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Vasoinhibitory effect of NP-252, a new dihydropyridine derivative, in canine cerebral artery
Life Sciences, Vol. 48, pp. 183-188 Printed in the U.S.A.
Pergamon Press
VASOINHIBITORY EFFECT OF NP-252, A NEW DIHYDROPYRIDINE DERIVATIVE, IN CANINE CEREBRAL ARTERY
Yoshinobu Akimoto, Kazuyoshi Kurahashi I, Hachlro Usui
and Motohatsu FuJiwara
and
iDepartment of Pharmacology, Faculty of Medicine Pharmacology Division, RI Center, Kyoto University, Kyoto 606, Japan (Received in final form November 8, 1990)
Summary The v a s ~ n h i b i t o r y e f f e c t of NP-252, a 1 , 4 - d i h y d r o p y r i d i n e derivative Ca a n t a g o n i s t , was examined i n c a n i n e c e r e b r a l a r t e r x 7 and t h i s e f f e c t was c ~ p a r e d with t h a t of n i f e d i p i n e . NP-252 (10 M) and n i f e d i p i n e ~ 1 0 M ~ n e a r l y a b o l i s h e d the c o n t r a c t i o n induce~ by a d d i t i o n of C a ' ' to ~ a ' ' - f r e e medium c o n t a i n i n g KC1. NP-252 (10 - v M) and nifedipine (I0 M) attenuated the contraction produced by thromboxane A 2 agonlst (STAp) in normal medium, and the resultant contractions were 22 % (n=6)~and 35 % (n=6) of the control contraction, respectively. The vasolnhlbltory effects of NP-252 were significantly stronger7than thos~ of nlfediplne in canine cerebral artery. NP-252 (i0- ++and i0M) dose-dependently attenuated nlfedlplne-reslstant Ca---contractlon in the presence of STAp in both canine cerebral and coronary arteries. The inhibitory e~f~ct of combined treatment wlth+~P-252 (I0 v M) and nitroglycerin (I0 M) on nifedlplne-resistant Ca -contractlon in the cerebral artery was additive. These results indicate that NP-252 possesses a stronger vasolnhlbltory effect than that of nifedlplne in canine cerebral artery.
Nifedlpine is the prototype dihydropyrldlne calcium antagonist, and is useful in the treatment of vasospastlc angina and essential hypertension (1,2,3,4,5,6). Hisaki e t a l . (7) synthesized n-octyl-2,6-dimethyl-3-cyclopropylaminocarbonyl-4-(o-nltrophenyl)-l,4-dihydropyridlne-5-carboxylate (NP-252) (Fig. i). They found that NP-252 had a potent vasodilatlng effect in various arterial preparations and showed a potent and long lasting antlhypertenslve effect in spontaneously hypertensive rats (7,8). In both cerebral and coronary circulation, thromboxane A 2 (TXAp) and serotonin act as spasmogenlc vasoactlve substances (9,10,11), and STA 2 is a stable TXA^z analogue (12). The present experiments were undertaken to compare cerebrovascular inhibitory effect of NP-252 with that of nlfedlplne in canine cerebral artery.
Methods Mongrel dogs (8-15 kg) of both sexes were anesthetized with sodium pentobarbltal (30 mg/kg, i.v.) and sacrificed by exsanguinatlon from the common carotid arteries. The brain was rapidly removed. The cerebral arteries (basilar and middle cerebral arteries) and coronary arteries were 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
184
Cerebrovasoinhibitlon
by NP-252
Vol. 48, No. 2, 1991
••'•NO
2 [~HNOC-~~COOC8 H17 CH3" "HN" "CH3 Fig. 1 Chemical structure of NP-252 isolated, and were cut into helical strips, 15 mm long and 1.5 mm wlde and 20 long and 2 mm wide, respectively. The strips were vertically fixed between hooks in an organ bath containing a nutrient solution, which was maintained at 3 7 + 0.5 o C and aerated with a mixture of 95% 02 and 5% COp. The composition of The solution was as follows (mM): NaCI, 120; KCI, 4.7; HgSO&, 1.2; KH2PO&, 1.2; CaCI^, 2.5; NaHCO^, 25 and glucose, I0. The pH of the solution was 7.4. z J The hook anchoring the upper end of the strip was connected to the lever of a force-dlsplacement transducer (NEC Sanei Instrument Co. Ltd., Tokyo, Japan). The resting tension was adjusted to 1.5 g. Before the experiments were begun, preparations were allowed to equilibrate for 90 mln in the organ bath, during which time the medium was replaced every 15 min and the tension was readjusted to 1.5 g. By the repetitive readjustments, the tension was reached to steady level and then experiments were started. Isometric contractions were recorded on an ink-writing oscillograph (NEC Sanei Instrument Co. Ltd., Tokyo, Japan). When the contractions induced by STA 2 (I0 -v M) in cerebral and coronary arteries were less t h a n ~ g and 1.6 g, respectively, the preparations were discarded. To obtain C a - i n d u c e d contraction u n ~ r deporalization by 70 mM KCI, the strips were incubated for 25 min in Ca -free medium, and then ~ I (70 raM) was introduced to the bath 15 min prior to the application of Ca . The test compounds, N ~ 2 5 2 and nifedipine, were added to the bath 25 min prior to the addition of Ca ++when their effect was to be studied. To obtain the nifedipine-r~istant Ca -induced contraction, the strips were incubate~ for 25 min in Ca -free medium containing EGTA (0. I mM) and nifedipine (I0 -v M). This procedure nearly abolished the KCI (70 mM)-induced contraction that is seen in normal medium. Then the T X ~ agonist, STAr was added to the bath 20 min prior to the application of Ca (2.5 mM). N~-252~_qr nitroglycerin was added to the bath 10 min before the addition of Ca . Nifedipine and ethylene glycol bis (8-aminoethyl e t h e r ) - N , N ' - t e t r a a c e t i c acid (Sigma Chemical Co., St. Louis, MO) were purchased from the company indicated. 9,11-epithio -ll,12-methano-TXAp (STAp) (Ono Pharmaceutical Co., Ltd., Osaka, Japan) and n-octyl-2,6-dimethgl-3-cgclopropylaminocarbonyl-4-(o-nitrophenyl)-l,4-dihydropyridlne-5-carboxylate (NP-252) (Nippon Shoji Kaisha, Ltd., Osaka, Japan) were kindly provided by the companies indicated. STA 2 was dissolved in distilled water. NP-252 and nifedlplne were dissolved in acetone and ethanol, respectively, to a concentration of I0- M. The final concentrations of ethanol and acetone in the tissue bath were under 0.1% (v/v) and 0.05% (v/v), respectively. These did not produce any change in resting tone of
Vol. 48, No. 2, 1991
Cerebrovasolnhlbltlon by NP-252
185
preparations. All data are expressed as mean ~ S.E. and statistical canoe was determined using Student's t-test.
slgnlfi-
Results ++ Effects of NP-252 on cerebral artery
the Ca
-contraction
and STA2-contractlons
in
canine
KCI (10-70 mM) caused a dose-dependent contraction of canine cerebral artery+and the concentration for the maximal contractile response was 70 m ~ In Ca -free medium containing KCI (70 ~ ) , the subsequent a p p l i ~ t l o n of Ca (2.5 mM) caused a tonic contraction (Ca''-contractlon). The ~ -cont~actlon was dose-dependently attenuated by the presence of NP-252 ~I0- v to I0 -° M) or nlfe~iplne (I0 -~- to I0-~ M) (Fig. ~ A ) . Both NP-252 (I0-" M) and nlfedlplne (I0 -~ M) nearly abolished the Cai~-contractlon. Th~ mean IC50 values of NP-252 and nlfedlplne were 1.4 X I0-- +~ and 5.8 X I0-- M, respectively. The vasolnhlbitory effect of NP-252 on Ca "-contractlon was 4 times stronger at its IC50 level than that of nlfediplne in canine cerebral artery. B
A 10~
.~ 5(
i
• NHed~oh~e
•
N~ed~ine
•
•
NP-252
NP-252
.~ .Q
j/
5(~
i
O Concentration of
drugs (-log M)
Concentration of drugs (-log
M)
Fig. 2 4+ Effects of NP-252 and nifedlpine on Ca -contraction (A) and on co~ractlon induced by STA? (B) in canine cereb~_~l arteries. Ca -contraction: the addltlo~ of CaCI? (2.5 mM) to Ca''-free medium containing K~I (70 mM) is regarded as I00 %. The contraction produced by STA 2 (I0-- M) is regarded as I00 %. Number of experiments were 7 to I0 and the mean with S.E. were presented. * indicates a significant difference against the value for nlfediplne.
186
Cerebrovasoinhibition by NP-252
Vol. 48, No. 2, 1991
STA 2 (i0 -II to 10 -7 M) induced a contraction of canine cerebral artery in a concentration-dependent manner, and the maximal contraction was obtained b~ STA~ at 10-- M. The inhibitory effects of NP-252 and nifedipine on STA^ ~I0-" M)-~nduced contraction were c o m ~ r e d . A~ shown in Fig. 2B, NP-252 (i~to I0-~ M) and nifedipine (3 X I0 ~ to I0 -~ M) attenuate~ the contractile response in concentration-dependent manner. NP-252 at I0 M caused a significantly greater attenuation of the contractile response than did nifedipine at I0-- M. Neither agent , however, abolish the contraction. The NP-252-resistant contraction (about 30 % of control) and nifedipine-resistant contraction (about 40 % of control) remained. The vasoinhibitory effect of NP-252 at its IC50 level was 7 times stronger than that of nifedipine. ++ Effect of NP-252 on nifedipine-resistant Ca -contraction in the presence STA 2 in canine cerebral and coronary arteries
of
In normal medium, the contractions induced by STA 2 (10 -8 M) in the cerebral and coronary arteries were 1.23 ± 0.06 g (n=26) and 2.07 ± 0.25 g (n~6), respectively, and these contractions were regarded~as 100%. In -8 Ca''-free medium containing EGTA (0. I mM) and nifedipine (I0 v M), STA?++(10 M) induced a slight tonic contraction and the subsequent a d d i t ~ n of C~ (2.5 mM) induced a large tonic c ~ t r a c t i o n (nifedipine-resistant Ca -contraction). The nifedipine-resist~nt Ca -contraction was about hal~ of the. contraction produced by STA9 (I0 -v M) in normal medium. NP-252 (I0-- a ~ i0-b M) concentration-dependently attenuated this nifedipine-resistant Ca -contraction. As shown in TABLE I, the effect of NP-252 in the coronary artery were g ~ a t e r than those in cerebral artery. In cerebral artery, nitroglycerin (i0 ~ M) attenuated the contraction more than NP-252 (I0 ~ M) did (Fig. 3). The both tr~_~tment with NP-252 and nitroglycerin produced an additive inhibition of the Ca"-contraction. In c ~ o n a r y artery, nitroglycerin (I0 -~ M) nearly abolished nifedipine-resistant C a - c o n t r a c t i o n 6 In the same experimental protocol, effects of nifedipine on NP-252 (I0- M)-resistant ~ontraction were examined in cerebral and coronary arteries. Nifedipine (10- M) in both arteries did not affect the contraction.
TABLE 1 Effects of NP-252 and nitroglycerin on nifedipine-resistant Ca ++contraction in canine cerebral and coronary arteries Cerebral artery Coronary artery (% inhibition) NP-252
I0-~ M I0 -b M
I0 ± 2 34 ± 2
(n=10) (n=10)
13 ± 3 44 ± 3
(n=6) (n=8)
Nitroglycerin
10-6 M
61 ± 4
(n=13)
97 ± i
(n=6)
Nitroglycerin + NP-252
I0-~ M i0 -b M
85 ± 2
(n=14)
II 8 Nifedipine-resistant Ca -contraction+in the presence o ~ S T A ~ (I0 M) was obtained by the addition of Ca (2.~gmM) to Ca''-fr~e medium containin~_~EGTA (0. i mM) and nifedipine (i0 v M). The nifedipine-resistant Ca -contractions in cerebral and coronary arteries were 0.62 ± 0.05 g (n=26) and 0.95 ± 0.13 g (n=16), respectively.
Vol. 48, No. 2, 1991
A: Control
Cerebrovasolnhlbltlon by NP-252
B: NP-252 (10-6 M)
C: Nitro (10-6 M)
187
D: NP-252 (10-6 M) Nitro (10-6 M)
•
~
,f
0.3g 10 rain
Fig. 3 Representative traces showing the inhibitory e f f e c ~ of NP-252 and nitroglycerin (Nitro) on nifedipine-resistant. Ca -contraction in canine cerebral artery. Nifedipine-resistant Ca1-~-contraction in t ~ presence of STA o ( 1 0 - v ~ ) ( O ) was obtained by the addition of Ca (2.5 mM) (A)6to Ca~T-free medium containing EGTA (0. I mM) 6and nifediplne (I0- M). A: control (wlthou~ agent). B: NP-252 (I0- M) was added a~ arrow. C: nltroglycein ~ 0 M) was added at arrow. D: NP-252 (I0 ~ M) and nitroglycerin (I0 ~ M) were added at arrow.
Discussion The vasoinhlbitory action of a newly synthesized dihydropyridlne derivative, NP-252, on canine cerebral artery was examined in comparison with nlfedlpine. In canine cerebral artery, NP-2 ~ a n d n i ~ d i p i n e abolished the contractile response to the addition of Ca to Ca -free medium in t ~ presence of KCI. The potency of the inhibitory action of NP-252 on the Ca contraction was greater than that of nifediplne. These results s ~ g e s t that NP-252 has a stronger inhibitory action on the voltage-operated C a channels (VOCs) than does nifedlpine in cerebral artery. It is known that TXA^ and serotonin are potent spasmogens in cerebral and coronary circu~atlon (9,10,11). We have proposed that the endothellum-derived contracting factor in canine cerebral artery is probably TXA 2 (13,14,15,16,17,18). In canine cerebral artery, TXA 2 agonlst, STA 2 elicited a strong contraction in a concentration-dependent manner. NP-252 and nifediplne concentration-dependently reduced the maxim~l contractile response induced by STA 2. In the presence of nlfedipine at I0- M, part of contraction remained. This suggests that the remaining ~ n t r a c t i o n (nifedlpine-resistant) was probably not due to mobiliz~_~ tion of C a through the VOCs and may have b e e n due to mobilization of C a via another mechanism (e.g. receptor-operated Ca channels, ROCs). Concernin~+to contractile mechanism of STA^, it was reported that the influxes of Ca activated by STA^ may occur wlt~ activation of th~_Freceptor operated Ca ~:'~ z channels ( R O C ) and partly of the voltage operated Ca channels (VOC)(12). Since nlfedip~ne-resistant contraction induced by norepinephrlne was de~ected, it was considered that not only VOCs but also ROCs may be involved in vascular contraction (19,20,21). NP-252 caused a more potent attenuation of the contraction produced by STA 2 than did nlfediplne. The inhibitory actions of NP-252 in the tested concentrations were significantly greater than those of nifedipine. In both cerebral and coronary arteries, NP-252 significantly attenuated nlfedlplne-resistant contraction. The inhibitory effect of NP-252 on the contraction was additive to that of nitroglycerin. These results suggest that NP-252 may have inhibitory effect on ROCs and/or weak TXA antagonistic effect, in addition to inhibitory effect on VOCs in cerebral an~ coronary arteries. Such inhibitory effect of NP-252 may be different from that of nitroglycerin.
188
Cerebrovasoinhibition by NP-252
Vol. 48, No. 2, 1991
Acknowledsements This study was supported in part by a Grant-in Aid for Scientific Research (61570101) from the Ministry of Education, Science and Culture and a Grant from the Smoking Research Foundation, Japan.
References I. G. GR N, and A. FLECKENSTEIN, Arzneim. Forsch. (Drug Res.) 22 334-344 (1972~ 2. A. FLECKENSTEIN, Ann. Rev. Pharmacol. Toxicol. 17 149-166 (1977) 3. D. BONADUCE, N. FERRARA, M, PETRETTA, E. ROMANO, M. POSTIGLIONE, F. RENGO, and M. CONDORELLI, Am. Heart J. 105 865-867 (1983) 4. P.W. SERRUYS, T.E.H. HOOGHOUDT, J.H.C. REIBER, C. SLAGER, R.W. BROWER, and P.G. HUGENHOLTZ, Br. Heart J. 49 227-241 (1983) 5. P.D. HENRY, Am. J. Cardiology 46 1047-1058 (1980) 6. E.M. SORKIN, S.P. CLISSOLD, and R.N. BROGDEN, Drugs 30 182-274 (1985) 7. M. HISAKI, K. KASHIMA, Y. SAKAMOTO, Y. OTA, M. HOJO, S. KATAYAMA and H. HATA, In the abstract of the 108th Meeting of Pharmaceutical Society of Japan, pp 684, Hiroshima, April (1988) 8. Y. AKIMOTO, K. KURAHASHI and M. FUJIWARA, Japan. J. Pharmacol. 49 216p
(1989) 9. D.J. BOULLIN, Cerebral vasospasm, Ed. D.J. Boullin, pp 111-142, John Wiley & Sons, Chichester (1980) I0. J.E. PEREZ, J.E. SAFFITZ, F.A. GUTIERREZ and P.D. HENRY, Cir. Res. 52 423-431 (1983) II. Y. YUI, K. SAKAGUCHI, T. SUSAWA, R. HATTORI, Y. TAKATSU, N. IKEDA and C. KAWAI, Advance in prostaslandin, thromboxane, and leukotriene research, Vol.15, Ed. O. HAYAISHI and S. YAMAMOTO, pp 485-486, Raven Press, New York
(1985) 12. M. FUJIOKA, T. NAGAO and H. KURIYAMA, Naunyn-Schmiedeberg's Arch. Pharmacol. 334 468-474 (1986) 13. H. USUI, K. KURAHASHI, K. ASHIDA, and M. FUJIWARA, IRCS Med. Sci. II 418-419 (1983) 14. H. USUI, M. FUJIWARA, T. TSUBOMURA, K. KURAHASHI, S. NOMURA and N. MIZUNO, Neural regulation of brain circulation, Ed. C. Owman and J.E. Hardebo, pp 261-272, Elsevier Science Publishers, Amsterdam (1986) 15. H. USUI, K. KURAHASHI, H. SHIRAHASE, K. FUKUI and M. FUJIWARA, Japan. J. Pharmacol. 44 228-231 (1987) 16. H. USUI, M. FUJIWARA, H. SHIRAHASE, K. KURAHASHI, S. OKAMOTO and S. MIYAMOTO, Neuronal messengers in vascular function, Ed. A. Nobin, C. Owman and B. Arneklo-Nobln, pp 537-547, Elsevier Science Publishers, Amsterdam
(1987) 17. H. SHIRAHASE, H. USUI, K. KURAHASHI, M. FUJIWARA, and K. FUKUI, J. Cardiovasc. Pharmacol. I0 517-522 (1987) 18. H. SHIRAHASE, H. USUI, K. MANABE, K. KURAHASHI, and M. FUJIWARA, J. Pharmacol. Exp. Ther. 247 701-705 (1988) 19. T.B. BOLTON, Physiol. Rev. 59 606-718 (1979) 20. C. VAN BREEMEN, P. AARONSON and R. LOUTZENHEIZER, Pharmacol. Rev. 30 167-208 (1979) 21. Y. AKIMOTO, K. KURAHASHI, H. USUI, M. FUJIWARA and S. SHIBATA, Japan. J. Pharmacol. 44 506-509 (1987)
Pergamon Press
VASOINHIBITORY EFFECT OF NP-252, A NEW DIHYDROPYRIDINE DERIVATIVE, IN CANINE CEREBRAL ARTERY
Yoshinobu Akimoto, Kazuyoshi Kurahashi I, Hachlro Usui
and Motohatsu FuJiwara
and
iDepartment of Pharmacology, Faculty of Medicine Pharmacology Division, RI Center, Kyoto University, Kyoto 606, Japan (Received in final form November 8, 1990)
Summary The v a s ~ n h i b i t o r y e f f e c t of NP-252, a 1 , 4 - d i h y d r o p y r i d i n e derivative Ca a n t a g o n i s t , was examined i n c a n i n e c e r e b r a l a r t e r x 7 and t h i s e f f e c t was c ~ p a r e d with t h a t of n i f e d i p i n e . NP-252 (10 M) and n i f e d i p i n e ~ 1 0 M ~ n e a r l y a b o l i s h e d the c o n t r a c t i o n induce~ by a d d i t i o n of C a ' ' to ~ a ' ' - f r e e medium c o n t a i n i n g KC1. NP-252 (10 - v M) and nifedipine (I0 M) attenuated the contraction produced by thromboxane A 2 agonlst (STAp) in normal medium, and the resultant contractions were 22 % (n=6)~and 35 % (n=6) of the control contraction, respectively. The vasolnhlbltory effects of NP-252 were significantly stronger7than thos~ of nlfediplne in canine cerebral artery. NP-252 (i0- ++and i0M) dose-dependently attenuated nlfedlplne-reslstant Ca---contractlon in the presence of STAp in both canine cerebral and coronary arteries. The inhibitory e~f~ct of combined treatment wlth+~P-252 (I0 v M) and nitroglycerin (I0 M) on nifedlplne-resistant Ca -contractlon in the cerebral artery was additive. These results indicate that NP-252 possesses a stronger vasolnhlbltory effect than that of nifedlplne in canine cerebral artery.
Nifedlpine is the prototype dihydropyrldlne calcium antagonist, and is useful in the treatment of vasospastlc angina and essential hypertension (1,2,3,4,5,6). Hisaki e t a l . (7) synthesized n-octyl-2,6-dimethyl-3-cyclopropylaminocarbonyl-4-(o-nltrophenyl)-l,4-dihydropyridlne-5-carboxylate (NP-252) (Fig. i). They found that NP-252 had a potent vasodilatlng effect in various arterial preparations and showed a potent and long lasting antlhypertenslve effect in spontaneously hypertensive rats (7,8). In both cerebral and coronary circulation, thromboxane A 2 (TXAp) and serotonin act as spasmogenlc vasoactlve substances (9,10,11), and STA 2 is a stable TXA^z analogue (12). The present experiments were undertaken to compare cerebrovascular inhibitory effect of NP-252 with that of nlfedlplne in canine cerebral artery.
Methods Mongrel dogs (8-15 kg) of both sexes were anesthetized with sodium pentobarbltal (30 mg/kg, i.v.) and sacrificed by exsanguinatlon from the common carotid arteries. The brain was rapidly removed. The cerebral arteries (basilar and middle cerebral arteries) and coronary arteries were 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc
184
Cerebrovasoinhibitlon
by NP-252
Vol. 48, No. 2, 1991
••'•NO
2 [~HNOC-~~COOC8 H17 CH3" "HN" "CH3 Fig. 1 Chemical structure of NP-252 isolated, and were cut into helical strips, 15 mm long and 1.5 mm wlde and 20 long and 2 mm wide, respectively. The strips were vertically fixed between hooks in an organ bath containing a nutrient solution, which was maintained at 3 7 + 0.5 o C and aerated with a mixture of 95% 02 and 5% COp. The composition of The solution was as follows (mM): NaCI, 120; KCI, 4.7; HgSO&, 1.2; KH2PO&, 1.2; CaCI^, 2.5; NaHCO^, 25 and glucose, I0. The pH of the solution was 7.4. z J The hook anchoring the upper end of the strip was connected to the lever of a force-dlsplacement transducer (NEC Sanei Instrument Co. Ltd., Tokyo, Japan). The resting tension was adjusted to 1.5 g. Before the experiments were begun, preparations were allowed to equilibrate for 90 mln in the organ bath, during which time the medium was replaced every 15 min and the tension was readjusted to 1.5 g. By the repetitive readjustments, the tension was reached to steady level and then experiments were started. Isometric contractions were recorded on an ink-writing oscillograph (NEC Sanei Instrument Co. Ltd., Tokyo, Japan). When the contractions induced by STA 2 (I0 -v M) in cerebral and coronary arteries were less t h a n ~ g and 1.6 g, respectively, the preparations were discarded. To obtain C a - i n d u c e d contraction u n ~ r deporalization by 70 mM KCI, the strips were incubated for 25 min in Ca -free medium, and then ~ I (70 raM) was introduced to the bath 15 min prior to the application of Ca . The test compounds, N ~ 2 5 2 and nifedipine, were added to the bath 25 min prior to the addition of Ca ++when their effect was to be studied. To obtain the nifedipine-r~istant Ca -induced contraction, the strips were incubate~ for 25 min in Ca -free medium containing EGTA (0. I mM) and nifedipine (I0 -v M). This procedure nearly abolished the KCI (70 mM)-induced contraction that is seen in normal medium. Then the T X ~ agonist, STAr was added to the bath 20 min prior to the application of Ca (2.5 mM). N~-252~_qr nitroglycerin was added to the bath 10 min before the addition of Ca . Nifedipine and ethylene glycol bis (8-aminoethyl e t h e r ) - N , N ' - t e t r a a c e t i c acid (Sigma Chemical Co., St. Louis, MO) were purchased from the company indicated. 9,11-epithio -ll,12-methano-TXAp (STAp) (Ono Pharmaceutical Co., Ltd., Osaka, Japan) and n-octyl-2,6-dimethgl-3-cgclopropylaminocarbonyl-4-(o-nitrophenyl)-l,4-dihydropyridlne-5-carboxylate (NP-252) (Nippon Shoji Kaisha, Ltd., Osaka, Japan) were kindly provided by the companies indicated. STA 2 was dissolved in distilled water. NP-252 and nifedlplne were dissolved in acetone and ethanol, respectively, to a concentration of I0- M. The final concentrations of ethanol and acetone in the tissue bath were under 0.1% (v/v) and 0.05% (v/v), respectively. These did not produce any change in resting tone of
Vol. 48, No. 2, 1991
Cerebrovasolnhlbltlon by NP-252
185
preparations. All data are expressed as mean ~ S.E. and statistical canoe was determined using Student's t-test.
slgnlfi-
Results ++ Effects of NP-252 on cerebral artery
the Ca
-contraction
and STA2-contractlons
in
canine
KCI (10-70 mM) caused a dose-dependent contraction of canine cerebral artery+and the concentration for the maximal contractile response was 70 m ~ In Ca -free medium containing KCI (70 ~ ) , the subsequent a p p l i ~ t l o n of Ca (2.5 mM) caused a tonic contraction (Ca''-contractlon). The ~ -cont~actlon was dose-dependently attenuated by the presence of NP-252 ~I0- v to I0 -° M) or nlfe~iplne (I0 -~- to I0-~ M) (Fig. ~ A ) . Both NP-252 (I0-" M) and nlfedlplne (I0 -~ M) nearly abolished the Cai~-contractlon. Th~ mean IC50 values of NP-252 and nlfedlplne were 1.4 X I0-- +~ and 5.8 X I0-- M, respectively. The vasolnhlbitory effect of NP-252 on Ca "-contractlon was 4 times stronger at its IC50 level than that of nlfediplne in canine cerebral artery. B
A 10~
.~ 5(
i
• NHed~oh~e
•
N~ed~ine
•
•
NP-252
NP-252
.~ .Q
j/
5(~
i
O Concentration of
drugs (-log M)
Concentration of drugs (-log
M)
Fig. 2 4+ Effects of NP-252 and nifedlpine on Ca -contraction (A) and on co~ractlon induced by STA? (B) in canine cereb~_~l arteries. Ca -contraction: the addltlo~ of CaCI? (2.5 mM) to Ca''-free medium containing K~I (70 mM) is regarded as I00 %. The contraction produced by STA 2 (I0-- M) is regarded as I00 %. Number of experiments were 7 to I0 and the mean with S.E. were presented. * indicates a significant difference against the value for nlfediplne.
186
Cerebrovasoinhibition by NP-252
Vol. 48, No. 2, 1991
STA 2 (i0 -II to 10 -7 M) induced a contraction of canine cerebral artery in a concentration-dependent manner, and the maximal contraction was obtained b~ STA~ at 10-- M. The inhibitory effects of NP-252 and nifedipine on STA^ ~I0-" M)-~nduced contraction were c o m ~ r e d . A~ shown in Fig. 2B, NP-252 (i~to I0-~ M) and nifedipine (3 X I0 ~ to I0 -~ M) attenuate~ the contractile response in concentration-dependent manner. NP-252 at I0 M caused a significantly greater attenuation of the contractile response than did nifedipine at I0-- M. Neither agent , however, abolish the contraction. The NP-252-resistant contraction (about 30 % of control) and nifedipine-resistant contraction (about 40 % of control) remained. The vasoinhibitory effect of NP-252 at its IC50 level was 7 times stronger than that of nifedipine. ++ Effect of NP-252 on nifedipine-resistant Ca -contraction in the presence STA 2 in canine cerebral and coronary arteries
of
In normal medium, the contractions induced by STA 2 (10 -8 M) in the cerebral and coronary arteries were 1.23 ± 0.06 g (n=26) and 2.07 ± 0.25 g (n~6), respectively, and these contractions were regarded~as 100%. In -8 Ca''-free medium containing EGTA (0. I mM) and nifedipine (I0 v M), STA?++(10 M) induced a slight tonic contraction and the subsequent a d d i t ~ n of C~ (2.5 mM) induced a large tonic c ~ t r a c t i o n (nifedipine-resistant Ca -contraction). The nifedipine-resist~nt Ca -contraction was about hal~ of the. contraction produced by STA9 (I0 -v M) in normal medium. NP-252 (I0-- a ~ i0-b M) concentration-dependently attenuated this nifedipine-resistant Ca -contraction. As shown in TABLE I, the effect of NP-252 in the coronary artery were g ~ a t e r than those in cerebral artery. In cerebral artery, nitroglycerin (i0 ~ M) attenuated the contraction more than NP-252 (I0 ~ M) did (Fig. 3). The both tr~_~tment with NP-252 and nitroglycerin produced an additive inhibition of the Ca"-contraction. In c ~ o n a r y artery, nitroglycerin (I0 -~ M) nearly abolished nifedipine-resistant C a - c o n t r a c t i o n 6 In the same experimental protocol, effects of nifedipine on NP-252 (I0- M)-resistant ~ontraction were examined in cerebral and coronary arteries. Nifedipine (10- M) in both arteries did not affect the contraction.
TABLE 1 Effects of NP-252 and nitroglycerin on nifedipine-resistant Ca ++contraction in canine cerebral and coronary arteries Cerebral artery Coronary artery (% inhibition) NP-252
I0-~ M I0 -b M
I0 ± 2 34 ± 2
(n=10) (n=10)
13 ± 3 44 ± 3
(n=6) (n=8)
Nitroglycerin
10-6 M
61 ± 4
(n=13)
97 ± i
(n=6)
Nitroglycerin + NP-252
I0-~ M i0 -b M
85 ± 2
(n=14)
II 8 Nifedipine-resistant Ca -contraction+in the presence o ~ S T A ~ (I0 M) was obtained by the addition of Ca (2.~gmM) to Ca''-fr~e medium containin~_~EGTA (0. i mM) and nifedipine (i0 v M). The nifedipine-resistant Ca -contractions in cerebral and coronary arteries were 0.62 ± 0.05 g (n=26) and 0.95 ± 0.13 g (n=16), respectively.
Vol. 48, No. 2, 1991
A: Control
Cerebrovasolnhlbltlon by NP-252
B: NP-252 (10-6 M)
C: Nitro (10-6 M)
187
D: NP-252 (10-6 M) Nitro (10-6 M)
•
~
,f
0.3g 10 rain
Fig. 3 Representative traces showing the inhibitory e f f e c ~ of NP-252 and nitroglycerin (Nitro) on nifedipine-resistant. Ca -contraction in canine cerebral artery. Nifedipine-resistant Ca1-~-contraction in t ~ presence of STA o ( 1 0 - v ~ ) ( O ) was obtained by the addition of Ca (2.5 mM) (A)6to Ca~T-free medium containing EGTA (0. I mM) 6and nifediplne (I0- M). A: control (wlthou~ agent). B: NP-252 (I0- M) was added a~ arrow. C: nltroglycein ~ 0 M) was added at arrow. D: NP-252 (I0 ~ M) and nitroglycerin (I0 ~ M) were added at arrow.
Discussion The vasoinhlbitory action of a newly synthesized dihydropyridlne derivative, NP-252, on canine cerebral artery was examined in comparison with nlfedlpine. In canine cerebral artery, NP-2 ~ a n d n i ~ d i p i n e abolished the contractile response to the addition of Ca to Ca -free medium in t ~ presence of KCI. The potency of the inhibitory action of NP-252 on the Ca contraction was greater than that of nifediplne. These results s ~ g e s t that NP-252 has a stronger inhibitory action on the voltage-operated C a channels (VOCs) than does nifedlpine in cerebral artery. It is known that TXA^ and serotonin are potent spasmogens in cerebral and coronary circu~atlon (9,10,11). We have proposed that the endothellum-derived contracting factor in canine cerebral artery is probably TXA 2 (13,14,15,16,17,18). In canine cerebral artery, TXA 2 agonlst, STA 2 elicited a strong contraction in a concentration-dependent manner. NP-252 and nifediplne concentration-dependently reduced the maxim~l contractile response induced by STA 2. In the presence of nlfedipine at I0- M, part of contraction remained. This suggests that the remaining ~ n t r a c t i o n (nifedlpine-resistant) was probably not due to mobiliz~_~ tion of C a through the VOCs and may have b e e n due to mobilization of C a via another mechanism (e.g. receptor-operated Ca channels, ROCs). Concernin~+to contractile mechanism of STA^, it was reported that the influxes of Ca activated by STA^ may occur wlt~ activation of th~_Freceptor operated Ca ~:'~ z channels ( R O C ) and partly of the voltage operated Ca channels (VOC)(12). Since nlfedip~ne-resistant contraction induced by norepinephrlne was de~ected, it was considered that not only VOCs but also ROCs may be involved in vascular contraction (19,20,21). NP-252 caused a more potent attenuation of the contraction produced by STA 2 than did nlfediplne. The inhibitory actions of NP-252 in the tested concentrations were significantly greater than those of nifedipine. In both cerebral and coronary arteries, NP-252 significantly attenuated nlfedlplne-resistant contraction. The inhibitory effect of NP-252 on the contraction was additive to that of nitroglycerin. These results suggest that NP-252 may have inhibitory effect on ROCs and/or weak TXA antagonistic effect, in addition to inhibitory effect on VOCs in cerebral an~ coronary arteries. Such inhibitory effect of NP-252 may be different from that of nitroglycerin.
188
Cerebrovasoinhibition by NP-252
Vol. 48, No. 2, 1991
Acknowledsements This study was supported in part by a Grant-in Aid for Scientific Research (61570101) from the Ministry of Education, Science and Culture and a Grant from the Smoking Research Foundation, Japan.
References I. G. GR N, and A. FLECKENSTEIN, Arzneim. Forsch. (Drug Res.) 22 334-344 (1972~ 2. A. FLECKENSTEIN, Ann. Rev. Pharmacol. Toxicol. 17 149-166 (1977) 3. D. BONADUCE, N. FERRARA, M, PETRETTA, E. ROMANO, M. POSTIGLIONE, F. RENGO, and M. CONDORELLI, Am. Heart J. 105 865-867 (1983) 4. P.W. SERRUYS, T.E.H. HOOGHOUDT, J.H.C. REIBER, C. SLAGER, R.W. BROWER, and P.G. HUGENHOLTZ, Br. Heart J. 49 227-241 (1983) 5. P.D. HENRY, Am. J. Cardiology 46 1047-1058 (1980) 6. E.M. SORKIN, S.P. CLISSOLD, and R.N. BROGDEN, Drugs 30 182-274 (1985) 7. M. HISAKI, K. KASHIMA, Y. SAKAMOTO, Y. OTA, M. HOJO, S. KATAYAMA and H. HATA, In the abstract of the 108th Meeting of Pharmaceutical Society of Japan, pp 684, Hiroshima, April (1988) 8. Y. AKIMOTO, K. KURAHASHI and M. FUJIWARA, Japan. J. Pharmacol. 49 216p
(1989) 9. D.J. BOULLIN, Cerebral vasospasm, Ed. D.J. Boullin, pp 111-142, John Wiley & Sons, Chichester (1980) I0. J.E. PEREZ, J.E. SAFFITZ, F.A. GUTIERREZ and P.D. HENRY, Cir. Res. 52 423-431 (1983) II. Y. YUI, K. SAKAGUCHI, T. SUSAWA, R. HATTORI, Y. TAKATSU, N. IKEDA and C. KAWAI, Advance in prostaslandin, thromboxane, and leukotriene research, Vol.15, Ed. O. HAYAISHI and S. YAMAMOTO, pp 485-486, Raven Press, New York
(1985) 12. M. FUJIOKA, T. NAGAO and H. KURIYAMA, Naunyn-Schmiedeberg's Arch. Pharmacol. 334 468-474 (1986) 13. H. USUI, K. KURAHASHI, K. ASHIDA, and M. FUJIWARA, IRCS Med. Sci. II 418-419 (1983) 14. H. USUI, M. FUJIWARA, T. TSUBOMURA, K. KURAHASHI, S. NOMURA and N. MIZUNO, Neural regulation of brain circulation, Ed. C. Owman and J.E. Hardebo, pp 261-272, Elsevier Science Publishers, Amsterdam (1986) 15. H. USUI, K. KURAHASHI, H. SHIRAHASE, K. FUKUI and M. FUJIWARA, Japan. J. Pharmacol. 44 228-231 (1987) 16. H. USUI, M. FUJIWARA, H. SHIRAHASE, K. KURAHASHI, S. OKAMOTO and S. MIYAMOTO, Neuronal messengers in vascular function, Ed. A. Nobin, C. Owman and B. Arneklo-Nobln, pp 537-547, Elsevier Science Publishers, Amsterdam
(1987) 17. H. SHIRAHASE, H. USUI, K. KURAHASHI, M. FUJIWARA, and K. FUKUI, J. Cardiovasc. Pharmacol. I0 517-522 (1987) 18. H. SHIRAHASE, H. USUI, K. MANABE, K. KURAHASHI, and M. FUJIWARA, J. Pharmacol. Exp. Ther. 247 701-705 (1988) 19. T.B. BOLTON, Physiol. Rev. 59 606-718 (1979) 20. C. VAN BREEMEN, P. AARONSON and R. LOUTZENHEIZER, Pharmacol. Rev. 30 167-208 (1979) 21. Y. AKIMOTO, K. KURAHASHI, H. USUI, M. FUJIWARA and S. SHIBATA, Japan. J. Pharmacol. 44 506-509 (1987)