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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 thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA AUGUST, 1990 @ JIN 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 be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of Obstetrics and Gynecology The University of British Columbia Vancouver, Canada Date September 10, 1990  DE-6 (2/88) A B S T R A C T A t r i a l n a t r i u r e t i c p e p t i d e (ANP) o f a t r i a l m y o c y t e o r i g i n , h a s b e e n shown t o p l a y a r o l e i n t h e d i u r e s i s , n a t r i u r e s i s , a n d a n t a g o n i s m o f a n g i o t e n s i n a n d v a s o p r e s s i n . H o w e v e r , i t i s now a p p a r e n t t h a t i n a d d i t i o n t o t h e p r o d u c t i o n o f t h e p e p t i d e i n t h e h e a r t a n d i n i t s r o l e i n f l u i d a n d e l e c t r o l y t e h o m e o s t a s i s , i t i s a l s o p r o d u c e d i n t h e c e n t r a l n e r v o u s s y s t e m p a r t i c i p a t i n g i n t h e r e g u l a t i o n o f p i t u i t a r y h o r m o n e s e c r e t i o n . 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 c e n t r a l a n d p e r i p h e r a l r o u t e s h a s b e e n shown t o i n h i b i t s e c r e t i o n o f l u t e i n i z i n g h o r m o n e (LH) i n t h e g o n a d e c t o m i z e d r a t m o d e l . A b e t t e r u n d e r s t a n d i n g o f t h e m o d u l a t o r y r o l e o f ANP on LH s e c r e t i o n a n d i t s p o s s i b l e m e c h a n i s m s w i l l a d d t o o u r k n o w l e d g e o f t h e e f f e c t s o f n e u r o p e p t i d e s on r e p r o d u c t i v e f u n c t i o n . B r a i n n a t r i u r e t i c p e p t i d e (BNP) i s a b i o a c t i v e p e p t i d e o f 26 a m i n o a c i d r e s i d u e s r e c e n t l y i d e n t i f i e d i n p o r c i n e b r a i n . T h e p e p t i d e e x e r t s p o t e n t d i u r e t i c - n a t r i u r e t i c a n d v a s o r e l a x a n t e f f e c t s , i n a m a n n e r s i m i l a r t o t h a t o f A N P . BNP h a s a r e m a r k a b l e h i g h s e q u e n c e h o m o l o g y t o A N P , e s p e c i a l l y i n t h e 17 a m i n o a c i d r i n g f o r m e d b y a n i n t r a m o l e c u l a r d i s u l f i d e l i n k a g e w h i c h i s r e q u i r e d f o r b i o l o g i c a l a c t i v i t y . T h e p r e s e n c e o f BNP w i t h ANP i n t h e m a m m a l i a n b r a i n a n d r e m a r k a b l e r e s e m b l a n c e i n t h e i r m o l e c u l a r s t r u c t u r e s a n d p h y s i o l o g i c a l f u n c t i o n s i m p l i e s t h a t BNP may a l s o e x e r t a n i n h i b i t o r y e f f e c t o n L H s e c r e t i o n l i k e A N P . T h i s r e s e a r c h f o c u s e d o n t h e e f f e c t s o f c e n t r a l l y a d m i n i s t e r e d ANP a n d BNP o n p u l s a t i l e L H s e c r e t i o n a n d t h e i r p o s s i b l e m e c h a n i s m s o f a c 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 f t e r t h i r d v e n t r i c l e i n f u s i o n o f ANP o r BNP, i n h i b i t i o n o f mean p l a s m a LH l e v e l , L H p u l s e a m p l i t u d e a n d p u l s e f r e q u e n c y was o b s e r v e d . I n s e a r c h i n g f o r t h e p o s s i b l e m e c h a n i s m s o f i n h i b i t o r y e f f e c t o f ANP o r BNP o n p u l s a t i l e LH s e c r e t i o n , t h e e f f e c t o f i n h i b i t i n g t h e e n d o g e n o u s o p i a t e s y s t e m w i t h n a l o x o n e o n t h e a c t i o n o f c e n t r a l l y a d m i n i s t e r e d ANP o r BNP was t e s t e d . A p p l i c a t i o n o f n a l o x o n e r e v e r s e d t h e i n h i b i t o r y e f f e c t o f ANP a n d BNP on mean p l a s m a L H l e v e l a n d L H p u l s e a m p l i t u d e , b u t i n t e r m s o f p u l s e f r e q u e n c y , n a l o x o n e t r e a t m e n t f a i l e d t o r e v e r s e t h e i n h i b i t o r y e f f e c t o f ANP o r B N P . I n s e p a r a t e e x p e r i m e n t s , p r e t r e a t m e n t w i t h p i m o z i d e , a d o p a m i n e r g i c r e c e p t o r b l o c k e r , p r e v e n t e d t h e i n h i b i t o r y a c t i o n o f ANP a n d BNP o n L H s e c r e t i o n . A f t e r i n f u s i o n o f ANP o r B N P , t h e r e were no s i g n i f i c a n t d e c r e a s e i n mean p l a s m a L H l e v e l , p u l s e a m p l i t u d e a n d p u l s e f r e q u e n c y i n t h e p i m o z i d e - p r e t r e a t e d i i i r a t s . I n summary, t h e p r e s e n t s t u d y shows t h a t b o t h ANP a n d BNP i n h i b i t p u l s a t i l e L H s e c r e t i o n , s u g g e s t i n g t h a t t h e i n h i b i t o r y e f f e c t s on L H s e c r e t i o n o n c e t h o u g h t t o b e m e d i a t e d b y ANP a l o n e may b e r e g u l a t e d t h r o u g h a d u a l m e c h a n i s m i n v o l v i n g b o t h ANP a n d B N P . F u r t h e r m o r e , t h e i n h i b i t o r y m e c h a n i s m s may i n v o l v e t h e i n t e r a c t i o n s o f ANP a n d BNP w i t h c e n t r a l o p i a t e s y s t e m a n d d o p a m i n e r g i c s y s t e m o n LH s e c r e t i o n . i v T A B L E OF CONTENTS PAGE A B S T R A C T i i T A B L E OF CONTENTS v L I S T OF F I G U R E S i x L I S T OF A B B R E V I A T I O N S x i i i ACKNOWLEDGEMENTS x i v 1. L I T E R A T U R E REVIEW 1 1 .1 A t r i a l N a t r i u r e t i c P e p t i d e (ANP) 1 1 . 1 . 1 M o r p h o l o g i c a l o v e r v i e w o f ANP 1 1 . 1 . 2 S t r u c t u r e o f ANP 3 1 . 1 . 3 B i o s y n t h e s i s o f ANP 5 1 . 1 . 4 P e r i p h e r a l h o m e o s t a s i s a c t i o n o f ANP 6 1 . 1 . 5 R e g u l a t i o n o f ANP s e c r e t i o n 7 1 . 1 . 6 M e c h a n i s m o f ANP a c t i o n 8 1 . 1 . 7 C e n t r a l a c t i o n s o f ANP 9 1 . 1 . 7 . 1 D i s t r i b u t i o n a n d b i n d i n g s i t e s o f 9 ANP i n t h e CNS 1 . 1 . 7 . 2 H y p o t h a l a m i c a c t i o n s o f ANP i n 11 i n h i b i t i n g p o s 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 1 . 1 . 7 . 3 H y p o t h a l a m i c a c t i o n s o f ANP i n 13 a l t e r i n g a n t e r i o r p i t u i t a r y v hormone secretion 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 secretion 14 in vivo 1.1.7.3.2 In h i b i t i o n of LH secretion i n vivo 15 1.2 Brain N a t r i u r e t i c Peptide 17 1.2.1 The structure of BNP 18 1.2.2 Biosynthesis of BNP 19 1.2.3 The d i s t r i b u t i o n of BNP 19 1.2.4 The physiology of BNP 21 1.3 Introduction to the r o l e of neuromediators i n 23 the LH regulation 1.3.1 Dopamine 2 3 1.3.2 Norepinephrine 24 1.3.3 Endogenous opioid peptides 27 2. EXPERIMENTAL 32 2.1 Experimental rationale and design 32 2.2 Materials and methods 36 2.3 Results 42 2.3.1 Eff e c t s of ANP and BNP infusion on 42 LH secretion 2.3.1.1 E f f e c t of sa l i n e infusion on LH 42 secretion 2.3.1.2 E f f e c t of 2nmol ANP infusion on 42 v i LH secretion 2.3.1.3 E f f e c t of 2nmol BNP infusion on 43 LH secretion 2.3.1.4 E f f e c t of 0.2nmol ANP infusion on 4 3 LH secretion 2.3.1.5 E f f e c t of 0.2nmol BNP infusion on 44 LH secretion 2.3.2 E f f e c t s of naloxone treatment on the 53 LH i n h i b i t o r y actions of i.e.v. infused ANP and BNP 2.3.2.1 E f f e c t s of naloxone treatment i n 53 control group 2.3.2.2 E f f e c t s of naloxone treatment i n 53 2nmol ANP group 2.3.2.3 E f f e c t s of naloxone treatment i n 54 2nmol BNP group 2.3.3 E f f e c t s of pimozide pretreatment on 61 the LH i n h i b i t o r y actions of i.e.v. infused ANP and BNP 2.3.3.1 E f f e c t s of pimozide pretreatment i n 61 control group 2.3.3.2 E f f e c t s of pimozide pretreatment i n 61 2nmol ANP group v i i 2 . 3 . 3 . 3 E f f e c t s o f p i m o z i d e p r e t r e a t m e n t i n 61 2nmol BNP g r o u p 3 . D I S C U S S I O N 70 4 . SUMMARY AND CONCLUSIONS 81 4 . 1 Summary 81 4 . 2 C o n c l u s i o n s 82 5 . R E F E R E N C E S 84 v i i i L I S T OF FIGURES PAGE F i g u r e 1. R e p r e s e n t a t i v e e x a m p l e o f L H r e l e a s e i n 46 o v x r a t s w i t h i . c . v i n f u s i o n o f s a l i n e . F i g u r e 2 . R e p r e s e n t a t i v e e x a m p l e o f LH r e l e a s e i n 47 o v x r a t s w i t h i . c . v i n f u s i o n o f 2 nmol A N P . F i g u r e 3 . R e p r e s e n t a t i v e e x a m p l e o f LH r e l e a s e i n 48 o v x r a t s w i t h i . c . v i n f u s i o n o f 2 nmol BNP. F i g u r e 4. R e p r e s e n t a t i v e e x a m p l e o f LH r e l e a s e i n 49 o v x r a t s w i t h i . c . v i n f u s i o n o f 0 .2 nmol A N P . F i g u r e 5 . R e p r e s e n t a t i v e e x a m p l e o f LH r e l e a s e i n 50 o v x r a t s w i t h i . c . v i n f u s i o n o f 0 .2 nmol B N P . F i g u r e 6. M e a n p l a s m a L H c o n c e n t r a t i o n i n t h e p r e - 51 a n d p o s t - A N P o r BNP i n f u s i o n p e r i o d . F i g u r e 7 . L H p u l s e a m p l i t u d e i n t h e p r e - a n d p o s t - A N P 52 o r BNP i n f u s i o n p e r i o d . i x F i g u r e 8 . LH p u l s e f r e q u e n c y i n t h e p r e - a n d p o s t - A N P 53 o r BNP i n f u s i o n p e r i o d . F i g u r e 9 . R e p r e s e n t a t i v e e x a m p l e o f L H r e l e a s e i n o v x 56 r a t s i n j e c t e d w i t h n a l o x o n e a f t e r i . c . v i n f u s i o n o f s a l i n e . F i g u r e 1 0 . R e p r e s e n t a t i v e e x a m p l e o f L H r e l e a s e i n o v x 57 r a t s i n j e c t e d w i t h n a l o x o n e a f t e r i . c . v i n f u s i o n o f 2 n m o l A N P . F i g u r e 1 1 . R e p r e s e n t a t i v e e x a m p l e o f L H r e l e a s e i n o v x 58 r a t s i n j e c t e d w i t h n a l o x o n e a f t e r i . c . v i n f u s i o n o f 2 n m o l B N P . F i g u r e 1 2 . Mean p l a s m a L H c o n c e n t r a t i o n i n t h e p r e - a n d 59 p o s t - A N P o r BNP i n f u s i o n a n d n a l o x o n e t r e a t m e n t p e r i o d . F i g u r e 1 3 . L H p u l s e a m p l i t u d e i n t h e p r e - a n d p o s t - A N P 60 o r BNP i n f u s i o n a n d n a l o x o n e t r e a t m e n t p e r i o d . F i g u r e 1 4 . L H p u l s e f r e q u e n c y i n t h e p r e - a n d p o s t - A N P 61 o r BNP i n f u s i o n a n d n a l o x o n e t r e a t m e n t p e r i o d . x Figure 15. Representative example of LH release i n 64 pimozide-pretreated ovx rats with i.c.v infusion of sal i n e . Figure 16. Representative example of LH release i n 65 pimozide-pretreated ovx rats with i.c.v i n f u s i o n of 2 nmol ANP. Figure 17. Representative example of LH release i n 66 pimozide-pretreated ovx rats with i.c.v infusion of 2 nmol BNP. Figure 18. Mean plasma LH concentration i n the pre- 67 and post-ANP or BNP infusion period with the pimozide pretreatment. Figure 19. LH pulse amplitude i n the pre- and post-ANP 68 or BNP infusion period with the pimozide pretreatment. Figure 20. LH pulse frequency i n the pre- and post-ANP 69 or BNP infusion period with the pimozide pretreatment. Figure 21. Photograph of representative f r o n t a l x i 70 sections i n the rat hypothalamus depicting the s i t e of 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 black t r i a n g l e . x i i L I S T O F A B B R E V I A T I O N S AHA a n t e r i o r h y p o t h a l a m i c a r e a ANP a t r i a l n a t r i u r e t i c p e p t i d e BNP b r a i n n a t r i u r e t i c p e p t i d e cGMP c y c l i c g u a n o s i n e 3'5'- m o n o p h o s p h a t e CNS c e n t r a l n e r v o u s s y s t e m DA d o p a m i n e EOP e n d o g e n o u s o p i o i d p e p t i d e s i . c . v . i n t r a c e r e b r o v e n t r i c u l a r L H l u t e i n i z i n g h o r m o n e LHRH l u t e i n i z i n g h o r m o n e r e l e a s i n g h o r m o n e MPOA m e d i a l p r e o p t i c a r e a mRNA m e s s e n g e r r i b o n u c l e i c a c i d NE n o r e p i n e p h r i n e n g n a n o g r a m n m o l n a n o m o l e OVX o v a r i e c t o m i z e d pANP p o r c i n e a t r i a l n a t r i u r e t i c p e p t i d e P R L p r o l a c t i n R I A a r a d i o i m m u n o a s s a y s . c s u b c u t a n e o u s l y SCN s u p r a c h i a s m a t i c n u c l e u s x n i ACKNOWLEDGEMENTS I w o u l d l i k e t o g r a t e f u l l y a c k n o w l e d g e my s u p e r v i s o r , D r . P e t e r L e u n g , f o r p r o v i d i n g me t h e o p p o r t u n i t y t o p u r s u e t h e s e s t u d i e s a n d f o r h i s c o n t i n u o u s s u p p o r t a n d e n c o u r a g e m e n t t h r o u g h o u t t h e t i m e s p e n t i n h i s l a b o r a t o r y . I a l s o w i s h t o a c k n o w l e d g e my c o l l e a g u e s , Hugo B e r g e n f o r h i s t e c h n i c a l a d v i c e , J i a n Wang a n d M . T . L i t t l e f o r t h e i r s u g g e s t i o n s t o my t h e s i s , M . R o d w a y , J . S t e e l e , a n d E u i - B a e J e u n g , who made my t i m e i n t h e l a b o r a t o r y a more e n j o y a b l e e x p e r i e n c e . L a s t , b u t n o t l e a s t , I t h a n k Y . P a n f o r h e r p a t i e n c e a n d e n c o u r a g e m e n t d u r i n g t h e f i n a l s t a g e s o f t h i s w o r k . x i v 1. L I T E R A T U R E REVIEW 1 .1 A T R I A L N A T R I U R E T I C P E P T I D E A t r i a l n a t r i u r e t i c p e p t i d e ( A N P ) , a l s o c a l l e d a t r i a l n a t r i u r e t i c f a c t o r ( A N F ) , i s a p e p t i d e o f a t r i a l m y o c y t e o r i g i n a n d h a s b e e n shown t o p l a y a r o l e i n t h e d i u r e s i s , n a t r i u r e s i s , a n d a n t a g o n i s m o f a n g i o t e n s i n a n d v a s o p r e s s i n a c t i o n s ( d e B o l d , 1 9 8 5 ; S a m s o n , 1 9 8 8 ) . H o w e v e r , i t i s now a p p a r e n t t h a t i n a d d i t i o n t o t h e p r o d u c t i o n o f t h e p e p t i d e i n t h e h e a r t a n d i n i t s r o l e i n f l u i d a n d e l e c t r o l y t e h o m e o s t a s i s , i t i s a l s o p r o d u c e d i n t h e c e n t r a l n e r v o u s s y s t e m a n d p l a y s a r o l e i n t h e r e g u l a t i o n o f p i t u i t a r y h o r m o n e s e c r e t i o n as w e l l ( J a c o b o w i t z e t a l . , 1 9 8 5 ; S h i b a s a k i e t a l . , 1986; Samson e t a l . , 1 9 8 8 a ) . T h e r e f o r e , a n o v e r v i e w o f t h e p h y s i o l o g y o f ANP i s i m p o r t a n t i n t h e u n d e r s t a n d i n g o f i t s s p e c i f i c r o l e i n p i t u i t a r y h o r m o n e s e c r e t i o n . 1 . 1 . 1 MORPHOLOGICAL OVERVIEW OF A N P : M o s t o f t h e c a r d i o c y t e s f o r m i n g t h e a t r i a l a n d v e n t r i c u l a r m u s c l e o f t h e m a m m a l i a n h e a r t a r e d i f f e r e n t i a t e d f o r m e c h a n i c a l w o r k . M o r p h o l o g i c a l l y t h i s i s e v i d e n t f r o m t h e i r l a r g e c o n t e n t o f c o n t r a c t i l e e l e m e n t s . H o w e v e r , i n 1956 , B . K i s c h p o i n t e d o u t a m o r p h o l o g i c a l d i f f e r e n c e b e t w e e n a t r i a l 1 a n d v e n t r i c u l a r c a r d i o c y t e s i n t h e h e a r t o f t h e g u i n e a p i g t h a t c o u l d n o t b e f i t t e d i n t o t h e f u n c t i o n a l f r a m e w o r k t h a t t h e h e a r t o n l y s e r v e s a s a m e c h a n i c a l pump ( K i s c h , 1 9 5 6 ) . He o b s e r v e d , a s o t h e r s d i d s u b s e q u e n t l y ( J a m i e s o n a n d P a l a d e , 1964; F e r r a n s e t a l . , 1 9 6 9 ) , t h a t a t r i a l c a r d i o c y t e s i n mammals , u n l i k e v e n t r i c u l a r c a r d i o c y t e s , h a v e m o r p h o l o g i c a l f e a t u r e s o f s e c r e t o r y c e l l s . T h e m o s t o b v i o u s e x p r e s s i o n o f t h i s d i f f e r e n t i a t i o n i s t h e p r e s e n c e o f m e m b r a n e - b o u n d s p e c i f i c a t r i a l g r a n u l e s w h i c h d i s p l a y a n e l e c t r o n - d e n s e c o r e a n d m e a s u r e 250 t o 500 n a n o m e t r e s . T h e s e g r a n u l e s a r e m o r e c o n c e n t r a t e d i n t h e c e n t r a l s a r c o p l a s m i c c o r e o f a t r i a l c a r d i o c y t e s . O f t e n , t h e g r a n u l e s a r e f o u n d a s s o c i a t e d w i t h a p r o m i n e n t G o l g i c o m p l e x f r o m w h i c h t h e y a r i s e . N u m e r o u s r o u g h e n d o p l a s m i c r e t i c u l u m a r e a l s o n o r m a l l y f o u n d . T h e s e f e a t u r e s o f t h e c e n t r a l s a r c o p l a s m i c c o r e o f t h e m a m m a l i a n a t r i a l c a r d i o c y t e a r e m o r e common i n t h e g e n e r a l p o p u l a t i o n o f c a r d i o c y t e s f o u n d i n t h e a u r i c l e . V a r i o u s m e t h o d s h a v e shown t h a t ANP i s s t o r e d w i t h i n s p e c i f i c a t r i a l g r a n u l e s ( d e B o l d e t a l . , 1 9 8 3 ; Grammer e t a l . , 1 9 8 3 ) . T h e n e t s p e c i f i c n a t r i u r e t i c a c t i v i t y o f a t r i a l e x t r a c t s i s h i g h e s t f o r a n i m a l s w i t h t h e h i g h e s t n u m b e r o f g r a n u l e s ( d e B o l d a n d S a l e r n o , 1 9 8 3 ) . T i s s u e f r a c t i o n a t i o n s t u d i e s show t h a t t h e h i g h e s t n a t r i u r e t i c a c t i v i t y i s a s s o c i a t e d w i t h f r a c t i o n s c o n t a i n i n g 2 p u r i f i e d g r a n u l e s ( d e B o l d e t a l . , 1 9 8 2 ) . I n a d d i t i o n , i m m u n o c y t o c h e m i c a l s t u d i e s c l e a r l y l o c a l i z e ANP w i t h i n t h e a t r i a l g r a n u l e s ( T a n a k a e t a l . , 1 9 8 4 ) . I n mammals t h e r e a r e v a r i a t i o n s i n t h e q u a n t i t y o f a t r i a l g r a n u l e s . R o d e n t s , f o r e x a m p l e , h a v e f a r m o r e a t r i a l g r a n u l e s t h a n l a r g e mammals s u c h a s c a t t l e , w h i c h h a v e v e r y few g r a n u l e s p e r c a r d i o c y t e . Human c a r d i o c y t e s h a v e a n i n t e r m e d i a t e c o n t e n t . I n r a t s , g r a n u l e c o n t e n t v a r i e s w i t h a g e . I t d o u b l e s b e t w e e n a g e s 6 a n d 10 weeks a n d , a l t h o u g h t h e number o f g r a n u l e s p l a t e a u s a t a d u l t h o o d , m o r p h o m e t r i c p r o c e d u r e s show s i g n i f i c a n t v a r i a t i o n s i n a n i m a l s o f t h e same a g e ( d e B o l d e t a l . , 1 9 8 3 ) . E x t r a c a r d i a c l o c a l i z a t i o n s o f ANP h a v e b e e n s u g g e s t e d , n o t a b l y i n t h e c e n t r a l n e r v o u s s y s t e m a n d k i d n e y ( S a k a m o t o e t a l . , 1 9 8 5 ; S a p e r e t a l . , 1 9 8 5 ) . H o w e v e r , q u a n t i t a t i v e l y t h e m a i n s t o r e o f ANP i n mammals i s t h e a t r i a a n d t h e amount o f ANP p r e c u r s o r m e s s e n g e r RNA i s h i g h e s t i n t h i s t i s s u e ( N a k a y a m a e t a l . , 1 9 8 4 ) . 1 . 1 . 2 STRUCTURE OF ANP W h i l e t h e p r e d o m i n a n t s t o r a g e f o r m o f ANP i n t h e a t r i a h a s b e e n i d e n t i f i e d a s a 1 5 , 0 0 0 - d a l t o n p r o - A N P , e a r l y b i o c h e m i c a l s t u d i e s r e s u l t e d i n t h e i s o l a t i o n o f n u m e r o u s l o w 3 m o l e c u l a r w e i g h t f r a g m e n t s p r o d u c e d b y t h e p r o t e o l y s i s o f p r o -ANP d u r i n g p u r i f i c a t i o n ( S e i d a h e t a l . , 1 9 8 4 ; F l y n n e t a l . , 1983) . E v i d e n c e o f t h e p r e s e n c e o f a h i g h m o l e c u l a r w e i g h t ANP i n t h e a t r i a a n d t h e s e q u e n c e a n a l y s i s o f h i g h m o l e c u l a r w e i g h t ANP f r o m a t r i a i d e n t i f i e d t h e p r e c u r s o r o f ANP a s a 1 2 6 - r e s i d u e p e p t i d e . ( C u r r i e e t a l . , 1 9 8 3 ; Grammer e t a l . , 1 9 8 3 ) . I s o l a t i o n f r o m r a p i d l y b o i l e d a t r i a , c u l t u r e d a t r i a l m y o c y t e s , o r p u r i f i e d a t r i a l g r a n u l e s a n d d i r e c t s e q u e n c e a n a l y s i s f i r m l y e s t a b l i s h e d t h a t i n t h e r a t , ANP e x i s t e d i n d e n s e s e c r e t o r y g r a n u l e s p r e d o m i n a n t l y i n t h i s 1 2 6 - r e s i d u e p r o - A N P f o r m ( M i y a t a e t a l . , 1985 ; G i b s o n e t a l . , 1 9 8 7 ; T h i b a u l t e t a l . , 1 9 8 7 ) . E x a m i n a t i o n o f p l a s m a ANP b y g e l f i l t r a t i o n o r h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y showed t h a t ANP c i r c u l a t e d i n a l o w m o l e c u l a r w e i g h t f o r m . ( S u g i y a m a e t a l . , 1 9 8 4 , M i y a t a e t a l . , 1 9 8 5 ) . P l a s m a ANP was p u r i f i e d , a n d i t s s e q u e n c e was d e t e r m i n e d a s t h e 2 8 - r e s i d u e p e p t i d e ( T h i b a u l t e t a l . , 1 9 8 5 ) . T h e a m i n o a c i d s e q u e n c e o f ANP i s a l m o s t i d e n t i c a l t h r o u g h o u t m a m m a l i a n s p e c i e s e x c e p t f o r p o s i t i o n 110 w h i c h i s i s o l e u c i n e i n mouse ( S e i d a h e t a l . , 1 9 8 4 ) , r a t ( N a p i e r e t a l . , 1984; A t l a s e t a l . , 1 9 8 4 ) , a n d r a b b i t ANP ( O i k a w a e t a l . , 1 9 8 5 ) , w h e r e a s human (Kangawa e t a l . , 1 9 8 4 ) , b o v i n e ( V l a s u k e t a l . , 1986; Ong e t a l . , 1987) a n d d o g ( O i k a w a e t a l . , 1985) 4 ANP h a v e m e t h i o n i n e i n t h i s p o s i t i o n . V a r i o u s c l e a v a g e a n d s y n t h e t i c m e t h o d s f o r i n t r o d u c i n g v a r i a t i o n s o f ANP s t r u c t u r e r e v e a l e d t h a t a d i s u l f i d e - l o o p e d s e q u e n c e o f 17 a m i n o a c i d s r i n g s t r u c t u r e i s e s s e n t i a l f o r A N P ' s b i o l o g i c a l f u n c t i o n s . O p e n i n g t h e r i n g s t r u c t u r e b y p r o t e o l y t i c c l e a v a g e o r r e d u c t i o n o f t h e d i s u l f i d e b r i d g e a l m o s t c o m p l e t e l y a b o l i s h e s i t s p h y s i o l o g i c a l a c t i v i t y ( M i s o n o e t a l . , 1 9 8 4 ) . 1 . 1 . 3 B I O S Y N T H E S I S OF ANP C l o n i n g a n d s e q u e n c e a n a l y s i s o f c o m p l e m e n t a r y DNA e n c o d i n g t h e ANP p r e c u r s o r h a v e p r o v i d e d a m o r e c o m p l e t e u n d e r s t a n d i n g o f a t r i a l n a t r i u r e t i c p e p t i d e s (Nakayama e t a l . , 1984; F l y n n e t a l . , 1 9 8 5 ) . F r o m t h e s e s t u d i e s i t h a s b e e n e s t a b l i s h e d t h a t ANP i s s y n t h e s i z e d i n a p r e p r o - h o r m o n e f o r m c o n t a i n i n g 152 a m i n o a c i d s i n t h e r a t a n d 151 a m i n o a c i d s i n t h e human. A h i g h d e g r e e o f h o m o l o g y e x i s t s b e t w e e n t h e r a t a n d human s e q u e n c e s . T h e s e p r e c u r s o r s c o n t a i n 2 4 - a n d 2 5 - a m i n o a c i d p u t a t i v e s i g n a l s e q u e n c e s , r e s p e c t i v e l y . T h e m a i n s t o r a g e f o r m o f ANP i n r a t a t r i a l h o m o g e n a t e s a n d i n i s o l a t e d g r a n u l e s i s a 1 2 6 - a m i n o a c i d p e p t i d e c a l l e d gamma r a t a t r i a l n a t r i u r e t i c p e p t i d e . I t i s d e r i v e d f r o m p r e p r o - A N P b y r e m o v a l o f t h e s i g n a l p e p t i d e a n d a r g i n i n e 5 r e s i d u e s 151 a n d 1 5 2 . T h e a b s e n c e o f t h e s e r e s i d u e s i n t h e p e p t i d e s s o f a r i s o l a t e d i n d i c a t e s e a r l y r e m o v a l a f t e r b i o s y n t h e s i s (Kanagawa e t a l . , 1 9 8 4 ) . T h e c i r c u l a t i n g f o r m o f r a t ANP i n p l a s m a i s a l p h a a t r i a l n a t r i u r e t i c p e p t i d e , a 2 8 - a m i n o a c i d p e p t i d e t h a t c o m p r i s e s r e s i d u e s 123 t o 150 o f p r e p r o - A N P . T h e c o n v e r s i o n o f p r o - A N P t o t h e a c t i v e f o r m s h o u l d t a k e p l a c e a t t h e t i m e o f i t s s e c r e t i o n f r o m t h e a t r i u m o r i m m e d i a t e l y t h e r e a f t e r ( G i b s o n e t a l . , 1 9 8 7 ; Imada e t a l . , 1 9 8 7 ) . W h i l e t h e c a r d i a c a t r i a a r e b y f a r t h e m a j o r s o u r c e o f A N P , many r e g i o n s o f t h e b r a i n , p i t u i t a r y , a d r e n a l m e d u l l a , l u n g , c a r d i a c v e n t r i c l e , a o r t i c a r c h , a n d r e n a l d i s t a l ( m e d u l l a r y ) c o l l e c t i n g d u c t c e l l s , a n d s p i n a l g a n g l i o n i c c e l l s seem t o c o n t a i n ANP i n s m a l l q u a n t i t i e s ( I n a g a m i e t a l . , 1 9 8 9 ) . I n some o f t h e s e t i s s u e s , e s p e c i a l l y i n r a t h y p o t h a l a m u s , t h e l o c a l b i o s y n t h e s i s h a s b e e n c l e a r l y d e m o n s t r a t e d b y i d e n t i f y i n g t h e l o c a l i z a t i o n o f ANP mRNA ( I n a g a m i e t a l . , 1 9 8 9 ) . 1 . 1 . 4 P E R I P H E R A L HOMEOSTATIC A C T I O N S OF ANP ANP e l i c i t s a v a r i e t y o f r e s p o n s e s w h i c h a r e d i r e c t e d t o w a r d t h e r e d u c t i o n o f b l o o d p r e s s u r e a n d b l o o d v o l u m e . I t i n h i b i t s r e n i n r e l e a s e , v a s o p r e s s i n r e l e a s e , a n d a l d o s t e r o n e 6 release. I t markedly stimulates 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 (increase of glomerular f i l t r a t i o n rate) and a d i r e c t tubular e f f e c t ( i n h i b i t i o n of sodium reuptake by inner medullary c o l l e c t i n g duct) (Mark, 1989). ANP administered into the cerebroventricles antagonizes central effects of angiotensin II i n water drinking, s a l t appetite, hypertension, and vasopressin release. I t also i n h i b i t s e l e c t r i c a l l y stimulated norepinephrine release from adrenergic nerve endings and neuronal a c t i v i t i e s of ganglia (David et a l . , 1989). ANP reduces vascular tone induced by vasoconstrictors such as norepinephrine, histamine, angiotensin I I , or vasopressin (Margaret et a l . , 1989). 1.1.5 REGULATION OF ANP SECRETION Plasma l e v e l s of ANP measured by radioimmunoassay vary widely from approximately 25 to 100 picograms/ml of plasma i n humans and 100 to 1000 picogram/ml in rats . Differences i n species, measuring techniques, and sampling protocols account for some of t h i s v a r i a b i l i t y (deBold, 1985). Studies indicate that the primary stimulus f o r a t r i a l stimulation of ANP secretion i s a t r i a l distension presumably due to elevation of venous blood pressure (Dietz, 1984; Ledsome et a l . , 1985). This can be caused by an elevation of 7 v e n o u s r e t u r n b l o o d d u e t o s a l t f e e d i n g ( S u g i y a m a e t a l . , 1 9 8 4 ) , h e a d down t i l t ( H o l l i s t e r e t a l . , 1986 ) , o r w a t e r i m m e r s i o n ( O g i h a r a e t a l . , 1 9 8 6 ) . P h a r m a c o l o g i c a l d o s e s o f v a s o c o n s t r i c t o r s s u c h a s e p i n e p h r i n e a n d v a s o p r e s s i n were shown t o s t i m u l a t e ANP r e l e a s e ( M a r g a r e t e t a l . , 1 9 8 9 ) . W h i l e t h i s may be due t o i n c r e a s e d b l o o d p r e s s u r e , t h e s t i m u l a t i o n c a n b e s e e n e v e n i n t i s s u e c u l t u r e c o n d i t i o n s ( S o n n e n b e r g a n d V e r e s s , 1984; M a n n i n g e t a l . , 1 9 8 5 ) . 1 . 1 . 6 MECHANISM OF ANP A C T I O N T h e e f f e c t s o f ANP m u s t b e m e d i a t e d b y r e c e p t o r - m e d i a t e d p r o c e s s e s . ANP i n f u s i o n i n r a t s was shown t o i n c r e a s e p l a s m a a n d u r i n a r y cGMP m a r k e d l y , r e f l e c t i n g a c t i v a t i o n o f g u a n y l a t e c y c l a s e (Hamet e t a l . , 1 9 8 4 ) . T h e g u a n y l a t e c y c l a s e a c t i v a t e d b y ANP was f o u n d t o b e t h e m e m b r a n e - b o u n d enzyme r a t h e r t h a n t h e c y t o s o l i c g u a n y l a t e c y c l a s e (Waldman e t a l . , 1984) . C y c l i c GMP p r o d u c t i o n i n r e s p o n s e t o ANP was f o u n d e x t e n s i v e l y i n n u m e r o u s t y p e s o f t i s s u e (Hamet e t a l . , 1 9 8 4 ) . R e c e n t l y , a s e c o n d t y p e o f ANP r e c e p t o r c h a r a c t e r i z e d b y i t s a b s e n c e o f g u a n y l a t e c y c l a s e a c t i v i t y h a s b e e n i d e n t i f i e d ( T a k a y a n a g i e t a l . , 1 9 8 7 ; L e i t m a n e t a l . , 1 9 8 8 ) . T h e e x a c t r o l e o f s u c h a r e c e p t o r i s n o t c l e a r . H o w e v e r , i t h a s b e e n s u g g e s t e d t h a t t h e c y c l a s e - f r e e r e c e p t o r i s a c l e a r a n c e r e c e p t o r whose f u n c t i o n 8 i s t o a b s o r b e x c e s s ANP a n d e i t h e r t o e l i m i n a t e i t f r o m c i r c u l a t i o n o r s t o r e a n d s l o w l y r e l e a s e i t ( M a a c k e t a l . , 1987) . 1 . 1 . 7 C E N T R A L ACTIONS OF ANP 1 . 1 . 7 . 1 D I S T R I B U T I O N AND BINDING S I T E S OF ANP I N THE CNS A t f i r s t i t was a r g u e d b y c a r d i o v a s c u l a r r e s e a r c h e r s t h a t a n y ANP d e t e c t e d w i t h i n t h e CNS m e r e l y r e f l e c t e d s e q u e s t r a t i o n f r o m p l a s m a . T h u s t h e i n i t i a l r e p o r t b y M o r i i e t a l . o f ANP p r e s e n c e i n l a r g e a r e a s o f t h e b r a i n , p a r t i c u l a r l y i n t h e h y p o t h a l a m u s ( M o r i i e t a l . , 1 9 8 5 ) , met w i t h much s k e p t i c i s m . H o w e v e r , l a t e r t h a t y e a r s e v e r a l g r o u p s r e p o r t e d t h e u n i q u e d i s t r i b u t i o n o f A N P - p o s i t i v e n e u r o n s w i t h i n t h e r a t b r a i n , s t r o n g l y s u g g e s t i n g l o c a l p r o d u c t i o n ( K a w a t a e t a l . , 1 9 8 5 ; S k o f i t s c h e t a l . , 1 9 8 5 ) . S t u d i e s showed t h a t t h e r e w e r e t h r e e m a j o r c o n c e n t r a t i o n s o f A N P - p o s i t i v e c e l l b o d i e s , t h e mos t a b u n d a n t b e i n g i n t h e w a l l s a d j a c e n t t o t h e v e n t r a l t h i r d v e n t r i c l e p a r t i c u l a r l y i n t h e r o s t r a l p a r t . T h e s e l a r g e , m o n o p o l a r A N P - p o s i t i v e n e u r o n s w e r e o b s e r v e d t o p r o j e c t t o t h e p a r v o c e l l u l a r p a r a v e n t r i c u l a r n u c l e u s , t h e p e r i v e n t r i c u l a r t h a l a m u s , t h e p r e o p t i c - s e p t a l r e g i o n s a n d t h e b e d n u c l e u s o f t h e s t r i a t e r m i n a l s ( S t a n d a e r t e t a l . , 1 9 8 5 ) . A s e c o n d c l u s t e r o f ANP c o n t a i n i n g n e u r o n s was d e t e c t e d i n t h e 9 l a t e r a l h y p o t h a l a m i c a r e a a n d a t h i r d i n b r a i n s t e m s t r u c t u r e s s u c h a s t h e p e r i p e d u n c u l o p o n t i n e , d o r s o l a t e r a l t e g m e n t a l , a n d p a r a b r a c h i a l n u c l e i a n d i n t h e n u c l e u s o f t h e t r a c t u s s o l i t a r i u s ( S t a n d a e r t e t a l . , 1 9 8 6 b ) . U n l i k e i n t h e h e a r t w h e r e t h e s t o r a g e f o r m o f ANP i s t h e 126 amino a c i d p r o h o r m o n e ( G l e m b o t s k i e t a l . , 1 9 8 5 ; N e e d l e m a n e t a l . , 1985) a n d f i n a l c l e a v a g e o c c u r s u p o n r e l e a s e ( M i c h e n e r e t a l . , 1 9 8 6 ) , i n t h e b r a i n t h e m a t u r e 28 a m i n o a c i d i s s t o r e d a f t e r c l e a v a g e f r o m t h e p r o h o r m o n e ( G l e m b o t s k i e t a l . , 1 9 8 5 ; S h i o n o e t a l . , 1 9 8 6 ) . S i n c e b r a i n ANP-mRNA t r a n s c r i p t s a r e s i m i l a r t o t h o s e i n t h e h e a r t , p o s t - t r a n s l a t i o n a l p r o c e s s i n g o f ANP i n t h e b r a i n a p p e a r s t o d i f f e r f r o m t h a t i n t h e h e a r t . T h e d i s t r i b u t i o n s o f s p e c i f i c ANP b i n d i n g s i t e s i n t h e r a t and g u i n e a p i g b r a i n s h a v e b e e n d e s c r i b e d . S i g n i f i c a n t b i n d i n g was d e t e c t e d i n t h e c e n t r a l s i t e s known t o be 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 a n d c a r d i o v a s c u l a r f u n c t i o n , s u c h a s c i r c u m v e n t r i c u l a r o r g a n s , i n p a r t i c u l a r t h e - s u b f o r n i c a l o r g a n ( Q u i r i o n e t a l . , 1984; S a a v e d r a e t a l . , 1986b; J o h n s o n , 1 9 8 5 ) . T h e i n t e n s i v e p r e s e n c e o f A N P - b i n d i n g s i t e s i n m e d i a n e m i n e n c e , s e p t u m , m e d i a l p r e o p t i c n u c l e u s , a n d a n t e r i o r p i t u i t a r y a l s o i n d i c a t e d a p o s s i b l e i n v o l v e m e n t o f ANP i n t h e h y p o t h a l a m i c c o n t r o l o f a n t e r i o r p i t u i t a r y h o r m o n e s e c r e t i o n ( Q u i r i o n e t a l . , 1 9 8 4 ) . 10 There are species differences i n the d i s t r i b u t i o n of ANP i n the CNS. Studies using RIA for ANP have shown that ANP-like immunoreactivity i s present i n the highest concentrations i n the hypothalamus and septum, followed by the midbrain and cerebral cortex i n rats (Morii et a l . , 1985). In the monkey brain, the highest concentration was found i n the midbrain followed by the pons and hippocampus. The ANP-like immunoreactivity concentration i n the dog brain was also the highest i n the midbrain (Fujino et a l . , 1987). As i s the case i n the peripheral, two classes of ANP receptors are present i n the brain, one thought to be involved i n clearance of the peptide, the other involved i n the expression of ANP's b i o l o g i c a l action (Leitman and Murad, 1987). 1.1.7.2 HYPOTHALAMIC ACTION OF THE ANP IN INHIBITING POSTERIOR PITUITARY HORMONE SECRETION The opposing actions of vasopressin and ANP i n the kidney and a b i l i t y of ANP to i n h i b i t vasopressin's action there suggested that ANP might a f f e c t vasopressin release i n CNS (Dillingham et a l . , 1986). Under basal conditions, ANP injected into the t h i r d cerebroventricle of conscious, unrestrained rats s i g n i f i c a n t l y i n h i b i t e d vasopressin 11 secretion. This e f f e c t i s s e l e c t i v e f o r vasopressin (oxytocin release was unrelated) and i s not mediated v i a an action of ANP on the vasopressin-containing nerve terminals i n the median eminence or neural lobe, but instead at the hypothalamic l e v e l , either d i r e c t l y on the dendrites or c e l l body of the vasopressin-producing neuron or on interneurons i n the v i c i n i t y (Samson et a l . , 1987). Crandall and Gregg also demonstrated t h i s vasopressin-inhibiting e f f e c t of ANP i n the hypothalamic neurohypophyseal explant, both under basal or angiotensin II-stimulated conditions, further supporting the hypothesis of a hypothalamic s i t e of action (Crandall and Gregg, 1986). These i n v i t r o r e s u l t s agree with the demonstration of the a b i l i t y of ANP pretreatment i n vivo to block angiotensin II-stimulated vasopressin secretion i n conscious rats (Yamada et a l . , 1986). The a b i l i t y of ANP to i n h i b i t vasopressin secretion and experimentally-induced water intake (Antunes et a l . , 1985) and s a l t preference (Antunes et a l . , 1986) might a l l be related 1 to the a b i l i t y of the peptides to antagonize the central action of angiotensin I I . In any event, these central actions seem well matched to many of the peripheral actions of ANP and suggest coordinated e f f e c t s that function p h y s i o l o g i c a l l y to 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 central actions of ANP unrelated to hydromineral balance (Standaert et a l . , 1986a; Samson et a l . , 1987). The presence of ANP binding s i t e s and ANP positive axon terminals i n regions seemingly unrelated to those functions and the presence of ANP responsive single neurons i n the medial preoptic and l a t e r a l septal areas( Wong et a l . , 1986.) suggest other possible neuromodulatory actions of the peptide. The presence of ANP immunoreactivity i n the external layer of the median eminence (Kawata et a l . , 1985b; Skofitsch et a l . , 1985) and ANP binding s i t e s in the p i t u i t a r y gland suggest delivery of centrally-derived ANP v i a the hypophyseal portal vessels to the anterior p i t u i t a r y where i t might exert some tropic action. However, studies f a i l e d to demonstrate a gradient of p o r t a l versus peripheral plasma ANP concentration i n the rat, a c r i t e r i o n s a t i s f i e d by a l l recognized hypophysiotropic agents (Samson and Bianchi, 1987). Although ANP w i l l stimulate guanylate cyclase a c t i v i t y i n cultured anterior p i t u i t a r y c e l l s , t h i s a c t i v i t y does not seem to be linked to any s i g n i f i c a n t e f f e c t on hormone in v i t r o (Simard et a l . , 1986). Several l i n e s of evidence suggested that 13 neither basal nor stimulated hormone secretion from cultured p i t u i t a r y c e l l s i s altered by ANP (Simard et al.,1986; Heisler et a l . , 1986) . Log doses of ANP ranging from one pico- to one micromolar f a i l e d to a l t e r the release of LH, PRL, thyroid stimulating hormone, f o l l i c l e stimulating hormone or growth hormone by c e l l s harvested from i n t a c t or gonadectomized rats (Samson and Bianchi, 1987). A d d i t i o n a l l y , the presence of ANP in the incubation medium f a i l e d to a l t e r the LH response of these c e l l s to LHRH, the growth hormone response to somatostatin or growth hormone-releasing hormone, the thyroid-stimulating hormone response to thyrotropin releasing hormone, or the a b i l i t y of dopamine to i n h i b i t PRL and a variety of PRL releasing factors to stimulate PRL release (Samson and Bianchi, 1987). These studies would suggest then that any observed e f f e c t of the peptide on p i t u i t a r y hormone secretion observed i n vivo must be exerted at the hypothalamic side of the hypothalamo-pituitary axis. 1.1.7.3.1 INHIBITION OF PROLACTIN SECRETION IN VIVO The a b i l i t i e s of ANP to i n t e r a c t with brain dopaminergic systems i n vivo (Nakao et a l . , 1986) and to 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 et a l . , 1986) suggested that ANP might play a role i n 14 the hypothalamic control of PRL secretion, since dopamine, produced i n tuberoinfundibular neurons, i s the -major hypothalamic PRL-release i n h i b i t i n g factor. No s i g n i f i c a n t e f f e c t s of bolus injected or peripherally infused ANP on PRL secretion were observed i n conscious rats (Samson and Bianchi, 1987). However, when the peptide was infused into the t h i r d v e n t r i c l e of the brain, a s i g n i f i c a n t i n h i b i t i o n of PRL secretion was observed. The e f f e c t was dose-related and r e l a t i v e l y long l a s t i n g (Samson and Bianchi, 1987). This action of ANP i s prevented by dopamine receptor blocker treatment and i s absent following i n h i b i t i o n of tyrosine hydroxylase a c t i v i t y , suggesting d i r e c t e f f e c t s of ANP on tuberoinfundibular dopamine neurons (Samson et a l . , 1988a). 1.1.7.3.2 INHIBITION OF LH SECRETION IN VIVO Brief elevation of c i r c u l a t i n g ANP l e v e l s e i t h e r by bolus i n j e c t i o n of the peptide or by acute volume expansion f a i l e d to s i g n i f i c a n t l y a l t e r LH release i n vivo (Samson et a l . , 1988). However, when plasma l e v e l s of ANP were elevated for 30 minutes or longer by either gradual and continued volume expansion or by intravenous infusion of the peptide at a dose which resulted i n a two to three f o l d elevation of c i r c u l a t i n g concentrations, plasma l e v e l s of LH f e l l 15 s i g n i f i c a n t l y (Samson et a l . , 1988; Standaert et a l . , 1986a). This e f f e c t was mediated c e n t r a l l y and not at the p i t u i t a r y l e v e l , because the LH secretory response to a subsequent bolus i n j e c t i o n of LHRH was not changed by the presence of elevated ANP (Samson et a l . , 1988). These r e s u l t s suggest that ANP acts e i t h e r as a r e s u l t of a central nervous system e f f e c t or some reflex-mediated i n h i b i t i o n of LH release. It i s unlikely that a peripheral reflex plays an important r o l e because other hypotensive agents f a i l e d to i n h i b i t LH release when given intravenously and because ANP s i g n i f i c a n t l y i n h i b i t e d LH secretion when injected into the t h i r d cerebroventricle (Samson et a l . , 1988). Furthermore, i n v i t r o studies have i d e n t i f i e d the median eminence as at least one possible s i t e of action of ANP, because the release of LHRH from median eminence explants i n v i t r o by catecholamines can be i n h i b i t e d i n a dose-related fashion by the presence of ANP i n the medium (Samson et a l . , 1988). In addition to a possible central action of ANP to antagonize catecholamine stimulation of LHRH release, ANP i n h i b i t i o n of LH secretion appears to be mediated i n part by an interaction with endogenous opiate systems, which are recognized to be physiologic i n h i b i t o r s of the hypothalamic 16 component of LH release (Bruni et a l . , 1977; Pang et a l . , 1977) . Pretreatment of rats with naloxone completely abolishes the LH i n h i b i t o r y action (Samson et a l . , 1988). The action of the endogenous opiate to i n h i b i t LH secretion i s thought to be expressed i n the preoptic and anterior hypothalamic regions (Fink, 1988). It should be noted that ANP-containing neuronal elements are present i n these regions and indeed terminal f i e l d s p o s i t i v e for ANP immunoactivity are present i n regions known to be important i n the central control of gonadotropin secretion (Quirion et a l . , 1984; Standaert et a l . , 1986b). Furthermore, i t has been demonstrated that the iontophoresed and micropressure-injected ANP can i n h i b i t the f i r i n g rate of single neurons in t h i s region (Wong et a l . , 1986). Thus, i n addition to possible median eminence actions of the peptides, r o s t r a l hypothalamic s i t e s might also be targets for the neuromodulatory actions of ANP. 1.2 BRAIN NATRIURETIC PEPTIDE -Brain n a t r i u r e t i c peptide (BNP) i s a novel bioactive peptide of 26 amino ac i d residues recently i d e n t i f i e d i n porcine brain (Sudoh et a l . , 1988a) and therefore there are only a few publications describing i t . It i s known that the peptide exerts 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 well 17 as a vasorelaxant e f f e c t , i n a manner s i m i l a r to that of ANP (Sudoh et a l . , 1988a). BNP has a remarkable high sequence homology to ANP, e s p e c i a l l y i n the 17 amino acid r i n g formed by an intramolecular d i s u l f i d e linkage which i s the central unit required f o r b i o l o g i c a l a c t i v i t y (Sudoh et a l . , 1988a; Sudoh et a l . , 1988b). The presence of BNP with ANP in mammalian brains and remarkable resemblance i n t h e i r molecular structures imply that BNP may share some physiological functions with ANP. 1.2.1 THE STRUCTURE OF BNP BNP has a remarkable high sequence homology to alpha-ANP, es p e c i a l l y i n the 17 amino acid r i n g formed by an intramolecular d i s u l f i d e linkage which i s the central unit required for b i o l o g i c a l a c t i v i t y . The highest homology i s observed when BNP i s compared to alpha-ANP(4-28), one of the brain forms of ANP. BNP-32, a N-terminal s i x amino acid extended form of "BNP, has been i d e n t i f i e d as the second form i n the BNP family i n porcine brain (Sudoh et a l . , 1988b). The comparison of the amino acid sequence of BNP and BNP-32 with alpha-ANP(4-28) and alpha-ANP(5-28), which are two major forms of ANP i n porcine brain (Ueda et a l . , 1987), are l i s t e d below. BNP-32: S-P-K-T-M-R-18 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 R-Y 1.2.2 BIOSYNTHESIS OF BNP As the sequence of pBNP i s not found i n the known sequence of the ANP, the peptide i s probably processed from i t s own precursor, g e n e t i c a l l y d i s t i n c t from the ANP precursor (Sudoh et a l . , 1989). Furthermore, the seven amino-acid changes observed between ANP and BNP are not convertible by a single nucleotide su b s t i t u t i o n , indicating that the genes encoding the two molecules diverged at an early stage (Sudoh et a l . , 1989). 1.2.3 THE DISTRIBUTION OF BNP The determination and comparison of regional d i s t r i b u t i o n of BNP and ANP have been made i n porcine brain. Concentrations of BNP are highest i n medulla-pons and the striatum among a l l the brain regions examined. BNP 19 concentrations of the respective regions can be summarized as follows: medulla-pons, striatum > hypothalamus, septum > midbrain-thalamus, cortex, olfactory bulbs > hippocampus > cerebellum (Ueda et a l . , 1988). Meanwhile, tissu e concentrations of ANP i n each region of porcine brain are simultaneously determined with the same t i s s u e extracts used for measurement of BNP concentrations. The highest concentration of ANP i s observed i n the o l f a c t o r y bulbs, and the second highest i n the hypothalamus. The medulla-pons and striatum do not contain as much ANP as BNP. Only the olfactory bulbs has comparable concentrations of BNP and ANP. Concentrations of ANP i n respective regions are i n the following order: Olfactory bulbs > hypothalamus > midbrain-thalamus > medulla-pons > septum > hippocampus, striatum. In terms of concentration of whole brain, BNP i s found to be about ten times higher than that of ANP (Ueda et a l . , 1988). This r e s u l t c l e a r l y shows that d i s t r i b u t i o n of BNP i s not p a r a l l e l to that of ANP, suggesting the p o s s i b i l i t y that the differences i n tissue concentration and d i s t r i b u t i o n of BNP and ANP r e f l e c t d i f f e r e n t physiological functions for these two n a t r i u r e t i c peptides. 20 1.2.4 THE PHYSIOLOGY OF BNP Intravenous injections of synthetic porcine BNP (pBNP) into anaesthetized rats resulted i n a remarkable increase i n excretion of urine and e l e c t r o l y t e s into urine, i n a manner very s i m i l a r to that e l i c i t e d by alpha-ANP (Sudoh et a l . , 1988). Furthermore, i n j e c t i o n of pBNP into anaesthetized rats causes a s i g n i f i c a n t decrease i n mean blood pressure, comparable to that induced by alpha-ANP at the same dose. Thus, pBNP i s as potent as alpha-ANP i n hypotensive and 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 . However, the rectum-relaxant a c t i v i t y of pBNP i s 3-4 times more potent than that of alpha-ANP, suggesting a unique feature i n the physiological s i g n i f i c a n c e of pBNP (Sudoh et a l . , 1988). In accordance with i t s peripheral function i n f l u i d and e l e c t r o l y t e homeostasis, BNP has been demonstrated to suppress both basal vasopressin secretion and All-induced vasopressin secretion i n euhydrated f r e e l y moving rats. The i n h i b i t o r y action of BNP on basal vasopressin i s comparable to that of alpha-ANP (Yamada et a l . , 1986). In addition, the time course and potency of the i n h i b i t o r y e f f e c t of BNP on All-induced vasopressin secretion are also comparable to t h e i r counterparts of ANP i n conscious rats (Yamada et a l . , 1986). Thus, c e n t r a l l y administered BNP and ANP appear to have 21 s i m i l a r e f f e c t s on vasopressin secretion i n conscious ra t s . BNP also exhibited equipotent i n h i b i t o r y actions on central All-induced water intake (Itoh et a l . , 1988), pressor response (Shirakami et a l . , 1988), when compared to ANP. These findings raise the p o s s i b i l i t y that some central actions of ANP are shared by BNP. It has been demonstrated that pBNP increases the i n t r a c e l l u l a r c y c l i c GMP contents i n the kidney e p i t h e l i a l c e l l l i n e . The stimulation by pBNP of c y c l i c GMP accumulation was comparable to that by alpha-ANP at the same concentrations (Iwata et a l . , 1989). Since these experiments were performed in the presence of the phosphodiesterase i n h i b i t o r , the increase i n c y c l i c GMP contents was probably due to ac t i v a t i o n of guanylate cyclase. The observation that the simultaneous addition of pBNP and alpha-ANP at the maximal e f f e c t i v e concentration had no additive e f f e c t on c y c l i c GMP contents suggests that both peptides interact with the same receptors (Iwata et a l . , 1989). 22 1.3 INTRODUCTION TO THE ROLE OF NEUROMEDIATORS IN LH REGULATION. 1.3.1 DOPAMINE There are two dopamine (DA)-producing neuronal groups that innervate regions of the preoptic-tuberal pathway (Lindvall and Bjorklund, 1978). A small group of 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 innervates the medial preoptic area (MPOA), anterior hypothalamic area (AHA), suprachiasmatic nucleus (SCN), and the p e r i v e n t r i c u l a r nucleus of the anterior hypothalamus (Lindvall and Bjorklund, 1978). In male rats, testosterone treatment i n h i b i t e d DA turnover i n these projections in the MPOA and AHA (Simpkins et a l . , 1980). A more caudally located c e l l group, the tuberoinfundibular DA system with terminations i n the v i c i n i t y of LHRH axons and terminals i n the median eminence (Fuxe et a l . , 1976), may play a role i n control of LHRH secretion. Although DA was found to excite LHRH release from the median eminence of male rats i n v i t r o (Negro-Vilar, et al.,1979), 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 produced l i t t l e stimulation of LH release i n vivo (Kalra et a l . , 1983) . A good deal of evidence agrees with the view that DA neurons may exert an i n h i b i t o r y influence on LH secretion (Gallo, 1980) and thereby mediate the negative feedback e f f e c t s of testosterone on LH release (Simpkins et a l . , 1980). Similar uncertainty e x i s t s as to the e f f e c t s of DA on LH release i n female r a t s . Activation of DA receptors i n OVX rats i n h i b i t e d LH release (Gallo, 1980), and DA turnover decreased preceding the preovulatory LH surge (Fuxe et a l . , 1976). Whereas these conform to an i n h i b i t o r y r o l e of DA, others have demonstrated the excitatory nature of DA on LH release. DA has been consistently shown to stimulate LHRH release i n v i t r o from the median eminence tissue of steroid-primed OVX rats (Negro-Vilar and Ojeda 1978). Also, i t has been reported a f t e r 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 of DA, LH release was stimulated in steroid-primed OVX rats (Vijayan and McCann, 1978) , whereas other studies have f a i l e d to substantiate these findings (Kalra and Gallo, 1983). Thus, these r e s u l t s can not reach a firm conclusion on the nature of DA p a r t i c i p a t i o n in the physiologic events that control LH release. 1.3.2 NOREPINEPHRINE Noradrenergic innervation of the hypothalamus i s e x t r i n s i c . Noradrenergic-containing terminals 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 the hypothalamus are projections from c e l l bodies i n the brainstem v i a the ventral and dorsal bundles (Dahlstrom et a l . , 1964; Palkovits, 1981). 24 Since the early observation that norepinephrine (NE) may stimulate ovulation i n the rabbit (Sawyer, 1975), attention has been directed toward de f i n i n g the role of NE i n LH release in the rat. Considerable evidence i s i n l i n e with the view that hypothalamic projections of NE-producing neurons i n the brainstem (Palkovits, 1981) may p a r t i c i p a t e i n the excitatory component (mediated p r i n c i p a l l y by alpha-adrenergic receptors (Kalra et a l . , 1983), and the i n h i b i t o r y component (mediated by alpha- and beta-adrenergic receptors (Caceres and Taleisnik, 1981; Caceres and Taleisnik, 1982), of the neural c i r c u i t r y c o n t r o l l i n g basal p u l s a t i l e and c y c l i c patterns of LH release. I n h i b i t i o n of noradrenergic a c t i v i t y by i n h i b i t i o n of NE synthesis (Negro-Vilar et a l . , 1982), blockade of alpha-adrenergic receptors (Weick, 1978), or destruction of the ventral noradrenergic bundle (Hancke et a l . , 1977) a l l r e s u l t i n suppression of p u l s a t i l e LH release i n the OVX r a t s . The stimulatory action of NE i s mediated v i a alpha-adrenergic receptors i n the hypothalamus and MPOA (Kalra and Gallo, 1983) . Studies have convincingly demonstrated that stimulation of LH release by NE may be the r e s u l t of hypersecretion of LHRH into the hypophyseal p o r t a l system (Krieg and Ching, 1982). That NE input i n the diencephalon may be important i n the preovulatory and steroid-induced discharge of 25 gonadotropins i s shown by the findings that pharmacological suppression of adrenergic neurotransmission blocked gonadotropin release, and restoration of adrenergic stimuli i n these rats reinstated the gonadotropin response (Kalra and McCann, 1974). Strong evidence implicates the en t i r e preoptic-tuberal pathway as the s i t e of inte r a c t i o n between NE and LHRH neurons (Kalra and McCann, 197 3). NE nerves terminate i n close proximity of dendrites, axons, and LHRH-containing perikarya in these areas (Jennes et a l . , 1982), and furthermore, LHRH and LH release induced by administration of NE res u l t s from act i v a t i o n of alpha-adrenergic receptors i n regions surrounding the t h i r d v e n t r i c l e (Kalra and Gallo, 1983; Krieg and Ching, 1982). Although observations i n which endogenous noradrenergic a c t i v i t y i s blocked are consistent with an ongoing stimulatory action of noradrenergic neurons on p u l s a t i l e LH release, the administration of NE into the t h i r d v e n t r i c l e causes i n h i b i t i o n (Dluzen et a l . , 1983). Intraventricular administration of NE or of adrenergic agonists suppressed the amplitude and frequency of p u l s a t i l e LH release i n OVX rats (Gallo and Drouva, 1979). Furthermore, e l e c t r i c a l stimulation of the major ascending noradrenergic pathway also i n h i b i t s p u l s a t i l e LH release i n OVX rats (Leung et a l . , 1981). Ta l e i s n i k and co-workers (Caceres and T a l e i s n i k , 1981; Caceres and Taleisnik, 1982) also have implicated i n h i b i t o r y NE inputs i n the preovulatory and ovarian steroid-induced LH release. The i n h i b i t i o n of LH release by NE may be mediated by beta-adrenergic receptors (Caceres and Taleisnik, 1981; Caceres and Taleisnik, 1982) , although these findings add another dimension to our understanding of noradrenergic control, the precise physiological s i g n i f i c a n c e of each of the two components i n governing LH release i s yet to be determined. 1.3.3 ENDOGENOUS OPIOID PEPTIDES Three major types of endogenous opioid peptides (EOP), each with a d i s t i n c t d i s t r i b u t i o n pattern i n the diencephalon, have been described i n the regulation of LH secretion (Richard et a l . , 1988). 1. Beta-endorphin i s the major product of proopiomelanocortin processing (Mains et a l . , 1977). I t i s mainly synthesized i n proopiomelanocortin c e l l s of the anterior and intermediate lobes of the p i t u i t a r y (Mains et a l . , 1977). I t i s also produced i n large amounts by neurons of the arcuate nucleus and p e r i v e n t r i c u l a r nucleus, which project to the median eminence, septal-MPOA and to a wide 27 spectrum of other brain structures (Finlay et a l . , 1981). The peptide i s highly active on delta and mu opiate receptors (Udenfriend and Meienhofer, 1984). Beta-endorphin i n h i b i t s p u l s a t i l e (Bruni et a l . , 1977) and preovulatory gonadotropin release (Ching, 1983). Presumably, these suppressive e f f e c t s are exerted by projections into the median eminence and septal-MPOA of beta-endorphin-producing c e l l s located i n the arcuate and p e r i v e n t r i c u l a r nucleus (Kalra, 1981). There has been considerable i n t e r e s t i n the suggestion that EOP may mediate the feedback e f f e c t s of gonadal stero i d s . Many l i n e s of evidence are i n accord with t h i s suggestion. Levels of beta-endorphin i n the hypophyseal portal blood fluctuate markedly during the menstrual cycle i n the monkey (Wardlaw et a l . , 1980; Weherenberg et a l . , 1982); ovariectomy reduced, whereas combined e s t r a d i o l and progesterone treatment restored, the beta-endorphin secretion rate to that observed during the l u t e a l phase of the monkey menstrual cycle (Wardlaw et a l . , 1982)-. Ovarian steroids have been shown to a l t e r beta-endorphin l e v e l s i n various regions of the diencephalon (Barden and Dupont, 1982; Barden et a l . , 1981). Beta-endorphin levels r i s e i n the median eminence and SCN and decrease i n the arcuate nucleus during the early phase of preovulatory LH release (Barden and Dupont, 1982; Barden et a l . , 1981). I t has 28 been showed that progesterone's a b i l i t y to c u r t a i l t r a n s i e n t l y or to suppress EOP release i n the preoptic-tuberal pathway may be the underlying mechanism f a c i l i t a t i n g LH release (Gabriel et a l . , 1983). 2. The two pentapeptides, methionine-enkephalin and leucine-enkephalin, show p r e f e r e n t i a l a f f i n i t y for delta-type opioid receptors and moderate a f f i n i t y for mu-receptors i n regions including the MPOA, AHA, SCN, p e r i v e n t r i c u l a r nucleus, and the median eminence (Watson et a l . , 1982). The r o l e of enkephalins i n regulation of gonadotropin secretion i s unclear. Whereas there was decreased LH release a f t e r systemic i n j e c t i o n of methionine-enkephalin (Bruni et a l . , 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 rats produced l i t t l e change i n p u l s a t i l e LH release (Leadman and Kalra, 1983). 3. Dynorphin i s mainly produced i n the paraventricular nucleus (Code and Fallon, 1986), and has a p r e f e r e n t i a l a f f i n i t y for kappa-type opioid receptors (Wood, 1982). - Intraventricular i n j e c t i o n s of dynorphin suppressed LH release, although the response was comparatively much smaller than that seen a f t e r beta-endorphin i n j e c t i o n (Leadman and Kalra, 1983). To understand the mode of EOP involvement i n regulation of LH secretion, opiate receptor agonists such as morphine 29 s u l f a t e and analogs of methione-enkephalin (Kalra, 1983) and opiate receptor antagonists such as naloxone have been extensively used (Van Vugt et a l . , 1982). Morphine suppressed LH release i n gonadectomized rats and the preovulatory LH surge and ovulation i n cy c l i n g rats (Johnson et a l . , 1982; Kalra, 1983). A major impact of opiates on gonadotropic secretion concerns the regulation of amplitude and the frequency of the p u l s a t i l e pattern of LH secretion. Administration of beta-endorphin reduces the LH p u l s a t i l i t y i n conscious, castrated rats (Van Vugt et a l . , 1982). I t i s noteworthy that continuous stimulation of opiate receptors with morphine i n h i b i t e d the LHRH accumulation induced by progesterone i n OVX estrogen-primed rats (Kalra and Simpkins, 1981), and that e l i c i t e d by testosterone i n orchidectomized rats (Gabriel et a l . , 1983). These findings suggest the EOP neurons may not only modulate LHRH release but they also may influence neurosecretory events modulated by steroids to promote LHRH accumulation i n the median eminence nerve terminals (Kalra et a l . , 1983). The opiate receptor antagonists naloxone stimulate LH release under a vari e t y of experimental conditions i n intact and gonadectomized steroid-pretreated rats (Kalra and Simpkins, 1981; L e i r i et a l . , 1979). The a b i l i t y of these 30 antagonists to stimulate LH release, perhaps by t r a n s i e n t l y displacing the EOP from t h e i r s i t e s i n the MPOA, median eminence and arcuate nucleus (Kalra, 1981), has led to the general b e l i e f that EOP may normally exert a tonic i n h i b i t o r y influence on LH release i n int a c t r a t s . Three subtypes of opiate receptors have been characterized: mu, delta and kappa. Opiate e f f e c t s on LH secretion are generally believed to involve the mu receptor subtype (Cicero et a l . , 1983; P f e i f f e r et a l . , 1983; Panerai et a l . , 1985). I t has been shown that 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 of the mu-agonist i n OVX rats produced a s i g n i f i c a n t suppression of LH secretion, while the d e l t a - and kappa-agonists had l i t t l e e f f e c t , leading to the conclusion that the mu-receptor i s the primary opiate receptor involved i n the regulation of LH secretion ( P f e i f f e r et a l . , 1983). The importance of endogenous opiates i n hypothalamic mechanisms c o n t r o l l i n g LH secretion seems highly dependent upon the endocrine condition of experimental animals (Bhanot et a l . , 1983; Piva et a l . , 1986). In gonadectomized r a t s , opiate receptor antagonists f a i l e d to e l i c i t LH release, implying decreased EOP tone i n these rats (Bhanot et a l . , 1983) . 31 2. EXPERIMENTAL 2.1 EXPERIMENTAL RATIONALE AND DESIGN The d i s t r i b u t i o n of brain neurons containing ANP-like immunoreactivity within the central nervous system (Kawata et a l . , 1985a), i n p a r t i c u l a r the intense innervation of the external layer of the median eminence and the presence of ANP-p o s i t i v e elements i n the preoptic-septal regions even i n the p i t u i t a r y gland (Jacobowitz et a l . , 1985), and the presence of ANP-binding s i t e s i n these tissues (Quirion et a l . , 1984.) indicate a possible involvement of ANP i n the hypothalamic control of anterior p i t u i t a r y hormone secretion. It has been reported that a six microgram dose of centrally-administered ANP can exert i n h i b i t o r y e f f e c t on mean plasma LH release 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 secretion (Samson et a l . , 1988b). However, i n t h i s report, the blood sampling i n t e r v a l s were too long to reveal the p u l s a t i l e pattern of LH release (at 0, 15, 30, 60. 90, 120 minutes) . As a r e s u l t , these investigators were unable to determine whether the i n h i b i t o r y e f f e c t of ANP on LH secretion was due to the decrease i n LH pulse amplitude or pulse frequency and the influence of naloxone on LH pulses. The f i r s t objective of t h i s study, therefore, i s to confirm and extend t h e i r r e s u l t s using shorter sampling i n t e r v a l (5 minutes) to ascertain the influence of ANP on the LH pulses. ANP has been reported to cause s i g n i f i c a n t decreases i n the l e v e l of dopamine and i t s metabolites i n the rat hypothalamus and septum (Nakao et a l . , 1986). Dopamine, i n turn, has been shown by some researchers to stimulate p u l s a t i l e LH secretion (Negro-Vilar et al.,1982) while others have reported an i n h i b i t o r y e f f e c t of dopamine on p u l s a t i l e LH release (Drouva and Gallo, 1976). Thus, i t i s of interest to examine the possible role of central dopaminergic system i n the ANP modulation of LH pulses i n t h i s study. Brain n a t r i u r e t i c peptide (BNP) i s a novel d i u r e t i c -n a t r i u r e t i c and vasorelaxant peptide o r i g i n a l l y isolated from porcine brain. BNP shares s t r u c t u r a l s i m i l a r i t y with that of ANP (Sudoh et a l . , 1988a). I t i s not known i f BNP a f f e c t s anterior p i t u i t a r y hormone secretion. Thus, the second major objective of t h i s study i s to determine i f BNP also exerts an i n h i b i t o r y e f f e c t on LH release. Female, Sprague-Dawley rats weighing 185-200 grams purchased from Charles River Canada, Inc. (Montreal, Canada) were i n d i v i d u a l l y caged i n a light-and temperature -controlled room (14-h l i g h t , 10-h dark; 22 ± 1 C e l s i u s ) . B i l a t e r a l ovariectomy was performed under sodium methohexital anaesthesia (50 mg/kg body weight), Two weeks l a t e r , each rat was implanted s t e r e o t a x i c a l l y with a 22-gauge guide cannula into the t h i r d brain v e n t r i c l e under sodium pentobarbital anaesthesia (Somnotol, 45 mg/kg body weight). Following a minimum 1-week recovery period, each rat was f i t t e d with an i n t r a - a t r i a l catheter. Two days l a t e r , a s a l i n e - f i l l e d polyethylene tubing was attached to the indwelling a t r i a l catheter and 50-microlitre blood samples were withdrawn at 5-minute i n t e r v a l s for three hours. Each animal remained i n i t s own cage during the bleeding procedure and each sample was replaced by an equal volume of 0.9% sal i n e . Blood samples were centrifuged and the plasma was stored at -2 0 Celsius u n t i l LH concentrations were measured by RIA. Experiments were designed into three s e r i e s : The f i r s t s e r i e s : Examining the e f f e c t of intracerebroventricular (i.c.v) administration of ANP or BNP on LH secretion i n ovariectomized (OVX) rats . At the midpoint of the blood-sampling period, each rat received an i.c . v infusion of saline containing 0.2 nmol, 2 nmol ANP or 0.2 nmol,2 nmol BNP to examine the modulatory e f f e c t of ANP or BNP on LH secretion at 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 the e f f e c t of blockade of the endogenous opiate system with naloxone on the action of ANP and BNP. 45 minutes a f t e r the beginning of the experiment, 2nmol ANP or 2nmol BNP was infused into the t h i r d v e n t r i c l e . Three boluses of 0.5 mg naloxone were then injected intravenously at 45, 75 and 105 minutes a f t e r i.c.v. infusion of 2nmol ANP or 2nmol BNP to examine whether naloxone could reverse the i n h i b i t o r y e f f e c t of ANP or 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 receptor blocker pimozide on the action of ANP or BNP. At the onset of the experiment, rats were injected with pimozide and at the midpoint of the experiment 2 nmol ANP or 2 nmol BNP was i.c.v. infused to examine whether pretreatment of pimozide could prevent the inh i b i t o r y e f f e c t of ANP or BNP. 35 2.2 MATERIALS AND METHODS Adult female Sprague-Dawley rats were i n d i v i d u a l l y caged in 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 ; 10-h dark, 22 ± 1 Celsius) and provided water and rat chow ad  libitum. One or two days a f t e r the rat arrived i n the animal holding f a c i l i t i e s , they were anaesthetized with sodium methohexital (B r i e t a l , E l i L i l l y ; 50 mg/kg of body weight) and ovariectomized (OVX). Two weeks following ovariectomy, s u r g i c a l cannulation of the t h i r d v e n t r i c l e was performed. The rat was anaesthetized with sodium pentobarbital (Somnotol, 45 mg/kg of body weight) and then placed i n a stereotaxic instrument with i t s head i n the f l a t - s k u l l p o sition as described by Paxinos and Watson (1982). The s k u l l was exposed and four holes were d r i l l e d , into which anchoring screws were secured. A small hole, the centre of which was 6.7 millimetre anterior to the interaural l i n e , was then d r i l l e d midsagltally i n the s k u l l . The dura mater was then exposed and a 22 gauge sta i n l e s s s t e e l cannula was lowered 9.0 millimetre into the brain. Placement of the cannula at t h i s p o s i t i o n resulted i n the t i p of the cannula i n the t h i r d v e n t r i c l e (Anterior: 6.7 millimetre anterior to the i n t e r a u r a l l i n e ; L ateral: midline, and Deep: 9.0 millimetre below the s k u l l surface). Dental cement (Hygenic Cold Cure) was placed around the cannula and the s k u l l screws, to secure the cannula to the s k u l l . To prevent leakage of cerebrospinal f l u i d and i n f e c t i o n of the t h i r d v e n t r i c l e , a 28 gauge stainless s t e e l s t y l e t t e exactly the same length as the cannula was inserted i n the cannula and i t s handle was cemented i n place u n t i l the day of the experiment. A f t e r v e n t r i c l e cannulation the rats were placed in individual cages and were given at l e a s t one week to recover from the surgery. On the day before the c o l l e c t i o n of blood samples the rats were anaesthetized b r i e f l y with B r i e t a l and a catheter made of s i l a s t i c tubing was inserted into the r i g h t jugular vein using the method described by Harms and Ojeda (1974). B r i e f l y , the r i g h t jugular vein was exposed and a curved 22-gauge needle was used to place the s i l a s t i c catheter into the vein. The catheter was s l i d into the vein and made to enter or approach the r i g h t atrium. The- catheter was then anchored onto the muscle under which the right jugular vein passes. The exposed end of the catheter was then passed underneath the skin and e x t e r i o r i z e d at the back of the neck and t i e d shut. The rats were then returned to t h e i r cages u n t i l the next day. In some experiments, the rats were reused a f t e r at lea s t one week recovery period. In these cases, the catheter was implanted i n the l e f t jugular vein (the surgical procedure was si m i l a r to that of implantation of catheter to the r i g h t jugular vein). On the day of the experiment the jugular catheter was connected to polyethylene tubing (PE-50) which had been f i l l e d with heparinized s a l i n e . Using t h i s catheter, blood samples were withdrawn with minimal disturbance to the rat . Before blood sampling was started, 200 m i c r o l i t r e heparin (1000 U/ml) was injected into the rats v i a the i n t r a - a t r i a l catheter to prevent 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 of whole blood was withdrawn from the rats v i a the i n t r a - a t r i a l catheter. The whole blood samples were placed into tubes on i c e . In a l l cases, 5 minutes before the s t a r t of infusion of ANP, BNP or saline into the t h i r d v e n t r i c l e , the 28 gauge s t y l e t t e was taken out of implanted 22 gauge cannula and an infusion s t y l e t t e was inserted inside the 22 gauge cannula such that the t i p of the infusion s t y l e t t e was i n the t h i r d v e n t r i c l e . In many cases cerebrospinal f l u i d was seen flowing out of the cannula. This was taken as evidence that the cannula was i n the t h i r d v e n t r i c l e . The infusion tubing had been previously connected to a Hamilton m i c r o l i t r e syringe i n a syringe pump. 38 The 2 m i c r o l i t r e volume was infused over a period of 2 minutes. Each animal remained i n i t s own cage during the bleeding procedure and each sample was replaced by an equal volume of 0.9% saline. A l l experiments were conducted i n conscious unrestrained animals. Blood samples were centrifuged immediately a f t e r experiments and the plasma was stored at -20 Celsius u n t i l LH concentrations were measured by radioimmunoassay (RIA). In s t u d i e s e x a m i n i n g t h e e f f e c t of intracerebroventricular (i.c.v.) administration of ANP or BNP on LH secretion i n ovariectomized rats, at the midpoint of the blood-sampling period (90 min), each rat received over two minutes an i.c.v infusion of 2 m i c r o l i t r e isotonic saline (control) alone or s a l i n e containing 0.2 nmol, 2 nmol ANP (rat ANP-28; Peninsula laboratories, Inc.,) or 0.2 nmol,2 nmol BNP (porcine BNP-26; Peninsula laboratories, Inc.,). -In another experiment the e f f e c t of blockade of the endogenous opiate system with naloxone on the action of i.c.v. administered ANP and BNP was tested i n OVX r a t . f o r t y - f i v e minutes a f t e r the beginning of the experiment, 2nmol ANP or 2nmol BNP was infused into the t h i r d v e n t r i c l e . Three boluses of 0.5 mg naloxone (Sigma, ST Louis) were then injected intravenously at 45, 75 and 105 minutes a f t e r i.c.v. infusion of 2nmol ANP or 2nmol BNP. In a separate experiment, dopaminergic receptor blocker pimozide (0.63 mg/kg, subcutaneously) was administered at the onset of the-experiment, and at the midpoint of the experiment 2 nmol ANP or 2 nmol BNP was i.c.v. infused. At the conclusion of each experiment, animals were anaesthetized with pentobarbital and infused with c r e s y l v i o l e t s t a i n into the t h i r d v e n t r i c l e . The rats were then perfused i n t r a c a r d i a l l y with s a l i n e followed by 10% formaldehyde solution i n saline. The brains were removed from the s k u l l s and l a t e r sectioned i n 50 micrometer to v e r i f y the location of the t h i r d v e n t r i c l e cannula. Plasma LH concentrations were measured with RIA k i t provided by the National Institute of A r t h r i t i s , Diabetes, and Digestive and Kidney Diseases, National Hormone and P i t u i t a r y Program. The assay was a s l i g h t modification of the double antibody RIA described by Niswender et a l . (1968). The reference preparation was NIADDK-rat-LH-RP-7. The interassay c o e f f i c i e n t of v a r i a t i o n was 14%, and the intraassay c o e f f i c i e n t of v a r i a t i o n was 10%. An adaptive threshold method was used to determine the time and amplitude of hormone pulses (Soules et a l . , 1987). 40 Mean plasma LH l e v e l s were determined by d i v i d i n g the sum of a l l the samples of one animal by the number of samples, pulse amplitude was the difference between the peak and nadir of each pulse. Mean plasma LH l e v e l , mean pulse amplitude and mean pulse frequency were calculated for the pre- and post-drug infusion period of each animal. Except i n the naloxone treatment group, pre- and post-infusion values were compared by means of a paired t - t e s t except where heterogeneity of variance was found, i n which case the Cochran Test was applied. In naloxone treatment group, s t a t i s t i c a l differences of the data were determined with one way analysis of variance followed by Scheffe's test. Values in the text represent the means ± standard error. 41 2.3 RESULTS 2.3.1 E f f e c t s of ANP and BNP infusion on LH secretion 2.3.1.1 E f f e c t s of saline infusion on LH secretion None of the four control animals receiving s a l i n e infusion showed a change i n mean plasma LH l e v e l (from 5.01 ± 0.51 ng/ml to 4.97 ± 0.33 ng/ml), pulse amplitude (4.76 ±0.23 ng/ml to 4.89 ± 0.22 ng/ml) and pulse frequency (from 3.50 ± 0.29 to 3.7 ± 0.41 number of pulses/90min) when the pre-infusion period values were compared to those of the post-infusion period, as shown in the representative example in Figure 1. 2.3.1.2 E f f e c t s of 2nmol ANP infusion on LH secretion In t h i s group (n=8), comparison of the pre-versus post-infusion mean plasma LH concentration revealed s i g n i f i c a n t decrease (from 5.10 ± 0.17 ng/ml to 4.08 ± 0.47 ng/ml, P <0.05). The LH pulse amplitude i n the post-infusion period was s i g n i f i c a n t l y lower than that i n the pre-infusion period (from 6.05 ± 0.81 ng/ml to 3.43 ± 0.80 ng/ml, P < 0.05). The number of LH pulses i n the post-infusion period was also s i g n i f i c a n t l y less than that in the pre-infusion period (from 4.38 ± 0.32 to 2.12 ± 0.39 number of pulses/90 min, P < 0.01), as shown i n the representative example i n Figure 2. 2.3.1.3 E f f e c t s of 2nmol BNP infusion on LH secretion In t h i s group (n=8), comparison of the pre-versus post-infusion mean plasma LH concentration revealed s i g n i f i c a n t decrease (from 5.75 ± 0.23 ng/ml to 3.79 ± 0.22 ng/ml, P < 0.01). The LH pulse amplitude i n the post-infusion period was s i g n i f i c a n t l y lower than that i n the pre-infusion period (from 6.03 ± 0.87 ng/ml to 2.37 ± 0.27 ng/ml, P < 0.01). The number of LH pulses i n the post-infusion period was also s i g n i f i c a n t l y less than that i n the pre-infusion period (from 5.14 ± 0.34 to 2.71 ± 0.28 number of pulses/90 min, P < 0.01), as shown in the representative example i n Figure 3. 2.3.1.4 Eff e c t s of 0.2nmol ANP infusion on LH secretion In t h i s group (n=8), there was no s i g n i f i c a n t decrease in mean plasma LH (from 5.27 ± 0.23 ng/ml to 4.88 ± 0.32 ng/ml), pulse amplitude (from 4.89 ± 0.62 ng/ml to 4.57 ± 0.40 ng/ml) and pulse frequency (from 3.87 ± 0.29 to 3.62 ± 0.36 number of pulses/90min) when pre-versus post-infusion periods were compared, as shown in the representative example i n Figure 4. 43 2.3.1.5 Effects of 0.2nmol BNP infusion on LH secretion In t h i s group (n=7) , no s i g n i f i c a n t decrease i n mean plasma LH (from 5.01 + 0.39 ng/ml to 4.34 ± 0.47 ng/ml), pulse amplitude (from 4.83 ± 0.47 ng/ml to 4.62 ± 0.37 ng/ml) and pulse frequency (from 3.85 ± 0.26 to 4.42 ± 0.29 number of pulses/90min) was observed, when pre-versus post-infusion periods compared, as shown i n the representative example i n Figure 5. The r e s u l t s of e f f e c t s of ANP and BNP infusion on LH secretion are summarized i n the Figure 6, 7 and 8. 44 Plasma LH (ng/ml) s a l i n e Figure l . Representative example of LH release i n ovx rats with i.c.v infusion of s a l i n e . Plasma LH (ng/ml) 12 i :  14 Plasma LH (ng/rnl) 0 I I I I I I I I I I I I I 1 I I 1 I I I I I I I I I I I I I I I 0 30 60 90 120 150 (min) 2nmolBNP Figure 3. Representative example of LH release i n ovx rats with i.c.v infusion, of 2 nmol BNP. 00 12 Plasma LH (ng/ml) i i i i 30 i i I i J 1—I 1 1—I—I L_J L_J I I I I L 60 90 120 150 (min) 0.2nmolANP Figure 4. Representative example of LH release i n ovx rats with i . c . v infusion of 0.2 nmol ANP. plasma LH (ng/ml) 12 i 5 h 4 pre-infusion post-infusion m 4 X X X X saline „ANP BNP , 0.2nmol O.znmol ANP 2n nmol o B N P , 2nmol Figure 6. Mean plasma LH concentration i n the pre- and post-ANP or BNP infusion period. * 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 of P < 0.05 and P < 0.01 separately. The "n" value for each group i s shown i n the panel. 8 7 6 -5 -4 3 2 0 pre-infusion post-infusion X X X ** saline 0.2nmol 0.] INP , !nmol ANP 2n nmol BNP 2ri nmol Figure 7. LH pulse amplitude i n the pre- and post-ANP or BNP infusion period. * 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 of P < 0.05 and P < 0.01 separately. The "n" value for each group i s shown i n the panel. to c 6 • MB E o CO CD V> D 4 o a> £ 2 a> co 0 pre-infusion post-infusion X saline X i X X X X _ ANP , 0.2nmol BNP O.znmol mol x X BNP 2n nmol Figure 8. LH pulse frequency i n the pre- and post-ANP or BNP infusion period. ** denotes s t a t i s t i c a l s i gnificance of P < 0.01. The "n" value for each group i s shown i n the panel. 2.3.2 Effects of naloxone treatment on the LH i n h i b i t o r y actions of i.c.v. infused ANP and BNP 2.3.2.1 Effects of naloxone treatment in control group In control group (n=5), treatment with naloxone (0.5 mg each time, i . v . bolus) at 45, 75 and 105 minutes a f t e r i.c.v. infusion of 0.9% sa l i n e had no s i g n i f i c a n t e f f e c t s on mean plasma LH le v e l (from 2.68 ± 0.54 ng/ml to 2.67 ± 0.62 ng/ml), pulse amplitude (5.66 ± 1.22 ng/ml to 5.88 ± 0.22 ng/ml) and pulse frequency (from 2.65 ± 0.21 to 2.81 ± 0.41 number of pulses/45 min), as shown i n the representative example i n Figure 9. 2.3.2.2 Effects of naloxone treatment i n 2nmol ANP group Treatment with naloxone (0.5 mg each time, i . v . bolus) at 45,75 and 105 minutes a f t e r i . c . v . infusion of 2nmol ANP reversed the i n h i b i t o r y e f f e c t s of ANP (n=7) on the mean plasma l e v e l (from 1.72 ± 0.42 ng/ml to 3.14 ± 0.44 ng/ml, P < 0.05). Application of naloxone also reversed the i n h i b i t o r y e f f e c t of ANP on LH pulse amplitude (from 2.11 ± 0.39 ng/ml to 5.04 ± 0.99 ng/ml, P < 0.05). In terms of pulse frequency, although there was a strong tendency that naloxone treatment may reverse the i n h i b i t o r y e f f e c t of ANP, naloxone treatment f a i l e d to a l t e r the pulse frequency s i g n i f i c a n t l y (from 1.57 ± 0.20 to 2.28 ± 0.24 number of pulses/45 min), as shown i n the representative example i n Figure 10. I t i s quite possible that when the number of experimental animals i n t h i s group i s increased, naloxone treatment w i l l reverse the i n h i b i t o r y e f f e c t of ANP on LH pulse frequency. 2.3.2.3 E f f e c t s of naloxone treatment i n 2nmol BNP group Treatment with naloxone (0.5 mg each time, i . v . bolus) at 45,75 and 105 minutes after i.c.v. i n f u s i o n of 2nmol BNP reversed the i n h i b i t o r y effects of BNP (n=7) on the mean plasma l e v e l (from 1.96 ± 0.29 ng/ml to 3.89 ± 0.48 ng/ml, P < 0.05). Administration of naloxone also reversed the i n h i b i t o r y e f f e c t s of BNP on LH pulse amplitude (from 2.09 ± 0.29 ng/ml to 5.40 ± 0.93 ng/ml, P < 0.05). In terms of pulse frequency, although there was a strong tendency that naloxone treatment may reverse the i n h i b i t o r y e f f e c t of BNP, naloxone treatment f a i l e d to a l t e r the pulse frequency s i g n i f i c a n t l y (from 1.71 ± 0.28 to 2.21 ± 0.10 number of pulses/45 min), as shown i n the representative example i n Figure 11. The e f f e c t s of naloxone treatment on the LH i n h i b i t o r y actions of i.c.v. infused ANP and BNP are summarized i n Figure 12, 13 and 14. 54 Plasma LH (ng/ml) 1 2 i 2 -0 J L J L 3 0 6 0 A saline 9 0 • naloxone J L J L J L J L 1 2 0 • naloxone 1 5 0 • naloxone (min) Figure 9. Representative example of LH release i n ovx rats injected with naloxone a f t e r i.c.v infusion of saline. 16 Plasma LH (ng/ml) (min) Figure 10. Representative example of LH release i n ovx rats injected with naloxone a f t e r i.c.v infusion of 2 nmol ANP. Plasma LH (ng/ml) 18 i 2nmolBNP naloxone naloxone naloxone Figure 11. Representative example of LH release i n ovx rats injected with naloxone a f t e r i.c.v infusion of 2 nmol BNP. 00 ^ 5 E 0) cd 0) 4 -3 -0 pre-treatment saline/ANP/BNP naloxone rx saline ANP BNP Figure 12. Mean plasma LH concentration i n the pre- and post-ANP or BNP infusion and naloxone treatment period. * denotes value 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 value a at P < 0.05. The "n" value for each group i s shown in the panel. . vo ^ 9 E o> 8 -r 6 Is CO a, 4 CO 1 0 pre-treatment sallne/ANP/BNP naloxone saline ANP BNP Figure 13. LH pulse amplitude i n the pre- and post-ANP or BNP infusion and naloxone treatment period. * denotes value 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 value a at P < 0.05. The "n" value for each group i s shown i n the panel. o (0 <D a O c § 2 jo a. 0 pre-treatment saline/ANP/BNP naloxone x X •to saline ANP BNP Figure 14. LH pulse frequency i n the pre- and post-ANP or BNP infusion and naloxone treatment period. * denotes value 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 at 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 value a and value b. Naloxone treatment does not reverse ANP's and BNP's inhi b i t o r y e f f e c t on LH pulse frequency. The "n" value for each group i s shown in the panel. 2.3.3 Effect of pimozide pretreatment on the LH i n h i b i t o r y actions of i.c.v. infused ANP and BNP 2.3.3.1 Ef f e c t of pimozide pretreatment i n control group None of four control animals receiving pimozide pretreatment showed a change i n mean plasma LH l e v e l (5.55 ± 0.38 ng/ml), pulse amplitude (4.19 ± 0.74 ng/ml) and frequency (4.76 ± 0.39 number of pulses/90min), compared with saline treatment group, as shown i n the representative example i n Figure 15. 2.3.3.2 Effects of pimozide pretreatment i n 2nmol ANP group Pretreatment of rats with pimozide (0.63 mg/kg, s.c) resulted in prevention of i n h i b i t o r y e f f e c t s of ANP on LH secretion from occurring. A f t e r infusion of 2nmol ANP (n=9), there were no s i g n i f i c a n t decreases i n mean plasma LH l e v e l (4.96 ± 0.24 ng/ml to 5.07 ± 0.43 ng/ml), pulse amplitude (from 4.26 ± 0.37 ng/ml to 4.36 ± 0.44 ng/ml) and pulse frequency (from 4^ .22 ± 0.27 to 3.77 ± 0.36 number of pulses/90 min), compared with pre-infusion period, as shown i n the representative example i n Figure 16. 2.3.3.3 Effects of pimozide pretreatment i n 2nmol BNP group 61 Pretreatment with pimozide (0.63 mg/kg, s.c) resulted i n prevention of i n h i b i t o r y e f f e c t s of BNP on LH secretion from occurring. When pre-infusion and post-infusion values were compared i n t h i s group (n=9), no s i g n i f i c a n t decreases i n mean plasma LH l e v e l (from 4.93 ± 0.65 ng/ml to 4.83 ± 0.66 ng/ml), pulse amplitude (from 5.02 ± 0.60 ng/ml to 4.56 ± 0.47 ng/ml) and pulse frequency (from 4.00 ± 0.37 to 3.88 ± 0.31 number of pulses/90 min) were observed, as shown i n the representative example i n Figure 17. The ef f e c t s of pimozide pretreatment on the LH in h i b i t o r y actions of i.c.v. infused ANP and BNP are summarized i n Figure 18, 19 and 20. At the conclusion of each experiment, animals were anaesthetized with pentobarbital and infused with cr e s y l v i o l e t s t a i n into the t h i r d v e n t r i c l e . The rats were then perfused i n t r a c a r d i a l l y with s a l i n e followed by 10% formaldehyde solution i n sa l i n e . The brains were removed from the s k u l l s and l a t e r sectioned i n 50 micrometer to v e r i f y the location of the t h i r d v e n t r i c l e cannula. The rats with cannula t i p s outside t h i r d v e n t r i c l e are not counted into the s t a t i s t i c s . Photograph of representative f r o n t a l sections i n the r a t hypothalamus depicting the s i t e of cannula implantation i n the t h i r d v e n t r i c l e i s shown i n Figure 21. 62 Plasma LH (ng/ml) 12 i 2 -J I I I L 0 • pimozide 30 60 -M— i-90 A saline I I I l 120 150 (min) Figure 15. Representative example of LH release i n pimozide-pretreated ovx rats with i.c.v infusion of sa l i n e . Plasma LH (ng/ml) 101 Figure 16. Representative example of LH release i n pimozide-pretreated ovx rats with i.c.v infusion of 2 nmol ANP. Plasma LH (ng/ml) 16 i ' 01 1 1 | 1 1 | 1 1 | 1 1 | 1 1 | 1 1 1 1 1 | 1 1 | 1 1 | 1 1 | 1 1 | 1 0 30 60 90 120 150 K*in) pimozide 2nmolBNP Figure 17. Representative example of LH release i n pimozide-pretreated ovx rats with i.c.v infusion of 2 nmol BNP. ^ 8 E ^> 7 w 6 -$ 0) 5 4 3 2 1 0 (0 0) 2 pimozide-treated rats C Z D pre-infusion r ~ ~ l post-infusion X saline x ANP BNP Figure 18. Mean plasma LH concentration i n the pre- and post-ANP or BNP infusion period with the pimozide pretreatment. The "n" value for each group i s shown i n the panel. as E 6 -•O 5 E * (0 0) 3 =) 4 1 2 —J 1 0 pimozide-treated rats pre-infusion post-infusion X saline T_-T ANP BNP Figure 1 9 . LH pulse amplitude i n the pre- and post-ANP or BNP infusion period with the pimozide pretreatment. The "n" value f o r each group i s shown in the panel. pimozide-treated rats pre-infusion post-infusion x x X saline ANP BNP Figure 20. LH pulse frequency i n the pre- and post-ANP or BNP infusion period with the pimozide pretreatment. The "n" value f o r each group i s shown i n the panel. t 01 0^ Figure 2 1 . Photograph of representative f r o n t a l sections i n the rat hypothalamus depicting the s i t e of 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 black t r i a n g l e . 3. DISCUSSION The presence of ANP within the central nervous system (Jacobowitz et a l . , 1985; Tanaka et a l . , 1984), p a r t i c u l a r l y that found within the diencephalon, and the detection of ANP-s p e c i f i c binding s i t e s i n the hypothalamus and p i t u i t a r y gland (Quirion et a l . , 1984), suggested the p o s s i b i l i t y that ANP may influence anterior p i t u i t a r y hormone secretion. The i n h i b i t i o n of p u l s a t i l e LH secretion reported i n the present study lends support to t h i s hypothesis. The decrease i n mean plasma LH le v e l could be attributed to the reduction of pulse amplitude and frequency. This i n h i b i t o r y e f f e c t occurred only i n high dose (2nmol) groups. Since the h a l f - l i f e of ANP i n plasma was short (1-3 min) (Yandle et a l . , 1986) and there was no data on h a l f - l i f e of ANP i n cerebrospinal f l u i d , whether or not f a i l u r e of acute infusion of ANP i n 0.2nmol dose group to a l t e r LH secretion could be attributed to the short h a l f - l i f e remained an open question. -The s i t e ( s ) of action of ANP within the hypothalamic-p i t u i t a r y axis are unknown. Infusion of ANP into the t h i r d v e n t r i c l e does not allow us to pinpoint the exact s i t e of action. However, several groups have suggested that ANP i s un l i k e l y to be acting at the l e v e l of the p i t u i t a r y gland 70 (Heisler et a l . , 1986; Simard et a l . , 1986). These researchers found that even ANP could activate guanylate cyclase a c t i v i t y in the p i t u i t a r y gland and ANP receptors were present i n the p i t u i t a r y gland (Quirion et a l . , 1984), they f a i l e d to detect any direct e f f e c t s of ANP on p i t u i t a r y hormone secretion. In agreement with these r e s u l t s , i t has been demonstrated that no s i g n i f i c a n t e f f e c t of ANP on basal or LHRH-stimulated LH secretion can be observed i n cultured anterior p i t u i t a r y c e l l s (Samson et a l . , 1988). Therefore i t seems l i k e l y that the two events (ANP-stimulated cGMP accumulation and hormone secretion) are unrelated. Due to the i n a b i l i t y of other researchers to demonstrate a dir e c t e f f e c t of ANP i n p i t u i t a r y c e l l cultures, i t i s reasonable to hypothesize that the ANP's actions, including i t s LH-inhibitory e f f e c t , are exerted at the l e v e l of the hypothalamus. The dense d i s t r i b u t i o n of ANP immunopositive fibres and ANP s p e c i f i c binding s i t e s i n preoptic-septal region provides the anatomical framework for such an action (Standaert et a l . , 1986b; Quirion et a l . , 1984). I t has been demonstrated that iontophoresis or micropressure i n j e c t i o n of synthetic ANP onto singl e neurons i n the preoptic-septal region of the rat (Wong et a l . , 1986), a region known to contain LHRH-positive c e l l bodies (Krey et a l . , 1983) and thought to be c r u c i a l i n the hypothalamic control of gonadotropin secretion (Kalra et a l . , 1985; Ramirez et a l . , 1984), resulted i n a predominantly i n h i b i t o r y e f f e c t on neuronal e x c i t a b i l i t y . Since ANP-positive f i b r e s and ANP receptors have been demonstrated i n t h i s region, i t i s possible that c e n t r a l l y administered ANP might exert i t s LH-inh i b i t o r y action in r o s t r a l hypothalamic structures. In addition, i t i s also possible that ANP can act at the median eminence l e v e l . The existence of intense ANP-positive f i b r e terminals i n the external layer of the median eminence (Kawata et a l . , 1985a; Skofitsch et a l . , 1985) supports such p o s s i b i l i t y . I f ANP acts i n vivo at the median eminence to i n h i b i t the release of LH by i n h i b i t i n g the release of endogenous LHRH, then the p i t u i t a r y LH response to any i . v . bolus i n j e c t i o n of exogenous LHRH during ANP infusion should not be altered. Indeed, infusion of ANP i n l e v e l s known to i n h i b i t LH secretion f a i l e d to block the p i t u i t a r y response _ to exogenous LHRH (Samson et a l . , 1988b). Although incubation of median eminence explants i n the presence of a wide dose range of ANP f a i l e d to a l t e r basal LHRH release, ANP was able to s i g n i f i c a n t l y i n h i b i t DA-stimulated LHRH release (Samson et a l . , 1988b), suggesting not only an e f f e c t of ANP at the median eminence l e v e l , but also an interaction of ANP with 72 brain catecholaminergic systems c o n t r o l l i n g neuropeptide secretion. De f i n i t e proof of the site(s) of action of ANP on LH secretion awaits further 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 secretion was predicted by the r e s u l t that l a t e r a l v e n t r i c u l a r administration of ANP e l i c i t e d decreased l e v e l s of catecholamine and i t s metabolites i n large regions of the brain, e s p e c i a l l y i n the hypothalamus and septum (Nakao et a l . , 1986), and by the report of the e f f e c t of ANP on dopamine-beta-hydroxylase a c t i v i t y i n c u l t u r e d pheochromocytoma c e l l s (Racz et a l . , 1986). Furthermore, strong evidence suggests that ANP acts at the hypothalamus to a l t e r dopamine release into the hypophyseal portal c i r c u l a t i o n , and hence to influence p i t u i t a r y hormone secretion. Third cerebroventricular infusion of ANP resulted i n profound i n h i b i t i o n of 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, 1987). Once again, since a d i r e c t p i t u i t a r y action of ANP on basal, secretagogue-stimulated, and DA-inhibited PRL release i n v i t r o could not be demonstrated (Samson et a l . , 1988a), the r e s u l t s suggested that the action of ANP to i n h i b i t PRL secretion i n vivo was 73 due to the release of DA, a major p r o l a c t i n i n h i b i t i n g factor, into the portal blood. S i m i l a r l y , i n our experiment, the in h i b i t o r y e f f e c t of ANP on LH secretion was completely blocked in the presence of the dopamine receptor blocker pimozide. Therefore i t i s possible, l i k e i n the case of PRL i n h i b i t i o n , that t h i s action of ANP to i n h i b i t LH secretion i n OVX rats i s due to the f a c i l i t a t i o n of DA release into the portal blood, since infusions of DA into the t h i r d v e n t r i c l e of OVX rats not treated with exogenous steroids resulted i n an i n h i b i t i o n of LH release i n many cases (Kalra et a l . , 1985; Ramirez et a l . , 1984). Another p o s s i b i l i t y i s that the action of ANP to i n h i b i t LH secretion i s due to modulation of DA turnover rate i n LHRH-containing nuclei, such as medial preoptic nucleus and septum, since s p e c i f i c ANP-binding s i t e s was i d e n t i f i e d i n these LHRH-containing nuclei thought to be c r u c i a l i n the hypothalamic control of LH secretion i n rats (Quirion et a l . , 1984). It i s i n t e r e s t i n g to compare our results with those reported on PRL. Pretreatment with the dopamine receptor antagonist, domperidone, resulted i n an elevation of PRL le v e l s that were r e s i s t a n t to the central action of ANP (Samson et a l . , 1988a). S i m i l a r l y , i n another s e r i e s of experiments once PRL had been inh i b i t e d by central 74 administration of ANP, domperidone treatment rapi d l y elevated PRL levels to those seen i n s a l i n e treated, domperidone-injected controls (Samson et a l . , 1988a). Thus i t appears that the mechanisms by which ANP i n h i b i t s LH and PRL secretions are s i m i l a r . The i n h i b i t o r y e f f e c t s of ANP on LH and PRL secretion -were considered to be s p e c i f i c because of f a i l u r e to observe s i g n i f i c a n t a l t e r a t i o n s i n growth hormone or thyroid stimulating hormone release a f t e r central administration of the peptide (Samson, 1988). In addition to a central i n h i b i t o r y action of ANP on LH secretion v i a dopaminergic system, ANP i n h i b i t i o n of LH secretion also appears to be mediated i n part by an interaction with endogenous opiate systems, which are recognized to be physiological i n h i b i t o r s of the hypothalamic component of LH release (Bruni et a l . , 1977; Pang et a l . , 1977). I t has been reported that ANP can modulate proopiomelanocortin-derived peptides (including beta-endorphin) secretion from both anterior and intermediate lobe -c e l l s of rat p i t u i t a r y i n v i t r o (Shibasaki, et a l . , 1986), indicating an interaction of ANP with central opioidergic system. Indeed, The present study shows that naloxone treatment a f t e r t h i r d v e n t r i c u l a r infusion of ANP reversed the i n h i b i t o r y e f f e c t of ANP on mean plasma LH secretion. This 75 r e s u l t i s i n agreement with that of Samson's group which shows that pretreatment of rats with naloxone completely abolished i n h i b i t o r y action of the maximum e f f e c t i v e i . v . dose of ANP on mean plasma LH secretion (Samson et a l . , 1988b). However in t h e i r experiment, the blood sampling i n t e r v a l s (at 0, 15, 30, 60. 90, 120 minutes) were too long to reveal the p u l s a t i l e pattern of LH release. Therefore, they were unable to determine whether t h i s i n h i b i t o r y e f f e c t of ANP on LH secretion was due to the decrease i n LH pulse amplitude or pulse frequency and the influence of naloxone on LH pulses. In our experiment, a shorter sampling i n t e r v a l (5 minutes) was employed to reveal the LH pulses. The r e s u l t c l e a r l y shows that the reversal of mean LH l e v e l by naloxone i s mainly due to the reversal of LH pulse amplitude. In terms of the s i t e of LH i n h i b i t i o n by endogenous opiates, i t i s generally considered to be exerted i n the preoptic and anterior hypothalamic regions (Fink, 1988). I t should be noted that ANP-containing neuronal elements are present i n these regions and indeed terminal f i e l d s p o s i t i v e for ANP immunoreactivity are present i n regions known to be important i n the central control of gonadotropin secretion (Jacobowitz et a l . , 1985). Three subtypes of opiate receptors have been characterized: mu, delta and kappa. Opiate e f f e c t s on LH 76 secretion are generally believed to involve the mu receptor subtype (Cicero et a l . , 1983; P f e i f f e r et a l . , 1983; Panerai et a l . , 1985). I t has been shown that i n t r a v e n t r i c u l a r inje c t i o n of the mu-agonist i n OVX rats produced a s i g n i f i c a n t suppression of 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 , leading to the conclusion that the mu-receptor i s the primary opiate receptor involved in the regulation of LH secretion ( P f e i f f e r et a l . , 1983). However, the observation that administration of bremazocine, a pr e f e r e n t i a l kappa agonists, i s as potent as morphine to lower LH secretion i n rats of both sex, suggested that both types of receptors could be involved i n mediating the response (Mark et a l . , 1983). In addition, the mu-receptor antagonist naloxone s i g n i f i c a n t l y antagonized the i n h i b i t i n g e f f e c t of morphine, but not that of bremazocine on LH secretion (mark et a l . , 1983). In the present study, naloxone has been shown to reverse the i n h i b i t o r y e f f e c t of ANP on LH secretion, thus the ANP's action should be exerted at least p a r t i a l l y v i a the opiate mu-receptor. Because naloxone could not completely reverse the ANP's action, i t i s possible that the other subtypes of opiate receptors may also mediate ANP's i n h i b i t o r y e f f e c t on LH secretion. BNP i s a newly i d e n t i f i e d peptide of 26 amino acid 77 residues, which has a remarkable homology to but i s d i s t i n c t from ANP i n structure. BNP e l i c i t s a v a r i e t y of responses very s i m i l a r to that of ANP, such as n a t r i u r e t i c - d i u r e t i c , hypotensive and chick rectum relaxant a c t i v i t i e s (Sudoh et a l . , 1988). Also i t has been shown that i * c . v . i n j e c t i o n of BNP s i g n i f i c a n t l y i n h i b i t e d basal arginine vasopressin secretion and the e f f e c t of BNP i s comparable to that of ANP (Yamada et a l . , 1986). In the present studies, BNP showed a response very sim i l a r to that of ANP, i . e . infusion of BNP i n h i b i t e d p u l s a t i l e LH secretion, naloxone reversed the i n h i b i t o r y e f f e c t of BNP on LH secretion, and t h i s i n h i b i t o r y e f f e c t was prevented by the pretreatment of pimozide. These re s u l t s suggest that the i n h i b i t o r y action of BNP on LH secretion once thought to be mediated by ANP may be regulated through a dual mechanism involving both ANP and BNP, and the mechanisms by which they i n h i b i t the p u l s a t i l e LH secretion are quite similar. Recent improvements i n neuropeptide characterization and l o c a l i z a t i o n methods have resulted i n an in-depth reappraisal of our understanding of neuroendocrine control. In addition to "conventional" neurotransmitters, over 30 peptides have recently been shown to regulate p i t u i t a r y reproductive 78 hormones (Richard et a l . , 1988). ANP and BNP are two new members of them. Like most of neuropeptides, ANP and BNP have been found i n s i g n i f i c a n t amounts i n the hypothalamus and produced l o c a l l y (Morii et a l . , 1985; Kawata et a l . , 1985; Skofitsch et a l . , 1985; Ueda et a l . , 1988; Sudoh et a l . , 1988). However, unlike most of neuropeptides which are released into the hypophyseal portal system and a f f e c t the anterior p i t u i t a r y adenohypophyseal c e l l s d i r e c t l y (Richard et a l . , 1988) , ANP seems mainly to act at the hypothalamic and median eminence l e v e l s (Samson et a l . , 1988b). In addition, the present study shows that ANP and BNP i n t e r a c t with other neurotransmission systems, l i k e opiate and dopaminergic systems, to exert t h e i r i n h i b i t o r y e f f e c t s on LH secretion. Like most hypothalamic peptides produced i n the preoptic-periventricular area, mapping studies based on neuron l a b e l l i n g methods have shown that median eminence i s only one of several projection s i t e s for ANP (Standaert et a l . , 1986b). Axons "of ANP-positive neurons also innervate several hypothalamic nuc l e i , i n p a r t i c u l a r medial preoptic area and paraventricular nucleus, as well as extrahypothalamic structures (Jacobowitz et a l . , 1985; Standaert et a l . , 1986b). This s t r a t e g i c p o s i t i o n accounts for i t r o l e i n modulating hormone regulation. 79 In general, ANP and BNP have much i n common with other neuropeptides i n regulating p i t u i t a r y reproductive hormones. 80 4. SUMMARY AND CONCLUSIONS 4.1 summary Over the past several years an abundance of information concerning the possible neuromodulatory actions of ANP has accumulated. Clear consensus ex i s t s that ANP can i n h i b i t many angiotensin II actions, p a r t i c u l a r l y s a l t and water intake and vasopressin secretion (David et a l . , 1989), which mirror ANP's peripheral roles i n maintenance of f l u i d and e l e c t r o l y t e homeostasis. More recently, evidence for the interaction of central ANP with brain hormones unrelated to f l u i d and e l e c t r o l y t e homeostasis has further expanded the possible roles for ANP i n hypothalamo-hypophyseal function, such as the inh i b i t o r y e f f e cts of ANP on LH and PRL secretion. The anatomical substrates for the actions of ANP on gonadotropin secretion are the presence of ANP-immunoreactivity i n neural pathways and hypothalamic s i t e s known to be important i n these functions (Jacobowitz et a l . , 1985). The i n h i b i t o r y e f f e c t of ANP on LH secretion seems s p e c i f i c , since s i m i l a r manipulations of ANP on thyroid-stimulating hormone or growth hormone resulted no s i g n i f i c a n t response (Samson, 1988). The present study demonstrates that t h i s i n h i b i t o r y e f f e c t i s also dose-related and reversible and that ANP's i n h i b i t o r y actions 81 on LH secretion may involve i n t e r a c t i o n s with central dopaminergic and opiate systems. As for suggestions for future work, i n order to estab l i s h a ph 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 for endogenous ANP or BNP i n the hypothalamic control of LH secretion one must demonstrate that the absence of neuronal ANP or BNP s i g n i f i c a n t l y a l t e r s reproductive a c t i v i t y . Infusion of ANP or BNP antibodies into the cerebroventricles or passive immunoneutralization techniques may help i n providing such information. 4.2 Conclusion The primary focus of the research presented here was i n establishing the modulatory roles of ANP and BNP i n LH secretion and t h e i r possible mechanisms. Two nmol ANP or BNP and 0.2 nmol ANP or BNP were infused into t h i r d v e n t r i c l e and t h e i r e f f e c t s on plasma LH concentrations were observed i n ovariectomized rats. In addition, naloxone and pimozide manipulations were employed to observe interactions of ANP or BNP with opiate and dopaminergic systems. The res u l t s presented i n t h i s report suggest that: 1. Both ANP and BNP can e l i c i t i n h i b i t o r y e f f e c t s on LH secretion implying that LH secretion can be modulated through 82 a dual mechanism involving both ANP and BNP; 2. 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