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Sigma receptors are highly concentrated in the rat pineal gland
15N
Bram ICe,earth ~0- ( 19901 158 -1¢~1)
BRES 23907
Sigma receptors are highly concentrated in the rat pineal gland Karl L R Jansen, Michael Dragunow and Richard L M Faull Department of Anatomy, Untversttv of Auckland, School of Medwme, Auckland (New Zealand) (Accepted 26 September 1989) Key words Autoradlographv, 1,3-dl-ortho-Tolyl-guamdlne, Pineal gland, cr Receptor
The dlstnbuuon of sigma (a) receptors m the rat brain was studied with autoradlography using [3H]l,3-dl-ortho-tolyl-guamdme ([3H]DTG) as a hgand The baghest concentration of ~r receptors was seen m the pineal gland, an area which has not been previously studied This result ~s of interest as both a receptors and the pineal gland have recently been shown to play a role not only m the nervous system but also m the ~mmune and endocnne systems Evidence has recently been presented that the sigma receptor may play a role in the nervous system and also the e n d o c n n e and immune systems 15 Halopertdol and some other antlpsychot~c drugs bind to o receptors, as do psychotomimeuc benzomorphan opiates, suggesting that the receptor may be mvolved m psychosis 14 Other studies have hnked the receptor to certain movement dtsorders I 16 Corticostero~ds and sex hormones have been shown to bind to o receptors 15, and these sites are found in high concentrations m the hypothalamic-pltuitary area 1°, m peripheral endocrme glands TM, and also on lymphocytes~5 19 The dtstrlbutlon of these sites m the rodent brain has been previously studted using ( + ) [3H]-3-(3-hydroxyphenyl)-N-(1-propyl)-plpendlne (3-PPP) 3 and [3H]SKF10,0477 as labels However, 3-PPP ts also a d o p a m m e type II receptor agontst 4 5 whtle SKF-10.047 also binds to the PCP site on the N-methyl-D-aspartate ( N M D A ) receptor 13 The more spectfic o-receptor hgand [3H]DTG17 has thus far been apphed to regions of the guinea pig 1° and cat brain 2, but has not been used to visuallse these sites in the rat CNS No report on the pmeal gland was included in these prevtous mvest~gattons 2 3 7 to In the present study, [3H]DTG was used to vlsuallse o receptors in the rat CNS using autoradtographic methods Wlstar rats (200-250 g) were killed by decapitation, their brams removed and lmmedmtely frozen wtth dry ice onto cryostat chucks 16~m sections were cut through the brain, with alternate brams being cut in the coronal plane o r the sagittal plane The sections were then mounted onto chrome/alum subbed slides, stored overnight at 4 °C and labelled according to the method of McLean and
Weber a°, except that secUons were premcubated in Tns-HC1 buffer for 1 h at 21 °C Briefly, following premcubation sections were incubated at 21 °C for 45 mm m 50 mM Trls-HCl (pH 7 4 at 21 °C), with 1 mg/ml bovine serum albumin (b s a ) and 4 nM [3H]DTG (specific actwity 52 3 C1/mmol, New England Nuclear) AdJacent sections were incubated with 10/~M halopendol or 10/~M D T G to assess non-specific binding Sections were then washed for 4 × 2 mln m 10 mM Tns HCI containing 1 mg/ml b s a (pH 7 4, 4 °C), dried, placed in X-ray cassettes w]th A m e r s h a m H y p e f f d m and appropriate standards for 8 weeks ° and subsequently developed and analysed as previously described 6 Sections were stained with Cresyl violet for histological examination and identification of anatomtcal areas In the resulting autoradlograms, the pineal gland was seen to have the highest levels of [3H]DTG binding m the rat brain (see Fig 1) The distribution m the rest of the brain was not markedly different from other studies, and over 95% of the binding was displaced by hatopendol indicating a htgh ratio of specific to non-specific bindmg High levels of binding were seen in the hypothalamtcp~tultary area, but these were less than those m the pineal gland In the hippocampus, [3H]DTG binding occurred over the granule and pyramidal cell layers Relatwely low levels were found in the basal gangha, m agreement w~th previous descriptions of o-receptor d~strlbutlons 3 1o The dense concentration of o-receptors m the pineal gland ~s of particular interest as the gland appears to have functions m the nervous endocrine and immune systems - - the 3 systems in whtch o receptors have been tmphcated The pmeal is, for example, revolved m the regulation of sex hormones ~2 which have been shown to
Correspondence K Jansen, Department of Anatomy, Umverslty of Auckland, School of Medlcme, Private Bag, Auckland, New Zealand 0006-8993/90/$03 50 © 1990 Elsevier Soence Pubhshers B V (Biomedical Division)
159 have
a high
affinity f o r
o r e c e p t o r s ~s
Secretion ot
m e l a t o n i n is r h y t h m i c a n d t h e h o r m o n e is i n v o l v e d m regulating
certain
rhythmic
processes
such
as
r e p r o d u c t i o n ~2 T h e o c c u r r e n c e o f t h e s e r e c e p t o r s in t h e p i n e a l p r o v i d e s f u r t h e r e v i d e n c e to s u p p o r t a p o s s i b l e role f o r t h e s e sites in e n d o c r i n e c o n t r o l
S~gma r e c e p t o r s
h a v e b e e n f o u n d m all rat e n d o c r i n e o r g a n s e x a m i n e d to d a t e ( p i t u i t a r y , a d r e n a l , testis, o v a r y a n d p l a c e n t a ) , with one study finding the highest levels overall to occur m the testis ~s T h e p i n e a l g l a n d can n o w be a d d e d to this hst T h e p i n e a l g l a n d also h a s a r o l e m t h e ~ m m u n e s y s t e m For
example,
melatonm
can
modulate
the
antibody
r e s p o n s e and antagonise the m l m u n o s u p p r e s s i v e effect of cortlcosterones
T h e r e is e v i d e n c e to s u g g e s t t h a t this
e f f e c t m a y b e m e d i a t e d b~ o p i a t e s '~ S i g m a r e c e p t o r s h a v e b e e n identified on l y m p h o c y t e s and may be mvolved m this p r o c e s s ~'
C
In c o n c l u s i o n , this s t u d y r o u n d a high c o n c e n t r a t m n o f o - r e c e p t o r s m t h e p i n e a l g l a n d , an a r e a n o t i n c l u d e d m
F
p r e v i o u s s t u d i e s , u s i n g a u t o r a d i o g r a p h y w~th [ ~ H ] D T G T h i s h n d i n g m a y h a v e c o n s i d e r a b l e s~gmficance b o t h t o r o u r u n d e r s t a n d i n g o t t h e s e r e c e p t o r s a n d ol t h e p i n e a l g l a n d a n d ~ts f u n c t i o n s
Fig 1 Autoradlographlc Vlsuallsanon of [~H]DTG binding to sigma receptors in (a) a coronal section and (c) a saglttal section of the rat brain, illustrating a high denslt~ of sigma receptors in the pineal bod,¢ (see arrows) (b) and (d) are the same sections stained with Cresvl ,dolet Bar = ~0 mm
This work was supported b~ the New Zealand Medical Research Council and the New Zealand Neurological Foundation Dr K Jansen is a training Iellow of the New Zealand Medical Research Council We are grateful to Dr Mark Sonders and Professor Eckard Weber 1or the DTG
1 Bowen, W D Walder, J M Yashar, A G , Matsumoto R R Walder F O and Lorden J F Altered halopendol-sensltlVe a receptors in the genetically dvstonlc (dt) rat Eur J Pharmacol 147 (1988) 153-154 2 Grayblel A M , Besson M J and Weber E Neuroleptlcsensitive binding sites in the nlgrostnatal system Evidence lor dffterentlal distribution ot sigma sites in the substantla mgra pars compacta ot the cat J Neurosci, 9 (1989) 326-338 3 Gundlach A k , Largent B L and Snyder, S H , Autoradlographic Iocallsatlon of sigma receptor binding sites in guinea pig and rat central nervous system with (+)?H-3-(3-hydrox'~phen'vl)-N-(1-propvl)-plperldme, J Neuroscl, 6 (19861 17571770 4 Hlorth S Carlsson A Wlkstrom H Lmdberg, P Stanchez D Hacksell U Ar',ldsson L E , Svensson, U and Nllsson J L G 3-PPP a new centrally acting DA-receptor agonlst with selectivity for autoreceptors Life & l , 28 (1981) 1225-1238 5 Hlorth S (lark D and Carlsson, A Lack ol functional evldenct, for the involvement o1 sigma opmte receptors in the acnons of the 3-PPP enannomers on central dopamlnerglc systems Discrepancies between m lttro and in vt~o obser,;atlons L1Je S u 37 (19851 673-684 6 Jansen K L R Dragunow, M and Faull R L M , [~H]glVclne binding sites NMDA and PCP receptors have similar dlStrlbunons in the human hlppocampus an autoradmgraphlc study Brain Research, 482 (19891 174-178 7 Largent, B L Gundlach A L and Snvder, S H Pharmacological and autoradlographlc discrimination ol sigma and phencychdlne receptor binding sites in brain with (+)-[~H]SKF10 047 (+)-[~H]-3-[Zl-h,~droxv phenvl]-N-(1-propyl) plperldlne
J Pharrnacol E,.p T h e r , 238 (1986) 739-748 8 Maestrom G J M Contl A and Plerpaoll, W Role of the pineal gland in immunity Circadian synthesis and release o1 melatonln modulates the antibody response and antagonlses the Immunosuppresslve effect ot cortlcosterone J N e u r o l m m u n o l 13 (1986) 19-~,0 9 Maestronl G J M Contl A and Plerpaoh W , qhe pineal gland and the circadian optaterglc, immunoregulatorv role ol melatonm Anti N I" Acad Sct 496 (19871 67-77 10 McLean S and Weber, E Autoradlographlc VlsUallsatlon of halopendol-sensltlVe sigma receptors m guinea-pig brain, Neuros~teme, 25 (1988) 259-269 11 Redman, J Armstrong S and Ng K T Free-running activity rh'~thms in the rat entrainment b'y melatonm S~_Ience 219 (1983) 1089-1(191 12 Relter R J , The pineal and its hormones in the control of reproduction in mammals Endocrine R m 1 (1980) 1//9-131 13 Sircar R Nmhtenhauser R Sere J R and Zukln S R , Characterlsatlon and autoradlographlc "~lsuallsatlon of (+)[~H]SKF ill 047 binding in rat and mouse brain further evidence for phencvhdlne/sigma opiate receptor commonaht,;, J Pharmacol Exp Ther , 2q7 (1986) 681-688 14 Sonders, M S Keana J F W and Weber E Phencvchdme and psychotomlmeIlC sigma opiates Recent insights into their biochemical and physiological sites of action Trends Neurosct 11 (1988) 37-40 15 Su T London E D and Jafte 1 H Steroid binding at o receptors suggests a link between endocrine nervous and immune systems Science, 240 (1988) 219-230 16 Walker J M Matsumoto R R Bowen W D Gans D C
16() Jones, K D and Walker F O ,Evtdence tor a role o1 halopertdol-sens~tl~e ~ 'optate' receptors m the motor etfects ot anti-psychotic drugs, Neurology, 38 (1988) 061-971 17 Weber, E , Sonders, M Quarum, M , McLean, D Pou, S and Keana, J F W 1 3-Dl(2-[5-~H]tolyl)guamdme a selective hgand that labels s~gma-type receptors for psychotomJmet~c opxates and anti-psychotic drugs Proc Natl A~ad ~,~t U S A 83 (1986) 8784-8788
18 Wolfe S A , Culp S G and De bouza, L B Sigma and phencychdme (PCP) receptors m rat endocrine organs, Sot Neuro~cl Abstr 14 (1988)1176 19 Wolfe, S A , Kulsakdmum, C Battagha, G , Jaffe, J H and De Souza, E B , Imtlal ~dent~ficat~on and characterisat~on of sigma receptors on human peripheral blood leukocytes, J Pharm Exp Ther, 247 (1988) 1114-1119
Bram ICe,earth ~0- ( 19901 158 -1¢~1)
BRES 23907
Sigma receptors are highly concentrated in the rat pineal gland Karl L R Jansen, Michael Dragunow and Richard L M Faull Department of Anatomy, Untversttv of Auckland, School of Medwme, Auckland (New Zealand) (Accepted 26 September 1989) Key words Autoradlographv, 1,3-dl-ortho-Tolyl-guamdlne, Pineal gland, cr Receptor
The dlstnbuuon of sigma (a) receptors m the rat brain was studied with autoradlography using [3H]l,3-dl-ortho-tolyl-guamdme ([3H]DTG) as a hgand The baghest concentration of ~r receptors was seen m the pineal gland, an area which has not been previously studied This result ~s of interest as both a receptors and the pineal gland have recently been shown to play a role not only m the nervous system but also m the ~mmune and endocnne systems Evidence has recently been presented that the sigma receptor may play a role in the nervous system and also the e n d o c n n e and immune systems 15 Halopertdol and some other antlpsychot~c drugs bind to o receptors, as do psychotomimeuc benzomorphan opiates, suggesting that the receptor may be mvolved m psychosis 14 Other studies have hnked the receptor to certain movement dtsorders I 16 Corticostero~ds and sex hormones have been shown to bind to o receptors 15, and these sites are found in high concentrations m the hypothalamic-pltuitary area 1°, m peripheral endocrme glands TM, and also on lymphocytes~5 19 The dtstrlbutlon of these sites m the rodent brain has been previously studted using ( + ) [3H]-3-(3-hydroxyphenyl)-N-(1-propyl)-plpendlne (3-PPP) 3 and [3H]SKF10,0477 as labels However, 3-PPP ts also a d o p a m m e type II receptor agontst 4 5 whtle SKF-10.047 also binds to the PCP site on the N-methyl-D-aspartate ( N M D A ) receptor 13 The more spectfic o-receptor hgand [3H]DTG17 has thus far been apphed to regions of the guinea pig 1° and cat brain 2, but has not been used to visuallse these sites in the rat CNS No report on the pmeal gland was included in these prevtous mvest~gattons 2 3 7 to In the present study, [3H]DTG was used to vlsuallse o receptors in the rat CNS using autoradtographic methods Wlstar rats (200-250 g) were killed by decapitation, their brams removed and lmmedmtely frozen wtth dry ice onto cryostat chucks 16~m sections were cut through the brain, with alternate brams being cut in the coronal plane o r the sagittal plane The sections were then mounted onto chrome/alum subbed slides, stored overnight at 4 °C and labelled according to the method of McLean and
Weber a°, except that secUons were premcubated in Tns-HC1 buffer for 1 h at 21 °C Briefly, following premcubation sections were incubated at 21 °C for 45 mm m 50 mM Trls-HCl (pH 7 4 at 21 °C), with 1 mg/ml bovine serum albumin (b s a ) and 4 nM [3H]DTG (specific actwity 52 3 C1/mmol, New England Nuclear) AdJacent sections were incubated with 10/~M halopendol or 10/~M D T G to assess non-specific binding Sections were then washed for 4 × 2 mln m 10 mM Tns HCI containing 1 mg/ml b s a (pH 7 4, 4 °C), dried, placed in X-ray cassettes w]th A m e r s h a m H y p e f f d m and appropriate standards for 8 weeks ° and subsequently developed and analysed as previously described 6 Sections were stained with Cresyl violet for histological examination and identification of anatomtcal areas In the resulting autoradlograms, the pineal gland was seen to have the highest levels of [3H]DTG binding m the rat brain (see Fig 1) The distribution m the rest of the brain was not markedly different from other studies, and over 95% of the binding was displaced by hatopendol indicating a htgh ratio of specific to non-specific bindmg High levels of binding were seen in the hypothalamtcp~tultary area, but these were less than those m the pineal gland In the hippocampus, [3H]DTG binding occurred over the granule and pyramidal cell layers Relatwely low levels were found in the basal gangha, m agreement w~th previous descriptions of o-receptor d~strlbutlons 3 1o The dense concentration of o-receptors m the pineal gland ~s of particular interest as the gland appears to have functions m the nervous endocrine and immune systems - - the 3 systems in whtch o receptors have been tmphcated The pmeal is, for example, revolved m the regulation of sex hormones ~2 which have been shown to
Correspondence K Jansen, Department of Anatomy, Umverslty of Auckland, School of Medlcme, Private Bag, Auckland, New Zealand 0006-8993/90/$03 50 © 1990 Elsevier Soence Pubhshers B V (Biomedical Division)
159 have
a high
affinity f o r
o r e c e p t o r s ~s
Secretion ot
m e l a t o n i n is r h y t h m i c a n d t h e h o r m o n e is i n v o l v e d m regulating
certain
rhythmic
processes
such
as
r e p r o d u c t i o n ~2 T h e o c c u r r e n c e o f t h e s e r e c e p t o r s in t h e p i n e a l p r o v i d e s f u r t h e r e v i d e n c e to s u p p o r t a p o s s i b l e role f o r t h e s e sites in e n d o c r i n e c o n t r o l
S~gma r e c e p t o r s
h a v e b e e n f o u n d m all rat e n d o c r i n e o r g a n s e x a m i n e d to d a t e ( p i t u i t a r y , a d r e n a l , testis, o v a r y a n d p l a c e n t a ) , with one study finding the highest levels overall to occur m the testis ~s T h e p i n e a l g l a n d can n o w be a d d e d to this hst T h e p i n e a l g l a n d also h a s a r o l e m t h e ~ m m u n e s y s t e m For
example,
melatonm
can
modulate
the
antibody
r e s p o n s e and antagonise the m l m u n o s u p p r e s s i v e effect of cortlcosterones
T h e r e is e v i d e n c e to s u g g e s t t h a t this
e f f e c t m a y b e m e d i a t e d b~ o p i a t e s '~ S i g m a r e c e p t o r s h a v e b e e n identified on l y m p h o c y t e s and may be mvolved m this p r o c e s s ~'
C
In c o n c l u s i o n , this s t u d y r o u n d a high c o n c e n t r a t m n o f o - r e c e p t o r s m t h e p i n e a l g l a n d , an a r e a n o t i n c l u d e d m
F
p r e v i o u s s t u d i e s , u s i n g a u t o r a d i o g r a p h y w~th [ ~ H ] D T G T h i s h n d i n g m a y h a v e c o n s i d e r a b l e s~gmficance b o t h t o r o u r u n d e r s t a n d i n g o t t h e s e r e c e p t o r s a n d ol t h e p i n e a l g l a n d a n d ~ts f u n c t i o n s
Fig 1 Autoradlographlc Vlsuallsanon of [~H]DTG binding to sigma receptors in (a) a coronal section and (c) a saglttal section of the rat brain, illustrating a high denslt~ of sigma receptors in the pineal bod,¢ (see arrows) (b) and (d) are the same sections stained with Cresvl ,dolet Bar = ~0 mm
This work was supported b~ the New Zealand Medical Research Council and the New Zealand Neurological Foundation Dr K Jansen is a training Iellow of the New Zealand Medical Research Council We are grateful to Dr Mark Sonders and Professor Eckard Weber 1or the DTG
1 Bowen, W D Walder, J M Yashar, A G , Matsumoto R R Walder F O and Lorden J F Altered halopendol-sensltlVe a receptors in the genetically dvstonlc (dt) rat Eur J Pharmacol 147 (1988) 153-154 2 Grayblel A M , Besson M J and Weber E Neuroleptlcsensitive binding sites in the nlgrostnatal system Evidence lor dffterentlal distribution ot sigma sites in the substantla mgra pars compacta ot the cat J Neurosci, 9 (1989) 326-338 3 Gundlach A k , Largent B L and Snyder, S H , Autoradlographic Iocallsatlon of sigma receptor binding sites in guinea pig and rat central nervous system with (+)?H-3-(3-hydrox'~phen'vl)-N-(1-propvl)-plperldme, J Neuroscl, 6 (19861 17571770 4 Hlorth S Carlsson A Wlkstrom H Lmdberg, P Stanchez D Hacksell U Ar',ldsson L E , Svensson, U and Nllsson J L G 3-PPP a new centrally acting DA-receptor agonlst with selectivity for autoreceptors Life & l , 28 (1981) 1225-1238 5 Hlorth S (lark D and Carlsson, A Lack ol functional evldenct, for the involvement o1 sigma opmte receptors in the acnons of the 3-PPP enannomers on central dopamlnerglc systems Discrepancies between m lttro and in vt~o obser,;atlons L1Je S u 37 (19851 673-684 6 Jansen K L R Dragunow, M and Faull R L M , [~H]glVclne binding sites NMDA and PCP receptors have similar dlStrlbunons in the human hlppocampus an autoradmgraphlc study Brain Research, 482 (19891 174-178 7 Largent, B L Gundlach A L and Snvder, S H Pharmacological and autoradlographlc discrimination ol sigma and phencychdlne receptor binding sites in brain with (+)-[~H]SKF10 047 (+)-[~H]-3-[Zl-h,~droxv phenvl]-N-(1-propyl) plperldlne
J Pharrnacol E,.p T h e r , 238 (1986) 739-748 8 Maestrom G J M Contl A and Plerpaoll, W Role of the pineal gland in immunity Circadian synthesis and release o1 melatonln modulates the antibody response and antagonlses the Immunosuppresslve effect ot cortlcosterone J N e u r o l m m u n o l 13 (1986) 19-~,0 9 Maestronl G J M Contl A and Plerpaoh W , qhe pineal gland and the circadian optaterglc, immunoregulatorv role ol melatonm Anti N I" Acad Sct 496 (19871 67-77 10 McLean S and Weber, E Autoradlographlc VlsUallsatlon of halopendol-sensltlVe sigma receptors m guinea-pig brain, Neuros~teme, 25 (1988) 259-269 11 Redman, J Armstrong S and Ng K T Free-running activity rh'~thms in the rat entrainment b'y melatonm S~_Ience 219 (1983) 1089-1(191 12 Relter R J , The pineal and its hormones in the control of reproduction in mammals Endocrine R m 1 (1980) 1//9-131 13 Sircar R Nmhtenhauser R Sere J R and Zukln S R , Characterlsatlon and autoradlographlc "~lsuallsatlon of (+)[~H]SKF ill 047 binding in rat and mouse brain further evidence for phencvhdlne/sigma opiate receptor commonaht,;, J Pharmacol Exp Ther , 2q7 (1986) 681-688 14 Sonders, M S Keana J F W and Weber E Phencvchdme and psychotomlmeIlC sigma opiates Recent insights into their biochemical and physiological sites of action Trends Neurosct 11 (1988) 37-40 15 Su T London E D and Jafte 1 H Steroid binding at o receptors suggests a link between endocrine nervous and immune systems Science, 240 (1988) 219-230 16 Walker J M Matsumoto R R Bowen W D Gans D C
16() Jones, K D and Walker F O ,Evtdence tor a role o1 halopertdol-sens~tl~e ~ 'optate' receptors m the motor etfects ot anti-psychotic drugs, Neurology, 38 (1988) 061-971 17 Weber, E , Sonders, M Quarum, M , McLean, D Pou, S and Keana, J F W 1 3-Dl(2-[5-~H]tolyl)guamdme a selective hgand that labels s~gma-type receptors for psychotomJmet~c opxates and anti-psychotic drugs Proc Natl A~ad ~,~t U S A 83 (1986) 8784-8788
18 Wolfe S A , Culp S G and De bouza, L B Sigma and phencychdme (PCP) receptors m rat endocrine organs, Sot Neuro~cl Abstr 14 (1988)1176 19 Wolfe, S A , Kulsakdmum, C Battagha, G , Jaffe, J H and De Souza, E B , Imtlal ~dent~ficat~on and characterisat~on of sigma receptors on human peripheral blood leukocytes, J Pharm Exp Ther, 247 (1988) 1114-1119