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KOB rats
Life Sciences, Vol. 50, pp. 1363-1369 Printed in the USA
CHANGES
Pergamon Press
IN R E S P O N S I V E N E S S OF THE AORTA TO GENETICALLY DIABETIC RATS: A STUDY
VASORELAXANT AGENTS IN W B N / K O B R A T S
IN
N o r i y u k i M i y a t a I, H i r o k o Y a m a u { a , K a t s u h a r u T s u c h ~ d a , S u s u m u Otomo, K a t s u o K a m a t a - a n d Y u t a k a K a s u y a R e s e a r c h C e n t e r , T a i s h o P h a r m a c e u t i c a l Co. Ltd., 1-403 Y o s h i n o - c h o , Ohmiya, S a i t a m a 330, J a p a n * D e p a r t m e n t of P h a r m a c o l o g y , S c h o o l of P h a r m a c y , H o s h i U n i v e r s i t y , 2 - 4 - 4 1 Ebara, S h i n a g a w a - k u , T o k y o 142, J a p a n (Received in final form February 25, 1992) Summary The e f f e c t s of v a r i o u s v a s o r e l a x a n t a g e n t s on a o r t a s f r o m c o n t r o l a n d g e n e t i c a l l y d i a b e t i c rats w e r e e x a m i n e d . The concentration-response c u r v e s for the i s o p r o t e r e n o l (ISO)induced r e l a x a t i o n of b o t h a o r t i c strips w i t h a n d w i t h o u t endothelium are s h i f t e d to the right in d i a b e t i c rats. T h e r e l a x a t i o n r e s p o n s e s of d i a b e t i c a o r t a to f o r s k o l i n a n d v a s o a c t i v e i n t e s t i n a l p e p t i d e did not d i f f e r f r o m t h o s e of c o n t r o l s . The r e l a x a t i o n r e s p o n s e s of d i a b e t i c a o r t a to c r o m a k a l i m and n i c o r a n d i l did not d i f f e r f r o m t h o s e of c o n t r o l s . T h e s e r e s u l t s i n d i r e c t l y i n d i c a t e that I S O - i n d u c e d r e l a x a t i o n r e s p o n s e s of the a o r t i c s t r i p s f r o m g e n e t i c a l l y d i a b e t i c rats d e c r e a s e d , and that this d e c r e a s e d r e l a x a t i o n r e s p o n s e of the s t r i p s to ISO m a y be due to d e c r e a s e d d e n s i t y or a f f i n i t y of b e t a a d r e n o c e p t o r s on the e n d o t h e l i u m and v a s c u l a r s m o o t h muscle. Vascular disease is one of the complicating features of diabetes m e l l i t u s in m a n (i). The r e a c t i v i t y of v a s c u l a r s m o o t h muscle from diabetic animals to contractile agents has been s t u d i e d e x t e n s i v e l y w i t h r e f e r e n c e to this p r o b l e m (2-5). W e h a v e previously reported that endothelium-dependent relaxation r e s p o n s e s of a o r t i c strips w e r e i m p a i r e d in both streptozotocin ( S T Z ) - i n d u c e d d i a b e t i c rats (6) a n d a l l o x a n - i n d u c e d d i a b e t i c rabbits (7). W e h a v e a l s o r e p o r t e d that i s o p r o t e r e n o l (ISO)-induced r e l a x a t i o n r e s p o n s e s of the a o r t i c s t r i p s are p a r t i a l l y d e p e n d e n t on the e x i s t e n c e of the e n d o t h e l i a l cells, and that the ISOinduced relaxation decreased in S T Z - i n d u c e d d i a b e t i c rats (8). The above-mentioned results, however, were obtained from chemically-induced d i a b e t i c animals. A particular s t r a i n of W i s t a r - d e r i v e d W B N rats has b e e n m a i n t a i n e d at the I n s t i t u t e of Pathology, Bonn University (Bonn, F.R.G.). S e v e r a l of these a n i m a l s w e r e b r o u g h t to J a p a n
I) To w h o m
all
correspondence
should
be a d d r e s s e d .
0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
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by Dr. Kobori in 1976 and b e c a m e k n o w n as W B N / K o b rats. A colony of these rats is being m a i n t a i n e d at the S h i z u o k a L a b o r a t o r y A n i m a l Center (Shizuoka, Japan). The main clinical sign of diabetes, g l u c o s e u r i a , was first d e t e c t e d at a b o u t 60 w e e k of age (9,10). P r e v i o u s l y , we h a v e r e p o r t e d that e n d o t h e l i u m - d e p e n d e n t relaxation responses of the aortic strips w e r e i m p a i r e d in a o r t i c s t r i p s from W B N / K o b rats (ii). However, there are no r e p o r t s of changes in r e a c t i v i t y of a r t e r i a l strips in r e s p o n s e to v a r i o u s v a s o r e l a x a n t a g e n t s u s i n g the m o d e l of g e n e t i c a l l y d i a b e t i c rats. The p u r p o s e of the p r e s e n t i n v e s t i g a t i o n was to e x a m i n e the effects of v a r i o u s v a s o r e l a x a n t a g e n t s on the reactivity of the aortas from a g e - m a t c h e d W i s t a r and W B N / K o b g e n e t i c a l l y d i a b e t i c rats.
Methods Animals
used
W B N / K o b rats (70-75 weeks) and age-matched Wistar rats were purchased from the S h i z u o k a L a b o r a t o r y A n i m a l C e n t e r (Shizuoka, Japan). All a n i m a l s w e r e fed s t a n d a r d chow. Serum glucose was measured e n z y m a t i c a l l y w i t h a H i t a c h i 7150 (Tokyo, Japan) a u t o analyser. Isolated
rat a o r t i c
strips
The rats w e r e s a c r i f i c e d by d e c a p i t a t i o n a n d a s e c t i o n of the thoracic aorta between the aortic a r c h and the d i a p h r a g m was r e m o v e d and p l a c e d in o x y g e n a t e d , modified Krebs Henseleit solution (KHS). The s o l u t i o n c o n s i s t e d of (mM) N a C I 118.0; KCI 4.7; N a H C O 3 25.0; CaCI 2 1.8; N a H 2 P O 4 1.2; M g S O 4 1.2; d e x t r o s e 11.0. In some e x p e r i m e n t s , the endothelium of the aortic strips was removed by g e n t l e r u b b i n g of the e n d o t h e l i a l s u r f a c e w i t h a d i s posable cotton applicatior. The a o r t a was c l e a n e d of l o o s e l y adhering fat and c o n n e c t i v e tissue and was cut into h e l i c a l s t r i p s 3 m m in w i d t h a n d 20 m m in length. The t i s s u e was placed in a well-oxygenated (95% 02 , 5% CO 2) b a t h of 10 ml KHS at 37°C w i t h one end c o n n e c t e d to a t i s s u e h o l d e r and the other to a forcedisplacement transducer (Nihon K o h d e n T B - 6 1 2 T ) . The t i s s u e was e q u i l i b r a t e d for 60 m i n under a r e s t i n g t e n s i o n of 1.0 g, which is optimal for i n d u c i n g the m a x i m a l c o n t r a c t i o n in e a c h vessel. D u r i n g this time the KHS in the tissue b a t h was r e p l a c e d e v e r y 20 min. Experimental
protocol
After e q u i l ~ b r a t i o n , each a o r t i c s t r i p was c o n t r a c t e d by treatment with 10-'M noradrenaline (NA). The p r e s e n c e of functional endothelial cells was c o n f i r m e d by d e m o n s t r a t i n g r e l a x a t i o n s to 10-~M acetylcholine (ACh). A o r t i c strips in w h i c h 80% r e l a x a t i o n s o c c u r e d w e r e tissues w i t h e n d o t h e l i u m . The r e m o v a l of e n d o t h e l i a l cells by r u b b i n g was c o n f i r m e d by the fact that tonic c o n t r a c t i o n of the a o r t a induced by NA was not affected by treatment with ACh. After the m a x i m a l tone was r e a c h e d each s a m p l e of t i s s u e was
VOl. 50, No. 18, 1992
Beta-adrenoceptors in Diabetes
1365
e x p o s e d to o n l y a s i n g l e r e l a x a n t agent. T h e r e l a x a t i o n r e s p o n s e s to ISO, f o r s k o l i n (FSK), vasoactive intestinal peptide (VIP), cromakalim, and nicorandil w e r e e x p r e s s e d in terms of t~e p e r c e n t a g e d e c r e a s e of the c o n t r a c t i l e force i n d u c e d by 10-'M NA. For the r e l a x a t i o n s t u d i e s , the a o r t i c s t r i p s w e r e p r e c o n t r a c t e d w i t h an e q u i e f f e c t i v e c o n c e n t r a t i o n of NA (10-'M). This conc e n t r a t i o n p r o d u c e d 8 0 - 9 0 % of the m a x i m a l r e s p o n s e . W h e n the NAi ~ d u c e d c o n t r a c t i o n r e a c h e d a p l a t e a u , ISO ( 1 0 - B - 1 0 - 5 M ) , F S K (10 - 8 - I 0 - 5 M ~ , VI E ( I 0 - 9 - I 0 - 6 M ) , c r o m a k a l i m (I0-8-I0-5M) and n i c o r a n d i l (10-u-10 - M) w e r e a d d e d in a c u m u l a t i v e m a n n e r .
0 Z 0 IO <
Z
O O
50
o~ Z
O X < -J ILl
rr 100
8
7
6
5
-LOG (ISQ) M
Fig.
1
Concentration-response c u r v e s for the r e l a x a t i o n r e s p o n s e s to ISO of a o r t i c s t r i p s w i t h e n d o t h e l i u m f r o m a g e - m a t c h e d W i s t a r ( 0 ) and W B N / K o b ( © ) rats and w i t h o u t e n d o t h e l i u m f r o m a g e - m a t c h e d c o n t r o l ( l ) and W B N / K o b 7 ( D ) rats. T h e a o r t i c s t r i p s w e r e p r e c o n t r a c t e d w i t h i0- M NA. V a l u e s r e p r e s e n t the m e a n of the r e s u l t s of 5 e x p e r i m e n t s , r e s p e c t i v e l y , w i t h the m e a n ~ S.E. i n d i c a t e d by v e r t i c a l lines. * p < 0.05; ** p < 0.01.
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Beta-adrenoceptors in Diabetes
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Drugs HA, VIP, ISO w e r e p u r c h a s e d f r o m S i g m a C h e m i c a l Co. (St. Louis, MO, USA). A C h c h l o r i d e was p u r c h a s e d f r o m D a i i c h i P h a r m a c e u t i c a l Co. (Tokyo, Japan). Cromakalim, FSK and nicorandil were synthesized in the T a i s h o R e s e a r c h L a b o r a t o r y . All concentrations a r e e x p r e s s e d as the f i n a l m o l a r c o n c e n t r a t i o n of the b a s e in the t i s s u e bath. Statistical
analysis
R e s u l t s are e x p r e s s e d as m e a n ± S.E.. S i g n i f i c a n c e was t e s t e d by the u n p a i r e d t test and c o n s i d e r e d s i g n i f i c a n t if p < 0.05 or p < 0.01. Results The c o n c e n t r a t i o n of g l u c o s e ± 20.3 in a g e - m a t c h e d control (n=10, p < 0.05).
in s e r u m was and WBN/Kob
116.5± rats,
4.5 a n d 385.0 respectively
W h e n a o r t i c s t r i p s w e r e p r e c o n t r a c t e d w i t h NA, ISO r e l a x e d the aortic strips with endothelium in a concentration-dependent manner. However, concentration-response curves for ISO were s h i f t e d to the r i g h t for a o r t i c s t r i p s f r o m WBN/Kob rats (Fig. i). In aortic strips without e n d o t h e l i a l cells, ISO r e l a x e d p r e c o n t r a c t e d a o r t a in a c o n c e n t r a t i o n - d e p e n d e n t manner, but the maximal relaxation in response to ISO was a l s o d e c r e a s e d s i g n i f i c a n t l y in W B N / K o b rats (Fig. i). F S K ( I 0 - 8 - I 0 - 5 M ) and V I P ( I 0 - 9 - I 0 - 6 M ) r e l a x e d the p r e c o n t r a c t e d aortic strips from both Wistar and WBN/Kob rats in a c o n c e n t r a t i o n - d e p e n d e n t manner. H o w e v e r , in c o n t r a s t to the effects of ISO, the c o n c e n t r a t i o n - d e p e n d e n t r e l a x a t i o n p r o d u c e d by FSK and VIP in a o r t i c s t r i p s w i t h o u t e n d o t h e l i u m from WBN/Kob rats d i d not d i f f e r f r o m that in the c o n t r o l s (Table i). C r o m a k a l i m ( I 0 - 8 - I 0 - 5 M ) and n i c o r a n d i l ( I 0 - 8 - I 0 - 5 M ) r e l a x e d the p r e c o n t r a c t e d a o r t i c s t r i p s in a c o n c e n t r a t i o n - d e p e n d e n t manner. However, the r e l a x a n t r e s p o n s e s to c r o m a k a l i m a n d n i c o r a n d i l w e r e not s i g n i f i c a n t l y d i f f e r e n t b e t w e e n v e s s e l s f r o m W B N / K o b a n d c o n trol rats (Figure 2, T a b l e i).
Discussion P r e v i o u s l y , we h a v e r e p o r t e d that I S O - i n d u c e d r e l a x a t i o n of rat aortic strips was partially d e p e n d e n t on the e x i s t e n c e of end o t h e l i a l c e l l s (8). This phenomenon was reconfirmed in the present experiments. We found that in both WBN/Kob rat a o r t a s w i t h a n d w i t h o u t endothelium, the c o n c e n t r a t i o n - r e s p o n s e c u r v e s for ISO-induced relaxation were shifted to the right. In S T Z - i n d u c e d d i a b e t i c rats, we h a v e p r e v i o u s l y reported that decreased relaxant responses of aorta (8) and rat s t o m a c h f u n d u s (12) to b e t a adrenoceptor-agonists.
Vol. 50, No. 18, 1992
Beta-adrenoceptors in Diabetes
0
1367
o
Q
Z
OC I,Z 0
nIZ
~ 5o
8
_o
Z
50
Z
_o ..I
QC IO0
100
-LOG (CROMAKALIM) M
-LOG (NIOORANDIL) M
Fig.
2
Concentration-response c u r v e s for the r e l a x a t i o n r e s p o n s e s to c r o m a k a l i m Cleft) of a o r t i c s t r i p s w i t h o u t e n d o t h e l i u m f r o m W i s t a r ( O , n=6) a n d W B N / K o b ( O , n=6) rats. Concentration-response c u r v e s for the r e l a x a t i o n r e s p o n s e s to n i c o r a n d i l (right) of a o r t i c s t r i p s w i t h o u t e n d o t h e l i u m f r o m W i s t a r (B , n=6) a n d W B N / K o b (D , n=~) rats. The a o r t i c s t r i p s w e r e p r e c o n t r a c t e d w i t h 10- I M n o r a d r e n a l i n e T h e r e s u l t s are e x p r e s s e d as m e a n ± S E M i n d i c a t e d by v e r t i c a l lines. F r o m the a b o v e - m e n t i o n e d r e p o r t s a n d the p r e s e n t data, it is proposed that the decrease in s i z e of the p o p u l a t i o n of b e t a a d r e n o c e p t o r s m a y be r e l a t e d to an i n c r e a s e in the circulating l e v e l s of c a t e c h o l a m i n e s . Of particular creased plasma p a t i e n t s (13).
Table aortic
i n t e r e s t in this c o n n e c t i o n catecholamine levels in
1 IC 50 or IC 25 v a l u e s for v a r i o u s v a s o r e l a x a n t s t r i p s f r o m W i s t a r and W B N / K o b rats.
Drugs
Wistar
FSK (I0-8-10-5M) IC ( 10 -7 ) 5
is the r e p o r t of inlong-term diabetic
_
_
IC25 (xl0-'M) Cromakalim ~I0-8-I0-5M) IC50 (xl0-'M) Nicorandil ~I0-8-10-5M) IC50 (xl0-UM)
agents
in
WBN/Kob
2 . 1 ± 0.2
(n=6)
2 . 5 ± 0.6
(n--6)
5.4+_ 1.6
(n=4)
4.1_+ 1.3
(n=4)
2 . 0 ± 0.3
(n--6)
1.6 + _ 0.3
(n=6)
1 . 4 ± 0.3
(n=6)
0 . 8 ± 0.3
(n=6)
animals.
FSK,
V a l u e s a r e e x p r e s s e d as m e a n ± SEM. n=number f o r s k o l i n ; VIP, v a s o a c t i v e i n t e s t i n a l p e p t i d e .
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The relaxant r e s p o n s e to F S K d i d not d i f f e r s i g n i f i c a n t l y between aortic strips without endothelium from control and WBN/Kob rats. Therefore, the d e c r e a s e d r e s p o n s i v e n e s s of a o r t i c s t r i p s f r o m W B N / K o b rats to ISO was not due to a l t e r e d a c t i v i t y of the a d e n y l a t e c y c l a s e in the v a s c u l a r s m o o t h m u s c l e . It has been r e p o r t e d that V I P c a u s e s r e l a x a t i o n in rat a o r t a and is a s s o c i a t e d i n c r e a s e d l e v e l s of c y c l i c AMP (14). We exa m i n e d the V I P - i n d u c e d r e l a x a t i o n in a o r t i c s t r i p s f r o m b o t h c o n trol a n d W B N / K o b rats, u s i n g V I P as a n o t h e r g e n e r a t o r of c y c l i c AMP. The r e l a x a n t r e s p o n s e to V I P did not differ significantly between aortic strips without endothelium from control and W B N / K o b rats. Thus, c o n s i d e r i n g the v a r i o u s r e l a x a n t a g e n t s used, we found that only the response mediated via the betaa d r e n o c e p t o r s was d e c r e a s e d in W B N / K o b rats. The relaxation response to cromakalim, a potassium channel opener, a n d n i c o r a n d i l , a nitrocompound with a potassium channel opening action, did not differ significantly between aortic s t r i p s w i t h o u t e n d o t h e l i u m f r o m c o n t r o l and W B N / K o b rats. Therefore, t h e s e r e s u l t s i n d i c a t e that the activity of the potassium c h a n n e l s in a o r t i c s t r i p s f r o m W B N / K o b rats is s i m i l a r to t h o s e in s t r i p s f r o m c o n t r o l rats. The relaxation-response curve for WBN/Kob rats in r e s p o n s e to s o d i u m n i t r o p r u s s i d e a n d a t r i a l n a t r i u r e t i c p e p t i d e did not d i f f e r s i g n i f i c a n t l y in a o r t i c s t r i p s f r o m c o n t r o l rats (ii). F r o m the p r e s e n t r e s u l t s of the effects of nicorandil, it is p a r t i a l l y s u g g e s t e d that the act i v i t y of the g u a n y l a t e c y c l a s e in a o r t i c s t r i p s d i d not differ f r o m that in c o n t r o l rats. On the o t h e r hand, we h a v e p r e v i o u s l y r e p o r t e d that c r o m a k a l i m - i n d u c e d r e l a x a t i o n of the a o r t i c s t r i p s without endothelium was decreased in rats with STZ-induced diabetes (15). To c l a r i f y the d i s c r e p a n c y b e t w e e n the a b o v e m e n t i o n e d r e p o r t s and the p r e s e n t data, and to determine the f u n c t i o n of p o t a s s i u m , f u t u r e i n v e s t i g a t i o n is n e c e s s a r y . In conclusion, we f o u n d that the d e c r e a s e d b e t a - a d r e n o c e p t o r m e d i a t e d r e l a x a t i o n r e s p o n s e of the a o r t i c strips from genetically diabetic rats. This observation is c o n s i s t e n t w i t h the findings obtained from rats with chemically-induced diabetes. Thus d i a b e t e s m e l l i t u s g e n e r a l l y p r o d u c e s a f u n c t i o n a l c h a n g e s in beta-adrenoceptors-mediated r e s p o n s e s in b l o o d v e s s e l s .
The authors wish nical assistance.
to thank M i s s
Kimiko
Kawarasaki
for her
tech-
References i. 2.
A. R. C H R I S T R I E B , Am. J. C a r d i o l . 32 5 9 2 - 6 0 6 (1973). D° K. A G R A W A L , and J. H. M C N E I L L , Can. J. P h y s i o l . P h a r m a c o l . 65 1 4 8 4 - 1 4 9 0 (1987). 3. R° J. HEAD, P. A. L O N G H U R S T , R. L. PANEK, a n d R. E. S T I T Z E L , Br. J. P h a r m a c o l . 91 2 7 5 - 2 8 6 (1987). 4. K. K A M A T A , N. MIYAT--A, a n d Y. KASUYA, J. P h a r m a c o b i o - D y n . ii 7 0 7 - 7 1 3 (1988). 5. T. ABIRU, K. KAMATA, N. M I Y A T A , a n d Y. K A S U Y A , Can. J. P h y s i o l . P h a r m a c o l . 68 8 8 2 - 8 8 8 (1990).
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6. 7. 8. 9. 10.
ii.
12. 13. 14. 15.
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K. K A M A T A , N. M I Y A T A , a n d Y. K A S U Y A , Br. J. P h a r m a c o l . 9 4 6 1 4 - 6 1 8 (1989). T. A B I R U , Y. W A T A N A B E , K. K A M A T A , N. M I Y A T A , a n d Y. K A S U Y A , Res. Comm. Chem. Pathol. P h a r m a c o l . 68 1 3 - 2 5 (1990). K. K A M A T A , N. M I Y A T A , Y. a n d K A S U Y A , J. P h a r m a c o l . Exp. Ther. 249 8 9 0 - 8 9 4 (1989). M. T S U C H I T A N I , T. S A E G U S A , I. N A R A M A , T. N I S H I K A W A , a n d T. GONDA, L a b o r a t o r y A n i m a l s . 19 2 0 0 - 2 0 7 (1985). K. N A K A M A , K. S H I C H I N O H E , K ? - K O B A Y A S H I , K. NAITO, O. U C H I D A , Y. Y A S U H A R A , a n d M. TOBE, Acta. D i a b e t e t o l o g i c a . L a t i n a 22 3 3 5 - 3 4 2 (1985). N. M I Y A T A , K. T S U C H I D A , S. O K U Y A M A , S. O T O M O , K. KZ:~ATA, a n d Y. K A S U Y A , Am. J. P h y s i o l . (Heart Circ. P h y s i o l ) (1992, in p r e s s ) . K. K A M A T A , A. S A K A M O T O , and Y. K A S U Y A , Jpn. J. P h a r m a c o l . 4/7 9 9 - 1 0 2 (1988). H. J. C H R I S T E N S E N , J. Clin. Invest. 51 7 7 9 - 7 8 7 (1972). P. S C H O E F F T E R , a n d J. C. S T O C L E T , Eur. J. P h a r m a c o l . 109 2 7 5 - 2 7 9 (1985). K. K A M A T A , N. M I Y A T A , a n d Y. K A S U Y A , Eur. J. P h a r m a c o l . 167 3 1 9 - 3 2 3 (1989).
CHANGES
Pergamon Press
IN R E S P O N S I V E N E S S OF THE AORTA TO GENETICALLY DIABETIC RATS: A STUDY
VASORELAXANT AGENTS IN W B N / K O B R A T S
IN
N o r i y u k i M i y a t a I, H i r o k o Y a m a u { a , K a t s u h a r u T s u c h ~ d a , S u s u m u Otomo, K a t s u o K a m a t a - a n d Y u t a k a K a s u y a R e s e a r c h C e n t e r , T a i s h o P h a r m a c e u t i c a l Co. Ltd., 1-403 Y o s h i n o - c h o , Ohmiya, S a i t a m a 330, J a p a n * D e p a r t m e n t of P h a r m a c o l o g y , S c h o o l of P h a r m a c y , H o s h i U n i v e r s i t y , 2 - 4 - 4 1 Ebara, S h i n a g a w a - k u , T o k y o 142, J a p a n (Received in final form February 25, 1992) Summary The e f f e c t s of v a r i o u s v a s o r e l a x a n t a g e n t s on a o r t a s f r o m c o n t r o l a n d g e n e t i c a l l y d i a b e t i c rats w e r e e x a m i n e d . The concentration-response c u r v e s for the i s o p r o t e r e n o l (ISO)induced r e l a x a t i o n of b o t h a o r t i c strips w i t h a n d w i t h o u t endothelium are s h i f t e d to the right in d i a b e t i c rats. T h e r e l a x a t i o n r e s p o n s e s of d i a b e t i c a o r t a to f o r s k o l i n a n d v a s o a c t i v e i n t e s t i n a l p e p t i d e did not d i f f e r f r o m t h o s e of c o n t r o l s . The r e l a x a t i o n r e s p o n s e s of d i a b e t i c a o r t a to c r o m a k a l i m and n i c o r a n d i l did not d i f f e r f r o m t h o s e of c o n t r o l s . T h e s e r e s u l t s i n d i r e c t l y i n d i c a t e that I S O - i n d u c e d r e l a x a t i o n r e s p o n s e s of the a o r t i c s t r i p s f r o m g e n e t i c a l l y d i a b e t i c rats d e c r e a s e d , and that this d e c r e a s e d r e l a x a t i o n r e s p o n s e of the s t r i p s to ISO m a y be due to d e c r e a s e d d e n s i t y or a f f i n i t y of b e t a a d r e n o c e p t o r s on the e n d o t h e l i u m and v a s c u l a r s m o o t h muscle. Vascular disease is one of the complicating features of diabetes m e l l i t u s in m a n (i). The r e a c t i v i t y of v a s c u l a r s m o o t h muscle from diabetic animals to contractile agents has been s t u d i e d e x t e n s i v e l y w i t h r e f e r e n c e to this p r o b l e m (2-5). W e h a v e previously reported that endothelium-dependent relaxation r e s p o n s e s of a o r t i c strips w e r e i m p a i r e d in both streptozotocin ( S T Z ) - i n d u c e d d i a b e t i c rats (6) a n d a l l o x a n - i n d u c e d d i a b e t i c rabbits (7). W e h a v e a l s o r e p o r t e d that i s o p r o t e r e n o l (ISO)-induced r e l a x a t i o n r e s p o n s e s of the a o r t i c s t r i p s are p a r t i a l l y d e p e n d e n t on the e x i s t e n c e of the e n d o t h e l i a l cells, and that the ISOinduced relaxation decreased in S T Z - i n d u c e d d i a b e t i c rats (8). The above-mentioned results, however, were obtained from chemically-induced d i a b e t i c animals. A particular s t r a i n of W i s t a r - d e r i v e d W B N rats has b e e n m a i n t a i n e d at the I n s t i t u t e of Pathology, Bonn University (Bonn, F.R.G.). S e v e r a l of these a n i m a l s w e r e b r o u g h t to J a p a n
I) To w h o m
all
correspondence
should
be a d d r e s s e d .
0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.
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Vol. 50, No. 18, 1992
by Dr. Kobori in 1976 and b e c a m e k n o w n as W B N / K o b rats. A colony of these rats is being m a i n t a i n e d at the S h i z u o k a L a b o r a t o r y A n i m a l Center (Shizuoka, Japan). The main clinical sign of diabetes, g l u c o s e u r i a , was first d e t e c t e d at a b o u t 60 w e e k of age (9,10). P r e v i o u s l y , we h a v e r e p o r t e d that e n d o t h e l i u m - d e p e n d e n t relaxation responses of the aortic strips w e r e i m p a i r e d in a o r t i c s t r i p s from W B N / K o b rats (ii). However, there are no r e p o r t s of changes in r e a c t i v i t y of a r t e r i a l strips in r e s p o n s e to v a r i o u s v a s o r e l a x a n t a g e n t s u s i n g the m o d e l of g e n e t i c a l l y d i a b e t i c rats. The p u r p o s e of the p r e s e n t i n v e s t i g a t i o n was to e x a m i n e the effects of v a r i o u s v a s o r e l a x a n t a g e n t s on the reactivity of the aortas from a g e - m a t c h e d W i s t a r and W B N / K o b g e n e t i c a l l y d i a b e t i c rats.
Methods Animals
used
W B N / K o b rats (70-75 weeks) and age-matched Wistar rats were purchased from the S h i z u o k a L a b o r a t o r y A n i m a l C e n t e r (Shizuoka, Japan). All a n i m a l s w e r e fed s t a n d a r d chow. Serum glucose was measured e n z y m a t i c a l l y w i t h a H i t a c h i 7150 (Tokyo, Japan) a u t o analyser. Isolated
rat a o r t i c
strips
The rats w e r e s a c r i f i c e d by d e c a p i t a t i o n a n d a s e c t i o n of the thoracic aorta between the aortic a r c h and the d i a p h r a g m was r e m o v e d and p l a c e d in o x y g e n a t e d , modified Krebs Henseleit solution (KHS). The s o l u t i o n c o n s i s t e d of (mM) N a C I 118.0; KCI 4.7; N a H C O 3 25.0; CaCI 2 1.8; N a H 2 P O 4 1.2; M g S O 4 1.2; d e x t r o s e 11.0. In some e x p e r i m e n t s , the endothelium of the aortic strips was removed by g e n t l e r u b b i n g of the e n d o t h e l i a l s u r f a c e w i t h a d i s posable cotton applicatior. The a o r t a was c l e a n e d of l o o s e l y adhering fat and c o n n e c t i v e tissue and was cut into h e l i c a l s t r i p s 3 m m in w i d t h a n d 20 m m in length. The t i s s u e was placed in a well-oxygenated (95% 02 , 5% CO 2) b a t h of 10 ml KHS at 37°C w i t h one end c o n n e c t e d to a t i s s u e h o l d e r and the other to a forcedisplacement transducer (Nihon K o h d e n T B - 6 1 2 T ) . The t i s s u e was e q u i l i b r a t e d for 60 m i n under a r e s t i n g t e n s i o n of 1.0 g, which is optimal for i n d u c i n g the m a x i m a l c o n t r a c t i o n in e a c h vessel. D u r i n g this time the KHS in the tissue b a t h was r e p l a c e d e v e r y 20 min. Experimental
protocol
After e q u i l ~ b r a t i o n , each a o r t i c s t r i p was c o n t r a c t e d by treatment with 10-'M noradrenaline (NA). The p r e s e n c e of functional endothelial cells was c o n f i r m e d by d e m o n s t r a t i n g r e l a x a t i o n s to 10-~M acetylcholine (ACh). A o r t i c strips in w h i c h 80% r e l a x a t i o n s o c c u r e d w e r e tissues w i t h e n d o t h e l i u m . The r e m o v a l of e n d o t h e l i a l cells by r u b b i n g was c o n f i r m e d by the fact that tonic c o n t r a c t i o n of the a o r t a induced by NA was not affected by treatment with ACh. After the m a x i m a l tone was r e a c h e d each s a m p l e of t i s s u e was
VOl. 50, No. 18, 1992
Beta-adrenoceptors in Diabetes
1365
e x p o s e d to o n l y a s i n g l e r e l a x a n t agent. T h e r e l a x a t i o n r e s p o n s e s to ISO, f o r s k o l i n (FSK), vasoactive intestinal peptide (VIP), cromakalim, and nicorandil w e r e e x p r e s s e d in terms of t~e p e r c e n t a g e d e c r e a s e of the c o n t r a c t i l e force i n d u c e d by 10-'M NA. For the r e l a x a t i o n s t u d i e s , the a o r t i c s t r i p s w e r e p r e c o n t r a c t e d w i t h an e q u i e f f e c t i v e c o n c e n t r a t i o n of NA (10-'M). This conc e n t r a t i o n p r o d u c e d 8 0 - 9 0 % of the m a x i m a l r e s p o n s e . W h e n the NAi ~ d u c e d c o n t r a c t i o n r e a c h e d a p l a t e a u , ISO ( 1 0 - B - 1 0 - 5 M ) , F S K (10 - 8 - I 0 - 5 M ~ , VI E ( I 0 - 9 - I 0 - 6 M ) , c r o m a k a l i m (I0-8-I0-5M) and n i c o r a n d i l (10-u-10 - M) w e r e a d d e d in a c u m u l a t i v e m a n n e r .
0 Z 0 IO <
Z
O O
50
o~ Z
O X < -J ILl
rr 100
8
7
6
5
-LOG (ISQ) M
Fig.
1
Concentration-response c u r v e s for the r e l a x a t i o n r e s p o n s e s to ISO of a o r t i c s t r i p s w i t h e n d o t h e l i u m f r o m a g e - m a t c h e d W i s t a r ( 0 ) and W B N / K o b ( © ) rats and w i t h o u t e n d o t h e l i u m f r o m a g e - m a t c h e d c o n t r o l ( l ) and W B N / K o b 7 ( D ) rats. T h e a o r t i c s t r i p s w e r e p r e c o n t r a c t e d w i t h i0- M NA. V a l u e s r e p r e s e n t the m e a n of the r e s u l t s of 5 e x p e r i m e n t s , r e s p e c t i v e l y , w i t h the m e a n ~ S.E. i n d i c a t e d by v e r t i c a l lines. * p < 0.05; ** p < 0.01.
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Drugs HA, VIP, ISO w e r e p u r c h a s e d f r o m S i g m a C h e m i c a l Co. (St. Louis, MO, USA). A C h c h l o r i d e was p u r c h a s e d f r o m D a i i c h i P h a r m a c e u t i c a l Co. (Tokyo, Japan). Cromakalim, FSK and nicorandil were synthesized in the T a i s h o R e s e a r c h L a b o r a t o r y . All concentrations a r e e x p r e s s e d as the f i n a l m o l a r c o n c e n t r a t i o n of the b a s e in the t i s s u e bath. Statistical
analysis
R e s u l t s are e x p r e s s e d as m e a n ± S.E.. S i g n i f i c a n c e was t e s t e d by the u n p a i r e d t test and c o n s i d e r e d s i g n i f i c a n t if p < 0.05 or p < 0.01. Results The c o n c e n t r a t i o n of g l u c o s e ± 20.3 in a g e - m a t c h e d control (n=10, p < 0.05).
in s e r u m was and WBN/Kob
116.5± rats,
4.5 a n d 385.0 respectively
W h e n a o r t i c s t r i p s w e r e p r e c o n t r a c t e d w i t h NA, ISO r e l a x e d the aortic strips with endothelium in a concentration-dependent manner. However, concentration-response curves for ISO were s h i f t e d to the r i g h t for a o r t i c s t r i p s f r o m WBN/Kob rats (Fig. i). In aortic strips without e n d o t h e l i a l cells, ISO r e l a x e d p r e c o n t r a c t e d a o r t a in a c o n c e n t r a t i o n - d e p e n d e n t manner, but the maximal relaxation in response to ISO was a l s o d e c r e a s e d s i g n i f i c a n t l y in W B N / K o b rats (Fig. i). F S K ( I 0 - 8 - I 0 - 5 M ) and V I P ( I 0 - 9 - I 0 - 6 M ) r e l a x e d the p r e c o n t r a c t e d aortic strips from both Wistar and WBN/Kob rats in a c o n c e n t r a t i o n - d e p e n d e n t manner. H o w e v e r , in c o n t r a s t to the effects of ISO, the c o n c e n t r a t i o n - d e p e n d e n t r e l a x a t i o n p r o d u c e d by FSK and VIP in a o r t i c s t r i p s w i t h o u t e n d o t h e l i u m from WBN/Kob rats d i d not d i f f e r f r o m that in the c o n t r o l s (Table i). C r o m a k a l i m ( I 0 - 8 - I 0 - 5 M ) and n i c o r a n d i l ( I 0 - 8 - I 0 - 5 M ) r e l a x e d the p r e c o n t r a c t e d a o r t i c s t r i p s in a c o n c e n t r a t i o n - d e p e n d e n t manner. However, the r e l a x a n t r e s p o n s e s to c r o m a k a l i m a n d n i c o r a n d i l w e r e not s i g n i f i c a n t l y d i f f e r e n t b e t w e e n v e s s e l s f r o m W B N / K o b a n d c o n trol rats (Figure 2, T a b l e i).
Discussion P r e v i o u s l y , we h a v e r e p o r t e d that I S O - i n d u c e d r e l a x a t i o n of rat aortic strips was partially d e p e n d e n t on the e x i s t e n c e of end o t h e l i a l c e l l s (8). This phenomenon was reconfirmed in the present experiments. We found that in both WBN/Kob rat a o r t a s w i t h a n d w i t h o u t endothelium, the c o n c e n t r a t i o n - r e s p o n s e c u r v e s for ISO-induced relaxation were shifted to the right. In S T Z - i n d u c e d d i a b e t i c rats, we h a v e p r e v i o u s l y reported that decreased relaxant responses of aorta (8) and rat s t o m a c h f u n d u s (12) to b e t a adrenoceptor-agonists.
Vol. 50, No. 18, 1992
Beta-adrenoceptors in Diabetes
0
1367
o
Q
Z
OC I,Z 0
nIZ
~ 5o
8
_o
Z
50
Z
_o ..I
QC IO0
100
-LOG (CROMAKALIM) M
-LOG (NIOORANDIL) M
Fig.
2
Concentration-response c u r v e s for the r e l a x a t i o n r e s p o n s e s to c r o m a k a l i m Cleft) of a o r t i c s t r i p s w i t h o u t e n d o t h e l i u m f r o m W i s t a r ( O , n=6) a n d W B N / K o b ( O , n=6) rats. Concentration-response c u r v e s for the r e l a x a t i o n r e s p o n s e s to n i c o r a n d i l (right) of a o r t i c s t r i p s w i t h o u t e n d o t h e l i u m f r o m W i s t a r (B , n=6) a n d W B N / K o b (D , n=~) rats. The a o r t i c s t r i p s w e r e p r e c o n t r a c t e d w i t h 10- I M n o r a d r e n a l i n e T h e r e s u l t s are e x p r e s s e d as m e a n ± S E M i n d i c a t e d by v e r t i c a l lines. F r o m the a b o v e - m e n t i o n e d r e p o r t s a n d the p r e s e n t data, it is proposed that the decrease in s i z e of the p o p u l a t i o n of b e t a a d r e n o c e p t o r s m a y be r e l a t e d to an i n c r e a s e in the circulating l e v e l s of c a t e c h o l a m i n e s . Of particular creased plasma p a t i e n t s (13).
Table aortic
i n t e r e s t in this c o n n e c t i o n catecholamine levels in
1 IC 50 or IC 25 v a l u e s for v a r i o u s v a s o r e l a x a n t s t r i p s f r o m W i s t a r and W B N / K o b rats.
Drugs
Wistar
FSK (I0-8-10-5M) IC ( 10 -7 ) 5
is the r e p o r t of inlong-term diabetic
_
_
IC25 (xl0-'M) Cromakalim ~I0-8-I0-5M) IC50 (xl0-'M) Nicorandil ~I0-8-10-5M) IC50 (xl0-UM)
agents
in
WBN/Kob
2 . 1 ± 0.2
(n=6)
2 . 5 ± 0.6
(n--6)
5.4+_ 1.6
(n=4)
4.1_+ 1.3
(n=4)
2 . 0 ± 0.3
(n--6)
1.6 + _ 0.3
(n=6)
1 . 4 ± 0.3
(n=6)
0 . 8 ± 0.3
(n=6)
animals.
FSK,
V a l u e s a r e e x p r e s s e d as m e a n ± SEM. n=number f o r s k o l i n ; VIP, v a s o a c t i v e i n t e s t i n a l p e p t i d e .
of
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The relaxant r e s p o n s e to F S K d i d not d i f f e r s i g n i f i c a n t l y between aortic strips without endothelium from control and WBN/Kob rats. Therefore, the d e c r e a s e d r e s p o n s i v e n e s s of a o r t i c s t r i p s f r o m W B N / K o b rats to ISO was not due to a l t e r e d a c t i v i t y of the a d e n y l a t e c y c l a s e in the v a s c u l a r s m o o t h m u s c l e . It has been r e p o r t e d that V I P c a u s e s r e l a x a t i o n in rat a o r t a and is a s s o c i a t e d i n c r e a s e d l e v e l s of c y c l i c AMP (14). We exa m i n e d the V I P - i n d u c e d r e l a x a t i o n in a o r t i c s t r i p s f r o m b o t h c o n trol a n d W B N / K o b rats, u s i n g V I P as a n o t h e r g e n e r a t o r of c y c l i c AMP. The r e l a x a n t r e s p o n s e to V I P did not differ significantly between aortic strips without endothelium from control and W B N / K o b rats. Thus, c o n s i d e r i n g the v a r i o u s r e l a x a n t a g e n t s used, we found that only the response mediated via the betaa d r e n o c e p t o r s was d e c r e a s e d in W B N / K o b rats. The relaxation response to cromakalim, a potassium channel opener, a n d n i c o r a n d i l , a nitrocompound with a potassium channel opening action, did not differ significantly between aortic s t r i p s w i t h o u t e n d o t h e l i u m f r o m c o n t r o l and W B N / K o b rats. Therefore, t h e s e r e s u l t s i n d i c a t e that the activity of the potassium c h a n n e l s in a o r t i c s t r i p s f r o m W B N / K o b rats is s i m i l a r to t h o s e in s t r i p s f r o m c o n t r o l rats. The relaxation-response curve for WBN/Kob rats in r e s p o n s e to s o d i u m n i t r o p r u s s i d e a n d a t r i a l n a t r i u r e t i c p e p t i d e did not d i f f e r s i g n i f i c a n t l y in a o r t i c s t r i p s f r o m c o n t r o l rats (ii). F r o m the p r e s e n t r e s u l t s of the effects of nicorandil, it is p a r t i a l l y s u g g e s t e d that the act i v i t y of the g u a n y l a t e c y c l a s e in a o r t i c s t r i p s d i d not differ f r o m that in c o n t r o l rats. On the o t h e r hand, we h a v e p r e v i o u s l y r e p o r t e d that c r o m a k a l i m - i n d u c e d r e l a x a t i o n of the a o r t i c s t r i p s without endothelium was decreased in rats with STZ-induced diabetes (15). To c l a r i f y the d i s c r e p a n c y b e t w e e n the a b o v e m e n t i o n e d r e p o r t s and the p r e s e n t data, and to determine the f u n c t i o n of p o t a s s i u m , f u t u r e i n v e s t i g a t i o n is n e c e s s a r y . In conclusion, we f o u n d that the d e c r e a s e d b e t a - a d r e n o c e p t o r m e d i a t e d r e l a x a t i o n r e s p o n s e of the a o r t i c strips from genetically diabetic rats. This observation is c o n s i s t e n t w i t h the findings obtained from rats with chemically-induced diabetes. Thus d i a b e t e s m e l l i t u s g e n e r a l l y p r o d u c e s a f u n c t i o n a l c h a n g e s in beta-adrenoceptors-mediated r e s p o n s e s in b l o o d v e s s e l s .
The authors wish nical assistance.
to thank M i s s
Kimiko
Kawarasaki
for her
tech-
References i. 2.
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