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Inhibition of pulsatile luteinizing hormone release by atrial natriuretic peptide and brain natriuretic… Zhang, Jin 1990

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INHIBITION OF PULSATILE LUTEINIZING HORMONE RELEASE BY ATRIAL NATRIURETIC PEPTIDE AND BRAIN NATRIURETIC PEPTIDE IN THE OVARIECTOMIZED RAT by JIN ZHANG M.Sc,  Zhejiang Medical University,  China  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE FACULTY OF MEDICINE DEPARTMENT OF OBSTETRICS AND GYNAECOLOGY  We accept t h i s t h e s i s as conforming to the required  standard  THE UNIVERSITY OF BRITISH COLUMBIA AUGUST, 1990 @  J I N ZHANG, 1990  In presenting  this thesis in partial fulfilment of the requirements for an advanced  degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may department  or by his or her representatives.  be granted by the head of my  It is understood that  copying or  publication of this thesis for financial gain shall not be allowed without my permission.  Department of  O b s t e t r i c s and Gynecology  The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  September 10, 1990  written  ABSTRACT  Atrial origin,  natriuretic  peptide  (ANP)  been  to  a  has  natriuresis, However, of  the  it  and is  antagonism  in  system  hormone  secretion.  peripheral  luteinizing better  the  knowledge  of  routes  its the  role  atrial in  angiotensin  the  and  in addition to  and is  in  also  in  its  diuresis,  vasopressin.  the  role  produced  the  myocyte  production  in in  regulation  fluid  the of  has  been  (LH)  in  of  the  the  to  inhibit  pituitary  mechanisms  of  rat  role  of  will  neuropeptides  central  secretion  gonadectomized  modulatory  possible effects  shown  and  central  A d m i n i s t r a t i o n o f ANP t h r o u g h b o t h  hormone  and  it  participating  understanding  secretion  of  heart  homeostasis,  nervous  and  play  now a p p a r e n t t h a t  peptide  electrolyte  shown  of  model.  A  ANP on LH  add  on  of  to  our  reproductive  function. Brain of  26  brain.  natriuretic  amino The  vasorelaxant  acid  residues  peptide  exerts  effects,  has  a remarkable high  the  17  linkage  amino  acid  which  is  peptide  ring  (BNP)  is  recently potent  a  bioactive  identified  sequence homology  required  for  porcine  diuretic-natriuretic  i n a manner s i m i l a r t o  formed by  in  peptide  an  to  that  of  A N P . BNP  ANP, e s p e c i a l l y  intramolecular  biological  and  in  disulfide  activity.  The  presence  of  remarkable  BNP  resemblance  physiological inhibitory  third  in  implies  of  that  LH p u l s e  brain  and  structures  and  BNP may  also  exert  an  l i k e ANP. the  effects  pulsatile  of  mammalian  molecular  on  action  infusion  LH l e v e l ,  the  LH s e c r e t i o n focused  mechanisms  in  their  A N P a n d BNP o n  ventricle  plasma  on  research  administered  ANP  functions  effect  This  possible  with  in  of  LH s e c r e t i o n  ovariectomized  ANP o r  BNP,  amplitude  centrally  and  and  rats.  inhibition pulse  their After  of  mean  frequency  was  observed. In effect  searching  of  ANP o r  for  the  action  centrally  Application and in  BNP o n terms  reverse In  of  of the  endogenous  plasma  pulse  opiate  system w i t h ANP  or  the  effect  naloxone BNP  was  inhibitory effect  LH l e v e l  and  LH p u l s e  frequency,  naloxone of  experiments,  A N P a n d BNP o n L H s e c r e t i o n . no  inhibitory  the  there  were  significant  pulse  amplitude and p u l s e  the  tested. o f ANP but  failed  to  pimozide,  a  ANP o r B N P .  pretreatment  After  decrease  in  with  inhibitory action  infusion mean  frequency i n the  i i i  of  on  amplitude,  treatment  dopaminergic receptor blocker, prevented the of  of  reversed  inhibitory effect  separate  mechanisms  LH s e c r e t i o n ,  administered  naloxone  mean  possible  BNP o n p u l s a t i l e  inhibiting of  the  of  ANP o r BNP,  plasma  LH  level,  pimozide-pretreated  rats. In BNP  summary,  inhibit  inhibitory mediated  the  pulsatile  effects by  present  ANP  on  LH  study  secretion,  LH s e c r e t i o n  alone  may  be  mechanism  involving  both  inhibitory  mechanisms  may i n v o l v e  BNP w i t h  central  opiate  shows  ANP  system  secretion.  iv  thought  regulated  the  both  suggesting  once  and  that  BNP.  ANP a n d  that  to  the  be  through  a  dual  Furthermore,  interactions  and dopaminergic  of  the  ANP a n d  system  on LH  T A B L E OF CONTENTS  PAGE  ABSTRACT  i i  TABLE  v  O F CONTENTS  LIST  OF FIGURES  ix  LIST  OF ABBREVIATIONS  xiii  ACKNOWLEDGEMENTS 1.  LITERATURE 1.1  xiv REVIEW  1  Atrial  Natriuretic  1.1.1  Morphological  1.1.2  Structure  1.1.3  Biosynthesis  1.1.4  Peripheral  homeostasis  1.1.5  Regulation  o f ANP s e c r e t i o n  1.1.6  Mechanism  1.1.7  Central  1.1.7.1  of  1 ANP  1  o f ANP  3  o f ANP  5 action  o f ANP  actions  6 7  o f ANP a c t i o n  8  o f ANP  9  and b i n d i n g  sites  of  9  i n t h e CNS  Hypothalamic inhibiting hormone  1.1.7.3  (ANP)  overview  Distribution ANP  1.1.7.2  Peptide  posterior  of  ANP i n  11  pituitary  secretion  Hypothalamic altering  actions  actions  anterior  v  of  ANP i n  pituitary  13  hormone 1.1.7.3.1  I n h i b i t i o n of p r o l a c t i n s e c r e t i o n in  1.1.7.3.2 1.2  secretion  vivo  I n h i b i t i o n of LH s e c r e t i o n  i n v i v o 15  B r a i n N a t r i u r e t i c Peptide  17  1.2.1  The s t r u c t u r e  18  1.2.2  B i o s y n t h e s i s o f BNP  19  1.2.3  The d i s t r i b u t i o n o f BNP  19  1.2.4  The p h y s i o l o g y o f BNP  21  o f BNP  1.3 I n t r o d u c t i o n t o t h e r o l e o f neuromediators i n the  2.  14  LH  23  regulation  1.3.1  Dopamine  23  1.3.2  Norepinephrine  24  1.3.3  Endogenous o p i o i d p e p t i d e s  27  EXPERIMENTAL  32  2.1  Experimental r a t i o n a l e and d e s i g n  32  2.2  Materials  36  2.3  Results 2.3.1  and methods  42 E f f e c t s o f ANP and BNP i n f u s i o n on LH  2.3.1.1  42  secretion  E f f e c t o f s a l i n e i n f u s i o n on LH  42  secretion 2.3.1.2  E f f e c t o f 2nmol ANP i n f u s i o n on vi  42  LH 2.3.1.3  E f f e c t o f 2nmol BNP i n f u s i o n on LH  2.3.1.4  2.3.2  43  secretion  E f f e c t o f 0.2nmol BNP i n f u s i o n on LH  43  secretion  E f f e c t o f 0.2nmol ANP i n f u s i o n on LH  2.3.1.5  secretion  44  secretion  E f f e c t s o f naloxone treatment on t h e  53  LH i n h i b i t o r y a c t i o n s o f i . e . v . infused 2.3.2.1  ANP and BNP  E f f e c t s o f naloxone treatment i n control  2.3.2.2  53  group  E f f e c t s o f naloxone treatment i n  53  2nmol ANP group 2.3.2.3  E f f e c t s o f naloxone treatment i n  54  2nmol BNP group 2.3.3  E f f e c t s o f pimozide pretreatment on the  61  LH i n h i b i t o r y a c t i o n s o f i . e . v .  i n f u s e d ANP and BNP 2.3.3.1  E f f e c t s o f pimozide pretreatment i n 61 control  2.3.3.2  group  E f f e c t s o f pimozide pretreatment i n 2nmol ANP group vii  61  2.3.3.3  Effects 2nmol  of  pimozide  pretreatment  in  61  BNP g r o u p  3.  DISCUSSION  70  4.  SUMMARY AND CONCLUSIONS  81  4.1  Summary  81  4.2  Conclusions  82  5.  REFERENCES  84  v i i i  LIST  Figure ovx  rats  Figure ovx  6.  Representative with  7.  i.c.v  with  i.c.v  i.c.v  with  i.c.v  with  i.c.v  or  BNP i n f u s i o n  of  of  amplitude  PAGE  LH r e l e a s e  in  46  saline.  of  LH r e l e a s e  in  47  in  48  in  49  in  50  pre-  51  2 nmol ANP.  of  LH r e l e a s e  2 nmol BNP.  of 0.2  of 0.2  LH r e l e a s e nmol A N P .  LH r e l e a s e nmol BNP.  LH c o n c e n t r a t i o n  BNP i n f u s i o n  LH p u l s e  of  example  infusion  Mean p l a s m a  of  example  infusion  Representative  of  example  infusion  Representative  of  example  infusion  Representative with  example  infusion  Representative  post-ANP  Figure or  5.  rats  Figure and  4.  rats  Figure ovx  3.  rats  Figure ovx  2.  rats  Figure ovx  1.  OF F I G U R E S  in  the  period.  in  period.  ix  the  p r e - and post-ANP  52  Figure or  8.  LH p u l s e  BNP i n f u s i o n  Figure rats  9.  frequency  the  pre-  and post-ANP  53  period.  Representative  injected  in  with  example  naloxone  after  of  LH r e l e a s e  i.c.v  in  infusion  ovx  56  of  saline.  Figure rats  10.  Representative  injected  with  example  naloxone  after  of  LH r e l e a s e  i.c.v  in  infusion  ovx  57  ovx  58  of  2 nmol ANP.  Figure rats  11.  Representative  injected  with  example  naloxone  after  of  LH r e l e a s e  i.c.v  in  infusion  of  2 nmol BNP.  Figure  12.  post-ANP  Figure or  13.  BNP i n f u s i o n  LH p u l s e  BNP i n f u s i o n  Figure or  or  Mean p l a s m a LH c o n c e n t r a t i o n  14.  amplitude  and naloxone  LH p u l s e  BNP i n f u s i o n  and n a l o x o n e  frequency  and naloxone  in  the  the  pre-  pre-  and  59  period.  and p o s t - A N P  60  period.  pre-  treatment  x  the  treatment  treatment  in  in  and post-ANP  period.  61  F i g u r e 15. R e p r e s e n t a t i v e example o f LH r e l e a s e i n  64  p i m o z i d e - p r e t r e a t e d ovx r a t s with i . c . v i n f u s i o n o f saline.  F i g u r e 16. R e p r e s e n t a t i v e example o f LH r e l e a s e i n  65  p i m o z i d e - p r e t r e a t e d ovx r a t s w i t h i . c . v i n f u s i o n o f 2 nmol  ANP.  F i g u r e 17. R e p r e s e n t a t i v e example o f LH r e l e a s e i n  66  p i m o z i d e - p r e t r e a t e d ovx r a t s with i . c . v i n f u s i o n o f 2 nmol  BNP.  F i g u r e 18. Mean plasma LH c o n c e n t r a t i o n i n the p r e -  67  and post-ANP or BNP i n f u s i o n p e r i o d with the pimozide pretreatment.  F i g u r e 19. LH p u l s e amplitude  i n the p r e - and post-ANP  or BNP i n f u s i o n p e r i o d w i t h the pimozide  F i g u r e 20. LH p u l s e frequency  pretreatment.  i n the p r e - and post-ANP  o r BNP i n f u s i o n p e r i o d with the pimozide  69  pretreatment.  F i g u r e 21. Photograph o f r e p r e s e n t a t i v e f r o n t a l xi  68  70  s e c t i o n s i n the r a t hypothalamus d e p i c t i n g the s i t e o f cannula t i p i n the t h i r d v e n t r i c l e . by a b l a c k  triangle.  xii  The s i t e i s marked  LIST  OF ABBREVIATIONS  AHA  anterior  hypothalamic  ANP  atrial  BNP  brain  cGMP  cyclic  CNS  central  DA  dopamine  EOP  endogenous  i.c.v.  intracerebroventricular  LH  luteinizing  hormone  LHRH  luteinizing  hormone  MPOA  medial  mRNA  messenger  NE  norepinephrine  ng  nanogram  nmol  nanomole  OVX  ovariectomized  pANP  porcine  PRL  prolactin  RIA  radioimmunoassay  s.c  subcutaneously  SCN  suprachiasmatic  natriuretic natriuretic guanosine nervous  peptide peptide monophosphate  3'5'system  opioid  preoptic  peptides  releasing  hormone  area  ribonucleic  atrial  area  acid  natriuretic  peptide  a  x n i  nucleus  ACKNOWLEDGEMENTS  I Dr.  would  Peter  Leung,  these studies throughout  thesis, time not  least, the  gratefully providing  spent  in  acknowledge  me  his  colleagues,  Hugo  Wang a n d M . T . L i t t l e  M . Rodway, J .  i n the  during  for  time  my  Jian  to  the  Steele,  Bergen for  thank  final  Y.Pan  stages  of  for  their  and E u i - B a e  her  this  xiv  for  pursue  encouragement  I  also his  wish  suggestions Jeung,  and  to  technical to  my  who made my  experience.  patience  work.  supervisor,  opportunity to  laboratory.  l a b o r a t o r y a more e n j o y a b l e I  my  and f o r h i s continuous s u p p o r t and  the  acknowledge advice,  like  Last,  but  encouragement  1.  1.1  ATRIAL NATRIURETIC Atrial  origin  factor  and  has  natriuresis, actions  and  the  heart  and  plays  secretion 1986;  a  physiology specific  1.1.1  in is  well et  of  role  ventricular  of  in  their  of  a  role  1988).  the  the  a l . ,  in  ANP i s  et  al.,  in  i n p i t u i t a r y hormone  the  work.  large content  B . K i s c h p o i n t e d out  the  and  the  and  myocyte diuresis,  vasopressin it  is  peptide  pituitary  1985;  the  system hormone  Shibasaki et  an  in  electrolyte  c e n t r a l nervous of  now  a l . ,  overview  of  the  understanding  of  its  secretion.  OF ANP:  cardiocytes of  the  However,  Therefore,  important  atrial  atrial in  fluid  regulation  1988a).  called  production of  role  (Jacobowitz  muscle  mechanical  peptide  angiotensin  Samson,  its  also  play of  MORPHOLOGICAL OVERVIEW Most  a  (ANP),  a l s o produced i n the  role  Samson  to  in addition to  it  as  shown  1985;  and  homeostasis,  is  antagonism  (deBold,  apparent that  peptide  (ANF),  been  REVIEW  PEPTIDE  natriuretic  natriuretic  for  LITERATURE  forming  mammalian h e a r t  Morphologically  of contractile  the are  this  elements.  is  and  differentiated evident  However,  a morphological difference  1  atrial  in  between  from 1956, atrial  and  ventricular  that the  could  not  heart  1964;  be  only  observed,  as  others  Ferrans  et  unlike  features  of  measure  prominent rough  atrial of  of  the  that  (deBold et  the  ANP a l . ,  is  with 1983).  the  highest  Tissue  natriuretic  in  morphological expression  of  membrane-bound  are  are  found  also  common  the  within  of  fractionation  in  the  granules  associated  2  a  These  mammalian population  methods  have  atrial  granules  The net  specific  highest for  (deBold  show t h a t with  with  found.  general  1983). is  atrial  Numerous  the  Various  extracts  studies  of  specific  a l . ,  of  associated  arise.  core  are  core  normally  auricle.  atrial  granules  sarcoplasmic  Grammer e t  is  of  These  sarcoplasmic  number  activity  Palade,  cardiocytes  obvious  from which they  in  natriuretic activity of  and  He  more  stored  1983;  1956).  nanometres.  more  found  that  core  reticulum  are  pig  which d i s p l a y an e l e c t r o n - d e n s e  granules  central  (Kisch,  have  presence  guinea  framework  atrial  The most  the  the  (Jamieson  that  central  complex  cardiocyte  cardiocytes  shown  the  endoplasmic  features  500  Often,  Golgi  is  pump  cardiocytes,  cells.  granules  in  cardiocytes.  1969),  of  functional  subsequently  al. ,  to  the  heart  did  ventricular  250  concentrated  into  the  a mechanical  secretory  atrial  in  as  differentiation  specific and  fitted  serves  mammals,  this  cardiocytes  and the  fractions  animals Salerno,  highest containing  purified  granules  (deBold  immunocytochemical atrial  granules In  atrial  granules.  granules few  than  age.  It  number  of  procedures age  (deBold  Extracardiac notably  i n the  1985;  Saper  main  store  of  et  1.1.2  the  quantity  far  more  which  content  adulthood,  variations  atrial  have  cardiocytes  of  very  have  varies  an with  although  the  morphometric  in animals  of  the  same  nervous  al.,  ANP h a v e  system  1985).  mammals  of  is  and k i d n e y  However, the  been  in  (Sakamoto  quantitatively  atria  highest  suggested,  and t h e this  et  the  amount  of  tissue  1984).  S T R U C T U R E O F ANP While  has  cattle,  at  localizations  ANP i n  a l . ,  ANP w i t h i n  6 a n d 10 w e e k s a n d ,  ANP p r e c u r s o r m e s s e n g e r RNA i s (Nakayamaet  addition,  1983).  central  al.,  as  granule  plateaus  a l . ,  In  the  have  Human  rats,  significant  in  example,  such  between ages  show  localize  variations  for  In  1982).  1984).  cardiocyte.  granules  et  are  mammals  content.  doubles  a l . ,  Rodents,  per  a l . ,  clearly  et  there  large  granules  intermediate  studies  (Tanaka  mammals  et  been  biochemical  the  predominant storage  identified studies  as  a  resulted  form  of  15,000-dalton i n the  3  isolation  ANP i n  the  pro-ANP, of  atria early  numerous  low  molecular weight fragments ANP  during  1983) in  purification  . Evidence of  the  atria  weight  and  ANP f r o m  126-residue 1983).  analysis dense  form  Thibault  et  (Currie rapidly  (Miyata et  of  liquid  1985).  determined The  as  the  form.  ANP was  acid  isoleucine  i n mouse  Atlas  1985), et  al.,  et  whereas 1986;  ANP a s  a  a l . , atrial  sequence  ANP e x i s t e d  in  126-residue  a l . ,  1987;  (Seidah  purified,  a l . , human  Ong e t  1984),  al.,  and  (Kangawa a l . ,  of  that  et  1987)  et  a l . ,  and  its  is  1984),  rat  rabbit  ANP  al.,  1984),  or  high  circulated  1984,  Miyata  sequence al.,  almost  for position  and dog  4  ANP  (Thibault et ANP  except  et  filtration  (Sugiyama  sequence  mammalian s p e c i e s  gel  showed  28-residue peptide  throughout  1984;  of  this  Gibson et  ANP b y  chromatography  Plasma  amino  molecular  direct  rat, in  a l . ,  cultured  and  predominantly  et  Grammer e t  atria,  the  pro-  w e i g h t ANP  high  1983;  in  1985;  of  precursor  al.,  that  plasma  i n a low m o l e c u l a r w e i g h t a l . ,  the  granules  a l . ,  Flynn  of  1987).  Examination  et  1984;  a high molecular  boiled  atrial  granules  a l . ,  proteolysis  analysis  et  established  a l . ,  performance  sequence  purified  firmly  et  identified  from  secretory  pro-ANP  the  peptide.  or  (Seidah  presence of  atria  Isolation  myocytes,  the  p r o d u c e d by t h e  1985).  identical  110  which  (Napier et (Oikawa  et  bovine  (Oikawa et  was  al.,  is  a l . , a l . ,  (Vlasuk 1985)  ANP h a v e  methionine  Various variations sequence ANP's  of  of  amino  biological  a l . ,  1.1.3  position.  and s y n t h e t i c  ANP s t r u c t u r e acids  cleavage  or  methods  revealed ring  functions.  almost completely et  this  cleavage  17  proteolytic  in  that  reduction  abolishes its  a  structure  Opening  Cloning encoding  the  Flynn  established  sequence  ANP  precursor  et  a l . ,  that  the  A high  human.  amino  analysis have  of  signal  isolated rat  activity  (Misono  prepro-ANP  by  these  in of  the  homology  sequences, form o f is  a  natriuretic  removal  of  the  in  has  and  151  a l . , been  prepro-hormone  form  amino  acids  between  the  24-  25-amino  exists  rat  and  atrial  126-amino  acid  peptide.  It  signal  5  complete  (Nakayama e t it  DNA  in rat  respectively. ANP  a  more  studies  in  rat  a  These p r e c u r s o r s c o n t a i n  granules  atrial  From  acids  The main s t o r a g e  gamma  bridge  complementary  provided  synthesized  degree  a n d human s e q u e n c e s . putative  1985).  ANP i s  152  in  by  OF ANP  and  containing  and  structure  disulfide  physiological  understanding of a t r i a l n a t r i u r e t i c peptides  acid  for  1984).  BIOSYNTHESIS  1984;  essential  ring  the  introducing  disulfide-looped is  the  of  for  is  peptide  homogenates  peptide  called  derived and  from  arginine  residues  151  and  peptides  so  far  biosynthesis The atrial  pro-ANP  of  its  to  et  a l . ,  many  lung,  the  the  hypothalamus, demonstrated (Inagami  1.1.4  et  a l . ,  PERIPHERAL ANP  toward  by  elicits  the  inhibits  the after  are  these  plasma  acid  take  or  a l . ,  small  in  is  alpha  peptide The  place  that  conversion at  immediately  the  thereafter  1987).  by  far  the  pituitary, arch,  major  source  adrenal and  quantities  renal  tissues,  (Inagami  especially  biosynthesis  has  distal  localization  cells  et  a l . ,  in  been of  of  medulla,  and s p i n a l g a n g l i o n i c  the  time  rat  clearly ANP  mRNA  1989).  HOMEOSTATIC ACTIONS a  reduction  renin  et  identifying  in  removal  prepro-ANP.  aortic  local  ANP  should  duct c e l l s ,  of  the  of  brain,  in  residues  early  28-amino  atrium  ventricle,  some  a  atria  the  ANP  rat  150  the  collecting  In  of  Imada  of  these  1984).  form  cardiac  contain  1989).  to  from  regions  (medullary)  a l . ,  form  123  of  indicates  peptide,  1987;  cardiac  to  et  active  secretion  While  seem  (Kanagawa  residues  of  absence  isolated  natriuretic  (Gibson  The  circulating  comprises  ANP,  152.  variety of  release,  of  blood  OF ANP  responses pressure  vasopressin  6  which  and  release,  are  blood and  directed  volume.  It  aldosterone  r e l e a s e . I t markedly s t i m u l a t e s d i u r e s i s and n a t r i u r e s i s by both a hemodynamic e f f e c t rate)  and  a  direct  ( i n c r e a s e o f glomerular  tubular  effect  filtration  (inhibition  of  sodium  reuptake by i n n e r medullary c o l l e c t i n g duct) (Mark, 1989). ANP administered  i n t o t h e c e r e b r o v e n t r i c l e s antagonizes  e f f e c t s o f a n g i o t e n s i n I I i n water d r i n k i n g , s a l t hypertension,  and  vasopressin  release.  It  central  appetite,  also  inhibits  e l e c t r i c a l l y s t i m u l a t e d norepinephrine r e l e a s e from a d r e n e r g i c nerve endings and neuronal al.,  1989).  ANP  vasoconstrictors  activities  reduces such  vascular  as  tone  (David e t  induced  norepinephrine,  angiotensin I I , or vasopressin  1.1.5  of ganglia  by  histamine,  (Margaret e t a l . , 1989).  REGULATION OF ANP SECRETION Plasma l e v e l s o f ANP measured by radioimmunoassay v a r y  widely from approximately  25 t o 100 picograms/ml o f plasma i n  humans and 100 t o 1000 picogram/ml i n r a t s . s p e c i e s , measuring techniques, f o r some o f t h i s v a r i a b i l i t y Studies  Differences i n  and sampling p r o t o c o l s account (deBold,  1985).  i n d i c a t e t h a t t h e primary s t i m u l u s  for atrial  s t i m u l a t i o n o f ANP s e c r e t i o n i s a t r i a l d i s t e n s i o n presumably due  to elevation  o f venous  blood  pressure  (Dietz,  1984;  Ledsome e t a l . , 1985). T h i s can be caused by an e l e v a t i o n o f 7  venous  return  1984),  head  immersion  blood  down  t i l t  (Ogihara  vasoconstrictors shown t o this can  be  1984;  GMP  et  et  1986).  (Sugiyama  a l . ,  1986  and  blood  culture  et  al.,  or  water  doses  vasopressin  (Margaret et  a l . ,  ),  Pharmacological  epinephrine  increased  of  a l . ,  pressure,  of  were  1989).  the  conditions  While  stimulation  (Sonnenberg  and  1985).  ANP m u s t b e m e d i a t e d  ANP i n f u s i o n  (Hamet e t  A N P was  types  type  guanylate  in  receptor  of  is  cyclase-free  rats  the  of  tissue  to  ANP r e c e p t o r  Leitman  et  not c l e a r . receptor  a l . ,  a  to  increase  activation  (Waldman e t ANP was et  a l . ,  has  been  1988).  guanylate  cyclase  activated  it  1984) .  exact  receptor  than  Cyclic  extensively  1984). by  rather  in  Recently,  its  a  absence  of  (Takayanagi role  of  whose  et  such  has been suggested t h a t  clearance  8  a l . ,  identified The  plasma  of  found  characterized  However, is  shown  receptor-mediated  membrane-bound enzyme  (Hamet  activity  by  The guanylate  cyclase  response  was  reflecting  1984).  in  cyclase  1987;  be  guanylate  production  second  a l . ,  found t o  cytosolic  numerous  al.,  as  u r i n a r y cGMP m a r k e d l y ,  cyclase  the  a l . ,  in tissue  effects  processes.  by  et  Manning  feeding  MECHANISM O F A N P A C T I O N The  and  salt  ANP r e l e a s e  to  seen even  Veress,  1.1.6  due  to  (Hollister  such  stimulate  may b e  due  a  the  function  is  to  absorb  circulation  excess  or  ANP a n d  store  and  either  slowly  to  release  eliminate  it  from  it  et  a l . ,  (Maack  1987) .  1.1.7  C E N T R A L A C T I O N S OF ANP  1.1.7.1  D I S T R I B U T I O N AND B I N D I N G  SITES  first  cardiovascular  At that  any  ANP  sequestration et  al.  it  of  argued  detected from  ANP  particularly  was  in  within  plasma.  presence the  by  the  Thus in  the  However,  reported  distribution  within  the  (Kawata e t  rat  there  cell  bodies, ventral  These  large,  project  to  brain,  a l . ,  that  the  unique  1985;  were the  nucleus  A second  most  strongly Skofitsch major  that  a l . ,  1985).  i n the  of  thalamus, the  cluster  stria  of  were  (Standaert  production  adjacent  nucleus,  regions et  a l . ,  to  part.  observed  o f ANP c o n t a i n i n g n e u r o n s w a s d e t e c t e d  9  showed  rostral  paraventricular  terminals  neurons  ANP-positive  walls  preoptic-septal  met  groups  Studies  p a r t i c u l a r l y i n the  the  brain,  several  local  Morii  1985),  ANP-positive  concentrations  parvocellular  by  the  a l . ,  year  monopolar A N P - p o s i t i v e neurons the  of  et  suggesting et  reflected  report  areas  of  abundant b e i n g  third ventricle  periventricular bed  three  later  researchers  merely  initial  (Morii  w i t h much s k e p t i c i s m . the  CNS  large  hypothalamus  O F A N P I N T H E CNS  and  to the the  1985). in  the  lateral  hypothalamic  such as  the  peripedunculopontine,  parabrachial solitarius  nuclei  i n the  et  in  heart  al.,  1985)  et  al.,  1986),  after  et  similar  ANP i n  and  binding  1986).  storage  in  brain  guinea  pig  the  mature  prohormone  the  of  brains  detected  in  tegmental,  of  the  to  form of  a l . ,  as  upon r e l e a s e 28  et  have the  sites  the  organ  1986b; J o h n s o n , sites  (Quirion  1985).  i n median eminence,  anterior  pituitary  also  ANP i n t h e  hypothalamic  secretion  (Quirion et  organs, et  al.,  The intensive septum,  a l . ,  of  1984).  10  1984;  a  to  of  be  function,  Saavedra  possible  the  Significant  particular  presence  anterior  in  known  medial preoptic  indicated control  in  are  heart.  sites  described.  central  1985;  processing  in  ANP b i n d i n g  been  stored  a l . ,  post-translational  specific  is  transcripts  from t h a t  the  (Michener  amino a c i d  ANP-mRNA  differ  circumventricular  - subfornical  tractus  Needleman  i m p o r t a n t i n h y d r o m i n e r a l b a l a n c e and c a r d i o v a s c u l a r such  and  ANP i s  1985;  (Glembotski  brain  heart,  appears  distributions  was  the  (Glembotski et  Since  structures  1986b).  where  brain  from the  those the  The rat  i n the  a l . ,  to  nucleus  and f i n a l c l e a v a g e occurs  cleavage  Shiono  i n b r a i n stem  dorsolateral  the  a l . ,  amino a c i d prohormone  et  of  and  (Standaert  Unlike 126  a r e a and a t h i r d  et  the al.,  ANP-binding nucleus,  involvement  pituitary  and of  hormone  There are s p e c i e s d i f f e r e n c e s i n the d i s t r i b u t i o n of i n the CNS.  S t u d i e s u s i n g RIA f o r ANP have shown t h a t ANP-like  immunoreactivity the  ANP  i s present  hypothalamus  and  i n the h i g h e s t c o n c e n t r a t i o n s i n  septum,  followed  by  the  midbrain  and  c e r e b r a l c o r t e x i n r a t s ( M o r i i e t a l . , 1985). In the monkey brain,  the  followed  highest by  the  immunoreactivity  concentration pons  i s the  receptors involved  are  found  i n the  midbrain  The  ANP-like  hippocampus.  c o n c e n t r a t i o n i n the dog b r a i n was  h i g h e s t i n the midbrain As  and  was  ( F u j i n o e t a l . , 1987).  case i n the p e r i p h e r a l , two present  a l s o the  in  the  brain,  i n c l e a r a n c e o f the peptide,  c l a s s e s of  one the  thought  other  to  ANP be  involved i n  the e x p r e s s i o n of ANP's b i o l o g i c a l a c t i o n (Leitman and  Murad,  1987).  1.1.7.2  HYPOTHALAMIC ACTION OF THE  ANP  IN  INHIBITING  POSTERIOR PITUITARY HORMONE SECRETION The kidney  opposing  and  ability  actions o f ANP  t h e r e suggested t h a t ANP CNS  of to  vasopressin inhibit  into  unrestrained  the rats  ANP  vasopressin's  in  the  action  might a f f e c t v a s o p r e s s i n r e l e a s e i n  ( D i l l i n g h a m e t a l . , 1986). Under b a s a l  injected  and  third  conditions,  cerebroventricle  significantly 11  inhibited  of  ANP  conscious, vasopressin  secretion. This e f f e c t i s selective f o r vasopressin r e l e a s e was ANP  on  the  median  unrelated)  and  i s not mediated v i a an a c t i o n of  vasopressin-containing  eminence  or  (oxytocin  neural  nerve  lobe,  the the  hypothalamic l e v e l , e i t h e r d i r e c t l y on the d e n d r i t e s o r  cell  in  the  vicinity  instead  in at  body o f the v a s o p r e s s i n - p r o d u c i n g  but  terminals  neuron or on  interneurons  (Samson e t a l . , 1987). C r a n d a l l  and  a l s o demonstrated t h i s v a s o p r e s s i n - i n h i b i t i n g e f f e c t  Gregg of  ANP  i n the hypothalamic neurohypophyseal e x p l a n t , both under b a s a l or a n g i o t e n s i n the h y p o t h e s i s Gregg,  1986).  II-stimulated conditions, further  of a hypothalamic s i t e of a c t i o n ( C r a n d a l l and These  in  vitro  demonstration of the a b i l i t y block  angiotensin  conscious  results  of ANP  II-stimulated  agree  pretreatment  vasopressin  with  the  i n v i v o to  secretion  in  r a t s (Yamada e t a l . , 1986).  The a b i l i t y of ANP experimentally-induced  t o i n h i b i t v a s o p r e s s i n s e c r e t i o n and  water i n t a k e (Antunes e t a l . , 1985)  s a l t preference  (Antunes e t a l . , 1986)  to  of  the  supporting  ability  the  peptides  to  and  might a l l be r e l a t e d  antagonize  the  1  central  a c t i o n o f a n g i o t e n s i n I I . In any event, these c e n t r a l a c t i o n s seem w e l l matched t o many of the p e r i p h e r a l a c t i o n s o f ANP  and  suggest c o o r d i n a t e d e f f e c t s t h a t f u n c t i o n p h y s i o l o g i c a l l y t o maintain  hydromineral  homeostasis. 12  1.1.7.3  HYPOTHALAMIC ACTIONS OF  ANP  IN  ALTERING ANTERIOR  PITUITARY HORMONE SECRETION Several l i n e s of evidence suggest c e n t r a l a c t i o n s of unrelated  to hydromineral balance  Samson et a l . , 1987). The ANP  (Standaert  presence of ANP  e t a l . , 1986a;  binding  sites  and  p o s i t i v e axon t e r m i n a l s i n regions seemingly u n r e l a t e d  those  functions  and  the  presence  of  ANP  ANP  responsive  to  single  neurons i n the medial p r e o p t i c and l a t e r a l s e p t a l areas( Wong e t a l . , 1986.) suggest other p o s s i b l e neuromodulatory a c t i o n s of the  peptide. The  presence o f  ANP  immunoreactivity  i n the  external  l a y e r of the median eminence (Kawata et a l . , 1985b; S k o f i t s c h et a l . , 1985)  and  ANP  binding  sites  i n the  suggest d e l i v e r y o f c e n t r a l l y - d e r i v e d ANP  pituitary  gland  v i a the hypophyseal  p o r t a l v e s s e l s t o the a n t e r i o r p i t u i t a r y where i t might exert some t r o p i c a c t i o n . However, s t u d i e s f a i l e d t o demonstrate a g r a d i e n t of p o r t a l v e r s u s p e r i p h e r a l plasma ANP in  the  rat,  a  hypophysiotropic ANP  will  criterion  agents (Samson and  stimulate  guanylate  anterior pituitary c e l l s , l i n k e d t o any et  satisfied  by  concentration  all  recognized  B i a n c h i , 1987). Although  cyclase  activity  t h i s a c t i v i t y does not  in  seem t o  s i g n i f i c a n t e f f e c t on hormone i n v i t r o  a l . , 1986).  Several  lines 13  of  evidence  cultured be  (Simard  suggested  that  n e i t h e r b a s a l nor s t i m u l a t e d  hormone s e c r e t i o n from c u l t u r e d  p i t u i t a r y c e l l s i s a l t e r e d by ANP (Simard e t al.,1986; H e i s l e r et a l . , 1986) . Log doses o f ANP r a n g i n g from one p i c o - t o one micromolar  failed  stimulating  to a l t e r  the release  hormone, f o l l i c l e  o f LH, PRL, t h y r o i d  stimulating  hormone o r growth  hormone by c e l l s harvested from i n t a c t o r gonadectomized  rats  (Samson and B i a n c h i , 1987). A d d i t i o n a l l y , t h e presence o f ANP i n the i n c u b a t i o n  medium f a i l e d t o a l t e r t h e LH response o f  these  LHRH,  cells  to  the  growth  hormone  response  to  somatostatin o r growth hormone-releasing hormone, the t h y r o i d s t i m u l a t i n g hormone response t o t h y r o t r o p i n r e l e a s i n g hormone, or the a b i l i t y o f dopamine t o i n h i b i t PRL and a v a r i e t y o f PRL releasing Bianchi,  factors 1987).  to  These  stimulate studies  PRL  would  release suggest  (Samson  and  then t h a t  any  observed e f f e c t o f the p e p t i d e on p i t u i t a r y hormone s e c r e t i o n observed i n v i v o must be e x e r t e d a t t h e hypothalamic s i d e o f the h y p o t h a l a m o - p i t u i t a r y a x i s .  1.1.7.3.1  INHIBITION OF PROLACTIN SECRETION IN VIVO  The a b i l i t i e s o f ANP t o i n t e r a c t w i t h b r a i n dopaminergic systems i n v i v o  (Nakao e t a l . , 1986) and t o i n h i b i t dopamine-  beta-hydroxylase a c t i v i t y i n pheochromocytoma c e l l s i n v i t r o (Racz e t a l . , 1986) suggested t h a t ANP might play a r o l e i n 14  the  hypothalamic c o n t r o l  produced  in  hypothalamic  of  PRL  secretion,  tuberoinfundibular  neurons,  PRL-release  since is  inhibiting factor.  dopamine, the  No  -major  significant  e f f e c t s of b o l u s i n j e c t e d or p e r i p h e r a l l y i n f u s e d ANP s e c r e t i o n were observed i n conscious r a t s (Samson and 1987). However, when the ventricle  of  secretion  was  relatively action  ANP  is  by  i n t o the  PRL  dose-related  and  receptor  effects  elevation  of  c i r c u l a t i n g ANP  p e p t i d e or by  levels  minutes  or  This  blocker tyrosine  of  ANP  on  longer  by  release  either  either  by  acute volume expansion in vivo  1988). However, when plasma l e v e l s o f ANP 30  1987).  i n h i b i t i o n of  direct  third  i n h i b i t i o n of  Bianchi,  dopamine  Bianchi,  LH SECRETION IN VIVO  f a i l e d t o s i g n i f i c a n t l y a l t e r LH  for  was  PRL  dopamine neurons (Samson e t a l . , 1988a).  b o l u s i n j e c t i o n of the  al.,  and  following  suggesting  INHIBITION OF  Brief  effect  (Samson  absent  infused  significant  The  prevented  activity,  tuberoinfundibular  1.1.7.3.2  a  lasting is  and  hydroxylase  brain,  observed.  long  of  treatment  the  p e p t i d e was  on  gradual  (Samson et  were and  elevated continued  volume expansion or by intravenous i n f u s i o n o f the p e p t i d e at a  dose which  circulating  resulted  i n a two  concentrations,  to  three  plasma 15  fold  levels  elevation of  LH  of fell  significantly  (Samson e t a l . , 1988; Standaert e t a l . , 1986a).  T h i s e f f e c t was mediated c e n t r a l l y and not a t t h e p i t u i t a r y l e v e l , because t h e LH s e c r e t o r y response t o a subsequent b o l u s i n j e c t i o n o f LHRH was not changed by the presence o f e l e v a t e d ANP (Samson e t a l . , 1988). These r e s u l t s suggest t h a t ANP a c t s e i t h e r as a r e s u l t of  a c e n t r a l nervous system e f f e c t o r some  inhibition reflex  o f LH r e l e a s e .  plays  an  reflex-mediated  I t i s unlikely that  important  role  because  other  agents f a i l e d t o i n h i b i t LH r e l e a s e when g i v e n and  because ANP  injected 1988).  into  significantly  the t h i r d  Furthermore,  hypotensive intravenously  i n h i b i t e d LH s e c r e t i o n  cerebroventricle  in vitro  a peripheral  studies  (Samson  have  median eminence as a t l e a s t one p o s s i b l e  et a l . ,  identified  site  when  the  of action of  ANP, because t h e r e l e a s e o f LHRH from median eminence e x p l a n t s i n v i t r o by catecholamines can be i n h i b i t e d i n a d o s e - r e l a t e d f a s h i o n by t h e presence o f ANP i n the medium (Samson e t a l . , 1988). In  addition  antagonize  to a possible  catecholamine  central action  stimulation  o f LHRH  o f ANP t o  release,  ANP  i n h i b i t i o n o f LH s e c r e t i o n appears t o be mediated i n p a r t by an  i n t e r a c t i o n with  recognized  endogenous  opiate  systems,  which a r e  t o be p h y s i o l o g i c i n h i b i t o r s o f t h e hypothalamic 16  component o f  LH  release  (Bruni  et  a l . , 1977;  Pang e t a l . ,  1977) . Pretreatment of r a t s w i t h naloxone completely a b o l i s h e s the LH i n h i b i t o r y a c t i o n (Samson e t a l . , 1988). The the endogenous o p i a t e t o  i n h i b i t LH  a c t i o n of  s e c r e t i o n i s thought  to  be expressed i n the p r e o p t i c and a n t e r i o r hypothalamic r e g i o n s (Fink, 1988). I t should be noted t h a t ANP-containing neuronal elements are  present  i n these  f i e l d s p o s i t i v e f o r ANP known t o be secretion  regions  and  indeed  immunoactivity are present  terminal  i n regions  important i n the c e n t r a l c o n t r o l of gonadotropin  ( Q u i r i o n et  a l . , 1984;  Standaert e t  Furthermore, i t has been demonstrated t h a t the and m i c r o p r e s s u r e - i n j e c t e d  a l . , 1986b). iontophoresed  ANP  can i n h i b i t the f i r i n g r a t e of  s i n g l e neurons i n t h i s r e g i o n  (Wong et a l . , 1986). Thus, i n  a d d i t i o n t o p o s s i b l e median eminence a c t i o n s of the rostral  hypothalamic  sites  neuromodulatory a c t i o n s of  1.2  might  also  be  targets  peptides, for  the  ANP.  BRAIN NATRIURETIC PEPTIDE Brain  natriuretic  peptide  of  porcine  brain  only a few  26  amino  peptide  acid  (BNP)  residues  is a  novel  recently  (Sudoh e t a l . , 1988a) and  bioactive  identified  in  therefore there  are  p u b l i c a t i o n s d e s c r i b i n g i t . I t i s known t h a t  the  peptide e x e r t s a potent d i u r e t i c - n a t r i u r e t i c a c t i v i t y as w e l l 17  as a v a s o r e l a x a n t e f f e c t , i n a manner s i m i l a r t o t h a t o f ANP (Sudoh e t a l . ,  1988a).  BNP has a remarkable h i g h  sequence  homology t o ANP, e s p e c i a l l y i n t h e 17 amino a c i d r i n g formed by an i n t r a m o l e c u l a r d i s u l f i d e unit required Sudoh  l i n k a g e which i s t h e c e n t r a l  forbiological activity  e t a l . , 1988b).  (Sudoh e t a l . ,  The presence o f BNP  with  1988a; ANP i n  mammalian b r a i n s and remarkable resemblance i n t h e i r molecular structures  imply  that  BNP  may  share  some  physiological  f u n c t i o n s w i t h ANP.  1.2.1  THE STRUCTURE OF BNP BNP has a remarkable h i g h sequence homology  ANP,  especially  intramolecular required  i n t h e 17 disulfide  for biological  amino  acid  l i n k a g e which activity.  ring  t o alpha-  formed  i s the c e n t r a l  The h i g h e s t  by an unit  homology i s  observed when BNP i s compared t o alpha-ANP(4-28), one o f the brain  forms  o f ANP.  BNP-32,  a N-terminal  s i x amino  acid  extended form o f "BNP, has been i d e n t i f i e d as t h e second form i n the BNP f a m i l y i n p o r c i n e b r a i n (Sudoh e t a l . ,  1988b). The  comparison o f t h e amino a c i d sequence o f BNP and BNP-32 with alpha-ANP(4-28) and alpha-ANP(5-28), which a r e two major forms of ANP i n p o r c i n e b r a i n (Ueda e t a l . , 1987), a r e l i s t e d below. BNP-32:  S-P-K-T-M-R18  D-S-G-C-F-G-R-R-L-D-R-I-G-S-L-S-G-L-G-C-N-V-L-R-R-Y BNP: D-S-G-C-F-G-R-R-L-D-R-I-G-S-L-S-G-L-G-C-N-V-L-R-R-Y alpha-ANP(4-28): R-S-S-C-F-G-G-R-M-D-R-I-G-A-Q-S-G-L-G-C-N-S-F  R-Y  alpha-ANP(5-28): S-S-C-F-G-G-R-M-D-R-I-G-A-Q-S-G-L-G-C-N-S-F  1.2.2  R-Y  BIOSYNTHESIS OF BNP As  the sequence  o f pBNP  i s not found  i n t h e known  sequence o f the ANP, the p e p t i d e i s probably p r o c e s s e d from i t s own p r e c u r s o r , g e n e t i c a l l y d i s t i n c t from the ANP p r e c u r s o r (Sudoh  e t a l . , 1989).  Furthermore,  the seven  amino-acid  changes observed between ANP and BNP are not c o n v e r t i b l e by a single nucleotide substitution,  i n d i c a t i n g t h a t t h e genes  encoding the two molecules d i v e r g e d a t an e a r l y stage (Sudoh et a l . ,  1.2.3  1989).  THE DISTRIBUTION OF BNP The  determination  and  comparison  of  regional  d i s t r i b u t i o n o f BNP and ANP have been made i n p o r c i n e b r a i n . Concentrations striatum  among  o f BNP a r e h i g h e s t a l l the  brain 19  i n medulla-pons regions  and t h e  examined.  BNP  c o n c e n t r a t i o n s of the r e s p e c t i v e r e g i o n s can be summarized as follows:  medulla-pons,  midbrain-thalamus, cerebellum  (Ueda  concentrations  striatum  cortex, et  of ANP  >  hypothalamus,  olfactory  al.,  bulbs  1988).  i n each r e g i o n  septum  > hippocampus >  Meanwhile, of  >  porcine  tissue  brain  are  simultaneously determined with the same t i s s u e e x t r a c t s used for  measurement  c o n c e n t r a t i o n of ANP  of  BNP  concentrations.  i s observed  The  highest  i n the o l f a c t o r y b u l b s ,  and  the second h i g h e s t i n the hypothalamus. The medulla-pons and s t r i a t u m do not c o n t a i n as much ANP bulbs  has  comparable  Concentrations  of  ANP  as BNP.  concentrations in  respective  f o l l o w i n g order: O l f a c t o r y bulbs  Only the o l f a c t o r y of  BNP  regions  and are  > hypothalamus >  ANP. in  the  midbrain-  thalamus > medulla-pons > septum > hippocampus, s t r i a t u m . In terms o f  c o n c e n t r a t i o n of whole b r a i n , BNP  about t e n times h i g h e r than t h a t of ANP This  result  clearly  be  of  BNP  i s not  suggesting the p o s s i b i l i t y t h a t the  d i f f e r e n c e s i n t i s s u e c o n c e n t r a t i o n and and ANP  to  (Ueda e t a l . , 1988).  shows t h a t d i s t r i b u t i o n  p a r a l l e l t o t h a t of ANP,  i s found  d i s t r i b u t i o n of  BNP  r e f l e c t d i f f e r e n t p h y s i o l o g i c a l f u n c t i o n s f o r these  two n a t r i u r e t i c p e p t i d e s .  20  1.2.4  THE  PHYSIOLOGY OF  BNP  Intravenous i n j e c t i o n s of s y n t h e t i c p o r c i n e BNP i n t o anaesthetized  r a t s r e s u l t e d i n a remarkable i n c r e a s e i n  e x c r e t i o n of u r i n e and very  similar  (pBNP)  to that  electrolytes elicited  by  i n t o u r i n e , i n a manner  alpha-ANP  (Sudoh e t a l . ,  1988). Furthermore, i n j e c t i o n of pBNP i n t o a n a e s t h e t i z e d r a t s causes  a  significant  comparable Thus,  to  pBNP  that  i s as  induced potent  diuretic-natriuretic  suggesting  a  by as  in  mean  blood  alpha-ANP a t the alpha-ANP  activity.  a c t i v i t y of pBNP i s 3-4 ANP,  decrease  in  pressure, same dose.  hypotensive  However, the  and  rectum-relaxant  times more potent than t h a t o f a l p h a -  unique  feature  in  the  physiological  s i g n i f i c a n c e of pBNP (Sudoh e t a l . , 1988). In accordance with i t s p e r i p h e r a l f u n c t i o n i n f l u i d  and  e l e c t r o l y t e homeostasis, BNP has been demonstrated t o suppress both b a s a l v a s o p r e s s i n s e c r e t i o n and A l l - i n d u c e d v a s o p r e s s i n secretion  i n euhydrated  a c t i o n of BNP  freely  moving  on b a s a l v a s o p r e s s i n  rats.  The  inhibitory  i s comparable t o t h a t of  alpha-ANP (Yamada e t a l . , 1986). In a d d i t i o n , the time course and  potency of the  vasopressin  secretion  c o u n t e r p a r t s of ANP Thus,  inhibitory  centrally  are  effect also  i n conscious  administered  on A l l - i n d u c e d  comparable  to  their  r a t s (Yamada e t a l . , 1986).  BNP 21  of BNP  and  ANP  appear  to  have  similar effects  on v a s o p r e s s i n s e c r e t i o n  i n conscious  rats.  BNP a l s o e x h i b i t e d equipotent i n h i b i t o r y a c t i o n s on c e n t r a l A l l - i n d u c e d water i n t a k e (Itoh e t a l . , 1988), p r e s s o r response (Shirakami e t a l . , 1988), when compared t o ANP. These f i n d i n g s raise  the p o s s i b i l i t y  t h a t some c e n t r a l  a c t i o n s o f ANP a r e  shared by BNP. It  has  intracellular  been  demonstrated  cyclic  GMP  contents  that  pBNP  increases the  i n t h e kidney  epithelial  c e l l l i n e . The s t i m u l a t i o n by pBNP o f c y c l i c GMP accumulation was comparable t o t h a t by alpha-ANP a t t h e same c o n c e n t r a t i o n s (Iwata e t a l . , 1989). Since these experiments in  t h e presence  o f the phosphodiesterase  were  performed  inhibitor,  the  i n c r e a s e i n c y c l i c GMP contents was probably due t o a c t i v a t i o n of  g u a n y l a t e c y c l a s e . The o b s e r v a t i o n t h a t t h e simultaneous  addition  o f pBNP  and alpha-ANP  a t t h e maximal  c o n c e n t r a t i o n had no a d d i t i v e e f f e c t on c y c l i c GMP  effective contents  suggests t h a t both p e p t i d e s i n t e r a c t w i t h t h e same r e c e p t o r s (Iwata e t a l . , 1989).  22  1.3  INTRODUCTION TO  THE  ROLE  OF  NEUROMEDIATORS  IN LH  (DA)-producing neuronal  groups  REGULATION. 1.3.1  DOPAMINE There are two dopamine  that  innervate  regions  ( L i n d v a l l and B j o r k l u n d ,  of  the preoptic-tuberal  pathway  1978). A small group o f DA c e l l s i n  the p e r i v e n t r i c u l a r n u c l e i i n n e r v a t e s the medial p r e o p t i c area (MPOA), nucleus  a n t e r i o r hypothalamic  area  (AHA),  suprachiasmatic  (SCN), and the p e r i v e n t r i c u l a r nucleus o f t h e a n t e r i o r  hypothalamus testosterone projections  (Lindvall  and B j o r k l u n d ,  treatment  inhibited  i n t h e MPOA and AHA  more c a u d a l l y l o c a t e d c e l l system with  terminations  1978). In male  DA  turnover  in  rats, these  (Simpkins e t a l . , 1980). A  group, t h e t u b e r o i n f u n d i b u l a r DA  i n the v i c i n i t y  o f LHRH axons and  t e r m i n a l s i n the median eminence (Fuxe e t a l . , 1976), may p l a y a r o l e i n c o n t r o l o f LHRH s e c r e t i o n . Although DA was found t o e x c i t e LHRH r e l e a s e from t h e median eminence o f male r a t s i n vitro  (Negro-Vilar, e t al.,1979),  produced l i t t l e al.,  intraventricular  injection  s t i m u l a t i o n o f LH r e l e a s e i n v i v o  (Kalra et  1983) . A good d e a l o f evidence  agrees with t h e view t h a t  DA neurons may e x e r t an i n h i b i t o r y i n f l u e n c e on LH s e c r e t i o n (Gallo,  1980) and thereby  mediate  the negative  feedback  e f f e c t s o f t e s t o s t e r o n e on LH r e l e a s e (Simpkins e t a l . , 1980).  S i m i l a r u n c e r t a i n t y e x i s t s as t o t h e e f f e c t s o f DA on LH r e l e a s e i n female r a t s . A c t i v a t i o n o f DA r e c e p t o r s i n OVX r a t s i n h i b i t e d LH r e l e a s e preceding  ( G a l l o , 1980), and DA t u r n o v e r decreased  the p r e o v u l a t o r y  LH  surge  (Fuxe  e t a l . , 1976).  Whereas these conform t o an i n h i b i t o r y r o l e o f DA, others have demonstrated t h e e x c i t a t o r y nature o f DA on LH r e l e a s e . DA has been c o n s i s t e n t l y shown t o s t i m u l a t e  LHRH r e l e a s e  from the median eminence t i s s u e o f s t e r o i d - p r i m e d  in vitro OVX  rats  ( N e g r o - V i l a r and Ojeda 1978). A l s o , i t has been r e p o r t e d a f t e r intraventricular  i n j e c t i o n o f DA, LH r e l e a s e  in steroid-primed  OVX r a t s ( V i j a y a n and McCann, 1978) , whereas  other  studies  have  failed  to substantiate  was  these  stimulated  findings  ( K a l r a and G a l l o , 1983). Thus, these r e s u l t s can not reach a firm  conclusion  on the nature  o f DA  p a r t i c i p a t i o n i n the  p h y s i o l o g i c events t h a t c o n t r o l LH r e l e a s e .  1.3.2  NOREPINEPHRINE Noradrenergic  innervation  e x t r i n s i c . Noradrenergic-containing  of  the  hypothalamus  is  t e r m i n a l s have a wide, but  uneven, d i s t r i b u t i o n throughout the hypothalamus  (Palkovits  et a l . , 1974). Terminals i n t h e hypothalamus a r e p r o j e c t i o n s from c e l l b o d i e s i n the brainstem v i a t h e v e n t r a l and d o r s a l bundles (Dahlstrom e t a l . , 1964; P a l k o v i t s , 1981). 24  Since the e a r l y o b s e r v a t i o n t h a t norepinephrine s t i m u l a t e o v u l a t i o n i n the  (NE)  may  r a b b i t (Sawyer, 1975), a t t e n t i o n  has been d i r e c t e d toward d e f i n i n g the r o l e of NE i n LH r e l e a s e i n the  r a t . Considerable  evidence  i s i n l i n e with  the  view  t h a t hypothalamic p r o j e c t i o n s o f NE-producing neurons i n the brainstem  ( P a l k o v i t s , 1981)  may  p a r t i c i p a t e i n the e x c i t a t o r y  component (mediated p r i n c i p a l l y by a l p h a - a d r e n e r g i c  receptors  (Kalra et a l . , 1983), and  (mediated  by  alpha-  and  the i n h i b i t o r y component  beta-adrenergic  T a l e i s n i k , 1981;  receptors  (Caceres  Caceres and T a l e i s n i k , 1982), o f the  and neural  c i r c u i t r y c o n t r o l l i n g b a s a l p u l s a t i l e and c y c l i c p a t t e r n s  of  LH r e l e a s e . I n h i b i t i o n o f n o r a d r e n e r g i c a c t i v i t y by i n h i b i t i o n of NE s y n t h e s i s ( N e g r o - V i l a r e t a l . , 1982), blockade o f a l p h a adrenergic  receptors  (Weick,  1978),  or  d e s t r u c t i o n of  v e n t r a l n o r a d r e n e r g i c bundle (Hancke e t a l . , 1977) i n suppression stimulatory receptors  of p u l s a t i l e LH  action  i n the  of  NE  r e l e a s e i n the OVX  i s mediated  hypothalamus  and  via  MPOA  the  a l l result rats.  The  alpha-adrenergic  (Kalra  and  Gallo,  1983) . S t u d i e s have c o n v i n c i n g l y demonstrated t h a t s t i m u l a t i o n of LH  r e l e a s e by NE  may  be  the  r e s u l t of h y p e r s e c r e t i o n  LHRH i n t o the  hypophyseal  1982). That NE  i n p u t i n the diencephalon  the  preovulatory  and  p o r t a l system  ( K r i e g and may  steroid-induced 25  of  Ching,  be important discharge  in of  gonadotropins  i s shown by t h e f i n d i n g s t h a t pharmacological  suppression  of  gonadotropin  adrenergic  neurotransmission  r e l e a s e , and r e s t o r a t i o n  blocked  of adrenergic  i n these r a t s r e i n s t a t e d t h e gonadotropin response  stimuli  (Kalra and  McCann, 1974). Strong evidence i m p l i c a t e s t h e e n t i r e p r e o p t i c - t u b e r a l pathway as t h e s i t e o f i n t e r a c t i o n between NE and LHRH neurons (Kalra  and McCann,  terminate  i n close  p r o x i m i t y o f d e n d r i t e s , axons, and LHRH-containing  perikarya  i n these areas and  197 3).  nerves  e t a l . , 1982), and furthermore,  LHRH  LH r e l e a s e induced by a d m i n i s t r a t i o n o f NE r e s u l t s  from  activation  (Jennes  NE  of  alpha-adrenergic  receptors  in  regions  surrounding the t h i r d v e n t r i c l e ( K a l r a and G a l l o , 1983; K r i e g and Ching,  1982).  Although o b s e r v a t i o n s i n which endogenous n o r a d r e n e r g i c a c t i v i t y i s blocked are c o n s i s t e n t w i t h an ongoing s t i m u l a t o r y a c t i o n o f n o r a d r e n e r g i c neurons on p u l s a t i l e LH r e l e a s e , t h e administration inhibition  of  NE  (Dluzen  into et  the  third  a l . , 1983).  ventricle  causes  Intraventricular  a d m i n i s t r a t i o n o f NE o r o f a d r e n e r g i c a g o n i s t s suppressed t h e amplitude  and frequency  o f p u l s a t i l e LH r e l e a s e i n OVX r a t s  ( G a l l o and Drouva, 1979). Furthermore, of t h e major ascending  e l e c t r i c a l stimulation  n o r a d r e n e r g i c pathway a l s o  inhibits  pulsatile  LH  release  in  OVX  rats  (Leung  et  a l . , 1981).  T a l e i s n i k and co-workers (Caceres and T a l e i s n i k , 1981; and T a l e i s n i k , 1982)  a l s o have i m p l i c a t e d i n h i b i t o r y NE i n p u t s  i n the p r e o v u l a t o r y and The  Caceres  o v a r i a n s t e r o i d - i n d u c e d LH r e l e a s e .  i n h i b i t i o n of LH r e l e a s e by NE may  be mediated by b e t a -  a d r e n e r g i c r e c e p t o r s (Caceres and T a l e i s n i k , 1981; Caceres and Taleisnik, dimension precise  1982) ,  although  these  findings  add  t o our understanding o f n o r a d r e n e r g i c c o n t r o l , physiological  significance  of  each  of  components i n governing LH r e l e a s e i s y e t t o be  1.3.3  another  the  the two  determined.  ENDOGENOUS OPIOID PEPTIDES Three major types of endogenous o p i o i d p e p t i d e s  (EOP),  each with a d i s t i n c t d i s t r i b u t i o n p a t t e r n i n the diencephalon, have been d e s c r i b e d i n the r e g u l a t i o n of LH s e c r e t i o n (Richard et a l . , 1.  1988). Beta-endorphin  proopiomelanocortin mainly  synthesized  a n t e r i o r and al., of  is  processing in  the (Mains  project  product  cells  i n t e r m e d i a t e l o b e s o f the p i t u i t a r y  arcuate  nucleus  and  27  of  the  (Mains  et  i n l a r g e amounts by neurons  periventricular  t o the median eminence,  of  e t a l . , 1977). I t i s  proopiomelanocortin  1977). I t i s a l s o produced  the  major  septal-MPOA  nucleus, and  which  t o a wide  spectrum o f o t h e r b r a i n s t r u c t u r e s ( F i n l a y e t a l . , peptide  i s highly  (Udenfriend pulsatile release are  active  on d e l t a  and Meienhofer,  (Bruni e t a l . ,  1984).  by  opiate receptors  Beta-endorphin  inhibits  1977) and p r e o v u l a t o r y gonadotropin  (Ching, 1983). Presumably,  exerted  and mu  1981). The  projections  into  these s u p p r e s s i v e e f f e c t s the median  eminence and  septal-MPOA o f beta-endorphin-producing c e l l s l o c a t e d i n the arcuate and p e r i v e n t r i c u l a r nucleus ( K a l r a , 1981). There has been c o n s i d e r a b l e i n t e r e s t  i n the s u g g e s t i o n t h a t  EOP may  mediate the feedback e f f e c t s o f gonadal s t e r o i d s . Many l i n e s of evidence  a r e i n a c c o r d with t h i s  beta-endorphin  i n t h e hypophyseal  suggestion. Levels of  portal  blood  fluctuate  markedly d u r i n g t h e menstrual c y c l e i n t h e monkey (Wardlaw e t al.,  1980; Weherenberg e t a l . , 1982);  whereas  combined  estradiol  and  ovariectomy  progesterone  r e s t o r e d , t h e beta-endorphin s e c r e t i o n r a t e t o t h a t  reduced, treatment observed  d u r i n g the l u t e a l phase o f the monkey menstrual c y c l e (Wardlaw et a l . , 1982)-. O v a r i a n s t e r o i d s have been shown t o a l t e r b e t a endorphin  levels  i n various  regions  of  t h e diencephalon  (Barden and Dupont, 1982; Barden e t a l . , 1981).  Beta-endorphin  l e v e l s r i s e i n t h e median eminence and SCN and decrease i n the arcuate  nucleus  during the early  phase  o f p r e o v u l a t o r y LH  r e l e a s e (Barden and Dupont, 1982; Barden e t a l . , 1981). I t has 28  been showed t h a t progesterone's a b i l i t y t o c u r t a i l t r a n s i e n t l y or t o suppress EOP r e l e a s e i n the p r e o p t i c - t u b e r a l pathway may be the u n d e r l y i n g mechanism f a c i l i t a t i n g LH r e l e a s e  (Gabriel  et a l . , 1983). 2.  The  two  pentapeptides,  leucine-enkephalin, opioid  receptors  and  moderate a f f i n i t y  t h e median eminence  enkephalins  in  and  show p r e f e r e n t i a l a f f i n i t y f o r d e l t a - t y p e  r e g i o n s i n c l u d i n g the MPOA, AHA, and  methionine-enkephalin  f o r mu-receptors  SCN, p e r i v e n t r i c u l a r n u c l e u s ,  (Watson e t a l . , 1982).  regulation  in  of  gonadotropin  The  r o l e of  secretion  is  u n c l e a r . Whereas t h e r e was decreased LH r e l e a s e a f t e r systemic injection  of  methionine-enkephalin  (Bruni  et  al.,  1977),  i n t r a v e n t r i c u l a r i n j e c t i o n s i n OVX r a t s produced l i t t l e change i n p u l s a t i l e LH r e l e a s e 3. Dynorphin nucleus  (Code  affinity  for  and  (Leadman and K a l r a ,  i s mainly produced Fallon,  kappa-type  - Intraventricular  1986), opioid  injections  of  1983).  i n the p a r a v e n t r i c u l a r and  has  a preferential  receptors  (Wood,  1982).  dynorphin  suppressed  LH  r e l e a s e , although the response was comparatively much s m a l l e r than  that  Kalra,  seen  a f t e r beta-endorphin  LH  and  1983). To understand the mode o f EOP  of  i n j e c t i o n (Leadman  secretion, opiate  receptor 29  involvement i n r e g u l a t i o n agonists  such as morphine  s u l f a t e and analogs o f methione-enkephalin opiate  receptor  e x t e n s i v e l y used LH  release  antagonists  i n gonadectomized  1983).  A  rats  i n cycling  major  impact  the regulation  frequency  of  pulsatile  the  with  morphine  been  (Johnson  et a l . ,  of  gonadotropic  amplitude  o f beta-endorphin reduces t h e LH  pulsatility  inhibited  of  and the secretion.  continuous  pattern  1982;  LH  i n conscious, c a s t r a t e d r a t s that  have  o f o p i a t e s on  concerns  noteworthy  naloxone  and t h e p r e o v u l a t o r y LH  rats  secretion  Administration  as  (Van Vugt e t a l . , 1982). Morphine suppressed  surge and o v u l a t i o n Kalra,  such  ( K a l r a , 1983) and  (Van Vugt e t a l . , stimulation  the LHRH  1982).  It is  of opiate receptors  accumulation  induced by  progesterone i n OVX estrogen-primed r a t s ( K a l r a and Simpkins, 1981), rats  and t h a t e l i c i t e d  by t e s t o s t e r o n e i n o r c h i d e c t o m i z e d  ( G a b r i e l e t a l . , 1983). These f i n d i n g s suggest the EOP  neurons may n o t o n l y modulate LHRH r e l e a s e but they a l s o may influence promote  neurosecretory LHRH  accumulation  terminals (Kalra e t a l . , The  events  modulated  i n the median  by  steroids  eminence  to  nerve  1983).  o p i a t e r e c e p t o r a n t a g o n i s t s naloxone  s t i m u l a t e LH  r e l e a s e under a v a r i e t y o f experimental c o n d i t i o n s i n i n t a c t and  gonadectomized  Simpkins,  1981; L e i r i  steroid-pretreated e t a l . , 1979). 30  rats  (Kalra  The a b i l i t y  and  o f these  antagonists  t o stimulate  displacing  t h e EOP  LH r e l e a s e , perhaps by t r a n s i e n t l y  from  eminence and arcuate  their  nucleus  sites  (Kalra,  i n the MPOA, median 1981), has l e d t o t h e  general b e l i e f t h a t EOP may normally e x e r t a t o n i c i n h i b i t o r y i n f l u e n c e on LH r e l e a s e i n i n t a c t r a t s . Three  subtypes  characterized:  mu,  of  delta  opiate  receptors  and kappa.  Opiate  have  been  e f f e c t s on LH  s e c r e t i o n a r e g e n e r a l l y b e l i e v e d t o i n v o l v e t h e mu  receptor  subtype ( C i c e r o e t a l . , 1983; P f e i f f e r e t a l . , 1983; Panerai et  al.,  1985).  I t has  been  shown  that  intraventricular  i n j e c t i o n o f the mu-agonist i n OVX r a t s produced a s i g n i f i c a n t suppression  o f LH s e c r e t i o n ,  while  the d e l t a -  and  kappa-  a g o n i s t s had l i t t l e e f f e c t , l e a d i n g t o the c o n c l u s i o n t h a t the mu-receptor  i s the primary o p i a t e  r e g u l a t i o n o f LH s e c r e t i o n The  importance  receptor  involved  ( P f e i f f e r e t a l . , 1983).  o f endogenous o p i a t e s  mechanisms c o n t r o l l i n g LH s e c r e t i o n  i n hypothalamic  seems h i g h l y  dependent  upon the endocrine c o n d i t i o n o f experimental animals et  a l . , 1983; P i v a  opiate implying  receptor  e t a l . , 1986).  antagonists  decreased  EOP tone  i n the  failed  In gonadectomized to e l i c i t  i n these  1983) .  31  (Bhanot  rats  LH  rats,  release,  (Bhanot e t a l . ,  2.  2.1  EXPERIMENTAL  EXPERIMENTAL RATIONALE AND The  DESIGN  d i s t r i b u t i o n of b r a i n neurons c o n t a i n i n g  ANP-like  immunoreactivity w i t h i n the c e n t r a l nervous system (Kawata e t al.,  1985a),  i n p a r t i c u l a r the  intense  innervation  of  the  e x t e r n a l l a y e r of the median eminence and the presence o f  ANP-  p o s i t i v e elements i n the p r e o p t i c - s e p t a l r e g i o n s p i t u i t a r y gland  (Jacobowitz e t a l . , 1985), and  even i n the the  presence  of ANP-binding s i t e s i n these t i s s u e s ( Q u i r i o n e t a l . , 1984.) indicate  a possible  involvement of ANP  i n the  hypothalamic  c o n t r o l of a n t e r i o r p i t u i t a r y hormone s e c r e t i o n . It  has  been  reported  c e n t r a l l y - a d m i n i s t e r e d ANP  that  a  six  microgram  dose  of  can e x e r t i n h i b i t o r y e f f e c t on mean  plasma LH r e l e a s e and naloxone can block i n h i b i t o r y e f f e c t of peripherally-administered  ANP  on LH s e c r e t i o n (Samson e t a l . ,  1988b). However, i n t h i s r e p o r t , the blood sampling i n t e r v a l s were too long t o r e v e a l the p u l s a t i l e p a t t e r n  o f LH  (at  result,  0,  15,  30,  60.  90,  120  minutes) . As  a  release these  i n v e s t i g a t o r s were unable t o determine whether the i n h i b i t o r y e f f e c t of ANP pulse  on LH  amplitude  naloxone on  LH  or  s e c r e t i o n was pulse  pulses.  The  due  frequency first  t o the decrease i n LH and  the  objective  of  influence this  of  study,  therefore, shorter  is  to  sampling  i n f l u e n c e of ANP ANP the  confirm  has  level  interval on the  LH  of  dopamine septum  turn,  shown  been  p u l s a t i l e LH s e c r e t i o n have r e p o r t e d an LH  release  to  examine the  i n the  ANP Brain  (5  their  minutes)  results  to  using  ascertain  the  pulses.  and  its  (Nakao e t by  some  metabolites  in  the  rat  a l . , 1986). Dopamine, researchers  to  in  stimulate  ( N e g r o - V i l a r e t al.,1982) w h i l e others  inhibitory  (Drouva and  effect  Gallo,  of  dopamine on  pulsatile  1976). Thus, i t i s of  p o s s i b l e r o l e of c e n t r a l  interest  dopaminergic system  modulation of LH p u l s e s i n t h i s study. natriuretic  n a t r i u r e t i c and  peptide  BNP  ANP  a l . , 1988a). I t  (Sudoh e t  (BNP)  is a  shares s t r u c t u r a l  diuretic-  s i m i l a r i t y with that  i s not  p i t u i t a r y hormone s e c r e t i o n .  known i f BNP Thus, the  o b j e c t i v e of t h i s study i s to determine i f BNP i n h i b i t o r y e f f e c t on LH Female,  novel  v a s o r e l a x a n t p e p t i d e o r i g i n a l l y i s o l a t e d from  porcine brain.  anterior  extend  been r e p o r t e d t o cause s i g n i f i c a n t decreases i n  hypothalamus and has  and  of  affects  second major  a l s o e x e r t s an  release.  Sprague-Dawley  rats  weighing  purchased from C h a r l e s R i v e r Canada, Inc.  185-200  grams  (Montreal, Canada)  were i n d i v i d u a l l y caged i n a l i g h t - a n d temperature  -controlled  room  (14-h l i g h t ,  ovariectomy anaesthesia  was  performed  ± 1 Celsius).  under  sodium  Bilateral  methohexital  s t e r e o t a x i c a l l y with a 22-gauge guide  the t h i r d  anaesthesia  22  (50 mg/kg body weight), Two weeks l a t e r , each r a t  was implanted into  10-h dark;  brain ventricle  (Somnotol,  under  cannula  sodium  pentobarbital  45 mg/kg body weight).  Following a  minimum 1-week recovery p e r i o d , each r a t was f i t t e d w i t h an intra-atrial  catheter.  polyethylene  tubing  Two  days  was a t t a c h e d  later,  a  saline-filled  t o the i n d w e l l i n g  atrial  c a t h e t e r and 5 0 - m i c r o l i t r e blood samples were withdrawn a t 5minute i n t e r v a l s f o r t h r e e hours.  Each animal remained i n i t s  own cage d u r i n g t h e b l e e d i n g procedure and each sample was r e p l a c e d by an equal volume o f 0.9% s a l i n e . Blood samples were c e n t r i f u g e d and the plasma was s t o r e d a t -2 0 C e l s i u s u n t i l LH concentrations designed The  were  measured  by  first  series:  Examining  were  the blood-sampling of saline  the  effect  of  ( i . c . v ) a d m i n i s t r a t i o n o f ANP o r BNP  on LH s e c r e t i o n i n o v a r i e c t o m i z e d  infusion  Experiments  into three s e r i e s :  intracerebroventricular  of  RIA.  period,  (OVX) r a t s . A t t h e midpoint  each  r a t received  an i . c . v  c o n t a i n i n g 0.2 nmol, 2 nmol ANP o r 0.2  nmol,2 nmol BNP t o examine the modulatory e f f e c t o f ANP o r BNP on LH s e c r e t i o n a t d i f f e r e n t dosage l e v e l s . 34  The second s e r i e s : Examining t h e e f f e c t o f blockade of the  endogenous o p i a t e system w i t h naloxone on t h e a c t i o n of  ANP and BNP. 45 minutes a f t e r the b e g i n n i n g o f the experiment, 2nmol ANP o r 2nmol BNP was i n f u s e d i n t o t h e t h i r d v e n t r i c l e . Three  boluses  of  0.5  mg  naloxone  were  then  injected  i n t r a v e n o u s l y a t 45, 75 and 105 minutes a f t e r i . c . v . of  infusion  2nmol ANP o r 2nmol BNP t o examine whether naloxone  could  r e v e r s e the i n h i b i t o r y e f f e c t o f ANP o r BNP. The t h i r d s e r i e s : Examining the e f f e c t of pretreatment of  rats  with dopaminergic  r e c e p t o r b l o c k e r pimozide on the  a c t i o n of ANP o r BNP. A t t h e onset o f t h e experiment, were  injected  experiment  with  pimozide  2 nmol ANP  and  at  t h e midpoint  o r 2 nmol BNP was  examine whether pretreatment of pimozide i n h i b i t o r y e f f e c t o f ANP o r BNP.  35  i.c.v.  rats  of the  infused to  c o u l d prevent the  2.2  MATERIALS AND METHODS A d u l t female Sprague-Dawley r a t s were i n d i v i d u a l l y caged  i n a l i g h t - and temperature- c o n t r o l l e d room (14-h l i g h t ; h  dark, 22 ± 1 C e l s i u s ) and p r o v i d e d water  l i b i t u m . One holding  10-  and r a t chow ad  or two days a f t e r the r a t a r r i v e d i n the animal  facilities,  they  were  a n a e s t h e t i z e d with  sodium  methohexital ( B r i e t a l , E l i L i l l y ; 50 mg/kg o f body weight) and ovariectomized Two of  the  (OVX).  weeks f o l l o w i n g ovariectomy, s u r g i c a l c a n n u l a t i o n third  ventricle  a n a e s t h e t i z e d with of  body weight)  with  i t s head  Paxinos  and  was  performed.  sodium p e n t o b a r b i t a l  The  rat  was  (Somnotol, 45 mg/kg  and then p l a c e d i n a s t e r e o t a x i c instrument i n the  Watson  flat-skull  (1982). The  position  skull  was  as  described  exposed  and  by  four  h o l e s were d r i l l e d , i n t o which a n c h o r i n g screws were secured. A s m a l l h o l e , the c e n t r e o f which was 6.7 m i l l i m e t r e a n t e r i o r to  the i n t e r a u r a l l i n e , was t h e n d r i l l e d m i d s a g l t a l l y i n the  skull.  The  dura  mater  was  s t a i n l e s s s t e e l cannula was b r a i n . Placement the  then  exposed  lowered 9.0  and  a  22  gauge  m i l l i m e t r e i n t o the  of the cannula a t t h i s p o s i t i o n r e s u l t e d i n  t i p o f the cannula i n the t h i r d v e n t r i c l e  (Anterior:  6.7  m i l l i m e t r e a n t e r i o r t o the i n t e r a u r a l l i n e ; L a t e r a l : m i d l i n e ,  and  Deep: 9.0 m i l l i m e t r e below the s k u l l  surface).  Dental  cement (Hygenic C o l d Cure) was placed around t h e cannula and the  skull  prevent  screws,  t o secure  the cannula  leakage o f c e r e b r o s p i n a l f l u i d  t o the s k u l l .  To  and i n f e c t i o n o f the  t h i r d v e n t r i c l e , a 28 gauge s t a i n l e s s s t e e l s t y l e t t e e x a c t l y the same l e n g t h as the cannula was i n s e r t e d i n t h e cannula and its  handle  was  cemented  i n place  until  t h e day  o f the  experiment. A f t e r v e n t r i c l e c a n n u l a t i o n t h e r a t s were p l a c e d in  individual  cages  and were given  at least  one week t o  recover from t h e s u r g e r y . On t h e day b e f o r e r a t s were a n a e s t h e t i z e d made o f s i l a s t i c vein using  the c o l l e c t i o n o f b l o o d b r i e f l y with  samples the  B r i e t a l and a c a t h e t e r  t u b i n g was i n s e r t e d i n t o t h e r i g h t  jugular  t h e method d e s c r i b e d by Harms and Ojeda  (1974).  B r i e f l y , the r i g h t j u g u l a r v e i n was exposed and a curved 22gauge needle was used t o p l a c e the s i l a s t i c c a t h e t e r i n t o the v e i n . The c a t h e t e r was s l i d i n t o the v e i n and made t o enter or approach t h e r i g h t atrium. The- c a t h e t e r was then anchored onto the muscle under which the r i g h t j u g u l a r v e i n passes. The exposed end o f t h e c a t h e t e r was then passed underneath the s k i n and e x t e r i o r i z e d a t t h e back o f t h e neck and t i e d  shut.  The r a t s were then r e t u r n e d t o t h e i r cages u n t i l t h e next day. In some experiments, t h e r a t s were reused  a f t e r a t l e a s t one  week  recovery  implanted similar  period.  In these  cases,  the catheter  was  i n t h e l e f t j u g u l a r v e i n (the s u r g i c a l procedure was to that  of implantation  of catheter  t o the r i g h t  jugular vein). On the day o f t h e experiment t h e j u g u l a r c a t h e t e r was connected t o p o l y e t h y l e n e t u b i n g (PE-50) which had been f i l l e d with h e p a r i n i z e d s a l i n e . Using were withdrawn with  t h i s catheter, blood  minimal d i s t u r b a n c e  t o the r a t .  samples Before  blood sampling was s t a r t e d , 200 m i c r o l i t r e h e p a r i n (1000 U/ml) was i n j e c t e d i n t o the r a t s v i a t h e i n t r a - a t r i a l prevent  catheter to  c l o t t i n g of the blood i n the tubing.  At 5-minute i n t e r v a l ,  50 m i c r o l i t r e o f whole blood was  withdrawn from t h e r a t s v i a t h e i n t r a - a t r i a l whole blood  samples were p l a c e d  c a t h e t e r . The  i n t o tubes on i c e . In a l l  cases,  5 minutes b e f o r e t h e s t a r t o f i n f u s i o n o f ANP, BNP o r  saline  into the t h i r d  taken  ventricle,  out of implanted  t h e 28 gauge s t y l e t t e was  22 gauge  cannula  and an i n f u s i o n  s t y l e t t e was i n s e r t e d i n s i d e t h e 22 gauge cannula  such t h a t  the t i p of the i n f u s i o n s t y l e t t e was i n t h e t h i r d  ventricle.  In many cases c e r e b r o s p i n a l f l u i d was seen f l o w i n g out o f t h e cannula.  T h i s was taken as evidence  t h a t t h e cannula  was i n  the t h i r d v e n t r i c l e . The i n f u s i o n t u b i n g had been p r e v i o u s l y connected t o a Hamilton m i c r o l i t r e s y r i n g e i n a s y r i n g e pump. 38  The  2 microlitre  volume  was  infused  over  a  period  of  2  minutes. Each animal remained i n i t s own cage d u r i n g the b l e e d i n g procedure and each sample was 0.9%  saline.  r e p l a c e d by an equal volume of  A l l experiments  unrestrained  animals.  were  Blood  conducted  samples  i n conscious  were  centrifuged  immediately a f t e r experiments and the plasma was s t o r e d a t 20  Celsius  until  radioimmunoassay In  LH  concentrations  were  measured  by  (RIA).  studies  examining  the  effect  of  i n t r a c e r e b r o v e n t r i c u l a r ( i . c . v . ) a d m i n i s t r a t i o n of ANP or BNP on LH s e c r e t i o n i n o v a r i e c t o m i z e d r a t s , a t t h e midpoint of the blood-sampling minutes  an  period  (90 min), each r a t r e c e i v e d  i.c.v infusion  of 2 m i c r o l i t r e  over  isotonic  two  saline  (control) alone o r s a l i n e c o n t a i n i n g 0.2 nmol, 2 nmol ANP ( r a t ANP-28; P e n i n s u l a l a b o r a t o r i e s , Inc.,) or 0.2 nmol,2 nmol BNP (porcine BNP-26; P e n i n s u l a l a b o r a t o r i e s , -In another  experiment  the  effect  Inc.,). of  blockade o f  the  endogenous o p i a t e system w i t h naloxone on t h e a c t i o n of i . c . v . administered ANP  and BNP  was  tested  i n OVX  rat. forty-five  minutes a f t e r the b e g i n n i n g o f the experiment, 2nmol ANP  or  2nmol BNP was i n f u s e d i n t o the t h i r d v e n t r i c l e . Three b o l u s e s of  0.5  mg  naloxone  (Sigma,  ST  Louis) were  then  injected  i n t r a v e n o u s l y a t 45, 75 and 105 minutes a f t e r i . c . v .  infusion  of 2nmol ANP o r 2nmol BNP. In a separate  experiment, dopaminergic r e c e p t o r  blocker  pimozide (0.63 mg/kg, subcutaneously) was administered  a t the  onset o f the-experiment, and a t t h e midpoint o f the experiment 2 nmol ANP o r 2 nmol BNP was i . c . v . At  the  conclusion  anaesthetized violet  with  stain  perfused  into  of  each  pentobarbital  infused.  experiment, and  infused  the t h i r d v e n t r i c l e .  intracardially  formaldehyde s o l u t i o n  with  animals with  The r a t s  saline  followed  were cresyl  were  then  by  10%  i n s a l i n e . The b r a i n s were removed from  the s k u l l s and l a t e r s e c t i o n e d i n 50 micrometer t o v e r i f y the l o c a t i o n o f the t h i r d v e n t r i c l e Plasma  LH  concentrations  cannula. were  measured with  RIA k i t  p r o v i d e d by t h e N a t i o n a l I n s t i t u t e o f A r t h r i t i s , Diabetes, and D i g e s t i v e and Kidney Diseases, N a t i o n a l Hormone and P i t u i t a r y Program. The assay was a s l i g h t m o d i f i c a t i o n o f the double antibody  RIA  described  by  Niswender  et  a l . (1968).  The  r e f e r e n c e p r e p a r a t i o n was NIADDK-rat-LH-RP-7. The i n t e r a s s a y coefficient  of  variation  was  14%,  and  the  intraassay  c o e f f i c i e n t o f v a r i a t i o n was 10%. An a d a p t i v e  t h r e s h o l d method was used t o determine the  time and amplitude o f hormone p u l s e s 40  (Soules e t a l . , 1987).  Mean plasma LH l e v e l s were determined  by d i v i d i n g t h e sum o f  a l l the samples o f one animal by t h e number o f samples, p u l s e amplitude  was t h e d i f f e r e n c e between the peak and n a d i r of  each p u l s e . Mean plasma LH l e v e l , mean pulse frequency  mean p u l s e amplitude  were c a l c u l a t e d  and  f o r t h e p r e - and p o s t -  drug i n f u s i o n p e r i o d o f each animal. Except infusion except case  i n t h e naloxone treatment  values  were compared by means o f a p a i r e d  where h e t e r o g e n e i t y  the Cochran  group,  group, p r e - and p o s t -  statistical  Test  was  t-test  o f v a r i a n c e was found,  i n which  a p p l i e d . In naloxone  treatment  d i f f e r e n c e s o f the data  were  determined  w i t h one way a n a l y s i s o f v a r i a n c e followed by S c h e f f e ' s t e s t . Values  i n the t e x t r e p r e s e n t t h e means ± s t a n d a r d  41  error.  2.3  RESULTS  2.3.1  E f f e c t s of ANP  2.3.1.1  and  BNP  i n f u s i o n on LH  E f f e c t s of s a l i n e i n f u s i o n on LH  None  of  the  four  control  secretion  animals  receiving  i n f u s i o n showed a change i n mean plasma LH ± 0.51  ng/ml t o  4.97  ±0.23 ng/ml t o 4.89 3.50  ± 0.29  to  ± 0.33 ± 0.22  3.7  ± 0.41  secretion  level  saline  (from  5.01  ng/ml),  pulse  amplitude  (4.76  ng/ml) and  pulse  frequency  (from  number of pulses/90min) when  the  p r e - i n f u s i o n p e r i o d v a l u e s were compared t o those of the p o s t infusion period, Figure  as  shown i n the  representative  example i n  1.  2.3.1.2  E f f e c t s of 2nmol ANP  i n f u s i o n on LH  secretion  In t h i s group (n=8), comparison o f the pre-versus p o s t infusion  mean plasma  decrease  (from  <0.05). was  The  5.10  LH  LH ±  0.17  ng/ml t o  revealed s i g n i f i c a n t 4.08  ± 0.47  ng/ml,  P  amplitude i n the p o s t - i n f u s i o n  period  s i g n i f i c a n t l y lower than t h a t i n the p r e - i n f u s i o n  period  (from 6.05  ± 0.81  number  LH  ng/ml t o 3.43  (from  ± 0.39  post-infusion  period  The  s i g n i f i c a n t l y l e s s than t h a t i n the p r e - i n f u s i o n p e r i o d t o 2.12  i n the  ng/ml, P < 0.05).  also  ± 0.32  pulses  ± 0.80  was  4.38  of  pulse  concentration  number o f pulses/90 min,  P < 0.01),  as shown i n the r e p r e s e n t a t i v e example i n F i g u r e 2.3.1.3  E f f e c t s of 2nmol BNP  2.  i n f u s i o n on LH s e c r e t i o n  In t h i s group (n=8), comparison of the p r e - v e r s u s i n f u s i o n mean plasma decrease 0.01).  (from  5.75  LH  concentration  ± 0.23  revealed  ng/ml t o 3.79  post-  significant  ± 0.22  ng/ml, P <  The LH p u l s e amplitude i n the p o s t - i n f u s i o n p e r i o d  s i g n i f i c a n t l y lower than t h a t i n the p r e - i n f u s i o n p e r i o d 6.03  ± 0.87  of  LH  ng/ml t o 2.37  pulses  in  ± 0.27  the  ng/ml, P < 0.01). The  post-infusion  period  (from number  was  also  s i g n i f i c a n t l y l e s s than t h a t i n the p r e - i n f u s i o n p e r i o d 5.14  ± 0.34  t o 2.71  ± 0.28  number of pulses/90 min,  as shown i n the r e p r e s e n t a t i v e example i n F i g u r e  2.3.1.4  E f f e c t s of 0.2nmol ANP  mean plasma  LH  (from  5.27  ± 0.23  ng/ml t o  ± 0.62  ng/ml) and  3.87  frequency  (from  number of pulses/90min) when pre-versus were  compared,  Figure  as  shown  i n the  4.  43  P < 0.01), 3.  no s i g n i f i c a n t decrease  ng/ml), p u l s e amplitude (from 4.89 pulse  (from  i n f u s i o n on LH s e c r e t i o n  In t h i s group (n=8), t h e r e was in  was  4.88  ng/ml t o 4.57  ± 0.29  t o 3.62  ±  0.32  ±  0.40  ±  0.36  p o s t - i n f u s i o n periods  representative  example  in  2.3.1.5  E f f e c t s of 0.2nmol BNP  In t h i s  group  (n=7) , no  plasma LH (from 5.01 amplitude pulse  + 0.39  (from 4.83  frequency  pulses/90min)  significant  ng/ml t o 4.34  ± 0.47  (from 3.85  was  i n f u s i o n on LH  observed,  ± 0.26 when  decrease  ± 0.47  ng/ml t o 4.62  secretion  ng/ml), p u l s e  ± 0.37  t o 4.42  ng/ml) and  ± 0.29  pre-versus  i n mean  number of  post-infusion  p e r i o d s compared, as shown i n the r e p r e s e n t a t i v e example i n F i g u r e 5. The  r e s u l t s of e f f e c t s of ANP  and  BNP  infusion  LH s e c r e t i o n are summarized i n the F i g u r e 6, 7 and  44  8.  on  Plasma LH (ng/ml)  saline Figure l . R e p r e s e n t a t i v e i n f u s i o n of s a l i n e .  example o f LH  r e l e a s e i n ovx r a t s  with i . c . v  12  Plasma LH (ng/ml) i  :  14  Plasma LH (ng/rnl)  0 0  I  I  I  I 30  I  I  I  I  I  I  I  I  I  60  1 I  I  1 I  90  I  I  I  I  I  I  I  I  I  120  I 150  I  I  I  I  (min)  2nmolBNP F i g u r e 3. R e p r e s e n t a t i v e i n f u s i o n , o f 2 nmol BNP.  example  o f LH r e l e a s e i n ovx r a t s  with i . c . v  12  Plasma LH (ng/ml)  00  i  i  i  i  i  30  i  I  60  i  J  1—I  90  1  1—I—I  L_J  L_J  120  I  150  I  I  I  L  (min)  0.2nmolANP F i g u r e 4. R e p r e s e n t a t i v e example o f LH r e l e a s e i n ovx r a t s i n f u s i o n o f 0.2 nmol ANP.  with i . c . v  plasma LH (ng/ml) 12 i  pre-infusion post-infusion  X  5h 4  m  X  X  X  4  saline  „ANP 0.2nmol  BNP , O.znmol  ANP nmol 2n  o  B N P  ,  2nmol  F i g u r e 6. Mean plasma LH c o n c e n t r a t i o n i n t h e p r e - and post-ANP o r BNP i n f u s i o n p e r i o d . * and ** denote s t a t i s t i c a l s i g n i f i c a n c e o f P < 0.05 and P < 0.01 s e p a r a t e l y . The "n" value f o r each group i s shown i n t h e p a n e l .  8  pre-infusion post-infusion  7 6 -  X  5 -  X X  4 3  **  2  0  saline  0.2nmol  INP , 0.] !nmol  ANP nmol 2n  BNP 2rinmol  F i g u r e 7. LH p u l s e amplitude i n the p r e - and post-ANP o r BNP i n f u s i o n p e r i o d . * and ** denote s t a t i s t i c a l s i g n i f i c a n c e o f P < 0.05 and P < 0.01 s e p a r a t e l y . The "n" v a l u e f o r each group i s shown i n t h e p a n e l .  •  c6 MB  E o  CO CD V>  D  x  pre-infusion post-infusion  4  X  X  X  X  X  o to  X  a>  X  £ 2  a> co  0  saline  i _ ANP , 0.2nmol  BNP O.znmol  mol  BNP nmol 2n  F i g u r e 8. LH p u l s e frequency i n the p r e - and post-ANP o r BNP i n f u s i o n p e r i o d . ** denotes s t a t i s t i c a l s i g n i f i c a n c e o f P < 0.01. The "n" v a l u e f o r each group i s shown i n the p a n e l .  2.3.2  Effects  o f naloxone  actions of i.c.v. infused 2.3.2.1  treatment on t h e LH  inhibitory  ANP and BNP  E f f e c t s o f naloxone treatment i n c o n t r o l  group  In c o n t r o l group (n=5), treatment w i t h naloxone (0.5 mg each time, i . v . b o l u s ) a t 45, 75 and 105 minutes a f t e r infusion  o f 0.9% s a l i n e  plasma LH l e v e l  had no s i g n i f i c a n t e f f e c t s  i.c.v. on mean  (from 2.68 ± 0.54 ng/ml t o 2.67 ± 0.62 ng/ml),  p u l s e amplitude (5.66 ± 1.22 ng/ml t o 5.88 ± 0.22 ng/ml) and pulse  frequency (from 2.65 ± 0.21 t o 2.81 ± 0.41 number o f  pulses/45 min), as shown  i n the representative  example i n  F i g u r e 9.  2.3.2.2  E f f e c t s o f naloxone treatment i n 2nmol ANP group  Treatment w i t h naloxone (0.5 mg each time, i . v . bolus) a t 45,75 and 105 minutes a f t e r reversed  the i n h i b i t o r y  plasma l e v e l  i.c.v. infusion  effects  o f ANP  o f 2nmol ANP  (n=7) on t h e mean  (from 1.72 ± 0.42 ng/ml t o 3.14 ± 0.44 ng/ml, P  < 0.05). A p p l i c a t i o n  o f naloxone a l s o reversed t h e i n h i b i t o r y  e f f e c t o f ANP on LH p u l s e amplitude (from 2.11 ± 0.39 ng/ml t o 5.04 ± 0.99 ng/ml, P < 0.05). In terms o f p u l s e frequency, although t h e r e was a s t r o n g tendency t h a t naloxone treatment may reverse t h e i n h i b i t o r y e f f e c t o f ANP, naloxone treatment  f a i l e d t o a l t e r the p u l s e frequency s i g n i f i c a n t l y (from ± 0.20  t o 2.28  ± 0.24  number of pulses/45 min),  1.57  as shown i n  the r e p r e s e n t a t i v e example i n F i g u r e 10. I t i s q u i t e p o s s i b l e t h a t when the number of experimental animals i n t h i s group i s increased,  naloxone  e f f e c t o f ANP  2.3.2.3  Treatment  reversed  the  0.05).  0.29  treatment  w i t h naloxone  (0.5 mg  inhibitory  the  inhibitory  (from 1.96  ± 0.29  e f f e c t s of BNP  ng/ml t o 5.40  ± 0.93  of  i n 2nmol BNP each time,  i.c.v.  effects  Administration  inhibitory  reverse  of naloxone  105 minutes a f t e r  plasma l e v e l <  will  on LH p u l s e frequency.  Effects  at 45,75 and  treatment  of  infusion BNP  naloxone  i . v . bolus) of 2nmol  (n=7)  ng/ml t o 3.89  group  BNP  on  the mean  ± 0.48  ng/ml, P  also  reversed  on LH p u l s e amplitude  the  (from 2.09  ±  ng/ml, P < 0.05). In terms o f p u l s e  frequency, a l t h o u g h t h e r e was a s t r o n g tendency t h a t  naloxone  treatment may  naloxone  treatment  r e v e r s e the i n h i b i t o r y e f f e c t o f BNP,  failed  (from 1.71  ± 0.28  t o a l t e r the p u l s e frequency t o 2.21 ± 0.10  significantly  number o f p u l s e s / 4 5 min), as  shown i n the r e p r e s e n t a t i v e example i n F i g u r e 11. The  effects  of naloxone  treatment on the LH i n h i b i t o r y a c t i o n s of i . c . v .  i n f u s e d ANP  and BNP  are summarized i n F i g u r e 12,  54  13 and  14.  Plasma LH (ng/ml) 12 i  2  -  J  0  L  J  30  L  J  60 A  saline  J  L  L  J  L  J  90  120  150  •  •  •  naloxone  Figure 9. R e p r e s e n t a t i v e example of LH r e l e a s e naloxone a f t e r i . c . v i n f u s i o n o f s a l i n e .  naloxone i n ovx r a t s  L  (min)  naloxone injected  with  16  Plasma LH (ng/ml)  (min)  Figure 10. R e p r e s e n t a t i v e example o f LH r e l e a s e naloxone a f t e r i . c . v i n f u s i o n of 2 nmol ANP.  i n ovx r a t s i n j e c t e d w i t h  Plasma LH (ng/ml) 18 i  2nmolBNP  naloxone  Figure 11. R e p r e s e n t a t i v e example o f LH r e l e a s e naloxone a f t e r i . c . v i n f u s i o n o f 2 nmol BNP.  naloxone  naloxone  i n ovx r a t s i n j e c t e d w i t h  ^  E  5  4 -  pre-treatment saline/ANP/BNP naloxone  0)  300  cd  0)  rx  0  saline  ANP  BNP  F i g u r e 12. Mean plasma LH c o n c e n t r a t i o n i n t h e p r e - and post-ANP o r BNP i n f u s i o n and naloxone treatment p e r i o d . * denotes v a l u e b i s s i g n i f i c a n t l y d i f f e r e n t from v a l u e a a t P < 0.05. The "n" v a l u e f o r each group i s shown i n t h e panel. .  ^  E  9  o> 8  r  -  pre-treatment sallne/ANP/BNP naloxone  6  Is vo  CO  a, 4 CO  1 0  saline  ANP  BNP  F i g u r e 13. LH p u l s e amplitude i n the p r e - and post-ANP o r BNP i n f u s i o n and naloxone treatment p e r i o d . * denotes v a l u e b i s s i g n i f i c a n t l y different from value a a t P < 0.05. The "n" v a l u e f o r each group i s shown i n t h e panel.  pre-treatment saline/ANP/BNP naloxone (0  <D  x  a O o  X  •to  c §2  jo a. 0  saline  ANP  BNP  F i g u r e 14. LH p u l s e frequency i n the p r e - and post-ANP o r BNP i n f u s i o n and naloxone treatment p e r i o d . * denotes v a l u e a i s s i g n i f i c a n t l y d i f f e r e n t from value b a t P < 0.05. Value ab i s not s i g n i f i c a n t l y d i f f e r e n t from v a l u e a and v a l u e b. Naloxone treatment does not r e v e r s e ANP's and BNP's i n h i b i t o r y e f f e c t on LH p u l s e frequency. The "n" v a l u e f o r each group i s shown i n the p a n e l .  2.3.3  E f f e c t o f pimozide  pretreatment  on t h e LH i n h i b i t o r y  a c t i o n s of i . c . v . i n f u s e d ANP and BNP 2.3.3.1  E f f e c t o f pimozide pretreatment  None  of  four  control  animals  i n c o n t r o l group receiving  pimozide  pretreatment showed a change i n mean plasma LH l e v e l 0.38 ng/ml), p u l s e amplitude (4.76  (4.19 ± 0.74 ng/ml) and frequency  ± 0.39 number o f pulses/90min),  treatment  group,  (5.55 ±  compared w i t h  saline  as shown i n t h e r e p r e s e n t a t i v e example i n  F i g u r e 15.  2.3.3.2  E f f e c t s o f pimozide pretreatment  Pretreatment o f r a t s w i t h pimozide i n prevention of i n h i b i t o r y  i n 2nmol ANP group  (0.63 mg/kg, s.c) r e s u l t e d  effects  o f ANP on LH s e c r e t i o n  from o c c u r r i n g . A f t e r i n f u s i o n o f 2nmol ANP (n=9), t h e r e were no s i g n i f i c a n t decreases i n mean plasma LH l e v e l ng/ml t o 5.07 ± 0.43 ng/ml), p u l s e amplitude  (4.96 ± 0.24  (from 4.26 ± 0.37  ng/ml t o 4.36 ± 0.44 ng/ml) and p u l s e frequency 0.27 t o 3.77 ± 0.36 number o f pulses/90 min),  (from 4^.22 ± compared with  p r e - i n f u s i o n p e r i o d , as shown i n t h e r e p r e s e n t a t i v e example i n F i g u r e 16.  2.3.3.3  E f f e c t s o f pimozide pretreatment i n 2nmol BNP group 61  Pretreatment w i t h pimozide  (0.63 mg/kg, s.c) r e s u l t e d i n  p r e v e n t i o n o f i n h i b i t o r y e f f e c t s o f BNP on LH s e c r e t i o n occurring. compared  When p r e - i n f u s i o n  i n this  group  mean plasma LH l e v e l  (n=9),  no s i g n i f i c a n t  v a l u e s were decreases i n  (from 4.93 ± 0.65 ng/ml t o 4.83 ± 0.66  ng/ml), p u l s e amplitude  (from 5.02 ± 0.60 ng/ml t o 4.56 ± 0.47  ng/ml) and p u l s e frequency number  and p o s t - i n f u s i o n  from  o f pulses/90  (from 4.00 ± 0.37 t o 3.88 ± 0.31  min) were  observed,  as shown  i n the  r e p r e s e n t a t i v e example i n F i g u r e 17. The e f f e c t s o f pimozide pretreatment  on the LH i n h i b i t o r y a c t i o n s o f i . c . v .  infused  ANP and BNP a r e summarized i n F i g u r e 18, 19 and 20. At  t h e c o n c l u s i o n o f each  anaesthetized violet  stain  perfused  with into  p e n t o b a r b i t a l and the t h i r d  intracardially  formaldehyde  experiment, infused  ventricle.  with  saline  animals with  were cresyl  The r a t s were followed  by  then 10%  s o l u t i o n i n s a l i n e . The b r a i n s were removed from  the s k u l l s and l a t e r s e c t i o n e d i n 50 micrometer t o v e r i f y t h e l o c a t i o n o f t h e t h i r d v e n t r i c l e cannula. The r a t s w i t h cannula tips  outside  statistics. the  rat  third  ventricle  Photograph hypothalamus  a r e n o t counted  into  the  of representative f r o n t a l sections i n depicting  the  site  of  cannula  i m p l a n t a t i o n i n t h e t h i r d v e n t r i c l e i s shown i n F i g u r e 21.  62  Plasma LH (ng/ml) 12 i  2 J  0 • pimozide  I  I  30  I  -M— i  L  60  90  I  I  120  I  l  150  (min)  A  saline  F i g u r e 15. R e p r e s e n t a t i v e example of LH r e l e a s e i n p i m o z i d e - p r e t r e a t e d ovx r a t s with i . c . v i n f u s i o n o f s a l i n e .  Plasma LH (ng/ml)  101  Figure 16. R e p r e s e n t a t i v e example o f LH r e l e a s e i n p i m o z i d e - p r e t r e a t e d ovx r a t s with i . c . v i n f u s i o n of 2 nmol ANP.  Plasma LH (ng/ml)  16 i  01  '  1  1  0 pimozide  |  1  1  |  1  30  1  |  1 1  |  1 1  60  |  1  1  1  1  1  |  1  90  1  |  1  1  120  |  1  1  |  1  |  1  150  1  K*in)  2nmolBNP  F i g u r e 17. R e p r e s e n t a t i v e example o f LH r e l e a s e i n p i m o z i d e - p r e t r e a t e d ovx r a t s with i . c . v i n f u s i o n o f 2 nmol BNP.  pimozide-treated rats  ^  8  ^>  7  w  6 -  E  $  C Z D pre-infusion r ~ ~ l post-infusion  X  0) 5  x  4  (0 0)  3  2  2 1  0  saline  ANP  BNP  F i g u r e 18. Mean plasma LH c o n c e n t r a t i o n i n t h e p r e - and post-ANP o r BNP i n f u s i o n p e r i o d with the pimozide pretreatment. The "n" v a l u e f o r each group i s shown i n the panel.  pimozide-treated rats  E 6 •O  5  pre-infusion post-infusion  X  T_-T  E * as  (0 0)  3  =) 4 1  2  —J  1  0  saline  ANP  BNP  F i g u r e 1 9 . LH p u l s e amplitude i n the p r e - and post-ANP o r BNP i n f u s i o n p e r i o d with the pimozide pretreatment. The "n" v a l u e f o r each group i s shown i n t h e p a n e l .  pimozide-treated rats pre-infusion post-infusion  x x  saline  X  ANP  BNP  F i g u r e 20. LH p u l s e frequency i n t h e p r e - and post-ANP o r BNP i n f u s i o n p e r i o d with t h e pimozide pretreatment. The "n" v a l u e f o r each group i s shown i n t h e p a n e l .  t  01 ^0  Figure 2 1 . Photograph o f r e p r e s e n t a t i v e f r o n t a l s e c t i o n s i n t h e r a t hypothalamus d e p i c t i n g t h e s i t e o f cannula t i p i n the t h i r d v e n t r i c l e . The s i t e i s marked by a b l a c k t r i a n g l e .  3. The  DISCUSSION  presence o f ANP w i t h i n the c e n t r a l nervous system  (Jacobowitz e t a l . , 1985; Tanaka e t a l . , 1984), p a r t i c u l a r l y t h a t found w i t h i n the diencephalon,  and t h e d e t e c t i o n o f ANP-  s p e c i f i c b i n d i n g s i t e s i n t h e hypothalamus and p i t u i t a r y gland ( Q u i r i o n e t a l . , 1984), suggested t h e p o s s i b i l i t y t h a t ANP may i n f l u e n c e a n t e r i o r p i t u i t a r y hormone s e c r e t i o n . The i n h i b i t i o n of p u l s a t i l e LH s e c r e t i o n r e p o r t e d i n the present study lends support  t o t h i s hypothesis.  The decrease  i n mean plasma LH  l e v e l c o u l d be a t t r i b u t e d t o the r e d u c t i o n o f p u l s e amplitude and  frequency. T h i s i n h i b i t o r y e f f e c t o c c u r r e d  only  i n high  dose (2nmol) groups. S i n c e the h a l f - l i f e o f ANP i n plasma was short on  (1-3 min) (Yandle e t a l . , 1986) and t h e r e was no data  half-life  failure  o f ANP  o f acute  i n cerebrospinal  i n f u s i o n o f ANP  a l t e r LH s e c r e t i o n  site(s)  whether o r not  i n 0.2nmol dose group t o  c o u l d be a t t r i b u t e d t o t h e s h o r t h a l f - l i f e  remained an open q u e s t i o n . The  fluid,  -  o f a c t i o n o f ANP w i t h i n  t h e hypothalamic-  pituitary  a x i s are unknown. I n f u s i o n o f ANP  ventricle  does not a l l o w  us t o p i n p o i n t  into the t h i r d  t h e exact  s i t e of  a c t i o n . However, s e v e r a l groups have suggested t h a t ANP i s unlikely  t o be a c t i n g a t the l e v e l 70  o f the p i t u i t a r y  gland  ( H e i s l e r e t a l . , 1986; found that even ANP  Simard e t a l . , 1986). These  could a c t i v a t e guanylate cyclase a c t i v i t y  i n the p i t u i t a r y gland p i t u i t a r y gland  and ANP  r e c e p t o r s were present  i n the  ( Q u i r i o n e t a l . , 1984), they f a i l e d t o d e t e c t  any d i r e c t e f f e c t s o f ANP  on p i t u i t a r y hormone s e c r e t i o n . In  agreement with these r e s u l t s , no  researchers  i t has  s i g n i f i c a n t e f f e c t of ANP  been demonstrated  on b a s a l  that  o r LHRH-stimulated  LH  s e c r e t i o n can be observed i n c u l t u r e d a n t e r i o r p i t u i t a r y c e l l s (Samson et a l . , 1988). T h e r e f o r e  i t seems l i k e l y t h a t the  events  accumulation  (ANP-stimulated  s e c r e t i o n ) are  cGMP  and  direct  effect  of  ANP  in pituitary  cell  t o demonstrate  cultures,  reasonable t o h y p o t h e s i z e t h a t the ANP's a c t i o n s , its  LH-inhibitory  hypothalamus. The fibres  and  ANP  region p r o v i d e s (Standaert  hormone  unrelated.  Due t o the i n a b i l i t y of other r e s e a r c h e r s a  two  effect,  are  exerted  dense d i s t r i b u t i o n specific  binding  at of  sites  the ANP in  i t is  including  level  of  the  immunopositive preoptic-septal  the anatomical framework f o r such an  action  e t a l . , 1986b; Q u i r i o n e t a l . , 1984). I t has  been  demonstrated t h a t i o n t o p h o r e s i s o r m i c r o p r e s s u r e i n j e c t i o n o f synthetic  ANP  onto  region  the  rat  contain  of  single (Wong e t  LHRH-positive  cell  neurons  in  a l . , 1986), bodies  (Krey  the a et  preoptic-septal region  known  a l . , 1983)  to and  thought  to  be  crucial  i n t h e hypothalamic  control  of  gonadotropin s e c r e t i o n  ( K a l r a e t a l . , 1985; Ramirez e t a l . ,  1984),  predominantly  resulted  neuronal receptors  in a  excitability. have  been  Since  inhibitory  ANP-positive  demonstrated  in this  effect  fibres  i n h i b i t o r y a c t i o n i n r o s t r a l hypothalamic  and ANP  region,  p o s s i b l e t h a t c e n t r a l l y a d m i n i s t e r e d ANP might e x e r t  on  i ti s i t s LH-  structures.  In a d d i t i o n , i t i s a l s o p o s s i b l e t h a t ANP can a c t a t t h e median eminence l e v e l . The e x i s t e n c e f i b r e terminals  i n the e x t e r n a l  of intense  ANP-positive  l a y e r o f t h e median eminence  (Kawata e t a l . , 1985a; S k o f i t s c h e t a l . , 1985) supports such possibility. inhibit  I f ANP a c t s  the r e l e a s e  i n vivo  o f LH  by  a t the median eminence t o inhibiting  the release  of  endogenous LHRH, then t h e p i t u i t a r y LH response t o any i . v . bolus i n j e c t i o n o f exogenous LHRH d u r i n g ANP i n f u s i o n should not  be a l t e r e d . Indeed, i n f u s i o n o f ANP i n l e v e l s known t o  i n h i b i t LH s e c r e t i o n f a i l e d t o b l o c k _  the p i t u i t a r y response  t o exogenous LHRH (Samson e t a l . , 1988b). Although of median eminence e x p l a n t s  incubation  i n t h e presence o f a wide dose  range o f ANP f a i l e d t o a l t e r b a s a l LHRH r e l e a s e , ANP was a b l e to s i g n i f i c a n t l y  inhibit  DA-stimulated LHRH r e l e a s e  (Samson  e t a l . , 1988b), suggesting n o t only an e f f e c t o f ANP a t t h e median eminence l e v e l ,  but a l s o 72  an i n t e r a c t i o n o f ANP w i t h  brain  catecholaminergic  systems  controlling  neuropeptide  secretion. D e f i n i t e proof  o f the  s i t e ( s ) of a c t i o n of ANP  on  LH  s e c r e t i o n awaits f u r t h e r experimentation. Our  observation  of  possible  i n t e r a c t i o n of  ANP  with  brain catecholaminergic  systems i n i n h i b i t i n g LH s e c r e t i o n was  predicted  result  by  the  administration catecholamine brain, al.,  of and  ANP  elicited  and  by  the  cells  report  dopamine  (Racz  circulation,  release  and  hence  of  the  et  into to  ventricular  al.,  levels  regions  septum effect  activity  strong evidence suggests t h a t ANP alter  in large  hypothalamus and  dopamine-beta-hydroxylase pheochromocytoma  lateral decreased  i t s metabolites  e s p e c i a l l y i n the 1986),  that  the  (Nakao et of  in 1986).  of  of  ANP  on  cultured Furthermore,  a c t s a t the hypothalamus to the  hypophyseal  influence  portal  pituitary  hormone  s e c r e t i o n . T h i r d c e r e b r o v e n t r i c u l a r i n f u s i o n of ANP  resulted  i n profound i n h i b i t i o n o f c i r c u l a t i n g PRL l e v e l s i n conscious, unrestrained  rats  (Samson and  Bianchi,  s i n c e a d i r e c t p i t u i t a r y a c t i o n o f ANP stimulated,  and  DA-inhibited  PRL  1987). Once  again,  on b a s a l , secretagogue-  release  i n v i t r o could  not  be demonstrated (Samson e t a l . , 1988a), the r e s u l t s suggested t h a t the a c t i o n of ANP  t o i n h i b i t PRL 73  secretion in vivo  was  due t o the r e l e a s e of DA, a major p r o l a c t i n i n h i b i t i n g f a c t o r , into  the  portal  inhibitory blocked  blood.  effect  i n the  of  Similarly,  ANP  presence  on of  LH the  i n our  experiment,  secretion dopamine  was  completely  receptor  pimozide. T h e r e f o r e i t i s p o s s i b l e ,  like  inhibition,  t o i n h i b i t LH  t h a t t h i s a c t i o n o f ANP  i n OVX r a t s i s due t o the  the  blocker  i n the case o f  PRL  secretion  f a c i l i t a t i o n of DA r e l e a s e i n t o the  p o r t a l blood, s i n c e i n f u s i o n s o f DA i n t o the t h i r d v e n t r i c l e of OVX  r a t s not t r e a t e d  w i t h exogenous s t e r o i d s r e s u l t e d i n  an i n h i b i t i o n of LH r e l e a s e i n many cases (Kalra e t a l . , 1985; Ramirez e t a l . , 1984). Another p o s s i b i l i t y i s t h a t the a c t i o n o f ANP  to i n h i b i t  turnover  rate  in  LH  secretion  i s due  LHRH-containing  t o modulation  nuclei,  such  of  as  medial  p r e o p t i c nucleus and septum, s i n c e s p e c i f i c ANP-binding was  i d e n t i f i e d i n these LHRH-containing  crucial  i n the hypothalamic  DA  sites  n u c l e i thought t o be  c o n t r o l of LH s e c r e t i o n  i n rats  (Quirion e t a l . , 1984). It reported  i s interesting on  antagonist, levels (Samson  that et  experiments  PRL.  to  Pretreatment  domperidone, were  PRL  our  with  resulted  resistant  a l . , 1988a). once  compare  to  the  been 74  the  i n an  Similarly, had  results  dopamine  action  another  inhibited  those  receptor  elevation  central in  with  of  PRL  of  ANP  series by  of  central  administration PRL  levels  of ANP,  to  those  domperidone treatment r a p i d l y seen  in  saline  treated,  elevated  domperidone-  i n j e c t e d c o n t r o l s (Samson e t a l . , 1988a). Thus i t appears t h a t the  mechanisms by  which ANP  i n h i b i t s LH  are  s i m i l a r . The  inhibitory effects  s e c r e t i o n w e r e c o n s i d e r e d t o be observe  thyroid  significant alterations  stimulating  administration  hormone  of the p e p t i d e  PRL  ANP  on  secretions LH  and  s p e c i f i c because of  -  to  of  and  in  growth  release  failure  hormone  after  via  secretion  also  interaction  dopaminergic appears  with  or  central  (Samson, 1988).  In a d d i t i o n t o a c e n t r a l i n h i b i t o r y a c t i o n of ANP secretion  PRL  system,  to  be  endogenous  ANP  i n h i b i t i o n of  mediated  opiate  on  in  part  systems,  by  which  LH LH an are  recognized t o be p h y s i o l o g i c a l i n h i b i t o r s of the hypothalamic component of 1977).  It  LH has  release  (Bruni  been  reported  proopiomelanocortin-derived  et  a l . , 1977; that  ANP  peptides  of r a t p i t u i t a r y i n v i t r o  indicating system.  an  interaction  Indeed,  The  of  present  with  study  e f f e c t of ANP  on  a l . , 1986),  central  opioidergic  shows  mean plasma LH 75  beta-  et  that  treatment a f t e r t h i r d v e n t r i c u l a r i n f u s i o n of ANP inhibitory  modulate  i n t e r m e d i a t e lobe -  (Shibasaki,  ANP  can  (including  endorphin) s e c r e t i o n from both a n t e r i o r and cells  Pang e t a l . ,  naloxone  reversed  secretion.  the This  r e s u l t i s i n agreement w i t h t h a t of Samson's group which shows t h a t pretreatment of r a t s w i t h naloxone  completely a b o l i s h e d  i n h i b i t o r y a c t i o n of the maximum e f f e c t i v e i . v . dose o f on mean plasma LH s e c r e t i o n i n t h e i r experiment,  ANP  (Samson et a l . , 1988b). However  the b l o o d sampling  intervals  (at 0,  15,  30, 60. 90, 120 minutes) were too l o n g t o r e v e a l the p u l s a t i l e pattern  of  determine  LH  release.  whether  s e c r e t i o n was  due  this  Therefore, inhibitory  t o the decrease  they  were  effect  of  unable ANP  to  on  LH  i n LH p u l s e amplitude  p u l s e frequency and the i n f l u e n c e of naloxone  or  on LH p u l s e s .  In our experiment, a s h o r t e r sampling i n t e r v a l (5 minutes) was employed t o r e v e a l the LH p u l s e s . The  result  clearly  t h a t the r e v e r s a l of mean LH l e v e l by naloxone t o the r e v e r s a l of LH p u l s e amplitude. of  LH  inhibition  considered hypothalamic  to  be  by  endogenous  exerted  regions  in  (Fink,  i s mainly  due  In terms of the  opiates,  the  shows  site  i t i s generally  preoptic  and  1988). I t should be  anterior noted  that  ANP-containing neuronal elements are present i n these r e g i o n s and indeed t e r m i n a l f i e l d s p o s i t i v e f o r ANP are p r e s e n t i n r e g i o n s known t o be c o n t r o l o f gonadotropin s e c r e t i o n Three characterized:  subtypes mu,  of  delta  important  76  i n the  (Jacobowitz e t a l . ,  opiate and  immunoreactivity  kappa.  receptors Opiate  have  effects  central 1985). been on  LH  s e c r e t i o n a r e g e n e r a l l y b e l i e v e d t o i n v o l v e the mu  receptor  subtype (Cicero e t a l . , 1983; P f e i f f e r e t a l . , 1983; Panerai et  a l . , 1985).  I t has  been  shown  that  intraventricular  i n j e c t i o n of the mu-agonist i n OVX r a t s produced a s i g n i f i c a n t suppression  o f LH  secretion,  while  the d e l t a -  and kappa-  agonists had no s i g n i f i c a n t e f f e c t , l e a d i n g t o t h e c o n c l u s i o n t h a t the mu-receptor i s the primary o p i a t e r e c e p t o r in  the r e g u l a t i o n o f LH s e c r e t i o n  However, the o b s e r v a t i o n  (Pfeiffer  that administration  a p r e f e r e n t i a l kappa a g o n i s t s ,  involved  e t a l . , 1983). o f bremazocine,  i s as potent as morphine t o  lower LH s e c r e t i o n i n r a t s o f both sex, suggested t h a t both types o f r e c e p t o r s c o u l d be i n v o l v e d i n mediating t h e response (Mark e t a l . , 1983). In a d d i t i o n , t h e mu-receptor  antagonist  naloxone s i g n i f i c a n t l y  e f f e c t of  antagonized the i n h i b i t i n g  morphine, but not t h a t o f bremazocine on LH s e c r e t i o n e t a l . , 1983). In t h e present  (mark  study, naloxone has been shown  t o reverse t h e i n h i b i t o r y e f f e c t o f ANP on LH s e c r e t i o n , thus the ANP's a c t i o n should be exerted a t l e a s t p a r t i a l l y v i a the opiate  mu-receptor.  reverse  the ANP's  Because action,  naloxone  could  i t i s possible  not completely that  the  other  subtypes o f o p i a t e r e c e p t o r s may a l s o mediate ANP's i n h i b i t o r y e f f e c t on LH s e c r e t i o n . BNP  i s a newly  identified 77  peptide  o f 26 amino  acid  residues,  which has  a remarkable homology t o but  from ANP  i n s t r u c t u r e . BNP  similar  to  that  hypotensive and  of  al.,  1988). A l s o  BNP  significantly  s e c r e t i o n and  e l i c i t s a v a r i e t y of responses very  ANP,  chick  such  as  natriuretic-diuretic,  rectum r e l a x a n t  i t has  is distinct  activities  been shown t h a t  inhibited  basal  the e f f e c t of BNP  (Sudoh  et  i * c . v . i n j e c t i o n of  arginine  vasopressin  i s comparable t o t h a t of  ANP  (Yamada et a l . , 1986). In similar  the to  pulsatile  present that  LH  studies,  of  ANP,  secretion,  BNP  i.e.  infusion  naloxone  e f f e c t of BNP  on LH s e c r e t i o n , and  prevented  the  by  showed  pretreatment  a of  reversed  the  of  pimozide.  may  be r e g u l a t e d  mechanism i n v o l v i n g both ANP  and  BNP,  inhibit  the  inhibited inhibitory  pulsatile  and  LH  These  was  results  on LH s e c r e t i o n once  thought t o be mediated by ANP  they  BNP  very  this inhibitory effect  suggest t h a t the i n h i b i t o r y a c t i o n o f BNP  which  response  the  through a dual mechanisms by  secretion  are  quite  similar. Recent improvements i n neuropeptide c h a r a c t e r i z a t i o n and l o c a l i z a t i o n methods have r e s u l t e d i n an in-depth r e a p p r a i s a l of our to  understanding of neuroendocrine c o n t r o l . In  "conventional"  recently  been  neurotransmitters,  shown  to  regulate 78  over  30  pituitary  addition  peptides  have  reproductive  hormones  (Richard  e t a l . , 1988).  ANP  and  BNP  are  members of them. L i k e most of neuropeptides, ANP been  found  produced  locally  Skofitsch 1988).  in significant  released  ( M o r i i e t a l . , 1985;  e t a l . , 1985;  However,  amounts i n the  most  i n t o the hypophyseal  anterior pituitary  of  adenohypophyseal  and  Kawata e t a l . , 1985; Sudoh  neuropeptides  portal  new  and BNP have  hypothalamus  Ueda e t a l . , 1988;  unlike  two  system  cells  and  et a l . ,  which  are  affect  the  directly  (Richard  et a l . , 1988) , ANP seems mainly t o a c t a t the hypothalamic and median eminence l e v e l s  (Samson e t a l . ,  the p r e s e n t study shows t h a t ANP neurotransmission systems,  systems,  to exert t h e i r  Like  most  1988b).  and BNP  like  opiate  In a d d i t i o n ,  i n t e r a c t w i t h other and  dopaminergic  i n h i b i t o r y e f f e c t s on LH  hypothalamic  peptides  secretion.  produced  in  the  p r e o p t i c - p e r i v e n t r i c u l a r area, mapping s t u d i e s based on neuron l a b e l l i n g methods have shown t h a t  median eminence i s o n l y one  of s e v e r a l p r o j e c t i o n s i t e s f o r ANP  (Standaert e t a l . ,  1986b).  Axons  also  several  "of  ANP-positive  hypothalamic n u c l e i , paraventricular  neurons  innervate  i n p a r t i c u l a r medial p r e o p t i c area and  nucleus,  as  well  as  extrahypothalamic  s t r u c t u r e s (Jacobowitz e t a l . , 1985; S t a n d a e r t e t a l . , This  strategic position  accounts  hormone r e g u l a t i o n . 79  for i t role  1986b).  i n modulating  In g e n e r a l , ANP and BNP have much i n common w i t h neuropeptides i n r e g u l a t i n g p i t u i t a r y r e p r o d u c t i v e  80  other  hormones.  4. 4.1  SUMMARY AND  CONCLUSIONS  summary Over the past s e v e r a l y e a r s an abundance of  concerning  the  possible  neuromodulatory a c t i o n s  accumulated. C l e a r consensus e x i s t s t h a t ANP angiotensin  II actions,  and v a s o p r e s s i n  particularly  salt  information of ANP  has  can i n h i b i t many and  water  intake  s e c r e t i o n (David e t a l . , 1989), which m i r r o r  ANP's p e r i p h e r a l r o l e s i n maintenance of f l u i d and e l e c t r o l y t e homeostasis. More r e c e n t l y , evidence f o r the central  ANP  electrolyte r o l e s f o r ANP inhibitory  with  brain  homeostasis  hormones has  i n t e r a c t i o n of  unrelated  further  to  expanded  the  fluid  and  possible  i n hypothalamo-hypophyseal f u n c t i o n , such as the effects  of  anatomical s u b s t r a t e s  ANP  on  LH  and  PRL  f o r the a c t i o n s of ANP  secretion.  The  on gonadotropin  s e c r e t i o n are the presence of ANP-immunoreactivity i n neural pathways and hypothalamic s i t e s known t o be important i n these f u n c t i o n s (Jacobowitz e t a l . , 1985). The ANP  on  LH  secretion  manipulations o f ANP  seems  i n h i b i t o r y e f f e c t of  specific,  since  similar  on t h y r o i d - s t i m u l a t i n g hormone or growth  hormone r e s u l t e d no s i g n i f i c a n t response (Samson, 1988).  The  present study demonstrates t h a t t h i s i n h i b i t o r y e f f e c t i s a l s o d o s e - r e l a t e d and r e v e r s i b l e and t h a t ANP's i n h i b i t o r y a c t i o n s 81  on  LH  secretion  may  involve  dopaminergic and o p i a t e As  ANP  demonstrate  BNP  f o r future  i n t h e hypothalamic c o n t r o l  significantly or  central  work,  i n order  to  a p h y s i o l o g i c a l l y s i g n i f i c a n t r o l e f o r endogenous  o r BNP  must  with  systems.  f o r suggestions  establish  interactions  that  the absence  a l t e r s reproductive  antibodies  into  immunoneutralization  o f LH s e c r e t i o n  o f neuronal  activity.  ANP  Infusion  the c e r e b r o v e n t r i c l e s  techniques may  help  or  or  one BNP  of ANP passive  i n providing  such  information.  4.2  Conclusion The  primary focus of the r e s e a r c h  establishing  t h e modulatory  roles  presented here was i n  o f ANP  and  BNP  i n LH  s e c r e t i o n and t h e i r p o s s i b l e mechanisms. Two nmol ANP o r BNP and  0.2 nmol ANP o r BNP were i n f u s e d i n t o t h i r d v e n t r i c l e and  their  e f f e c t s on plasma LH c o n c e n t r a t i o n s were observed i n  ovariectomized  rats.  In  addition,  naloxone  and  pimozide  m a n i p u l a t i o n s were employed t o observe i n t e r a c t i o n s o f ANP o r BNP  with  opiate  and  dopaminergic  systems.  The  results  presented i n t h i s r e p o r t suggest t h a t : 1. 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