Endocrine Research Communications
ISSN: 0093-6391 (Print) (Online) Journal homepage: http://www.tandfonline.com/loi/ierc19
Interactions of Bovine Thyrotropin and Human Chorionic Gonadotropin with Adenylate Cyclase in Bovine Thyroid Membranes S. M. Amir, H. Uchimura & S. H. Ingbar To cite this article: S. M. Amir, H. Uchimura & S. H. Ingbar (1978) Interactions of Bovine Thyrotropin and Human Chorionic Gonadotropin with Adenylate Cyclase in Bovine Thyroid Membranes, Endocrine Research Communications, 5:3, 239-247, DOI: 10.1080/07435807809083757 To link to this article: http://dx.doi.org/10.1080/07435807809083757
Published online: 07 Aug 2009.
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Date: 12 May 2016, At: 09:02
ENDOCRINE RESEARCH COMMUNICATIONS, 5 ( 3 ) , 239-247 ( 1 9 7 8 )
INTERACTIONS OF BOVINE THYROTROPIN AND HUtvlAN CHORIONIC GONADOTROPIN WITH ADEMYLATE CYCLASE IN BOVINE THYROID MEMBRANES
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S.I.1. A m i r , H. Uchimura and S.H. I n g b a r Thorndike L a b o r a t o r y o f Harvard Medical School and t h e Department o f Medicine, Beth I s r a e l H o s p i t a l Boston, Massachusetts 02215
List of Abbreviations bTSH, bovine t h y r o t r o p i n ; hCG, human c h o r i o n i c gonadotropin; hCG-c, crude human c h o r i o n i c gonadotropin; hCG-p, p u r i f i e d human c h o r i o n i c gonadotropin; c y c l i c AMP, adenosine 3 ' ,5'-monophosphate; ITP, i n o s i n e 5 ' - t r i p h o s p h a t e ; BSA, b o v i n e serum a l b u min.
Abstract Previous s t u d i e s have shown t h a t crude p r e p a r a t i o n s o f human c h o r i o n i c gonadotropin b i n d t o b o v i n e t h y r o i d membranes, d i s p l a c e 1251 - l a b e l e d b o v i n e t h y r o t r o p i n therefrom, and a r e weak agonists t h e r e i n w i t h respect t o the a c t i v a t i o n o f adenylate cyclase. The p r e s e n t s t u d i e s r e v e a l t h a t c o n c e n t r a t i o n s o f c h o r i o n i c g o n a d o t r o p i n s u f f i c i e n t t o e l i c i t a maximal a g o n i s t i c r e sponse o f a d e n y l a t e c y c l a s e a r e s t r o n g l y a n t a g o n i s t i c t o t h e s t i m u l a t o r y a c t i o n o f b o v i n e t h y r o t r o p i n i n t h e t h y r o i d membrane system. T h i s e f f e c t i s r e m i n i s c e n t o f t h e i n h i b i t o r y e f f e c t s o f crude human c h o r i o n i c gonadotropin on o t h e r extragonadal t i s s u e s i n v i t r o , and, l i k e them, appears t o be mediated by some f a c t o r m t h e r t h a n human c h o r i o n i c gonadotropin i t s e l f , s i n c e h i g h l y p u r i f i e d human c h o r i o n i c gonadotropin was w i t h o u t e f f e c t . Recent s t u d i e s have r e v e a l e d t h a t crude p r e p a r a t i o n s o f human c h o r i o n i c gonadotropin e x e r t a v a r i e t y o f b i o l o g i c a l e f f e c t s 239 Copyright 0 1979 by Marcel Dekker, Inc. All Rights Reserved. Neither this work nor any part may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, or by any information storage and retrieval system, without permission in writing from the publisher.
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AMIR, UCHIMURA, AND INGBAR
-in v i t r o t h a t appear t o be unrelated t o t h e i r c l a s s i c a l gonadotropic actions.
In cultured lymphocytes, f o r example, crude hCG
has been found t o i n h i b i t both phytohemagglutinin-induced t r a n s formation and the mixed lymphocyte reaction (1-3).
Other s t u d i e s
have shown t h a t crude hCG i n h i b i t s t h e uptake and incorporation of amino acids by cultured f i b r o b l a s t s ( 4 ) .
In both types of
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systems, t h e i n h i b i t o r y a c t i v i t y of crude hCG preparations appeared t o r e s i d e i n some f a c t o r ( s ) o t h e r than hCG, s i n c e highl y p u r i f i e d hCG was without i n h i b i t o r y e f f e c t . We now report an additional in vitro i n h i b i t o r y e f f e c t of crude hCG on an extragonadal t i s s u e .
Crude hCG was found t o in-
hi b i t the stimulation of adenylate cyclase LATP pyrophosphatelyase (cyclizing),EC4.6.1a a c t i v i t y produced by bovine thyrotropin i n bovine thyroid membranes.
Like t h e i n h i b i t o r y e f f e c t s
mentioned above, t h e antagonist e f f e c t of crude hCG on t h e stimul a t o r y e f f e c t of bTSH could not be reproduced using a highly purif i e d hCG preparation. Materials and Methods Bovine TSH was p u r i f i e d from a preparation supplied by National I n s t i t u t e of A r t h r i t i s , Metabolism and Digestive Diseases (MIH-TSH-B8) (3.49 i . u . / m g ) ,
by methods described e a r l i e r ( 5 , 6 ) .
P a r t i a l l y purified bTSH (Thytropafi was a l s o obtained from
Armour Pharmaceutical Company. 13,450 i.u./mg;
Purified hCG (hCG-p) ( C R 121 ,
CR 119, 11,600 i.u./mg), hCG-cX (0.81 i.u./nlg),
and hCG-p (40.3 i.u./mg) were supplied by t h e Center f o r Popula-
24 1
EXTRAGONADAL ACTION O F hCG
t i o n Research, N a t i o n a l I n s t i t u t e o f C h i l d H e a l t h and Human Development o f t h e N a t i o n a l I n s t i t u t e s o f Health. ( a p p r o x i m a t e l y 2,500 i.u./mg)
Crude hCG (hCG-c)
was purchased from Cal biochem
L a b o r a t o r i e s , San Diego, C a l i f o r n i a . Bovine t h y r o i d plasma membranes were prepared as d e s c r i b e d p r e v i o u s l y and s t o r e d f r o z e n a t -60 C u n t i l used ( 6 ) .
Adenylate
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c y c l a s e a c t i v i t y i n b o v i n e t h y r o i d membranes was assessed by meas u r i n g t h e i n c r e a s e i n c y c l i c AMP c o n c e n t r a t i o n i n t h e i n c u b a t i o n medium t h a t o c c u r r e d d u r i n g 15 min p e r i o d s o f i n c u b a t i o n a t 37 C, u s i n g r a d i o i m u n o a s s a y f o r c y c l i c AMP measurement.
The assay mix-
t u r e c o n t a i n e d 50 ug o f membrane p r o t e i n i n 0.25 m l 25 HC1 b u f f e r , pH 7.6,
mM
Tris-
c o n t a i n i n g 0.1% BSA, 0.05 m14 ITP, 1.5 mM
Tris-ATP, 0.3 mg/ml c r e a t i n e phosphokinase, 10 mM phosphocreatine, 3
mM
rlgC12, 1 mM methyl i s o b u t y l x a n t h i n e (MIX) and v a r i o u s con-
c e n t r a t i o n s o f bTSH and/or hCG. P r o t e i n c o n c e n t r a t i o n i n t h e t h y r o i d membranes was d e t e r mined a c c o r d i n g t o t h e method o f Lowry e t a l . , f o l l o w i n g s o l u b i l i z a t i o n i n 1 N NaOH f o r 2-5 min a t 100 C ( 7 ) .
Bovine serum albunin
was employed as t h e standard.
Both bTSH and hCG-c s t i m u l a t e d a d e n y l a t e c y c l a s e i n b o v i n e t h y r o i d membranes i n a dose-dependent manner ( F i g . 1).
Maximal
c y c l i c AMP a c c u m u l a t i o n induced by bTSH was noted a t a concentra-
AMIR, UCHIMURA, AND INGBAR
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242
FIGURE 1 E f f e c t o f crude hCG (hCG-c) on a d e n y l a t e c y c l a s e s t i m u l a t i o n by bTSH i n bovine t h y r o i d membranes. L e f t ; bovine t h y r o i d membranes were i n c u b a t e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f bTSH a l o n e (-), o r w i t h bTSH i n combination w i t h a f i x e d conc e n t r a t i o n o f hCG-c (3,200 i.u/ml) (b--d).Right; b o v i n e t h y r o i d membranes were i n c u b a t e d w i t h i n c r e a s i n g c o n c e n t r a t i o n s of R e s u l t s a r e expressed as t h e mean of hCG-c a l o n e (M). d u p l i c a t e d e t e r m i n a t i o n s . For d e t a i l s , see under M a t e r i a l s and Methods.
t i o n o f approximately 300 m.i.u./ml
(2.7 X 10-'M).'
c e n t r a t i o n s had an i n h i b i t o r y e f f e c t .
H i g h e r con-
The e x t e n t o f maximum
s t i m u l a t i o n o f a d e n y l a t e c y c l a s e by hCG-c was much lower, and r e 'Molar c o n c e n t r a t i o n s o f bTSH, hCG and hCG s u b u n i t s have been c a l c u l a t e d based on t h e f o l l o w i n g assumptions: p u r i f i e d bTSH has a b i o a c t i v i t y o f 40 i.u/mg and a m o l e c u l a r w e i g h t of 28,000; p u r i f i e d hCG i s assumed t o have a b i o a c t i v i t y o f 13,450 iu./mg and a m o l e c u l a r w e i g h t o f 37,000; m o l e c u l a r w e i g h t v a l u e s of 15,000 and 22,000 have been used f o r hCG-o( and hCG-p, respectively.
243
EXTRAGONADAL ACTION OF hCG
l a t i v e l y l a r g e amounts of the hormone were required t o e l i c i t Maximal response t o hCG-c was demonstrated a t a concentration of 3,200 i.u./ml (6.4 X 10- 6M) (Fig. 1 ) . No f u r -
this effect.
t h e r increase i n c y c l i c AMP concentration was measured when a higher concentration (4,000 i.u./ml; 8.0 X 10-6M) was added t o t h e medium.
Preparations of hCG-c obtained from several sources
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showed considerable variation i n t h e i r a b i l i t y t o stimu a t e adenylate cyclase, a n d t h e r e f o r e a preparation from a s ngle source was employed i n a l l experiments reported here. When t h e maximally e f f e c t i v e dose of hCG-c (3,200 i.u./ml; 6.4 X 10-6b1) was combined with increasing concentrations of bTSH, values f o r c y c l i c AMP concentration remained nearly t h e same as those found in the absence of bTSH.
The i n h i b i t o r y e f f e c t of
hCG-c on TSH-dependent a c t i v a t i o n of adenylate cyclase was evi-
d e n t a t t h e e n t i r e range of bTSH concentrations t e s t e d (Fig. l ) . Exhaustive d i a l y s i s of hCG-c f a i l e d t o diminish i t s i n h i b i t o r y effect. In separate experiments, t h e e f f e c t s of hCG-p
e i t h e r alone
o r i n combination with bTSH, were a l s o assessed ( F g. 2 ) .
A clear
stimulation o f adenylate cyclase by bTSH was again demonstrated. However, i n c o n t r a s t t o the findings with hCG-c, hCG-p in conc e n t r a t i o n s as high as 4,000 i.u./ml
(8.0 X lO-%) f a i l e d both t o
stimulate adenylate cyclase and t o i n h i b i t the stimulatory e f f e c t of bTSH.
The e f f e c t s of hCG-o( and hCG-p were a l s o t e s t e d .
When
added a t a fixed concentration of 280 u g h 1 (hCG-M, 1.9 X 10% 5 1 hCG-p, 1.3 X 10- M) to t h e two highest bTSH concentrations used (300 m.i.u. and 400 m.i.u./ml;
2.7 X 10-7M and 3.6 X 10-7M),
AMIR, UCHIMURA, AND INGBAR
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244
I
a
r P0 u)
0.8-
1
hCG-p
-
bTSH
W
E
0'41 0
,
0
>
80
,
I
,
A
,
160 240 320 400
bTSH (mlU/ml)
FIGURE 2 E f f e c t o f p u r i f i e d hCG (hCG-p) (CR 121, 13,450 i.u./mg) on adenyl a t e c y c l a s e s t i m u l a t i o n by bTSH i n b o v i n e t h y r o i d membranes. L e f t ; b o v i n e t h y r o i d membranes were i n c u b a t e d e i t h e r w i t h i n c r e a s i n g c o n c e n t r a t i o n s o f bTSH a l o n e (-), o r w i t h bTSH t o g e t h e r w i t h a f i x e d c o n c e n t r a t i o n o f hCG-p (3,200 i.uJm1) Right; b o v i n e t h y r o i d membranes were i n c u b a t e d w i t h (m). hCG-p a l o n e (&--A). Results a r e expressed as t h e mean o f du p l i c a t e d e t e r m i n a t i o n s . For d e t a i l s , see under M a t e r i a l s and Methods.
n e i t h e r s u b u n i t e x h i b i t e d any i n h i b i t o r y e f f e c t on t h e s t i m u l a t i o n o f a d e n y l a t e c y c l a s e o b t a i n e d w i t h bTSH a l o n e ( d a t a n o t shown).
245
EXTRAGONADAL A C T I O N OF h C G
Discussion We have p r e v i o u s l y demonstrated t h e presence o f a t h y r o i d s t i m u l a t i n g f a c t o r i n p r e p a r a t i o n s o f crude hCG, as judged by t h e i r a b i l i t y t o i n h i b i t the binding o f
lZ5I -bTSH t o b o v i n e
t h y r o i d membranes and t o a c t i v a t e a d e n y l a t e c y c l a s e t h e r e i n
(8, 9 ) .
We were, however, unable t o show a s i m i l a r e f f e c t when
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h i g h l y p u r i f i e d p r e p a r a t i o n s o f hCG were employed, s u g g e s t i n g t h a t t h e t h y r o i d - s t i m u l a t i n g a c t i v i t y i n crude hCG i s n o t hCG itself.
We have now demonstrated t h a t crude hCG, i n a d d i t i o n t o
b e i n g a weak a g o n i s t w i t h r e s p e c t t o t h y r o i d s t i m u l a t i o n , i s a p o t e n t a n t a g o n i s t t o t h e s t i m u l a t o r y a c t i o n o f bTSH.
Purified
hCG, i n c o n t r a s t , had n e i t h e r t h e a g o n i s t n o r t h e a n t a g o n i s t e f f e c t , and s u b u n i t s o f hCG a l s o had no a n t a g o n i s t a c t i o n .
I t i s n o t p o s s i b l e a t p r e s e n t t o conclude whether a s i n g l e f a c t o r p r e s e n t i n crude hCG has b o t h a g o n i s t and a n t a g o n i s t prope r t i e s o r whether t h e two p r o p e r t i e s r e s i d e i n separate e n t i t i e s . I t seems c l e a r , however, t h a t n e i t h e r p u r i f i e d hCG n o r e i t h e r o f
i t s subunits i s responsible f o r t h i s action. The a n t a g o n i s t a c t i v i t y o f crude hCG w i t h r e s p e c t t o t h y r o i d s t i m u l a t i o n i s r e m i n i s c e n t o f i n h i b i t o r y p r o p e r t i e s d i s p l a y e d by c r u d e hCG i n o t h e r systems.
F o r example, crude hCG i s an
i n h i b i t o r of phytohemagglutinin-induced t r a n s f o r m a t i o n o f lymphoc y t e s and o f t h e mixed lymphocytes r e a c t i o n , w h i l e pure hCG i s n o t (1-3).
S i m i l a r l y , Golbus and S i i t e r i have shown t h a t crude,
246
AMIR, UCHIMURA, AND INGBAR
b u t n o t p u r i f i e d , hCG i s i n h i b i t o r y t o t h e uptake and i n c o r p o r a t i o n o f amino a c i d s by c u l t u r e d f i b r o b l a s t s ( 4 ) . I t i s p r e s e n t l y u n c e r t a i n whether t h e t h y r o t r o p i n antago-
n i s t p r e s e n t i n crude hCG i s i d e n t i c a l t o one o r b o t h o f t h e i n h i b i t o r y f a c t o r s a c t i v e i n these o t h e r systems o r whether t h e l a t t e r two f a c t o r s a r e i d e n t i c a l t o one a n o t h e r .
Resolution o f
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t h i s q u e s t i o n w i l l r e q u i r e p u r i f i c a t i o n o f a t l e a s t one i n h i b i t o r y f a c t o r and t e s t i n g o f i t i n t h e o t h e r systems. Acknowledgements T h i s work was supported by g r a n t No. AM-18416 f r o m t h e N a t i o n a l I n s t i t u t e o f A r t h r i t i s , Metabolism and D i g e s t i v e D i s eases, N a t i o n a l I n s t i t u t e s o f Health.
The a u t h o r s a r e i n d e b t e d
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