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Antihypertensive compounds with potent protein kinases inhibitory activity
Life Sciences, Vol. 44, pp. 1351-1362 Printed in the U.S.A.
ANTIHYPERTENSIVE
M i t s u g u Hachisu,
COMPOUNDS INHIBITORY
Toyokazu
Pergamon Press
WITH POTENT ACTIVITY
Hiranuma,
PROTEIN
Masao Koyama
KINASES
and M a s a j i
P h a r m a c e u t i c a l R e s e a r c h L a b o r a t o r i e s , Meiji Seika Kaisha M o r o o k a - c h o , Kohoku-ku, Y o k o h a m a 222, J a p a n
Sezaki
Ltd.,
(Received in final form March 3, 1989)
Summary
A new i n d o r o c a r b a z o l e a n t i b i o t i c SF2370 has been found in the culture b r o t h of A c t i n o m a d u r a sp.; it has also b e e n found to have the a c t i v i t i e s of antihyp e r t e n s i o n and diuresis. Derivatives of SF2370, NA0344, NA0345 and NA0346, when injected (0.i-I mg/kg i.v.) significantly lowered the blood p r e s s u r e of anesthetized normotensive rats and showed a long lasting a n t i h y p e r t e n s i v e action. In the case of p.o. a d m i n i s t r a t i o n to c o n s c i o u s r e s t r a i n e d SHRs, m e a s u r e m e n t of blood p r e s s u r e by the p l e t h y s m o g r a p h i c m e t h o d i n d i c a t e d that 10 m g / k g of the c o m p o u n d s was enough to lower the blood pressure; the a n t i h y p e r t e n s i v e act i v i t i e s were found to r e m a i n more than 12 hrs after oral a d m i n i s t r a t i o n . By s t u d y i n g of the mode of antih y p e r t e n s i v e action, these c o m p o u n d s dose d e p e n d e n t l y relaxed the isolated aortic p r e p a r a t i o n c o n t r a c t e d by norepinephrine. In addition, it was found that protein kinase C a c t i v i t y of mice brain was i n h i b i t e d by the compounds; the IC50 values were in the range of 0.062 - 0.20 MM. Moreover, a superprecipitation of a c t o m y o s i n from smooth m u s c l e of c h i c k e n g i z z a r d was inhibited by the compounds, having an IC50 values of 0.31 0.72 ~M.
In 1985, a new i n d o r o c a r b a z o l e a n t i b i o t i c SF2370 was found in the culture broth of A c t i n o m a d u r a sp.,(l, 2) and it was also found that the c o m p o u n d provides a n t i h y p e r t e n s i v e and d i u r e t i c action. To o b t a i n more potent c o m p o u n d s for a n t i h y p e r t e n s i v e action, several new compounds of NA d e r i v a t i v e s were derived from SF2370. The structually related compound of s t a u r o s p o r i n e providing antihyp e r t e n s i v e a c t i v i t y was r e p o r t e d to inhibit p r o t e i n kinase C activity in a non-competitive manner w i t h r e s p e c t to p h o s p h o l i p i d s in 1986(3). On the other hand, it is reported(4, 5) that a c t i v a t i o n of p r o t e i n kinase C in the smooth muscle cells led to the m a i n t e n a n c e of the s u s t a i n e d phase of smooth m u s c l e c o n t r a c t i o n w i t h phospho0024-3205/89 $3.00 + .00 Copyright (c) 1989 Pergamon Press plc
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rylation of c a l d e s m o n and a s m a l l n u m b e r of low-molecular-weight c y t o s o l i c p r o t e i n s . T h e r e f o r e , we s t u d i e d the a n t i h y p e r t e n s i v e eff e c t s of NA d e r i v a t i v e s and t h e i r m o d e of a c t i o n . The d a t a s u g g e s t s t h a t one of the m o d e s of a n t i h y p e r t e n s i v e a c t i o n is due to the v a s cular smooth muscle relaxation, but the r e l a x a t i o n is less r e l a t e d to the i n h i b i t i o n of p r o t e i n k i n a s e C a c t i v i t y .
Materials
and
Methods
M e a s u r e m e n t of b l o o d p r e s s u r e in a n e s t h e t i z e d n o r m o t e n s i v e r a t s M a l e S D - r a t s ( S h i z u o k a Lab. A n i m a l s , S h i z u o k a ) , w e i g h i n g 4 0 0 - 4 5 0 g, w e r e a n e s t h e t i z e d w i t h p e n t o b a r b i t a l Na ( A b b o t Labs., U . S . A . ) . The b l o o d p r e s s u r e was m e a s u r e d f r o m the c e r v i c a l artery with a pressure t r a n s d u c e r ( N i h o n K o h d e n M P U 0 . 5 A ) and a c a r r i e r a m p l i f i e r ( N i h o n K o h d e n RP-5) and r e c o r d e d on a p o l y g r a p h ( N i h o n K o h d e n R M - 8 5 ) . The heart rate was also recorded with a tachometer (Nihon Kohden RT-5) converted from blood pressure pulses. C o m p o u n d s w e r e i n j e c t e d into the f e m o r a l v e i n i n s e r t i o n w i t h a p o l y e t h y l e n e c a n n u l a e .
M e a s u r e m e n t of b l o o d p r e s s u r e in c o n s c i o u s s o ~ a n e o u s l y hyper~ t e n s i v e r a t s (SHRs) For the m e a s u r e m e n t of the b l o o d p r e s s u r e in SHRs, two m e t h o d s w e r e e m p l o y e d ; one was the d i r e c t m e t h o d ( 6 ) w h i c h m e a s u r e d the b l o o d p r e s s u r e f r o m the m e d i a n c o c c y g e a l a r t e r y , and the o t h e r one is the t a i l cuff m e t h o d ( 7 ) . SHRs(Charles River Japan Inc.), 14-18 w e e k s of age w e r e u s e d t h r o u g h o u t the e x p e r i m e n t . C o m pounds were administered orally. Direct method T h i s e x p e r i m e n t a l p r o c e d u r e is d e s c r i b e d in ano t h e r p u b l i c a t i o n (6). In short, SHRs were anesthetized lightly w i t h e t h y l e t h e r ( S h o w a e t h e r Co.), and a p o l y e t h y l e n e c a t h e t e r was inserted into the median coccygeal a r t e r y to m e a s u r e the blood pressure. The a n i m a l s w e r e r e s t r a i n e d in a w i r e m e s h cage. The blood pressure a n d the h e a r t rate were monitored using a similar procedure, a n d the s a m e e q u i p m e n t was u s e d as in the c a s e of the m e a s u r e m e n t of b l o o d p r e s s u r e in a n e s t h e t i z e d n o r m o t e n s i v e r a t s described previously.
T a i l cuff m e t h o d SHRs w e r e w a r m e d in a c h a m b e r at 3 7 ° C for 5 min, and the b l o o d p r e s s u r e was m e a s u r e d i n d i r e c t l y at the tail using a plethysmographic p u l s e m o n i t o r (Ueda w o r k s USM-105). The v a l u e s w e r e m e a s u r e d f i v e t i m e s at o n e - h o u r i n t e r v a l s b e g i n n i n g one h o u r a f t e r d r u g a d m i n i s t r a t i o n , and the m e a n of the f i v e v a l u e s was estimated as the b l o o d pressure value. The b l o o d pressure was m e a s u r e d b e f o r e a n d 2, 5, 8, 12 and 24 hrs a f t e r the a d n i m i s t r a t i o n of drug.
R e l a x a t i o n of the i s o l a t e d t h o r a c i c a o r t a f r o m g u i n e a - p i g Male Hartley clean g u i n e a - p i g s ( S h i z u o k a Lab. A n i m a l s , Shizuoka) were s t u n n e d on the h e a d a n d bled. The t h o r a c i c a o r t a was h e l i c a l l y cut and s u s p e n d e d in a Magnus tube filled with Tyrode's s o l u t i o n and continuously aerated. The t e n s i o n w a s r e c o r d e d on a m i n i p o l y g r a p h (Nihon K o h d e n R M - 6 1 0 0 ) t h r o u g h F D - p i c k up ( N i h o n Kohden TB-611). The a o r t a was c o n t r a c t e d w i t h 0.1 ~M n o r e p i n e p h r i n e and t h e n the compounds were cumulatively a d d e d to the M u g n u s t u b e to t e s t the relaxing action.
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I s o l a t i o n of p r o t e i n kinase C and assay of the a c t i v i t y Prepara t i o n of p r o t e i n kinase C was f o l l o w e d by the m e t h o d of K i k k a w a et al.(8). In brief, 150 brains of mice, about 45 g, were h o m o g e n i z e d w i t h a potter h o m o g e n i z e r in 50 mM of T r i s - H C l buffer at pH 8.0 containing 0.25 M sucrose, 2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM e t h y l e n e g l y c o l - b i s - ( b e t a - a m i n o e t h y l ether) N , N , N ' , N ' t e t r a a c e t i c acid (EGTA), 2 mM p h e n y l m e t h y l s u l f o n y l f l u o r i d e (PMSF), 50 mM 2 - m e r c a p t o e t h a n o l and 1 % glycerol. The h o m o g e n a t e was centrifuged w i t h 15,000 x g for i0 min and the s u p e r n a t a n t was collected. The s u p e r n a t a n t was r e - c e n t r i f u g e d w i t h 100,000 x g for 60 min, and the s u p e r n a t a n t was c o l l e c t e d for the chromatography. After the s u p e r n a t a n t was a d s o r b e d on to the dimethylamino ethyl (DEAE) c e l l u l o s e column w h i c h was e q u i l i b r a t e d w i t h 20 mM of TrisHCI buffer at pH 8.0 containing 2 mM EDTA, 10 mM EGTA, 50 mM 2-mercaptoethanol and 10% glycerol, protein, kinase C was eluted with 0-0.3 M s o d i u m chloride gradient. F r a c t i o n s w i t h high p r o t e i n kinase C a c t i v i t y were c o l l e c t e d and c o n c e n t r a t e d to about 2 mlo
P r o t e i n kinase C a c t i v i t y was a s s a y e d by m e a s u r i n g the incorpoation of 32p into calf thymus HI histone. The r e a c t i o n mixture (0.25 ml) contained 40 ~ M of T r i s - H C l buffer at pH 7.5, 5 mM of MgCI2, 20 ~M of CaCI 2 i0 UM of [ r - 3 2 p ] A T P (I00 - 200 cpm/pmol, A m e r s h a m Int. plc), ~ M of p h o s p h a t i d y l serine, 1 ~ g / m l 12-0t e t r a d e c a n o y l p h o r b o l 1 3 - a c e t a t e ( T P A ) in d i m e t h y l s u l f o x i d e , 80 ~ i of inhibitor and I0 ~ I enzyme solution. A f t e r i n c u b a t i o n for 3 min at 30 ° C, the r e a c t i o n was stopped by the a d d i t i o n of 25 % trichloroacetic acid (TCA). The acid p r e c i p i t a t e d m a t e r i a l s were collected on a n i t r o c e l l u r o s e m e m b r a n e filter and were w a s h e d w i t h 15 ml 25 % TCA. The r a d i o a c t i v i t y of 3 2 p - l a b e l e d samples was d e t e r m i n e d using a liquid s c i n t i l l a t i o n counter (Beckman LS-3800).
260
P r e p a r a t i o n of native a c t o m y o s i n and assay of the s u p e r p r e c i p i ration P r e p a r a t i o n of native a c t o m y o s i n was m o s t l y f o l l o w e d by the method of Kohama et al (9). In brief, the smooth m u s c l e of c h i c k e n gizzard was h o m o g e n i z e d in 4 times volume of 0.4 M KCI containing 20 mM T r i s - H C l ( p H 7.5), 10 mM ATP, 1 mM A z i d e and 0.1 M d i i s o p r o p y l f l u o r o p h o s p h a t e ( D F P ) . The h o m o g e n a t e was then c e n t r i f u g e d at 15,000 x g for 10 min. Five mM ATP was added to the s u p e r n a t a n t and it was r e - c e n t r i f u g e d at 80,000 x g for 30 min. The s u p e r n a t a n t of 80,000 x g c e n t r i f u g a t i o n was d i a l y s e d for 3 hrs with 9 times volume of 1 mM NaHCO 3 c o n t a i n i n g 1 mM MgCl 2 and 0.1 mM d i t h i o t h r e i t o l (DTT). The p r e c i p i t a t e d a c t o m y o s i n by the d i a l y s i s was s u s p e n d e d in the 50 mM KCI c o n t a i n i n g 0.i mM NaHCO 3 and I mM MgCI 2 and then c e n t r i f u g e d at 8,000 x g for 10 min. This p r o c e d u r e was r e p e a t e d two or three times to w a s h the actomyosin. A l l steps were p e r f o r m e d at 0 - 4°C.
Superprecipitation was induced at room t e m p e r a t u r e (25 ° C) by mixing 1 mM ATP w i t h an i n c u b a t i o n m i x t u r e c o n t a i n i n g a p p r o x i m a t e l y 0.5 m g / m l of native a c t o m y o s i n , 20 mM T r i s - H C l ( p H 7.5), 8 mM MgCI 2, 50 mM KCI and a 0.1 mM Ca z+ or a 0.1 mM E G T A and m o n i t o r e d w i t h a spectrophotometer at 660 nm (9). The i n h i b i t i n g study of various c o m p o u n d s was p e r f o r m e d under the c o n d i t i o n of 0.1 mM Ca z+.
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Compounds used Nicardipine was purchased from Muromachi Chemical Co.. NA c o m p o u n d s were derived in our l a b o r a t o r y f r o m S F 2 3 7 0 (2) (Fig. i). Norepinephrine injection (Sankyo Pharm Co.) w a s used.
S t a t i s t i c s D a t a w e r e e x p r e s s e d as the m e a n of the mean. The s t a t i s t i c a l a n a l y s i s was the a p v a l u e of less t h a n 0.05 was a c c e p t e d as significant,
H N
± the s t a n d a r d e r r o r S t u d e n t ' s t - t e s t and being statistically
0 R " COOCH 3
SF2370
R'CH2N(CH3) 2
NA0344
R-CH2NHCH3
NA0345
R--CH2NH 2
NA0346
H3C-"
Structure
FIG of
i. NA
compounds
Results
E f f e c t of NA c o m p o u n d s on b l o o d p r e s s u r e and h e a r t rate in anesthetized normotensive r a t s F i g u r e 2 s h o w s the t y p i c a l r e c o r d i n g of the action of N A 0 3 4 5 and nicardipine. With an intravenous i n j e c t i o n (i.v.) of NA c o m p o u n d s , the b l o o d p r e s s u r e was l o w e r e d , taking 2 - 3 min for the m a x i m u m response; the e f f e c t w a s longlasting. The h e a r t r a t e i n c r e a s e d s l i g h t l y or s o m e t i m e s d i d not change. Nicardipine l o w e r e d the b l o o d p r e s s u r e immediately after injection and blood pressure c o n t i n u e d to be d e p r e s s e d for more t h a n 60 min. The h e a r t r a t e d e c r e a s e d p a r a l l e l to the b l o o d p r e s sure f a l l w i t h a h i g h d o s e of n i c a r d i p i n e , w h e r e a s a low dose of nicardipine increased the h e a r t rate. N A 0 3 4 4 at 1 m g / k g showed m a x i m u m r e s p o n s e at 2.5 m i n a f t e r the injection with a blood pressure f a l l of 55 mmHg; the effect was almost restored at 10 m i n after d o s i n g . H o w e v e r , N A 0 3 4 6 at 1 m g / k g t o o k a s i m i l a r a m o u n t of time f o r the m a x i m u m r e s p o n s e w i t h the b l o o d p r e s s u r e f a l l of 30 mmHg , and the m a g n i t u d e of the r e s p o n s e s h o w e d a l m o s t no change u n t i l 30 m i n a f t e r d o s i n g . N A 0 3 4 5 s h o w e d an i n t e r m e d i a t e pattern between NA0344 a n d N A 0 3 4 6 in the b l o o d p r e s s u r e f a l l (50 mmHg); h o w e v e r , it s i g n i f i c a n t l y ( p < 0.05) l o w e r e d the b l o o d p r e s s u r e m o r e t h a n 60 m i n (Fig. 3).
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Antihypertension of Kinase Inhibitor
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NA compounds dose d e p e n d e n t l y l o w e r e d the b l o o d p r e s s u r e to the extent of 0.1-1.0 m g / k g i.v., w h e n the v a l u e s w e r e taken at the m a x i m u m response. The p o t e n c y of NA0345 and N A 0 3 4 4 were a l m o s t the same, and N A 0 3 4 6 was s o m e w h a t w e a k e r t h a n the p r e v i o u s two compounds. N i c a r d i p i n e was stronger than these compounds. NA compounds i n c r e a s e d s l i g h t l y or did not a f f e c t the heart rate, w h e r e a s n i c a r d i p i n e d e c r e a s e d the heart rate at a high dose (Fig. 4).
A
3 rain mmHg 200
m
m
0 Lbeatslmln 500 F
NA0345 1 mglkg I.v.
10
30
60 mln
B mmHg
maim beatslr.ln 500 F
t Nlcardlplne 0.3 mglkg I.v.
10
30
60 rain
FIG 2 Typical recording of the blood p r e s s u r e and the heart rate of a n e s t h e t i z e d n o r m o t e n s i v e rats a f t e r the inject i o n of 1 m g / k g NA0345 (A) and 0.3 m g / k g n i c a r d i p i n e (B). Upper panel i n d i c a t e s blood p r e s s u r e and lower panel i n d i c a t e s heart rate in A and B, r e s p e c t i v e l y .
Effect of NA d e r i v a t i v e s a d m i n i s t e r e d p.o. on the blood pressure of SHRs D i r e c t method: N A 0 3 4 4 and NA0345 at 10 m g / k g lowered the b l o o d p r e s s u r e w i t h the m a x i m u m r e s p o n s e noted m o r e than 5 hrs after oral administration. The same dose of NA0346 showed the weakest e f f e c t on the blood p r e s s u r e fall; the m a x i m u m r e s p o n s e was 3 hrs a f t e r dosing, and the m a g n i t u d e was 13 mmHg in m e a n blood p r e s s u r e fall. These compounds increased the heart rate but not significantly. N i c a r d i p i n e lowered the b l o o d p r e s s u r e 30 mmHg in mean blood p r e s s u r e at 2 hrs after the administration and the e f f e c t c o n t i n u e d more than 5 hrs. The heart rate was s i g n i f i c a n t l y i n c r e a s e d 3-5 hrs after d o s i n g (Fig. 5, 6).
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Antihypertension of Kinase Inhibitor
NA0344
I mg/kg
I v . ( .-4
Vol. 44, No. 19, 1989
)
,.o1 .....................
E 1oo ao so
so
~ 35o
3SO 300
NAO345 I mg/kg I v ( n=7 )
~
NIc&rdlptne 0 3 m g l k g I.v. ( n * S )
leo
120
1oo
~/
•/ ~
120
~
~e eo
'°°1 300
t O
5
~O
20 30 T i m e , mln
'°°
45
60
250
0
5
tO
20 30 T i m e , min
45
60
FIG 3 Effect of NA c o m p o u n d s on the blood p r e s s u r e and the heart rate of a n e s t h e t i z e d n o r m o t e n s i v e rats. Systolic: s y s t o l i c blood pressure, Diastolic: d i a s t o l i c blood pressure. The n u m b e r s in p a r e n t h e s e s indicate numbers of experiments. Each point represents mean+SE of 4-7 e x p e r i m e n t s performed. *p<0.05, **p<0.01, c o m p a r e d with the v a l u e s before drug injection.
A
B
o, E E
0-
+40
• • •
NAO344 NA0345 NA0346
O
Nlce, r d I D l n e
~_ E
+20
oJ
~- - 2 0
-20. o: **
co
"~
-40
u .~ - 4 0
o
>. ~n
¢ = -60'
-80
c'~ - l O C
c
g
-60
O.1
0.3 Dose
(mglkghv.)
1.O
0.1
0.3 Dose
1.O
(mg/kg].v.)
FIG 4 Dose-response of NA c o m p o u n d s on the maximum systolic blood p r e s s u r e changes and the heart rate changes in ane s t h e t i z e d n o r m o t e n s i v e rats. A: change of s y s t o l i c blood pressure, B: change of heart rate. E a c h point r e p r e s e n t s m e a n + S E of 4-7 e x p e r i m e n t s performed. *p<0.05, **p<0.01, c o m p a r e d w i t h the v a l u e s before drug injection.
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Antihypertension of Kinase Inhibitor
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~l rain
mmHg BP 2oor~ 100L--
i
beatllmln 400F~
300t-
i
Cont.
1
2
:t
NA0346 lOm~ko
3
4
5 hrs
p.o.
FIG
5
Typical recording of the blood pressure and the heart rate m e a s u r e d from the c o c c y g e a l artery in r e s t r a i n e d c o n c i o u s SHR after oral a d m i n i s t r a t i o n of 10 m g / k g NA0345. BP: blood pressure, HR: heart rate.
NA0344 10 mglkg P.o. ( n=3 ) A
180]
180
NA0348 10 mg/kg p.o. ( n=3 )
160
laot
140
140
! 120
*--
E 400
i
~'~,.
120
DilIION{
!
4001
I
350 3OO
mg/kg p.o~ ( n=3 ) 200, 180
t
Nicardipine 10 mg/kg p.o. ( n=3 )
180~ 180
!,
1-
160 140
140 120
120 400
.ooI
350 3OO
.
~: 350 0
1
2 3 Time . hours
4
300
5
FIG
0
1
2 Time ,
3
4
5
hours
6
Effect of NA c o m p o u n d s on the blood p r e s s u r e and the heart rate of r e s t r a i n e d concious SHR a f t e r oral a d m i n i s tration. The n u m b e r s in p a r e n t h e s e s indicate n u m b e r s of experiments. Systolic: s y s t o l i c blood pressure, Diastolic: d i a s t o l i c blood pressure. E a c h point r e p r e s e n t s m e a n i S E of 3 e x p e r i m e n t s performed. *p<0.05, **p<0.01, c o m p a r e d w i t h the v a l u e s before drug a d m i n i s t r a t i o n .
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T a i l cuff method: NA c o m p o u n d s of 10 m g / k g p.o. significantly lowered the systolic b l o o d p r e s s u r e in SHRs, and the maximum r e s p o n s e was 8-12 hrs a f t e r oral administration. NA0345 showed significant ( p < 0 . 0 5 ) b l o o d p r e s s u r e f a l l e v e n at 24 hrs a f t e r the a d m i n i s t r a t i o n . The o r d e r of p o t e n c y was N A 0 3 4 5 = NA0344 > NA0346. The o r d e r w a s the same as in the c a s e of the d i r e c t m e t h o d of b l o o d pressure measurement. N i c a r d i p i n e at 10 m g / k g p.o. s h o w e d maximum r e s p o n s e at 2 hrs a f t e r a d m i n i s t r a t i o n and it s i g n i f i c a n t l y ( p < 0 . 0 5 ) l o w e r e d the b l o o d p r e s s u r e u n t i l 8 hrs a f t e r d o s i n g (Fig. 7).
200
Vehicle ( n=8 )
180 J 2001
NA0344 10 mg/kg p.o. ( n=4 )
~ 160] ~~ E
16o]
I"
2001
NA0345 10 mg/kg p,o, ( n=4 )
~"
180
16o
.p.
L-- -~-~-~'"
140 .~ 0
°
~
2001
10 rnglkg p.o. ( n---4 )
N ~ 0 3 4 6
16o.
½1
~
.
160' 200"
NIcerdlplne
10 mg/kg p.o. ( n=4 )
160-
14°'
6
2
i
e
1'2
"
2"4
Time , hours
FIG 7 Effect of NA c o m p o u n d s on the systolic blood pressure m e a s u r e d w i t h the t a i l cuff m e t h o d in S H R a f t e r o r a l adm i n i s t r a t i o n . The n u m b e r s in p a r e n t h e s e s i n d i c a t e n u m b e r s of experiments. Each point represents mean±SE of 6-4 experiments performed. *p<0.05, **p<0.01, compared with the v a l u e s b e f o r e d r u g a d m i n i s t r a t i o n .
Effect of NA c o m p o u n d s on the c o n t r a c t i o n of norepinephrine in the t h o r a c i c a o r t a The t h o r a c i c a o r t a c o n t r a c t e d w i t h 0.1 ~ M n o r e p i n e p h r i n e , and t h e n 1 - 30 p g / m l of the c o m p o u n d s was cumulatively a p p l i e d in the M a g n u s tube. N A 0 3 4 4 and NA0345 dose-dependently r e l a x e d t h e a o r t a but N A 0 3 4 6 and S F 2 3 7 0 did not relax the a o r t a o v e r the r a n g e of the c o n c e n t r a t i o n s (Fig. 8).
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Antihypertension of Kinase Inhibitor
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120 A
a~ 1 1 0 C
100
0 .m 0
90
i--
ro
0
80 70 60 50
•
NA0344 ( n = 3 )
•
NA0346 ( n=4 )
I*
|
1
-6
10
|
-5
10
-4
Concentration ( g/ml )
FIG 8 Dose-response of NA compounds on the thoracic aorta c o n t r a c t e d w i t h 0.1 ~M n o r e p i n e p h r i n e . Each point repres e n t s m e a n + S E of 3-4 e x p e r i m e n t s p e r f o r m e d . * p < 0 . 0 5 , comp a r e d w i t h the v a l u e s b e f o r e d r u g a p p l i c a t i o n .
P r o t e i n k i n a s e C i n h i b i t i o n by NA c o m p o u n d s NA c o m p o u n d s inh i b i t e d the a c t i v i t y of p r o t e i n k i n a s e C isolated from mice brain in the p r e s e n c e or a b s e n s e of 1 2 - O - t e t r a d e c a n o y l p h o r b o l 13-acetate (TPA). The IC50 v a l u e s are s h o w n in T a b l e I. The a c t i v i t y of protein kinase C activated by T P A was 1.5 - 2.5 t i m e s more sensit i v e l y i n h i b i t e d by NA c o m p o u n d s t h a n n o n - a c t i v a t e d protein kinase C. The p o t e n c y of NA c o m p o u n d s to i n h i b i t p r o t e i n k i n a s e C was n e a r l y equal, and the o r d e r was NA0346 = NA0345 > SF2370 > NA0344. The o r d e r of the p o t e n c y was the s a m e in the a b s e n c e and p r e s e n c e of TPA.
Inhibition of s u p e r p r e c i p i t a t i o n of n a t i v e a c t o m y o s i n by NA compounds NA c o m p o u n d s i n h i b i t e d the s u p e r p r e c i p i t a t i o n of native a c t o m y o s i n i s o l a t e d f r o m the s m o o t h m u s c l e of c h i c k e n g i z z a r d . The IC50 v a l u e s are s h o w n in T a b l e I. The o r d e r of the p o t e n c y of NA compounds to inhibit the s u p e r p r e c i p i t a t i o n of actomyosin was N A 0 3 4 4 > N A 0 3 4 5 > N A 0 3 4 6 >> S F 2 3 7 0 . The d i f f e r e n c e of the potency was a b o u t t w i c e w h e n c o m p a r e d a m o n g the NA c o m p o u n d s , w h e r e a s the p o t e n c y of S F 2 3 7 0 was 10 - 20 t i m e s less t h a n NA c o m p o u n d s .
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TABLE I The IC50 v a l u e s of NA c o m p o u n d s on p r o t e i n k i n a s e C in the p r e s e n c e and in the a b s e n c e of T P A and on the sup e r p r e c i p i t a t i o n of a c t o m y o s i n .
Compound
SF2370 NA0344 NA0345 NA0346
protein IC50 T P A (+) 0.092 0.114 0.068 0.062
kinase (~M) TPA
C
superprecipitation IC50 (~M)
(-)
0.24 0.26 0.ii 0.09
7.60 0.31 0.57 0.72
A s s a y s w e r e c o n d u c t e d as p r e v i o u s l y d e s c r i b e d w i t h the a d d i t i o n of NA c o m p o u n d s in a p p r o p r i a t e c o n c e n t r a t i o n s . TPA:12-O-tetradecanoylphorbol 13-acetate
Discussion
NA compounds dose dependently l o w e r e d the b l o o d p r e s s u r e of normotensive rats injected intravenously. The b l o o d pressure of SHRs was a l s o l o w e r e d w i t h p o t e n t and long l a s t i n g a c t i o n by oral administration. In p a r t i c u l a r , 10 m g / k g of N A 0 3 4 5 d e m o n s t r a t e d an antihypertensive effect even 24 hrs a f t e r o r a l a d m i n i s t r a t i o n . In the c a s e of i n t r a v e n o u s i n j e c t i o n , the o r d e r of p o t e n c y w h e n compared with the v a l u e of maximum blood pressure fall was N A 0 3 4 4 N A 0 3 4 5 > N A 0 3 4 6 , and the o r d e r of p o t e n c y when compared w i t h the d u r a t i o n of a c t i o n was NA0346 ~ NA0345 > NA0344. In the c a s e of oral administration, the o r d e r of p o t e n c y was NA0344 k NA0345 > NA0346. I n d e e d , N A 0 3 4 4 had a p o t e n t but s h o r t - l i v e d e f f e c t on the b l o o d p r e s s u r e f a l l a f t e r i n t r a v e n o u s i n j e c t i o n . On the o t h e r hand, NA0346 had w e a k b l o o d p r e s s u r e f a l l e f f e c t w i t h a long duration. NA0345 was an i n t e r m e d i a t e t y p e a f t e r the intravenous injection, and t h u s N A 0 3 4 5 s h o w e d a p o t e n t and long l a s t i n g antihypertensive e f f e c t on SHR by o r a l a d m i n i s t r a t i o n . S F 2 3 7 0 at 100 m g / k g lowered the b l o o d p r e s s u r e of SHR by o r a l a d m i n i s t r a t i o n at a b o u t the same degree to i0 m g / k g NA c o m p o u n d s (10). The p o t e n c y of SF2370 was a b o u t 10 t i m e s less t h a n t h a t of NA c o m p o u n d s w i t h the o r a l administration. D a t a of S F 2 3 7 0 i n j e c t e d i n t r a v e n o u s l y was not obtained b e c a u s e of its s o l u b i l i t y in w a t e r . NA c o m p o u n d s o n l y s l i g h t l y and temporarily i n c r e a s e d the h e a r t r a t e a f t e r the i n t r a v e n o u s injection in n o r m o t e n s i v e rats. The h e a r t r a t e of SHR also increased s l i g h t l y by NA c o m p o u n d s a d m i n i s t e r e d p.o.. N i c a r d i p i n e t e m p o r a r i l y increased the h e a r t r a t e w i t h l o w e r d o s e s and d e c r e a s e d the heart r a t e w i t h h i g h e r d o s e s in n o r m o t e n s i v e r a t s i n j e c t e d i n t r a v e n o u s l y . Oral administration of n i c a r d i p i n e m a r k e d l y i n c r e a s e d the heart rate.
N A 0 3 4 4 and N A 0 3 4 5 but not N A 0 3 4 6 r e l a x e d the i s o l a t e d aortic p r e p a r a t i o n c o n t r a c t e d by 0.i ~ M n o r e p i n e p h r i n e o v e r the d o s e r a n g e of 3-30 ~ g / m l . As the o n s e t of the r e l a x i n g a c t i o n w a s v e r y slow, the a c t i o n m a y o c c u r a f t e r c o m p o u n d s p a s s t h r o u g h the cytoplasmic m e m b r a n e . As N A 0 3 4 6 p o s s e s s e s a f r e e a m i n o a c i d g r o u p , it m a y have m o r e d i f f i c u l t y p e n e t r a t i n g the c y t o p l a s m i c m e m b r a n e t h a n the o t h e r t w o c o m p o u n d s , d e m o n s t r a t i n g no r e l a x a t i o n . The o r d e r was p a r a l l e l
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to the a c t i v i t y of m a x i m u m blood p r e s s u r e fall after i.v. injection. Therefore, it may be c o n s i d e r e d that the a n t i h y p e r t e n s i v e effect of NA c o m p o u n d s is due to the v a s o d i l a t i n g action. The blood pressure was lowered to a m a x i m u m w i t h i n 2 min after the intravenous i n j e c t i o n of NA compounds. The r e s u l t s also support a direct action of NA c o m p o u n d s to the v a s c u l a r tone, t h o u g h the NA compounds provide a d i u r e t i c a c t i o n c o m p a r a b l e to hydroflumethiazide (I0).
NA c o m p o u n d s inhibited the p r o t e i n kinase C a c t i v i t y in the condition of a c t i v a t e d or n o n - a c t i v a t e d (basal) by TPA at 0.01-0.3 ~ M (Table I). A c t i v a t e d p r o t e i n kinase C a c t i v i t y was more sensitively inhibited by the c o m p o u n d s than basal activity of protein kinase C was, and the dose of c o m p o u n d s r e q u i r e d to inhibit activ a t e d p r o t e i n kinase C was about half the dose of the basal p r o t e i n kinase C i n h i b i t i o n in each compound. The order of potency was NA0346 ~ NA0345 > SF2370 > NA 0344. The order b e t w e e n relaxation of the aorta and p r o t e i n kinase C i n h i b i t i o n was a n t i p a r a l l e l . As the sorce of p r o t e i n kinase C was from mice brain, inhibition of protein kinase C by NA c o m p o u n d s m i g h t not correspond with the relaxation of v a s c u l a r smooth muscle by the compounds. In the m e c h a n i s m of the s u s t a i n e d phase of smooth muscle contraction, i.e. caldesmon and a small number of low-molecular-weight cytosolic p r o t e i n s are p h o s p h o r y l a t e d by the p r o t e i n kinase C and the muscle tone is m a i n t a i n e d (4, 5). The NA c o m p o u n d s may inhibit the sustained phase of v a s c u l a r c o n t r a c t i o n t h r o u g h p r o t e i n kinase C inhibition and d e m o n s t r a t e a long lasting antihypertensive effect. As s t a u r o s p o l i n e w h i c h p r o v i d e s the a n t i h y p e r t e n s i v e effect is also r e p o r t e d to inhibit the p r o t e i n kinase C a c t i v i t y (3), the role of p r o t e i n kinase C in the v a s c u l a r c o n t r a c t i o n seems to be noteworthy.
The superprecipitation of a c t o m y o s i n from smooth muscle of chicken gizzard was inhibited by the NA compounds. The potency difference was about twice among the NA c o m p o u n d s and the potency of SF2370 was 10 - 20 times less than NA compounds (Table I). W h e n the superprecipitation of a c t o m y o s i n was inhibited by the NA compounds, m y o s i n was not p h o s p h o r y l a t e d w h e n studied by the ureap o l y a c r y l - a m i d e e l e c t r o p h o r e s i s of the a c t o m y o s i n (data not shown), s u g g e s t i n g that m y o s i n light chain kinase m i g h t be i n h i b i t e d by NA compounds. Kase et al.(ll, 12) r e p o r t e d that K 2 5 2 a ( S F 2 3 7 0 ) and its derivatives inhibited v a r i o u s kinases such as p r o t e i n kinase A, protein kinase G and M L C K in a d d i t i o n to p r o t e i n kinase C with somewhat d i f f e r e n t IC50 values. The order of the potency between r e l a x a t i o n of the v a s c u l a r smooth muscle c o n t r a c t e d by norepinephrine and the i n h i b i t i o n of s u p e r p r e c i p i t a t i o n of actomyosin from the smooth muscle was parallel. The IC50 values for superprecipitation of a c t o m y o s i n was 3 - 12 times larger than that for p r o t e i n kinase C a c t i v a t e d by TPA. The results, however, suggest the possibility that r e l a x a t i o n of the s m o o t h muscle by NA compounds is due to the i n h i b i t i o n of M L C K a c t i v i t y r a t h e r than the i n h i b i t i o n of p r o t e i n kinase C activity. The s m o o t h muscle contraction at the t o n i c phase is e x p l a i n e d byAthe phosphorylation of myosin t h r o u g h a c t i v a t i o n of M L C K w i t h Ca z+ and calmoduline (13, 14). Therefore, i n h i b i t i o n of M L C K a c t i v i t y by NA c o m p o u n d s might play a i m p o r t a n t role for the r e l a x a t i o n of v a s c u l a r smooth muscle, t h o u g h the i n h i b i t i o n of s u p e r p r e c i p i t a t i o n of a c t o m y o s i n by the NA c o m p o u n d s is w e a k e r t h a n that of p r o t e i n kinase C activity.
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In conclusion, NA c o m p o u n d s demonstrated a potent and long lasting a n t i h y p e r t e n s i v e e f f e c t t h r o u g h v a s o d i l a t i n g action. The v a s o d i l a t i n g a c t i o n by the NA c o m p o u n d s may involve the i n h i b i t i o n of s m o o t h muscle c o n t r a c t i o n m e c h a n i s m s t h r o u g h protein kinases. Further studies are n e c e s s a r y to e l u c i d a t e the mode by which NA c o m p o u n d s exert their v a s o d i l a t i n g action. As the NA c o m p o u n d s also have a d i u r e t i c action, the c o n t r i b u t i o n of this e f e e c t to the fall the b l o o d p r e s s u r e r e q u i r e s f u r t h e r study.
Acknowledqements We are i n d e b t e d to Dr. S h i n i c h i Kondo, Pharmaceutical Labs., M e i j i Seika K a i s h a Ltd., for his i n v a l u a b l e s u g g e s t i o n s and contributions to help c o m p l e t e this work. We also thankful to Dr. Kazuhiro Kohama, Dept of P h a r m a c o l o g y , Tokyo U n i v e r s i t y , for his k i n d n e s s in t e a c h i n g us the p r e p a r a t i o n and assay for s u p e r p r e c i p i r a t i o n of a c t o m y o s i n of c h i c k e n g i z z a r d s m o o t h muscle.
References I)
2)
3)
4) 5) 6)
7) 8) 9) 10) 11)
12) 13) 14)
M. SEZAKI, T. SASAKI, T. NAKAZAWA, U. TAKEDA, M. IWATA, T. WATANABE, M. KOYAMA, F. KAI, T. S H O M U R A and M. KOJIMA, J. A n t i b i o t i c s , 38, 1437-1439 (1985). T. SASAKI, M. KOYAMA, M. SEZAKI, Y. TAKEUCHI, Y. KODAMA, K. KAWAMURA, A. KASUYA, A. ITAI and Y. IITAKA, Jpn. J. A n t i b i o t i c s , 39, 653-654 (1986). T. Tamaoki, H. Nomoto, I. Takahashi, Y. Kato, M. M o r i m o t o and F. Tomita, Biochem, Biophys, Res., Commun., 135, 397-402 (1986). J.V. S M A L L and D.O. DeMEYES Jr., J. Cell Biol., 102, 210-220 (1986). S. PARK and H. RASMUSSEN, J. Biol. Chem., 261, 15734-15739 (1986). M. HACHISU, T. TSURUOKA, H. TAKAHASHI, H. ASAOKA, Y. SEKIZAWA, T. K O E D A and S. INOUYE, J. Pharmacobio-Dyn., 6, 922-931 (1983). T. KOEDA, M. HACHISU, T. N I I Z A T O and Y. SEKIZAWA, Pharmacometrics, 17, 569-573 (1979). U. KIKKAWA, Y. TAKAI, R. MINAKUCHI, S. I N O H A R A and Y. NISHIZUKA, J. Biol. Chem., 257, 1 3 3 4 1 - 1 3 3 4 8 (1982). K. Kohama, K. Saida, M. H i r a t a and S. Ebashi, Jpn. J. Pharmacol. 45, 253-260 (1986). M. Hachisu, T. Hiranuma, S. Sagawa, M. K o y a m a and M. Nishio, Jpn. J. Pharmacol., 43, 295p (1987). H. KASE, K. IWAHASHI, S. NAKANISHI, Y. MATSUDA, K. YAMADA, M. TAKAHASHI, C. MUNAKATA, A. SATO and M. KANEKO, Biochem. Biophys Res. Commun., 142, 436-440 (1987). S. NAKANISHI, H. KASE, S. N A K A M U R A and Y. NONOMURA, Jpn. J. P h a r m a c o l . , 43, 169p (1987). P.F. DILLON, M.O. A K S O Y and S.P. DRISKA, Science 211, 495-497 (1981). K.E. KAMM and J.T. STULL, Annu. Rev. Pharmacol. Toxicol., 25, 593-620 (1985).
ANTIHYPERTENSIVE
M i t s u g u Hachisu,
COMPOUNDS INHIBITORY
Toyokazu
Pergamon Press
WITH POTENT ACTIVITY
Hiranuma,
PROTEIN
Masao Koyama
KINASES
and M a s a j i
P h a r m a c e u t i c a l R e s e a r c h L a b o r a t o r i e s , Meiji Seika Kaisha M o r o o k a - c h o , Kohoku-ku, Y o k o h a m a 222, J a p a n
Sezaki
Ltd.,
(Received in final form March 3, 1989)
Summary
A new i n d o r o c a r b a z o l e a n t i b i o t i c SF2370 has been found in the culture b r o t h of A c t i n o m a d u r a sp.; it has also b e e n found to have the a c t i v i t i e s of antihyp e r t e n s i o n and diuresis. Derivatives of SF2370, NA0344, NA0345 and NA0346, when injected (0.i-I mg/kg i.v.) significantly lowered the blood p r e s s u r e of anesthetized normotensive rats and showed a long lasting a n t i h y p e r t e n s i v e action. In the case of p.o. a d m i n i s t r a t i o n to c o n s c i o u s r e s t r a i n e d SHRs, m e a s u r e m e n t of blood p r e s s u r e by the p l e t h y s m o g r a p h i c m e t h o d i n d i c a t e d that 10 m g / k g of the c o m p o u n d s was enough to lower the blood pressure; the a n t i h y p e r t e n s i v e act i v i t i e s were found to r e m a i n more than 12 hrs after oral a d m i n i s t r a t i o n . By s t u d y i n g of the mode of antih y p e r t e n s i v e action, these c o m p o u n d s dose d e p e n d e n t l y relaxed the isolated aortic p r e p a r a t i o n c o n t r a c t e d by norepinephrine. In addition, it was found that protein kinase C a c t i v i t y of mice brain was i n h i b i t e d by the compounds; the IC50 values were in the range of 0.062 - 0.20 MM. Moreover, a superprecipitation of a c t o m y o s i n from smooth m u s c l e of c h i c k e n g i z z a r d was inhibited by the compounds, having an IC50 values of 0.31 0.72 ~M.
In 1985, a new i n d o r o c a r b a z o l e a n t i b i o t i c SF2370 was found in the culture broth of A c t i n o m a d u r a sp.,(l, 2) and it was also found that the c o m p o u n d provides a n t i h y p e r t e n s i v e and d i u r e t i c action. To o b t a i n more potent c o m p o u n d s for a n t i h y p e r t e n s i v e action, several new compounds of NA d e r i v a t i v e s were derived from SF2370. The structually related compound of s t a u r o s p o r i n e providing antihyp e r t e n s i v e a c t i v i t y was r e p o r t e d to inhibit p r o t e i n kinase C activity in a non-competitive manner w i t h r e s p e c t to p h o s p h o l i p i d s in 1986(3). On the other hand, it is reported(4, 5) that a c t i v a t i o n of p r o t e i n kinase C in the smooth muscle cells led to the m a i n t e n a n c e of the s u s t a i n e d phase of smooth m u s c l e c o n t r a c t i o n w i t h phospho0024-3205/89 $3.00 + .00 Copyright (c) 1989 Pergamon Press plc
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rylation of c a l d e s m o n and a s m a l l n u m b e r of low-molecular-weight c y t o s o l i c p r o t e i n s . T h e r e f o r e , we s t u d i e d the a n t i h y p e r t e n s i v e eff e c t s of NA d e r i v a t i v e s and t h e i r m o d e of a c t i o n . The d a t a s u g g e s t s t h a t one of the m o d e s of a n t i h y p e r t e n s i v e a c t i o n is due to the v a s cular smooth muscle relaxation, but the r e l a x a t i o n is less r e l a t e d to the i n h i b i t i o n of p r o t e i n k i n a s e C a c t i v i t y .
Materials
and
Methods
M e a s u r e m e n t of b l o o d p r e s s u r e in a n e s t h e t i z e d n o r m o t e n s i v e r a t s M a l e S D - r a t s ( S h i z u o k a Lab. A n i m a l s , S h i z u o k a ) , w e i g h i n g 4 0 0 - 4 5 0 g, w e r e a n e s t h e t i z e d w i t h p e n t o b a r b i t a l Na ( A b b o t Labs., U . S . A . ) . The b l o o d p r e s s u r e was m e a s u r e d f r o m the c e r v i c a l artery with a pressure t r a n s d u c e r ( N i h o n K o h d e n M P U 0 . 5 A ) and a c a r r i e r a m p l i f i e r ( N i h o n K o h d e n RP-5) and r e c o r d e d on a p o l y g r a p h ( N i h o n K o h d e n R M - 8 5 ) . The heart rate was also recorded with a tachometer (Nihon Kohden RT-5) converted from blood pressure pulses. C o m p o u n d s w e r e i n j e c t e d into the f e m o r a l v e i n i n s e r t i o n w i t h a p o l y e t h y l e n e c a n n u l a e .
M e a s u r e m e n t of b l o o d p r e s s u r e in c o n s c i o u s s o ~ a n e o u s l y hyper~ t e n s i v e r a t s (SHRs) For the m e a s u r e m e n t of the b l o o d p r e s s u r e in SHRs, two m e t h o d s w e r e e m p l o y e d ; one was the d i r e c t m e t h o d ( 6 ) w h i c h m e a s u r e d the b l o o d p r e s s u r e f r o m the m e d i a n c o c c y g e a l a r t e r y , and the o t h e r one is the t a i l cuff m e t h o d ( 7 ) . SHRs(Charles River Japan Inc.), 14-18 w e e k s of age w e r e u s e d t h r o u g h o u t the e x p e r i m e n t . C o m pounds were administered orally. Direct method T h i s e x p e r i m e n t a l p r o c e d u r e is d e s c r i b e d in ano t h e r p u b l i c a t i o n (6). In short, SHRs were anesthetized lightly w i t h e t h y l e t h e r ( S h o w a e t h e r Co.), and a p o l y e t h y l e n e c a t h e t e r was inserted into the median coccygeal a r t e r y to m e a s u r e the blood pressure. The a n i m a l s w e r e r e s t r a i n e d in a w i r e m e s h cage. The blood pressure a n d the h e a r t rate were monitored using a similar procedure, a n d the s a m e e q u i p m e n t was u s e d as in the c a s e of the m e a s u r e m e n t of b l o o d p r e s s u r e in a n e s t h e t i z e d n o r m o t e n s i v e r a t s described previously.
T a i l cuff m e t h o d SHRs w e r e w a r m e d in a c h a m b e r at 3 7 ° C for 5 min, and the b l o o d p r e s s u r e was m e a s u r e d i n d i r e c t l y at the tail using a plethysmographic p u l s e m o n i t o r (Ueda w o r k s USM-105). The v a l u e s w e r e m e a s u r e d f i v e t i m e s at o n e - h o u r i n t e r v a l s b e g i n n i n g one h o u r a f t e r d r u g a d m i n i s t r a t i o n , and the m e a n of the f i v e v a l u e s was estimated as the b l o o d pressure value. The b l o o d pressure was m e a s u r e d b e f o r e a n d 2, 5, 8, 12 and 24 hrs a f t e r the a d n i m i s t r a t i o n of drug.
R e l a x a t i o n of the i s o l a t e d t h o r a c i c a o r t a f r o m g u i n e a - p i g Male Hartley clean g u i n e a - p i g s ( S h i z u o k a Lab. A n i m a l s , Shizuoka) were s t u n n e d on the h e a d a n d bled. The t h o r a c i c a o r t a was h e l i c a l l y cut and s u s p e n d e d in a Magnus tube filled with Tyrode's s o l u t i o n and continuously aerated. The t e n s i o n w a s r e c o r d e d on a m i n i p o l y g r a p h (Nihon K o h d e n R M - 6 1 0 0 ) t h r o u g h F D - p i c k up ( N i h o n Kohden TB-611). The a o r t a was c o n t r a c t e d w i t h 0.1 ~M n o r e p i n e p h r i n e and t h e n the compounds were cumulatively a d d e d to the M u g n u s t u b e to t e s t the relaxing action.
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I s o l a t i o n of p r o t e i n kinase C and assay of the a c t i v i t y Prepara t i o n of p r o t e i n kinase C was f o l l o w e d by the m e t h o d of K i k k a w a et al.(8). In brief, 150 brains of mice, about 45 g, were h o m o g e n i z e d w i t h a potter h o m o g e n i z e r in 50 mM of T r i s - H C l buffer at pH 8.0 containing 0.25 M sucrose, 2 mM ethylenediaminetetraacetic acid (EDTA), 10 mM e t h y l e n e g l y c o l - b i s - ( b e t a - a m i n o e t h y l ether) N , N , N ' , N ' t e t r a a c e t i c acid (EGTA), 2 mM p h e n y l m e t h y l s u l f o n y l f l u o r i d e (PMSF), 50 mM 2 - m e r c a p t o e t h a n o l and 1 % glycerol. The h o m o g e n a t e was centrifuged w i t h 15,000 x g for i0 min and the s u p e r n a t a n t was collected. The s u p e r n a t a n t was r e - c e n t r i f u g e d w i t h 100,000 x g for 60 min, and the s u p e r n a t a n t was c o l l e c t e d for the chromatography. After the s u p e r n a t a n t was a d s o r b e d on to the dimethylamino ethyl (DEAE) c e l l u l o s e column w h i c h was e q u i l i b r a t e d w i t h 20 mM of TrisHCI buffer at pH 8.0 containing 2 mM EDTA, 10 mM EGTA, 50 mM 2-mercaptoethanol and 10% glycerol, protein, kinase C was eluted with 0-0.3 M s o d i u m chloride gradient. F r a c t i o n s w i t h high p r o t e i n kinase C a c t i v i t y were c o l l e c t e d and c o n c e n t r a t e d to about 2 mlo
P r o t e i n kinase C a c t i v i t y was a s s a y e d by m e a s u r i n g the incorpoation of 32p into calf thymus HI histone. The r e a c t i o n mixture (0.25 ml) contained 40 ~ M of T r i s - H C l buffer at pH 7.5, 5 mM of MgCI2, 20 ~M of CaCI 2 i0 UM of [ r - 3 2 p ] A T P (I00 - 200 cpm/pmol, A m e r s h a m Int. plc), ~ M of p h o s p h a t i d y l serine, 1 ~ g / m l 12-0t e t r a d e c a n o y l p h o r b o l 1 3 - a c e t a t e ( T P A ) in d i m e t h y l s u l f o x i d e , 80 ~ i of inhibitor and I0 ~ I enzyme solution. A f t e r i n c u b a t i o n for 3 min at 30 ° C, the r e a c t i o n was stopped by the a d d i t i o n of 25 % trichloroacetic acid (TCA). The acid p r e c i p i t a t e d m a t e r i a l s were collected on a n i t r o c e l l u r o s e m e m b r a n e filter and were w a s h e d w i t h 15 ml 25 % TCA. The r a d i o a c t i v i t y of 3 2 p - l a b e l e d samples was d e t e r m i n e d using a liquid s c i n t i l l a t i o n counter (Beckman LS-3800).
260
P r e p a r a t i o n of native a c t o m y o s i n and assay of the s u p e r p r e c i p i ration P r e p a r a t i o n of native a c t o m y o s i n was m o s t l y f o l l o w e d by the method of Kohama et al (9). In brief, the smooth m u s c l e of c h i c k e n gizzard was h o m o g e n i z e d in 4 times volume of 0.4 M KCI containing 20 mM T r i s - H C l ( p H 7.5), 10 mM ATP, 1 mM A z i d e and 0.1 M d i i s o p r o p y l f l u o r o p h o s p h a t e ( D F P ) . The h o m o g e n a t e was then c e n t r i f u g e d at 15,000 x g for 10 min. Five mM ATP was added to the s u p e r n a t a n t and it was r e - c e n t r i f u g e d at 80,000 x g for 30 min. The s u p e r n a t a n t of 80,000 x g c e n t r i f u g a t i o n was d i a l y s e d for 3 hrs with 9 times volume of 1 mM NaHCO 3 c o n t a i n i n g 1 mM MgCl 2 and 0.1 mM d i t h i o t h r e i t o l (DTT). The p r e c i p i t a t e d a c t o m y o s i n by the d i a l y s i s was s u s p e n d e d in the 50 mM KCI c o n t a i n i n g 0.i mM NaHCO 3 and I mM MgCI 2 and then c e n t r i f u g e d at 8,000 x g for 10 min. This p r o c e d u r e was r e p e a t e d two or three times to w a s h the actomyosin. A l l steps were p e r f o r m e d at 0 - 4°C.
Superprecipitation was induced at room t e m p e r a t u r e (25 ° C) by mixing 1 mM ATP w i t h an i n c u b a t i o n m i x t u r e c o n t a i n i n g a p p r o x i m a t e l y 0.5 m g / m l of native a c t o m y o s i n , 20 mM T r i s - H C l ( p H 7.5), 8 mM MgCI 2, 50 mM KCI and a 0.1 mM Ca z+ or a 0.1 mM E G T A and m o n i t o r e d w i t h a spectrophotometer at 660 nm (9). The i n h i b i t i n g study of various c o m p o u n d s was p e r f o r m e d under the c o n d i t i o n of 0.1 mM Ca z+.
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Compounds used Nicardipine was purchased from Muromachi Chemical Co.. NA c o m p o u n d s were derived in our l a b o r a t o r y f r o m S F 2 3 7 0 (2) (Fig. i). Norepinephrine injection (Sankyo Pharm Co.) w a s used.
S t a t i s t i c s D a t a w e r e e x p r e s s e d as the m e a n of the mean. The s t a t i s t i c a l a n a l y s i s was the a p v a l u e of less t h a n 0.05 was a c c e p t e d as significant,
H N
± the s t a n d a r d e r r o r S t u d e n t ' s t - t e s t and being statistically
0 R " COOCH 3
SF2370
R'CH2N(CH3) 2
NA0344
R-CH2NHCH3
NA0345
R--CH2NH 2
NA0346
H3C-"
Structure
FIG of
i. NA
compounds
Results
E f f e c t of NA c o m p o u n d s on b l o o d p r e s s u r e and h e a r t rate in anesthetized normotensive r a t s F i g u r e 2 s h o w s the t y p i c a l r e c o r d i n g of the action of N A 0 3 4 5 and nicardipine. With an intravenous i n j e c t i o n (i.v.) of NA c o m p o u n d s , the b l o o d p r e s s u r e was l o w e r e d , taking 2 - 3 min for the m a x i m u m response; the e f f e c t w a s longlasting. The h e a r t r a t e i n c r e a s e d s l i g h t l y or s o m e t i m e s d i d not change. Nicardipine l o w e r e d the b l o o d p r e s s u r e immediately after injection and blood pressure c o n t i n u e d to be d e p r e s s e d for more t h a n 60 min. The h e a r t r a t e d e c r e a s e d p a r a l l e l to the b l o o d p r e s sure f a l l w i t h a h i g h d o s e of n i c a r d i p i n e , w h e r e a s a low dose of nicardipine increased the h e a r t rate. N A 0 3 4 4 at 1 m g / k g showed m a x i m u m r e s p o n s e at 2.5 m i n a f t e r the injection with a blood pressure f a l l of 55 mmHg; the effect was almost restored at 10 m i n after d o s i n g . H o w e v e r , N A 0 3 4 6 at 1 m g / k g t o o k a s i m i l a r a m o u n t of time f o r the m a x i m u m r e s p o n s e w i t h the b l o o d p r e s s u r e f a l l of 30 mmHg , and the m a g n i t u d e of the r e s p o n s e s h o w e d a l m o s t no change u n t i l 30 m i n a f t e r d o s i n g . N A 0 3 4 5 s h o w e d an i n t e r m e d i a t e pattern between NA0344 a n d N A 0 3 4 6 in the b l o o d p r e s s u r e f a l l (50 mmHg); h o w e v e r , it s i g n i f i c a n t l y ( p < 0.05) l o w e r e d the b l o o d p r e s s u r e m o r e t h a n 60 m i n (Fig. 3).
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Antihypertension of Kinase Inhibitor
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NA compounds dose d e p e n d e n t l y l o w e r e d the b l o o d p r e s s u r e to the extent of 0.1-1.0 m g / k g i.v., w h e n the v a l u e s w e r e taken at the m a x i m u m response. The p o t e n c y of NA0345 and N A 0 3 4 4 were a l m o s t the same, and N A 0 3 4 6 was s o m e w h a t w e a k e r t h a n the p r e v i o u s two compounds. N i c a r d i p i n e was stronger than these compounds. NA compounds i n c r e a s e d s l i g h t l y or did not a f f e c t the heart rate, w h e r e a s n i c a r d i p i n e d e c r e a s e d the heart rate at a high dose (Fig. 4).
A
3 rain mmHg 200
m
m
0 Lbeatslmln 500 F
NA0345 1 mglkg I.v.
10
30
60 mln
B mmHg
maim beatslr.ln 500 F
t Nlcardlplne 0.3 mglkg I.v.
10
30
60 rain
FIG 2 Typical recording of the blood p r e s s u r e and the heart rate of a n e s t h e t i z e d n o r m o t e n s i v e rats a f t e r the inject i o n of 1 m g / k g NA0345 (A) and 0.3 m g / k g n i c a r d i p i n e (B). Upper panel i n d i c a t e s blood p r e s s u r e and lower panel i n d i c a t e s heart rate in A and B, r e s p e c t i v e l y .
Effect of NA d e r i v a t i v e s a d m i n i s t e r e d p.o. on the blood pressure of SHRs D i r e c t method: N A 0 3 4 4 and NA0345 at 10 m g / k g lowered the b l o o d p r e s s u r e w i t h the m a x i m u m r e s p o n s e noted m o r e than 5 hrs after oral administration. The same dose of NA0346 showed the weakest e f f e c t on the blood p r e s s u r e fall; the m a x i m u m r e s p o n s e was 3 hrs a f t e r dosing, and the m a g n i t u d e was 13 mmHg in m e a n blood p r e s s u r e fall. These compounds increased the heart rate but not significantly. N i c a r d i p i n e lowered the b l o o d p r e s s u r e 30 mmHg in mean blood p r e s s u r e at 2 hrs after the administration and the e f f e c t c o n t i n u e d more than 5 hrs. The heart rate was s i g n i f i c a n t l y i n c r e a s e d 3-5 hrs after d o s i n g (Fig. 5, 6).
1356
Antihypertension of Kinase Inhibitor
NA0344
I mg/kg
I v . ( .-4
Vol. 44, No. 19, 1989
)
,.o1 .....................
E 1oo ao so
so
~ 35o
3SO 300
NAO345 I mg/kg I v ( n=7 )
~
NIc&rdlptne 0 3 m g l k g I.v. ( n * S )
leo
120
1oo
~/
•/ ~
120
~
~e eo
'°°1 300
t O
5
~O
20 30 T i m e , mln
'°°
45
60
250
0
5
tO
20 30 T i m e , min
45
60
FIG 3 Effect of NA c o m p o u n d s on the blood p r e s s u r e and the heart rate of a n e s t h e t i z e d n o r m o t e n s i v e rats. Systolic: s y s t o l i c blood pressure, Diastolic: d i a s t o l i c blood pressure. The n u m b e r s in p a r e n t h e s e s indicate numbers of experiments. Each point represents mean+SE of 4-7 e x p e r i m e n t s performed. *p<0.05, **p<0.01, c o m p a r e d with the v a l u e s before drug injection.
A
B
o, E E
0-
+40
• • •
NAO344 NA0345 NA0346
O
Nlce, r d I D l n e
~_ E
+20
oJ
~- - 2 0
-20. o: **
co
"~
-40
u .~ - 4 0
o
>. ~n
¢ = -60'
-80
c'~ - l O C
c
g
-60
O.1
0.3 Dose
(mglkghv.)
1.O
0.1
0.3 Dose
1.O
(mg/kg].v.)
FIG 4 Dose-response of NA c o m p o u n d s on the maximum systolic blood p r e s s u r e changes and the heart rate changes in ane s t h e t i z e d n o r m o t e n s i v e rats. A: change of s y s t o l i c blood pressure, B: change of heart rate. E a c h point r e p r e s e n t s m e a n + S E of 4-7 e x p e r i m e n t s performed. *p<0.05, **p<0.01, c o m p a r e d w i t h the v a l u e s before drug injection.
Vol. 44, No. 19, 1989
Antihypertension of Kinase Inhibitor
1357
~l rain
mmHg BP 2oor~ 100L--
i
beatllmln 400F~
300t-
i
Cont.
1
2
:t
NA0346 lOm~ko
3
4
5 hrs
p.o.
FIG
5
Typical recording of the blood pressure and the heart rate m e a s u r e d from the c o c c y g e a l artery in r e s t r a i n e d c o n c i o u s SHR after oral a d m i n i s t r a t i o n of 10 m g / k g NA0345. BP: blood pressure, HR: heart rate.
NA0344 10 mglkg P.o. ( n=3 ) A
180]
180
NA0348 10 mg/kg p.o. ( n=3 )
160
laot
140
140
! 120
*--
E 400
i
~'~,.
120
DilIION{
!
4001
I
350 3OO
mg/kg p.o~ ( n=3 ) 200, 180
t
Nicardipine 10 mg/kg p.o. ( n=3 )
180~ 180
!,
1-
160 140
140 120
120 400
.ooI
350 3OO
.
~: 350 0
1
2 3 Time . hours
4
300
5
FIG
0
1
2 Time ,
3
4
5
hours
6
Effect of NA c o m p o u n d s on the blood p r e s s u r e and the heart rate of r e s t r a i n e d concious SHR a f t e r oral a d m i n i s tration. The n u m b e r s in p a r e n t h e s e s indicate n u m b e r s of experiments. Systolic: s y s t o l i c blood pressure, Diastolic: d i a s t o l i c blood pressure. E a c h point r e p r e s e n t s m e a n i S E of 3 e x p e r i m e n t s performed. *p<0.05, **p<0.01, c o m p a r e d w i t h the v a l u e s before drug a d m i n i s t r a t i o n .
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T a i l cuff method: NA c o m p o u n d s of 10 m g / k g p.o. significantly lowered the systolic b l o o d p r e s s u r e in SHRs, and the maximum r e s p o n s e was 8-12 hrs a f t e r oral administration. NA0345 showed significant ( p < 0 . 0 5 ) b l o o d p r e s s u r e f a l l e v e n at 24 hrs a f t e r the a d m i n i s t r a t i o n . The o r d e r of p o t e n c y was N A 0 3 4 5 = NA0344 > NA0346. The o r d e r w a s the same as in the c a s e of the d i r e c t m e t h o d of b l o o d pressure measurement. N i c a r d i p i n e at 10 m g / k g p.o. s h o w e d maximum r e s p o n s e at 2 hrs a f t e r a d m i n i s t r a t i o n and it s i g n i f i c a n t l y ( p < 0 . 0 5 ) l o w e r e d the b l o o d p r e s s u r e u n t i l 8 hrs a f t e r d o s i n g (Fig. 7).
200
Vehicle ( n=8 )
180 J 2001
NA0344 10 mg/kg p.o. ( n=4 )
~ 160] ~~ E
16o]
I"
2001
NA0345 10 mg/kg p,o, ( n=4 )
~"
180
16o
.p.
L-- -~-~-~'"
140 .~ 0
°
~
2001
10 rnglkg p.o. ( n---4 )
N ~ 0 3 4 6
16o.
½1
~
.
160' 200"
NIcerdlplne
10 mg/kg p.o. ( n=4 )
160-
14°'
6
2
i
e
1'2
"
2"4
Time , hours
FIG 7 Effect of NA c o m p o u n d s on the systolic blood pressure m e a s u r e d w i t h the t a i l cuff m e t h o d in S H R a f t e r o r a l adm i n i s t r a t i o n . The n u m b e r s in p a r e n t h e s e s i n d i c a t e n u m b e r s of experiments. Each point represents mean±SE of 6-4 experiments performed. *p<0.05, **p<0.01, compared with the v a l u e s b e f o r e d r u g a d m i n i s t r a t i o n .
Effect of NA c o m p o u n d s on the c o n t r a c t i o n of norepinephrine in the t h o r a c i c a o r t a The t h o r a c i c a o r t a c o n t r a c t e d w i t h 0.1 ~ M n o r e p i n e p h r i n e , and t h e n 1 - 30 p g / m l of the c o m p o u n d s was cumulatively a p p l i e d in the M a g n u s tube. N A 0 3 4 4 and NA0345 dose-dependently r e l a x e d t h e a o r t a but N A 0 3 4 6 and S F 2 3 7 0 did not relax the a o r t a o v e r the r a n g e of the c o n c e n t r a t i o n s (Fig. 8).
Vol. 44, No. 19, 1989
Antihypertension of Kinase Inhibitor
1359
120 A
a~ 1 1 0 C
100
0 .m 0
90
i--
ro
0
80 70 60 50
•
NA0344 ( n = 3 )
•
NA0346 ( n=4 )
I*
|
1
-6
10
|
-5
10
-4
Concentration ( g/ml )
FIG 8 Dose-response of NA compounds on the thoracic aorta c o n t r a c t e d w i t h 0.1 ~M n o r e p i n e p h r i n e . Each point repres e n t s m e a n + S E of 3-4 e x p e r i m e n t s p e r f o r m e d . * p < 0 . 0 5 , comp a r e d w i t h the v a l u e s b e f o r e d r u g a p p l i c a t i o n .
P r o t e i n k i n a s e C i n h i b i t i o n by NA c o m p o u n d s NA c o m p o u n d s inh i b i t e d the a c t i v i t y of p r o t e i n k i n a s e C isolated from mice brain in the p r e s e n c e or a b s e n s e of 1 2 - O - t e t r a d e c a n o y l p h o r b o l 13-acetate (TPA). The IC50 v a l u e s are s h o w n in T a b l e I. The a c t i v i t y of protein kinase C activated by T P A was 1.5 - 2.5 t i m e s more sensit i v e l y i n h i b i t e d by NA c o m p o u n d s t h a n n o n - a c t i v a t e d protein kinase C. The p o t e n c y of NA c o m p o u n d s to i n h i b i t p r o t e i n k i n a s e C was n e a r l y equal, and the o r d e r was NA0346 = NA0345 > SF2370 > NA0344. The o r d e r of the p o t e n c y was the s a m e in the a b s e n c e and p r e s e n c e of TPA.
Inhibition of s u p e r p r e c i p i t a t i o n of n a t i v e a c t o m y o s i n by NA compounds NA c o m p o u n d s i n h i b i t e d the s u p e r p r e c i p i t a t i o n of native a c t o m y o s i n i s o l a t e d f r o m the s m o o t h m u s c l e of c h i c k e n g i z z a r d . The IC50 v a l u e s are s h o w n in T a b l e I. The o r d e r of the p o t e n c y of NA compounds to inhibit the s u p e r p r e c i p i t a t i o n of actomyosin was N A 0 3 4 4 > N A 0 3 4 5 > N A 0 3 4 6 >> S F 2 3 7 0 . The d i f f e r e n c e of the potency was a b o u t t w i c e w h e n c o m p a r e d a m o n g the NA c o m p o u n d s , w h e r e a s the p o t e n c y of S F 2 3 7 0 was 10 - 20 t i m e s less t h a n NA c o m p o u n d s .
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TABLE I The IC50 v a l u e s of NA c o m p o u n d s on p r o t e i n k i n a s e C in the p r e s e n c e and in the a b s e n c e of T P A and on the sup e r p r e c i p i t a t i o n of a c t o m y o s i n .
Compound
SF2370 NA0344 NA0345 NA0346
protein IC50 T P A (+) 0.092 0.114 0.068 0.062
kinase (~M) TPA
C
superprecipitation IC50 (~M)
(-)
0.24 0.26 0.ii 0.09
7.60 0.31 0.57 0.72
A s s a y s w e r e c o n d u c t e d as p r e v i o u s l y d e s c r i b e d w i t h the a d d i t i o n of NA c o m p o u n d s in a p p r o p r i a t e c o n c e n t r a t i o n s . TPA:12-O-tetradecanoylphorbol 13-acetate
Discussion
NA compounds dose dependently l o w e r e d the b l o o d p r e s s u r e of normotensive rats injected intravenously. The b l o o d pressure of SHRs was a l s o l o w e r e d w i t h p o t e n t and long l a s t i n g a c t i o n by oral administration. In p a r t i c u l a r , 10 m g / k g of N A 0 3 4 5 d e m o n s t r a t e d an antihypertensive effect even 24 hrs a f t e r o r a l a d m i n i s t r a t i o n . In the c a s e of i n t r a v e n o u s i n j e c t i o n , the o r d e r of p o t e n c y w h e n compared with the v a l u e of maximum blood pressure fall was N A 0 3 4 4 N A 0 3 4 5 > N A 0 3 4 6 , and the o r d e r of p o t e n c y when compared w i t h the d u r a t i o n of a c t i o n was NA0346 ~ NA0345 > NA0344. In the c a s e of oral administration, the o r d e r of p o t e n c y was NA0344 k NA0345 > NA0346. I n d e e d , N A 0 3 4 4 had a p o t e n t but s h o r t - l i v e d e f f e c t on the b l o o d p r e s s u r e f a l l a f t e r i n t r a v e n o u s i n j e c t i o n . On the o t h e r hand, NA0346 had w e a k b l o o d p r e s s u r e f a l l e f f e c t w i t h a long duration. NA0345 was an i n t e r m e d i a t e t y p e a f t e r the intravenous injection, and t h u s N A 0 3 4 5 s h o w e d a p o t e n t and long l a s t i n g antihypertensive e f f e c t on SHR by o r a l a d m i n i s t r a t i o n . S F 2 3 7 0 at 100 m g / k g lowered the b l o o d p r e s s u r e of SHR by o r a l a d m i n i s t r a t i o n at a b o u t the same degree to i0 m g / k g NA c o m p o u n d s (10). The p o t e n c y of SF2370 was a b o u t 10 t i m e s less t h a n t h a t of NA c o m p o u n d s w i t h the o r a l administration. D a t a of S F 2 3 7 0 i n j e c t e d i n t r a v e n o u s l y was not obtained b e c a u s e of its s o l u b i l i t y in w a t e r . NA c o m p o u n d s o n l y s l i g h t l y and temporarily i n c r e a s e d the h e a r t r a t e a f t e r the i n t r a v e n o u s injection in n o r m o t e n s i v e rats. The h e a r t r a t e of SHR also increased s l i g h t l y by NA c o m p o u n d s a d m i n i s t e r e d p.o.. N i c a r d i p i n e t e m p o r a r i l y increased the h e a r t r a t e w i t h l o w e r d o s e s and d e c r e a s e d the heart r a t e w i t h h i g h e r d o s e s in n o r m o t e n s i v e r a t s i n j e c t e d i n t r a v e n o u s l y . Oral administration of n i c a r d i p i n e m a r k e d l y i n c r e a s e d the heart rate.
N A 0 3 4 4 and N A 0 3 4 5 but not N A 0 3 4 6 r e l a x e d the i s o l a t e d aortic p r e p a r a t i o n c o n t r a c t e d by 0.i ~ M n o r e p i n e p h r i n e o v e r the d o s e r a n g e of 3-30 ~ g / m l . As the o n s e t of the r e l a x i n g a c t i o n w a s v e r y slow, the a c t i o n m a y o c c u r a f t e r c o m p o u n d s p a s s t h r o u g h the cytoplasmic m e m b r a n e . As N A 0 3 4 6 p o s s e s s e s a f r e e a m i n o a c i d g r o u p , it m a y have m o r e d i f f i c u l t y p e n e t r a t i n g the c y t o p l a s m i c m e m b r a n e t h a n the o t h e r t w o c o m p o u n d s , d e m o n s t r a t i n g no r e l a x a t i o n . The o r d e r was p a r a l l e l
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to the a c t i v i t y of m a x i m u m blood p r e s s u r e fall after i.v. injection. Therefore, it may be c o n s i d e r e d that the a n t i h y p e r t e n s i v e effect of NA c o m p o u n d s is due to the v a s o d i l a t i n g action. The blood pressure was lowered to a m a x i m u m w i t h i n 2 min after the intravenous i n j e c t i o n of NA compounds. The r e s u l t s also support a direct action of NA c o m p o u n d s to the v a s c u l a r tone, t h o u g h the NA compounds provide a d i u r e t i c a c t i o n c o m p a r a b l e to hydroflumethiazide (I0).
NA c o m p o u n d s inhibited the p r o t e i n kinase C a c t i v i t y in the condition of a c t i v a t e d or n o n - a c t i v a t e d (basal) by TPA at 0.01-0.3 ~ M (Table I). A c t i v a t e d p r o t e i n kinase C a c t i v i t y was more sensitively inhibited by the c o m p o u n d s than basal activity of protein kinase C was, and the dose of c o m p o u n d s r e q u i r e d to inhibit activ a t e d p r o t e i n kinase C was about half the dose of the basal p r o t e i n kinase C i n h i b i t i o n in each compound. The order of potency was NA0346 ~ NA0345 > SF2370 > NA 0344. The order b e t w e e n relaxation of the aorta and p r o t e i n kinase C i n h i b i t i o n was a n t i p a r a l l e l . As the sorce of p r o t e i n kinase C was from mice brain, inhibition of protein kinase C by NA c o m p o u n d s m i g h t not correspond with the relaxation of v a s c u l a r smooth muscle by the compounds. In the m e c h a n i s m of the s u s t a i n e d phase of smooth muscle contraction, i.e. caldesmon and a small number of low-molecular-weight cytosolic p r o t e i n s are p h o s p h o r y l a t e d by the p r o t e i n kinase C and the muscle tone is m a i n t a i n e d (4, 5). The NA c o m p o u n d s may inhibit the sustained phase of v a s c u l a r c o n t r a c t i o n t h r o u g h p r o t e i n kinase C inhibition and d e m o n s t r a t e a long lasting antihypertensive effect. As s t a u r o s p o l i n e w h i c h p r o v i d e s the a n t i h y p e r t e n s i v e effect is also r e p o r t e d to inhibit the p r o t e i n kinase C a c t i v i t y (3), the role of p r o t e i n kinase C in the v a s c u l a r c o n t r a c t i o n seems to be noteworthy.
The superprecipitation of a c t o m y o s i n from smooth muscle of chicken gizzard was inhibited by the NA compounds. The potency difference was about twice among the NA c o m p o u n d s and the potency of SF2370 was 10 - 20 times less than NA compounds (Table I). W h e n the superprecipitation of a c t o m y o s i n was inhibited by the NA compounds, m y o s i n was not p h o s p h o r y l a t e d w h e n studied by the ureap o l y a c r y l - a m i d e e l e c t r o p h o r e s i s of the a c t o m y o s i n (data not shown), s u g g e s t i n g that m y o s i n light chain kinase m i g h t be i n h i b i t e d by NA compounds. Kase et al.(ll, 12) r e p o r t e d that K 2 5 2 a ( S F 2 3 7 0 ) and its derivatives inhibited v a r i o u s kinases such as p r o t e i n kinase A, protein kinase G and M L C K in a d d i t i o n to p r o t e i n kinase C with somewhat d i f f e r e n t IC50 values. The order of the potency between r e l a x a t i o n of the v a s c u l a r smooth muscle c o n t r a c t e d by norepinephrine and the i n h i b i t i o n of s u p e r p r e c i p i t a t i o n of actomyosin from the smooth muscle was parallel. The IC50 values for superprecipitation of a c t o m y o s i n was 3 - 12 times larger than that for p r o t e i n kinase C a c t i v a t e d by TPA. The results, however, suggest the possibility that r e l a x a t i o n of the s m o o t h muscle by NA compounds is due to the i n h i b i t i o n of M L C K a c t i v i t y r a t h e r than the i n h i b i t i o n of p r o t e i n kinase C activity. The s m o o t h muscle contraction at the t o n i c phase is e x p l a i n e d byAthe phosphorylation of myosin t h r o u g h a c t i v a t i o n of M L C K w i t h Ca z+ and calmoduline (13, 14). Therefore, i n h i b i t i o n of M L C K a c t i v i t y by NA c o m p o u n d s might play a i m p o r t a n t role for the r e l a x a t i o n of v a s c u l a r smooth muscle, t h o u g h the i n h i b i t i o n of s u p e r p r e c i p i t a t i o n of a c t o m y o s i n by the NA c o m p o u n d s is w e a k e r t h a n that of p r o t e i n kinase C activity.
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In conclusion, NA c o m p o u n d s demonstrated a potent and long lasting a n t i h y p e r t e n s i v e e f f e c t t h r o u g h v a s o d i l a t i n g action. The v a s o d i l a t i n g a c t i o n by the NA c o m p o u n d s may involve the i n h i b i t i o n of s m o o t h muscle c o n t r a c t i o n m e c h a n i s m s t h r o u g h protein kinases. Further studies are n e c e s s a r y to e l u c i d a t e the mode by which NA c o m p o u n d s exert their v a s o d i l a t i n g action. As the NA c o m p o u n d s also have a d i u r e t i c action, the c o n t r i b u t i o n of this e f e e c t to the fall the b l o o d p r e s s u r e r e q u i r e s f u r t h e r study.
Acknowledqements We are i n d e b t e d to Dr. S h i n i c h i Kondo, Pharmaceutical Labs., M e i j i Seika K a i s h a Ltd., for his i n v a l u a b l e s u g g e s t i o n s and contributions to help c o m p l e t e this work. We also thankful to Dr. Kazuhiro Kohama, Dept of P h a r m a c o l o g y , Tokyo U n i v e r s i t y , for his k i n d n e s s in t e a c h i n g us the p r e p a r a t i o n and assay for s u p e r p r e c i p i r a t i o n of a c t o m y o s i n of c h i c k e n g i z z a r d s m o o t h muscle.
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