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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 p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of  requirements f o r an advanced degree at the  the  University  o f B r i t i s h Columbia, I agree t h a t  the L i b r a r y s h a l l make  it  and  f r e e l y a v a i l a b l e for reference  study.  I  further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may  be  department or by h i s o r her  granted by  the head o f  representatives.  my  It i s  understood t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r 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 The U n i v e r s i t y of B r i t i s h 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 Date  c  c  / ~> /na\  May  29,  1986  Columbia  (Pathology, Pharmacology Frvysio logy and Biochemistry)  ABSTRACT OF THESIS Alpha-2 adrenoceptors in Brown Adipose Tissue of Infant Rats. by M. IQBAL SHAIKH This thesis consists of five chapters. The first chapter deals with general background and introduction. Each of the subsequent chapters are divided into sections. The first section deals with pharmacological characterization of <*2~ oceptors using various ligands. The second section pertains to the study of binding characteristics of *<2~ ^ P following chemical sympathectomy by 6-hydroxydopamine and chronic blockade of °<2 ^ P by yohimbine injections. The third section deals with the study of guanylate cyclase system in relation to ©<2 enoceptors 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-dayold rats. Finally cyclic GMP production in obese and lean mice in relation to ©^-adrenoceptors stimulation was discussed in the fifth section. adren_  ac  -ac  rerioce  renoce  tors  tors  -adr  3  Binding characteristics of ©^-antagonists ([ H]-RX-781094, [ H]-yohimbine, [3 H]-rauwolscine) and agonists ([ 3 H]-clon3  3  idine, [ H]-norepinephrine) to o^-adrenoceptors on isolated plasma membrane fragments from brown adipose tissue were studied. The binding of [^K]-yohimbine was rapid,saturable and reversible. Yohimbine, (-)-epinephrine, -ii-  and in  clonidine that  «<2  order  ^  _ a <  r e n o c e  showed ing  displaced of  [ H ] - y o h i m b i n e from  potency  Ptors.  as would  A Scatchard  (B  ) of  0.15  be e x p e c t e d  plot  an e q u i l i b r i u m c o n s t a n t  capacity  i t s binding  3  (^d)  of of  pmol/mg  of  binding  yohimbine  to  binding  18 nM a n d t o t a l  protein.  sites  bind-  Binding  of  max [  3  3 and [ H ] - c l o n i d i n e  HJ-RX781094  rapid, on  stable,  saturable  the binding  presence  o f more  of  the  or  yohimbine  and  of  (-)[  than  one b i n d i n g  6 5 . 8 nM r e s p e c t i v e l y ,  ephrine  probably  site  with  (71.3  of  fmol/mg  ephrine 1.4  nolol a  shared  and B  supported  binding  analysis  using  (-)-epinephrine  revealed of  site;  i n much  In a d d i t i o n ,  Binding  of  of  (^-epin-  lower  the  number  two  coefficient  positively H]-dihydroalpre-  and n o r e p i n e p h r i n e  5 0 nM a n d 10 nM r e s p e c t i v e l y  (p^-site)  and B 3  of  0.19  and 0 . 5 pmol/mg  prenolol ing  binding  either  atypical rats.  It  could  a relative  nature  of  protein. also  However,  (-)[  non-specificity  of  that -  H]-dihydroal-  the ligand  t h e p l a s m a membranes i i i -  with  max  be d i s p l a c e d by y o h i m b i n e  t h e / 3 ^ - a d r e n o c e p t o r s i n brown  i s suggested  other  (-)-norepin-  a Hill  (-)[  o f . 6 0 . 4 nM  0 . 2 2 and 0 . 2 4  binding  the presence  sites.  a Kd  and  was d i s p l a c e a b l e by p r a c t o l o l of  Scatchard  and was s p e c i f i c f o r only.  Studies the  yohimbine  o n e common  of  indicated  values  m a x  6 4 . 5 nM w a s p r e s e n t  protein)  further  cooperative  site.  binding  Norepinephrine,  and yohimbine  binding.  H]-norepinephrine  as the d i s p l a c i n g agent,  protein.  a similar pattern  and r e v e r s i b l e  H]-norepinephrine  pmol/mg  of  (-)[  showed  suggest-  or  fat from  an  of  infant  actively  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 adrenoceptor sub-type.  3  Binding studies carried out with [ H]-yohimbine on membranes isolated from brown fat of chemically sympathectomized 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 H i 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 pretreatment resulted in more than two-fold increase in the number of binding sites for both [ 3H]-yohimbine and (-)[ 3 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 -iv-  blocker. Incubation (0.2-20>M) cyclic of  GMP  the  of  into trend  ceptor  seen.  response  The  For  to  brown  receptor-protein  The  data  of  supported  brown  fat  is  the  from  of  tissue  concentration one-week-old  c l o n i d i n e and  c o u l d be b l o c k e d by cyclic  incubation,  and  stimulation  fat  0 . 5 - I^MM o f  tissue  clonidine  clonidine  elevation  occurred at  response  When  pieces with  dependent  occurred at  yohimbine.  i n response  was  tissue  rats.  incubation.  releaseable  fat  peak  response  incubation with elevated  The  one-month-old  peak  5 minutes  brown  a dose- and;time-'  content.  for  20  rats,  showed  of  bound  the  GMP was  prior  concentration, separated  fraction,  hypothesis  linked  3 -  a  similar  that  (directly  or  o^-reindirect-  2+  ly  perhaps  via  Ca  Earlier response upon  of  acute  compared  )  to  guanylate  in vivo  brown  fat  to  control  dependent  increase.  where  fragments  from  showed  concentration suggested  that  in  GMP  cyclic  • i n the  lean  two-fold  tissue  to  shown a  catecholamine  mates  and  which in  increase  in  non-stimulated  i n obese  mice.  -v-  to  injections  as  were  is  a dose-  stimulated  controls. by  the  and  experiments  cyclic  respond  mice  mice  vitro  the  defective  i n obese  showed  lean mice  capability  production  had  activation.  production  Preliminary  obese  compared  GMP  and  litter  time-  clonidine,  experiments  cyclic  c o l d exposure  cyclase  an  GMP This increase  same a s  that  where with  Forskolin glycerol  release  week-old  rats.  acid  and  Isobutylmethylxanthine  in isolated adipocytes Clonidine,  of g l y c e r o l r e l e a s e  by  clonidine  and  by  yohimbine.  was  antagonized  regulatory  protein  inhibitory  e f f e c t of  itory  e f f e c t of  inhibitory the role  (Ni)  by  protein  anti-lipolytic  (Ni).  on  E  and  2  was  Inactivation  This  toxin  indicated  suggested  state  of  - vi  activity  -  of  inhibitory  abolished  that,  of  that  the the  i s mediated  e f f e c t o f © ^ - a d r e n o c e p t o r s may  i n c o n t r o l l i n g the  Inhibition  concentration-dependent  glycerol release  I t was  one-  nicotinic  glycerol release.  pertussis  clonidine.  clonidine  f r o m brown f a t o f  prostaglandin  showed i n h i b i t o r y e f f e c t s on  stimulated  fat  perhaps, have a cells.  inhibvia  ACKNOWLEDGEMENTS  I  would  J.P.  Skala  helpful to  like  Dr.  Dr.  J.  W.L.  interest  assistance Special I  there I  thanks  were  love  I for  taking  education.  Dr.  Burton  friends  support,  I would  dedicate  this  reached  to h i s  -vii-  L.  Easthope,. about  indeed".  I  this  whom  wish  him.  e s p e c i a l l y my m o t h e r , of higher  goals  Without  their  education.  t o my y o u n g e r  and commitment  I am  me t e c h n i c a l  like  for the pursuit  have never  thesis  Peter  is a friend  of  reading  work.  and persons  level  for  Dr.  and comments.  for giving  this  a r e due t o  (Physiology),  (Biochemistry)  t o my p a r e n t s ,  the highest  his sincerity  of  My t h a n k s  Pederson  suggestions  i n need  and encouragement of  Committee,^in  a r e d u e t o my f r i e n d  grateful  and  o f my D o c t o r a l  the course  a few more  environment  indebted  necessary  c a n s a y , "A f r i e n d  Dr.  am p e r s o n a l l y  and D r .  work.  to  I  (Pharmacology),  during  attainment and  out t h i s  and thanks  the s u i t a b l e  t o M r s . W. R o d r i g u e z  am f o r e v e r  their  me w i t h  i n my g r a d u a t e  and making  thankful  my a p p r e c i a t i o n  Chairman  (Zoology),  my t h e s i s  all  to carry  Dunn,  Diamond  Phillips  also  for providing  advice  personal  to express  for and help  far.  brother, family.  M. Z a f a r  Shaikh,  TABLE  OF  CONTENTS Page  ABSTRACT  i i  ACKNOWLEDGEMENTS  v i i  LIST  OF  xi  LIST  OF T A B L E S  FIGURES  xiv  CHAPTERS 1.  G E N E R A L BACKGROUND AND I N T R O D U C T I O N 1.1  Brown  Adipose  1.2  Adrenergic  1.3  Receptor 1.3.1 1.3.2  2.  Tissue  1  Receptors  4  Ligand  Binding  5  Saturation Experiments Competition or Labelled Ligand Displacement Experiments  1.4  The B e t a - a d r e n e r g i c  1.5  The A l p h a - a d r e n e r g i c  1.6  Thesis  Pathway  7 13 21  Pathway  Main O b j e c t i v e  23 33  M A T E R I A L S AND METHODS  34  2.1  Materials  34  2.2  Animals  35  2.3  Methods 2.3.1  6-hydroxydopamine of  2.3.2 2.3.3 2.3.4  2.3.5  pre-treatment  the r a t  . P l a s m a Membrane  36 Preparations...  Ligand Binding Assays E f f e c t of o 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 t o t a l and 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 of brown f a t f r a g m e n t s from o n e week-old rats E f f e c t of a g o n i s t s i n v i t r o on 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 fragments: from obese mice and t h e i r l e a n l i t t e r mates  - v i i i -  36 38  39  44  Page 2.3.6  2.3.7  2.3.8 3.  45  E f f e c t of c l o n i d i n e on f o r s k o lin-stimulated lipolysis in adi p o c y t e s from brown f a t o f a d u l t hamsters  46  Data A n a l y s i s  46  RESULTS 3.1  3.2  3.3  3.4  3.5  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 lated l i p o l y s i s i n adipocytes from brown f a t o f 1 - w e e k - o l d r a t s by c l o n i d i n e  _  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 o f ^--adrenoceptors binding sites i n isol a t e d p l a s m a membrane f r a g m e n t s f r o m b r own 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  E f f e c t of 6-hydroxydopamine and c h r o n i c yohimbine p r e - t r e a t m e n t on t h e b i n d i n g characteristics of ^-adrenoceptors i n i s o l a t e d p l a s m a membrane f r a g m e n t s from brown f a t o f i n f a n t r a t s  58  E f f e c t of adrenergic 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 c o n t e n t s o f r a t brown a d i p o s e t i s s u e . . .  74  Inhibition of forskolin-stimulated 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 o f l ^ w e e k - o l d r a t s by c l o n i d i n e  85  E f f e c t o f o ^ - a g o n i s t and ^ . - 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 fragments from obese mice and t h e i r unaffected littermates  97  DISCUSSION 4.1  4.2  4.3  102  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 o f ©< - a d r e n o c e p t o r s i n p l a s m a membrane f r a g m e n t s o f b r o w n f a t from 7 - d a y - o l d r a t s  106  E f f e c t o f c h e m i c a l sympathectomy and c h r o n i c yohimbine pre-treatment on b i n d i n g characteristics of ^-adrenoceptors i n brown f a t o f 7 - d a y - o l d r a t s  114  E f f e c t o f adrenergic 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 c o n t e n t s i n brown a d i p o s e t i s s u e o f 7day-old rats  120  -ix-  Page 4.4  4.5  5.  E f f e c t o f o<_-agonist on 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 a d i p o c y t e s from 7 - d a y - o l d r a t s  128  E f f e c t o f adrenergic a g o n i s t and antago 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 brown f a t fragments from obese mice and u n a f f e c t e d l i t t e r - m a t e s  131  CONCLUSION  134  BIBLIOGRAPHY  137  APPENDIX  158  VITAE  .16J-.  -x-  LIST  OF  FIGURES  Figure 1A  Page 3  S p e c i f i c b i n d i n g of [ H]-yohimbine t o plasma membrane 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 of o n e - w e e k - o l d r a t s 3  IB  2A  I n h i b i t i o n of [ H]-yohimbine b i n d i n g c r e a s i n g concentrations of yohimbine epinephrine  by i n and ( - ) -  S p e c i f i c b i n d i n g of [3Hj-RX-781094 t o plasma membrane 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 one-week-old rats  49  49  51  3  2B  2C  2D  3A  S p e c i f i c b i n d i n g of [ H ] - c l o n i d i n e t o plasma membrane 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 one-week-old rats  51  3  S p e c i f i c binding of [ H]-norepinephrine to p l a s m a membrane 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 of one-week-old r a t s i n the presence of 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  S p e c i f i c binding of [ H]-norepinephrine t o . 1 p l a s m a membrane 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 of one-week-old r a t s i n the presence of 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  52  3  Scatchard p l o t of [ H]-norepinephrine binding t o b r o w n f a t p l a s m a membrane f r a g m e n t s from 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  54  3B  Hill  54  4A  Displacement of s p e c i f i c binding of [ ^ - n o r e p i n e p h r i n e t o brown f a t p l a s m a membrane fragments from o n e - w e e k - o l d 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 epinephrine or p r a c t o l o l . . .  4B  plot 3  3  57  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 by increasing concentrations of epinephrine i n the p r e s e n c e o f 1 0 ~ 3 M p r a c t o l o l and by i n c r e a s i n g concentrations of p r a c t o l o l i n the presence of 10 M epinephrine 59 _ 5  -xi-  Figure 5A  5B  6  7A,B  7C  8  9A,B  9C  Page  (-)[ 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 p l a s m a membrane 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 .  59  3  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 to brown f a t p l a s m a membrane 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  59  E f f e c t of c h e m i c a l sympathectomy of 5 - d a y - o l d 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 d o s e o f 6 h y d r o x y d o p a m i n e on 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 membrane fragments i s o l a t e d 3 days l a t e r  63  E f f e c t of c h e m i c a l sympathectomy of 5 - d a y - o l d r a t s on t h e s p e c i f i c b i n d i n g o f [3H]-norepine p h r i n e t o b r o w n f a t p l a s m a membrane i s o l a t e d 3 days l a t e r  66  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 membrane fragments from brown f a t of sympathectomized 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  69  3  Displacement of [ 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 membrane f r a g m e n t s i s o l a t e d f r o m brown f a t of 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 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 epinephrine or yohimbine  71  E f f e c t of 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 on t h e b i n d i n g of [ 3 H ] - y o h i m b i n e and [ 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 membrane f r a g m e n t s i s o l a t e d 7 days l a t e r  72  3  S c a t c h a r d a n a l y s i s of [ 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 membrane f r a g m e n t s i s o l a t e d from c h r o n i c a l l y yohimbine p r e - t r e a t e d r a t s . .  75  3  9D  10  11  S c a t c h a r d a n a l y s i s of [ 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 membrane f r a g m e n t s f r o m o n i c a l l y yohimbine p r e - t r e a t e d r a t s  chr-  75  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 c o n t e n t s i n brown a d i p o s e t i s s u e of o n e - m o n t h - o l d r a t s  78  E f f e c t of c l o n i d i n e and y o h i m b i n e i n v i t r o 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 from 7 - d a y - o l d r a t s  82  - x i i -  on  Figure 12  13A,B  13C  Page E f f e c t o f c l o n i d i n e i n v i t r o on t o t a l , free 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 of brown f a t from 7 - d a y - o l d r a t s  86  E f f e c t of c o l d e x p o s u r e and 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 brown a d i p o s e t i s s u e of g e n e t i c a l l y obese mice  98  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 brown f a t fragments of g e n e t i c a l l y obese mice and t h e i r l e a n l i t t e r mates  100  - x i i i -  LIST OF TABLES  Page  Table 1.  E f f e c t of p r o s t a g l a n d i n (PGE2) 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 d g l y c e r o l r e l e a s e i n adipocytes i s o l a t e d from brown adipose t i s s u e of 7-day-old r a t s  2A.  E f f e c t of increasing concentrations of c l o n i d i n e 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 a d i pocytes i s o l a t e d from brown f a t of 7-day-old r a t s .  2B.  R e v e r s a l o f c l o n i d i n e e f f e c t on f o r s k o l i n stimul a t e d g l y c e r o l r e l e a s e i n adipocytes i s o l a t e d from brown f a t of 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 o f yohimbine  3.  E f f e c t o f c l o n i d i n e and n i c o t i n i c a c i d on i s o butylmethylxanthine stimulated g l y c e r o l release i n adipocytes i s o l a t e d from brown f a t o f one-weekold rats  4.  E f f e c t of p e r t u s s i s t o x i n pre-treatment on i n h i b i t i o n of forskolin-stimulated 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 and PGE i n r a t a d i p o c y t e s  90  91  92  93  94  9  5A  E f f e c t o f c l o n i d i n e on f o r s k o l i n and i s o b u t y l 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 i n adipocytes i s o l a t e d from brown f a t o f a d u l t hamsters  5B.  E f f e c t o f p r o s t a g l a n d i n (PGE ) 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 d g l y c e r o l r e l e a s e i n adipocytes i s o l a t e d from brown f a t o f a d u l t hamsters  95  2  -xiv-  96  CHAPTER 1 GENERAL  1.1  BACKGROUND AND  Brown A d i p o s e  Tissue  Brown a d i p o s e  t i s s u e has been  thermogenic  organ  hibernators  [70,  in non-shivering 8 4 ] , but also  the  r a t [ 5 6 , 5 7 , 181 ] ,  38,  91, 153, 160,  obesity  161,  scheme  activity  causes release  granules  which binds  thermogenesis,  increased  to ^-adrenergic  activation  (e.g.,  by  increasing intracellular cyclic kinase  activity  hormone-sensitive  lipase  [26, 155].  sensitive  lipase  breakdown  of  The  fatty  acids  CoA  which enters forms  triglycerides  into  acetyl  the mitochondria, CoA.  The a c e t y l  and r e s u l t s  The  r e l e a s e d NADH++ H + e n t e r phosphorylation  p o s s e s s e s some u n i q u e  described below.  then  which,  c y c l i c AMP-  stimulates of  a  hormone-  acids  and g l y c e r o l .  into  fatty  a n d v i a y 3 - a n d wCoA e n t e r s  electron  results  by f a c i l i t a t i n g the  i n formation  generate  This  activate  so r e l e a s e d are a c t i v a t e d  mitochondria  drion  fatty  generally  cyclase)  Activation  increases l i p o l y s i s  in  oxidative  which  genetic  pre-synaptic.  receptors.  AMP,  [5, 36,  sympathetic  from  adenylate  protein  of  to the  of norepinephrine  cyclases  types  of  ATP.  acyloxida-  t h e TCA c y c l e protons  transport  and unusual  (NADH++H+).  c h a i n and by  The brown f a t  mitochon-  c h a r a c t e r i s t i c s which are  I n the conventional chemiosmotic t h e o r y , t h e r e i s an  -1-  in  such as  thermogenesis  According  major  not only  in non-hibernators  in  dependent  of  thermogenesis  in diet-induced  [39, 68, 69, 7 5 , 1 9 9 ] . of  i m p l i c a t e d as the  2 0 3 ] and i n c e r t a i n  accepted  tion  INTRODUCTION  obligatory piratory to  the  chain  outer  gradient. in  coupling  FoFl  phosphate. automatic  As  a feedback  (1) d e c r e a s e d decreased gradient  coupled  respiratory  proton  decreased  of  inhibition  the  proton  flow  inner  hypotheses several (i)  ways  Rapid either  in  Although  the no  hydrolyzing arsenate their (ii)  which  extra  (£>Go)  the  cell  exhibits  cellular  respiration. respiratorj proton  the  ATP  and  ATP  an  demand  This  leads  chain;  (2)  to  electrochemical  mitochondrial  respiratory In  of  ATP  oligomycin  Thermogenesis  energy  coupling  outer  cytoplasm  at  free  channel  ADP  the  mechanisms  action  the  H+  from  membrane;  synthetase,  ATP  chain  general might  or  the  (4)  and  (5)  to  a l l  be  central there  yet  to  its  synthesis  mitochondrial  been  prevent  are  enhanced.  intramitocltondrial  have do  is  terms,  subsequent  physiological  and  the  H+  ATP  thermogenesis  hydrolysis  matrix  synthesis.  thermogenesis. i n which  through  (3) e n h a n c e d  ATP  mitochondrial  a decreased  in  res-  of  through  mitochondrial  of  back  upon  and  re-entry  decreased mitochondrial The  this  down t h e  an e l e c t r o c h e m i c a l  synthesis  extrusion;  across  the  synthetase),  control,  electron  from  flow  (ATP  a result  H+  transfer  generating  ions  ATPase m o l e c u l e the  of  thus  hydrogen  causes  electron  pumping  medium,  As  released  causes  and  between  matrix. ATP  described,  ATP  synthesis  by  synthetase.  can a l s o  be  mitochondrial  -2-  enhanced ATPases  by (in  shivering actomyosin  in  complex) hydrolyze ATP and produce heat. (iii)  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 will 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 nonthermogenic 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 ^mv ^ so as to lowerAu + to 150-175 mv. According to Nicholls [139,140] this +  +  H  has one important consequence: this lowered potential is thermodynamically 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 cell [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-  mitochondrial synthetase  matrix.  path  and  proton  It  has been p u r i f i e d 156].  of  for  model has  adaptive  genic the  understood  circuit  it  unclear.  of  of  pathway So  Secondly,  there  the  circuit  short  is  [72,  are and  the  15].  components  the  interaction  no p l a u s i b l e  during  regulation no  degree  141,  can e x p l a i n  capacity  acute  same  mitochondria  far,  which  thermogenic and  of  ATP-  direct  pathways  the  chronic  cold  of  in  and  thermo-  evidence  for  intact  cells  in  tissues.  1.2  the  Adrenergic  Receptors  Basing  c o n c l u s i o n on  his  catecholamines,  tion  of  adrenergic  Subsequently, beta  suggested  of the  alpha-2,  using  Ahlquist receptors  Lands  receptors  a spectrum  and  not  brown-fat  short is  s h o r t - c i r c u i t s the  mitochondria  from hamster still  thus  development  i n normal  thermogenesis  episodes.  operation  the  been c o n s t r u c t e d  regulation  diet-induced  of  the  nucleotides with  vitro  or  as  However,  responsible  protein  prevents  of  [112,  gradient  This  et  al.  into  beta  [2]  the first  into  [107]  different  this  [109,  and  subclassifica-  beta  on t h e  186]. alpha  s u g g e s t i o n was  techniques  and  of  sub-types.  suggested s u b c l a s s i f i c a t i o n  a n d fi^ b a s e d  agonists  effects  proposed  alpha  s u b c l a s s i f i c a t i o n of and  diverse  selectivity  Langer  [108]  receptors  subsequently  of  into  alpha-1  confirmed  p h a r m a c o l o g i c a l methods  [16,  186,213 ] . Several  selective  alpha-1,  -4-  alpha-2,  beta-1,  and  beta-2  agonists during  and a n t a g o n i s t s  the past  a rather  few y e a r s .  Dobutamine  s e l e c t i v e yS^-adrenoceptor  Metoprolol selective  and p r a c t o l o l a r e w e l l ^-adrenoceptor  Terbutaline, as  either existed  SalbutamoL  a d r  nal  the e x p l o r a t i o n  function,  other  as w e l l  biochemical  into ultimate  showed a s t a t e namely gical  [125] , w h i l e  of r a t h e r  [59, 1 3 7 ] .  c l o n i d i n e as s e l e c t i v e  211].  receptors  ligands  greatly  d e t a i l s of receptor of the receptors  facilistructure, to  i n t r a n s l a t i n g the receptor  physiologic  and d e n s i t y  rather  selective labelled  as t h e r e l a t i o n  components  u s e d as a  [66,  of adrenergic  of molecular  classified  response.  sig-  Such s t u d i e s a d d i t i o n a l l y  of the receptor  receptor  states,  and t h e p h y s i o l o -  state of the t i s s u e .  Receptor Liqand Since  radioligand 2  of h i g h l y  as  [164, 2 1 7 ] .  o f dynamic r e l a t i o n s h i p between  affinity  1.3  c^ -  agents  enoceptor antagonist  that bind v a r i o u s . c l a s s e s  introduced  known examples  blocking  The a v a i l a b i l i t y  and  agonist  [30]. Yohimbine i s e x t e n s i v e l y  s e l e c t i v e cj<2~  tated  has been  introduced  M e t h y l n o r e p i n e p h r i n e have.been  s e l e c t i v e ^.,-agonist  ^2~agonist  o r have been  r e c e  P  t o r s  a significant  binding '  Binding  this  studies part  part  transmitter  t h e s i s deals  with  involving characterization of  of the general  with the background information radioactive  of t h i s  introduction w i l l  pertaining  deal  t o the binding  of the  t o membranes f r o m t h e a p p r o p r i a t e  target  organ, and  the theory behind i t .  i n most i n s t a n c e s  The  neurotransmitter  c o n s i s t s of at l e a s t two  the r e c o g n i t i o n or b i n d i n g  s i t e and  receptor  d i s t i n c t components:  some second p o r t i o n of  the  r e c e p t o r which " t r a n s l a t e s " t r a n s m i t t e r r e c o g n i t i o n i n t o a second messenger, u s u a l l y an  alteration in  accumulation of a c y c l i c n u c l e o t i d e . discussed  The  i n t h i s t h e s i s are p r i m a r i l y  °<2-receptors.  Adrenergic  receptors  among d i f f e r e n t t i s s u e s .  ion  permeability  group of  or  receptors  adrenergic  are w i d e l y d i s t r i b u t e d  These d i f f e r e n t r e c e p t o r  subtypes  can  not o n l y 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 b a s i s of doseresponse i n v a r i o u s t i s s u e s using antagonists,  but  s e l e c t i v e agonists  can a l s o be c h a r a c t e r i z e d  using 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 . a b i l i t y o f new  simple,  can study the i n i t i a l  s e n s i t i v e , and  undertaking a b i n d i n g or a new  criterion,  However, i n tissue led  of whether or a physiological  important t h i n g i n attempt-  i d e n t i f i c a t i o n i s t h a t f a i l u r e to meet o n l y  even when a l l others  on the i d e n t i f i c a t i o n . binding  receptors.  can a l l too e a s i l y be  The  are met,  can  with the development of new  fulfilled,  one  cast severe doubt  A l s o , even when a l l c r i t e r i a  study appear t o be  the p o s s i b i l i t y  f o r the remains,  c l a s s e s o f more s p e c i f i c drugs  -6-  one  neurotransmitter  s i t e r e a l l y represents  or a p h a r m a c o l o g i c a l r e c e p t o r . receptor  avail-  s p e c i f i c procedures,  i n s u f f i c i e n t a t t e n t i o n t o the q u e s t i o n  not a p a r t i c u l a r b i n d i n g  ing  Thus with the  study, e s p e c i a l l y i n v o l v i n g a new  r a d i o a c t i v e l i g a n d , one  a s t r a y by  pharmacologically  or r e c o g n i t i o n stage o f  or drug i n t e r a c t i o n with b i o l o g i c a l  and  and  sophisticated disprove  the  technology for  techniques,  that  identification.  there  are  a number  of  2.  Presence  3.  Rapid  4.  Specificity.  of  high  binding  1.3.1  a specific  later  light  of  present  must  be  fulfilled  receptor.  kinetics. The  more  be  potent  topics  that  the  a set  of  and  For  in and  tubes the  of  following  only  for  distinct  analogues. L-isomers  Binding  the  the  s i m p l e way and  of the  containing  amount  saturable  of  the  ligands  range  -7-  [30, to  of  used  usually  drug  site  193  be sites  sites  to  study  refers  or  are  binding  to  a  dis-  lower.  demonstrating  the  1.3.2  affinity"  in  saturability  non-radioactive  tissue.If  radioactivity  binding  to  binding  number  "high  term  nanomolar  1.3.1  references  a finite  reversible  l a b e l l e d drug  lowers  reversible  be  receptors,  constant  a  D-isomers.  Sections  What c o n s t i t u t e s  on c o n t e x t .  add  the  in  requirement  there  A common  the  hormone  and K i n e t i c s  presented  discussed in  affinity.  sociation  addition  exhibit  Experiments  A minimum  is  than  of  may  s t e r e o s p e c i f i c , the  binding  neurotransmitter  to  site  must  224].  depends  binding  The  extensively  high  sites.  a set  Saturation  specific  binding  towards  The  form  the  c r i t e r i a that  affinity  Saturability  to  in  will  specificity  being  200,  data  Saturability  5.  is  However,  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 of 1.  of  additional  the  bound,  exists.  latter then  Since  a  there  is  a very l i m i t e d number o f 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 on most  t i s s u e membranes, one should be able t o r e a d i l y s a t u r a t e the number o f d i s p l a c e a b l e b i n d i n g s i t e s .  T h i s i s examined by  measuring the b i n d i n g 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 r a d i o l i g a n d i n t h e presence o f a f i x e d c o n c e n t r a t i o n o f unlabelled ligand. constant  When the d i s p l a c e a b l e b i n d i n g becomes  as a f u n c t i o n o f r a d i o l i g a n d c o n c e n t r a t i o n , s a t u r a t i o n  is indicated.  In a p l o t between l i g a n d c o n c e n t r a t i o n s  and t h e  f r a c t i o n bound, i t i s i n d i c a t e d by t h e p l a t e a u i n g o f the s p e c i f i c b i n d i n g component. Although t h e measurement o f b i n d i n g i s s t r a i g h t f o r w a r d , the i n t e r p r e t a t i o n o f data r e q u i r e s mathematical a n a l y s i s o f t h e experimental  data and c o n v e r s i o n o f b i n d i n g parameters t o t h e  Langmuir isotherm.  The equation  i s d e r i v e d by the a p p l i c a t i o n  of law o f mass a c t i o n t o drug r e c e p t o r 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] r  i  a  =  where K i s t h e a s s o c i a t i o n constant  c  :[• RL ] The  above equation  receptors  can be expressed  i n terms o f f r a c t i o n o f  (r) occupied by t h e l a b e l l e d l i g a n d .  [RL]+[R] = B  B m a x  max t o t a l receptor concentration L = Ligand c o n c e n t r a t i o n R = Unoccupied r e c e p t o r s RL= Ligand occupied r e c e p t o r s .  -8-  =  multiplying  by  [L]  [RL]  [L]  + [R]  [RL]  [L]  +  or  [L]  RL  ; R L .}  [ R L ] ( [ L ]+ Kd  = Bmax[L]  £R] ^ )  =  B  M  = A  X  B  max  [ L ]  [ L ]  [RL]=-max [L]+Kd  or  [RL] _ [ L ] Bmax [L]+Kd  _  1" 1+Kd  where  [T]  Which  i s -a h y p e r b o l i c  Langmuir The  a saturation  clearly curve  specifically.  Thus  and i s a l s o  This  explains  i f the ligand equation  as t h e  to f a c i l i t a t e  i s binding  behind  the c a l c u l a t i o n of B  [L']+Kd or + [RL]Kd = Bmax[L]  -9-  obtaining  to the receptors  can be transformed  = v*  [RL][L]  known  the rational  [L]  [RL]  i s f r a c t i o n of t o t a l r e c e p t o r s o c c u p i e d by the r a d i o l i g a n d .  isotherm.  equation  function  curve  [RL] Bmax  m a x  to a  linear  and K d .  Dividing [ R L ]  by [ L ] +  [ R L ] .  K  =  D  B  M  A  X  [ L ]  or [ R L ]  max  B  =  I L T  Since the  ~  K  in  the  ligand,  [RL]  gives  3  _  =  [ R L ] K  D  of  B  +  M  A  K  D  studies  X  D  i t  l a b e l l e d ligand which  and one  free  R L  ligand binding  fraction  receptors  C  already  a plot *  of  a straight  knows  Bound Free  the  line with  is  quite  a slope  of - -  B  max«  This  is  neurotransmitter classic  mass  binding  studies,  represents  receptor-ligand  action  laws,  has  yielded  negative  cooperative  particularly agonist.  common  if  plot  behavior.  competing  agent  binding  sequential  site  one). the  a given  behavior,  In  so t h a t  ligand-binding  with the  to  above the  equation.  -10-  intercerpt Although  studies  radioligand  most  follow  from  its  generally  apparent  Hill  slope  plot  behavior  i s between  detect equation, labelled  systems  model,  bound  and X  anomalous  competition  the  competes  (i.e.,  This  c a n be u s e d  to  neurotransmitter-receptor-  interactions  than  anomalous  one  anomalous  less  Hill  of  bound  d  interaction  i n many  displacement  receptor  significantly  a Scatchard  measure  of  specifically  o  to  specifically  K  a s  easy  concentration  _t  [RL] [L]  is  and  as  is  antagonist  "quantitate"  suppose ligand at  c o u l d be adapted  that  such a  more to  and  than a  dl j=±  K  R  + L  -  (R  L)1  ^2 (R _ L )  (R  -  L)1  + L  4  (R  -  L)2  + L  K t 4 ^ ( R-  -  (R so t h a t B  _ _ JD —  the  y  L)n_1  n=theoretical  receptor the  the  net  c a n be  written  ligand-binding  number  molecule,  that  of  Ligand receptor complex a t s i t e 2.  as  the  fLln = —*— J t . l r T ,n Kd+[LJ  max  Hill yield  B  [ L ]J n  = BKj d  (B  -B)  max1  x  binding  constant  max B  =  is  the  be  transformed  may  ^  -B)  line.  Hill  Kd  per  composed  of  interaction is  altered  equation ^  Thus  , „1 K d -B)  1  + ,  which  sites  this  yields  B[L]n  = log  and  In  step.  which  (B h log[L] = - l o g — ^ (B  to  equation  a straight +  degree  (K^)  above  equation  to  the  isotherm.  ligand-binding  = a composite  determine  each d i s c r e t e  Rearranging  log  =  n  i n t r i n s i c d i s s o c i a t i o n constant  factors  [L L ]J n  equation  )  .^.  E  Kj-+[L]n  model  The  L  _  [L]n  max  describes  B = B  ( R  SdH-(R _  Scatchard  which  at  L)3  + L  = Ligand receptor complex a t s i t e 1  (R -  2  -  log  n log  -11-  [L]  to  a logarithmic  form  B A graph Log  of  K,  log ^  is  ^ v e r s u s l o g [L] is a straight line. max B a b c i s s a v a l u e where l o g 7= 0 < max~ }  the  T  a  Thus The  B  n log slope of  n~ - c a l l e d plot  the  the  where  blank)  line  Hill  with  The  coefficient  (nH)-. or  concentration  A  similar  competition  of  in .  the The  radioligand absence data  are  binding  of  experi-  radiolabelled  increasing concentrations  specific  as B  gives  in displacement  occurring  taken  log  a fixed  incubated  ligands.  is  J. =  c a n be u s e d  ments is  [ L  B  any  then  of  unlabelled  (total  as  log  [displacer]  = 1.0,  the  system  system  is  positively  system  is  either  heterogenous plot data, give  set  is widely often rise  to  experimental  the  is  to  yield  the  Hill  non-cooperative; cooperative,  when  a n d when  negatively  cooperative  of  sites.  used  binding to  analyze  transformation  problems,  the of  especially  error.  -12-  plot.  or  When  the  n^Cl.O, n.  the  represents the  radioligand  if  data  a  Scatchard  binding  Scatchard the  nH  njj>1.0,  Although  the  agents log — B^ -B max  m a x  versus  minus  displacing  plotted  ligand  equation  contain  an  may  This e r r o r i n c r e a s e s d r a m a t i c a l l y at low b i n d i n g l e v e l s . such circumstances,  the Scatchard  p l o t may  l i n e a r and t h i s c o u l d g i v e the impression b i n d i n g to m u l t i p l e  sites.  1.3.2  or L a b e l l e d Ligand  Competition By performing  displacement  Under  appear t o be nont h a t the l i g a n d i s  Displacement Experiments  experiments one  can  accumulate  a g r e a t d e a l of a d d i t i o n a l supplementary i n f o r m a t i o n about 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 l i g a n d t o i t s r e c e p t o r s .  This  i s p a r t i c u l a r l y t r u e i n cases where the d i r e c t measurement of l i g a n d b i n d i n g to a r e c e p t o r i s not f e a s i b l e because the a f f i n i t y of the l i g a n d f o r r e c e p t o r i s low and  the maximal  r e c e p t o r c o n c e n t r a t i o n a c c e s s i b l e i n v i t r o i s a l s o low. i f one would l i k e t o measure the a f f i n i t y  o f a low  Thus,  affinity  l i g a n d to a r e c e p t o r , i t can be done by measuring i t s a b i l i t y displace a 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.  b a s i c p r i n c i p l e of displacement a n a l y s i s of c o m p e t i t i v e  the low a f f i n i t y  experiments i s d e r i v e d from  the h i g h a f f i n i t y r a d i o a c t i v e l y  1^ with the d i s s o c i a t i o n constant  and  a g o n i s t H with the d i s s o c i a t i o n constant  then [Rl  [R ] T  = CR] + C R l i ] = CR]  The  antagonism, i n drug r e c e p t o r r e a c t i o n s .  Thus i f the r e c e p t o r binds l a b e l l e d antagonist  to  [111  + —  CR]  + Cij.] -  CR]  Cij]  C  R I  i]  Ki  -13-  = [Rii]  KJJ,  Where [ I i ] i s the c o n c e n t r a t i o n o f f r e e r a d i o a c t i v e l y  labelled  a n t a g o n i s t and [ftp] the t o t a l r e c e p t o r c o n c e n t r a t i o n which equals the sum o f f r e e r e c e p t o r c o n c e n t r a t i o n [R] and the a n t a g o n i s t bound r e c e p t o r [RI^] when H i s absent. circumstance [Rli]  the f r a c t i o n o f occupied  Under such  r e c e p t o r s ( r ) i s equal t o  _ [IiVKi Ki  I f the low a f f i n i t y l i g a n d H i s present, t h e r e a r e two a n t a g o n i s t s , one low a f f i n i t y a n t a g o n i s t H, and the second h i g h a f f i n i t y l a b e l l e d a n t a g o n i s t competing f o r the same r e c e p t o r sites .  One o f t h e important  parameters, i n assays i n v o l v i n g  competition experiments, i s the c a l c u l a t i o n o f I C 5 0  values  (the c o n c e n t r a t i o n o f a n o n - r a d i o a c t i v e drug t h a t i n h i b i t s 50% of the r a d i o a c t i v e drug's b i n d i n g t o a s p e c i f i c s i t e ) which can be used t o c a l c u l a t e the apparent e q u i l i b r i u m d i s s o c i a t i o n constant o f the n o n - r a d i o a c t i v e drug f o r the r a d i o a c t i v e b i n d i n g site.  I t i s assumed t h a t a t I C 5 0 value o n e - h a l f o f the  b i n d i n g s i t e s o r i g i n a l l y occupied by the r a d i o a c t i v e drug a r e occupied by the n o n - r a d i o a c t i v e drug, the K  d  and i t c o u l d be taken as  o f the n o n - r a d i o a c t i v e drug. T h i s d e f i n i t i o n a p p l i e s  o n l y when the drug i s f r e e t o i n t e r a c t with the r e c e p t o r i n t h e absence of any o t h e r i n t e r f e r i n g drug.  In the case o f  competition experiments, t h e r e i s always r a d i o a c t i v e drug present The  i n c o n c e n t r a t i o n s s u f f i c i e n t t o b i n d t o the r e c e p t o r .  r a d i o a c t i v e drug i n t e r f e r e s with n o n - r a d i o a c t i v e drug  -14-  binding with  just  as  non-radioactive  radioactive  equation  drug  assumes  binding  a new v a l u e  drug [29,  as  binding  200,  224  interferes  ].  2  [I ]  radioactive under  the  receptor these  ligand  new  2  r 1  t I  ~  T  the  with the  1+  the  2  a  tions  of  receptor-ligand  and  I  t^  L  [RH] low  is  of  free  complex  respectively  concentration  affinity  ligand.  receptor  given  ^s  the  the  of  the  Under  o c c u p i e d by  the  by:  i  ] / K  2  c o n c e n  fraction  antagonist ;  ~ [R ]  ^ie  t  situation,  conditions,  [RI ]  a r e  and  complexed  radioactive  +  K  [R*2^  *  a n c  2  above  [R][H] ^  9  Where  the  follows:  [R][I ] [ R ] = [R] + [ R I ] + [RH] = [R] + — T  Thus  [ I ] [H] 2  +  —  Dividing  r  1  by  r  gives  I. r1  K. 1 1 ^ K.  r  I2  in  the  9  2 ^ ^ ^ i ^ K  I  concentrations  gives  A  At  the  active  ligand  of  above  the  plot  a typical  ment.  the  in  of  is  equation  and  in  curve  seen  where  d i s p l a c e d by gives  -15-  a  the  as a f u n c t i o n  concentration I2  t*2]  a n d  absence  s.igmoidal  equation  the  of  fraction  occupied  H.  of —  displacement  gives  H  presence  pectively.  This  [I ]  f  r  e  e  a n t a c  [H]  or  of log  competition  50% o f H,  e  antagonist  presence  of  in  r  by  ~^-=  the |  receptors  bound and  J H  o n  ist  res[H]  experiradio-  rearrangement  0 = Yi—j  5  [ H ]  K  H  ~[I~  T  ]  Since  [ H  The above e q u a t i o n K  1 + r  r  l  =  i n general  'total  [ I ] or  = tHlfree  =  [  H  [H]  ]  becomes  i  i [ l ] K  2  K.  [H]~ K  H  =  1 [H] I+:K  H  (  1  +  [ I  2  3  }  K.  l At  IC50 when 50% o f t h e l i g a n d  equation  H  K  = T  [ H ] , the  by  becomes  0  [ H ]  i s displaced  5  T  ^  K.  +  ™  ^0.5 50 K „ = K , of n o n - l a b e l l e d H d . . . antagonist K . = K , of l a b e l l e d d -m drug I = concentration of l a b e l l e d drug  m  =  1  I  I C  1  2  The above e q u a t i o n between K  D  apparent  indicates  of non-labelled  of l a b e l l e d drug u s i n g Even a f t e r  identified  a very  Scatchard  receptors  receptors ticular  exists  site.  calculated  binding into  some o f t h e s e  are:  -16-  assays  there are  consideration  a p a r t i c u l a r c l a s s of  f o r a certain neurotransmitter  F o r example,  from  successfully  be t a k e n  that  relationship  analysis.  ligands  must  b e f o r e one c a n s a y t e n t a t i v e l y  drug  have b e e n  using.radio-labelled  a number o f p a r a m e t e r s t h a t  important  at a par-  considerations  (i)  Distribution There  are  two  respect: istics  of  Binding  Sites  questions  which  First,  whether  i s present  neurotransmitter  are  known  to  is  receptors.  tional  to  It  is  be p r e s e n t  relevance  to  exist.  less at  the  the  characterthe  whether  pharmacological  to  s i t e s where  response  for  Secondly,  stimulation  likely  this  receptors  p h y s i o l o g i c a l or  associated with  in  similar  s i t e s where  parameter  receptors  of  other  functional,  of  binding  important  at  a certain  ade  are  or  find it  block-  a class  has  no  of  funcr-  associated with  that  tissue. (ii)  Density The  of  density  peripheral tissue [184,  (iii)  Receptors of  adrenergic  receptors  tissues generally  weight 223].  has  been  density,  a c c o m p a n i e d by  suggests  binding  to  in brain  and  tend t o f a l l w i t h i n 0.1  (approximately It  both  2.5  pmoles/mg  suggested a low  acceptor  that  affinity or  too  in  pmol/mg  protein) large  binding,  non-receptor  a probably  sites.  Saturability Although of  the  sites, been  saturability  been mentioned  minimal c r i t e r i a for examples  shown  specific  that  sites  abound  a n d may  However,  the  several  of  in  s t i l l  this  earlier  s p e c i f i c binding  the  a l i g a n d may  affinity. use  has  literature  to  where  bind  to  show  saturability  problem  acceptor  as  one  receptor it  or  has non-  and  high  c a n be c i r c u m v e n t e d  by  l i g a n d s each 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-  particular binding (iv)  class of receptors  data  i n various  ways.  Quantitative Correlation In o r d e r  to i d e n t i f y  radioligand a specific a  strong  a specific  i n a homogenate type  binding  site  for a  o r membrane f r a c t i o n  of receptor,  i t i s necessary  quantitative correlation  affinities  of a s e r i e s of agonists  between  the value  studies  and t h a t d e t e r m i n e d by p h a r m a c o l o g i c a l  [132].  F o r example, e x c e l l e n t c o r r e l a t i o n s h a v e  as d e t e r m i n e d by r a d i o l i g a n d b i n d i n g  between  i  n  in vitro  evident  cyclase a c t i v i t y  and  for [ H]-dihydroalprenolol  i s more l i k e l y  t o see s u c h as  experiments,  such c o r r e l a t i o n  between  studies been  s t i m u l a t i o n and b l o c k a d e o f ^ - r e c e p t o r -  adenylate  of c o m p e t i t i o n t h o u g h one  of  and a n t a g o n i s t s f o r  sites  stimulated  as  t o show  binding  reported  in vitro  vivo pharmacological (v)  and a n a l y s i s o f t h e  binding  measurements [132],  Al-  correlation may  experiments  n o t be and i n -  experiments.  Stereospecificity Although s t e r e o s p e c i f i c i t y the  most  useful pharmacological  receptor  binding,  absolute  criterion  opiates and  has been mentioned  in identifying  i t does n o t n e c e s s a r i l y p r o v e of receptor  have been shown t o b i n d  cerebrosides  criteria  a s one o f  identification. to glass fiber  stereospecifically  -18-  [114, 1 8 3 ] .  t o be an In  fact,  filters  (vi)  R e l a t i o n s h i p Between B i n d i n g T h e r e a r e a number o f o t h e r the b i n d i n g of a c l a s s agonist  o f r e c e p t o r s may be d i f f e r e n t  d i f f e r e n t l y with  the r e a l  T h e s e and o t h e r Burt,  of receptors  receptors  must be some p h a r m a c o l o g i c a l responsiveness Furchgott,  series  should  In h i s study obtained  of the t i s s u e  of ^-adrenergic  of r a b b i t , t h o r a c i c  aorta  dihydroergocryptine  smooth m u s c l e .  points  out that  in  -19-  of t h i s  r e c e p t o r s on  strips  pharmacological  by t h e b i n d i n g o f [ H ] -  However, T a l l a r i d a ,  many  binding  was  t o membrane f r a c t i o n s  uterus  of a  testing.  as d e t e r m i n e d by  methods and t h a t d e t e r m i n e d  from  the a f f i n i t i e s  an e x c e l l e n t c o r r e l a t i o n  between  F o r example,  obtained  d e t e r m i n e d by p h a r m a c o l o g i c a l  the  there  that the a f f i n i t i e s  and a n t a g o n i s t s  c o r r e l a t e with  discussed Finally,  i n a tissue,  to the neurotransmitter.  of agonists  studies  i n question.  s t u d i e s suggest  evidence  1978 [ 6 1 ] s u g g e s t e d  Iti s  t o employ  have been e x t e n s i v e l y  o f whether l i g a n d b i n d i n g  presence of appropriate  series  sites.  1978 [ 2 9 ] and Yamamura, 1978 [ 2 2 4 ] .  regardless  labelled  behave  know what c r i t e r i a  class  criteria  affinities  or i n case o f  c l a s s e s of binding  t h a t one s h o u l d  to estimate  by  affect  for a  a l i g a n d b i n d i n g may  changes i n temperature  binding, with  imperative  f a c t o r s w h i c h may  s t u d i e s - namely a p p a r e n t b i n d i n g  or antagonist;  multiple  and R e s p o n s e  systems,  other  of r a b b i t 1981 [ 1 9 3 ]  than  smooth m u s c l e  affinities Tallarida First  by  theory  based  on  constants  to the presence  assays  constants are not  present  at the  to l i g a n d  assays  situation  of spare  partial  on  of a  the  labelled  becomes more  r e c e p t o r s , i n many  realized  that in  i n both the  same as  the  plot  of dose-response  competitive occupies  antagonist w i l l  equal  167,  210],  from  has  Thirdly,  in  Finally,  some  occur  full  produced of  o n l y when t h e  by  a agonist  receptor population  r e g a r d l e s s of the  binding procedures,  a  assumptions.  i n the presence  involved, dissociation  -20-  con-  pharmacological  responses  p r o p o r t i o n s of the  disadvantages  obtained  a b s e n c e and  dis-  curve using e i t h e r  assumes t h a t e q u a l  a g o n i s t i n the  tissues.  or s e v e r a l other  E a c h o f t h e s e methods  Thus, S c h i l d  complex  the c o n c e n t r a t i o n of  receptor s i t e s .  of a S c h i l d  are  in vitro  a g o n i s t o r a n t a g o n i s t t o compete w i t h a  agonist.  of  pharmacological  to c a l c u l a t e  i n v i v o and  binding assays,  make use  modifications  and  inhibition  t h e c o n c e n t r a t i o n s employed  experiments  60,  behind i t .  obtained pharmacologically  The  i t s h o u l d be  sociation  trast  determine  procedures.  three p o s s i b l e reasons  of c o m p e t i t i v e  responses.  Secondly,  [  p o s s i b l e to  by. a n o n - l a b e l l e d compound. E s t i m a t e s  dissociation  an  be  precise pharmacological  p o i n t s out  substance  drug  not  of a l l , the b i n d i n g s t u d i e s are based  simple  due  i t may  drawbacks  constants  along with  those  obtained  from  .procedures receptor  1.4  in vitro  and i n v i v o  do add t o o u r f u r t h e r  characterization  pharmacological  understanding of  and i d e n t i f i c a t i o n .  The B e t a - a d r e n e r g i c Pathway While  i t i s clear  that  stimulation  o f the beta  a d r e n o c e p t o r s by n o r e p i n e p h r i n e i n brown a d i p o s e principal  stimulus f o r n o n - s h i v e r i n g thermogenesis  the d e t a i l s  adipocytes  The f i r s t  Two a p p r o a c h e s  the c h a r a c t e r i s t i c s pharmacological a r e used  [25],  upon brown  membrane  h a v e b e e n t a k e n t o s t u d y and  of adrenergic receptors.  analysis,  where s e l e c t e d  t o study t h e i r  several biological  responses  membrane a d e n y l a t e c y c l a s e  relative  (e.g., l i p o l y t i c  activity,  (1) C o n v e n t i o n a l  a g o n i s t s and  effects  identify  antagon-  and p o t e n c i e s on effects,  variations  plasma  in cyclic  AMP  o f t h e a d i p 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 t u r n o v e r ) ,  (2) The s e c o n d  approach  techniques to i d e n t i f y The  first  i s t h e use o f r a d i o l i g a n d b i n d i n g and c h a r a c t e r i z e  attempts  a d r e n o c e p t o r s were b a s e d  to identify  putative receptors. fatcell  t o p l a s m a membranes by A p r i l l e ,  and M a r i n e t t i ,  1974 [ 9 8 ] .  due t o t h e f a c t  beta  on t h e s t u d y o f t h e b i n d i n g o f [ H ] -  catecholamines  tioned  (NST)  step i n norepinephrine action  i s i t s b i n d i n g t o a p p r o p r i a t e plasma  receptors.  content  i s the  o f t h e m o l e c u l a r mechanisms a r e y e t t o be c o m p l e t e l y  understood.  ists  tissue  that  1974 [ 4 ] ; K o r e t z  However, t h e s e s t u d i e s h a v e b e e n tritiated  s i m u l t a n e o u s l y b i n d t o a membrane  -21-  catecholamines can  catechol-binding protein  ques-  related  to catechol-O-methyl  selective  beta  transferase.  Availability  the  (-)[  3  HBI,  [24,214].  decribed  ceptors  that  capacity.  from  sub-types. brown  of  brown  Studies  one of h i g h  Rothwell of  into  gardless  by A r c h  ephrine  show  that  protein  lipase.  show  there  between  cyclic  [49],  whereas 4 9  purification  activation  kinase  adenylate  activation  within  indicated that  8,  rat  46, 93]. Re-  through  cyclase,  or linear  o f no r e p o r t s  norepin-  of  studies  hormonealso  relationship  [97].  The  and then  former  declines  f o r a t l e a s t 60 of a s u c c e s s f u l  triglyceride  carried  c y c l i c AMP  and a c t i v a t i o n  elevated  -422-  membranes  b o t h a n d  the reported  of hormone-sensitive  2 0 nM)  reported  a l l the studies  10 t o 20 m i n u t e s  We a r e a w a r e  +  c h a r a c t e r i s t i c s a n d do  and l i p o l y s i s  remaines  (K^ = 1 0 1  i n microsomal  of yg-receptor  Interestingly,  lipolysis  ].  of  adreno-  3 . 5 + 0 . 7 nM)  recently  [8]  of ^-adrenoceptors,  AMP g e n e r a t i o n  t o be maximal  [I  atypical  i s no s t r a i g h t f o r w a r d  tends  minutes  show  [3H]-  of beta  (K^=  contain  e t a l . (1984)  to activation  sensitive that  affinity  p^- a n d fi^- s u b c l a s s i f i c a t i o n [ 7 ,  leads  generation,  states  receptors  plasma  [64], using  low a f f i n i t y  rats  demonstrated  i n rat adipocyte  et a l . [162]  f a t of adult  of the nature  to date  of  adrenergic  fat ^-adrenoceptors  fall  investigators  two a f f i n i t y  and the other  the population  isolated  out  the presence  low capacity,  yS-adrenoceptors  G i u d i c e l l i et a l . (1982a)  in rat fat cells:  and h i g h e r  not  [ H]-DHA v a r i o u s  presence of beta-l-adrenoceptors  membranes  and  Using  highly  3 H]-dihydroalprenolol ('[ H ] - D H A ) 3 (+_) [ H ] - h y d r o x y b e n z y l i s o p r o t e r -  antagonists  and b e t a a d r e n e r g i c a g o n i s t 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 3 selectively.  of  lipase  from  brown  adipose  tissue.  AMP-dependent  protein  fat  has  so f a r  been  concentration  ratio"  and  brown or  fat  fatty  rate  has  been  acids.  is  or  1.5  adrenergic  consumption  or  if  the  and  the  protein  kinase  [177],  Lipolysis  r e l e a s e of cases the  either  released  fatty  the  acids  the  "activity in  glycerol  lipolysis of  brown  could  glycerol are  oxi-  brown  role  of  by  phentolamine  in  fat  cell  in  the  l i p o l y s i s and  early  as  1977  effective This  cause  whereas  70%  and  [85].  the  -23-  was  a  suggested  Selective could  in  depolarize was  eliciting  sensitive  c<-adrenergic  i n h i b i t i o n of  [84].  oxygen  L--phenylephrine  was n o t  a complete  depolarization  receptor  norepinephrine  depolarization  propranolol, could  than  in  thermogenesis,  agonists  [55].  of  plays  increase  e<-adrenergic  membrane  activation  adipocytes  oC- o r ^ - a d r e n e r g i c  depolarization  -rine-induced  of as  fat  part  subsequently  be more  inhibition  pathway  i n brown  and H o r o w i t z  depolarization.  induced  in  a c o r r e l a t i o n between  AMP,  i n both  a dominating  concentrations  to  e.g.,  the  cyclic  lipolytic activity  norepinephrine-mediated  additional  by H o r w i t z  shown  by  l i n k i n g the  significant proportions  receptors  quantitatively  rat  the  glycerol release  Alpha-adrenergic  possible  to  cyclic  assayed  if  evidence  re-esterified.  Although  the  of of  rephosphorylated  dised  kinase  However,  be u n d e r e s t i m a t e d  only  indirect,  tissue  the  The  to  antagonist  norepinephrine-  i n h i b i t i o n of  A possible  link  phenylephbetween  ot-receptor  activation,  Na  transmembrane  transport,  and r e -  2+ distribution Nedergaard  of  and L i n d b e r g  [^H]-prazosin fat be  [127]  i n t r a c e l l u l a r Ca  to crude  and r a t brown  o f oC^  [138].  Direct  membrane fat  radioligand  fractions  [154]  by  showed  from  binding  hamster  of  brown  the e^-receptors  to  sub-type.  Mohell  et  a l . [128]  also  adrenergically  mediated  total  heat  production  this  receptor  measured  stimulation  of  characteristic However,  was p r o p o s e d  process  i n brown class  an  alpha-  about  fat  the  the  associated  response"  with  [129].  alpha-l-mediated  enhancement  20% t o  and t h a t  had been  "phosphatidylinositol  turnover  that  contributed  a r e l a t i o n s h i p between  idylinositol  showed  and i n c r e a s e  phosphatin  cytosolic  2+ Ca  i s y e t t o be f u l l y  conclusion  c a n be drawn  cellular  events  membrane  associated with Skala  brown  fat  activity  cyclic  steady-state activity stages periods  of  of of  suggested  and Knight  [ 178]  cyclic  cyclic  brown high  of  of  in vivo link  GMP p r o d u c t i o n protein  cyclic  GMP-dependent  f a t development  the cell  protein  studies  in rat  b e t w e e n c<-  and i n c r e a s e d  kinase.  GMP a n d t h e  seemed  between  across the  kinase  Since  the  high  increased during  to correspond  proliferative  activity  of  t h a t oC-adrenoceptor  mediated  stimulation  -24-  final  e*^-receptors.  a possible  GMP-dependent  concentration  of a message  in their  of  no  the relationship  stimulation  evidence  stimulation,  of  concerning  Therefore,  and the t r a n s m i s s i o n  presented  adrenergic  understood.  the tissue, of  early  with i t was cyclic  GMP  system might maturation cyclase  have  of  Ca  ted is  also  [152].  [126]  systems  or  suggested  as  about  the  of  regulation  such a l i n k  Exton have  [40],  in  [47],  a feedback  [143],  the  by  studies  spleen c e l l s  also  in  indirectly via  processes  c e l l u l a r response available  a role  existence  Several  and and  The  directly  developmental  atocytes some  was  + +  and P o i n t e r the  tissue.  activation  cellular  in  been p l a y i n g  the  regulator very  cyclase is modulated.  The  most  to  levels  fractions. in various  tissues,  soluble  and p a r t i c u l a t e  similar  proteins. Reports  in  other  role  in  [178]  lines  trophic  effect  existed  receptors  of  the  of  norepinephrine the  diverged in  the  affinities  the  its  component  25-  in  media-  above,  from  soluble  and  or  cyclic  [143].  part-  the  the represent  GMP  Skala  link  system  in  trophic The  Knight  addition  Thus  the  effect  the  to  possof  phenotype  relative  determined  and  between  fat.  level.  GMP  regulatory  system  the  not  cyclase  and  on brown  p l a s m a membrane, of  homogenate  a possible  receptor  hep-  information  a possible  c a l o r i g e n i c and at  of  few m e n t i o n e d  guanylate  indicate  presence  GMP  receptor  whether  and d i f f e r e n t i a t i o n  the  that  adrenoceptors and  i s unknown  a c t i v i t i e s of  seemed t o  [171]  can increase c y c l i c  system and © ( - a d r e n e r g i c r e c e p t o r  norepinephrine  tion  it  of  cyclase in  many h o r m o n e s  -adrenergic  suggested  GMP  ibility  the  on  t i s s u e growth  had  cyclic the  cell  guanylate  Although  Schultz  l i t t l e  by w h i c h g u a n y l a t e  iculate  intra-  proliferation  than  have  in  implicated cyclic  other  t i s s u e s tends  guanylate  as a second messenger  However,  mechanisms,  to  changes  [52],  of  propor effect  of  of norepinephrine on brown fat.  It, therefore, seemed appropriate  to search for the presence of o( 2~ eceptors in actively prolir  ferating brown adipose tissue of infant rats and provide additional support by in vitro experiments for the proposed link between the cyclic GMP system and the ^-adrenergic system. We have used several approaches in an attempt to demonstrate 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^-adrenoceptors in isolated membrane fragments were studies following chem-. ical sympathectomy and chronic blockade of o< ^adrenoceptors by yohimbine.  (iii)  Experiments were carried out to test the poss-  i b i l i t y that cyclic GMP participates in the o< -adrenergic effector 2  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 first 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< -ligands. 2  (ii) In the second part we approached the problem in an indirect way.  It has been shown that the particular class of recep-  -26-  tors set  c a n be "up" o r  of  "down"  regulated  physiologic or pathologic  based on the i n s u l i n  receptor  adrenergic  receptors  [215,  drugs  the a b i l i t y  have  tors  in tissues.  that  of  active  Hormone o r d r u g appear to  t h e hormone  it  has been  titating [34].  shown  Studies  receptors  that  of  [172]  and  of  i n receptor  their  own r e c e p -  has been observed  the ambient  concentration  i t s receptors number  responsiveness  and  is  of  [194].  affinity  or  sensitivity of  rabb-  to cause p o s t - j u n c t i o n a l  super-sensitivity  of  nic-  [202]  tissue  and c a r d i o v a s c u l a r of  frog  erythrocytes  to s i g n i f i c a n t l y reduce  rats  particular  pre-treatment  receptors  Similarly,  pattern  i n tissue  a  t h a t a w i d e v a r i e t y o f hormones and  For example,reserpine  exposure  shown  healthy  caused  changes  upon  the tissue.  dopamine  a n d t h e number  changes  as w e l l .  Chronic  adrenergic into  molecule  of  the density  r e l a t i o n between  membrane  has been  to regulate  related  to mediate  67]  showed  In general,the  an i n v e r s e  biological  states  [62,  216]  depending  [123].  resulted  in vitro  to  to  (-)-isoproterenol  t h e number  Pheochromocytomas  norepinephrine  of  functional  placed  interscapularly  i n diminished B-adrenoceptor  incubation  of  a concentration-dependent  platelet decrease  membranes  beta-  number  with  [182].  clonidine  of ^ - a d r e n o c e p t o r  binding  3 sites vation  for  [ H]-yohimbine  has been  c^-adrenergic  purely [157].  alpha  of  i s beta  [185] ,  in cardiacc^-  epinephrine  i n nature  In contrast,  epinephrine  i n rat brain  was r e p o r t e d  The e f f e c t  On t h e o t h e r  t o be a s s o c i a t e d w i t h  receptors  sympathectomy 222].  shown  [19].  hand,  and a s i m i l a r  [124]  effect  and jB-adrenoceptors  on estrogen  the progesterone  due t o a d e c r e a s e  dener-  a n i n c r e a s e d number  primed  due t o an i n c r e a s e d d e n s i t y  during  sympathetic  phase  rabbit of  of of  [221,  Uterus i s  «x-receptors  the effect  i n the density  of  of o(-rec-  eptors  [215]. Chronic  associated with blockade in  rat  of  blockade  beta-adrenergic  an i n c r e a s e i n  dopaminergic  brain  of  has  been  receptor  receptors  shown  to  by  lead  number  the  to  receptors [48,  is  also  65].  antagonist  Chronic  haloperidol  an i n c r e a s e d number  of  dop-  3 aminergic  receptors  Mukherjee  et  al.  occupancy  of  receptor  ness  of  the  if  chemical  be  then  perhaps  seen i n  We,  enoceptors problem.  brown  sites  lead  to  from  in  form of  of  brown  number.  1)  or  other  tissues,  changes  Two  Chemical using  [11,  infant  approaches  has been  if  were of  on i n f a n t  changes.  such  sympathetic  in  to  nerve  Administration  involutory  were  exposed  to  daily  a hormone  -28-  to  of  the  this  a  single  nerve  ter-  changes  in  administration  receptor  and/or  terminals  second s e r i e s of  chronically block  will  ^"adr-  study  in  rats  This  experimental  changes  undertaken  the  by  affinity  and r e s u l t In  brown  either  destroy  2)  suggested  ^2-adrenoceptors  adaptive  study  the  responsive-  above,  to  198].  of  of  in  well.  shown  197,  to  the  environment  associated with  176,  Binding  present  tissue  6-hydroxydopamine.  (an ^ - a n t a g o n i s t ) iii)  decided to  axons  in  in ^-adrenoceptors  sympathectomy  6-hydroxydopamine  fat  of  were  [28].  changes  mentioned  chronic blockade  was  that  alterations  compensatory  fat  binding  a m p l i f i c a t i o n system as  modulation  and p r e - t e r m i n a l  iments, imbine  demonstrated  therefore,  performed  minals  any  result  the  manipulation  dose of  have  sympathectomy  density.  was  131]  mediated  results  [ H]-haloperidol  functional ^-adrenoceptors  adipocytes,  would  [13 0 ,  receptor  These that  as measured  of  experyoh-  the © ^ - a d r e n o c e p t o r s . sites  is  not  suff-  icient to e l i c i t specific cellular responses. The hormonal sig^ nal can be deciphered only when passed on via an effector system(s). In the third part of our study, experiments were designed 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) whether 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 testing the activation of cyclic GMP-dependent protein kinase. An ^activation ratio" assay similar to that reported for cyclic AMPdependent protein kinase [177] could not be adopted for the nondissociating cyclic GMP-dependent enzyme. Instead, an alternative approach, a modification [151] of the Dufau et al.[51] technique, 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 GMPdependent protein kinase is the only protein known to specifically bind i t s cyclic GMP inside a c e l l . Therefore, the absolute amount of protein-bound cyclic GMP was considered to be indicative of the activated enzyme.  -29-  (iv) Although ligand binding is used to sub-classify adrenergic receptors into their sub-types, i t is always complementary 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 referred 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 monocytes, although not producing a biological response, is quantitatively reflective of the degree of insulin resistance and hyperinsulinemia of the host. Presence of c( -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 receptor 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): guanine-nucleotide-binding protein complex (N) and the catalytic moiety 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 attenuate cyclic AMP levels through i t s interaction with the guanine2  -30-  nucleotide-binding is tus  protein  f a c i l i t a t e d by of  * 2  P  - r e c e  seven-day-old hibitory  GTP.  rats,  effect  (sub-maximal  lic  of  AMP  a n ADP  inactivation  if  e*2~  r e c e  from brown in  t o r  fat  al.  basis  [87] , of  and  ity  77].  [74,  bolic  41  experiments  12,  these ted the  animals  [201].  32,  result  A number  73,  m o l e c u l a r mechanism  sta-  tissue  studies  reduce  shown of  Ni  were  or  by  that  inhibitory  on  of  the  in-  abolish  decrease this  [18]  the.,  in  cyc-  results  from  leading  to  its  input  to  adenylate  c a r r i e d out  to  investigate  of  lipolysis  was m e d i a t e d  obese  used  in by  adipocytes  Ni  118,  model  in  a wide  175,  as  investigators the  studies  studies  variety  and  on  reported  which have  is  by  Hummel  metabolic  thermogenic  on  diabetic  endocrine  increased  226].  metabolic  -31-  of  mice  hyperinsulinemia  188,  a mutation  of  in  (ob/ob)  receptor-mediated  hyperlipidemia  from of  C  forskolin-stimulated  mediated  hyperglycemia,  to  N and  functional  adipose  on  to  genetically  present  hyperphagia,  [6,  are  rats  regulation,  resistance, cold  shown  have been w i d e l y  such as  brown  of  fat.  of  mice  the  additional  inhibition  as an e x p e r i m e n t a l  defects,  from  component  one-week-old  The  examine  been  preventing  discovery they  has  kDa  stimulated  body w e i g h t  response  It  from white  Since  been  actions  .  thus  of  adipocytes  (v) et  and  binding  lipolysis.  has  of  The  (clonidine)  s  whose  s e r i e s of P  on  toxin  ribosylation  to  c a r r i e d out  stimulation)  [37]  (N).  adipocytes  n  a<  hormones  A  order  °<2~ 3 i- t  formation  cyclase.  In  we  of  Pertussis effect  i-  t o r s  complex  obesor  with  insulin  sensitivity  These d i s o r d e r s recessively  attempted defect  to  met-  in  inheri-  elucidate  i n obese  mice  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 mitchondria [68, 69, 75, 82].  Several studies [14, 15, 44, 106] i n -  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 indicating 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 lipolytic 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. -32-  We stud-  i e d t h i s problem i n obese and lean mice to see i f c y c l i c GMP concentrations  i n brown f a t were i n f l u e n c e d by c o l d exposure  and  stimulation.  Preceptor  1.6  Finally,  the main objectives  o f t h i s study can be summ-  a r i z e d as f o l l o w s : (i)  To i d e n t i f y and c h a r a c t e r i z e i n brown f a t o f developing  (ii)  °<2 brenergic -ac  receptors  rats,  To g a i n a d d i t i o n a l i n f o r m a t i o n  about the r e c e p t o r  state  as a f u n c t i o n o f modulation o f i t s environment (e.g. changes i n d e n s i t y and a f f i n i t y r e s u l t i n g from denervat i o n o r c h r o n i c blockade o f r e c e p t o r s ) . (iii)  To show t h a t <=< -receptors are indeed f u n c t i o n a l 2  i.ei  they show i n h i b i t o r y e f f e c t on /B-adrenergic  lated  g l y c e r o l r e l e a s e as r e p o r t e d  i n adipocytes  receptors stimufrom  white f a t i n human, hamsters and r a t s , (iv)  To show t h a t  <*2  - r e c e  P  t o r s  a  r  e  a l s o i n v o l v e d i n some  y e t u n i d e n t i f i e d way i n a c t i v a t i o n o f the g u a n y l a t e cyclase (v)  system.  To c a r r y o u t p r e l i m i n a r y s t u d i e s t o i n v e s t i g a t e i f c y c l i c GMP c o n c e n t r a t i o n s  i n brown f a t o f obese mice a r e  i n f l u e n c e d by c o l d exposure o r ^ - r e c e p t o r  -33-  stimulation.  CHAPTER  2  M A T E R I A L S AND METHODS  2.1  Materials Clonidine  hydrochloride,  1-norepinephrine, dopamine, tol  were  MO.  H  a l l obtained  from Sigma C h e m i c a l Company,  St.  w  a  s  r  Ont.,  Forskolin  chem,  San D i e g o ,  c  e  v  CA.,  e  MA),  and were  of  were  3 [ H] those  grade  and M e t o p r o l o l were For c y c l i c Nuclear  provided  Liqands:  H(N)]-norepinephrine  dihydroalprenolol  filters  were  (N.J.  Bedford,  l-[ring,  ring-  H]-clonidine  [Methyl->3H]-yohimbine  (75^0 -  Ci/mmol);  — propyl-  (61.1 C i / m m o l ) ;  80.5 Ci/mmol);  - 3 4 -  (pore  Corporation.  ( 3 9 . 7 and 104.8 Ci/mmol);  hydrochloride  a l l were  H]  MA.  3 [Benzene  3  USA)  f r o m Whatman (18.8  [  Other  ( 4 8 . 4 and 5 2 . 9 Ci/mmol);  (79.5 Ci/mmol);  hydrochloride  and c y c l i c  HAWP M i l l i p o r e F i l t e r s  fiber  H]-rauwolscine  cyclic  i n NEN R I A k i t s .  GF/C g l a n m i c r o  3  s u p p l i e d by  GMP a n d  S c i e n t i f i c Company  purity.  Calbio-  R I A k i t was u s e d  from M i l l i p o r e C o r p o r a t i o n ,  l-[7-  Mississauga,  purchased from  AMP ( 3 1 . 3 C i / m m o l e )  l-[ring]2,5,6-3H]-norepinephrine [Methyl-  were  Louis,  collagenase  Biochemical,  pharmacy.  from F i s h e r  reagent  0 . 4 5 ^m) were  Dobutamine  t h e New E n g l a n d  used were  as a g i f t ;  from Worthington  cyclic  Ci/mmole)  chemicals  from Hassle  d  Hospital  determinations,  Whatman  i  and P e r t u s s i s t o x i n  General  (NEN-Boston,  size  e  was o b t a i n e d  (9.1  6-hydroxydithiothrei-  35/25  Vancouver  pyrocatechol,  (-)  isoproterenol hydrochloride,  1)  GMP  1-epinephrine,  serum a l b u m i n ,  alprenolol,  (type  AMP  (-)  bovine  . H(N)]-  obtained  from  New  obtained  from  Amersham  2.2  England  [  H]  RX-781094  was  Radiochemicals.  Animals Albino Wistar  at  Nuclear.  room t e m p e r a t u r e  were  fed  dated by per hr  Purina  and  light/  dark  interscapular cessed  as  smears. left  Memorial were  in  were  male or  in  fed  Laboratories,  12  light-dark  hr  ad l i b i t u m . least  one week  carbon moved lary  The  dioxide from  minced w i t h  for  7  days  ob/ob  and  at  5 weeks  at  in  12  hr-12  at  brown and  ages  of  weighing NJ)  kept  The  cells  80 g at  given this  adipose  trimmed  prepared  -35-  hr  of  age.  as  They  light-dark The  mice  16  weeks.  (Charles 25°C w i t h  food  and  schedule then  a  extraneous described  hr-  at  killed  and  12  water  for  t i s s u e was  cervical of  normal  Jackson  approximately  sub-scapular, was  the  libitum.  a n i m a l s were  and brown  fat  ad  kept  and at  from  60 -  were  schedule  a n i m a l s were  narcosis,  and w a t e r  the  the  pro-  their  ME  Chow  and  12/12  section.  obtained  23°C  animals  23° C i n  rapidly  Harbor,  use.  at  was  (BAT)  were  eight  tissue  Lakeview,  scissors  mothers  to  Bar  lighting  The  reduced  kept  c y c l e : , andc.  Pregnancies  were  interscapular,  regions.  light-dark  decapitated  hamsters  before  were  They were  ob/+)  experiments  River  hr  ad l i b i t u m .  C57BL/6J/6J  Purina  Male Golden  hr-12  Methods  i n p l a s t i c cages  and were used  the  throughout.  animals were  adipose  Laboratories,  kept  cycle  (+/+  12  their  c y c l e . _ The  described  used  Newborns  with  brown  controls  in  and w a t e r  Obese m i c e lean  were  ( 2 3 ° C)  Chow  vaginal  litter  rats  by  reaxiltissue, subsequently  To (male  prepare  breeder  and  Laboratories,  cells  from brown  female)  were  obtained  Lakeview,  New  Jersey.  kept  together  for  a week  maintained  at  25°C with  given  and w a t e r  male were  food  separated until  from  infant  remain w i t h  the  their  adipose  2.3.1  For  injected  base  Following  yohimbine  with  They were  2.3.2 rats  were  After  a week  and  a  single  cage.  the  in  They  after  females  separate  They were  were and were  cages  allowed  which  rats  River  l i g h t i n g schedule  and put  days, were  to  they  used as a s o u r c e  pre-treatment,  kept  at  the  sacrificed  for  dissected,  the  dark  killed  weight  and  experiment,  rats.  saline  body  Randomly  served  as  of  on day  8.  rats  were  i.p.)  was  for  For injected 7  days.  21.  preparation: their  trimmed  the  14-day-old  mg/kg body  on day  50 m g / k g  i n t r a p e r i t o n e a l dose  a n i m a l s were  (10  decapitated,  in  5-day-old  injected with  pre-treatment,  yohimbine  -20°C  immediately before  a single  P l a s m a membrane  rapidly  rats)  littermates  6-hydroxydopamine,  daily  7  intraperitoneally into  controls.  chronic  for  [176]  i n 0.9% NaCl  interchanged  females  i n a breeder  delivered.  6-hydroxydopamine  free  dissolved  Two  rat  adult  tissue.  Methods  of  mother  60 y o u n g  2.3  weight  male breeder  tissue,  from Charles  light-dark  ad l i b i t u m .  a n i m a l s were  (approximately brown  12:12  adipose  of  60 -  70  seven-day-old  i n t e r s c a p u l a r brown extraneous -36-  fat  t i s s u e s and  was immersed  in  ice-cold  tinuously (6  -  bubbled  with  bicarbonate  No.  fication  3 silk  cloth  of t h e method  2  scissors  (Nitex  2  .  Pooled  were  A modi-  e t a l . [117]  fragments.  soaked overnight  i n imM EDTA, water  double  before  use.  c a r r i e d o u t a t 0°  finely  minced  homogenization containing  1 mM M g C l 2 ,  homogenized Elvehjem separated the  homogenizer.  3 0 mM N a C l ,  the Teflon  The p r o c e d u r e  was l a y e r e d buffer  over  interfacial  band  o f membranes  pelleted  by c e n t r i f u g a t i o n  centrifugation. oc-receptors, EDTA  washed  membrane  were  7.4  and  in a glass  10 m l o f 4 1 % o f  Potter-  3 times 10 m l o f  sucrose  i n the  a t 9 5 , 0 0 0 x g ( 3 3 , 0 0 0 rpm) The  white  were  a t 9 5 , 0 0 0 x g f o r 20 m i n u t e s .  The  buffer,  using  i d e n t i c a l r e - s u s p e n s i o n and  preparation  t o remove  preparations  pH  i n i c e cold water.  In the case of assays  i n 5 0 mM T r i s  buffer,  and t h e membranes  twice  t h e membrane  of the  w a s c o l l e c t e d i n 15% s u c r o s e  i n the homogenization  were  pestle  ultracentrifuge.  solution  membranes  i n 10 v o l u m e s  was r e p e a t e d  and c e n t r i f u g e d  1 h r i n a Beckman L B - 5 5  - 4°C.  1 mM d i t h i o t h r e i t o l )  by 1 t o 2 m i n . o f c o o l i n g  homogenate  distilled  ( 1 0 nM s o d i u m p h o s p h a t e  by 8 s t r o k e s o f  homogenization for  t i s s u e was s u s p e n d e d  buffer  was  A l l plastic  and r i n s e d t e n t i m e s w i t h  The  fat  through  pH 7 . 0  A l l p r o c e d u r e s were  con-  brown  Co., Switzerland).  d e s c r i b e d by Maeda  tubes  pH 7 . 4  and f i l t e r e d  t o i s o l a t e t h e p l a s m a membrane  and c e l l u l o s e n i t r a t e  buffer,  9 5 % Q> + 5% CO  7 g) was m i n c e d w i t h  Nitex  used  Krebs-Ringer  involving  was r e w a s h e d w i t h  membrane-bound  suspended  -37-  binding  ions  to 2 . 5 mM  and GTP.  i n 5 0 mM T r i s - H C l ,  pH  A l l  7.4  containing^mM  for  binding  contents  2.3.3  Binding thawed  centration enized and  in  0.1  (Brinkman  0 . 5 ml of 3  -  before  glass  mg/ml  MgCl2,  Plasma  membrane  according  of  appropriate  pH 7 . 4 binding  buffer.  above  specific  of  pH 7 . 4 ,  incubation  media  binding  of  experiments.  be  proportional  tration used  over  a range  of  i n a standard  perature  (also  yielding  most  terminated  also were  assay.  ligands  After  by a d d i n g  0 . 8 ml of  -38-  i n 5 0 mM N a -  for  of to  concen-  and t h u s ,  0.08 tem-  experiments  the incubations washing  the  was f o u n d  20 m i n . a t r o o m  ice-cold  10 mM  in a series  protein  0 . 4 mg/ml  results),  and  10 mM M g C l 2 .  the binding  e s t a b l i s h e d by p r e l i m i n a r y consistent  10 mM M g C l 2  t o be o p t i m a l  t h e membrane  0.05 -  of  determined  was a s s a y e d  found  In these, to  incubated  containing  containing  the respective  preliminary directly  pH 7 . 4  [3H]-RX-781094  buffer,  incubation  [ H ] - y o h i m b i n e was  o u t i n 50 mM g l y c y l g l y c i n e , and b i n d i n g  two up  Binding was  containing of  with  were  3 and [ H ] - c l o n i d i n e  buffer,  rehomog-  i n Eppendorf NY)  a l . [116].  to a con-  and g e n t l y  Westbury,  et  preparations  use and d i l u t e d  incubation  -20 C and used  t o Lowry  membrane  samples  Instruments,  ascorbic acid  K-phosphate  was  weeks.  t i s s u e homogenizer  Duplicate  5 0 mM T r i s - H C l  at  4 . 0 mg p r o t e i n / m l  H]-norepinephrine  carried  The  1.6  stored  Aliquots  immediately of  1 - 3  frozen,  determined  Assays:  down s t r o k e s .  (-)-[ in  were  i n a hand-held  tubes  rapidly  assays within  protein  were  MgCl2  as  were  buffer  0 . 2 mg  ( 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 fiber filters. 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 LS9000 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 nonlabelled 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 computer-predicted plot of BCTIC computer program for equilibrium binding assays (Med. 40 and Med.79) was selected as the"representative" 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 oneweek-old rats was pooled, minced with scissors and filtered on Nitex silk f i l t e r 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-  Incubation o Tissue in  a total  pH  7.4,  ine  fragments  volume  of  (.0.1  1 ml of  which contained  serum albumin  0 . 2 g)  were  Krebs-Ringer  half  type V ) ,  -  of  incubated  at  bicarbonate  recommended  Ca  + +  ,  37 C  buffer,  3% B S A  (bov-  5 mM g l u c o s e a n d 1 mM 3 - i s o b u t y l -  methylxanthine. All  samples were  95% C>2 + 5% C 0 2 w i t h yohimbine, min.  in  metoprolol,  Reactions  ice-water  bation  AMP  levels  (1976)[150]  stopped  effects  i n canine used  0.1  by a 3  on N i t e x  the incubation  fragments  silk  from  the  c l o t h under  antagonists  therefore,  slices.  pressure  hypothalamus. considered i t  high concentrations on brown  of fat  in  of  appropriate  fragments.  ( 1 9 8 0)  cyclic  Shartner pressor  associated with In view  slices  norepineph-  and  to block  incu-  suction.  et a l .  increase  Philippu  flasks  antagonize  to antagonize  antagonists  -40-  to  Yamashita  1 mM y o h i m b i n e  i n blood  anterior  agonists  of  on TSH-stimulated  mM t o  studies  of  by immersing  the tissue  thyroid  of  ects  min. followed  1 0 0 uM y o h i m b i n e  stimulation  ively  ^  on the t i s s u e .  using  and i n c r e a s e  we,  o r  (prazosin,  20 uM c l o n i d i n e o r 0 . 5 - 5 0 /iM d o b u t -  concentrations  response  reported  ^  of  c a r r i e d out on i s o l a t e d t i s s u e p i e c e s and  inhibitory  ponse  were  using high  agonists  rine  0.5 -  i n an atmosphere  0 . 4 mM a n t a g o n i s t s  35/25^  medium by f i l t r a t i o n  report  [225]  H  and s e p a r a t i n g  Studies  the  or without  incubation with  amine.  pre-incubated  electrical  these to use  to antagonize  res-  reported relatthe  eff-  E x t r a c t i o n Procedures Incubation media.  T r i c h l o r a c e t i c a c i d (40% w/v)  added to g i v e a f i n a l c o n c e n t r a t i o n of 10%;  was  tubes were v o r t o  exed and  c e n t r i f u g e d a t 3,000 X g f o r 15 minutes at 4 C.  c l e a r supernatant  was  n e u t r a l i z e d with  fuged a t 3,000 X g f o r 15 minutes and  The  s o l i d CaCO^/ r e c e n t r i evaporated to dryness a t  60°C. T i s s u e fragments from each i n c u b a t i o n f l a s k were immediately p l a c e d i n 1 ml of TIM 7.4;  mercaptoethanol 0.1  mM,  buffer  ( T r i s - H C l 20 mM,  3-isobutylmethylxanthine  pH  1  mM)  and homogenized u s i n g Brinkman Instrument P o l y t r o n . F i n a l volume of each homogenate was mination  adjusted  to 2.5 ml. For the  of t o t a l t i s s u e c y c l i c n u c l e o t i d e content,  of the homogenate was c o n c e n t r a t i o n of 10%  d e p r o t e i n i z e d u s i n g 40% TCA and  deter-  an a l i q u o t  to g i v e  t r e a t e d i d e n t i c a l l y as the  final  deprotein-  i z e d i n c u b a t i o n media samples. I n t r a - c e l l u l a r protein-bound order to separate  c y c l i c nucleotides.  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  i n t o the " f r e e " f r a c t i o n and  nucleotides  the f r a c t i o n bound to the  u l a t o r y subunit o f the r e p r e s e n t a t i v e p r o t e i n kinase tein-bound"  f r a c t i o n ) , we  by Podesta e t a l . [151].  In  reg-  ("pro-  have adopted the method d e s c r i b e d T h i s method i s based upon the  ob-  s e r v a t i o n o f Dufau e t a l . [51] t h a t the r e c e p t o r p r o t e i n with c y c l i c AMP  bound to i t i s r e t a i n e d by the HAWP M i l l i p o r e  t e r s of 0.45  pm  fil-  pore s i z e , whereas the unbound c y c l i c AMP  f r e e l y pass through.  can  1 ml a l i q u o t s of the above d e s c r i b e d  t i s s u e homogenate were spun a t 10,000 X g f o r 10 minutes, -41-  the  pellet discarded, and the supernatant diluted with further 2 ml of TIM buffer and filtered through Millipore filters. The filters 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 minutes. After evaporation to dryness, these eluates were used for bound ' cyclic nucleotide determinations. 1  Control experiments for bound cyclic nucleotide determinations. These were carried out to determine Ci) whether unbound cyclic nucleotides re-associate with free receptor protein 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 cycl 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 centrifuged as described above. 0.2 ml aliquots of the supernatant were pre-incubated for 10 to 20 mino 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-methyl3 xanthine and 22 nM [ H] cyclic nucleotide. Non-specific binding was determined in parallel incubation in the presence of 10 juM non-labelled cyclic nucleotides  Cyclic lated  n u c l e o t i d e bound  to  receptor  p r o t e i n was  iso-  by m i l l i p o r e f i l t r a t i o n as d e s c r i b e d a b o v e .  filters vials,  were  transferred to  dried for  1 hour,  oxyethanol.  After  cocktail  g of  (10  plastic  scintillation  and d i s s o l v e d i n  a d d i t i o n of  10 m l o f  2.5-diphenyloxazol  bis-[4-methyl-5-phenyloxazol-2yl]  The  2 ml of  2-mth-  scintillation  and 0 . 3  benzene  g of  1.4  in a litre  of  3  (ii)  Toluene,  H-radioactivity  tillation  counter with  Measurements of For in  ogenate  an e f f i c i e n c y of  d i s s o c i a t i o n of  t h i s purpose, the  was c o u n t e d i n  brown  adipose  p r e s e n c e and absence o f was  then c e n t r i f u g e d at  a liquid  scin-  20%.  bound c y c l i c t i s s u e was  nucleotide. homogenized  mercaptoethanol. 10,000 x  The  g for  10  hom-  minutes.  o  The  s u p e r n a t a n t was  intervals  of  time  described  above  then  (0,  for  15,  incubated at 30 m i n u t e s )  4 C for  various  and p r o c e s s e d as  i n t r a c e l l u l a r p r o t e i n bound  cyclic  nucleotide.  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 homogenate was incubated with 300 p i of 80 mM-potassium phosphate -43-  b u f f e r pH 6.5, c o n t a i n i n g 10 mM magnesium a c e t a t e ; 1 mM 2-mercaptoethanol;  0.1 mM 3 - i s o b u t y l - l - m e t h y l x a n t h i n e and 2 nM  3 o [ H ] - c y c l i c n u c l e o t i d e incubated f o r 30 minutes a t 2-4 C f o r f r e e receptor s i t e s  (non-exchange  conditions). Non-specific binding  was determined i n p a r a l l e l i n c u b a t i o n s i n the presence o f 10 uM non-labelled c y c l i c nucleotides. the  The r e s t o f the procedure was  same as d e s c r i b e d i n c y c l i c n u c l e o t i d e a s s o c i a t i o n w i t h f r e e  r e c e p t o r p r o t e i n experiments. C y c l i c GMP and C y c l i c AMP Assays. c o n s t i t u t e d i n sodium-acetate b u f f e r  Dry samples were r e -  (pH 6.2, 50 mM)  and a f t e r  c e n t r i f u g a t i o n a t 3,000 x g f o r 15 minutes to remove a d d i t i o n a l t r a c e s o f CaCO^/ supernatants were used f o r RIA assays o f c y c l i c nucleotides.  The method o f S t e i n e r e t a l . [187] was used w i t h  a d d i t i o n o f a c e t y l a t i o n step d e s c r i b e d by Harper e t a l . [ 7 1 ] . New England N u c l e a r (NEN) Boston, MA assay k i t s were used.  The Sam-  p l e s i n d u p l i c a t e were counted i n S e a r l e Instrument gamma c o u n t e r and standard curves were prepared f o r each s e t o f d e t e r m i n a t i o n s . P r o t e i n was assayed by the method o f Lowry e t a l . [116] with bovine serum albumin as a standard.  2.3.5  E f f e c t of agonists i n vitro on c y c l i c GMP c o n t e n t s i n brown f a t fragments from obese mice and t h e i r l e a n l i t t e r mates. T o t a l brown adipose t i s s u e from 35 obese and 35 c o n t r o l  mice  (normal l e a n l i t t e r mates) was pooled, minced,  N i t e x # 30 f i l t e r ,  and d i v i d e d i n t o a l i q u o t s  o f approximately  100 mg each t o be used f o r the subsequent i n v i t r o The r e s t o f b a s i c methodology  f i l t e r e d on  incubations.  f o r t i s s u e i n c u b a t i o n and c y c l i c  -44-  nucleotides  extraction  and a s s a y s was  the  same a s  described  earlier.  2.3.6  Inhibition adipocytes clonidine. The  removed ary  brown  from  the  regions,  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 from brown f a t of 1 - w e e k - o l d r a t s adipose  interscapular,  trimmed  of  t i s s u e p i e c e s were  buffer  (appendix  10  mg/ml  C)  1-week-old  subscapular,  was  cervical  i n Krebs-Ringer  6 mg/ml  serum a l b u m i n  rats  and  axill-  t i s s u e and minced w i t h  suspended  containing  bovine  from  extraneous  Minced  and  tissue  in by  type  scissors.  bicarbonate  crude b a c t e r i a l  collagenase  V and  in  incubated  a  a shaking water period, moved  the  and  adipocyte  filter.  W/V)  37  C for  plastic vial  the  carbonate (.4%  at  The  buffer  7.4  by  were  serum albumin  adjusted  to  after  The  s u s p e n s i o n was  cell  room the  temprature. adipocytes  Krebs-Ringer cells Ringer  were  The  were  bicarbonate  p e n s i o n was adipocyte  taken  of  washed w i t h  for  the  The  at  was  pH  assay of  cell  re-suspended  in  of  and  40  a  coarse  by  of of  four  mg/ml  protein  was  type  V.  5 minutes  at  aspiration,  washings,  fxl o f  and  albumin-free  albumin-free 100  b i -  t h i s medium  25-30 ml of  Following  and an a l i q u o t  through  g for  removed  volume  re-  Following  serum a l b u m i n  50 x  another  digestion  i n Krebs-Ringer  mM C a C l 2  bovine  buffer.  i n measured  s u s p e n s i o n was  V.  centrifuged  buffer  4 minutes.  filtered  1.5  type  infranatant  suspended  every  the  s u s p e n s i o n was  re-suspended  addition  bicarbonate  During  tissue  hand  containing  bovine 7.4  minutes.  s u s p e n s i o n was  cells  pH  15  containing  shaken v i g o r o u s l y  digestion Nitex  bath  the  Krebs-  cell  content.  an e q u a l volume  sus-  The of  another  Krebs-Ringer  (8% W/V) onate  BSA.  buffer  a control  containing  albumin-free amount o f  In  were  buffer  experiment  BSA  Krebs-Ringer  a.lbumin  adipocytes  bicarbonate  ( t y p e V)  containing the  buffer  left  after  these  stored  under  an atmosphere  mg/ml  Krebs-Ringer  was w a s h e d  bicarbonate  80  four  to  washings.  times  with  determine  The  of  bicarb-  the  isolated  brown  02~C02(95:5%)  in  o  a slowly before  shaking water  use.  forskolin)  Various were  bath  pipetted  transferred  into  adjusted  to  then  content  the  paper for  under  with  into  of  These were  pertussis  drugs  tubes. were  and  an a l i q u o t  The  volume  of  incubated  2 00 u l  aliquot  of  set  of  (0.5  c l o n i d i n e i n h i b i t i o n of  the  W i e l a n d method  experiments, mg/ml)  for  the  yohimbine,  cell  15  to  suspension  each tube  for  millipore  minutes  subsequent of  through  the  45  (clonidine,  filtered A  toxin  of  approximately  p l a s t i c tubes,  tubes  g l y c e r o l by  another  C for  30 minutes  these  suction.  assay of In  to  1 ml.  37  combinations  a preincubation period was  at  was  minutes  0 . 4 5 pm  filtrate  and  filter  was  removed  [212]. cells  3^ h o u r s ,  were  and  forskolin-stimulated  incubated  the  response  lipolysis  was  re-analyzed.  2.3.7  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 - 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 from brown f a t of adult hamsters . Adipocytes  the  procedure  old  rats.  2.3.8  Data  from  adult  hamsters  as d e s c r i b e d f o r  were  adipocytes  prepared  for  brown  according fat  of  1-week-  Analysis  Statistical  a n a l y s i s of  -46-  the  data  was  done  using  to  Student  ' t ' test wherever appropriate. Each of the paired designed experiment was analyzed by paired 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-hydroxydopamine 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]. 1  -47-  CHAPTER 3 RESULTS 3.1  Identification and characterization of ©^-adrenoceptors 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 irenoceptor binding characteristics of rat cortex. The -aG  binding characteristics of o < . 2  -rece  P  tors  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. binding, assessed from addition of 10  The non-specific  (-)epinephrine or  yohimbine, was directly proportional to free ligand concentration.  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 (K ) of 18 nM and maximum number d  -48-  Fig.  1 A. S p e c i f i c  binding  o f [ H]-yohimbine  t o plasma  membrane  fragments i s o l a t e d  week-old  r a t s 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  concentration (•-•) Inset:  f r o m brown f a t o f o n e -  i n t h e p r e s e n c e o f 10 ^iM y o h i m b i n e  and 10 AUM e p i n e p h r i n e Scatchard  plot  was d e t e r m i n e d u s i n g  (o-o) .  f o r yohimbine  linear  data.  Slope  regression analysis  (r = 0.96). B. I n h i b i t i o n  of [ H]-yohimbine  concentrations  (o-o)•  o f yohimbine  Each p o i n t  determinations  binding (•-•)  represents  by i n c r e a s i n g  and  epinephrine  t h e mean o f t r i p l i c a t e  i n one r e p r e s e n t a t i v e e x p e r i m e n t .  -49-  of b i n d i n g  sites  regression  coefficient,  a  by  (Bmax  y o h i m b i n e was  equimolar b a s i s  ) ' o f 156 fmol/mg / a pf r o t e i n v( l i n e a r r = 0.96).  greater  than t h a t  a highly  [ 5 0 ] , has p r e v i o u s l y  functional  receptors 3  i n brain  o f [ H]-RX-781094  f u n c t i o n of i n c r e a s i n g 10 /tM y o h i m b i n e .  reached  (-)-epinephrine  sites  been used  [45].  F i g . 2A shows t h e s p e c i f i c  t o brown f a t p l a s m a membranes a s a  The s p e c i f i c  binding  o f 20 fmol/mg p r o t e i n  t o be t e m p e r a t u r e  10 nM.  dependent.  Lowering  a compound  binding.  Clonidine,  a partial  ©^-adrenoceptor a g o n i s t  have determined  clonidine binding  s a t u r a t i o n was Since  principal from  reached  synaptic  i n vivo  i n f a n t r a t s , we, 3  teristics  approximately  was  ws found  incubation the s p e c i f i c group,  possesses  Saturation  poorly  binding  reproducible.  The a p p a r e n t number  25  nM.  of (-)-norepinephrine  i s the  a c t i n g upon brown f a t membranes studied  o f (-)[ H ] - n o r e p i n e p h r i n e Initial -50-  of  50 fmol/mg p r o t e i n and  at approximately  therefore,  membranes f r o m brown f a t .  binding  number  i n t h e p r e s e n c e o f 10 /uM  ( F i g . 2B).  release  stimulus  the  activity.  We  half  and h a l f s a t u r a t i o n  of imidazoline  c l o n i d i n e t o brown f a t membranes was  s i t e s was  showed an a p p a r e n t  eliminated  of  y o h i m b i n e o r 10 /*M c l o n i d i n e  i n the presence of  The s p e c i f i c  t e m p e r a t u r e t o 4°C a l m o s t c o m p l e t e l y  binding  to characterize  ligand concentration  at approximately  specific  on  s e l e c t i v e c^-adrenoceptor  antagonist  of b i n d i n g  by  (Fig. IB).  RX-781094,  binding  Displacement of the l i g a n d  the binding  charac-  t o i s o l a t e d plasma experiment  showed t h e  X o  10  20  30  40  50  [3H] RX 781094 (nM)  20  40  60  80  100  [3H] Clonidine (nM) Fig  2.  A.  Specific  b i n d i n g o f [ H ] - R X 781094 t o p l a s m a  membrane  fragments  week-old  r a t s i n t h e p r e s e n c e o f 10 p-M y o h i m b i n e  3  isolated  f r o m brown f a t o f o n e as  a function of increasing ligand concentration. Results  a r e t h e 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.  Specific  binding  of [^HUclonidine to i d e n t i c a l  membrane 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  y o h i m b i n e (•-•) o r 10 /*M c l o n i d i n e ( A - A ) . R e s u l t s a r e shown a s t h e mean + S.E.M. o f 4 e x p e r i m e n t s d o n e i n duplicate.  -51-  a.  20  60  120  240  g i g . 2 C. S p e c i f i c b i n d i n g of [ H]-norepinephrine to plasma membrane framents i s o l a t e d from brown f a t of oneweek-old r a t s i n the presence of 20 /iM norepinephrine and 20 /iM c l o n i d i n e (•-•) or 20 /JM c l o n i d i n e  (o-o).  Each point i s the mean of d u p l i c a t e determinations from two experiments. D . S p e c i f i c b i n d i n g of [ H]-norepinephrine t o plasma membrane fragments i s o l a t e d from brown f a t of oneveek-old r a t s i n the presence of 10 /iM epinephrine (•-•) or 100  yohimbine  (o-O).  Each p o i n t i s  the mean + S.E.M. o f d u p l i c a t e determinations from four experiments.  -52-  b i n d i n g was  rapid,  stable  (-)-norepinephrine,  20 ^ M  100  yohimbine In  (Fig.  and d i s p l a c e a b l e i n clonidine,  2C a n d  10  the  presence  of  (-)-epinephrine  20 juM  or  D).  equilibrium binding  studies  (Fig.  3A),  the  specific  3 binding  of  fragments  the was  (-)[  H]-norepinephrine  reversible  and  to  plasma  saturable.  Both  membrane  (-)-epinephrine  and  3 yohimbine  displaced  sites  in  their  effects.  were  either  one  of  (-)[  alone  When  together  combined  the  H]-norepinephrine  a concentration-dependent  used  their  (-)[  and  10 pM  to  effect  alone.  determined  10 /uK ( - ) - e p i n e p h r i n e  in  binding,  the  between the  than the  presence  presence  of  the  effect  of  specific  binding  of  epinephrine  10 /AM  a combination  of  y o h i m b i n e was d e s c r i b e d a s a 3 yohimbine s e n s i t i v e component of (-)[ H]-norepinephrine binding (Fig. 3A). A S c a t c h a r d a n a l y s i s of the s p e c i f i c b i n d i n g of 3 (-)[ 10 /M 60.4  and  in  in  H]-norepinephrine  30% h i g h e r  and  equipotent yohimbine  was  difference  binding  10 /iM  (-)[  only  its  and were  (-)-epinephrine  H]-norepinephrine that  manner  displace  The  from  H]-norepinephrine (-)-epinephrine nM a n d  65.8  10  (Fig or  3A  inset)  in  the  10 /*M y o h i m b i n e  nM a n d B  values  of  presence  revealed  0.22  and  Hill  plot  Kd  of  either  values  0.24  of  pmol/mg  max protein  (r  exhibited  = 0.96,  coefficients  (-)-norepinephrine even  at  respectively. 1.41  (Fig.  displacement 3A  and  probably  the © ^ - r e c e p t o r s .  specific plot  0.8)  inset)  of with  2B)  1.43  thus  indicating that  has more  than  one  A Scatchard 3 (-)[  (Fig.  a n a l y s i s of  H]-norepinephrine  approximately -53-  binding  the  site  yohimbine  showed  same K ,  the  a  (64.5  linear nM)  Fig.  3 A. 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  brown f a t plasma membrane old  rats  i n the presence  and  10 ^-M ( - ) - e p i n e p h r i n e  fragments o f 10  placement by  subtracting  ephrine from of  i n the presence  that  10  using  linear  i n the presence  of a  o f t h e same d a t a .  determinations Inset: Scat-  S l o p e s were  regression analysis,  and y o h i m b i n e a l o n e  specific Hill (•-•)  component  plot  determined  ( r = 0.96, 0.8 of (-)-epine-  and t h e y o h i m b i n e -  respectively.  of the data  i n p a n e l A f o r yohimbine  or (-)-epinephrine  displacing  combination  yohimbine.  0.77) f o r b i n d i n g i n t h e p r e s e n c e  phrine  calculated 3  o f 10 JLK ( - ) - e p i n e p h r i n e  one r e p r e s e n t a t i v e e x p e r i m e n t . plot  dis-  b i n d i n g of [ H]-norepin-  a r e t h e mean o f d u p l i c a t e  chard  and  B.  determined  to specific  ( - ) - e p i n e p h r i n e and 100 /*M  Results from  the s p e c i f i c  (•-•)  (-)[ ^ - n o r e -  ( A - A ) was  by yohimbine o n l y  one-week-  yohimbine 3  (o-o).  pinephrine binding sensitive  from  agent.  -54-  (o-o) as t h e l i g a n d  c  *5 o a a»  A  B/F 3  E 2>  2  o E  1  o CO  100  50 100 Bound (fmol/mg proteinL*  CL  0  C  *EL 50 0  w O Z IE" CO  10  20  30  1  40  ,, 1  50  10 Concentration (nM)  [3H] Norepinephrine (nM)  -55-  100  but  a much l o w e r  ) (71 f m o l / m g p r o t e i n ) (r max C a t e c h o l a m i n e s s u c h as ( - ) - e p i n e p h r i n e  ephrine  are  mixed  norepinephrine it  c o u l d be  We h a v e  (B  agonists.  s e e n when  a result  further  of  competition  investigated binds  In  experiments  competition  to  displaced  As  shown  than  0.77).  and  binding was  both  used  jB-receptors  4A),  both  range  (-)[  to  displace  (-)[  H]-  H]-  i n brown  binding  (-)-Epinephrine  a lower  of  fat.  (-)-epinephrine  H]-norepinephrine  manner.  (-)-norepin3  o<- a n d y f l - r e c e p t o r s . 3  p o s s i b i l i t y that  (Fig. 3  at  at  o<- a n d  (-)[  practolol  of  was  in  a  more  concentration.  (-)-epinephrine e x h i b i t e d a dose-dependent 3 d i s p l a c e m e n t of (-)[ H]-norepinephrine even i n the p r e s e n c e -3 3 o f 10 M practolol. Conversely, (-)[ H]-norepinephrine could still  in Fig  the  both  concentration-dependent effective  specific  (-)-epinephrine  norepinephrine  and p r a c t o l o l  The  =  be displaced  practolol When  4B,  the  even data  equation  [35], In  infant viously  in in  to  a concentration-dependent  the  presence  Fig.  the  order  rats  in  4B w e r e  of  analyzed  apparent to  that  compare of  adult  described experiments  10  of our  (-)-epinephrine.  by  Cheng  membrane  [24]  by  M  66 nM f o r  animals,  manner  and  Prussoff  e p i n e p h r i n e was o b t a i n e d .  preparation  we h a v e  on b i n d i n g  repeated of  from pre-  ^-adrenergic  3 ligand  (-)[  as  competing  the  saturable  H]-dihydroalprenolol agent  (Fig.  arid r e v e r s i b l e w i t h  at  approximately  to  free  ligand  10  nM.  using  5A).  The  binding  half-maximal  Non-specific  concentration  10 juM n o r e p i n e p h r i n e  and  rapid,  saturation  b i n d i n g was  specific  -56-  was  binding  observed  proportional represented  Fig.  4 A.  Displacement epinephrine  B.  of  specific  (-)[  ^-nor-  t o brown f a t p l a s m a membrane f r o m  week-old  r a t s by  labelled  (-)-epinephrine  Displacement increasing  binding of  of  increasing concentrations  (-)[  (O-o)  and  concentrations  of  practolol  H]-norepinephrine  onenon(•-•).  binding  of n o n - l a b e l l e d  by  (-)-epi-  _ 3  nephrine and  by  i n the  presence of  10  M practolol  i n c r e a s i n g c o n c e n t r a t i o n of  (o-o)  non-labelled  —5 practolol  i n the  presence of  (n-a).  -57-  10  M  (-)-epinephrine  25 10  60% of t h e t o t a l .  nM a n d a B  Scatchard  o f 0 . 5 5 pmol/mg  max  analysis protein  revealed  (r  of  to  f a t p l a s m a membranes  i n the presence  or a combination  of  (-)[  0 . 1 9 pmol/mg  protein  cJihydroalprenolol bine  only  binding  i n the presence  yohimbine. obtained  binding  by s u b t r a c t i n g  10 /*M p r a c t o l o l of  by Scatchard  alone  10 ^ M o f p r a c t o l o l  A Kd , o f 4 0 nM a n d B m a x o f analysis  5-day-old  1 served  l i t t e r s were  as c o n t r o l s  The a n i m a l s i n t h e second g r o u p a single-dose  i n j e c t i o n of  the effect  on brown cribed  (-)[3H]-  of  112  f m o l //m g»  3 (-)[  yohim-  its specific from  that  a n d 100 fp r o t e i n  H]-dihydroalprenolol (r = 0 . 8 ) .  E f f e c t of 6-hydroxydopamine and c h r o n i c yohimbine pretreatment on 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 i n i s o l a t e d p l a s m a membrane f r a g m e n t s from i n t e r s c a p u l a r brown a d i p o s e t i s s u e of i n f a n t rats. The  Since  (r = 0.96).  s p e c i f i c a l l y d i s p l a c e a b l e by yohimbine  3.2  Group  of  10 /uM  1 0 0 >xM  s p e c i f i c a l l y d i s p l a c e a b l e by  i n the presence  2  was  binding  of either  the binding i n the presence 5 0 nM ( / S l - s i t e ) a n d B max  ( F i g . 5B i n s e t )  ( F i g . 5B) was d e t e r m i n e d  determined  H]-dihydroalprenolol  10 #M p r a c t o l o l w i t h  yohimbine. A Scatchard a n a l y s i s of of p r a c t o l o l gave a K , of p r a c t o l o l * d of  Fig  3  the s p e c i f i c binding  practolol  of  = 0.88).  5B s h o w s brown  a  into  three  and was i n j e c t e d  with  s a l i n e on day 5.  were  and Hahn  6-hydroxydopamine  and a c t i v i t y [176],  by  by  on day 5. 6-hydroxydopamine  has already  no a t t e m p t  -58-  groups.  c h e m i c a l l y sympathectomized  of c h e m i c a l sympathectomy  f a t composition  by S k a l a  divided  been  w a s made  to  desstudy  Fig.  5  A.  (-)[  H]-dihydroalprenolol  binding  p l a s m a membrane f r a g m e n t s Total  binding  competing the  specific  binding  (•—•)  between t h e  binding.  Scatchard  determined  B.  Specific in  the  by  Results  binding  yohimbine  the  site).  specific  specific  was  linear  by  practolol  and  data.  Slope  (-)[  H]-dihydroal-  was  from  plot  in  that  of  binding.  regression  by  calculated  binding  +  the determined  1 0 0 ALM (-)[  H]-  Slope analysis  displaceable  yohimbine-specific  -59-  0.88).  (•—o)  displaceable  for binding  respectively).  was  a n a l y s i s (r =  1 0 /uM p r a c t o l o l  0.82  one  H]-dihydroalprenolol  Inset: Scatchard  and  from  the  dihydroalprenolol  (r = 0 . 9 6 ,  and  done i n d u p l i c a t e s .  1 0 M-K p r a c t o l o l  d e t e r m i n e d by  shown a r e  This binding  presence of  yohimbine.  non-  selectively  s u b t r a c t i n g the  in  the  the  1 0 /t*M p r a c t o l o l 3  (•—•).  presence of in  (-)[  binding  binding  of  and  regression  of  presence of  prenolol  by  plot  linear  ( P^-receptors  (A--A) b i n d i n g  c a l c u l a t e d as total  rats..  any  1 0 /AM ( - ) - n o r e p i n e p h r i n e ;  r e p r e s e n t a t i v e experiment Inset:  absence of  agent; n o n - s p e c i f i c  difference specific  from o n e - w e e k - o l d  (4k—•) i n t h e  presence of  t o brown f a t  component  c  *5  o k.  a  a> E "o £  c *3 o a. cn E  B/F 50  X .  25  -i—i  -o e  u  100 2 0 0 3 0 0  3  Bound (fmol/mg protein)  o ca « 1200  B/F 30 20  x X N .  o E  10r  -o e  0  o ca  Bound (fmol/mg  X 20  3  60  100  140 protein)  o c a o 160 o w 120 -o  x 0  10  20  80 40 0  30  [ 3 HJ  [ 3 H ] D i h y d r o a l p r e n o l o l (nM)  -60-  X  50  100  D i h y d r o a l p r e n o l o l (nM)  the  brown f a t c o m p o s i t i o n o r a c t i v i t y .  The  5-day-old animals  used  i n t h e p r e s e n t e x p e r i m e n t s d i d n o t show any s i g n i f i c a n t  side  effects.  rats)  The  two  weeks o f age,  showed t h a t  younger  administration. diately  after  animals.  7 days  bine-injected tained  was  younger  25% o f t h e  o f 30 a n i m a l s , 1 d i e d day o f t r e a t m e n t . improved  after  on t h e s e c o n d The  animals'  the i n i t i a l  i n t h e i r body w e i g h t ,  noted.  older  three  as compared  Brown a d i p o s e t i s s u e  were l i g h t e r i n c o l o r  and  pads  of  attempt  to carry  position  o u t any  detailed  s a m p l e s , we  analysis  Fig.  B show t h e e f f e c t s o f c h e m i c a l 3  tomy w i t h 6-hydroxydopamine on membrane f r a g m e n t s 8-day-old r a t s . saline  isolated In  ( panel A  both )  and  -61-  the  con-  com-  sympathec-  binding  interscapular  the. c o n t r o l  that  of experiments.  [ H]-yohimbine  from  to  no  group.  of the t i s s u e  i n the p r e s e n t s e r i e s  and  and  d i d not  or a c t i v i t y 6A  days,  yohim-  apparently  more t r i g l y c e r i d e s t h a n t h o s e o f t h e c o n t r o l  day  tol-  a l l animals appeared w e l l ,  of the s c a r c i t y of t h e t i s s u e  with  of the  imme-  that  Because  of  appeared  developed i n l e s s  of i n j e c t i o n s  rats  tremor  yohimbine  e f f e c t s were o b s e r v e d ;  difference  controls,  the  side  on t h e f o u r t h  significant  tolerated  fewer  erance of the i n j e c t i o n s and a f t e r  the animals  experiments  i n the m a j o r i t y  weeks o f age,  (of group I I I  commenced u n t i l  preliminary  animals poorly  From a t o t a l  1 died  because  the i n j e c t i o n s  twitching  animals.  n o t be  S e i z u r e s and m u s c u l a r  A t two  muscular  of  treatment w i t h yohimbine  on t h e o t h e r hand, c o u l d  reached  and  chronic  to  plasma  brown f a t  group  experimental  injected  group  (panel  B) t h e  (as  a s s e s s e d by  was  directly  to  at  the  the  50 n M .  difference  nM  gave  The  (Fig.  (B-  6A).  (K^)  of  14  155  to  those 3  binding  of  animals  (Fig.  75  [  fmol/mg  protein.  in  inset,  again  gave  a  value  of  B  m a x  It such  as  is  nM.  known  epinephrine  therefore,  characteristics brane The  preparation  number  and  affinity  of  and  as  to  180  fmol/mg  approximately in  the  inset  are  con-  sites  almost  iden-  Specific  sympathectomized  number  plot  of  binding  was  of  sites  reached  the  a  data, value  at shown  of  8.6  protein.  compared  to  for  antagonists, exhibit  agonists  a higher  the ^ - r e c e p t o r s  information  H]-norepinephrine  isolated  specific binding  from  treatment  fmol/mg  of  binding  half-saturation  additional 3  (-)[  of  up  apparent  section 2.1.  membranes  a linear  45  (taken  a dissociation  values  of  an  shown  with  These  Woolf  that,  sought of  = 0.91)  and n o r e p i n e p h r i n e  of stereoselectivity We,  The  at  [94,220]  an a p p a r e n t The  binding  sites  reached  results to  showed  10  nM a n d  plot  protein.  approximately the  was  (r  in  yohimbine)  concentration  showed  specific binding  H]-yohimbine  fmol/mg  ligand  group  a maximum  recorded  H]-yohimbine  non-labelled  free  control  Woolf  [  and n o n - s p e c i f i c b i n d i n g )  saturation  nM a n d  6B)  the  total the  The  10 ^.M  of  s p e c i f i c yohimbine  relationship  ) of  tical  of  half  a linear  stant  from  of  to  [ H]-yohimbine  protein. 18  The  between  p l a s m a membranes of  addition  proportional  least  number  non-specific binding  to  on t h e the  degree  [ 204,207  binding  identical  mem-  from the sympathectomized animals. 3 ( - ) [ H ] - n o r e p i n e p h r i n e t o p l a s m a mem-  -62-  ].  Fig.  6  E f f e c t o f c h e m i c a l sympathectomy  of 5-day-old  by a s i n g l e  (50 mg/kg  weight) ing  intraperitoneal  dose  o f [ H]-yohimbine isolated  was d e t e r m i n e d yohimbine  t o brown f a t p l a s m a  3 days  later.  a t 25 C.  Each p o i n t  experiment.  Inset:  Woolf  s l o p e s were d e t e r m i n e d  non-labelled  f r o m one r e p r e s e n t a -  plot  of the data.  by l i n e a r r e g r e s s i o n  Results from  of the ligand  6-hydroxydopamine  -63-  saline-injected  binding  to  rats.  t o membranes  injected  analysis  respectively).  Shows 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 membranes f r o m c o n t r o l  B.  binding  i s t h e mean o f two  ( r = 0.91 and 0.94 f o r p a n e l A and B  A.  membrane  Specific  i n t h e p r e s e n c e o f 10 /M  d e t e r m i n a t i o n s done i n d u p l i c a t e  The  body  o f 6-hydroxydopamine on t h e s p e c i f i c b i n d -  fragments  tive  rats  rats.  [ HJ Yohimbine (nM)  [ H J Y o h i m b i n e (nM)  3  3  -64-  branes  from  both  reversible  and  control  and  saturable  by  experimental  a n i m a l s was  10 /tM ( - ) - e p i n e p h r i n e .  rapid,  The  3 specific from  binding  control  epinephrine range  of  of  (-)[  animals ranged  the  (Fig.  from  ligand  H]-norepinephrine 7A)  in  50% o f  the  the  to  plasma  presence of  total  concentration  to  binding  25% a t  the  membranes  10 jtM (-)in  the  lower  higher  range.  Scatchard a n a l y s i s of these b i n d i n g data gave 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 r p m o l / m ga ' d max 3 protein. to  However,  the  p l a s m a membrane  pamine  pretreated  fragments animals  characteristics  under  absolute  of  amount  p l a s m a membranes in  the  (-)[  of  than  in  ranged  from  66% o f  ligand  concentration  plot  of  the  data  coefficient was  of  analyzed  ence  of  the  two  these  data  gave  animals.  total  to  60% i n  the  in  an  regression  (r)  of  0.8.  site  a  showed  The x  of  low  a K, d  animals  affinity  3 pmol/mg  a Hill  was  the  -65-  lower  range  the it  A  of  Scatchard  curve  with  Scatchard  showed  inset).  The  36 nM a n d a B  the  a plot  pres-  higher of  0.65  max site  exhibited  protein.  coefficient  binding  range.  When  to  significantly  specific  7B,  of  The  pretreated  [159],  (Fig.  unusual  conditions.  exponential  Rosenthal  components  some  binding  higher  by  6-hydroxydo-  specific  The  binding  H]-norepinephrine  of  experimental  fit  protein. m  showed  (-)-epinephrine  according to  B  7B)  (-)[  fat  6-hydroxydopamine  the  binding  2 0 0 nM a n d a  brown  best  affinity binding 3 -1 pmol/mg  same  control  was  from  of  H]-norepinephrine  10  higher  binding  (Fig.  the  from  presence  specific  of  1.5  Further (Fig.  a  of  analysis 7B  inset)  of  Fig.  7  Effect  of  single  intraperitoneal  weight) of  sympathectomy  Specific  binding  to  isolated  was  representative  A. S h o w s to  the  injected data.  Slope  gression  Results  was  the  body  specific fat  binding  plasma  later. the  point  presence is  in duplicates  of  the  specific  from  Inset:  analysis  of  in  Each  isolated  rats.  3 days  a  the  from  experiment.  results  membranes  brown  determined  2 determinations  r a t s by  (50 mg/kg on t h e  10 juM ( - ) - e p i n e p h r i n e .  mean, o f  B.  dose  (-)[3H]-norepinephrine fragments  one  5-day-old  of 6-hydroxydbpamine  membrane  of  of  control  Scatchard  determined (r  =  by  binding saline  plot  of  linear  the  re-  0.93).  specific binding  to  mem-  o branes treated of  the  i s o l a t e d from rats. data.  Left The  6-hydroxydopamine inset:  curve  exponential  fitting  (r  inset:  plot  the  Hill  -66-  of  Scatchard  was  plot  determined  = 0.8). same  pre-  Right data.  by  [ H]Norepinephrine (nM)  [3H] Norepinephrine (nM)  3  -67-  indicating  positive  norepinephrine  cooperative  interaction  in  (-)[  H]-  binding. 3 7 C s h o w s the s p e c i f i c b i n d i n g o f  Fig. presence  1 0 pM  of  a combination  8 shows  the  (-)[3H]-norepinephrine  from brown dopamine,  fat by  yohimbine. peting  placement each  binding  to  8-day-old-rats,  of  different  that  curves  in  and  curve  concentrations was m o r e  the  presence  of  for  the  displacement isolated 6-hydroxy-  i n j e c t e d by  (-)-epinephrine  potent  binding  of  1 0 /uM p r a c t o l o l .  p l a s m a membranes  previously  (-)[3H]-norepinephrine  than  or  yohimbine  sites.  The  in  binding  comdis-  showed a s t e e p s l o p e , and t h e y were n o n - p a r a l l e l t o  other. Plasma  day-old for  competition  (-)-Epinephrine  for  and  1 0 ju-H ( - ) - e p i n e p h r i n e  of  Fig. of  (-)-epinephrine  [ H]-norepinephrine i n the  rats  membrane  pre-treated  seven days) were  RX-781094  and  used  The  at  Binding brane  for  specific  approximately experiments  fragments  15  as  demonstrated  the  binding  nM a n d  repeated  from  brown  yohimbine  fat  ^-ligands showed  i n the  number  and h a l f  1 0 nM r e s p e c t i v e l y ( F i g .  with  [3Hj-yohimbine  reversible  and  using  on  9 B and A ) .  pmol/mg  (P<0.01)  than  protein the  B-  (r  = 0.8).  This  calculated in  -68-  was  significantly  control  animals  mem-  binding in  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 of b i n d i n g3 d a t a ( F i g . 9 C ) r e v e a l e d a K , o f 6 6 . 7 nM and a B m _ of d max 0.35  of  satura-  identical  saturable  [3H]-  presence of  an apparent  150 fmol/mg p r o t e i n  of 2 1 -  (lOmg/kgi.p.  equilibrium binding studies  9 0 and  s p e c i f i c b i n d i n g s i t e s of  isolated  chronically with  [3H]-rauwolscine  1 0 fiM y o h i m b i n e .  tion  fragments  higher  (mean  =  the these  Fig.  7C  [ H]-norepinephrine isolated Total was  from  b i n d i n g t o p l a s m a membrane  brown f a t o f s y m p a t h e c t o m i z e d  (•-•), n o n - s p e c i f i c  determined  (V-T ; v-v  i n t h e a b s e n c e and  ( - ) - e p i n e p h r i n e and  ) binding  presence  ii.M ( - ) - e p i n e p h r i n e o r a c o m b i n a t i o n  rats.  of  of  10  10 juK  practolol respectively. 3  Specific  b i n d i n g of  presence  of  in  10 /tM  the presence  of  [ H]-norepinephrine  (-)-epinephrine 10 fM  (-)-epinephrine  10 /iK p r a c t o l o l ( a - o ) was corresponding non-specific  -69-  (•-•)  determined  by  b i n d i n g from  i n the and  that  and subtracting the  total.  -70-  Fig.  8  Displacement  of  [ H]-norepinephrine  t o p l a s m a mebrane f r a g m e n t s fat  of  binding  isolated  f r o m brown  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  the mean o f t r i p l i c a t e  -71-  (•-*).  Each p o i n t i s  determinations.  Fig.  9  Effect  of c h r o n i c yohimbine  (10 mg/kg body w e i g h t binding  i . p . f o r 7 d a y s ) on t h e  o f o ^ - a n t a g o n i s t s t o plasma  fragments old  pre-treatment  isolated  rats.  membrane  f r o m brown f a t o f 21-day-  Each p o i n t  i s t h e mean 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 from a r e p r e s e n t a t i v e  A.  Shows t o t a l  experiment.  (•-©) and t h e s p e c i f i c 3  binding the B.  (O-O) o f [ H ] - r a u w o l s c i n e i n  p r e s e n c e o f 10 pK  Shows t h e t o t a l (•-•) of  yohimbine.  (•-•), n o n - s p e c i f i c  and t h e s p e c i f i c  binding  [ H]-RX-781094 t o i d e n t i c a l 3  preparations  (O-O) membrane  i n t h e p r e s e n c e o f 10 /iM  yohimbine.  -72-  -73-  0.152  pmol/mg  protein).  The  same b i n d i n g  experiments  3 were  repeated  either  (-)[  H]-norepinephrine  10 /tM ( - ) - e p i n e p h r i n e  placing from  using  agent .  The  experimental  ratio  or  of  compared  was  5-fold  higher  at  the  low  and  3-fold  higher  at  the  high  (Fig.  9D)  protein B  max  indicated  for  o f  3.3  the  control  ligand  Scatchard  (r  .98)  =  for  and  a  yohimbine  and  disbinding  animals  concentration  nM a n d B _ of max  the  with  0.4  produced  analysis 3.0  of  (r  =  was  quite  AMP.  had  of  pmol/mg  66.7  nM  and  0.98).  a decrease  control  in  ( 2 . 0 ^M)  was  effect  Pre-incubation  mM a n t a g o n i s t s  d  ^^5/25  values  resulted  the e  c  (Fig. in  of  in  r  e  levels a  s  e  10A).  d  cyclic  -74-  in Fig  AMP  ( 2 . 0 pJA)  the  production  one  hour  and/^2-H35y25)  nucleotides,  l e v e l more of  stimu-  clonidine  pieces for  10A,  cyclic in  ^-metoprolol,  Addition  dose-dependent  shown  effective  cyclic  incubated .  ( 2 . juM) ,  stimulating  tissue  of  were  stimulating  Both  ((^-yohimbine,  in  As  effective  GMP.  rats  clonidine  (2/x.M).  cyclic  slight  1-month o l d ( 5 . /*.M) ,  salbutamol  production  ^-antagonist  clonidine  and  and c l o n i d i n e  and p h e n y l e p h r i n e cyclic  from  phenylephrine  (50 ^,M)  production  lating  the  of  as the  non-specific  from  range.  fragments  of  (50 ^ M )  dobutamine AMP  fat  presence  doubamine  of  that  ligand  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 c o n t e n t s of r a t brown adipose t i s s u e . Brown  in  protein  to  68.3  (-)-epinephrine Pm°l/m9  3 , 6  to  range  a K, of d  the  10 ^Jtf y o h i m b i n e  specific  as  as  0.2,  elevation  of  2,  than  half  20/tM  cyclic  GMP  "Fig.  9C  Scatchard binding from  to  for  in  fat 7  of  days  weight).  determined the  [  H]-yohimbine  as  with  The the  absence  that  done  in  m i n e d by l i n e a r  regression  specific  binding  parations. as t h e those  The  in  the  in  of  a  was  b i n d i n g was in  presence  the of  (•-•)  or  10 jtM ( - ) - e p i n e p h r i n e  Each  point  is  the  done  in  linear  of  mean  deteranalysis.  membrane  bine  duplicate.  binding  = 0.88)  binding  the  the  Slope (r  was  presence  is  identical  of  mg/kg  H]-norepinephrine  specific  difference obtained  (-)[  to  (10  in duplicate  experiment.  analysis  the  point  representative  Scatchard  of  isolated  injected  binding  difference  Each  2 determinations  rats  yohimbine  specific  and  specific  fragments  21-day-old  10 /*M y o h i m b i n e .  D  of  p l a s m a membrane  brown  daily body  analysis  mean o f Slope  regression analysis  -75-  2  predetermined  absence  10  and  yohim(•-•).  determinations  was (r  determined  by  = 0.98  both).  for  L°HJYohimbine B o u n d ( f m o l / m g protein)  -76-  [ H]NE 3  (pmol/mg  Bound protein)  level. was  No  seen  with  such e f f e c t  (Fig  10B  left  5,  and  elevation  of  cyclic  of  1 hour  However, of  0.4  amine  in  AMP  presence  of  effective  production  (Fig  Fig of  isolated  from  cyclic  GMP  fat  total  content  was  tissue  produce  pieces with  cyclic  GMP  rats.  of  effect  0.4 of  nucleotide  be  GMP  concentrations  (released  i n brown  in  the  media  fragments  A dose-dependent  elevation  0.5  -  a peak 1.0  response  for  (Fig.  concentrations  in  occurring  Pre-incubation  /uM.  1 hour  completely  11B).  non-stimulated  -77-  to  on c y c l i c  various  mM y o h i m b i n e  and  with  fat  clonidine  stimulated  pre-incubated  incubation  effect  level  seen w i t h of  were  dobutpanel).  an ^ - a n t a g o n i s t ,  + intracellular)  7-day-old  presence  of  right  AMP.  panel).  effect  cyclic  the  effect  fragments  blocking clonidine right  cyclic  in  lower  by  (/^-antagonist)  dobutamine  of  fragments  Pre-  prazosin  ^35/25  level  followed  yohimbine,  the  MM o f  stimulating  fat  panel).  50 ,«.M o f  10B  incubated  dose-dependent  0.4  the  (Fig.  antagonists  of  Both  the  brown  clonidine concentration  the  brown  concentration  fragments  (©^-antagonist) ,  of  upper  AMP  middle  with  in  at  abolished  tissue  blocked  shows  incubation  lower  increase  where  10B  11A  c l o n i d i n e on  during  the  an  showed  in  fat  showed  10B  production  experiments  cyclic  incubation with  pre-incubation  0.5ytf.M c l o n i d i n e most  by  produced  on c y c l i c  the  (Fig  yohimbine  mM m e t o p r o l o l  Similar  Brown  adipose  followed  (/^-agonist)  panel).  AMP  brown  (o^-antagonist), for  fat  50 JJM. d o b u t a m i n e  0.5,  incubation  on brown  far  cells in  generally  excess  of  that  Fig  10  Effect  of  in vitro tissue  adrenergic on c y c l i c  of  1-month-old  and  pooled  in  aliquots  of  of  agonists  and  ume o f (pH  1.0  7.4).  -  0.2  on a N i t e x  nucleotides  were  the  Results  is  minations.  Vertical  first  set  the  of  of  agonist  effect  of  antagonist  Upper  left  different 20.0 /uM)  and  in  bars  respectively.  10%  combinations a total  vol-  buffer  cooling were  in  ice-  separated  suction.  Cyclic  trichloracetic RIA  as  to  four  any  left  second set  of  con-  drug.  separate  S.E.M.  from  described  as percent  absence of  indicate  the  deter-  from  the  shows  the  shows  mean.  the  only.  left  concentrations fat  by  under  three  columns  only;  lower  on brown  the  incubated  bicarbonate  expressed  mean o f  effect  by  and  C in  t r i p l i c a t e by  are  four  37  tissue  cleaned,  various o  fragments  filter  determined  Each v a l u e  The  tissue  in  region,  stopped  extracted  determined  values  at  adipose  adipose  30 r a t s )  g with  were  immediately  trol  to  Krebs-Ringer  and  and  Brown  antagonists  Reactions  i n methods.  B.  ( f r o m up  cold water,  acid  A.  rats.  antagonists  i n brown  interscapular  0.1  ml of  and  nucleotides  was d i s s e c t e d f r o m minced  agonists  panel of  cyclic  Lower m i d d l e  -78-  shows  clonidine GMP panel  and  the  effect  (0.2,  2.0,  cyclic  AMP  shows  the  of and levels  effect  of  different and  50 fM)  clonidine prior is  c o n c e n t r a t i o n s of dobutamine on  c y c l i c AMP  (0.5 ^M)  presence  shown i n t h e  and  levels.  The  dobutamine  of u p p e r and  tively.  -79-  (0.5, effect  (50 pM)  /  and  lower  right  5.0 of  i n the antagonists  panel  respec-  Figure 0 A 1  200 c o o  6! 100  o a.  0f»  =• 200 c o o a. <  100  EL  0f» HH  P h e n y l e p h r i n e ($.o*M)  Clonidine  (2. O»N)  0 o b u l a m i n e ( S 0 . 0 »M)  <*>  Prazosin  Ofj  Yohimbine  P> M e i o p r o l o l 33/J3  (400/tM) (40o*M)  (4oo„n) < 400  »»)  S c l b u t o m o l ( 2 .o ^M)  S t a t i s t i c a l significance was determined using Paired " t " t e s t . * P < ** P <  0.05 0.02  -80-  Figure  10 B  200 i-  c o *3  '00  O  o c o  Dobutamine 0.5 5.0 50.0  200 Clonidine  MM.  Oobutamina  **  (5Q  jjjuj)  **  0.2 2.0 20 K9  o.  <  \s/  100  s// / / / /// / / / \ /ss  k  t77  /7/  /// /// /// /// /// /// ,///.  ///.  ///•,  ///\ /// ///  y//' '//,  sss ss/  'ss,  '//,\  '///  '//, •/// /// /// /// /// /// ss/  /// /// /// /// ///, ///\ , ///, ss/ sss. ssy  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 ' ' ^ **P P  < n'no < 0.02  ' ??* V l (non-stimulated c y c l i c nucleotide production). Bt3  bed  s  tesa  -81-  t  ,!  ^  Fig.  11  Effect  of clonidine  cyclic  GMP  old  rats.  of  i n methods.  either  0.4 mM  Samples were  of  yohimbine  o f any d r u g .  eight  Vertical  followed  in mM)  of d i f f e r e n t  i n the  i s t h e mean  out i n t r i p l i c a t e .  t h e SEM.  Panel A  concentrations  P a n e l B shows t h e e f f e c t o f  (0.5 /tM) on s a m p l e s  the absence  Results  of controls  Each v a l u e  bars represent  clonidine.  clonidine  pre-incu-  by a 3 m i n u t e  (0.5 /M).  experiments c a r r i e d  shows t h e e f f e c t of  o u t as  i n the p r e s e n c e o r i n t h e absence  e x p r e s s e d as p e r c e n t  absence  on  i n brown f a t f r a g m e n t s f r o m 7-day-  incubation with clonidine are  in vitro  I n c u b a t i o n s were c a r r i e d  described bated  and y o h i m b i n e  pre-incubated  or presence o f yohimbine  and t h e e f f e c t  o f yohimbine  -82-  (0.4 mM)  (0.4 alone.  Concentration  ( 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 "  test.  * 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 production.  -83-  needed  for  which  complete  i s bound  to  physiologically the  functional  determined  a protein  on  the  brown of  receptor.  fat  and  was  0.1  dissociation  of  cyclic  specificity sites.  In  of  of  the  -  to  kinase  cyclic  tissue.  GMP-  151  on  We,  15  -  40% u n d e r bound showed  is  of  about  therefore, which  the  the  cyclic  also  is  bound  binding  Dufau  AMP  The  bound  25% o f  the  total  appropriate  fraction  of  site  cyclic  105  al. of  fraction and  was  stimulation.  a dose-response to  et  content  GMP.  50 ^.M c o r r e s p o n d e d  is  dependent  .]  the  that  information  receptor  cyclic  was  experiments  experiments bound  of  nucleotide,  the  nucleotide  cyclic  nucleotide  bound  2-mercaptoethanol.  in  the  of  cyclic  out  of  of  carried  with  carried  presence  cyclic  were  nucleotide  cyclic  dissociation  binding  30 of  fraction  AMP  under  curve  -  160%  of  the  value). Control  unbound  applied  to  some  the  protein  tested  stimulation  (isoproterenol control  This  . modification[  values  isoproterenol  of  the  intracellular nucleotide  nucleotides  increase  absolute  state  is  protein  and p r o v i d e s  of  first  then  cyclic  to  A  It  GMP-dependent  component  kinase.  both  found The  fraction  technique  activation.  important,  regulatory  protein [51]  cyclic  activation  the  to  kinase  at  free  to  check  reassociation  receptor binding  protein to  its  nucleotide  4 ° C up  to  Re-association  nucleotide  was  experiments showed  following  -84-  in  no  any  of  receptor  observed  30 m i n u t e s  for  and  dissociation experiments,  2-mercaptoethanol  3 [ H] c y c l i c  out  no in  the  presence  carried  increase  . incubation  in for  out the  various  i n t e r v a l s of  between f r e e c y c l i c the  experimental  time thus nucleotide  conditions  experiments c a r r i e d out nucleotide  binding  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  to  and  used  the  receptor  i n our  to determine  i t s receptor  protein  assays.  the  Preliminary  specificity  protein  under  indicated  of  cyclic  binding  3  of  [ H]  cyclic  indicating ing  on  the  12  cyclic  rats.  and  specific  saturable  cyclic  nucleotide  GMP  level  clonidine  (0.5 pM)  i n brown f a t f r a g m e n t s f r o m determinations  incubation  media, the  separated  protein-bound  thus bind-  GMP  f r a c t i o n s of  3-4  fraction  3.4  Clonidine  produced  GMP.  nucleotide  The  then d e c l i n i n g g r a d u a l l y . ,  The  1,  forskolin  isolated  stimulating effect by  t o p r o t e i n was  25  the  content.  shown i n T a b l e  in adipocytes  was  concentration  in by  lipolysis  a  response  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 a d i p o c y t e s f r o m brown f a t o f 1-week-old r a t s clonidine .  GMP  out  releaseable  of  As  inhibited  fragments  - 30%  tissue cyclic  bound  7-day-  tissue  cyclic  m i n u t e s and  in-  were c a r r i e d  intracellular,  a peak o f c y c l i c  o c c u r r i n g between cyclic  fraction.  e l e v a t i o n of  protein-bound  rats.  of  nucleotide  the  total  effect  Cyclic  monophasic w i t h  The  number o f  shows t h e  time- dependent and  r a p i d , r e v e r s i b l e and  sites.  vitro  on  t o be  a finite  Fig.  old  AMP  prostaglandin  E  9  of and  -85-  (1.25 ^M)  f r o m brown f a t o f f o r s k o l i n was nicotinic  stimulated 1-week-old  partially  acid.  Table  2A  Fig.  12  Effect GMP  o f c l o n i d i n e (0.5 ^M)  i n f r a g m e n t s o f brown f a t f r o m  rats.  Brown a d i p o s e  interscapular minced w i t h 0.1  - 0.2  clonidine at  in vitro  tissue  was  scissors  suction.  isolated  and p o o l e d  i n a l i q u o t s of  was  added and t h e i n c u b a t i o n s were  by f i l t r a t i o n  on a N i t e x f i l t e r  nucleotides content  (O-o)  determined  and t h e s e p a r a t e d  by RIA.  of c y c l i c of c y c l i c  i n methods.  bars  (•-•),  GMP  means o f two t o f i v e (vertical  under  o f the incubatissue  fragments  a c i d and  In a d d i t i o n t o t h e t o t a l GMP  of values  the  ( D - D ) was  determined  determinations  the  percent  out  f o r each of the time  SEM)  periods  -86-  as  represent the in triplicate  and a r e e x p r e s s e d  in control  clonidine.  tissue  protein-bound  R e s u l t s shown  represent  stopped  T i s s u e p i e c e s were  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  described  cleaned,  F o l l o w i n g a 60 m i n u t e p r e - i n c u b a t i o n ,  Cyclic  fraction  from  g.  t i o n media  content  cyclic  7-day-old  r e g i o n o f n e a r l y 60 r a t s ,  1, 2, 3, 5, and 10 m i n u t e s .  separated  on  incubations  as  carried  i n t h e absence o f  - 8 7 -  shows  the  effect  of  forskolin  (0.625  ^M)  adipocytes.  increasing concentrations stimulated  Clonidine  tration-dependent was  antagonized  The  maximum  by  of  adipocytes  and h a m s t e r  brown  antagonizes  upon  cyclase.  was  still  35%  (Table  on  isolated capable 3).  (Table  (inhibitory  GTP  cyclase  for  cyclase is  protein)  forskolin of via  their  is believed  stimulatory  rat  of to  of  to  (Table  in  2B).  white  ].  Isobutyl-  of  adenosine  experiments, stimulate  brown  (l.>uM)  important  action  l i p o l y s i s by  sensitive  fat,  where  lipolysis  in  clonidine  as much a s  15  -  isobutylmethylxanthine  i n h i b i t i o n by  or  acts  adenylate an as y e t  to  inhibitory by  a direct  cyclase  by  action. not  thought  and Ns  shows b i p h a s i c r e g u l a t i o n quire  an  [58,169  set  clonidine  yohimbine;  formation  used  effect  of  a concen-  nicotinic  3) .  Adenylate Ni  was  inhibiting  also  is  inhibitory  another  of  on  isolated  in  yohimbine  AMP  7-day-old  Simulatory  l i p o l y s i s was  acid  the  In  from  of  5  adipocytes  isobutylmethylxanthine  adipocytes  at  l i p o l y s i s and c y c l i c  adenylate  effect  released adenosine  methylxanthine  100 /M  inhibitory  achieved  Endogenously regulator  The  in  glycerol release  increasing concentrations  antagonism  clonidine  glycerol release  inhibited  manner.  of  [173]  unidentified  to  coupled  (stimulating  GTP,  require  the  protein.  It  action by  on an  mechanism  -88-  to  protein).  because both  Forskolin  or  be  the  both It  proteins  stimulation  of  presence  of  either  has  suggested  the  been  catalytic  indirect [22,  action  189].  adenylate  that  sub-unit mediated  Thus,  re-  the  activation by  inputs  of adenylate  to  We,  from B o r d e t e l l a therefore,  action  As shown  t o x i n was  pertussis selectively  considered  i t of i n t e r e s t  o f r a t brown f a t a d i p o -  associated  of alpha-2 agonist  with a decrease  ( c l o n i d i n e ) on  i n T a b l e 4, brown a d i p o c y t e s  rats pre-incubated with pertussis  toxin  from  with  lipolysis. treated  f o r s k o l i n showed  The b a s a l  cells  was  of l i p o l y s i s  a minor  lipolysis.  Clonidine,  Ineffective  in inhibiting  lipolysis. 7-day-  subsequently  1.5 x t h e n o r m a l r a t e o f  1.3 x t h e n o r m a l ,  probably maintains  ;  rate  i n the  (0.5 mg/ml) f o r  3% h o u r s t o 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 N i and stimulated  affected  (Ns) o r i n h i b i t o r y ( N i )  i f such pre-treatment  by p e r t u s s i s  inhibitory  old  toxin  Ni p r o t e i n .  investigate  cytes  by f o r s k o l i n c a n be  a f f e c t i n g the s t i m u l a t o r y  protein. Pertussis blocks  cyclase  in pertussis  suggesting Ni  i n h i b i t o r y input  i n a concentration the l i p o l y t i c  toxin  protein  on t h e r a t e o f  o f 1 / i M t o 5/tM,  was  r e s p o n s e o f brown  adipocytes to f o r s k o l i n . Interestingly, peated using effect  of c l o n i d i n e  stimulated contrary, an  adipocytes  when t h e same e x p e r i m e n t s were r e f r o m h a m s t e r brown f a t , no i n h i b i t o r y  on f o r s k o l i n o r  glycerol release prostaglandin  E  2  was  isobutylmethylxanthine  observed  and n i c o t i n i c  (Table acid  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  / i n h a m s t e r a d i p o c y t e s a s shown  i n table  -89-  5B.  5A).  still  On t h e showed  glycerol  release  Table  2  Drug ( s )  Glycerol release >c.mol/mg p r o t e i n  B a s a l (4)  0.165 +•0.015 * 0.295 +•0.007  Forskolin  ( 1.25  uM) (4)  Forskolin  + PGE  2  0.1 mg/ml (4)  Forskolin  + PGE  2  0.5 mg/ml (4)  Forskolin  + PGE  2  1.0 mg/ml (4)  ***  Forskolin  + PGE  2  5.0 mg/ml (4)  ****0.189 + 0.015  Forskolin  + nicotinic  a c i d (40 ^M) ( 2 )  ** 0.210 + 0.005 0.25 + 0.027 0.22 + 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 < 0.01 for forskolin + Vs forskolin stim. glycerol release. 0.05 for forskolin + vs forskolin stim. glycerol release. 0.05 for forskolin + vs. forskolin stimulated glycerol release.  * * * * *  1. E f f e c t o f P r o t a g l a n d i n ( P G E ) 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 f r o m 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 v a l u e i s t h e mean +SEM o f f o u r e x p e r i m e n t s done in duplicate.  * * * *  P  P G E  P  <  P G E o  P  <  P G E  -90-  2  2  Table  2A.  Drug  (s)  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 of c l o n i d i n e on 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 mean 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 two 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 ..• the b a s a l non-stimulated g l y c e r o l l e v e l .  Glycerol release /_mol/mg p r o t e i n . Expt.  Basal Forskolin  (0.625  /tM)  # 1  Expt.  # 2  0. 5 8 9  (100)  1. 0 1 4  (100)  2. 5 8 9  (439. 56%)  5. 286  (521. 30%)  1. 1 1 5  (189. 30%)  1. 6 7 1  (164. 79%)  Forskolin  +  clonidine  (5  Forskolin  +  clonidine  (1.0  AM)  1. 4 1 4  (240. 06%)  2. 4 4 7  (241. 32%)  Forskolin  +  clonidine  (0.5  ^M)  1. 1 7 5  (199. 49%)  2. 9 4 8  (290. 73%)  Forskolin  +  clonidine  (0.1  /*M)  1. 8 9 2  (321. 22%)  3. 0 6 1  (301. 87%) .  jtM)  -91-  Table  Drug  2B.  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 / t 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 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 - w e e k - o l d 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 o f y o h i m b i n e . R e s u l t s a r e t h e mean 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 show t h e r e s u l t s e x p r e s s e d as 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 .  Glycerol release //jnol/mg p r o t e i n  (s)  Expt. Basal  # 1  Expt.  # 2  0.340  (100)  0.152(100)  0.564  (165.88%)  0.257(169.07%)  Forskolin  (0.625/u.M)  Forskolin  + clonidine  (1.0 KM)  0.350  (102.94%)  0.187(123.03%)  Forskolin  + clonidine ( 1 . 0 /»-M)  + yohimbine (O.5X10"9M)  0.389  (114.4%)  0.199  + c l o n i d i n e + yohimbine (1.0 (0.5X10-8M)  0.369  (108.53%)  0.389  (114.41%)  0.427  (125.59%)  0.466  (137.06%)  Forskolin Forskolin  + clonidine + ( 1 . 0 A<-M)  Forskolin  (1.0  Forskolin  fiM)  '0.245  (161.18%)  0.212  (139.47%)  yohimbine  (0. 5x  + clonidine +  (130.92%)  10~ M) 7  yohimbine  (0.5x  10  _ £ >  M)  + c l o n i d i n e + yohimbine (1.0 ^M) ( 5 . Ox iom)  0.221 (145.39%)  Table  Drug  3.  E f f e c t of c l o n i d i n e ( o<-,-agonist) and n i c o t i n i c a c i d on i s o b u t y l m e t h y l x a n t h i n e (MIX)-stimulated 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 brown 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 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 two 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 show t h e r e s u l t s e x p r e s s e d as t h e p e r c e n t of the 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 level.  Glycerol release ^mol/mg p r o t e i n  (s)  Expt.  # 1  Expt.  0.463  # 2.  .(100%)  Basal  0.459  (100%)  MIX  0.610  (132.9%)  0.780(168.47%)  ( 1 0 0 /_M)  MIX + c l o n i d i n e  ( 1 A M)  0.510  (111.11%)  0.483 (104.32%)  MIX + c l o n i d i n e  ( 0 . 5 p-U)  0 . 6 0 3 (131.37%)  0. 591 (127.64%)  0 . 5 0 8 (110.67%)  0.605 (130.67%)  MIX + n i c o t i n i c a c i d  (40 ftM)  -93-  Table  4  Drug  (s)  E f f e c t of 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 on 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 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 and PGE2 i n r a t a d i p o c y t e s . Brown 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 were i s o l a t e d as d e s c r i b e d i n methods and 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 3 7 ° 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 was m e a s u r e d i n t h e f i l t e r a t e by 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 mean o f t h e p e r c e n t o f b a s a l (non-stimulated) g l y c e r o l l e v e l f o r each experiment.  Control group  Basal  0. 403+0. 085  Forskolin  (1.25  Forskolin  +  Forskolin  A  M)  * * 0. 755+0. 101  clonidine (5 A M ) + clonidine (1.0 ^ M )  Forskolin  + PGE  Forskolin  + PGE2  Statistical  ^mol/mg  Pertussis toxin p r e - t r e a t e d group protein  (100%) (202.58+27 .14%)  0. 566+0 .147 ***0. 902+0 .242  "kick  (160.19+6.42%)  0. 456+0 ..173 (112.38+28 .19%)  0. 884+ 0.207 (159.07 + 4.17%)  0. 581+0. 251  (125.21+37 .97%)  1. 071+  0.144  (162.85+4.17%)  (136.18+9. 15%)  1. 019+  0.203  (154.64+5.68%)  (134.22+10 .82%)  1. 03 + 0.302  2 ( 0 . 5 ^ ) 0. 535+0. 101 (0.1/<M) 0. 592+0. 098  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 *  * P < 0.01 f o r s k o l i n * * P < 0.02 f o r s k o l i n * * * P <0.05 f o r s k o l i n forskolin  (100%)  test  + 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 . Vs b a s a l g l y c e r o l r e l e a s e . + c l o n i d i n e Vs 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 Vs b a s a l g l y c e r o l release i n p e r t u s s i s t o x i n pre-treated animals. -94-  (125.52+16.31%)  Table 5A Effect of clonidine (c< 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, 2  -  Glycerol release A-mol/mg protein  Drug (s) Basal (4) Forskolin Forskolin Forskolin Forskolin Forskolin  0.278 + 0.015  (1.25 A M ) (4) + clonidine (5.0 A M ) + clonidine (1.0 A M ) + clonidine (0.5 A M ) + clonidine (0.1 A ) M  Basal (2) M I X (100 (2) M I X + clonidine ( 5. A M ) (2) MIX + clonidine (1.0 A M ) ( 2 ) M I X + clonidine (0.5AM)(2) MIX + clonidine.(0.1 A M ) ( 2 ) A M )  -95-  0.306 + 0.024  (4) (4) (4) ( )  0.287 + 0.041 0.309 + 0.035 0.304 + 0.035 0. 300 .+ 0.034  4  Expt. # 1  Expt. # 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  Table 5B  Effect of prostaglandin (PGE^) and nicotinic acid on forskolinstimulated glycerol release in adipocytes isolated from brown fat of adult hamsters. Each value is the mean of duplicate determinations from one to two experiments. Number in the parenthesis show the results expressed as the percent of the basal non-stimulated glycerol level.  Drug (s)  Glycerol release ^.mol/mg protein Expt. #1  .:. ExPt. # 2.  Basal (2) Forskolin (1 .25 AM) (2) Forskolin + PGE (5 mg/ml) (2) Forskolin + PGE (1 mg/ml) (1)  0. 209 0. 279 (133.49% ) 0. 21 7 (1 03.80% ) 0. 233 (11 1 . 4 8 * )  Forskolin  +  PGE (0.1 mg/ml)(1)  0. 263 (125.84% )  Forskolin  +  nicotinic acid (40 ju.M)  0. 233  2  2  2  (111.1.4  %  )  0. 363 0. 457 0. 297  3.5  Effect of o<2- and >S — agonists in vitro on cyclic GMP content in brown fat fragments from obese (ob/ob, 2month-old) mice and their unaffected littermates. n  We in our,initial 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 pronounced 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 exhibited 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 ©<.agonist and ^ ~agonist. Cyclic GMP production in the presence of clonidine ( ©<-agonist) in obese mice was nearly the same as that observed in unaffected control littermates. However, a lower basal cyclic GMP concentration was seen in the brown fat of obese mice than their lean littermates'. 2  /  1  2  -97-  Fig. 13A !  Effect  of cold  content  i n brown a d i p o s e  obese m i c e  (C57BL/6J-ob,  littermates represent 3 - 4  F i g . 13 B  exposure  tissue  GMP  of g e n e t i c a l l y  2-month-old).  s e r v e d as c o n t r o l s .  Lean  V e r t i c a l bars  t h e +SEM o f 6 - 8 d e t e r m i n a t i o n s on  animals.  C: a n i m a l s  Effect  of norepinephrine  weight,  i . p . ) on c y c l i c  adipose  tissue  served  (6°C) on c y c l i c  a t room  (0.1 mg/100 g body GMP c o n t e n t  o f obese m i c e .  as c o n t r o l s .  temperature.  Lean  V e r t i c a l bars  i n brown littermates represent  t h e +SEM o f 6 - 8 d e t e r m i n a t i o n s on 3 - 4 animals.  C: a n i m a l s  injected  -98-  with  saline  only.  - 9 9 -  Fig.  13  C  Effect  of  cyclic  GMP  of  obese  clonidine  (0.5  jiM)  in vitro  c o n c e n t r a t i o n s i n brown  (ob/ob,  2 months  old)  littermates.  Tissue  f r o m up  20 m i c e w e r e  incubated  aliquots  of  0.1  -  0.2  0 . 5 pM c l o n i d i n e o r were the  f i l t e r under  extracted  expressed mean o f single  were  by  10%  four  three  suction.  pooled  minutes  separated  RIA. and  with  Reactions  water,  and  immediately  Cyclic  nucleotides  trichloracetic acid  as pmol/mg p r o t e i n , to  the  ( i n  ice-cold  i n t r i p l i c a t e by  two  fragments  fragments  50 /uM d o b u t a m i n e .  t i s s u e fragments  determined  a  for  s t o p p e d by c o o l i n g i n  on a N i t e x were  g)  fat  m i c e and  unaffected to  on  and  Results  each value  separate determinations  experiment.  -100-  are is from  the  CLONIDINE  0  DOBUTAMINE (5.0  •101-  0 . 5 « M  *M)  CHAPTER  4  DISCUSSION  4.  A hormone can have m u l t i p l e e f f e c t s  A tissue  or c e l l s  under d i f f e r e n t the  respond  both  circumstances.  same hormone  Norepinephrine  the acute  stimulation  long-term  of c a l o r i g e n i c  adipocytes  and  components  for  adrenoceptors  the r e l a t i v e  preferentially  to d i f f e r e n t  of n o r e p i n e p h r i n e  different animal's  physiological growth.  The  of utmost s i g n i f i c a n c e effects  t o two  regulation  mediated  that  effector  effects  birth  Therefore,  the  important and  [18 0 ] .  can  the  binding in vivo  be m o d u l a t e d  under  stages of  of norepinephrine development.  functional  an  are  These  role  of  capacity  one  norepinephrine  exerted v i a c y c l i c  ^ - a d r e n e r g i c s t i m u l a t i o n was  -102-  effects  when t h e brown f a t r e a c h e s  The  of t i s s u e m a t u r a t i o n  of i t s  different  systems,  during p e r i n a t a l  i t s differentiation  weeks a f t e r  Norepinephrine  the f i n a l  c o n d i t i o n s or d i f f e r e n t  trophic  tissue  on w h i c h r e c e p t o r s a r e  assuming  stimulation  become p a r t i c u l a r y  t h e peak o f  initiates  affinity  determine  Depending  o c c u p i e d , and  i s the  i n t h e p l a s m a membrane o f brown  n o r e p i n e p h r i n e may  are l i n k e d  effects  It  f u n c t i o n of  p r o p o r t i o n s and  of a d r e n e r g i c s t i m u l a t i o n .  from  efferents  o r b o t h c<- and y f l - r e c e p t o r s .  tissue.  differently  a d a p t i v e changes i n brown f a t [ 1 8 0 ] .  phenotype of the  sites  nerve  or  released  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 .  can b i n d t o e i t h e r  in  to the  s t o r a g e g r a n u l e s of the sympathetic  principal  and  can  in a c e l l  suggested  by  GMP-  Skala  and  Knight  as e a r l y  as  1979  supported  section  4.3). ©^-adrenergic m e d i a t i o n of the also  our  T h i s proposed  further  p r o c e s s was  by  [178].  in vitro  observed  Thus i t a p p e a r e d  that  from  system  to s e a r c h f o r the presence and j8^-  o f p^-,  shown i n membranes f r o m [24,  .127,162],  ligand  a d r e n o c e p t o r s had  4.1  brown  excellent  t h e brown a d i p o s e  in section  [146], adipose  model  of ©^-adrenoceptors.  we  The  previously  tissue  i n brown f a t o f o n e - w e e k - o l d  the presence rats  been  of a d u l t  d i s c u s s the r e s u l t s  b i n d i n g study demonstrating  ceptors  an  in  proliferative  proliferating  r a t s would be  was  (discussed  i n other eukaryotic c e l l s  tissue  presence  experiments  the a c t i v e l y  one-week-old  hypothesis  of  rats  of  our  c^^-adreno-  using highly  specific  o^-ligands. Studies  of adrenoceptors  help  to e l u c i d a t e  that  a hormone c a n have m u l t i p l e  cells  can  respond  circumstances. a hormone c a n molecules  cell  under  divergence of these m u l t i p l e  affinities  which e f f e c t  unit.  actions  conditions.  response  level: one  units the  would  cellular  t h i s mechanism a  i n accordance  Experimental modulation  -103-  of  relative  ( s ) would p r e d o m i n a t e By  same  different  of these d i s c r i m i n a t o r  t o t h e same h o r m o n a l s t i m u l i . i t s own  The  fact  The  p r o b a b l y b i n d t o more t h a n  "discriminator/effector"  can modify  vailing  in a cell.  o c c u r a l r e a d y a t t h e p l a s m a membrane  then determine response  effects  t o a hormone d i f f e r e n t l y  The  abundance and/or  can  a b a s i c mechanism r e s p o n s i b l e f o r t h e  o f t h e hormone c a n  type of the  i n brown a d i p o c y t e s  w i t h the  of the  pre-  tissue  conditions the  receptors  relevance the  by c h e m i c a l  of the receptor  determined  binding  o f our s t u d y  the binding  system  and c h r o n i c  sites  (discussed  f o r the t i s s u e .  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 -  i n 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  of norepinephrine  i s not s u f f i c i e n t responses.  in itself  the adipocytes. AMP  associated  to  phatidic  to e l i c i t  (s) that  sites  specific sub-cellular  t h e message c a n be  I t h a s been known f o r many y e a r s  with  t h e n a t u r e o f t h e c*^-adrenoceptor  the turnover  acid.  i n the adrenergic  s t i m u l a t i o n of yfl-adrencceptors.  system, ^ - a d r e n o c e p t o r  increase  to the o^-receptor  i s t h e s e c o n d messenger  i s known a b o u t effector  exposure t o  I t i s o n l y when t h e h o r m o n a l s i g n a l s a r e p a s s e d  on v i a e f f e c t o r s y s t e m  cyclic  chemical  ( an e < 2 - - n t a g o n i s t ) .  Binding  by  In  i n s e c t i o n 4 . 2 ) , we  sympathectomy by 6-hydroxydopamine o r by c h r o n i c yohimbine  blockade of  h a s been used t o d e t e r m i n e t h e f u n c t i o n a l  second p a r t  ceptor  sympathectomy  This  deciphered that pathway  Much l e s s associated  s t i m u l a t i o n h a s been shown  o f p h o s p h a t i d y l i n o s i t o l and p h o s -  probably  i s associated  with  an i n f l u x  2+ o f Ca lease  through o f bound  associated  'gates'  intracellular  e f f e c t o r system  [178-180] had i m p l i c a t e d mediator during  i n the f a t c e l l 2+ Ca  membrane o r w i t h r e -  . c< 2 ~ a d r e n o c e p t o r and i t s  i s the least  the c y c l i c  of responses associated  GMP  with  understood.  s y s t e m as t h e p o s s i b l e  ©V-receptor  brown f a t g r o w t h and m a t u r a t i o n .  have a l s o s u g g e s t e d  involvement  of c y c l i c  -104-  Skala  Several GMP  stimulation other  studies  i n the develop-  mental [126]  processes and  to note was  [40]  i n the p r o l i f e r a t i o n  spleen c e l l s  [47].  In t h i s  observed  and  in tissue  rats  seemed t o be  cyclic  g r o w t h and  ©^-adrenoceptor  GMP  are  a very suitable  and  lation  and  cyclic  r a t s by  GMP  a series  Thus,  implicated  processes both  t o p l a y some  effector  infant  of  system.  the We  between ©(-adrenoceptor  accumulation of i n v i t r o  i n brown f a t o f  experiments  further stimu-  one-week-  (discussed  in  4.3). The  inhibitory  and  cyclic  has  been e x t e n s i v e l y  AMP  o f our  isolated  c e p t o r s and  It  on  7-day-old  complements our demonstrates  characteristics adipocytes  [ 3 , 27,  investigated  stimulation from  o f «*2~adrenoceptors on  i n human and  studied  s t u d y , we  4.4)  action  accumulation  o^-adrenoceptor  section  [146].  model f o r s t u d y  i t s associated  the h y p o t h e s i s of a l i n k  cytes  i t is interesting  m a t u r a t i o n , and brown f a t o f  tested  part  context  i n other eukaryotic c e l l s  role  section  hepatocytes  t h a t ©c^-adrenergic m e d i a t i o n o f p r o l i f e r a t i v e  ©^-adrenoceptors  old  of  similar  ligand  that  101-103].  the  these  adipocytes In the  inhibitory  lipolysis rats.  hamster  lipolysis  fourth  action  of  i n brown f a t a d i p o -  This study  (discussed  binding studies r e c e p t o r s show  to ^ - a d r e n o c e p t o r s  on  e<2  in - a d r e n o  ~  physiological  reported in  from white f a t . i s known t h a t  thermogenesis  defective  i s associated  brown a d i p o s e  with obesity  tissue  i n a number  of  d i f f e r e n t types o f obese animals. Our i n i t i a l i n vivo study, showed time-depen-  -105-  pendent from  changes  l e a n mice  i n the c y c l i c following  administration. exhibited similar  cold  stimuli.  considered  obese  mice  was  exposure  GMP  GMP  during  mice  c o n c e n t r a t i o n when e x p o s e d  to further  concentration  in vitro  (6°C) and n o r e p i n e p h r i n e  cyclic  and o b e s i t y .  i n f l u e n c e d by c o l d  o f brown f a t  brown f a t o f obese  suggested that  i t of i n t e r e s t  i f cyclic  stimulation  This  i n thermogenesis  study  concentration  Contrary to that,  no change i n c y c l i c  have a r o l e  GMP  pursue  GMP  We,  system  to  could  therefore,  this  problem to  i n brown f a t f r a g m e n t s o f exposure  and <?<-receptor  w i t h ©<.- and J3-  incubation  adrenergic  agonists.  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 o f c<..-adrenoceptors i n p l a s m a membrane f r a g m e n t s o f brown f a t from 7 - d a y - o l d r a t s . The  p r e s e n c e o f p^-  p r e v i o u s l y been shown of a d u l t  rats  o^-  and  fi^-  i n membranes f r o m brown a d i p o s e  [ 2 4 , 127, 162, 1 9 J ] .  s t u d y had y e t b e e n u n d e r t a k e n w h i c h  in this  f r o m brown f a t o f i n f a n t binding  characteristics  subclass. used  Highly  membrane  rats  demonstrated  the presence  The main o b j e c t i v e o f  i s t o show t h a t  contain  binding  membranes  adrenoceptors which  show  compatible w i t h those expected of °<2  selective  to study t h e i r  part  tissue  However, no l i g a n d  o f .^-.-adrenoceptors i n r a t brown f a t . our s t u d y d i s c u s s e d  adrenoceptors has  binding  - a n t a g o n i s t s and a g o n i s t s characteristics  to isolated  were plasma  fragments. One o f t h e g r e a t e s t  problems  -106-  we  encountered  -  i n our  binding  s t u d i e s was  the s c a r c i t y  brown f a t o f s e v e n - d a y - o l d we  were u n a b l e  and  perform  to carry  statistical  The  of the t i s s u e  rats.  Because of t h i s  treatment  of a l l our  method d e s c r i b e d by Maeda e t a l . [ 1 1 7 ] . prepared  from a v a r i e t y yields  of  assays.  shown t h a t  not a l t e r  In a d d i t i o n ,  fication  for equilibrium have  membranes  other c e l l affinity  n  l i 9  d  a  n  d  s  a  n  d  types and  214  ].  to  of these  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  and  characterization  made t o t e s t  ceptors using  the p u r i t y studies  specifi-  carried  c o r t e x and  similar  i n view  does  plasma  one-week-old r a t b r a i n  r e p o r t e d [24,163,  Initial  and  from  a  have  technique  Svoboda e t a l . [ 1 9 2 ]  showed b i n d i n g c h a r a c t e r i s t i c s v e r y  considered  the  Ligand binding studies  using selective  already  was  suited  by membranes f r o m  the  t h e p l a s m a membranes  t h e maximum b i n d i n g c a p a c i t y ,  membrane f r a g m e n t s fat  according to  have shown t h a t well  experiments  data.  the minor c o n t a m i n a t i o n of i s o l a t e d  of the a d r e n o c e p t o r s .  out by  difficulty  These a u t h o r s  characterized  t i s s u e s and  from brown f a t c e l l s  city  and  p l a s m a membrane f r a g m e n t s  binding  the  out e x t e n s i v e m e t h o d o l o g i c a l  p l a s m a membranes were p r e p a r e d  successfully  from  o f a d r e n o c e p t o r s , and  brown  those studies  we  identino  attempt  o f our membrane p r e p a r a t i o n s .  undertaken  to characterize ©^-adreno-  [ H ] - y o h i m b i n e , [ H]-RX-781094, [ H ] - c l o n i d i n e 3  3  [ H]-norepinephrine  that  t h e b i n d i n g was  tion  occurring  ( F i g . 1,  reversible  at l i g a n d  3  2A, and  B,  2C,  and  D)  showed  saturable with half  c o n c e n t r a t i o n s of approximately  -107-  satura15  nM  (yohimbine), tical  10 nM ( R X - 7 8 1 0 9 4 )  treatment  obtain ited  of  analysis  of  assays.  plot  (Fig.  of  t h e membrane  Nevertheless,  was p e r f o r m e d .  Scatchard  1A)  adipocytes  f r o m human a b d o m i n a l  value  m a x  (156 f m o l / m g  r e p o r t e d by Tharpe for  human w h i t e  18 nM o f  those  24 0 nM [ 6 6 ]  protein)  and L e f k o w i t z  fat adipocytes  kinetic  from  and those  reported  t i s s u e 4 - 5 nM  [19 5 ] .  (145  from  hamster  fmol/mg  The t e m p r a t u r e  in  [195].  w a s i n t h e same r a n g e  i n 19 81  lim-  yohimbine  reported  SC a d i p o s e  only  for a very  i n some i n s t a n c e s ,  was between  f a t pad a d i p o c y t e s  B  protein  The c a l c u l a t e d  epididymal  The  Statis-  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  s u f f i c i e n t amount  number  a n d 25 nM ( c l o n i d i n e ) .  as  protein) dependence  3 and r e v e r s i b i l i t y o f dicates  that  receptor binding agent  the binding  sites.  (Fig.  2B)  of  binding  sites  i s at  The l o w e r  using yohimbine  agonist  these  the s p e c i f i c  compared t o  Both  clonidine  i n -  oi^-  H]-clonidine displacing  clonidine i s a partial  a n d may n o t o n l y  than yohimbine  [  c l o n i d i n e as the  could r e f l e c t the fact  as w e l l  [ H]-RX-781094  the p h y s i o l o g i c a l l y active 3  saturation of  i m i d a z o l i n e group  sites  binding of  but a different  have  different  affinity  for  the  [66],  (-)-epinephrine  and yohimbine  were  equipotent  in  3 displacing  (-)[  H]-norepinephrine  from  i t s binding  sites  (Fig.  3 3A).  When  (-)[  H]-epinephrine  and yohimbine  were  used alone  to  3 d i s p l a c e (-)[ H]-norepinephrine from i t s binding s i t e s , a 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 and 0 . 2 4 pmol/mg max protein indicate  r e s p e c t i v e l y were that  both  obtained.  (-)-epinephrine -108-  Similar  and & m a x  and yohimbine  values  are competing  with  (-)[ H ] - n o r e p i n e p h r i n e b i n d i n g f o r t h e  same r e c e p t o r w i t h s i m i l a r problem  further,  affinities.  a combination  To i n v e s t i g a t e  of yohimbine  this  and ( - ) - e p i n -  3  e p h r i n e was u s e d  to displace  (-)[ H ] - n o r e p i n e p h r i n e  from i t s  3 binding  sites.  The s p e c i f i c  ephrine  i n the presence  e p i n e p h r i n e was of e i t h e r is  and t h e y  the s p e c i f i c  bine  shared  epinephrine. 1.4 a l s o  f o r yohimbine density detect  single Both  finding  value site  the presence  non-sharing binding i t showed n e a r l y  site  t h e same  (71 fmol/mg p r o t e i n ) . Y o h i m -  with norepinephrine but not w i t h coefficients  of approximately  o f more t h a n one b i n d i n g  binding site  makes  site  of the other r e c e p t o r s i t e s - single  displacing  low  i t more d i f f i c u l t  i t seems t o be masked by t h e r e l a t i v e l y  ligand  one When  and e p i n e p h r i n e ( F i g . 3 B ) . The r e l a t i v e l y  higher density using  second  of the second since  B  Identical H i l l  support  of t h i s  one common b i n d i n g s i t e .  b i n d i n g o f the second  this  i n the presence  and e p i n e p h r i n e b i n d t o more t h a n  share at l e a s t  nM) b u t a l o w e r  o f y o h i m b i n e and  interpretation  was a n a l y z e d by S c a t c h a r d a n a l y s i s (64.5  o f (-)[ ^ - n o r e p i n -  30% h i g h e r t h a n  A possible  that both yohimbine  site,  of a combination  approximately  one a l o n e .  displacement  to  much  i n experiments  agent.  ( - ) - e p i n e p h r i n e and ( - ) - n o r e p i n e p h r i n e a r e known  t o be mixed  adrenoceptor  b o t h theoi-  and fS-  agonists, that  adrenoceptor  i s , they b i n d to  subclasses.  However, i n  contrast  to norepinephrine, epinephrine exhibits  affinity  f o r the  sites  than  f o r t h e J3 - s i t e s  -109-  a higher in in vitro  K  d  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  4A) , t h e higher displacement of C-) - [ H]-norepinephrine  (Fig.  (-)-epinephrine  i n a lower  practolol  suggests  competing  f o r two  t h e e*-  for by  and jS-  the r e s u l t s  can  displace,  that  o f Fig.4B  Conversely,  analysis that  using  has  even  constant  (K^)  t h e Cheng and  that  are likely  supported  (-)-epinephrine  (-) ^ ^ - n o r e p i n -  i n the presence  of  prac-  i n F i g . 3A  u s i n g the  (-)-epinephrine  o f 66  obtained  from  S i m i l a r °^2~  specificity  An  [35] was  the  Thus  rats  from  f o r epinephrine  Scatchard  at  i n the of  i n several other  almost  it-seems  norepinephrine  7-day-old  p r e v i o u s l y been r e p o r t e d  nM  same l i g a n d .  competes w i t h  i n brown f a t o f  of e p i n e p h r i n e .  Prussoff equation  of yohimbine  range.  This i s  manner,  even i n t h e p r e s e n c e  to the  centration  practolol  p r a c t o l o l displaced [ H]-norepinephrine  dissociation  o^-receptors  indicate  i t s binding sites  binding sites  identical  which  than  o f r e c e p t o r s - most  i n a dose-dependent  tolol.  calculated  types  and  receptors respectively.  from  apparent  (-)-epinephrine  different  ephrine  its  range of c o n c e n t r a t i o n  by  the  lower  con-  epinephrine studies  [204,  205,206 ] . 3  Saturation binding studies carried dihydroalprenolol and  10  as t h e  practolol  showed r a p i d ,  10 nM  and  10 ^u-M  using  reversible  ( F i g . 5A).  ( n o r e p i n e p h r i n e ) was  The  -110-  binding to a  dissociation  similar  (-)[  H]-  (-)-norepinephrine  as t h e n o n - l a b e l l e d d i s p l a c i n g  s a t u r a b l e and  number o f r e c e p t o r s of  ligand  out  agents finite  constant  to that reported  by  (K^)  Williams  et  tissue.  With  as t h a t  al•  [214]  practolol,  reported  membrane  in rat  by  adipocytes  the  of  Bukowiecki  fragments  from  adult  et  from  50 nM w a s al.for  rat  parametrial  brown  nearly  the  same  (-)-propranolol fat  in  The B  [24].  max value  of  0.19  membrane  Svoboda  10  cells  et or  cells).  preparations  al• 46  rats  This  demand rats  and  for  is  for  the / ^ - a d r e n o c e p t o r s  somewhat  higher  i n hamster  fmol/mg  protein  brown  c o u l d be  due  to  the  associated with Yohimbine,  an  need  i n brown  (92  fat  fmol/ protein  of  [180]«  norepin-  differen-  cold-acclimated  was  quite  our  Increased  increased ^-adrenoceptors o^-antagonist,  in  one-week-  growth,  capacity fat  for  in  our  reported  2 mg m e m b r a n e  thermogenesis  increased functional  in  of ^ - a d r e n o c e p t o r s  i n c r e a s e d brown  thermogenesis  those  cells  accentuated  non-shivering by  than fat  assuming  i n c r e a s e d number  accompanied  tiation,  was  [191]  ephrine-stimulated old  protein  preparations  by  /10  pmol/mg  adult  number  [24].  effective  3  in  displacing  of  39 n M .  for  This  yohimbine  ments (34  from  nM)  by  has been This  (-)[  H]-dihydroalprenolol  value  is  binding  used  et  kidney al.  extensively  displacement  of  (-)[  rather  s u r p r i s i n g because  ligand  i s not  or  to  the  5B)  reported  with  absolutely  the yfl-adrenoceptors  [  (48  nM)  20], in 3  and  calf  frag-  cerebral  cortex  ^-adrenoceptors.  H]-dihydroalprenolol would  specific  i n brown  Kd  (-)[3H]-d ihydroalprenolol  characterizing  this  a  value  t o © ^ - a d r e n o c e p t o r s i n membrane  guinea-pig Brodde  close  (Fig.  for  fat  -111-  indicate  of  the  by that  yohimbine either  the  ^-adrenoceptors,  infant  rats  have  was  '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 binding site for [ H]-DHA [41,133, 166']. Its high affinity binding site is sensitive to displacement by ^-agonists and antagonists, whereas the second, low affinity site is occupied by ^-antagonists [ 133, 166]. Bukowiecki et a l . in their study were unable to see any dis3 placement of (-)[ H]-DHA binding to partially purified membrane fragments from brown fat of adult rats by phentolamine or phenoxybenzamine. 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 system on brown fat plasma membrane are expressed fully only during the early ontogenic stages of animal's l i f e . 3  -112-  The  presence  fat  of  adult rats  has  been  the  forskolin-  and  isoproterenol-  of  respiration It  of  in isolated  adrenoceptor  suggested  which the  conditions  teristics  of the  brown a d i p o c y t e s thus  ephrine the  the  to note  brown  indicated induced  by  suppression  lipolysis  t h a t Kunos and changes  observed  i n v i v o may  by  react  Ishac i n «*^-  them on  could also  c o u l d be  system  to  modulated  and  isolated  charac-  i n p l a s m a membrane  t o an  J3^-  altered  change d u r i n g o n t o g e n i c response  [100]  adaptive  [100] . I t i s p o s s i b l e t h a t t h e  adrenoceptor  and  [190].  of  development,  identical  norepin-  a c c o r d i n g t o the  age  of  animals. In summary, t h e m a j o r f i n d i n g s  study (i)  the  c o u l d r e p r e s e n t a new  tissue  intracellular  stimulus  in  that r e c i p r o c a l  in vitro,  metabolic  and  indirectly  s t i m u l a t e d responses,  hepatocytes  mechanism by  even  adipocytes  is interesting  have r e c e n t l y  rat  u -.-adrenoceptors  of t h i s  p a r t of  our  are: 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  Binding agonists fat  of  and  t o p l a s m a membrane f r a g m e n t s f r o m  1-week-old  as e x p e c t e d  rats  show b i n d i n g  brown  characteristics  of b i n d i n g to s p e c i f i c ex_-adrenoceptor  sites. 3  (ii)  Binding of  (-)[ H ] - n o r e p i n e p h r i n e  presence  o f more t h a n  ephrine,  yohimbine  shared  one  one  and  indicated  binding site.  (-)-epinephrine  common b i n d i n g s i t e ;  -113-  the  the  Norepinprobably  other  site  seemed  t o be p r e s e n t i n much l o w e r number and was yohimbine  specific for  and n o r e p i n e p h r i n e o n l y .  3  (iii)  (-)[ H ] - n o r e p i n e p h r i n e bound t o b o t h °<- and/3adrenoceptors.  B i n d i n g t o © c - a d r e n o c e p t o r s was 2  d i s p l a c e a b l e by y o h i m b i n e in  lower  and by  (-)-epinephrine  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 Site  for ^-adrenoceptors.  other than  I t was  this  postulated that  sites.  similar  The s e c o n d  cated b i n d i n g to s i t e s  due t o  to those  site  indi-  the /^-adrenoceptor.  b i n d i n g component  the ^ - a d r e n o c e p t o r .  was  .......  E f f e c t o f c h e m i c a l sympathectomy and 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 on 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 brown f a t o f 7 - d a y - o l d r a t s . Ligand b i n d i n g experiments  show t h a t binding  the p r o p e r t i e s  sites  carried  of f u n c t i o n a l l y  c a n be a l t e r e d  considered  i t a p p r o p r i a t e to study  of ^ - a d r e n o c e p t o r s In t h i s  s e c t i o n we  following  rats  active  agent.  the b i n d i n g  in vivo  tissues  adrenoceptor  We,  and by  therefore,  characteristics  experimental  modulations.  discuss the trend 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 ^ - a d r e n o c e p t o r s infant  out i n other  by c h e m i c a l sympathectomy  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  bine  two b i n d i n g  1 showed b i n d i n g c h a r a c t e r i s t i c s  reported  4.2  showed  following  i n membranes i s o l a t e d  f r o m brown  c h e m i c a l sympathectomy  administration.  -114 —  f a t of  or c h r o n i c yohim-  6-hydroxydopamine was used to achieve total sympathectomy of brown fat nerve terminals, while a chronic blockade of c* -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 binding 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 ^" 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]. How3 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 ex3 2  -a(  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]-norepinephrine compared to our earlier reported value of 0.22 pmol/mg protein. This discrepancy could-115be due to the use of a different handling group of and animals exposed toIt more extensive to who salinewereinjections. has  also  been  suggested  (especially the  brane  of  [30].  ions  It  is  preparations  compounds  the  binding  can v a r y  brane  fragments.  brane  preparations  s u c h as Na+, possible  the  of  [  H]-agonists  tooi2~receptors  catecholamines)  presence  a s GTP  that  Mg++,  that  in  different  Therefore,  the  reported  and  nucleotides  of  ions  preparations  were  always  mem-  and/or  of  and  by  such  purified  these  experimental  here  influenced  partially  concentrations  between  is  the  mem-  control  isolated  mem-  simul-  taneously . Binding isolated  from  of  brown  characteristics distinct of  a much  for  ligand.  of  of  showed  larger The  to  sympathectomized  compatible  sites  whereas  H]-norepinephrine  fat  populations  binding  the  (-)[  with  the  binding  rats  presence  sites.  former  of  sites  were  of  at  high  exhibited  probably  membranes  showed  A smaller  c h a r a c t e r i s t i c s of  number  plasma  least  two  proportion  affinity  sites,  very  affinity  identical  low to  the  small  3  number  of  high  affinity  binding  sites  revealed  in  the  [  H]3  yohimbine  binding  norepinephrine tomized increase low  was much h i g h e r  seemed  classified  The  specific binding  animals  affinity  experiments.  to  be  receptor  as mixed  due  to  agonists  amount  membranes  than  entirely  sites  absolute  in  to  (Fig.  the  the  7B).  (i.e.,  from  of  sympathec-  controls.  appearance  This of  Catecholamines  they  bind  to  (-)[  the are  b o t h «K-  3  ^-receptors).  The  higher in  degree  specific  binding  seen  Fig.  7B  specific  binding  to ^ - r e c e p t o r s .  of  [  could The  -116-  H]-norepinephrine also  result  from  specific binding  of  and  H]-  (-)[  H]-norepinephrine  was  the  same a s  that  (-)-epinephrine that  the  were  of  and  majority t h e ©<In  the  epinephrine ephrine  of  in  the  the  presence  10 /*M p r a c t o l o l 3 the  (-)[  competition  of  (-)-epinephrine  a combination  (Fig.  yohimbine  experiments  7C)  sites  sympathectomized  animals  in  was  These  more  (Fig.  could displace  binding  yohimbine.  10 ptM  of  thus  H]-norepinephrine  its  (-)-Epinephrine  presence  of  10 /*M  indicating  binding  sites  type.  and  from  in  results  (-)[  i n membranes  are  at  to  (-)-  ^-norepinbrown  fat  dependent  lower  similar  both  from  a concentration  effective  8) 3  of  manner.  concentrations  those  in  than  saturation  analysis  ( F i g . 7A, B) w h e r e a much h i g h e r d e g r e e o f a b s o l u t e 3 of [ 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 the presence 3  amount of  10 pM  epinephrine  yohimbine  (Fig.  relatively sites for  6A,  higher  o c c u p i e d by  parallel results  B) .  discussed  compared  to  by  U'Prichard  effectiveness  of  agonists  other  agonists  [204,205  and y o h i m b i n e  binding  courses.  observed Studies  (-)-epinephrine  norepinephrine  was  This in  sites.  The  observation  s e c t i o n 4.1  two is  [  and  report  Snyder  competing  ].  suggests  antagonist  The  for  steep  binding  slope  cooperativity  curves  exhibited  i n agreement  with  H]-  in nonthe  indicating displacement  of  and  least  3 [  H]-norepinephrine  one n o n - o v e r l a p p i n g Chronic  by  yohimbine  epinephrine  from  at  site.  blockade  repeated  administration  increase  i n the  number  of of  of  the ^ - a d r e n o c e p t o r s high  doses  <x9-binding -117-  of  in vivo  yohimbine  sites  i n brown  by  showed fat  an  plasma  membranes  isolated  from  21-day-old  rats. Equilibrium binding s t u 3  dies c a r r i e d out with<* -specif i c l i g a n d s [ H]-rauwolscine 2  thyl-yohimbine,  (_<-me-  [ H ] - y o h i m b i n e and [ H ] - R X 7 8 1 0 9 4 ( F i g . 9A, B, C) 3  3  i n d i c a t e d an i n c r e a s e i n t h e a p p a r e n t number o f s p e c i f i c b i n d i n g as compared  to binding  on membranes  from non-treated  sites  rats (Fig.  3  2A, 6 A ) . 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 b i n d i n g ( F i g . 9C) i n d i c a t e d b o t h an i n c r e a s e i n t h e number o f b i n d i n g s i t e s (B  0.35 pmol/mg  max  66.7  nM) compared  protein)  and a d e c r e a s e  in affinity  (K, o  to that  in controls  (B  ( F i g . 6A).  The b i n d i n g  o f (-)[ H ] - n o r e p i n e p h r i n e  m a x  0 . 1 5 pmol/mg  protein  3  and to but  K  14 nM)  d  identical  membrane  a B value max  identical  almost  values  were o b t a i n e d competing  preparations double t h a t  agent.  These r e s u l t s  affinity  binding  sites  affinity  and h i g h  yohimbine  administration.  following  a chronic  density lungs  i n human  following The  regulation  receptors;  or epinephrine suggest  binding  blockade  sites  lymphocytes  of the receptor  as t h e high  o f low  chronic  receptor  density  and r a t h e a r t  reported  ^-adrenoceptor  v e n t r i c l e and  administration  [1, 21].  f o r such a compensating  phenotype  i s n o t known.  (a) de novo s y n t h e s i s  (b) r e v e r s a l o f d o w n - r e g u l a t i o n  -118-  binding  following  of higher  10 d a y s o f p r o p r a n o l o l  Almost  of small,  of the ©^-adrenoceptors  t o the observations  include:  served  a shift  The i n c r e a s e d  mechanism r e s p o n s i b l e  explanations  of the s p e c i f i c  o f ©^.-.-receptors t o a p o p u l a t i o n  density  value  of c o n t r o l animals.  (K^ 68.3 and 66.7 nM)  whether yohimbine  here i s s i m i l a r  showed an unchanged  The p o s s i b l e  of additional exerted  by e n d o g -  enous c a t e c h o l a m i n e s ; the  antagonist-occupied  Ishac an  and  [100]  receptors.  activation  or  r e c e p t o r phenotype or to adapt  their  metabolic  of  their  the  receptor  inhibition.  calorigenic  effects  of  that  the  classification. can  be  Such a change  in  the  allow  infant  the  animals  to the  prevailing  recent  reports  These  sites  and  contains with  a finding are  and  its  results adreno©^-sub-  number a n d / o r b i n d i n g  receptor  [78]  agonists  phenotype  compatible  influenced experimentally,  haps i n d i c a t e s t h a t t h e s e  tissues  [ 8 , 31,147 ] ,  important.  characteristics  Furthermore,  o r g a n s and  some a d r e n e r g i c  brown f a t a d r e n o c e p t o r  brown f a t o f  c e p t o r s which e x h i b i t  erties  their  o f brown f a t i n o b e s i t y  developmental r e g u l a t i o n s are very indicate  or  i n o b e s i t y treatments  s t u d i e s of the  and  mechanism: namely  In view of the  hormonal s e n s i t i v i t y  of  populations  i t s m o d u l a t i o n may  conditions.  a n a l o g u e s used  detailed  another  i n v i v o hormonal r e s p o n s i v e n e s s  or o t h e r  of d i m i n i s h e d  r a t e of d e g r a d a t i o n  In a d d i t i o n , Kunos  have r e c e n t l y p r o p o s e d  i n t e r c o n v e r s i o n of d i s t i n c t  reciprocal  and  (c) a slower  prop-  which  per-  physiologically  active. Summary: (i)  Chemical an  sympathectomy by  i n c r e a s e i n the  number o f b i n d i n g  affinity sites  days  a decrease  isolated  showed in  the  t o brown f a t  from 7-day-old  rats  later. 3  (ii)  and  of yohimbine  p l a s m a membrane f r a g m e n t s two  6-hydroxydopamine  T o t a l number o f  (-)[  • H]-norepinephrme binding -119-  sites  to ^ - a d r e n o c e p t o r s isolated than  from  that  sympathectomized  i n the  characteristics least The  two  compatible  i n the  seemed t o be affinity  controls.  distinct  increase  i n p l a s m a membrane  due  animals  The  was  binding  with  populations total  fragments  the  higher  showed  presence of  of b i n d i n g  sites.  number o f b i n d i n g  entirely  to the  at  sites  appearance of  low  sites. 3  (iii)  ( - ) - E p i n e p h r 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 its  (iv)  binding  Chronic peated  to ^ - a d r e n o c e p t o r s  blockade  resulted  i n changes i n t h e  cX^-adrenoceptors  4.3  mediating  specific  the  l o n g been u s e d by  and  as m o d e l s i n t h e  density  of  and a n t a g o n i s t s i n v i t r o i n brown a d i p o s e t i s s u e  their  effector  systems  e l u c i d a t i o n of the  neurotransmitters  the ^ - a d r e n e r g i c  lipolysis  v i a the  signal  to the  tissue, cell  a major r o l e  -120-  :  their  cyclic  AMP  to a c t i v a t e  system of p r o t e i n k i n a s e s . Although  pathway o f brown f a t p l a y s  have  mechanisms  communicate  In brown a d i p o s e  transmits  ergic  and  f o r yohimbine.  receptors  messages.  re-  yohimbine  p h y s i o l o g i c a l a c t i o n s of catecholamines  w h i c h hormones and  intracellular  i n v i v o by  doses of  affinity  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 on c y c l i c n u c l e o t i d e c o n t e n t s of 7 - d a y - o l d r a t s . The  -  of o ^ - a d r e n o c e p t o r s  a d m i n i s t r a t i o n of h i g h  from  in  the  ^-adren-  non-shivering  thermogenesis [85]  [179],  had suggested  production  of  a minor  by brown  pathway b e s i d e s  Horwitz  fat.  Jungas  now  shown  tributes brown  [218]. that  role  as e a r l y  of ^(.-adrenoceptors  The p r e s e n c e  acting  i n t e r a c t i o n was a l s o  Pharmacological  studies  20% t o t h e t o t a l  f a t o f hamsters.  production  no e n t i r e l y  can be a s c r i b e d t o effector three  system  t h e c<-adrenergic  i s even  hypotheses  have  been  of  "second messengers"  in  brown  flux in  into  the c e l l  then  stimulate  hypothesis such  understood.  advanced  GMP s y s t e m  tion.  Pointer  for  various  the formulation  the  formation stimulation  an i n c r e a s e d N a +  sites  [52]  "second  releases Ca  activates  The t h i r d  as the mediator  [152]  of  (^-adrenoceptors  h a d shown  -121-  from  protein  result intra-  kinase  suggests  of ^-adrenoceptor  accumulation of  + +  messengers"  + +  hypothesis  Ca  The s e c o n d  and d i a c y l g l y c e r o l as a This  role  years,  ^-adrenoceptor  activities. of  path-  o<-adrenergic-  In recent  stimulation  cellular  in  satisfactory  The i n c r e a s e d c y t o s o l i c  fat.  con-  heat  brown  d i a c y l g l e r c o l [143],  cyclic  need  of  stimulation.  storage  process  The  [138] has reported  as i n o s i t o l triphosphate  cellular  have  production  to explain of  by Wise  as a r e s u l t  suggests  of ex^-receptor  via  from  pathway.  as a r e s u l t  f a t . Nedergaard  adipocytes  may  less  modulator  the B-adrenergic  the body's  thermogenesis,  alternate  [127-129]  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 during  heat  suggested  measured heat  While i t i s c l e a r t h a t  1977  in  as a  an 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  about  as  of a second,  the ^-adrenergic-pathway  the lipase-substrate  and  and H o r o w i t z  C the  stimula-  cyclic  GMP i n  isolated  rat  ulation,  although  any in  liver  Fain  fat  cells  from  rat  on  their  based  ence of  a link  accumulation tested the  by  cyclic  in  cyclic  GMP  kinase, cyclic kinase.  that  chosen,  tides  a  "free"  determinations.  absolute sidered  GMP  amount to  be  GMP-dependent [151]  of  the  that  and  the  a cell  indicative  Dufau  only  et  bound  of  the  the  cyclic  for  An  of  the  on  This  a l .  technique  to  cyclic  to  to  total  develop pro-  a  nucleothe  kinase,  was  was,  RIA  specifically  activated  method  of  approach  nucleotide of  protein  AMP-dependent  prior  protein  amount  total  GMP-dependent  known  cyclic  of  [177]  the  fraction  -122-  GMP  protein  indirect  kinase. [51]  in vitro  fraction  cyclic  protein  is  pres-  further  GMP-dependent  Knight  a separation  protein  protein  and  the  a "bound"  the  cyclic  was  portion  GMP-dependent  used  and  and  fat.  successful.  namely  inside  for  Skala  proposed  antagonists  cyclic  Skala  lipolysis  s e c t i o n we d i s c u s s  relevant  cyclic  assay  not  Since  of  and  estimate  s i m i l a r to were  this  w a s made  ratio"  kinase  cyclic  In  to  and  tissue.  hypothesis  to  by  notice  unable  stimulation  brown  to  were  [178],  which binds  attempts  therefore,  bind  of  stim-  adipose  The  agonists  activates  Earlier  into  ft-  ^-adrenoceptor  accumulation  study  fat.  contents  that  "activation  protein  in vivo  the physiologically  is  kinase,  parametrial  of  [53]  GMP  experiments.  an a t t e m p t GMP  cyclic  brown  and  nucleotide Since  tein  rat  oi-  of  a result  between ^ - a d r e n e r g i c  in vitro  effect  as  and B u t c h e r  c o r r e l a t i o n between  Knight,  an  cells  the  was  con-  cyclic  modification  w h i c h had  shown  a  good c o r r e l a t i o n intracellular  cyclic  protein kinases adrenocortical The  between hormone c o n c e n t r a t i o n ,  and  AMP  bound t o t h e  from  rats.  was  first  technique  AMP  response a s s o c i a t e d with  and  p r o t e i n bound  fractions  in  preceptor then  of  isolated  a p p l i e d to separate  and  of  regulatory sub-unit  corticosterone production  cells  amount  cyclic  stimulation into  a p p l i e d to, c y c l i c  free GMP.  3  Initial  s a t u r a t i o n experiments c a r r i e d  out w i t h  [ H]-cyclic  3 AMP  showed b i n d i n g  tein  of  [ H]-cyclic  from brown f a t t o be  concentrations  the  cyclic at  protein-bound AMP  with  which the  determined The  the  T h e r e was  cyclic  reflected  the  production  r a n g e o f 0.5,  Prior  metoprolol  ergic cyclic  than  or  blocked  the  effect  confirm  the  s t i m u l a t i o n and  free  temperature  Therefore,  (4°G)  the  values  situation. AMP  ( F i g . 10A), 5.0,  was  dissociation  a s s o c i a t i o n of the  was  not  but  o n l y most  50 /uM  of  with  produced a graded pieces with  conres0.4  50 y«.M d o b u t a m i n e ( F i g .  a s s o c i a t i o n between  the  pro-  dobutamine i n a  i n c u b a t i o n o f brown f a t t i s s u e  These r e s u l t s receptor  1%  brown f a t f r a g m e n t s were i n c u b a t e d  centration  high  This binding  p r o t e i n at the  in vivo  pro-  d i s p l a c e a b l e by  AMP.  less  or  of c y c l i c  (^-agonist)  10B).  AMP  receptor  dobutamine  mM  s a t u r a b l e and  intracellular  e x p e r i m e n t s were p e r f o r m e d .  nounced when t h e  ponse.  to  of n o n - l a b e l l e d c y c l i c  temperature dependent. of  AMP  tissue  /^-adren-  concentration  of  most e f f e c t i v e  in  AMP. On  the  other  h a n d , c l o n i d i n e was  -123-  stimulating  the p r o d u c t i o n of c y c l i c  concentration-dependent dose-response old  r a t s was  c u r v e on  of  0.5 /_M fat  i n the former  clonidine, while  clonidine.  i n one-week-old ity  [179,  180].  yohimbine GMP  found  B).  t o 40% u n d e r  15  protein  also  that  brown while  The  bound and  occurring  and  stimulation  and  by  cyclic was  c u r v e s were  at 3 minutes  of  GMP  i t increased  was  to  These i n v i t r o  30  experi-  hypothesis of a l i n k  a c t i v a t i o n of guanylate  these receptors  sep-  the f r a c t i o n  bound f r a c t i o n o f c y c l i c  t o 25% o f t h e t o t a l ,  demonstrated  blocked  media, time-dependent  ments s u p p o r t e d the e a r l i e r p r o p o s e d  and  be  stimulation,  clonidine . stimulation.  -^-adrenoceptor  The  i t occurred at  S i m i l a r l y , when t h e t o t a l  incubation  ( F i g . 12).  t o be  may  e f f e c t o f c l o n i d i n e was  o b t a i n e d w i t h maximum r e s p o n s e incubation  case  i n v o l u t i v e phase  as a r e s u l t o f c l o n i d i n e  i n the  one-month-  one-week-old r a t s .  explanation  i s i n the  into intracellular,  released  from  from  i t i s a t t h e peak o f i t s f u n c t i o n a l a c t i v -  This  ( F i g 11A,  produced,  arated  rats  I n t e r e s t i n g l y , t h e clonidine  i n the l a t t e r  rats  showed a  c a s e o c c u r r e d a t 20 /xM c o n c e n t r a -  A possible  i n one-month-old  and  brown f a t f r a g m e n t s  d i f f e r e n t than that  peak r e s p o n s e tion  response.  GMP  between cyclase  t o show c h a r a c t e r i s t i c s  of © ^ - s u b - t y p e . Although induced pineal  several  accumulation [144]  the p r e c i s e  as  other studies  of c y c l i c  GMP  i n the p a r o t i d  a r e s u l t of s t i m u l a t i o n  nature of the l i n k  have shown  catecholamine[219]  of ^ - a d r e n o c e p t o r s ,  between - ^ - - a d r e n o c e p t o r s  -124-  and  etim-  ulation ical of  and  guanylate cyclase  s i g n i f i c a n c e is unclear.  the  cytosolic/  the  and  and  (cell-free cortex.  preparation)  The  e f f e c t was  Although a v a r i e t y of unsaturated  fatty  On  soluble the  hand,  of guanylate cyclase [110]. in was  1978  [111]  presented  c y t o s o l i c guanylate  independent  of  evidence cyclase  in rabbit  the  renal  presence of  n i t r o p r u s s i d e could  enzyme was  a c t i v a t e d by  has  poly-, stimulate only  activation  a l s o b e e n s u g g e s t e d -by L e v i l l i e r s  detergent-dispersed t o be  calcium.  catecholamine.  i n d i r e c t mechanisms f o r t h e  B o t h c y t o s o l i c and  reported  enzyme i n brown f a t i s  catecholamine  and  question  a c t i v a t i o n of  membrane bound g u a n y l a t e c y c l a s e ;  form of  other  i s the  a g e n t s i n c l u d i n g ascorbate»  acids  b o t h c y t o s o l i c and  by  i n the  physiolog-  both.  coworkers,  d i r e c t a c t i v a t i o n of  its  unresolved  whether t h i s  membrane-bound, o r  Liang  the  Also  m o l e c u l a r mechanisms i n v o l v e d  guanylate cyclase  for  a c t i v a t i o n and  membrane bound g u a n y l a t e  p l a s m a membranes f r o m r a t  highly  s e n s i t i v e to  et" a l . 1 9 7 8  cyclase  adipocytes  a c t i v a t i o n by  low  2+ concentrations appears that  of d i v a l e n t c a t i o n s 2+  Ca  may  hormonal c o n t r o l of Mey  and  Vanhoutte  response by  as  guanylate  [122]  an  Ca  intermediate  cyclase  i n the  have shown t h a t  . in a  suggested  adrenergic  both alpha^that  the  and  be  -125-  triggered  adrenoceptors.  c o n t r a c t i l e r e s p o n s e by the  putative  the c o n t r a c t i l e  alpha^-  s t i m u l i depended more on  Thus, i t  adipose t i s s u e .  i n i s o l a t e d v e n o u s smooth m u s c l e c o u l d  a c t i v a t i o n of  I t was  act  s u c h as  influx  of  alpha^extra-  cellular  Ca  while o^-adrenoceptors  stimulation  caused  . . . a relatively ions.  greater  Studies  2+  mobilization of c e l l u l a r  by Z a v o i c o and F e i n s t e i n  that  stimulation of ^ - a d r e n o c e p t o r s ++ . . f r e e Ca i n p l a t e l e t s ._<2"" 9 a d r e n e r  l c :  stores  [ 2 2 7 ] have  Ca  + +  s i g n a l l i n g may be i n v o l v e d  b o t h oCj^- and o<2- a d r e n o c e p t o r The cyclase  influx  existence  and c y c l i c  cells.  effector  [88].  i n a c t i v a t i o n of  systems.  i s also unclear. o f a temporal GMP  level  London e t a l . [ 1 1 5 ]  r e l a t i o n s h i p between  i n synchronized  Tetrahymena  s t u d y have d e m o n s t r a t e d 2+ by Ca -calmodulin. This 2+  of guanylate cyclase  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 o f Ca On t h e o t h e r  hand, o t h e r  studies  suggest  produced  as a r e s u l t o f p h o s p h o l i p a s e  activate  protein kinase  phosphorylate ignore  activation, tion  calcium  Kudo e t a l . [ 9 9 ] , i n t h e i r  activation  not  Thus, i t  p r e c i s e mechanisms o f a c t i v a t i o n o f g u a n y l a t e  by c a l c i u m  reported  reported  increases i n t r a c e l l u l a r . ++ r e g u l a t i o n o f Ca  transport i n r a b b i t i n t e s t i n e h a s been d e s c r i b e d seems t h a t  o f Ca  of hormone-sensitive  regulator"  C a c t i v a t i o n , could  cyclase  described  of free  l i p a s e [208],  fatty  could  [228].  p o s s i b l e mechanisms o f g u a n y l a t e  s u c h as by r e l e a s e  EGTA.  diacylglycerol,  C [ 7 9 , 1.43], w h i c h i n t u r n  and a c t i v a t e g u a n y l a t e  other  with  One  can-  cyclase  a c i d s by a c t i v a -  o r by t h e " f e e d b a c k  i n w h i t e f a t by Ho and S u t h e r l a n d  [9,  80]. In the  s p i t e of the obvious  guanylate cyclase  lack  of detailed studies  s y s t e m i n brown f a t , t h e -126-  on  information  obtained  so  some r o l e , The  high  far  in  seems  the  to  indicate  regulation  steady-state  activity  of  served  by  and K n i g h t  upon of  cold  high  cyclic  acclimation  of  s y s t e m may  during seems  the to  play  processes  cyclic  GMP,  [180].  and  the  protein  kinase  ob-  perinatal  period  and  GMP-dependent  [178]  proliferative  the  developmental  concentration  higher  Skala  of  that  correspond  with  periods  activity.  Summary;  (i)'  Incubation old  rats  of  in vitro  an e l e v a t i o n was  blocked  Clonidine, and of The  in  fat  fragments  with ^ - a g o n i s t  the  cyclic  AMP  concentration-dependent.  onist, (ii)  brown  timebrown  an  this  fat  effect  was  fractions, of both  the  content.  produced  The a  produced in  isolated  a  response ^-antag-  concentration-  cyclic from  GMP  When  separated  the  into  total free  c l o n i d i n e produced fractions.  tissue  and  content  7-day-old  c l o n i d i n e c o u l d be b l o c k e d by  an o < 2 - a n t a g o n i s t . content  dobutamine  Metoprolol,  elevation  fragments  of  one-month-  effect,  o^-agonist,  dependent  from  yohimbine,  cyclic  protein  rats.  GMP  bound  a time-dependent  elevation  4.4  E f f e c t of "<2~ 9 iforskolin-stimulated lipolysis i n brown f a t a d i p o c y t e s i s o l a t e d from 7 - d a y - o l d r a t s . a  Robison alpha  s t  o  n  e t a l . [158] o r i g i n a l l y  e f f e c t s of  adenylate  o n  cyclase  ghosts  activity.  Subsequently  [ 2 7 ] , human p l a t e l e t  adipocyte ghosts this  inhibitory  that  c a t e c h o l a m i n e s were associated with a decrease i n  c y c l a s e by a l p h a c a t e c h o l a m i n e s was cell  had p r o p o s e d  inhibition  r e p o r t e d i n human f a t  lysates  [ 9 0 ] and h a m s t e r  [ 3 ] . S a b o l and N i r e n b e r g effect  of adenylate  [165] s u g g e s t e d  o f a l p h a c a t e c h o l a m i n e s was  mediated  through a l p h a 2 ~ r e c e p t o r s . Subsequent c e l l s have c l e a r l y alpha 2  linked duced  shown t h a t  adrenoceptors  to a decrease  the s t i m u l a t i o n of  inhibits  lipolysis.  in intracellular  by an i n h i b i t i o n  activity  cyclic  It  is  AMP  levels  o f p l a s m a membrane a d e n y l a t e  cyclase  objective  of t h i s  part  of our study  i s . t o show,  t h e stimulation o f ^-adrenoceptors i n brown adipocytes from one-week-  old r a t s show i n h i b i t o r y e f f e c t on forskolin-stimulated glycerol as 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 In our s t u d y the  inhibitory  effect  high concentrations.  NG  f a t o f hamsters  ( T a b l e 1) on i s o l a t e d of p r o s t a g l a n d i n E  ju.K) s t i m u l a t e d l i p o l y s i s  tion  pro-  [33, 101-103]. The  that  s t u d i e s w i t h human and h a m s t e r w h i t e f a t  ranged  from  A similar  2  and  man.  brown a d i p o c y t e s , on f o r s k o l i n  20 t o 35% from  degree  release  of  (1.25  low t o  inhibi-  by © ^ - a g o n i s t s was r e p o r t e d by S a b o l and N i r e n b e r g i n  108-15 n e u r o b l a s t o m a  forskolin—stimulated  x hybrid  lipolysis  cells  [165].  I n h i b i t i o n of  by p r o s t a g l a n d i n E  -128-  9  and  nicotinic  a c i d was v e r y  hamster e p i d i d y m a l Stimulation adipocytes  juM)  could  clonidine  adipocytes  to that  of g l y c e r o l release  be i n h i b i t e d  by c l o n i d i n e .  concentration-dependent  were r e p o r t e d  (Table  These e f f e c t s  2A) and c o u l d of yohimbine  hibiting  lipolysis  acid  3)-  isobutyl-  inhibitory  of i n -  stimulated  g l y c e r o l release, could  to the phosphodiesterase  glycerol release,  inhibitory  Similar inhibitory  isobutyl-methylxanthine-stimulated  -129-  by n i c o t i n i c  a c t i o n o f c l o n i d i n e on  to f o r s k o l i n  stimulated  that  a c t i o n i s not mediated  Isobutyl-methylxanthine  A low i n h i b i t o r y  study,  by 100 juM o f  capable  stimulated  methylxanthine^  by  In our  were s t i m u l a t e d  isobutyl-methylxanthine  due  [58,169-].  r e l e a s e was 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  (Table  rats  accumulation  by as much as 15 t o 30%, i n d i c a t i n g  adenosine r e c e p t o r s .  glycerol  AMP  inhibitor).  c l o n i d i n e was s t i l l  _<2-adrenoceptor-stimulated via  and c y c l i c  (a. p h o s p h o d i e s t e r a s e  isobutyl-methylxanthine,  (Table  t o be an  and h a m s t e r brown a d i p o c y t e s  when i n f a n t r a t brown a d i p o c y t e s  be  from a d u l t  o f endogenous a d e n o s i n e a r e b l o c k e d  methylxanthine  was  [190].  regulator of l i p o l y s i s  i n white adipocytes  (0.625  inhibition  i n brown f a t a d i p o c y t e s  by S u n d i n and F a i n  brown f a t  of f o r s k o l i n  Endogenous a d e n o s i n e h a s been r e p o r t e d important  reported i n  in isolated  This  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  Similar results  already  [168] ( T a b l e 1 ) .  by s u b - m a x i m a l c o n c e n t r a t i o n s  antagonized 2B).  similar  compared  p o s s i b l y be  a c t i o n of i s o b u t y l -  effects lipolysis  o f c l o n i d i n e on have been  reported  in  hamster  epididymal  It on t h e  has  been  catalytic  activation affected  of  by  proteins.  sub-unit  adenylate  inputs  ADP-ribosylation  toxin not  stimulated (PGE2)  of  This  pertussis  by  clonidine,  we w e r e  toxin  effect  that  to  show  adipocytes  any  of  effect  on  cytes.  Similar  and  fat  can block  also and  the  Ni  in  (Table  exerted  Interestingly,  action  of  clonidine  stimulated adult  lipolysis  hamsters showed  (Table an  l i p o l y s i s i n hamster by  the  protein  input  nicotinic acid  reported  for  forskolin  exerted  of  agonist.  from  [37].  abolished  inhibitory  a l l  prostaglandin  release  inactivation  be  causes  adipocytes  inhibitory  r e s u l t s were  pertussis  brown  completely  forskolin-stimulated  Thus,  inhibitory  cyclase  Clonidine  and  [173].  or  thus  b a s a l but  isolated  directly  adenylate  isobutylmethylxanthine  However, p r o s t a g l a n d i n E^  McMahon  and  inhibitory adipo-  Schimmel  120]. In  forskolin release  Ni  an o < ^ - a d r e n o c e p t o r  in  [119,  Bordetella  abolished the  or  5) .  the  cyclase  stimulatory  of  was  acts  f o r s k o l i n can s t i l l  on g l y c e r o l  toxin  on f o r s k o l i n fat  from  lipolysis.  toxin  unable  brown  the  increased the  indicates  by  c y c l a s e by  a subunit  pertussis  forskolin  adenylate  s u p p r e s s i o n of  of  inhibitory  absence 4) .  rate  of  pre-treatment  only  [168].  that  affecting  of  receptor-mediated  3^ h o u r s  proposed  Pertussis  Pertussis  adipocytes  in  conclusion, this and  study  demonstrates  isobutylmethylxanthine  isolated  adipocytes  from  -130-  that  stimulated brown  fat  of  both  glycerol one-week-old  rats.  Clonidine  nicotinic  acid  ( an  of  dependent.  Effect  increasing  the  abolished din by  E2.  glycerol of  the  clonidine  inhibitory  This  suggests  probably  similar  r e l e a s e by  inhibitory  clonidine results  which  already  adipocytes  [54,  101,  activity  4.5  of  via  have  fat  of  and  *<2  o n  i  s t  ) •  toxin  - a d r e n o c e  adenylate  P  t o r s  cyclase,  protein and  (Ni)  human  The  anti-lipolytic in  9  prostaglan-  for  a role  by  _ a n t a  pertussis  clonidine  inhibitory  103].  concentration  (an e » < 2  by  stimulation  release  hamster effect  c o n t r o l l i n g the  state  cells.  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 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 fragments 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 Brown  d e f e c t (s) [  may  brown  of  reported  102,  of c<2-a<lrenoceptors  (Ni)  and  2  antagonized  yohimbine  i n h i b i t i o n of  i s mediated  that  of  was  ALM)  E_  glycerol  c l o n i d i n e was  (1  effects  in  stimulated  protein  that  to  of  forskolin  concentration  of  prostaglandin  -  inhibited  Inhibition  Inactivation  <x_,-agonist),  77].  fat  genetically  in ^-adrenoceptor  They have  efficiency. rapidly  of  They  obese  stimulated  a low m e t a b o l i c are  hypothermic,  extremely and  (ob/ob)  die  rate  about  shows  thermogenic and  sensitive  in  mice  a high to  three  metabolic  cold, hours  response  become when  kept  6  at  4 C.  pare obese  the and  The  objective  responses lean mice  the oc_-adrenoceptor  of  this  o f ..brown to and  see  part  fat if  cyclic  the GMP  -131-  of  our  cyclic  study  GMP  d e f e c t (s) systems  was  to  production extended'to  as w e l l .  In  comin  initial mice,  in vivo  experiments,  following  cold  administration change from  in  tended by  their  control  increase  brown  cyclic  fat  GMP  to  In  of  7-day  ceptor study c  summary, ^ 2  shows  neurotransmitter  the a  d  r  e  n  o  P  The  t  r  in  following  GMP  the  presence  in norepinephrine  of  of  cold  be  of  mediated  mice.  of  due  to  results,  brown  Lack  some  of and  defect(s)  a link  fat  presence  f r o m brovm between Tn  ^"adreno-  characteristics manipulations. implicated in and  the  long  calorigenic  fat  addition,  and ^ - a d r e n o c e p t o r s  -132-  obese  d e s c r i b e s the  been  tissue  lower  fat.  binding  has  a  exposure  production.  brown  B^-  response  adipocytes  n  w a s  was  lean mice.  experimental  norepinephrine  ( e^2-a9'onist)  in vivo  and  presence  cyclic  changes  *  s  production  our  following  o  GMP  ex-  unlike  presented  e  were  difference  the  fat  time-dependent  of  study c  a  cyclic  no  brown  fat  on brown  the  and  in  possibly  to  brown  sites  c a l o r i g e n i c response  response. role  in vivo  and  stimulation  <2~adrenoceptors  acute  _  study,  ob/ob  exhibited  showed  13 . C ) ,  in  i n obese  i n j e c t i o n may  old rats  also  protein  of  experiments  only  a difference  binding  who  clonidine The  preliminary  production  the  functional  by  i.p.),  contrast  these  (Fig.  lean mice.  production  norepinephrine  1 of  and  demonstrate  GMP  proximal  When  stimulation  in vitro  cyclic  cyclic  and B ) .  in  mice  tissue  norepinephrine  weight  mates)  experiments  upon  and  contents  concentration/mg  our  failed-to  GMP  adipose  (6 C)  mg/100 g body  litter  A  i n obese GMP  Thus,  (lean  in vitro fat  exposure  cyclic  (Fig.13  to  similar  (0.1  brown  of The both  term and  the  trophic  their  response  has  been w e l l documented.  which  shows  the  mechanisms  norepinephrine.  and c y c l i c  proliferation. it  i s assumed  membrane entity. nities  of  of  effects  that  of  are  different  binding  in vivo  associated  may  have  a role  the  finding  proportion for  sites  effector  of  are  sites  norepinephrine  response, atively the  one  high  brown  and g r o w t h ially  explain  finding older  [119]  rats  i s more  or  the  in  is  the  and  our  peak  of  in its  Perhaps  [24]  and McMahon  -133-  that  the systems,  and  during  the  different binding  trophic  presence of infant  rats  relwhen  proliferation  this  may a l s o  and  <X - . - a d r e n o c e p t o r s  hamsters.  which  tissue's  the  study.  l a c k of  upon  final  effector  change  the  a f f i -  the  oi -adrenoceptor  upon  if  dynamic  and the  assuming  demonstrate  the  a  adrenoceptors  If  effects  at  Buckowiecki's of  on  and  plasma  determine  of ^^"-d-renoceptors  tissue  shown  animal.  l i k e l y to  proportion  adipose as  of  is  different  can d i f f e r  stages  rat  Depending  o c c u p i e d and  norepinephrine  systems  the  phenotype  norepinephrine  l i n k e d to  developmental mediate  its  in  been  ^-adreno-  be e x p l a i n e d  a developing of  that  with  has  t i s s u e growth  phenotype  stimulation.  preferentially  effects  of  associated  suggested in  undertaken  lipolysis  can best  adrenoceptor  adipocytes  been  i n h i b i t i o n of  GMP  present  yet  response  been  adrenergic  receptors  the  trophic  also  components  the  of  has  relative  its  has  It  Our  brown  The  study  -.-adrenergic  known f o r seme t i m e . ceptors  No  part-  Schimmel's  i n brown  fat  of  CHAPTER 5 CONCLUSION  The  principal  f i n d i n g s of t h i s  investigation  a r e as  follows: 1.  Ligand  binding studies carried  selective o^-ligands membrane tissue  out using h i g h l y  and p a r t i a l l y  purified  f r a g m e n t s o f i n t e r s c a p u l a r brown  f r o m one-week-old  characteristics  rats  plasma  adipose  exhibited binding  as would be e x p e c t e d  of binding to  o^-adrenoceptors. 2.  Norepinephrine,  (-)-epinephrine,  bound t o more t h a n tors.  and 3.  on ^ - a d r e n o c e p -  the other  d e n s i t y was s p e c i f i c  yohimbine  site  shared  present  one com-  i n much  f o r (-)-norepinephrine  only. 3  Binding  o f (-)[ 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  binding  sites.  by  either  site  Site  practolol  prenolol  either  binding  ( ^ - s i t e ) c o u l d be  to displacement  occupied The s e c o n d  by y o h i m b i n e ,  the n o n - s p e c i f i c i t y  or binding  Y o h i m b i n e bound  1  or (-)-epinephrine.  was s e n s i t i v e  indicating  4.  site  Y o h i m b i n e and ( - ) - e p i n e p h r i n e  mon b i n d i n g s i t e ; lower  one b i n d i n g  and y o h i m b i n e  of d i h y d r o a l -  o f dihydroalprenolo to o^-adrenoceptors.  t o a s m a l l e r number o f h i g h  s i t e s i n membrane  fragments  isolated  brown f a t o f 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  -134-  affinity from  animals  than  it  d i d t o membrane  animals  of  fragments  i s o l a t e d from  control  t h e same a g e .  3  The  (-)[  H]-norepinephrine  membranes high  isolated  and low a f f i n i t y  binding  s i t e s more  Chronic  yohimbine  than for  two-fold  than  whereas  unchanged. that  brown  ceptors, could  and t h a t  with  time-  dependent  3  fat  The  rats  of  brown  change cyclic  supported  hypothesis  of  and  GMP p r o d u c t i o n  sites  remained  possessed ©^-adrenoreceptors and by  t o an ^ - b l o c k e r . from  clonidine resulted  and t o t a l  a link  sites  i t was c o n c l u d e d  one-week-old i n d o s e - and  increases in tissue cyclic  experiments  cyclic  rats  fragments  a time-dependent  vitro  studies,  c h a r a c t e r i s t i c s of these  s  more  of binding  for norepinephrine  developing  Stimulation  fraction  in a  of ©^-adrenoceptor binding  of young  on  of  sympathectomy.  resulted  i n t h e number  on t h e s e  <=<2~ ^ ^ ^  trations. showed  that  o f brown a  after  revealed  number  by c h e m i c a l sympathectomy  exposure  Incubation  bound  f a t of  be a l t e r e d  chronic  rats  Based  doubled  fat  rats  and [ H ] - n o r e p i n e p h r i n e .  f o r yohimbine  decreased  The t o t a l  administration  [ H]-yohimbine  t o brown  sympathectomized  sites.  increase  3  affinity  in  from  binding  GMP c o n c e n -  f a t b y 0 . 5 /M c l o n i d i n e in intracellular, GMP l e v e l s . earlier  between oC-receptor i n brown  protein  These  proposed stimulation  f a t of  one-week-old  Clonidine release  inhibited forskolin-stimulated  i n isolated adipocytes Yohimbine  glycerol  f r o m brown f a t o f one-  week-old  rats.  antagonized  this inhibitory  effect.  The 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  were  abolished  by p r e - t r e a t m e n t  o f brown f a t a d i p o c y t e s  pertussis  toxin. Clonidine  showed no e f f e c t on  cytes  i s o l a t e d f r o m brown f a t o f a d u l t  adipo-  hamsters.  In v i v o  s t u d y on g e n e t i c a l l y o b e s e mice showed a  blunted  cyclic  (4°C)  GMP  production  or norepinephrine a d m i n i s t r a t i o n .  when t h e brown f a t f r a g m e n t s incubated similar lean  following  mice.  of-cyclic  GMP  cold  However,  production  they  showed  to those i n  The only d i f f e r e n c e was a lower basal GMP/mg p r o t e i n  exposure  f r o m obese mice were  i n the presence o f c l o n i d i n e ,  l e v e l s of c y c l i c  with  concentration  i n brown f a t o f o b e s e  mice.  Bibliography Aarons, R.D., Molinoff, P.B. (1982) o f jQ-adrenergic receptors in rat lung after chronic treatment. J . 221, 439-443. Ahlquist, Am. J .  R.P. 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In order to compare our methodology and the binding characteristics of our membrane preparations to that already reported in literature, we carried out i n i t i a 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]-rauwolsmax 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  KdaL = 500 nM • • • • i  B  Kd • 21.8 rBmax = 0.205 50  pmol /mg . protein,  150  BOUND (Fmol  -160-  /  m g  250 pr  ot«)  APPENDIX B Krebs-Ringer Bicarbonate Buffer  1. 2. 3. 4. 5. 6. 7.  NaCl KC1 KH P0 MgS0 .7H 0 NaHCOCaCl Glucose 2  118.5mM 4.8mM 1.18mM 1.18mM 24.9mM 1.25mM 5mM  4  4  2  2  -161-  APPENDIX C Modified Krebs-Ringer Bicarbonate Buffer, Buffer used in Experiments of Section 2.3.6 1  -  23. 4. 5. 6. 7. 8.  NaCl.  118.5mM •  KCl.t  MgS0 KH P0 NaHC0 HEPES Cacl Glucose 4  2  4  3  2  -162-  4.8mM 1.18mM 1.18mM 11.14mM 30mM 2.5mM lOmM  -163-  APPENDIX D L i g a n d s and t h e Drugs u s e d i n t h e b i n d i n g Ligands o r Drugs u s e d  experiments.  Structure  H-ClOnidine C l o n i d i n e hydrochlor i d e , [Benzene R i n g -  M.H. Sp . a c t i v i t y  Approx. Approx. Approx. Approx.  266.(  M.U.  Alpha-2 agonist [ R e f s . 30,190]  Remarks  K_(Yohimb.) from B from s a t u r . K d ( c l o n i d . ) from B from s a t u r . m a x  m a X  S a t u r . c u r v e = 35nM c u r v e =40 fmol/mg p r o t . S a t u r . c u r v e = 30nM. c u r v e =50 fmol/mg p r o t .  266.6 61.1 Ci/mmol  OH  K<j ( e p i n e p h r i n e ) from S c a t . p l o t = 60.4 nM B a x ( e p i n e p h ) from S c a t , p l o t = 0.22 pmol/mg p r o t d ( y o h i m b . ) f r o m S c a t , p l o t = 65.8 nM B^ax(yohimb.) from S c a t , p l o t = 0.24 pmol/mg p r o t  H-Norepinephrine N o r e p i n e p h r i n e , Levo[Ring-2,5,6- H]3  m  K  3  Mixed a g o n i s t « * 2 / « i » P l . P2 ( R e f s . 1 8 4 , 204, 207].  H0CKCHHH  205-  2  M.W. Sp a c t i v i t y  2  169.2 52.9 Ci/mmol 4 8.4 Ci/mmol  H-Rauwolscine R a u w o l s c i n e , [MethylH ] o r (et-yohimbine) 3  3  oc.-antagonist [ R S f s . 2 3 , 30, 31,148  M.W. 354.4 S p . a c t i v i t y 79.5 Ci/mmol  Hill  c o e f f i c i e n t from H i l l  p l o t = 1.5  -164APPENDIX D Liqands Liqands or Drugs u s e d  and  t h e Drugs u s e d  i n the b i n d i n g  experiments.  Structure  H-RX-781094 (l,4-[6,7-3H]benzodioxan-2-yl)-2-imidazoline hydrochloride 3  J  o<.2-antagonist i R e f s . 30,45,50]  Remarks  13  -Approx. K<j (yohimb.) f r o m S a t u r . c u r v e = 10 nM -Approx. B from s a t u r . c u r v e =20 fmol/mg p r o t . m a X  H.HCl  M.W. 2 4 4 Sp.activity  56.1  H-yohimbine Yohimbine, [Methyl3H]ot2~antagonist [Refs.20,30,66,81, 163 ] .  Ci/mmol  -Approx. K<j (yohimb.) from S a t u r . c u r v e =15 nM -Approx. B from S a t u r . curve=125 fmol/mg -K (Yohimb.) from S c a t , p l o t = 18 nM -Bmax f r o m S c a t , p l o t = 156 fmol/mg p r o t . m a  d  M.W. Sp. H-DHA Dihydroalprenolol hydrochloride, levoIRing,propylor Dihydroalprenolol  hydrochlor ide,levo-  354.3  H.U. J M . t  activity  7 5 . 0 Ci/mmol  ^ ^"^NCV^  „ NjW^lttio,^ fr^l-WjcSjOi,  R.K.  w.t  a;.i  -  Approx. K ^ ( n o r e p i . ) f r o m S a t u r . c u r v e = 10 nM A p p r o x . B ( n o r e p i . ) f r o m satur.curve=0.45fmol/mg K ( n o r e p i . ) from S c a t , p l o t = 10 nM prot. Bmax^ - ?*-'' S c a t , p l o t =0.55 fmol/mg.prot. m a x  d  1101  6  f  r  o  m  - K ( p r a c t o l o l ) from S c a t , p l o t = 50 _ B j a a x f p r a c t . ) from S c a t , p l o t »0.19 d  nM pmol/mg.prot.  lpropyl-1,2,3-3H]M.W. 287.8 |(yohimb.) f r o m S c a t , p l o t = 4 0 nM i l - a n t a g o n i s t [ R e f s . Sp. a c t i v i t y 1 0 4 . 8 Ci/mmol. B ax(yohimb.) from S c a t . p l o t = 0 . 1 1 2  24,41,48, 133, 214].  J  3 9 . 7 Ci/mmol  ro  pmol/mg.prot.  -165-  APPENDIX D Ligands Ligands or Drugs u s e d  and  t h e Drugs used i n the b i n d i n g  Structure  H-RX-781094 (l,4-[6,7-3H]benzodioxan-2-y1)-2-imidazoline hydrochloride  Remarks  J  0 0^  I |  <rf.2-antagonist [ R e f s . 30,45,50]  M.W.  Sp.activity  experiments.  -Approx. K<j(yohimb.) from S a t u r . c u r v e = 10 nM -Approx. B-^JJ f r o m s a t u r . c u r v e =20 fmol/mg p r o t .  I  H.HC  244 56.1  Ci/mmol  H-yohimbine Yohimbine, [Methyl3 ]oC2 tagonist (Refs.20,30,66,81, ].  -Approx. K ( y o h i m b . ) from S a t u r . c u r v e =15 nM -Approx. B-gjj from S a t u r . curve=125 fmol/mg p r o t . -K<j (Yohimb.) from S c a t , p l o t = 18 nM m a x from S c a t , p l o t = 156 fmol/mg p r o t . d  H  - B  _ a n  Sp. H-DHA Dihydroalprenolol hydrochloride, levo[Ring,propyl- H(N))3  or  Dihydroalprenolol hydrochloride,levo[propyl-l,2,3-3H]^ 1 - a n t a g o n i s t [Refs 24,41,48, ].  M.W. 354.3 a c t i v i t y 75.0  Ci/mmol  Approx. K ^ f n o r e p i . ) f r o m S a t u r . c u r v e = 10 nM Approx.B x(norepi.)from satur.curve=0.45fmol/mg K ^ t n o r e p i . ) from S c a t , p l o t = 10 nM prot. B ( n o r e p i . ) from S c a t , p l o t =0.55 fmol/mg.prot. m a  OCKJCHC^NMA . HO  (WW* « _ Sp.  M.W. 287.8 a c t i v i t y 104.8 39.7  m a x  K _ ( p r a c t o l o l ) from S c a t , p l o t = 50 B - i a x ( p r a c t . ) f r o m S c a t , p l o t =0.19  nM pmol/mg.prot.  |- K_(yohimb.) from S c a t , p l o t = 40 nM Ci/mmol. B x ( y o h i m b . ) from S c a t . plot=0.112 pmol/mg.prot. Ci/mmol m a  -166APPENDIX E L i q a n d s and t h e Drugs u s e d I n t h e b i n d i n g e x p e r i m e n t s . (6-hydroxydopamine and y o h i m b i n e p r e t r e a t e d a n i m a l s ) . Ligands or Drugs u s e d  Structure  Remarks  H-yohimbine o<2-antagonist)  Control animals: -Approx. K,j(yohimb.) f r o m S a t u r . c u r v e = 18 nM -Approx. Bmax f r o m S a t u r . c u r v e = 180 fmol/mg p r o t . -K (yohimb.) f r o m Woolf p l o t = 14 nM - B ( y o h i m b . ) from Woolf p l o t = 155 fmol/mg p r o t . d  m a x  6-hydroxydopamine p r e - t r e a t e d  rats.  -Approx. K. (yohimb.) .from S a t u r . c u r v e => 10 nM -Approx. B _ (yohimb.) from S a t u r . c u r v e = 75 fmol/mg -K<j (yohimb.) f r o m Woolf p l o t = 8.6 nM prot. - B ( y o h i m b . ) from Woolf p l o t = 45 fmol/mg p r o t . a x  m a x  M.W.  354.4  C h r o n i c yohimbine p r e - t r e a t e d r a t s . - K,j (yohimb.) f r o m S c a t , p l o t = 66.7 nM Bmax (yohimb.) from S c a t , p l o t = 0.35 pmol/mg  prot.  Control animals; K(j ( e p i n e p h r i n e ) from S c a t , p l o t = 68 nM B ( e p i n e p h r i n e ) from S c a t , p l o t = 1.39 pmol/mg  H-Norepinephrine (Mixed a g o n i s t )  m a x  prot.  6-hydroxydopamine p r e t r e a t e d a n i m a l s : K,j ( e p i n e p h r i n e ) low a f f i n i t y s i t e from S c a t plot=36nM B ( e p i . ) low a f f i n i t y s i t e = 0.65 pmol/mg p r o t . K ( e p i . ) h i g h a f f i n i t y s i t e from S c a t . p l o t = 200 nM Bmax^ Pi*' i g a f f i n i t y s i t e = 3 pmol/mg p r o t . m a x  d  e  H0CHCH NH 2  M.W.  169.2  2  Hill  n  h  c o e f f i c i e n t from H i l l  plot  =1.5  C h r o n i c yohimbine p r e - t r e a t e d a n i m a l s : ( e p i . ) from S c a t , p l o t = 68 nM max ( e p i . ) from S c a t , p l o t = 3 pmol/mg p r o t . K<j (yohimb.) f r o m S c a t , p l o t = 66.7 nM max (yohimb.) from S c a t , p l o t = 3 . 6 pmol/mg. p r o t . B  B  -167-  APPENDIX E L i g a n d s and t h e D r u g s u s e d i n t h e b i n d i n g e x p e r i m e n t s Ligands o r Drugs u s e d  Epinephrine (Mixed a g o n i s t ) [Refs: 205-207].  Practolol (B_-antagonist)  Dobutamine (B,-agonist)  Structure  Remarks  A l p h a - 2 > Alpha-1> Beta-, > Beta-2  PUBLICATIONS 1.  S h a i k h , I . M., C a n n o n d e R o d r i g u e z , W., and S k a l a , J . P . (1986) " *2~ d g 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 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 I s o l a t e d Membrane F r a g m e n t s " . (Submitted). <  0  A  r e n e r  : L C  2.  S h a i k h , I . M., C a n n o n 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 o f 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 Y o h i m bine A d m i n i s t r a t i o n . " (Submitted).  3.  S h a i k h , I . M. , Schimmel, R . J . , S k a l a , J . P . (1 9 8 6 ) " °< - 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 " . (In 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 Adipose T i s s u e o f I n f a n t Rats. (In p r e p a r a t i o n ) .  5.  D i a m o n d , 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 I n d u c e d C o n t r a c t i o n a n d 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 . 2 3 , 45-48.  6.  K e e s e y , J . , S h a i k h , I . M., W o l f g r a m , F . , Chow, A. ( 1 9 7 6 ) A b l o c k a d e o f N o r m a l M a m m a l i a n A c e t y l c h o l i n e R e c e p t o r s by Myasthenia g r a v i s s e r a . A n n . N.Y. A c a d . S c i . 274 , 2 4 4 - 2 5 2 .  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 o r d i n a t i o n C o m p l e x e s o f Osmium w i t h C u r a r i f o r m J . M e d i c i n a l Chem. 1_8, 1088-1094.  8.  S h a i k h , I.M., T h o m p s o n , N i c o t i n e on R a t G a s t r i c S o c . 13, 178-184  9.  Pharmacology of I o n i c C o o r d i n a t e d Complexes of 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.  Seasonal V a r i a t i o n s i n Tissue L i p i d s i n Uromastix 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 .  Bifunctional Activity.  Co-  J.H. (1970) A c u t e and C h r o n i c Effects S e c r e t i o n . Proc. Western Pharmacol.  hardwicki i  ABSTRACTS.  1.  S k a l a , J.P., S h a i k h , I.M., and Cannon de R o d r i g u e z , W. (1985) " The e< - 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 D e v e l o p i n g R a t s " . C l i n . I n v e s t . Med. 8^, A 170.  2.  S k a l a , J.P., S h a i k h , I.M.,and Cannon de R o d r i g u e z , W. (1985) " N o r e p i n e p h r i n e E f f e c t s upon Brown A d i p o c y t e s of I n f a n t R a t s A r e M e d i a t e d v i a B o t h <*- and fS-Adrenergic Receptors." F e d . P r o c . 44, 1160.  3.  S k a l a , J.P., S h a i k h , M.I., and Cannon de R o d r i g u e z , W. (1985) " The ^ - 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 c y t e s o f D e v e l o p i n g R a t s . " P a e d . Res. 1_9, 163A.  4.  S k a l a , J.P., and S h a i k h , I.M. (1984) " The t o r s i n Brown A d i p o s e T i s s u e of N e o n a t a l Rats'.' IV. Pan. Biochem. C o n g r e s s P r o c . No. 276.  5.  S k a l a , J.P., and S h a i k h , t o r s i n Brown A d i p o c y t e s  6.  S K a l a , J . P . and S h a i k h , I.M. (1984) " C a t e c h o l a m i n e E f f e c t s Brown A d i p o s e T i s s u e o f Newborn R a t s A r e M e d i a t e d Partially v i a the ©^""Adrenergic R e c e p t o r s . " F e d . P r o c . 4_3. 1581  7.  S k a l a , J . P . , S h a i k h , I.M., and V a c e k , L. (1983) " C y c l i c N u c l e o 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 o f Brown F a t i n G e n e t i c a l l y Obese M i c e . " F e d . P r o c . 4_2, 1849.  8.  9.  «*-Adrenergic  RecepAm.  I.M. (1984) " The °< - A d e r e n e r g i c Recepof Developing Rats. P a e d . Res. 18, 145A upon  S k a l a , J . P . , S h a i k h , I.M., and V a c e k , L. ( 1 9 8 3 ) . " C y c l i c - GMP'Participates i n - A d r e n e r g i c R e s p o n s e o f Brown A d i p o s e T i s s u e o f Young R a t s . P e d . Res. 1_7, 172. Diamond, J . , and S h a i k h , I.M. (1980) E f f e c t s o f H y d r a l a z i n e and D-600 on T e n s i o n and P h o s p h o r y l a s e A c t i v i t y i n R a b b i t A o r t a " . F e d . P r o c . 3j) ( 3 7 ) , 1176.  

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