r o t e i n kinase C. A higher c o n c e n t r a t i o n (1 /zM) of D P B a n d P D B u i n d u c e d less relaxation. F u r t h e r m o r e , 1 ~ M D P B inhibited the r e l a x a t i o n due to c a r b a c h o l . This result is consistent with the p r e v i o u s r e p o r t showing that D P B ( W e i n h e i m e r et al., 1986) a n d P D B u (Lewis a n d H e n d e r s o n , 1987) i n h i b i t e d the E D R F - i n d u c e d relaxation. T h e d u a l effect of p h o r b o l esters is consistent with the findings rep o r t e d b y D e Nucci et al. (1988) that a c t i v a t i o n of
210 protein kinase C by a diacylglycerol kinase inhibitor increases the basal release of EDRF followed
by inhibition, possibly by activating the negative feedback control of the receptor-coupled synthesis and/or release of EDRF. Since 1 /~M DPB also inhibited
the relaxation
due
to nitroprusside,
a
cyclic GMP-mediated relaxation mechanism in s m o o t h m u s c l e cells m a y a l s o b e i n h i b i t e d b y t h e
activation of protein kinase C. F r o m t h e s e r e s u l t s , it is s u g g e s t e d t h a t t h e a c t i v a t i o n o f p r o t e i n k i n a s e C in t h e e n d o t h e l i a l cells r e s u l t s in t h e i n c r e a s e d s y n t h e s i s o r r e l e a s e o f EDRF whereas the activation of protein kinase C in t h e s m o o t h m u s c l e cells r e s u l t s i n c o n t r a c t i o n ,
Acknowledgements We are grateful to Dr. Hiroshi Ozaki of our department for his suggestions. This work was supported in part by a Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
References Castagna, M., Y. Takai, K. Kaibuchi, U. Kikkawa and Y. Nishizuka, 1982, Direct activation of calcium-activated
phospholipid-dependent protein kinase by tumor-promoting phorbol esters, J. Biol. Chem. 257, 7847. De Nucci, G., R.J. Gryglewski, T.D. Warner and J.R. Vane, 1988, Receptor-mediated release of endothelium-derived relaxing factor and prostacycling from bovine aortic endothelial cells is coupled, Proc. Natl. Acad. Sci. U.S.A., 85, 2334. Furchgott, R.F. and J.V. Zawadzki, 1980), The obligatory role of endothelial cells in the relaxation of arterial smooth muscle by acetylcholine, Nature 288, 373. Lewis, M.J. and A.H. Henderson, 1987, A phorbol ester inhibits the release of endothelium-derived relaxing factor, European J. Pharmacol. 137. 167. Martin, W., G.M. Villani, D. Jothianandan and R.F. Furchgott, 1985, Selective blockade of endothelium-dependent and glyceryl trinitrate-induced relaxation by hemoglobin and by methylene blue in the rabbit aorta, J. Pharmacol. Exp. Ther. 232, 708. Nagase, H., H. Karaki and N. Urakawa, 1987, Palytoxin-induced endothelium-dependent relaxation in the isolated rat aorta, Naunyn-Schiedeb. Arch. Pharmacol. 335, 575. Rasmussen, H., J. Forder, I. Kojima and A. Scribine, 1984. TPA-induced contraction of isolated rabbit vascular smooth muscle, Biochem. Biophys. Res. Commun. 122, 776. Toda, N., 1984, Endothelium-dependent relaxation induced by angiotensin 2 and histamine in isolated arteries of dog, Br. J. Pharmacol. 81,301. Weinheimer, G., B. Wagner and H. O~wald, 1986, Interference of phorbol esters with endothelium-dependent vascular smooth muscle relaxation, European J. Pharmacol. 130, 319.
207
Elsevier EJP 20593
Short communication
Phorbol ester-induced release of endothelium-derived relaxing factor Kiyoshi S a k a t a a n d Hideaki K a r a k i Department of Veterinary Pharmacology, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan Received 26 January 1990, accepted 13 February 1990
The effects of a phorbol ester, 12-deoxyphorbol 13-isobutyrate (DPB), on contraction of isolated vascular smooth muscle of rat aorta were examined. DPB (100 nM-1 ~tM) induced a concentration-dependent contraction in resting aorta. DPB (3-100 nM) also induced a concentration-dependent relaxation of the norepinephrine-induced contraction. The contractile effect of DPB was potentiated whereas the relaxant effect was inhibited by endothelium removal or by methylene blue, suggesting that lower concentrations of DPB release endothelium-derived relaxing factor to inhibit contraction in rat aorta. Smooth muscle (vascular); Aorta (rat); Phorboi ester; EDRF (endothelium-derived relaxing factor); Protein kinase C
1. Introduction
2. Materials and methods
Phorbol esters, the activators of protein kinase C (Castagna et ai., 1982), have been widely used to examine the role of this protein kinase in biological systems. It has been shown that phorbol esters induce contraction in isolated blood vessels (Rasmussen et al., 1984). Furthermore, phorbol esters inhibit the vasodilatation caused by endothelium-derived relaxing factor (EDRF) (Weinheimer et al., 1986; Lewis and Henderson, 1987; De Nucci et al., 1988). In the present experiments with isolated rat aorta we found that phorbol ester has dual effects, a vasodilator effect at lower concentrations which is dependent on endothelium of the blood vessel and a vasoconstrictor effect at higher concentrations which is attenuated in the presence of endothelium,
Male Wistar rats, weighing about 250 g, were stunned and bled. The thoracic aorta was dissected out and strips, width 2-3 mm and length 5-8 mm, were prepared. In some experiments, the endothelium was removed by gently rubbing the intimal surface with a finger moistened with physiological salt solution (Furchgott and Zawadzki, 1980). Each muscle strip was attached to a holder under a resting tension of 0.5 g was equilibrated for 60 min in the bathing solution. Contractile tension was recorded isometrically. It was verified for each preparation that 1/~M carbachol induced almost complete (more than 80%) relaxation of the 100 nM norepinephrine-induced contraction in order to check the functional integrity of the endothelium. In the muscle strips from which the endothelium had been rubbed off, the 1 ptM
Correspondence to: H. Karaki, Department of Veterinary Pharmacology, Faculty of Agriculture, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan.
carbachol-induced relaxation was less than 10% of the norepinephrine-induced contraction. The concentration of phorbol ester required to induce a half-maximum effect (ECs0) was determined by cumulative addition of phorbol ester. The results were expressed as means 5= S.E.M. At least four
0014-2999/90/$03.50 © 1990 Elsevier Science Publishers B.V. (Biomedical Division)
2O8 muscle strips were used to calculate a m e a n value. Statistical significance was e x a m i n e d using Stud e n t ' s t-test a n d a P value less than 0.01 was taken as showing a significant difference. T h e physiological salt solution ( p H 7.4, 3 7 ° C ) c o n t a i n e d ( m M ) 136.9 NaCI, 5.4 KC1, 1.5 CaC12, 1.0 MgCI 2, 23.8 N a H C O 3 a n d 5.5 glucose. H i g h - K solution was m a d e b y substituting the a p p r o p r i a t e a m o u n t of NaCI with KC1. These solutions were s a t u r a t e d with a 95% 0 2 a n d 5% C O 2 mixture. T h e drugs used were 1 - n o r e p i n e p h r i n e bitartrate, m e t h y l e n e blue, s o d i u m n i t r o p r u s s i d e ( W a k o Pure C h e m icals, Osaka), c a r b a m y l c h o l i n e chloride (carbachol, T o k y o Kasei, Tokyo), i n d o m e t h a c i n (Sigma, St. Louis, M O ) , 1 2 - d e o x y p h o r b o l 1 3 - i s o b u t y r a t e (DPB)., p h o r b o l 12,13-dibutyrate (PDBu), a n d p h o r b o l 12, 1 3 - d i d e c a n o a t e ( P D D ) ( F u n a k o s h i , Tokyo).
3. Results A s shown in fig. l a , D P B i n d u c e d a c o n c e n t r a t i o n - d e p e n d e n t increase in muscle tone with an ECs0 of 183.2 nM. E n d o t h e l i u m removal or 1 # M m e t h y l e n e blue (5-min p r e i n c u b a t i o n ) shifted the c o n c e n t r a t i o n - r e s p o n s e curve for D P B to the left w i t h o u t c h a n g i n g the m a x i m u m contractile tension. The ECso value in the absence of e n d o thelium was 54.6 n M a n d that in the presence of m e t h y l e n e blue was 55.0 nM. As shown in fig 2a, 100 n M D P B relaxed the s u s t a i n e d c o n t r a c t i o n i n d u c e d b y 100 n M n o r e p i n e p h r i n e . T h e i n h i b i t o r y effect increased as the D P B c o n c e n t r a t i o n was increased from 3 to 100 n M (fig. l b ) . A further increase in the D P B c o n c e n t r a t i o n to 1 /xM decreased the i n h i b i t o r y effect. T h e n o r e p i n e p h r i n e - i n d u c e d c o n t r a c t i o n was p o t e n t i a t e d in the absence of e n d o t h e l i u m (fig. 2b) or in the presence of 1 /zM m e t h y l e n e blue (fig. 2c) as r e p o r t e d previously ( N a g a s e et al., 1987). T h e a d d i t i o n of 100 n M D P B d i d not relax, b u t instead p o t e n t i a t e d to n o r e p i n e p h r i n e - i n d u c e d c o n t r a c t i o n in these muscles (fig. 2b a n d c). T h e effect on n i t r o p r u s s i d e was not altered b y e n d o thelium removal or b y a brief p r e i n c u b a t i o n ( < 10 min) with m e t h y l e n e blue (fig. 2b a n d c). H o w ever, p r o l o n g e d i n c u b a t i o n with m e t h y l e n e blue
~ot~.~÷) .wth~.~b~ ~.--~~
200 ~ "~ lo0
o 10o b ~ / / ~ , a o t ~ , ~ + ) .~ 50 .~
9
8
r
6
Fig. 1. Contractile and relaxant effects of DPB in rat aorta. (a) Contractile effect. (@) Aorta with endothelium; (©) aorta without endothelium; (11) aorta with endothelium which was pretreated with 1 /tM methylene blue for 5 min. Carbachol (1 ~M) relaxed the 100 nM norepinephrine-induced contraction by 90.6+ 2.6% (n = 8) in the presence of endothelium and by 5.0+4.4% (n = 4) in the absence of endothelium, as shown in fig. 2a and b. DPB was added cumulatively when the muscle tension reached a steady level (usually within 20 min). The ECso value for DPB was 183.2 nM (95% confidence limit: 181.6-184.8nM, n =4) in the presence of endothelium, 54.6 nM (95% confidence limit: 54.3-54.8 nM, n = 4) in the absence of endothelium, and 55.0 nM (95% confidence limit: 54.7-55.2 nM, n = 4) in the presence of methylene blue. The muscle tension induced by the 65.4 mM K+-induced contraction was taken as 100%. (b) Relaxant effect of DPB in the norepinephrine-stimulated aorta with endothelium. DPB was added during the 100 nM norepinephrine-induced sustained contraction. A concentration-response curve was obtained from a single application of DPB, as shown in fig. 2a. The ECs0 value for the vasodilator effect of DPB was 25.6 nM (95% confidence limit: 14.5-48.0nM, n =4).
( > 60 min) inhibited the effect of n i t r o p r u s s i d e ( d a t a not shown). In contrast, 1 0 / ~ M i n d o m e t h a cin did not change the v a s o d i l a t o r effect of D P B (n = 4, d a t a not shown). In a o r t a with e n d o t h e l i u m , 10, 30 a n d 100 n M P D B u relaxed the 100 n M n o r e p i n e p h r i n e - i n d u c e d c o n t r a c t i o n b y 12.9 + 6.3, 32.7 + 10.9 a n d 8.4 + 3.3%, respectively (n = 4 each). A higher c o n c e n t r a t i o n of P D B u (1 ~tM) i n d u c e d a c o n t r a c -
209
a. Endothelium(+)
i
i n d u c e d c o n t r a c t i o n b y 1 /~M c a r b a c h o l o r 1 /tM s o d i u m n i t r o p r u s s i d e was i n h i b i t e d in the presence
i
!~";~:k....~_~ Nitr°prusside ]
/f"~'~k
J. . . . .
2rain
J
. . . . . . . . . .
.
.
Norepinephrlne
of 1 /tM D P B (n = 2 each, d a t a not shown). 03°
. . . .
.
4. Discussion
Norepinephrine
Carbachol
DPB
Nitroprusstde
b. Endotbelium(-) . "\
'
I ,
I
-J/
-~ . . . . . . , Norepinephrine
_ _ ~ --~"~------~ _
,. . . . . . . . . . Norepinephrlne
Carbachol
0.3g ,
DPB
Nitroprusside
, c. Endotheliumt+)
i~! l _.:. . ~. . . . . .
Norepinephrine
L.__l
Carbachol
~
f
I
i
'k
~ _~_i . . . . . . . . . . . . . Methylene blue L Noreplnephrine ,
0.3g _----=____ , ,
DPB
Fig. 2. Effects of 1 /IM carbachol (CCh), 100 nM DPB and 1 #M sodium nitroprusside on the contraction induced by 100 nM norepinephrine (NE) in aorta with (a) and without endothelium (b). In the presence of endothelium, carbachol, DPB and nitroprusside relaxed the norepinephrine-induced contraction by 92.5 4-2.5% (n = 4), 79.8 4-5.9% (n = 4) and 100% (n = 4), respectively. In the absence of endothelium, neither carbachol nor DPB induced relaxation although nitroprusside completely inhibited the contraction. In (c), I p.M methylene blue was added 2 rain before the addition of norepinephrine to the aorta with endothelium. DPB did not relax the muscle in the presence of methylene blue but nitroprusside inhibited the contraction.
tion in a o r t a with or without e n d o t h e l i u m . In contrast, P D D (10 nM-1 # M ) d i d not relax the n o r e p i n e p h r i n e - i n d u c e d c o n t r a c t i o n nor d i d it ind u c e a c o n t r a c t i o n in a o r t a with or without e n d o thelium (n = 4 each, d a t a not shown), T h e relaxation of the 100 n M n o r e p i n e p h r i n e -
In the isolated rat aorta, 100 nM-1 /xM D P B showed a c o n t r a c t i l e effect which was p o t e n t i a t e d b y endothelium removal. This result led us to e x a m i n e t h e effect of D P B on e n d o t h e l i u m . W h e n a d d e d d u r i n g the s u s t a i n e d c o n t r a c t i o n i n d u c e d b y n o r e p i n e p h r i n e , 3-100 n M D P B i n d u c e d conc e n t r a t i o n - d e p e n d e n t relaxation. W h e n the e n d o thelium was removed, the v a s o d i l a t o r effect was abolished, suggesting that D P B acts on the e n d o thelium. It has been shown that e n d o t h e l i u m releases p r o s t a g l a n d i n s (Toda, 1984). In the p r e s e n t experiments, however, the D P B - i n d u c e d relaxation was not affected b y i n d o m e t h a c i n , suggesting that release of the inhibitory prostaglandins was not involved. Furchgott and Zawadzki (1980) rep o r t e d that m u s c a r i n i c agonists act on the vascular e n d o t h e l i u m a n d release E D R F . A f t e r a brief i n c u b a t i o n ( < 10 min) with m e t h y l e n e blue, a selective i n h i b i t o r of E D R F - m e d i a t e d r e l a x a t i o n ( M a r t i n et al., 1985), the effects of D P B a n d c a r b a c h o l were inhibited a l t h o u g h the effect of n i t r o p r u s s i d e was i n h i b i t e d o n l y after a p r o l o n g e d i n c u b a t i o n ( > 60 rain). M e t h y l e n e blue also augm e n t e d the c o n t r a c t i o n s i n d u c e d by DPB. These results suggest that D P B releases E D R F . A n o t h e r p h o r b o l ester, PDBu, also i n d u c e d rel a x a t i o n at 10-100 n M a n d c o n t r a c t i o n at 1 / x M in a o r t a with e n d o t h e l i u m . In contrast, a p h o r b o l ester which does not activate p r o t e i n kinase C, P D D ( C a s t a g n a et al., 1982), was ineffective. These results suggest that the effects of D P B a n d P D B u are a t t r i b u t a b l e to the a c t i v a t i o n of p r o t e i n kinase C. A higher c o n c e n t r a t i o n (1 /zM) of D P B a n d P D B u i n d u c e d less relaxation. F u r t h e r m o r e , 1 ~ M D P B inhibited the r e l a x a t i o n due to c a r b a c h o l . This result is consistent with the p r e v i o u s r e p o r t showing that D P B ( W e i n h e i m e r et al., 1986) a n d P D B u (Lewis a n d H e n d e r s o n , 1987) i n h i b i t e d the E D R F - i n d u c e d relaxation. T h e d u a l effect of p h o r b o l esters is consistent with the findings rep o r t e d b y D e Nucci et al. (1988) that a c t i v a t i o n of
210 protein kinase C by a diacylglycerol kinase inhibitor increases the basal release of EDRF followed
by inhibition, possibly by activating the negative feedback control of the receptor-coupled synthesis and/or release of EDRF. Since 1 /~M DPB also inhibited
the relaxation
due
to nitroprusside,
a
cyclic GMP-mediated relaxation mechanism in s m o o t h m u s c l e cells m a y a l s o b e i n h i b i t e d b y t h e
activation of protein kinase C. F r o m t h e s e r e s u l t s , it is s u g g e s t e d t h a t t h e a c t i v a t i o n o f p r o t e i n k i n a s e C in t h e e n d o t h e l i a l cells r e s u l t s in t h e i n c r e a s e d s y n t h e s i s o r r e l e a s e o f EDRF whereas the activation of protein kinase C in t h e s m o o t h m u s c l e cells r e s u l t s i n c o n t r a c t i o n ,
Acknowledgements We are grateful to Dr. Hiroshi Ozaki of our department for his suggestions. This work was supported in part by a Grant-in Aid for Scientific Research from the Ministry of Education, Science and Culture, Japan.
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