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Effect of urea-treatment on agonist binding affinity of the muscarinic acetylcholine receptor
Life Sciences, Vol. 33, pp. 963-970 Printed in the U.S.A.
EFFECT
OF
Pergamon Press
UREA-TREATMENT ON AGONIST BINDING AFFINITY OF THE MUSCARINIC ACETYLCHOLINE RECEPTOR
Kazuo Matsumoto,
Shuji Uchida, Hiroshi Higuchi, Atsushi Mizushima and Hiroshi Yoshida
Department of Pharmacology I, Osaka University School of Medicine, Nakanoshima, Kita-ku, Osaka 530, Japan (Received in final form June 16, 1983) SUMMARY Urea-treatment of the microsome fraction of the heart of guinea-pigs caused selective reduction in the apparent affinity of an agonist (carbachol), but not an antagonist (atropine), to muscarinic acetylcholine receptors (mAChR), measured as inhibition of binding of ~H-quinuclidinyl benzilate (3H-QNB). This effect was similar to that of Gpp(NH)p. The effects of urea-treatment and Gpp(NH)p were not additive. On the other hand, treatment of the mierosome fraction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) increased the apparent affinity of agonist, but not antagonist. The effect of DTNB predominated over those of urea-treatment and Gpp(NH)p, when these treatments were combined with DTNB. The specific binding of radiolabeled antagonists has been used to characterize mAChR in several tissues (1-5). The binding of antagonists to mAChR follows the law of mass action (6), so long as the binding is performed in physiological buffer systems (7). On the other hand, the binding of agonists to mAChR, measured as inhibition of binding of a radiolabeled antagonist, is more complicated and deviates from the law of mass action (4,8,9). Investigations of the competitive inhibition of 3H-antagonist binding by agonists have provided evidence which suggests the existence of two major populations of mAChR having different affinities for agonists and equal affinity for antagonists (8). A third minor population of superhigh affinity sites for agonists has also been described (8). Guanine nucleotides selectively reduce the apparent affinity for agonist binding in many receptor systems, such as serotonergic (i0), beta-adrenergic (Ii), alpha-adrenergic (12), dopaminergic (13) and opiate (14) receptor systems. This effect of guanine nucleotides has also been demonstrated on mAChR in several tissues (15-26). In addition to the effect of guanine nucleotides on agonist binding, recent observations in the muscarinic cholinergic (7,19,23,26), dopaminergic (27) and beta-adrenergic (28) receptor systems indicate that guanine nucleotides increase the apparent affinity for antagonist binding. 0024-3205/83 $3.00 +.00 Copyright (c) 1983 Pergamon Press Ltd.
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R e c e n t l y we r e p o r t e d that r a d i a t i o n i n a c t i v a t i o n by g a m m a - r a y s (24) and t r y p s i n - t r e a t m e n t (25) of g u i n e a - p i g ileum m i c r o s o m e f r a c t i o n c a u s e d s e l e c t i v e r e d u c t i o n in the a p p a r e n t a f f i n i t y of agonist, but not a n t a g o n i s t , to mAChR, and r e d u c e d the e f f e c t of G p p ( N H ) p on the a f f i n i t y of a g o n i s t to mAChR. This p a p e r r e p o r t s that u r e a - t r e a t m e n t of g u i n e a - p i g heart m i c r o s o m e f r a c t i o n d i s p l a y e d a s i m i l a r e f f e c t to r a d i a t i o n i n a c t i v a t i o n and trypsin-treatment. ~TERIALS
AND M E T H O D S
i. M a t e r i a l s [3~H] (±)QNB (5.0 Ci/mmol) was o b t a i n e d from RCC A m e r s h a m . other c h e m i c a l s w e r e of h i g h e s t p u r i t y a v a i l a b l e c o m m e r c i a l l y .
All
2. P r e p a r a t i o n of h e a r t m i c r o s o m e f r a c t i o n The w h o l e hearts from 250-400 g male g u i n e a - p i g s , freed of c o n n e c t i v e tissue, g r e a t vessels, b l o o d and fat, w e r e h o m o g e n i z e d in 5 v o l u m e s of ice cold 50 m M T r i s - H C l b u f f e r (pH 7.4). The m i c r o s o m e f r a c t i o n was p r e p a r e d by c e n t r i f u g a t i o n of the i0,000 xg s u p e r n a t a n t at i00,000 xg for 60 min. The m i c r o s o m e f r a c t i o n was s u s p e n d e d in 50 m M T r i s - H C l b u f f e r (pH 7.4) and stored at -80°C until use. F r e e z i n g the f r a c t i o n at -80°C for 4 w e e k s did not a f f e c t the Kd and Bmax of QNB binding. T h e r e f o r e , we used the p r e p a r a t i o n s frozen w i t h i n 4 weeks. 3. U r e a - t r e a t m e n t The m i c r o s o m e f r a c t i o n of w h o l e heart, c o n t a i n i n g a p p r o x i m a t e l y i0 mg of protein, was i n c u b a t e d w i t h 4 M urea in 25 m M T r i s - H C l , 50 m M NaCI, 0.5 m M MgCI2, pH 7.4, at 0°C for 30 min. A similar v o l u m e of d i s t i l l e d water, i n s t e a d of urea, was a d d e d to the c o n t r o l tubes. The tubes w e r e c e n t r i f u g e d at 100,000 xg for 30 min. The p e l l e t s o b t a i n e d at this step were s u s p e n d e d in 50 m M T r i s - H C l b u f f e r (pH 7.4) and c e n t r i f u g e d at i00,000 xg for 30 min in o r d e r to w a s h the free urea. This w a s h i n g p r o c e d u r e , w h i c h c o n s i s t s of s u s p e n s i o n in fresh b u f f e r and c e n t r i f u g a t i o n , was r e p e a t e d three times. The final p e l l e t s w e r e s u s p e n d e d in 50 m M T r i s - H C l b u f f e r (pH 7.4) and u s e d for b i n d i n g assays. 4. G p p ( N H ) p and DTNB t r e a t m e n t s and 3H-QNB b i n d i n g assay U n t r e a t e d or u r e a - t r e a t e d p r e p a r a t i o n s of h e a r t m i c r o s o m e f r a c t i o n w e r e i n c u b a t e d w i t h 100 u M G p p ( N H ) p or I ~ 4 DTNB for i0 min at 37°C, and then kept on ice. T h e n v a r i o u s c o n c e n t r a t i o n s of c a r b a c h o l or atro ine w e r e a d d e d and b i n d i n g of 3H-QNB was s t a r t e d H-QNB. The b a s i c m e d i u m for b i n d i n g a s s a y by a d d i n g 1.2 n M c o n s i s t e d of i00 m M NaCI, 50 m M T r i s - H C l (pH 7.4), 1 m M M g C ~ and a b o u t I00 ug p r o t e i n in 2 ml. The b i n d i n g of 3H-QNB was a s s a y e d at 37°C for 60 min. For e s t i m a t i o n of n o n s p e c i f i c binding,° 0.01 m M a t r o p i n e was a d d e d to the medium. O t h e r d e t a i l s w e r e as p r e v i o u s l y d e s c r i b e d (29). 5. P r o t e i n d e t e r m i n a t i o n P r o t e i n was d e t e r m i n e d
by the m e t h o d
of Lowry
et al.(30).
6. A n a l y s i s of b i n d i n g data The IC50 v a l u e s for c a r b a c h o l and a t r o p i n e w e r e c o r r e c t e d for the r a d i o l i g a n d o c c u p a n c y shift a c c o r d i n g to I ~ 0 c o r = and K i = I ~ 0 / ( I + L / K ) r e s p e c t i v e l y , w h e r e K and L r e p r e s e n t the d i s s o c i a t i o n c o n s t a n t and the c o n c e n t r a t i o n of H-QNB respectively
IC50/(I+L/K)
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Urea Effect on Muscarinic Receptor
965
~41). Ki s h o u l d be u s e d ol~ly in the c a s e t h a t the llill c o e f f i c i e n t for i n h i b i t o r is not s i g n i f i c a n t l y d i f f e r e n t T h e r e f o r e we u s e d I C 5 0 c o r to c o z r e c t the IC v a l u e s for carbachol. 50
RESULT
AND
from
i.
DISCUSSION
U r e a - t r e a t m e n t r e d u c e a the a p p a r e n t a f f i n i t y of c a r b a c h o l to m A C h R in g u i n e a - p i g h e a r t m i c r o s o m e f r a c t i o n (Fig. i) . P r e t r e a t m e n t of h e a r t m i c r o s o m e f r a c t i o n w i t h u r e a e n h a n c e d the b i n d i n g s of 3 H - Q N B in the p r e s e n c e of i00 u M c a r b a c h o l in the u n t r e a t e d m i c r o s o m e f r a c t i o n (Fig. 2). T h i s e z f e c t of p r e t r e a t m e n t w i t h u r e a d e p e n d e d on the c o n c e n t r a t i o n of urea, and w a s m a x i m a l on p r e t r e a t m e n t w i t h 4 M of u r e a for 30 min at 0°C (Fig. 2). T h e r e f o r e , u r e a - t r e a t m e n t was p e z f o r m e d u n d e r t h e s e c o n d i t i o n s for the r e s u l t s in Fig. i.
C) Contro] • GPp(NH)p G Ureq
lO0
~ •
DTNB
GPp(NHlP+UFeQ
• DTNB+Gpp(NH)D v DTNB+UFeO
50
\
c--
Carbachol
\
N Atropine
o_-# 8
7
6
5
D
t
4
3
2
-logOigood ] Fig.
1
E f f e c t s of D T N B , G p p ( N H ) p and u r e a - t r e a t m e n t on the c o m p e t i t i v e i n h i b i t i o n of 3 H - Q N B b i n d i n g by a t r o p i n e a n d c a r b a c h o l in the h e a r t m i c r o s o m e f r a c t i o n . DTNB, Gpp(NH)p and urea-treatment and 3H-QNB binding were p e r f o r m e d as d e s c r i b e d in " M e t h o d s " . The results shown are t a k e n f r o m a r e p r e s e n t a t i v e e x p e r i m e n t and s i m i l a r r e s u l t s w e r e o b t a i n e d in 3 s e p a r a t e e x p e r i m e n t s , w i t h e a c h p o i n t d o n e in t r i p l i c a t e .
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Urea Effect on Muscarinic Receptor
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Gpp(NH)p • (+) [] ( - )
| m
[.,,,] (M* Fig.
2
E f f e c t s of p r e t r e a t m e n t w i t h v a r i o u s c o n c e n t r a t i o n s of urea for 30 m i n at 0°C on the s p e c i f i c b i n d i n g of ~ - Q N B in the p r e s e n c e of c a r b a c h o l w i t h or w i t h o u t G p p ( N H ) p to the h e a r t m i c r o s o m e fraction. G u i n e a - p i g h e a r t m i c r o s o m e f r a c t i o n was i n c u b a t e d for 30 m i n at 0°C w o t h urea at the indicated concentrations. Then the w a s h i n g p r o c e d u r e , w h i c h c o n s i s t s of s u s p e n s i o n in fresh b u f f e r and c e n t r i fugation, w a s r e p e a t e d three times. The final p e l l e t s w e r e s u s p e n d e d in 50 m M T r i s - H C l (pH 7.4) and w e r e i n c u b a t e d w i t h ( m ) or w i t h o u t ( Q ) i00 uM G p p ( N H ) p , and then i00 u M c a r b a c h o l was added, and H-QNB binding was started, as d e s c r i b e d u n d e r "Methods". The p o i n t s shown are m e a n s of t r i p l i c a t e d e t e r m i n a t i o n s in two replicate experiments. B o u n d QNB in the a b s e n c e of c a r b a c h o l at each t r e a t m e n t was taken as i00 %.
Fig. 1 shows the e f f e c t s of DTNB, G p p ( N H ) p and u r e a - t r e a t m e n t on the c o m p e t i t i v e i n h i b i t i o n of 3 H - Q N B b i n d i n g by the a n t a g o n i s t a t r o p i n e and the a g o n i s t c a r b a c h o l . The e f f e c t s of these t r e a t m e n t s on 3 H - Q N B b i n d i n g w e r e s t u d i e d in s a t u r a t i o n e x p e r i m e n t s , and it was found that these t r e a t m e n t s did not a f f e c t the d i s s o c i a t i o n c o n s t a n t of 3 H - Q N B b i n d i n g in h e a r t m i c r o s o m e f r a c t i o n s i g n i f i c a n t l y (Table i) . F u r t h e r m o r e , these t r e a t m e n t s had no e f f e c t s on i n h i b i t i o n by a t r o p i n e of 3 H - Q N B b i n d i n g (Fig. 1 and T a b l e 2). In c o n t r a s t , they had clear e f f e c t s on i n h i b i t o n by c a r b a c h o l of ~ - Q N B b i n d i n g (Fig. 1 and T a b l e 2). The a p p a r e n t a f f i n i t y of c a r b a c h o l to m A C h R was e n h a n c e d by DTNB and r e d u c e d by
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Urea Effect on Muscarinic Receptor
G p p ( N H ) p (Fig. i). These reports (15-26, 31-33).
results
Table
are c o n s i s t e n t
with
967
previous
1
Effects of DTNB, G p p ( N H ) p and u r e a - t r e a t m e n t on the b i n d i n g p a r a m e t e r s of 3 H - Q N B in the heart m i c r o s o m e fraction. Equilibrium b i n d i n g m e a s u r e m e n t s were made at five d i f f e r e n t c o n c e n t r a t i o n s of 3H-QNB, spaced b e t w e e n 0.25 and 2.0 nM. Data were a n a l y z e d a c c o r d i n g to the m e t h o d of S c a t c h a r d and are p r e s e n t e d as the means f SD of 3 separate experiments, each done in triplicate.
KD Control Gpp (NH) p DTNB G p p ( N H ) p + DTNB Urea Urea + G p p ( N H ) p Urea + DTNB
Bma x ( p m o l e s / m g
(nM)
0.56 0 59 0 56 0 56 0 56 0 59 0 57
0.29 ± 0.06 0 30 ± 0.04 0 29 ± 0.08 0 30 0 31
± 0.08 ± 0.06
0 30 f 0.07 0 31 i 0.06
Table
+_ f ± ± f f ±
0 0 0 0 0 0 0
protein) 15 17 15 16 16 19 16
2
Effects of DTNB, G p p ( N H ) p and u r e a - t r e a t m e n t on the b i n d i n g p a r a m e t e r s of a t r o p i n e and c a r b a c h o l in the heart m i c r o s o m e fraction. Atropine Hill coefficient
Carbachol Ki (nM)
Hill coefficient
Control
1.02±0.07
4.58±0.70
d0.51±0.06
Gpp(NHJp Urea Gpp(NH)p+Urea DTNB
1.00i0.02 1.02±0.04 N.D. 1.03f0.04
4.49±0.46 4.43±0.66 N.D. c5.62±1.20
a0.78±0.07 a0. a0.77±0.07 77±0.08 c0.67±0.09
DTNB+Gpp(NH)p
N.D.
N.D.
c0.68f0.ii
DTNB+Urea
N.D.
N.D.
b0.70±0.09
IC50co r
(uM)
e4.36±0.55 b18.4 i6.3 !~9.1 ±6.5 1.8 f9.7 ad0.55±0.25 ad0.55±0.35 0.49f0.18
a b c S i g n i f i c a n t l y d i f f e r e n t from control (a p<0.01 b p<0.05, c ~<0.l) d _ S i g n i f i c a n t l y d i f f e r e n t from u r e a - t r e a t m e n t d p<0.01, e p<0.05) ( The S t u d e n t s ' s t-test was used to test significance. Data are p r e s e n t e d as the means ± SD of 3 separate experiments, each done in triplicate. N.D. : not d e t e r m i n e d U r e a - t r e a t m e n t d e c r e a s e d the a p p a r e n t a f f i n i t y of agonist, but not antagonist, to m A C h R (Fig. I). After u r e a - t r e a t m e n t , the Hill c o e f f i c i e n t (34) for c a r b a c h o l and IC50ce r of c a r b a c h o l w e r e i n c r e a s e d (Table 2). U r e a - t r e a t m e n t reduced the effect of G p p ( N H ) p on the b i n d i n g a f f i n i t y of agonist to m A C h R (Fig. 1 and
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T a b l e 2). The e f f e c t s of G p p ( N H ) p and urea t r e a t m e n t s on a g o n i s Z b i n d i n g a f f i n i t y to m A C h R w e r e similar and were not a d d i t i v e under the c o n d i t i o n s that gave m a x i m a l e f f e c t s (Fig. i and Table 2). ~loreover, w h e n c o m b i n e d w i t h DTNB, the e f f e c t s of G P P ( N H ) p and urea t r e a t m e n t s w e r e both o v e r c o m e by that of DTNB (Fig. 1 and Table 2). These r e s u l t s i n d i c a t e that Gpp(NI{)p and urea t r e a t m e n t s have the same t a r g e t or that there is some o v e r l a p in their t a r g e t areas. Recently, E h l e r t et al (19) and B i r d s a l l et al (33) have s u g g e s t e d that d e c r e a s e in a g o n i s t b i n d i n g a f f i n i t y for m A C h R c a u s e d by g u a n i n e n u c l e o t i d e s is m e d i a t e d t h r o u g h the i n t e r a c t i o n of m A C h R w i t h G T P - b i n d i n g p r o t e i n (G-protein). More recently, our study on r a d i a t i o n i n a c t i v a t i o n s u g g e s t e d i) that G p p ( N H ) p b o u n d to an o t h e r subunit than the l i g a n d - b i n d i n g subunit, 2) that the b i n d i n g of G p p ( N H ) p to this s u b u n i t c a u s e d d i s s o c i a t i o n of this g u a n i n e n u c l e o t i d e - b i n d i n g s u b u n i t from the l i g a n d - b i n d i n g subunit and 3) that the d i s s o c i a t e d l i g a n d - b i n d i n g s u b u n i t showed low a f f i n i t y to the a g o n i s t (24). Our p r e v i o u s study also s u g g e s t e d that t r y p s i n - t r e a t m e n t d e s t r o y e d the G - p r o t e i n linked w i t h m A C h R or split the part of m A C h R that is r e s p o n s i b l e for a t t a c h m e n t of G - p r o t e i n , and c o n s e q u e n t l y c a u s e d the low a f f i n i t y state of m A C h R to a g o n i s t (25). The p r e s e n t r e s u l t s s u g g e s t that u r e a - t r e a t m e n t acts at some of a series of steps i n v o l v e d in the linkage b e t w e e n m A C h R and G - p r o t e i n . U r e a - t r e a t m e n t has the e f f e c t to s o l u b i l i z e or d e n a t u r e m e m b r a n e - b o u n d proteins. U r e a - t r e a t m e n t may have s o l u b i l i z e d the G - p r o t e i n l i n k e d w i t h mAChR, but our trials to s o l u b i l i z e the G - p r o t e i n by u r e a - t r e a t m e n t r e s u l t e d in failure. T h e r e f o r e , we suppose that u r e a - t r e a t m e n t p r o b a b l y d e n a t u r e d the G - p r o t e i n or the part of m A C h R linked w i t h G - p r o t e i n , and c o n s e q u e n t l y c a u s e d the low a f f i n i t y state of m A C h R to agonist. U r e a - t r e a t m e n t p r o b a b l y b r o u g h t the u n c o u p l i n g b e t w e e n m A C h R and G - p r o t e i n , and r e s u l t e d in the low a f f i n i t y state of m A C h R to agonist. The low a f f i n i t y state of r e c e p t o r to a g o n i s t c a u s e d by u n c o u p l i n g b e t w e e n r e c e p t o r and G - p r o t e i n is also r e p o r t e d in m u s c a r i n i c c h o l i n e r g i c (35), b e t a - a d r e n e r g i c (36) and d o p a m i n e r g i c (37) r e c e p t o r systems. S a l v a t e r a (38) r e p o r t e d that t r e a t m e n t of rat b r a i n w i t h 6 M urea d e c r e a s e d the n u m b e r of 3 H - Q N B b i n d i n g sites. We also found that t r e a t m e n t of g u i n e a - p i g h e a r t m i c r o s o m e f r a c t i o n w i t h 4 M urea d e c r e a s e d the n u m b e r of 3 H - Q N B b i n d i n g sites and m e m b r a n e b o u n d p r o t e i n s to the same degree, and c o n s e q u e n t l y did not a f f e c t the d e n s i t y of 3 H - Q N B b i n d i n g sites. The e f f e c t of DTNB t r e a t m e n t on a g o n i s t b i n d i n g a f f i n i t y to m A C h R p r e d o m i n a t e d over that of G p p ( N H ) p , u r e a - t r e a t m e n t (Fig. 1 and T a b l e 2) and t r y p s i n - t r e a t e n t (25). A r o n s t a m et al. p r o p o s e d that N - e t h l m a l e i m i d e (NEM} t r e a t m e n t i n c r e a s e d a g o n i s t b i n d i n g a f f i n i t y by c o n v e r t i n g m A C h R from a state of low a g o n i s t a f f i n i t y to a "stable" state of high a g o n i s t affinity, w h e r e a s both states had the same high a f f i n i t y for a n t a g o n i s t (39). We suppose that DTNB t r e a t m e n t e n h a n c e d a g o n i s t b i n d i n g a f f i n i t y for m A C h R in a s i m i l a r m a n n e r to N E M treatment. Our study on r a d i a t i o n i n a c t i v a t i o n i n d i c a t e d that the m o l e c u l a r size of the unit for the D T N B e f f e c t was a little larger than that of the r e c e p t o r s u b u n i t and that of the G p p ( N H ) p e f f e c t was m u c h larger than that of the r e c e p t o r s u b u n i t (24). P e r h a p s the a c t i o n of DTNB is m e d i a t e d t h r o u g h an i n t e r a c t i o n b e t w e e n m A C h R and a n o t h e r m e m b r a n e c o m p o n e n t d i s t i n c t from G - p r o t e i n .
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Urea Effect on Muscarinic Receptor
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We u s e d p l u s / m i n u s QNB at the c o n c e n t r a t i o n of ].2 nM, w h i c h was several times h i g h e r than the Kd. Under these c o n d i t i o n s , b o t h p o s i t i v e and n e g a t i v e s t e r e o i s o m e r s will c o n t r i b u t e to c o m p e t i t i o n curves (40). Yhis limits the q u a n t i t a t i v e u t i l i t y of these very c o m p l e x e x p e r i m e n t s . This t e c h n i c a l l i m i t a t i o n w o u l d limit the q u a n t i t a t i v e i n t e r p r e t a t i o n of our results but p r o b a b l y have no b e a r i n g on the q u a l i t a t i v e analysis. A c k n o w l e d g e m e n t : We thank Mrs. M i e k o N a k a m u r a for a s s i s t a n c e p r e p a r a t i o n of this m a n u s c r i p t . This w o r k was s u p p o r t e d in part by a G r a n t - i n - A i d for S c i e n t i f i c R e s e a r c h from M i n i s t r y o~ E d u c a t i o n , S c i e n c e and C u l t u r e of Japan.
in
References 1. N.J.M. B I R D S A L L and E.C. IIUL~, J. Neurochem. 27 7-16 (1976) 2. H.I. YA~LA24URA and S.H. SNYDER, Proc. Natl. Acad-~. Sci. 71 1 7 2 5 - 1 7 2 9 (]974) 3. A.J BEI.D, S. VAN DEN HOBEN, C.A. W O U T E R S E and M.A.P. ZEGERS, Europ. J. Pharmacol. 30 360-363 (1975) 4. R.E. HRUSKA, R. WHITE, J. AZAII ant] H.I. Y A ~ M U R A ~ B r a i n Res. 148 493-498 (1978) 5. H.I Y~iURA and S.H. SNYDER, Mol. Pharmacol. i0 861-867 (1974) 6. E.C HULIdE, N.J.M. BIRDSALL, A.S.V. B U R G E N and P. MAHTA, Mol. P h a r m a c o l . 14 737-750 (1978) 7 E C HULME, C.P. BERRIE, N.J.M. B I R D S A L L and A.S.V. BURGEN, Europ. O. Pharmacol. 73 137-142 (1981) 8 N J M. B I R D S A L L , A . S . V _ _ B U R G E N and E.C. HULME, Mol. Pharmacol. 14 723-736 (1978) 9 E C HULME, A.S.V. B U R G E N and N.J.M. BIRDSALL, INSERM. 50 49-70 {1976) 10 S J PEROUTKA, R.M. L E B O V I T Z and S.H. SNYDER, Mol. Pharmacol. 16 700-708 (1979) ii L T W I L L I A M S and R.J. LEFKOWITZ, J. Biol. Chem. 252 7 2 0 7 - 7 2 1 3 (1977) 12 D C U ' P R I C H A R D and S.H. SNYDER, J. Biol. Chem. 253 3 4 4 4 - 3 4 5 2 (1978) 13. I C R E E S E and S.H. SNYDER, Europ. J. Pharmacol. 50 459-461 (1978) 14. S R. C H I L D E R S and S.H. SNYDER, Life Sci. 23 759-762 (1978) 15. C . P . B E R R I E , N.J.M. BIRDSALL, A.S.V. B U R G E N and E.C. HULKS, Biochem. Biophys. Res. Commun. 87 1000-1005 (1979) ]6 L.B. R O S E N B E R G E R , W.R. R O E S K E and H.I. YAMAMURA, Europ. J. Pharmacol. 14 179-180 (1979) 17. L.B. R O S E N B E R G E R , H.I. Y A M ~ 4 U R A and W.R. ROESKE, J. Biol. Chem. 255 820-823 (1980) 18 F.J. EHLERT, W.R. ROESKE, L.B. R O S E N B E R G E R and II.I. YAMAMURA, Life Sci. 26 245-252 {1980) 19. F.J. EHLERT]--W.R. R O E S K E and H.I. Y A ~ M U R A , J. Supramol. Struct. 14 149-162 (1980) 20. M. S O K O L O V S K Y , D. G U R W I T Z and R. GALRON, Biochem. Biophys. Res. Commun. 94 487-492 (1980) 21. J.W. WEI and P~-V. SULAKHE, N a u n y n - S c h m i e d e b e r g ' s Arch. P h a r m a c o l . 314 51-59 (1980) 22. F. EHLERT, L. R O S E N B E R G E R , W. R O E S K E and H. YAMAMURA, Soc. N e u r o s c i . A b s t r a c t s 5 401 (1979)
970
23 24 25 26 27 28 29 30 31 32 33
34. 35. 36. 37. 38. 39. 40. 41.
Urea Effect on Muscarinic Receptor
Vol. 33, No. i0, 1983
F.J. EHLERT, W.R. R O E S K E and H.I. YA~[AMUF~, Fed. Proc. 40 153-159 (1981) S. UCHIDA, K. ~TSUI,IOTO, K. TAKEYASU, H. HIGUCHI and H. YOSHIDA, Life Sci. 31 201-209 (1982) K. ~ T S U M O T O , S. UCHIDA, K. T A K E Y A S U , H. HIGUCHI and H. YOSHIDA, Life Sci. 31 2il-220 (1982) E. B U R G I S S E R , A. DE LEAN and R.J. LEFKOWITZ, Proc. Natl. Acad. Sci. U S A 79 1 7 3 2 - 1 7 3 6 (1982) A. DE LEAN~--B.F. K i L P A T R I C K and M.G. CARON, E n d o c r i n o l . ii0 1 0 6 4 - 1 0 6 6 (1982) B.B. W O L F E and T.K. HARDEN, J. C y c l i c Nucl. Res. 7 303-312 (1981) S. UCHIDA, K. T A K E Y A S U , T. ~.~TSUDA and H. YOSHIDA, Life Sci. 24 1 8 0 5 - 1 8 1 2 (]979) O.H. LOWRY, N.J. R O S E B R O U G H , A.L. F A R R and R.J. RANDAL, J. Biol. Chem. 193 265-275 (1951) S. UCHIDA, K. T A K E Y A S U , S. I C H I D A and H. YOSHIDA, Japan. J. Pharmacol. 28 853-862 (1978) R.S. A R O N S T A M ana M.E. E L D E F R A W I , Biochem. Pharmacol. 28 701-703 (1979) N.J.M. BIRDSALL, C.P. BERRIE, A.S.V. B U R G E N and E.C. HULME, in R e c e p t o r s for N e u r o t r a n s m i t t e r s and P e p t i d e Hormones, (G.Pepeu, M.J. K u h a r and S.J. Enna eds.) Raven Press, New York,pp.107-116 (1980) A.V. HILL, J. Physiol. 39 361-373 (1909) D. G U R W I T Z and M. S O K O L O V S K Y , Biochem. Biophys. Res. Commun. 94 493-500 (1980) T.K. HARDEN, Y. F. SU and J.P. PERKINS, J. C y c l i c Nucl. Res. 5 99-106 (1979) M.W. H A M B L I N and I. CREESE, Mol. Pharmacol. 21 52-56 (1982) P.M. S A L V A T E R R A , Biochim. Biophys. A c t a 601 78-91 (1980) R.S. A R O N S T A M , L.G. A B O O D and W. HOSS, Mol. Pharmacol. 14 575-586 (1978) E. B U R G I S S E R , A.A. HANCOCK, R.J. L E F K O W I T Z and A . D E LEAN, Mol. Pharmacol. 19 205-216 (1981) R. HAMMER, C.--P. BERRIE, N.J.M. BIRDSALL, A.S.V. B U R G E N and E.C. HULME, N a t u r e 283 90-92 (1980)
EFFECT
OF
Pergamon Press
UREA-TREATMENT ON AGONIST BINDING AFFINITY OF THE MUSCARINIC ACETYLCHOLINE RECEPTOR
Kazuo Matsumoto,
Shuji Uchida, Hiroshi Higuchi, Atsushi Mizushima and Hiroshi Yoshida
Department of Pharmacology I, Osaka University School of Medicine, Nakanoshima, Kita-ku, Osaka 530, Japan (Received in final form June 16, 1983) SUMMARY Urea-treatment of the microsome fraction of the heart of guinea-pigs caused selective reduction in the apparent affinity of an agonist (carbachol), but not an antagonist (atropine), to muscarinic acetylcholine receptors (mAChR), measured as inhibition of binding of ~H-quinuclidinyl benzilate (3H-QNB). This effect was similar to that of Gpp(NH)p. The effects of urea-treatment and Gpp(NH)p were not additive. On the other hand, treatment of the mierosome fraction with 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) increased the apparent affinity of agonist, but not antagonist. The effect of DTNB predominated over those of urea-treatment and Gpp(NH)p, when these treatments were combined with DTNB. The specific binding of radiolabeled antagonists has been used to characterize mAChR in several tissues (1-5). The binding of antagonists to mAChR follows the law of mass action (6), so long as the binding is performed in physiological buffer systems (7). On the other hand, the binding of agonists to mAChR, measured as inhibition of binding of a radiolabeled antagonist, is more complicated and deviates from the law of mass action (4,8,9). Investigations of the competitive inhibition of 3H-antagonist binding by agonists have provided evidence which suggests the existence of two major populations of mAChR having different affinities for agonists and equal affinity for antagonists (8). A third minor population of superhigh affinity sites for agonists has also been described (8). Guanine nucleotides selectively reduce the apparent affinity for agonist binding in many receptor systems, such as serotonergic (i0), beta-adrenergic (Ii), alpha-adrenergic (12), dopaminergic (13) and opiate (14) receptor systems. This effect of guanine nucleotides has also been demonstrated on mAChR in several tissues (15-26). In addition to the effect of guanine nucleotides on agonist binding, recent observations in the muscarinic cholinergic (7,19,23,26), dopaminergic (27) and beta-adrenergic (28) receptor systems indicate that guanine nucleotides increase the apparent affinity for antagonist binding. 0024-3205/83 $3.00 +.00 Copyright (c) 1983 Pergamon Press Ltd.
964
Urea Effect on Muscarinic Receptor
Vol. 33, No. i0, 1983
R e c e n t l y we r e p o r t e d that r a d i a t i o n i n a c t i v a t i o n by g a m m a - r a y s (24) and t r y p s i n - t r e a t m e n t (25) of g u i n e a - p i g ileum m i c r o s o m e f r a c t i o n c a u s e d s e l e c t i v e r e d u c t i o n in the a p p a r e n t a f f i n i t y of agonist, but not a n t a g o n i s t , to mAChR, and r e d u c e d the e f f e c t of G p p ( N H ) p on the a f f i n i t y of a g o n i s t to mAChR. This p a p e r r e p o r t s that u r e a - t r e a t m e n t of g u i n e a - p i g heart m i c r o s o m e f r a c t i o n d i s p l a y e d a s i m i l a r e f f e c t to r a d i a t i o n i n a c t i v a t i o n and trypsin-treatment. ~TERIALS
AND M E T H O D S
i. M a t e r i a l s [3~H] (±)QNB (5.0 Ci/mmol) was o b t a i n e d from RCC A m e r s h a m . other c h e m i c a l s w e r e of h i g h e s t p u r i t y a v a i l a b l e c o m m e r c i a l l y .
All
2. P r e p a r a t i o n of h e a r t m i c r o s o m e f r a c t i o n The w h o l e hearts from 250-400 g male g u i n e a - p i g s , freed of c o n n e c t i v e tissue, g r e a t vessels, b l o o d and fat, w e r e h o m o g e n i z e d in 5 v o l u m e s of ice cold 50 m M T r i s - H C l b u f f e r (pH 7.4). The m i c r o s o m e f r a c t i o n was p r e p a r e d by c e n t r i f u g a t i o n of the i0,000 xg s u p e r n a t a n t at i00,000 xg for 60 min. The m i c r o s o m e f r a c t i o n was s u s p e n d e d in 50 m M T r i s - H C l b u f f e r (pH 7.4) and stored at -80°C until use. F r e e z i n g the f r a c t i o n at -80°C for 4 w e e k s did not a f f e c t the Kd and Bmax of QNB binding. T h e r e f o r e , we used the p r e p a r a t i o n s frozen w i t h i n 4 weeks. 3. U r e a - t r e a t m e n t The m i c r o s o m e f r a c t i o n of w h o l e heart, c o n t a i n i n g a p p r o x i m a t e l y i0 mg of protein, was i n c u b a t e d w i t h 4 M urea in 25 m M T r i s - H C l , 50 m M NaCI, 0.5 m M MgCI2, pH 7.4, at 0°C for 30 min. A similar v o l u m e of d i s t i l l e d water, i n s t e a d of urea, was a d d e d to the c o n t r o l tubes. The tubes w e r e c e n t r i f u g e d at 100,000 xg for 30 min. The p e l l e t s o b t a i n e d at this step were s u s p e n d e d in 50 m M T r i s - H C l b u f f e r (pH 7.4) and c e n t r i f u g e d at i00,000 xg for 30 min in o r d e r to w a s h the free urea. This w a s h i n g p r o c e d u r e , w h i c h c o n s i s t s of s u s p e n s i o n in fresh b u f f e r and c e n t r i f u g a t i o n , was r e p e a t e d three times. The final p e l l e t s w e r e s u s p e n d e d in 50 m M T r i s - H C l b u f f e r (pH 7.4) and u s e d for b i n d i n g assays. 4. G p p ( N H ) p and DTNB t r e a t m e n t s and 3H-QNB b i n d i n g assay U n t r e a t e d or u r e a - t r e a t e d p r e p a r a t i o n s of h e a r t m i c r o s o m e f r a c t i o n w e r e i n c u b a t e d w i t h 100 u M G p p ( N H ) p or I ~ 4 DTNB for i0 min at 37°C, and then kept on ice. T h e n v a r i o u s c o n c e n t r a t i o n s of c a r b a c h o l or atro ine w e r e a d d e d and b i n d i n g of 3H-QNB was s t a r t e d H-QNB. The b a s i c m e d i u m for b i n d i n g a s s a y by a d d i n g 1.2 n M c o n s i s t e d of i00 m M NaCI, 50 m M T r i s - H C l (pH 7.4), 1 m M M g C ~ and a b o u t I00 ug p r o t e i n in 2 ml. The b i n d i n g of 3H-QNB was a s s a y e d at 37°C for 60 min. For e s t i m a t i o n of n o n s p e c i f i c binding,° 0.01 m M a t r o p i n e was a d d e d to the medium. O t h e r d e t a i l s w e r e as p r e v i o u s l y d e s c r i b e d (29). 5. P r o t e i n d e t e r m i n a t i o n P r o t e i n was d e t e r m i n e d
by the m e t h o d
of Lowry
et al.(30).
6. A n a l y s i s of b i n d i n g data The IC50 v a l u e s for c a r b a c h o l and a t r o p i n e w e r e c o r r e c t e d for the r a d i o l i g a n d o c c u p a n c y shift a c c o r d i n g to I ~ 0 c o r = and K i = I ~ 0 / ( I + L / K ) r e s p e c t i v e l y , w h e r e K and L r e p r e s e n t the d i s s o c i a t i o n c o n s t a n t and the c o n c e n t r a t i o n of H-QNB respectively
IC50/(I+L/K)
Vol. 33, No. i0, 1983
Urea Effect on Muscarinic Receptor
965
~41). Ki s h o u l d be u s e d ol~ly in the c a s e t h a t the llill c o e f f i c i e n t for i n h i b i t o r is not s i g n i f i c a n t l y d i f f e r e n t T h e r e f o r e we u s e d I C 5 0 c o r to c o z r e c t the IC v a l u e s for carbachol. 50
RESULT
AND
from
i.
DISCUSSION
U r e a - t r e a t m e n t r e d u c e a the a p p a r e n t a f f i n i t y of c a r b a c h o l to m A C h R in g u i n e a - p i g h e a r t m i c r o s o m e f r a c t i o n (Fig. i) . P r e t r e a t m e n t of h e a r t m i c r o s o m e f r a c t i o n w i t h u r e a e n h a n c e d the b i n d i n g s of 3 H - Q N B in the p r e s e n c e of i00 u M c a r b a c h o l in the u n t r e a t e d m i c r o s o m e f r a c t i o n (Fig. 2). T h i s e z f e c t of p r e t r e a t m e n t w i t h u r e a d e p e n d e d on the c o n c e n t r a t i o n of urea, and w a s m a x i m a l on p r e t r e a t m e n t w i t h 4 M of u r e a for 30 min at 0°C (Fig. 2). T h e r e f o r e , u r e a - t r e a t m e n t was p e z f o r m e d u n d e r t h e s e c o n d i t i o n s for the r e s u l t s in Fig. i.
C) Contro] • GPp(NH)p G Ureq
lO0
~ •
DTNB
GPp(NHlP+UFeQ
• DTNB+Gpp(NH)D v DTNB+UFeO
50
\
c--
Carbachol
\
N Atropine
o_-# 8
7
6
5
D
t
4
3
2
-logOigood ] Fig.
1
E f f e c t s of D T N B , G p p ( N H ) p and u r e a - t r e a t m e n t on the c o m p e t i t i v e i n h i b i t i o n of 3 H - Q N B b i n d i n g by a t r o p i n e a n d c a r b a c h o l in the h e a r t m i c r o s o m e f r a c t i o n . DTNB, Gpp(NH)p and urea-treatment and 3H-QNB binding were p e r f o r m e d as d e s c r i b e d in " M e t h o d s " . The results shown are t a k e n f r o m a r e p r e s e n t a t i v e e x p e r i m e n t and s i m i l a r r e s u l t s w e r e o b t a i n e d in 3 s e p a r a t e e x p e r i m e n t s , w i t h e a c h p o i n t d o n e in t r i p l i c a t e .
966
Urea Effect on Muscarinic Receptor
100
Vol. 33, No. i0, 1983
Gpp(NH)p • (+) [] ( - )
| m
[.,,,] (M* Fig.
2
E f f e c t s of p r e t r e a t m e n t w i t h v a r i o u s c o n c e n t r a t i o n s of urea for 30 m i n at 0°C on the s p e c i f i c b i n d i n g of ~ - Q N B in the p r e s e n c e of c a r b a c h o l w i t h or w i t h o u t G p p ( N H ) p to the h e a r t m i c r o s o m e fraction. G u i n e a - p i g h e a r t m i c r o s o m e f r a c t i o n was i n c u b a t e d for 30 m i n at 0°C w o t h urea at the indicated concentrations. Then the w a s h i n g p r o c e d u r e , w h i c h c o n s i s t s of s u s p e n s i o n in fresh b u f f e r and c e n t r i fugation, w a s r e p e a t e d three times. The final p e l l e t s w e r e s u s p e n d e d in 50 m M T r i s - H C l (pH 7.4) and w e r e i n c u b a t e d w i t h ( m ) or w i t h o u t ( Q ) i00 uM G p p ( N H ) p , and then i00 u M c a r b a c h o l was added, and H-QNB binding was started, as d e s c r i b e d u n d e r "Methods". The p o i n t s shown are m e a n s of t r i p l i c a t e d e t e r m i n a t i o n s in two replicate experiments. B o u n d QNB in the a b s e n c e of c a r b a c h o l at each t r e a t m e n t was taken as i00 %.
Fig. 1 shows the e f f e c t s of DTNB, G p p ( N H ) p and u r e a - t r e a t m e n t on the c o m p e t i t i v e i n h i b i t i o n of 3 H - Q N B b i n d i n g by the a n t a g o n i s t a t r o p i n e and the a g o n i s t c a r b a c h o l . The e f f e c t s of these t r e a t m e n t s on 3 H - Q N B b i n d i n g w e r e s t u d i e d in s a t u r a t i o n e x p e r i m e n t s , and it was found that these t r e a t m e n t s did not a f f e c t the d i s s o c i a t i o n c o n s t a n t of 3 H - Q N B b i n d i n g in h e a r t m i c r o s o m e f r a c t i o n s i g n i f i c a n t l y (Table i) . F u r t h e r m o r e , these t r e a t m e n t s had no e f f e c t s on i n h i b i t i o n by a t r o p i n e of 3 H - Q N B b i n d i n g (Fig. 1 and T a b l e 2). In c o n t r a s t , they had clear e f f e c t s on i n h i b i t o n by c a r b a c h o l of ~ - Q N B b i n d i n g (Fig. 1 and T a b l e 2). The a p p a r e n t a f f i n i t y of c a r b a c h o l to m A C h R was e n h a n c e d by DTNB and r e d u c e d by
Vol. 33, No. i0, 1983
Urea Effect on Muscarinic Receptor
G p p ( N H ) p (Fig. i). These reports (15-26, 31-33).
results
Table
are c o n s i s t e n t
with
967
previous
1
Effects of DTNB, G p p ( N H ) p and u r e a - t r e a t m e n t on the b i n d i n g p a r a m e t e r s of 3 H - Q N B in the heart m i c r o s o m e fraction. Equilibrium b i n d i n g m e a s u r e m e n t s were made at five d i f f e r e n t c o n c e n t r a t i o n s of 3H-QNB, spaced b e t w e e n 0.25 and 2.0 nM. Data were a n a l y z e d a c c o r d i n g to the m e t h o d of S c a t c h a r d and are p r e s e n t e d as the means f SD of 3 separate experiments, each done in triplicate.
KD Control Gpp (NH) p DTNB G p p ( N H ) p + DTNB Urea Urea + G p p ( N H ) p Urea + DTNB
Bma x ( p m o l e s / m g
(nM)
0.56 0 59 0 56 0 56 0 56 0 59 0 57
0.29 ± 0.06 0 30 ± 0.04 0 29 ± 0.08 0 30 0 31
± 0.08 ± 0.06
0 30 f 0.07 0 31 i 0.06
Table
+_ f ± ± f f ±
0 0 0 0 0 0 0
protein) 15 17 15 16 16 19 16
2
Effects of DTNB, G p p ( N H ) p and u r e a - t r e a t m e n t on the b i n d i n g p a r a m e t e r s of a t r o p i n e and c a r b a c h o l in the heart m i c r o s o m e fraction. Atropine Hill coefficient
Carbachol Ki (nM)
Hill coefficient
Control
1.02±0.07
4.58±0.70
d0.51±0.06
Gpp(NHJp Urea Gpp(NH)p+Urea DTNB
1.00i0.02 1.02±0.04 N.D. 1.03f0.04
4.49±0.46 4.43±0.66 N.D. c5.62±1.20
a0.78±0.07 a0. a0.77±0.07 77±0.08 c0.67±0.09
DTNB+Gpp(NH)p
N.D.
N.D.
c0.68f0.ii
DTNB+Urea
N.D.
N.D.
b0.70±0.09
IC50co r
(uM)
e4.36±0.55 b18.4 i6.3 !~9.1 ±6.5 1.8 f9.7 ad0.55±0.25 ad0.55±0.35 0.49f0.18
a b c S i g n i f i c a n t l y d i f f e r e n t from control (a p<0.01 b p<0.05, c ~<0.l) d _ S i g n i f i c a n t l y d i f f e r e n t from u r e a - t r e a t m e n t d p<0.01, e p<0.05) ( The S t u d e n t s ' s t-test was used to test significance. Data are p r e s e n t e d as the means ± SD of 3 separate experiments, each done in triplicate. N.D. : not d e t e r m i n e d U r e a - t r e a t m e n t d e c r e a s e d the a p p a r e n t a f f i n i t y of agonist, but not antagonist, to m A C h R (Fig. I). After u r e a - t r e a t m e n t , the Hill c o e f f i c i e n t (34) for c a r b a c h o l and IC50ce r of c a r b a c h o l w e r e i n c r e a s e d (Table 2). U r e a - t r e a t m e n t reduced the effect of G p p ( N H ) p on the b i n d i n g a f f i n i t y of agonist to m A C h R (Fig. 1 and
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T a b l e 2). The e f f e c t s of G p p ( N H ) p and urea t r e a t m e n t s on a g o n i s Z b i n d i n g a f f i n i t y to m A C h R w e r e similar and were not a d d i t i v e under the c o n d i t i o n s that gave m a x i m a l e f f e c t s (Fig. i and Table 2). ~loreover, w h e n c o m b i n e d w i t h DTNB, the e f f e c t s of G P P ( N H ) p and urea t r e a t m e n t s w e r e both o v e r c o m e by that of DTNB (Fig. 1 and Table 2). These r e s u l t s i n d i c a t e that Gpp(NI{)p and urea t r e a t m e n t s have the same t a r g e t or that there is some o v e r l a p in their t a r g e t areas. Recently, E h l e r t et al (19) and B i r d s a l l et al (33) have s u g g e s t e d that d e c r e a s e in a g o n i s t b i n d i n g a f f i n i t y for m A C h R c a u s e d by g u a n i n e n u c l e o t i d e s is m e d i a t e d t h r o u g h the i n t e r a c t i o n of m A C h R w i t h G T P - b i n d i n g p r o t e i n (G-protein). More recently, our study on r a d i a t i o n i n a c t i v a t i o n s u g g e s t e d i) that G p p ( N H ) p b o u n d to an o t h e r subunit than the l i g a n d - b i n d i n g subunit, 2) that the b i n d i n g of G p p ( N H ) p to this s u b u n i t c a u s e d d i s s o c i a t i o n of this g u a n i n e n u c l e o t i d e - b i n d i n g s u b u n i t from the l i g a n d - b i n d i n g subunit and 3) that the d i s s o c i a t e d l i g a n d - b i n d i n g s u b u n i t showed low a f f i n i t y to the a g o n i s t (24). Our p r e v i o u s study also s u g g e s t e d that t r y p s i n - t r e a t m e n t d e s t r o y e d the G - p r o t e i n linked w i t h m A C h R or split the part of m A C h R that is r e s p o n s i b l e for a t t a c h m e n t of G - p r o t e i n , and c o n s e q u e n t l y c a u s e d the low a f f i n i t y state of m A C h R to a g o n i s t (25). The p r e s e n t r e s u l t s s u g g e s t that u r e a - t r e a t m e n t acts at some of a series of steps i n v o l v e d in the linkage b e t w e e n m A C h R and G - p r o t e i n . U r e a - t r e a t m e n t has the e f f e c t to s o l u b i l i z e or d e n a t u r e m e m b r a n e - b o u n d proteins. U r e a - t r e a t m e n t may have s o l u b i l i z e d the G - p r o t e i n l i n k e d w i t h mAChR, but our trials to s o l u b i l i z e the G - p r o t e i n by u r e a - t r e a t m e n t r e s u l t e d in failure. T h e r e f o r e , we suppose that u r e a - t r e a t m e n t p r o b a b l y d e n a t u r e d the G - p r o t e i n or the part of m A C h R linked w i t h G - p r o t e i n , and c o n s e q u e n t l y c a u s e d the low a f f i n i t y state of m A C h R to agonist. U r e a - t r e a t m e n t p r o b a b l y b r o u g h t the u n c o u p l i n g b e t w e e n m A C h R and G - p r o t e i n , and r e s u l t e d in the low a f f i n i t y state of m A C h R to agonist. The low a f f i n i t y state of r e c e p t o r to a g o n i s t c a u s e d by u n c o u p l i n g b e t w e e n r e c e p t o r and G - p r o t e i n is also r e p o r t e d in m u s c a r i n i c c h o l i n e r g i c (35), b e t a - a d r e n e r g i c (36) and d o p a m i n e r g i c (37) r e c e p t o r systems. S a l v a t e r a (38) r e p o r t e d that t r e a t m e n t of rat b r a i n w i t h 6 M urea d e c r e a s e d the n u m b e r of 3 H - Q N B b i n d i n g sites. We also found that t r e a t m e n t of g u i n e a - p i g h e a r t m i c r o s o m e f r a c t i o n w i t h 4 M urea d e c r e a s e d the n u m b e r of 3 H - Q N B b i n d i n g sites and m e m b r a n e b o u n d p r o t e i n s to the same degree, and c o n s e q u e n t l y did not a f f e c t the d e n s i t y of 3 H - Q N B b i n d i n g sites. The e f f e c t of DTNB t r e a t m e n t on a g o n i s t b i n d i n g a f f i n i t y to m A C h R p r e d o m i n a t e d over that of G p p ( N H ) p , u r e a - t r e a t m e n t (Fig. 1 and T a b l e 2) and t r y p s i n - t r e a t e n t (25). A r o n s t a m et al. p r o p o s e d that N - e t h l m a l e i m i d e (NEM} t r e a t m e n t i n c r e a s e d a g o n i s t b i n d i n g a f f i n i t y by c o n v e r t i n g m A C h R from a state of low a g o n i s t a f f i n i t y to a "stable" state of high a g o n i s t affinity, w h e r e a s both states had the same high a f f i n i t y for a n t a g o n i s t (39). We suppose that DTNB t r e a t m e n t e n h a n c e d a g o n i s t b i n d i n g a f f i n i t y for m A C h R in a s i m i l a r m a n n e r to N E M treatment. Our study on r a d i a t i o n i n a c t i v a t i o n i n d i c a t e d that the m o l e c u l a r size of the unit for the D T N B e f f e c t was a little larger than that of the r e c e p t o r s u b u n i t and that of the G p p ( N H ) p e f f e c t was m u c h larger than that of the r e c e p t o r s u b u n i t (24). P e r h a p s the a c t i o n of DTNB is m e d i a t e d t h r o u g h an i n t e r a c t i o n b e t w e e n m A C h R and a n o t h e r m e m b r a n e c o m p o n e n t d i s t i n c t from G - p r o t e i n .
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We u s e d p l u s / m i n u s QNB at the c o n c e n t r a t i o n of ].2 nM, w h i c h was several times h i g h e r than the Kd. Under these c o n d i t i o n s , b o t h p o s i t i v e and n e g a t i v e s t e r e o i s o m e r s will c o n t r i b u t e to c o m p e t i t i o n curves (40). Yhis limits the q u a n t i t a t i v e u t i l i t y of these very c o m p l e x e x p e r i m e n t s . This t e c h n i c a l l i m i t a t i o n w o u l d limit the q u a n t i t a t i v e i n t e r p r e t a t i o n of our results but p r o b a b l y have no b e a r i n g on the q u a l i t a t i v e analysis. A c k n o w l e d g e m e n t : We thank Mrs. M i e k o N a k a m u r a for a s s i s t a n c e p r e p a r a t i o n of this m a n u s c r i p t . This w o r k was s u p p o r t e d in part by a G r a n t - i n - A i d for S c i e n t i f i c R e s e a r c h from M i n i s t r y o~ E d u c a t i o n , S c i e n c e and C u l t u r e of Japan.
in
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