Life Sciences, Vol. 51, pp. PL 165-170 Printed in the USA

Pergamon Press

PHARMACOLOGY Accelerated

LETTERS

Communication

NEPHRECTOMY AND A NEWLY IDENTIFIED BINDING SITE A N G I O T E N S I N II IN THE R A T A D R E N A L C O R T E X Keifu

Song*,

N a o t a k a Shiota, H i d e k i and M i z u o M i y a z a k i

FOR

Okunishi

D e p a r t m e n t of P h a r m a c o l o g y , O s a k a M e d i c a l C o l l e g e 2-7, D a i g a k u - m a c h i , T a k a t s u k i , 569, J A P A N (Submitted July 28, 1992; accepted August 21, 1992; received in final form August 31, 1992) A b s t r a c t : A n g i o t e n s i n II (Ang II) b i n d i n g s i t e s in a d r e n a l g l a n d s of nephrectomized rats were investigated by in vitro autoradiography using 125I-[Sarl,Ile8]Ang II as l i g a n d s . A n g II b i n d i n g s i t e w a s i n c r e a s e d to 1 6 1 % in t h e c o r t e x a n d d e c r e a s e d to 67 % i n t h e m e d u l l a 48 h a f t e r n e p h r e c t o m y . In t h e m e d u l l a , the A T 2 a n t a g o n i s t ( P D 1 2 3 1 7 7 , 5 ~M) i n h i b i t e d s p e c i f i c b i n d i n g b y 90 % w h e r e a s t h e A T 1 a n t a g o n i s t (DuP753, 5 ~M) i n h i b i t e d b y o n l y i0 %. In c o n t r a s t , in t h e cortex, n e i t h e r D u P 7 5 3 (5 ~M) n o r P D 1 2 3 1 7 7 (5 ~M) s u b s t a n t i a l l y inhibited the binding. Binding in the presence of either the AT 1 or AT 2 antagonist was abolished by the simultaneous p r e s e n c e of b o t h a n t a g o n i s t s . These results suggest the presence of a new Ang II binding site with unique pharmacological properties and differing from currently known subtypes o f A n g II r e c e p t o r s , in the adrenal cortex after nephrectomy.

Introduction AT 1 and AT 2 subtypes of Ang II r e c e p t o r s have been identified using new peptide and non-peptide A n g II r e c e p t o r antagonists (1,2). A T 1 r e c e p t o r is b e l i e v e d to b e r e s p o n s i b l e for k n o w n a c t i o n s o f A n g II, w h e r e a s f u n c t i o n a l s i g n i f i c a n c e of AT 2 receptor is at present unclear. Iwai and Inagami (3) r e p o r t e d that the expression level of the m-RNA of AT 1 receptors in the a d r e n a l c o r t e x w a s d e c r e a s e d in b i l a t e r a l l y n e p h r e c t o m i z e d rats. It i s w e l l k n o w n t h a t t o t a l n u m b e r o f A n g II r e c e p t o r s of rat a d r e n a l c o r t e x is i n c r e a s e d a f t e r n e p h r e c t o m y (4). A c o m p l i c a t e d regulation of Ang II receptor subtypes may occur after nephrectomy. We investigated and differentiated A n g II r e c e p t o r binding in the rat adrenal cortex of nephrectomized rats. We obtained e v i d e n c e o f a h e r e t o f o r e n o t i d e n t i f i e d A n g II b i n d i n g s i t e i n t h e a d r e n a l c o r t e x of n e p h r e c t o m i z e d rats. Methods Tissue *To whom

preparation:

Male

correspondence

wistar

should

rats weighing

250-300

be a d d r e s s e d .

0024-3205/92 $5.00 + .00 Copyright © 1992 Pergamon Press Ltd All rights reserved.

g

were

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Nephrectomy and Ang II Receptors

Vol. 51, No. 18, 1992

bilaterally n e p h r e c t o m i z e d (n = 6) or s h a m o p e r a t e d (n = 5), a n d were decapitated 48 h l a t e r . T h e a d r e n a l glands were quickly r e m o v e d , s n a p f r o z e n in i s o p e n t a n e - d r y ice (-40 °C), a n d s t o r e d at - 8 0 °C. C o n s e c u t i v e sections 20 ~ M t h i c k n e s s were cut on a cryostat at -20 °C a n d t h a w - m o u n t e d onto gelatin-coated slides, dehydrated for 2 h under reduced pressure at 4 °C a n d u s e d f o r incubation. Quantitative i__nnv i t r o a u t o r a d i o g r a p h y : A n g II r e c e p t o r binding sites were labelled as d e s c r i b e d elsewhere (5). B r i e f l y , the s e c t i o n s w e r e i n c u b a t e d in i0 m M s o d i u m p h o s p h a t e b u f f e r ( p H 7.4) containing 150 m M NaCI, 5 m M N a 2 E D T A , 0.2 % b o v i n e s e r u m a l b u m i n and 0.4 mM bacitracin w i t h 0.2 ~ C i / m l of 1 2 5 I - [ S a r l , I l e 8 ] A n g II (specific activity; 2200Ci/mmol). Non-specific bindinq was determined in t h e p r e s e n c e of 0.5 ~ M u n l a b e l l e d [sarl,Ile~]Ang II. A f t e r incubation, the sections were washed with ice-cold b u f f e r , l o a d e d i n t o X - r a y c a s s e t t e s a n d e x p o s e d to RX X - r a y f i l m for 1.5 days. A set of 125-I r a d i o a c t i v i t y s t a n d a r d w a s i n c l u d e d . After exposure, the films were developed with Fuji Rendol developer a t 18 °C f o r 5 m i n a n d t h e o p t i c a l density was quantitated w i t h an M C I D i m a g e a n a l y s i s s y s t e m ( I m a g i n g R e s e a r c h Inc. O n t a r i o , C a n a d a ) . The r a d i o a c t i v i t y s t a n d a r d s w e r e f i t t e d to a calibration c u r v e by c o m p u t e r to c o n v e r t o p t i c a l d e n s i t y v a l u e s of e a c h p i x e l i n t o d p m of 125-I r a d i o l i g a n d / m m 2. Differentiation o f A n g If r e c e p t o r b i n d i n g sites: To d i f f e r e n t i a t e Ang II r e c e p t o r binding sites, four compounds were used. [Sarl,Ile8]Ang II is a p o t e n t a n d s p e c i f i c a n t a g o n i s t of A n g II w h i c h b l o c k s a l l k n o w n A n g II r e c e p t o r s . 2-n-butyl-4-chloro-5hydroxymethyl-l-[2'(iH-tetrazol-5-yl)biphenyl-4-yl)methyl] imidaz o l e ( D u P 7 5 3 ) is s p e c i f i c n o n - p e p t i d e AT 1 a n t a g o n i s t and 1-[(4amino-3-methylphenyl)methyl]-5-(diphenyl acetyl )-4,5,6,7-tetrahydro-iH-imidazo[4,5-c]pyridine-6-carboxylic acid (PD123177) and nicotinic acid-Tyr-(Ne-benzyloxy-carbonyl-Arg)Lys-His-Pro-Ile-OH (CGP42112B) are non-peptide and peptide AT 2 antagonist respectively. A T 1 b i n d i n g w a s d e t e r m i n e d as t h a t i n h i b i t e d b y an e x c e s s of D u P 7 5 3 (5 ~M) a n d AT 2 b i n d i n g as t h a t i n h i b i t e d b y an e x c e s s of P D 1 2 3 1 7 7 (5 ~M) or C G P 4 2 1 1 2 B (i ~M). Characteristics of Ang I I receptor binding sites: Binding characteristics were assessed by competition studies. For this purpose, unlabelled [Sarl,Ile8]Ang II, D u P 7 5 3 , PD123177, and CGP42112B w e r e a d d e d at t h e c o n c e n t r a t i o n r a n g e of i0 p M to 5 p M to t h e i n c u b a t i o n m e d i u m c o n t a i n i n g 0.2 ~Ci of 125i_[Sar,~l,ile8]Ang II. A n g II r e c e p t o r binding sites were also assessed by the susceptibility to increased concentrations of dithiothreitol (DTT). Statistical significance was examined by the unpaired S t u d e n t ' s t-test. Results Non-specific binding, measured in t h e p r e s e n c e o f [ S a r I, I l e 8 ] A n g II (0.5 ~M), w a s v i r t u a l l y u n d e t e c t a b l e at a l l s i t e s on the autoradiographs. Thus results represent specific binding. A S s h o w n in FIG. IA, IE, a n d T A B L E i, n e p h r e c t o m y l e d to an i n c r e a s e in A n g II b i n d i n g in t h e c o r t e x and a decrease in t h e medulla. In c o n t r o l rats, AT 1 b i n d i n g w a s e q u i v a l e n t to 80 % of t o t a l c o r t i c a l b i n d i n g a n d I0 % of t o t a l m e d u l l a r y b i n d i n g (FIG. IB, IC, T A B L E i). A T 1 b i n d i n g d e t e r m i n e d in this manner agreed well with binding p e r s i s t i n g in the p r e s e n c e of 5 ~ M of P D 1 2 3 1 7 7 or 1 ~M of CGP42112B (FIG. IB, IC, T A B L E i). A T 2 b i n d i n g was

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Nephrectomy and Ang II Receptors

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equivalent t o 20 % of t h e t o t a l c o r t i c a l b i n d i n g a n d 90 % of t h e total medullary binding. (FIG. IB, IC, T A B L E 1 ) . AT 2 binding determined in t h i s m a n n e r a g r e e d w e l l w i t h b i n d i n g p e r s i s t i n g in the presence of 5 ~M of DuP753 (FIG. IB, IC, T A B L E i). B y contrast, in nephrectomized rats, AT 1 b i n d i n g inhibited by an e x c e s s of D u P 7 5 3 a c c o u n t e d for o n l y 21 % of t h e t o t a l c o r t i c a l b i n d i n g a n d t h i s b i n d i n g did not a g r e e w i t h b i n d i n g p e r s i s t i n g in t h e p r e s e n c e of an e x c e s s of P D 1 2 3 1 7 7 or C G P 4 2 1 1 2 B (FIG. IF, IG, TABLE i). A T 2 b i n d i n g inhibited by an excess of PD123177 and CGP42112B accounted for o n l y 22 % of t h e t o t a l c o r t i c a l binding and this binding did not agree with binding persisting in the p r e s e n c e of an e x c e s s of D u P 7 5 3 (FIG. IF, IG, T A B L E i). T h e s u m of binding persisting in the presence of AT 1 antagonist and AT 2 antagonist exceeded total cortical binding b y 60 %. In t h e medulla, the relationship between subtypes and total binding was preserved, as in the c o n t r o l rats (FIG. IF, IG, T A B L E i). T h e AT 1 or A T 2 a n t a g o n i s t e a c h a l o n e f a i l e d to i n h i b i t 1 2 5 I - [ S a r l , I l e 8 ] A n g II b i n d i n g b u t w h e n b o t h a n t a g o n i s t s were simultaneously in t h e incubation, binding was potently inhibited (FIG. IH). These o b s e r v a t i o n s w e r e r e p r o d u c i b l e u s i n g C G P 4 2 1 1 2 B i n s t e a d of P D 1 2 3 1 7 7 ( T A B L E i). W i t h r e g a r d to AT 2 r e c e p t o r binding, it w a s increased f r o m 42 ± 15 d p m / m m 2 to 145 ± 35 d p m / m m 2 in t h e c o r t e x . W h i l e in the medulla, AT~ binding was decreased f r o m 371 ± 13 d p m / m m 2 to 242 ± 22 d p m / m m ~

i¸i ~

• 2

FIG. 1 Ang II b i n d i n g to sections of rat adrenal glands demonstrated b y in v i t r o a u t o r a d i o g r a p h y using 125I[sarl,Ile8]Ang II as the r a d i o l i g a n d . A n g II b i n d i n g to control r a t s is s h o w n f r o m A to D a n d n e p h r e c t o m i z e d r a t s f r o m E to H. A a n d E s h o w t o t a l b i n d i n g . B and F s h o w b i n d i n g in t h e p r e s e n c e of D u P 7 5 3 (5 ~M). C a n d G s h o w b i n d i n g in the p r e s e n c e of P D 1 2 3 1 7 7 (5 NM). D a n d H s h o w b i n d i n g in t h e p r e s e n c e of b o t h D u P 7 5 3 (5 pM) a n d PD123177 (5 N M ) . W h i t e represents high density of b i n d i n g w h i l e b l a c k i n d i c a t e s b a c k g r o u n d levels.

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Nephrectomy and Ang II Receptors

TABLE

vol. 51, No. 18, 1992

1

125I-[Sarl,Ile8]Ang

II Binding

to the A d r e n a l

Gland.

Control (dpm/mm 2 )

Nephrex (dpm/mm 2 )

Cortex Total 420 ± 20 B i n d i n g i n h i b i t e d by DuP753 (5 ~M) 336 ± 20

Medulla 414 ± 13

PD12317

(5 pM)

CGP42112A

(i ~M)

2

138 ± 15"

Medulla 277 ± 26* 35 ±

8

74 ±

7

371

± 13

145 ± 14"

242

± 22*

82 ±

8

376

± 21

158

250

± 12"

Binding persisting 1 2 3 1 7 7 (5 ~M)

in

the

presence

1

16 ±

16 ± Binding persisting C G P 4 2 1 1 2 B (i ~M)

in 20 ±

Binding

35 ±

Cortex 647 ± 22*

in t h e p r e s e n c e 333 ±

the 4

of

both

1

presence 16 ±

± 18" DuP753

38 ± of

4

both 26 ±

of DTT (5 mM) 8 521 ± 14

650

3*

(5

15 ±

DuP753 4

± 12"

~M)

(5

~M)

and

1

±

7*

R e s u l t s w e r e e x p r e s s e d as m e a n ± s t a n d a r d error. * p < 0 . 0 1 compared with sham-operated control by the unpaired S t u d e n t ' s t-test.

B/B0 (%) 120

B/Bo (%) CTR

100

100

80

80

60

60

40

40

20

20

0 -11

-10

-9 -8 -7 log(drug[M])

Nephrex

120

-6

-5

0 -11

-10

-9 -8 -7 log(drug[M])

PD

2

14 ±

407

and

,

,

-6

-5

FIG. 2 B i n d i n g i s o t h e r m s s h o w i n g the i n h i b i t i o n of 1 2 5 I - [ S a r I, I l e 8 ] A n g II b i n d i n g to the a d r e n a l c o r t e x b y u n l a b e l l e d [ S a r I, I l e 8 ] A n g II a n d s u b t y p e a n t a g o n i s t s . D u P 7 5 3 ; Q, P D 1 2 3 1 7 7 ; • , C G P 4 2 1 1 2 B ; [] , [ s a r l , I l e S ] A n g II; O .

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Nephrectomy and Ang II Receptors

PL-169

A s s h o w n in FIG. 2A a n d 2B, t h e p o t e n c y of [ S a r l , I l e 8 ] A n g II w a s s i m i l a r at t h e a d r e n a l c o r t e x in b o t h g r o u p s w i t h I C 5 0 2 . 6 - 3 . 8 nM. P D 1 2 3 1 7 7 a n d C G P 4 2 1 1 2 B e x h i b i t e d a w e a k i n h i b i t i o n of A n g II b i n d i n g , e v e n at a h i g h dose. D u P 7 5 3 i n h i b i t e d A n g II r e c e p t o r binding in dose dependent manner in the cortex of control with I C 5 0 1 9 0 n M w h e r e a s , in t h e c o r t e x of n e p h r e c t o m i z e d rat, D u P 7 5 3 inhibited binding of only a small portion that derived from AT 1 receptors. Effect of increased concentrations o f D T T o n A n g II b i n d i n g was also examined (FIG. 3). In t h e m e d u l l a o f b o t h g r o u p s , DTT enhanced binding, in a biphasic manner (FIG. 3B), findings c o n s i s t e n t w i t h p r e v i o u s o b s e r v a t i o n s (1,2). In t h e a d r e n a l c o r t e x of s h a m - o p e r a t e d r a t s (FIG. 3A), D T T d e c r e a s e d t h e b i n d i n g . O n t h e o t h e r hand, D T T w a s v i r t u a l l y i n e f f e c t i v e in t h e a d r e n a l c o r t e x of n e p h r e c t o m i z e d rat.

siso (%)

B/B0 (%) 120

Cortex

140

Medulla

100 [~-~ 8O

-o

120

6O

o

4O 2O 0 0

, 2

, 4

, 6

, 8

i 10

, 100 12 0

, 2

-Iog(DTT[mM])

Effect of Control;O,

i 4

, 6

i 8

, 10

I

12

-Iog(DTT[mM])

FIG. 3 DTT on the binding to Nephrectomy;Q.

the

adrenal

glands.

Discussion In rat adrenal cortex, bilateral nephrectomy led to development of a new binding s i t e f o r A n g II w h i c h was not affected by either AT 1 and AT 2 antagonists each alone. When both a n t a g o n i s t s w e r e p r e s e n t s i m u l t a n e o u s l y in t h e i n c u b a t i o n s , A n g II binding was potently inhibited. An allosteric effect may be i n v o l v e d i n t h e i n h i b i t i o n o f b i n d i n g to t h e n e w l y d e v e l o p e d A n g II b i n d i n g sites. In vitro differentiation of the mouse neuroblastoma-rat g l i o m a h y b r i d c e l l line, N G - I 0 8 - 1 5 , w i t h d i m e t y l s u l p h o x i d e and low serum induced AT 2 receptor subtype (6) and also in vitro differentiation o f t h e m o u s e n e u r o b l a s t o m a cells, N e u r o - A 2 cells, w i t h P G 1 i n d u c e d t h e n e w b i n d i n g s i t e s f o r A n g II (7). P o t a s s i u m loading to rats developed hyperplasia of the zona glomerulosa of t h e a d r e n a l g l a n d s a n d i n c r e a s e d A n g II r e c e p t o r n u m b e r (8). High concentration of plasma potassium due to nephrectomy may be r e s p o n s i b l e f o r i n c r e a s e a n d i n d u c t i o n of t h e n e w b i n d i n g s i t e for A n g II s h o w n in t h e p r e s e n t study.

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Nephrectomy and Ang II Receptors

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Consistent with the findings of Iwai and Inagami (3) , c o r t i c a l A T 1 r e c e p t o r b i n d i n g d e t e r m i n e d as t h a t i n h i b i t e d b y a n e x c e s s of A T 1 a n t a g o n i s t w a s r e d u c e d to a p p r o x i m a t e l y 30 % of t h e control. After nephrectomy, the number of adrenal A n g II r e c e p t o r s w a s u n c h a n g e d for 12 h and t h e n i n c r e a s e d p r o g r e s s i v e l y o v e r t h e n e x t 24 h a n d r e a c h e d a p l a t e a u w h i c h w a s m a i n t a i n e d o v e r t h e n e x t 8 h (4). A newly developed Ang I I receptor binding can explain this discrepancy. A n e w A n g II b i n d i n g s i t e w a s i d e n t i f i e d i n N e u r o - A 2 c e l l s , w h e n e x a m i n i n g t h e s u s c e p t i b i l i t y to AT 1 and AT 2 a n t a g o n i s t s , a n d DTT decreased A n g II b i n d i n g to these cells (7). T h e o v e r a l l e f f e c t o f D T T o n A n g II b i n d i n g v a r i e d w i t h t h e r a t i o o f A T 1 a n d AT 2 receptors, the newly identified A n g II b i n d i n g site is probably n o t a f f e c t b y DTT, b e c a u s e this binding is m u c h m o r e predominant. In t h i s sense, n e w b i n d i n g s i t e f o r A n g II w h i c h w e identified differs from that observed on Neuro A2 cells. The effect of simultaneous presence of both antagonists was not e x a m i n e d in t h e i r study. T h i s is a p p a r e n t l y the first demonstration that shows the d e v e l o p m e n t o f a n e w b i n d i n g s i t e for A n g II a n d a c h a n g e in A n g II r e c e p t o r s u b t y p e s f o l l o w i n g t r e a t m e n t in vivo.

Acknowledgments D u P 7 5 3 a n d P D 1 2 3 1 7 7 w e r e s y n t h e s i z e d by Dr. K. M i y a k e a n d M. Matsukura ( T s u k u b a R e s e a r c h L a b o r a t o r i e s , E i s a i Co. Ltd. T s u k u b a , Japan). We are grateful to them for the gifts of DuP753 and PD123177 and also Dr. M. d e G a s p a r o (Ciba-Geigy, Basle, S w i t z e r l a n d ) for t h e g i f t of C G P 4 2 1 1 2 B . References i. 2.

3. 4. 5. 6.

7. 8.

S. W H I T E B R E A D , M. M E L E , B. K A M B E R a n d M. d e G A S P A R O . B i o c h e m . B i o p h y s . Res. Commun. 163: 2 8 4 - 2 9 1 (1989). A.T. CHIU, W.F. HEBLIN, D . E . M c C A L L , R.J. A R D E C K Y , D.J. CARIN, J.V. D U N C I A , L.J. PEASE, P.C. WONG, R.R. W E X L E R , A.L. J O H N S O N a n d P . B . M . W . M . T I M M E R M A N S . B i o c h e m . B i o p h y s . Res. C o m m u n . 165: 1 9 6 - 2 0 3 (1989). N. IWAI, a n d T. INAGAMI. B i o c h e m . B i o p h y s . Res. C o m m u n . 182: 1 0 9 4 - 1 0 9 9 (1992). G. A G U I L E R A , A. S C H I R A R , A. B A U K A L . a n d K . J . C A T T . Nature 289:507-509 (1981). K. S O N G , A . M . A L L E N , G. P A X I N O S and F.A.O. MENDELSOHN. J. Comp. N e u r o l o g y 316: 4 6 7 - 4 8 4 (1992). S.E. B R Y S O N , P. W A R B U R T O N , H.P. W I N T E R S G I L L , G.M. D R E W , A.T. M I C H E L , S.G. B A L L and A.J. B A L M F O R T H . Eur. J. P h a r m a c o l . 225: 1 1 9 - 1 2 7 (1992) S. C H A K I a n d T. INAGAMI. B i o c h e m . B i o p h y s . Res. C o m m u n . 182: 3 8 8 - 3 9 4 (1992). J. D O U G L A S and K.J. CATT. J. C l i n . Invest. 103: 834-843 (1975).