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Characterization of melatonin binding sites in the Harderian gland and median eminence of the rat
Life Sciences, Vol. 48, pp. 1165-1171 Printed in the U.S.A.
Pergamon Press
C H A R A C T E R I Z A T I O N OF MI~.&TONIN B I N D I N G S I T E S IN T H E H A R D E R I A N G L A N D A N D MEDIAN EMINENCE OF T H E R A T
M.A
L o p e z - G o n z a l e z , J . R . C a l v e , A . R u b l e , R. G o b e r n a and J.M. Guerrero*
T h e U m v e r s l t y of S e w U e S c h o o l of M e d l c m e , D e p a r t m e n t of M e d i c a l B 1 o e h e n u s t r y a n d M o l e c u l a r B i o l o g y , A v d a S a n c h e z P1zjuan 4, 4 1 0 0 9 - S e v l l l a , S p m n (Recelved in flnal form January 16, 1991) S,,mm~ l
T h e c h a r a c t e r l z a t l o n of s p e c i f i c m e l a t o n m b i n d i n g s i t e s m t h e H a r d e r m n g l a n d ( H G ) a n d m e d i a n e r m n e n e e (ME) of t h e r a t w a s s t u d i e d u s i n g [ 1 2 5 1 ] m e l a t o n m B i n d i n g of m e l a t o m n to m e m b r a n e c r u d e p r e p a r a t l o n s of b o t h t l s s u e s w a s d e p e n d e n t on time a n d t e m p e r a t u r e . T h u s , maximal b i n d i n g w a s o b t m n e d a t 37° C a f t e r 30-60 m m l n e u b a t l o n . B i n d i n g w a s a l s o d e p e n d e n t on protein concentration (up to 1.5 mg/ml). The specific binding of [ 1 2 5 1 ] m e l a t o n m w a s s a t u r a b l e , e x h l b l t m g o n l y o n e c l a s s of b i n d i n g s i t e s in b o t h t i s s u e s . T h e d 3 s s o e l a t l o n c o n s t a n t s ( K d ) w e r e 170 a n d 190 pM f o r ME a n d H G , r e s p e c t i v e l y . T h e e o n c e n t r a t l o n of t h e b i n d i n g s i t e s m ME w a s 8 f m o l ] m g p r o t e l n , a n d in t h e HG 4 f m o l J m g p r o t e i n . I n c o m p e t i t i o n s t u d i e s , b i n d i n g of [ 1 2 5 1 ] m e l a t o m n t o ME o r HG w a s l n h l b l t e d b y i n c r e a s i n g c o n c e n t r a t i o n o f n a t i v e m e l a t o n m ; 50% i n l u b l t l o n w a s o b s e r v e d a t a b o u t 702 a n d 422 nM f o r ME a n d HG, r e s p e c t i v e l y AddltlonaUy, the [1251]melatonm b l n d m g t o t h e c r u d e m e m b r a n e s w a s n o t a f f e c t e d b y t h e a d d l t l o n of d i f f e r e n t drugs such as norepmephrlne, lSOproterenol, phenylephrme, propranolol, or prazosln T h e r e s u l t s c o n f i r m t h e p r e s e n c e of m e l a t o n m b i n d i n g s l t e s m m e d i a n e r m n e n c e a n d s h o w , f o r t h e f i r s t t i m e , t h e e x i s t e n c e of m e l a t o n l n b i n d i n g s i t e s in t h e H a r d e r l a n g l a n d T h e p i n e a l h o r m o n e m e l a t o n l n t r a n s d u e e s t h e e f f e c t o f p h o t o p e r l o d on t h e neuroendocrme system and thus regulates seasonal rhythms, notably reproduetlon (1,2). The means by whlch melatomn brings about these diverse effects are not well k n o w n . H o w e v e r , t h e i n t r o d u c t l o n of a l u g h - a f f l m t y , h i g h s p e c i f i c l i g a n d , [ 1 2 5 1 ] m e l a t o n m h a s r e s u l t e d i n t h e l d e n t i f l c a t l o n of p u t a t i v e s l t e s of a c t l o n . H i g h affinity melatonm receptors have been ehar~acterlzed m the rabblt and chicken r e t i n a , p r o v i d i n g e w d e n c e f o r a l o c a l s l t e of a e t l o n , a s m e l a t o n m i s p r o d u c e d i n the retina (3,4) Melatonm-bmdmg sltes have also been identlfled m the rat (59 ) , h a m s t e r ( 9 - 1 1 ) , s h e e p ( 1 2 , 1 3 ) , a n d h u m a n (14) h y p o t h a l a m u s , r e v e a h n g c e n t r a l s i t e s of a c t i o n f o r m e l a t o m n o f p i n e a l Orlgun. T h e s e s t u d l e s i d e n t 3 f i e d t h e suprachlasmatlc nucleus and median ermnence reguons as contmnlng binding sites. Nevertheless, using autoradlography at the hght nueroseope level, the melatonln b m d l n g p r e v i o u s l y a s c r i b e d t o m e d i a n e m i n e n c e h a s s h o w n t o b e s p e c i f i c to t h e p a r s t u b e r a l i s of t h e p i t u i t a r y m t h e r a t a n d s h e e p ( 7 , 8 , 1 2 , 1 3 ) .
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0024-3205/91 $3.00 +.00 Copyright (c) 1991 Pergamon Press plc
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Rat Harderlan Gland Melatonin Receptors
Vol. 48, No. 12, 1991
I n t h e p r e s e n t s t u d y , we c h a r a c t e m z e d m e l a t o n m b i n d i n g s i t e s i n a c r u d e m e m b r a n e f r a c t i o n i s o l a t e d from r a t H a r d e m a n g l a n d a n d m e d i a n e n u n e n c e . T h e H a r d e m a n g l a n d is a t u b u l o a l v e o l a r g l a n d l o c a t e d w l t t u n t h e o c u l a r o r b i t of m a n y t e r r e s t r i a l s p e c i e s t h a t it m a y s e r v e a s a n e x t r a r e t m a l p h o t o t r a n s d u c e r ( 1 5 , 1 6 ) . M o r e o v e r , H a r d e m a n g l a n d is m u c h h k e t h e p i n e a l g l a n d i n b e i n g c a p a b l e of s y n t h e s i z i n g m e l a t o n m . T h e e n z y m e s n e c e s s a r y f o r m e l a t o n m p r o d u c t i o n , 1. e . , Na c e t y l t r a n s f e r a s e a n d h y d r o x y u n d o l e - O - m e t h y l t r a n s f e r a s e , a r e b o t h f o u n d to b e active in the rodent Hardeman gland (17,18). Furthermore, melatonm synthesis i n t h e r a t H a r d e m a n g l a n d is c h a r a c t e r i z e d b y c i r c a d i a n r h y t h m s ( 1 9 , 2 0 ) . H o w e v e r , t h e r o l e f o r m e l a t o n m i n t h e H a r d e m a n g l a n d r e m m n s to b e e s t a b h s h e d We h a v e f u r t h e r i n v e s t i g a t e d t h e p r e s e n c e of m e l a t o n m b i n d i n g s i t e s i n t h e r a t Hardeman gland using [125I]melatonm, and comparing the results with those o b t m n e d i n med3an e r m n e n c e . O u r r e s u l t s c o n f i r m t h e p r e s e n c e of m e l a t o n m b i n d i n g sites in median ermnence and provide the first exudence that melatonm can d i r e c t l y i n t e r a c t w i t h s t r u c t u r e s from t h e r a t H a r d e m a n g l a n d . Material a n d M e t h o d s
A d u l t male W l s t a r r a t s b o r n i n o u r a n i m a l f a c l h t y w e r e u s e d . A n i m a l s r e c e i v e d food a n d w a t e r a d h b l t u m a n d w e r e e x p o s e d to a n a u t o m a t i c a l l y r e g u l a t e d h g h t d a r k (LD) c y c l e of 1 4 : 1 0 ; t h e h g h t s w e r e t u r n e d off d m l y f r o m 2000 t h r o u g h 0600 h . T h e a n i m a l s w e r e k i l l e d b y d e c a p i t a t i o n b e t w e e n 1600 a n d 1800 h a n d t h e i r m e d i a n e n u n e n c e s (ME) a n d H a r d e m a n g l a n d s (HG) w e r e r a p i d l y d i s s e c t e d . T h e t i s s u e s w e r e h o m o g e n i z e d i n 50 mM T m s - H C l , pH 7 4, w i t h 4 mM CaCl2 a n d c e n t r i f u g e d a t 30.000 x g f o r 30 n u n . P e l l e t s ( c r u d e m e m b r a n e s ) w e r e w a s h e d twice wuth h o m o g e n i z a t i o n b u f f e r a n d t h e n r e s u s p e n d e d . C r u d e m e m b r a n e s w e r e s t o r e d at -80 C u n t i l a s s a y P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d b y t h e m e t h o d of L o w r y (21) u s i n g b o v i n e s e r u m a l b u n u n a s s t a n d a r d . F o r b i n d i n g r e a c t i o n s , c r u d e m e m b r a n e s ( u p to 1.5 mg p r o t e m / m l ) w e r e i n c u b a t e d w i t h [ 1 2 5 I ] m e l a t o n m (50-800 pM) m 50 mM T m s - H C I , pH 7 . 4 , c o n t m m n g 4 mM CaC12 m a t o t a l r e a c t i o n v o l u m e of 100 ~l. T h e n u x t u r e was i n c u b a t e d m a s h a k e r b a t h a t t h e m d m a t e d t e m p e r a t u r e s a n d t i m e s . B o u n d [ 1 2 5 ] m e l a t o n m was s e p a r a t e d from u n b o u n d r a d i o a c t i v i t y b y f i l t r a t i o n t h r o u g h Whatman GF]B g l a s s fiber filters under negative pressure S p e c i f i c b i n d i n g was c a l c u l a t e d as t h e d i f f e r e n c e b e t w e e n t o t a l a n d n o n s p e c i f m b i n d i n g ( b i n d i n g i n t h e p r e s e n c e of 10 p_M m e l a t o n m ) . F o r p h a r m a c o l o g i c a l c h a r a c t e r i z a t i o n , some s a m p l e s w e r e i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s of d i f f e r e n t d r u g s . Melatonm, (-)-moproterenol, L-phenylephrme, (-)-norepmephrme, DLp r o p r a n o l o l , a n d p r a z o s m w e r e p u r c h a s e d f r o m Sigma C h e n u c a l Co. ( S t . L o u m , MO, U S A ) . [ 1 2 5 I ] N a was p u r c h a s e d from t h e R a d l o c h e n n c a l C e n t r e ( A m e r s h a m , U K ) . M e l a t o m n was l o d m a t e d b y t h e m e t h o d of V a k k u m et al ( 2 2 ) , a n d p u r i f m d b y f i l t r a t i o n t h r o u g h a s l h c a gel c o l u m n ( 2 3 ) . [ 1 2 5 I ] m e l a t o m n (2175 Cl/mmol) was g r e a t e r t h a n 95% p u r e a n d i t was s t a b l e f o r a t l e a s t 2 m o n t h s . O t h e r r e a g e n t s w e r e of a n a l y t m a l g r a d e Unless otherwuse specified, r e s u l t s are e x p r e s s e d as the mean + s t a n d a r d e r r o r (SEM) of e x p e m m e n t s p e r f o r m e d m t m p h c a t e . S a t u r a U o n s t u d m s w e r e a n a l y z e d u s i n g t h e EBDA/LIGAND p r o g r a m ( 2 4 ) . Results
F i g u r e 1 d e p m t s t h e tlme c o u r s e of s p e c i f m b m c h n g of [ 1 2 5 I ] m e l a t o n m to e l t h e r m e d l a n e n u n e n c e ( t o p ) o r H a r d e r l a n g l a n d ( b o t t o m ) c r u d e m e m b r a n e s a t 0, 15 o r 37°C. T h e r a t e of b i n d i n g was more r a p i d a t 37°C c o m p a r e d w l t h t h a t a t 0 o r 4°C. A t 37oC, b i n d i n g r e a c h e d maxlmal p e a k l e v e l s a t 60 a n d 30 n u n f o r m e d l a n e n u n e n c e
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Rat Harderlan Gland Melaton±n Receptors
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a n d H a r d e m a n g l a n d , r e s p e c t i v e l y . B i n d i n g r e m m n e d s t a b l e f o r a t l e a s t 30 n u n , decreasing sigmfmantly after 2 hour mcubat]on. Therefore, further expemments w i t h e i t h e r t m s u e w e r e p e r f o r m e d a t 37oC f o r 60 n u n to p r o w d e a m a m m a l b m d a n g . T h e r e was a p r o p o r t i o n a l r e l a t l o n s t u p b e t w e e n t h e e x t e n t of b i n d i n g of [ 1 2 5 I ] m e l a t o m n to r a t h e r m e d i a n e r m n e n c e o r H a r d e m a n g l a n d a n d t h e p r o t e i n c o n c e n t r a t ] o n m t h e i n c u b a t i o n n u x t u r e u p to 1 5 m g / m l ( F i g . 2 ) . Followung e x p e m m e n t s w e r e p e r f o r m e d w i t h 1 m g / m l of p r o t e i n c o n c e n t r a t i o n .
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Time c o u r s e of speclfm binding of [125I]melatonm to medmn eminence or Hardeman gland crude membranes. M e m b r a n e s (1 0 m g / m l ) were incubated with [ 125I ] m e l a t o n m ( 150 pM) a t 0 ( I - - a ) , 15 (&--A), o r 3 7 ° ( S - - S ) C; t h e n , ]ncubat]on was interrupted at the indicated times. Each p o i n t ]s t h e m e a n _+ SEM of 3 experiments performed m tr]phcate.
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mg.~ [ 125I ] m e l a t o m n b i n d i n g to e i t h e r median enunence (O--O) or Hardeman gland (O--O) crude membranes as a f u n c t ] o n of p r o t e i n concentration. M e m b r a n e s ( u p to I. 5 mg/ml) were incubated wlth [125I]melatonm at 37 ° C f o r 60 n u n . Each p o i n t m t h e m e a n _+ SEM of 3 experzments performed in
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Rat Harderian Gland Melatonin Receptors
Vol. 48, No. 12, 1991
S a t u r a b l h t y of [ 1 2 5 I ] m e l a t o n m b i n d i n g t o t h e m e m b r a n e s f r o m m e d i a n e n u n e n c e a n d H a r d e m a n g l a n d w a s d e t e r n u n e d u s i n g a r a n g e o f l i g a n d c o n c e n t r a t i o n f r o m 25 to 1000 pM ( F i g . 3 ) . S p e c i f i c b i n d i n g r e a c h e d i t s p l a t e a u a t 400 pM [ 125I ] m e l a t o n m . S c a t c h a r d a n a l y m s (25) i n d i c a t e d a s i n g l e c l a s s of b i n d i n g s i t e s . C o m p u t e r - a s m s t e d a n a l y s i s of t h e d a t a y m l d e d a K d of 170 + 40 pM i n m e d i a n e m i n e n c e a n d 190 _+ 50 pM in H a r d e r ] a n g l a n d . T h e b i n d i n g c a p a c i t y in t h e m e d i a n e n u n e n c e p r e p a r a t i o n w a s 8 . 0 + 1 , 2 f m o l / m g p r o t e i n ; in t h e H a r d e r ] a n g l a n d preparatmn it was 4 0 + 0.8 fmol/mg protein. I n c o m p e t , h o n s t u d i e s ( F i g . 4 ) , t h e s p e c i f i c b i n d i n g of [ 1 2 5 I ] m e l a t o n m to m e d i a n e n u n e n c e o r H a r d e r ] a n g l a n d w a s l n t u b ] t e d b y i n c r e a s i n g c o n c e n t r a t i o n of n a t i v e m e l a t o m n , 50% m t u b ] t ] o n was o b s e r v e d a t a b o u t 702 a n d 422 nM f o r m e d m n e r m n e n c e a n d H a r d e r ] a n g l a n d , r e s p e c t i v e l y . A d d i t i o n a l l y , t h e [ 125I ] m e l a t o n m b i n d i n g t o t h e c r u d e m e m b r a n e s w a s n o t a f f e c t e d b y t h e a d d i t i o n of d i f f e r e n t drugs that may (or not) modify rat pineal melatonm production Thus, table I shows that neither norepmephr]ne, ]soproterenol (a g-adrenerg]c agomst), phenylephr]ne (an a-adrenerg]c agomst), propranolol (a B-adrenergm blocker), nor prazosm (an a-adrenergm blocker) modified melatonm binding.
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Fig. 3 C o n c e n t r a t i o n d e p e n d e n c e of [125I ] m e l a t o n m b i n d i n g . M e d i a n e n u n e n c e ( O - - O ) o r H a r d e r ] a n g l a n d ( O - - O ) c r u d e m e m b r a n e s (1 0 m g / m l ) w e r e i n c u b a t e d w i t h [ 1 2 5 I ] m e l a t o n m ( u p to 1 nM) a t 37oC f o r 60 nun E a c h p o i n t is t h e m e a n + SEM of 3 e x p e r ] m e n t s p e r f o r m e d m t r ] p h c a t e . I n s e t : S c a t c h a r d a n a l y m s of t h e d a t a . Discussion
Melatonm-bmdmg rotes have been mmnly descr]bed m a relatively few m o r p h o l o g m a U y a n d f u n c t i o n a l l y d m t m c t a r e a s of t h e b r a i n , p l t m t a r y , a n d r e t i n a (3-14). The present study, however, revealed, for the first time, specffm [ 125I ] m e l a t o n m b i n d i n g m t h e r a t H a r d e r ] a n g l a n d , a t u b u l o a l v e o l a r g l a n d l o c a t e d w ] t t n n t h e o c u l a r o r b i t of m a n y r o d e n t s . A s m u l a r s t u d y w a s d e v e l o p e d m m e d i a n e n u n e n c e w h e r e i t h a s p r e v i o u s l y b e e n i n d i c a t e d a s a t a r g e t rote f o r m e l a t o n m .
Vol. 48, No. 12, 1991
Rat Harderian Gland Melatonin Receptors
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Inh]bition of [ 125I ] m e l a t o n i n b m d m g to m e d m n e m i n e n c e ( O -O) or H a r d e r l a n gland (O--O) crude membranes by unlabeled melatomn. M e m b r a n e s (1 0 m g l m l ) were incubated with [ 125I ] m e l a t o m n (150 pM) and unlabeled melatonm ( u p to 10 p.M) a t 37oC f o r 60 n u n Each p o i n t m t h e mean + SEM of 3 experiments performed m tmphcate.
- LOG MELATONIN CONCENTRATION(M)
Table I. Competlt]on of unlabeled melatonm (MEL), norepmephrme (NE), isoproterenol ( ISO), phenylephrme (PHE), propranolol (PRO), and prazosm (PRA) for [1251]melatonm binding rotes in m e d m n eminence (ME) and Harderlan gland (HG) crude membranes. Membranes (1.0 mg/ml) were incubated ~uth [125I]melatomn at 37oC for 60 nun m the absence ( C O N ) or presence of the drugs (1 p.M).
CON MEL NE ISO PHE PRO PRA
ME
HG
100 <5 94 112 92 100 98
I00 <5 98 103 101 91 93
Results are expressed as the percentage of maxnnal binding m the absence of the drugs. Other two experiments gave mnnlar results.
T h e binding charactermt]cs of the Harderlan gland or medlan ennnence for [1251]melatonm were exannned using crude membranes. [1251]melatomn b m c h n g sites m both median enunence and H a r d e r m n gland were found to exbablt propert]es such as dependence on tLrne, temperature, m e m b r a n e concentration, saturablhty, and specfflmty m the lnteraclnon with the hormone. Only one class of lugh-afflmty b m c h n g sites were found in both t]ssues. Thus, the K d of the m e d m n enunence binding rotes descrlbed in tlus article (170 pM) is in the range of serum melatonln concentratlon, and mnnlar to that desembed b y other authors m the rat (5,6,9,26). Interestingly, Harderian gland binding rotes also exlublt a
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l u g h a f f m l t y (190 p M ) , a n d p r o b a b l y r e c o g n l z e n o t o n l y m e l a t o n m c o n t m n e d in t h e gland (17-20) but also serum melatonin. Melatonm concentration m rodent blood a t n l g h t a r e m t h e p l e o m o l a r to low n a n o m o l a r r a n g e ( 2 7 , 2 8 ) , c o m p a r a b l e t o t h e K d v a l u e s f o r h l g h - a f f l m t y [ 1251 ] m e l a t o n m b m c h n g s l t e s d e t e r n u n e d m t l u s s t u d y . I n c o m p e t l t l o n s t u d l e s , [ 1251 ] m e l a t o n l n b l n d m g was m l u b l t e d b y u n l a b e l e d m e l a t o n m . A s d e s c r i b e d b y o t h e r a u t h o r s , a d r e n e r g u e a g o m s t s a n d b l o c k e r s d i d n o t mod3fy [ 1251 ] m e l a t o n l n b i n d i n g ( 9 ) . T h e p r e s e n c e of h l g h - a f f m l t y [ 1 2 5 I ] m e l a t o m n b i n d i n g s l t e s m m e d m n e n n n e n c e c o n f i r m s e a r h e r o b s e r v a t l o n s t h a t i s o n e of t h e m a j o r s l t e s of [ 1 2 5 1 ] m e l a t o n l n In rodent brmn (5-13,26). The medlan enunenee m critically and mtlmately revolved m h y p o t h a l a r m c - h y p o p h y s l a l f u n c t l o n , mak3ng i t t e m p t i n g t o s p e c u l a t e t h a t t l u s r e g i o n i s a p r l m e s l t e f o r m e l a t o m n e f f e c t s on r e p r o d u c t l o n . I n f a c t , m e l a t o n m influences the actlwty of t h e h y p o t h a l a n n c - p l t u l t a r y - g o n a d a l axm and tlmes s e a s o n a l c h a n g e s m r e p r o d u c t i v e f u n e t l o n ( 1 , 2 7 ) . T h u s , m e l a t o m n r e c e p t o r s in t h e m e d i a n e n n n e n c e m a y p l a y a n i m p o r t a n t r o l e m m e d m t m g m e l a t o m n e f f e c t s on reproductlve function. On t h e o t h e r h a n d , t h e p r e s e n c e of [ 1 2 5 1 ] m e l a t o n m b i n d i n g s i t e s m H a r d e r l a n g l a n d a r e a l s o d e s c r i b e d . H a r d e r l a n g l a n d i s b e h e v e d to p l a y a r o l e a s a n extraretmal phototransducer. Thus, removing Harderlan glands m neonatal rats p r e v e n t h g h t - l n d u c e d e f f e c t s on p i n e a l l n d o l e a n u n e m e t a b o h s m ( 1 5 , 1 6 ) . M o r e o v e r , there is a close relatlonslup between Harderlan gland and plneal gland, since Harderlan gland, hke pineal gland, is also capable of synthesizing melatomn, and it conteuns the enzymes necessary for melatonm productlon (17-20). In the last f e w y e a r s , some r e s u l t s h a v e s h o w n n e w s m u l a r l t l e s b e t w e e n p i n e a l g l a n d a n d H a r d e r l a n g l a n d s i n c e n y c t o h e m e r a l v a r m t l o n s m t y p e II t h y r o x i n e 5 ' - d e l o d m a s e , e n z y m e t h a t c o n v e r t s t h y r o x i n e i n t r n o d o t h y r o n m e , h a v e b e e n i d e n t l f l e d in b o t h g l a n d s ( 2 8 - 3 0 ) . A l t h o u g h t h e r o l e f o r m e l a t o n m m t h e H a r d e r l a n g l a n d reme3ns to b e e s t a b h s h e d , p i n e a l g l a n d s e e m s to i n f l u e n c e t h e m e l a t o n l n p r o d u c i n g c a p a c i t y of t h e H a r d e r m n g l a n d . T h u s , p m e a l e c t o m y h a s b e e n s h o w n to e l e v a t e m e l a t o n m l e v e l s m t h e r a t H a r d e ~ a n g l a n d w i t h o u t i n t e r f e r i n g w]th t h e e l r c a d m n r h y t h m (19). Hence the [125I]melatonm binding sltes m the Harderlan gland can medlate t h e e f f e c t of m e l a t o n l n l n b 3 b l t l n g t h e H a r d e r l a n g l a n d m e l a t o m n p r o d u c t l o n . I n c o n c l u s l o n , H a r d e r l a n g l a n d c r u d e m e m b r a n e s e x l u b l t s p e c i f i c [ 1251 ] m e l a t o n m b i n d i n g s l t e s w i t h s i m i l a r c h a r a c t e r l s t l c s t o m e l a t o m n r e c e p t o r s In m e d l a n enunence. Tins receptor may recognize not only melatonln contained in the gland b u t a l s o s e r u m m e l a t o n m , a n d m a y med3ate t h e e f f e c t of m e l a t o m n on H a r d e r l a n gland metabohsm.
Acknowledgments S u p p o r t e d b y g r a n t s f r o m t h e C o n s e l e r i a d e Educac16n y C1encla d e la J u n t a d e A n d a l u c l a ( S E T . 8 8 ) a n d F o n d o d e I n v e s t l g a c l o n e s S a m t a r l a s d e la S e g u r l d a d Soelal (90/0688).
References 1. L T A M A R K I N , N . G . L E F E B V R E , C.W. H O L L I S T E R , a n d B . D . GOLDMAN, E n d o c r i n o l o g y 101 631-634 (1977) 2. R . J . R E I T E R , E n d o c r i n e R e v . 1 109-131 (1980) 3. M . L . D U B O C O V I C H , J . P h a r m a e o l . E x p . T h e r . 234 395-401 (1985) 4. M . L . D U B O C O V I C H , a n d J . S T A K A H A S H I , P r o c . N a t l . A c a d . SCl. U . S . A 84 3916-3920 (1987) 5. J . V A N E C E K , A . P A V L I K , a n d H. I L L N E R O V A , B r m n R e s . 435 359-362 (1987) 6. J . V A N E C E K , J . N e u r o c h e m . 5_! 1436-1440 (1988)
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7. L.M. WILLIAMS, and P.J. MORGAN, J. Endoclunol. 119 RI-R3 (1988) 8. L.M. WILLIAMS, J Molec. Endocrmol. 3 71-75 (1989) 9. D R. WEAVER, S.C. RIVKEES, and S.M. REPPERT, J. Neuroscl. 9 2581-2590 (1989) 10. M.J. DUNCAN, J S TAKAHASHI,and M. DUBOCOVICH, Endoclunology125 1011-1018 (1989) 11.L.M. WILLIAMS, P.J. MORGAN, M.H. HASTINGS, W. LAWSON, G. DAVIDSON, and H.E. HOWWELL, J. Neuroendocrmol. 1 315-320 (1989) 12. M.M. R E V I E R S , J.P. R A V A U L T , Y TILLET, and J. PI~LLETIER, Neuroscl. Lett. 100 89-93 (1989) 13. P J M O R G A N , L.M. WILLIAMS, G D A V I D S O N , W L A W S O N , and E. H O W E L L , J. Neuroendocrlnol. 1 1-4 (1989) 14. S.M R E P P E R T , D.R. W E A V E R , S.A R I V K E E S , and E.G. S T O P A Science 242 78-81 (1988) 15. J. O L C E S E , and A. W E S C H E , Comp. B1ochem Physlol. 93A 655-665 (1989) 16. L W E T T E R B E R G , E. G E L L E R , and A Y U W I L E R , Science 168 884-885 (1970) 17. B V I V I E N - R O E L S , P P E V E T , M P. D U B O I S , J A R E N D T , and G.M. B R O W N , Cell Tins. Res 217 105-115 (1981) 18.A M E N E N D E Z - P E L A E Z , C. S A N T A N A , K A. H O W E S , I S A B R Y , and R.J. REITER, J. Pineal Res. 5 293-300 19. R.J. REITER, B A R I C f f A R D S O N , S.A M A T T H E W S , S.J L A N E , and B.N. FERGUSON, Life Sin. 32 1229-1236 (1983) 20. B.H. W H I T E , K. M O S H E R , and S. B I N K L E Y , J Pineal Res. 1 129-138 (1984) 21 O H. L O W R Y , N.J R O S E N B R O U G H , A.L. F A R R , and R.J. RANDALl,, J Biol. C h e m 193 265-275 (1951) 22 O. V A K K U R I , J L E P P A L U O T O , and O. V U O L T E E N A H O , Acta Endocrmol. 106 152-158 (1984) 23.L.P NILES, D S P I C K E R I N G , and B.G. S A Y E R , B1ochem. B1ophys. Res C o m m u n . 147 949-956 (1987) 24. G . A M C P H E R S O N , J Pharm. Methods 14 213-228 (1985) 25. G. S C A T C H A R D , Ann. N. Y Acad. Sin. 51 660-672 (1949) 26. Jo VANECEK, N e u r o e n d o c r m o l o g y 48 201-203 (1988) 2 7 . L . TAMARKIN, C . J . BAIRD, a n d O . F . X . ALMEIDA, Smence 227 714-720 (1985) 26. J M. GUERRERO, M. PUIG-DOMINGO, G M. VAUGHAN, and R.J. REITER, Life Scl. 41 1179-1185 (1987) 29 J.M. GUERRERO, M. PUIG-DOMINGO, and R.J. REITER, Endocrinology122 236-241 (1988) 30. C. OSUNA, A. RUBIO, R. GOBERNA, and J.M. GUERRERO, L i f e Scl. 4_66 1945-1951 (1990)
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L o p e z - G o n z a l e z , J . R . C a l v e , A . R u b l e , R. G o b e r n a and J.M. Guerrero*
T h e U m v e r s l t y of S e w U e S c h o o l of M e d l c m e , D e p a r t m e n t of M e d i c a l B 1 o e h e n u s t r y a n d M o l e c u l a r B i o l o g y , A v d a S a n c h e z P1zjuan 4, 4 1 0 0 9 - S e v l l l a , S p m n (Recelved in flnal form January 16, 1991) S,,mm~ l
T h e c h a r a c t e r l z a t l o n of s p e c i f i c m e l a t o n m b i n d i n g s i t e s m t h e H a r d e r m n g l a n d ( H G ) a n d m e d i a n e r m n e n e e (ME) of t h e r a t w a s s t u d i e d u s i n g [ 1 2 5 1 ] m e l a t o n m B i n d i n g of m e l a t o m n to m e m b r a n e c r u d e p r e p a r a t l o n s of b o t h t l s s u e s w a s d e p e n d e n t on time a n d t e m p e r a t u r e . T h u s , maximal b i n d i n g w a s o b t m n e d a t 37° C a f t e r 30-60 m m l n e u b a t l o n . B i n d i n g w a s a l s o d e p e n d e n t on protein concentration (up to 1.5 mg/ml). The specific binding of [ 1 2 5 1 ] m e l a t o n m w a s s a t u r a b l e , e x h l b l t m g o n l y o n e c l a s s of b i n d i n g s i t e s in b o t h t i s s u e s . T h e d 3 s s o e l a t l o n c o n s t a n t s ( K d ) w e r e 170 a n d 190 pM f o r ME a n d H G , r e s p e c t i v e l y . T h e e o n c e n t r a t l o n of t h e b i n d i n g s i t e s m ME w a s 8 f m o l ] m g p r o t e l n , a n d in t h e HG 4 f m o l J m g p r o t e i n . I n c o m p e t i t i o n s t u d i e s , b i n d i n g of [ 1 2 5 1 ] m e l a t o m n t o ME o r HG w a s l n h l b l t e d b y i n c r e a s i n g c o n c e n t r a t i o n o f n a t i v e m e l a t o n m ; 50% i n l u b l t l o n w a s o b s e r v e d a t a b o u t 702 a n d 422 nM f o r ME a n d HG, r e s p e c t i v e l y AddltlonaUy, the [1251]melatonm b l n d m g t o t h e c r u d e m e m b r a n e s w a s n o t a f f e c t e d b y t h e a d d l t l o n of d i f f e r e n t drugs such as norepmephrlne, lSOproterenol, phenylephrme, propranolol, or prazosln T h e r e s u l t s c o n f i r m t h e p r e s e n c e of m e l a t o n m b i n d i n g s l t e s m m e d i a n e r m n e n c e a n d s h o w , f o r t h e f i r s t t i m e , t h e e x i s t e n c e of m e l a t o n l n b i n d i n g s i t e s in t h e H a r d e r l a n g l a n d T h e p i n e a l h o r m o n e m e l a t o n l n t r a n s d u e e s t h e e f f e c t o f p h o t o p e r l o d on t h e neuroendocrme system and thus regulates seasonal rhythms, notably reproduetlon (1,2). The means by whlch melatomn brings about these diverse effects are not well k n o w n . H o w e v e r , t h e i n t r o d u c t l o n of a l u g h - a f f l m t y , h i g h s p e c i f i c l i g a n d , [ 1 2 5 1 ] m e l a t o n m h a s r e s u l t e d i n t h e l d e n t i f l c a t l o n of p u t a t i v e s l t e s of a c t l o n . H i g h affinity melatonm receptors have been ehar~acterlzed m the rabblt and chicken r e t i n a , p r o v i d i n g e w d e n c e f o r a l o c a l s l t e of a e t l o n , a s m e l a t o n m i s p r o d u c e d i n the retina (3,4) Melatonm-bmdmg sltes have also been identlfled m the rat (59 ) , h a m s t e r ( 9 - 1 1 ) , s h e e p ( 1 2 , 1 3 ) , a n d h u m a n (14) h y p o t h a l a m u s , r e v e a h n g c e n t r a l s i t e s of a c t i o n f o r m e l a t o m n o f p i n e a l Orlgun. T h e s e s t u d l e s i d e n t 3 f i e d t h e suprachlasmatlc nucleus and median ermnence reguons as contmnlng binding sites. Nevertheless, using autoradlography at the hght nueroseope level, the melatonln b m d l n g p r e v i o u s l y a s c r i b e d t o m e d i a n e m i n e n c e h a s s h o w n t o b e s p e c i f i c to t h e p a r s t u b e r a l i s of t h e p i t u i t a r y m t h e r a t a n d s h e e p ( 7 , 8 , 1 2 , 1 3 ) .
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I n t h e p r e s e n t s t u d y , we c h a r a c t e m z e d m e l a t o n m b i n d i n g s i t e s i n a c r u d e m e m b r a n e f r a c t i o n i s o l a t e d from r a t H a r d e m a n g l a n d a n d m e d i a n e n u n e n c e . T h e H a r d e m a n g l a n d is a t u b u l o a l v e o l a r g l a n d l o c a t e d w l t t u n t h e o c u l a r o r b i t of m a n y t e r r e s t r i a l s p e c i e s t h a t it m a y s e r v e a s a n e x t r a r e t m a l p h o t o t r a n s d u c e r ( 1 5 , 1 6 ) . M o r e o v e r , H a r d e m a n g l a n d is m u c h h k e t h e p i n e a l g l a n d i n b e i n g c a p a b l e of s y n t h e s i z i n g m e l a t o n m . T h e e n z y m e s n e c e s s a r y f o r m e l a t o n m p r o d u c t i o n , 1. e . , Na c e t y l t r a n s f e r a s e a n d h y d r o x y u n d o l e - O - m e t h y l t r a n s f e r a s e , a r e b o t h f o u n d to b e active in the rodent Hardeman gland (17,18). Furthermore, melatonm synthesis i n t h e r a t H a r d e m a n g l a n d is c h a r a c t e r i z e d b y c i r c a d i a n r h y t h m s ( 1 9 , 2 0 ) . H o w e v e r , t h e r o l e f o r m e l a t o n m i n t h e H a r d e m a n g l a n d r e m m n s to b e e s t a b h s h e d We h a v e f u r t h e r i n v e s t i g a t e d t h e p r e s e n c e of m e l a t o n m b i n d i n g s i t e s i n t h e r a t Hardeman gland using [125I]melatonm, and comparing the results with those o b t m n e d i n med3an e r m n e n c e . O u r r e s u l t s c o n f i r m t h e p r e s e n c e of m e l a t o n m b i n d i n g sites in median ermnence and provide the first exudence that melatonm can d i r e c t l y i n t e r a c t w i t h s t r u c t u r e s from t h e r a t H a r d e m a n g l a n d . Material a n d M e t h o d s
A d u l t male W l s t a r r a t s b o r n i n o u r a n i m a l f a c l h t y w e r e u s e d . A n i m a l s r e c e i v e d food a n d w a t e r a d h b l t u m a n d w e r e e x p o s e d to a n a u t o m a t i c a l l y r e g u l a t e d h g h t d a r k (LD) c y c l e of 1 4 : 1 0 ; t h e h g h t s w e r e t u r n e d off d m l y f r o m 2000 t h r o u g h 0600 h . T h e a n i m a l s w e r e k i l l e d b y d e c a p i t a t i o n b e t w e e n 1600 a n d 1800 h a n d t h e i r m e d i a n e n u n e n c e s (ME) a n d H a r d e m a n g l a n d s (HG) w e r e r a p i d l y d i s s e c t e d . T h e t i s s u e s w e r e h o m o g e n i z e d i n 50 mM T m s - H C l , pH 7 4, w i t h 4 mM CaCl2 a n d c e n t r i f u g e d a t 30.000 x g f o r 30 n u n . P e l l e t s ( c r u d e m e m b r a n e s ) w e r e w a s h e d twice wuth h o m o g e n i z a t i o n b u f f e r a n d t h e n r e s u s p e n d e d . C r u d e m e m b r a n e s w e r e s t o r e d at -80 C u n t i l a s s a y P r o t e i n c o n c e n t r a t i o n was d e t e r m i n e d b y t h e m e t h o d of L o w r y (21) u s i n g b o v i n e s e r u m a l b u n u n a s s t a n d a r d . F o r b i n d i n g r e a c t i o n s , c r u d e m e m b r a n e s ( u p to 1.5 mg p r o t e m / m l ) w e r e i n c u b a t e d w i t h [ 1 2 5 I ] m e l a t o n m (50-800 pM) m 50 mM T m s - H C I , pH 7 . 4 , c o n t m m n g 4 mM CaC12 m a t o t a l r e a c t i o n v o l u m e of 100 ~l. T h e n u x t u r e was i n c u b a t e d m a s h a k e r b a t h a t t h e m d m a t e d t e m p e r a t u r e s a n d t i m e s . B o u n d [ 1 2 5 ] m e l a t o n m was s e p a r a t e d from u n b o u n d r a d i o a c t i v i t y b y f i l t r a t i o n t h r o u g h Whatman GF]B g l a s s fiber filters under negative pressure S p e c i f i c b i n d i n g was c a l c u l a t e d as t h e d i f f e r e n c e b e t w e e n t o t a l a n d n o n s p e c i f m b i n d i n g ( b i n d i n g i n t h e p r e s e n c e of 10 p_M m e l a t o n m ) . F o r p h a r m a c o l o g i c a l c h a r a c t e r i z a t i o n , some s a m p l e s w e r e i n c u b a t e d w i t h v a r y i n g c o n c e n t r a t i o n s of d i f f e r e n t d r u g s . Melatonm, (-)-moproterenol, L-phenylephrme, (-)-norepmephrme, DLp r o p r a n o l o l , a n d p r a z o s m w e r e p u r c h a s e d f r o m Sigma C h e n u c a l Co. ( S t . L o u m , MO, U S A ) . [ 1 2 5 I ] N a was p u r c h a s e d from t h e R a d l o c h e n n c a l C e n t r e ( A m e r s h a m , U K ) . M e l a t o m n was l o d m a t e d b y t h e m e t h o d of V a k k u m et al ( 2 2 ) , a n d p u r i f m d b y f i l t r a t i o n t h r o u g h a s l h c a gel c o l u m n ( 2 3 ) . [ 1 2 5 I ] m e l a t o m n (2175 Cl/mmol) was g r e a t e r t h a n 95% p u r e a n d i t was s t a b l e f o r a t l e a s t 2 m o n t h s . O t h e r r e a g e n t s w e r e of a n a l y t m a l g r a d e Unless otherwuse specified, r e s u l t s are e x p r e s s e d as the mean + s t a n d a r d e r r o r (SEM) of e x p e m m e n t s p e r f o r m e d m t m p h c a t e . S a t u r a U o n s t u d m s w e r e a n a l y z e d u s i n g t h e EBDA/LIGAND p r o g r a m ( 2 4 ) . Results
F i g u r e 1 d e p m t s t h e tlme c o u r s e of s p e c i f m b m c h n g of [ 1 2 5 I ] m e l a t o n m to e l t h e r m e d l a n e n u n e n c e ( t o p ) o r H a r d e r l a n g l a n d ( b o t t o m ) c r u d e m e m b r a n e s a t 0, 15 o r 37°C. T h e r a t e of b i n d i n g was more r a p i d a t 37°C c o m p a r e d w l t h t h a t a t 0 o r 4°C. A t 37oC, b i n d i n g r e a c h e d maxlmal p e a k l e v e l s a t 60 a n d 30 n u n f o r m e d l a n e n u n e n c e
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a n d H a r d e m a n g l a n d , r e s p e c t i v e l y . B i n d i n g r e m m n e d s t a b l e f o r a t l e a s t 30 n u n , decreasing sigmfmantly after 2 hour mcubat]on. Therefore, further expemments w i t h e i t h e r t m s u e w e r e p e r f o r m e d a t 37oC f o r 60 n u n to p r o w d e a m a m m a l b m d a n g . T h e r e was a p r o p o r t i o n a l r e l a t l o n s t u p b e t w e e n t h e e x t e n t of b i n d i n g of [ 1 2 5 I ] m e l a t o m n to r a t h e r m e d i a n e r m n e n c e o r H a r d e m a n g l a n d a n d t h e p r o t e i n c o n c e n t r a t ] o n m t h e i n c u b a t i o n n u x t u r e u p to 1 5 m g / m l ( F i g . 2 ) . Followung e x p e m m e n t s w e r e p e r f o r m e d w i t h 1 m g / m l of p r o t e i n c o n c e n t r a t i o n .
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Time c o u r s e of speclfm binding of [125I]melatonm to medmn eminence or Hardeman gland crude membranes. M e m b r a n e s (1 0 m g / m l ) were incubated with [ 125I ] m e l a t o n m ( 150 pM) a t 0 ( I - - a ) , 15 (&--A), o r 3 7 ° ( S - - S ) C; t h e n , ]ncubat]on was interrupted at the indicated times. Each p o i n t ]s t h e m e a n _+ SEM of 3 experiments performed m tr]phcate.
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mg.~ [ 125I ] m e l a t o m n b i n d i n g to e i t h e r median enunence (O--O) or Hardeman gland (O--O) crude membranes as a f u n c t ] o n of p r o t e i n concentration. M e m b r a n e s ( u p to I. 5 mg/ml) were incubated wlth [125I]melatonm at 37 ° C f o r 60 n u n . Each p o i n t m t h e m e a n _+ SEM of 3 experzments performed in
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Rat Harderian Gland Melatonin Receptors
Vol. 48, No. 12, 1991
S a t u r a b l h t y of [ 1 2 5 I ] m e l a t o n m b i n d i n g t o t h e m e m b r a n e s f r o m m e d i a n e n u n e n c e a n d H a r d e m a n g l a n d w a s d e t e r n u n e d u s i n g a r a n g e o f l i g a n d c o n c e n t r a t i o n f r o m 25 to 1000 pM ( F i g . 3 ) . S p e c i f i c b i n d i n g r e a c h e d i t s p l a t e a u a t 400 pM [ 125I ] m e l a t o n m . S c a t c h a r d a n a l y m s (25) i n d i c a t e d a s i n g l e c l a s s of b i n d i n g s i t e s . C o m p u t e r - a s m s t e d a n a l y s i s of t h e d a t a y m l d e d a K d of 170 + 40 pM i n m e d i a n e m i n e n c e a n d 190 _+ 50 pM in H a r d e r ] a n g l a n d . T h e b i n d i n g c a p a c i t y in t h e m e d i a n e n u n e n c e p r e p a r a t i o n w a s 8 . 0 + 1 , 2 f m o l / m g p r o t e i n ; in t h e H a r d e r ] a n g l a n d preparatmn it was 4 0 + 0.8 fmol/mg protein. I n c o m p e t , h o n s t u d i e s ( F i g . 4 ) , t h e s p e c i f i c b i n d i n g of [ 1 2 5 I ] m e l a t o n m to m e d i a n e n u n e n c e o r H a r d e r ] a n g l a n d w a s l n t u b ] t e d b y i n c r e a s i n g c o n c e n t r a t i o n of n a t i v e m e l a t o m n , 50% m t u b ] t ] o n was o b s e r v e d a t a b o u t 702 a n d 422 nM f o r m e d m n e r m n e n c e a n d H a r d e r ] a n g l a n d , r e s p e c t i v e l y . A d d i t i o n a l l y , t h e [ 125I ] m e l a t o n m b i n d i n g t o t h e c r u d e m e m b r a n e s w a s n o t a f f e c t e d b y t h e a d d i t i o n of d i f f e r e n t drugs that may (or not) modify rat pineal melatonm production Thus, table I shows that neither norepmephr]ne, ]soproterenol (a g-adrenerg]c agomst), phenylephr]ne (an a-adrenerg]c agomst), propranolol (a B-adrenergm blocker), nor prazosm (an a-adrenergm blocker) modified melatonm binding.
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Fig. 3 C o n c e n t r a t i o n d e p e n d e n c e of [125I ] m e l a t o n m b i n d i n g . M e d i a n e n u n e n c e ( O - - O ) o r H a r d e r ] a n g l a n d ( O - - O ) c r u d e m e m b r a n e s (1 0 m g / m l ) w e r e i n c u b a t e d w i t h [ 1 2 5 I ] m e l a t o n m ( u p to 1 nM) a t 37oC f o r 60 nun E a c h p o i n t is t h e m e a n + SEM of 3 e x p e r ] m e n t s p e r f o r m e d m t r ] p h c a t e . I n s e t : S c a t c h a r d a n a l y m s of t h e d a t a . Discussion
Melatonm-bmdmg rotes have been mmnly descr]bed m a relatively few m o r p h o l o g m a U y a n d f u n c t i o n a l l y d m t m c t a r e a s of t h e b r a i n , p l t m t a r y , a n d r e t i n a (3-14). The present study, however, revealed, for the first time, specffm [ 125I ] m e l a t o n m b i n d i n g m t h e r a t H a r d e r ] a n g l a n d , a t u b u l o a l v e o l a r g l a n d l o c a t e d w ] t t n n t h e o c u l a r o r b i t of m a n y r o d e n t s . A s m u l a r s t u d y w a s d e v e l o p e d m m e d i a n e n u n e n c e w h e r e i t h a s p r e v i o u s l y b e e n i n d i c a t e d a s a t a r g e t rote f o r m e l a t o n m .
Vol. 48, No. 12, 1991
Rat Harderian Gland Melatonin Receptors
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Inh]bition of [ 125I ] m e l a t o n i n b m d m g to m e d m n e m i n e n c e ( O -O) or H a r d e r l a n gland (O--O) crude membranes by unlabeled melatomn. M e m b r a n e s (1 0 m g l m l ) were incubated with [ 125I ] m e l a t o m n (150 pM) and unlabeled melatonm ( u p to 10 p.M) a t 37oC f o r 60 n u n Each p o i n t m t h e mean + SEM of 3 experiments performed m tmphcate.
- LOG MELATONIN CONCENTRATION(M)
Table I. Competlt]on of unlabeled melatonm (MEL), norepmephrme (NE), isoproterenol ( ISO), phenylephrme (PHE), propranolol (PRO), and prazosm (PRA) for [1251]melatonm binding rotes in m e d m n eminence (ME) and Harderlan gland (HG) crude membranes. Membranes (1.0 mg/ml) were incubated ~uth [125I]melatomn at 37oC for 60 nun m the absence ( C O N ) or presence of the drugs (1 p.M).
CON MEL NE ISO PHE PRO PRA
ME
HG
100 <5 94 112 92 100 98
I00 <5 98 103 101 91 93
Results are expressed as the percentage of maxnnal binding m the absence of the drugs. Other two experiments gave mnnlar results.
T h e binding charactermt]cs of the Harderlan gland or medlan ennnence for [1251]melatonm were exannned using crude membranes. [1251]melatomn b m c h n g sites m both median enunence and H a r d e r m n gland were found to exbablt propert]es such as dependence on tLrne, temperature, m e m b r a n e concentration, saturablhty, and specfflmty m the lnteraclnon with the hormone. Only one class of lugh-afflmty b m c h n g sites were found in both t]ssues. Thus, the K d of the m e d m n enunence binding rotes descrlbed in tlus article (170 pM) is in the range of serum melatonln concentratlon, and mnnlar to that desembed b y other authors m the rat (5,6,9,26). Interestingly, Harderian gland binding rotes also exlublt a
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Rat Harderlan Gland Melatonln Receptors
Vol. 48, No. 12, 1991
l u g h a f f m l t y (190 p M ) , a n d p r o b a b l y r e c o g n l z e n o t o n l y m e l a t o n m c o n t m n e d in t h e gland (17-20) but also serum melatonin. Melatonm concentration m rodent blood a t n l g h t a r e m t h e p l e o m o l a r to low n a n o m o l a r r a n g e ( 2 7 , 2 8 ) , c o m p a r a b l e t o t h e K d v a l u e s f o r h l g h - a f f l m t y [ 1251 ] m e l a t o n m b m c h n g s l t e s d e t e r n u n e d m t l u s s t u d y . I n c o m p e t l t l o n s t u d l e s , [ 1251 ] m e l a t o n l n b l n d m g was m l u b l t e d b y u n l a b e l e d m e l a t o n m . A s d e s c r i b e d b y o t h e r a u t h o r s , a d r e n e r g u e a g o m s t s a n d b l o c k e r s d i d n o t mod3fy [ 1251 ] m e l a t o n l n b i n d i n g ( 9 ) . T h e p r e s e n c e of h l g h - a f f m l t y [ 1 2 5 I ] m e l a t o m n b i n d i n g s l t e s m m e d m n e n n n e n c e c o n f i r m s e a r h e r o b s e r v a t l o n s t h a t i s o n e of t h e m a j o r s l t e s of [ 1 2 5 1 ] m e l a t o n l n In rodent brmn (5-13,26). The medlan enunenee m critically and mtlmately revolved m h y p o t h a l a r m c - h y p o p h y s l a l f u n c t l o n , mak3ng i t t e m p t i n g t o s p e c u l a t e t h a t t l u s r e g i o n i s a p r l m e s l t e f o r m e l a t o m n e f f e c t s on r e p r o d u c t l o n . I n f a c t , m e l a t o n m influences the actlwty of t h e h y p o t h a l a n n c - p l t u l t a r y - g o n a d a l axm and tlmes s e a s o n a l c h a n g e s m r e p r o d u c t i v e f u n e t l o n ( 1 , 2 7 ) . T h u s , m e l a t o m n r e c e p t o r s in t h e m e d i a n e n n n e n c e m a y p l a y a n i m p o r t a n t r o l e m m e d m t m g m e l a t o m n e f f e c t s on reproductlve function. On t h e o t h e r h a n d , t h e p r e s e n c e of [ 1 2 5 1 ] m e l a t o n m b i n d i n g s i t e s m H a r d e r l a n g l a n d a r e a l s o d e s c r i b e d . H a r d e r l a n g l a n d i s b e h e v e d to p l a y a r o l e a s a n extraretmal phototransducer. Thus, removing Harderlan glands m neonatal rats p r e v e n t h g h t - l n d u c e d e f f e c t s on p i n e a l l n d o l e a n u n e m e t a b o h s m ( 1 5 , 1 6 ) . M o r e o v e r , there is a close relatlonslup between Harderlan gland and plneal gland, since Harderlan gland, hke pineal gland, is also capable of synthesizing melatomn, and it conteuns the enzymes necessary for melatonm productlon (17-20). In the last f e w y e a r s , some r e s u l t s h a v e s h o w n n e w s m u l a r l t l e s b e t w e e n p i n e a l g l a n d a n d H a r d e r l a n g l a n d s i n c e n y c t o h e m e r a l v a r m t l o n s m t y p e II t h y r o x i n e 5 ' - d e l o d m a s e , e n z y m e t h a t c o n v e r t s t h y r o x i n e i n t r n o d o t h y r o n m e , h a v e b e e n i d e n t l f l e d in b o t h g l a n d s ( 2 8 - 3 0 ) . A l t h o u g h t h e r o l e f o r m e l a t o n m m t h e H a r d e r l a n g l a n d reme3ns to b e e s t a b h s h e d , p i n e a l g l a n d s e e m s to i n f l u e n c e t h e m e l a t o n l n p r o d u c i n g c a p a c i t y of t h e H a r d e r m n g l a n d . T h u s , p m e a l e c t o m y h a s b e e n s h o w n to e l e v a t e m e l a t o n m l e v e l s m t h e r a t H a r d e ~ a n g l a n d w i t h o u t i n t e r f e r i n g w]th t h e e l r c a d m n r h y t h m (19). Hence the [125I]melatonm binding sltes m the Harderlan gland can medlate t h e e f f e c t of m e l a t o n l n l n b 3 b l t l n g t h e H a r d e r l a n g l a n d m e l a t o m n p r o d u c t l o n . I n c o n c l u s l o n , H a r d e r l a n g l a n d c r u d e m e m b r a n e s e x l u b l t s p e c i f i c [ 1251 ] m e l a t o n m b i n d i n g s l t e s w i t h s i m i l a r c h a r a c t e r l s t l c s t o m e l a t o m n r e c e p t o r s In m e d l a n enunence. Tins receptor may recognize not only melatonln contained in the gland b u t a l s o s e r u m m e l a t o n m , a n d m a y med3ate t h e e f f e c t of m e l a t o m n on H a r d e r l a n gland metabohsm.
Acknowledgments S u p p o r t e d b y g r a n t s f r o m t h e C o n s e l e r i a d e Educac16n y C1encla d e la J u n t a d e A n d a l u c l a ( S E T . 8 8 ) a n d F o n d o d e I n v e s t l g a c l o n e s S a m t a r l a s d e la S e g u r l d a d Soelal (90/0688).
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