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Alpha-2 adrenoreceptors in brown adipose tissue of infant rats Shaikh, Muhammad Iqbal 1986

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ALPHA-2 ADRENOCEPTORS IN BROWN ADIPOSE TISSUE OF INFANT RATS BY MUHAMMAD IQBAL SHAIKH B.Sc, (Honors) The University of Karachi, 1967. M.Sc, The University of Karachi, 1968. M.S., The University of California, Los-Angeles, 1975, A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS OF THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Interdisciplinary Studie.s) [Pathology, Pharmacology Physiology,Biochemistry] We accept this thesis as conforming to the required standard. TEE UNIVERSITY OF BRITISH COLUMBIA* May 1986 © Muhammad Iqbal Shaikh, 198 6 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by h i s or her representatives. It i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of I n t e r d i s c i p l i n a r y Studies (Pathology, Pharmacology Frvysio logy and Biochemistry) The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date May 29, 1986 c c / ~> /na\ ABSTRACT OF THESIS Alpha-2 adrenoceptors in Brown Adipose Tissue of Infant Rats. by M. IQBAL SHAIKH This thesis consists of five chapters. The f irst chapter deals with general background and introduction. Each of the subsequent chapters are divided into sections. The f irst section deals with pharmacological characterization of <*2~adren_ oceptors using various ligands. The second section pertains to the study of binding characteristics of *<2~ a c^ r e n o c eP t o r s follow-ing chemical sympathectomy by 6-hydroxydopamine and chronic blockade of °<2 - a c^ r e r i o c eP t o r s by yohimbine injections. The third section deals with the study of guanylate cyclase system in re-lation to ©<2-adrenoceptors stimulation in brown fat fragments of 7-day-old rats. The fourth section is devoted to the study of the physiological response associated with the stimulation of ^-adrenoceptors in isolated adipocytes from brown fat of 7-day-old rats. Finally cyclic GMP production in obese and lean mice in relation to ©^ -adrenoceptors stimulation was discussed in the fifth section. 3 Binding characteristics of ©^ -antagonists ([ H]-RX-78-3 3 3 1094, [ H]-yohimbine, [ H]-rauwolscine) and agonists ([ H]-clon-3 idine, [ H]-norepinephrine) to o^-adrenoceptors on isolated plas-ma membrane fragments from brown adipose tissue were studied. The binding of [^ K]-yohimbine was rapid,saturable and reversible. Yohimbine, (-)-epinephrine, - i i -a n d c l o n i d i n e d i s p l a c e d [ 3 H ] - y o h i m b i n e f r o m i t s b i n d i n g s i t e s i n t h a t o r d e r o f p o t e n c y a s w o u l d b e e x p e c t e d o f b i n d i n g t o « < 2 _ a < ^ r e n o c e P t o r s . A S c a t c h a r d p l o t o f y o h i m b i n e b i n d i n g s h o w e d a n e q u i l i b r i u m c o n s t a n t ( ^ d ) o f 18 nM a n d t o t a l b i n d -i n g c a p a c i t y ( B ) o f 0 . 1 5 p m o l / m g p r o t e i n . B i n d i n g o f max 3 3 [ H J - R X 7 8 1 0 9 4 a n d [ H ] - c l o n i d i n e s h o w e d a s i m i l a r p a t t e r n o f r a p i d , s t a b l e , s a t u r a b l e a n d r e v e r s i b l e b i n d i n g . S t u d i e s o n t h e b i n d i n g o f ( - ) [ H ] - n o r e p i n e p h r i n e i n d i c a t e d t h e p r e s e n c e o f m o r e t h a n o n e b i n d i n g s i t e . S c a t c h a r d a n a l y s i s o f t h e ( - ) [ H ] - n o r e p i n e p h r i n e b i n d i n g u s i n g ( - ) - e p i n e p h r i n e o r y o h i m b i n e a s t h e d i s p l a c i n g a g e n t , r e v e a l e d a K d o f . 6 0 . 4 nM a n d 6 5 . 8 nM r e s p e c t i v e l y , a n d B m a x v a l u e s o f 0 . 2 2 a n d 0 . 2 4 p m o l / m g p r o t e i n . N o r e p i n e p h r i n e , y o h i m b i n e a n d ( ^ - e p i n -e p h r i n e p r o b a b l y s h a r e d o n e common b i n d i n g s i t e ; t h e o t h e r s i t e w i t h o f 6 4 . 5 nM w a s p r e s e n t i n m u c h l o w e r n u m b e r ( 7 1 . 3 f m o l / m g p r o t e i n ) a n d w a s s p e c i f i c f o r ( - ) - n o r e p i n -e p h r i n e a n d y o h i m b i n e o n l y . I n a d d i t i o n , a H i l l c o e f f i c i e n t o f 1 . 4 f u r t h e r s u p p o r t e d t h e p r e s e n c e o f t w o p o s i t i v e l y c o o p e r a t i v e b i n d i n g s i t e s . B i n d i n g o f ( - ) [ H ] - d i h y d r o a l p r e -n o l o l w a s d i s p l a c e a b l e b y p r a c t o l o l a n d n o r e p i n e p h r i n e w i t h m a x a o f 5 0 nM a n d 10 nM r e s p e c t i v e l y ( p ^ - s i t e ) a n d B 3 o f 0 . 1 9 a n d 0 . 5 p m o l / m g p r o t e i n . H o w e v e r , ( - ) [ H ] - d i h y d r o a l -p r e n o l o l b i n d i n g c o u l d a l s o b e d i s p l a c e d b y y o h i m b i n e s u g g e s t -i n g e i t h e r a r e l a t i v e n o n - s p e c i f i c i t y o f t h e l i g a n d o r a n a t y p i c a l n a t u r e o f t h e / 3 ^ - a d r e n o c e p t o r s i n b r o w n f a t o f i n f a n t r a t s . I t i s s u g g e s t e d t h a t t h e p l a s m a m e m b r a n e s f r o m a c t i v e l y - i i i -proliferating brown fat of infant rats possess both B -^ and ^"Adrenoceptors. The physiological in vivo agonist (-)-nor-epinephrine may exert its effects via both or either adreno-ceptor sub-type. 3 Binding studies carried out with [ H]-yohimbine on membranes isolated from brown fat of chemically sympath-ectomized infant rats showed smaller number of high affinity yohimbine binding sites when compared to those isolated from control (saline-injected) rats of the same age. The 3 (-)[ H]-norepinephrine binding to identical membrane preparations revealed the presence of both high (K^  = 36 nM) and low (K^  = 200 nM) affinity binding sites; with a Hi l l coefficient of 1.5. The total number of norepinephrine binding sites more than doubled after sympathectomy; this increase was caused by emergence of low affinity sites. Chronic yohimbine pre-treatment resulted in more than two-fold increase in the 3 3 number of binding sites for both [ H]-yohimbine and (-)[ H]-norepinephrine. The affinity of o^-adrenoceptors for yohimbine binding sites decreased whereas that for norepinephrine remained unchanged. These results not only confirm the presence of ^-adrenoceptors in brown fat of developing rats but also indicate that the binding characteristics of these receptors can be altered by chemical sympathectomy and by chronic exposure of infant rats to an <* -receptor - i v -b l o c k e r . I n c u b a t i o n o f b r o w n f a t t i s s u e p i e c e s w i t h c l o n i d i n e ( 0 . 2 - 2 0 > M ) s h o w e d a d o s e - a n d ; t i m e - ' d e p e n d e n t e l e v a t i o n o f t i s s u e c y c l i c GMP c o n t e n t . T h e p e a k r e s p o n s e o c c u r r e d a t t h e c o n c e n t r a t i o n o f 20 f o r o n e - m o n t h - o l d r a t s . F o r b r o w n f a t f r o m o n e - w e e k - o l d r a t s , t h e p e a k r e s p o n s e o c c u r r e d a t 0.5 - I^ MM o f c l o n i d i n e a n d 3 -5 m i n u t e s o f i n c u b a t i o n . T h e r e s p o n s e c o u l d b e b l o c k e d b y p r i o r i n c u b a t i o n w i t h y o h i m b i n e . When t i s s u e c y c l i c GMP c o n c e n t r a t i o n , e l e v a t e d i n r e s p o n s e t o c l o n i d i n e i n c u b a t i o n , w a s s e p a r a t e d i n t o r e l e a s e a b l e a n d r e c e p t o r - p r o t e i n b o u n d f r a c t i o n , a s i m i l a r t r e n d w a s s e e n . T h e d a t a s u p p o r t e d t h e h y p o t h e s i s t h a t o ^ - r e -c e p t o r s t i m u l a t i o n o f b r o w n f a t i s l i n k e d ( d i r e c t l y o r i n d i r e c t -2 + l y p e r h a p s v i a C a ) t o g u a n y l a t e c y c l a s e a c t i v a t i o n . E a r l i e r i n v i v o e x p e r i m e n t s h a d s h o w n a d e f e c t i v e r e s p o n s e o f b r o w n f a t c y c l i c GMP p r o d u c t i o n i n o b e s e m i c e u p o n a c u t e c o l d e x p o s u r e a n d c a t e c h o l a m i n e i n j e c t i o n s a s c o m p a r e d t o c o n t r o l l i t t e r m a t e s w h i c h s h o w e d a d o s e - a n d t i m e - d e p e n d e n t i n c r e a s e . P r e l i m i n a r y i n v i t r o e x p e r i m e n t s w h e r e w h e r e f r a g m e n t s f r o m o b e s e a n d l e a n m i c e w e r e s t i m u l a t e d w i t h c l o n i d i n e , s h o w e d t w o - f o l d i n c r e a s e i n t h e c y c l i c GMP c o n c e n t r a t i o n c o m p a r e d t o n o n - s t i m u l a t e d c o n t r o l s . T h i s s u g g e s t e d t h a t t i s s u e c a p a b i l i t y t o r e s p o n d b y a n i n c r e a s e i n c y c l i c GMP p r o d u c t i o n i n o b e s e m i c e i s t h e same a s t h a t • i n t h e l e a n m i c e . - v -F o r s k o l i n and Isobutylmethylxanthine sti m u l a t e d g l y c e r o l r e l e a s e i n i s o l a t e d a d i p o c y t e s from brown f a t of one-week-old r a t s . C l o n i d i n e , p r o s t a g l a n d i n E 2 and n i c o t i n i c a c i d showed i n h i b i t o r y e f f e c t s on g l y c e r o l r e l e a s e . I n h i b i t i o n of g l y c e r o l r e l e a s e by c l o n i d i n e was concentration-dependent and was antagonized by yohimbine. I n a c t i v a t i o n of i n h i b i t o r y r e g u l a t o r y p r o t e i n (Ni) by p e r t u s s i s t o x i n a b o l i s h e d the i n h i b i t o r y e f f e c t of c l o n i d i n e . T h i s i n d i c a t e d that the i n h i b -i t o r y e f f e c t of c l o n i d i n e on g l y c e r o l r e l e a s e i s mediated v i a i n h i b i t o r y p r o t e i n ( N i ) . I t was suggested t h a t , perhaps, the a n t i - l i p o l y t i c e f f e c t of ©^-adrenoceptors may have a r o l e i n c o n t r o l l i n g the s t a t e of a c t i v i t y of f a t c e l l s . - v i -ACKNOWLEDGEMENTS I w o u l d l i k e t o e x p r e s s my a p p r e c i a t i o n a n d t h a n k s t o D r . J . P . S k a l a f o r p r o v i d i n g me w i t h t h e s u i t a b l e e n v i r o n m e n t a n d h e l p f u l a d v i c e t o c a r r y o u t t h i s w o r k . I am p e r s o n a l l y i n d e b t e d t o D r . W . L . D u n n , C h a i r m a n o f my D o c t o r a l C o m m i t t e e , ^ i n t a k i n g p e r s o n a l i n t e r e s t i n my g r a d u a t e e d u c a t i o n . My t h a n k s a r e d u e t o D r . J . D i a m o n d ( P h a r m a c o l o g y ) , D r . P e d e r s o n ( P h y s i o l o g y ) , D r . P h i l l i p s ( Z o o l o g y ) , a n d D r . B u r t o n ( B i o c h e m i s t r y ) f o r r e a d i n g my t h e s i s a n d m a k i n g n e c e s s a r y s u g g e s t i o n s a n d c o m m e n t s . I am a l s o t h a n k f u l t o M r s . W. R o d r i g u e z f o r g i v i n g me t e c h n i c a l a s s i s t a n c e d u r i n g t h e c o u r s e o f t h i s w o r k . S p e c i a l t h a n k s a r e d u e t o my f r i e n d P e t e r L . E a s t h o p e , . a b o u t whom a l l I c a n s a y , "A f r i e n d i n n e e d i s a f r i e n d i n d e e d " . I w i s h t h e r e w e r e a f e w m o r e f r i e n d s a n d p e r s o n s l i k e h i m . I am f o r e v e r g r a t e f u l t o my p a r e n t s , e s p e c i a l l y my m o t h e r , f o r t h e i r l o v e a n d e n c o u r a g e m e n t f o r t h e p u r s u i t o f h i g h e r g o a l s a n d a t t a i n m e n t o f t h e h i g h e s t l e v e l o f e d u c a t i o n . W i t h o u t t h e i r h e l p a n d s u p p o r t , I w o u l d h a v e n e v e r r e a c h e d t h i s f a r . I d e d i c a t e t h i s t h e s i s t o my y o u n g e r b r o t h e r , M . Z a f a r S h a i k h , f o r h i s s i n c e r i t y a n d c o m m i t m e n t t o h i s f a m i l y . - v i i -T A B L E OF CONTENTS P a g e A B S T R A C T i i ACKNOWLEDGEMENTS v i i L I S T OF F I G U R E S x i L I S T OF T A B L E S x i v C H A P T E R S 1 . GENERAL BACKGROUND AND I N T R O D U C T I O N 1 . 1 B r o w n A d i p o s e T i s s u e 1 1 . 2 A d r e n e r g i c R e c e p t o r s 4 1 . 3 R e c e p t o r L i g a n d B i n d i n g 5 1 . 3 . 1 S a t u r a t i o n E x p e r i m e n t s 7 1 . 3 . 2 C o m p e t i t i o n o r L a b e l l e d L i g a n d D i s p l a c e m e n t E x p e r i m e n t s 13 1 . 4 T h e B e t a - a d r e n e r g i c P a t h w a y 21 1 . 5 T h e A l p h a - a d r e n e r g i c P a t h w a y 2 3 1 . 6 T h e s i s M a i n O b j e c t i v e 3 3 2 . M A T E R I A L S AND METHODS 34 2 . 1 M a t e r i a l s 34 2 . 2 A n i m a l s 3 5 2 . 3 M e t h o d s 2 . 3 . 1 6 - h y d r o x y d o p a m i n e p r e - t r e a t m e n t o f t h e r a t 36 2 . 3 . 2 . P l a s m a M e m b r a n e P r e p a r a t i o n s . . . 36 2 . 3 . 3 L i g a n d B i n d i n g A s s a y s 38 2 . 3 . 4 E f f e c t o f o c ^ - a g o n i s t s a n d a n t a g -o n i s t s i n v i t r o o n t o t a l a n d r e -c e p t o r - b o u n d c y c l i c GMP c o n t e n t s o f b r o w n f a t f r a g m e n t s f r o m o n e -w e e k - o l d r a t s 39 2 . 3 . 5 E f f e c t o f a g o n i s t s i n v i t r o o n c y c l i c GMP c o n t e n t s i n b r o w n f a t f r a g m e n t s : f r o m o b e s e m i c e a n d t h e i r l e a n l i t t e r m a t e s 44 - v i i i -P a g e 2 . 3 . 6 I n h i b i t i o n o f f o r s k o l i n - s t i m u -l a t e d l i p o l y s i s i n a d i p o c y t e s f r o m b r o w n f a t o f 1 - w e e k - o l d r a t s b y c l o n i d i n e 4 5 2 . 3 . 7 E f f e c t o f c l o n i d i n e o n f o r s k o -l i n - s t i m u l a t e d l i p o l y s i s i n a d -i p o c y t e s f r o m b r o w n f a t o f a d -u l t h a m s t e r s 4 6 2 . 3 . 8 D a t a A n a l y s i s 4 6 3 . R E S U L T S _ 3 . 1 I d e n t i f i c a t i o n a n d c h a r a c t e r i z a t i o n o f ^ - - a d r e n o c e p t o r s b i n d i n g s i t e s i n i s o -l a t e d p l a s m a m e m b r a n e f r a g m e n t s f r o m b r -o w n a d i p o s e t i s s u e o f i n f a n t r a t s 48 3 . 2 E f f e c t o f 6 - h y d r o x y d o p a m i n e a n d c h r o n i c y o h i m b i n e p r e - t r e a t m e n t o n t h e b i n d i n g c h a r a c t e r i s t i c s o f ^ - a d r e n o c e p t o r s i n i s o l a t e d p l a s m a m e m b r a n e f r a g m e n t s f r o m b r o w n f a t o f i n f a n t r a t s 58 3 . 3 E f f e c t o f a d r e n e r g i c a g o n i s t s a n d a n t a -g o n i s t s i n v i t r o o n c y c l i c n u c l e o t i d e c o n t e n t s o f r a t b r o w n a d i p o s e t i s s u e . . . 74 3 . 4 I n h i b i t i o n o f f o r s k o l i n - s t i m u l a t e d l i p -o l y s i s i n a d i p o c y t e s f r o m b r o w n f a t o f l ^ w e e k - o l d r a t s b y c l o n i d i n e 8 5 3 . 5 E f f e c t o f o ^ - a g o n i s t a n d ^ . - a g o n i s t i n v i t r o o n c y c l i c GMP c o n t e n t s i n b r o w n f a t f r a g m e n t s f r o m o b e s e m i c e a n d t h e i r u n a f f e c t e d l i t t e r m a t e s 97 4 . D I S C U S S I O N 1 0 2 4 . 1 I d e n t i f i c a t i o n a n d c h a r a c t e r i s t i c s o f b i n d i n g s i t e s o f ©< - a d r e n o c e p t o r s i n p l a s m a m e m b r a n e f r a g m e n t s o f b r o w n f a t f r o m 7 - d a y - o l d r a t s 1 0 6 4 . 2 E f f e c t o f c h e m i c a l s y m p a t h e c t o m y a n d c h -r o n i c y o h i m b i n e p r e - t r e a t m e n t o n b i n d i n g c h a r a c t e r i s t i c s o f ^ - a d r e n o c e p t o r s i n b r o w n f a t o f 7 - d a y - o l d r a t s 114 4 . 3 E f f e c t o f a d r e n e r g i c a g o n i s t s a n d a n t a -g o n i s t s i n v i t r o o n c y c l i c n u c l e o t i d e c o n t e n t s i n b r o w n a d i p o s e t i s s u e o f 7 -d a y - o l d r a t s 1 2 0 - i x -P a g e 4 . 4 E f f e c t o f o < _ - a g o n i s t o n f o r s k o l i n - s t i m u -l a t e d l i p o l y s i s i n b r o w n f a t a d i p o c y t e s f r o m 7 - d a y - o l d r a t s 1 2 8 4 . 5 E f f e c t o f a d r e n e r g i c a g o n i s t a n d a n t a g -o n i s t i n v i t r o o n c y c l i c GMP c o n t e n t s i n b r o w n f a t f r a g m e n t s f r o m o b e s e m i c e a n d u n a f f e c t e d l i t t e r - m a t e s 1 3 1 5 . C O N C L U S I O N 1 3 4 B I B L I O G R A P H Y 1 3 7 A P P E N D I X 1 5 8 VITAE .16J-. -x-L I S T OF F I G U R E S F i g u r e P a g e 3 1A S p e c i f i c b i n d i n g o f [ H ] - y o h i m b i n e t o p l a s m a m e m b r a n e f r a g m e n t s i s o l a t e d f r o m b r o w n f a t o f o n e - w e e k - o l d r a t s 4 9 3 IB I n h i b i t i o n o f [ H ] - y o h i m b i n e b i n d i n g b y 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 y o h i m b i n e a n d ( - ) -e p i n e p h r i n e 4 9 2A S p e c i f i c b i n d i n g o f [ 3 H j - R X - 7 8 1 0 9 4 t o p l a s m a m e m b r a n e f r a g m e n t s i s o l a t e d f r o m b r o w n f a t o f o n e - w e e k - o l d r a t s 51 3 2B S p e c i f i c b i n d i n g o f [ H ] - c l o n i d i n e t o p l a s m a m e m b r a n e f r a g m e n t s i s o l a t e d f r o m b r o w n f a t o f o n e - w e e k - o l d r a t s 51 3 2C S p e c i f i c b i n d i n g o f [ H ] - n o r e p i n e p h r i n e t o p l a s m a m e m b r a n e f r a g m e n t s i s o l a t e d f r o m b r o w n f a t o f o n e - w e e k - o l d r a t s i n t h e p r e s e n c e o f 10 /*M n o r e p i n e p h r i n e o r 10 /LM c l o n i d i n e 52 3 2D S p e c i f i c b i n d i n g o f [ H ] - n o r e p i n e p h r i n e t o . 1 p l a s m a m e m b r a n e f r a g m e n t s i s o l a t e d f r o m b r o w n f a t o f o n e - w e e k - o l d r a t s i n t h e p r e s e n c e o f 10 e p i n e p h r i n e o r 10 *tM y o h i m b i n e 5 2 3 3A S c a t c h a r d p l o t o f [ H ] - n o r e p i n e p h r i n e b i n d i n g t o b r o w n f a t p l a s m a m e m b r a n e f r a g m e n t s f r o m o n e - w e e k - o l d r a t s i n t h e p r e s e n c e o f 10 y o h i m b i n e o r 10 piii e p i n e p r h i n e 5 4 3B H i l l p l o t 5 4 3 4A D i s p l a c e m e n t o f s p e c i f i c b i n d i n g o f [ ^ - n o r -e p i n e p h r i n e t o b r o w n f a t p l a s m a m e m b r a n e f r a g -m e n t s f r o m o n e - w e e k - o l d r a t s b y 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 e p i n e p h r i n e o r p r a c t o l o l . . . 57 3 4B D i s p l a c e m e n t o f [ H ] - n o r e p i n e p h r i n e b i n d i n g b y 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 e p i n e p h r i n e i n t h e p r e s e n c e o f 1 0 ~ 3 M p r a c t o l o l a n d b y 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 p r a c t o l o l i n t h e p r e s e n c e o f 1 0 _ 5 M e p i n e p h r i n e 5 9 - x i -F i g u r e P a g e 5A ( - ) [ H ] - d i h y d r o a l p r e n o l o l b i n d i n g t o b r o w n f a t p l a s m a m e m b r a n e f r a g m e n t s f r o m o n e - w e e k - o l d r a t s i n t h e p r e s e n c e o f 10 ^ M n o r e p i n e p h r i n e . 5 9 3 5B S p e c i f i c b i n d i n g o f [ H ] - d i h y d r o a l p r e n o l o l t o b r o w n f a t p l a s m a m e m b r a n e f r a g m e n t s i n t h e p r e s e n c e o f 10 /M. p r a c t o l o l o r 10 « M y o h i m b i n e 5 9 6 E f f e c t o f c h e m i c a l s y m p a t h e c t o m y o f 5 - d a y - o l d r a t s b y a s i n g l e i n t r a p e r i t o n e a l d o s e o f 6 -h y d r o x y d o p a m i n e o n t h e s p e c i f i c b i n d i n g o f [ ^ H ] - y o h i m b i n e t o b r o w n f a t p l a s m a m e m b r a n e f r a g m e n t s i s o l a t e d 3 d a y s l a t e r 6 3 7 A , B E f f e c t o f c h e m i c a l s y m p a t h e c t o m y o f 5 - d a y - o l d r a t s o n t h e s p e c i f i c b i n d i n g o f [ 3 H ] - n o r e p i n -e p h r i n e t o b r o w n f a t p l a s m a m e m b r a n e i s o l a t e d 3 d a y s l a t e r 6 6 3 7C [ H ] - n o r e p i n e p h r i n e b i n d i n g t o p l a s m a m e m b r a n e f r a g m e n t s f r o m b r o w n f a t o f s y m p a t h e c t o m i z e d r a t s i n t h e p r e s e n c e o f 10 ^ M e p i n e p h r i n e a n d 10 A M p r a c t o l o l 6 9 3 8 D i s p l a c e m e n t o f [ H ] - n o r e p i n e p h r i n e b i n d i n g t o p l a s m a m e m b r a n e f r a g m e n t s i s o l a t e d f r o m b r o w n f a t o f 6 - h y d r o x y d o p a m i n e p r e - t r e a t e d 8 -d a y - o l d r a t s b y i n c r e a s i n g c o n c e n t r a t i o n o f e p i n e p h r i n e o r y o h i m b i n e 71 9 A , B E f f e c t o f c h r o n i c y o h i m b i n e p r e - t r e a t m e n t o n t h e b i n d i n g o f [ 3 H ] - y o h i m b i n e a n d [ 3 H ] - R X -7 8 1 0 9 4 t o b r o w n f a t p l a s m a m e m b r a n e f r a g m e n t s i s o l a t e d 7 d a y s l a t e r 72 3 9C S c a t c h a r d a n a l y s i s o f [ H ] - y o h i m b i n e s p e c i f i c b i n d i n g t o p l a s m a m e m b r a n e f r a g m e n t s i s o l a t e d f r o m c h r o n i c a l l y y o h i m b i n e p r e - t r e a t e d r a t s . . 7 5 3 9D S c a t c h a r d a n a l y s i s o f [ H ] - n o r e p i n e p h r i n e b i n d i n g t o p l a s m a m e m b r a n e f r a g m e n t s f r o m c h r -o n i c a l l y y o h i m b i n e p r e - t r e a t e d r a t s 7 5 10 E f f e c t o f a d r e n e r g i c a g o n i s t s a n d a n t a g o n i s t s i n v i t r o o n c y c l i c n u c l e o t i d e c o n t e n t s i n b r o w n a d i p o s e t i s s u e o f o n e - m o n t h - o l d r a t s 78 11 E f f e c t o f c l o n i d i n e a n d y o h i m b i n e i n v i t r o o n c y c l i c GMP c o n t e n t s i n b r o w n f a t f r a g m e n t s f r o m 7 - d a y - o l d r a t s 8 2 - x i i -F i g u r e P a g e 12 E f f e c t o f c l o n i d i n e i n v i t r o o n t o t a l , f r e e a n d b o u n d c y c l i c GMP c o n t e n t s i n f r a g m e n t s o f b r o w n f a t f r o m 7 - d a y - o l d r a t s 86 1 3 A , B E f f e c t o f c o l d e x p o s u r e a n d n o r e p i n e p h r i n e a d m i n i s t r a t i o n o n c y c l i c GMP c o n t e n t s o f b r o w n a d i p o s e t i s s u e o f g e n e t i c a l l y o b e s e m i c e 9 8 13C E f f e c t o f c l o n i d i n e i n v i t r o o n c y c l i c GMP c o n c e n t r a t i o n s i n b r o w n f a t f r a g m e n t s o f g e n e t i c a l l y o b e s e m i c e a n d t h e i r l e a n l i t t e r m a t e s 1 0 0 - x i i i -LIST OF TABLES Table Page 90 1. E f f e c t of prostaglandin (PGE2) and n i c o t i n i c acid on f o r s k o l i n stimulated g l y c e r o l release in adipocytes i s o l a t e d from brown adipose tissue of 7-day-old rats 2A. E f f e c t of increasing concentrations of clonidine on f o r s k o l i n stimulated g l y c e r o l release i n a d i - 91 pocytes i s o l a t e d from brown fat of 7-day-old r a t s . 2B. Reversal of clonidine e f f e c t on f o r s k o l i n stimu-lated g l y c e r o l release i n adipocytes i s o l a t e d 92 from brown f a t of one-week-old rats by increasing concentrations of yohimbine 3. E f f e c t of clonidine and n i c o t i n i c acid on i s o -butylmethylxanthine stimulated g l y c e r o l release 93 i n adipocytes i s o l a t e d from brown f a t of one-week-old rats 4 . E f f e c t of pertussis toxin pre-treatment on i n h i b i -t ion of forskolin-stimulated g l y c e r o l release by 94 clonidine and PGE 9 i n r a t adipocytes 5A E f f e c t of clonidine on f o r s k o l i n and i s o b u t y l -methylxanthine stimulated glycer o l release i n 95 adipocytes i s o l a t e d from brown fat of adult hamsters 5B. E f f e c t of prostaglandin (PGE2) and n i c o t i n i c acid on f o r s k o l i n stimulated g l y c e r o l release i n 96 adipocytes i s o l a t e d from brown f a t of adult hamsters - x i v -CHAPTER 1 G E N E R A L BACKGROUND AND I N T R O D U C T I O N 1 . 1 B r o w n A d i p o s e T i s s u e B r o w n a d i p o s e t i s s u e h a s b e e n i m p l i c a t e d a s t h e m a j o r t h e r m o g e n i c o r g a n i n n o n - s h i v e r i n g t h e r m o g e n e s i s n o t o n l y i n h i b e r n a t o r s [ 7 0 , 8 4 ] , b u t a l s o i n n o n - h i b e r n a t o r s s u c h a s t h e r a t [ 5 6 , 5 7 , 1 8 1 ] , i n d i e t - i n d u c e d t h e r m o g e n e s i s [ 5 , 3 6 , 3 8 , 9 1 , 1 5 3 , 1 6 0 , 1 6 1 , 2 0 3 ] a n d i n c e r t a i n t y p e s o f g e n e t i c o b e s i t y [ 3 9 , 6 8 , 6 9 , 7 5 , 1 9 9 ] . A c c o r d i n g t o t h e g e n e r a l l y a c c e p t e d s c h e m e o f t h e r m o g e n e s i s , i n c r e a s e d s y m p a t h e t i c a c t i v i t y c a u s e s r e l e a s e o f n o r e p i n e p h r i n e f r o m p r e - s y n a p t i c . g r a n u l e s w h i c h b i n d s t o ^ - a d r e n e r g i c r e c e p t o r s . T h i s r e s u l t s i n a c t i v a t i o n o f c y c l a s e s ( e . g . , a d e n y l a t e c y c l a s e ) w h i c h , b y i n c r e a s i n g i n t r a c e l l u l a r c y c l i c A M P , a c t i v a t e c y c l i c A M P -d e p e n d e n t p r o t e i n k i n a s e a c t i v i t y w h i c h t h e n s t i m u l a t e s a h o r m o n e - s e n s i t i v e l i p a s e [ 2 6 , 1 5 5 ] . A c t i v a t i o n o f h o r m o n e -s e n s i t i v e l i p a s e i n c r e a s e s l i p o l y s i s b y f a c i l i t a t i n g t h e b r e a k d o w n o f t r i g l y c e r i d e s i n t o f a t t y a c i d s a n d g l y c e r o l . T h e f a t t y a c i d s s o r e l e a s e d a r e a c t i v a t e d i n t o f a t t y a c y l -CoA w h i c h e n t e r s t h e m i t o c h o n d r i a , a n d v i a y 3 - a n d w- o x i d a -t i o n f o r m s a c e t y l C o A . T h e a c e t y l C o A e n t e r s t h e T C A c y c l e i n m i t o c h o n d r i a a n d r e s u l t s i n f o r m a t i o n o f p r o t o n s ( N A D H + + H + ) . T h e r e l e a s e d N A D H + + H + e n t e r e l e c t r o n t r a n s p o r t c h a i n a n d b y o x i d a t i v e p h o s p h o r y l a t i o n g e n e r a t e A T P . T h e b r o w n f a t m i t o c h o n -d r i o n p o s s e s s e s s o m e u n i q u e a n d u n u s u a l c h a r a c t e r i s t i c s w h i c h a r e d e s c r i b e d b e l o w . I n t h e c o n v e n t i o n a l c h e m i o s m o t i c t h e o r y , t h e r e i s a n - 1 -o b l i g a t o r y c o u p l i n g b e t w e e n e l e c t r o n t r a n s f e r d o w n t h e r e s -p i r a t o r y c h a i n a n d p u m p i n g o f H + f r o m t h e m i t o c h o n d r i a l m a t r i x t o t h e o u t e r m e d i u m , t h u s g e n e r a t i n g a n e l e c t r o c h e m i c a l H + g r a d i e n t . A s h y d r o g e n i o n s f l o w b a c k t h r o u g h t h e H + c h a n n e l i n F o F l A T P a s e m o l e c u l e ( A T P s y n t h e t a s e ) , t h e f r e e e n e r g y (£>Go) r e l e a s e d c a u s e s t h e c o u p l e d s y n t h e s i s o f A T P f r o m ADP a n d p h o s p h a t e . A s a r e s u l t o f t h i s c o u p l i n g t h e c e l l e x h i b i t s a n a u t o m a t i c r e s p i r a t o r y c o n t r o l , a d e c r e a s e d c e l l u l a r A T P d e m a n d c a u s e s a f e e d b a c k i n h i b i t i o n u p o n r e s p i r a t i o n . T h i s l e a d s t o (1) d e c r e a s e d e l e c t r o n f l o w i n t h e r e s p i r a t o r j c h a i n ; (2) d e c r e a s e d p r o t o n e x t r u s i o n ; (3) e n h a n c e d p r o t o n e l e c t r o c h e m i c a l g r a d i e n t a c r o s s t h e i n n e r a n d o u t e r m i t o c h o n d r i a l m e m b r a n e ; (4) d e c r e a s e d p r o t o n r e - e n t r y t h r o u g h t h e A T P s y n t h e t a s e , a n d (5) d e c r e a s e d m i t o c h o n d r i a l A T P s y n t h e s i s . T h e m i t o c h o n d r i a l r e s p i r a t o r y c h a i n i s c e n t r a l t o a l l h y p o t h e s e s o f t h e r m o g e n e s i s . I n g e n e r a l t e r m s , t h e r e a r e s e v e r a l w a y s i n w h i c h t h e r m o g e n e s i s m i g h t b e e n h a n c e d . ( i ) R a p i d h y d r o l y s i s o f A T P s u b s e q u e n t t o i t s s y n t h e s i s e i t h e r i n t h e c y t o p l a s m o r t h e m i t o c h o n d r i a l m a t r i x . A l t h o u g h n o p h y s i o l o g i c a l i n t r a m i t o c l t o n d r i a l A T P h y d r o l y z i n g m e c h a n i s m s h a v e y e t b e e n d e s c r i b e d , a r s e n a t e a n d o l i g o m y c i n d o p r e v e n t A T P s y n t h e s i s b y t h e i r a c t i o n a t A T P s y n t h e t a s e . ( i i ) T h e r m o g e n e s i s c a n a l s o b e e n h a n c e d b y s h i v e r i n g i n w h i c h e x t r a m i t o c h o n d r i a l A T P a s e s ( i n a c t o m y o s i n -2-complex) hydrolyze ATP and produce heat. (i i i) The third possibility is the presence of a specific pathway in the mitochondrial energy transduction system prior to ATP synthetase. Such an alternate electron transfer pathway wi l l allow 'uncoupled' respiration to occur at a sufficient rate to raise the temprature. The study of brown-fat mitochondria prepared from animals adapted for thermogenesis, showed presence of an alternate pathr way which in the presence of GDP-binding protein (thermogenin) allowed extrusion of protons into the matrix. The chemiosmotic theory predicts two states for brown-fat mitochondrion. A non-thermogenic state when protons are not shuttled across the inner mitochondrial membrane, 1^*^ + (proton electrochemical potential) is some 220 mv and the respiration is stoichiometrically coupled with ATP synthesis (as in normal mitochondria). A second state in which the leak conductance (CmH+) across the inner mitochondrial membrane is increased above 5 nmol H+ min "^mg m^v ^ so as to lowerAu + to 150-175 mv. According to Nicholls [139,140] this H has one important consequence: this lowered potential is thermo-dynamically competent to achieve maximal rates of thermogenesis and s t i l l retain some capacity for synthesis of ATP to f u l f i l l the needs of the cel l [142], A 32,000 molecular weight protein capable of binding purine nucleotide (GDP) and present on the inner membrane of brown adipose mitochondria allows the reentry of protons into the -3-m i t o c h o n d r i a l m a t r i x . T h i s p r o t e i n t h u s s h o r t - c i r c u i t s t h e A T P -s y n t h e t a s e p a t h a n d p r e v e n t s t h e d e v e l o p m e n t o f same d e g r e e o f p r o t o n g r a d i e n t a s i n n o r m a l m i t o c h o n d r i a [ 7 2 , 1 4 1 , 1 5 ] . I t h a s b e e n p u r i f i e d f r o m h a m s t e r b r o w n - f a t m i t o c h o n d r i a [ 1 1 2 , 1 5 6 ] . H o w e v e r , s t i l l n o t u n d e r s t o o d a r e t h e c o m p o n e n t s r e s p o n s i b l e f o r t h e s h o r t c i r c u i t p a t h w a y a n d t h e i n t e r a c t i o n o f n u c l e o t i d e s w i t h i t i s u n c l e a r . S o f a r , n o p l a u s i b l e i n  v i t r o m o d e l h a s b e e n c o n s t r u c t e d w h i c h c a n e x p l a i n t h e c h r o n i c a d a p t i v e r e g u l a t i o n o f t h e r m o g e n i c c a p a c i t y d u r i n g c o l d a n d d i e t - i n d u c e d t h e r m o g e n e s i s a n d a c u t e r e g u l a t i o n o f t h e r m o -g e n i c e p i s o d e s . S e c o n d l y , t h e r e i s n o d i r e c t e v i d e n c e f o r t h e o p e r a t i o n o f t h e s h o r t c i r c u i t p a t h w a y s i n i n t a c t c e l l s o r t i s s u e s . 1 . 2 A d r e n e r g i c R e c e p t o r s B a s i n g h i s c o n c l u s i o n o n t h e d i v e r s e e f f e c t s o f t h e c a t e c h o l a m i n e s , A h l q u i s t [ 2 ] f i r s t p r o p o s e d s u b c l a s s i f i c a -t i o n o f a d r e n e r g i c r e c e p t o r s i n t o a l p h a a n d b e t a s u b - t y p e s . S u b s e q u e n t l y , L a n d s e t a l . [ 1 0 7 ] s u g g e s t e d s u b c l a s s i f i c a t i o n o f b e t a r e c e p t o r s i n t o a n d fi^ b a s e d o n t h e s e l e c t i v i t y o f a s p e c t r u m o f b e t a a g o n i s t s [ 1 0 9 , 1 8 6 ] . L a n g e r [ 1 0 8 ] s u g g e s t e d t h e s u b c l a s s i f i c a t i o n o f a l p h a r e c e p t o r s i n t o a l p h a - 1 a n d a l p h a - 2 , a n d t h i s s u g g e s t i o n w a s s u b s e q u e n t l y c o n f i r m e d u s i n g d i f f e r e n t t e c h n i q u e s a n d p h a r m a c o l o g i c a l m e t h o d s [ 1 6 , 1 8 6 , 2 1 3 ] . S e v e r a l s e l e c t i v e a l p h a - 1 , a l p h a - 2 , b e t a - 1 , a n d b e t a - 2 -4-agonists and an t a g o n i s t s e i t h e r e x i s t e d or have been i n t r o d u c e d during the past few years. Dobutamine has been i n t r o d u c e d as a r a t h e r s e l e c t i v e yS^-adrenoceptor a g o n i s t [164, 2 1 7 ] . Met o p r o l o l and p r a c t o l o l are w e l l known examples of r a t h e r s e l e c t i v e ^ - a d r e n o c e p t o r b l o c k i n g agents [59, 1 3 7 ] . T e r b u t a l i n e , SalbutamoL Methylnorepinephrine have.been c l a s s i f i e d as s e l e c t i v e ^.,-agonist [125] , while c l o n i d i n e as s e l e c t i v e ^ 2 ~ a g o n i s t [30]. Yohimbine i s e x t e n s i v e l y used as a r a t h e r s e l e c t i v e cj<2~ a d renoceptor a n t a g o n i s t [66, 211]. The a v a i l a b i l i t y of h i g h l y s e l e c t i v e l a b e l l e d l i g a n d s that bind v a r i o u s . c l a s s e s of a d r e n e r g i c r e c e p t o r s g r e a t l y f a c i l i -t a t e d the e x p l o r a t i o n of molecular d e t a i l s of r e c e p t o r s t r u c t u r e , and f u n c t i o n , as w e l l as the r e l a t i o n of the r e c e p t o r s t o other b i o c h e m i c a l components i n t r a n s l a t i n g the r e c e p t o r s i g -n a l i n t o u l t i m a t e p h y s i o l o g i c response. Such s t u d i e s a d d i t i o n a l l y showed a s t a t e of dynamic r e l a t i o n s h i p between r e c e p t o r s t a t e s , namely a f f i n i t y and d e n s i t y of the r e c e p t o r and the p h y s i o l o -g i c a l s t a t e of the t i s s u e . 1.3 Receptor Liqand B i n d i n g Since a s i g n i f i c a n t p a r t of t h i s t h e s i s d e a l s w i t h r a d i o l i g a n d b i n d i n g s t u d i e s i n v o l v i n g c h a r a c t e r i z a t i o n of c ^ 2 - r e c e P t o r s ' t h i s p a r t of the g e n e r a l i n t r o d u c t i o n w i l l d e a l with the background i n f o r m a t i o n p e r t a i n i n g t o the b i n d i n g of the r a d i o a c t i v e t r a n s m i t t e r to membranes from the a p p r o p r i a t e t a r g e t organ, and the theory behind i t . The neurotransmitter receptor in most instances consists of at l e a s t two d i s t i n c t components: the recognition or binding s i t e and some second portion of the receptor which "translates" transmitter recognition into a second messenger, usually an alteration in ion permeability or accumulation of a c y c l i c nucleotide. The group of receptors discussed i n t h i s thesis are p r i m a r i l y adrenergic °<2-receptors. Adrenergic receptors are widely d i s t r i b u t e d among d i f f e r e n t tissues. These d i f f e r e n t receptor subtypes can not only be d i f f e r e n t i a t e d p h y s i o l o g i c a l l y on the basis of dose-response i n various tissues using s e l e c t i v e agonists and antagonists, but can also be characterized pharmacologically using highly s e l e c t i v e l a b e l l e d ligands. Thus with the a v a i l -a b i l i t y of new simple, sensitive, and s p e c i f i c procedures, one can study the i n i t i a l or recognition stage of neurotransmitter or drug i n t e r a c t i o n with b i o l o g i c a l receptors. However, in undertaking a binding study, e s p e c i a l l y involving a new tissue or a new radioactive ligand, one can a l l too e a s i l y be led astray by i n s u f f i c i e n t attention to the question of whether or not a p a r t i c u l a r binding s i t e r e a l l y represents a physiological or a pharmacological receptor. The important thing i n attempt-ing receptor i d e n t i f i c a t i o n i s that f a i l u r e to meet only one c r i t e r i o n , even when a l l others are met, can cast severe doubt on the i d e n t i f i c a t i o n . Also, even when a l l c r i t e r i a for the binding study appear to be f u l f i l l e d , the p o s s i b i l i t y remains, with the development of new classes of more s p e c i f i c drugs and -6-s o p h i s t i c a t e d t e c h n i q u e s , t h a t a d d i t i o n a l d a t a w i l l l a t e r d i s p r o v e t h e i d e n t i f i c a t i o n . H o w e v e r , i n t h e l i g h t o f p r e s e n t t e c h n o l o g y t h e r e a r e a n u m b e r o f c r i t e r i a t h a t m u s t b e f u l f i l l e d f o r a s u c c e s s f u l i d e n t i f i c a t i o n o f a s p e c i f i c r e c e p t o r . 1 . S a t u r a b i l i t y 2 . P r e s e n c e o f h i g h a f f i n i t y b i n d i n g s i t e s . 3 . R a p i d b i n d i n g k i n e t i c s . 4 . S p e c i f i c i t y . T h e b i n d i n g s i t e may e x h i b i t a d i s t i n c t s p e c i f i c i t y t o w a r d s a s e t o f h o r m o n e a n a l o g u e s . 5 . T h e b i n d i n g m u s t b e s t e r e o s p e c i f i c , t h e L - i s o m e r s b e i n g m o r e p o t e n t t h a n t h e D - i s o m e r s . S a t u r a b i l i t y a n d K i n e t i c s o f B i n d i n g 1 . 3 . 1 S a t u r a t i o n E x p e r i m e n t s T h e t o p i c s p r e s e n t e d i n S e c t i o n s 1 . 3 . 1 t o 1 . 3 . 2 a r e e x t e n s i v e l y d i s c u s s e d i n t h e f o l l o w i n g r e f e r e n c e s [ 3 0 , 1 9 3 2 0 0 , 2 2 4 ] . A m i n i m u m r e q u i r e m e n t f o r t h e b i n d i n g t o b e s p e c i f i c i s t h a t t h e r e b e o n l y a f i n i t e n u m b e r o f b i n d i n g s i t e s o f h i g h a f f i n i t y . W h a t c o n s t i t u t e s " h i g h a f f i n i t y " s i t e s d e p e n d s o n c o n t e x t . F o r r e v e r s i b l e l i g a n d s u s e d t o s t u d y n e u r o t r a n s m i t t e r r e c e p t o r s , t h e t e r m u s u a l l y r e f e r s t o a d i s -s o c i a t i o n c o n s t a n t i n t h e n a n o m o l a r r a n g e o r l o w e r . A common a n d s i m p l e w a y o f d e m o n s t r a t i n g s a t u r a b i l i t y i s t o a d d t h e l a b e l l e d d r u g a n d t h e d r u g i n n o n - r a d i o a c t i v e f o r m t o a s e t o f t u b e s c o n t a i n i n g t h e t i s s u e . I f t h e l a t t e r a d d i t i o n l o w e r s t h e a m o u n t o f r a d i o a c t i v i t y b o u n d , t h e n a r e v e r s i b l e a n d s a t u r a b l e b i n d i n g s i t e e x i s t s . S i n c e t h e r e i s -7-a very limited number of neurotransmitter receptors on most tissue membranes, one should be able to readily saturate the number of displaceable binding s i t e s . This i s examined by measuring the binding of increasing concentrations of radioligand i n the presence of a fixed concentration of unlabelled ligand. When the displaceable binding becomes constant as a function of radioligand concentration, saturation i s indicated. In a plot between ligand concentrations and the fr a c t i o n bound, i t i s indicated by the plateauing of the s p e c i f i c binding component. Although the measurement of binding i s straightforward, the i n t e r p r e t a t i o n of data requires mathematical analysis of the experimental data and conversion of binding parameters to the Langmuir isotherm. The equation i s derived by the app l i c a t i o n of law of mass action to drug receptor combination . [224 ] . Thus, b[L]+a[R] = c[RL] The d i s s o c i a t i o n constant (K^) = [L]k[R] a = where K i s the association constant r i c :[• RL ] The above equation can be expressed i n terms of f r a c t i o n of receptors (r) occupied by the l a b e l l e d ligand. [RL]+[R] = B m a x Bmax = t o t a l receptor concentration L = Ligand concentration R = Unoccupied receptors RL= Ligand occupied receptors. -8-m u l t i p l y i n g b y [ L ] [ R L ] [ L ] + [ R ] [ L ] = B m a x [ L ] [ R L ] [ L ] + R L } £ R ] ^ = B m a x [ L ] ; R L . o r [ R L ] ( [ L ] + K d ) = B M A X [ L ] [ R L ] = - m a x [ L ] + K d o r [ R L ] _ [ L ] _ 1" w h e r e [ R L ] i s f r a c t i o n o f t o t a l Bmax [ L ] + K d 1+ K d B m a x r e c e p t o r s o c c u p i e d b y [ T ] t h e r a d i o l i g a n d . W h i c h i s -a h y p e r b o l i c c u r v e a n d i s a l s o k n o w n a s t h e L a n g m u i r i s o t h e r m . T h e e q u a t i o n c l e a r l y e x p l a i n s t h e r a t i o n a l b e h i n d o b t a i n i n g a s a t u r a t i o n c u r v e i f t h e l i g a n d i s b i n d i n g t o t h e r e c e p t o r s s p e c i f i c a l l y . T h i s e q u a t i o n c a n b e t r a n s f o r m e d t o a l i n e a r f u n c t i o n t o f a c i l i t a t e t h e c a l c u l a t i o n o f B m a x a n d K d . T h u s [RL] = v*[L] [ L ' ] + K d o r [ R L ] [ L ] + [ R L ] K d = B m a x [ L ] -9-D i v i d i n g b y [ L ] [ R L ] + [ R L ] . K D = B M A X [ L ] o r [ R L ] = B m a x ~ C R L 3 = _ [ R L ] + B M A X I L T K D K D K D S i n c e i n t h e l i g a n d b i n d i n g s t u d i e s i t i s q u i t e e a s y t o m e a s u r e t h e f r a c t i o n o f l a b e l l e d l i g a n d w h i c h i s s p e c i f i c a l l y b o u n d t o r e c e p t o r s a n d o n e a l r e a d y k n o w s t h e c o n c e n t r a t i o n o f f r e e l i g a n d , a p l o t o f B o u n d [ R L ] _ t o s p e c i f i c a l l y b o u n d * F r e e [ L ] [ R L ] g i v e s a s t r a i g h t l i n e w i t h a s l o p e o f - - a n d X i n t e r c e r p t K d a s B m a x « T h i s i s r e p r e s e n t s a S c a t c h a r d e q u a t i o n . A l t h o u g h m o s t n e u r o t r a n s m i t t e r r e c e p t o r - l i g a n d i n t e r a c t i o n s t u d i e s f o l l o w c l a s s i c m a s s a c t i o n l a w s , i n m a n y n e u r o t r a n s m i t t e r - r e c e p t o r -b i n d i n g s t u d i e s , d i s p l a c e m e n t o f a g i v e n r a d i o l i g a n d f r o m i t s r e c e p t o r h a s y i e l d e d a n o m a l o u s b e h a v i o r , g e n e r a l l y a p p a r e n t a s n e g a t i v e c o o p e r a t i v e i n t e r a c t i o n s ( i . e . , H i l l p l o t s l o p e s i g n i f i c a n t l y l e s s t h a n o n e ) . T h i s a n o m a l o u s b e h a v i o r i s p a r t i c u l a r l y common i f t h e c o m p e t i t i o n i s b e t w e e n a n t a g o n i s t a n d a g o n i s t . H i l l p l o t c a n b e u s e d t o d e t e c t a n d " q u a n t i t a t e " s u c h a n o m a l o u s b e h a v i o r . I n t h e a b o v e e q u a t i o n , s u p p o s e t h a t a c o m p e t i n g a g e n t c o m p e t e s w i t h t h e l a b e l l e d l i g a n d a t m o r e t h a n o n e b i n d i n g s i t e s o t h a t t h e s y s t e m s c o u l d b e a d a p t e d t o a s e q u e n t i a l l i g a n d - b i n d i n g m o d e l , -10-K d l R + L j = ± ( R - L ) 1 ( R - L ) 1 + L 4 ^ 2 y ( R _ L ) 2 ( R - = L i g a n d r e c e p t o r complex a t s i t e 1 K (R - L ) 2 + L t 4 ^ ( R - L ) 3 ( R _ E . ^ . = L i g a n d r e c e p t o r complex a t s i t e 2. ( R - L ) n _ 1 + L S d H - ( R _ L ) n s o t h a t t h e S c a t c h a r d e q u a t i o n c a n b e w r i t t e n a s B [ L ] n _ _ max JD — K j - + [ L ] n w h i c h d e s c r i b e s t h e n e t l i g a n d - b i n d i n g i s o t h e r m . I n t h i s m o d e l n = t h e o r e t i c a l n u m b e r o f l i g a n d - b i n d i n g s i t e s p e r r e c e p t o r m o l e c u l e , = a c o m p o s i t e c o n s t a n t c o m p o s e d o f t h e i n t r i n s i c d i s s o c i a t i o n c o n s t a n t ( K ^ ) a n d i n t e r a c t i o n f a c t o r s t h a t d e t e r m i n e t h e d e g r e e t o w h i c h K d i s a l t e r e d a t e a c h d i s c r e t e b i n d i n g s t e p . R e a r r a n g i n g t h e a b o v e e q u a t i o n y i e l d s B f L l n = = —*— J w h i c h i s t h e H i l l e q u a t i o n B t . l r T , n ^ max K d + [ L J T h e H i l l e q u a t i o n may b e t r a n s f o r m e d t o a l o g a r i t h m i c f o r m t o y i e l d a s t r a i g h t l i n e . T h u s B [ L ] n = B K j + B [ L ] n m a x 1 J d [ L ] n ( B - B ) , L J x max „1 B = K d ( B - B ) 1 h l o g [ L ] = - l o g — ^ + l o g ( B - B ) , l o g ^ = l o g - n l o g [ L ] -11-B A g r a p h o f l o g ^ ^ v e r s u s l o g [ L ] i s a s t r a i g h t l i n e . max B L o g K , i s t h e a b c i s s a v a l u e w h e r e l o g 7 - - T = 0 a <Bmax~ B} T h u s n l o g [ L J. = l o g T h e s l o p e o f t h e l i n e g i v e s n~ - c a l l e d t h e H i l l c o e f f i c i e n t ( n H ) - . A s i m i l a r p l o t c a n b e u s e d i n d i s p l a c e m e n t o r c o m p e t i t i o n e x p e r i -m e n t s w h e r e a f i x e d c o n c e n t r a t i o n o f r a d i o l a b e l l e d l i g a n d i s 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 u n l a b e l l e d l i g a n d s . T h e s p e c i f i c r a d i o l i g a n d b i n d i n g ( t o t a l m i n u s b l a n k ) o c c u r r i n g i n t h e a b s e n c e o f a n y d i s p l a c i n g a g e n t s i s t a k e n a s B . T h e d a t a a r e t h e n p l o t t e d a s l o g - — m a x B ^ - B max v e r s u s l o g [ d i s p l a c e r ] t o y i e l d t h e H i l l p l o t . When n H = 1.0, t h e s y s t e m i s n o n - c o o p e r a t i v e ; w h e n njj>1.0, t h e s y s t e m i s p o s i t i v e l y c o o p e r a t i v e , a n d w h e n n ^ C l . O , t h e n. s y s t e m i s e i t h e r n e g a t i v e l y c o o p e r a t i v e o r r e p r e s e n t s a h e t e r o g e n o u s s e t o f b i n d i n g s i t e s . A l t h o u g h t h e S c a t c h a r d p l o t i s w i d e l y u s e d t o a n a l y z e t h e r a d i o l i g a n d b i n d i n g d a t a , o f t e n t h e t r a n s f o r m a t i o n o f t h e S c a t c h a r d e q u a t i o n may g i v e r i s e t o p r o b l e m s , e s p e c i a l l y i f t h e d a t a c o n t a i n a n e x p e r i m e n t a l e r r o r . -12-This error increases dramatically at low binding l e v e l s . Under such circumstances, the Scatchard p l o t may appear to be non-li n e a r and t h i s could give the impression that the ligand i s binding to multiple s i t e s . 1.3.2 Competition or Labelled Ligand Displacement Experiments By performing displacement experiments one can accumulate a great deal of additional supplementary information about the binding c h a r a c t e r i s t i c s of the ligand to i t s receptors. This i s p a r t i c u l a r l y true in cases where the d i r e c t measurement of ligand binding to a receptor i s not f e a s i b l e because the a f f i n i t y of the ligand for receptor i s low and the maximal receptor concentration accessible i n v i t r o i s also low. Thus, i f one would l i k e to measure the a f f i n i t y of a low a f f i n i t y ligand to a receptor, i t can be done by measuring i t s a b i l i t y to displace a high a f f i n i t y r adioactively l a b e l l e d antagonist. The basic p r i n c i p l e of displacement experiments i s derived from analysis of competitive antagonism, i n drug receptor reactions. Thus i f the receptor binds the high a f f i n i t y r a d i o a c t i v e l y l a b e l l e d antagonist 1^ with the d i s s o c i a t i o n constant and the low a f f i n i t y agonist H with the d i s s o c i a t i o n constant K J J , then [Rl [111 [R T] = CR] + CRli] = CR] + — CR] + Cij.] - C R I i ] Ki CR] C i j ] = [ R i i ] -13-Where [ I i ] i s the concentration of free r a d i o a c t i v e l y l a b e l l e d antagonist and [ftp] the t o t a l receptor concentration which equals the sum of free receptor concentration [R] and the antagonist bound receptor [RI^] when H i s absent. Under such circumstance the f r a c t i o n of occupied receptors (r) i s equal to [ R l i ] _ [ I i V K i Ki If the low a f f i n i t y ligand H i s present, there are two antagonists, one low a f f i n i t y antagonist H, and the second high a f f i n i t y l a b e l l e d antagonist competing for the same receptor s i t e s . One of the important parameters, i n assays involving competition experiments, i s the c a l c u l a t i o n of IC50 values (the concentration of a non-radioactive drug that i n h i b i t s 50% of the radioactive drug's binding to a s p e c i f i c s i t e ) which can be used to calculate the apparent equilibrium d i s s o c i a t i o n constant of the non-radioactive drug for the radioactive binding s i t e . It i s assumed that at IC50 value one-half of the binding s i t e s o r i g i n a l l y occupied by the radioactive drug are occupied by the non-radioactive drug, and i t could be taken as the K d of the non-radioactive drug. This d e f i n i t i o n applies only when the drug i s free to interact with the receptor i n the absence of any other i n t e r f e r i n g drug. In the case of competition experiments, there i s always radioactive drug present i n concentrations s u f f i c i e n t to bind to the receptor. The radioactive drug i n t e r f e r e s with non-radioactive drug -14-b i n d i n g j u s t a s n o n - r a d i o a c t i v e d r u g b i n d i n g i n t e r f e r e s w i t h r a d i o a c t i v e d r u g b i n d i n g [ 2 9 , 2 0 0 , 2 2 4 ] . T h u s t h e a b o v e e q u a t i o n a s s u m e s a new v a l u e a s f o l l o w s : [ R ] [ I 9 ] [R][H] [R T] = [R] + [ R I 2 ] + [RH] = [R] + — K + L ^ W h e r e [ I 2 ] a n c * [R * 2 ^ a r e t ^ i e c o n c e n t ^ a t i o n s o f t h e f r e e r a d i o a c t i v e l i g a n d a n d t h e r e c e p t o r - l i g a n d c o m p l e x r e s p e c t i v e l y u n d e r t h e new s i t u a t i o n , a n d [RH] i s t h e c o n c e n t r a t i o n o f t h e r e c e p t o r c o m p l e x e d w i t h t h e l o w a f f i n i t y l i g a n d . U n d e r t h e s e c o n d i t i o n s , t h e f r a c t i o n o f r e c e p t o r o c c u p i e d b y t h e r a d i o a c t i v e a n t a g o n i s t I 2 ^ s g i v e n b y : [ R I 2 ] ; t I 2 ] / K i r 1 ~ [ R T ] ~ 1 + [ I 2 ] + [H] — D i v i d i n g r 1 b y r g i v e s I. r 1 K. [ I 9 ] T h i s d i s p l a c e m e n t e q u a t i o n r 1 1 ^ I 2 ^ ^ ^ i ^ g i v e s t h e f r a c t i o n o f r e c e p t o r s K. K H o c c u p i e d b y a n t a g o n i s t I 2 i n t h e p r e s e n c e o f H . a n d t * 2 ] a r e f r e e a n t a c J o n i s t c o n c e n t r a t i o n s i n t h e a b s e n c e a n d i n t h e p r e s e n c e o f H r e s -p e c t i v e l y . A p l o t o f — a s a f u n c t i o n o f [ H ] o r l o g [ H ] g i v e s a t y p i c a l s . i g m o i d a l c u r v e s e e n i n c o m p e t i t i o n e x p e r i -m e n t . A t t h e c o n c e n t r a t i o n w h e r e 50% o f t h e b o u n d r a d i o -a c t i v e l i g a n d I 2 i s d i s p l a c e d b y H , ~ ^ - = | a n d r e a r r a n g e m e n t o f t h e a b o v e e q u a t i o n g i v e s -15-[ H ] 0 5 K H = Yi—j  T~[I~ ] Since i n g e n e r a l [I] or [H] [ H ' t o t a l = t H l f r e e = [ H ] The above equation becomes 1 + K i r l = K i = 1 r [ l 2 ] [ H ] ~ [H] K . K H I + : K H ( 1 + [ I 2 3 } K . l At IC50 when 50% of the l i g a n d i s d i s p l a c e d by [ H ] , the equation becomes [ H ] 0 5 K H = T T ^ ™ m ^ 0 . 5 = I C 5 0 K . + 1 K „ = K , of n o n - l a b e l l e d I H d . . . a n t a g o n i s t K . = K , of l a b e l l e d 1 d -m drug I 2 = c o n c e n t r a t i o n of l a b e l l e d drug The above equation i n d i c a t e s a very important r e l a t i o n s h i p between apparent of n o n - l a b e l l e d drug c a l c u l a t e d from K D of l a b e l l e d drug u s i n g Scatchard a n a l y s i s . Even a f t e r r e c e p t o r s have been s u c c e s s f u l l y i d e n t i f i e d u s i n g . r a d i o - l a b e l l e d l i g a n d s b i n d i n g assays t h e r e are a number of parameters t h a t must be taken i n t o c o n s i d e r a t i o n before one can say t e n t a t i v e l y t h a t a p a r t i c u l a r c l a s s of r e c e p t o r s e x i s t s f o r a c e r t a i n n e u r o t r a n s m i t t e r at a par-t i c u l a r s i t e . For example, some of these c o n s i d e r a t i o n s are: -16-( i ) D i s t r i b u t i o n o f B i n d i n g S i t e s T h e r e a r e t w o q u e s t i o n s w h i c h a r e i m p o r t a n t i n t h i s r e s p e c t : F i r s t , w h e t h e r b i n d i n g o f s i m i l a r c h a r a c t e r -i s t i c s i s p r e s e n t a t o t h e r s i t e s w h e r e r e c e p t o r s f o r t h e n e u r o t r a n s m i t t e r a r e k n o w n t o e x i s t . S e c o n d l y , w h e t h e r a c e r t a i n f u n c t i o n a l , p h y s i o l o g i c a l o r p h a r m a c o l o g i c a l p a r a m e t e r i s a s s o c i a t e d w i t h t h e s t i m u l a t i o n o r b l o c k -a d e o f r e c e p t o r s . I t i s l e s s l i k e l y t o f i n d a c l a s s o f r e c e p t o r s t o b e p r e s e n t a t s i t e s w h e r e i t h a s n o funcr-t i o n a l r e l e v a n c e t o t h e r e s p o n s e a s s o c i a t e d w i t h t h a t t i s s u e . ( i i ) D e n s i t y o f R e c e p t o r s T h e d e n s i t y o f a d r e n e r g i c r e c e p t o r s b o t h i n b r a i n a n d i n p e r i p h e r a l t i s s u e s g e n e r a l l y t e n d t o f a l l w i t h i n 0 . 1 pmol/mg t i s s u e w e i g h t ( a p p r o x i m a t e l y 2 . 5 p m o l e s / m g p r o t e i n ) [ 1 8 4 , 2 2 3 ] . I t h a s b e e n s u g g e s t e d t h a t t o o l a r g e a d e n s i t y , a c c o m p a n i e d b y a l o w a f f i n i t y b i n d i n g , p r o b a b l y s u g g e s t s b i n d i n g t o a c c e p t o r o r n o n - r e c e p t o r s i t e s . ( i i i ) S a t u r a b i l i t y A l t h o u g h s a t u r a b i l i t y h a s b e e n m e n t i o n e d e a r l i e r a s o n e o f t h e m i n i m a l c r i t e r i a f o r s p e c i f i c b i n d i n g t o r e c e p t o r s i t e s , e x a m p l e s a b o u n d i n t h e l i t e r a t u r e w h e r e i t h a s b e e n s h o w n t h a t a l i g a n d may b i n d t o a c c e p t o r o r n o n -s p e c i f i c s i t e s a n d may s t i l l s h o w s a t u r a b i l i t y a n d h i g h a f f i n i t y . H o w e v e r , t h i s p r o b l e m c a n b e c i r c u m v e n t e d b y t h e u s e o f s e v e r a l l i g a n d s e a c h h a v i n g h i g h s p e c i f i c i t y f o r a -17-p a r t i c u l a r c l a s s of r e c e p t o r s and a n a l y s i s of the b i n d i n g data i n v a r i o u s ways. ( i v ) Q u a n t i t a t i v e C o r r e l a t i o n In order to i d e n t i f y a s p e c i f i c b i n d i n g s i t e f o r a r a d i o l i g a n d i n a homogenate or membrane f r a c t i o n as a s p e c i f i c type of r e c e p t o r , i t i s necessary t o show a strong q u a n t i t a t i v e c o r r e l a t i o n between the va l u e of a f f i n i t i e s of a s e r i e s of a g o n i s t s and a n t a g o n i s t s f o r b i n d i n g s i t e s as determined by r a d i o l i g a n d b i n d i n g s t u d i e s and that determined by ph a r m a c o l o g i c a l s t u d i e s [132]. For example, e x c e l l e n t c o r r e l a t i o n s have been r e p o r t e d between s t i m u l a t i o n and blockade of ^ - r e c e p t o r -s t i m u l a t e d adenylate c y c l a s e a c t i v i t y and measurements of c o m p e t i t i o n f o r [ H ] - d i h y d r o a l p r e n o l o l [132], A l -though one i s more l i k e l y to see such as c o r r e l a t i o n i n i n v i t r o experiments, such c o r r e l a t i o n may not be evident between i n v i t r o b i n d i n g experiments and i n - v i v o p h a r m a c o l o g i c a l experiments. (v) S t e r e o s p e c i f i c i t y Although s t e r e o s p e c i f i c i t y has been mentioned as one of the most u s e f u l pharmacological c r i t e r i a i n i d e n t i f y i n g r e c e p t o r b i n d i n g , i t does not n e c e s s a r i l y prove to be an absolute c r i t e r i o n of r e c e p t o r i d e n t i f i c a t i o n . In f a c t , o p i a t e s have been shown to b i n d t o g l a s s f i b e r f i l t e r s and c e r e b r o s i d e s s t e r e o s p e c i f i c a l l y [114, 183]. -18-( v i ) R e l a t i o n s h i p Between B i n d i n g and Response There are a number of other f a c t o r s which may a f f e c t the b i n d i n g s t u d i e s - namely apparent b i n d i n g a f f i n i t i e s of a c l a s s of r e c e p t o r s may be d i f f e r e n t f o r a l a b e l l e d a g o n i s t or a n t a g o n i s t ; a l i g a n d b i n d i n g may behave d i f f e r e n t l y with changes i n temperature or i n case of m u l t i p l e b i n d i n g , with c l a s s e s of b i n d i n g s i t e s . I t i s imperative t h a t one should know what c r i t e r i a to employ to estimate the r e a l c l a s s of r e c e p t o r s i n q u e s t i o n . These and other c r i t e r i a have been e x t e n s i v e l y d i s c u s s e d by B u r t , 1978 [29] and Yamamura, 1978 [224]. F i n a l l y , r e g a r d l e s s of whether l i g a n d b i n d i n g s t u d i e s suggest the presence of a p p r o p r i a t e r e c e p t o r s i n a t i s s u e , t h e r e must be some phar m a c o l o g i c a l evidence of the t i s s u e responsiveness to the n e u r o t r a n s m i t t e r . For example, Fu r c h g o t t , 1978 [61] suggested t h a t the a f f i n i t i e s o f a s e r i e s of a g o n i s t s and a n t a g o n i s t s obtained from b i n d i n g s t u d i e s should c o r r e l a t e w i t h the a f f i n i t i e s of t h i s s e r i e s determined by ph a r m a c o l o g i c a l t e s t i n g . In h i s study an e x c e l l e n t c o r r e l a t i o n was obtained between of ^ - a d r e n e r g i c r e c e p t o r s on s t r i p s of r a b b i t , t h o r a c i c a o r t a as determined by ph a r m a c o l o g i c a l methods and that determined by the b i n d i n g of [ H]-d i h y d r o e r g o c r y p t i n e t o membrane f r a c t i o n s of r a b b i t uterus smooth muscle. However, T a l l a r i d a , 1981 [193] p o i n t s out that i n many systems, other -19-than smooth muscle i t may not be p o s s i b l e to determine a f f i n i t i e s by p r e c i s e pharmacological procedures. T a l l a r i d a p o i n t s out three p o s s i b l e reasons behind i t . F i r s t of a l l , the b i n d i n g s t u d i e s are based on the simple theory of c o m p e t i t i v e i n h i b i t i o n of a l a b e l l e d substance by. a n o n - l a b e l l e d compound. Estimates of d i s s o c i a t i o n constants obtained p h a r m a c o l o g i c a l l y are based on responses. The s i t u a t i o n becomes more complex due to the presence of spare r e c e p t o r s , i n many t i s s u e s . Secondly, i t should be r e a l i z e d t h a t i n p h a r m a c o l o g i c a l assays the c o n c e n t r a t i o n s employed to c a l c u l a t e d i s -s o c i a t i o n constants i n both i n v i v o and i n v i t r o experiments are not the same as the c o n c e n t r a t i o n of drug present at the r e c e p t o r s i t e s . T h i r d l y , i n con-t r a s t to l i g a n d b i n d i n g assays, the pharmacological assays make use of a S c h i l d p l o t or s e v e r a l other m o d i f i c a t i o n s of dose-response curve u s i n g e i t h e r a p a r t i a l a g o n i s t or a n t a g o n i s t to compete wi t h a f u l l a g o n i s t . Each of these methods has some assumptions. Thus, S c h i l d assumes t h a t equal responses produced by an a g o n i s t i n the absence and i n the presence of a c o m p e t i t i v e a n t a g o n i s t w i l l occur only when the a g o n i s t occupies equal p r o p o r t i o n s of the r e c e p t o r p o p u l a t i o n [ 60, 167, 210], F i n a l l y , r e g a r d l e s s of the drawbacks and disadvantages i n v o l v e d , d i s s o c i a t i o n c onstants obtained from b i n d i n g procedures, along w i t h those -20-obtained from i n v i t r o and i n v i v o p h a r m a c o l o g i c a l .procedures do add to our f u r t h e r understanding of re c e p t o r c h a r a c t e r i z a t i o n and i d e n t i f i c a t i o n . 1.4 The Bet a - a d r e n e r g i c Pathway While i t i s c l e a r t h a t s t i m u l a t i o n of the beta adrenoceptors by norepinephrine i n brown adipose t i s s u e i s the p r i n c i p a l stimulus f o r n o n - s h i v e r i n g thermogenesis (NST) [25], the d e t a i l s of the molecular mechanisms are yet to be completely understood. The f i r s t step i n no r e p i n e p h r i n e a c t i o n upon brown adipocytes i s i t s b i n d i n g to a p p r o p r i a t e plasma membrane r e c e p t o r s . Two approaches have been taken t o study and i d e n t i f y the c h a r a c t e r i s t i c s of a d r e n e r g i c r e c e p t o r s . (1) C o n v e n t i o n a l pharmacological a n a l y s i s , where s e l e c t e d a g o n i s t s and antagon-i s t s are used to study t h e i r r e l a t i v e e f f e c t s and p o t e n c i e s on s e v e r a l b i o l o g i c a l responses (e.g., l i p o l y t i c e f f e c t s , plasma membrane adenylate c y c l a s e a c t i v i t y , v a r i a t i o n s i n c y c l i c AMP content of the adip o c y t e s and p h o s p h a t i d y l i n o s i t o l turnover), (2) The second approach i s the use of r a d i o l i g a n d b i n d i n g techniques to i d e n t i f y and c h a r a c t e r i z e p u t a t i v e r e c e p t o r s . The f i r s t attempts to i d e n t i f y f a t c e l l beta adrenoceptors were based on the study of the b i n d i n g of [ H]-catecholamines to plasma membranes by A p r i l l e , 1974 [ 4 ] ; K o r e t z and M a r i n e t t i , 1974 [98]. However, these s t u d i e s have been ques-t i o n e d due to the f a c t t h a t t r i t i a t e d catecholamines can simultaneously b i n d to a membrane c a t e c h o l - b i n d i n g p r o t e i n -21-r e l a t e d t o c a t e c h o l - O - m e t h y l t r a n s f e r a s e . A v a i l a b i l i t y o f h i g h l y 3 3 s e l e c t i v e b e t a a n t a g o n i s t s ( - ) [ H ] - d i h y d r o a l p r e n o l o l ('[ H ] - D H A ) 3 a n d b e t a a d r e n e r g i c a g o n i s t (+_) [ H ] - h y d r o x y b e n z y l i s o p r o t e r -3 e n o l ( [ H ] - H B l ) made i t p o s s i b l e t o l a b e l y S - a d r e n o c e p t o r s 3 s e l e c t i v e l y . U s i n g [ H ] - D H A v a r i o u s i n v e s t i g a t o r s d e m o n s t r a t e d t h e p r e s e n c e o f b e t a - l - a d r e n o c e p t o r s i n r a t a d i p o c y t e p l a s m a m e m b r a n e s [ 2 4 , 2 1 4 ] . G i u d i c e l l i e t a l . ( 1 9 8 2 a ) [ 6 4 ] , u s i n g [ 3 H ] -H B I , d e c r i b e d t h e p r e s e n c e o f t w o a f f i n i t y s t a t e s o f b e t a a d r e n o -c e p t o r s i n r a t f a t c e l l s : o n e o f h i g h a f f i n i t y ( K ^ = 3 . 5 + 0 . 7 nM) a n d l o w c a p a c i t y , a n d t h e o t h e r o f l o w a f f i n i t y ( K ^ = 1 0 1 + 2 0 nM) a n d h i g h e r c a p a c i t y . R o t h w e l l e t a l . [ 1 6 2 ] r e c e n t l y r e p o r t e d t h a t t h e p o p u l a t i o n o f a d r e n e r g i c r e c e p t o r s i n m i c r o s o m a l m e m b r a n e s i s o l a t e d f r o m b r o w n f a t o f a d u l t r a t s c o n t a i n b o t h a n d s u b - t y p e s . S t u d i e s b y A r c h e t a l . ( 1 9 8 4 ) [ 8 ] i n d i c a t e d t h a t r a t b r o w n f a t ^ - a d r e n o c e p t o r s s h o w a t y p i c a l c h a r a c t e r i s t i c s a n d d o n o t f a l l i n t o p^- a n d fi^- s u b c l a s s i f i c a t i o n [ 7 , 8 , 4 6 , 9 3 ] . R e -g a r d l e s s o f t h e n a t u r e o f ^ - a d r e n o c e p t o r s , a l l t h e s t u d i e s c a r r i e d o u t t o d a t e s h o w t h a t a c t i v a t i o n o f y g - r e c e p t o r t h r o u g h n o r e p i n -e p h r i n e l e a d s t o a c t i v a t i o n o f a d e n y l a t e c y c l a s e , c y c l i c AMP g e n e r a t i o n , p r o t e i n k i n a s e a c t i v a t i o n a n d a c t i v a t i o n o f h o r m o n e -s e n s i t i v e l i p a s e . I n t e r e s t i n g l y , t h e r e p o r t e d s t u d i e s a l s o s h o w t h a t t h e r e i s n o s t r a i g h t f o r w a r d o r l i n e a r r e l a t i o n s h i p b e t w e e n c y c l i c AMP g e n e r a t i o n a n d l i p o l y s i s [ 9 7 ] . T h e f o r m e r t e n d s t o b e m a x i m a l w i t h i n 10 t o 20 m i n u t e s a n d t h e n d e c l i n e s [ 4 9 ] , w h e r e a s l i p o l y s i s r e m a i n e s e l e v a t e d f o r a t l e a s t 6 0 m i n u t e s [ I 4 9 ] . We a r e a w a r e o f n o r e p o r t s of a s u c c e s s f u l p u r i f i c a t i o n o f h o r m o n e - s e n s i t i v e t r i g l y c e r i d e l i p a s e f r o m - 4 2 2 -b r o w n a d i p o s e t i s s u e . T h e o n l y e v i d e n c e l i n k i n g t h e c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e t o t h e l i p o l y t i c a c t i v i t y i n b r o w n f a t h a s s o f a r b e e n i n d i r e c t , e . g . , a c o r r e l a t i o n b e t w e e n t h e t i s s u e c o n c e n t r a t i o n o f c y c l i c A M P , t h e p r o t e i n k i n a s e " a c t i v i t y r a t i o " a n d t h e r a t e o f g l y c e r o l r e l e a s e [ 1 7 7 ] , L i p o l y s i s i n b r o w n f a t h a s b e e n a s s a y e d b y t h e r e l e a s e o f e i t h e r g l y c e r o l o r f a t t y a c i d s . H o w e v e r , i n b o t h c a s e s t h e l i p o l y s i s c o u l d b e u n d e r e s t i m a t e d i f s i g n i f i c a n t p r o p o r t i o n s o f t h e g l y c e r o l i s r e p h o s p h o r y l a t e d o r i f t h e r e l e a s e d f a t t y a c i d s a r e o x i -d i s e d o r r e - e s t e r i f i e d . 1 . 5 A l p h a - a d r e n e r g i c p a t h w a y A l t h o u g h n o r e p i n e p h r i n e - m e d i a t e d a c t i v a t i o n o f a d r e n e r g i c r e c e p t o r s i n b r o w n f a t a d i p o c y t e s p l a y s q u a n t i t a t i v e l y a d o m i n a t i n g p a r t i n t h e i n c r e a s e i n o x y g e n c o n s u m p t i o n a n d s u b s e q u e n t l y i n l i p o l y s i s a n d t h e r m o g e n e s i s , a p o s s i b l e a d d i t i o n a l r o l e o f e < - a d r e n e r g i c r e c e p t o r w a s s u g g e s t e d b y H o r w i t z a n d H o r o w i t z a s e a r l y a s 1977 [ 8 5 ] . S e l e c t i v e c o n c e n t r a t i o n s o f oC- o r ^ - a d r e n e r g i c a g o n i s t s c o u l d d e p o l a r i z e t h e r a t b r o w n f a t c e l l m e m b r a n e [ 5 5 ] . L - - p h e n y l e p h r i n e w a s s h o w n t o b e m o r e e f f e c t i v e t h a n n o r e p i n e p h r i n e i n e l i c i t i n g d e p o l a r i z a t i o n . T h i s d e p o l a r i z a t i o n w a s n o t s e n s i t i v e t o i n h i b i t i o n b y p r o p r a n o l o l , w h e r e a s t h e c < - a d r e n e r g i c a n t a g o n i s t p h e n t o l a m i n e c o u l d c a u s e 70% i n h i b i t i o n o f n o r e p i n e p h r i n e -i n d u c e d d e p o l a r i z a t i o n a n d a c o m p l e t e i n h i b i t i o n o f p h e n y l e p h -- r i n e - i n d u c e d d e p o l a r i z a t i o n [ 8 4 ] . A p o s s i b l e l i n k b e t w e e n -23-o t - r e c e p t o r a c t i v a t i o n , Na t r a n s m e m b r a n e t r a n s p o r t , a n d r e -2 + d i s t r i b u t i o n o f i n t r a c e l l u l a r C a w a s p r o p o s e d b y N e d e r g a a r d a n d L i n d b e r g [ 1 3 8 ] . D i r e c t r a d i o l i g a n d b i n d i n g o f [ ^ H ] - p r a z o s i n t o c r u d e m e m b r a n e f r a c t i o n s f r o m h a m s t e r b r o w n f a t [ 1 2 7 ] a n d r a t b r o w n f a t [ 1 5 4 ] s h o w e d t h e e ^ - r e c e p t o r s t o b e o f oC^ s u b - t y p e . M o h e l l e t a l . [ 1 2 8 ] a l s o s h o w e d t h a t a n a l p h a -a d r e n e r g i c a l l y m e d i a t e d p r o c e s s c o n t r i b u t e d a b o u t 2 0 % t o t h e t o t a l m e a s u r e d h e a t p r o d u c t i o n i n b r o w n f a t a n d t h a t t h e s t i m u l a t i o n o f t h i s r e c e p t o r c l a s s h a d b e e n a s s o c i a t e d w i t h c h a r a c t e r i s t i c " p h o s p h a t i d y l i n o s i t o l r e s p o n s e " [ 1 2 9 ] . H o w e v e r , a r e l a t i o n s h i p b e t w e e n a l p h a - l - m e d i a t e d p h o s p h a t -i d y l i n o s i t o l t u r n o v e r e n h a n c e m e n t a n d i n c r e a s e i n c y t o s o l i c 2+ C a i s y e t t o b e f u l l y u n d e r s t o o d . T h e r e f o r e , n o f i n a l c o n c l u s i o n c a n b e d r a w n c o n c e r n i n g t h e r e l a t i o n s h i p b e t w e e n t h e c e l l u l a r e v e n t s a n d t h e t r a n s m i s s i o n o f a m e s s a g e a c r o s s t h e c e l l m e m b r a n e a s s o c i a t e d w i t h s t i m u l a t i o n o f e * ^ - r e c e p t o r s . S k a l a a n d K n i g h t [ 178] i n t h e i r i n v i v o s t u d i e s i n r a t b r o w n f a t p r e s e n t e d e v i d e n c e o f a p o s s i b l e l i n k b e t w e e n c<-a d r e n e r g i c s t i m u l a t i o n , c y c l i c GMP p r o d u c t i o n a n d i n c r e a s e d a c t i v i t y o f c y c l i c G M P - d e p e n d e n t p r o t e i n k i n a s e . S i n c e t h e h i g h s t e a d y - s t a t e c o n c e n t r a t i o n o f c y c l i c GMP a n d t h e i n c r e a s e d a c t i v i t y o f c y c l i c G M P - d e p e n d e n t p r o t e i n k i n a s e d u r i n g e a r l y s t a g e s o f b r o w n f a t d e v e l o p m e n t s e e m e d t o c o r r e s p o n d w i t h p e r i o d s o f h i g h p r o l i f e r a t i v e a c t i v i t y o f t h e t i s s u e , i t w a s s u g g e s t e d t h a t o C - a d r e n o c e p t o r m e d i a t e d s t i m u l a t i o n o f c y c l i c GMP - 2 4 -s y s t e m m i g h t h a v e b e e n p l a y i n g a r o l e i n t h e r e g u l a t i o n o f m a t u r a t i o n o f t i s s u e . T h e e x i s t e n c e o f s u c h a l i n k t o g u a n y l a t e c y c l a s e a c t i v a t i o n d i r e c t l y o r i n d i r e c t l y v i a c h a n g e s i n i n t r a -c e l l u l a r C a + + w a s a l s o s u g g e s t e d b y E x t o n [ 5 2 ] , S c h u l t z [ 1 7 1 ] a n d P o i n t e r [ 1 5 2 ] . S e v e r a l s t u d i e s h a v e i m p l i c a t e d c y c l i c GMP i n t h e d e v e l o p m e n t a l p r o c e s s e s [ 4 0 ] , i n t h e p r o l i f e r a t i o n o f h e p -a t o c y t e s [ 1 2 6 ] a n d s p l e e n c e l l s [ 4 7 ] , a s a s e c o n d m e s s e n g e r i n some s y s t e m s a n d a l s o a s a f e e d b a c k r e g u l a t o r o f r e c e p t o r m e d i a -t e d c e l l u l a r r e s p o n s e [ 1 4 3 ] , H o w e v e r , v e r y l i t t l e i n f o r m a t i o n i s a v a i l a b l e a b o u t t h e m e c h a n i s m s , o t h e r t h a n f e w m e n t i o n e d a b o v e , b y w h i c h g u a n y l a t e c y c l a s e i s m o d u l a t e d . T h e h o m o g e n a t e f r o m m o s t t i s s u e s t e n d s t o h a v e g u a n y l a t e c y c l a s e i n s o l u b l e a n d p a r t -i c u l a t e f r a c t i o n s . A l t h o u g h m a n y h o r m o n e s c a n i n c r e a s e c y c l i c GMP l e v e l s i n v a r i o u s t i s s u e s , i t i s u n k n o w n w h e t h e r o r n o t t h e s o l u b l e a n d p a r t i c u l a t e a c t i v i t i e s o f g u a n y l a t e c y c l a s e r e p r e s e n t s i m i l a r p r o t e i n s . R e p o r t s o n - a d r e n e r g i c r e c e p t o r s a n d c y c l i c GMP s y s t e m i n o t h e r c e l l l i n e s s e e m e d t o i n d i c a t e a p o s s i b l e r e g u l a t o r y r o l e i n t i s s u e g r o w t h a n d d i f f e r e n t i a t i o n [ 1 4 3 ] . S k a l a a n d K n i g h t [ 1 7 8 ] h a d s u g g e s t e d t h e p r e s e n c e o f a p o s s i b l e l i n k b e t w e e n c y c l i c GMP s y s t e m a n d © ( - a d r e n e r g i c r e c e p t o r s y s t e m i n a d d i t i o n t o t h e t r o p h i c e f f e c t o f n o r e p i n e p h r i n e o n b r o w n f a t . T h u s t h e p o s s -i b i l i t y e x i s t e d t h a t t h e c a l o r i g e n i c a n d t h e t r o p h i c e f f e c t o f n o r e p i n e p h r i n e d i v e r g e d a t t h e r e c e p t o r l e v e l . T h e p h e n o t y p e o f t h e a d r e n o c e p t o r s i n t h e p l a s m a m e m b r a n e , t h e r e l a t i v e p r o p o r t i o n a n d t h e a f f i n i t i e s o f i t s c o m p o n e n t d e t e r m i n e d t h e e f f e c t 2 5 -of norepinephrine on brown fat. It, therefore, seemed appropriate to search for the presence of o( 2~receptors in actively proli -ferating brown adipose tissue of infant rats and provide addi-tional support by in vitro experiments for the proposed link bet-ween the cyclic GMP system and the ^-adrenergic system. We have used several approaches in an attempt to demons-trate the presence of ©^ -adrenoceptors in brown fat of 7-day-old rats. (i) The binding characteristics of ^ -adrenoceptors on i s -olated plasma membrane fragments from brown fat of one-week-old rats were determined using selective ( X j l i g a n d s . (ii) In the second part, changes in the binding characteristics of eK^-adreno-ceptors in isolated membrane fragments were studies following chem-. ical sympathectomy and chronic blockade of o< ^adrenoceptors by yohimbine. ( i i i ) Experiments were carried out to test the poss-ib i l i ty that cyclic GMP participates in the o<2-adrenergic effector pathway. (iv) Isolated adipocytes from brown fat of one-week-old rats were used to study the effect of (^-adrenoceptor stimulation on forskolin- and isobutylmethylxanthine-stimulated lipolysis. (v) Brown fat fragments from obese mice and normal littermates were used to study the effect of o^-agonist and antagonists on tissue concentrations of cyclic GMP. (i) In the f irst part of our study we investigated the binding characteristics of oc -^adrenoceptors i n membrane fragments from the brown fat of one-week-old rats using selective o<2-ligands. (ii) In the second part we approached the problem in an indirect way. It has been shown that the particular class of recep--26-t o r s c a n b e " u p " o r " d o w n " r e g u l a t e d d e p e n d i n g u p o n a p a r t i c u l a r s e t o f p h y s i o l o g i c o r p a t h o l o g i c s t a t e s o f t h e t i s s u e . S t u d i e s b a s e d o n t h e i n s u l i n r e c e p t o r [ 6 2 , 6 7 ] d o p a m i n e r e c e p t o r s [ 1 7 2 ] a n d a d r e n e r g i c r e c e p t o r s [ 2 1 5 , 2 1 6 ] s h o w e d t h a t a w i d e v a r i e t y o f hormones and d r u g s h a v e t h e a b i l i t y t o r e g u l a t e t h e d e n s i t y o f t h e i r o w n r e c e p -t o r s i n t i s s u e s . I n g e n e r a l , t h e p a t t e r n t h a t h a s b e e n o b s e r v e d i s t h a t o f a n i n v e r s e r e l a t i o n b e t w e e n t h e a m b i e n t c o n c e n t r a t i o n o f b i o l o g i c a l a c t i v e m o l e c u l e a n d t h e n u m b e r o f i t s r e c e p t o r s [ 1 9 4 ] . H o r m o n e o r d r u g r e l a t e d c h a n g e s i n r e c e p t o r n u m b e r a n d a f f i n i t y a p p e a r t o m e d i a t e c h a n g e s i n t i s s u e r e s p o n s i v e n e s s o r s e n s i t i v i t y t o t h e h o r m o n e a s w e l l . F o r e x a m p l e , r e s e r p i n e p r e - t r e a t m e n t o f r a b b -i t h a s b e e n s h o w n t o c a u s e p o s t - j u n c t i o n a l s u p e r - s e n s i t i v i t y o f n i c -t i t a t i n g m e m b r a n e [ 2 0 2 ] a n d c a r d i o v a s c u l a r t i s s u e t o n o r e p i n e p h r i n e [ 3 4 ] . C h r o n i c e x p o s u r e o f f r o g e r y t h r o c y t e s t o ( - ) - i s o p r o t e r e n o l h a s b e e n s h o w n t o s i g n i f i c a n t l y r e d u c e t h e n u m b e r o f f u n c t i o n a l b e t a -a d r e n e r g i c r e c e p t o r s [ 1 2 3 ] . P h e o c h r o m o c y t o m a s p l a c e d i n t e r s c a p u l a r l y i n t o h e a l t h y r a t s r e s u l t e d i n d i m i n i s h e d B - a d r e n o c e p t o r n u m b e r [ 1 8 2 ] . S i m i l a r l y , i n v i t r o i n c u b a t i o n o f p l a t e l e t m e m b r a n e s w i t h c l o n i d i n e c a u s e d a c o n c e n t r a t i o n - d e p e n d e n t d e c r e a s e o f ^ - a d r e n o c e p t o r b i n d i n g 3 s i t e s f o r [ H ] - y o h i m b i n e [ 1 9 ] . O n t h e o t h e r h a n d , s y m p a t h e t i c d e n e r -v a t i o n h a s b e e n s h o w n t o b e a s s o c i a t e d w i t h a n i n c r e a s e d n u m b e r o f c ^ - a d r e n e r g i c r e c e p t o r s i n r a t b r a i n [ 1 8 5 ] , a n d a s i m i l a r e f f e c t o f s y m p a t h e c t o m y w a s r e p o r t e d i n c a r d i a c c ^ - a n d j B - a d r e n o c e p t o r s [ 2 2 1 , 2 2 2 ] . T h e e f f e c t o f e p i n e p h r i n e o n e s t r o g e n p r i m e d r a b b i t U t e r u s i s p u r e l y a l p h a i n n a t u r e d u e t o a n i n c r e a s e d d e n s i t y o f « x - r e c e p t o r s [ 1 5 7 ] . I n c o n t r a s t , d u r i n g t h e p r o g e s t e r o n e p h a s e t h e e f f e c t o f e p i n e p h r i n e i s b e t a [ 1 2 4 ] d u e t o a d e c r e a s e i n t h e d e n s i t y o f o ( - r e c -e p t o r s [ 2 1 5 ] . C h r o n i c b l o c k a d e o f b e t a - a d r e n e r g i c r e c e p t o r s i s a l s o a s s o c i a t e d w i t h a n i n c r e a s e i n r e c e p t o r n u m b e r [ 4 8 , 6 5 ] . C h r o n i c b l o c k a d e o f d o p a m i n e r g i c r e c e p t o r s b y t h e a n t a g o n i s t h a l o p e r i d o l i n r a t b r a i n h a s b e e n s h o w n t o l e a d t o a n i n c r e a s e d n u m b e r o f d o p -3 a m i n e r g i c r e c e p t o r s a s m e a s u r e d [ H ] - h a l o p e r i d o l b i n d i n g [ 2 8 ] . M u k h e r j e e e t a l . [13 0 , 1 3 1 ] h a v e d e m o n s t r a t e d t h a t c h a n g e s i n t h e o c c u p a n c y o f r e c e p t o r s i t e s l e a d t o a l t e r a t i o n s i n t h e r e s p o n s i v e -n e s s o f t h e r e c e p t o r m e d i a t e d a m p l i f i c a t i o n s y s t e m a s w e l l . T h e s e r e s u l t s f r o m o t h e r t i s s u e s , m e n t i o n e d a b o v e , s u g g e s t e d t h a t i f f u n c t i o n a l ^ - a d r e n o c e p t o r s w e r e p r e s e n t o n i n f a n t b r o w n a d i p o c y t e s , t h e n a n y m o d u l a t i o n o f t i s s u e e n v i r o n m e n t e i t h e r b y c h e m i c a l s y m p a t h e c t o m y o r c h r o n i c b l o c k a d e o f ^ 2 - a d r e n o c e p t o r s w o u l d p e r h a p s r e s u l t i n c o m p e n s a t o r y a d a p t i v e c h a n g e s . T h i s w i l l b e s e e n i n t h e f o r m o f c h a n g e s i n ^ - a d r e n o c e p t o r s a f f i n i t y a n d / o r d e n s i t y . W e , t h e r e f o r e , d e c i d e d t o s t u d y i f s u c h e x p e r i m e n t a l m a n i p u l a t i o n o f b r o w n f a t w a s a s s o c i a t e d w i t h c h a n g e s i n ^ " a d r -e n o c e p t o r s n u m b e r . Two a p p r o a c h e s w e r e u n d e r t a k e n t o s t u d y t h i s p r o b l e m . 1) C h e m i c a l s y m p a t h e c t o m y o f s y m p a t h e t i c n e r v e t e r m i n a l s w a s p e r f o r m e d u s i n g 6 - h y d r o x y d o p a m i n e . A d m i n i s t r a t i o n o f a s i n g l e d o s e o f 6 - h y d r o x y d o p a m i n e h a s b e e n s h o w n t o d e s t r o y t h e n e r v e t e r -m i n a l s a n d p r e - t e r m i n a l a x o n s a n d r e s u l t i n i n v o l u t o r y c h a n g e s i n b r o w n f a t [ 1 1 , 1 7 6 , 1 9 7 , 1 9 8 ] . 2) I n t h e s e c o n d s e r i e s o f e x p e r -i m e n t s , i n f a n t r a t s w e r e e x p o s e d t o d a i l y a d m i n i s t r a t i o n o f y o h -i m b i n e ( a n ^ - a n t a g o n i s t ) t o c h r o n i c a l l y b l o c k t h e © ^ - a d r e n o c e p t o r s . i i i ) B i n d i n g o f a h o r m o n e t o r e c e p t o r s i t e s i s n o t s u f f -- 2 8 -icient to e l ic i t specific cellular responses. The hormonal sig^ nal can be deciphered only when passed on via an effector sys-tem(s). In the third part of our study, experiments were desi-gned to study the effector system(s) linked to ^-adrenoceptor stimulation. This study was an extension of the earlier reported in vivo study by Skala and Knight [178] suggesting a link between <*-adrenergic stimulation and the guanylate cyclase system. The hypothesis was further tested with three main objectives, 1) whether this relationship also held true for in vitro incubation of brown fat fragments with <<-agonists, 2) what was the possible nature of «K-receptor sub-types in this stimulation, and 3) whe-ther cyclic GMP generated activated cyclic GMP-dependent protein kinase. Although questions 1) and 2) were relatively simple to test, there were some inherent difficulties associated with tes-ting the activation of cyclic GMP-dependent protein kinase. An ^activation ratio" assay similar to that reported for cyclic AMP-dependent protein kinase [177] could not be adopted for the non-dissociating cyclic GMP-dependent enzyme. Instead, an alterna-tive approach, a modification [151] of the Dufau et al.[51] tech-nique, was used to separate the total cyclic nucleotide into "free" and a "protein bound" fraction of the nucleotide prior to their estimations by radioimmunoassays. Appropriate cyclic GMP-dependent protein kinase is the only protein known to specifi-cally bind its cyclic GMP inside a ce l l . Therefore, the absolute amount of protein-bound cyclic GMP was considered to be indica-tive of the activated enzyme. -29-(iv) Although ligand binding is used to sub-classify adrenergic receptors into their sub-types, i t is always complem-entary to the biological response associated with each receptor sub-type. In fact, i t has been suggested that the term "receptor" of any sub-type be reserved for those situations in which both binding and biological response are observed. A hormone binding site detected in the absence of a biological response to be ref-erred to as "acceptor" [17]. The opponents of this terminology consider i t to be highly arbitrary and biased by limitations of the experiments. For example, binding of insulin to human circulating mono-cytes, although not producing a biological response, is quantitati-vely reflective of the degree of insulin resistance and hyperins-ulinemia of the host. Presence of c(2-receptors has been demonstrated in hamster white adipocytes [33], in human white fat cells [103] and indi-r cated in adult brown adipose tissue [89, 190]. ©^ -adrenergic rece-ptor stimulation has been implicated in adenylate cyclase inhib^ ition and insulin release inhibition [135, 136]. Hormonal stimulation or inhibition of adenylate cyclase has been shown to involve the interaction of at least three major components: the hormone or neurotransmitter receptors(s): guan-ine-nucleotide-binding protein complex (N) and the catalytic moi-ety of adenylate cyclase (C). Interaction of agonist with receptor leads to interaction with a stimulatory (Ns) or inhibitory (Ni) regulatory complex which in turn modulates the activity of the catalytic component. An occupied receptor may increase or atten-uate cyclic AMP levels through its interaction with the guanine--30-n u c l e o t i d e - b i n d i n g p r o t e i n c o m p l e x ( N ) . T h e b i n d i n g o f N a n d C i s f a c i l i t a t e d b y G T P . I n o r d e r t o e x a m i n e t h e f u n c t i o n a l s t a -t u s o f * 2 - r e c e P t o r s i - n a d i p o c y t e s f r o m b r o w n a d i p o s e t i s s u e o f s e v e n - d a y - o l d r a t s , we c a r r i e d o u t a d d i t i o n a l s t u d i e s o n t h e i n -h i b i t o r y e f f e c t o f °<2~a<3oni-st ( c l o n i d i n e ) o n f o r s k o l i n - s t i m u l a t e d ( s u b - m a x i m a l s t i m u l a t i o n ) l i p o l y s i s . P e r t u s s i s t o x i n h a s b e e n s h o w n t o r e d u c e o r a b o l i s h t h e . , e f f e c t o f h o r m o n e s w h o s e a c t i o n s a r e m e d i a t e d b y d e c r e a s e i n c y c -l i c AMP f o r m a t i o n [ 3 7 ] . I t h a s b e e n s h o w n t h a t t h i s r e s u l t s f r o m a n ADP r i b o s y l a t i o n o f 4 1 k D a c o m p o n e n t o f N i [ 1 8 ] l e a d i n g t o i t s i n a c t i v a t i o n a n d t h u s p r e v e n t i n g i n h i b i t o r y i n p u t t o a d e n y l a t e c y c l a s e . A s e r i e s o f e x p e r i m e n t s w e r e c a r r i e d o u t t o i n v e s t i g a t e i f e * 2 ~ r e c e P t o r s t i m u l a t e d i n h i b i t i o n o f l i p o l y s i s i n a d i p o c y t e s f r o m b r o w n f a t o f o n e - w e e k - o l d r a t s w a s m e d i a t e d b y N i a s r e p o r t e d i n a d i p o c y t e s f r o m w h i t e f a t . (v) S i n c e d i s c o v e r y o f g e n e t i c a l l y o b e s e ( o b / o b ) m i c e b y H u m m e l e t a l . [ 8 7 ] , t h e y h a v e b e e n w i d e l y u s e d i n s t u d i e s o n m e t a b o l i c b a s i s o f b o d y w e i g h t r e g u l a t i o n , r e c e p t o r - m e d i a t e d t h e r m o g e n i c r e s p o n s e a n d a s a n e x p e r i m e n t a l m o d e l i n s t u d i e s o n d i a b e t i c o b e s -i t y [ 7 4 , 7 7 ] . T h e m i c e p r e s e n t a w i d e v a r i e t y o f e n d o c r i n e o r m e t -b o l i c d e f e c t s , s u c h a s h y p e r g l y c e m i a , h y p e r i n s u l i n e m i a w i t h i n s u l i n r e s i s t a n c e , h y p e r p h a g i a , h y p e r l i p i d e m i a a n d i n c r e a s e d s e n s i t i v i t y t o c o l d [ 6 , 1 2 , 3 2 , 7 3 , 1 1 8 , 1 7 5 , 1 8 8 , 2 2 6 ] . T h e s e d i s o r d e r s i n t h e s e a n i m a l s r e s u l t f r o m a m u t a t i o n w h i c h i s r e c e s s i v e l y i n h e r i -t e d [ 2 0 1 ] . A n u m b e r o f i n v e s t i g a t o r s h a v e a t t e m p t e d t o e l u c i d a t e t h e m o l e c u l a r m e c h a n i s m o f t h e m e t a b o l i c d e f e c t i n o b e s e m i c e - 3 1 -leading to energy conservation and increased metabolic efficiency. It has been suggested that brown adipose tissue of ob/ob mice exhibit alterations at several sites: one at the level of ^-rec-eptor, one at the level of lipolytic response, one at the level of fatty acid activation or B-oxidation [10, 175 J, one at the level of defective proton conductance pathway in brown fat mit-chondria [68, 69, 75, 82]. Several studies [14, 15, 44, 106] in -dicated that the adenylate cyclase activity of the obese mice adipose tissue did not respond to stimulation by ^-adrenergic agonists; however, NaF stimulation and sensitivity was found to be normal [43, 44, 106]. A possible defect at the GTP binding to the regulatory site of adenylate cyclase was suggested [14]. The . lipolytic response to dibutyryl cyclic AMP in white adipocytes from obese and lean mice was shown to be similar, probably indic-ating a normal hormone-sensitive lipase and protein kinase [13, 175]. Phosphodiesterase was reported to be abnormally elevated in obese mice compared to control lean mice [13, 92, 175], but addition of phosphodiesterase inhibitor failed to restore the lipo-lytic rate to normal after hormonal stimulation [42, 175]. Mild cold acclimation [82, 83] and chronic T4 treatment is said to improve the defective functioning of brown adipose tissue [76, 77, 196]. Skala and Knight [178] in their study demonstrated that cold exposure and norepinephrine injection produced an immediate rise in brown fat concentration of cyclic GMP. This suggested that cyclic GMP could play some role in the mechanism by which brown adipocytes increase their rate of heat production. We stud--32-ied this problem i n obese and lean mice to see i f c y c l i c GMP concentrations i n brown fat were influenced by cold exposure and Preceptor stimulation. 1.6 F i n a l l y , the main objectives of thi s study can be summ-arized as follows: (i) To i d e n t i f y and characterize °<2-acbrenergic receptors i n brown f a t of developing rats, ( i i ) To gain a d d i t i o n a l information about the receptor state as a function of modulation of i t s environment (e.g. changes i n density and a f f i n i t y r e s u l t i n g from denerva-ti o n or chronic blockade of receptors). ( i i i ) To show that <=<2-receptors are indeed functional receptors i . e i they show i n h i b i t o r y e f f e c t on /B-adrenergic stimu-lated g l y c e r o l release as reported i n adipocytes from white f a t i n human, hamsters and r a t s , (iv) To show that < * 2 - r e c e P t o r s a r e also involved i n some yet u n i d e n t i f i e d way i n activation of the guanylate cyclase system. (v) To carry out preliminary studies to investigate i f cyc-l i c GMP concentrations i n brown f a t of obese mice are influenced by cold exposure or ^-receptor stimulation. -33-C H A P T E R 2 M A T E R I A L S AND METHODS 2 . 1 M a t e r i a l s C l o n i d i n e h y d r o c h l o r i d e , b o v i n e s e r u m a l b u m i n , ( - ) 1 - n o r e p i n e p h r i n e , ( - ) 1 - e p i n e p h r i n e , p y r o c a t e c h o l , 6 - h y d r o x y -d o p a m i n e , a l p r e n o l o l , i s o p r o t e r e n o l h y d r o c h l o r i d e , d i t h i o t h r e i -t o l w e r e a l l o b t a i n e d f r o m S i g m a C h e m i c a l C o m p a n y , S t . L o u i s , MO. H 3 5 / 2 5 w a s r e c e i v e d f r o m H a s s l e a s a g i f t ; c o l l a g e n a s e ( t y p e 1) w a s o b t a i n e d f r o m W o r t h i n g t o n B i o c h e m i c a l , M i s s i s s a u g a , O n t . , F o r s k o l i n a n d P e r t u s s i s t o x i n w e r e p u r c h a s e d f r o m C a l b i o -c h e m , S a n D i e g o , C A . , D o b u t a m i n e a n d M e t o p r o l o l w e r e s u p p l i e d b y V a n c o u v e r G e n e r a l H o s p i t a l p h a r m a c y . F o r c y c l i c GMP a n d c y c l i c AMP d e t e r m i n a t i o n s , t h e New E n g l a n d N u c l e a r R I A k i t w a s u s e d 3 3 ( N E N - B o s t o n , M A ) , c y c l i c [ H ] AMP ( 3 1 . 3 C i / m m o l e ) a n d c y c l i c [ H ] GMP ( 9 . 1 C i / m m o l e ) w e r e t h o s e p r o v i d e d i n NEN R I A k i t s . O t h e r c h e m i c a l s u s e d w e r e f r o m F i s h e r S c i e n t i f i c C o m p a n y ( N . J . U S A ) a n d w e r e o f r e a g e n t g r a d e p u r i t y . HAWP M i l l i p o r e F i l t e r s ( p o r e s i z e 0 . 4 5 ^ m ) w e r e f r o m M i l l i p o r e C o r p o r a t i o n , B e d f o r d , M A . W h a t m a n G F / C g l a n m i c r o f i b e r f i l t e r s w e r e f r o m W h a t m a n C o r p o r a t i o n . L i q a n d s : l - [ 7 - H ( N ) ] - n o r e p i n e p h r i n e ( 1 8 . 8 C i / m m o l ) ; l - [ r i n g ] 2 , 5 , 6 - 3 H ] - n o r e p i n e p h r i n e ( 4 8 . 4 a n d 5 2 . 9 C i / m m o l ) ; . 3 — [ M e t h y l - H ] - r a u w o l s c i n e ( 7 9 . 5 C i / m m o l ) ; l - [ r i n g , p r o p y l - H ( N ) ] -d i h y d r o a l p r e n o l o l h y d r o c h l o r i d e ( 3 9 . 7 a n d 1 0 4 . 8 C i / m m o l ) ; -3 [ B e n z e n e r i n g - H ] - c l o n i d i n e h y d r o c h l o r i d e (61.1 C i / m m o l ) ; [ M e t h y l - > 3 H ] - y o h i m b i n e ( 7 5 ^ 0 - 8 0 . 5 C i / m m o l ) ; a l l w e r e - 3 4 -o b t a i n e d f r o m New E n g l a n d N u c l e a r . [ H ] R X - 7 8 1 0 9 4 w a s o b t a i n e d f r o m A m e r s h a m R a d i o c h e m i c a l s . 2 . 2 A n i m a l s A l b i n o W i s t a r r a t s w e r e u s e d t h r o u g h o u t . T h e y w e r e k e p t a t r o o m t e m p e r a t u r e ( 2 3 ° C) i n 12 h r - 1 2 h r l i g h t - d a r k c y c l e : , a n d c . w e r e f e d P u r i n a Chow a n d w a t e r a d l i b i t u m . P r e g n a n c i e s w e r e d a t e d b y v a g i n a l s m e a r s . N e w b o r n s w e r e r e d u c e d t o e i g h t a n i m a l s p e r l i t t e r a n d l e f t w i t h t h e i r m o t h e r s f o r 7 d a y s a t 23° C i n 12/12 h r l i g h t / d a r k c y c l e . _ T h e a n i m a l s w e r e d e c a p i t a t e d a n d t h e i n t e r s c a p u l a r b r o w n a d i p o s e t i s s u e ( B A T ) w a s r a p i d l y p r o -c e s s e d a s d e s c r i b e d i n t h e M e t h o d s s e c t i o n . O b e s e m i c e m a l e C 5 7 B L / 6 J / 6 J o b / o b a n d t h e i r n o r m a l l e a n c o n t r o l s (+/+ o r o b / + ) w e r e o b t a i n e d f r o m t h e J a c k s o n M e m o r i a l L a b o r a t o r i e s , B a r H a r b o r , ME a t 5 w e e k s o f a g e . T h e y w e r e k e p t i n p l a s t i c c a g e s a t 2 3 ° C i n 12 h r - 1 2 h r l i g h t - d a r k c y c l e a n d w e r e f e d P u r i n a C h o w a n d w a t e r a d l i b i t u m . T h e m i c e w e r e u s e d i n e x p e r i m e n t s a t t h e a g e s o f a p p r o x i m a t e l y 16 w e e k s . M a l e G o l d e n h a m s t e r s w e i g h i n g 6 0 - 8 0 g ( C h a r l e s R i v e r L a b o r a t o r i e s , L a k e v i e w , N J ) w e r e k e p t a t 2 5 ° C w i t h a 12 h r -12 h r l i g h t - d a r k l i g h t i n g s c h e d u l e a n d g i v e n f o o d a n d w a t e r a d l i b i t u m . T h e a n i m a l s w e r e k e p t a t t h i s s c h e d u l e f o r a t l e a s t o n e w e e k b e f o r e u s e . T h e a n i m a l s w e r e t h e n k i l l e d b y c a r b o n d i o x i d e n a r c o s i s , a n d b r o w n a d i p o s e t i s s u e w a s r e -m o v e d f r o m i n t e r s c a p u l a r , s u b - s c a p u l a r , c e r v i c a l a n d a x i l -l a r y r e g i o n s . T h e b r o w n f a t w a s t r i m m e d o f e x t r a n e o u s t i s s u e , m i n c e d w i t h s c i s s o r s a n d c e l l s p r e p a r e d a s d e s c r i b e d s u b s e q u e n t l y - 3 5 -To p r e p a r e c e l l s f r o m b r o w n a d i p o s e t i s s u e , a d u l t r a t s ( m a l e b r e e d e r a n d f e m a l e ) w e r e o b t a i n e d f r o m C h a r l e s R i v e r L a b o r a t o r i e s , L a k e v i e w , New J e r s e y . Two f e m a l e s a n d a s i n g l e m a l e w e r e k e p t t o g e t h e r f o r a w e e k i n a b r e e d e r c a g e . T h e y w e r e m a i n t a i n e d a t 2 5 ° C w i t h 1 2 : 1 2 l i g h t - d a r k l i g h t i n g s c h e d u l e a n d g i v e n f o o d a n d w a t e r a d l i b i t u m . A f t e r a w e e k t h e f e m a l e s w e r e s e p a r a t e d f r o m t h e m a l e b r e e d e r r a t a n d p u t i n s e p a r a t e c a g e s u n t i l i n f a n t a n i m a l s w e r e d e l i v e r e d . T h e y w e r e a l l o w e d t o r e m a i n w i t h t h e i r m o t h e r f o r 7 d a y s , a f t e r w h i c h t h e y ( a p p r o x i m a t e l y 6 0 y o u n g r a t s ) w e r e u s e d a s a s o u r c e f o r b r o w n a d i p o s e t i s s u e . 2 . 3 M e t h o d s 2 . 3 . 1 F o r 6 - h y d r o x y d o p a m i n e p r e - t r e a t m e n t , 5 0 m g / k g b o d y w e i g h t o f f r e e b a s e [ 1 7 6 ] k e p t a t - 2 0 ° C i n t h e d a r k a n d d i s s o l v e d i n 0 . 9 % N a C l i m m e d i a t e l y b e f o r e t h e e x p e r i m e n t , w a s i n j e c t e d i n t r a p e r i t o n e a l l y i n t o 5 - d a y - o l d r a t s . R a n d o m l y i n t e r c h a n g e d l i t t e r m a t e s i n j e c t e d w i t h s a l i n e s e r v e d a s c o n t r o l s . F o l l o w i n g a s i n g l e i n t r a p e r i t o n e a l d o s e o f 6 - h y d r o x y d o p a m i n e , t h e a n i m a l s w e r e k i l l e d o n d a y 8 . F o r c h r o n i c y o h i m b i n e p r e - t r e a t m e n t , 1 4 - d a y - o l d r a t s w e r e i n j e c t e d d a i l y w i t h y o h i m b i n e ( 1 0 m g / k g b o d y w e i g h t i . p . ) f o r 7 d a y s . T h e y w e r e s a c r i f i c e d o n d a y 2 1 . 2 . 3 . 2 P l a s m a m e m b r a n e p r e p a r a t i o n : 6 0 - 7 0 s e v e n - d a y - o l d r a t s w e r e d e c a p i t a t e d , t h e i r i n t e r s c a p u l a r b r o w n f a t w a s r a p i d l y d i s s e c t e d , t r i m m e d o f e x t r a n e o u s t i s s u e s a n d i m m e r s e d - 3 6 -i n i c e - c o l d K r e b s - R i n g e r b i c a r b o n a t e b u f f e r , pH 7 . 4 c o n -t i n u o u s l y b u b b l e d w i t h 9 5 % Q>2 + 5% CO 2 . P o o l e d b r o w n f a t ( 6 - 7 g ) w a s m i n c e d w i t h s c i s s o r s a n d f i l t e r e d t h r o u g h N i t e x N o . 3 s i l k c l o t h ( N i t e x C o . , S w i t z e r l a n d ) . A m o d i -f i c a t i o n o f t h e m e t h o d d e s c r i b e d b y M a e d a e t a l . [ 1 1 7 ] w a s u s e d t o i s o l a t e t h e p l a s m a m e m b r a n e f r a g m e n t s . A l l p l a s t i c a n d c e l l u l o s e n i t r a t e t u b e s w e r e s o a k e d o v e r n i g h t i n i m M E D T A , pH 7 . 0 a n d r i n s e d t e n t i m e s w i t h d o u b l e d i s t i l l e d w a t e r b e f o r e u s e . A l l p r o c e d u r e s w e r e c a r r i e d o u t a t 0° - 4 ° C . T h e f i n e l y m i n c e d t i s s u e w a s s u s p e n d e d i n 10 v o l u m e s o f t h e h o m o g e n i z a t i o n b u f f e r ( 1 0 nM s o d i u m p h o s p h a t e b u f f e r , pH 7 . 4 c o n t a i n i n g 1 mM M g C l 2 , 30 mM N a C l , 1 mM d i t h i o t h r e i t o l ) a n d h o m o g e n i z e d b y 8 s t r o k e s o f t h e T e f l o n p e s t l e i n a g l a s s P o t t e r -E l v e h j e m h o m o g e n i z e r . T h e p r o c e d u r e w a s r e p e a t e d 3 t i m e s s e p a r a t e d b y 1 t o 2 m i n . o f c o o l i n g i n i c e c o l d w a t e r . 10 m l o f t h e h o m o g e n a t e w a s l a y e r e d o v e r 10 m l o f 4 1 % o f s u c r o s e i n t h e h o m o g e n i z a t i o n b u f f e r a n d c e n t r i f u g e d a t 9 5 , 0 0 0 x g ( 3 3 , 0 0 0 r p m ) f o r 1 h r i n a B e c k m a n L B - 5 5 u l t r a c e n t r i f u g e . T h e w h i t e i n t e r f a c i a l b a n d o f m e m b r a n e s w a s c o l l e c t e d i n 15% s u c r o s e s o l u t i o n i n t h e h o m o g e n i z a t i o n b u f f e r , a n d t h e m e m b r a n e s w e r e p e l l e t e d b y c e n t r i f u g a t i o n a t 9 5 , 0 0 0 x g f o r 20 m i n u t e s . T h e m e m b r a n e s w e r e w a s h e d t w i c e u s i n g i d e n t i c a l r e - s u s p e n s i o n a n d c e n t r i f u g a t i o n . I n t h e c a s e o f a s s a y s i n v o l v i n g b i n d i n g t o o c - r e c e p t o r s , t h e m e m b r a n e p r e p a r a t i o n w a s r e w a s h e d w i t h 2 . 5 mM EDTA i n 50 mM T r i s t o r e m o v e m e m b r a n e - b o u n d i o n s a n d G T P . A l l m e m b r a n e p r e p a r a t i o n s w e r e s u s p e n d e d i n 50 mM T r i s - H C l , p H - 3 7 -7 . 4 c o n t a i n i n g ^ m M M g C l 2 r a p i d l y f r o z e n , s t o r e d a t - 2 0 C a n d u s e d f o r b i n d i n g a s s a y s w i t h i n 1 - 3 w e e k s . P l a s m a m e m b r a n e p r o t e i n c o n t e n t s w e r e d e t e r m i n e d a c c o r d i n g t o L o w r y e t a l . [ 1 1 6 ] . 2 . 3 . 3 B i n d i n g A s s a y s : A l i q u o t s o f m e m b r a n e p r e p a r a t i o n s w e r e t h a w e d i m m e d i a t e l y b e f o r e u s e a n d d i l u t e d t o a c o n -c e n t r a t i o n o f 1 . 6 - 4 . 0 mg p r o t e i n / m l a n d g e n t l y r e h o m o g -e n i z e d i n a h a n d - h e l d g l a s s t i s s u e h o m o g e n i z e r w i t h t w o u p a n d d o w n s t r o k e s . D u p l i c a t e s a m p l e s i n E p p e n d o r f i n c u b a t i o n t u b e s ( B r i n k m a n I n s t r u m e n t s , W e s t b u r y , NY) w e r e i n c u b a t e d i n 0 . 5 m l o f a p p r o p r i a t e i n c u b a t i o n b u f f e r . B i n d i n g o f 3 3 ( - ) - [ H ] - n o r e p i n e p h r i n e a n d [ H ] - c l o n i d i n e w a s d e t e r m i n e d i n 5 0 mM T r i s - H C l b u f f e r , pH 7 . 4 c o n t a i n i n g 10 mM M g C l 2 a n d 0 . 1 m g / m l a s c o r b i c a c i d b i n d i n g o f [ H ] - y o h i m b i n e w a s c a r r i e d o u t i n 50 mM g l y c y l g l y c i n e , p H 7 . 4 c o n t a i n i n g 10 mM M g C l 2 , a n d b i n d i n g o f [ 3 H ] - R X - 7 8 1 0 9 4 w a s a s s a y e d i n 5 0 mM N a -K - p h o s p h a t e b u f f e r , pH 7 . 4 , a l s o c o n t a i n i n g 10 mM M g C l 2 . T h e a b o v e i n c u b a t i o n m e d i a w e r e f o u n d t o b e o p t i m a l f o r t h e s p e c i f i c b i n d i n g o f t h e r e s p e c t i v e l i g a n d s i n a s e r i e s o f p r e l i m i n a r y e x p e r i m e n t s . I n t h e s e , t h e b i n d i n g w a s f o u n d t o b e d i r e c t l y p r o p o r t i o n a l t o t h e m e m b r a n e p r o t e i n c o n c e n -t r a t i o n o v e r a r a n g e o f 0 . 0 5 - 0 . 4 m g / m l a n d t h u s , 0 . 0 8 - 0 . 2 mg w a s u s e d i n a s t a n d a r d a s s a y . A f t e r 20 m i n . a t r o o m t e m -p e r a t u r e ( a l s o e s t a b l i s h e d b y p r e l i m i n a r y e x p e r i m e n t s a s y i e l d i n g m o s t c o n s i s t e n t r e s u l t s ) , t h e i n c u b a t i o n s w e r e t e r m i n a t e d b y a d d i n g 0 . 8 m l o f i c e - c o l d w a s h i n g b u f f e r - 3 8 -( 50 mM Tris-HCl or 50 mM glycylglycine, pH 7.4 containing 10 mM MgC^/ 3 mM pyrocatechol and 0.1 mg/ml ascorbic acid) f o l l -owed by rapid vacuum filtration through Whatman GF/C glass f ib-er f i l ters. Filters were washed 4 times with 12 ml aliquots of the washing buffer, suspended in Biofluor (New England Nuclear, Boston, MA) and their radioactivity assessed in a Beckman LS-9000 liquid scintillation counter at 45% efficiency. The specific binding of each ligand was determined by taking a difference of the total binding in the absence of non-labelled drug and the non-specific binding in the presence of more than 100 X of the non-labelled competing drug. Generally 2-4 ligand binding experiments were carried out. Each experiment was done in duplicate or triplicate. Duplicate determinations varied between 3-15% and their mean was used in calculating the specific binding. The experiment with optimal range of ligand concentrations and a l l values fitting the comp-uter-predicted plot of BCTIC computer program for equilibrium binding assays (Med. 40 and Med.79) was selected as the"represen-tative" o r "typical"experiment [121, 134] . 2.3.4 Effect of ^-agonists and antagonists in vitro on total, free and receptor-bound cyclic GMP contents of brown fat fragments from one-week-old rats.  A total of approximately 3.0- 3.5 g of BAT from 4 0 one-week-old rats was pooled, minced with scissors and filtered on Nitex silk f i l ter under suction. These tissue pieces were then divided into aliquots of approximately 100-200 mg each to be used for the in vitro incubations. -39-I n c u b a t i o n o T i s s u e f r a g m e n t s ( . 0 . 1 - 0 . 2 g ) w e r e i n c u b a t e d a t 37 C i n a t o t a l v o l u m e o f 1 m l o f K r e b s - R i n g e r b i c a r b o n a t e b u f f e r , p H 7 . 4 , w h i c h c o n t a i n e d h a l f o f r e c o m m e n d e d C a + + , 3% B S A ( b o v -i n e s e r u m a l b u m i n t y p e V ) , 5 mM g l u c o s e a n d 1 mM 3 - i s o b u t y l -m e t h y l x a n t h i n e . A l l s a m p l e s w e r e p r e - i n c u b a t e d i n a n a t m o s p h e r e o f 95% C>2 + 5% C 0 2 w i t h o r w i t h o u t 0 . 4 mM a n t a g o n i s t s ( p r a z o s i n , y o h i m b i n e , m e t o p r o l o l , H 3 5 / 2 5 ^ ^ o r ^ m i n . f o l l o w e d b y a 3 m i n . i n c u b a t i o n w i t h 0 . 5 - 20 uM c l o n i d i n e o r 0 . 5 - 5 0 /iM d o b u t -a m i n e . R e a c t i o n s w e r e s t o p p e d b y i m m e r s i n g t h e i n c u b a t i o n f l a s k s i n i c e - w a t e r a n d s e p a r a t i n g t h e t i s s u e f r a g m e n t s f r o m t h e i n c u -b a t i o n m e d i u m b y f i l t r a t i o n o n N i t e x s i l k c l o t h u n d e r s u c t i o n . S t u d i e s c a r r i e d o u t o n i s o l a t e d t i s s u e p i e c e s a n d s l i c e s r e p o r t u s i n g h i g h c o n c e n t r a t i o n s o f a n t a g o n i s t s t o a n t a g o n i z e t h e a g o n i s t s r e s p o n s e o n t h e t i s s u e . Y a m a s h i t a e t a l . ( 1 9 8 0) [ 2 2 5 ] r e p o r t e d u s i n g 1 0 0 uM y o h i m b i n e t o a n t a g o n i z e n o r e p i n e p h -r i n e i n h i b i t o r y e f f e c t s o n T S H - s t i m u l a t e d i n c r e a s e i n c y c l i c AMP l e v e l s i n c a n i n e t h y r o i d s l i c e s . P h i l i p p u a n d S h a r t n e r ( 1 9 7 6 ) [ 1 5 0 ] u s e d 0 . 1 mM t o 1 mM y o h i m b i n e t o b l o c k p r e s s o r r e s -p o n s e a n d i n c r e a s e i n b l o o d p r e s s u r e a s s o c i a t e d w i t h e l e c t r i c a l s t i m u l a t i o n o f a n t e r i o r h y p o t h a l a m u s . I n v i e w o f t h e s e r e p o r t e d s t u d i e s w e , t h e r e f o r e , c o n s i d e r e d i t a p p r o p r i a t e t o u s e r e l a t -i v e l y h i g h c o n c e n t r a t i o n s o f a n t a g o n i s t s t o a n t a g o n i z e t h e e f f -e c t s o f a g o n i s t s o n b r o w n f a t f r a g m e n t s . - 4 0 -Extraction Procedures Incubation media. T r i c h l o r a c e t i c acid (40% w/v) was added to give a f i n a l concentration of 10%; tubes were vort-o exed and centrifuged at 3,000 X g for 15 minutes at 4 C. The clear supernatant was neutralized with s o l i d CaCO^/ r e c e n t r i -fuged at 3,000 X g for 15 minutes and evaporated to dryness at 60°C. Tissue fragments from each incubation flask were immediately placed i n 1 ml of TIM buffer (Tris-HCl 20 mM, pH 7.4; mercaptoethanol 0.1 mM, 3-isobutylmethylxanthine 1 mM) and homogenized using Brinkman Instrument Polytron. F i n a l volume of each homogenate was adjusted to 2.5 ml. For the deter-mination of t o t a l tissue c y c l i c nucleotide content, an aliquot of the homogenate was deproteinized using 40% TCA to give f i n a l concentration of 10% and treated i d e n t i c a l l y as the deprotein-ized incubation media samples. I n t r a - c e l l u l a r protein-bound c y c l i c nucleotides. In order to separate the t o t a l i n t r a c e l l u l a r c y c l i c nucleotides into the "free" f r a c t i o n and the f r a c t i o n bound to the reg-ulatory subunit of the representative protein kinase ("pro-tein-bound" f r a c t i o n ) , we have adopted the method described by Podesta et a l . [151]. This method i s based upon the ob-servation of Dufau et a l . [51] that the receptor protein with c y c l i c AMP bound to i t i s retained by the HAWP M i l l i p o r e f i l -ters of 0.45 pm pore siz e , whereas the unbound c y c l i c AMP can f r e e l y pass through. 1 ml aliquots of the above described tissue homogenate were spun at 10,000 X g for 10 minutes, the -41-pellet discarded, and the supernatant diluted with further 2 ml of TIM buffer and filtered through Millipore f i l ters. The fi lters were rewashed with further 2 x 2 ml of the TIM buffer dried, and transferred into tubes containing 0.8 ml of TIM . buffer. They were heated in boiling water bath for 10 min-utes. After evaporation to dryness, these eluates were used for 1 bound ' cyclic nucleotide determinations. Control experiments for bound cyclic nucleotide determinations. These were carried out to determine Ci) whether un-bound cyclic nucleotides re-associate with free receptor pro-tein and (ii) whether bound cyclic nucleotides dissociate into free cyclic nucleotides and free receptor protein, during the course of experimental procedures used for isolating bound cyc-l i c nucleotides. (i) Measurement of association of free cyclic nucleotide  with free receptor protein. For this, brown adipose tissue was homogenized in TIM buffer and centri-fuged as described above. 0.2 ml aliquots of the supernatant were pre-incubated for 10 to 20 min-o utes at 4 C, followed by a second incubation of 30 minutes with 300 pi of 80 mM potassium phosphate buffer, pH 6.5, containing 10 mM magnesium acetate, 1 mM 2-mercaptoethanol, 0.1 mM 3-isobutyl-l-methyl-3 xanthine and 22 nM [ H] cyclic nucleotide. Non-speci-f ic binding was determined in parallel incubation in the presence of 10 juM non-labelled cyclic nucleotides C y c l i c n u c l e o t i d e b o u n d t o r e c e p t o r p r o t e i n w a s i s o -l a t e d b y m i l l i p o r e f i l t r a t i o n a s d e s c r i b e d a b o v e . T h e f i l t e r s w e r e t r a n s f e r r e d t o p l a s t i c s c i n t i l l a t i o n v i a l s , d r i e d f o r 1 h o u r , a n d d i s s o l v e d i n 2 m l o f 2 - m t h -o x y e t h a n o l . A f t e r a d d i t i o n o f 10 m l o f s c i n t i l l a t i o n c o c k t a i l (10 g o f 2 . 5 - d i p h e n y l o x a z o l a n d 0 . 3 g o f 1 . 4 b i s - [ 4 - m e t h y l - 5 - p h e n y l o x a z o l - 2 y l ] b e n z e n e i n a l i t r e o f 3 T o l u e n e , H - r a d i o a c t i v i t y w a s c o u n t e d i n a l i q u i d s c i n -t i l l a t i o n c o u n t e r w i t h a n e f f i c i e n c y o f 2 0 % . ( i i ) M e a s u r e m e n t s o f d i s s o c i a t i o n o f b o u n d c y c l i c n u c l e o t i d e . F o r t h i s p u r p o s e , b r o w n a d i p o s e t i s s u e w a s h o m o g e n i z e d i n t h e p r e s e n c e a n d a b s e n c e o f m e r c a p t o e t h a n o l . T h e h o m -o g e n a t e w a s t h e n c e n t r i f u g e d a t 1 0 , 0 0 0 x g f o r 10 m i n u t e s . o T h e s u p e r n a t a n t w a s t h e n i n c u b a t e d a t 4 C f o r v a r i o u s i n t e r v a l s o f t i m e ( 0 , 1 5 , 30 m i n u t e s ) a n d p r o c e s s e d a s d e s c r i b e d a b o v e f o r i n t r a c e l l u l a r p r o t e i n b o u n d c y c l i c n u c l e o t i d e . Measurement of free and cyclic nucleotide-bound receptor sites. These experiments were carried out to determine the specificity of cyclic nucleotide binding to its receptor protein. Tissue fragments were incubated with 50 pM dobutamine and 0.5 uM clonidine and processed as described above for bound cyclic nucleotide samples. After homogenization, the homogenate was filtered through cheese cloth to remove fat. 200 p.1 of homo-genate was incubated with 300 pi of 80 mM-potassium phosphate - 4 3 -buffer pH 6.5, containing 10 mM magnesium acetate; 1 mM 2-mercaptoethanol; 0.1 mM 3-isobutyl-l-methylxanthine and 2 nM 3 o [ H]-cyclic nucleotide incubated for 30 minutes at 2-4 C f o r free receptor s i t e s (non-exchange conditions). Non-specific binding was determined i n p a r a l l e l incubations i n the presence of 10 uM non-labelled c y c l i c nucleotides. The rest of the procedure was the same as described i n c y c l i c nucleotide association with free receptor protein experiments. C y c l i c GMP and C y c l i c AMP Assays. Dry samples were re-constituted i n sodium-acetate buffer (pH 6.2, 50 mM) and a f t e r centrifugation at 3,000 x g for 15 minutes to remove a d d i t i o n a l traces of CaCO^/ supernatants were used for RIA assays of c y c l i c nucleotides. The method of Steiner et a l . [187] was used with addition of acetylation step described by Harper et al.[71 ] . The New England Nuclear (NEN) Boston, MA assay k i t s were used. Sam-ples i n duplicate were counted i n Searle Instrument gamma counter and standard curves were prepared for each set of determinations. Protein was assayed by the method of Lowry et a l . [116] with bovine serum albumin as a standard. 2.3.5 E f f e c t of agonists i n v i t r o on c y c l i c GMP contents i n brown f a t fragments from obese mice and t h e i r lean l i t t e r mates.  Total brown adipose tissue from 35 obese and 35 control mice (normal lean l i t t e r mates) was pooled, minced, f i l t e r e d on Nitex # 30 f i l t e r , and divided into aliquots of approximately 100 mg each to be used for the subsequent i n v i t r o incubations. The rest of basic methodology for tissue incubation and c y c l i c -44-n u c l e o t i d e s e x t r a c t i o n a n d a s s a y s w a s t h e s a m e a s d e s c r i b e d e a r l i e r . 2 . 3 . 6 I n h i b i t i o n o f F o r s k o l i n - s t i m u l a t e d l i p o l y s i s i n a d i p o c y t e s f r o m b r o w n f a t o f 1 - w e e k - o l d r a t s b y c l o n i d i n e .  T h e b r o w n a d i p o s e t i s s u e f r o m 1 - w e e k - o l d r a t s w a s r e m o v e d f r o m t h e i n t e r s c a p u l a r , s u b s c a p u l a r , c e r v i c a l a n d a x i l l -a r y r e g i o n s , t r i m m e d o f e x t r a n e o u s t i s s u e a n d m i n c e d w i t h s c i s s o r s . M i n c e d t i s s u e p i e c e s w e r e s u s p e n d e d i n K r e b s - R i n g e r b i c a r b o n a t e b u f f e r ( a p p e n d i x C) c o n t a i n i n g 6 m g / m l c r u d e b a c t e r i a l c o l l a g e n a s e a n d 10 m g / m l b o v i n e s e r u m a l b u m i n t y p e V a n d i n c u b a t e d i n a a s h a k i n g w a t e r b a t h a t 37 C f o r 1 5 m i n u t e s . D u r i n g t h e d i g e s t i o n p e r i o d , t h e p l a s t i c v i a l c o n t a i n i n g t i s s u e s u s p e n s i o n w a s r e -m o v e d a n d s h a k e n v i g o r o u s l y b y h a n d e v e r y 4 m i n u t e s . F o l l o w i n g d i g e s t i o n t h e a d i p o c y t e s u s p e n s i o n w a s f i l t e r e d t h r o u g h a c o a r s e N i t e x f i l t e r . T h e c e l l s w e r e r e - s u s p e n d e d i n K r e b s - R i n g e r b i -c a r b o n a t e b u f f e r p H 7 . 4 c o n t a i n i n g 1 . 5 mM C a C l 2 a n d 40 m g / m l (.4% W/V) b o v i n e s e r u m a l b u m i n t y p e V . T h e pH o f t h i s m e d i u m w a s a d j u s t e d t o 7 . 4 a f t e r a d d i t i o n o f b o v i n e s e r u m a l b u m i n t y p e V . T h e c e l l s u s p e n s i o n w a s c e n t r i f u g e d a t 50 x g f o r 5 m i n u t e s a t r o o m t e m p r a t u r e . T h e i n f r a n a t a n t w a s r e m o v e d b y a s p i r a t i o n , a n d t h e a d i p o c y t e s w e r e w a s h e d w i t h a n o t h e r 2 5 - 3 0 m l o f a l b u m i n - f r e e K r e b s - R i n g e r b i c a r b o n a t e b u f f e r . F o l l o w i n g f o u r w a s h i n g s , t h e c e l l s w e r e s u s p e n d e d i n m e a s u r e d v o l u m e o f a l b u m i n - f r e e K r e b s -R i n g e r b i c a r b o n a t e b u f f e r a n d a n a l i q u o t o f 1 0 0 fxl o f c e l l s u s -p e n s i o n w a s t a k e n f o r t h e a s s a y o f c e l l p r o t e i n c o n t e n t . T h e a d i p o c y t e s u s p e n s i o n w a s r e - s u s p e n d e d i n a n e q u a l v o l u m e o f a n o t h e r K r e b s - R i n g e r b i c a r b o n a t e b u f f e r c o n t a i n i n g 80 m g / m l (8% W/V) B S A . I n a c o n t r o l e x p e r i m e n t t h e K r e b s - R i n g e r b i c a r b -o n a t e b u f f e r c o n t a i n i n g BSA ( t y p e V) w a s w a s h e d f o u r t i m e s w i t h a l b u m i n - f r e e K r e b s - R i n g e r b i c a r b o n a t e b u f f e r t o d e t e r m i n e t h e a m o u n t o f a . l b u m i n l e f t a f t e r t h e s e w a s h i n g s . T h e i s o l a t e d b r o w n a d i p o c y t e s w e r e s t o r e d u n d e r a n a t m o s p h e r e o f 0 2 ~ C 0 2 ( 9 5 : 5 % ) i n o a s l o w l y s h a k i n g w a t e r b a t h a t 37 C f o r a p p r o x i m a t e l y 4 5 m i n u t e s b e f o r e u s e . V a r i o u s c o m b i n a t i o n s o f d r u g s ( c l o n i d i n e , y o h i m b i n e , f o r s k o l i n ) w e r e p i p e t t e d i n t o p l a s t i c t u b e s , a n d s u b s e q u e n t t o a p r e i n c u b a t i o n p e r i o d o f 3 0 m i n u t e s a n a l i q u o t o f c e l l s u s p e n s i o n w a s t r a n s f e r r e d i n t o t h e s e t u b e s . T h e v o l u m e o f e a c h t u b e w a s a d j u s t e d t o 1 m l . T h e s e t u b e s w e r e i n c u b a t e d f o r 1 5 m i n u t e s a n d t h e n t h e c o n t e n t w e r e f i l t e r e d t h r o u g h m i l l i p o r e 0 . 4 5 pm f i l t e r p a p e r u n d e r s u c t i o n . A 2 00 u l a l i q u o t o f t h e f i l t r a t e w a s r e m o v e d f o r a s s a y o f g l y c e r o l b y t h e W i e l a n d m e t h o d [ 2 1 2 ] . I n a n o t h e r s e t o f e x p e r i m e n t s , t h e c e l l s w e r e i n c u b a t e d w i t h p e r t u s s i s t o x i n ( 0 . 5 m g / m l ) f o r 3 ^ h o u r s , a n d t h e r e s p o n s e t o c l o n i d i n e i n h i b i t i o n o f f o r s k o l i n - s t i m u l a t e d l i p o l y s i s w a s r e - a n a l y z e d . 2 . 3 . 7 E f f e c t o f c l o n i d i n e o n f o r s k o l i n - s t i m u l a t e d g l y c e r o l r e l e a s e i n a d i p o c y t e s i s o l a t e d f r o m b r o w n f a t o f a d u l t h a m s t e r s .  A d i p o c y t e s f r o m a d u l t h a m s t e r s w e r e p r e p a r e d a c c o r d i n g t o t h e p r o c e d u r e a s d e s c r i b e d f o r a d i p o c y t e s f o r b r o w n f a t o f 1 - w e e k -o l d r a t s . 2 . 3 . 8 D a t a A n a l y s i s S t a t i s t i c a l a n a l y s i s o f t h e d a t a w a s d o n e u s i n g S t u d e n t - 4 6 -' t ' test wherever appropriate. Each of the paired designed experiment was analyzed by paired 1 t * test using Stat 1-029 program of Hewlett Packard calculator as developed by Ostle [14 5]. The statistical significance of the ligand binding experiments to plasma membrane fragments from brown fat of 6-hydroxydop-amine and yohimbine pre-treated animals was not determined because of insufficient experiments. However, the general trends in these binding, experiments were very similar to those already reported in other tissues [l23, 182, 185]. -47-CHAPTER 3 RESULTS 3.1 Identification and characterization of ©^ -adreno-ceptors binding sites in isolated plasma membrane fragments from brown adipose tissue of infant rats. Yohimbine a specific ©^ -adrenergic antagonist has been extensively used as a radioligand in characterization of ot.^-adrenergic receptors in rat brain cortex [66,163]. In order to ascertain the conditions of our assays we have repeated the experiments of Rouot et al.[163] using the methodology as described by U'Prichard and Snyder [204] for determining the c*2-aGirenoceptor binding characteristics of rat cortex. The binding characteristics of o< .2 - r e c e P t o r s i n r a t brain cortex were of the same order of magnitude as reported by Rouot[163] and Perry and U'Prichard [ 148] (see appendix A). Fig. 1A shows results from a representative experiment on [3Hlyohimbine binding to the plasma membrane fraction isolated from brown fat of one-week-old rats. The non-specific binding, assessed from addition of 10 (-)epinephrine or yohimbine, was directly proportional to free ligand concentr-ation. Specific binding ranged from 18% to 50% at high and low concentrations of the ligand, respectively, and binding showed apparent saturation at approximately 30 nM of [3H]yohimbine. Scatchard analysis indicated a single class of binding sites with a dissociation constant (Kd) of 18 nM and maximum number -48-F i g . 1 A. S p e c i f i c b i n d i n g of [ H]-yohimbine t o plasma membrane fragments i s o l a t e d from brown f a t of one-week-old r a t s as a f u n c t i o n of i n c r e a s i n g l i g a n d c o n c e n t r a t i o n i n the presence of 10 ^ iM yohimbine (•-•) and 10 AUM e p i n e p h r i n e (o-o) . Inse t : Scatchard p l o t f o r yohimbine data. Slope was determined u s i n g l i n e a r r e g r e s s i o n a n a l y s i s (r = 0.96). B. I n h i b i t i o n of [ H]-yohimbine b i n d i n g by 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 yohimbine (•-•) and ep i n e p h r i n e (o-o)• Each p o i n t r e p r e s e n t s the mean of t r i p l i c a t e d e t erminations i n one r e p r e s e n t a t i v e experiment. - 4 9 -of b i n d i n g s i t e s (B ) of 156 fmol/mg p r o t e i n ( l i n e a r a max ' / a f v r e g r e s s i o n c o e f f i c i e n t , r = 0.96). Displacement of the l i g a n d by yohimbine was g r e a t e r than t h a t by (-)-epinephrine on equimolar b a s i s ( F i g . IB). RX-781094, a h i g h l y s e l e c t i v e c^-adrenoceptor antago n i s t [50], has p r e v i o u s l y been used to c h a r a c t e r i z e f u n c t i o n a l r e c e p t o r s i n b r a i n [ 4 5 ] . F i g . 2A shows the s p e c i f i c 3 b i n d i n g of [ H]-RX-781094 to brown f a t plasma membranes as a f u n c t i o n of i n c r e a s i n g l i g a n d c o n c e n t r a t i o n i n the presence of 10 /tM yohimbine. The s p e c i f i c b i n d i n g showed an apparent number of b i n d i n g s i t e s of 20 fmol/mg p r o t e i n and h a l f s a t u r a t i o n ws reached at approximately 10 nM. The s p e c i f i c b i n d i n g was found to be temperature dependent. Lowering the i n c u b a t i o n temperature to 4°C almost completely e l i m i n a t e d the s p e c i f i c b i n d i n g . C l o n i d i n e , a compound of i m i d a z o l i n e group, possesses a p a r t i a l ©^-adrenoceptor ag o n i s t a c t i v i t y . S a t u r a t i o n b i n d i n g of c l o n i d i n e to brown f a t membranes was p o o r l y r e p r o d u c i b l e . We have determined c l o n i d i n e b i n d i n g i n the presence of 10 /uM yohimbine or 10 /*M c l o n i d i n e ( F i g . 2B). The apparent number of s p e c i f i c b i n d i n g s i t e s was approximately 50 fmol/mg p r o t e i n and h a l f s a t u r a t i o n was reached at approximately 25 nM. Since s y n a p t i c r e l e a s e of (-)-norepinephrine i s the p r i n c i p a l i n v i v o s t i m u l u s a c t i n g upon brown f a t membranes from i n f a n t r a t s , we, t h e r e f o r e , s t u d i e d the b i n d i n g c h a r a c -3 t e r i s t i c s of (-)[ H]-norepinephrine to i s o l a t e d plasma membranes from brown f a t . I n i t i a l experiment showed the -50-X o 10 20 30 40 [3H] RX 781094 (nM) 50 F i g 2. A. 20 40 60 80 100 [3H] Clonidine (nM) S p e c i f i c b i n d i n g o f [ 3H]-RX 781094 t o plasma membrane fragments i s o l a t e d from brown f a t o f one-week-old r a t s i n the presence o f 10 p-M yohimbine as a f u n c t i o n o f i n c r e a s i n g l i g a n d c o n c e n t r a t i o n . R e s u l t s a r e the mean o f t r i p l i c a t e d e t e r m i n a t i o n s i n a s i n g l e experiment. B. S p e c i f i c b i n d i n g o f [^HUclonidine t o i d e n t i c a l membrane fragments i n the presence of 10 /*.M yohimbine (•-•) or 10 /*M c l o n i d i n e ( A - A ) . R e s u l t s a re shown as the mean + S.E.M. o f 4 experiments done i n d u p l i c a t e . -51-a. 20 60 120 240 gig. 2 C. Specific binding of [ H]-norepinephrine to plasma membrane framents isolated from brown fat of one-week-old rats i n the presence of 20 /iM norepinephrine and 20 /iM clonidine (•-•) or 20 /JM clonidine (o-o). Each point i s the mean of duplicate determinations from two experiments. D . Specific binding of [ H]-norepinephrine to plasma membrane fragments isolated from brown f a t of one-veek-old rats i n the presence of 10 /iM epinephrine (•-•) or 100 yohimbine (o-O). Each point i s the mean + S.E.M. of duplicate determinations from four experiments. -52-b i n d i n g w a s r a p i d , s t a b l e a n d d i s p l a c e a b l e i n t h e p r e s e n c e o f 20 juM ( - ) - n o r e p i n e p h r i n e , 20 ^ M c l o n i d i n e , 10 ( - ) - e p i n e p h r i n e o r 100 y o h i m b i n e ( F i g . 2C a n d D ) . I n e q u i l i b r i u m b i n d i n g s t u d i e s ( F i g . 3 A ) , t h e s p e c i f i c 3 b i n d i n g o f t h e ( - ) [ H ] - n o r e p i n e p h r i n e t o p l a s m a m e m b r a n e f r a g m e n t s w a s r e v e r s i b l e a n d s a t u r a b l e . B o t h ( - ) - e p i n e p h r i n e a n d 3 y o h i m b i n e d i s p l a c e d ( - ) [ H ] - n o r e p i n e p h r i n e f r o m i t s b i n d i n g s i t e s i n a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r a n d w e r e e q u i p o t e n t i n t h e i r e f f e c t s . W h e n 10 pM ( - ) - e p i n e p h r i n e a n d 10 /iM y o h i m b i n e w e r e u s e d t o g e t h e r t o d i s p l a c e ( - ) [ H ] - n o r e p i n e p h r i n e b i n d i n g , t h e i r c o m b i n e d e f f e c t w a s o n l y 3 0 % h i g h e r t h a n t h e e f f e c t o f e i t h e r o n e a l o n e . T h e d i f f e r e n c e b e t w e e n t h e s p e c i f i c b i n d i n g o f t h e ( - ) [ H ] - n o r e p i n e p h r i n e i n t h e p r e s e n c e o f 10 /AM e p i n e p h r i n e a l o n e a n d t h a t d e t e r m i n e d i n t h e p r e s e n c e o f a c o m b i n a t i o n o f 10 /uK ( - ) - e p i n e p h r i n e a n d 10 y o h i m b i n e w a s d e s c r i b e d a s a 3 y o h i m b i n e s e n s i t i v e c o m p o n e n t o f ( - ) [ H ] - n o r e p i n e p h r i n e b i n d i n g ( F i g . 3 A ) . A S c a t c h a r d a n a l y s i s o f t h e s p e c i f i c b i n d i n g o f 3 ( - ) [ H ] - n o r e p i n e p h r i n e ( F i g 3A i n s e t ) i n t h e p r e s e n c e o f e i t h e r 10 /M ( - ) - e p i n e p h r i n e o r 10 /*M y o h i m b i n e r e v e a l e d K d v a l u e s o f 6 0 . 4 nM a n d 6 5 . 8 nM a n d B v a l u e s o f 0 . 2 2 a n d 0 . 2 4 p m o l / m g max p r o t e i n ( r = 0 . 9 6 , 0 . 8 ) r e s p e c t i v e l y . H i l l p l o t ( F i g . 2 B ) e x h i b i t e d c o e f f i c i e n t s 1 . 4 1 a n d 1 . 4 3 t h u s i n d i c a t i n g t h a t t h e ( - ) - n o r e p i n e p h r i n e p r o b a b l y h a s m o r e t h a n o n e b i n d i n g s i t e e v e n a t t h e © ^ - r e c e p t o r s . A S c a t c h a r d a n a l y s i s o f y o h i m b i n e 3 s p e c i f i c d i s p l a c e m e n t o f ( - ) [ H ] - n o r e p i n e p h r i n e s h o w e d a l i n e a r p l o t ( F i g . 3A i n s e t ) w i t h a p p r o x i m a t e l y t h e s a m e K , ( 6 4 . 5 nM) - 5 3 -F i g . 3 A. S p e c i f i c b i n d i n g of (-)[ H]-norepinephrine to brown f a t plasma membrane fragments from one-week-o l d r a t s i n the presence of 10 yohimbine (•-•) 3 and 10 -^M (-)-epinephrine ( o - o ) . (-)[ ^ - n o r e -p i n e p h r i n e b i n d i n g s e n s i t i v e t o s p e c i f i c d i s -placement by yohimbine o n l y ( A - A ) was c a l c u l a t e d 3 by s u b t r a c t i n g the s p e c i f i c b i n d i n g of [ H]-norepin-ephrine i n the presence of 10 JLK (-)-epinephrine from t h a t determined i n the presence of a combination of 10 (-)-epinephrine and 100 /*M yohimbine. R e s u l t s are the mean of d u p l i c a t e determinations from one r e p r e s e n t a t i v e experiment. Inset: Scat-chard p l o t of the same da t a . Slopes were determined u s i n g l i n e a r r e g r e s s i o n a n a l y s i s , ( r = 0.96, 0.8 and 0.77) f o r b i n d i n g i n the presence of (-)-epine-p h r i n e and yohimbine alone and the yohimbine -s p e c i f i c component r e s p e c t i v e l y . B. H i l l p l o t of the data i n pane l A f o r yohimbine (•-•) or (-)-epinephrine (o-o) as the l i g a n d d i s p l a c i n g agent. -54-c *5 o a a» E 2> o E o CO CL 0 C *EL 0 w O Z I E " CO 100 50 B/F 3 A 2 1 50 100 Bound (fmol/mg proteinL* 1 ,, 1 10 20 30 40 50 [3H] Norepinephrine (nM) 10 100 Concentration (nM) -55-b u t a m u c h l o w e r ( B ) ( 7 1 f m o l / m g p r o t e i n ) ( r = 0 . 7 7 ) . max C a t e c h o l a m i n e s s u c h a s ( - ) - e p i n e p h r i n e a n d ( - ) - n o r e p i n -3 e p h r i n e a r e m i x e d a g o n i s t s . T h e s p e c i f i c b i n d i n g o f ( - ) [ H ] -n o r e p i n e p h r i n e s e e n w h e n ( - ) - e p i n e p h r i n e w a s u s e d t o d i s p l a c e i t c o u l d b e a r e s u l t o f c o m p e t i t i o n a t b o t h o<- a n d y f l - r e c e p t o r s . 3 We h a v e f u r t h e r i n v e s t i g a t e d t h e p o s s i b i l i t y t h a t ( - ) [ H ] -n o r e p i n e p h r i n e b i n d s t o b o t h o<- a n d j B - r e c e p t o r s i n b r o w n f a t . I n c o m p e t i t i o n e x p e r i m e n t s ( F i g . 4 A ) , b o t h ( - ) - e p i n e p h r i n e 3 a n d p r a c t o l o l d i s p l a c e d ( - ) [ H ] - n o r e p i n e p h r i n e b i n d i n g i n a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r . ( - ) - E p i n e p h r i n e w a s m o r e e f f e c t i v e t h a n p r a c t o l o l a t a l o w e r r a n g e o f c o n c e n t r a t i o n . A s s h o w n i n F i g 4 B , ( - ) - e p i n e p h r i n e e x h i b i t e d a d o s e - d e p e n d e n t 3 d i s p l a c e m e n t o f ( - ) [ H ] - n o r e p i n e p h r i n e e v e n i n t h e p r e s e n c e - 3 3 o f 10 M p r a c t o l o l . C o n v e r s e l y , ( - ) [ H ] - n o r e p i n e p h r i n e c o u l d s t i l l b e d i s p l a c e d i n a c o n c e n t r a t i o n - d e p e n d e n t m a n n e r b y p r a c t o l o l e v e n i n t h e p r e s e n c e o f 10 M ( - ) - e p i n e p h r i n e . When t h e d a t a i n F i g . 4B w e r e a n a l y z e d b y C h e n g a n d P r u s s o f f e q u a t i o n [ 3 5 ] , t h e a p p a r e n t o f 66 nM f o r e p i n e p h r i n e was o b t a i n e d . I n o r d e r t o c o m p a r e o u r m e m b r a n e p r e p a r a t i o n f r o m i n f a n t r a t s t o t h a t o f a d u l t a n i m a l s , we h a v e r e p e a t e d p r e -v i o u s l y d e s c r i b e d e x p e r i m e n t s [ 2 4 ] o n b i n d i n g o f ^ - a d r e n e r g i c 3 l i g a n d ( - ) [ H ] - d i h y d r o a l p r e n o l o l u s i n g 10 juM n o r e p i n e p h r i n e a s t h e c o m p e t i n g a g e n t ( F i g . 5 A ) . T h e b i n d i n g w a s r a p i d , s a t u r a b l e a r i d r e v e r s i b l e w i t h h a l f - m a x i m a l s a t u r a t i o n o b s e r v e d a t a p p r o x i m a t e l y 10 n M . N o n - s p e c i f i c b i n d i n g w a s p r o p o r t i o n a l t o f r e e l i g a n d c o n c e n t r a t i o n a n d s p e c i f i c b i n d i n g r e p r e s e n t e d -56-F i g . 4 A. Displacement of s p e c i f i c b i n d i n g of (-)[ ^ - n o r -e p i n e p h r i n e to brown f a t plasma membrane from one-week-old r a t s by 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 non-l a b e l l e d (-)-epinephrine (O-o) and p r a c t o l o l (•-•). B. Displacement of (-)[ H]-norepinephrine b i n d i n g by 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 n o n - l a b e l l e d ( - ) - e p i -_ 3 nephrine i n the presence of 10 M p r a c t o l o l (o-o) and by i n c r e a s i n g c o n c e n t r a t i o n of n o n - l a b e l l e d —5 p r a c t o l o l i n the presence of 10 M (-)-epinephrine (n-a). -57-25 - 6 0 % o f t h e t o t a l . S c a t c h a r d a n a l y s i s r e v e a l e d a o f 10 nM a n d a B o f 0 . 5 5 p m o l / m g p r o t e i n ( r = 0 . 8 8 ) . F i g max 3 5B s h o w s t h e s p e c i f i c b i n d i n g o f ( - ) [ H ] - d i h y d r o a l p r e n o l o l t o b r o w n f a t p l a s m a m e m b r a n e s i n t h e p r e s e n c e o f e i t h e r 10 /uM p r a c t o l o l o r a c o m b i n a t i o n o f 10 #M p r a c t o l o l w i t h 1 0 0 >xM y o h i m b i n e . A S c a t c h a r d a n a l y s i s o f t h e b i n d i n g i n t h e p r e s e n c e o f p r a c t o l o l g a v e a K , o f p r a c t o l o l 5 0 nM ( / S l - s i t e ) a n d B * d m a x o f 0 . 1 9 p m o l / m g p r o t e i n ( F i g . 5B i n s e t ) ( r = 0 . 9 6 ) . ( - ) [ 3 H ] -c J i h y d r o a l p r e n o l o l b i n d i n g s p e c i f i c a l l y d i s p l a c e a b l e b y y o h i m -b i n e o n l y ( F i g . 5 B ) w a s d e t e r m i n e d b y s u b t r a c t i n g i t s s p e c i f i c b i n d i n g i n t h e p r e s e n c e o f 10 /*M p r a c t o l o l a l o n e f r o m t h a t d e t e r m i n e d i n t h e p r e s e n c e o f 10 ^ M o f p r a c t o l o l a n d 1 0 0 y o h i m b i n e . A K , o f 4 0 nM a n d B o f 112 f m o l / m g p r o t e i n 2 d max / » f 3 w a s o b t a i n e d b y S c a t c h a r d a n a l y s i s o f ( - ) [ H ] - d i h y d r o a l p r e n o l o l b i n d i n g s p e c i f i c a l l y d i s p l a c e a b l e b y y o h i m b i n e ( r = 0 . 8 ) . 3 . 2 E f f e c t o f 6 - h y d r o x y d o p a m i n e a n d c h r o n i c y o h i m b i n e p r e t r e a t m e n t o n t h e b i n d i n g c h a r a c t e r i s t i c s o f ^ - a d r e n o c e p t o r s i n i s o l a t e d p l a s m a m e m b r a n e f r a g m e n t s f r o m i n t e r s c a p u l a r b r o w n a d i p o s e t i s s u e o f i n f a n t r a t s . T h e 5 - d a y - o l d l i t t e r s w e r e d i v i d e d i n t o t h r e e g r o u p s . G r o u p 1 s e r v e d a s c o n t r o l s a n d w a s i n j e c t e d w i t h s a l i n e o n d a y 5 . The a n i m a l s i n t h e s e c o n d g r o u p w e r e c h e m i c a l l y s y m p a t h e c t o m i z e d b y a s i n g l e - d o s e i n j e c t i o n o f 6 - h y d r o x y d o p a m i n e o n d a y 5 . S i n c e t h e e f f e c t o f c h e m i c a l s y m p a t h e c t o m y b y 6 - h y d r o x y d o p a m i n e o n b r o w n f a t c o m p o s i t i o n a n d a c t i v i t y h a s a l r e a d y b e e n d e s -c r i b e d b y S k a l a a n d H a h n [ 1 7 6 ] , n o a t t e m p t w a s made t o s t u d y - 5 8 -F i g . 5 A. (-)[ H ] - d i h y d r o a l p r e n o l o l b i n d i n g to brown f a t plasma membrane fragments from one-week-old r a t s . . T o t a l b i n d i n g (4k—•) i n the absence of any competing agent; n o n - s p e c i f i c (A--A) b i n d i n g i n the presence of 10 /AM (-)-norepinephrine; and s p e c i f i c b i n d i n g (•—•) c a l c u l a t e d as the d i f f e r e n c e between the t o t a l and the non-s p e c i f i c b i n d i n g . R e s u l t s shown are from one r e p r e s e n t a t i v e experiment done i n d u p l i c a t e s . I n s e t : Scatchard p l o t of the d a t a . Slope was determined by l i n e a r r e g r e s s i o n a n a l y s i s (r = 0 . 8 8 ) . B. S p e c i f i c b i n d i n g of (-)[ H ] - d i h y d r o a l p r e n o l o l i n the presence of 10 /t*M p r a c t o l o l (•—o) 3 ( P^-receptors b i n d i n g s i t e ) . (-)[ H ] - d i h y d r o a l -p r e n o l o l b i n d i n g s e l e c t i v e l y d i s p l a c e a b l e by yohimbine ( • — • ) . T h i s b i n d i n g was c a l c u l a t e d by s u b t r a c t i n g the s p e c i f i c b i n d i n g i n the presence of 10 M-K p r a c t o l o l from t h a t determined i n the presence of 10 /uM p r a c t o l o l + 100 ALM yohimbine. I n s e t : Scatchard p l o t of (-)[ H]-d i h y d r o a l p r e n o l o l s p e c i f i c b i n d i n g . Slope was determined by l i n e a r r e g r e s s i o n a n a l y s i s (r = 0 . 9 6 , and 0.82 f o r b i n d i n g d i s p l a c e a b l e by p r a c t o l o l and y o h i m b i n e - s p e c i f i c component r e s p e c t i v e l y ) . -59-c *5 o k. a a> E "o £ - o e 3 o ca « 1 2 0 0 B/F 5 0 2 5 X . - i — i u 1 0 0 2 0 0 3 0 0 B o u n d ( f m o l / m g p r o t e i n ) 0 10 2 0 3 0 [ 3 H ] D i h y d r o a l p r e n o l o l (nM) c * 3 o a . cn E o E - o e 3 o ca o c a o o w - o x 1 6 0 120 8 0 4 0 B/F 3 0 2 0 1 0 r 0 x X N . X 2 0 6 0 100 1 4 0 B o u n d ( f m o l / m g p r o t e i n ) X 0 5 0 1 0 0 [ 3 H J D i h y d r o a l p r e n o l o l (nM) -60-the brown f a t composition or a c t i v i t y . The 5-day-old animals used i n the present experiments d i d not show any s i g n i f i c a n t side e f f e c t s . The c h r o n i c treatment with yohimbine (of group I I I r a t s ) on the other hand, could not be commenced u n t i l the animals reached two weeks of age, because p r e l i m i n a r y experiments showed that younger animals p o o r l y t o l e r a t e d the yohimbine a d m i n i s t r a t i o n . S e i z u r e s and muscular tremor appeared imme-d i a t e l y a f t e r the i n j e c t i o n s i n the m a j o r i t y of the younger animals. At two weeks of age, fewer s i d e e f f e c t s were observed; muscular t w i t c h i n g developed i n l e s s that 25% of the o l d e r animals. From a t o t a l of 30 animals, 1 died on the second day and 1 died on the f o u r t h day of treatment. The animals' t o l -erance of the i n j e c t i o n s improved a f t e r the i n i t i a l three days, and a f t e r 7 days of i n j e c t i o n s a l l animals appeared w e l l , and no s i g n i f i c a n t d i f f e r e n c e i n t h e i r body weight, as compared to that of c o n t r o l s , was noted. Brown adipose t i s s u e pads of yohim-b i n e - i n j e c t e d r a t s were l i g h t e r i n c o l o r and ap p a r e n t l y con-t a i n e d more t r i g l y c e r i d e s than those of the c o n t r o l group. Because of the s c a r c i t y of the t i s s u e samples, we d i d not attempt to c a r r y out any d e t a i l e d a n a l y s i s of the t i s s u e com-p o s i t i o n or a c t i v i t y i n the present s e r i e s of experiments. F i g . 6A and B show the e f f e c t s of chemical sympathec-3 tomy with 6-hydroxydopamine on [ H]-yohimbine b i n d i n g to plasma membrane fragments i s o l a t e d from i n t e r s c a p u l a r brown f a t of 8-day-old r a t s . In both the. c o n t r o l group i n j e c t e d with s a l i n e ( p a n e l A ) and the experimental -61-g r o u p ( p a n e l B) t h e n o n - s p e c i f i c b i n d i n g o f [ H ] - y o h i m b i n e ( a s a s s e s s e d b y t h e a d d i t i o n o f 10 .^M n o n - l a b e l l e d y o h i m b i n e ) w a s d i r e c t l y p r o p o r t i o n a l t o t h e f r e e l i g a n d c o n c e n t r a t i o n u p t o a t l e a s t 50 n M . T h e s p e c i f i c y o h i m b i n e b i n d i n g ( t a k e n a s t h e d i f f e r e n c e b e t w e e n t o t a l a n d n o n - s p e c i f i c b i n d i n g ) t o p l a s m a m e m b r a n e s f r o m t h e c o n t r o l g r o u p s h o w e d a n a p p a r e n t n u m b e r o f [ H ] - y o h i m b i n e s p e c i f i c b i n d i n g s i t e s o f 180 f m o l / m g p r o t e i n . T h e h a l f s a t u r a t i o n w a s r e a c h e d a t a p p r o x i m a t e l y 18 nM ( F i g . 6 A ) . T h e W o o l f p l o t [ 9 4 , 2 2 0 ] s h o w n i n t h e i n s e t g a v e a l i n e a r r e l a t i o n s h i p ( r = 0 . 9 1 ) w i t h a d i s s o c i a t i o n c o n -s t a n t ( K ^ ) o f 14 nM a n d a m a x i m u m n u m b e r o f b i n d i n g s i t e s (B- ) o f 155 f m o l / m g p r o t e i n . T h e s e v a l u e s a r e a l m o s t i d e n -t i c a l t o t h o s e r e c o r d e d i n r e s u l t s o f s e c t i o n 2 . 1 . S p e c i f i c 3 b i n d i n g o f [ H ] - y o h i m b i n e t o m e m b r a n e s f r o m s y m p a t h e c t o m i z e d a n i m a l s ( F i g . 6 B ) s h o w e d a n a p p a r e n t n u m b e r o f b i n d i n g s i t e s o f 7 5 f m o l / m g p r o t e i n . T h e h a l f - s a t u r a t i o n w a s r e a c h e d a t a p p r o x i m a t e l y 10 n M . T h e W o o l f t r e a t m e n t o f t h e d a t a , s h o w n i n t h e i n s e t , a g a i n g a v e a l i n e a r p l o t a n d a v a l u e o f 8 . 6 nM a n d a B m a x v a l u e o f 4 5 f m o l / m g p r o t e i n . I t i s k n o w n t h a t , c o m p a r e d t o a n t a g o n i s t s , a g o n i s t s s u c h a s e p i n e p h r i n e a n d n o r e p i n e p h r i n e e x h i b i t a h i g h e r d e g r e e o f s t e r e o s e l e c t i v i t y a n d a f f i n i t y f o r t h e ^ - r e c e p t o r s [ 2 0 4 , 2 0 7 ] . W e , t h e r e f o r e , s o u g h t a d d i t i o n a l i n f o r m a t i o n o n t h e b i n d i n g 3 c h a r a c t e r i s t i c s o f ( - ) [ H ] - n o r e p i n e p h r i n e t o t h e i d e n t i c a l mem-b r a n e p r e p a r a t i o n i s o l a t e d f r o m t h e s y m p a t h e c t o m i z e d a n i m a l s . 3 T h e s p e c i f i c b i n d i n g o f (-)[ H ] - n o r e p i n e p h r i n e t o p l a s m a m e m -- 6 2 -F i g . 6 E f f e c t of chemical sympathectomy of 5-day-old r a t s by a s i n g l e i n t r a p e r i t o n e a l dose (50 mg/kg body weight) of 6-hydroxydopamine on the s p e c i f i c b i n d -i n g of [ H]-yohimbine to brown f a t plasma membrane fragments i s o l a t e d 3 days l a t e r . S p e c i f i c b i n d i n g was determined i n the presence of 10 /M n o n - l a b e l l e d yohimbine at 25 C. Each p o i n t i s the mean of two deter m i n a t i o n s done i n d u p l i c a t e from one r e p r e s e n t a -t i v e experiment. I n s e t : Woolf p l o t of the data. The s l o p e s were determined by l i n e a r r e g r e s s i o n a n a l y s i s (r = 0.91 and 0.94 f o r panel A and B r e s p e c t i v e l y ) . A. Shows the r e s u l t s of the s p e c i f i c b i n d i n g to membranes from c o n t r o l s a l i n e - i n j e c t e d r a t s . B. R e s u l t s of the l i g a n d b i n d i n g t o membranes from 6-hydroxydopamine i n j e c t e d r a t s . -63-[ 3HJ Yohimbine (nM) [ 3 HJYohimbine (nM) -64-b r a n e s f r o m b o t h c o n t r o l a n d e x p e r i m e n t a l a n i m a l s w a s r a p i d , r e v e r s i b l e a n d s a t u r a b l e b y 10 /tM ( - ) - e p i n e p h r i n e . T h e 3 s p e c i f i c b i n d i n g o f ( - ) [ H ] - n o r e p i n e p h r i n e t o p l a s m a m e m b r a n e s f r o m c o n t r o l a n i m a l s ( F i g . 7 A ) i n t h e p r e s e n c e o f 10 jtM (-)-e p i n e p h r i n e r a n g e d f r o m 5 0 % o f t h e t o t a l b i n d i n g i n t h e l o w e r r a n g e o f t h e l i g a n d c o n c e n t r a t i o n t o 2 5 % a t t h e h i g h e r r a n g e . S c a t c h a r d a n a l y s i s o f t h e s e b i n d i n g d a t a g a v e a l i n e a r p l o t ( r = 0 . 9 3 ) w i t h a K , o f 6 8 . 0 3 nM a n d a B o f 1 . 3 9 p m o l / m g ' d max r a 3 p r o t e i n . H o w e v e r , t h e s p e c i f i c b i n d i n g o f ( - ) [ H ] - n o r e p i n e p h r i n e t o p l a s m a m e m b r a n e f r a g m e n t s f r o m b r o w n f a t o f 6 - h y d r o x y d o -p a m i n e p r e t r e a t e d a n i m a l s ( F i g . 7 B ) s h o w e d some u n u s u a l c h a r a c t e r i s t i c s u n d e r t h e same e x p e r i m e n t a l c o n d i t i o n s . T h e a b s o l u t e a m o u n t o f ( - ) [ H ] - n o r e p i n e p h r i n e s p e c i f i c b i n d i n g t o p l a s m a m e m b r a n e s f r o m 6 - h y d r o x y d o p a m i n e p r e t r e a t e d a n i m a l s i n t h e p r e s e n c e o f 10 ( - ) - e p i n e p h r i n e w a s s i g n i f i c a n t l y h i g h e r t h a n i n t h e c o n t r o l a n i m a l s . T h e s p e c i f i c b i n d i n g r a n g e d f r o m 6 6 % o f t h e t o t a l b i n d i n g i n t h e l o w e r r a n g e o f l i g a n d c o n c e n t r a t i o n t o 6 0 % i n t h e h i g h e r r a n g e . A S c a t c h a r d p l o t o f t h e d a t a w a s b e s t f i t b y a n e x p o n e n t i a l c u r v e w i t h a c o e f f i c i e n t o f r e g r e s s i o n ( r ) o f 0 . 8 . When t h e S c a t c h a r d p l o t w a s a n a l y z e d a c c o r d i n g t o R o s e n t h a l [ 1 5 9 ] , i t s h o w e d t h e p r e s -e n c e o f t w o b i n d i n g c o m p o n e n t s ( F i g . 7 B , i n s e t ) . T h e h i g h e r a f f i n i t y b i n d i n g s i t e s h o w e d a K , o f 36 nM a n d a B o f 0 . 6 5 -1 3 d max p m o l / m g p r o t e i n . T h e l o w a f f i n i t y s i t e e x h i b i t e d a o f 2 0 0 nM a n d a B m a x o f 3 p m o l / m g p r o t e i n . F u r t h e r a n a l y s i s o f t h e s e d a t a g a v e a H i l l c o e f f i c i e n t o f 1 . 5 ( F i g . 7B i n s e t ) -65-F i g . 7 E f f e c t o f s y m p a t h e c t o m y o f 5 - d a y - o l d r a t s b y a s i n g l e i n t r a p e r i t o n e a l d o s e ( 5 0 m g / k g b o d y w e i g h t ) o f 6 - h y d r o x y d b p a m i n e o n t h e s p e c i f i c b i n d i n g o f ( - ) [ 3 H ] - n o r e p i n e p h r i n e t o b r o w n f a t p l a s m a m e m b r a n e f r a g m e n t s i s o l a t e d 3 d a y s l a t e r . S p e c i f i c b i n d i n g w a s d e t e r m i n e d i n t h e p r e s e n c e o f 10 juM ( - ) - e p i n e p h r i n e . E a c h p o i n t i s t h e m e a n , o f 2 d e t e r m i n a t i o n s i n d u p l i c a t e s f r o m o n e r e p r e s e n t a t i v e e x p e r i m e n t . A. S h o w s t h e r e s u l t s o f t h e s p e c i f i c b i n d i n g t o m e m b r a n e s i s o l a t e d f r o m c o n t r o l s a l i n e i n j e c t e d r a t s . I n s e t : S c a t c h a r d p l o t o f t h e d a t a . S l o p e w a s d e t e r m i n e d b y l i n e a r r e -g r e s s i o n a n a l y s i s ( r = 0 . 9 3 ) . B . R e s u l t s o f t h e s p e c i f i c b i n d i n g t o mem-o b r a n e s i s o l a t e d f r o m 6 - h y d r o x y d o p a m i n e p r e -t r e a t e d r a t s . L e f t i n s e t : S c a t c h a r d p l o t o f t h e d a t a . T h e c u r v e w a s d e t e r m i n e d b y e x p o n e n t i a l f i t t i n g ( r = 0 . 8 ) . R i g h t i n s e t : H i l l p l o t o f t h e s a m e d a t a . - 6 6 -[3H]Norepinephrine (nM) [3H] Norepinephrine (nM) -67-i n d i c a t i n g p o s i t i v e c o o p e r a t i v e i n t e r a c t i o n i n ( - ) [ H ] -n o r e p i n e p h r i n e b i n d i n g . 3 F i g . 7 C s h o w s t h e s p e c i f i c b i n d i n g o f [ H] - n o r e p i n e p h r i n e i n t h e p r e s e n c e o f 1 0 pM ( - ) - e p i n e p h r i n e a n d t h a t i n t h e p r e s e n c e o f a c o m b i n a t i o n o f 1 0 ju-H ( - ) - e p i n e p h r i n e a n d 1 0 /uM p r a c t o l o l . F i g . 8 s h o w s t h e c o m p e t i t i o n c u r v e f o r t h e d i s p l a c e m e n t o f ( - ) [ 3 H ] - n o r e p i n e p h r i n e b i n d i n g t o p l a s m a m e m b r a n e s i s o l a t e d f r o m b r o w n f a t o f 8 - d a y - o l d - r a t s , p r e v i o u s l y i n j e c t e d b y 6 - h y d r o x y -d o p a m i n e , b y d i f f e r e n t c o n c e n t r a t i o n s o f ( - ) - e p i n e p h r i n e o r y o h i m b i n e . ( - ) - E p i n e p h r i n e w a s m o r e p o t e n t t h a n y o h i m b i n e i n c o m -p e t i n g f o r ( - ) [ 3 H ] - n o r e p i n e p h r i n e b i n d i n g s i t e s . T h e b i n d i n g d i s -p l a c e m e n t c u r v e s s h o w e d a s t e e p s l o p e , a n d t h e y w e r e n o n - p a r a l l e l t o e a c h o t h e r . P l a s m a m e m b r a n e f r a g m e n t s i s o l a t e d f r o m b r o w n f a t o f 2 1 -d a y - o l d r a t s p r e - t r e a t e d c h r o n i c a l l y w i t h y o h i m b i n e ( l O m g / k g i . p . f o r s e v e n d a y s ) w e r e u s e d f o r e q u i l i b r i u m b i n d i n g s t u d i e s u s i n g [ 3 H ] -R X - 7 8 1 0 9 4 a n d [ 3 H ] - r a u w o l s c i n e a s t h e ^ - l i g a n d s i n t h e p r e s e n c e o f 1 0 fiM y o h i m b i n e . T h e s p e c i f i c b i n d i n g s h o w e d a n a p p a r e n t n u m b e r o f s p e c i f i c b i n d i n g s i t e s o f 9 0 a n d 1 5 0 f m o l / m g p r o t e i n a n d h a l f s a t u r a -t i o n a t a p p r o x i m a t e l y 1 5 nM a n d 1 0 nM r e s p e c t i v e l y ( F i g . 9 B a n d A ) . B i n d i n g e x p e r i m e n t s r e p e a t e d w i t h [ 3 H j - y o h i m b i n e o n i d e n t i c a l mem-b r a n e f r a g m e n t s d e m o n s t r a t e d r e v e r s i b l e a n d s a t u r a b l e b i n d i n g i n t h e p r e s e n c e o f 10/KM n o n - l a b e l l e d y o h i m b i n e . S c a t c h a r d a n a l y s i s o f t h e s e b i n d i n g d a t a ( F i g . 9 C ) r e v e a l e d a K , o f 6 6 . 7 n M a n d a B m _ o f 3 d max 0 . 3 5 p m o l / m g p r o t e i n ( r = 0 . 8 ) . T h i s w a s s i g n i f i c a n t l y h i g h e r ( P < 0 . 0 1 ) t h a n t h e B- c a l c u l a t e d i n c o n t r o l a n i m a l s ( m e a n = -68-F i g . 7C [ H]-norepinephrine b i n d i n g to plasma membrane i s o l a t e d from brown f a t of sympathectomized r a t s . T o t a l (•-•), n o n - s p e c i f i c (V-T ; v-v ) b i n d i n g was determined i n the absence and presence of 10 ii.M (-)-epinephrine or a combination of 10 juK (-)-epinephrine and p r a c t o l o l r e s p e c t i v e l y . 3 S p e c i f i c b i n d i n g of [ H]-norepinephrine i n the presence of 10 /tM (-)-epinephrine (•-•) and t h a t i n the presence of 10 fM (-)-epinephrine and 10 /iK p r a c t o l o l (a-o) was determined by s u b t r a c t i n g c orresponding n o n - s p e c i f i c b i n d i n g from the t o t a l . -69--70-F i g . 8 Displacement of [ H]-norepinephrine b i n d i n g to plasma mebrane fragments i s o l a t e d from brown f a t of 6-hydroxydopamine p r e t r e a t e d 8-day-old r a t s by i n c r e a s i n g c o n c e n t r a t i o n of (-)-epin-ephrine (o-o) or yohimbine (•-*). Each p o i n t i s the mean 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 . -71-F i g . 9 E f f e c t of c h r o n i c yohimbine pre-treatment (10 mg/kg body weight i . p . f o r 7 days) on the b i n d i n g of o ^ - a n t a g o n i s t s to plasma membrane fragments i s o l a t e d from brown f a t of 21-day-o l d r a t s . Each p o i n t i s the mean of d u p l i c a t e d e t e r m i n a t i o n s from a r e p r e s e n t a t i v e experiment. A. Shows t o t a l (•-©) and the s p e c i f i c 3 b i n d i n g (O-O) of [ H]- ra u w o l s c i n e i n the presence of 10 pK yohimbine. B. Shows the t o t a l (•-•), n o n - s p e c i f i c (•-•) and the s p e c i f i c b i n d i n g (O-O) of [ 3H]-RX-781094 to i d e n t i c a l membrane p r e p a r a t i o n s i n the presence of 10 /iM yohimbine. -72--73-0 . 1 5 2 p m o l / m g p r o t e i n ) . T h e s a m e b i n d i n g e x p e r i m e n t s 3 w e r e r e p e a t e d u s i n g ( - ) [ H ] - n o r e p i n e p h r i n e a s t h e l i g a n d a n d e i t h e r 10 /tM ( - ) - e p i n e p h r i n e o r 10 J^tf y o h i m b i n e a s t h e d i s -p l a c i n g a g e n t . T h e r a t i o o f s p e c i f i c t o n o n - s p e c i f i c b i n d i n g f r o m e x p e r i m e n t a l a s c o m p a r e d t o t h a t f r o m c o n t r o l a n i m a l s w a s 5 - f o l d h i g h e r a t t h e l o w r a n g e o f l i g a n d c o n c e n t r a t i o n a n d 3 - f o l d h i g h e r a t t h e h i g h r a n g e . S c a t c h a r d a n a l y s i s ( F i g . 9D) i n d i c a t e d a K , o f 6 8 . 3 nM a n d B _ o f 3 . 0 p m o l / m g d max p r o t e i n f o r ( - ) - e p i n e p h r i n e ( r = . 9 8 ) a n d a o f 6 6 . 7 nM a n d B m a x o f 3 , 6 P m ° l / m 9 p r o t e i n f o r y o h i m b i n e ( r = 0 . 9 8 ) . 3 . 3 E f f e c t o f a d r e n e r g i c a g o n i s t s a n d a n t a g o n i s t s i n v i t r o o n c y c l i c n u c l e o t i d e c o n t e n t s o f r a t b r o w n a d i p o s e t i s s u e . B r o w n f a t f r a g m e n t s f r o m 1 - m o n t h o l d r a t s w e r e i n c u b a t e d . i n t h e p r e s e n c e o f p h e n y l e p h r i n e ( 5 . /*.M) , c l o n i d i n e ( 2 . juM) , d o u b a m i n e ( 5 0 ^ M ) a n d s a l b u t a m o l ( 2 / x . M ) . A s s h o w n i n F i g 1 0 A , d o b u t a m i n e ( 5 0 ^ , M ) w a s q u i t e e f f e c t i v e i n s t i m u l a t i n g c y c l i c AMP p r o d u c t i o n a n d c l o n i d i n e ( 2 . 0 ^ M ) w a s e f f e c t i v e i n s t i m u -l a t i n g t h e p r o d u c t i o n o f c y c l i c G M P . B o t h c l o n i d i n e ( 2 . 0 pJA) a n d p h e n y l e p h r i n e h a d s l i g h t e f f e c t i n s t i m u l a t i n g t h e p r o d u c t i o n o f c y c l i c A M P . P r e - i n c u b a t i o n o f t i s s u e p i e c e s f o r o n e h o u r w i t h 0 . 4 mM a n t a g o n i s t s ( ( ^ - y o h i m b i n e , ^ - m e t o p r o l o l , a n d / ^ 2 - H 3 5 y 2 5 ) p r o d u c e d a d e c r e a s e i n t h e l e v e l s o f c y c l i c n u c l e o t i d e s , ^ - a n t a g o n i s t ^^5/25 d e c r e a s e d c y c l i c AMP l e v e l m o r e t h a n h a l f t h e c o n t r o l v a l u e s ( F i g . 1 0 A ) . A d d i t i o n o f 0 . 2 , 2 , 2 0 / t M c l o n i d i n e r e s u l t e d i n d o s e - d e p e n d e n t e l e v a t i o n o f c y c l i c GMP -74-" F i g . 9C S c a t c h a r d a n a l y s i s o f [ H ] - y o h i m b i n e s p e c i f i c b i n d i n g t o p l a s m a m e m b r a n e f r a g m e n t s i s o l a t e d f r o m b r o w n f a t o f 2 1 - d a y - o l d r a t s i n j e c t e d d a i l y f o r 7 d a y s w i t h y o h i m b i n e ( 1 0 m g / k g b o d y w e i g h t ) . T h e s p e c i f i c b i n d i n g w a s d e t e r m i n e d a s t h e d i f f e r e n c e o f t h e b i n d i n g i n t h e a b s e n c e a n d t h a t i n t h e p r e s e n c e o f 10 /*M y o h i m b i n e . E a c h p o i n t i s t h e m e a n o f 2 d e t e r m i n a t i o n s d o n e i n d u p l i c a t e i n 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 . S l o p e w a s d e t e r -m i n e d b y l i n e a r r e g r e s s i o n ( r = 0 . 8 8 ) a n a l y s i s . D S c a t c h a r d a n a l y s i s ( - ) [ H ] - n o r e p i n e p h r i n e s p e c i f i c b i n d i n g t o i d e n t i c a l m e m b r a n e p r e -p a r a t i o n s . T h e s p e c i f i c b i n d i n g w a s d e t e r m i n e d a s t h e d i f f e r e n c e o f b i n d i n g i n t h e a b s e n c e a n d t h o s e o b t a i n e d i n t h e p r e s e n c e o f 10 y o h i m -b i n e ( • - • ) o r 10 jtM ( - ) - e p i n e p h r i n e ( • - • ) . E a c h p o i n t i s t h e m e a n o f 2 d e t e r m i n a t i o n s d o n e i n d u p l i c a t e . S l o p e w a s d e t e r m i n e d b y l i n e a r r e g r e s s i o n a n a l y s i s ( r = 0 . 9 8 f o r b o t h ) . - 7 5 -L°HJYohimbine B o u n d ( f m o l / m g p r o t e i n ) [ 3 H ] N E B o u n d ( p m o l / m g p r o t e i n ) -76-l e v e l . No s u c h e f f e c t o n b r o w n f a t c y c l i c AMP c o n c e n t r a t i o n w a s s e e n ( F i g 10B l e f t p a n e l ) . B r o w n f a t f r a g m e n t s i n c u b a t e d w i t h 0 . 5 , 5 , a n d 5 0 JJM. d o b u t a m i n e s h o w e d d o s e - d e p e n d e n t e l e v a t i o n o f c y c l i c AMP ( F i g 10B l o w e r m i d d l e p a n e l ) . P r e -i n c u b a t i o n o f b r o w n a d i p o s e t i s s u e w i t h 0 . 4 MM o f p r a z o s i n ( o ^ - a n t a g o n i s t ) , y o h i m b i n e ( © ^ - a n t a g o n i s t ) , ^ 3 5 / 2 5 ( / ^ - a n t a g o n i s t ) f o r 1 h o u r f o l l o w e d b y i n c u b a t i o n w i t h 5 0 ,«.M o f d o b u t a m i n e ( / ^ - a g o n i s t ) p r o d u c e d a n i n c r e a s e i n t h e l e v e l o f c y c l i c A M P . H o w e v e r , p r e - i n c u b a t i o n o f b r o w n f a t f r a g m e n t s i n t h e p r e s e n c e o f 0 . 4 mM m e t o p r o l o l b l o c k e d t h e s t i m u l a t i n g e f f e c t o f d o b u t -a m i n e o n c y c l i c AMP p r o d u c t i o n ( F i g . 10B l o w e r r i g h t p a n e l ) . S i m i l a r e x p e r i m e n t s w h e r e b r o w n f a t f r a g m e n t s w e r e p r e - i n c u b a t e d i n t h e p r e s e n c e o f a n t a g o n i s t s f o l l o w e d b y i n c u b a t i o n w i t h 0 . 5 y t f . M c l o n i d i n e s h o w e d y o h i m b i n e , a n ^ - a n t a g o n i s t , t o b e m o s t e f f e c t i v e i n b l o c k i n g c l o n i d i n e e f f e c t o n c y c l i c GMP p r o d u c t i o n ( F i g 1 0 B u p p e r r i g h t p a n e l ) . F i g 11A s h o w s t h e e f f e c t o f v a r i o u s c o n c e n t r a t i o n s o f c l o n i d i n e o n t o t a l c y c l i c GMP l e v e l ( r e l e a s e d i n t h e m e d i a d u r i n g i n c u b a t i o n + i n t r a c e l l u l a r ) i n b r o w n f a t f r a g m e n t s i s o l a t e d f r o m 7 - d a y - o l d r a t s . A d o s e - d e p e n d e n t e l e v a t i o n i n t h e c y c l i c GMP c o n t e n t w a s s e e n w i t h a p e a k r e s p o n s e o c c u r r i n g a t c l o n i d i n e c o n c e n t r a t i o n o f 0 . 5 - 1 . 0 /uM. P r e - i n c u b a t i o n o f t i s s u e p i e c e s w i t h 0 . 4 mM y o h i m b i n e f o r 1 h o u r c o m p l e t e l y a b o l i s h e d t h e e f f e c t o f c l o n i d i n e ( F i g . 1 1 B ) . B o t h s t i m u l a t e d a n d n o n - s t i m u l a t e d c e l l s g e n e r a l l y p r o d u c e c y c l i c n u c l e o t i d e c o n c e n t r a t i o n s f a r i n e x c e s s o f t h a t - 7 7 -F i g 10 E f f e c t o f a d r e n e r g i c a g o n i s t s a n d a n t a g o n i s t s i n v i t r o o n c y c l i c n u c l e o t i d e s i n b r o w n a d i p o s e t i s s u e o f 1 - m o n t h - o l d r a t s . B r o w n a d i p o s e t i s s u e w a s d i s s e c t e d f r o m i n t e r s c a p u l a r r e g i o n , c l e a n e d , m i n c e d a n d p o o l e d ( f r o m u p t o 30 r a t s ) a n d i n c u b a t e d i n a l i q u o t s o f 0 . 1 - 0 . 2 g w i t h v a r i o u s c o m b i n a t i o n s o o f a g o n i s t s a n d a n t a g o n i s t s a t 37 C i n a t o t a l v o l -ume o f 1 . 0 m l o f K r e b s - R i n g e r b i c a r b o n a t e b u f f e r ( p H 7 . 4 ) . R e a c t i o n s w e r e s t o p p e d b y c o o l i n g i n i c e -c o l d w a t e r , a n d t h e t i s s u e f r a g m e n t s w e r e s e p a r a t e d i m m e d i a t e l y o n a N i t e x f i l t e r u n d e r s u c t i o n . C y c l i c n u c l e o t i d e s w e r e e x t r a c t e d b y 10% t r i c h l o r a c e t i c a c i d a n d d e t e r m i n e d i n t r i p l i c a t e b y R I A a s d e s c r i b e d i n m e t h o d s . R e s u l t s a r e e x p r e s s e d a s p e r c e n t o f c o n -t r o l v a l u e s d e t e r m i n e d i n t h e a b s e n c e o f a n y d r u g . E a c h v a l u e i s t h e m e a n o f t h r e e t o f o u r s e p a r a t e d e t e r -m i n a t i o n s . V e r t i c a l b a r s i n d i c a t e S . E . M . f r o m t h e m e a n . A . T h e f i r s t s e t o f f o u r c o l u m n s f r o m l e f t s h o w s t h e e f f e c t o f a g o n i s t o n l y ; t h e s e c o n d s e t s h o w s t h e e f f e c t o f a n t a g o n i s t o n l y . B . U p p e r l e f t a n d l o w e r l e f t p a n e l s h o w s t h e e f f e c t o f d i f f e r e n t c o n c e n t r a t i o n s o f c l o n i d i n e ( 0 . 2 , 2 . 0 , a n d 20.0 /uM) o n b r o w n f a t c y c l i c GMP a n d c y c l i c AMP l e v e l s r e s p e c t i v e l y . L o w e r m i d d l e p a n e l s h o w s t h e e f f e c t o f -78-d i f f e r e n t c o n c e n t r a t i o n s of dobutamine (0.5, 5.0 and 50 fM) on c y c l i c AMP l e v e l s . The e f f e c t of c l o n i d i n e (0.5 ^M) and dobutamine (50 pM) i n the p r i o r presence of / and a n t a g o n i s t s i s shown i n the upper and lower r i g h t panel r e s p e c -t i v e l y . -79-200 c o o 6! 100 o =• 200 c o o a. < 100 Figure 1 0 A a. 0f» 0f» EL HH Pheny lephr ine ($.o*M) Clon id ine ( 2 . O » N ) <*> P r a z o s i n (400 / tM) Ofj Yoh imb ine ( 4 0 o * M ) P> M e i o p r o l o l (4oo„n) 0obulamine(S0.0 »M) S c l b u t o m o l ( 2 .o M^) 33/J3 < 400 » » ) S t a t i s t i c a l significance was determined using Paired " t " test. * P < 0 . 0 5 * * P < 0 . 0 2 - 8 0 -Figure 1 0 B 200 i-c o *3 '00 O o 200 c o K 9 Clonidine 0.2 2.0 20 o. < 100 t77 /7/ /// /// /// /// /// /// ,///. ///. ///•, ///\ /// /// ks// / / / /// / / / \ / s s s s s ss/ /// /// /// /// ///, ///, sss. \s/ y//' '//, 'ss, '//,\ '/// '//, •/// /// /// /// /// /// ss/ , ///\ ss/ ssy Dobutamine 0.5 5.0 50.0 MM. ** O o b u t a m i n a (5Q jjjuj) ** S t a t i s t i c a l significance was d e t e r m i n e d ' ' ^ t ,! ^ **P < n'no ' Bt3??*bedVs tesal (non-stimulated P < 0.02 cyclic nucleotide production). -81-F i g . 11 E f f e c t of c l o n i d i n e and yohimbine i n v i t r o on c y c l i c GMP i n brown f a t fragments from 7-day-o l d r a t s . Incubations were c a r r i e d out as d e s c r i b e d i n methods. Samples were p r e - i n c u -bated e i t h e r i n the presence or i n the absence of 0.4 mM yohimbine f o l l o w e d by a 3 minute i n c u b a t i o n w i t h c l o n i d i n e (0.5 /M). R e s u l t s are expressed as percent of c o n t r o l s i n the absence of any drug. Each v a l u e i s the mean of e i g h t experiments c a r r i e d out i n t r i p l i c a t e . V e r t i c a l bars re p r e s e n t the SEM. Panel A shows the e f f e c t of d i f f e r e n t c o n c e n t r a t i o n s of c l o n i d i n e . Panel B shows the e f f e c t of c l o n i d i n e (0.5 /tM) on samples pr e - i n c u b a t e d i n the absence or presence of yohimbine (0.4 mM) and the e f f e c t of yohimbine (0.4 mM) alone. -82-C o n c e n t r a t i o n ( pM ) S t a t i s t i c a l s i g n i f i c a n c e was d e t e r m i n e d u s i n g P a i r e d " t " t e s t . * P < 0 . 0 5 C l o n i d i n e s t i m u l a t e d V s b a s a l n o n - s t i m u l a t e d c-GMP p r o d u c t i o n . -83-n e e d e d f o r c o m p l e t e k i n a s e a c t i v a t i o n . I t i s t h e f r a c t i o n w h i c h i s b o u n d t o c y c l i c G M P - d e p e n d e n t p r o t e i n k i n a s e t h a t i s p h y s i o l o g i c a l l y i m p o r t a n t , a n d p r o v i d e s s o m e i n f o r m a t i o n a b o u t t h e f u n c t i o n a l a c t i v a t i o n s t a t e o f t h e t i s s u e . W e , t h e r e f o r e , a l s o d e t e r m i n e d t h e f r a c t i o n o f i n t r a c e l l u l a r n u c l e o t i d e w h i c h i s b o u n d t o a p r o t e i n r e c e p t o r . T h i s p r o t e i n r e c e p t o r i s t h e b i n d i n g s i t e o n t h e r e g u l a t o r y c o m p o n e n t o f c y c l i c G M P - d e p e n d e n t p r o t e i n k i n a s e . A . m o d i f i c a t i o n [ 1 5 1 .] o f t h e D u f a u e t a l . [ 5 1 ] t e c h n i q u e w a s f i r s t t e s t e d o n t h e c y c l i c AMP c o n t e n t o f b r o w n f a t a n d t h e n a p p l i e d t o c y c l i c G M P . T h e b o u n d f r a c t i o n o f b o t h c y c l i c n u c l e o t i d e s w a s 15 - 2 5 % o f t h e t o t a l a n d w a s f o u n d t o i n c r e a s e t o 30 - 4 0 % u n d e r a p p r o p r i a t e s t i m u l a t i o n . T h e a b s o l u t e v a l u e s o f t h e b o u n d f r a c t i o n o f c y c l i c AMP u n d e r i s o p r o t e r e n o l s t i m u l a t i o n s h o w e d a d o s e - r e s p o n s e c u r v e ( i s o p r o t e r e n o l 0 . 1 - 5 0 ^.M c o r r e s p o n d e d t o 1 0 5 - 160% o f t h e c o n t r o l v a l u e ) . C o n t r o l e x p e r i m e n t s w e r e c a r r i e d o u t t o c h e c k f o r a n y d i s s o c i a t i o n o f b o u n d c y c l i c n u c l e o t i d e , r e a s s o c i a t i o n o f u n b o u n d c y c l i c n u c l e o t i d e w i t h f r e e r e c e p t o r p r o t e i n a n d s p e c i f i c i t y o f t h e c y c l i c n u c l e o t i d e b i n d i n g t o i t s r e c e p t o r s i t e s . I n c y c l i c n u c l e o t i d e d i s s o c i a t i o n e x p e r i m e n t s , n o d i s s o c i a t i o n o f b o u n d c y c l i c n u c l e o t i d e w a s o b s e r v e d i n e x p e r i m e n t s c a r r i e d o u t a t 4 ° C u p t o 30 m i n u t e s i n t h e p r e s e n c e o f 2 - m e r c a p t o e t h a n o l . R e - a s s o c i a t i o n e x p e r i m e n t s c a r r i e d o u t i n t h e p r e s e n c e o f 2 - m e r c a p t o e t h a n o l s h o w e d n o i n c r e a s e i n t h e 3 . b i n d i n g o f [ H] c y c l i c n u c l e o t i d e f o l l o w i n g i n c u b a t i o n f o r - 8 4 -v a r i o u s i n t e r v a l s of time thus i n d i c a t i n g l a c k of r e - a s s o c i a t i o n between f r e e c y c l i c n u c l e o t i d e and the r e c e p t o r p r o t e i n under the experimental c o n d i t i o n s used i n our assays. P r e l i m i n a r y experiments c a r r i e d out to determine the s p e c i f i c i t y of c y c l i c n u c l e o t i d e b i n d i n g to i t s r e c e p t o r p r o t e i n i n d i c a t e d b i n d i n g 3 of [ H] c y c l i c AMP to be r a p i d , r e v e r s i b l e and s a t u r a b l e thus i n d i c a t i n g a f i n i t e number of s p e c i f i c c y c l i c n u c l e o t i d e b i n d -ing s i t e s . F i g . 12 shows the e f f e c t of c l o n i d i n e (0.5 pM) i n -v i t r o on c y c l i c GMP l e v e l i n brown f a t fragments from 7-day-o l d r a t s . C y c l i c n u c l e o t i d e determinations were c a r r i e d out on the i n c u b a t i o n media, the separated t i s s u e fragments and the protein-bound f r a c t i o n . C l o n i d i n e produced a time- dependent e l e v a t i o n of i n t r a c e l l u l a r , r e l e a s e a b l e and protein-bound f r a c t i o n s of c y c l i c GMP. The response was monophasic with a peak of c y c l i c n u c l e o t i d e c o n c e n t r a t i o n o c c u r r i n g between 3 - 4 minutes and then d e c l i n i n g g r a d u a l l y . , The c y c l i c GMP f r a c t i o n bound to p r o t e i n was 25 - 30% of the t o t a l t i s s u e c y c l i c GMP content. 3.4 I n h i b i t i o n of f o r s k o l i n - s t i m u l a t e d l i p o l y s i s i n a d i p o c y t e s from brown f a t of 1-week-old r a t s by c l o n i d i n e .  As shown i n Table 1, f o r s k o l i n (1.25 ^M) s t i m u l a t e d l i p o l y s i s i n a d i p o c y t e s i s o l a t e d from brown f a t of 1-week-old r a t s . The s t i m u l a t i n g e f f e c t of f o r s k o l i n was p a r t i a l l y i n h i b i t e d by p r o s t a g l a n d i n E 9 and n i c o t i n i c a c i d . Table 2A -85-F i g . 12 E f f e c t of c l o n i d i n e (0.5 ^M) i n v i t r o on c y c l i c GMP i n fragments of brown f a t from 7-day-old r a t s . Brown adipose t i s s u e was i s o l a t e d from i n t e r s c a p u l a r r e g i o n of n e a r l y 60 r a t s , cleaned, minced wi t h s c i s s o r s and pooled i n a l i q u o t s of 0.1 - 0.2 g. F o l l o w i n g a 60 minute p r e - i n c u b a t i o n , c l o n i d i n e was added and the i n c u b a t i o n s were stopped at 1, 2, 3, 5, and 10 minutes. T i s s u e pieces were separated by f i l t r a t i o n on a N i t e x f i l t e r under s u c t i o n . C y c l i c n u c l e o t i d e s content of the incuba-t i o n media (O-o) and the separated t i s s u e fragments were e x t r a c t e d i n 10% t r i c h l o r a c e t i c a c i d and determined by RIA. In a d d i t i o n t o the t o t a l t i s s u e content of c y c l i c GMP (•-•), the protein-bound f r a c t i o n of c y c l i c GMP ( D - D ) was determined as d e s c r i b e d i n methods. R e s u l t s shown r e p r e s e n t the means of two to f i v e d e t e r m i n a t i o n s i n t r i p l i c a t e ( v e r t i c a l bars represent SEM) and are expressed as the percent of valu e s i n c o n t r o l i n c u b a t i o n s c a r r i e d out f o r each of the time p e r i o d s i n the absence of c l o n i d i n e . - 8 6 -- 8 7 -s h o w s t h e e f f e c t o f 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 c l o n i d i n e o n f o r s k o l i n ( 0 . 6 2 5 ^ M ) s t i m u l a t e d g l y c e r o l r e l e a s e i n i s o l a t e d a d i p o c y t e s . C l o n i d i n e i n h i b i t e d g l y c e r o l r e l e a s e i n a c o n c e n -t r a t i o n - d e p e n d e n t m a n n e r . T h e i n h i b i t o r y e f f e c t o f c l o n i d i n e (l.>uM) w a s a n t a g o n i z e d b y 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 y o h i m b i n e ; T h e m a x i m u m a n t a g o n i s m a c h i e v e d a t 5 y o h i m b i n e ( T a b l e 2 B ) . E n d o g e n o u s l y r e l e a s e d a d e n o s i n e i s a n i m p o r t a n t r e g u l a t o r o f l i p o l y s i s a n d c y c l i c AMP f o r m a t i o n i n w h i t e a d i p o c y t e s a n d h a m s t e r b r o w n a d i p o c y t e s [ 5 8 , 1 6 9 ] . I s o b u t y l -m e t h y l x a n t h i n e a n t a g o n i z e s t h e i n h i b i t o r y a c t i o n o f a d e n o s i n e u p o n a d e n y l a t e c y c l a s e . I n a n o t h e r s e t o f e x p e r i m e n t s , w h e r e 1 0 0 /M i s o b u t y l m e t h y l x a n t h i n e w a s u s e d t o s t i m u l a t e l i p o l y s i s i n a d i p o c y t e s i s o l a t e d f r o m 7 - d a y - o l d r a t b r o w n f a t , c l o n i d i n e w a s s t i l l c a p a b l e o f i n h i b i t i n g l i p o l y s i s b y a s m u c h a s 15 -3 5 % ( T a b l e 3 ) . S i m u l a t o r y e f f e c t o f i s o b u t y l m e t h y l x a n t h i n e o n l i p o l y s i s w a s a l s o s e n s i t i v e t o i n h i b i t i o n b y n i c o t i n i c a c i d ( T a b l e 3 ) . A d e n y l a t e c y c l a s e i s t h o u g h t t o b e c o u p l e d t o b o t h N i ( i n h i b i t o r y p r o t e i n ) a n d N s ( s t i m u l a t i n g p r o t e i n ) . I t s h o w s b i p h a s i c r e g u l a t i o n b y G T P , b e c a u s e b o t h t h e p r o t e i n s r e -q u i r e GTP f o r t h e i r a c t i o n . F o r s k o l i n 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 i s b e l i e v e d n o t t o r e q u i r e t h e p r e s e n c e o f e i t h e r s t i m u l a t o r y o r i n h i b i t o r y p r o t e i n . I t h a s b e e n s u g g e s t e d t h a t f o r s k o l i n a c t s b y a d i r e c t a c t i o n o n t h e c a t a l y t i c s u b - u n i t o f a d e n y l a t e c y c l a s e [ 1 7 3 ] o r b y a n i n d i r e c t a c t i o n m e d i a t e d v i a a n a s y e t u n i d e n t i f i e d m e c h a n i s m [ 2 2 , 1 8 9 ] . T h u s , t h e - 8 8 -a c t i v a t i o n of adenylate c y c l a s e by f o r s k o l i n can be a f f e c t e d by inputs a f f e c t i n g the s t i m u l a t o r y (Ns) or i n h i b i t o r y (Ni) p r o t e i n . P e r t u s s i s t o x i n from B o r d e t e l l a p e r t u s s i s s e l e c t i v e l y b l o c k s Ni p r o t e i n . We, t h e r e f o r e , considered i t of i n t e r e s t to i n v e s t i g a t e i f such pre-treatment of r a t brown f a t a d i p o -cytes by p e r t u s s i s t o x i n was a s s o c i a t e d with a decrease i n the i n h i b i t o r y a c t i o n of alpha-2 ag o n i s t ( c l o n i d i n e ) on l i p o l y s i s . As shown i n Table 4, brown adi p o c y t e s from 7-day-o l d r a t s p r e - i n c u b a t e d with p e r t u s s i s t o x i n (0.5 mg/ml) f o r 3% hours to i n a c t i v a t e i n h i b i t o r y p r o t e i n Ni and subsequently s t i m u l a t e d with f o r s k o l i n showed 1.5 x the normal r a t e of l i p o l y s i s . The b a s a l r a t e of l i p o l y s i s i n p e r t u s s i s t o x i n t r e a t e d c e l l s was 1.3 x the normal, suggesting Ni p r o t e i n probably maintains a minor i n h i b i t o r y input on the r a t e of l i p o l y s i s . C l o n i d i n e , i n a c o n c e n t r a t i o n of 1 /iM to 5/tM, was ; I n e f f e c t i v e i n i n h i b i t i n g the l i p o l y t i c response o f brown ad i p o c y t e s to f o r s k o l i n . I n t e r e s t i n g l y , when the same experiments were r e -peated u s i n g a d i p o c y t e s from hamster brown f a t , no i n h i b i t o r y e f f e c t of c l o n i d i n e on f o r s k o l i n or i s o b u t y l m e t h y l x a n t h i n e s t i m u l a t e d g l y c e r o l r e l e a s e was observed (Table 5A). On the c o n t r a r y , p r o s t a g l a n d i n E 2 and n i c o t i n i c a c i d s t i l l showed an i n h i b i t o r y e f f e c t on f o r s k o l i n - s t i m u l a t e d g l y c e r o l r e l e a s e / i n hamster a d i p o c y t e s as shown i n t a b l e 5B. - 8 9 -Table 1. E f f e c t of P r o t a g l a n d i n (PGE 2) and n i c o t i n i c a c i d on f o r s k o l i n - s t i m u l a t e a g l y c e r o l r e l e a s e i n a d i p o c y t e s i s o l a t e d from i n t e r s c a p u l a r brown adipose t i s s u e of seven-day-old r a t s . Each value i s the mean +SEM of f o u r experiments done i n d u p l i c a t e . Drug (s) Basa l (4) F o r s k o l i n ( 1.25 uM) (4) F o r s k o l i n + PGE 2 0.1 mg/ml (4) F o r s k o l i n + PGE 2 0.5 mg/ml (4) F o r s k o l i n + PGE 2 1.0 mg/ml (4) F o r s k o l i n + PGE 2 5.0 mg/ml (4) F o r s k o l i n + n i c o t i n i c a c i d (40 ^M) (2) G l y c e r o l r e l e a s e >c.mol/mg p r o t e i n 0.165 +•0.015 * 0.295 +•0.007 ** 0.210 + 0.005 0.25 + 0.027 *** 0.22 + 0.015 ****0.189 + 0.015 0.186 Statistical analysis of the data was done using Student's t test. * P< 0.01 for forskolin stimulated-glycerol release Vs basal * * P < 0.01 for forskolin + P G E 2 Vs forskolin stim. glycerol release. * * * P < 0.05 for forskolin + P G E o vs forskolin stim. glycerol release. * * * * P < 0.05 for forskolin + P G E 2 vs. forskolin stimulated glycerol release. -90-T a b l e 2 A . E f f e c t o f 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 c l o n i d i n e o n f o r s k o l i n -s t i m u l a t e d ( o . 6 2 5 /*-M) g l y c e r o l r e l e a s e i n i s o l a t e d a d i p o c y t e s f r o m b r o w n f a t o f 7 - d a y - o l d r a t s . R e s u l t s a r e t h e m e a n o f d u p l i c a t e d e t e r m i n a t i o n s f r o m t w o r e p r e s e n t a t i v e e x p e r i m e n t s . V a l u e s i n t h e p a r e n t h e s i s s h o w t h e r e s u l t s e x p r e s s e d a s t h e p e r c e n t . o f ..• t h e b a s a l n o n - s t i m u l a t e d g l y c e r o l l e v e l . D r u g ( s ) G l y c e r o l r e l e a s e / _ m o l / m g p r o t e i n . E x p t . # 1 E x p t . # 2 B a s a l 0. 589 (1 0 0 ) 1. 014 ( 1 0 0 ) F o r s k o l i n ( 0 . 6 2 5 /tM) 2. 589 ( 4 3 9 . 56%) 5 . 286 ( 5 2 1 . 30%) F o r s k o l i n + c l o n i d i n e (5 jtM) 1. 1 1 5 ( 1 8 9 . 30%) 1. 671 ( 1 6 4 . 7 9 % ) F o r s k o l i n + c l o n i d i n e ( 1 . 0 AM) 1. 414 ( 2 4 0 . 06%) 2. 447 ( 2 4 1 . 32%) F o r s k o l i n + c l o n i d i n e ( 0 . 5 ^M) 1. 1 7 5 ( 1 9 9 . 49%) 2. 948 ( 2 9 0 . 7 3 % ) F o r s k o l i n + c l o n i d i n e ( 0 . 1 /*M) 1. 892 ( 3 2 1 . 22%) 3. 0 6 1 ( 3 0 1 . 8 7 % ) . - 9 1 -T a b l e 2 B . R e v e r s a l o f c l o n i d i n e ( 1 . 0 jxM.) e f f e c t o n f o r s k o l i n ( 0 . 6 2 5 /tM)-s t i m u l a t e d g l y c e r o l r e l e a s e i n a d i p o c y t e s i s o l a t e d f r o m b r o w n f a t o f 1 - w e e k - o l d r a t s b y 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 y o h i m b i n e . R e s u l t s a r e t h e m e a n o f t h e d u p l i c a t e d e t e r m i n a t i o n s f r o m t w o e x p e r i m e n t s . V a l u e s i n t h e p a r e n t h e s i s s h o w t h e r e s u l t s e x p r e s s e d a s t h e p e r c e n t o f t h e b a s a l n o n - s t i m u l a t e d g l y c e r o l l e v e l . G l y c e r o l r e l e a s e / / j n o l / m g p r o t e i n E x p t . # 1 E x p t . # 2 0 . 3 4 0 ( 1 0 0 ) 0 . 1 5 2 ( 1 0 0 ) 0 . 5 6 4 ( 1 6 5 . 8 8 % ) 0 . 2 5 7 ( 1 6 9 . 0 7 % ) 0 . 3 5 0 ( 1 0 2 . 9 4 % ) 0 . 1 8 7 ( 1 2 3 . 0 3 % ) 0 . 3 8 9 ( 1 1 4 . 4 % ) 0 . 1 9 9 ( 1 3 0 . 9 2 % ) 0.369 (108.53%) ' 0 . 2 4 5 (161.18%) 0 . 3 8 9 ( 1 1 4 . 4 1 % ) 0 . 2 1 2 ( 1 3 9 . 4 7 % ) 0 . 4 2 7 ( 1 2 5 . 5 9 % ) 0.466 (137.06%) 0.221 (145.39%) D r u g ( s ) B a s a l F o r s k o l i n ( 0 . 6 2 5 / u.M) F o r s k o l i n + c l o n i d i n e ( 1 . 0 K M ) F o r s k o l i n + c l o n i d i n e + y o h i m b i n e ( 1 . 0 /»-M) ( O . 5 X 1 0 " 9 M ) F o r s k o l i n + c l o n i d i n e + y o h i m b i n e ( 1 . 0 ( 0 . 5 X 1 0 - 8 M ) F o r s k o l i n + c l o n i d i n e + y o h i m b i n e ( 1 . 0 A<-M) ( 0 . 5 x 1 0~ 7M) F o r s k o l i n + c l o n i d i n e + y o h i m b i n e ( 1 . 0 fiM) ( 0 . 5 x 1 0 _ £ >M) F o r s k o l i n + c l o n i d i n e + y o h i m b i n e ( 1 . 0 ^ M ) ( 5 . Ox iom) T a b l e 3 . E f f e c t o f c l o n i d i n e ( o < - , - a g o n i s t ) a n d n i c o t i n i c a c i d o n i s o b u t y l m e t h y l x a n t h i n e ( M I X ) - s t i m u l a t e d g l y c e r o l r e l e a s e i n a d i p o c y t e s i s o l a t e d f r o m b r o w n f a t o f s e v e n - d a y - o l d r a t s . E a c h v a l u e i s t h e m e a n o f d u p l i c a t e d e t e r m i n a t i o n s f r o m t w o e x p e r i m e n t s . V a l u e s i n t h e p a r e n t h e s i s s h o w t h e r e s u l t s e x p r e s s e d a s t h e p e r c e n t o f t h e b a s a l n o n - s t i m u l a t e d g l y c e r o l l e v e l . D r u g ( s ) G l y c e r o l r e l e a s e ^ m o l / m g p r o t e i n E x p t . # 1 E x p t . # 2 . B a s a l 0 . 4 5 9 ( 1 0 0 % ) 0 . 4 6 3 . ( 1 0 0 % ) M I X ( 1 0 0 /_M) 0 . 6 1 0 ( 1 3 2 . 9 % ) 0 . 7 8 0 ( 1 6 8 . 4 7 % ) M I X + c l o n i d i n e ( 1 A M) 0 . 5 1 0 ( 1 1 1 . 1 1 % ) 0 . 4 8 3 ( 1 0 4 . 3 2 % ) M I X + c l o n i d i n e ( 0 . 5 p-U) 0 . 6 0 3 ( 1 3 1 . 3 7 % ) 0 . 5 9 1 ( 1 2 7 . 6 4 % ) M I X + n i c o t i n i c a c i d (40 ftM) 0 . 5 0 8 ( 1 1 0 . 6 7 % ) 0 . 6 0 5 ( 1 3 0 . 6 7 % ) - 9 3 -T a b l e 4 E f f e c t o f p e r t u s s i s t o x i n p r e - t r e a t m e n t o n i n h i b i t i o n o f f o r s k o l i n - s t i m u l a t e d g l y c e r o l r e l e a s e b y c l o n i d i n e a n d P G E 2 i n r a t a d i p o c y t e s . B r o w n f a t a d i p o c y t e s f r o m s e v e n - d a y - o l d r a t s w e r e i s o l a t e d a s d e s c r i b e d i n m e t h o d s a n d i n c u b a t e d i n p e r t u s s i s t o x i n f r o m B o r d e t e l l a p e r t u s s i s (0.5 m g / m l ) . F o l l o w i n g a p r e -i n c u b a t i o n p e r i o d o f 3% h o u r s , c e l l s w e r e i n c u b a t e d a t 37°C w i t h f o r s k o l i n (1.25 /cM) a n d v a r i o u s c o n c e n t r a t i o n s o f c l o n i d i n e a n d PGE2 i n a t o t a l v o l u m e o f 1.0 m l . R e a c t i o n s w e r e s t o p p e d i m m e d i a t e l y b y f i l t r a t i o n o n a m i l l i p o r e f i l t e r u n d e r s u c t i o n . G l y c e r o l w a s m e a s u r e d i n t h e f i l t e r a t e b y W i e l a n d m e t h o d . R e s u l t s a r e t h e m e a n + SEM f r o m f o u r e x p e r i m e n t s d o n e i n d u p l i c a t e . N u m b e r s i n t h e p a r e n t h e s i s a r e t h e m e a n o f t h e p e r c e n t o f b a s a l ( n o n - s t i m -u l a t e d ) g l y c e r o l l e v e l f o r e a c h e x p e r i m e n t . D r u g ( s ) C o n t r o l P e r t u s s i s t o x i n g r o u p p r e - t r e a t e d g r o u p ^ m o l / m g p r o t e i n B a s a l 0. 403+0. 085 (100%) 0. 566+0 .147 (100%) F o r s k o l i n (1.25 A M ) * * 0. 755+0. 101 (202.58+27 .14%) ***0. 902+0 .242 (160.19+6.42%) F o r s k o l i n "kick + c l o n i d i n e (5 A M ) 0. 456+0 . .173 (112.38+28 .19%) 0. 884+ 0.207 (159.07 + 4.17%) F o r s k o l i n + c l o n i d i n e (1.0 ^ M ) + P G E 2 ( 0 . 5 ^ ) 0. 581+0. 251 (125.21+37 .97%) 1. 071+ 0.144 (162.85+4.17%) F o r s k o l i n 0. 535+0. 101 (136.18+9. 15%) 1. 019+ 0.203 (154.64+5.68%) F o r s k o l i n + P G E 2 (0.1/<M) 0. 592+0. 098 (134.22+10 .82%) 1. 03 + 0.302 (125.52+16.31%) S t a t i s t i c a l s i g n i f i c a n c e o f t h e d a t a was d e t e r m i n e d u s i n g P a i r e d ' t * t e s t * P < 0.01 f o r s k o l i n + PG E 2 V s f o r s k o l i n s t i m u l a t e d g l y c e r o l r e l e a s e . * * P < 0.02 f o r s k o l i n Vs b a s a l g l y c e r o l r e l e a s e . * * * P <0.05 f o r s k o l i n + c l o n i d i n e V s f o r s k o l i n s t i m u l a t e d g l y c e r o l r e l e a s e o r f o r s k o l i n V s b a s a l g l y c e r o l r e l e a s e i n p e r t u s s i s t o x i n p r e - t r e a t e d a n i m a l s . -94-Table 5A Effect of clonidine (c<2-agonist) on forskolin and isobutylmethylxanthine stimulated glycerol release in adipocytes isolated from brown fat of adult hamsters. Each value is the mean of duplicate determinations from two to four experiments, Drug (s) Glycerol release A-mol/mg protein Basal (4) Forskolin (1.25 A M ) (4) Forskolin + clonidine (5.0 A M ) (4) Forskolin + clonidine (1.0 A M ) (4) Forskolin + clonidine (0.5 A M ) (4) Forskolin + clonidine (0.1 A M ) ( 4 ) 0.278 + 0.015 0.306 + 0.024 0.287 + 0.041 0.309 + 0.035 0.304 + 0.035 0. 300 .+ 0.034 Expt. # 1 Expt. # 2, Basal (2) M I X (100 A M ) (2) M I X + clonidine ( 5. A M ) (2) M I X + clonidine (1.0 AM ) ( 2 ) M I X + clonidine (0.5AM)(2) M I X + clonidine.(0.1 AM ) ( 2 ) 0.275 0. 357 0.373 o.378 0.392 0.386 0.314 0.375 0.394 0.375 0.375 0.353 -95-Table 5B Effect of prostaglandin (PGE^ ) and nicotinic acid on forskolin-stimulated glycerol release in adipocytes isolated from brown fat of adult hamsters. Each value is the mean of duplicate deter-minations from one to two experiments. Number in the parenthesis show the results expressed as the percent of the basal non-sti-mulated glycerol level. Drug (s) Glycerol release .^mol/mg protein Expt. #1 .:. ExPt. # 2. Basal (2) 0. 209 0. 363 Forskolin (1 .25 AM) (2) 0. 279 (133.49% ) 0. 457 Forskolin + PGE2 (5 mg/ml) (2) 0. 21 7 (1 03.80% ) 0. 297 Forskolin + PGE2 (1 mg/ml) (1) 0. 233 (11 1 . 4 8 * ) Forskolin + PGE2 (0.1 mg/ml)(1) 0. 263 (125.84% ) Forskolin + nicotinic acid (40 ju.M) 0. 233 ( 1 1 1 . 1 . 4 % ) 3.5 Effect of o<2- and >Sn — agonists in vitro on cyclic GMP content in brown fat fragments from obese (ob/ob, 2-month-old) mice and their unaffected littermates.  We in our, init ial in vivo experiments, determined cyclic GMP content of brown adipose tissue of ob/ob mice after cold (6°C) exposure and norepinephrine administration (0.1 mg/100 g body weight i.p.) and compared to identical determinations carried out on unaffected littermates. A pro-nounced difference was observed in the profile of cyclic GMP content of brown adipose tissue of obese mice and their lean littermates. Although the brown fat of control mice showed a time- dependent increase in cyclic GMP content on cold exposure and norepinephrine injection, the brown fat of obese mice ex-hibited no change (Fig. 13A and B). In a series of in vitro experiments, cyclic GMP content of fragments of brown fat from obese mice and their unaffected littermates was determined in the presence of ©<.2-agonist and /^1~agonist. Cyclic GMP production in the presence of clonidine ( ©<2-agonist) in obese mice was nearly the same as that observed in unaffected control littermates. However, a lower basal cyclic GMP concen-tration was seen in the brown fat of obese mice than their lean littermates'. -97-Fig. !13A E f f e c t of c o l d exposure (6°C) on c y c l i c GMP content i n brown adipose t i s s u e of g e n e t i c a l l y obese mice (C57BL/6J-ob, 2-month-old). Lean l i t t e r m a t e s served as c o n t r o l s . V e r t i c a l bars r e p r e s e n t the +SEM of 6 - 8 determinations on 3 - 4 animals. C: animals at room temperature. F i g . 13 B E f f e c t of norepinephrine (0.1 mg/100 g body weight, i.p.) on c y c l i c GMP content i n brown adipose t i s s u e of obese mice. Lean l i t t e r m a t e s served as c o n t r o l s . V e r t i c a l bars represent the +SEM of 6 - 8 determinations on 3 - 4 animals. C: animals i n j e c t e d w i t h s a l i n e o n l y . -98-- 9 9 -F i g . 1 3 C E f f e c t o f c l o n i d i n e ( 0 . 5 jiM) i n v i t r o o n c y c l i c GMP c o n c e n t r a t i o n s i n b r o w n f a t f r a g m e n t s o f o b e s e ( o b / o b , 2 m o n t h s o l d ) m i c e a n d t h e u n a f f e c t e d l i t t e r m a t e s . T i s s u e f r a g m e n t s p o o l e d f r o m u p t o 20 m i c e w e r e i n c u b a t e d ( i n a l i q u o t s o f 0 . 1 - 0 . 2 g ) f o r t h r e e m i n u t e s w i t h 0 . 5 p M c l o n i d i n e o r 50 /uM d o b u t a m i n e . R e a c t i o n s w e r e s t o p p e d b y c o o l i n g i n i c e - c o l d w a t e r , a n d t h e t i s s u e f r a g m e n t s w e r e s e p a r a t e d i m m e d i a t e l y o n a N i t e x f i l t e r u n d e r s u c t i o n . C y c l i c n u c l e o t i d e s w e r e e x t r a c t e d b y 10% t r i c h l o r a c e t i c a c i d a n d d e t e r m i n e d i n t r i p l i c a t e b y R I A . R e s u l t s a r e e x p r e s s e d a s p m o l / m g p r o t e i n , a n d e a c h v a l u e i s t h e m e a n o f t w o t o f o u r s e p a r a t e d e t e r m i n a t i o n s f r o m a s i n g l e e x p e r i m e n t . - 1 0 0 -CLONIDINE 0 . 5 « M 0 DOBUTAMINE ( 5 . 0 * M ) •101-CHAPTER 4 D I S C U S S I O N 4. A hormone can have m u l t i p l e e f f e c t s i n a c e l l or t i s s u e . A t i s s u e or c e l l s can respond to the same hormone d i f f e r e n t l y under d i f f e r e n t circumstances. Norepinephrine r e l e a s e d from the storage granules of the sympathetic nerve e f f e r e n t s i s the p r i n c i p a l a g o n i s t a c t i n g upon brown a d i p o c y t e s . I t i n i t i a t e s both the acute s t i m u l a t i o n of c a l o r i g e n i c f u n c t i o n of t i s s u e and long-term adaptive changes i n brown f a t [180]. Norepinephrine can bind to e i t h e r or both c<- and yfl-receptors. T h e r e f o r e , the phenotype of the adrenoceptors i n the plasma membrane of brown adipocytes and the r e l a t i v e p r o p o r t i o n s and a f f i n i t y of i t s components f o r norepinephrine may determine the f i n a l e f f e c t s of a d r e n e r g i c s t i m u l a t i o n . Depending on which r e c e p t o r s are p r e f e r e n t i a l l y occupied, and assuming t h a t d i f f e r e n t b i n d i n g s i t e s are l i n k e d to d i f f e r e n t e f f e c t o r systems, the i n v i v o e f f e c t s of norepinephrine s t i m u l a t i o n can be modulated under d i f f e r e n t p h y s i o l o g i c a l c o n d i t i o n s or d i f f e r e n t stages of an animal's growth. The t r o p h i c e f f e c t s of norepinephrine are of utmost s i g n i f i c a n c e during p e r i n a t a l development. These e f f e c t s become p a r t i c u l a r y important when the brown f a t reaches the peak of i t s d i f f e r e n t i a t i o n and f u n c t i o n a l c a p a c i t y one to two weeks a f t e r b i r t h [18 0]. The r o l e of norepinephrine i n r e g u l a t i o n of t i s s u e maturation e x e r t e d v i a c y c l i c GMP-mediated ^ - a d r e n e r g i c s t i m u l a t i o n was suggested by Skala and -102-Knight as e a r l y as 1979 [178]. T h i s proposed hypothesis was f u r t h e r supported by our i n v i t r o experiments ( d i s c u s s e d i n s e c t i o n 4.3). ©^-adrenergic mediation of the p r o l i f e r a t i v e process was a l s o observed i n other e u k a r y o t i c c e l l s [146], Thus i t appeared that the a c t i v e l y p r o l i f e r a t i n g brown adipose t i s s u e from one-week-old r a t s would be an e x c e l l e n t model system to search f o r the presence of ©^-adrenoceptors. The presence of p^-, and j8^- adrenoceptors had p r e v i o u s l y been shown i n membranes from the brown adipose t i s s u e of a d u l t r a t s [24, .127,162], i n s e c t i o n 4.1 we d i s c u s s the r e s u l t s of our l i g a n d b i n d i n g study demonstrating the presence of c^^-adreno-ceptors i n brown f a t of one-week-old r a t s u s i n g h i g h l y s p e c i f i c o ^ - l i g a n d s . Studies of adrenoceptors i n brown a d i p o c y t e s can help to e l u c i d a t e a b a s i c mechanism r e s p o n s i b l e f o r the f a c t that a hormone can have m u l t i p l e e f f e c t s i n a c e l l . The same c e l l s can respond to a hormone d i f f e r e n t l y under d i f f e r e n t circumstances. The divergence of these m u l t i p l e a c t i o n s of a hormone can occur a l r e a d y at the plasma membrane l e v e l : molecules of the hormone can probably bind to more than one type of the " d i s c r i m i n a t o r / e f f e c t o r " u n i t . The r e l a t i v e abundance and/or a f f i n i t i e s of these d i s c r i m i n a t o r u n i t s would then determine which e f f e c t (s) would predominate the c e l l u l a r response to the same hormonal s t i m u l i . By t h i s mechanism a c e l l can modify i t s own response i n accordance with the p r e -v a i l i n g c o n d i t i o n s . Experimental modulation of the t i s s u e -103-c o n d i t i o n s by chemical sympathectomy and c h r o n i c blockade of the r e c e p t o r s has been used to determine the f u n c t i o n a l relevance of the receptor b i n d i n g s i t e s f o r the t i s s u e . In the second p a r t of our study ( d i s c u s s e d i n s e c t i o n 4.2), we determined the b i n d i n g c h a r a c t e r i s t i c s of the a l p l i ^ - a d r e n o -ceptor system i n brown f a t of i n f a n t r a t s f o l l o w i n g chemical sympathectomy by 6-hydroxydopamine or by c h r o n i c exposure to yohimbine ( an e<2--ntagonist). B i n d i n g of norepinephrine to the o ^ - r e c e p t o r s i t e s i s not s u f f i c i e n t i n i t s e l f to e l i c i t s p e c i f i c s u b - c e l l u l a r responses. I t i s only when the hormonal s i g n a l s are passed on v i a e f f e c t o r system (s) that the message can be deciphered by the a d i p o c y t e s . I t has been known f o r many years t h a t c y c l i c AMP i s the second messenger i n the a d r e n e r g i c pathway a s s o c i a t e d w i t h s t i m u l a t i o n of yfl-adrencceptors. Much l e s s i s known about the nature of the c*^-adrenoceptor a s s o c i a t e d e f f e c t o r system, ^ - a d r e n o c e p t o r s t i m u l a t i o n has been shown to i n c r e a s e the turnover of p h o s p h a t i d y l i n o s i t o l and phos-p h a t i d i c a c i d . T h i s probably i s a s s o c i a t e d with an i n f l u x 2 + of Ca through 'gates' i n the f a t c e l l membrane or with r e -2 + lease of bound i n t r a c e l l u l a r Ca . c< 2~adrenoceptor and i t s a s s o c i a t e d e f f e c t o r system i s the l e a s t understood. Skala [178-180] had i m p l i c a t e d the c y c l i c GMP system as the p o s s i b l e mediator of responses a s s o c i a t e d w i t h ©V-receptor s t i m u l a t i o n during brown f a t growth and mat u r a t i o n . S e v e r a l other s t u d i e s have a l s o suggested involvement of c y c l i c GMP i n the develop--104-mental processes [40] i n the p r o l i f e r a t i o n of hepatocytes [126] and spleen c e l l s [47]. In t h i s context i t i s i n t e r e s t i n g to note that ©c^-adrenergic mediation of p r o l i f e r a t i v e p r ocesses was observed i n other e u k a r y o t i c c e l l s [146]. Thus, both ©^-adrenoceptors and c y c l i c GMP are i m p l i c a t e d to p l a y some r o l e i n t i s s u e growth and maturation, and brown f a t of i n f a n t r a t s seemed to be a very s u i t a b l e model f o r study of the ©^-adrenoceptor and i t s a s s o c i a t e d e f f e c t o r system. We f u r t h e r t e s t e d the h y p o t h e s i s of a l i n k between ©(-adrenoceptor s t i m u -l a t i o n and c y c l i c GMP accumulation i n brown f a t of one-week-o l d r a t s by a s e r i e s of i n v i t r o experiments ( d i s c u s s e d i n s e c t i o n 4.3). The i n h i b i t o r y a c t i o n of «*2~adrenoceptors on l i p o l y s i s and c y c l i c AMP accumulation i n human and hamster a d i p o c y t e s has been e x t e n s i v e l y s t u d i e d [3, 27, 101-103]. In the f o u r t h part of our study, we i n v e s t i g a t e d the i n h i b i t o r y a c t i o n of o ^ - a d r e n o c e p t o r s t i m u l a t i o n on l i p o l y s i s i n brown f a t a d i p o -cytes i s o l a t e d from 7-day-old r a t s . T h i s study ( d i s c u s s e d i n s e c t i o n 4.4) complements our l i g a n d b i n d i n g s t u d i e s on e < 2 - a d r e n o ~ ceptors and demonstrates t h a t these r e c e p t o r s show p h y s i o l o g i c a l c h a r a c t e r i s t i c s s i m i l a r to ^ - a d r e n o c e p t o r s r e p o r t e d i n ad i p o c y t e s from white f a t . I t i s known t h a t d e f e c t i v e brown adipose t i s s u e thermogenesis i s a s s o c i a t e d with o b e s i t y i n a number of different types of obese animals. Our i n i t i a l i n vivo study, showed time-depen--105-pendent changes i n the c y c l i c GMP c o n c e n t r a t i o n of brown f a t from lean mice f o l l o w i n g c o l d exposure (6°C) and norepinephrine a d m i n i s t r a t i o n . Contrary to t h a t , brown f a t of obese mice e x h i b i t e d no change i n c y c l i c GMP c o n c e n t r a t i o n when exposed to s i m i l a r s t i m u l i . T h i s suggested t h a t c y c l i c GMP system c o u l d have a r o l e i n thermogenesis and o b e s i t y . We, t h e r e f o r e , considered i t of i n t e r e s t to f u r t h e r pursue t h i s problem to study i f c y c l i c GMP c o n c e n t r a t i o n i n brown f a t fragments of obese mice was i n f l u e n c e d by c o l d exposure and <?<-receptor s t i m u l a t i o n d u r i n g i n v i t r o i n c u b a t i o n with ©<.- and J3- a d r e n e r g i c a g o n i s t s . 4.1 I d e n t i f i c a t i o n and c h a r a c t e r i s t i c s o f b i n d i n g s i t e s of c<..-adrenoceptors i n plasma membrane fragments of brown f a t from 7-day-old r a t s . The presence of p^- o^- and fi^- adrenoceptors has p r e v i o u s l y been shown i n membranes from brown adipose t i s s u e of a d u l t r a t s [24, 127, 162, 19J ]. However, no l i g a n d b i n d i n g study had yet been undertaken which demonstrated the presence of .^-.-adrenoceptors i n r a t brown f a t . The main o b j e c t i v e of our study d i s c u s s e d i n t h i s p a r t i s to show that membranes from brown f a t of i n f a n t r a t s c o n t a i n adrenoceptors which show b i n d i n g c h a r a c t e r i s t i c s compatible w i t h those expected of °<2 -s u b c l a s s . H i g h l y s e l e c t i v e - a n t a g o n i s t s and a g o n i s t s were used to study t h e i r b i n d i n g c h a r a c t e r i s t i c s t o i s o l a t e d plasma membrane fragments. One of the g r e a t e s t problems we encountered i n our -106-b i n d i n g s t u d i e s was the s c a r c i t y of the t i s s u e from the brown f a t of seven-day-old r a t s . Because of t h i s d i f f i c u l t y we were unable to c a r r y out e x t e n s i v e m e t h o d o l o g i c a l experiments and perform s t a t i s t i c a l treatment of a l l our data. The plasma membranes were prepared a c c o r d i n g to the method d e s c r i b e d by Maeda et a l . [117]. These authors have s u c c e s s f u l l y prepared and c h a r a c t e r i z e d the plasma membranes from a v a r i e t y of t i s s u e s and have shown t h a t the technique y i e l d s plasma membrane fragments w e l l s u i t e d f o r e q u i l i b r i u m b i n d i n g assays. In a d d i t i o n , Svoboda et a l . [192] have shown t h a t the minor contamination of i s o l a t e d membranes from brown f a t c e l l s by membranes from other c e l l types does not a l t e r the maximum b i n d i n g c a p a c i t y , a f f i n i t y and s p e c i f i -c i t y of the adrenoceptors. Ligand b i n d i n g s t u d i e s c a r r i e d out by u s i n g s e l e c t i v e a n d l i 9 a n d s a n d plasma membrane fragments from one-week-old r a t b r a i n c o r t e x and brown f a t showed b i n d i n g c h a r a c t e r i s t i c s very s i m i l a r to those already r e p o r t e d [24,163, 214 ]. i n view of these s t u d i e s we considered our membrane p r e p a r a t i o n a p p r o p r i a t e f o r i d e n t i -f i c a t i o n and c h a r a c t e r i z a t i o n of adrenoceptors, and no attempt was made to t e s t the p u r i t y of our membrane p r e p a r a t i o n s . I n i t i a l s t u d i e s undertaken to c h a r a c t e r i z e ©^-adreno-ceptors using [ 3H]-yohimbine, [ 3H]-RX-781094, [ 3 H ] - c l o n i d i n e and [ H]-norepinephrine ( F i g . 1, 2A, B, 2C, and D) showed that the b i n d i n g was r e v e r s i b l e and s a t u r a b l e w i t h h a l f s a t u r a -t i o n o c c u r r i n g at l i g a n d c o n c e n t r a t i o n s of approximately 15 nM -107-( y o h i m b i n e ) , 10 nM ( R X - 7 8 1 0 9 4 ) a n d 25 nM ( c l o n i d i n e ) . S t a t i s -t i c a l t r e a t m e n t o f r e s u l t s w a s d i f f i c u l t b e c a u s e we c o u l d o n l y o b t a i n s u f f i c i e n t a m o u n t o f t h e m e m b r a n e p r o t e i n f o r a v e r y l i m -i t e d n u m b e r o f a s s a y s . N e v e r t h e l e s s , i n s o m e i n s t a n c e s , k i n e t i c a n a l y s i s w a s p e r f o r m e d . T h e c a l c u l a t e d 18 nM o f y o h i m b i n e f r o m S c a t c h a r d p l o t ( F i g . 1A) w a s b e t w e e n t h o s e r e p o r t e d f r o m h a m s t e r e p i d i d y m a l f a t p a d a d i p o c y t e s 24 0 nM [ 6 6 ] a n d t h o s e r e p o r t e d i n a d i p o c y t e s f r o m h u m a n a b d o m i n a l SC a d i p o s e t i s s u e 4 - 5 nM [ 1 9 5 ] . T h e B m a x v a l u e ( 1 5 6 f m o l / m g p r o t e i n ) w a s i n t h e s a m e r a n g e a s r e p o r t e d b y T h a r p e a n d L e f k o w i t z i n 19 81 ( 1 4 5 f m o l / m g p r o t e i n ) f o r h u m a n w h i t e f a t a d i p o c y t e s [19 5 ] . T h e t e m p r a t u r e d e p e n d e n c e 3 a n d r e v e r s i b i l i t y o f t h e s p e c i f i c b i n d i n g o f [ H ] - R X - 7 8 1 0 9 4 i n -d i c a t e s t h a t t h e b i n d i n g i s a t t h e p h y s i o l o g i c a l l y a c t i v e oi^-3 r e c e p t o r s i t e s . T h e l o w e r s a t u r a t i o n o f c l o n i d i n e [ H ] - c l o n i d i n e b i n d i n g u s i n g y o h i m b i n e c o m p a r e d t o c l o n i d i n e a s t h e d i s p l a c i n g a g e n t ( F i g . 2B) c o u l d r e f l e c t t h e f a c t c l o n i d i n e i s a p a r t i a l a g o n i s t o f i m i d a z o l i n e g r o u p a n d m a y n o t o n l y h a v e d i f f e r e n t b i n d i n g s i t e s t h a n y o h i m b i n e b u t a d i f f e r e n t a f f i n i t y f o r t h e t h e s e s i t e s a s w e l l [ 6 6 ] , B o t h ( - ) - e p i n e p h r i n e a n d y o h i m b i n e w e r e e q u i p o t e n t i n 3 d i s p l a c i n g ( - ) [ H ] - n o r e p i n e p h r i n e f r o m i t s b i n d i n g s i t e s ( F i g . 3 3 A ) . When ( - ) [ H ] - e p i n e p h r i n e a n d y o h i m b i n e w e r e u s e d a l o n e t o 3 d i s p l a c e ( - ) [ H ] - n o r e p i n e p h r i n e f r o m i t s b i n d i n g s i t e s , a o f 6 0 . 4 n M a n d 6 5 . 8 n M a n d B ^ v a l u e s o f 0 . 2 2 a n d 0 . 2 4 p m o l / m g m a x p r o t e i n r e s p e c t i v e l y w e r e o b t a i n e d . S i m i l a r a n d & m a x v a l u e s i n d i c a t e t h a t b o t h ( - ) - e p i n e p h r i n e a n d y o h i m b i n e - 1 0 8 -are competing with (-)[ H]-norepinephrine b i n d i n g f o r the same re c e p t o r with s i m i l a r a f f i n i t i e s . To i n v e s t i g a t e t h i s problem f u r t h e r , a combination of yohimbine and (-)-epin-3 ephrine was used to d i s p l a c e (-)[ H]-norepinephrine from i t s 3 bi n d i n g s i t e s . The s p e c i f i c displacement of (-)[ ^ - n o r e p i n -ephrine i n the presence of a combination of yohimbine and epinephrine was approximately 30% h i g h e r than i n the presence of e i t h e r one a l o n e . A p o s s i b l e i n t e r p r e t a t i o n of t h i s f i n d i n g i s t h a t both yohimbine and epinephrine b i n d t o more than one s i t e , and they share at l e a s t one common b i n d i n g s i t e . When the s p e c i f i c b i n d i n g of the second non-sharing b i n d i n g s i t e was analyzed by Scatchard a n a l y s i s i t showed n e a r l y the same K d (64.5 nM) but a lower B value (71 fmol/mg p r o t e i n ) . Yohim-bine shared t h i s second s i t e with n o r e p i n e p h r i n e but not w i t h ep i n e p h r i n e . I d e n t i c a l H i l l c o e f f i c i e n t s of approximately 1.4 a l s o support the presence of more than one b i n d i n g s i t e f o r yohimbine and epinephrine ( F i g . 3B). The r e l a t i v e l y low d e n s i t y of the second b i n d i n g s i t e makes i t more d i f f i c u l t to detect s i n c e i t seems to be masked by the r e l a t i v e l y much higher d e n s i t y of the other r e c e p t o r s i t e s i n experiments using s i n g l e l i g a n d - s i n g l e d i s p l a c i n g agent. Both (-)-epinephrine and (-)-norepinephrine are known to be mixed adrenoceptor a g o n i s t s , t h a t i s , they b i n d to both theoi- and fS- adrenoceptor s u b c l a s s e s . However, i n c o n t r a s t to n o r e p i n e p h r i n e , epinephrine e x h i b i t s a h i g h e r a f f i n i t y f o r the s i t e s than f o r the J3 - s i t e s i n i n v i t r o -109-assays and b i n d i n g s t u d i e s [105]. In c o m p e t i t i o n experiments 3 ( F i g . 4A) , the higher displacement of C-) - [ H]-norepinephrine by (-)-epinephrine i n a lower range of c o n c e n t r a t i o n than p r a c t o l o l suggests t h a t (-)-epinephrine and p r a c t o l o l are competing f o r two d i f f e r e n t types of r e c e p t o r s - most l i k e l y f o r the e*- and jS- r e c e p t o r s r e s p e c t i v e l y . T h i s i s supported by the r e s u l t s of Fig.4B which i n d i c a t e t h a t (-)-epinephrine can d i s p l a c e , i n a dose-dependent manner, (-) ^ ^ - n o r e p i n -ephrine from i t s b i n d i n g s i t e s even i n the presence of p r a c -t o l o l . Conversely, p r a c t o l o l d i s p l a c e d [ H]-norepinephrine from i t s b i n d i n g s i t e s even i n the presence of e p i n e p h r i n e . An apparent d i s s o c i a t i o n constant (K^) of 66 nM f o r e p i n e p h r i n e c a l c u l a t e d u s i n g the Cheng and P r u s s o f f e q u a t i o n [35] was almost i d e n t i c a l to the of yohimbine o b t a i n e d from the Scatchard a n a l y s i s i n F i g . 3A using the same l i g a n d . Thus it-seems t h a t (-)-epinephrine competes with n o r e p i n e p h r i n e at the o ^ - r e c e p t o r s i n brown f a t of 7-day-old r a t s i n the lower con-c e n t r a t i o n range. S i m i l a r °^2~ s p e c i f i c i t y of epinephrine has p r e v i o u s l y been r e p o r t e d i n s e v e r a l other s t u d i e s [204, 205,206 ]. 3 S a t u r a t i o n b i n d i n g s t u d i e s c a r r i e d out u s i n g (-)[ H]-d i h y d r o a l p r e n o l o l as the l i g a n d and 10 u^-M (-)-norepinephrine and 10 p r a c t o l o l as the n o n - l a b e l l e d d i s p l a c i n g agents showed r a p i d , s a t u r a b l e and r e v e r s i b l e b i n d i n g to a f i n i t e number of r e c e p t o r s ( F i g . 5A). The d i s s o c i a t i o n constant (K^) of 10 nM (norepinephrine) was s i m i l a r t o t h a t r e p o r t e d by -110-W i l l i a m s e t a l • [ 2 1 4 ] i n r a t a d i p o c y t e s f r o m p a r a m e t r i a l t i s s u e . W i t h p r a c t o l o l , t h e o f 50 nM w a s n e a r l y t h e s a m e a s t h a t r e p o r t e d b y B u k o w i e c k i e t a l . f o r ( - ) - p r o p r a n o l o l i n m e m b r a n e f r a g m e n t s f r o m a d u l t r a t b r o w n f a t [ 2 4 ] . T h e B max v a l u e o f 0 . 1 9 p m o l / m g p r o t e i n f o r t h e / ^ - a d r e n o c e p t o r s i n o u r m e m b r a n e p r e p a r a t i o n s w a s s o m e w h a t h i g h e r t h a n t h o s e r e p o r t e d b y S v o b o d a e t a l • [ 1 9 1 ] i n h a m s t e r b r o w n f a t c e l l s ( 9 2 f m o l / 10 c e l l s o r 4 6 f m o l / m g p r o t e i n a s s u m i n g 2 mg m e m b r a n e p r o t e i n / 1 0 c e l l s ) . T h i s i n c r e a s e d n u m b e r o f ^ - a d r e n o c e p t o r s i n o u r p r e p a r a t i o n s c o u l d b e d u e t o t h e a c c e n t u a t e d n e e d f o r n o r e p i n -e p h r i n e - s t i m u l a t e d n o n - s h i v e r i n g t h e r m o g e n e s i s i n o n e - w e e k -o l d r a t s a c c o m p a n i e d b y i n c r e a s e d b r o w n f a t g r o w t h , d i f f e r e n -t i a t i o n , a n d i n c r e a s e d f u n c t i o n a l c a p a c i t y [ 1 8 0 ] « I n c r e a s e d d e m a n d f o r t h e r m o g e n e s i s i n b r o w n f a t o f c o l d - a c c l i m a t e d a d u l t r a t s i s a s s o c i a t e d w i t h i n c r e a s e d ^ - a d r e n o c e p t o r s n u m b e r [ 2 4 ] . Y o h i m b i n e , a n o ^ - a n t a g o n i s t , w a s q u i t e e f f e c t i v e 3 i n d i s p l a c i n g ( - ) [ H ] - d i h y d r o a l p r e n o l o l ( F i g . 5 B ) w i t h a K d o f 39 n M . T h i s v a l u e i s c l o s e t o t h e r e p o r t e d v a l u e f o r y o h i m b i n e b i n d i n g t o © ^ - a d r e n o c e p t o r s i n m e m b r a n e f r a g -m e n t s f r o m g u i n e a - p i g k i d n e y ( 4 8 nM) a n d c a l f c e r e b r a l c o r t e x ( 3 4 nM) b y B r o d d e e t a l . [ 2 0 ] , ( - ) [ 3 H ] - d i h y d r o a l p r e n o l o l h a s b e e n u s e d e x t e n s i v e l y i n c h a r a c t e r i z i n g ^ - a d r e n o c e p t o r s . 3 T h i s d i s p l a c e m e n t o f ( - ) [ H ] - d i h y d r o a l p r e n o l o l b y y o h i m b i n e w a s r a t h e r s u r p r i s i n g b e c a u s e t h i s w o u l d i n d i c a t e t h a t e i t h e r t h e l i g a n d i s n o t a b s o l u t e l y s p e c i f i c f o r t h e ^ - a d r e n o c e p t o r s , o r t h e y f l - a d r e n o c e p t o r s i n b r o w n f a t o f i n f a n t r a t s h a v e - 1 1 1 -'atypical or "hybrid" characteristics similar to those observed in lipolytic responses of rat adipocytes to adrenergic agonists [7, 8, 46]. Detailed analysis of the binding characteristics 3 of [ H]- DHA to membrane fractions from adult rat hepatocytes and adipocytes from epididymal tissue show more than one bind-ing site for [3H]-DHA [41,133, 166']. Its high affinity bind-ing site is sensitive to displacement by ^-agonists and anta-gonists, whereas the second, low affinity site is occupied by ^-antagonists [ 133, 166]. Bukowiecki et a l . in their study were unable to see any dis-3 placement of (-)[ H]-DHA binding to partially purified membrane fragments from brown fat of adult rats by phentolamine or phen-oxybenzamine. A possible explanation could be that the isolation and purification technique used by these authors is different 3 from that used in our study. [ H]-DHA is a lipophilic ligand and its binding characteristics to sites other than the B-site may differ according to membrane isolation and purification methods. A more plausible explanation may lie in the age difference of the animals used in our study and those used by Bukowiecki et al . Brown fat of one-week-old rats is approaching the peak of its growth, differentiation and functional capacity. Adrenergic stimulation exerted via ©^ -adrenoceptors is known to participate in regulations of proliferative activity of other cell types [146] . Hence, perhaps the characteristics of the adrenoceptor sys-tem on brown fat plasma membrane are expressed fully only during the early ontogenic stages of animal's l i fe . - 1 1 2 -The presence of u -.-adrenoceptors even i n the brown f a t of a d u l t r a t s has been i n d i r e c t l y i n d i c a t e d by s u p p r e s s i o n of the f o r s k o l i n - and i s o p r o t e r e n o l - induced l i p o l y s i s and r e s p i r a t i o n i n i s o l a t e d adipocytes [190]. I t i s i n t e r e s t i n g to note t h a t Kunos and Ishac [100] have r e c e n t l y suggested that r e c i p r o c a l changes i n «*^- and J3^-adrenoceptor s t i m u l a t e d responses, observed by them on i s o l a t e d r a t hepatocytes i n v i t r o , c ould r e p r e s e n t a new adaptive mechanism by which the t i s s u e i n v i v o may r e a c t to a l t e r e d m etabolic c o n d i t i o n s [100] . I t i s p o s s i b l e t h a t the charac-t e r i s t i c s of the adrenoceptor system i n plasma membrane of brown adipocytes c o u l d a l s o change d u r i n g ontogenic development, and thus the i n t r a c e l l u l a r response to an i d e n t i c a l n o r e p i n -ephrine s t i m u l u s c o u l d be modulated a c c o r d i n g to the age of the animals. In summary, the major f i n d i n g s of t h i s p a r t of our study are: (i) B i n d i n g of h i g h l y s e l e c t i v e o ^ - a n t a g o n d s t s and a g o n i s t s to plasma membrane fragments from brown f a t of 1-week-old r a t s show b i n d i n g c h a r a c t e r i s t i c s as expected of b i n d i n g to s p e c i f i c e x _ - a d r e n o c e p t o r s i t e s . 3 ( i i ) B i n d i n g of (-)[ H]-norepinephrine i n d i c a t e d the presence of more than one b i n d i n g s i t e . Norepin-ephrine, yohimbine and (-)-epinephrine probably shared one common b i n d i n g s i t e ; the other s i t e seemed -113-to be p r e s e n t i n much lower number and was s p e c i f i c f o r yohimbine and norepinephrine o n l y . 3 ( i i i ) (-)[ H]-norepinephrine bound to both °<- and/3-adrenoceptors. B i n d i n g to ©c 2-adrenoceptors was d i s p l a c e a b l e by yohimbine and by (-)-epinephrine i n lower c o n c e n t r a t i o n ranges. 3 (iv) (-)[ H]-d i h y d r o a l p r e n o l o l showed two b i n d i n g s i t e s . S i t e 1 showed b i n d i n g c h a r a c t e r i s t i c s s i m i l a r to those rep o r t e d f o r ^ - a d r e n o c e p t o r s . The second s i t e i n d i -cated b i n d i n g to s i t e s other than the /^-adrenoceptor. I t was p o s t u l a t e d t h a t t h i s b i n d i n g component was due to the ^ - a d r e n o c e p t o r . .... 4.2 E f f e c t of chemical sympathectomy and c h r o n i c yohim-bine pre-treatment on b i n d i n g c h a r a c t e r i s t i c s of ^ ^ - a d r e n o c e p t o r s i n brown f a t of 7-day-old r a t s . Ligand b i n d i n g experiments c a r r i e d out i n other t i s s u e s show that the p r o p e r t i e s of f u n c t i o n a l l y a c t i v e adrenoceptor b i n d i n g s i t e s can be a l t e r e d by chemical sympathectomy and by a c h r o n i c exposure to r e c e p t o r b l o c k i n g agent. We, t h e r e f o r e , considered i t a p p r o p r i a t e to study the b i n d i n g c h a r a c t e r i s t i c s of ^ - a d r e n o c e p t o r s f o l l o w i n g i n v i v o experimental modulations. In t h i s s e c t i o n we d i s c u s s the t r e n d i n b i n d i n g c h a r a c t e r i s t i c s o f the o^-adrenoceptors i n membranes i s o l a t e d from brown f a t o f i n f a n t r a t s f o l l o w i n g chemical sympathectomy or c h r o n i c yohim-bine a d m i n i s t r a t i o n . -114 — 6-hydroxydopamine was used to achieve total sympath-ectomy of brown fat nerve terminals, while a chronic blockade of c*2-receptors was produced by daily intraperitoneal injections of yohimbine in high concentrations. Chemical sympathectomy by a single dose administration of 6-hydroxydopamine has been shown to destroy adrenergic nerve endings supplying brown fat [176, 197, 198], Equilibrium bind-ing studies carried out on brown fat plasma membrane fragments from chemically sympathectomized 5-day-old rats resulted in higher affinity of yohimbine for its binding sites (Fig. 6A) to °< 2 - a (^" renoceptors 3 days later. Similar changes in the affinity and/or density of both <X- and jB-adrenoceptors have been reported to occur in rat brain after sympathectomy [81, 185, -206, 209]. How-3 ever a lowered number of total binding sites of [ H]-yohimbine (155 fmol/mg protein in the controls compared to 45.5 fmol/mg protein in. experimental animals) was rather surprising as we ex-3 pected an increase in the specific binding of [ H]-yohimbine f o l l -owing chemical sympathectomy. A of 66.8 nM for (-)-epinephrine (Fig. 7A) obtained 3 from (-)[ H]-norepinephrine binding to brown fat plasma membranes from control animals was similar to a of 60.4 nM reported in our earlier section. However, a significantly higher B (1.39 max 3 pmol/mg protein was obtained in the binding of (-)[ H]-norepineph-rine compared to our earlier reported value of 0.22 pmol/mg protein. This discrepancy could be due to the use of a different group of animals who were exposed to more extensiv  handling d to saline injections. It has -115-a l s o b e e n s u g g e s t e d t h a t t h e b i n d i n g o f [ H ] - a g o n i s t s ( e s p e c i a l l y c a t e c h o l a m i n e s ) t o o i 2 ~ r e c e p t o r s i s i n f l u e n c e d b y t h e p r e s e n c e o f i o n s s u c h a s N a + , Mg++, a n d n u c l e o t i d e s s u c h a s GTP [ 3 0 ] . I t i s p o s s i b l e t h a t i n p a r t i a l l y p u r i f i e d mem-b r a n e p r e p a r a t i o n s t h e c o n c e n t r a t i o n s o f t h e s e i o n s a n d / o r c o m p o u n d s c a n v a r y b e t w e e n d i f f e r e n t p r e p a r a t i o n s o f t h e mem-b r a n e f r a g m e n t s . T h e r e f o r e , t h e e x p e r i m e n t a l a n d c o n t r o l mem-b r a n e p r e p a r a t i o n s r e p o r t e d h e r e w e r e a l w a y s i s o l a t e d s i m u l -t a n e o u s l y . B i n d i n g o f ( - ) [ H ] - n o r e p i n e p h r i n e t o p l a s m a m e m b r a n e s i s o l a t e d f r o m b r o w n f a t o f s y m p a t h e c t o m i z e d r a t s s h o w e d c h a r a c t e r i s t i c s c o m p a t i b l e w i t h t h e p r e s e n c e o f a t l e a s t t w o d i s t i n c t p o p u l a t i o n s o f b i n d i n g s i t e s . A s m a l l e r p r o p o r t i o n o f b i n d i n g s i t e s s h o w e d c h a r a c t e r i s t i c s o f h i g h a f f i n i t y s i t e s , w h e r e a s a m u c h l a r g e r n u m b e r o f s i t e s e x h i b i t e d v e r y l o w a f f i n i t y f o r t h e l i g a n d . T h e f o r m e r w e r e p r o b a b l y i d e n t i c a l t o t h e s m a l l 3 n u m b e r o f h i g h a f f i n i t y b i n d i n g s i t e s r e v e a l e d i n t h e [ H ] -3 y o h i m b i n e b i n d i n g e x p e r i m e n t s . T h e a b s o l u t e a m o u n t o f ( - ) [ H ] -n o r e p i n e p h r i n e s p e c i f i c b i n d i n g t o m e m b r a n e s f r o m s y m p a t h e c -t o m i z e d a n i m a l s w a s m u c h h i g h e r t h a n i n t h e c o n t r o l s . T h i s i n c r e a s e s e e m e d t o b e d u e e n t i r e l y t o t h e a p p e a r a n c e o f t h e l o w a f f i n i t y r e c e p t o r s i t e s ( F i g . 7 B ) . C a t e c h o l a m i n e s a r e c l a s s i f i e d a s m i x e d a g o n i s t s ( i . e . , t h e y b i n d t o b o t h « K - a n d 3 ^ - r e c e p t o r s ) . T h e h i g h e r d e g r e e o f [ H ] - n o r e p i n e p h r i n e s p e c i f i c b i n d i n g s e e n i n F i g . 7B c o u l d a l s o r e s u l t f r o m s p e c i f i c b i n d i n g t o ^ - r e c e p t o r s . T h e s p e c i f i c b i n d i n g o f -116-( - ) [ H ] - n o r e p i n e p h r i n e i n t h e p r e s e n c e o f 10 ptM ( - ) - e p i n e p h r i n e w a s t h e same a s t h a t i n t h e p r e s e n c e o f a c o m b i n a t i o n o f 10 /*M ( - ) - e p i n e p h r i n e a n d 10 /*M p r a c t o l o l ( F i g . 7 C ) t h u s i n d i c a t i n g 3 t h a t t h e m a j o r i t y o f t h e ( - ) [ H ] - n o r e p i n e p h r i n e b i n d i n g s i t e s w e r e o f t h e ©<- t y p e . I n t h e c o m p e t i t i o n e x p e r i m e n t s ( F i g . 8 ) b o t h ( - ) -3 e p i n e p h r i n e a n d y o h i m b i n e c o u l d d i s p l a c e ( - ) [ ^ - n o r e p i n -e p h r i n e f r o m i t s b i n d i n g s i t e s i n m e m b r a n e s f r o m b r o w n f a t o f s y m p a t h e c t o m i z e d a n i m a l s i n a c o n c e n t r a t i o n d e p e n d e n t m a n n e r . ( - ) - E p i n e p h r i n e w a s m o r e e f f e c t i v e a t l o w e r c o n c e n t r a t i o n s t h a n y o h i m b i n e . T h e s e r e s u l t s a r e s i m i l a r t o t h o s e i n s a t u r a t i o n a n a l y s i s ( F i g . 7 A , B ) w h e r e a m u c h h i g h e r d e g r e e o f a b s o l u t e 3 a m o u n t o f [ H ] - n o r e p i n e p h r i n e s p e c i f i c b i n d i n g i n t h e p r e s e n c e 3 o f 10 pM e p i n e p h r i n e w a s o b s e r v e d c o m p a r e d t o a n t a g o n i s t [ H ] -y o h i m b i n e ( F i g . 6 A , B ) . S t u d i e s b y U ' P r i c h a r d a n d S n y d e r r e p o r t r e l a t i v e l y h i g h e r e f f e c t i v e n e s s o f a g o n i s t s c o m p e t i n g f o r b i n d i n g s i t e s o c c u p i e d b y o t h e r a g o n i s t s [ 2 0 4 , 2 0 5 ] . T h e s t e e p s l o p e f o r ( - ) - e p i n e p h r i n e a n d y o h i m b i n e s u g g e s t s c o o p e r a t i v i t y i n n o r e p i n e p h r i n e b i n d i n g s i t e s . T h e t w o c u r v e s e x h i b i t e d n o n -p a r a l l e l c o u r s e s . T h i s o b s e r v a t i o n i s i n a g r e e m e n t w i t h t h e r e s u l t s d i s c u s s e d i n s e c t i o n 4 . 1 i n d i c a t i n g d i s p l a c e m e n t o f 3 [ H ] - n o r e p i n e p h r i n e b y y o h i m b i n e a n d e p i n e p h r i n e f r o m a t l e a s t o n e n o n - o v e r l a p p i n g s i t e . C h r o n i c b l o c k a d e o f t h e ^ - a d r e n o c e p t o r s i n v i v o b y r e p e a t e d a d m i n i s t r a t i o n o f h i g h d o s e s o f y o h i m b i n e s h o w e d a n i n c r e a s e i n t h e n u m b e r o f < x 9 - b i n d i n g s i t e s i n b r o w n f a t p l a s m a - 1 1 7 -membranes i s o l a t e d from 2 1 - d a y - o l d r a t s . E q u i l i b r i u m b i n d i n g s t u -3 d i e s c a r r i e d out with<* 2-specif i c l i g a n d s [ H]-rauwolscine (_<-me-thyl-yohimbine, [ 3H]-yohimbine and [ 3H]-RX781094 ( F i g . 9A, B, C) i n d i c a t e d an i n c r e a s e i n the apparent number of s p e c i f i c b i n d i n g s i t e s as compared to b i n d i n g on membranes from non-treated r a t s ( F i g . 3 2A, 6A). Scatchard a n a l y s i s of [ H]-yohimbine b i n d i n g ( F i g . 9C) i n d i c a t e d both an in c r e a s e i n the number of bi n d i n g s i t e s (B 0.35 pmol/mg p r o t e i n ) and a decrease i n a f f i n i t y (K, max o 66.7 nM) compared to that i n c o n t r o l s (B m a x 0 . 1 5 pmol/mg p r o t e i n 3 and K d 14 nM) ( F i g . 6A). The b i n d i n g of (-)[ H]-norepinephrine to i d e n t i c a l membrane p r e p a r a t i o n s showed an unchanged value but a B v a l u e almost double t h a t of c o n t r o l animals. Almost max i d e n t i c a l v a l u e s (K^ 68.3 and 66.7 nM) of the s p e c i f i c b i n d i n g were obtained whether yohimbine or epi n e p h r i n e served as the competing agent. These r e s u l t s suggest a s h i f t of s m a l l , h i g h a f f i n i t y b i n d i n g s i t e s of ©^.-.-receptors to a p o p u l a t i o n of low a f f i n i t y and h i g h d e n s i t y b i n d i n g s i t e s f o l l o w i n g c h r o n i c yohimbine a d m i n i s t r a t i o n . The i n c r e a s e d r e c e p t o r d e n s i t y f o l l o w i n g a c h r o n i c blockade of the ©^-adrenoceptors r e p o r t e d here i s s i m i l a r to the o b s e r v a t i o n s of higher ^ - a d r e n o c e p t o r d e n s i t y i n human lymphocytes and r a t heart v e n t r i c l e and lungs f o l l o w i n g 10 days of p r o p r a n o l o l a d m i n i s t r a t i o n [1, 21]. The mechanism r e s p o n s i b l e f o r such a compensating r e g u l a t i o n of the rec e p t o r phenotype i s not known. The p o s s i b l e e x p l a n a t i o n s i n c l u d e : (a) de novo s y n t h e s i s of a d d i t i o n a l r e c e p t o r s ; (b) r e v e r s a l of down-regulation exerted by endog--118-enous catecholamines; and (c) a slower r a t e of degradation of the a n t a g o n i s t - o c c u p i e d r e c e p t o r s . In a d d i t i o n , Kunos and Ishac [100] have r e c e n t l y proposed another mechanism: namely an i n t e r c o n v e r s i o n of d i s t i n c t r e c e p t o r p o p u l a t i o n s or t h e i r r e c i p r o c a l a c t i v a t i o n or i n h i b i t i o n . Such a change i n the r e c e p t o r phenotype or i t s modulation may allow organs and t i s s u e s to adapt t h e i r i n v i v o hormonal responsiveness to the p r e v a i l i n g metabolic or other c o n d i t i o n s . In view of the recent r e p o r t s of diminished hormonal s e n s i t i v i t y of brown f a t i n o b e s i t y [78] and of the c a l o r i g e n i c e f f e c t s of some adr e n e r g i c a g o n i s t s and t h e i r analogues used i n o b e s i t y treatments [8, 31,147 ], d e t a i l e d s t u d i e s of the brown f a t adrenoceptor phenotype and i t s developmental r e g u l a t i o n s are very important. These r e s u l t s i n d i c a t e that the brown f a t of i n f a n t animals c o n t a i n s adreno-ceptors which e x h i b i t c h a r a c t e r i s t i c s compatible with ©^-sub-c l a s s i f i c a t i o n . Furthermore, the number and/or b i n d i n g prop-e r t i e s can be i n f l u e n c e d e x p e r i m e n t a l l y , a f i n d i n g which per-haps i n d i c a t e s t h a t these r e c e p t o r s i t e s are p h y s i o l o g i c a l l y a c t i v e . Summary: (i) Chemical sympathectomy by 6-hydroxydopamine showed an i n c r e a s e i n the a f f i n i t y and a decrease i n the number of b i n d i n g s i t e s of yohimbine to brown f a t plasma membrane fragments i s o l a t e d from 7-day-old r a t s two days l a t e r . 3 • ( i i ) T o t a l number of (-)[ H]-norepinephrme b i n d i n g s i t e s -119-to ^ - a d r e n o c e p t o r s i n plasma membrane fragments i s o l a t e d from sympathectomized animals was higher than t h a t i n the c o n t r o l s . The b i n d i n g showed c h a r a c t e r i s t i c s compatible with the presence of at l e a s t two d i s t i n c t p o p u l a t i o n s of b i n d i n g s i t e s . The i n c r e a s e i n the t o t a l number of b i n d i n g s i t e s seemed to be due e n t i r e l y to the appearance of low a f f i n i t y s i t e s . 3 ( i i i ) (-)-Epinephrine d i s p l a c e d (-) [ H]-norepinephrine from i t s b i n d i n g to ^ - a d r e n o c e p t o r s -( i v ) Chronic blockade of o^-adrenoceptors i n v i v o by r e -peated a d m i n i s t r a t i o n of h i g h doses of yohimbine r e s u l t e d i n changes i n the a f f i n i t y and d e n s i t y of cX^-adrenoceptors f o r yohimbine. 4 . 3 E f f e c t of a d r e n e r g i c a g o n i s t s and a n t a g o n i s t s i n v i t r o on c y c l i c n u c l e o t i d e contents i n brown adipose t i s s u e of 7-day-old r a t s .  The s p e c i f i c r e c e p t o r s and t h e i r e f f e c t o r systems mediating the p h y s i o l o g i c a l a c t i o n s o f catecholamines have : long been used as models i n the e l u c i d a t i o n of the mechanisms by which hormones and n e u r o t r a n s m i t t e r s communicate t h e i r i n t r a c e l l u l a r messages. In brown adipose t i s s u e , c y c l i c AMP t r a n s m i t s the ^ - a d r e n e r g i c s i g n a l to the c e l l to a c t i v a t e l i p o l y s i s v i a the system of p r o t e i n k i n a s e s . Although the ^-adren-e r g i c pathway of brown f a t p l a y s a major r o l e i n n o n - s h i v e r i n g -120-t h e r m o g e n e s i s [ 1 7 9 ] , H o r w i t z a n d H o r o w i t z a s e a r l y a s 1 9 7 7 [ 8 5 ] h a d s u g g e s t e d a m i n o r r o l e o f ^ ( . - a d r e n o c e p t o r s i n h e a t p r o d u c t i o n b y b r o w n f a t . T h e p r e s e n c e o f a s e c o n d , a l t e r n a t e p a t h w a y b e s i d e s t h e ^ - a d r e n e r g i c - p a t h w a y a c t i n g a s a m o d u l a t o r o f t h e l i p a s e - s u b s t r a t e i n t e r a c t i o n w a s a l s o s u g g e s t e d b y W i s e a n d J u n g a s [ 2 1 8 ] . P h a r m a c o l o g i c a l s t u d i e s [ 1 2 7 - 1 2 9 ] h a v e now s h o w n t h a t a n a l p h a - a d r e n e r g i c a l l y m e d i a t e d p r o c e s s c o n -t r i b u t e s a b o u t 2 0 % t o t h e t o t a l m e a s u r e d h e a t p r o d u c t i o n i n b r o w n f a t o f h a m s t e r s . W h i l e i t i s c l e a r t h a t t h e B - a d r e n e r g i c p a t h -way i s m a i n l y r e s p o n s i b l e t o f u l f i l l t h e b o d y ' s n e e d f o r h e a t p r o d u c t i o n d u r i n g t h e r m o g e n e s i s , n o e n t i r e l y s a t i s f a c t o r y r o l e c a n b e a s c r i b e d t o t h e c<-adrenergic p a t h w a y . T h e o < - a d r e n e r g i c -e f f e c t o r s y s t e m i s e v e n l e s s u n d e r s t o o d . I n r e c e n t y e a r s , t h r e e h y p o t h e s e s h a v e b e e n a d v a n c e d t o e x p l a i n t h e f o r m a t i o n o f " s e c o n d m e s s e n g e r s " a s a r e s u l t o f ^ - a d r e n o c e p t o r s t i m u l a t i o n i n b r o w n f a t . N e d e r g a a r d [ 1 3 8 ] h a s r e p o r t e d a n i n c r e a s e d N a + f l u x i n t o t h e c e l l a s a r e s u l t o f s t i m u l a t i o n o f ( ^ - a d r e n o c e p t o r s i n a d i p o c y t e s f r o m b r o w n f a t . T h e i n c r e a s e d c y t o s o l i c C a + + may t h e n s t i m u l a t e v a r i o u s c e l l u l a r a c t i v i t i e s . T h e s e c o n d h y p o t h e s i s s u g g e s t s t h e f o r m u l a t i o n o f " s e c o n d m e s s e n g e r s " s u c h a s i n o s i t o l t r i p h o s p h a t e a n d d i a c y l g l y c e r o l a s a r e s u l t o f e x ^ - r e c e p t o r s t i m u l a t i o n . T h i s r e l e a s e s C a + + f r o m i n t r a -c e l l u l a r s t o r a g e s i t e s [ 5 2 ] a c t i v a t e s p r o t e i n k i n a s e C v i a d i a c y l g l e r c o l [ 1 4 3 ] , T h e t h i r d h y p o t h e s i s s u g g e s t s t h e c y c l i c GMP s y s t e m a s t h e m e d i a t o r o f ^ - a d r e n o c e p t o r s t i m u l a -t i o n . P o i n t e r [ 1 5 2 ] h a d s h o w n a c c u m u l a t i o n o f c y c l i c GMP i n - 1 2 1 -i s o l a t e d r a t l i v e r c e l l s a s a r e s u l t o f ^ - a d r e n o c e p t o r s t i m -u l a t i o n , a l t h o u g h F a i n a n d B u t c h e r [ 5 3 ] w e r e u n a b l e t o n o t i c e a n y c o r r e l a t i o n b e t w e e n c y c l i c GMP a c c u m u l a t i o n a n d l i p o l y s i s i n f a t c e l l s f r o m r a t p a r a m e t r i a l a d i p o s e t i s s u e . S k a l a a n d K n i g h t , b a s e d o n t h e i r i n v i v o s t u d y [ 1 7 8 ] , p r o p o s e d t h e p r e s -e n c e o f a l i n k b e t w e e n ^ - a d r e n e r g i c s t i m u l a t i o n a n d c y c l i c GMP a c c u m u l a t i o n i n r a t b r o w n f a t . T h e h y p o t h e s i s w a s f u r t h e r t e s t e d b y i n v i t r o e x p e r i m e n t s . I n t h i s s e c t i o n we d i s c u s s t h e e f f e c t o f oi- a n d ft- a g o n i s t s a n d a n t a g o n i s t s i n v i t r o o n c y c l i c n u c l e o t i d e c o n t e n t s o f b r o w n f a t . S i n c e t h e p h y s i o l o g i c a l l y r e l e v a n t p o r t i o n o f t o t a l c y c l i c GMP i s t h a t w h i c h b i n d s t o c y c l i c G M P - d e p e n d e n t p r o t e i n k i n a s e , a n a t t e m p t w a s made t o e s t i m a t e t h e f r a c t i o n o f t o t a l c y c l i c GMP t h a t a c t i v a t e s c y c l i c G M P - d e p e n d e n t p r o t e i n k i n a s e . E a r l i e r a t t e m p t s b y S k a l a a n d K n i g h t [ 1 7 7 ] t o d e v e l o p a n " a c t i v a t i o n r a t i o " a s s a y f o r t h e c y c l i c G M P - d e p e n d e n t p r o -t e i n k i n a s e , s i m i l a r t o t h a t u s e d f o r c y c l i c A M P - d e p e n d e n t p r o t e i n k i n a s e w e r e n o t s u c c e s s f u l . An i n d i r e c t a p p r o a c h w a s , t h e r e f o r e , c h o s e n , n a m e l y a s e p a r a t i o n o f t h e c y c l i c n u c l e o -t i d e s i n t o a " f r e e " a n d a " b o u n d " f r a c t i o n p r i o r t o t h e R I A d e t e r m i n a t i o n s . S i n c e t h e o n l y p r o t e i n k n o w n t o s p e c i f i c a l l y b i n d c y c l i c GMP i n s i d e a c e l l i s t h e p r o t e i n k i n a s e , t h e a b s o l u t e a m o u n t o f p r o t e i n b o u n d c y c l i c n u c l e o t i d e w a s c o n -s i d e r e d t o b e i n d i c a t i v e o f t h e a m o u n t o f a c t i v a t e d c y c l i c G M P - d e p e n d e n t p r o t e i n k i n a s e . T h i s m e t h o d w a s a m o d i f i c a t i o n [ 1 5 1 ] o f t h e D u f a u e t a l . [ 5 1 ] t e c h n i q u e w h i c h h a d s h o w n a - 1 2 2 -good c o r r e l a t i o n between hormone c o n c e n t r a t i o n , amount of i n t r a c e l l u l a r c y c l i c AMP bound to the r e g u l a t o r y sub-unit of p r o t e i n k i n ases and c o r t i c o s t e r o n e p r o d u c t i o n i n i s o l a t e d a d r e n o c o r t i c a l c e l l s from r a t s . The technique was f i r s t a p p l i e d to separate c y c l i c AMP response a s s o c i a t e d with p r e c e p t o r s t i m u l a t i o n i n t o f r e e and p r o t e i n bound f r a c t i o n s and then a p p l i e d to, c y c l i c GMP. 3 I n i t i a l s a t u r a t i o n experiments c a r r i e d out w i t h [ H ] - c y c l i c 3 AMP showed b i n d i n g of [ H ] - c y c l i c AMP to i n t r a c e l l u l a r p r o -t e i n from brown f a t to be s a t u r a b l e and d i s p l a c e a b l e by h i g h c o n c e n t r a t i o n s of n o n - l a b e l l e d c y c l i c AMP. T h i s b i n d i n g was temperature dependent. There was l e s s than 1% d i s s o c i a t i o n of the protein-bound c y c l i c AMP or a s s o c i a t i o n of the f r e e c y c l i c AMP w i t h the r e c e p t o r p r o t e i n at the temperature (4°G) at which the experiments were performed. T h e r e f o r e , the v a l u e s determined r e f l e c t e d the i n v i v o s i t u a t i o n . The p r o d u c t i o n of c y c l i c AMP was not only most p r o -nounced when the brown f a t fragments were incubated w i t h dobutamine ( ^ - a g o n i s t ) ( F i g . 10A), but dobutamine i n a con-c e n t r a t i o n range of 0.5, 5.0, or 50 /uM produced a graded r e s -ponse. P r i o r i n c u b a t i o n of brown f a t t i s s u e p i e c e s with 0.4 mM metoprolol blocked the e f f e c t of 50 y«.M dobutamine ( F i g . 10B). These r e s u l t s c o n f i r m the a s s o c i a t i o n between /^-adren-e r g i c r e c e p t o r s t i m u l a t i o n and the t i s s u e c o n c e n t r a t i o n of c y c l i c AMP. On the other hand, c l o n i d i n e was most e f f e c t i v e i n -123-s t i m u l a t i n g the p r o d u c t i o n of c y c l i c GMP and showed a concentration-dependent response. I n t e r e s t i n g l y , the clonidine dose-response curve on brown f a t fragments from one-month-o l d r a t s was d i f f e r e n t than that from one-week-old r a t s . The peak response i n the former case occurred at 20 /xM concentra-t i o n of c l o n i d i n e , w h ile i n the l a t t e r case i t occurred at 0.5 /_M c l o n i d i n e . A p o s s i b l e e x p l a n a t i o n may be that brown f a t i n one-month-old r a t s i s i n the i n v o l u t i v e phase while i n one-week-old r a t s i t i s at the peak of i t s f u n c t i o n a l a c t i v -i t y [179, 180]. T h i s e f f e c t of c l o n i d i n e was blocked by yohimbine ( F i g 11A, B). S i m i l a r l y , when the t o t a l c y c l i c GMP produced, as a r e s u l t of c l o n i d i n e s t i m u l a t i o n , was sep-arated i n t o i n t r a c e l l u l a r , p r o t e i n bound and the f r a c t i o n r e l e a s e d i n the i n c u b a t i o n media, time-dependent curves were obtained with maximum response o c c u r r i n g at 3 minutes of i n c u b a t i o n ( F i g . 12). The bound f r a c t i o n of c y c l i c GMP was found to be 15 to 25% of the t o t a l , and i t i n c r e a s e d t o 30 to 40% under c l o n i d i n e . s t i m u l a t i o n . These i n v i t r o e x p e r i -ments supported the e a r l i e r proposed h y p o t h e s i s of a l i n k between -^-adrenoceptor s t i m u l a t i o n and a c t i v a t i o n of guanylate c y c l a s e and a l s o demonstrated these r e c e p t o r s to show c h a r a c t e r i s t i c s of ©^-sub-type. Although s e v e r a l other s t u d i e s have shown catecholamine-induced accumulation of c y c l i c GMP i n the p a r o t i d [219] and p i n e a l [144] as a r e s u l t of s t i m u l a t i o n of ^ - a d r e n o c e p t o r s , the p r e c i s e nature of the l i n k between -^--adrenoceptors etim--124-u l a t i o n and guanylate c y c l a s e a c t i v a t i o n and i t s p h y s i o l o g -i c a l s i g n i f i c a n c e is u n c l e a r . A l s o unresolved i s the q u e s t i o n of the molecular mechanisms i n v o l v e d i n the a c t i v a t i o n of guanylate c y c l a s e and whether t h i s enzyme i n brown f a t i s c y t o s o l i c / membrane-bound, or both. Liang and coworkers, 1978 [111] presented evidence f o r the d i r e c t a c t i v a t i o n of c y t o s o l i c guanylate c y c l a s e ( c e l l - f r e e p r e p a r a t i o n ) by catecholamine i n r a b b i t r e n a l c o r t e x . The e f f e c t was independent of the presence of c a l c i u m . Although a v a r i e t y of agents i n c l u d i n g ascorbate» poly-, unsaturated f a t t y a c i d s and n i t r o p r u s s i d e could s t i m u l a t e both c y t o s o l i c and membrane bound guanylate c y c l a s e ; o n l y the s o l u b l e form of enzyme was a c t i v a t e d by catecholamine. On the other hand, i n d i r e c t mechanisms f o r the a c t i v a t i o n of guanylate cyclase has a l s o been suggested -by L e v i l l i e r s et" al.1978 [110]. Both c y t o s o l i c and membrane bound guanylate c y c l a s e i n d e t e r g e n t - d i s p e r s e d plasma membranes from r a t a d i p o c y t e s was r e p o r t e d to be h i g h l y s e n s i t i v e to a c t i v a t i o n by low 2+ c o n c e n t r a t i o n s of d i v a l e n t c a t i o n s such as Ca . Thus, i t 2 + appears that Ca may act as an i n t e r m e d i a t e i n a p u t a t i v e hormonal c o n t r o l of guanylate c y c l a s e i n the adipose t i s s u e . Mey and Vanhoutte [122] have shown t h a t the c o n t r a c t i l e response i n i s o l a t e d venous smooth muscle could be t r i g g e r e d by a c t i v a t i o n of both a l p h a ^ - and a l p h a ^ - adrenoceptors. I t was suggested t h a t the c o n t r a c t i l e response by a l p h a ^ -a d r e n e r g i c s t i m u l i depended more on the i n f l u x of e x t r a --125-c e l l u l a r Ca while o^-adrenoceptors s t i m u l a t i o n caused . . . 2+ a r e l a t i v e l y g r e a t e r m o b i l i z a t i o n of c e l l u l a r s t o r e s of Ca i o n s . S t u d i e s by Zavoico and F e i n s t e i n [227] have r e p o r t e d that s t i m u l a t i o n of ^ - a d r e n o c e p t o r s i n c r e a s e s i n t r a c e l l u l a r ++ . . . ++ f r e e Ca i n p l a t e l e t s . _<2"" a d r e n e r9 l c : r e g u l a t i o n of Ca transport in rabbit i n t e s t i n e has been d e s c r i b e d [88]. Thus, i t seems t h a t C a + + s i g n a l l i n g may be i n v o l v e d i n a c t i v a t i o n of both oCj^- and o<2- adrenoceptor e f f e c t o r systems. The p r e c i s e mechanisms of a c t i v a t i o n of guanylate c y c l a s e by c a l c i u m i s a l s o u n c l e a r . London et a l . [115] report e d e x i s t e n c e of a temporal r e l a t i o n s h i p between c a l c i u m i n f l u x and c y c l i c GMP l e v e l i n synchronized Tetrahymena c e l l s . Kudo et a l . [99], i n t h e i r study have demonstrated 2+ a c t i v a t i o n of guanylate c y c l a s e by Ca -ca l m o d u l i n . T h i s 2+ a c t i v a t i o n was r e v e r s i b l e by c h e l a t i o n of Ca with EGTA. On the other hand, other s t u d i e s suggest d i a c y l g l y c e r o l , produced as a r e s u l t of phospholipase C a c t i v a t i o n , c o u l d a c t i v a t e p r o t e i n k i n a s e C [79, 1.43], which i n t u r n c o u l d phosphorylate and a c t i v a t e guanylate c y c l a s e [228]. One can-not ignore other p o s s i b l e mechanisms of guanylate c y c l a s e a c t i v a t i o n , such as by r e l e a s e of f r e e f a t t y a c i d s by a c t i v a -t i o n of hormone-sensitive l i p a s e [208], or by the "feedback r e g u l a t o r " d e s c r i b e d i n white f a t by Ho and Sutherland [9, 80]. In s p i t e of the obvious l a c k of d e t a i l e d s t u d i e s on the guanylate c y c l a s e system i n brown f a t , the i n f o r m a t i o n -126-o b t a i n e d s o f a r s e e m s t o i n d i c a t e t h a t t h e s y s t e m may p l a y some r o l e , i n t h e r e g u l a t i o n o f d e v e l o p m e n t a l p r o c e s s e s [ 1 8 0 ] . T h e h i g h s t e a d y - s t a t e c o n c e n t r a t i o n o f c y c l i c G M P , a n d t h e h i g h e r a c t i v i t y o f c y c l i c G M P - d e p e n d e n t p r o t e i n k i n a s e o b -s e r v e d b y S k a l a a n d K n i g h t d u r i n g t h e p e r i n a t a l p e r i o d a n d u p o n c o l d a c c l i m a t i o n [ 1 7 8 ] s e e m s t o c o r r e s p o n d w i t h p e r i o d s o f h i g h p r o l i f e r a t i v e a c t i v i t y . S u m m a r y ; ( i ) ' I n c u b a t i o n o f b r o w n f a t f r a g m e n t s f r o m o n e - m o n t h -o l d r a t s i n v i t r o w i t h ^ - a g o n i s t d o b u t a m i n e p r o d u c e d a n e l e v a t i o n i n t h e c y c l i c AMP c o n t e n t . T h e r e s p o n s e w a s c o n c e n t r a t i o n - d e p e n d e n t . M e t o p r o l o l , a ^ - a n t a g -o n i s t , b l o c k e d t h i s e f f e c t , ( i i ) C l o n i d i n e , a n o ^ - a g o n i s t , p r o d u c e d a c o n c e n t r a t i o n -a n d t i m e - d e p e n d e n t e l e v a t i o n i n c y c l i c GMP c o n t e n t o f b r o w n f a t f r a g m e n t s i s o l a t e d f r o m 7 - d a y - o l d r a t s . T h e e f f e c t o f c l o n i d i n e c o u l d b e b l o c k e d b y y o h i m b i n e , a n o < 2 - a n t a g o n i s t . W h e n t h e t o t a l t i s s u e c y c l i c GMP c o n t e n t w a s s e p a r a t e d i n t o f r e e a n d p r o t e i n b o u n d f r a c t i o n s , c l o n i d i n e p r o d u c e d a t i m e - d e p e n d e n t e l e v a t i o n o f b o t h t h e f r a c t i o n s . 4.4 E f f e c t of " < 2 ~ a 9 o n i - s t o n f o r s k o l i n - s t i m u l a t e d l i p o l y s i s i n brown f a t adipocytes i s o l a t e d from 7-day-old r a t s . Robison et a l . [158] o r i g i n a l l y had proposed t h a t alpha e f f e c t s of catecholamines were associated with a decrease in adenylate c y c l a s e a c t i v i t y . Subsequently i n h i b i t i o n of adenylate c y c l a s e by alpha catecholamines was r e p o r t e d i n human f a t c e l l ghosts [27], human p l a t e l e t l y s a t e s [90] and hamster adipocyte ghosts [ 3 ] . Sabol and Ni r e n b e r g [165] suggested t h i s i n h i b i t o r y e f f e c t of alpha catecholamines was mediated through a l p h a 2 ~ r e c e p t o r s . Subsequent s t u d i e s with human and hamster white f a t c e l l s have c l e a r l y shown that the s t i m u l a t i o n of a l p h a 2 - adrenoceptors i n h i b i t s l i p o l y s i s . I t i s l i n k e d to a decrease i n i n t r a c e l l u l a r c y c l i c AMP l e v e l s pro-duced by an i n h i b i t i o n of plasma membrane adenylate c y c l a s e a c t i v i t y [33, 101-103]. The o b j e c t i v e of t h i s p a r t of our study i s . to show, that the stimulation of ^ -adrenoceptors in brown adipocytes from one-week-old rats show inhibitory effect on forskolin-stimulated glycerol r e l e a s e as r e p o r t e d i n a d i p o c y t e s from white f a t o f hamsters and man. In our study (Table 1) on i s o l a t e d brown a d i p o c y t e s , the i n h i b i t o r y e f f e c t of p r o s t a g l a n d i n E 2 on f o r s k o l i n (1.25 ju.K) s t i m u l a t e d l i p o l y s i s ranged from 20 to 35% from low to high c o n c e n t r a t i o n s . A s i m i l a r degree of i n h i b i -t i o n by ©^-agonists was r e p o r t e d by Sabol and Nirenberg i n NG 108-15 neuroblastoma x h y b r i d c e l l s [165]. I n h i b i t i o n of f o r s k o l i n — s t i m u l a t e d l i p o l y s i s by p r o s t a g l a n d i n E 9 and -128-n i c o t i n i c a c i d was very s i m i l a r to that a l r e a d y r e p o r t e d i n hamster epididymal adipocytes [168] (Table 1). S t i m u l a t i o n of g l y c e r o l r e l e a s e i n i s o l a t e d brown f a t adipocytes by sub-maximal c o n c e n t r a t i o n s of f o r s k o l i n (0.625 juM) could be i n h i b i t e d by c l o n i d i n e . T h i s i n h i b i t i o n was c l o n i d i n e concentration-dependent (Table 2A) and c o u l d be antagonized by 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 yohimbine (Table 2B). S i m i l a r r e s u l t s i n brown f a t ad i p o c y t e s from a d u l t r a t s were reported by Sundin and F a i n [190]. Endogenous adenosine has been r e p o r t e d to be an important r e g u l a t o r of l i p o l y s i s and c y c l i c AMP accumulation i n white a d i p o c y t e s and hamster brown adip o c y t e s [58,169-]. These e f f e c t s of endogenous adenosine are blocked by i s o b u t y l -methylxanthine (a. phosphodiesterase i n h i b i t o r ) . In our study, when i n f a n t r a t brown adip o c y t e s were s t i m u l a t e d by 100 juM of is o b u t y l - m e t h y l x a n t h i n e , c l o n i d i n e was s t i l l capable of i n -h i b i t i n g l i p o l y s i s by as much as 15 to 30%, i n d i c a t i n g t h a t _<2-adrenoceptor-stimulated i n h i b i t o r y a c t i o n i s not mediated v i a adenosine r e c e p t o r s . Isobutyl-methylxanthine s t i m u l a t e d g l y c e r o l r e l e a s e was a l s o s e n s i t i v e to i n h i b i t i o n by n i c o t i n i c a c i d (Table 3)- A low i n h i b i t o r y a c t i o n of c l o n i d i n e on is o b u t y l - m e t h y l x a n t h i n e s t i m u l a t e d g l y c e r o l r e l e a s e , compared to f o r s k o l i n s t i m u l a t e d g l y c e r o l r e l e a s e , c o u l d p o s s i b l y be due to the phosphodiesterase i n h i b i t o r y a c t i o n of i s o b u t y l -methylxanthine^ S i m i l a r i n h i b i t o r y e f f e c t s of c l o n i d i n e on i s o b u t y l - m e t h y l x a n t h i n e - s t i m u l a t e d l i p o l y s i s have been r e p o r t e d -129-i n h a m s t e r e p i d i d y m a l a d i p o c y t e s [ 1 6 8 ] . I t h a s b e e n p r o p o s e d t h a t f o r s k o l i n a c t s d i r e c t l y o n t h e c a t a l y t i c s u b - u n i t o f a d e n y l a t e c y c l a s e [ 1 7 3 ] . T h u s , a c t i v a t i o n o f a d e n y l a t e c y c l a s e b y f o r s k o l i n c a n s t i l l b e a f f e c t e d b y i n p u t s a f f e c t i n g t h e s t i m u l a t o r y o r i n h i b i t o r y p r o t e i n s . P e r t u s s i s t o x i n f r o m B o r d e t e l l a p e r t u s s i s c a u s e s A D P - r i b o s y l a t i o n o f a s u b u n i t o f N i a n d t h u s c a n b l o c k a l l r e c e p t o r - m e d i a t e d s u p p r e s s i o n o f a d e n y l a t e c y c l a s e [ 3 7 ] . P e r t u s s i s t o x i n p r e - t r e a t m e n t o f b r o w n f a t a d i p o c y t e s f o r 3 ^ h o u r s n o t o n l y i n c r e a s e d t h e b a s a l b u t a l s o t h e f o r s k o l i n s t i m u l a t e d r a t e o f l i p o l y s i s . C l o n i d i n e a n d p r o s t a g l a n d i n ( P G E 2 ) i n h i b i t o r y e f f e c t o n g l y c e r o l r e l e a s e e x e r t e d i n t h e a b s e n c e o f p e r t u s s i s t o x i n w a s c o m p l e t e l y a b o l i s h e d ( T a b l e 4 ) . T h i s i n d i c a t e s t h a t t h e i n a c t i v a t i o n o f N i p r o t e i n b y p e r t u s s i s t o x i n a b o l i s h e d t h e i n h i b i t o r y i n p u t e x e r t e d b y c l o n i d i n e , a n o < ^ - a d r e n o c e p t o r a g o n i s t . I n t e r e s t i n g l y , we w e r e u n a b l e t o s h o w a n y i n h i b i t o r y a c t i o n o f c l o n i d i n e o n f o r s k o l i n o r i s o b u t y l m e t h y l x a n t h i n e s t i m u l a t e d l i p o l y s i s i n b r o w n f a t a d i p o c y t e s i s o l a t e d f r o m a d u l t h a m s t e r s ( T a b l e 5) . However, p r o s t a g l a n d i n E^ a n d n i c o t i n i c a c i d s h o w e d a n i n h i b i t o r y e f f e c t o n f o r s k o l i n - s t i m u l a t e d l i p o l y s i s i n h a m s t e r a d i p o -c y t e s . S i m i l a r r e s u l t s w e r e r e p o r t e d b y M c M a h o n a n d S c h i m m e l [ 1 1 9 , 1 2 0 ] . I n c o n c l u s i o n , t h i s s t u d y d e m o n s t r a t e s t h a t b o t h f o r s k o l i n a n d i s o b u t y l m e t h y l x a n t h i n e s t i m u l a t e d g l y c e r o l r e l e a s e i n i s o l a t e d a d i p o c y t e s f r o m b r o w n f a t o f o n e - w e e k - o l d - 1 3 0 -r a t s . C l o n i d i n e ( a n < x _ , - a g o n i s t ) , p r o s t a g l a n d i n E_ a n d - 2 n i c o t i n i c a c i d i n h i b i t e d f o r s k o l i n s t i m u l a t e d g l y c e r o l r e l e a s e I n h i b i t i o n o f g l y c e r o l r e l e a s e b y c l o n i d i n e w a s c o n c e n t r a t i o n d e p e n d e n t . E f f e c t o f c l o n i d i n e (1 A L M ) w a s a n t a g o n i z e d b y i n c r e a s i n g t h e c o n c e n t r a t i o n o f y o h i m b i n e ( a n e » < 2 _ a n t a 9 o n i s t ) • I n a c t i v a t i o n o f i n h i b i t o r y p r o t e i n ( N i ) b y p e r t u s s i s t o x i n a b o l i s h e d t h e i n h i b i t o r y e f f e c t s o f c l o n i d i n e a n d p r o s t a g l a n -d i n E 2 . T h i s s u g g e s t s t h a t s t i m u l a t i o n o f * < 2 - a d r e n o c e P t o r s b y c l o n i d i n e r e s u l t s i n i n h i b i t i o n o f a d e n y l a t e c y c l a s e , w h i c h p r o b a b l y i s m e d i a t e d v i a i n h i b i t o r y p r o t e i n ( N i ) s i m i l a r t o t h a t a l r e a d y r e p o r t e d f o r h u m a n a n d h a m s t e r a d i p o c y t e s [ 5 4 , 1 0 1 , 1 0 2 , 1 0 3 ] . T h e a n t i - l i p o l y t i c e f f e c t o f c < 2 - a < l r e n o c e p t o r s may h a v e a r o l e i n c o n t r o l l i n g t h e s t a t e o f a c t i v i t y o f b r o w n f a t c e l l s . 4 . 5 E f f e c t o f a d r e n e r g i c a g o n i s t s a n d a n t a g o n i s t s i n v i t r o o n c y c l i c GMP c o n t e n t i n b r o w n f a t f r a g m e n t s f r o m o b e s e ( o b / o b ) m i c e a n d u n a f f e c t e d l i t t e r m a t e s j(°k/r B r o w n f a t o f g e n e t i c a l l y o b e s e ( o b / o b ) m i c e s h o w s d e f e c t ( s ) i n ^ - a d r e n o c e p t o r s t i m u l a t e d t h e r m o g e n i c r e s p o n s e [ 7 7 ] . T h e y h a v e a l o w m e t a b o l i c r a t e a n d a h i g h m e t a b o l i c e f f i c i e n c y . T h e y a r e e x t r e m e l y s e n s i t i v e t o c o l d , b e c o m e r a p i d l y h y p o t h e r m i c , a n d d i e i n a b o u t t h r e e h o u r s w h e n k e p t 6 a t 4 C . T h e o b j e c t i v e o f t h i s p a r t o f o u r s t u d y w a s t o c o m -p a r e t h e r e s p o n s e s o f . . b r o w n f a t c y c l i c GMP p r o d u c t i o n i n o b e s e a n d l e a n m i c e t o s e e i f t h e d e f e c t ( s ) e x t e n d e d ' t o t h e o c _ - a d r e n o c e p t o r a n d c y c l i c GMP s y s t e m s a s w e l l . I n -131-i n i t i a l i n v i v o e x p e r i m e n t s , b r o w n a d i p o s e t i s s u e o f o b / o b m i c e , f o l l o w i n g c o l d e x p o s u r e (6 C ) a n d n o r e p i n e p h r i n e a d m i n i s t r a t i o n ( 0 . 1 m g / 1 0 0 g b o d y w e i g h t i . p . ) , e x h i b i t e d n o c h a n g e i n t h e i r c y c l i c GMP c o n t e n t s i n c o n t r a s t t o b r o w n f a t f r o m c o n t r o l ( l e a n l i t t e r m a t e s ) m i c e who s h o w e d a t i m e - d e p e n d e n t i n c r e a s e ( F i g . 1 3 A a n d B ) . When t h e s e e x p e r i m e n t s w e r e e x -t e n d e d t o i n v i t r o e x p e r i m e n t s ( F i g . 13 . C ) , c y c l i c GMP p r o d u c t i o n b y b r o w n f a t u p o n s t i m u l a t i o n b y c l o n i d i n e ( e ^ 2 - a 9 ' o n i s t ) w a s s i m i l a r i n o b e s e a n d l e a n m i c e . T h e o n l y d i f f e r e n c e w a s a l o w e r c y c l i c GMP c o n c e n t r a t i o n / m g p r o t e i n i n b r o w n f a t o f o b e s e m i c e . T h u s , o u r i n v i t r o p r e l i m i n a r y s t u d y , u n l i k e o u r i n v i v o r e s u l t s , f a i l e d - t o d e m o n s t r a t e a d i f f e r e n c e i n t h e r e s p o n s e o f b r o w n f a t c y c l i c GMP p r o d u c t i o n i n o b e s e a n d l e a n m i c e . L a c k o f c y c l i c GMP p r o d u c t i o n i n v i v o f o l l o w i n g c o l d e x p o s u r e a n d n o r e p i n e p h r i n e i n j e c t i o n may p o s s i b l y b e d u e t o some d e f e c t ( s ) p r o x i m a l t o t h e b i n d i n g s i t e s o n b r o w n f a t . I n s u m m a r y , t h e s t u d y p r e s e n t e d d e s c r i b e s t h e p r e s e n c e 1 o f f u n c t i o n a l ^ 2 _ a d r e n o c e P t o r s * n a d i p o c y t e s f r o m brovm f a t of 7 - d a y o l d r a t s a n d t h e p r e s e n c e o f a l i n k b e t w e e n ^ " a d r e n o -c e p t o r s t i m u l a t i o n a n d c y c l i c GMP p r o d u c t i o n . T n a d d i t i o n , t h e s t u d y a l s o s h o w s c h a n g e s i n t h e b i n d i n g c h a r a c t e r i s t i c s o f c < 2 ~ a d r e n o c e p t o r s f o l l o w i n g e x p e r i m e n t a l m a n i p u l a t i o n s . T h e n e u r o t r a n s m i t t e r n o r e p i n e p h r i n e h a s b e e n i m p l i c a t e d i n b o t h a c u t e c a l o r i g e n i c r e s p o n s e o f b r o w n f a t a n d t h e l o n g t e r m t r o p h i c r e s p o n s e . T h e p r e s e n c e o f B^- a n d ^ - a d r e n o c e p t o r s a n d t h e i r r o l e i n n o r e p i n e p h r i n e m e d i a t e d t i s s u e c a l o r i g e n i c r e s p o n s e -132-h a s b e e n w e l l d o c u m e n t e d . No s t u d y h a s y e t b e e n u n d e r t a k e n w h i c h s h o w s t h e m e c h a n i s m s o f t r o p h i c r e s p o n s e a s s o c i a t e d w i t h n o r e p i n e p h r i n e . - . - a d r e n e r g i c i n h i b i t i o n o f l i p o l y s i s h a s b e e n known f o r seme t i m e . I t h a s a l s o b e e n s u g g e s t e d t h a t ^ - a d r e n o -c e p t o r s a n d c y c l i c GMP may h a v e a r o l e i n t i s s u e g r o w t h a n d p r o l i f e r a t i o n . O u r p r e s e n t f i n d i n g c a n b e s t b e e x p l a i n e d i f i t i s a s s u m e d t h a t t h e a d r e n o c e p t o r p h e n o t y p e i n t h e p l a s m a m e m b r a n e o f b r o w n a d i p o c y t e s o f a d e v e l o p i n g r a t i s a d y n a m i c e n t i t y . T h e r e l a t i v e p r o p o r t i o n o f i t s p h e n o t y p e a n d t h e a f f i -n i t i e s o f i t s c o m p o n e n t s f o r n o r e p i n e p h r i n e d e t e r m i n e t h e f i n a l e f f e c t s o f t h e a d r e n e r g i c s t i m u l a t i o n . D e p e n d i n g u p o n w h i c h r e c e p t o r s a r e p r e f e r e n t i a l l y o c c u p i e d a n d a s s u m i n g t h a t t h e d i f f e r e n t b i n d i n g s i t e s a r e l i n k e d t o d i f f e r e n t e f f e c t o r s y s t e m s , t h e i n v i v o e f f e c t s o f n o r e p i n e p h r i n e o n a d r e n o c e p t o r s a n d t h e a s s o c i a t e d e f f e c t o r s y s t e m s c a n d i f f e r a n d c h a n g e d u r i n g d i f f e r e n t d e v e l o p m e n t a l s t a g e s o f t h e a n i m a l . I f oi -adrenoceptor b i n d i n g s i t e s m e d i a t e n o r e p i n e p h r i n e e f f e c t s u p o n t h e t i s s u e ' s t r o p h i c r e s p o n s e , o n e i s m o r e l i k e l y t o d e m o n s t r a t e t h e p r e s e n c e o f r e l -a t i v e l y h i g h p r o p o r t i o n o f ^ ^ " - d - r e n o c e p t o r s i n i n f a n t r a t s w h e n t h e b r o w n a d i p o s e t i s s u e i s a t t h e p e a k o f i t s p r o l i f e r a t i o n a n d g r o w t h a s s h o w n i n o u r s t u d y . P e r h a p s t h i s may a l s o p a r t -i a l l y e x p l a i n B u c k o w i e c k i ' s [ 2 4 ] a n d M c M a h o n a n d S c h i m m e l ' s f i n d i n g [ 1 1 9 ] o f t h e l a c k o f <X - . - a d r e n o c e p t o r s i n b r o w n f a t o f o l d e r r a t s o r h a m s t e r s . -133-CHAPTER 5 CONCLUSION The p r i n c i p a l f i n d i n g s of t h i s i n v e s t i g a t i o n are as f o l l o w s : 1. Ligand b i n d i n g s t u d i e s c a r r i e d out using h i g h l y s e l e c t i v e o ^ - l i g a n d s and p a r t i a l l y p u r i f i e d plasma membrane fragments of i n t e r s c a p u l a r brown adipose t i s s u e from one-week-old r a t s e x h i b i t e d b i n d i n g c h a r a c t e r i s t i c s as would be expected of b i n d i n g to o^-a d r e n o c e p t o r s . 2. Norepinephrine, (-)-epinephrine, and yohimbine bound to more than one b i n d i n g s i t e on ^ - a d r e n o c e p -t o r s . Yohimbine and (-)-epinephrine shared one com-mon b i n d i n g s i t e ; the other s i t e present i n much lower d e n s i t y was s p e c i f i c f o r (-)-norepinephrine and yohimbine only. 3 3 . B i n d i n g of (-)[ H ] - d i h y d r o a l p r e n o l o l e x h i b i t e d two b i n d i n g s i t e s . S i t e 1 ( ^ - s i t e ) c o u l d be occupied by e i t h e r p r a c t o l o l or (-)-epinephrine. The second s i t e was s e n s i t i v e to displacement by yohimbine, i n d i c a t i n g e i t h e r the n o n - s p e c i f i c i t y of d i h y d r o a l -p r e n o l o l or bi n d i n g of dihydroalprenolo to o^-adrenoceptors. 4. Yohimbine bound t o a s m a l l e r number of high a f f i n i t y b i n d i n g s i t e s i n membrane fragments i s o l a t e d from brown f a t of c h e m i c a l l y sympathectomized animals than -134-i t d i d t o m e m b r a n e f r a g m e n t s i s o l a t e d f r o m c o n t r o l a n i m a l s o f t h e s a m e a g e . 3 T h e ( - ) [ H ] - n o r e p i n e p h r i n e b i n d i n g t o b r o w n f a t m e m b r a n e s i s o l a t e d f r o m s y m p a t h e c t o m i z e d r a t s r e v e a l e d h i g h a n d l o w a f f i n i t y s i t e s . T h e t o t a l n u m b e r o f b i n d i n g s i t e s m o r e t h a n d o u b l e d a f t e r s y m p a t h e c t o m y . C h r o n i c y o h i m b i n e a d m i n i s t r a t i o n r e s u l t e d i n a m o r e t h a n t w o - f o l d i n c r e a s e i n t h e n u m b e r o f b i n d i n g s i t e s 3 3 f o r [ H ] - y o h i m b i n e a n d [ H ] - n o r e p i n e p h r i n e . T h e a f f i n i t y f o r y o h i m b i n e o f © ^ - a d r e n o c e p t o r b i n d i n g s i t e s d e c r e a s e d w h e r e a s t h a t f o r n o r e p i n e p h r i n e r e m a i n e d u n c h a n g e d . B a s e d o n t h e s e s t u d i e s , i t was c o n c l u d e d t h a t b r o w n f a t o f d e v e l o p i n g r a t s p o s s e s s e d © ^ - a d r e n o -c e p t o r s , a n d t h a t c h a r a c t e r i s t i c s o f t h e s e r e c e p t o r s c o u l d b e a l t e r e d b y c h e m i c a l s y m p a t h e c t o m y a n d b y c h r o n i c e x p o s u r e o f y o u n g r a t s t o a n ^ - b l o c k e r . I n c u b a t i o n o f b r o w n f a t f r a g m e n t s f r o m o n e - w e e k - o l d r a t s w i t h <=<2~a^on^s^ c l o n i d i n e r e s u l t e d i n d o s e - a n d t i m e - d e p e n d e n t i n c r e a s e s i n t i s s u e c y c l i c GMP c o n c e n -t r a t i o n s . S t i m u l a t i o n o f b r o w n f a t b y 0 . 5 /M c l o n i d i n e s h o w e d a t i m e - d e p e n d e n t c h a n g e i n i n t r a c e l l u l a r , p r o t e i n b o u n d f r a c t i o n a n d t o t a l c y c l i c GMP l e v e l s . T h e s e i n v i t r o e x p e r i m e n t s s u p p o r t e d e a r l i e r p r o p o s e d h y p o t h e s i s o f a l i n k b e t w e e n o C - r e c e p t o r s t i m u l a t i o n a n d c y c l i c GMP p r o d u c t i o n i n b r o w n f a t o f o n e - w e e k - o l d C l o n i d i n e i n h i b i t e d f o r s k o l i n - s t i m u l a t e d g l y c e r o l r e l e a s e i n i s o l a t e d a d i p o c y t e s from brown f a t of one-week-old r a t s . Yohimbine antagonized t h i s i n h i b i t o r y e f f e c t . The i n h i b i t o r y e f f e c t s of c l o n i d i n e were a b o l i s h e d by pre-treatment of brown f a t adipocytes with p e r t u s s i s t o x i n . C l o n i d i n e showed no e f f e c t on adipo-c y t e s i s o l a t e d from brown f a t of a d u l t hamsters. In v i v o study on g e n e t i c a l l y obese mice showed a blunted c y c l i c GMP p r o d u c t i o n f o l l o w i n g c o l d exposure (4°C) or norepinephrine a d m i n i s t r a t i o n . However, when the brown f a t fragments from obese mice were incubated i n the presence of c l o n i d i n e , they showed s i m i l a r l e v e l s of c y c l i c GMP p r o d u c t i o n to those i n l e a n mice. The only difference was a lower basal concentration o f - c y c l i c GMP/mg p r o t e i n i n brown f a t of obese mice. B i b l i o g r a p h y A a r o n s , R . D . , M o l i n o f f , P . B . ( 1 9 8 2 ) C h a n g e s i n t h e d e n s i t y o f jQ-adrenergic r e c e p t o r s i n r a t l y m p o c y t e s , h e a r t a n d l u n g a f t e r c h r o n i c t r e a t m e n t . J . P h a r m a c o l . E x p . T h e r 2 2 1 , 4 3 9 - 4 4 3 . A h l q u i s t , R . P . ( 1 9 4 8 ) S t u d y o f t h e A d r e n o t r o p i c R e c e p t o r s A m . J . P h y s i o l . 1 5 3 , 5 8 6 - 6 0 0 . A k t o r i e s , K . , S c h u l t z , G . a n d J a k o b s , K . H . ( 1 9 7 9 ) I n h i b i t i o n o f h a m s t e r f a t c e l l a d e n y l a t e c y c l a s e b y p r o s t a g l a n d i n -s' a n d e p i n e p h r i n e : r e q u i r e m e n t f o r GTP a n d s o d i u m i o n s . F E B S L e t t . 1 0 7 , 1 0 0 - 1 0 4 . A p r i l l e , J . R . , L e f k o w i t z , R . K . , a n d W a r s h a w , J . ( 1 9 7 4 ) 3 H - n o r e p i n e p h r i n e b i n d i n g a n d l i p o l y s i s b y i s o l a t e d f a t c e l l s . B i o c h e m . B i o p h y s . A c t a 3 7 3 > 5 0 2 - 5 1 3 . A p t e l b a u m , M . J . , B o t s c a n o n , J . , a n d L a c a t i s , D . ( 1 9 7 1 ) E f f e c t o f c a l o r i e r e s t r i c t i o n a n d e x c e s s i v e c a l o r i e i n t a k e o n e n e r g y e x p e n d i t u r e . A m . J . C l i n . N u t r . 2 4 , 1 4 0 5 - 1 4 0 9 . A r c h , R . S . , J . ( 1 9 8 3 ) D e f e c t i v e t h e r m o r e g u l a t o r y t h e r m o -g e n e s i s i n g e n e t i c a l l y o b e s e ( o b / o b ) m i c e i s n o t d u e t o i n s e n s i t i v i t y i n n o r a d r e n a l i n e , p p . 3 5 - 3 9 , E n v i r o n -m e n t s , D r u g s , a n d T h e r m o r e g u l a t i o n s , L o m a x , P . e d . S . K a r g e r , B a s e l . A r c h , J . R . S . , A i n s w o r t h , A . T . , C a w t h o r n e , M . A . , P i e r c y , V . , S e n n i t t , M . V . , T h o d y , V . E . , W i l s o n , C , W i l s o n , S . ( 1 9 8 4 ) A t y p i c a l / ^ - a d r e n o c e p t o r o n b r o w n a d i p o c y t e s a s t a r g e t f o r a n t i - o b e s i t y d r u g s . N a t u r e 3 0 9 , 1 6 3 - 1 6 5 . A r c h , J . R . S . , A i n s w o r t h , A . T . , C a w t h o r n e , M . A . , P i e r c y , V . , S e n n i t t , M . V . , T h o d y , V . E . , W i l s o n , C , W i l s o n , S . ( 1 9 8 4 ) A t y p i c a l / S - a d r e n o c e p t o r o n b r o w n a d i p o c y t e s a s t a r g e t f o r a n t i o b e s i t y d r u g s . I n t . J . O b e s . 8_, S u p p . 1 , 1 - 1 1 . A s a k a w a , T . , R u i z , J . S n y d e r , R . ; S c h e i n b a u m , I . , R u s s e l l , R . T . , a n d H o , R . J . ( 1 9 7 5 ) E l e v a t i o n o f c y c l i c GMP l e v e l s i n a d i p o c y t e s b y a n e n d o g e n o u s f a c t o r . F e d . P r o c . . 3 4 , 6 1 6 . A s s i m a c o p o u l o s - J e a n e t , F . , G i a c o b i n o , J . - P . , S e y d o u x , J . , G i r a r d i e r , L . , a n d J e a n r e n a u d , B . ( 1 9 8 2 ) A l t e r a t i o n s o f b r o w n a d i p o s e t i s s u e i n g e n e t i c a l l y o b e s e ( o b / o b ) m i c e , I I . S t u d i e s o f ^ - a d r e n e r g i c r e c e p t o r s a n d f a t t y a c i d d e g r a d a t i o n . E n d o c r i n o l . 1 1 0 , 4 3 9 - 4 4 3 . -137-11 B a r n a r d , T . ( 1 9 7 4 ) E f f e c t o f 6 - h y d r o x y d o p a m i n e o n b r o w n a d i p o s e t i s s u e a d r e n e r g i c i n n e r v a t i o n a n d a d i p o c y t e s s t r u c t u r e . I n p r o c e e d i n g o f t h e s y m p o s i u m , D e p r e s s e d m e t a b o l i s m a n d c o l d t h e r m o g e n e s i s , h e l d i n P r a g u e , O c t . 9 - 1 2 . C h a r l e s U n i v e r s i t y . 12 B a t e s , M . W . , M a y e r , J . a n d N a u s s , S . F . ( 1 9 5 5 ) F a t m e t a b o l -i s m i n t h r e e f o r m s o f e x p e r i m e n t a l o b e s i t y . F a t t y a c i d t u r n o v e r . A m . J . P h y s i o l . 1 8 0 , 3 0 9 - 3 1 4 . 13 . , B e g i n - H e i c k , N . , a n d H e i c k , H . M . C . ( 1 9 7 7 ) I n c r e a s e d r e -s p o n s e o f a d i p o s e t i s s u e o f t h e o b / o b m o u s e t o t h e a c t i o n o f a d r e n a l i n e a f t e r t r e a t m e n t w i t h t h y r o x i n e . C a n . J . P h y s i o l . P h a r m a c o l . 5_5, 1 3 2 0 - 1 3 2 9 . 14 B e g i n - H e i c k , N . ( 1 9 8 0 ) A d e n y l a t e c y c l a s e i n l e a n a n d o b e s e ( o b / o b ) m o u s e e p i d i d y m a l w h i t e a d i p o c y t e s . C a n . J . B i o -c h e m . 5 8 , 1 0 3 3 - 1 0 3 8 . 15 , B e g i n - H e i c k , N . , H e i c k , H . M . C . ( 1 9 8 2 ) A d e n y l a t e c y c l a s e a c t i v i t y i n b r o w n a d i p o s e t i s s u e o f t h e g e n e t i c a l l y o b e s e ( o b / o b ) m o u s e . C a n . J . B i o c h e m . C e l l . B i o l . 6 0 , 9 1 0 - 9 1 6 . 16 • , B e r t h e l s e n , S . , a n d P e t t m g e r , W . A . ( 1 9 7 7 ) A f u n c t i o n a l b a s i s f o r c l a s s i f i c a t i o n o f < x - a d r e n e r g i c r e c e p t o r s . L i f e S c i . 2_1, 5 9 5 - 6 0 0 . 17 B i r n b a u m e r , L . , P o h l , S . L . , a n d K a u f m a n n , A . J . ( 1 9 7 4 ) R e c e p t o r s a n d a c c e p t o r s : A n e c e s s a r y d i s t i n c t i o n i n h o r m o n e b i n d i n g s t u d i e s . A d v . C y c l i c N u c l e o t i d e R e s . 4 , 2 3 9 - 2 8 1 . 18 B o k o c k , G . M . , K a t a d a , T . , N o r t h u p , J . K . H e w l e t t , E . L . a n d G i l m a n , A . G . ( 1 9 8 3 ) I d e n t i f i c a t i o n o f t h e p r e d o m -i n a n t s u b s t r a t e f o r A D P - r i b o s y l a t i o n b y i s l e t a c t i v a t i n g p r o t e i n . J . B i o l . C h e m . 2 5 8 , 2 0 7 2 - 2 0 7 5 . 19 B r o d d e , O . E . , A n l a u f , M . , G r a b e n , N . , a n d B o c k , K . D . ( 1 9 8 2 ) I n v i t r o a n d i n v i v o d o w n r e g u l a t i o n o f h u m a n p l a t e l e t s © { - - a d r e n o c e p t o r s b y c l o n i d i n e . E u r . J . C l i n . P h a r m a c o l . 2 3 : ( 5 ) , 4 0 3 - 4 0 9 . 20 3 B r o d d e , O . E . , E y m e r , T . , a n d A r r o y o , J . ( 1 9 8 3 ) : H - y o h i m -b i n e b i n d i n g t o g u i n e a - p i g k i d n e y a n d c a l f c e r e b r a l c o r t e x m e m b r a n e s : C o m p a r i s o n w i t h h u m a n p l a t e l e t s . A r c h . I n t . d e P h a r m a c o d y n . T h e r . 2 6 6 ( 2 ) , 2 0 8 - 2 2 0 . 1 3 8 -21 B r o d d e , 0 . - E . , D a u l , A . , S t u k a , N . , O ' H a r a , N . , a n d B o y -c h a r d , U . ( 1 9 8 5 ) E f f e c t o f ^ - a d r e n o c e p t o r a n t a g o n i s t a d m i n i s t r a t i o n o n / ^ - - a d r e n o c e p t o r d e n s i t y i n human l y m p h o c y t e s . T h e r o l e o f t h e " i n t r i n s i c s y m p a t h o m i m e t r i c a c t i v i t y " ; N a u n y n S c h m i e d e b e r g s A r c h . P h a r m a c o l . 3 2 8 ( 4 ) , 4 1 7 - 2 2 . 22 B r o o k e r , G . , P e d o n e , C , a n d B a r o v s k y , K . ( 1 9 8 3 ) S e l e c t i v e r e d u c t i o n o f f o r s k o l i n - s t i m u l a t e d c y c l i c AMP a c c u m u l a -t i o n b y i n h i b i t o r s o f p r o t e i n s y n t h e s i s . S c i e n c e , 2 2 0 , 1 1 6 9 - 1 1 7 0 . 2 3 B r u c e , P . B . , a n d U ' P r i c h a r d , D . C . ( 1 9 8 1 ) [ 3 H ] - R a u w o l s c i n e ( © { - y o h i m b i n e ) : A s p e c i f i c a n t a g o n i s t r a d i o l i g a n d f o r b r a i n © { . - - a d r e n e r g i c r e c e p t o r s . E u r . J . P h a r m a c o l . 7 6 , 4 6 1 - 6 4 . 24 B u k o w i e c k i , L . , F o l e a , N . , V a l l i e r e s , J . , a n d L e b l a n c , J . ( 1 9 7 8 ) / 9 - a d r e n e r g i c r e c e p t o r s i n b r o w n a d i p o s e t i s s u e . C h a r a c t e r i z a t i o n a n d a l t e r a t i o n s d u r i n g a c c l i m a t i o n o f r a t s t o c o l d . E u r . J . B i o c h e m . 9_2, 1 8 9 - 1 9 6 . 25 B u k o w i e c k i , L . , F o l l e a , N . , P a r a d i s , A . , a n d C o l l e t , A . ( 1 9 8 0 ) S t e r e o s p e c i f i c s t i m u l a t i o n o f b r o w n a d i p o c y t e r e s p i r a t i o n b y c a t e c h o l a m i n e s v i a ^ . . - a d r e n o c e p t o r s . A m . J . P h y s i o l . 2 3 8 , E 5 5 2 - E 5 6 3 . 26 B u k o w i e c k i , L . J . ( 1 9 8 4 ) M e c h a n i s m s o f s t i m u l u s - c a l o r i g e n e s i s c o u p l i n g i n b r o w n a d i p o s e t i s s u e . C a n . J . B i o c h e m . C e l l . B i o l . 62 ( 7 ) , 6 2 3 - 6 3 0 . 27 B u r n s , T . W . , a n d L a n g l e y , P . E . ( 1 9 7 5 ) T h e e f f e c t o f a l p h a a n d b e t a a d r e n e r g i c r e c e p t o r s s t i m u l a t i o n o n a d e n y l a t e c y c l a s e a c t i v i t y o f h u m a n a d i p o c y t e s . J . C y c l i c N u c l e o -t i d e R e s . 1_, 3 2 1 - 2 8 . 28 B u r t , D . R . , C r e e s e , I . , S n y d e r , S . H . ( 1 9 7 7 ) A n t i s c h i z o -p h r e n i c d r u g s : c h r o n i c t r e a t m e n t e l e v a t e d o p a m i n e r e c e p t o r b i n d i n g i n b r a i n . S c i e n c e , 1 9 6 , 3 2 6 - 3 3 2 . 29 B u r t , R . 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( 1 9 7 8 ) R e l e a s e o f f a t t y c i d s f r o m a d i p o s e t i s s u e i n g e n e t i c a l l y ( o b / o b ) m i c e . F E B S l e t t . 9 0 , 1 3 2 - 1 3 4 . 33 C a r p e n e , C . , B e r l a n , M . , a n d L a f o n t a n / M . ( 1 9 8 3 ) L a c k o f F u n c t i o n a l a n t i l i p o l y t i c a l p h a 2 - a d r e n o c e p t o r i n r a t f a t c e l l : c o m p a r i s o n w i t h h a m s t e r a d i p o c y t e s . C o m p . B i o c h e m . P h y s i o l . 7 4 C , 4 1 - 5 . 34 C a r r i e r , 0 . ( 1 9 7 5 ) R o l e o f c a l c i u m i n p o s t - j u n c t i o n a l s u p e r s e n s i t i v i t y . F e d . P r o c . 3_4, 1 9 7 5 - 1 9 8 0 . 3 5 C h e n g , Y . , a n d W . H . P r u s o f f ( 1 9 7 3 ) R e l a t i o n s h i p b e t w e e n t h e i n h i b i t i o n c o n s t a n t ( k i ) a n d t h e c o n c e n t r a t i o n o f i n h i b i t o r w h i c h c a u s e s 5 0 % i n h i b i t i o n o f a n e n z y m a t i c r e a c t i o n . B i o c h e m . 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Rev. 18, 629-659. 2 0 3Tulp, O.L., Gregory, M.H., and Danforth, E.W. (1982) Characteristics of diet-induced brown adipose tissue growth and thermogenesis in rats. Life Sci. 30, 1525-1530, 2 0 4U'Prichard, D.C, and Snyder, S.H. (1977) [3H]-epinephrine and [3H]-norepinephrine binding to (*-noradrenergic receptors in calf brain membranes. Life Sci. 20_, 527-534. 2 0 5U'Prichard, D.C, and Snyder, S.H. (1977b) Binding of [3H]-catecholamines to ^-adrenergic receptor sites in calf brain. J . Biol. Chem. 252, 6450-6460. 20 6 U'Prichard, D.C, Bechtel, B., Rouot, B., and Snyder, S.H. (1978) Multiple apparent alpha-noradrenergic receptor binding sites in rat brain: effect of 6-hydroxydopamine. Mol. Pharmacol. 1_6, 47-60. 207 U'Prichard, D.C, Snyder, S.H. (1978) Catecholamine binding to _<-receptors in rat brain: enhancement by reserpine. Eur. J . Pharmacol. _5_1, 145-155. 208 Walach, D., and Pastau, I. (1976) Stimulation of Guanylate Cyclase of Fibroplasts by free fatty acids. J . Biol. 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(1976) Adipocytes /^-adrenergic receptors: Identification and subcellular localization by (-)[3H]-dihydroalprenolol . binding. J . Biol. Chem. 251, 3096-3104. -155-215 Williams, L.T., and Lefkowitz, R.J. (1977) Regulation of rabbit myometrial alpha adrenergic receptors by estrogen and progesterone. J . Clin. Invest. 60, 815-8. 2 1 6Williams, L.T., and Lefkowitz, R.J. (1977b) Thyroid hormone regulation of Ji -adrenergic receptor number. J . Biol. Chem. 252, 2787-2795. 217 Williams, R.S., and Bishop, T. (1981) Selectivity of dobuta-mine for adrenergic receptor sub-types: in vitro analysis by radioligand binding. J . Clin. Invest. 67, 1703-1711. 218 Wise, L.S., and Jungas, R.L. (1978) Evidence for a dual mechanism of lipolysis activation by epinephrine in rat adipose tissue. J . Biol. Chem. 253, 2624-2627. Wojcik, J.D., Graud, R.J., and Kimberg, D.V. (1975) Amylase Secretion by rabbit Parotid Gland, Role of" Cyclic AMP. Biochim. Biophys. 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(1980) Effects of -adrenergic action on cyclic AMP levels in canine thyroid slices. Life Sci. 27, 1127-1130 2 2 6 Yen, T.T.T., and Steinmetz, J.A. (1972) Lipolysis of genet-ically obese and/or hyperglycemic mice with reference to insulin response of adipose tissue. Horm. Metab. Res.. 4, 331-337. -156-? 2 7 2 + Zavoico, G.B., and Feinstein, M.B. (1984) Cytoplasmic Ca in platelets is controlled by cyclic AMP: antagonism between stimulators and inhibitors of adenylate cyclase. Biochim. Biophys. Res. Commun. 120, 579-585. 228 Zwiller, J . , Revel, M.O., and Malyiya, A.N. (1984) Protein Kinase c catalyzes phosphorylation of guanylate cyclase in vitro. J . Biol. Chem. 260 (3), 1350-1353. -157-APPENDIX A [ H]-Rauwolscine and [ H]-norepinephrine have been extensively used to characterize ^^-adrenoceptors in rat brain. In order to compare our methodology and the binding characteristics of our membrane preparations to that already reported in literature, we carried out in i t ia l ligand binding experiments on membrane fragments isolated from rat brain cortex. The dissociation constant (K )^ and 3 maximum binding capacity/mg protein (B ) for [ H]-rauwols-max 3 cine [23] and [ H]-norepinephrine [2(71] calculated from these experiments were in agreement with the values already reported. Fig. 16 Shows binding of [ H]-rauwolscine to membranes isolated from the cortex of one-week-old rat brain. Specific binding (•-•) was determined by taking the difference of total binding (•-•) in the absence of 10 -^M yohimbine and that of non-specific binding (o-o) in the presence of '10 /u.K yohimbine. Each point is the mean of duplicate determinations from one representative experiment. Inset: Scatchard plot of the data. Fig. 17 Scatchard plot of (-)[ H]-norepinephrine specific binding in the presence of 10 /_M epinephrine and atenolol from one representative experiment. -158-9 B KdaL = 500 nM • • • • i Kd rBmax = • 21.8 0.205 pmol /mg . protein, 50 150 2 5 0 BOUND (Fmol / m g prot«) -160-APPENDIX B Krebs-Ringer Bicarbonate Buffer 1. NaCl 118.5mM 2. KC1 4.8mM 3. KH2P04 1.18mM 4. MgS04.7H20 1.18mM 5. NaHCO- 24.9mM 6. CaCl2 1.25mM 7. Glucose 5mM -161-APPENDIX C Modified Krebs-Ringer Bicarbonate Buffer, Buffer used in Experiments of Section 2.3.6 1- NaCl. 118.5mM 2- KCl.t • 4.8mM 3. MgS04 1.18mM 4. KH2P04 1.18mM 5. NaHC03 11.14mM 6. HEPES 30mM 7. Cacl 2 2.5mM 8. Glucose lOmM -162--163-APPENDIX D Ligands and the Drugs used i n the bin d i n g experiments. Ligands o r  Drugs used H-ClOnidine C l o n i d i n e hydrochlo- r i d e , [Benzene Ring-Alpha-2 a g o n i s t [Refs. 30,190] S t r u c t u r e Sp 3H-Norepinephrine Norepinephrine, Levo- [Ring-2,5,6- 3H]-Mixed a g o n i s t «*2/«i » P l . P2 (Refs.184, 204, 205-207]. M . U . 2 6 6 . ( M.H. 266.6 . a c t i v i t y 61.1 Ci/mmol OH Sp H0CKCH2HH2 M.W. 169.2 a c t i v i t y 52.9 Ci/mmol 4 8.4 Ci/mmol Remarks Approx. K_(Yohimb.) from Satur.curve = 35nM Approx. B m a x from sat u r . curve =40 fmol/mg p r o t . Approx. Kd(clonid.) from Satur.curve = 30nM. Approx. B m a X from satur. curve =50 fmol/mg p r o t . K<j (epinephrine) from S c a t . p l o t = 60.4 nM B max(epineph) from Scat, p l o t = 0.22 pmol/mg pro t Kd(yohimb.)from Scat, p l o t = 65.8 nM B^ax(yohimb.) from Scat, p l o t = 0.24 pmol/mg prot H i l l c o e f f i c i e n t from H i l l plot= 1.5 3H-Rauwolscine Rauwolscine, [Methyl-3H] or (et-yohimbine) oc.-antagonist [RSfs.23, 30, 31,148 M.W. 354.4 S p . a c t i v i t y 79.5 Ci/mmol -164-APPENDIX D Liqands o r  Drugs used Liqands and the Drugs used i n the binding experiments.  S t r u c t u r e Remarks 3H-RX-781094 (l,4-[6,7-3H]benzo-d i o x a n - 2 - y l ) - 2 - i m i -d a z o l i n e h y d r o c h l o r i d e o<.2-antagonist i R e f s . 30,45,50] 13 J H . H C l M.W. 2 4 4 S p . a c t i v i t y 56.1 Ci/mmol H-yohimbine Yohimbine, [Methyl-3H]-ot2~antagonist [Refs.20,30,66,81, 163 ]. H.U. J M . t M.W. 3 5 4 . 3 Sp. a c t i v i t y 7 5 . 0 Ci/mmol -Approx. K<j (yohimb.) from Satur. curve = 10 nM -Approx. B m a X from satur. curve =20 fmol/mg pr o t . -Approx. K<j (yohimb.) from Satur.curve =15 nM -Approx. B m a from Satur. curve=125 fmol/mg -K d (Yohimb.) from Scat, p l o t = 18 nM -Bmax from Scat, p l o t = 156 fmol/mg prot. H-DHA D i h y d r o a l p r e n o l o l ^ h y d r o c h l o r i d e , l e v o - ^ " ^ N C V ^ R.K. w.t - Approx. K^(norepi.)from Satur. curve = 10 nM „ - A p p r o x . B m a x ( n o r e p i . ) f r o m satur.curve=0.45fmol/mg NjW l^ttio,^  - K d ( n o r e p i . ) from Scat, p l o t = 10 nM p r o t . fr^l-WjcSjOi, - Bmax^1101-6?*-'' f r o m Scat, p l o t =0.55 fmol/mg.prot. - K d ( p r a c t o l o l ) from Scat, p l o t = 50 nM a;.i _ Bjaaxfpract.) from Scat, p l o t »0.19 pmol/mg.prot. IRing,propyl-or D i h y d r o a l p r e n o l o l hydrochlor ide,levo-l p r o p y l - 1 , 2 , 3 - 3 H ] - M.W. 2 8 7 . 8 |- (yohimb.) from Scat, p l o t = 4 0 nM i l - a n t a g o n i s t [Refs. Sp. a c t i v i t y 1 0 4 . 8 Ci/mmol. B r oax(yohimb.) from Scat. p l o t = 0 . 1 1 2 pmol/mg.prot. 2 4 , 4 1 , 4 8 , 1 3 3 , 2 1 4 ] . J 3 9 . 7 Ci/mmol -165-APPENDIX D Ligands or  Drugs used Ligands and the Drugs used i n the b i n d i n g experiments.  S t r u c t u r e Remarks I H.HC JH-RX-781094 (l,4-[6,7-3H]benzo-dioxan-2-y1)-2-imi-d a z o l i n e h y d r o c h l o r i d e <rf.2-antagonist I [Refs. 30,45,50] | M.W. 244 S p . a c t i v i t y 56.1 Ci/mmol 0 0 ^ H-yohimbine Yohimbine, [Methyl-3 H ] -o C 2 _ a n t a g o n i s t (Refs.20,30,66,81, ]. M.W. 354.3 Sp. a c t i v i t y 75.0 Ci/mmol -Approx. K<j(yohimb.) from Satur. curve = 10 nM -Approx. B-^JJ from satur. curve =20 fmol/mg pro t . -Approx. K d(yohimb.) from Satur.curve =15 nM -Approx. B-gjj from Satur. curve=125 fmol/mg prot. -K<j (Yohimb.) from Scat, p l o t = 18 nM - Bmax from Scat, p l o t = 156 fmol/mg prot. H-DHA D i h y d r o a l p r e n o l o l h y d r o c h l o r i d e , l e v o -[Ring,propyl- 3H(N))-or D i h y d r o a l p r e n o l o l h y d r o c h l o r i d e , l e v o -[propyl-l,2,3-3H]-^1-antagonist [Refs 24,41,48, ]. Approx. K^fnorepi.)from Satur. curve = 10 nM Approx.B m ax(norepi.)from satur.curve=0.45fmol/mg K^tnorepi.) from Scat, p l o t = 10 nM p r o t . B m a x ( n o r e p i . ) from Scat, p l o t =0.55 fmol/mg.prot. K _ ( p r a c t o l o l ) from Scat, p l o t = 50 nM B-iax(pract.) from Scat, p l o t =0.19 pmol/mg.prot. M.W. 287.8 |- K_(yohimb.) from Scat, p l o t = 40 nM Sp. a c t i v i t y 104.8 Ci/mmol. B m ax(yohimb.) from Scat. plot=0.112 pmol/mg.prot. 39.7 Ci/mmol O C K J C H C ^ N M A . H O (WW* «_ -166-APPENDIX E Liqands and the Drugs used In the binding experiments. (6-hydroxydopamine and yohimbine p r e t r e a t e d animals). Ligands or  Drugs used H-yohimbine o<2-antagonist) S t r u c t u r e M.W. 3 5 4 . 4 Remarks Co n t r o l animals: -Approx. K,j(yohimb.) from Satur. curve = 18 nM -Approx. Bmax from Satur. curve = 180 fmol/mg p r o t . -K d (yohimb.) from Woolf p l o t = 14 nM -B m a x(yohimb.) from Woolf p l o t = 155 fmol/mg p r o t . 6-hydroxydopamine p r e - t r e a t e d r a t s . -Approx. K. (yohimb.) .from Satur. curve => 10 nM -Approx. B _ a x (yohimb.) from Satur. curve= 75 fmol/mg -K<j (yohimb.) from Woolf p l o t = 8.6 nM prot. -B m a x(yohimb.) from Woolf p l o t = 45 fmol/mg p r o t . Chronic yohimbine p r e - t r e a t e d r a t s . - K,j (yohimb.) from Scat, p l o t = 66.7 nM Bmax (yohimb.) from Scat, p l o t = 0.35 pmol/mg prot . H-Norepinephrine (Mixed agonist) H 0 C H C H 2 N H 2 M.W. 169.2 C o n t r o l animals; K(j (epinephrine) from Scat, p l o t = 68 nM B m a x ( e p i n e p h r i n e ) from Scat, p l o t = 1.39 pmol/mg p r o t . 6-hydroxydopamine p r e t r e a t e d animals: K,j (epinephrine) low a f f i n i t y s i t e from Scat plot=36nM B m a x (epi.) low a f f i n i t y s i t e = 0.65 pmol/mg prot . K d (epi.) high a f f i n i t y s i t e from Scat. plot= 200 nM Bmax^ ePi*' n i g h a f f i n i t y s i t e = 3 pmol/mg p r o t . H i l l c o e f f i c i e n t from H i l l p l o t =1.5 Chronic yohimbine p r e - t r e a t e d animals: (epi.) from Scat, p l o t = 68 nM Bmax (epi.) from Scat, p l o t = 3 pmol/mg p r o t . K<j (yohimb.) from Scat, p l o t = 66.7 nM Bmax (yohimb.) from Scat, p l o t =3.6 pmol/mg. prot . -167-APPENDIX E Ligands and the Drugs used i n the binding experiments Ligands o r S t r u c t u r e Remarks Drugs used Epinephrine (Mixed agonist) [Refs: 205-207]. Practolol (B_-antagonist) Alpha-2 > Alpha-1> Beta-, > Beta-2 Dobutamine (B,-agonist) PUBLICATIONS 1. S h a i k h , I. M., Cannon de R o d r i g u e z , W., and S k a l a , J.P. (1986) " < * 2 ~ A d r e n e r g : L C R e c e p t o r s i n Brown A d i p o s e T i s s u e o f I n f a n t Rats- I. I d e n t i f i c a t i o n and C h a r a c t e r i s t i c s of B i n d i n g S i t e s of I s o l a t e d Membrane F r a g m e n t s " . ( S u b m i t t e d ) . 2. S h a i k h , I . M., Cannon de R o d r i g u e z , W., and S k a l a , J.P. (1986) " © ^ " A d r e n e r g i c R e c e p t o r s i n Brown A d i p o s e T i s s u e of I n f a n t R a t s . I I . E f f e c t s o f C h e m i c a l Sympathectomy and C h r o n i c Yohim-b i n e A d m i n i s t r a t i o n . " ( S u b m i t t e d ) . 3. S h a i k h , I . M. , Schimmel, R.J., S k a l a , J.P. (1 986) " °< - A d r e n e r g i c R e c e p t o r s i n Brown A d i p o s e T i s s u e o f I n f a n t R a t s . I I I . E f f e c t on L i p o l y s i s " . ( I n p r e p a r a t i o n ) . 4. S h a i k h , I . M., S k a l a , J.P. (1986) " S t i m u l a t i o n o f © ^ - A d r e n o -c e p t o r s i s L i n k e d t o G u a n y l a t e c y c l a s e A c t i v a t i o n i n Brown A d i p o s e T i s s u e o f I n f a n t R a t s . ( I n p r e p a r a t i o n ) . 5. Diamond, J . , S h a i k h , I.M. (1980) E f f e c t o f H y d r a l a z i n e and D-600 on KCL In d u c e d C o n t r a c t i o n and P h o s p h o r y l a s e A c t i v a t i o n i n R a b b i t A o r t a . W e s t e r n P h a r m a c o l o g i c a l S o c i e t y . 23, 45-48. 6. Keesey, J . , S h a i k h , I . M., Wolfgram, F., Chow, A. (1976) A b l o c k a d e of Normal Mammalian A c e t y l c h o l i n e R e c e p t o r s by M y a s t h e n i a g r a v i s s e r a . Ann. N.Y. A c a d . S c i . 274 , 244-252. 7. T a y l o r , D.B., S h a i k h , I.M. (1975) S y n t h e s i s o f B i f u n c t i o n a l Co-o r d i n a t i o n Complexes o f Osmium w i t h C u r a r i f o r m A c t i v i t y . J . M e d i c i n a l Chem. 1_8, 1088-1094. 8. S h a i k h , I.M., Thompson, J.H. (1970) A c u t e and C h r o n i c E f f e c t s N i c o t i n e on Rat G a s t r i c S e c r e t i o n . P r o c . W e s t e r n P h a r m a c o l . Soc. 13, 178-184 9. P h a r m a c o l o g y o f I o n i c C o o r d i n a t e d Complexes o f T r a n s i t i o n E l e m e n t s . M.S. T h e s i s . P h a r m a c o l o g y . UCLA S c h o o l o f M e d i c i n e . 10. 0 S e a s o n a l V a r i a t i o n s i n T i s s u e L i p i d s i n U r o m a s t i x M.S. Biochem. t h e s i s . U n i v e r s i t y of K a r a c h i . h a r d w i c k i i ABSTRACTS. 1. Skala, J.P., Shaikh, I.M., and Cannon de Rodriguez, W. (1985) " The e< -Adrenergic Receptors i n Brown Adipose T i s s u e of Developing Rats". C l i n . Invest. Med. 8^, A 170. 2. Skala, J.P., Shaikh, I.M.,and Cannon de Rodriguez, W. (1985) " Norepinephrine E f f e c t s upon Brown Adipocytes of Infant Rats Are Mediated v i a Both <*- and fS-Adrenergic Receptors." Fed. Proc. 44, 1160. 3. Skala, J.P., Shaikh, M.I., and Cannon de Rodriguez, W. (1985) " The ^ - A d r e n e r g i c Receptors i n Brown Adipocytes of Developing Rats." Paed. Res. 1_9, 163A. 4. Skala, J.P., and Shaikh, I.M. (1984) " The «*-Adrenergic Recep-t o r s i n Brown Adipose T i s s u e of Neonatal Rats'.' IV. Pan. Am. Biochem. Congress Proc. No. 276. 5. Skala, J.P., and Shaikh, I.M. (1984) " The °< -Aderenergic Recep-to r s i n Brown Adipocytes of Developing Rats. Paed. Res. 18, 145A 6. SKala, J.P. and Shaikh, I.M. (1984) " Catecholamine E f f e c t s upon Brown Adipose T i s s u e of Newborn Rats Are Mediated P a r t i a l l y v i a the ©^""Adrenergic Receptors." Fed. Proc. 4_3. 1581 7. Skala, J.P., Shaikh, I.M., and Vacek, L. (1983) " C y c l i c Nucleo-t i d e and A d r e n e r g i c S t i m u l a t i o n of Brown Fat i n G e n e t i c a l l y Obese Mice." Fed. Proc. 4_2, 1849. 8. Skala, J.P., Shaikh, I.M., and Vacek, L. (1983). " C y c l i c - GMP'Participates i n -Adrenergic Response of Brown Adipose T i s s u e of Young Rats. Ped. Res. 1_7, 172. 9. Diamond, J . , and Shaikh, I.M. (1980) E f f e c t s of H y d r a l a z i n e and D-600 on Tension and Phosphorylase A c t i v i t y i n Rabbit Aorta". Fed. Proc. 3j) (37), 1176. 

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